Note: Descriptions are shown in the official language in which they were submitted.
,^- WO93/21218 h 1~ ~ D 3 3 PCT/GBg3/00808
-- 1 --
SYNTHETIC POLYPEPTIDES DERIVED FROM THE HIV ENVELOPE GLYCOPROTEIN.
The present invention relates to synthetic
polypeptides. It particuiarly relates to synthetic
polypeptides which emulate the three-dimensional
structures and/or electrostatic surfaces and/or other
physical, chemical and structural properties of specific
regions of viral envelope proteins. It is of particular
initerest to the desi~n of vaccines, immunologically
active therapeutic agents, diagnostics and other medical
or scientific agents in relation to the Human
Immunodeficiency Virus (HIV) known to be the ca~sati~e
agent of Acquired Immune Deficiency Syndrome (AIDS).
15 . Within the last decade AIDS has emerged as an :;~
important medical problem throughout the world and there
is currently an urgent need for agents for khe study, i;
: ~iagnosis, treatment and/or prevention of infection by
~: the HI~, the causative agent of the disease. With the
20 availabi1ity of the amino acid sequences of proteins ~.
~: ~ : produced by the HIV l and HIV 2 viruses (see~ for
~example, ~atner, L. et al., Nature 313r 277 (1985); .
Meusing, M.A. et al., Nature 313, 450 (19~5); Wain- .:
~obson, S. et al., Cell 40, 9 ~l985)), it has been .~`
:: 25 po~sible to devise synthetic polypeptides which emulatei
the~antigenic properties of the viral envelope proteins~
: An object of the present invention is the :~
development of synthetic poly~eptides which can ~licit
the production of antibodies to the HIV virus, and most
preferably neutralising antibodies, that is, antibodies
which pr~vent infection by and/or limit thei~pread of ! ii
the ~IY YirUS by passive or active immunisation.
Passive immuni~ation with such antibodies may constitute
an effective means of treatment of AIDS patients thus
controlling the spread of the virus within and between
individuals and hence slow or halt the ~ro~ress of the
disease.
:: . o~r invention provides a synthetic polypeptide
'.
W~93/21218 PCT/GB93/00808 f~,
2~ ~Q3~
-- 2 ~
havin~ at least one antigenic property of the en~elope
protein of at least one strain of Human Immunadeficiency
Virus (HIV), said polypeptide consisting substantially
o~ an amino acid sequence of formula (I):-
, ~ ;.
X~R1-Leu-R2-Leu-Thr-Val-Trp-Gly-R3-Lys-Y
wherein ~1 is selected from Gln-Gln R4-R5, Gln-F~-
R~ R4~Rs~ Rs or R1 is absent;
R2 is an amino acid residue ~elected from
Gln, Lys, Glu or Arg;
: R3 is an amino acid re~idue selecte~ from
Ile, Thr or Ala; ,
~4 iS His or Glu;
~ R5 is Leu or Met; and ~ ~
: : : X and Y may each independently be absent ~.
~' ~ or independently be one o~ moré~amino
acid res~idues~
. No~mally,~hen R1 is Gln-Gln-R4-R5 and X and Y are ~-
2~:~ present, X~and Y are not homologous with the natural
enYelope protein:sequenre. More particularly/ when X
and/or~Y~are present and R1 is as defined above X and Y
do::~not pro~ide or ~orm part of an antigenic property of
the~envelope~p~otein of at lea:st one strain o~Human
25 ~:Immunod~ficien:cy~irus. : ~ ;
Peptides accordin~to formula I above wi~hout X and
Y being~present will of course be use~ul,: for èxam~le,
in~the~production of antibodies to the HIV. Such
: peptides will be:especially effectivs when coniugated to
~; ;:30 a ~arrier~im~lecu~l.e.~ ~, Howeve~, when !x~ or Y: are:~resent ~
hey may be an~ length but preferably less than~20:iamino .
acids~,~more~preferably less than 10, eg. 3 to 6O It
will~of course ba ,appreciat2d that the s~u~nce~ -
: :according to formula I may constitute a pr~tein~with~X
35~ and ~ béing major portions of the protein with the
ntigenic sequence being, for exa~ple, part of an
: exposed loop on a globular protein.
: . ' . ' ' '
: ~ ".
~,;. WO93/2121B 21 ~. 8 0 3 ~ PCT/CB93/00~08
' ':':'
-- 3
Preferably, if R1 is present, R5 is either Leu or
Met, more preferably Leu, and R~ if present, is His or
Glu, more preferably His. R2 is preferably Gln, Lys or
Glu, more preferably Gln and R3 is preferably Ile. ~
Preferably in a sequence according to formula (I), ';:
R1 is Gln-Gln-R4-R5 or is absent and when present, R4 is
preferably His and Rs is Leu. Also it is preferable that
R2 is Leu and R3 is Gln.
