Indian Patents
Title of Invention

A LIPOPEPTIDE
Abstract The present invention provides a lipopeptide comprising a polypeptide conjugated to one or more lipid moieties wherein: (i) said polypeptide comprises an amino acid sequence that comprises: (a) the amino acid sequence of a T helper cell (Th) epitope and the amino acid sequence of a B cell epitope, wherein said amino acid sequences are different; and (b) one or more internal lysine residues or internal lysine analog residues for covalent attachment of each of said lipid moieties via the epsilon group or terminal side-chain group of said lysine or lysine analog; and (ii) each of said one or more lipid moieties is covalently attached to an epsilon-amino group of said one or more internal lysine residues or to a terminal side chain group of said one or more internal lysine analog residues.
Full Text A LIPOPEPTIDE
Field of the invention
The present invention relates generally to the field of immunology, and more
particularly to reagents for generating antibody and/or cellular responses to a
peptide immunogen, and methods for using said reagents for enhancing the
immune response of a subject, or for the vaccination of a subject. Even more
specifically, the present invention relates to novel lipopeptides having
enhanced immunogenic activity, formulations and vaccine compositions
comprising said lipopeptides, such as, for example, in combination with a
pharmaceutically acceptable carrier or excipient,, and to methods for making
and using the formulations and vaccine compositions of the invention.
Background to the invention
1. General
This specification contains amino acid sequence information prepared using
Patentln Version 3.1, presented herein after the Abstract. Each sequence is
identified in the sequence listing by the numeric indicator followed by the
sequence identifier (e.g. 1, 2, etc). The length of each sequence
and source organism are indicated by information provided in the numeric
indicator fields and , respectively. Sequences referred to in the
specification are defined by the term "SEQ ID NO:", followed by the sequence
identifier (eg. SEQ ID NO: 1 refers to the sequence designated as 1).
As used herein the term "derived from" shall be taken to indicate that a
specified integer may be obtained from a particular source albeit not
necessarily directly from that source.

Throughout this specification, unless the context requires otherwise, the word
"comprise", or variations such as "comprises" or "comprising", will be
understood to imply the inclusion of a stated step or element or integer or group
of steps or elements or integers but not the exclusion of any other step or
element or integer or group of elements or integers.
Those skilled in the art will appreciate that the invention described herein is
susceptible to variations and modifications other than those specifically
described. It is to be understood that the invention includes all such variations
and modifications. The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this specification,
individually or collectively, and any and all combinations or any two or more of
said steps or features.
The present invention is not to be limited in scope by the specific examples
described herein. Functionally-equivalent products, compositions and methods
are clearly within the scope of the invention, as described herein.
All the references cited in this application are specifically incorporated by
reference herein.
The present invention is performed without undue experimentation using,
unless otherwise indicated, conventional techniques of molecular biology,
microbiology, virology, recombinant DNA technology, peptide synthesis in
solution, solid phase peptide synthesis, and immunology. Such procedures
are described, for example, in the following texts that are incorporated by
reference:
1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratories, New York, Second Edition (1989),
whole of Vols l, Il, and III;

2. DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed.,
1985), IRL Press, Oxford, whole of text;
3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984)
IRL Press, Oxford, whole of text, and particularly the papers therein by
Gait, pp1-22; Atkinson et al., pp35-81; Sproat et a/., pp 83-115; and Wu
et al.,pp 135-151;
4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J.
Higgins, eds., 1985) IRL Press, Oxford, whole of text;
5. Animal Cell Culture: Practical Approach, Third Edition (John R.W.
Masters, ed., 2000), ISBN 0199637970, whole of text;

6. Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press,
Oxford, whole of text;
7. Perbal, B., A Practical Guide to Molecular Cloning (1984);
8. Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic
Press, Inc.), whole of series;
9. J.F. Ramalho Ortigao, "The Chemistry of Peptide Synthesis" In:
Knowledge database of Access to Virtual Laboratory website
(Interactiva, Germany);
10. Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R.L. (1976).
Biochem. Blophys. Res. Commun. 73 336-342
11. Merrifield, R.B. (1963). J. Am. Chem. Soc. 85, 2149-2154.

12. Barany, G. and Merrifield, R.B. (1979) in The Peptides (Gross, E. and
Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York.
13. Wünsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls
Methoden der Organischen Chemie (Muler, E., ed.), vol. 15, 4th edn.,
Parts 1 and 2, Thieme, Stuttgart.
14. Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Veriag,
Heidelberg.
15. Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide
Synthesis, Springer-Veriag, Heidelberg.

16. Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474.
17. Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C.
Blackwell, eds., 1986, Blackwell Scientific Publications).
Description of the related art
Immunotherapy or vaccination are attractive for the prophylaxis or therapy of a
wide range of disorders, such as, for example, certain infectious diseases, or
cancers. However, the application and success of such treatments are limited
in part by the poor immunogenicity of the target antigen. Many peptides,
glycopeptides, lipids, lipopeptides, carbohydrates etc., are poorly immunogenic.
Several techniques are used to enhance the immune response of a subject to a
peptide immunogen.
It is known to utilize an adjuvant formulation that is extrinsic to the peptide
immunogen (i.e. it is mixed with the immunogen prior to use), such as, for
example, complete Freund's adjuvant (CFA), to enhance the immune response
of a subject to a peptide immunogen. However, many of the adjuvants
currently available are too toxic for use in humans, or simply ineffective.
Moreover, adjuvants of this type require prior formulation with the peptide
immunogen immediately before administration, such formulations often having
a tow solubility or being insoluble.
Lipopeptides, wherein a lipid moiety that is known to act as an adjuvant Is
covalently coupled to a peptide immunogen, may be capable of enhancing the
immunogenicity of an otherwise weakly immunogenic peptide in the absence of
an extrinsic adjuvant [Jung et a/., Angew Chem, Int Ed Engl 10, 872, (1985);
Martinon et a/., J Immunol 149, 3416, (1992); Toyokuni et a/., J Am Chem Soc
116, 395, (1994); Deprez, ef a/., J Med Chem 38, 459, (1995); and Sauzet et
a/., Vaccine 13, 1339, (1995); Benmohamed et al., Eur. J. Immunol. 27, 1242,
(1997); Wiesmuller et a/., Vaccine 7, 29, (1989); Nardln et a/., Vaccine 16, 590,

(1998); Benmohamed, et al. Vaccine 18, 2843, (2000); and Obert, et al.,
Vaccine 16, 161, (1998)]. Suitable lipopeptides show none of the harmful side
effects associated with adjuvant formulations, and both antibody and cellular
responses have been observed against lipopeptides.
Several different fatty acids are known for use in lipid moieties. Exemplary fatty
acids include, but are not limited to, palmitoyl, myristoyl, stearoyl and decanoyl
groups or, more generally, any C2 to C30 saturated, monounsaturated, or
polyunsaturated fatty acyl group is thought to be useful.
The lipoamino acid N-palmitoyl-S-[2,3-6/s(palmitoyloxy)propyl]cysteine, also
known as Pam3Cys or Pam3Cys-OH (Wiesmuller et al., Z. Physiol.Chem. 364
(1983), p593), is a synthetic version of the N-terminal moiety of Braun's
lipoprotein that spans the inner and outer membranes of Gram negative
bacteria. Pam3Cys has the structure of Formula (I):

United States Patent No. 5, 700, 910 to Metzger et al (December 23, 1997)
describes several N-acyl-S-(2-hydroxyalkyl)cysteines for use as intermediates
in the preparation of lipopeptides that are used as synthetic adjuvants, B
lymphocyte stimulants, macrophage stimulants, or synthetic vaccines. Metzger
et al. also teach the use of such compounds as intermediates in the synthesis

of Pam3Cys-OH (Wiesmuller ef a/., Z. Physiol.Chem. 364, p593, 1983), and of
lipopeptides that comprise this lipoamino acid or an analog thereof at the N-
termlnus.
Par3Cys has been shown to be capable of stimulating virus-specific cytotoxic
T lymphocyte (CTL) responses against influenza virus-infected cells (Deres ef
a/., Nature 342, 561, 1989) and to elicit protective antibodies against foot-and-
mouth disease (Wiesmuller ef a/., Vaccine 7, 29, 1989; United States Patent
No. 6,024.964 to Jung ef a/., February 15, 2000) when coupled to the
appropriate epitopes.
Recently, Pam2Cys (also known as dipalmitoyl-S-glyceryl-cysteine or S-{2.3-
bis(palmitoyloxy)propyl}cysteine, an analogue of Pam3Cys, has been
synthesised (Metzger, J. W., A. G. Beck-Sickinger, M. Loleit, M. Eckert, W. G.
Bessler, and G. Jung. 1995. J Pept Sci 1:184.) and been shown to correspond
to the lipid moiety of MALP-2, a macrophage-activating lipopeptide isolated
from mycoplasma (Sacht, G., A. Marten, U. Deiters, R. Sussmuth, G. Jung, E.
Wingender. and P. F. Muhlradt. 1998. Eur J Immunol 28:4207: Muhlradt, P. F..
M. Kiess, H. Meyer, R. Sussmuth. and G. Jung. 1998. Infect Immun 66:4804:
Muhlradt. P. F.. M. Kiess, H. Meyer, R. Sussmuth, and G. Jung. 1997. J Exp
Med 185:1951). Pam2Cys has the structure of Formula (II):


Pam2Cys is reported to be a more potent stimulator of splenocytes and
macrophages than Pam3Cys (Metzger et al., J Pept. Set 1,184,1995; Muhlradt
et al., J Exp Med 185,1951,1997; and Muhlradt et a/., Infect Immun 66, 4804.
1998).
Generation of an antibody response against a given antigen requires the
generation of a strong T helper cell response. Accordingly, it is desirable to
administer an antigen in conjunction with at least one T-helper cell epitope
(Vitiello et a/., J. Clin. Invest. 95, 341-349, 1995; Livingston et al., J. Immunol.
159, 1383-1392, 1997). However, because T helper cell responses are
provided by CD4* T-cells that recognize fragments of peptide antigens in
context of MHC class II molecules on the surface of antigen presenting cells
(APCs), most of the processed forms of peptide antigens are only presented by
one or a few alleles of MHC haplotypes. This causes the T helper response to
a given antigenic peptide to be strictly under genetic control of an individual.
To avoid large genetic variation in the immune responses of a given population
of individuals to an antigen, an antigen is administered in conjunction with a
large protein having a range of T helper epitopes.
Alternatively, promiscuous or permissive T-helper epitope-containing peptides
are administered in conjunction with the antigen. Promiscuous or permissive T-
helper epitope-containing peptides are presented in the context of a vast
majority of MHC class II haplotypes, such that they Induce strong CD4* T
helper responses in the majority of an outbred human population. Examples of
promiscuous or permissive T-helper epitopes are tetanus toxoid peptide,
Plasmodium falcipamm pfg27, lactate dehydrogenase, and HIVgp120
(Contreas et al., Infect Immun, 66, 3579-3590, 1998; Gaudebout et a/., J.
A.I.D.S. Human Retrovirol 14, 91-101, 1997; Kaumaya et al, J. Mol. Recog. 6,
81-94, 1993; and Fern and Good J. Immunol. 148, 907-913, 1992). Ghosh ef

a/., Immunol 104, 5&-66, 2001 and International Patent Application No.
PCT/AUOO/00070 (WO 00/46390) also describe T-helper epitopes from the
fusion protein of Canine Distemper Virus (CDV-F). Certain promiscuous T-
helper epitopes induce strong B cell responses to a given antigen, and can
bypass certain haplotype restricted immune responses (Kaumaya et a/., J. Mol.
Recog. 6, 81-94,1993).
Routinely, a vaccine preparation will comprise a mixture of polypeptides
comprising the T-helper cell epitope and antigenic epitope, however it is also
known to administer a single polypeptide comprising both the T-helper epitope
and the antigenic epitope (eg. Ghosh and Jackson, Int. Immunol. 11, 1103,
1999).
Summary of the Invention
In work leading up to the present invention, the inventors sought to produce
highly immunogenic lipopeptides having a lipid moiety and a polypeptide moiety
comprising both a T helper epitope and an antigenic B cell epitope against
which an immune response is desired. The lipopeptides of the invention have
the lipid moiety attached via the terminal side chain amino group of an internal
lysine, or an internal lysine analog such as, for example, ornithine,
diaminopropionic acid, or diaminobutyric acid, in the polypeptide moiety. This
is distinct from the N-terminal attachments, or C-terminal attachments (Grass-
Masse etal. Vaccine, 14,375,1996), described previously.
Accordingly, by positioning said one or more lysine residues) or lysine analog
residue(s) at predetermined locations within the polypeptide during peptide
synthesis, the attachment site of the lipid is readily specified. Thus, the
positioning of the lipid moiety in the lipopeptide Is targeted to enhance the utility
of the end-product for vaccine or adjuvant formulations.

Surprisingly, the inventors have found that attachment of the lipid moiety via the
side-chain epsilon-amino group of an internal lysine residue or the terminal
side-chain group of an internal lysine analog residue positioned between the
amino acid sequences of the T helper epitope and the antigen, enhances the
solubility of the lipopeptide product in many cases.
One advantage provided by the lipopetides of the present invention is that they
are sufficiently immunogenic such that it is generally not necessary to include
an extrinsic adjuvant in vaccine formulations comprising these lipopeptides.
The present invention clearly encompasses the attachment of a lipid moiety via
the epsilon-amino group of an internal lysine residue or the terminal side-chain
group of an internal lysine analog residue present in the amino acid sequence
of the T helper epitope or the antigen, the only requirement being that the lipid
moiety is not attached to the N-terminus or the C-terminus of the peptide. As
exemplified herein, the inventors have clearly shown that, for example, the lipid
may be attached to the epsilon amino group of an internal lysine residue within
the T-helper epitope without loss of the ability of the subject lipopeptides in
generating an immune response, compared to a lipopeptide wherein the lipid is
added to the epsilon amino group of a lysine positioned between the T-helper
epitope and the B-cell epitope.
By "internal" means at a location other than the N-terminus or the C-terminus of
a polypeptide comprising a T helper epitope and antigenic B cell epitope.
Preferably, the lipid moiety is attached to the peptide moiety via the epsilon
amino group of a lysine residue or the terminal side-chain group of an internal
lysine analog residue positioned between the amino acid sequences of the T
helper epitope and the antigenic B cell epitope

As will be known to the skilled person, solubility of an antigen is highly
desirable for producing vaccine formulations on a commercial basis. In this
respect, the inventors have found that the most effective lipopeptides of the
invention are highly soluble. The relative ability of the lipopeptides of the
invention to induce an antibody response in the absence of external adjuvant
was reflected by their ability to upregulate the surface expression of MHC class
II molecules on immature dendritic cells (DC).
As exemplified herein, the structure of the lipid moiety is not essential to activity
of the resulting lipopeptide, as lipid moieties comprising palmitic acid, lauric
acid, stearic acid or octanoic acid can be used without loss of immunogenicity.
Accordingly, the present invention is not to be limited by the structure of the
lipid moiety, unless specified otherwise, or the context requires otherwise.
Similarly, the addition of multiple lipid moieties to the peptide moiety, although
generally not required, is also encompassed by the invention, unless specified
otherwise or the context requires otherwise. As exemplified herein, the addition
of multiple lipid moieties to the peptide moiety, such as, for example, to a
position within the T-heiper epitope, and to a position between the T-helper
epitope and the B-cell epitope, does not adversely affect the ability of the
lipopeptide to stimulate IgG production compared to a peptide having only a
single lipid moiety attached.
It will be apparent from the preceding that the polypeptide is synthesized
conveniently as a single amino acid chain, thereby requiring no post-synthesis
modification to incorporate both epitopes.
Optionally, an amino acid spacer is added at either side of the internal lysine or
lysine analog to which the lipid moiety is to be attached, such as, for example,
between the T-helper and B-cell epitopes.

As exemplified herein, the present inventors produced the lipopeptide of the
invention by coupling the lipid moiety to an exposed epsilon-amino group of an
internal lysine residue positioned between the T-helper and B-cell epitopes in
the synthetic peptide moiety, with or without a spacer. Particularly preferred
spacers in this context consist of serine dimers, trimers, teramers, etc.
A spacer of any conventional type can also be added between the lipid moiety
and the polypeptide moiety. Particularly preferred spacers in this context
consist of arginine or serine dimers, trimers, teramers, etc. Alternatively, a 6-
aminohexanoic acid spacer can be used.
Alternative spacers are also contemplated. For example, a spacer may be
added to the exposed epsilon amino group of an internal lysine or to the
terminal side-chain group of an internal lysine analog before addition of the lipid
moiety.
Alternatively, a lipoamino acid of Formula (III) or (IV) may be added directly to
the epsilon amino group of the internal lysine residue or to the terminal side-
chain group of the internal lysine analog.
Also exemplified herein, the lipopeptide of the present invention induces the
production of a high titer antibody against the B cell epitope moiety when
administered to an animal subject, without any requirement for an adjuvant to
achieve a similar antibody titer. This utility is supported by the enhanced
maturation of dendritic cells following administration of the subject lipopeptides
(i.e. enhanced antigen presentation compared to lipopeptides having N-
terminally coupled lipid).

Also exemplified herein, a lipopeptide of the present invention comprising an
antigenic B cell epitope of LHRH is capable of inducing infertility in a mouse
model representative of other mammals in which infertility is to be induced. The
sustained production of antibodies against LHRH achieved by the lipopeptides
of the invention demonstrates the general utility of the subject lipopeptides in
inducing humoral immunity and as an active agent in a vaccine preparation.
Also exemplified herein, a lipopeptide of the present invention comprising an
antigenic B cell epitope of the M protein of Group A Streptococcus (herein
"GAS") is capable of inducing protection in a mouse model representative of
humans and other mammals in which vaccination against GAS is indicated.
The data provided herein indicate that the lipopeptides of the present invention
are capable of inducing a sustained production of antibodies against GAS (both
serum IgG, and salivary and faecal IgA), and the opsonization of GAS, and the
survival of animals against a subsequent GAS challenge. These data
demonstrate the general utility of the subject lipopeptides in inducing humoral
immunity and as an active agent in a vaccine preparation against GAS.
Also exemplified herein, a lipopeptide of the present invention comprising an
antigenic B cell epitope of gastrin ("pentagastrin") is capable of inducing the
sustained production of antibodies against gastrin and/or cholecystekinin in a
mouse model of other mammals in which inhibition of gastric acid secretion is
indicated. The data provided herein demonstrate the general utility of the
subject lipopeptides in inducing humoral immunity against gastrin and
immunoneutralization of gastrin, to thereby block secretion of gastric acid, in an
animal suffering from hypergastrinemia, Zollinger-Ellison syndrome, gastric
ulceration or duodenal ulceration due to excessive and unregulated secretion of
gastric acid, or to reduce or prevent the formation of gastrin-dependent tumours
in the pancreas or duodenum (i.e. the prophylaxis and/or therapy of
gastrinoma).

As will be clear to those skilled in the art, the nature of the T-helper and B cell
epitopes is not critical in the context of the present invention. The novel
approach of attaching the lipid moiety to the epsilon amino group of one or
more internal lysine residues or lysine analogue residues within the polypeptide
portion of the construct has broad application. Accordingly, based on the
results presented herein, it will be understood that a wide range of T-helper and
B cell epitopes can be used in the lipopeptide constructs.
In fact, the broad range of applications exemplified herein indicate the
generality of the lipopeptides of the present invention in the prophylaxis and
therapy of a number of different conditions in humans and other mammals in
which the generation of an immune response against an antigenic B cell
epitope is indicated. Accordingly, the present invention is not to be limited to
the treatment of any specific condition, ailment or disease state.
accompanying
Brief description of the accompanying drawings:
Figure 1 is a representation of the structures of synthetic peptides and
lipopeptides (left) and the relative solubilities of a sample of those peptides and
lipopeptides in saline solution (right). Peptides were designated as follows:
(i) [Th] consisting of a CD4* T-helper epitope from the light chain of
influenza virus haemagglutinin (SEQ ID NO: 1) or peptide P25 from
CDV-F (SEQ ID NO: 24);
(ii) [B] consisting of a B cell epitope consisting of residues 1-10 of LHRH
(SEQ ID NO: 2) or residues 2-10 of LHRH (SEQ ID NO: 3) or residues 6-
10 of LHRH (SEQ ID NO: 4), a B cell epitope of the M protein of Group A
Streptococcus ("peptide J14"; SEQ ID NO: 101); or a B cell epitope of

gastrin contained within the C-terminai 5 residues of gastrin (i.e.,
"pentagastrin"; SEQ ID NO: 102);
(Hi) [Th]-[B] consisting of a polypeptide having (i) and (ii) (e.g., SEQ ID NOs:
5,103,104,105,107,109 or 111); and
(iv) [Th]-Lys-[B] consisting of a polypeptide having (i) and (ii) separated by a
lysine residue (e.g., SEQ ID NOs: 7, 9,13,106,108,110, or 112).
Lipopeptides were designated as follows:
(i) Pam3Cys-[Th-[B] consisting of a lipid of the Formula (I) conjugated to
the N-terminus of peptide |Th]-[B] supra (i.e. to the N-terminus of, for
example, any one of SEQ ID NOs: 5,103,104,105, 107,109 or 111);
(ii) Pam3Cys-Ser-Ser-[Th]-[B] consisting of a lipoamino acid of the Formula
(III) conjugated to the N-terminus of peptide [Th]-[B] supra (i.e. to the N-
terminus of, for example, any one of SEQ ID NOs: 5, 103,104, 105, 107,
109 or 111);
(iii) Pam2Cys-[Th]-|B] consisting of a lipid of the Formula (II) conjugated to
the N-terminus of peptide [Th]-[B] supra (i.e. to the N-terminus of, for
example, any one of SEQ ID NOs: 5,103,104,105,107,109 or 111);
(iv) Pam2Cys-Ser-Ser-[Th]-[B] consisting of a lipid of the Formula (IV)
conjugated to the N-terminus of peptide [Th]-[B] supra (i.e. to the N-
terminus of, for example, any one of SEQ ID NOs: 5,103,104,105,107,
109 or 111);
(v)[Th]-Lys(Pam3Cys)-[B] consisting of peptide [Th]-Lys-[B] (e.g., any one
of SEQ ID NOs: 7, 9, 13, 106, 108, 110, or 112) and a lipid of the
Formula (I) conjugated to the epsilon-amino group of the internal lysine
(Lys) of said peptide;

(vi) [Th]-Lys(Pam2Cys)-[B] consisting of peptide [Th]-Lys-[B] (e.g., any one
of SEQ ID NOs: 7, 9, 13, 106, 108, 110, or 112) and a lipid of the
Formula (II) conjugated to the epsilon-amino group of the internal lysine
(Lys) of said peptide; and
(vii) [Th]-Lys(Pam2Cys-Ser-Ser)-[B] consisting of peptide [Th]-Lys-[B] (e.g.,
any one of SEQ ID NOs: 7, 9, 13, 106, 108, 110, or 112) conjugated
serially via the epsilon amino group of the internal lysine (Lys) to a
serine homodimer (i.e. Ser-Ser) and then a lipid of the Formula (II).
Thus, to produce this branched lipopeptide, the two serine residues were
added to the epsilon-amino group of the lysine residue before the lipid
moiety was attached.
Relative solubility of the peptides and lipopeptides based upon the influenza
virus haemagglutinin T-helper epitope (SEQ ID NO: 1) and the LHRH 1-10 B-
cell epitope (SEQ ID NO: 2) is indicated at the right of the figure, ranging from
low solubility (-) to high solubility (++++).
Figure 2 is a photographic representation showing the solubilities of
lipopeptides designated [Th]-Lys(Pam2Cys-Ser-Ser)-[B] (left) and Pam2Cys-
Ser-Ser-[Th]-[B] (right) in Figure 1, wherein the polypeptide moieties have the
amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 5,
respectively. Both solutions are approximately 1 mg/ml lipopeptide in saline
solution. The enhanced clarity of the solution comprising lipopeptide [Th]-
Lys(Pam2Cys-Ser-Ser)-[B] is indicative of its higher solubility compared to
lipopeptide Pam2Cys-Ser-Ser-|Th]-[B].
Figure 3 is a graphical representation showing the anti-LHRH antibody titers
obtained using each of the peptides and lipopeptides shown in Figure 1,
wherein the polypeptide moieties have the amino acid sequences set forth in

SEQ ID NO: 5 or SEQ ID NO: 7. A negative control lipopeptide designated
Pam3Cys-Ser-Lys4 consisted of the lipid of Formula (I) conjugated to the N-
terminus of a peptide having the amino acid sequence Ser-Lys-Lys-Lys-Lys
(SEQ ID NO: 17). All peptides and lipopeptides were administered sub-
cutaneously (s.c.) in saline for both primary inoculation (open circles) and
secondary inoculations (closed circles). The two non-lipidated peptides [Th]-
Lys-[B] and [Th]-[B] were administered in complete Freund's adjuvant (CFA) for
the primary inoculations, and in incomplete Freund's adjuvant (IFA) for the
secondary inoculations. For administration of the peptide [Th]-[B] in
combination with the lipopeptide Pam3Cys-S-Lys4, peptide was dissolved in
saline and mixed with the lipopeptide in 1:1 or 1:5 molar ratio as indicated. The
dose of peptide and lipopeptide immunogens administered was 20 nmole. In all
cases, control groups of animals received saline emulsified in CFA for priming
and saline emulsified in IFA for the secondary inoculation.
Figure 4 is a graphical representation showing anti-LHRH antibody titers (log10)
on the ordinate for each anti-LHRH antibody isotype (i.e. IgM, IgA, lgG1,
lgG2a, lgG2b, lgG3, and total Ig) (abscissa) obtained or elicited during
secondary antibody responses following inoculation with the lipopeptide [Th]-
Lys(Pam2Cys-Ser-Ser)-[B] (SEQ ID NO: 7). Mice were bled 2 weeks after
receiving the second dose of the lipopeptide vaccine administered in saline
either subcutaneously (open squares) or intranasally (closed squares) in saline.
Figure 5 is a graphical representation showing the relative abilities of peptides
and lipopeptides shown in Figure 1 (i.e. SEQ ID NO: 5 or SEQ ID NO: 7) to
enhance the expression of MHC class II molecules on the surface of dendritic
cells. Peptides and lipopeptides are indicated in each panel according to the
nomenclature of Figure 1. For each peptide or lipopeptide , 8x104 D1 cells
were exposed to 4.5 fmole of peptide or lipopeptide and incubated overnight.

