Note: Descriptions are shown in the official language in which they were submitted.
~2~ V{)
A~TIGENIC MODIFICATION OF POLYPEPTIDES
This invention relates to antigenic modification of polypeptides. More
particularly, this invention relates to specific techniques for modification of
polypeptides, to antigens for provoking the formation, by the immune system of an
animal, of antibodies to exogenous proteins e.g. proteins found in viruses and other
05 pathogenic organisms, and to processes for fertility control using chemically-
modified antigens.
In our British patent No. 1,567,764 and in our U.S. patent No. 4,302,386
it is disclosed that
hormones or other proteins found naturally in an animal can be chemically modified
10 outside the body of the animal (a term which is used herein to include human beings)
so that, when injected into the animal, the modified hormones or other proteins
provoke the formation of antibodies which react not only with the chemically-
modi~led hormone or other protein but also with the unmodified hormone or other
protein found naturally in the animal, thereby reducing the level of the natural15 hormone or other protein in the body of the animal. Instead of using the entire
hormone or other protein, the aforementioned patents also disclose the use of
natural or synthetic fragments of such proteins in this so-called "isoimmlmization~'
technique.
The aforementioned patents are mainly concerned with the application of their
20 isoimmunoization technique in controlling fertility, so that the protein which is
modified is a reproducti~re hormone, such as Follicle Stimulating Hormone (FSH),Leutinising Hormone (LH), Human Placental Lactogen (llPL), ~luman Prolactin and
Human Chorionic Gonadotropin (HCG), or a peptide having an amino acid sequence
corresponding to a part of one of these hormones. However, the aforementioned
25 patents also disclose the applicability of the isoimmunization technique to other
protein hormones, for example:
1. Gastrin, for the treatment of Zollinger-Ellison Syndrome;
2. Angiotension 11, for the treatment of hypertension;
~ 3. Growth hormone and somatomedian, for the treatment of diabetes and
associated micro and macro vascular diseases;
4. Parathyroid hormone for the treatment of kidney stones;
5. Insulin and glucagon, for the treatment of hyperinsulinoma;
6. Thyroid stimulating hormone, for the treatment of hyperthyroidism;
7. Secretin, for the trea~ment Or irritable bowel syndrome.
1','~ ~;
The ~forementioned patents also disclose the use of rnodified polypeptides derived
from chorionic gonadotropin, LH or FSH, or a frugment thereof, for use in treating
certain carcinomas.
The main technique of chemical modification disclosed in the aforementioned
ûS patents is coupling of the hormone, other than protein or fragment thereof to a
large ~'carrier" molecule such as diphtheria toxoid, tetanus toxoid or a synthetic
polymer; these carrier molecules are not endogenous to the animal to be treated.The uforementioned patents also describe polymerization of the hormone, other
protein, or frsgment thereof by reaction with a bifunctional organic reagent (for
10 example a bi-functional imidoester such as dimethyl adipimidate, dimethyl sube~
imidate or diethyl malonimidate) or dimerization by oxidation of a thiol group to
form a disulfide bridge. All these modes of chemical modification have dis-
advantages. Dimerization of the hormone, other protein or fragment ViQ a disulfide
bridge does not introduce exogenous material into the animal being treated but,
15 since the chemically-modified antigen udministered to the ~nimal is only a dimer of
a hormone, other protein or fragment which is not itself immunogenic to the animal,
such dimers are rarely successful in provoking useful levels of antibodies. The
modified polypeptides produced using carriers contain a very high proportion of non-
endogenous materiul, and will usually provoke the formation of substantial quan-
20 tities of an~ibodies to the carrier as well as to the hormone, other protein orfra~nt ther~of. Altho~h the forrnation of antibodies to the carrier may
sometimes be useful (for example, a vaccine based upon 8 HCG frugment coupled todiphtheria toxoid and intended for fertility control has the incidental advantage ot
also conferring protection against diphtheria), there are mnny occasions upon which
25 it is not desirable to provoke the formation of relatively large quantities of
antibodies to the carrier; for example, if one wishes to use a vaccine containing e
modified polypeptide to tre~t B patient with a c~rcinoma or a serious viral infection,
it may be desirable to avoid overstraining t~le patient's immune system by
ch011enging it not only with the hormone, other protein or fragment thereof to which
30 antibodies ure desired, ~ut slso with the carrier.
In theory, perhups the most promising of the modification techniques disclosed
in the aforementioned patents is polymerization of the hormone, other protein orfragment thereof by reaction with a bifunctional reagent. This technique has, intheory, the advuntages of introducing a relutively small proportion Or non-
35 cndogenous ma~erial into the animal bcing treuted (and even this relatively small
~s proportion of non-endogenous materinl can bc choscn so that it is not strongly
o~
immunogenic), while still providing a modified polypeptide large enough to be
stron~ly immunogenic. Unfortunately, experiments have proved that straight-
forward application of the bifunctional org~nic reagent polymerization technique to
most hormones, other proteins or fragments thereof of practical interest produces
05 very complicated mixtures of modified polypeptides having correspondingly com-
plicated immunogenic properties. Furthermore, the immunogenic properties of the
polymerized polypeptides thus produced are not readily reproduceable, but such
reproduceability is essential in any material intended for pharmaceutical use, since
the necessary tests of safety and efficiency cannot be performed on non-repro-
10 duceable material.
We have now found (though this knowledge is not in the published literature)that the reason for the very complicated immunogenic properties and the lack of
reproduceability present in polymers produced by the bifunctional organic reagent
polymerization technique is that, notwithstanding the use of a bifunctional reagent,
15 extensive cross-linking of the hormone, other protein fragment thereof occurs, such
crosslinking presumably being due to the presence of free amino, thiol, carboxyland perhaps other groups (the exact groups involved depending of course upon which
groups the bifunctional organic reagent is designed to react with) at non-terminal
positions on the hormone, protein or fragment thereof. Such cross-linking produces
20 branching and three-dimensional structure in the resultant polymers. Not only does
the relatively random cross-linking thus produced render the structure of the
polymers themselves unpredictable and non-reproduceable, but such cross-linking
may well alter the tertiary structure and shape of the hormone, other protein orfragment thereof being polymerized, thus affecting its immunogenic properties.
Accordingly, we have concluded that to produs~e useful modified polypeptides
by the bifunctional organic reagent polymerization technique, it is essential tooperate in such a way that coupling of the polypeptide fragments being polymerized
occurs only at or near the terminals of the fragments, thus producing a true linear
polymer substantially free of non-linear polymers of the fragments.