Preferred forms of polypeptide of formula (I) ,:
according to the invention consist of the sequences:'
'' '
Seq. X.D. No: l
X~Gln-Gln-~is-Leu-Leu-Gln-Leu-Th~-Val-Trp-~ly-Ile-Lys-Y ~:
(Ia); and :
:
Seq. I.D. No: 2
X-Leu-Gln-Leu-Thr-Val-Trp-Gly Ile-Lys-Y (Ib)
' ' "
~: wherein X and Y are as defined above aIthough it is :~
; 20 preferred that if X or Y are present they are relatively : ;:
short se~uences. Pre~erably, X is absent and Y is 2 or
: 3 residues long, e.g. Gly-Cys or Gly-Cys-Ala. With .
regard to se~uences Ia and Ib above, it is preferred that X
is~absent and Y is Gly-Cys in sequence Ia and i5 Gly-Cys or
.25 Gly-Cys-Ala in seguence Ib; such C-terminal extensions
; provide alternate sites for coupling:to a Garrier~
Polypep~ides acoording to formula I resem~le
certain 2pitopic portions of HIV l en~Jelope protein. ::
A~other preferred form of polypeptide accordi~g to
30 the invention- consists substantially of an amino acid - ;
segu~nce:of formulal~tII~
X R1-Leu-Arg-Leu-Thr-Val-Trp-G1y-R3-Lys Y (~I3
' . ~
:
: 35 wherein R1 is selected from Gln-Gln-Glu-R~, Gln-
. Glu-~, Glu~ or ~1 i5 absent;
R3 is Thr ~r Alai
R5 is Met or Leu; and
: X and Y are as defined above. --
CANCELLEO / ~NNUL~
WO~3/2121~ 2 1 18 ~ 3 3 PC~/GBg3/00808 ~
-- 4
Preferably, RS, if present, is Met and R3 is
preferably Thr. ~lso, it is preferable that R1 is Gln-
Gln-Glu-Me~ or absent.
Preferred forms of polypeptide according to formula
II consist of the sequences~
Seq. I.D. No: 3 ~.`
X-~ln-Gln-Glu-Met-Leu~Arg-Leu-Thr-Val-Trp-Gly~Thr-Lys-Y
(IIa); and
Sec~. I . D:. No: 4 .
X-Leu-Arg-Leu-Thr-V~l-Trp-Gly-Thr-Lys-Y (IIb). .-
..
Preferably, X is absent and Y is 2 or 3 residues
long, e.g. Çly-Cys~ o~ Gly-Cys-Ala.
Polypeptides according to formula II are similar to
ertain epi~op~s o~ HIV 2 envelope proteins.
Preferred polypeptide seyuences according to the ~:~
:20~ invention~were chosen on the basis~of their
topographical similarity to one or more antigenic .:
determinants of the ~HIV envelope proteins. For example,~
a~n:ant~igenic determinant to which a given polypeptide
was;:;o}iginally designed to be an analogue may also:~how~
2~ topo~xaphical similarity to one or more other regions of :~
the~HIV envelope~proteins possibly due to duplication of
ancestral genes, or because the polypeptide is an : ,~
ana1~gue~of a dis~onti~uous dete ~ inant, or because the
polypeptides~have been designed to be polyvalent. ~A ~;: 30 ~discontinuous epitope may be vie~ed as ~eing composed of
clbsely opposed sequential epitopes ~hich m~,y be ~f
antigenic signi~icanc~ in their own right and a
polyva1ent polypeptide may con~ain two or more::~
continuous or discontinuous) determinan~ analogu s in a
35 single polypeptide chain, thus providing a means ~o ~ :
simultaneously elicit the production of a range of
a ffl ibodies which will recognise two or more dete~minants
on the HIV en~elope proteins.
Peptides according to the invention may be
~ :
WO93~21218 21 1~ IJ 3 ~ PCT/GB93/00808
-- 5
synthesised for example using either standard 9-
fluorenyl methoxycar~onyl (F-Moc~ chemistry (see, for
example, Atherton, E. and Sheppard, R. C. (1985) J.
Chem. Soc. Chem. Comm. 165) or standard
butyloxycarbonate tT-Boc) chemistry. The correctness of
the structure and the level of purity, which will :
normally be in excess of 85%, should be carefully checked.
Various chromatographic analyses, including high
per~ormance liquid chromatography, and spectrographic
analyses, may for example be employed for this purpose.
All the sequences herein are stated using the
standard I.U.P.A.C. three-letter-code ab~reviations for
amino acid residues d fined as follows: Gly-Glycine,
Ala-Alanine, Val-Valine, Leu-Leucine, Ile-Isoleucine,
Ser-Serine, Thr-Threonine, Asp-Aspartic acid, Glu-
Glutamic acid, Asn-Asparagine, Gln-Glutamine, Lys-
: : Lyslne, ~is-Histidine t Arg-Arginine, Phe-Phenylalanine t
::~ Tyr-Tyrosine, Trp-Tryptophan, Cys-Cysteine, ~et-
Methionine and Pro-Proline.
For therapeutic applications, polypeptides
; according to the i~vention or antibodies thereto may be
administered on their own or with other agents such as
3'-azido-3l-deo~ythymidine (AZT) ~zi~ovudine), which
acts at a dif$erent level by inter~ering with the
25 ~rep1ication of the genetic material of the virus, and/or ;;~
. ~ HIV~-pro~ease inhibitors, which block:the action of an
~ .
:: ~ : enzyme~ essential to~ the development of the virus.
Polypeptides according to the invention ~ay be used
~: to raise antibodies which will cross-react with envelope
~proteins produced by a wide range of ~IV l and/or HIV 2
s~rains. Our ana~yses have shown t~at, since the
: conformational/topographic/electrostatic properti~s of
polypeptides according to ~he in~ention are such that
they are highly likely to Plicit the;produc~ion of
antibodies which will cross-react with HIV envelope
proteins from several or many strains, fur~her
advantages may arise from combining se~eral variant
polypeptides in a larger polypeptide. Such a ~-
-:
WO93/Z1218 2 1 1 ~ ~ 3 3 6 - PCT/GB93/00808 f~-
polypeptide may have the general formula (III):
[La~F}m~[~~G]n~Lc (III)
wherein F and G may each independently be a polypeptide
according to any one of For~ul~e I to IIb, L is a
linking sequence, a, b and c are each independently O or
l and m and n are each positive numbers e.g. ~etween l
and lO inclusive. ~ is preferably a short,
conformationally flexible section of polypeptide chain
such as, for example and without limit (Seq. I.D. No: 5
Gly-Gly-Gly-Gly-Gly, (Seq. I.D. No: 6) Gly-Pro-Gly-Pro-
Gly-Pro or (Seq. I.D. No: 7) Gly-Ser-Ala-Gly-Ser-Gly- ~
Ala. It shouId be clear that each repeat ma~ optionally ~:
have a different variant of a polypeptide according to
the invention.