The cells were collected and the MHC class II molecules expression was
determined by flow cytometry after staining with FITC-conjugated anti-l-Ek,d
monoclonal antibody. About 3x104 D1 cells were analyzed for each sample.
Data shown are for a representative of four independent experiments, and
indicate enhanced staining with monoclonal antibody (i.e. enhanced D1 cell
maturation) following administration of lipopeptides, particularly lipopeptide
[Th]-Lys(Pam2Cys-Ser-Ser)-[B] which induced a D1 maturation rate
approaching the level observed for D1 cells challenged with lipopolysaccharide
(LPS). Data obtained using the non-lipidated peptide [Th]-Lys-[B] are
substantially the same as for D1 cells incubated in medium without any added
peptide, lipopeptide or LPS, indicating a spontaneous maturation rate of about
26%.
Figure 6 is a graphical representation showing anti-LHRH antibody responses
elicited by lipidated (Th]-Lys(Pam2Cys)-[B] in which [Th] consists of CD4+ T cell
epitope from the light chain of influenza haemagglutinin (SEQ ID NO: 1) and [B]
is LHRH 1-10 (SEQ ID NO: 2) or LHRH 6-10 (i.e. the C-terminal 5 residues of
LHRH; SEQ ID NO: 4), with or without a serine spacer (Ser-Ser) positioned
between the lipid and peptide moieties. Lipopeptide [Th]-Lys(Pam2Cys)-
GlyLeuArgProGly is structurally similar to [Th]-Lys(Pam2Cys)-[B], however this
lipopeptide comprises SEQ ID NO: 4 in place of SEQ ID NO: 2.
Figure 7 is a representation showing structural data, HPLC and mass spectra
data for different lipopeptide constructs based on the T helper epitope P25
(SEQ ID NO: 24) and LHRH 2-10 (SEQ ID NO: 3), wherein the peptide moiety
has the amino acid sequence set forth in SEQ ID NO: 9 and the lipid moiety is
selected from the group consisting of: (i) Pam2Cys; (ii) Ste2Cys; (III) Lau2Cys;
and (iv) Oct2Cys. Different spacers were also positioned between the lipid

moiety and the peptide moiety, as follows: (i) Ser-Ser, two serine residues; (ii)
Arg-Arg, two arginine residues; and (iii) Ahx, 6-aminohexanoic acid. Structures
of the lipopeptides are indicated in the left column; HPLC chromatograms for
each lipopeptide are indicated in the middle column: and mass spectra are
shown in the right column of the figure.
Figure 8 is a graphical representation showing the immunogenicity of those
lipopeptides indicated in the legend to Figure 7 having a Ser-Ser spacer
between the peptide and the lipid moiety and wherein the lipid moiety is
selected from the group consisting of: (i) Pam2Cys; (ii) Ste2Cys; (iii) Lau2Cys;
and (iv) Oct2Cys. Groups of BALB/c mice (6-8 weeks old) were inoculated
subcutaneously with 20 nmoles of peptide immunogens for both primary and
secondary vaccinations. All lipopeptides were administered in saline. The non
lipidated peptide [Th]-Lys-[B] was administered in CFA as a control. Sera were
obtained from blood taken at 4 weeks following the primary vaccination (open
circles) and 2 weeks following the secondary vaccination (closed circles).
Figure 9 is a graphical representation showing immunogenicity of lipopeptide
immunogens from Figure 7 having different spacers positioned between the
lipid and peptide moieties, in particular spacers consisting of serine
homodimers (Ser-Ser), arginine homodimers (Arg-Arg), or 6-aminohexanoic
acid (Ahx). Groups of BALB/c mice (6-8 weeks old) were inoculated
subcutaneously with 20 nmoles of peptide immunogens for both primary and
secondary vaccinations. All lipopeptides were administered in saline. The non
lipidated peptide [Th]-Lys-[B] was administered in CFA as a control. Sera were
obtained from blood taken at 4 weeks following the primary vaccination (open
circles) and 2 weeks following the secondary vaccination (closed circles).

Figure 10 is a graphical representation showing quality control data for a
lipopeptide construct [Th](Pam2Cys-Ser-Ser)-[B] in which the lipid moiety is
pendant from the epsilon-amino group of an internal lysine residue (Lys-14)
within the helper T cell epitope of the peptide set forth in SEQ ID NO: 103. The
structures of the lipopeptide is indicated in the left column; an HPLC
chromatogram for the lipopeptide is indicated in the middle column; and mass
spectra data are shown in the right column of the figure.
Figure 11 is a graphical representation showing immunogenicity of the
lipopeptide immunogen described in the legend to Figure 10, compared to a
lipopeptide immunogen having the lipid moiety added to an internal lysine
residue positioned between the T-helper epitope and the B-cell epitope (i.e.,
the lipid moiety is added to the amino acid sequence set forth in SEQ ID NO: 9,
which differs from SEQ ID NO: 103 in having an internal lysine added between
the T-helper and B-cell epitopes). A control non-lipidated peptide having the
amino acid sequence set forth in SEQ ID NO: 9 (i.e., [Th]-Lys-[B]) was also
used as a control. Groups of BALB/c mice (6-8 weeks old) were inoculated
subcutaneously with 20 nmoles of peptide immunogens for both primary and
secondary vaccinations. All lipopeptides were administered in saline. The non
lipidated control peptide [Th]-Lys-[B] was administered in CFA. Sera were
obtained from blood taken at 4 weeks following the primary vaccination (open
circles) and 2 weeks following the secondary vaccination (closed circles). The
lipopeptide construct [Th](Pam2Cys-Ser-Ser)-[B] has the lipid moiety attached
to the epsilon-amino group of a lysine residue (Lys-14) within the helper T cell
epitope. The lipopeptide construct [Th]-Lys(Pam2Cys-Ser-Ser)-[B] has the lipid
attached to the epsilon-amino group of a lysine residue placed between the two
peptide epitopes.

Figure 12 is a graphical representation showing the ability of a lipopeptide
comprising the T-helper epitope P25 (SEQ ID NO: 24) and a Group A
Streptococcus B cell epitope ("J14"; SEQ ID NO: 101) and having the amino
acid sequence of SEQ ID NO: 106, and one or two lipid moieties to elicit serum
IgG in mice. The lipoamino acid moiety Pam2Cys-Ser-Ser was added to an
internal lysine positioned between the T-helper epitope and the B-cell epitope
in all lipopeptides tested. In the Hpopeptide [Th]-Lys(Pam2Cys-Ser-Ser)-[J14],
this is the only lipid moiety, whereas in the Hpopeptide Pam2Cys-Ser-Ser-|Th]-
Lys(Pam2Cys-Ser-Ser)-[J14], an additional lipoamino acid moiety Pam2Cys-
Ser-Ser was added to the N-terminal amino group of the T-helper epitope.
Other immunogens were as follows: J14, non-lipidated peptide consisting of the
J14 B-cell epitope-containing peptide (SEQ ID NO: 101); [Th]-[J14], a non-
lipidated peptide consisting of the T-helper epitope (SEQ ID NO: 24) and the
J14 peptide (SEQ ID NO: 101) and having the amino acid sequence of SEQ ID
NO: 106; a lipidated peptide consisting of the T-helper epitope (SEQ ID NO:
24) and the LHRH B-cell epitope-containing peptide (SEQ ID NO: 3) and
having the amino acid sequence of SEQ ID NO: 9; and phosphate-buffered
saline (PBS). Female outbred Quackenbush mice 4-6 weeks old (15/group)
were inoculated intranasally with 60ug of peptide-based vaccine in a total
volume of 30µl PBS. Mice received three doses of vaccine at 21-day intervals.
Seven days following the final dose mice were bled from the tail vein and J14-
specific serum IgG was determined. Mice that received either J14-containing
lipopeptides had significantly higher (P control groups.
Figure 13 is a graphical representation showing the opsonisation capability of
antisera elicited by the non-lipidated peptides and lipopeptides indicated in the

legend to Figure 12. Female outbred Quackenbush mice 4-6 weeks old
(15/group) were inoculated intranasally with 60ug of peptide-based vaccine in a
total volume of 30ul PBS. Mice received three doses of vaccine at 21-day
intervals. Indirect bacteriacidal assays were performed to determine the ability
of sera from immunized mice to opsonise or "kill" the M1 GAS strain in vitro.
Sera collected from mice immunized with either J14-containing lipopeptides
were capable of significant (P from animals immunized with control peptides or lipopeptides or PBS.
Figure 14 is a graphical representation showing the ability of the non-lipidated
peptides and lipopeptides indicated in the legend to Figure 12 to elicit salivary
IgA in mice. Female outbred Quackenbush mice 4-6 weeks old (15/group) were
inoculated intranasally with 60ug of each peptide-based vaccine in a total
volume of 30µl PBS. Mice received three doses of vaccine at 21-day intervals.
Eight days following the final dose saliva was collected from individual mice
and the average J14-specific salivary IgA antibody titres were determined by
standard ELISA. The mice inoculated with either J14-containing lipopeptides
had significantly (P immunized with control peptides or control lipopeptides or PBS.
Figure 15 is a graphical representation showing the ability of the non-lipidated
J14-containing peptides and J14-containing lipopeptides indicated in the
legend to Figure 12 to elicit fecal IgA in mice. Female outbred Quackenbush
mice 4-6 weeks old (15/group) were inoculated intranasally with 60ug of
peptide-based vaccine in a total volume of 30µl PBS. Mice received three
doses of vaccine at 21-day intervals. Fecal IgA was determined 6 days
following the last dose of antigen. Only mice inoculated with mono-lipidated
J14-containing peptide, wherein the lipid moiety was positioned between the T-

helper epitope and the B-cell epitope (i.e., [Th]-Lys(Pam2Cys-Ser-Ser)-[J14])
had significant (P Figure 16 is a graphical representation showing the ability of mice to survive
challenge with bacteria following inoculation with the non-lipidated peptides and
lipopeptides indicated in the legend to Figure 12. Two weeks after the last
dose of antigen, mice were challenged intranasally with M1 GAS strain and
survival determined at various time points afterwards. Mice inoculated with
mono-lipidated J14-containing peptide, wherein the lipid moiety was positioned
between the T-helper epitope and the B-cell epitope (i.e., [Th]-Lys(Pam2Cys-
Ser-Ser)-[J14]) demonstrated the best survival following challenge.
Figure 17 is a graphical representation showing the immunogenicity of
lipopeptide immunogens based on gastrin. Groups (5 animals/group) of BALB/c
mice (6-8 weeks of age) were inoculated subcutaneously in the base of tail with
20nmoles of peptide immunogens. The peptides used were Gastrin-17 (SEQ
ID NO: 113); [P25]-Lys-[PentaGastrin] (SEQ ID NO: 110) in which PentaGastrin
is the C-terrninal sequence GWMDF of gastrin as set forth in (SEQ ID NO:
102); and [P25]-Lys(Pam2Cys-Ser-Ser)-[PentaGastrin] (SEQ ID NO: 110 with
lipid added to an internal lysine residue). All lipopeptides were administered in
PBS and the non-lipidated peptides were administered in CFA. The negative
control was saline emulsified with CFA. Sera were obtained from animals 4
weeks after immunisation and at the same time the animals received a second
similar dose of antigen. Mice were bled a second time 2 weeks after receiving
the second dose of antigen and antibodies capable of reacting with the peptide
gastrin-17 sequence detected by ELISA. The results are expressed as the titre
of anti-gastrin-17 antibodies.

Detailed description of the preferred embodiments
Upopeptides
One aspect of the invention provides an isolated lipopeptide comprising a
polypeptide conjugated to one or more lipid moieties wherein:
(i) said polypeptide comprises an amino acid sequence that
comprises:
(a) the amino acid sequence of a T helper cell (Th) epitope and
the amino acid sequence of a B cell epitope, wherein said
amino acid sequences are different; and
(b) one or more internal lysine residues or internal lysine analog
residues for covalent attachment of each of said lipid moieties
via the epsilon-amino group or terminal side-chain group of
said lysine or lysine analog; and
(ii) each of said one or more lipid moieties is covalently attached
directly or indirectly to an epsilon-amino group of said one or
more internal lysine residues or to a terminal side-chain group of
said internal lysine analog residues.
As used herein, the term "lipopeptide" means any non-naturally occurring
composition of matter comprising one or more lipid moieties and one or more
amino acid sequences that are directly or indirectly conjugated, said
composition of matter being substantially free of non-specific non-conjugated
lipid or protein.
By "directly" means that a lipid moiety and an amino acid sequence are not
separated by a spacer molecule.

By "indirectly" means that a lipid moiety and an amino acid sequence are
separated by a spacer comprising one or more carbon-containing molecules,
such as, for example, one or more amino acid residues.
The amino acid sequence may be of any length, constrained by the
requirement for functionality of both the T-helper epitope and the B cell epitope.
As used herein, the term "internal lysine residue" means a lysine residue in the
polypeptide comprising both the T-helper epitope and the B-cell epitope,
wherein said lysine is not the N-terminal amino acid residue or the C-terminal
residue of said polypeptide. This means that the internal lysine residue to which
the lipid moiety is attached is a residue that is present in the amino acid
sequence of the T helper cell epitope or the amino acid sequence of the
antigen. The internal lysine residue may also be distinct from the T-helper
epitope or the B-cell epitope, in which case it must link these two epitopes of
the polypeptide.
Similarly, the term "internal lysine analog residue" means a lysine analog
residue in the polypeptide comprising both the T-helper epitope and the B-cell
epitope, wherein said lysine analog is not the N-terminal amino acid residue or
the C-terminal residue of said polypeptide. The criteria for establishing whether
or not a lysine residue is "internal" shall apply mutatis mutandis to determining
whether or not a lysine analog is internal.
By "lysine analog" is meant a synthetic compound capable of being
incorporated into the internal part of a peptide that has a suitable side-group to
which the lipid moiety can be coupled, including an amino acid analog or non-
naturally occurring amino acid having such an amino side group. Preferred
lysine analogs include compounds of the following general Formula (V):


wherein n is an integer from 0 to 3 and wherein X is a terminal side-chain group
of said internal lysine analog residue selected from the group consisting of NH,
O and S. More preferably, n is an integer having a value from 1 to 3. More
preferably, X is an amino group and the lysine analog is a diamino compound.
In a particularly preferred embodiment, the lysine analog is selected from the
group consisting of 2,3 diaminopropionic acid (Dpr), 2,4-diaminobutyric acid
(Dab) and 2,5-diaminovaleric acid [i.e. ornithine (Orn)].
Those skilled in the art will know the meaning of the term "epsilon-amino
group".
The term "terminal side-chain group" means a substituent on the side chain of a
lysine analog the is distal to the alpha-carbon of said analog, such as, for
example, a beta-amino of Dpr, gamma-amino of Dab, or delta-amino of Orn.
The inventors have found that the most effective lipopeptides are highly
soluble. The relative ability of the lipopeptides of the invention to induce an
antibody response in the absence of external adjuvant was reflected by their
ability to upregulate the surface expression of MHC class II molecules on
immature dendritic cells (DC), particularly D1 cells as described by Winzler et al
J Exp Med 185,317,1997).
As will be known to those skilled in the art, the epsilon amino group of lysine is
the terminal amino group of the side chain of this amino acid. Use of the
epsilon amino group of lysine or the terminal side-chain group of a lysine
analog for cross-linkage to the lipid moiety facilitates the synthesis of the

polypeptide moiety as a co-linear amino acid sequence incorporating both the
T-helper epitope and the B cell epitope. There is a clear structural distinction
between a lipopeptide wherein lipid is attached via the epsilon amino group of a
lysine residue or the terminal side-chain group of a lysine analog and a
lipopeptide having the lipid attached via an alpha amino group of lysine, since
the latter-mentioned lipopeptides can only have the lipid moiety conjugated to
an N-terminal residue.
Accordingly, it is particularly preferred for at least one internal lysine residue or
internal lysine analog to which the lipid moiety is attached to be positioned
within the polypeptide moiety so as to separate the immunologically-functional
epitopes. For example, the internal lysine residue or internal lysine analog
residue may act as a spacer and/or linking residue between the epitopes.
Naturally, wherein the internal lysine or internal lysine analog is positioned
between the T-helper epitope and the B cell epitope, the lipid moiety will be
attached at a position that is also between these epitopes, albeit forming a
branch from the amino acid sequence of the polypeptide. Preferably, a single
internal lysine residue or internal lysine analog is used to separate B cell and T-
helper epitopes (e.g., any one of SEQ ID NOs: 7, 9,13,106,108,110, or 112),
in which case the lipid moiety is attached via the epsilon amino group of a
lysine residue or the terminal side-chain group of a lysine analog positioned
between the amino acid sequences of the T helper epitope and the antigenic B
cell epitope.
The epsilon amino group of the internal lysine or the terminal side-chain group
of a lysine analog can be protected by chemical groups which are orthogonal to
those used to protect the alpha-amino and side-chain functional groups of other
amino acids. In this way, the epsilon amino group of lysine or the terminal side-
chain group of a lysine analog can be selectively exposed to allow attachment
of chemical groups, such as lipid-containing moieties, specifically to the epsilon
amino group or the terminal side-chain group as appropriate.

For peptide syntheses using Fmoc chemistry, a suitable orthogonally protected
epsilon group of lysine is provided by the modified amino acid residue Fmoc-
Lys(Mtt)-OH {Nα-Fmoc-Nε-4-methyItrityl-L-lysine). Similar suitable
orthogonally-protected side-chain groups are available for various lysine
analogs contemplated herein, eg. Fmoc-Orn(Mtt)-OH (Nα-Fmoc-Nδ-4-
methyltrityl-L-Ornithine), Fmoc-Dab(Mtt)-OH (Nα-Fmoc-Nγ-4-methyftrityl-L-
diaminobutyric acid) and Fmoc-Dpr(Mtt)-OH (Nα-Fmoc-Nβ-4-methyltrityl-L-
diaminopropionic acid). The side-chain protecting group Mtt is stable to
conditions under which the Fmoc group present on the alpha amino group of
lysine or a lysine analog is removed but can be selectively removed with 1 %
trifluoroacetic acid in dichloromethane. Fmoc-Lys(Dde)-OH (Nα-Fmoc-Nδ-1-
(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-L-lysine) or Fmoc-Lys(ivDde)-
OH {Nα-Fmoc-Nε-1-(4,4-dirnethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-
L-lysine) can also be used in this context, wherein the Dde side-chain
protecting groups is selectively removed during peptide synthesis by treatment
with hydrazine.
For peptide syntheses using Boc chemistry, Boc-Lys(Fmoc)-OH can be used.
The side-chain protecting group Fmoc can be selectively removed by treatment
with piperidine or DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene) but remains in
place when the Boc group is removed from the alpha terminus using
trifluoroacetic acid.
The optimum distance between the T-helper epitope and the B dell epitope,
and consequently, the precise positioning and number of internal lysine or
lysine analog residues in the lipopeptide of the invention, is readily determined
empirically, for each combination of T helper epitopes, B cell epitopes, and
lipids. In the case of synthetic peptides and polypeptides, the limitations of the
synthesis methodology used to prepare the polypeptides may, in part,

determine the separation between the T-helper epitope and the B cell epitope
that is achievable, and the number and positioning of internal lysine or lysine
analog residue(s).
Preferably, the T helper epitope and B cell epitope are separated by at least
one or two or three or four or five amino acid residues including a single
internal lysine residue or lysine analog residue.
The present invention clearly contemplates the addition of multiple lipid
moieties to the polypeptide moiety. To achieve this, the polypeptide may
include multiple internal lysine residues or multiple internal lysine analog
residues or a combination thereof. Steric hindrance may occur in the addition
of lipid if multiple internal lysine or lysine analog residues are positioned more
closely together, thereby producing a mixture of end-products, or a reduced
yield.
Relevant to this consideration is the fact that it is not necessary for the entire
amino acid sequence comprising the T-helper epitope or the entire amino acid
sequence comprising the B cell epitope to have an immune function.
Accordingly, the said amino acid sequences, whilst comprising said epitopes
may have additional sequence not possessing T-helper cell activity or a B cell
epitope. Where such additional sequences include one or more internal lysine
or lysine analog residues, the terminal side-chain groups of such residues may
serve as attachment sites for the lipid moiety. Naturally, it is essential to retain
T-helper function and B cell epitope function.
The positioning of the internal lysine residue or internal lysine analog for
attachment of the lipid moiety should also be selected such that attachment of
the lipid moiety does not interfere with the immune function of the T-helper
epitope or the B cell epitope in a subject to whom the lipopeptide is

administered. For example, depending upon the selection of lipid moiety, the
attachment of said lipid within the B cell epitope may sterically hinder antigen
presentation.
A generalized preferred form of the lipopeptide of the invention, wherein the
internal lysine or internal lysine analog is positioned between the T-helper and
B-cell epitopes is provided by the general Formula (VI).
Formula (VI):

wherein:
epitope is a T-helper epitope or B-cell epitope;
A is either present or absent and consists of an amino acid spacer
of about 1 to about 6 amino acids in length;
n is an integer having a value of 1,2,3, or 4;
X is a terminal side-chain group selected from the group consisting
of NH, O and S and preferably consisting of NH;
Y is either present of absent and consists of a spacer of about 1 to
about 6 amino acids in length, wherein it is preferred for said
spacer to comprise arginine, serine or 6-aminohexanoic acid; and
Z is a lipid moiety, preferably a lipoamino acid moiety selected from
the group consisting of Pam2Cys, Pam3Cys, Ste2Cys, Lau2Cys,
and Octroys.