It has also been found that the isoimmunization technique described in the
af orem entioned patent csn usefully be extended to proteins which are not endo-genous nor substantially immunogenic to the animal to be treated. Finally, it has
been found that modified antigens for use in fertility control can be produced by
chemical modification of zona pellucida or sperm antigens.
A~cordingly, in one aspect this invention defines a modified polypeptide for
provoking the formation, in the body of an animal, of antibodies to a protein, the
.` f:
' ' ~ .
. : ~
o~
-4-
modified polypeptide being char~cterized in that it comprises a linear polymer ofpolypeptide fragments, each of the fragments, in its monomeric form, being
substanti~lly non-immunogenic to the anim~l and having a molecular structure
similar to Q frQgment of the protein to w~iich ~ntibodies are to be provoked, the
05 linear polymer, after administration into the body of the animal, having a gre~ter
capacity to provoke the formation of the antibodies than the protein, the linetlr
polymer being substantially free of non-linear polymers of the fragments.
In another aspect, this invention provides Q method for producing a line~r
polymeric polypeptide for provoking the formation, in the body of an animQl, of
10 antibodies to one or more proteins which is substantially non-immunogenic to the
animal, the method being characterized by:
(a) procuring a first peptide h~ving a molecular structure similar to Q
fragment of the or one of the proteins;
(b) reacting this first peptide with a bifunction~l coupling reagent while
15 the first peptide is in ~ form having only a single site capable of reacting with the
coupling reagent, this site being at or adjacent one of the termini of the firstpeptide, thereby c~using one of the functional groups of the coupling reagent toreact with the one site on the peptide;
(c) reacting the product of step (b) with a second peptide having fl
20 molecular structure similar to a frsgment of the or one of the proteins while the
second peptide is in a form having only n single site capable of reacting with the
other functional group of the coupling reagent, this site being at or adjacent one of
the termini of the second peptide, thereby forming a dimeric peptide wherein thefirst and second peptide ~re interconnencted vi~ a residue of the coupling reagent;
(d) reacting the resultant peptide with a bifunctional coupling reagent
while the peptide is in a form h~ving only e single site capable of reacting with the
coupling reagent, this site being at or adjacent one of the termini of the peptide,
thereby causing one of the function~l groups of the coupling reagent to re~ct with
the one site on the peptide;
(e) rencting the product of step (d) with ~ further peptide having a
molecular structure simil~r to Q fragment Df the or one of the proteins while this
further pepUde is in a form having only Q single site capable of rencting with the
other functionfll group of the coupling reagent, this site being at or adjecent one of
the termini of the further peptide, thereby torming a polymeric peptide wherein thc
35 first, second and further peptides are interconnected via residues of the coupling
raagent;
~`
.,
,
~x~ o~
(f) repeating steps (d) and (e) until the desired polymer length has been
achieved.
In another aspect, this invention provides a method for producing a modified
polypeptide for provoking the formation, in the body of an animal, of antibodies to a
05 protein which is substantially non-immunogenic to the animal, the method being
characterized by:
a. procuring a first peptide having a molecular structure similar to a
fragment of the protein, the first peptide not having an unblocked thiol
group and having an unblocked amino group only at its N-terminal but no
other unblocked amino group;
b. reacting the first peptide with an amino group activating agent, thereby
producing an activated amino group at the N-terminal of the first
peptide;
c. reacting the activated first peptide with a second peptide having a
molecular structure similar to Q fragment of the protein, the second
peptide having a C-terminal cysteine bearing an unblocked thiol group
but not having any other unblocked thiol groups, thereby coupling the N-
terminal of the first peptide to the C-terminal of the second peptide;
d. reacting the resultant compound in a form having an unblocked amino
group at its N-terminal but no other unblocked amino groups, and no
unblocked thiol group, with an amino-group activating agent, thereby
producing an activated amino-group at the N-terminal of the resultant
compound;
e. reacting the activated compound produced in step (d) with a further
peptide having a molecular structure similar to a fragment of the
protein, this further peptide having a C-terminal cys$eine bearing an
unblocked thiol group, but not having any other unblocked thiol groups,
thereby coupling the activated N-terminal of the reactivated compound
produced in step (d) to the C-terminal of the further peptide; and
f. repeating steps (d) and (e) until the desired polymer length has been
achieved.
In another aspect, this invention provides an anti~en for provoking the
formation, in the body of an animal, of antibodies to a protein which is not
endogenous nor substantially immunogenic to the animal, characteri~ed in that the
35 antigen comprises the protein, or a peptide having a sequence corresponding to at
least part of the sequence of the protein, which protein or peptide has been
~.~
,,
. . ~:
-: : -
:
. ,
~2~ 0()V
--6--
chemically modified outside the body of the animal, the antigen having a greatercapacity to provoke the form~tion of the antibodies than the protein in its
unmodified form.
In another aspect, this invention provides a process for preparing an antigen ofns the invention, which process is characterized by:
procuring the protein which is not endogenous or immunogenic to the animal,
or the peptide having a sequence corresponding to at least part of the sequence of
the protein; and
chemically modifying the protein or peptide outside the body of the animal,
10 thereby producing the antigen according to claim 3.
In another aspect, this invention provides a modified antigen for use in
fertility control in an animal characterized in that it comprises an antigen derived
from the zona pellucid~ or from sperm, or a peptide having a sequence corres-
ponding to at leQst part of the sequence of such a zona pellucida or sperm antigen,
15 which antigen or peptide has been chemically modified outside the body of theanimal, the modified antigen, after administration into the body of the animal,
having a greater capacity to provoke the formation of antibodies than the
unmodified antigen from which it is derived.
~inally, this inv~ntion provides a vaccine for provoking the formation, in the
20 body of an animal, of antibodies to a protein, this vaccine being characterized in
that it comprises a modified polypeptide, antigen or modified antigen of the
invention and a vehicle comprising a mixture of mannide monooleate with squalaneand/cr squalene.
As already mentioned, the linear polymeric modified polypeptides of the
25 invention comprise inert polymers of polypeptide fragments substantially free of
non-linear polymers. In saying that the polypeptide fragments used in the linearpolymeric polypeptide of the invention have a molecular structure similar to a
fragment of the protein to which antibodies are to be provoked, we do not
necessarily imply that the entire amino acid sequence of each fragment must
30 correspond exactly to part of the protein to which antibodies are to be provoked; for
example, in certain cases certain substitutions of amino acids may be possible
without affecting the immunogenic character of the fragment. For example, our
aforementioned U.S. p~tent No. 4,302,386 describes a polypeptide, designated
Structure (IX), which is notionally derived from the beta subunit of HCG but in
35 which the cysteine residue at the 110 position is replaced by alpha-aminoblltyric acid.