~ : Polyvalent dete~minant analogues as defined by
Formula III are referred to as pseudohomopolyvalent,
wherein~variants~ o essentially the same determinant ;~
~analogue~are repeated in a single polypeptide chain. In
~ addition, simple homopolyvalent polypeptide immunogens, ~
: which:contain multiple copies of the same variant of one
: of:th~ det~.rmi~ant~analogue according to any one of
ormulae I to IIb~, would also be expected ta be~
25~ effe~tive, and are a1so included within the~scope of the
present inven~ion. ;~
Pseudohomopolyva~lent immunogenic polypeptides are
:;; expected to be particularly valuable as vaccines, where
they should elicit the production of a range of :~
(neutralisin~) antibodies with a similar ~ut
non-identical unde~lying spec`ificity, which ~etween them ..
would cross-react with envelope protein from a wider
range of HIV strains, and would~thus be more e~fective
at conferring protective immunity. There would also be
35 :: ad~antages in constructing heteropolyYalent polypeptides
: which contain one or more copies, in any order, of one
~ o~ the polypeptides according to the present invention
-~ and one or more other polypeptide analogues of
determinant analogues. Such polypeptides, which are
:,
:; , "
~ WO93/21218 ~ 1 3 ~.3~ ~ 3 PCT/G893/0080~
. ., :
provided for in the present invention, have the general
formula (I~):
"
Ld-[G-L]m F-~L-G]n-Le (IV)
;
wherein F is a polypeptide according to any one of
Formulae I to IIb, G is a polypeptide according to any
on~ of Formulae I to IIb or other sequence, m and n are
each positive numbers e.g. between l and lO inclu~ive,
and d and e are each independently 0 or l. I'L" is
preferably a short, conformationally flexible section vf
polypeptide chain:such as, for example and without
limit (Seq, X~D. No: 5) Gly-Gly-Gly-Gly-Gly, (Seq. I.D.
No: 6) Gly-Pro~Gly-Pro-Gly-Pro or (Seq. I,D. No: 7) Gly- ;
15 Ser-Ala~Gly Ser-Gly-Ala. .:
It is to be understood that any antigenically
; significant subfragmen~s and/or antigenically
significant variants of the above-identified polypeptide :~
: sequences which rétain the general form and function of
` 20 the parent polypeptide are included within the scope of
this invention. In particular, the substitution of any
of:thè:specific residues:by residues having comparable
: ` conformational and/or physical properties, including
. substitution by rare amino acids ~e.g. D-stexeoisomers~
:25 ` or s~nthetic amino acid analogues, is included:O For
:example~, substituti~n of a residue by another in ~he
same~Set, as defin d below, is included within the ambit
of the in~entionl Set 1 - Ala, Val, Leuf Ile, Phe, ~yr,
Trp and Met; Set 2 - Ser, Thr, Asn and Gln; Set~3 - Asp
: 30 and 1u; Set 4 - Lys, His and Arg; 5et 5 - ~sn and Asp;
~: - Set 6 - ~lu and ~In; Set 7 - Gly, Ala, Pro, ~erland Thr.
D-stereoisomers of a1l amino acid types, may be
: :subs~itutéd, for example, D-Phe, D-Tyr and D-T~p.
In preferred embodim~nts of the inven~ion, X and Y
~if present may independently include one or~more
se~ments of protein sequence with the ability to act as
a T-cell epitope. For example, segments of amino acid
: sequence of the general formula 1-2-3-4, where 1 is Gly ::
` or a charged amino acid (e~g. Lys, His, Arg, Asp or
,
fJ 1 ,,~
WO93/21218 PCT/GB93/00808 ~ ;
-- 8
Glu), 2 is a hydrophobic amino acid (e.g. Ile, Leu, Val,
Met, Tyr, Phe, Trp, Ala), 3 is either a hydrophobic
amino acid (as defined above) or an uncharged polar
amino acid (e.g. Asnj Ser, Thr, Pro, Gln, Gly), and 4 is
5 a polar amino acid (e.g~ Lys, ~rg, His, ~lu, Asp, Asn, ~:
Gln, Ser, Thr, Pro), appear to act as T-cell epitopes in ~ ~
at least some instances (Rothbard, J.B. & Taylor, W.~. :
(1988). A sequence pattern in common to T-cell
epitopes. The EMBO Journal 7(l): 93-lO0). Similarly
segments can be of the sequence 1'-2'-3'-41_51, wherein
1' is equivalent to 1 as defined earlier, 2' to 2, 3'
and 4' to 3, and 5' to 4 (ibid). Both forms are
included wi~hin the scope of the present invention and
one or more T-cell epitopes (preferably less than five)
may be incorporated into a polypeptide according to any
one of formulae I to IIb. Therefore each epitope may be
of the type defined above ox may be of other structure
and may be separated by spacer segments of any length or
composition (preferably less than five amino acid
20 residues in lengthj and comprise for example residues `~
selected from Gly, Ala, Pro, Asn, Thr, Ser or
polyfun~tional linkers such as non-~ amino acids. It is
: possible for a C- or N-terminal linker to represent a
:complete protein, thus obviating the possible need for
~conjuga~ion to a carrier protein~
:; : Also included within the scope of this invention
~: ~ are derivati~es of the polypeptide according to formula .`
; I in which X sr ~ are or include a "r~tro inverso" amino
acid, i.e. a bifunctional amine ha~ing a functional ~;
group corresponding to an amino acid. For example an
analogùe according ~o the in~entioniand containing a :
retro i~verso amino acid may have the formula:
R ~
Al - N - C - N - A2
H H H
:
.~- WO93/21218 21 ~ 0 3 3 PCT/GB93/00B08
g _ ,
where R is any funotional group, e.g. a glycine
side chain, and Al and A2 are preferably each a copy of :.
one of the analogues defined herein (but not necessarily
the same) a~tached by its N- or C-terminal end~ T-cell
epitopes may optionally be included as discussed
earlier.