Those skilled in the art will be aware that Ste2Cys is also known as S-[2,3-
bis(stearoyloxy)propyl]cysteine or distearoyl-S-glyceryl-cysteine; that Lau2Cys
is also known as S-[2,3-bis(lauroyloxy)propyl]cysteine or dilauroyl-S-glyceryl-
cysteine); and that Oct2Cys is also known as S-[2,3-
bis(octanoyloxy)propyl]cysteine or dioctanoyl-S-glyceryl-cysteine).
The T-helper epitope is any T-helper epitope known to the skilled artisan for
enhancing an immune response in a particular target subject (i.e. a human
subject, or a specific non-human animal subject such as, for example, a rat,
mouse, guinea pig, dog, horse, pig, or goat). Preferred T-helper epitopes
comprise at least about 10-24 amino acids in length, more generally about 15
to about 20 amino acids in length.
Promiscuous or permissive T-helper epitopes are particularly preferred as
these are readily synthesized chemically and obviate the need to use longer
polypeptides comprising multiple T-helper epitopes.
Examples of promiscuous or permissive T-helper epitopes suitable for use in
the lipopeptides of the present invention are selected from the group consisting
of:
(i) a rodent or human T-helper epitope of tetanus toxoid peptide (TTP),
such as, for example amino acids 830-843 of TTP (Panina-Bordignon et
a/., Eur. J. Immun. 19, 2237-2242,1989);
(ii) a rodent or human T-helper epitope of Plasmodium falciparum pfg27;
(iii) a rodent or human T-helper epitope of lactate dehydrogenase;
(iv) a rodent or human T-helper epitope of the envelope protein of HIV or
HIVgp120 (Berzofsky et al., J. Clin. Invest. 88, 876-884,1991);

(v) a synthetic human T-helper epitope (PADRE) predicted from the amino
acid sequence of known anchor proteins (Alexander et ai, Immunity 1,
751-761,1994);
(vi) a rodent or human T-helper epitope of measles virus fusion protein (MV-
F; Muller et a/., Mol. Immunol. 32, 37-47, 1995; Partidos et ai, J. Gen.
Virol., 71, 2099-2105, 1990);
(vii) a T-helper epitope comprising at least about 10 amino acid residues of
canine distemper virus fusion protein (CDV-F) such as, for example,
from amino acid positions 148-283 of CDV-F (Ghosh et a/., Immunol.
104, 58-66,2001; International Patent Publication No. WO 00/46390);
(viii) a human T-helper epitope derived from the peptide sequence of
extracellular tandem repeat domain of MUC1 mucin (US Patent
Application No. 0020018806);
(ix) a rodent or human T-helper epitope of influenza virus haemagglutinin
(IV-H)(Jackson et al. Virol. 198,613-623,1994;; and
(x) a bovine or camel T-helper epitope of the VP3 protein of foot and mouth
disease virus (FMDV-01 Kaufbeuren strain), comprising residues 173 to
176 of VP3 or the corresponding amino acids of another strain of FMDV.
As will be known to those skilled in the art, a T-helper epitope may be
recognised by one or more mammals of different species. Accordingly, the
designation of any T-helper epitope herein is not to be considered restrictive
with respect to the immune system of the species in which the epitope is
recognised. For example, a rodent T-helper epitope can be recognised by the
immune system of a mouse, rat, rabbit, guinea pig, or other rodent, or a human
or dog.
More preferably, the T-helper epitope will comprise an amino acid sequence
selected from the group consisting of:
(i) GALNNRFQIKGVELKS from IV-H (SEQ ID NO: 1);




The T-helper epitopes disclosed herein are included for the purposes of
exemplification only. Using standard peptide synthesis techniques known to the
skilled artisan, the T-helper epitopes referred to herein are readily substituted
for a different T-helper epitope to adapt the lipopeptide of the invention for use
in a different species. Accordingly, additional T-helper epitopes known to the
skilled person to be useful in eliciting or enhancing an immune response in a
target species are not to be excluded.
Additional T-helper epitopes may be identified by a detailed analysis, using in
vitro T-cell stimulation techniques of component proteins, protein fragments
and peptides to identify appropriate sequences (Goodman and Sercarz, Ann.
Rev. Immunol., 1, 465, (1983); Berzofsky, In: "The Year in Immunology, Vol. 2"
page 151, Karger, Basel, 1986; and Livingstone and Fathman, Ann. Rev.
Immunol., 5, 477,1987).
The B cell epitope is conveniently derived from the amino acid sequence of an
immunogenic protein, lipoprotein, or glycoprotein of a virus, prokaryotic or
eukaryotic organism, including but not limited to an antigen derived from a

mammalian subject or a bacterium, fungus, protozoan, or parasite that infects
said subject. Idiotypic and anti-idiotypic B cell epitopes against which an
immune response is desired are specifically included, as are lipid-modified B
cell epitopes. Alternatively, the B cell epitope may be a carbohydrate antigen,
such as, for example, an ABH blood group antigen, transplantation antigen (eg.
Gal alpha1-3Gal beta1-4GlcNAc; Sandrin et al., Proc. Natl. Acad. Sci. USA 90,
11391-11395, 1993; Galili et a/., Proc. Natl. Acad. Sci. USA 84, 1369-1373,
1987; Schofield et al., Nature 418:785-789,2002) or a conjugate thereof.
The B-cell epitope will be capable of eliciting the production of antibodies when
administered to a mammal, preferably neutralizing antibody, and more
preferably, a high titer neutralizing antibody.
Shorter B cell epitopes are preferred, to facilitate peptide synthesis.
Preferably, the length of the B cell epitope will not exceed about 30 amino acids
in length. More preferably, the B cell epitope sequence consists of about 25
amino acid residues or less, and more preferably less than 20 amino acid
residues, and even more preferably about 5-20 amino acid residues in length.
Preferably, peptides will assume a conformation that mimics the conformation
of the native polypeptide from which the B cell epitope is derived.
Preferred B cell epitopes from parasites are those associated with leishmania,
malaria, trypanosomiasis, babesiosis, or schistosomiasis, such as, for example
a B cell epitope selected from the group consisting of:
(i) a B cell epitope of Plasmodium falciparum (NANP) 3 (Good et al, J. Exp.
Med. 164, 655 1986);
(ii) a B cell epitope of Circumsporozoa (Good et al., Protein Sci., 235, 1059,
1987);

(iii) a B cell epitope comprising amino acid residues 326-343 of Leishmania
donovani Repetitive Peptide (Liew et al., J. Exp. Med. 172,1359 (1990));
(iv) a B cell epitope of Toxoplasma gondii P30 surface protein (Darcy et al.,
J. Immunol. 149, 3636 (1992)); and
(v) a B cell epitope of Schistosoma mansoni Sm-28GST antigen
(Wolowxzuk et al., J. Immunol 746:1987 (1991)).
Preferred virus-specific B cell epitopes are derived from and/or capable of
generating antibodies against Rotaviruses, Herpes viruses, Corona viruses,
Picornaviruses (eg. Apthovirus), Respiratory Synctial virus, Influenza Virus,
Parainfluenza virus, Adenovirus, Pox viruses, Bovine herpes virus Type I,
Bovine viral diarrhea virus, Bovine rotaviruses, Canine Distemper Virus (CDV),
Equine Rhinitis A Virus (ERAV); Equine Rhinitis B Virus (ERBV); Foot and
Mouth Disease Virus (FMDV), Measles Virus (MV), Human Immunodeficiency
Viruses (HIV), Feline Immunodeficiency Viruses (FIV), Epstein-Barr virus
(EBV), or hepatitis virus, and the like. Suitable viral B cell epitopes include, but
are not limited to epitopes selected from the group consisting of:
(i) HIV gp120 V3 loop, amino acid residues 308-331 (Jatsushita et al., J.
Virol. 62,2107(1988));
(ii) HIV gp120 amino acid residues 428-443 (Ratner et al., Nature 313:277
(1985));
(iii) HIV gp120 amino acid residues 112-124 (Berzofsky et al., Nature 334,
706 (1988));
(iv) a B cell epitope of HIV Reverse transcriptase (Hosmalin et al. Proc. Natl
Acad. Sci.(USA) 67, 2344 (1990));
(v) Influenza virus nucleoprotein amino acid residues 335-349 (Townsend et
al. Cell 44, 959(1986));
(vi) Influenza virus nucleoprotein amino acid residues 366-379 (Townsend et
al. Cell 44, 959 (1986));

(vii) Influenza virus hemagglutinin amino acid residues 48-66 (Mills et al., J.
Exp. Med. 163, 1477(1986));
(viii) Influenza virus hemagglutinin amino acid residues 111-120 (Hackett et
al., J. Exp. Med 158, 294 (1983));
(ix) Influenza virus hemagglutinin amino acids 114-131 (Lamb and Green,
Immunology 50, 659 (1983));
(x) Epstein-Barr LMP amino acid residues 43-53 (Thorley-Lawson et al.,
Proc. Natl Acad. Sci. (USA) 84, 5384 (1987));
(xi) Hepatitis B virus surface antigen amino acid residues 95-109 (Milich et
al., J. Immunol. 134, 4203 (1985));
(xii) Hepatitis B virus surface antigen amino acid residues 140-154;
(xiii) Hepatitis B virus Pre-S antigen amino acid residues 120-132 (Milich et
al., J. Exp. Med. 164, 532 (1986));
(xiv) Herpes simplex virus gD protein amino acid residues 5-23 (Jayaraman et
al., J. Immunol. 151, 5777 (1993));
(xv) Herpes simplex virus gD protein amino acid residues 241-260 (Wyckoff
et al., Immunobiol., 177,134 (1988));
(xvi) Rabies glycoprotein amino acid residues 32-44 (MacFarlan et al., J.
Immunol. 133, 2748 (1984));
(xvii) The major FMDV epitope comprising at least amino acid residues 134-
168 or 137-160 or residues 142-160 or residues 137-162 or residues
145-150 of the VP1 capsid protein of FMDV serotype O1. or the
corresponding amino acid residues of another serotype, such as, for
example, serotypes A, C, SAT1, SAT2, SAT3, or ASIA1 (US Patent Nos.
5,864,008 and 6,107,021); and
(xviii) The hypervariable region-1 (HVR1) of the E2 protein of hepatitis C virus
(HCV) variant AD78 (Zibert et al., J. Virol. 71, 4123-4127,1997).
Preferred bacteria-specific B cell epitopes are derived from and/or capable of
generating antibodies against Pasteurella, Actinobacillus, Haemophilus, Listeria

monocytogenes, Mycobacterium, Staphylococcus, E. coli, Shigella, and the
like. Suitable bacterial B cell epitopes include, but are not limited to epitopes
selected from the group consisting of:
(i) Mycobacterium tuberculosis 65Kd protein amino acid residues 112-126
(Lamb et al., EMBO J., 6,1245 (1987));
(ii) M. tuberculosis 65Kd protein amino acid residues 163-184 (Lamb et a/.,
EMBO J., 6,1245(1987));
(iii) M. tuberculosis 65Kd protein amino acid residues 227-243 (Lamb et a/.,
EMBO J., 6,1245(1987));
(iv) M. tuberculosis 65Kd protein amino acid residues 242-266 (Lamb et aL,
EMBO J., 6,1245(1987));
(v) M. tuberculosis 65Kd protein amino acid residues 437-459 (Lamb et ah,
EMBO J., 6,1245(1987));
(vi) M. tuberculosis ESAT-6 protein residues 3-15 (Morten et ah, Infect.
Immun. 66, 717-723,1998);
(vii) M. tuberculosis ESAT-6 protein residues 40-62 (Morten et ah, Infect
Immun. 66, 717-723,1998);
(viii) Mycobacterium scrofulaceum alpha-antigen residues 279-290 (Mikiko et
al., Microb. Path. 23, 95-100,1997);
(ix) Staphylococcus aureus nuclease protein amino acid residues 61-80
(Finnegan et al., J. Exp. Med. 164,897(1986));
(x) a B cell epitope of Escherichia coli heat stable enterotoxin (Cardenas et
ah, Infect. Immunity 61, 4629 (1993));
(xi) a B cell epitope of Escherichia coli heat labile enterotoxin (Clements ef
ah, Infect. Immunity 63, 685 (1986));
(xii) a B cell epitope of Shigella sonnei form I antigen (Formal et ah, Infect.
Immunity 34, 746 (1981));
(xiii) a B cell epitope from Group A Streptococcus , preferably derived from
the M protein, more preferably from the C-terminal half of the M protein
and more preferably a minimum, helical, non-host-cross-reactive peptide

derived from the conserved C-terminal half of the M protein and
comprising a non-M-protein peptide designed to maintain helical folding
and antigenicity displayed within said minimum, helical, non-host-cross-
reactive peptide. For example, the non-M-protein peptide (eg peptide
J14) can be linked to one or more serotypic M protein peptides using
chemistry that enables the immunogen to display all the individual
peptides pendant from an alkane backbone, thereby conferring excellent
immunogenicity and protection (US Pat. No. 6,174,528; Brandt et al.,
Nat Med. 6:455-459,2000);
(xiv) a B cell epitope of the Cholera toxin B subunit (CTB), such as, for
example described by Kazemi and Finkelstein Mol. Immunol. 28, 865-
876,1991;
(xv) a B cell epitope of a protein of Bacillus anthracis (anthrax), such as, for
example, a B cell epitope derived from a protein of the outer exosporium
of anthrax such as the 250 kDa glycoprotein (Sylvestre et al., In: Proc.
4th Int. Conf. Anthrax, St John's College Annapolid, Mayland, CA June
10-13,2001, Abstract 31B; and
(xvi) a B cell epitope from a protein of tetanus, such as, for example, the
tetanus toxoid protein.
Preferred B cell epitopes from mammalian subjects are derived from and/or
capable of generating antibodies against a tumor antigen. Tumor antigens are
usually native or foreign antigens, the expression of which is correlated with the
development, growth, presence or recurrence of a tumor. In as much as tumor
antigens are useful in differentiating abnormal from normal tissue, they are
useful as a target for therapeutic intervention. Tumor antigens are well known
in the art. Indeed, several examples are well-characterized and are currently
the focus of great interest in the generation of tumor-specific therapies. Non-
limiting examples of tumor antigens are carcinoembryonic antigen (CEA),

prostate specific antigen (PSA), melanoma antigens (MAGE, BAGE, GAGE),
and mucins, such as MUC-1.
Alternatively, a preferred B cell epitope from a mammalian subject is derived
from zona pellucida protein such as ZP3 (Chamberlin and Dean Proc. Natl.
Acad. Sci.( USA) 87, 6014-6018, 1990) or ZP3a (Yurewicz et al., Biochim.
Biophys. Acta 1174, 211-214, 1993)] of humans or other mammals such as
pigs. Particuarly preferred B cell epitopes within this category include amino
acid residues 323-341 of human ZP3 (Chamberlin and Dean Proc. Natl. Acad.
Sci.(USA) 87, 6014-6018, 1990); amino acid residues 8-18 or residues 272-283
or residues 319-330 of porcine ZP3a (Yurewicz et al., Biochim. Biophys. Acta
1174, 211-214, 1993).
Further preferred B cell epitopes from a mammalian subject are derived from
and/or capable of generating antibodies against a peptide hormone, such as,
for example, a satiety hormone (eg. leptin), a digestive hormone (eg. gastrin),
or a reproductive peptide hormone [eg. luteinising hormone-releasing hormone
(LHRH), follicle stimulating hormone (FSH), luteinising hormone (LH), human
chorionic gonadotropin (hCG; Carlsen et al., J. Biol. Chem. 248, 6810-6827,
1973), or alternatively, a hormone receptor such as, for example, the FSH
receptor (Kraaij et al., J. Endocrinol. 158,127-136,1998). Particuarly preferred
B cell epitopes within this category include the C-terminal portion (CTP) of b-
hCG that is antigenically non cross-reactive with LH (Carlsen et al., J. Biol.
Chem. 248, 6810-6827,1973).
In a particularly preferred embodiment, a peptide comprising a B-cell epitope
will comprise an amino acid sequence selected from the group consisting of:
(i) EHWSYGLRPG derived from LHRH (herein referred to as "LHRH 1-10";
SEQ ID NO: 2);





(xviii) A sequence from the M. tuberculosis ESAT-6 protein selected from the
group consisting of: EQQWNFAGIEAAA (SEQ ID NO: 97) and
AAAWGGSGSEAYQGVQQKWDATA (SEQ ID NO: 98).
(xix) GGPTRTIGGSQAQTASGLVSMFSVGPSQK (SEQ ID NO: 99) from
HCV;
(xx) KFQDAYNAAGGH (SEQ ID NO: 100) from M. scrofulaceum alpha
antigen;
(xxi) KQAEDKVKASREAKKQVEKALEQLEDKVK (SEQ ID NO: 101) from the
M protein of group A Streptococcus (i.e., peptide designated herein as
"J14"); and
(xxii) GWMDF (SEQ ID NO: 102) from gastrin (i.e., pentagastrin consisting of
the C-terminal five amino acid residues of gastrin).
It will be apparent from the preceding description that the polypeptide moiety of
the subject lipopeptide is synthesized conveniently as a single amino acid
chain, thereby requiring no post-synthesis modification to incorporate both
epitopes.
A polypeptide moiety which comprises a highly immunogenic B cell epitope of
LHRH (eg. SEQ ID NO: 2 or 3 or 4) linked either to a T-helper epitope of
influenza virus hemagglutinin (eg. SEQ ID NO: 1) or a T-helper epitope of CDV-
F (eg. SEQ ID NO: 20, 24, 26, or 44) is particularly preferred, such as, for
example, a polypeptide comprising an amino acid sequence selected from the
group consisting of:



In a particularly preferred embodiment, the LHRH epitope (i.e. LHRH1-10 as
set forth in SEQ ID NO: 2; LHRH 2-10 as set forth in SEQ ID NO: 3; or LHRH
6-10 as set forth in SEQ ID NO: 4) is positioned such that the C-terminal
glycine residue is exposed or not internal. Accordingly, the configuration set
forth in any one of SEQ ID Nos: 5, 7, or 9-16 is particularly preferred.
In one exemplified embodiment, LHRH 1-10 is conjugated to the T-helper
epitope of influenza virus haemagglutinin (i.e., SEQ ID NO: 1) as described by
the sequence set forth in SEQ ID NO: 5 or 7, and LHRH 2-10 or LHRH 6-10 is
conjugated to a T-helper epitope of CDV-F (i.e., SEQ ID NO: 24) as described
by the sequence set forth in SEQ ID NO: 9, 13, 103 or 104. Other
combinations are clearly possible and encompassed by the present invention.
In an alternative embodiment, a polypeptide moiety which comprises a highly
immunogenic B cell epitope of the M protein of Group A streptococcus (eg. the
J14 peptide set forth in SEQ ID NO: 101) linked to a T-helper epitope of CDV-F
(eg. SEQ ID NO: 24) or influenza virus haemagglutinin (e.g., SEQ ID NO: 1) is
particularly preferred, such as, for example, a polypeptide comprising an amino
acid sequence selected from the group consisting of:
(i) KLIPNASLIENCTKAELKQAEDKVKASREAKKQVEKALEQLEDKYK
(SEQ ID NO: 105);


The skilled artisan will readily be able to synthesize additional polypeptide
moieties to those exemplified herein for use in the subject lipopeptides, by
substituting the T-helper epitope and/or the B cell epitope of any one of SEQ ID
Nos: 5-16 or any one of SEQ ID Nos: 103-112 with another T-helper epitope or
B cell epitope, such as, for example a T-helper epitope set forth in any one of
SEQ ID Nos: 18-56, or a B cell epitope set forth in any one of SEQ ID Nos: 57-
102. Moreover, the selection of appropriate T-helper epitope and B cell
combinations will be apparent to the skilled artisan from the disclosure provided
herein, according to the target species and the antigen against which an
immune response is sought.

The amino acid sequences of the polypeptide moities described herein,
including those exemplified polypeptides set forth in SEQ ID Nos: 5-16 and
SEQ ID Nos: 103-112, may be modified for particular purposes according to
methods well known to those of skill in the art without adversely affecting their
immune function. For example, particular peptide residues may be derivatized
or chemically modified in order to enhance the immune response or to permit
coupling of the peptide to other agents, particularly lipids. It also is possible to
change particular amino acids within the peptides without disturbing the overall
structure or antigenicity of the peptide. Such changes are therefore termed
"conservative" changes and tend to rely on the hydrophilicity or polarity of the
residue. The size and/or charge of the side chains also are relevant factors in
determining which substitutions are conservative.
It is well understood by the skilled artisan that, inherent in the definition of a
biologically functional equivalent protein or peptide, is the concept that there is
a limit to the number of changes that may be made within a defined portion of
"the molecule and still result in a molecule with an acceptable level of equivalent
biological activity. Biologically functional equivalent peptides are thus defined
herein as those peptides in which specific amino acids may be substituted.
Particular embodiments encompass variants that have one, two, three, four,
five or more variations in the amino acid sequence of the peptide. Of course, a
plurality of distinct proteins/peptides with different substitutions may easily be
made and used in accordance with the invention.
Those skilled in the art are well aware that the following substitutions are
permissible conservative substitutions (i) substitutions involving arginine, lysine
and histidine; (ii) substitutions involving alanine, glycine and serine; and (iii)
substitutions involving phenylalanine, tryptophan and tyrosine. Peptides
incorporating such conservative substitutions are defined herein as biologically
functional equivalents.