In particular, when it is desired to provoke the formation of antibodies to an
,
.. ~............................................................. .
. ~ .,' :, .
.... ~ ............... ::
. . , -, . ~ ,~ ' .
:' :~ ' ', -
~2~
--7--
endogenous protein, the polypeptide fragments used to form the appropriate
modified polypeptide of the invention must have a molecular structure similar to a
fragment of the endogenous protein which antibodies are lo be provoked, this does
not exclude the possibility thnt such fragments might actually be derived from a05 protein in a different species, since mnny proteins are either identical between
species or differ from one another so little in amino ncid sequence that considerable
cross-reactivity exists between antibodies to the corresponding proteins in the two
species. For example, as men~ioned below, zon~ pellucida enzymes from a pig will,
when injected into humans, produce antibodies which display considernble activity
10 against human zona antigens. Accordingly, for example, if one wishes to form a
modified polypeptide for provoking the formation of antibodies, in humans, to zona
pellucida untigens, appropriate polypeptide fragments may be prepared from zona
pellucida antigens of pigs. Also, the fragments used in the linear polymeric
polypeptides of the invention may incorporate sequences of amino acids having no15 counterpart in the sequence of the protein from which the Eragment is notionally
derived. Again, for example, our aforementioned U.S. patent No. 4,302,386
describes certain polypeptide fragments, designated Structure (IY), (VIII), (IX), (X)
and (XIV) which are notionally derived from the beta subunit of HCG but which
incorpornte spncer sequences comprising multiple proline residues.
It may at first appear surprising thAt 8 linear polymer of a polypeptide, the
monomeric form of which is effectively non-immunogenic to an animal9 can be
immunogenic to the same animal. It is believed (though the invention is in no way
limited by this belief) that the increase in immunogenicity upon polymerization is
due to the incrense in physical size of the molecule, which enables the molecule to
25 be recognized much more easily by the animal's immune system. It can be shownthat at least some monomeric polypeptides ars very weakly immunogenic and cause
the animal's immune system to produce detectable quantities of antibodies, whichquantities, however are much too small to be effective. Immune systems are not
well-adapted to recognize molecules as small ns the small polypeptides when the
30 polypeptides are present In polymeric form.
It will be Qpparent to those skilled in the art that the polypeptide fragments
used in the linear polymeric polypeptides of the invention may be either nfltural (i.e.
derived from natural proteins) or mny be produced synthetically. Obviously, a
synthetic polypeptide will perîorm in the same manner ns A nnturally occuring one in
35 as much as the immune system of the animal to which the modified polypeptide is
administered will react in exactly the same way to both. Also the frngments within
,
.:
. :
: ~
q OOl()
--8--
each linear polymer may be the same or different and need not necessarily be
derived from the same protein.
The linear polymeric polypeptides of the invention may be use~ to provoke the
formation of antibodies to both endogenous and non-endogenous proteins by using
05 appropriate polypeptide fragments to form the polymers. (The term "endogenous" is
useà herein to denote a protein which is native to the species to be treated,
regardless of whether the antigen is endogenous to the particular individual animal
being treated. Thus, for example, for present purposes a porcine sperm antigen is
regarded as being endogenous to a sow even though obviously such a sperm antigen10 will not normally be present in the body of a sow.) Further details OI the use of non-
endogenous proteins fragments of which may be utilized in the linear polymeric
polypeptides of the invention is given below in connection with the discussion of the
antigens of the invention. As regards endogenous proteins, fragments of which may
be used to form the linear polymeric polypeptides of the invention, in general it may
15 be stated that such endogenous proteins may include any of the protein.s mentioned
in our aforernentioned U.S. patent No. ~,302,386, as described above. Incidentally,
it should be noted that the use of the linear polymeric polypeptides of the invention
based upon growth hormone and/or somatomedian is not confined to diabetic
patients. Thus, the linear polymeric polypeptides of the invention based upon these
20 two hormones may be used to treat non-diabetic patients, such as persons suffering
from acromegaly, who have excessive levels of growth hormone and/or somato-
median.
Particularly preferred linear polymeric polypeptides of the invention are those
formed from fragments having a molecular structure similar to a fragment of
25 Human Chorionic Gonadotropin, and in particular similar to a fragment of the beta-
subunit thereof. Two particularly preferred fragments for use in the linear
polymeric polypeptides of the ihvention are:
As~As~Pr~Ar~Phe-Gln-As~Ser-5er-Se~Ser-Lys-Ala-Pro-Pr~
Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pr~Gly-Pro-Ser-Asp-Thr-
Pro-Ile-Leu-Pro-Gln-Cys (hereinafter designated fragment A); and
As~E~is-Pro-Le~Th~Cys-Asp-As~Pr~Arg-Phe-Gln-Asp-Ser-Se~
Ser-Ser-Lys-Ala-Pr~Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-
Pro-Gyl-Pro-Ser-As~Th~Pr~Ile-Leu-Pro-Gl~Cys.
Other HC~derived fragments usable in the linear polymeric polypeptides of the
35 invention include those of Structure (Il~(VIII), ~VIIIa) and (IX~(XIV) as set forth
below, further details of the immunological nature of these fragments being given in
the aforementioned U.S. patent No. 4,30~,386:
~ '`I
- 9 -
As~As~Pr~Ar~Phe-Gln-As~Ser-Ser-Ser-Ser-Lys-Ala-Pr~Pr~
Pro-Ser-Leu-Pro-Ser-Pro--Ar~Leu-Pr~Gly-Pro-Ser -As~Thr-
Pro-Ile-Leu-Pro-Gln
Structure (II)
05 Gln-As~Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-
Pro-Ser-Ar~Leu-Pro-Gly~Pro-Ser-As~Thr-Pro-Ile-Leu-Pro-Gln
Structure (III)
Cys-Pro-Pro-Pro-Pro-Pro-Pro-Ser-As~Thr-Pro-Ile-Leu-Pro-
Gln
Structure (IV)
As~As~Pro-Ar~Phe-Gln-As~Ser-Pro-Pro-Pro-Pro-Pro-Pr~Cys
Structure (V)
Phe-Gln-As~Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-
Ser-Pro-Ser Ar~Leu-Pro-Gly-Pro-Ser-As~Thr-Pro-Ile-Leu-
Pro-Gln
Structure (VI)
As~As~Pro-Ar~Phe-Gln-As~Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-
Pro-Ser -Leu-Pro-Ser
Structure (VII) ~ .