Re-tro-in~erso modification of peptides involves the
reversal of one or more peptide bonds to create
analogues more resistant than the original molecule to
enzymatic degradation and offer one convenient route to
the generation of branched immunogens which contain a ~;
high concentration of epitope for a medium to large
immunogen. The use of these compounds in large-scale ;~
solution synthesis of retro-inverso analogues of short- ~:
chain biologically ac~ive peptides has great potential.
It should be noted that analogues incorporating
retro in~erso amino acid derivatives cannot be made
directly using a recombinant DNA system. However, the
basic analogues can, and they can then be purified an~
chemically linked to the retro-inverso amino-acids using
standard peptide~organic chemistry. A practical and
convenient novel procedure for the solid-phase synthesis
on polyamide-type resin of retro-inverso peptides has
~ :~ been described recently [Gazerro, H., Pinori, M. &
;~ ~ 25 Verdini, A.S. (199Oj. A new general procedure for~the ~.
- solid-phase synthesis:of retro-inverso peptidPs. In
'Innovation and Perspecti~es in Solid Phase Synthesis~
Ed. Roger Epton.::~ SPCC (UK~ Ltd, Birmingham, UK].
The polypeptides are opkionally linked to a carrier
molecule, either ~hrough chemical groups within the
polypeptidés the~selves'~or through additional amino l i :
`acids added at eikher the C- or N-terminus, and which :~
may ba separated ~rom the polypeptides themselves or
surrounded by one or more addikional amino acids, in
3S order to render them optimal ~or their immunological
function. ~ Many linkages are suitable and include fo~
xample use of the side chains of Tyr, Cys and Lys
residues~ Suitable carriers include, for example,
WO93/21218 2 11. ~ O ~ ~ PCT/GB93/00808 r~
- 10 -- .
purified protein derivative of tuberculin (PPD), tetanus
toxoid, cholera toxin and i~s B subunit, ovalbumin,
bo~ine serum albumin, soybean trypsin inhibitor, muramyl
dipeptide and analogues thereoff and Braun's lipoprotein
although other suitable carriers will be readily
apparent to the skilled person. For example, multiple ,
ankigen peptides may be used such as those comprising a
polylysyl core, e.g. heptalysyl, bearing reactive amin~
termini. Polypeptide antigens according to the
in~ention may be reacted wi~h, or synthesised on, the
amino termini and different polypeptide antigens may be
reacted with the same core or carrier.
When using PPD as a carrier for polypeptides
according to the invention, a higher titre of antibodies
is achieved if the recipient of the polypeptide-PPD
conjugate is already tuberculin sensitive, e.g. by
vlr~ue of earlier BCG vaccination. In the case of a
human vaccine it is worth noting that in the UK and many
:other Gountries the population is routinely offered BCG
:: 20 vaccination and is therefore largely PPD-sensitive.
;: Hence PPD is e~pected to be a preferred carrier for use
in such countries.
The mode of coup~ing the polypeptide to th oarrier ~.
will depend on the nature of the materials to be
: 2S coup1ed~ For example, a lysine residue in the carrier
may be coupled to a C-terminal or other cysteine residue
in a polypeptide by treatment with N-
~~maleimidobutyryloxy-succinim~de (Kitagawal T. & Ackawa,
: ~ T. ~1976) 3. Biochem. 79, 233). Other coupling
reactions and reagents have been described in the
iiterature. ; ~
The polypeptides, either alone or linked to a
carrier moleculP, may be administered by any route (eg
parenteral, nasal, oralg rectal, intra-vaginal), with or
3S without the use of conventional adjuvants (such as
aluminium hydroxide or, when not ~or administration to
:: humans, FreundCs complete or incsmplete adjuvants~ :
- and/or other i~munopotentiating agents. The invention
.
h 11~ 0~ ~
. WO93/21218 PCT/GB93/00808
-- 11 - . .
also includes formulation of polypeptides according to
the inven~ion in slow-release formsl such as a sub-
dermal implant or depot comprising, for example,
liposomes (Allison, A.C. & Gregoriadis, G. (1974) Nature
(London) 25~, 252) or biodegradable microcapsules
manufactured ~rom co-polymers of lactic acid and
glycolic acids (Gresser, J. D. and Sanderson, J. E.
(1984) in l'Biopolymer Controlled Release Systems" pp
127-138, Ed. D. L. Wise).
It is to be understood that the polypeptides
according to the invention may be synthesised by any
conventional method, either directly us.ing manual or
automated peptide synthesis techniques as mentioned
above, or indirectly by ~NA or DNA synthesis and
conventional techn.i~ues of molecular biology and genetic
engineering. Such techniques may be used to produce
hybrid:proteins containing one or more of the
polypeptides inser~ed into another polypeptide se~uence.