The importance of the hydropathic amino acid index in conferring interactive
biological function on a protein is generally understood in the art (Kyte &
Doolittle, J. Mol. Biol. 157, 105-132, 1982). It is known that certain amino acids
may be substituted for other amino acids having a similar hydropathic index or
score and still retain a similar biological activity The hydropathic index of amino
acids also may be considered in determining a conservative substitution that
produces a functionally equivalent molecule. Each amino acid has been
assigned a hydropathic index on the basis of their hydrophobicity and charge
characteristics, as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8);
glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3);
proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-
3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In making changes
based upon the hydropathic index, the substitution of amino acids whose
hydropathic indices are within .+/- 0.2 is preferred. More preferably, the
substitution will involve amino acids having hydropathic indices within .+/- 0.1,
and more preferably within about +/- 0.05.
It is also understood in the art that the substitution of like amino acids is made
effectively on the basis of hydrophilicity, particularly where the biological
functional equivalent protein or peptide thereby created is intended for use in
immunological embodiments, as in the present case (e.g. US Patent No.
4,554,101), As detailed in US Patent No. 4,554,101, the following hydrophilicity
values have been assigned to amino acid residues: arginine (+3.0); lysine
(+3.0); aspartate (+3.0 +/- 0.1); glutamate (+3.0 +/- 0.1); serine (+0.3);
asparagine (+0.2); glutamine,(+0.2); glycine (0); threonine (-0.4); proline (-0.5
+/- 0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5);
tryptophan (-3.4). In making changes based upon similar hydrophilicity values,

it is preferred to substitute amino acids having hydrophilicity values within
about +/- 0.2 of each other, more preferably within about +/- 0.1, and even
more preferably within about +/- 0.05.
Having identified peptides suitable for use as immunogens, it also is
contemplated that other sterically similar compounds may be formulated to
mimic the key portions of the peptide structure. Such compounds, which may
be termed peptidomimetics, may be used in the same manner as the peptides
of the invention and hence are also functional equivalents. The generation of a
structural functional equivalent may be achieved by the techniques of modeling
and chemical design known to those of skill in the art. It will be understood that
all such sterically similar constructs fall within the scope of the present
invention.
Another method for determining the "equivalence" of modified peptides involves
a functional approach. For example, a given peptide is used to generate
monoclonal or polyclonal antibodies. These antibodies can then, in turn, be
used to screen libraries of degenerate peptides that include thousands or
hundreds of thousands of other peptides, thereby identifying structures that are,
at least to a certain extent, immunologically equivalent. Of course, these
structures may bear some primary sequence homology to the peptide used to
generate the antibodies, but they also may be quite different.
The polypeptide moiety is readily synthesized using standard techniques, such
as the Merrifield method of synthesis (Merrifieid, J Am Chem Soc, 85,:2149-
2154,1963) and the myriad of available improvements on that technology (see
e.g., Synthetic Peptides: A User's Guide, Grant, ed. (1992) W.H. Freeman &
Co., New York, pp. 382; Jones (1994) The Chemical Synthesis of Peptides,
Clarendon Press, Oxford, pp. 230.); Barany, G. and Merrifield, R.B. (1979) in
The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic

Press, New York; Wünsch, E., ed. (1974) Synthese von Peptiden in Houben-
Weyls Methoden der Organischen Chemie (Müler, E., ed.), vol. 15, 4th edn.,
Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide
Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984)
The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M.
(1985) Int. J. Peptide Protein Res. 25, 449-474.
The lipid moiety may comprise any C2 to C30 saturated, monounsaturated, or
polyunsaturated linear or branched fatty acyl group, and preferably a fatty acid
group selected from the group consisting of: palmitoyl, myristoyl, stearoyl,
lauroyl, octanoyl, and decanoyl.
Lipoamino acids are particularly preferred lipid moieties within the present
context. As used herein, the term "lipoamino acid" refers to a molecule
comprising one or two or three or more lipids covalently attached to an amino
acid residue, such as, for example, cysteine or serine or lysine or an analog
thereof. In a particularly preferred embodiment, the lipoamino acid comprises
cysteine and optionally, one or two or more arginine or serine residues, or
alternatively, 6-aminohexanoic acid.
The lipid moiety is preferably a compound having a structure of General
Formula (VII):


wherein:
(i) X is selected from the group consisting of sulfur, oxygen, disulfide (-S-S-
), methylene (-CH2-), and amino (-NH-);
(ii) m is an integer being 1 or 2;
(iii) n is an integer from 0 to 5;
(iv) R1 is selected from the group consisting of hydrogen, carbonyl (-CO-),
and R'-CO- wherein R' is selected from the group consisting of alkyl
having 7 to 25 carbon atoms, alkenyl having 7 to 25 carbon atoms, and
alkynyl having 7 to 25 carbon atoms, wherein said alkyl, alkenyl or
alkynyl group is optionally substituted by a hydroxyl, amino, oxo, acyl, or
cycloalkyl group;
(v) R2 is selected from the group consisting of R'-CO-O-, R'-O-, R'-O-CO,
R'-NH-CO-, and R'-CO-NH-, wherein R' is selected from the group
consisting of alkyl having 7 to 25 carbon atoms, alkenyl having 7 to 25
carbon atoms, and alkynyl having 7 to 25 carbon atoms, wherein said
alkyl, alkenyl or alkynyl group is optionally substituted by a hydroxyl,
amino, oxo, acyl, or cycloalkyl group; and
(vi) R3 is selected from the group consisting of R'-CO-O-, R'-O-, R'-O-CO-,

R'-NH-CO-, and R'-CO-NH-, wherein R' is selected from the group
consisting of alkyl having 7 to 25 carbon atoms, alkenyl having 7 to 25
carbon atoms, and alkynyl having 7 to 25 carbon atoms, wherein said
alkyl, alkenyl or alkynyl group is optionally substituted by a hydroxyl,
amino, oxo, acyl, or cycloalkyl group
and wherein each of R1, R2 and R3 are the same or different.
Depending upon the substituent, the lipid moiety of general structure VII may
be a chiral molecule, wherein the carbon atoms directly or indirectly covalently
bound to integers R1 and R2 are asymmetric dextrorotatory or levorotatory (i.e.
an R or S) configuration.
Preferably, X is sulfur; m and n are both 1; R1 is selected from the group
consisting of hydrogen, and R'-CO-, wherein R' is an alkyl group having 7 to 25
carbon atoms; and R2 and R3 are selected from the group consisting of R'-CO-
O-, R'-O-, R'-O-CO-, R'-NH-CO-, and R'-CO-NH-, wherein R' is an alkyl group
having 7 to 25 carbon atoms.
Preferably, R' is selected from the group consisting of. palmitoyl, myristoyl,
stearoyl, lauroyl, octanoyl, and decanoyl. More preferably, R' is selected from
the group consisting of: palmitoyi, stearoyl, lauroyl, and octanoyl, and decanoyl.
Each integer R' in said lipid moiety may be the same or different.
In a particularly preferred embodiment, X is sulfur, m and n are both 1; R1 is
hydrogen or R'-CO- wherein R' is selected from the group consisting of:
palmitoyi, stearoyl, lauroyl, and octanoyl; and R2 and R3 are each R'-CO-O-
wherein R' is selected from the group consisting of: palmitoyi, stearoyl, lauroyl,
and octanoyl. Particularly preferred compounds wherein R' is palmitoyi are
shown by Formula (I) and Formula (II) supra.


wherein:
(i) R4 is selected from the group consisting of: (i) an aipha-acyl-fatty acid
residue consisting of between about 7 and about 25 carbon atoms; (ii)
an alpha-alkyl-beta-hydroxy-fatty acid residue; (iii) a beta-hydroxy ester
of an alpha-alkyl-beta-hydroxy-fatty acid residue wherein the ester group
is preferably a straight chain or branched chain comprising more than 8
carbon atoms; and (iv) a lipoamino acid residue; and
(ii) R5 is hydrogen or the side chain of an amino acid residue.
Preferably, R4 consists of between about 10 and about 20 carbon atoms, and
more preferably between about 14 and about 18 carbon atoms.
Optionally, wherein R4 is a lipoamino acid residue, the side-chain of the
integers R4 and R5 can form a covalent linkage. For example, wherein R4
comprises an amino acid selected from the group consisting of lysine, ornithine,
glutamic acid, aspartic acid, a derivative of lysine, a derivative of ornithine, a
derivative of glutamic acid, and a derivative of aspartic acid, then the side chain
of that amino acid or derivative is covalently attached, by virtue of an amide or
ester linkage, to R5.
Preferably, the structure set forth in General Formula VIII is a lipid moiety
selected from the group consisting of: N,N'-diacyllysine; N.N'-diacylornithine;
di(monoalkyl)amide or ester of glutamic acid; di(monoalkyl)amide or ester of

aspartic acid; a N,O-diacyl derivative of serine, homoserine, or threonine; and a
N,S-diacyl derivative of cysteine or homocysteine.
Amphipathic molecules, particularly those having a hydrophobicity not
exceeding the hydrophobicity of Pam3Cys (Formula (I)) are also preferred.
The lipid moieties of Formula (I), Formula (II), Formula (VI) or Formula (VIII) are
further modified during synthesis or post-synthetically, by the addition of one or
more spacer molecules, preferably a spacer that comprises carbon, and more
preferably one or more amino acid residues. These are conveniently added to
the lipid structure via the terminal carboxy group in a conventional
condensation, addition, substitution, or oxidation reaction. The effect of such a
spacer molecule is to separate the lipid moiety from the polypeptide moiety to
reduce steric hindrance effects that might otherwise reduce immunogenicity of
the lipopeptide product.
Arginine or serine dimers, trimers, tetramers, etc, or alternatively, 6-
aminohexanoic acid, are particularly preferred for this purpose.
Preferably, such spacers include a terminal protected amino acid residue to
facilitate the later conjugation of the modified lipoamino acid to the polypeptide.
Exemplary modified lipoamino acids produced according to this embodiment
are presented as Formulae (HI) and (IV), which are readily derived from
Formulae (I) and (II), respectively by the addition of a serine homodimer. As
exemplified herein, Pam3Cys of Formula (I), or Pam2Cys of Formula (II) is
conveniently synthesized as the lipoamino acids Pam3Cys-Ser-Ser of Formula
(III), or Pam2Cys-Ser-Ser of Formula (IV) for this purpose.


As an alternative to the addition of a spacer to the lipid moiety, the spacer may
be added to the epsilon amino group of the internal lysine residue or to the
terminal side-chain group of a lysine analog in the polypeptide moiety, either as
a short peptide, such as, for example an arginine or serine homodimer,
homotrimer, homotetramer, etc, or alternatively, by the sequential addition of
amino acid residues, thereby producing a branched polypeptide chain. This
approach takes advantage of the modified nature of the epsilon amino group on
the internal lysine residue or to the terminal side-chain group of a lysine analog,
as appropriate, to achieve specificity in the addition of the spacer. Naturally, to
avoid sequential spacer addition, the terminal amino acid residue of the spacer

should preferably be protected, such that de-protection can facilitate
conjugation of the lipid moiety to the branched polypeptide.
Alternatively, the spacer may be added to a non-modified epsilon amino group
of the polypeptide by conventional nucleophilic substitution reaction. However,
it is preferred to follow this approach if the polypeptide has an amino acid
sequence comprising a single internal lysine or lysine analog residue and a
blocked N-terminus.
The lipid moiety is prepared by conventional synthetic means, such as, for
example, the methods described in US Patent Nos. 5,700,910 and 6,024,964,
or alternatively, the method described by Wiesmuller et al., Hoppe Seylers Zur
Physiol. Chem. 364, 593 (1983), Zeng et al., J. Pept. Sci 2,66 (1996), Jones et
al., Xenobiotica 5, 155 (1975), or Metzger et al., Int. J. Pept.Protein Res. 38,
545 (1991). Those skilled in the art will be readily able to modify such methods
to achieve the synthesis of a desired lipid for use conjugation to a polypeptide.
Combinations of different lipids are also contemplated for use in the
lipopeptides of the invention. For example, one or two myristoyl-containing
lipids or lipoamino acids are attached via internal lysine or lysine analog
residues to the polypeptide moiety, optionally separated from the polypeptide
by a spacer. Other combinations are not excluded.
The lipopeptides of the invention are readily modified for diagnostic purposes.
For example, it is modified by addition of a natural or synthetic hapten, an
antibiotic, hormone, steroid, nucleoside, nucleotide, nucleic acid, an enzyme,
enzyme substrate, an enzyme inhibitor, biotin, avidin, polyethylene glycol, a
peptidic polypeptide moiety (e.g. tuftsin, polylysine), a fluorescence marker
(e.g. FITC, RITC, dansyl, luminol or coumarin), a bioluminescence marker, a

spin label, an alkaloid, biogenic amine, vitamin, toxin (e.g. digoxin, phalloidin,
amanitin, tetrodotoxin), or a complex-forming agent.
As exemplified herein, highly immunogenic and soluble lipopeptides are
provided comprising Pam3Cys of Formula (I), or Pam2Cys of Formula (II) or
Ste2Cys or Lau2Cys or Oct2Cys conjugated via the epsilon amino group of an
internal lysine residue of a polypeptide that comprises: (i) the amino acid
sequence of a CD4+ T-helper epitope derived from the light chain of influenza
virus hemagglutinin (Jackson et at. Virol. 198, 613-623, 1994; i.e. amino acid
sequence GALNNRFQIKGVELKS; SEQ ID NO:1) or a peptide derived from the
CDV-F protein (SEQ ID NO: 24); (ii) a B-cell epitope-containing peptide
comprising an amino acid sequence selected from the group consisting of the
amino acid sequence of luteinising hormone-releasing hormone (LHRH; Fraser
et at., J. Endocrinol. 63, 399 (1974); Fraser and Baker, J. Endocrinol. 77, 85
(1978); i.e. "LHRH 1-10", amino acid sequence EHWSYGLRPG; SEQ ID NO:
2; "LHRH 2-10", amino acid sequence HWSYGLRPG; SEQ ID NO: 3; or "LHRH
6-10", amino acid sequence GLRPG; SEQ ID NO: 4), Group A Streptococcus
(GAS) M protein (i.e., SEQ ID NO; 101), and pentagastrin (i.e., SEQ ID NO:
102); (iii) a lysine residue positioned between said CD4+ T-helper epitope and
said B-cell epitope; and optionally (iv) a lysine residue positioned within said
CD4* T-helper epitope.
Preparation of lipopeptides
A second aspect of the invention provides a method of producing a lipopeptide
comprising:
(i) producing a polypeptide comprising an amino acid sequence that
comprises:
(a) the amino acid sequence of a T helper cell (Th) epitope and the
amino acid sequence of a B cell epitope, wherein said amino acid
sequences are different; and

(b) one or more internal lysine residues or internal lysine analog
residues; and
(iii) covalently attaching each of said one or more lipid moieties directly or
indirectly to an epsilon-amino group of said one or more internal lysine
residues or to the terminal side-chain group of said one or more internal
lysine analog residues so as to produce a lipopeptide having the lipid
moiety attached to the epsilon amino group of said internal lysine
residue or having the lipid moiety attached to the terminal side-chain
group of said internal lysine analog residue.
Preferably, the method further comprises production of the lipid moiety.
Conventional chemical syntheses referred to herein are the preferred means
for producing the polypeptide moiety and the lipid moiety.
Preferably, the internal lysine residue or internal lysine analog is modified by
selective removal of a blocking group (eg: Mtt) from the terminal side-chain
group, particularly from the terminal side-chain amino group, so as to permit the
addition of an amino acid residue, a spacer or lipid moiety, including a
lipoamino acid, at that position.
For attachment of the lipid to the polypeptide, it is convenient for the functional
groups of the polypeptide to be protected in a manner known in the art of
peptide synthesis, to ensure that no undesirable reactions at those groups
takes place at a significant reaction rate.
By known coupling processes, the polypeptide is synthesized on a solid or
soluble carrier, such as a polymer (for example Merrifield resin) and made
available for conjugation to a spacer, amino acid, or lipid. For example, the
epsilon amino group of the internal lysine or the terminal side-chain group of an

internal lysine analog is protected by one of a number of protecting groups.
Blocking groups (also called protecting groups or masking groups) are used to
protect the amino group of the amino acid having an activated carboxyl group
that is involved in the coupling reaction, or to protect the carboxyl group of the
amino acid having an acylated amino group that is involved in the coupling
reaction. For coupling to occur, a blocking group must be removed without
disrupting a peptide bond, or any protecting group attached to another part of
the peptide.
For solid phase peptide synthesis, blocking groups that are stable to the
repeated treatments necessary for removal of the amino blocking group of the
growing peptide chain and for repeated amino acid couplings, are used for
protecting the amino acid side-chains. Additionally, the peptide-resin
anchorage that protects the C-terminus of the peptide must be protected
throughout the synthetic process until cleavage from the resin is required.
Accordingly, by the judicious selection of orthogonally protected alpha-amino
acids, lipids and/or amino acids are added at desired locations to a growing
peptide whilst it is still attached to the resin.
Preferred amino blocking groups are easily removable but sufficiently stable to
survive conditions for the coupling reaction and other manipulations, such as,
for example, modifications to the side-chain groups. Preferred amino blocking
groups are slected from the group consisting of: (i) a benzyloxycarbonyl group
(Z or carbobenzoxy) that is removed easily by catalytic hydrogenation at room
temperature and ordinary pressure, or using sodium in liquid ammonia and
hydrobromic acid in acetic acid; (ii) a t-Butoxycarbonyl group (Boc) that is
introduced using t-butoxycarbonyl azide or di-tert-butyldicarbonate and
removed using mild acid such as, for example, trifluoroacetic acid (50% TFA in
dichloromethane), or HCI in acetic acid/dioxane/ethylacetate; (iii) a 9-
fluorenylmethyloxycarbonyl group (Fmoc) that is cleaved under mildly basic,

non-hydrolytic conditions, such as, for example, using a primary or secondary
amine (eg. 20% piperidine in dimethyl formamide); (iv) a 2-(4-biphenylyl)
propyl(2)oxycarbonyl group (Bpoc); (v) a 2-nitro-phenylsulfenyl group (Nps);
and (vi) a dithia-succionyl group (Dts).
Side chain-protecting groups will vary for the functional side chains of the
amino acids forming the peptide being synthesized. Side-chain protecting
groups are generally based on the Bzl group or the tBu group. Amino acids
having alcohols or carboxylic acids in the side-chain are protected as Bzl
ethers, Bzl esters, cHex esters, tBu ethers, or tBu esters. Side-chain protection
of Fmoc amino acids requires blocking groups that are ideally base stable and
weak acid (TFA) labile. For example, the epsilon-amino group of lysine is
protected using Mtt (eg. Fmoc-lysine(Mtt)-OH). Alternatively, a halogenated
benzyl derivative such as CIZ is used to protect the lysine side chain should
enhanced acid stability be required. The thiol group of Cystine, the imidazole
of Histidine, or guanidino group of Arginine, generally require specialised
protection. Many different protecting groups for peptide synthesis have been
described (see The Peptides, Gross et al., eds., Vol. 3, Academic Press, New
York, 1981).
The two most widely used protection strategies are the Boc/Bzl- and the
Fmoc/tBu-strategies. In Boc/Bzl, Boc is used for amino protection and the side-
chains of the various amino acids are protected using Bzl- or cHex-based
protecting groups. A Boc group is stable under catalytic hydrogenation
conditions and is used orthogonally along with a Z group for protection of many
side chain groups. In Fmoc/tBu, Fmoc is used for amino protection and the
side-chains are protected with tBu-based protecting groups.
Peptides are lipidated by methods well known in the art. Standard
condensation, addition, substitution or oxidation (e.g. disulfide bridge formation

or amide bond formation between a terminal amino group on the internal lysine
or internal lysine analog with the carboxy terminal group of an incoming amino
acid or peptide or lipoamino acid) reactions result in the addition of lipid to the
polypeptide.
In an alternative embodiment, a peptide of the present invention for use as an
immunogen is produced by chemoselective ligation or chemical conjugation.
Such methods are well-known in the art, and allow for the individual peptide
components to be produced by chemical or recombinant means, followed by
their chemoselective ligation in an appropriate configuration or conformation or
order (eg. Nardin et ai, Vaccine 16, 590 (1998); Nardin et al., J. Immunol. 166,
481 (2001); Rose et al., Mol. Immunol. 32, 1031 (1995); Rose et al, Bioconjug.
Chem 7, 552 (1996); and Zeng et a/., Vaccine 18, 1031 (2000), which are
incorporated herein by reference).
Lipopeptide formulations
The lipopeptide is coveniently formulated in a pharmaceutically
acceptable excipient or diluent, such as, for example, an aqueous
solvent, non-aqueous solvent, non-toxic excipient, such as a salt,
preservative, buffer and the like. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oil and injectable
organic esters such as ethyloleate. Aqueous solvents include water,
alcoholic/aqueous solutions, saline solutions, parenteral vehicles such
as sodium chloride, Ringer's dextrose, etc. Preservatives include
antimicrobial, anti-oxidants, chelating agents and inert gases. The pH
and exact concentration of the various components the pharmaceutical
composition are adjusted according to routine skills in the art.
The addition of an extrinsic adjuvant to the lipopeptide formulation, although
generally not required, is also encompassed by the invention. Such extrinsic

adjuvants include all acceptable immunostimuiatory compounds such as, for
example, a cytokine, toxin, or synthetic composition. Exemplary adjuvants
include IL-1, IL-2, BCG, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-
isoglutamine (thur-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP
11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-
alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine
(CGP) 1983A, referred to as MTP-PE), lipid A, MPL and RIBI, which contains
three components extracted from bacteria, monophosphoryl lipid A, trehalose
dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween
80 emulsion.
It may be desirable to co-administer biologic response modifiers (BRM) with the
lipopeptide, to down regulate suppressor T cell activity. Exemplary BRM's
include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA,
USA); Indomethacin (IND; 150 mg/d) (Lederle, NJ, USA); or low-dose
Cyclophosphamide (CYP; 75, 150 or 300 mg/m.sup.2) (Johnson/Mead, NJ,
USA).
Use of the lipopeptide in immunization
The novel lipopeptides of the invention differ in essential aspects from known
lipopeptide conjugates of antigens in their enhanced solubility and
immunogenicity, and their ability to elicit immune repsonses without the
administration of additional adjuvant. Accordingly, a particular utility of the
lipopeptides of the present invention is in the fields of antibody production,
synthetic vaccine preparation, diagnostic methods employing antibodies and
antibody ligands, and immunotherapy for veterinary and human medicine.
More particularly, the lipopeptide of the present invention induces the specific
production of a high titer antibody against the B cell epitope moiety when

administered to an animal subject, without any requirement for an adjuvant to
achieve a similar antibody titer. This utility is supported by the enhanced
maturation of dendritic cells following administration of the subject lipopeptides
(i.e. enhanced antigen presentation compared to lipopeptides having N-
terminally coupled lipid).
Accordingly, a third aspect of the invention provides a method of eliciting the
production of antibody against an antigenic B cell epitope comprising
administering an isolated lipopeptide comprising a polypeptide conjugated to
one or more lipid moieties to said subject for a time and under conditions
sufficient to elicit the production of antibodies against said antigenic B cell
epitope, wherein:
(i) said polypeptide comprises:
(a) the amino acid sequence of a T helper cell (Th) epitope and
the amino acid sequence of a B cell epitope, wherein said
amino acid sequences are different; and
(b) one or more internal lysine residues or internal lysine analog
residues for covalent attachment of each of said lipid moieties
via an epsilon-amino group of said internal lysine or via a
terminal side-chain group of said internal lysine analog; and
(ii) each of said one or more lipid moieties is covalently attached
directly or indirectly to an epsilon-amino group of said one or
more internal lysine residues or to a terminal side-chain group of
said one or more internal lysine analog residues.
The effective amount of lipopeptide used in the production of antibodies varies
upon the nature of the immunogenic B cell epitope, the route of administration,
the animal used for immunization, and the nature of the antibody sought. All
such variables are empirically determined by art-recognized means.

Reference herein to antibody or antibodies includes whole polyclonal and
monoclonal antibodies, and parts thereof, either alone or conjugated with other
moieties. Antibody parts include Fab and F(ab)2 fragments and single chain
antibodies. The antibodies may be made in vivo in suitable laboratory animals,
or, in the case of engineered antibodies (Single Chain Antibodies or SCABS,
etc) using recombinant DNA techniques in vitro.
In accordance with this aspect of the invention, the antibodies may be
produced for the purposes of immunizing the subject, in which case high titer or
neutralizing antibodies that bind to the B cell epitope will be especially
preferred. Suitable subjects for immunization will, of course, depend upon the
immunizing antigenic B cell epitope. It is contemplated that the present
invention will be broadly applicable to the immunization of a wide range of
animals, such as, for example, farm animals (e.g. horses, cattle, sheep, pigs,
goats, chickens, ducks, turkeys, and the like), laboratory animals (e.g. rats,
mice, guinea pigs, rabbits), domestic animals (cats, dogs, birds and the like),
feral or wild exotic animals (e.g. possums, cats, pigs, buffalo, wild dogs and the
like) and humans.
Alternatively, the antibodies may be for commercial or diagnostic purposes, in
which case the subject to whom the lipopeptide is administered will most likely
be a laboratory or farm animal. A wide range of animal species are used for the
production of antisera. Typically the animal used for production of antisera is a
rabbit, a mouse, rat, hamster, guinea pig, goat, sheep, pig, dog, horse, or
chicken. Because of the relatively large blood volume of rabbits, a rabbit is a
preferred choice for production of polyclonal antibodies. However, as will be
known to those skilled in the art, larger amounts of immunogen are required to
obtain high antibodies from large animals as opposed to smaller animals such
as mice. In such cases, it will be desirable to isolate the antibody from the
immunized animal.