As~As~Pro-Ar~Phe-Gln-As~Ser-Pro-Pro-Pro-Cys-Pro-Pro-Pr~
Ser-As~Thr-Pro-ne-Leu-Pro-Gln
~Structure (VIII)
As~As~Pr~Ar~Phe-Gln-As~Ser-Pro-Pro-Pro-Pro-Pro-Pro-Cys-
; ~ Pro-Pro-Pro-Pro~Pro-Pro-Se~As~Thr~Pro-Ile-Leu-Pr~Gln
~ ~ ~ 25 Structure (VI[Ia)
:
-' :
L~ V
-10-
As~His-Pro-Leu-Thr-Aba-As~As~Pro-Ar~Phe-Gln-As~Ser-Ser-
Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu
-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Pro-Pro-Pro-Pr~
Pro-Pro-Cys
05 Structure (IX)
Asp-AsE~Pro-Ar~Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-
Pro-Ser-Leu-Pr~Ser-Pro-Ser-Arg~Leu-Pr~Gly-Pro-Ser-Asp-Th~
Pro-Ile-Leu-Pro-Gln-Pro-Pro-Pro-Pro-Pro-Pro-Cys
Structure (X)
As~Asp-Pro-Ar~Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pr~
Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pr~Gly-Pro-Ser-Asp-Thr-
Pro-Tle-Leu-Pro-Gln-Cys
Structure (XI)
Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-
Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pr~Ser-
Asp-Thr-Pro-Il~Leu-Pro-Gln
Structure (XII)
Asp-His-Pro-Leu-Thr-Aba-As~As~Pro-Ar~Phe-Gln-Asp-Ser-Ser-Ser-
Se~Lys-Ala-Pr~Pr~Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-
Gly-Pro-Ser-Asp-Thr-Pro-lle-Leu-Pro-Gln-Cys
Structure (XIII)
Cys-Pro-Pro-Pro-Pro-Pro-Pro-Asp-As~Pro-Ar~Phe-Gln-As~Ser-
Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Ar~
Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln
Structure (XIY)
Obviously, if it is desired to use one of the above peptides which lacks a C-
: terminal cysteine as a second or later fragment in preparing the linear polymeric
peptides of the invention, it will be necessary to add a C-terminal cysteine to the
peptide; appropriate methods for doing so are of course well known to those skilled
30 in the field of polypeptide synthesis. Also, some of the above peptides will of course
reguire blocking of non terminal amino and/or thio groups before use.
.. . .
~............................... .
gL2~ 0()~)
The linesr polymeric polypeptides of the invention based upon HCG-derived
fragmenes are usable in exactly the same manner as the corresponding modified
polypeptides in which the same fragments are coupled to carriers, as described in
our aforementioned U.S. patent No. 4,302,386. Moreoever, since this patent was
ns written, further dis~overies have been made con-~erning the aSsociQtion between
HCG and immunologically similar materials and certain carCinOmQS. It appears
(though the invention is in no way limited by this belief) that certain carcinomas
exude chorionic gonadotropin or ~n immunologically-similar material on their
surfaces, thereby presenting to the immune system of the host animal a surface,
10 which, superficially, appears to be formed of material endogenous to the hostanimal, and which is thus relatively non-immunogenic. Because of this known
association between certain carcinomas and chorionic gonadotropin or chorionic
gonadotropin-like materials, the HCG-derived linear polymeric polypeptides of the
invention flre useful for the treatment of HCG and chorionic gonadotropin-
15 associated carcinomas. The same linear polymeric polypeptides of the inventionare also of course useful for fertility control, as described in the ~forementioned
U.S. patent.
The fr~gments used in the linear polymeric polypeptides of the invention may
also include fr~gments of the zona pellucids and sperm antigens discussed in detail
20 below.
As alre~dy mentioned, the line~r polymeric peptides of the invention are
produced by polymerizing fragments of the protein to which antibodies are to be
provoked rather than the entire protein itself. This use of fragments r~ther than an
entire proteins hes important advantages (and similar advnntages are sec~red by
25 the use of rragments in the ~ntigens and modified antigens of the invention
discuæed in det~il below). It is well recogni~ed by those skilled in immunology (see
e.g. W.R. Jones, "Immunological Fertility Regulation", Blackwell Scientific Pub-lications, Vic~oria, Austrnlia (1982) (the entire disclosure of this work is herein
incorporated by reference), pages 11 et seq.) that one of the greatest potential30 hazards Or a vaccine, especially a contraceptive vaccine, is cross-reactivity with
no~tnrget ~ntigens, producing what is essentially an artificiall~induced aut~
immune disease capable of causing immunopathological lesions in, and/o; loss of
function of, the tisues carrying the crossreactive substances. Two possible
mechanisms for such cross-re~ctivity are:
(a) Presence of shared antigenic determinallts; a complex protein may
contain components (amin~acid sequences) identical to those present in
fl non-target antigens;
. . ~ .
.
~L2 ti L~ 0~3 ~J
--12-
(b) steric overlap between non-identical but structurally relnted parts of the
protein and no~target antigens.
Obviously, the chnnges pvsed by both these modes of cross-reactivity may be
lessened by using, in the linear polymeric polypeptides, nntigens end modified
05 antigens of the invention, a fregment of Q complex protein rnther thnn the whole
protein. Since the fragment has a simpler sequence thnn the protein from which it
is derived, there is less chance of shared nntigenic dete~mi~r~s or sterie overlnp
with non-tnrget proteins. In particular, eross-reactions c~n often be avoided byusing frngments derived from n portion of the target protein (i.e. the protein to
10 which nntibodies ~re to be provoked) which is not similar in sequence to the non-
target but cross-renctive protein. ln particular, as described in our nforementioned
U.S. patent No. 4,302386, one of the major problems in provoking nntibodies to HCG
is cross-reactivity of HCG antibodies with LH, this cross-reactivity being at least
largely due to identity of amino acid sequence between LH and the 1-110 amino acid
15 sequence of the bet~ subunit of HCG. Accordingly, when it is desired to form an
HC~derived lline~r polymeric polypeptide of the invention, the fragment used is
prefernbly one having n molecular structure similar to pnrt or all of the 111-145
sequence of the bet~ subunit of HCG, since it is only this 111-145 sequence of bet~-
HCG which difers from the corresponding sequence of LH. However, rese~rch
20 indieates th~t the fr~ments used in the linear polymeric polypeptides of the
invention mny contain sequences corresponding to the 101-110 sequence of beta-
HCG which is comsnon ~o bet~-HCG ~nd LH without inducing the formation of
~ntibodies re~ctive to LH. Thus, one can use in the linear polymeric polypeptides of
the invention frQgments conteining part of or all of this common 101-110 sequence
25 without c~using cross-re~ctivity with LH. For ex~mple, Structure (U) ~bove
represents the 111-145 ~mino acid sequence of betn-HCG. If desired, A fragment
having the 101-145 ~mino ~cid sequence of betn-HCG could be substituted for the
ragment oî Stuucture (Il) in the linenr polymeric polypeptides of the inventionwithout subst~nti~lly eftecting the activity of the linenr polymers ~nd without
30 c~using er~s-reactivity with LH.