Ano~her aspect of the presen~ invention therefore
~20 provides a DNA molecule coding for at least one
synthetic~polypeptide according to the invention,
pre:ferabIy incorporated into a suitable expression
vector~replicable in microorganisms or in mammalian,
ins~ct, p~lant, fungal or other cells. The DNA may also
25 ~ be;part:;:of the DNA:se~uence for a longer product:e.g.
. ~ the polypeptldes~ may be expressed as parts of other
prote~in into which they have been inserted by geneti~
engineering. On2 practical guide to ~ch techniques i5
'Molecular cloning: a }aboratory manuali' by Sambrook, ~:
J., Fritsch, E.F. and Maniatis, T. (2nd ~dition, 1989j.
:i Polypeptides according to the`ihven~ion may be usedi ~:
either alone or linked to an appropriate carrier, as.
(a) ~Peptide vacGines~ for use to prevent infec ion ~y
one or more strain of HIV; ~
lb) ~s ligands in assays of, for example, s~ra from HIV
: posi~ive patients; : -
:; ~(c) As quality control agents in testing, for example,
binding levels of antibodies raised against the
~'.
:
WO93/21218 2 ~ 1 8 0 ~ 3 PCT/CB93/00808
- 12 -
polypeptides;
~d) As immunogenic agents for the generation of
monoclonal or polyclonal antibodies by immunisation of
an appropriate animal, such antibodies being of use for
5 (i) the scientific study of the HIV virus, (ii) as ~:
diagnostic agents, e.g. as part of histochemical . ~ :
reagents, (iii) for the passive immunisation of ~IV
patlents, either as a treakment for AIDS in itself, or
in combination with other agents such as, for example
AZT and/or HIV protease inhibitors, and (iv) as a means
of targeting other agents ~e.g. AZT or HIV protease
inhibitors) to HIV infected cells expressing HIV
envelope proteins on their surfaces, such agents either .
being Iinked covalently or otherwise associated, e.g. a~
in liposomes containing such agents and incorporating
antibodie~ raised against any of the antigenic
: ~ :polypeptides~ The invention further provides for
gene~ically en~ineered forms or sub-components,
espec:ially VH rPgions ~ of antibodies raised against the
~:20 polypeptides, and of~humanised forms of antibodies
: :initially raised against the polypeptides in other ~.
animals, using techni~ues described in the literature;
and~
e~The:treatment of HIU infections,~e~ither by
~displ:acing the binding of HIV virus:to human or animal
cells::or by disturbing the three-dimensional
organisation of the virus in vivo;~as well~as aiding the
scientif ic study of HIV viruses in vi~ro. ::
In respect of~detection and diagnosis,~of HI~ or
30 antibodies against HIV, the skilled person will be aware `~
of a~varie~y of~imm~unoassay techniques known i~ ~he a~t j ;
inte ~ ~ a, sandwich assay, ~ompetitive and non-
competitive assays and the~use of direct and indirect
labelling. - ~;
35 ~ A further aspect of the inYention provides a kit~ ~ ;
for detecting HIV or antibodies against HIV which
comprises at least one synthetic polypeptide according
to the invention.
~ .
~: '
A ~ WO 93t21218 ~ 3 t PCT/GB93/008U8
The preparation of polyclonal or monoclonal
antibodies, humanised forms of such antibodies (see, for
example, Thompson K. M. et al (1986) Immunology 58, 157-
160), single domain antibodies (see, for example, Ward,
E. S., Gussow, D., Griffiths, A. D., Jones, P. and
Winter, G. (l~9) Nature 341, 544-546), and antibodies .
which might cross the blood-brain barrier, which bind
specifically to a synthetic polypeptide according to the
present invention, may be carried out by conventional
means and such antibodies are considered to form part o~
this invention. Antibodies according to the invention
are, inter alia, of use in a method of diagnosing ;
mammalian HIV infection which comprises incubating a
.. . .
sample of tissue or body fluid of mammal with an ~;
15 effective amount of antibody as described herein and :-
determining whether, and if desired the extent to which
and/or rate at which, cross-r~action between said sample
and said antibody occurs. Diagnostic kits which contain ~:~
~: at lea~t one of said antibodies also form part of this
: ;,
O invention.
A ~urther aspect of the invention pro~ides
synthetic polypeptides ~or use in therapy or:prophy1axis -:
of mammalian HIV infection and/or stimulating the
mammalian immune: system and/or blocking the:cellular
25~ receptors for t:e HIV virus and for the preparation of
m~dicaments suitab~le for such uses. Also included are
pharm~ceutical compositions containing, as active~
ingredient, at least ~one poly~eptide ~r
polypeptide-carrier conjugate as described herein:in
30 ~association with one or more pharmaceutically acceptabl~ ~;
a;d~uvant~lcarrier~and/or1excipient. iThe:compo~it~lons may
be formulated for oral, rectal, nasal or especially -:
parenteral administration (including intra-C~S
administration).
The invention further provides a method ~f therapy
: : or prophylaxis of mammalian HIY infection and/or of
stimulating the mammalian immune system and/or of
~; blocking the cellular receptors for the HIV virus, which
: :
WO93/21218 h 1 1 3 0 ~ 3 PCT/GB93/00808 ~c~
comprises administering an effective amount of a
polypeptide as hereinbefore defined, either in isolation
or in combination with other agents for the treatment of
AIDS such as AZT and/or inhibitors of the HIV protease.