Preferably, the antibody is a high titer antibody. By "high titer" means a
sufficiently high titer to be suitable for use in diagnostic or therapeutic
applications. As will be known in the art, there is some variation in what might
be considered "high titer". For most applications a titer of at least about 103-104
is preferred. More preferably, the antibody titer will be in the range from about
104 to about 105 , even more preferably in the range from about 105 to about
106.
More preferably, in the case of B cell epioptes from pathogens, viruses or
bacteria, the antibody is a neutralizing antibody (i.e. it is capable of neutralizing
the infectivity of the organism fro which the Bcell epitope is derived).
To generate antibodies, the lipopeptide, optionally formulated with any suitable
or desired carrier, adjuvant, BRM, or pharmaceutically acceptable excipient, is
conveniently administered in the form of an injectable composition. Injection
may be intranasal, intramuscular, sub-cutaeous, intravenous, intradermal,
intraperitoneal, or by other known route. The lipopeptides of the present
invention have demonstrated efficacy when administered Intranasally. For
intravenous injection, it is desirable to include one or more fluid and nutrient
replenishers. Means for preparing and characterizing antibodies are well
known in the art. (See, e.g., ANTIBODIES: A LABORATORY MANUAL, Cold
Spring Harbor Laboratory, 1988, incorporated herein by reference).
The efficacy of the lipopeptide in producing an antibody is established by
immunizing an animal, for example, a mouse, rat, rabbit, guinea pig, dog,
horse, cow, goat or pig, with a formulation comprising the lipopeptide, and then
monitoring the immune response to the B cell epitope, as described in the
Examples. Both primary and secondary immune responses are monitored.

The antibody titer is determined using any conventional immunoassay, such as,
for example, ELISA, or radio immunoassay.
The production of polyclonal antibodies may be monitored by sampling blood of
the immunized animal at various points following immunization. A second,
booster injection, may be given, if required to achieve a desired antibody titer.
The process of boosting and titering is repeated until a suitable titer is
achieved. When a desired level of immunogenicity is obtained, the immunized
animal is bled and the serum isolated and stored, and/or the animal is used to
generate monoclonal antibodies (Mabs).
For the production of monoclonal antibodies (Mabs) any one of a number of
well-known techniques may be used, such as, for example, the procedure
exemplified in US Patent No. 4,196,265, incorporated herein by reference.
For example, a suitable animal will be immunized with an effective amount of
the lipopeptide of the invention and under conditions sufficient to stimulate
antibody producing cells. Rodents such as mice and rats are preferred
animals, however, the use of rabbit, sheep, or frog cells is also possible. The
use of rats may provide certain advantages, but mice are preferred, with the
BALB/c mouse being most preferred as the most routinely used animal and one
that generally gives a higher percentage of stable fusions.
Following immunization, somatic cells capable of producing antibodies,
specifically B lymphocytes (B cells), are selected for use in the MAb generating
protocol. These cells may be obtained from biopsied spleens, tonsils or lymph
nodes, or from a peripheral blood sample. Spleen cells and peripheral blood
cells are preferred, the former because they are a rich source of antibody-
producing cells that are in the dividing plasmablast stage, and the latter
because peripheral blood is easily accessible. Often, a panel of animals will

have been immunized and the spleen of animal with the highest antibody titer
removed. Spleen lymphocytes are obtained by homogenizing the spleen with a
syringe. Typically, a spleen from an immunized mouse contains approximately
5 x 107 to 2 x 108 lymphocytes.
The B cells from the immunized animal are then fused with cells of an immortal
myeloma cell, generally derived from the same species as the animal that was
immunized with the lipopeptide formulation. Myeloma cell lines suited for use
in hybridoma-producing fusion procedures preferably are non-antibody-
producing, have high fusion efficiency and enzyme deficiencies that render
then incapable of growing in certain selective media which support the growth
of only the desired fused cells, or hybridomas. Any one of a number of
myeloma cells may be used and these are known to those of skill in the art
(e.g. murine P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO,
NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0; or rat R210.RCY3,
Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LlCR-LON-HMy2
and UC729-6). A preferred murine myeloma cell is the NS-1 myeloma cell line
(also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS
Human Genetic Mutant Cell Repository under Accession No. GM3573.
Alternatively, a murine myeloma SP2/0 non-producer cell line which is 8-
azaguanine-resistant is used.
To generate hybrids of antibody-producing spleen or lymph node cells and
myeloma cells, somatic cells are mixed with myeloma cells in a proportion
between about 20:1 to about 1:1, respectively, in the presence of an agent or
agents (chemical or electrical) that promote the fusion of cell membranes.
Fusion methods using Sendai virus have been described by Kohler and
Milstein, Nature 256, 495-497,1975; and Kohler and Milstein, Eur. J. Immunol.
6,511-519,1976. Methods using polyethylene glycol (PEG), such as 37% (v/v)

PEG, are described in detail by Gefter et at., Somatic Cell Genet. 3, 231-236,
1977. The use of electrically induced fusion methods is also appropriate.
Hybrids are amplified by culture in a selective medium comprising an agent that
blocks the de novo synthesis of nucleotides in the tissue culture media.
Exemplary and preferred agents are aminopterin, methotrexate and azaserine.
Aminopterin and methotrexate block de novo synthesis of both purines and
pyrimidines, whereas azaserine blocks only purine synthesis. Where
aminopterin or methotrexate is used, the media is supplemented with
hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where
azaserine is used, the media is supplemented with hypoxanthine.
The preferred selection medium is HAT, because only those hybridomas
capable of operating nucleotide salvage pathways are able to survive in HAT
medium, whereas myeloma cells are defective in key enzymes of the salvage
pathway, (e.g., hypoxanthine phosphoribosyl transferase or HPRT), and they
cannot survive. B cells can operate this salvage pathway, but they have a
limited life span in culture and generally die within about two weeks.
Accordingly, the only cells that can survive in the selective media are those
hybrids formed from myeloma and B cells.
The amplified hybridomas are subjected to a functional selection for antibody
specificity and/or titer, such as, for example, by immunoassay (e.g.
radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay,
dot immunobinding assay, and the like).
The selected hybridomas are serially diluted and cloned into individual
antibody-producing cell lines, which clones can then be propagated indefinitely
to provide MAbs. The cell lines may be exploited for MAb production in two
basic ways. A sample of the hybridoma is injected, usually in the peritoneal

cavity, into a histocompatible animal of the type that was used to provide the
somatic and myeloma cells for the original fusion. The injected animal develops
tumors secreting the specific monoclonal antibody produced by the fused cell
hybrid. The body fluids of the animal, such as serum or ascites fluid, can then
be tapped to provide MAbs in high concentration. The individual cell lines could
also be cultured in vitro, where the MAbs are naturally secreted into the culture
medium from which they are readily obtained in high concentrations. MAbs
produced by either means may be further purified, if desired, using filtration,
centrifugation and various chromatographic methods such as HPLC or affinity
chromatography.
Monoclonal antibodies of the present invention also include anti-idiotypic
antibodies produced by methods well-known in the art. Monoclonal antibodies
according to the present invention also may be monoclonal heteroconjugates,
(i.e., hybrids of two or more antibody molecules). In another embodiment,
monoclonal antibodies according to the invention are chimeric monoclonal
antibodies. In one approach, the chimeric monoclonal antibody is engineered
by cloning recombinant DNA containing the promoter, leader, and variable-
region sequences from a mouse anti-PSA producing cell and the constant-
region exons from a human antibody gene. The antibody encoded by such a
recombinant gene is a mouse-human chimera, its antibody specificity is
determined by the variable region derived from mouse sequences. Its isotype,
which is determined by the constant region, is derived from human DNA.
In another embodiment, monoclonal antibodies according to the present
invention is a "humanized" monoclonal antibody, produced by techniques well-
known in the art. That is, mouse complementary determining regions ("CDRs")
are transferred from heavy and light V-chains of the mouse Ig into a human V-
domain, followed by the replacement of some human residues in the framework
regions of their murine counterparts. "Humanized" monoclonal antibodies in

accordance with this invention are especially suitable for use in in vivo
diagnostic and therapeutic methods.
As stated above, the monoclonal antibodies and fragments thereof according to
this invention are multiplied according to in vitro and in vivo methods well-
known in the art. Multiplication in vitro is carried out in suitable culture media
such as Dulbecco's modified Eagle medium or RPMI 1640 medium, optionally
replenished by a mammalian serum such as fetal calf serum or trace elements
and growth-sustaining supplements, e.g., feeder cells, such as normal mouse
peritoneal exudate cells, spleen cells, bone marrow macrophages or the like. In
vitro production provides relatively pure antibody preparations and allows
scale-up to give large amounts of the desired antibodies. Techniques for large
scale hybridoma cultivation under tissue culture conditions are known in the art
and include homogenous suspension culture, (e.g., in an airlift reactor or in a
continuous stirrer reactor or immobilized or entrapped cell culture).
Large amounts of the monoclonal antibody of the present invention also may
be obtained by multiplying hybridoma cells in vivo. Cell clones are injected into
mammals which are histocompatible with the parent cells, (e.g., syngeneic
mice, to cause growth of antibody-producing tumors. Optionally, the animals
are primed with a hydrocarbon, especially oils such as Pristane
(tetramethylpentadecane) prior to injection.
In accordance with the present invention, fragments of the monoclonal antibody
of the invention are obtained from monoclonal antibodies produced as
described above, by methods which include digestion with enzymes such as
pepsin or papain and/or cleavage of disulfide bonds by chemical reduction.
The monoclonal conjugates of the present invention are prepared by methods
known in the art, e.g., by reacting a monoclonal antibody prepared as

described above with, for instance, an enzyme in the presence of a coupling
agent such as glutaraldehyde or periodate. Conjugates with fluorescein
markers are prepared in the presence of these coupling agents, or by reaction
with an isothiocyanate. Conjugates with metal chelates are similarly produced.
Other moieties to which antibodies may be conjugated include radionuclides
such as, for example, 3H, 125l, .32P, .35S, 14C, 51Cr, 36CI, 57Co, 58Co, 59Fe, 75Se,
and 152Eu. Radioactively labeled monoclonal antibodies of the present
invention are produced according to well-known methods in the art. For
instance, monoclonal antibodies are iodinated by contact with sodium or
potassium iodide and a chemical oxidizing agent such as sodium hypochlorite,
or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal
antibodies according to the invention may be labeled with technetium" by
ligand exchange process, for example, by reducing pertechnate with stannous
solution, chelating the reduced technetium onto a Sephadex column and
applying the antibody to this column or by direct labeling techniques, (e.g., by
incubating pertechnate, a reducing agent such as SNCI2, a buffer solution such
as sodium-potassium phthalate solution, and the antibody).
Any immunoassay may be used to monitor antibody production by the
lipopeptide formulations. Immunoassays, in their most simple and direct sense,
are binding assays. Certain preferred immunoassays are the various types of
enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA)
known in the art. Immunohistochemical detection using tissue sections is also
particularly useful. However, it will be readily appreciated that detection is not
limited to such techniques, and Western blotting, dot blotting, FACS analyses,
and the like may also be used.
Most preferably, the assay will be capable of generating quantitative results.

For example, antibodies are tested in simple competition assays. A known
antibody preparation that binds to the B cell epitope and the test antibody are
incubated with an antigen composition comprising the B cell epitope, preferably
in the context of the native antigen. "Antigen composition" as used herein
means any composition that contains some version of the B cell epitope in an
accessible form. Antigen-coated wells of an ELISA plate are particularly
preferred. In one embodiment, one would pre-mix the known antibodies with
varying amounts of the test antibodies (e.g., 1:1,1:10 and 1:100) for a period of
time prior to applying to the antigen composition. If one of the known antibodies
is labeled, direct detection of the label bound to the antigen is possible;
comparison to an unmixed sample assay will determine competition by the test
antibody and, hence, cross-reactivity. Alternatively, using secondary antibodies
specific for either the known or test antibody, one willbe able to determine
competition.
An antibody that binds to the antigen composition will be able to effectively
compete for binding of the known antibody and thus will significantly reduce
binding of the latter. The reactivity of the known antibodies in the absence of
any test antibody is the control. A significant reduction in reactivity in the
presence of a test antibody is indicative of a test antibody that binds to the B
cell epitope (i.e., it cross-reacts with the known antibody).
In one exemplary ELISA, the antibodies against the B cell epitope are
immobilized onto a selected surface exhibiting protein affinity, such as a well in
a polystyrene microtiter plate. Then, a composition containing the B cell epitope
is added to the wells. After binding and washing to remove non-specifically
bound immune complexes, the bound epitope may be detected. Detection is
generally achieved by the addition of a second antibody that is known to bind to
the B cell epitope and is linked to a detectable label. This type of ELISA is a
simple "sandwich ELISA". Detection may also be achieved by the addition of

said second antibody, followed by the addition of a third antibody that has
binding affinity for the second antibody, with the third antibody being linked to a
detectable label.
Induction of sterility
An appropriately configured lipopeptide of the present invention comprising an
antigenic B cell epitope of a reproductive hormone or a hormone receptor is
capable of inducing infertility in a subject.
Accordingly, a further aspect of the invention provides a method of inducing
infertility in a subject comprising administering to said subject an isolated
lipopeptide comprising a polypeptide conjugated to one or more lipid moieties,
wherein:
(i) said polypeptide comprises:
(a) the amino acid sequence of a T helper cell (Th) epitope and
the amino acid sequence of a B cell epitope of a reproductive
hormone or hormone receptor, and wherein said amino acid
sequences are different; and
(b) one or more internal lysine residues or internal lysine analog
residues for covalent attachment of each of said lipid moieties
via an epsilon-amino group of said internal lysine or via a
terminal side-chain group of said internal lysine analog; and
(ii) each of said one or more lipid moieties is covalently attached
directly or indirectly to an epsilon-amino group of said one or
more internal lysine residues or to a terminal side-chain group of
said one or more internal lysine analog residues; and
(iii) said lipopeptide is administered for a time and under conditions
sufficient to elicit a humoral immune response against said
antigenic B cell epitope.

The lipopeptides may be administered in the form of any convenient lipopeptide
formulation as described herein.
By "humoral immune response" means that a secondary immune response is
generated against the B cell epitope sufficient to prevent oogenesis,
spermatogenesis, fertilization, implantation, or embryo development..
Preferably, the humoral immunity generated includes a sustained level of
antibodies against the B cell epitope in the subject. By a "sustained level of
antibodies" is meant a sufficient level of circulating antibodies against the B cell
epitope to prevent oogenesis, spermatogenesis, fertilization, implantation, or
embryo development.
Preferably, antibodies levels are sustained for at least a single reproductive
cycle of an immunized female subject, and more preferably for at least about
six months or 9 months or 12 months or 2 years.
Preferably, the B cell epitope is derived from the amino acid sequence of
luteinising hormone-releasing hormone (LHRH), follicle stimulating hormone
(FSH), luteinising hormone (LH), human chorionic gonadotropin (hCG), a zona
pellucida protein such as ZP3, or a FSH receptor ZP3a of humans or other
mammals, such as pigs.
Particularly preferred B cell epitopes within this category include the C-terminal
portion (CTP) of p-hCG; amino acid residues 323-341 of human ZP3; amino
acid residues 8-18 or residues 272-283 or residues 319-330 of porcine ZP3a.
Even more preferably, the B ceil epitope comprises an amino acid sequence
selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID
NO: 4, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ

ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO:
84.
The T-helper epitope preferably comprises an amino acid sequence selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO: 20, SEQ ID NO: 24,
SEQ ID NO: 26 and SEQ ID NO: 44, however any one of SEQ ID Nos: 1 or 18-
56 can be used.
In a particularly preferred embodiment of the invention, the T-helper epitope
comprises an amino acid sequence as set forth in any one of SEQ ID NO: 1,
SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 44, and the
B-cell epitope comprises an amino acid sequence of LHRH as set forth in SEQ
ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4. In accordance with such a
preferred embodiment, the polypeptide comprises an amino acid sequence
selected from the group consisting of SEQ ID Nos: 5-16, 103 or 104. Also in
accordance with this preferred embodiment, it is preferred (albeit not essential)
that the lipid moiety comprise a lipoamino acid selected from the group
consisting of: (i) Pam2Cys; (ii) Ste2Cys; (iii) Lau2Cys; and (iv) Oct2Cys.
The sustained production of antibodies against LHRH achieved by the
lipopeptides of the invention demonstrates the general utility of the subject
lipopeptides as an active agent in a vaccine preparation for inducing sterility, or
as a contraceptive agent.
Accordingly, a further aspect of the invention provides a contraceptive agent
comprising a pharmaceutically acceptable diluent and a lipopeptide comprising
an isolated polypeptide conjugated to one or more lipid moieties wherein:
(i) said polypeptide comprises:
(a) the amino acid sequence of a T helper cell (Th) epitope and
the amino acid sequence of a B cell epitope of a reproductive

hormone or hormone receptor, wherein said amino acid
sequences are different; and
(b) one or more internal lysine residues or internal lysine analog
residues for covalent attachment of each of said lipid moieties
via an epsilon-amino group of said internal lysine or via a
terminal side-chain group of said internal lysine analog; and
(ii) each of said one or more lipid moieties is covalently attached
directly or indirectly to an epsilon-amino group of said one or
more internal lysine residues or to a terminal side-chain group of
said one or more internal lysine analog residues.
The vaccine/contraceptive agent of the invention may comprise one or more
carriers or excipients or other agents as described herein above under
"lipopeptide formulations".
Similarly, administration of the subject vaccine/contraceptive agent is achieved
by means described herein above. Preferably, the subject is a human, or an
animal subject such as, for example, a farm animal, laboratory animal,
domestic animal, feral animal or wild exotic animal.
Immunization against Group A Steptococcus
Group A streptococcus (GAS) is the bacterial agent of relatively mild illnesses
such as, for example, "strep throat," and impetigo, as well as rarer severe and
even life-threatening diseases such as, for example, necrotizing faciitis and
streptococcal toxic shock syndrome. Severe, sometimes life-threatening, GAS
disease may occur when bacteria get into parts of the body where bacteria
usually are not found, such as the blood, muscle, or the lungs, an infection
termed "invasive GAS disease". Two of the most severe forms of invasive GAS
disease are necrotizing fasciitis and Streptococcal Toxic Shock Syndrome
(STSS). Necrotizing fasciitis destroys muscles, fat, and skin tissue. STSS

causes blood pressure to drop rapidly and organs (e.g., kidney, liver, lungs) to
fail. About 20% of patients with necrotizing fasciitis and more than half with
STSS die. About 10%-15% of patients with other forms of invasive group A
streptococcal disease die. There were about 9,400 cases of invasive GAS
disease in the United States alone in 1999.
invasive GAS infections generally occur when the bacteria get past the
defenses of the person who is infected, such as, for example, when a person
has sores or other breaks in the skin that allow the bacteria to get into the
tissue, or when the person's ability to fight off the infection is decreased
because of chronic illness or an illness that affects the immune system,
incuding HIV/AIDS. Also, some virulent strains of GAS are more likely to cause
severe disease than others. People suffering from chronic illnesses like cancer,
diabetes, and kidney dialysis, and those who use medications such as steroids
have a higher risk.
As exemplified herein, an appropriately configured lipopeptide of the present
invention comprising an antigenic B cell epitope of a Group A streptococcus
antigen, preferably protein M, is capable of immunizing an animal host against
GAS, and more particularly inducing serum IgG, saliva IgA and fecal IgA
against the M protein of GAS, and also providing a protective immune response
against a subsequent challenge by GAS thereby reducing GAS-induced
mortality.
Accordingly, a further aspect of the invention provides a method of inducing an
immune response against a Group A streptococcus antigen in a subject
comprising administering to said subject an isolated lipopeptide comprising a
polypeptide conjugated to one or more lipid moieties, wherein:
(iv) said polypeptide comprises:

(b) the amino acid sequence of a T helper cell (Th) epitope and
the amino acid sequence of a B cell epitope of a Group A
streptococcus antigen, wherein said amino acid sequences
are different; and
(c) one or more internal lysine residues or internal lysine analog
residues for covalent attachment of each of said lipid moieties
via an epsilon-amino group of said internal lysine or via a
terminal side-chain group of said internal lysine analog; and
(v) each of said one or more lipid moieties is covalently attached
directly or indirectly to an epsilon-amino group of said one or
more internal lysine residues or to a terminal side-chain group of
said one or more internal lysine analog residues; and
(vi) said lipopeptide is administered for a time and under conditions
sufficient to elicit a humoral immune response against said
antigenic B cell epitope.
The lipopeptides may be administered in the form of any convenient lipopeptide
formulation as described herein.
By "humoral immune response" means that a secondary immune response is
generated against the B cell epitope sufficient to induce serum IgG, saliva IgA
or fecal IgA against a peptide comprising the B-cell epitope, or alternatively or
in addition, providing a protective immunity against a subsequent challenge
with Group A streptococcus.
Preferably, the humoral immunity generated includes a sustained level of
antibodies against the B cell epitope in the subject. By a "sustained level of
antibodies" is meant a sufficient level of circulating antibodies against the B cell
epitope to prevent the spread of infection by a Group A streptococcus following

a subsequently challenge, and/or reduce morbidity or mortality in a subject that
is subsequently challenged with a Group A streptococcus.
Preferably, antibodies levels are sustained for at least about six months or 9
months or 12 months or 2 years.
Preferably, the B cell epitope is derived from the amino acid sequence of the M
protein of Group A streptococcus.
Particularly preferred B cell epitopes within this category include a peptide that
comprises the amino acid sequence set forth in SEQ ID NO: 101.
The T-helper epitope preferably comprises an amino acid sequence selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO: 20, SEQ ID NO: 24,
SEQ ID NO: 26 and SEQ ID NO: 44, however any one of SEQ ID Nos: 1 or 18-
56 can be used.
In a particularly preferred embodiment of the invention, the T-helper epitope
comprises an amino acid sequence as set forth in SEQ ID NO: 24 and the B-
cell epitope comprises an amino acid sequence set forth in SEQ ID NO: 101.
In accordance with such a preferred embodiment, the polypeptide comprises
an amino acid sequence selected from the group consisting of SEQ ID Nos:
105-108. Also in accordance with this preferred embodiment, it is preferred
(albeit not essential) that the lipid moiety comprise a lipoamino acid of Formula
(I) or (II), however any lipid as described herein will be useful.
The sustained production of antibodies against the J14 peptide achieved by the
lipopeptides of the invention demonstrates the general utility of the subject
lipopeptides as an active agent in a vaccine preparation for providing protective
immunity against Group A streptococcus.

Accordingly, a further aspect of the invention provides a vaccine against Group
A streptococcus comprising a pharmaceutically acceptable diluent and a
lipopeptide comprising an isolated polypeptide conjugated to one or more lipid
moieties wherein:
(iii) said polypeptide comprises:
(b) the amino acid sequence of a T helper cell (Th) epitope and
the amino acid sequence of a B cell epitope of a Group A
streptococcus antigen, wherein said amino acid sequences
are different; and
(c) one or more internal lysine residues or internal lysine analog
residues for covalent attachment of each of said lipid moieties
via an epsilon-amino group of said internal lysine or via a
terminal side-chain group of said internal lysine analog; and
(iv) each of said one or more lipid moieties is covalently attached
directly or indirectly to an epsilon-amino group of said one or
more internal lysine residues or to a terminal side-chain group of
said one or more internal lysine analog residues.
The vaccine of the invention may comprise one or more carriers or excipients
or other agents as described herein above under "lipopeptide formulations".
Similarly, administration of the subject vaccine is achieved by means described
herein above, preferably by an intranasal route. Preferably, the subject is a
human, or an animal subject such as, for example, a farm animal, laboratory
animal, domestic animal, feral animal or wild exotic animal.
Inhibition or prevention of excessive and unregulated gastric acid secretion
Gastrin is known to stimulate gastric acid secretion by parietal cells, an activity
mediated by binding of gastrin to gastrin receptors or cholecystekinin receptors.