Polymerizstion of the fragments to form the linear polymeric poly~eptides of
the invention may be effected in any manner for coupling peptide frngments to form
linear polymers thereof kslown ~o those skilled in the art. Thc linenr polymcriepolypeptides oi the invention may ~e divided into two distinct types. In the firs~
35 type, the individunl peptide fr~gments ore linkcd head-to-t~U by pcptide linkngcs, so
that the wholc polymer compriscs solely the frngmcnts thcmsclvcs nnd docs not
-
ov
13-
contain Any extraneous m~terial. Although such pure polymers do have the
advant~ge of not introducing any extraneous material into the body of the animalbeing treated, they are usually too expensive to be practical, since the necesary
fragments (whether produced by total synthesis or cleavage of Q n~tural protein) are
05 themselves very expensive Elnd substantW losses occur during the polymerization
process. Furthermore, the head-to-tail coupling of the fragments~ without any
intervening residues, may produce immunological determinants which have no
counterpart in the unpolymerized fragment. For example, if the fragment described
above, comprising the 105-145 sequence of HCG, is polymerized by means of peptide
10 linkages, a sequence:
Pro-Ile-Leu-Pro-Gln-As~His-Pro-Leu-Thr
will be produced at each junction between adjacent fragments, and this sequence
may provoke the formation of antibodies which would not be produced by the
fragment itself, and which may be undesirable. In colloquial terms, since there is no
15 "punctuationl' to tell the immune system of the recipient animal where one fr~gment
begins and another ends, the animal's immune system may inadvertently start
reading at the wrong residue and produce unwanted antibodies by running the
sequences of adjacent fragments together. For this reason, in general, we do notrecommend the use of linear polymers in which the fragments are connected by
20 peptide polymers, though of course such linear polymers may be useful in certain
instances.
Various methods of coupling polypeptide fragments via peptide bonds are
known to those skille~ in the art. For example, one fragment to be coupled may
have its C-termin~l carboxyl group blocked (e.g. by astearification) and be re~cted
25 with the other fragment, which has its N-terminal amino group blocked, but its
carboxyl group activnted by means of an activating agent. Obviously, blocking ofnon-terminal amino and carboxyl groups may be necessary- Also, aswell knowrl to
those skilled in this field, it may be advantageous to attach one end of the
polymer being produced to a support, such as polystyrine resin support, the polymer
30 only being detached from the support after polymerization is completed.
In the second type of linear polymer polypeptide of theinvention~the
peptide fragments are connected to one another by means of residues derived from a
bifunctional reagent used to effect polymerization of the fragments, so thAt thefinal linear polymer is an alternating linear polymer of polypeptide fr~gments and
35 coupling reagent residues. Although this type of polymer necessarily introduces
some extr~neous m~teri~l into the nnimal being treated, the proportion of extrA
',~ J
:_.
, ', : ~,
: . `.
- ~ ' : ' .~ , , :
:-, , ,:
~6~
--14-
neous material can be made consi~erably lower than it would be if the fragments
were coupled to a large carrier, such as diph~heria toxoid. The coupling reagent,
- which is necessarily a bifunctional coupling reagent to produce a true linear
polymer, can be chosen so that the resides it leaves in the polymer are not strongly
05 immunogenic (so that they do not plas!e the strain on the immune system of the
recipient animal that, for example, a large carrier molecule such as diphtheria
toxoid would) and the presence of these residues in the polymer has the advantage of
substantially eliminQting false immunological determinants produced by conjunction
of the head of one fragment with the tail of an adjacent fragment, as discussed
1 0 above.
To ensure that a true linear polymer is produced during the polymerization
process, one terminal of a first polypeptide fragment is reacted with the bi-
functional coupling reagent so that the coupling reagent reacts with a group present
at or adjacent one terminal of the fragment; for example, the coupling reagent may
15 react with a N-terminal amino group, a C-terminal carboxyl grou~ or a free thiol
group present on a C-terminal cysteine. Obviously, the nature of the coupling
reagent used determines what group on the peptide reacts. In order to avoid any
cross-linking and to ensure a reproduceable product, it is important that only one
site on the first fragment be available for reaction with the coupling reagent so that
20 the coupling reagent can only attach to the first fragment at this one site. As those
skilled in this field are aware, if it is desired to use a fragment containing more than
one group which could react with the coupling reagent, the excess sites may be
blocked by attaching suitable protective groups thereto. The product formed by
reaction of the first fragment with the coupling reagent is then reacted with a
25 second fragment (which may be the same or different from the first fragment)
having a single site available to react with the second reactive group of the
bifunctional bicoupling reagent, thereby coupling the first and second fragments by
a residue derived from the coupling reagent. Following any necessary purification
of this dimeric product, it is then reacted with a further portion of a coupling agent
30 which may be the same or ~ifferent reagent from that used to effect the firstcoupling), thereby reacting the free terminal of either the first or second fragment
with the coupling reagent. Naturally, it is important to ensure that only one site on
the dimer is available for coupling to the coupling reagent, and as will be apparent
to those skilled in the art, blocking or unblocking of potential reactive groups on the
35 dimeric polypeptide may be necessary. The product of the reaction of the dimeric
polypeptide with the coupling reagent is then reacted with a third fragment hanng
.
. . .
.~.
:
- .,
,
o~
only a single site av~ilable for reaction with the remaining reactive group of the
coupling reagent, thereby producing a linear polymer containing three polypeptide
fragments. Obviously, this process can be repeated until the desired size of lineur
polymer has been produced.