The following examples are intended to illustrate
the invention and are not limiting in any way. ~ :
ExamPl e ~
A C-terminally extended form of peptide Ib of
lO sequence (Seq. I.D. No. 8) ~:;
Leu-Gln-Leu~Thr-Val-Trp-Gly-Ile-Lys-Gly-Cys-Ala :~
: was synthesised using standard solid-phase F~moc ~;
methodologies (Atherton, E. and Sheppard, RoC~ ~ 1985;
J.Chem. Soc~ Comm. 165-166). The C-terminal Gly-Cys-Ala ~
was introduced to provide an alternative coupling site ::
to the carrier. The peptide was cleaved from the resin
in the presence of tri~luoroacetic acid and subsequent
purification of peptide wa5 achieved by gel filtration,
ion exchange chromatography and reverse-phase high
: : per~ormance li~uid chromatoyraphy. The purity of the
resultant peptide was in excess of 85%. The C-terminal ;.
aIanin was included to assist the conjugation.~ The
peptid was di5sol~ed in phosphate-buffered saline ~PBS;
5mg~ml:) and mixed with an equal ~olume of ovalbumin~ -
(5mg/'mI~ prior to the:addition of glutaraldehyde to a
final con entration of 0.1%(w/v)~ The conjugate mixture
::
5 allowed to s~and for 30 minutes prior to
em~lsification with Freund's adjuvant. Each sheep (5
animals'in éach group) was;immunised~with the eguivatentl ;
of 250 ~g of peptide in Freund's Complete Adjuvant
(F~ ach animal was given a boo5ter injection at 2
weeks and then again at 5-6 weeks with a similar amount
3S ~ of peptide in Freund's Incomplete AdjuYant (F A). Blood
~: : samples were taken 7-lO days after the final booster
injecti~n and assayed for binding to HIV gpl60 en~elope
~ protein.
; ~wo HIV-l strains were used in the study. One of
.
h . ~ ;,t,~
,.-. . WO 93/21218 PCT/GBg3/00808
-- 15 --
these was a fully~characterised, well-recognised and
widely~used HIV-l strain named GB8 available from Dr G~
Farrarr, Centre for Applied Microbiology and Research .
(CAMR), Porton Down, Salisbury, UK and discussed in
AIDS 1: 147-150 (1987). Th~ other was the RF strain of
HIV-1 used in the syncytium inhibition assay. RF strain
of HIV-1 is known from Science 224: 497~500 (1984).
Sequences from two or more HIV-1 isolates from a single
individual have been obtained and the capabil.ities of
the test materials to inhibit growth and replication of
these ~irus strains wexe assessed comparatively. The
isolates were obtained from Dr~ G. Farrarr under
designations GB8A, GB8B and ~B8D.
Cel1 lines ~9, C8166 and JM are known to support
replication or show syncytium formation when infected
with HI~-1 'laboratory strains' of GB8 or RF.
) Assa~s of inhibition of live HIV-I antl-~eptide
. antisera :
Three standard assay procedures have been used to assess :,
the inhibition of live HIV-l by anti-peptide antisera. :~
; ~ : : , ,
i) Neut:ralisation assay
: ~ ~
: Thi5 a6say measures the capability of~the anti-peptide
anti6era ~o prevent attachment and infection of
su6ce~tible ceIls by live, ce}l-free virus. The
measurement of this is determined by syncytium counts.
: 30
Tèst~antisera wer~ inactiivated ~y heat at 56~C for 30 ;!
; :minutes, diluted appropriately in RPMI 1~40 and
incubated with an equal volume of HIV-l conta~ning fluid
.
of known titre for 30 minutes at room temperature ~RT) .
A CD4~ cell line (JM) at a known concentration and
highly susceptible to infection with HIV-l GB8 strain
: was exposed to the HIV-l antiserum mixture, left for 2
hours at 37C, washed, resuspended in cell culture
medium, incu~ated and assessed by syncytium cvunts at
~; ~
WO93/21218 2 1 ~ 8 0 ~ ~3 PCT/GB93/00808 ,~
- 16 ~
specified times. Neukralising activity of the antisera ~;
was assessed by reduction in syncytium counts compared
to controls.
,~
S Controls included with each batch of tests were known
negatîve and positive sera replacing the test antiserum .
in an otherwi~e identical procedure.
(ii) Inhibition Assay
,
This assay de-termines the ability of the anti-peptide
antisera to inhibit replication of HIV-l subsequent to
cellular infection and measures levels of extracellular
and intracellular HIV~l p24 antigen. In addition,
1~ syncytium detection and enumeration are perfo~med. ~;
A highly susceptible CD4+ cell line was exposed to HIV-l
at a known multiplirity of infection and incubated ~or 2
hours at 37C. The ceIls were then washed 3 times with
: 20 ~PMI 1640 and an equal volume of appropriately diluted,
: heat-inactivated test antiserum was added. The infected
cell serum suspension was incubated for l h~our at 37C,
amL~ed microscopically and the culture medlum sampled
: :at~specified time-points subsequently (at days 3 and 5~ ;
~or measurement of p24 antigen. Intracellular p24
~ntigen lavels wers determined at similar time points
~ollowing lysis o~ the cells using appropr}ate lysis
bu fer. Samples to be tested.for HIV l p24 antigen by
; ELISA were stored until the assays were performed.
Syncytium counts were measured on day 5.
~on~rols for each batch of tests included known negative
,,
and po~itiVQ sera replacing the test antiserum in an
otherwise identica1 procedur
Syncytium assay
This as ay determinPs the capability of anti-peptide
antisera to prevent spread of live HIV-l from infected
J~3
WO93/21218 PCT/GB93/00808
- 17 -
to uninfected cells and to prevent fusion between cells
mediated through reactivity between the virus ~:
glycoprotein (gpl60) and the CD4 molecule. The
measurement is carried out by syncytium detection and
5 enumeratio~O ~:
A known concentration of an HIV-l producer ~CD4~) cell
line supporting active virus replication (H9 cells
chronically infected with HIV-l RF strain) was washed 3 ~;
; 10 times and mixed with the antiserum under test used at
specified dilutions. After incubation for 30 minutes at
37C these cells were mixed in specified pxoportions :~
~: with an indicator CD~+ cell line (C8166), highly
susceptible to HIV-l infection and syncytium induction.