The terminal four-to-five amino acid residues of gastrin provide the same
receptor specificity and activity as the full-length protein. The terminal five
amino acid residues of gastrin are termed "pentagastrin". Unregulated gastrin
expression or secretion causes hypergastrinemia, which can lead to Zollinger-
Ellison syndrome, the formation of gastric and duodenal ulcers, or gastrinoma
in the pancreas or duodenum, as a consequence of excessive and unregulated
gastric acid secretion. Immunoneutralization of gastrin using antibodies against
gastrin is also known to block secretion of gastric acid in response to
intragastric secretion of gastrin peptides.
As exemplified herein, an appropriately configured lipopeptide of the present
invention comprising an antigenic B cell epitope of a gastrin peptide is capable
of immunizing an animal host against gastrin or an effect of excessive gastrin
production in a mouse model of other mammals in which inhibition of gastric
acid secretion is indicated. The data provided herein demonstrate the general
utility of the subject lipopeptides in inducing humoral immunity against gastrin
and immunoneutralization of gastrin, to thereby block secretion of gastric acid,
in an animal suffering from hypergastrinemia, Zollinger-Ellison syndrome,
gastric ulceration or duodenal ulceration due to excessive and unregulated
secretion of gastric acid, or to reduce or prevent the formation of gastrin-
secreting tumors in the pancreas or duodenum (i.e. the prophylaxis and/or
therapy of gastrinoma).
Accordingly, a further aspect of the invention provides a method of inducing an
immune response against a gastrin peptide in a subject comprising
administering to said subject an isolated lipopeptide comprising a polypeptide
conjugated to one or more lipid moieties, wherein:
(vii) said polypeptide comprises:
(c) the amino acid sequence of a T helper cell (Th) epitope and
the amino acid sequence of a B cell epitope of a gastrin

peptide antigen, wherein said amino acid sequences are
different; and
(d) one or more internal lysine residues or internal lysine analog
residues for covalent attachment of each of said lipid moieties
via an epsilon-amino group of said internal lysine or via a
terminal side-chain group of said internal lysine analog; and
(viii) each of said one or more lipid moieties is covalently attached
directly or indirectly to an epsilon-amino group of said one or
more internal lysine residues or to a terminal side-chain group of
said one or more internal lysine analog residues; and
(ix) said lipopeptide is administered for a time and under conditions
sufficient to elicit a humoral immune response against said
antigenic B cell epitope.
The lipopeptides may be administered in the form of any convenient lipopeptide
formulation as described herein.
By "humoral immune response" means that a secondary immune response is
generated against the B cell epitope sufficient to induce serum IgG against a
gastrin peptide comprising the B-cell epitope.
Preferably, the humoral immunity generated includes a sustained level of
antibodies against the B cell epitope in the subject. By a "sustained level of
antibodies" is meant a sufficient level of circulating antibodies against the B cell
epitope to prevent excessive or unregulated gastric acid secretion in response
to gastrin.
Preferably, antibodies levels are sustained for at least about six months or 9
months or 12 months or 2 years.

Preferably, the B cell epitope is contained within a pentagastrin peptide.
Particularly preferred B cell epitopes within this category include a peptide that
comprises the amino acid sequence set forth in SEQ ID NO: 102, however the
full length gastrin protein or any immunogenic fragment thereof comprising a B-
cell epitope may also be used.
The T-helper epitope preferably comprises an amino acid sequence selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO: 20, SEQ ID NO: 24,
SEQ ID NO: 26 and SEQ ID NO: 44, however any one of SEQ ID Nos: 1 or 18-
56 can be used.
In a particularly preferred embodiment of the invention, the T-helper epitope
comprises an amino acid sequence as set forth in SEQ ID NO: 24 and the B-
cell epitope comprises an amino acid sequence set forth in SEQ ID NO: 102.
In accordance with such a preferred embodiment, the polypeptide comprises
an amino acid sequence selected from the group consisting of SEQ ID Nos:
109-112. Also in accordance with this preferred embodiment, it is preferred
(albeit not essential) that the lipid moiety comprise a lipoamino acid of Formula
(I) or (II), however any lipid as described herein will be useful.
The sustained production of antibodies against pentagastrin or gastrin that is
achieved by the lipopeptides of the invention demonstrates the general utility of
the subject lipopeptides as an active agent in a vaccine preparation for
reducing an adverse effect of gastrin in a subject in need thereof.
Accordingly, a further aspect of the invention provides a vaccine against a
disease or condition induced by excessive gastrin secretion in a subject
comprising a pharmaceutically acceptable diluent and a lipopeptide comprising
an isolated polypeptide conjugated to one or more lipid moieties wherein:
(v) said polypeptide comprises:

(c) the amino acid sequence of a T helper cell (Th) epitope and
the amino acid sequence of a B cell epitope of a gastrin
peptide antigen, wherein said amino acid sequences are
different; and
(d) one or more internal lysine residues or internal lysine analog
residues for covalent attachment of each of said lipid moieties
via an epsilon-amino group of said internal lysine or via a
terminal side-chain group of said internal lysine analog; and
(vi) each of said one or more lipid moieties is covalently attached
directly or indirectly to an epsilon-amino group of said one or
more internal lysine residues or to a terminal side-chain group of
said one or more internal lysine analog residues.
The vaccine of the invention may comprise one or more carriers or excipients
or other agents as described herein above under "lipopeptide formulations".
Similarly, administration of the subject vaccine is achieved by means described
herein above. Preferably, the subject is a human.
The present invention is further described with reference to the following non-
limiting examples and the drawings.
EXAMPLE 1
Materials and Methods
Chemicals
Unless otherwise stated chemicals were of analytical grade or its equivalent.
N,N'-dimethylformamide (DMF), piperidine, trifluoroacetic acid (TFA),
O'benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU),
1-hydroxybenzotriazole (HOBt) and diisopropylethylamine (DIPEA) and
diisopropylcarbodiimide (DIPCDI) were obtained from Auspep Pty. Ltd.,

Melbourne, Australia and Sigma-Aldrich Pty. Ltd., Castle Hill, Australia.
O'benzotriazole-N,N,N',N'-etramethyluronium tetrafluoroborate (TBTU) was
obtained from Bachem, (Bachem AG, Switzerland). Dichloromethane (DCM)
and diethylether were from Merck Pty Ltd. (Kilsyth, Australia). Phenol and
triisopropylsilane (TIPS) were from Aldrich (Milwaulke, Wl) and
trinitrobenzylsulphonic acid (TNBSA) and diaminopyridine (DMAP) from Fluka;
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was obtained from Sigma and
palmitic acid was from Fluka.
Synthesis of lipid moieties of Formulae (I)
Pam3Cys was prepared according to the method described by Weismuller et
al., Hoppe Seylers Z Physiol Chem 364, 593 (1983), as modified according to
the method described by Zeng et al, J Pept Sci 2, :66 (1996). The lipoamino
acid Pam3Cys is coupled to the exposed epsilon-amino group of lysine
according to the procedure described by Zeng et a/, (supra). Briefly, a 2-fold
excess of Pam3Cys, TBTU and HOBt was dissolved in DCM and a 3-fold
excess of DIPEA added. This solution was then added to the resin-bound
peptide to generate the lipopeptide.
Synthesis of lipid moieties of Formulae (II)
Pam2Cys and its derivative Fmoc-Pam2Cys-OH were prepared according to the
methods described by Jones et ai, Xenobiotica 5, 155 (1975) and Metzger et
at., Int J Pept Protein Res 38, 545 (1991).
Synthesis of lipopeptides
Pam2Cys, Ste2Cys, Oct2Cys, or Lau2Cys were coupled to peptide using a
variation of the methods described by Jones et ai, Xenobiotica 5, 155 (1975)
and Metzger et al, Int J Pept Protein Res 38, 545 (1991).

I. Synthesis of S-(2,3-Dihydroxypropyl)cysteine:
Triethylamine (6 g, 8.2 ml, 58 mmoles) was added to L-cysteine hydrochloride
(3 g, 19 mmole) and 3-bromo-propan-1,2-diol (4.2 g, 2.36 ml, 27 mmole) in
water and the homogeneous solution kept at room temperature for 3 days. The
solution was reduced in vacuo at 40°C to a white residue which was boiled with
methanol (100ml), centrifuged and the residue dissolved in water (5ml). This
aqueous solution was added to acetone (300ml) and the precipitate isolated by
centrifugation. The precipitate was purified by several precipitations from water
with acetone to give S-(2,3-dihydroxypropyl)cysteine as a white amorphous
powder (2.4 g, 12.3 mmol, 64.7%).
II. Synthesis of N-Fluorenylmethoxycarbonyl-S-(2,3-dihydroxypropyl)
cysteine (Fmoc-Dhc-OH):
S-(2,3-dihydroxypropyl)cysteine (2.45 g, 12.6 mmole) was dissolved in 9%
sodium carbonate (20 ml). A solution of fluorenylmefhoxycarbonyl-N-
hydroxysuccinimide (3.45 g, 10.5 mmole) in acetonitrile (20 ml) was added and
the mixture stirred for 2 h, then diluted with water (240 ml), and extracted with
diethyl ether (25 ml x 3). The aqueous phase was acidified to pH 2 with
concentrated hydrochloric acid and was then extracted with ethyl acetate (70
ml x 3). The extract was washed with water (50 ml x 2) and saturated sodium
chloride solution (50 ml x 2), dried over sodium sulfate and evaporated to
dryness. Recrystalisation from ether and ethyl acetate at -20°C yielded a
colourless powder (2.8 g, 6.7 mmole, 63.8%).
III. Coupling of Fmoc-Dhc-OH to resin-bound peptide:
Fmoc-Dhc-OH (100mg, 0.24 mmole) was activated in DCM and DMF (1:1, v/v,
3 ml) with HOBt (36 mg, 0.24 mmole) and DICI (37 ul, 0.24 mmol) at 0 °C for 5
min. The mixture was then added to a vessel containing the resin-bound
peptide (0.04 mmole, 0.25g amino-peptide resin). After shaking for 2 h the
solution was removed by filtration and the resin was washed with DCM and

DMF (3 x 30 ml each). The reaction was monitored for completion using the
TNBSA test. If necessary a double coupling was performed.
IVa. Palmitoylation of the two hydroxy groups of the Fmoc-Dhc-peptide resin:
Palmitic acid (204 mg, 0.8 mmole), DICI (154 ul, 1 mmole) and DMAP (9.76
mg, 0.08 mmole) were dissolved in 2 ml of DCM and 1 ml of DMF. The resin-
bound Fmoc-Dhc-peptide resin (0.04 mmole, 0.25 g) was suspended in this
solution and shaken for 16 h at room temperature. The solution was removed
by filtration and the resin was then washed with DCM and DMF thoroughly to
remove any residue of urea. The removal of the Fmoc group was accomplished
with 2.5% DBU (2 x 5mins).
IVb. Stearovlation of the two hydroxy groups of the Fmoc-Dhc-peptide resin:
Stearic acid (about 0.8 mmole), DICI (154 ul, 1 mmole) and DMAP (9.76 mg,
0.08 mmole) were dissolved in 2 ml of DCM and 1 ml of DMF. The resin-bound
Fmoc-Dhc-peptide resin (0.04 mmole, 0.25 g) was suspended in this solution
and shaken for 16 h at room temperature. The solution was removed by
filtration and the resin was then washed with DCM and DMF thoroughly to
remove any residue of urea. The removal of the Fmoc group was accomplished
with 2.5% DBU (2 x 5mins).
IVc. Lauroylation of the two hydroxy groups of the Fmoc-Dhc-peptide resin:
Lauric acid (about 0.8 mmole), DICI (154 ul, 1 mmole) and DMAP (9.76 mg,
0.08 mmole) were dissolved in 2 ml of DCM and 1 ml of DMF. The resin-bound
Fmoc-Dhc-peptide resin (0.04 mmole, 0.25 g) was suspended in this solution
and shaken for 16 h at room temperature. The solution was removed by
filtration and the resin was then washed with DCM and DMF thoroughly to
remove any residue of urea. The removal of the Fmoc group was accomplished
with 2.5% DBU (2 x 5mins).

IVd. Octanoylation of the two hydroxy groups of the Fmoc-Phc-peptide resin:
Octanoic acid (about 0.8 mmole), DICI (154 ul, 1 mmole) and DMAP (9.76 mg,
0.08 mmole) were dissolved in 2 ml of DCM and 1 ml of DMF. The resin-bound
Fmoc-Dhc-peptide resin (0.04 mmole, 0.25 g) was suspended in this solution
and shaken for 16 h at room temperature. The solution was removed by
filtration and the resin was then washed with DCM and DMF thoroughly to
remove any residue of urea. The removal of the Fmoc group was accomplished
with 2.5% DBU (2 x 5mins).
Peptide Synthesis
The general procedure used for the peptide synthesis has been described by
Jackson et al, Vaccine 18, 355 (1999). To enable lipid attachment between
the CD4+ T cell epitope and B-cell epitope, Fmoc-lysine(Mtt)-OH was inserted
at a point between the two epitopes in the approximate centre of the resin-
bound peptide. If lipid was to be added to another position within the peptide,
such as, for example, the Lys-14 residue of SEQ ID NO: 24, then the Fmoc-
lysine(Mtt)-OH was also inserted at that position. Following completion of
peptide synthesis the Mtt group was removed by continual flow washing with
1% TFA in dichloromethane over a period of 30-45 mins to expose the epsilon
amino group of the lysine residue. Two serine residues were coupled to this
epsilon amino group in the case where two serine residues were used as
spacer. Alternatively, two arginine residues were coupled to this epsilon amino
group in the case where two arginine residues were used as spacer.
Alternatively, 6-aminohexanoic acid was coupled to this epsilon amino group.
The subsequent coupling of the lipid moiety, such as, for example, Pam3Cys,
Pam2Cys, Ste2Cys, Oct2Cys, or Lau2Cys was described above.
All resin-bound peptide constructs were cleaved from the solid phase support
with reagent B (88% TFA, 5% phenol, 2% TIPS, 5% water) for 2 hr, and

purified by reversed phase chromatography as described by Zeng ef al.,
Vaccine 18, 1031 (2000).
Analytical reversed phase high pressure liquid chromatography (RP-HPLC)
was carried out using a Vydac C4 column (4.6 x 250 mm) installed in a Waters
HPLC system and developed at a flow rate of 1ml/min using 0.1% TFA in H2O
and 0.1% TFA in CH3CN as the limit solvent. All products presented as a
single major peak on analytical RP-HPLC and had the expected mass when
analysed by MALDI-TOF mass spectrometry on a Bruker BIFLEX instrument
equipped with delayed ion extraction. The final quantitation of the immunogens
was done by measuring the absorption at 280 nm exploiting the presence of a
tryptophan and a tyrosine residue in the peptide constructs (molar extinction
coefficient of 6.6 x103).
To investigate the effect of serine by incorporating two residues between the
peptide and lipid moieties of the Pam3Cys-containing peptides and Pam2Cys-
containing peptides, two serine residues were added sequentially to the peptide
prior to covalent attachment of the lipid moiety (the structures of which are
shown in Figure 1). Summaries of their characteristics, carried out by analytical
RP-HPLC and mass spectrometry, are presented in Tables 1 and 2.
• Immunization protocols
Groups of five female BALB/c mice, 6 to 8 weeks old, were inoculated at day 0
and again on day 28. Alternatively, female outbred Quackenbush mice, 4-6
weeks old, were immunized intranasally and provided with boosts as per the
primary immunization at 21-day intervals. For subcutaneous (s.c.) inoculations
(100 µl volume per dose), lipopeptlde constructs were prepared in saline and
non-lipidated peptides formulated as an emulsion in an equal volume of
complete Freund's adjuvant (CFA) for the primary injection or incomplete
Freund's adjuvant for the secondary inoculation. For intranasal (i.n.)

inoculations, 50 ul of peptide in saline were applied to the nares of mice
anaesthetised with penthrane for inhalation. Sera were prepared from blood
taken at 4 weeks after the primary inoculation and two weeks after the
secondary inoculation, or altenatively, from tail bleeds seven days following the
final immunization.
Enzyme-linked immunosorbent assays (ELISA)
ELISA assays were carried out on serum samples as described essentially by
Ghosh et al., Int Immun. 11, 1103, (1999), using the immunizing antigen (eg.,
LHRH, J14 or pentagastrin) as the coating antigen. The titres of antibody are
expressed as the reciprocal of the highest dilution of serum to achieve an OD
of 0.2, which represents approximately 5 times the background binding in the
absence of antibody. The isotype of antibodies specific for LHRH or J14 was
determined using rabbit antisera directed against mouse IgM, lgG1, lgG2a,
lgG2b, lgG3 or IgA (ICN Pharmaceuticals Inc., Costa Mesa, CA) as previously
described by Ghosh et al., Int Immun. 11,1103, (1999).
Fertility studies
After being inoculated with peptide immunogen and following exposure to male
mice, female mice were tested for their ability to drop litters. A group of female
mice immunized with saline in CFA was used as a control. A male mouse was
introduced into a cage in which two or three female mice were kept and male
mice rotated between each cage to expose each group of female mice to every
male. Males and females were kept together for a total of 3 weeks at the end of
which time the males were removed and the females kept under observation.





Dendritic cell culture
Dendritic cells (DC) were cultured in medium based on complete IDDM. This
consisted of Iscove's Modified Dulbecco's Medium (IMDM) containing 25 mM
HEPES and without alpha-thioglycerol or L-glutamine (JRH Bioscience,
Lenexa, USA), supplemented with 10% (v/v) heat inactivated (56°C, 30 min)
foetal calf serum (CSL Ltd., Parkville, Victoria, Australia), gentamicin (24
ug/mL), giutamine (2 mM), sodium pyruvate (2 mM), penicillin (100 lU/mL),
streptomycin (180 µg/mL) and 2-mercaptoethanol (0.1 mM). For DC generation
complete IMDM was further supplemented with 30% supernatant from cultured
NIH/3T3 cells and 5% GM-CSF in the form of a supernatant from Ag8653 cells
transfected with the GM-CSF gene (DC medium).
The culture method for immature dendritic cells was adapted from Winzler et
a/., J. Exp Med. 185, 317 (1997). Spleen cells from a BALB/c mouse were
seeded at 1.5 x 10 cells per 55 mm dish (Techno-Plas, S.A., Australia) in 3 ml
DC medium and incubated at 37°C with 5% CO2. All the equipment used for
culturing was pyrogen free. The medium was changed every 4 days and all
cells returned to the dish. On day 12, both suspended and weakly adherent
cells were collected by forcefully pipetting and then aspirating the medium. The
procedure was repeated with 2 ml of PBS. The remaining strongly adherent
cells were discarded. The collected cells were pelleted by centrifugation and
reseeded into a new dish. Cells were subsequently maintained on a 4 day
alternating cycle of media change and passage. After 1 month of continuous
culturing, the floating and semi-adherent cells took on the appearance and
staining characteristics of immature DC and are referred to as D1 cells. Under
these passage conditions the majority of cultured D1 cells maintain an
immature phenotype characterized by an intermediate expression level of cell
surface MHC class II molecules.

Flow cytometric analysis of D1 cells
D1 cells (1 x 10s cells per sample) were seeded in a new Petri dish with 1 mL
of DC media and incubated with 0.0045 nmole of lipopeptide, dissolved in
complete IMDM medium. Lipopolysaccharide purified from E. coli serotype
0111:B4 (Difco, Detroit, Michigan, USA, a kind gift from Dr. E. Margot Anders,
Department of Microbiology and Immunology, University of Melbourne) was
used at 5 ng/mL as a positive control for DC maturation. After overnight
incubation, the cells were harvested and washed once with PBS with 1% FCS.
To prevent non-specific binding to FCγRII/lll, the cells were pre-incubated with
20 µL of normal mouse serum for 5 mins at room temperature. The cells were
then exposed to FITC-conjugated monoclonal antibody 14-4-4S (lgG2a . anti-l-
Ek,d ; Ozato et a/., J. Immunol., 124, 533,1980) for 30 min on ice. Monoclonal
antibody 36/1 (Brown et ai, Arch Virol 114, 1 1990), which is specific for the
hemagglutinin of influenza virus, was used as an isotype control. All antibodies
were used at 2.5 ng/mL The samples were washed once with PBS containing
1% FCS and fixed with PBS containing 4% paraformaldehyde on ice for 15
minutes. Flow cytometry analysis was performed using a FACSort (Becton
Dickinson, San Jose, USA) and the data were analysed using FlowJo software
(Tree Star, Inc., San Carios.CA, USA)

EXAMPLE 2
Studies on lipopeptides comprising LHRH B cell epitopes
Solubility properties of lipopeptides comprising LHRH
Visual inspection of the different lipopeptide preparations comprising LHRH
showed that they differed markedly in their solubilities (Figure 2). Enhanced
solubility was most evident in those cases where lipid was attached between
the two epitopes at the approximate centre of the molecule. The lipopeptides
designated [Th]-Lys(Panri2Cys)-[B] and [Th]-Lys(Pam3Cys)-[B] were soluble in
saline at concentrations of at least 8 mg/ml (no higher concentrations were
examined), whereas constructs in which lipid was attached to the N-terminus of
the sequence formed opalescent solutions at concentrations as low as 0.25
mg/ml.
Efforts to further enhance the solubility of peptides with N-terminally linked lipid
by the incorporation of two hydrophilic serine residues between the lipid and
peptide moieties (i.e. Pam2Cys-Ser-Ser-[Th]-[B] and Pam3Cys-Ser-Ser-[Th]-
[B]), proved unsuccessful. In fact the lipopeptide Pam3Cys-Ser-Ser-[Th]-[B] was
so insoluble that it could not be purified by RP-HPLC under conditions used for
the other lipopeptides. We considered that the insoluble nature of this construct
would prevent it from being considered as a viable proposition for manufacture
as a vaccine.
Immunogenicity of lipopeptides comprising LHRH B cell epitopes
The three lipopeptides designated Pam2Cys-Ser-Ser-rTh]-|B], [Th]-
Lys(Pam2Cys)-[B] and [Th]-Lys(Pam3Cys)-[B], when administered s.c. in saline
induced high levels of anti-LHRH antibody. In fact, antibody titres induced after
two doses of these lipopeptides were similar to those obtained with [Th]-[B] or
[Th]-Lys-IB] when administered in CFA (Figure 3). The titres of anti-LHRH
antibodies in sera of mice that had received Pam3Cys-Ser-Ser-[Th]-[B] or
Pam2Cys-[Th]-[B] were slightly lower. The two soluble lipopeptides [Th]-

Lys(Pam2CysHB], [Th]-Lys(Pam3CysHB] induced 10 to 100-fold higher levels
of anti-LHRH antibody following the primary inoculation than did the other less
soluble lipopeptide constructs. Two groups of five mice receiving [Th]-[B]
admixed with Pam3Cys-Ser-(Lys)4 in the ratio 1:1 or 1:5 did not elicit significant
levels of anti-LHRH antibody, a finding that contrasts with other results reported
using Pam3Cys-Ser-(Lys)4as an adjuvant (Jung, G., and W. G. Bessler. (1995)
In: "Immunological recognition of peptides in medicine and biology", N. D.
Zegers, W. J. A. Boersma, and E. Claassen, eds.. CRC Press, Boca, New
York, London, Tokyo, p. 159).
The results of the fertility study carried out two weeks after the second
inoculation with the various lipopeptides are shown in Table 3.
None of the mice that received either of the two soluble lipopeptide constructs,
[Th]-Lys(Pam2Cys)-[B] or [Th]-Lys(Pam3Cys)-[B], administered in saline or the
two non-lipidated constructs [Th]-[B] or [Th]-Lys-[B] administered in CFA,
became pregnant. One mouse from the group that received Pam2Cys-Ser-Ser-
[Th]-[B], and two animals from the groups that received Pam3Cys-Ser-Ser-[Th]-
[B] or Pam2Cys-[ThHB] dropped litters. All members of control groups of mice
that received saline in CFA or the peptide [Th]-[B] co-admixed with Pam3Cys-S-
(Lys)4 dropped litters.
Antibody levels were followed up to 7 months after the second dose of peptide
vaccine. The titres of anti-LHRH antibody present in lipopeptide-primed mice
and in mice primed with non lipidated peptide administered in CFA decrease
between 4 and 20 fold during a 26 week period. Three months following the
secondary inoculation a fertility study carried out on all mice yielded similar
results to the 2 week post-immunization trial. Mice that had received the
soluble lipopeptides, [Th]-Lys(Pam2CysHB] or [Th]-Lys(Pam3Cys)-[B], in saline
or the non-lipidated [Th]-[B] and [Th]-Lys-[B] in CFA were still infertile.