05 It will be apparent to those skilled in this field that the bifunctional coupling
reagents used to prepare the linear polymeric polypeptides of the invention should
be asymmetric i.e. they should have two functional groups which react with
different groups on the fragments being polymerized, since, for example, if one
attempted to react a bifunctional bicoupling reagent having two functional groups
10 which both reacted with amino groups with a first fragment having a single amino
group, st least some of the first fragment would be dimerized via a residue derived
from the bifunctional bicoupling reagent. Such dimerization may in theory be
avoided by using a very large excess of the coupling reagent, but in practice it is
undesirable to run the risk of producing even a small proportion of dimer. Similarly,
15 in later stages of the polymerization process, it will be even more undesirable to use
symmetric coupling reagents, thereby running the risk of dimerizing the partially
formed polymers already produced. In the preferred process for producing the linear
polymeric polypeptides in the invention already described, the polymer chain is
begun with a first peptide having no unblocked thiol group and having an unblocked
20 amino group only at its N-terminal (peptides containing thiol groups and/or amino
groups other than at the N-terminal may of course be used if all these thiol andamino groups are blocked with any conventional blocking agent). This first peptide
is then reacted with an amino group nctivating agent, a preferred activating agent
for this purpose being 6-maleimido caproic acyl N-hydroxy succinimide ester (MCS);
25 reaction of the peptide with this reagent is optimally effected ~t a pH of 6.6). The
activating agent reacts with the amino group at the N-terminal of the first peptide
to form an activ2ted form of the first peptide; in the case of MCS, it is the ester
portion of the reagent which reacts with the N-terminal group of the peptide. It is
normally ~hen necessary to remove excess activating agent before continuing the
30 preparative process. Once the excess activating agent is removed, the activated
first peptide is reacted with a second peptide having a C-terminal cysteine in areduced state (i.e. having an unblocked 3-thiol group), thereby causing coupling of
the N-terminal of the activated first peptide to the C-terminal Or the second
peptide viaan activating sgent residue. Desirably, the resultant dimer is purified as
35 described in more detail below. Next, the dimer is again reacted with an amin~
group activating agent and then with a second portion of the second pcptide or with
~ . .
. , : ,, :
01~3~)
--16 -
a third peptide, thereby producing a trimer. This procedure is repeated until the
desired chain length has been achieved.
In order to secure reproduceable responses from the immune systems o~
treated animals, it is important that the linear polymeric polypeptides of the
05 invention be used in the form pure polymers in which all the molecules contain t}ie
same number of fragments. To achieve such pure polymers, effective purification
should be used after each polymerization step of the polymerization process.
Because of the close chemical similarity between polymers containing difIerent
numbers of fragments, chemical purification is ineffective, so purification must be
10 effected by physical methods. Gel filtration may be used if desired, but our
preferred purification method is reverse-phase, high-pressure liquid chroma-
tography, preferably using a molecular sieve as the solid phase.
In this method of forming linear polymers, the first and second peptides may
be identical in chemical configuration except that in the first peptide the C-
15 terminal cysteine hus a blocked thiol group. The first two preferred fragments forforming linear polymeric polypeptides of the invention to form antibodies to HCG
mentioned above may be described as (l~l-145)-Cys and (105-145~Cys, where the
figures refer to the amino acid sequence in the beta subunit of HCG. It will be
appreciated that, when these fragments are to be used in forming linear polymeric
20 polypeptides of the invention by the method just described, the lysine residue at
position 122 must have its amino group blocked and, in the case of the (105-145~Cys
fragment, the non-terminal cysteine at position 110 must have its thiol group
blocked, preferably with an acetamidomethyl group.
The linear polymeric peptides of the invention preferably contain from about 4
25 to about 14 fragments.
The modified antigens of the invention produced by chemically modifying non-
endogenous proteins wi~l now be described in more detail. As those skilled in the art
are aware, there are numerous pathogens and similar materials known which are not
endogenous to animals, which are capable of produ~ing harmful effects in the
30 animal's body but which are not immunogenic to the animal, in the sense that
introduction of the pathogen or other material into the body of the animal fails to
elicit from the animal's irnmune system production of the quantity of appropriate
antibodies necessary for the animal's irnmune system to destroy the pathogen or
similar material. ~or example, the Herpes simplex Type Il virus is capable of
35 producing a number of harmful effects in man, including the production of painful
lesions in the genit~ areas. ~lthough this virus has, like most viruses, protein
:
-,
': ~ ' .' ~:.
.. ~
~ - ~ - . :.
~4(~3~3
--17-
included in its structure, the viral protein is not strongly immunogenic in rnost
human beings, so that only about 5096 of infected human beings produce antibodies
to the virus. This lack of immune response to the virus by many human beings
means that the virus can remain in the infected human beings for at least several
05 years, and this persistence of the virus in the infected individuals not only causes
those individuals to suffer recurrent attacks of the painful symptoms caused by the
virus, but also renders them long-term carriers of the virus. This persistence of the
virus in infected individuals is one of the factors largely responsible for the
epidemic proportions which ~ _ simplex infections have reached in several
10 countries. By prep~ring an antigen OI this invention derived from a protein having a
sequence similar to that of at least part of the sequence of a Herpes simplex viral
protein, it is possible to stimulate the human immune system so as to render it
capable of producing large quantities of antibodies to the Herpes simplex virus. Not
only should this stimulation of the immune system reduce the occurrence of
15 symptoms associated with Herpes simplex infection, but it should help to control the
spread of the virus. Similarly, the immune response of humans and other animals to
viruses such as colds, influenza and other viruses can be increased by preparingmodified antigens of the invention based upon peptides having sequences corres-
ponding to viral proteins of the appropriate virus. If, as appears likely, a virus is
20 responsible for acquired immune deficiency syndrome (AIDS) a modified antigen of
this invention could also be used to produce immunity to this disease.
In general, the methods used for preparing the antigens of the invention based
upon non-endogenous materials, such as viral proteins or peptides corresponding to
parts thereof, are the same as those used for modifying endogenous proteins or
25 fragments thereof, as described in our aforementioned U.S. patent No. 4,302~386.
However, it will be appreciated that the preferred methods used for modifying non-
endogenous materials may differ in certain respects from those used for modifying
endogenous peptides. Since, in general, the non-endogenous peptide will provoke at
least a limited immune response from the animal to which the antigen is to be
30 administered, the requirements for modification of the non-endogenous protein or
peptide to produce an antigen of the invention will tend to be less stringent than
those for modification of an endogenous and completely non-immunogenic protein or
peptide. However~ since the non-endogenous protein or peptide is being modified to
increase its immlmogenic effect in the animal into which it is to be administered, in
35 gener~l it will still be desirable that the carrier used to modify the non-endogenous
protein or peptide (if chemical modification is to be effected by coupling the non-
. .: .
- -
:.. ,;. :: ..
-`
.
., .
.. . . . .
~,~, ' '~
~ ~ .
~6~0~3
-18-
endogenous protein or peptide to n carrier, rather forming a linear polymericpolypeptide of the invention) to produce the antigen of the invention be a materialwhich itself provokes ~ strong response from the animal's immune system. For
example, the carrier may be a bacterial toxoid such as diphtheria toxoid or tetanus
oS toxoid.