These cells were observed dail~ for syncy~ium formation~
Controls for each batch of tests included known positive ~.
and negative sera replacin~ the test serum in an
,.
otherwise identical procedure.
; 20
. RESULTS
Neutralisation:assay
25: ~A number of sera raised to peptide Ib~exhibited in vitrQ
ne~tralising activity against HIV-l GB8 strains~ Table
: 1 indicates greater acti~ity of serum 17 ayains~ all GB8
sequential isolates. The assay demonstrates that the
sera neutralise~cell-free virus.
: : :
`
:'
' ~ . ' ' '
~::: '
,~.
W093/~l2l8 21 1 ~ 0 3 3 PCT/GB93tO0808 ~
- 18 -
Table 1 : Anti-peptide sheep sera showing in v}tro
neutralising activity against HIV-1 GB8 strains
~ ~ . __ .
Serum Serum Neutralising activity*
against HIV-l strain :.
~ _ Dilution _ _ _ _ _ ~ ~.
Lab No. GB8 GB8AGB8B GB~D ;
. Tested
, - , _ ~ _ _ _
17 1:5 4/72(g4) 0/98(100)3/80(96) 19/77(75) .~;
1:10 13/72(~2) 14/98(86)22/8~(72) 21/77(73)
~7 1:5 15/85(~2)4/48(9~) 5/60(92) :
1:10 49/85(-)27/48(-) 10/~(83)
17 ~:5 2/51(96)
. 1:10 17/51(67)
21 1:5 10/68(85)18/59(69) ~/36(78)
1:10 . 28/68(59)18/59(69) 22/36(-)
37 1:5 ~7s~99) S/48(90) 23/59~61)
1:10 4~/7s( ) 15/~(69) 39/59(-)
37 : 1:5 15/58(74) 16/60(73)
1:10 32/58(-) 54/60(-)
37 : 1:5 29/7Z(60) 23/48(52)
: 1:10 68/72(-) 25/48(-)
~ :
__ ~ ~
* - mean no. of synctia in test/mean no. of syncytia in
control C% reducti:on)
) = reduction < 50%
:
: Replication inhibition assay
Sera 17 and 37 show comparable activity in ~rms of
reduction in syncytium counts at day 5 in the
replication inhibition assay as demonstrated in Table 2,
: Furth r, replication is preYented as indicated by p~4
antigen determination. It should be noted that the sera
use~ in this assay were not diluted.
.
,:
WO 93J21218 PCI'/GB~3/00808
- 19 - '
_ _ . _ _ _ . _ ~ ~:
~;
. ~ ~ h
..`:
?- :~ ~ 0 .'
V n~ ~ ~o ~ ~ ,"~'
~ a o u~ ~ ~ o o ~ O ~ : ~
u~ ~ ~r u~ :"
m ~ ~ ~ ~ :
,~ ~ V ,~
~ -- _ . ;'
U~
u~ ,1 ,1 o o In ~ r
:~ ~ ~r ~r o ~1 t`
'I O :1 ~ r- co ~ u~ r
~ ~ ~ r co
:1~ ~ X
0.~ ~ ~ . . _ _ _
3 ~ ~ ~
.,, 0 ~ ~ ~o ~ ~
Q p,, ~ : ~ o ~ m t`
.
~ ~ ~ ~ :
_ _ ~ , _ ~1
a3 0 h .C
4 ~1 . S Ln ~ ~O ~ 0
~ ~ ~) ~ o ~
; ~:: ;: U V ~ v O ::
: $~ ::~ _ ~ ~ ~ :
~ X ~ ~
:~ ~ _ _ ~
~ ~; N ~ ~ 5 ; ~S) t~ ~ o ~ ~ -;
~,) o ~ o ~ r~
.8 .~ :h ` ! ~ i
E-~ ~ H C .;
,.' ~ . ~ ~.
'' ;., '` E z ~ ~ o ¦ o
~z L ,~ `
.
.
W093/21218 ~ 3 c~ PCT/GB93/OOB08
- 20 -
Syncytium inhibition assay
Table 3 gi~es data for serum 17 in the syncytium
inhibition assay. A dilution of 1:100 reduced the
inhibiting activity of the serum to a little less than
50%~ This test suggests how even diluting the serum may
prevenk cell fusion and hence spread of live HIV.
,
Table 3: Syncytium inhibiting activing o f anti-peptide
: against HIV-l ~ strain H9-RF) in vitro
_ ... .
Serum S~rum .
Syncytium-inhibiting act.ivity
_ Dilutionagainst H9-RF
Lab No. Tested
__ _ _ _ _ .__ _.
17 l.50 3/101 (97)*
1017 1:2S 12/51 (77)
:So: ~ 16/51 (69)
~: : ~:100 27/51 (47)
_ ~
: ~,
15 ~ *`= Mean number of syncytia in test/Mean number of ::
s~ncytia in control ~% reduction)
` ~ : ~ ' ;'
~ ~ '
:: ~
?.e W093/21218 2 ~ J 3 3 PCT/GB93/OOB08 .~;
: - 21 -
Exam~le 2
ELISA reactlvi~ _f HIV-2 positive human se_a with
pePtides accordinq to formulae IIa and IIb
A C-terminally extended peptide of formula XIa having
the sequence (Seq. I.D. No: 9)
Gln Gln-Glu-Met-Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-
Lys-Gly Cys (2~)
:
and a C-terminally extended peptide of formula IIb
having the sequence (Seq. I.D. No: lO)
:,
Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Gly-Cys (2B)
were synthesised and:purified as described in Example l.