Pam2Cys is a more potent adjuvant than Pam2Cys
The results presented in Figure 3 and Table 2 indicate that the two branched
lipopeptides [Th]-Lys(Pam2Cys)-[B] and [Th]-Lys(Pam3Cys)-[B] were not only
more soluble but also elicited higher antibody titres, particularly in the primary
antibody response, than did the immunogens Pam2Cys-|Th]-[B], Pam2Cys-Ser-
Ser-[Th]-[B] and Pam3Cys-Ser-Ser-[Th]-[B].
To examine this further, we investigated the effect of decreasing the dose on
the immunogenicity of [Th]-Lys(Pam2Cys)-[B] and |Th]-Lys(Pam3Cys)-[B]. At
doses of 10 nmole and 1 nmole, [Th]-Lys(Pam2Cys)-[B] induced higher
antibody titres than did [Th]-Lys(Pam3Cys)-[B] (Table 4). A more striking
difference was observed in the mating trial; 1 of 5 and 0 of 5 mice receiving 10
and 1 nmole [Th]-Lys(Pam2Cys)-[B], respectively, dropped litters whereas 3 of
5 and 5 of 5 mice receiving [Th]-Lys(Pam3Cys)-[B] at these doses dropped
litters (Table 4). These results indicate that Pam2Cys-containing peptides are
better immunogens than Pam2Cys- containing peptides.
The effect of including two additional serine residues into the Pam2Cys-
containing immunogens had little or no effect on the fertility status of animals
although there was an improvement in the antibody titres that were generated
following the second dose (Table 4).



Systemic antibody responses following intranasal (i.n.) immunization
We inoculated [Th]-Lys(Pam2Cys-Ser-Ser)-[B] and Pam2Cys-Ser-Ser-[Th]-[B]
in saline by the intranasal route. The same vaccines were also inoculated by
the subcutaneous route and the systemic anti-LHRH antibody responses were
measured. The solution used for inoculation of [Th]-Lys(Pam2Cys-Ser-Ser)-[B]
was clear and the one for Pam2Cys-Ser-Ser-[Th]-[B] was opalescent indicating
solubility differences between the two preparations.
Following two intranasal inoculations, each of the vaccines induced similar
titres of serum anti-LHRH antibodies which were slightly lower than those
induced following subcutaneous inoculation (Table 5). The more soluble [Th]-
Lys(Pam2Cys-Ser-Ser)-[B] induced significantly higher levels of anti-LHRH
antibody 4 weeks after a single dose than did the less soluble Pam2Cys-Ser-
Ser-[Th]-[B] (p = 0.00007); in fact this was similar to the result obtained
following subcutaneous inoculation. The fertility trial showed that two intranasal
inoculations of [Th]-Lys(Pam2Cys-Ser-Ser)-[B] prevented all mice from
becoming pregnant in contrast to those animals receiving Pam2Cys-Ser-Ser-
[Th]-[B] intranasally in which 3 of 5 mice became pregnant.
A comparison of the longevity of the responses induced by the two constructs
when administered by the two different routes is also shown in Table 5. Twenty
six weeks following the second dose of vaccine the levels of antibody in all
mice had dropped below those observed 2 weeks after receiving the second
dose. The decrease in anti-LHRH antibody in the group that received |Th]-
Lys(Pam2Cys-Ser-Ser)-[B] subcutaneously, however, was much less apparent
again indicating the superiority of a configuration in this context wherein
Pam2Cys-Ser-Ser is attached at the approximate centre of the molecule.



We also determined the titres of individual antibody isotypes that were directed
towards LHRH and obtained from animals following two subcutaneous or
intranasal doses of the soluble lipopeptide [Th]-Lys(Pam2Cys-Ser-Ser)-[B]
(Figure 4). Intranasal inoculation appeared to induce higher levels of lgG3,
lgG2b and possibly IgM than did subcutaneous inoculation even though the
amount of total Ig induced by intranasal inoculation was less.
Exposure of DC to peptides and lipopeptides induce different levels of cell
surface MHC class II molecules
The priming of naive CD4+ T ceils in secondary lymphoid organs by dendritic
cells is preceded by maturation of DC upon exposure to antigen. This
maturation is characterised by up-regulation of MHC products and co-
stimulatory molecules on the DC surface. We therefore determined whether
the various peptides and lipopeptides could differentially activate dendritic cells
in an attempt to explain the different immunogenic properties of these vaccine
candidates.
The results of experiments in which a line of immature DC, D1 cells, were
exposed to peptides, stained for surface expression of MHC class II molecules
then analysed by flow cytometry, demonstrated that |Th]-Lys(Pam2Cys-Ser-
Ser)-[B] was the most effective and Pam2Cys-[ThHB] was the least effective in
causing maturation of DC (Figure 5). The ability of [Th]-Lys(Pam2Cys-Ser-
Ser)-[B] to up-regulate class II expression approached that of bacterial
lipopolysaccharide (LPS) and Pam2Cys-Ser-Ser[Th]-[B] and [Th]-
Lys(Pam2Cys)-[B] displayed intermediate levels of activation. The non-
lipidated peptide was unable to induce maturation of D1 cells greater than the
26% which occurs spontaneously in culture. The ability of the lipopeptides to
induce the maturation of D1 cells was concentration-dependent (data not
shown). The relative abilities of these lipopeptides to induce maturation of D1
cells directly reflected their ability to induce antibody, providing a possible
mechanism for differences in immunogenicity.

Antibody responses to the C-terminal pentapeptide of LHRH
As shown in Figure 6, approximately equivlaent antibody responses are elicited
by lipidated [Th]-Lys(Pam2CysHB] in which [Th] consists of CD4+ T cell epitope
from the light chain of influenza hemagglutinin (SEQ ID NO: 1) and [B] is LHRH
1-10 (SEQ ID NO: 2) or LHRH 6-10 (i.e., the last C-terminal 5 residues of
LHRH; SEQ ID NO: 4), with or without a serine spacer (Ser-Ser) positioned
between the lipid and peptide moieties. These data support the proposition
that the usefulness of the lipopeptides is not limited to any specific amino acid
sequence being used as the immunizing antigen.
Lipids other than Pam2Cys are useful in the lipopeptide constructs
Groups of BALB/c mice (6-8 weeks old) were inoculated subcutaneously with
20 nmoles of the peptide immunogens shown in Figure 7, comprising the lipid
moieties Pam2Cys; Ste2Cys; Lau2Cys; or Oct2Cys conjugated to the amino acid
sequence set forth in SEQ ID NO: 9 (i.e. a peptide comprising the CDV-F T-
helper epitope of SEQ ID NO: 24 conjugated to LHRH 2-10 as set forth in SEQ
ID NO: 3, with an internal lysine residue positioned between these epitopes),
for both primary and secondary vaccinations. Peptide structures are shown in
Figure 7. All lipopeptides were administered in saline. The non lipidated
peptides was administered in CFA as a control. Sera were obtained from blood
taken at 4 weeks following the primary vaccination and 2 weeks following the
secondary vaccination.
Data shown in Figure 8 indicate that strong primary and secondary antibody
responses can be obtained when the Pam2Cys moiety is substituted for
another lipid moiety in the lipopeptide constructs.

Different spacers can be used to separate lipid from peptide in the iipopeptides
Groups of BALB/c mice (6-8 weeks old) were inoculated subcutaneously with
20 nmoles of the peptide immunogens shown in Figure 7, comprising the lipid
moiety Pam2Cys conjugated to the amino acid sequence set forth in SEQ ID
NO: 9 and separated therefrom using a spacer consisting of a serine
homodimer, arginine homodimer or 6-aminohexanoic acid. Peptide structures
are shown in Figure 7. All Iipopeptides were administered in saline. The non
lipidated peptides was administered in CFA as a control. Sera were obtained
from blood taken at 4 weeks following the primary vaccination and 2 weeks
following the secondary vaccination.
Data shown in Figure 9 indicate that strong primary and secondary antibody
responses can be obtained when the Pam2Cys moiety is separated from the
peptide moiety in the lipopeptide constructs using a variety of different spacers.
The lipid moiety can be attached to an internal lysine residue within the T-
helper epitope
To determine the stringency of a requirement for positioning of the internal
lysine residue to which the lipid moiety is attached, we also studied the
immunogenicity of a lipopeptide construct wherein the lipid was attached to an
internal lysine residue within the T-helper epitope. Groups of BALB/c mice (o
weeks and 4 weeks old) were inoculated subcutaneously with 20 nmoles of the
peptide immunogens comprising the lipid moiety Pam2Cys conjugated to the
amino acid sequence set forth in SEQ ID NO: 9 between the T-helper epitope
and B-cell epitope, or alternatively, conjugated to the amino acid sequence set
forth in SEQ ID NO: 103 at position lys-14 within the T-helper epitope. Peptide
structures are shown in Figures 7 and 10. All Iipopeptides were administered in
saline. The non lipidated peptide was administered in CFA as a control. Sera
were obtained from blood taken at 4 weeks following the primary vaccination
and 2 weeks following the secondary vaccination.

Data shown in Figure 11 indicate that strong antibody responses are obtainable
using lipopeptides wherein the lipid moiety is attached to either position,
suggesting that strict placement of the internal lysine and, as a consequence,
the lipid moiety, is not essential to immunogenicity.
Discussion
In this study we describe the assembly of a variety of lipopeptide immunogens
composed of a CD4+ T cell epitope, the self peptide LHRH which includes one
or more B cell epitopes and Pam3Cys or Pam2Cys.
Without placing any strict requirement on the need for approximate central
positioning of the lipid, we found that the solubility of the resulting vaccine was
greatly improved by placing lipids in the approximate centre of the peptide
immunogen between the T cell epitope and LHRH instead of at the more usual
position at the N-terminus. A clear solution in saline at the concentration
required for inoculation could easily be obtained with these branched
structures. In contrast, the immunogens in which the lipid was coupled at the N-
terminus were less soluble, giving a cloudy or opalescent solution in saline.
Investigation of the antibody responses and subsequent fertility trials indicated
that the water-soluble lipopeptides induced higher antibody titres 4 weeks after
the primary inoculation and were also more efficient in preventing pregnancy
than were the less soluble lipopeptides where lipid was attached to the N-
terminus. A water-soluble self-adjuvanting vaccine has clear advantages over
partially soluble or insoluble material allowing for simplification of the
manufacturing process and also more accurate metering of dose.
Investigations into thte stringency of a requirement for positioning the lipid
moiety indicated that some fexibility is possible, since antibody responses were
also observed in immunized animals when the lipid was positioned within the T-
helper epitope, rather than between the T-helper epitope and the B-cell
epitope.

Investigations into the effects of varying the lipopeptide dose indicated that
Pam2Cys-containing lipopeptides are better immunogens than are Pam2Cys-
containing peptides. However, other lipidopeptides were also useful in
generating strong antibody responses, such as, for example, Ste2Cys-
containing lipopeptides, Lau2Cys-containing lipopeptides, and Oct2Cys-
containing lipopeptides.
We found in the present study that insertion of two serine residues or two
arginine residues between the lipid moiety and the peptide sequence increased
the potency of the resulting Pam2Cys-containing immunogens. When lipid is
attached to the N-terminus, the two serine residues could either be acting as an
inert spacer between the lipid and the peptide sequence or as an extension of
the T helper cell epitope and perhaps modulating immunological activity. In
those cases where lipid is coupled to the epsilon-amino group of a lysine
residue at the centre of the molecule, the two serine residues or two arginine
residues are acting as a spacer, because the inert spacer, 6-aminohexanoic
acid achieved similar results.
We also found that the immunogenicity of lipopeptide constructs was not
dependent upon the specific amino acid sequence of the T-helper epitope or
the B-cell epitope used, indicating general utility of the approach taken to
producing a wide range of lipopeptides against different antigenic B-cell
epitopes and in a number of different animal hosts.
It is understood that macrophages are stimulated by microbial products which
bind to cell surface receptors; the signal resulting from this binding event is
transmitted via Toll-like receptors and results in the production of pro-
inflammatory cytokines and chemokines. These receptors are also present in
populations of DC, and, when engaged, transmit signals for cellular maturation

and migration as well as for the production of molecules required for efficient
antigen presentation.
The various synthetic lipopeptide vaccines used in this study were found to
induce the up-regulation of class II MHC molecules, a marker used to assess
DC maturation, on the surface of immature DC. In contrast, the non-lipidated
peptide construct was unable to cause maturation of DC indicating that the lipid
moiety is responsible for the effect. The hierarchy of lipopeptide-induced
maturation of DC reflects the hierarchy of immunogenicity exhibited by the
peptide constructs implies that the ability of the vaccine to interact with and
Induce maturation of DC leads to a better immune response, possibly by
increasing the efficiency of CD4+ T cell priming by DC that have been signalled
to mature and migrate to the draining lymph node.
The lipopeptides can trigger an immune response in the absence of additional
adjuvant and can therefore be delivered by non-parenteral routes. We
therefore investigated the antibody response following intranasal inoculation of
Pam2Cys-containing peptides. The results obtained here showed that
intranasal inoculation of [Th]-Lys(Pam2Cys-Ser-SerHB] or Pam2Cys-Ser-Ser-
[Th]-[B] induced lower titres of systemic anti-LHRH antibody than those induced
by inoculation by the subcutaneous route and also that the isotype profiles of
immunoglobulins were different. Intranasal inoculation of the soluble lipopeptide
[Th]-Lys(Pam2Cys-Ser-Ser)-[B] induced higher levels of lgG2b and lgG3, but
lower levels of lgG1 and lgG2a compared to subcutaneous immunization. This
may indicate that the two routes of immunization result in the induction of
somewhat different subsets of T cells providing help for antibody production
which may, in part, be due to the different populations of DCs encountered at
different sites. It may also reflect a preference that dendritic cells have for
molecules with unusual geometries.

Intranasal inoculation of the water-soluble peptide construct [Th]-Lys(Pam2Cys-
Ser-Ser)-[B] induced significantly higher anti-LHRH antibody titres 4 weeks
after the first dose of vaccine than did insoluble Pam2Cys-Ser-Ser-[Th]-[B].
Fertility trials carried out with these mice demonstrated that only intranasal
inoculation with [Th]-Lys(Pam2Cys-Ser-Ser)-[B] was able to totally prevent
reproduction. Although similar antibody titres were apparent in both groups of
mice following the second dose of antigen, high titres of antibody were only
elicited during the primary response to [Th]-Lys(Pam2Cys-Ser-Ser)-[B]. It is
therefore possible that for an immunocontraceptive vaccine to be effective, the
time for which high titres of antibody are present is an important determinant of
efficacy.
Taken together, the measurements of antibody titres and the results of the
fertility trials demonstrate that placement of Pam2Cys between the B cell
epitope and the T helper epitope, at the approximate centre of a totally
synthetic peptide vaccine increases the solubility and also the immunogenicity
of the vaccine. This improved immunogenicity is further improved by the
introduction of two serine residues between the lipid and the peptide sequence
of these branched peptide vaccines. The finding that incorporation of lipid, self-
adjuvanting moieties into different positions of peptide-based vaccines
profoundly alters physical, immunogenic and biological properties provides
another strategy for successful vaccine design.

EXAMPLE 3
Studies on lipopeptides comprising a B cell epitope from the
M protein of Group A streptococcus
The effect of multiple lipids
To test whether or not immunogenicicty of the lipopeptides was dependent
upon the number of lipids conjugated to the peptides, and to demonstrate that
effective lipopeptides could be formulated against different antigenic B-cell
epitope-containing peptides, we produced lipopeptides comprising a peptide
moiety that comprises the CDV-F P25 T-helper epitope and a Group A
Streptococcus B cell epitope J14 (i.e. the peptide moiety has the amino acid
sequence of SEQ ID NO: 105), and one or two lipid moieties. The lipoamino
acid moiety Pam2Cys-Ser-Ser was added to an internal lysine positioned
between the T-helper epitope and the B-cell epitope and, in one construct, an
additional lipoamino acid moiety Pam2Cys-Ser-Ser was also added to an N-
terminal lysine in the T-helper epitope.
Female outbred Quackenbush mice 4-6 weeks old (15/group) were inoculated
intranasally with 60ug of peptide-based vaccine in a total volume of 30ul PBS.
Mice received three doses of vaccine at 21-day intervals. Fecal IgA was
determined 6 days following the last dose of antigen. Seven days following the
final dose mice were bled from the tail vein and J14-specific serum IgG was
determined. Indirect bacteriocidal assays were also performed to determine
the ability of sera from immunized mice to opsonise or "kill" the M1 GAS strain
in vitro. Eight days following the final dose saliva was collected from individual
mice and the average J14-specific salivary IgA antibody titres were determined
by standard ELISA. Two weeks after the last dose of antigen, mice were
challenged intranasally with M1 GAS strain and survival determined at various
time points afterwards.

Data in Figure 12 indicate that significant (P elicited using either lipopeptide compared to non-lipidated peptides or PBS,
indicating that the lipopeptide constructs are not dependent upon the selecton
of T-helper or B-cell epitope, and that lipopeptides comprising single or multiple
lipid moieties can be used to elicit high serum IgG levels following intranasal
immunization.
Data presented in Figure 13 also indicate that sera collected from mice
immunized with J14-containing lipopeptides having one or two lipid moieties
were also capable of significant (P collected from animals immunized with control non-lipidated peptides or PBS.
Data presented in Figure 14 indicate that mice inoculated J14-containing
lipopeptides having one or two lipid moieties had significantly (P saliva IgA titres than the control groups that were immunized control non-
lipidated peptides or PBS. However, the monolipidated peptide was far
superior than the bi-lipidated peptide in inducing saliva IgA levels by intranasal
administration.
Interestingly, only mice inoculated with mono-lipidated J14-containing peptide,
wherein the lipid moiety was positioned between the T-helper epitope and the
B-cell epitope (i.e., [Th]-Lys(Pam2Cys-Ser-Ser)-[J14]) had significant (P faecal IgA titres at 6 days following final immunization, compared to PBS or
non-lipidated peptide (Figure 15). This may be a consequence of timing, since
fecal IgA was determined before saliva IgA or serum IgG levels were
determined. Alternatively, it may be a consequence of the intranasal
administration route. Other explanations cannot be excluded at present.
As shown in Figure 16, mice inoculated with with mono-lipidated J14-containing
peptide, wherein the lipid moiety was positioned between the T-helper epitope
and the B-cell epitope (i.e., [Th]-Lys(Pam2Cys-Ser-Ser)-[J14]) also
demonstrated the best survival following intransal challenge with GAS,

compared to the bi-lipidated peptide or non-lipidated peptide. However, some
protective immunity was conferred by both the bi-lipidated peptide and non-
lipidated peptide compared to the J14 peptide alone or PBS.
In summary, the data presented in Examples 2 and 3 indicate that the
lipopeptide formulations of the present invention are broadly applicable to
inducing strong antibody responses in animals, particularly murine models, with
a variety of T-helper epitopes and B-cell epitopes. Additionally, the lipopeptide
formulations are particularly suited to intranasal administration, since strong
IgG and IgA responses are obtained by this route. However, our data indicate
that, at least for J14 immunogens, mono-lipidated peptides may serve as better
mucosal adjuvants than lipopeptides comprising multiple lipid moieties.

EXAMPLE 4
Studies on lipopeptides comprising a B cell epitope from gastrin
The immunogenicity of lipopeptide immunogens based on gastrin was
determined. Female BALB/c mice were inoculated subcutaneously in the base
of the tail with 20nmoles of peptide or lipopeptide immunogens. All
lipopeptides were administered in PBS and the non-lipidated peptides were
administered in CFA. Saline emulsified with CFA was used as a negative
control. The peptides used were Gastrin-17 (sequence
EGPWLEEEEEAYGWMDF; SEQ ID NO: 113); [P25Kys-[PentaGastrin] (SEQ
ID NO: 110) in which PentaGastrin is the C-terminai sequence GWMDF of
gastrin-17 (i.e., SEQ ID NO: 102); and [P25]-Lys(Pam2Cys-Ser-Ser}-
[PentaGastrin]. 4 weeks after immunisation sera was obtained from the
animals and at the same time they received a second similar dose of antigen.
Mice were bled a second time a further 2 week after the second dose of
antigen and antibodies capable of reacting with the peptide gastrin-17
sequence determined in ELISA.
As shown in Figure 17, mice inoculated with Gastrin-17 in CFA contained levels
of anti-Gastrin-17 antibodies equivalent to the negative control of Saline in
CFA. While immunisation with the non-lipidated peptide [P25]-Lys-
[PentaGastrin] elicited very low levels of anti-Gastrin-17 antibodies, mice
challenged with the lipopeptide [P25]-Lys(Pam2Cys-Ser-Ser)-[PentaGastrin]
demonstrated high antibody titres similar to that elicited after immunisation with
the peptide in CFA. These data again illustrate that the lipopeptide
formulations of the present invention are broadly applicable to inducing strong
antibody responses in animals, with a variety of T-helper epitopes and B-cell
epitopes.