The antigens of the invention are prepared by chemically modifying Q protein
which is not endogenous or immunogenic to the animal to be treated, or a peptidehaving a sequence corresponding to at least part of the sequence of the protein, this
chemical modification being effected outside the body of the animal. The chemical
10 modification m~y be effected by coupling the protein or peptide to a carrier, by
polymerizing the peptide to form a linear polymeric polypeptide of the invention or
by any other chemical modification technique which will make the protein or
peptide sufficiently immunogenic. Preferred techniques for chemical modificationof the protein or peptide by coupling to a carrier include the following (further
15 details of optimum techniques for each of the following coupling reactions are given
in our aforementioned ll.S. patent No. 4,302,386):
a. Treating a protein or peptide having a sulfhydryl group thereon with an
activator of the structure:
~--X~ --X--CON~
wherein X represents a non-reacting group comprising substitutedor
unsubstituted phenyl or Cl-10 alkylene moieties, or a combinAtion there-
of, or an amino acid chain, so as to cause reaction of the maleiimide
group of the activator with the sulfhydryl group on the protein or
peptide; and treating the resulting activated protein or peptide at
slightly Mlkaline pH with a carrier moiety biologically foreign to the
animal and selected haYing a size sufficient to elicit antibody response
following the administration thereof into the body of the animal.
b. Treating, under neutral or acid conditions, a carrier not hnving M
sulfhy~yl group but having an amino group with an activ~tor as defined
in the preceding parsgraph, so as to cause reaction of the aCtivAtor with
.
~,
~ .
,
~: :. .. : : - :
. ~ . .
::
:
: . ..
: . ~.. . . .
~L2 ~ Lq ~
--19--
the amino group on the c~rrier, the carrier being biologically foreign to
the animal to be treated and having a size sufficient to elicit antibody
response following administration thereof into the body of the animal;
and treating the resulting activated carrier with the protein or peptide,
05 which must have a sulfhydryl group thereon, thereby reacting the
maleiimide group of the activator with the sulfhydryl group on the
protein or peptide.
c. Treating a carrier biologica~ly foreign to the animPl to be treated and
having Rn amino group with an activator present as Rn active ester of
chloro-, dichlor~, brom~ or iodo- acetic ~ci~ so as to cause reaction of
the activator with the amino group of the carrier and treating the
resulting activated c~rrier with the protein or peptide, which must hàve
a sulfhydryl group thereon, thereby reacting the activated carrier with
the sulfhydryl group of the protein or peptide.
d. Treating ~ protein or peptide that does not have a sulfhydryl group but
has an amino group with an activator present RS an active ester of chlor-
, dichlor~, bromo- or iodo- acetic acid, and treating the resulting
halomethyl Qlkylating group containing moiety with a sulfhydryl group-
containing carrier biologically foreign to the animal to be treated so as
to react the sulfhydryl group of the carrier with the halomethyl
alkylating group.
It will be noted that several of the preferred coupling techniques described
above require the presence of a sulfhydryl group in the protein or peptide.
It is well known to those skilled in the art that, in mQny natural proteins
25 containing cysteine residues, these residues are not presene in their thio form
containing a free-SH group; instead pairs of cysteine residues are lined by means of
disulfide ~ridges to form cystine. Such disulfide bridges are very important in
determining the conformation of the protein. In most cases, the disulfide bridges
present in the natural form of the protein are eRsily reduced to pairs of -SH groups
30 by means of mild reducing agents under conditions which leave the remaining parts
of the protein molecule unchanged. Accordingly, when it is desired to produce free
-SH groups in proteins in order to carry out the ~oupling reactions discussed ~bove,
one conver~ient way of providing such fre~SH groups may be to cleave disulfide
bridges naturally present in the protein or other polypeptide which is desired to
;; 35 couple.
: ~r~
~3
- ~ : : ,. : : :
,. : ~ ~ - - ~ . . .
- ~
~2~
-20-
The generation of free-SH by reduction of disulfide bridges in naturally
occurring forms a~proteins may also affect the cross-reuctivity of the antibodies
produced when a modified polypeptide produced from the protein is injected into an
animal. Frequently, an antibody recognizes its corresponding antigen not only by05 the amino acid sequence in the antigen but also by the conormation (shape) of the
antigen. Accordingly, an antibody which ~jinds very strongly to a protein or other
polypeptide in its natural conformntion may bind much less strongly, if at all, to the
same protein or polypeptide whose conformation has been drastically altered by
breaking disulfide bridges therein. Accordingly, the breaking of disulfide bridges in
10 proteins or other polypeptides may provide a basis for reducing the cross-reactivity
between antibodies to antigens having the same amino acid sequence along parts of
the molecule.
As mentioned above, this invention extends to chemically modified antigens
derived from zona pellucida or sperm antigens, or peptides having a sequence
15 corresponding to at least part of the sequence of such a zona pellucida or sperm
antigen. These modified antigens of the invention are useful for fertility control. It
is known that antigens from the zona pellucida (the outer covering of the ovum)
when injected into female primates produce antibodies having anti-fertilization
effects, including prevention of sperm attachment to, and penetration of, the zona
20 pellucida of the unfertilized ovum, and prevention of dispersal of the zona pellucida
of the fertilized ovum prior to implantation (such dispersal of the zona apparenUy
being an essentisl prerequisite for implantation). See e.g. W.R. Jones, "Immuno-logical Fertility Regulation", Blackwell Scientific Publications, Victoria, Australia
(1982), pages 160 et seq. Such anti-fertility effects are believed to be due to
25 formation of an antibody-antigen precipitate on the zona, this precipitate rendering
the zons unable to undergo its normal sperm-binding reaction and also rendering the
zona insensitive to the action of the protreases normally responsible for dispersal of
the zona.
An anti-fertility vaccine based upon zona antigens is especially attractive
30 because zona antigens appear to be relatively free of side-effects on other tissues
and because methods have been developed for producing swine zona antigens in large
quantities; swine ~nd human zona antigens show very good cross-reactivity. As inthe case of anti-fertility vaccines based upon the beta-HCG antigens discussed
~bove and in our aforementioned U.S. patent No. 4,302,386, better results will be
35 obtained by modifying n :~ona antigen or a fragment thereof to produce a modified
antigen of this invention.
~,, ,
~: : ' ,:
.
3L"~ Lq~O ~3(3
-21-
Several antigens, especiaLly sperm enzymes, known to exist in sperm, may be
usèd in the modified antigens of this invention; see W.R. Jones, op. cit., pages 133 et
seq. The most promising such antigen is the lactate hydrogenase known as LDH-C4
or LDH-~. Although of course lactate dehydrogenases are present in other tissues,
05 LDH-C4 is distinct from other lactate dehydrogenase isoenzymes and appears to be
sperm-specific. Moreover, the enzyme is not strongly species specific, and methods
for its isolation and purification are known. Again, the best results will be obtained
by modifying LDH-C4 or a fragment thereof to produce R modified polypeptide of
this invention. Several natural peptide fragments of LDH-C~ have been prepared,
10 sequenced and shown to bind to antibodies against the parent molecule. (See E.