WE CLAIM:
1. A lipopeptide comprising a polypeptide conjugated to one or more lipid
moieties such as herein described wherein:
(i) said polypeptide comprises an amino acid sequence that comprises:
(a) the amino acid sequence of a T helper cell (Th) epitope such as
herein described and the amino acid sequence of a B cell epitope, such
as herein described wherein said amino acid sequences are different;
and
(b) one or more internal lysine residues or internal lysine analog
residues for covalent attachment of each of said lipid moieties via the
epsilon-amino group or terminal side-chain group of said lysine or lysine
analog; and
(ii) each of said one or more lipid moieties is covalently attached to an
epsilon-amino group of said one or more internal lysine residues or to a
terminal side-chain group of said one or more internal lysine analog residues.
2. The lipopeptide as claimed in claim 1 wherein the lipid is attached to the
epsilon-amino group of a lysine residue.
3. The lipopeptide as claimed in claim 1 or 2 wherein the internal lysine
residue to which a lipid moiety is attached is positioned between the Th epitope
and the B cell epitope.
4. The lipopeptide as claimed in claim 1 or 2 wherein the internal lysine
residue to which a lipid moiety is attached is positioned within the Th epitope.
5. The lipopeptide as claimed in claim 1 or 2 comprising two lipid moieties.
6. The lipopeptide as claimed in claim 5 wherein an internal lysine residue
to which a lipid moiety is attached is positioned between the Th epitope and

the B cell epitope and an internal lysine residue to which a lipid moiety is
attached is positioned within the Th epitope.
7. The lipopeptide as claimed in any one of claims 1 to 6 wherein the lipid
moiety has a structure of General Formula (VII):

wherein:
(i) X is selected from the group consisting of sulfur, oxygen, disulfide (-S-
S-), and methylene (-CH2-), and amino (-NH-);
(ii) m is an integer being 1 or 2;
(iii) n is an integer from 0 to 5;
(iv) R1 is selected from the group consisting of hydrogen, carbonyl (-CO-),
and R'-CO- wherein R' is selected from the group consisting of alkyl
having 7 to 25 carbon atoms, alkenyl having 7 to 25 carbon atoms, and
alkynyl having 7 to 25 carbon atoms, wherein said alkyl, alkenyl or
alkynyl group is optionally substituted by a hydroxyl, amino, oxo, acyl, or
cycloalkyl group;
(v) R2 is selected from the group consisting of R'-CO-O-, R'-O-, R'-O-CO-,
R'-NH-CO-, and R'-CO-NH-, wherein R' is selected from the group
consisting of alkyl having 7 to 25 carbon atoms, alkenyl having 7 to 25
carbon atoms, and alkynyl having 7 to 25 carbon atoms, wherein said
alkyl, alkenyl or alkynyl group is optionally substituted by a hydroxyl,
amino, oxo, acyl, or cycloalkyl group; and

(vi) R3 is selected from the group consisting of R'-CO-O-, R'-O-, R'-O-CO-,
R'-NH-CO-, and R'-CO-NH-, wherein R' is selected from the group
consisting of alkyl having 7 to 25 carbon atoms, alkenyl having 7 to 25
carbon atoms, and alkynyl having 7 to 25 carbon atoms, wherein said
alkyl, alkenyl or alkynyl group is optionally substituted by a hydroxyl,
amino, oxo, acyl, or cycloalkyl group
and wherein each of R1, R2 and R3 are the same or different.
8. The lipopeptide as claimed in claim 7 wherein X is sulfur; m and n are
both 1; R1 is selected from the group consisting of hydrogen, and R'-CO-,
wherein R' is an alkyl group having 7 to 25 carbon atoms; and R2 and R3 are
selected from the group consisting of R'-CO-O-, R'-O-, R'-O-CO-, R'-NH-CO-,
and R'-CO-NH-, wherein R' is an alkyl group having 7 to 25 carbon atoms.
9. The lipopeptide as claimed in claim 8 wherein R' is selected from the
group consisting of: palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, and
decanoyl.
10. The lipopeptide as claimed in claim 9 wherein R' is selected from the
group consisting of: palmitoyl, stearoyl, lauroyl, and octanoyl, and decanoyl.
11. The lipopeptide as claimed in any one of claims 7 to 10 wherein the lipid
is contained within a lipoamino acid moiety selected from the group consisting
of: Pam2Cys, Pam3Cys, Ste2Cys, Lau2Cys, and Oct2Cys.
12. The lipopeptide as claimed in claim 11 wherein the lipoamino acid
moiety is selected from the group consisting of Pam2Cys, Ste2Cys, Lau2Cys,
and Oct2Cys.
13. The lipopeptide as claimed in claim 11 wherein the lipoamino acid
moiety has the structure of Formula (II):



14. The lipopeptide as claimed in any one of claims 1 to 6 wherein the lipid
moiety has the following General Formula (VIII):

wherein:
(i) R4 is selected from the group consisting of: (i) an alpha-acyl-fatty acid
residue consisting of between about 7 and about 25 carbon atoms; (ii)
an alpha-alkyl-beta-hydroxy-fatty acid residue; (iii) a beta-hydroxy ester
of an alpha-alkyl-beta-hydroxy-fatty acid residue; and (iv) a lipoamino
acid residue; and
(ii) R5 is hydrogen or the side chain of an amino acid residue.
15. The lipopeptide as claimed in any one of claims 1 to 14 wherein the lipid
moiety is separated from the peptide moiety by a spacer.
16. The lipopeptide as claimed in claim 15 wherein the spacer comprises
arginine, serine or 6-aminohexanoic acid.
17. The lipopeptide as claimed in claim 15 or 16 wherein the spacer
consists of a serine homodimer.

18. The lipopeptide as claimed in claim 15 or 16 wherein the spacer
consists of an arginine homodimer.
20. The lipopeptide as claimed in claim 15 or 16 wherein the spacer
consists of 6-aminohexanoic acid.
21. The lipopeptide as claimed in any one of claims 1 to 20 wherein the
internal lysine or internal lysine analog is nested within a synthetic amino acid
sequence having low immunogenicity.
22. The lipopeptide as claimed in any one of claims 1 to 21 wherein the T-
helper epitope is a T-helper epitope of influenza virus haemagglutinin or a T-
helper epitope of canine distemper virus F (CDV-F) protein.
23. The lipopeptide as claimed in claim 22 wherein the a T-helper epitope of
influenza virus haemagglutinin comprises the amino acid sequence set forth in
SEQ ID NO: 1 or SEQ ID NO: 18.
24. The lipopeptide as claimed in claim 23 wherein the a T-helper epitope of
influenza virus haemagglutinin comprises the amino acid sequence set forth in
SEQ ID NO: 1.
25. The lipopeptide as claimed in claim 22 wherein the T-helper epitope of
CDV-F protein comprises the amino acid sequence set forth in SEQ ID NO: 24.
26. The lipopeptide as claimed in any one of claims 1 to 25 wherein the B
cell epitope is from an immunogenic protein, lipoprotein, or glycoprotein of a
virus.
27. The lipopeptide as claimed in any one of claims 1 to 25 wherein the B
cell epitope is from an immunogenic protein, lipoprotein, or glycoprotein of a
prokaryotic organism.

28. The lipopeptide as claimed in claim 27 wherein the B cell epitope is from
the M protein of Group A streptococcus.
29. The lipopeptide as claimed in claim 28 wherein the B cell epitope
comprises the amino acid sequence set forth in SEQ ID NO: 101.
30. The lipopeptide as claimed in any one of claims 1 to 25 wherein the B
cell epitope is from an immunogenic protein, lipoprotein, or glycoprotein of a
eukaryotic organism.
31. The lipopeptide as claimed in claim 30 wherein the eukaryotic organism
is a parasite.
32. The lipopeptide as claimed in claim 30 wherein the eukaryotic organism
is a mammal.
33. The lipopeptide as claimed in claim 32 wherein the B cell epitope is from
a peptide hormone of a mammal.
34. The lipopeptide as claimed in claim 33 wherein the peptide hormone is
a digestive hormone or a reproductive peptide hormone.
35. The lipopeptide as claimed in claim 34 wherein the digestive hormone is
gastrin or pentagastrin.
36. The lipopeptide as claimed in claim 35 comprising the amino acid
sequence set forth in SEQ ID NO: 102 or SEQ ID NO: 113.
37. The lipopeptide as claimed in claim 34 wherein the reproductive
hormone is luteinising hormone-releasing hormone (LHRH) or a fragment
thereof.

38. The lipopeptide as claimed in claim 31 comprising the amino acid
sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4.
39. The lipopeptide as claimed in any one of claims 1 to 38 wherein the
polypeptide comprises an amino acid sequence selected from the group
consisting of:
a polypeptide comprising an amino acid sequence selected from the group
consisting of:
(xv) GALNNRFQIKGVELKSEHWSYGLRPG (SEQ ID NO: 5);
(xvi) GALNNRFQIKGVELKSKEHWSYGLRPG (SEQ ID NO: 7);
(xvii) KLIPNASLIENCTKAELKHWSYGLRPG (SEQ ID NO: 9);
(xviii) KLIPNASLIENCTKAELKGLRPG (SEQ ID NO: 13);
(xix) KLIPNASLIENCTKAELHWSYGLRPG (SEQ ID NO: 103);
(xx) KLIPNASLIENCTKAELGLRPG (SEQ ID NO: 104);
(xxi) KLIPNASLIENCTKAELKQAEDKVKASREAKKQVEKALEQLEDKVK
(SEQ ID NO: 105);
(xxii) KLIPNASLIENCTKAELKKQAEOKVKASREAKKQVEKALEQLEOKVK
(SEQ ID NO: 106);
(xxiii) GALNNRFQIKGVELKSKQAEDKVKASREAKKQVEKALEQLEDKVK
(SEQ ID NO: 107);
(xxiv) GALNNRFQIKGVELKSKKQAEDKVKASREAKKQVEKALEQLEOKVK
(SEQ ID NO: 108);
(xxv) KLIPNASLIENCTKAELGWMDF (SEQ ID NO: 109);
(xxvi) KLIPNASLIENCTKAELKGWMDF (SEQ ID NO: 110);
(xxvii) GALNNRFQIKGVELKSGWMDF (SEQ ID NO: 111); and
(xxviii) GALNNRFQIKGVELKSKGWMOF (SEQ ID NO: 112).
40. The lipopeptide as claimed in any one of claims 1 to 39 capable of
upregulating the surface expression of MHC class II molecules on immature
dendritic cells (DC).

41. The lipopeptide as claimed in claim 40 wherein the DC are D1 cells.
42. A lipopeptide comprising a polypeptide conjugated to one or more lipid
moieties wherein:
(i) said polypeptide comprises an amino acid sequence that comprises:
(a) the amino acid sequence of a T helper cell (Th) epitope and the
amino acid sequence of a B cell epitope, wherein said amino acid
sequences are different; and
(b) one or more internal lysine residues for covalent attachment of each
of said lipid moieties via the epsilon-amino group of said one or more
lysine residues;
(ii) each of said one or more lipid moieties is covalently attached to an
epsilon-amino group of said one or more internal lysine residues; and
(iii) said lipopeptide has the general Formula (VI):

epitope is a T-helper epitope or B-cell epitope;
A is either present or absent and consists of an amino acid spacer
of 1 to 6 amino acids in length;
n is an integer having a value of 1, 2, 3, or 4;
X is a terminal side-chain group selected from the group consisting
of NH, O and S;
Y is either present of absent and consists of a spacer of 1 to 6
amino acids in length, wherein said spacer comprises arginine,
serine or 6-aminohexanoic acid; and

Z is a lipoamino acid moiety selected from the group consisting of
Pam2Cys, Pam3Cys, Ste2Cys, Lau2Cys, and Oct2Cys.
43. The lipopeptide as claimed in claim 42 wherein A is absent.
44. The lipopeptide as claimed in claim 43 wherein the B cell epitope
comprises the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO:
3 or SEQ ID NO:4.
45. The lipopeptide as claimed in claim 43 wherein: (i) the B cell epitope
comprises the amino acid sequence set forth in SEQ ID NO: 101; (ii) Y is
present and consists of a serine homodimer; and (iii) Z consists of Pam2Cys.
46. The lipopeptide as claimed in claim 45 wherein the T helper epitope
comprises the amino acid sequence set forth in SEQ ID NO: 24 and wherein a
lipid moiety is attached to the polypeptide via the epsilon-amino group of a
lysine residue within SEQ ID NO: 24.
47. The lipopeptide as claimed in claim 45 wherein the lipid moiety is
attached to the polypeptide via Lys-14 of SEQ ID NO: 24.
48. The lipopeptide as claimed in claim 43 wherein: (i) the B cell epitope
comprises the amino acid sequence set forth in SEQ ID NO: 102; (ii) Y is
present and consists of a serine homodimer; and (iii) Z consists of Pam2Cys.
49. The lipopeptide as claimed in any one of claims 42 to 48 capable of
upregulating the surface expression of MHC class II molecules on immature
dendritic cells (DC).
50. The lipopeptide as claimed in claim 49 wherein the DC are D1 cells.
51. A method of producing a lipopeptide comprising:

(i) producing a polypeptide comprising an amino acid sequence that
comprises:
(a) the amino acid sequence of a T helper cell (Th) epitope and the
amino acid sequence of a B cell epitope, wherein said amino acid
sequences are different; and
(b) one or more internal lysine residues or internal lysine analog
residues; and
(ii) covalently attaching each of said one or more lipid moieties directly or
indirectly to an epsilon-amino group of said one or more internal lysine
residues or to the terminal side-chain group of said one or more internal
lysine analog residues so as to produce a lipopeptide having the lipid
moiety attached to the epsilon amino group of said internal lysine
residue or having the lipid moiety attached to the terminal side-chain
group of said internal lysine analog residue.
52. The method as claimed in claim 51 wherein the polypeptide is
synthesized by a chemical synthesis means.
53. The method as claimed in claim 51 or 52 comprising producing the lipid
moiety.
54. The method as claimed in claim 53 comprising synthesizing the lipid
moiety as a lipoamino acid.
55. The method as claimed in claim 54 comprising adding a spacer to the
amino acid moiety of the lipoamino acid.
56. The method as claimed in claim 55 wherein the lipid comprises an
arginine homodimer or serine homodimer or 6-aminohexanoic acid .
57. The method as claimed in claim 55 or 56 comprising adding the spacer
to the lipoamino acid via the terminal carboxy group in a process that

comprises performing a condensation, addition, substitution, or oxidation
reaction.
58. The method as claimed in any one of claims 55 to 57 wherein the spacer
comprises a terminal protected amino acid residue to facilitate conjugation of
the lipoamino acid to a polypeptide.
59. The method as claimed in claim 58 comprising de-protecting the terminal
protected amino acid of the spacer and conjugating the lipoamino acid to a
polypeptide.
60. The method as claimed in claim 54 comprising adding a spacer to a non-
modified epsilon amino group of the polypeptide in a process comprising
performing a nucleophilic substitution reaction.
61. The method as claimed in claim 60 wherein the polypeptide has an
amino acid sequence comprising a single internal lysine or lysine analog
residue and a blocked N-terminus.
62. The method as claimed in claim 60 or 61 wherein the lipid comprises an
arginine homodimer or serine homodimer or 6-aminohexanoic acid .
63. A composition comprising the lipopeptide as claimed in any one of
claims 1 to 50 and a pharmaceutically acceptable excipient or diluent.
64. The composition as claimed in claim 63 comprising a biologic response
modifier (BRM).
65. A composition for eliciting the production of antibody against an
antigenic B cell epitope in a subject comprising the lipopeptide as claimed in
any one claims 1 to 50 or the composition as claimed in claim 63 or 64
wherein said composition is administrable for a time and under conditions

sufficient to elicit the production of antibodies against said antigenic B cell
epitope.
66. A composition as claimed in claim 65 wherein the lipopeptide is
administered intranasally to the subject.
67. A composition as claimed in claim 66 wherein the lipopeptide is
administered to the subject by injection.
68. A composition as claimed in any one of claims 65 to 67 for eliciting the
production of high titer antibodies.
69. A composition as claimed in any one of claims 65 to 68 wherein the
antigenic B cell epitope is from a pathogen and wherein said composition is for
generating neutralizing antibodies against the pathogen.
70. A composition as claimed in any one of claims 65 to 69 for producing a
monoclonal antibody against the antigenic B cell epitope.
71. A composition for inducing infertility in a subject comprising a lipopeptide
comprising a polypeptide conjugated to one or more lipid moieties, wherein:
(i) said polypeptide comprises:
(a) the amino acid sequence of a T helper cell (Th) epitope and the
amino acid sequence of a B cell epitope of a reproductive
hormone or hormone receptor, and wherein said amino acid
sequences are different;
(b) one or more internal lysine residues or internal lysine analog
residues for covalent attachment of each of said lipid moieties via
an epsilon-amino group of said internal lysine or via a terminal
side-chain group of said internal lysine analog; and
(c) each of said one or more lipid moieties is covalently attached
directly or indirectly to an epsilon-amino group of said one or

more internal lysine residues or to a terminal side-chain group of
said one or more internal lysine analog residues; wherein
(ii) said composition is administrable for a time and under conditions
sufficient to elicit a humoral immune response against said antigenic B
cell epitope.
72. The composition as claimed in claim 71 wherein the lipopeptide is
administered in combination with a pharmaceutically acceptable excipient or
diluent.
73. The composition as claimed in claim 71 or 72 wherein a secondary
immune response is generated against the B cell epitope sufficient to prevent
oogenesis, spermatogenesis, fertilization, implantation, or embryo
development in the subject.
74. The composition as claimed in any one of claims 71 to 73 wherein
antibody levels are sustained for at least a single reproductive cycle of an
immunized female subject.
75. The composition as claimed in any one of claims 71 to 74 wherein the B
cell epitope is derived from the amino acid sequence of luteinising hormone-
releasing hormone (LHRH).
76. The composition as claimed in claim 75 wherein the B cell epitope
comprises the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO:
3 or SEQ ID NO:4.
77. The composition as claimed in any one of claims 71 to 76 wherein the
T-helper epitope comprises an amino acid sequence as set forth in SEQ ID
NO: 1 or SEQ ID NO: 24.

78. The composition as claimed in any one of claims 71 to 77 wherein the
lipid moiety comprises a lipoamino acid selected from the group consisting of:
(i) Pam2Cys; (ii) Ste2Cys; (iii) Lau2Cys; and (iv) Oct2Cys.
79. A contraceptive agent comprising the lipopeptide as claimed in any one
of claims 1 to 50 wherein the B cell epitope is from a reproductive hormone or
hormone receptor.
80. A contraceptive agent comprising the lipopeptide as claimed in claim 44.
81. The lipopeptide as claimed in claim 44 in the preparation of a
contraceptive reagent for reducing fertility in an animal subject.
82. A composition for inducing an immune response against a Group A
streptococcus antigen in a subject comprising a lipopeptide comprising a
polypeptide conjugated to one or more lipid moieties, wherein:
(i) said polypeptide comprises:
(a) the amino acid sequence of a T helper cell (Th) epitope and the
amino acid sequence of a B cell epitope of a Group A
streptococcus antigen, and wherein said amino acid sequences
are different;
(b) one or more internal lysine residues or internal lysine analog
residues for covalent attachment of each of said lipid moieties via
an epsilon-amino group of said internal lysine or via a terminal
side-chain group of said internal lysine analog; and
(c) each of said one or more lipid moieties is covalently attached
directly or indirectly to an epsilon-amino group of said one or
more internal lysine residues or to a terminal side-chain group of
said one or more internal lysine analog residues; wherein
(ii) said composition is administrable for a time and under conditions
sufficient to elicit a humoral immune response against said antigenic B
cell epitope.

83. The composition as claimed in claim 82 wherein the lipopeptide is
administered in combination with a pharmaceutically acceptable excipient or
diluent.
84. The composition as claimed in claim 82 or 83 wherein a secondary
immune response is generated against the B cell epitope sufficient to prevent
the spread of infection by a Group A streptococcus and/or reduce morbidity or
mortality in a subject following a subsequent challenge with a Group A
streptococcus.

85. The composition as claimed in any one of claims 82 to 84 wherein the
B cell epitope is derived from the amino acid sequence of the M protein of
Group A streptococcus.
86. The composition as claimed in claim 85 wherein the B cell epitope
comprises the amino acid sequence set forth in SEQ ID NO: 101.
87. The composition as claimed in any one of claims 82 to 86 wherein the
T-helper epitope comprises an amino acid sequence as set forth in SEQ ID
NO: 1 or SEQ ID NO: 24.
88. The composition as claimed in any one of claims 82 to 87 wherein the
lipid moiety comprises Pam2Cys.
89. A vaccine comprising the lipopeptide as claimed in any one of claims 1
to 50 wherein the B cell epitope is from the M protein of Group A
streptococcus.
90. A vaccine comprising the lipopeptide as claimed in claim 45.

91. The lipopeptide as claimed in claim 45 in the preparation of a
contraceptive reagent for reducing fertility in an animal subject.
92. A composition for inducing an immune response against a gastrin
peptide in a subject comprising a lipopeptide comprising a polypeptide
conjugated to one or more lipid moieties, wherein:
(i) said polypeptide comprises:
(a) the amino acid sequence of a T helper cell (Th) epitope and the
amino acid sequence of a B cell epitope of a gastrin polypeptide
antigen, and wherein said amino acid sequences are different;
(b) one or more internal lysine residues or internal lysine analog
residues for covalent attachment of each of said lipid moieties via
an epsilon-amino group of said internal lysine or via a terminal
side-chain group of said internal lysine analog; and
(c) each of said one or more lipid moieties is covalently attached
directly or indirectly to an epsilon-amino group of said one or
more internal lysine residues or to a terminal side-chain group of
said one or more internal lysine analog residues; wherein
(ii) said composition is administrable for a time and under conditions
sufficient to elicit a humoral immune response against said antigenic B
cell epitope.
93. The composition as claimed in claim 92 wherein the lipopeptide is
administered in combination with a pharmaceutically acceptable excipient or
diluent.
94. The composition as claimed in claim 92 or 93 wherein a secondary
immune response is generated against the B cell epitope sufficient to prevent
or block secretion of gastric acid in an animal in need thereof.

95. The composition as claimed in claim 94 wherein the animal suffers from
a condition selected from the group consisting of hypergastrinemia, Zollinger-
Ellison syndrome, gastric ulceration, duodenal ulceration and gastrinoma.
96. The composition as claimed in any one of claims 92 to 95 wherein the
B cell epitope is derived from the amino acid sequence of pentagastrin.
97. The composition as claimed in claim 96 wherein the B cell epitope
comprises the amino acid sequence set forth in SEQ ID NO: 102.
98. The composition as claimed in any one of claims 92 to 97 wherein the
T-helper epitope comprises an amino acid sequence as set forth in SEQ ID
NO: 24.
99. The composition as claimed in any one of claims 92 to 98 wherein the
lipid moiety comprises Pam2Cys.
100. A vaccine comprising the lipopeptide as claimed in any one of claims 1
to 50 wherein the B cell epitope is from a gastrin polypeptide.
101. A vaccine comprising the lipopeptide as claimed in claim 46.
102. The lipopeptide as claimed in claim 46 in the preparation of a
contraceptive reagent for reducing fertility in an animal subject.
103. The composition as claimed in any one of claims 65 to 70 wherein the
antibody comprises an immunoglobulin selected from the group consisting of
IgM, IgA, and IgG.
104. The composition as claimed in claim 103 wherein the immunoglobulin is
IgM.

105. The composition as claimed in claim 103 wherein the immunoglobulin is
IgA.
106. The composition as claimed in claim 103 wherein the immunoglobulin is
IgG.
107. The composition as claimed in claim 106 wherein the IgG is selected
from the group consisting of lgG1, lgG2a, lgG2b, and lgG3.

The present invention provides a lipopeptide comprising a polypeptide
conjugated to one or more lipid moieties wherein:
(i) said polypeptide comprises an amino acid sequence that comprises:
(a) the amino acid sequence of a T helper cell (Th) epitope and the
amino acid sequence of a B cell epitope, wherein said amino acid
sequences are different; and
(b) one or more internal lysine residues or internal lysine analog
residues for covalent attachment of each of said lipid moieties via the
epsilon group or terminal side-chain group of said lysine or lysine
analog; and
(ii) each of said one or more lipid moieties is covalently attached to an
epsilon-amino group of said one or more internal lysine residues or to a
terminal side chain group of said one or more internal lysine analog residues.

Documents:

278-KOLNP-2005-FORM-27.pdf

278-kolnp-2005-granted-abstract.pdf

278-kolnp-2005-granted-assignment.pdf

278-kolnp-2005-granted-claims.pdf

278-kolnp-2005-granted-correspondence.pdf

278-kolnp-2005-granted-description (complete).pdf

278-kolnp-2005-granted-drawings.pdf

278-kolnp-2005-granted-examination report.pdf

278-kolnp-2005-granted-form 1.pdf

278-kolnp-2005-granted-form 18.pdf

278-kolnp-2005-granted-form 3.pdf

278-kolnp-2005-granted-form 5.pdf

278-kolnp-2005-granted-gpa.pdf

278-kolnp-2005-granted-reply to examination report.pdf

278-kolnp-2005-granted-sequence listing.pdf

278-kolnp-2005-granted-specification.pdf


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Patent Number 228050
Indian Patent Application Number 278/KOLNP/2005
PG Journal Number 05/2009
Publication Date 30-Jan-2009
Grant Date 28-Jan-2009
Date of Filing 25-Feb-2005
Name of Patentee THE COUNCIL OF THE QUEENSLAND INSTITUTE OF MEDICAL RESEARCH
Applicant Address 300 HERSTON ROAD, HERSTON, QUEENSLAND 4029
Inventors:
# Inventor's Name Inventor's Address
1 JACKSON DAVID 74 WOODVILLE STREET, NORTH BALWYN, VICTORIA 3104
2 ZENG WEIGUANG 46 MERCANTILE PARADE, KENSINGTON, VICTORIA 3031
PCT International Classification Number C07K 9/00
PCT International Application Number PCT/AU2003/001018
PCT International Filing date 2003-08-12
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/402,838 2002-08-12 U.S.A.