Goldburg, "LDH-X as a sperm-specific antigen", in T. Wegmann and T.J. Gill (eds.~,
Reproductive Immunology, Oxford University Press, 1981).
Although theoretically an anti-fertility vaccine based on sperm antigens might
15 be useful in males, the likelihood of testicular damage renders it more likely that
such a vaccine will find its utility in females. It is known that circulating antibodies
in the female bloodstream do penetrate the genital fluids; for example experiments
in baboons with vaccines based upon the peptide of Structure (XII) above conjugated
with tetanus toxoid have shown the presence of HCG antibodies in the genital fluids.
20 However, one possible problem with any vaccine based on sperm antigens is
maintaining a sufficiently high antibody level in female genital fluids to complex
,with the large amounts of sperm involved.
The techniques used for chemical modification of zona pellucida or sperm
antigens, or peptides derived therefrom, to form the modified antigens of the
25 invention include all the techniques discussed in our aforementioned ~.~. patent No
4,302,386 and also those techniques discussed above. Thus, the antigen or peptide
may be coupled to a carrier, such as tetanus toxoid or diphtheria toxoid, or may be
polymerized to form a linear polymeric polypeptide of the invention. The preferred
te¢hniques for forming such linear polymeric polypeptides have already been
30 discussed above, while the preferred techniques for coupling the antigens or peptides
to carriers are the same as those for coupling non-endogenous proteins or peptides,
as already discussed above.
~ inally, as mentioned above, this invention extends to a vaccine containing a
modified p~lypeptide, antigen or modified antigen of the invention and a vehicle35 comprising a mixture of mannide monooleate with squalane and/or squalene. It has
been found that this ~ehicle has the effect of increasing the quantity of antibodies
, : : :
oov
provoked by the linear polymeric polypeptide, antigen or modified antigen of theinvention when the vaccine is administered to an animal. To further increase thequantity of antibodies provoked by administration of the vaccine, it is advantageous
to include in the vaccine an immunological adjuvant. The term 'ladjuvant" is used in
05 its normal meaning to one skilled in the art of immunology, namely as meaning a
substance which will elevate the total immune response of the animal to which the
vaccine is administered i.e. the adjuvant is a non-specific immuno-stimul~tor.
Preferred adjuvants are muramyl dipeptides, especially:
NAc~nor Mur-L.Ala-D.isoGln;
NAc-Mur-(6-0-stearoyl~L.Ala-D.isoGln; or
N Glycol-M ur-L.~Abu-D.isoGln
The vaccines of this invention may be administered parenterally to the animals to be
protected; the usual modes of administration of the vaccine being intramuscular and
sub-cutaneous injections. The quantity of vaccine to be employed will of course
15 vary depending upon various factors, including the condition being treated and its
severity. However, in general, unit doses of 0.1-50 mg. in large mammals admin-
istered with one to five times the intervals of 1 to 5 weeks provide satisfactory
results. Primary immunization may also be fo~lowed by llbooster'l immunization at 1
to 12 month intervals.
To prepare the vaccines of the invention, it is convenient to first mix the
linear polymeric polypeptide, antigen or modified antigen of the invention with the
muramyl dipeptide (or other adjuvant) and then to emulsify the resultant mixture in
the mannide monooleate/squalene or squalane vehicle. Squalene is preferred to
squalane for use in the vaccines of the invention, and preferably about ~ parts by
25 volllme of squalene and/or squalane are used per part by volume of mannide
m onooleate.
The animals to be treated with the linear polymeric polypeptides, antigens,
modified antigens and vaccines of this invention include both humans and other
animal species.
The following example is now given, though by way of illustration only, to
show details of the preparation and use of a linear polymeric polypeptide of theinvention.
EXAMPLE
Fragment A described above (a fragment having an amino acid sequence
35 corresponding to the 105-I45 sequence of beta-llCG, with a cysteine residue added to
the C-terminal ther~of) was polymerized to form a hexamer. A first portion of
3~
-23-
fragment A had both its thiol groups (on the non-terminal cysteine at the position
corresponding to position 110 of beta-HCG, and on its C--terminal cysteine) and its
non-terminal amino group (on the lysine residue at the position corresponding toposition 122 in beta-HCG) blocked. This blocked form of Fragment A was reacted
05 with the bifunctional organic coupling reagent (or amino group activating agent)
MCS in a buffered aqueous solution at pH 6.6, thereby reacting the ester portion of
the MCS with the N-terminal amino group of the first portion of Fragment A. The
resultant product was then reacted with a second portion of Fragment A, which was
used in the same form as the first portion of Fragment A except that the C-terminal
10 cysteine bore an unblocked thiol group, thereby reacting the remaining functional
group of the MCS with the free thiol group on the second portion of Fragment A and
producing a dimer in which the N-terminal of the first portion of Fragment A wascoupled to the C-terminal of the second portion of Fragment A via an MCS residue.
This dimer was then purified by gel filtration. The polymerization was then
15 repeated in the same manner until a hex~mer of Fragment A had been produced.
Because the purification following each polymerization step was effected by gel
filtration rather than by reverse-phase, high-pressure liquid chromatography, the
hexamer was undoubtedly somewhat impure and contaminated by traces of penta-
mer9 tetramer etc., so that the results in the animal tests results described below
20 could not be expected to be as good as would be produced using a pure hexamer.
To test the effectiveness of this hexameric polypeptide in provoking the
formation of antibodies to HCG, the hexamer was formed into a vaccine using
Complete Freunds' Adjuvant and injected into five rabbits. Each rabbit was giventhree injections of the vaccine intramuscularly at 3 week intervals, each injection
25 containing 0.5 mg. of the hexamer. Starting three weeks after the first injection,
each rabbit was bled weekly and the level of antibodies to HCG in the blood
determined. The following average values of antibody level were found (the figures
in parenthesis represent the confidence limits i.e. average + or - standard error):
:-
- .
'
v
--24--
TABLE
Weeks After First Injection Antibody concentration (moles/liters x 10 10)
3 5 (2-7)
18 (13-22)
30 (21-39)
6 45 (31)-60)
7 58 (32-83)
8 61 (34-86)
9 77 (~0-108)
110 (53->125)
11 100 (50->125)
12 79 (30-1l9)
13 53 (22-83)
The, the above results show that even the crude hexamer preparation used in
15 these experiments was much more strongly immunogenic than the very we~kly
immunogenic fragment from which it was derived.
.
: .
.
-
~ ' ' ' .'' ' ,
: :. ,
.