: The peptid~s were then used in an ELISA with ~IV
positive human:sera. Peptid~ "lOA" (disclosed in ;~
~Example l of our cop~nding application~published as
W09l/OOgO3) is a known to be an immunodominan~ peptide
related to the HIV envelope protein and is known to :.
recognise substantially all HIV l infected individuals.
This peptide was used to investigate cross-reacti~ity
: 25~;and confir~l the suitability of the protocoI.
LISA Protocol ~ ~
100 ~1 of coating buffer, containing 20:~g/~1 of
:~30 ~ peptide, was added to ach well of the ELISA plates ::~
yne~ech) and the plates incubated overnight at 4~Co
2. Plates were washed once in Trls bufferled sali~e and
: : : dried thoroughly.~ :
3.~ 200 ~l of blocking buf~er was added to each well,
the plat~s shaken~for 10 seconds and:incubated for l
hour ~t 37C.
4. The:sera to be tested were diluted l/lO0 or 1~200
using dilution buffer. O.l ml was added to each well,
: and the pla~es incubated-for l hour at 37~C. This was
done in triplicate for each sample.
.
w~ g3/2.2l8 2 1 1 ~ ~ 3 3 P~/GB93/00808 f~
-- 2 2 ~
5. The plates were washed three times for 2 minutes
with washing buffer.
6. 100 ~l of alkaline phosphatase conjugated goat
anti-human IgG (l/2000 in dilution buffer) was added to
each well and plates incubated for 1 hour at 37C.
7. The wells were washed as in (5).
~. 50 ~l of alkaline phosphatase buffer was added to
each well followed by 50 ~l of substrate (10 mg/ml in
water~ and the plates incubated for 15 mins at 37C.
9. The absoxbence was read immediately at 4~4 nm.
The human sera samples tested were as follows: .
20 known HIV-2 po~itive sera that had previously
been shown to exhibit no cross-reactivity with HIV-l
sera. All twenty patien~s came from the Ivory Coast.
5 know~ HIV-2 positiye sera that had previously ;
been shown to exhibit no cross-reactivity with HIV-l ,::
sera. ~ll fi~e patients came ~rom Africa but not the
Ivory Coast.;
7 known H~V 1 positive sera that had been shown to
exhibit no cross-reactivity with HIV-2 sera. .
lO~known HI~ negative sera samples from age and sex
matched subjects.:
he control:contained no serum, but the total
; :~ 25 volume was~ maintalned~by addition of~0:.1 ml of dilution .~: :buffer.
It~is clear ~rom the results in Table 4 below ~hat :
No~cross reactivity between HIV-l and HIV~2
:peptides was observed, that is HIV-2 sera did not
~recognîse the HIV l peptide and HIV-l sera did not
; recognise the HIV-2 peptides;
'
2)~ No peptide specific activity was found in the HIV
~: : negati~e samples;
35:
3) There is a distinction in reacti~ities between sera
: of patients who came from the Ivory Coast, and-patients
: who ¢ame from other parts 9f the African continent; and
4) The HIV-l~ serum samples had detectable antibodies
: :,
.
,~ û 3
...~ WO93/21218 ~ 3 PCT/GB93/00808
- 23 -
to the HIV-l (lOA) peptide.
Table 4 : Reactivity of HIV positive sera with peptides
2A, 2B and lOA.
_ . _ __
Reactivities
Number of positive sera/sample size
_ _ ... ..
5ample panel 2A 2B lOA (reference)
~ _
HIV-2 positive
Ivo-y Coast 0/20 0/20 0/20
~ _ ~ _ .
HIV-2 positive
Mon-Ivory Coast 5/5 5/5 0/5
__ . __ _
HIV-l positive ~/7 0/7 6/7
_
HI~ negati~e 0/lO - 0/lO 0/lO
_._ . _ ._ _
~s exp~cted, no posi tlve resul ts occurre d with the control.
,:
These results show that HIV-2+ sera from a number
of patients contain antibodies which react specifically
with the HIV-2 peptides according to the formulae IIa
and IIb of the invention~
2~ Example 3 . ~ :
-~
Immunisation of mice with ~_Eep ide accordinq to ~ormula
:.Ia_con~uqated to a carrier Protein
~ . : :
.
. .
; 25 ~A C-te~minally extended according to formula Ia ha~ing
~ ~the~se~uence (Seq. I.D~ No~
; ~ ~Gln-Gln-~is-Leu-Leu-Gln-Leu-Thr Val-Trp-
: - : : Gly-Ile-Lys-Gly-Cys (lA)
was synthesised and purified as des~ribed i~ Example l.
Con;u ation ~ ;
The peptide was conjugated to ovalbumin using elther
glut~raldehyde or MBS (M-maleimidobenzoyl-N-hydroxy-
~: succinimide ester) as the coupling agent.
:: :
Glutaraldehy~de : The peptide was dissolved in PB5 (5
~ .
'
WO93/2121~ 2 1 1 ~ ~ 3 3 PCT/GB93/0080X ~;
- 24 -
mg/ml~ and ovalbumi~ added to give a molar ratio of 50
amino acids of carrier per l mole of peptide. The total
~olume was adjusted ~o 2 ml with PBS. 2 ml o~ 0.2%
solution of glutaraldehyde in PBS was slowly added to
the stirring mixture and the solution left stirring at
room temperature for l hour. The mixture was dialysed
against large volumes of PBS overnight and stored at
-20C for not longer than 3 weeks.
MBS : O.l ml of a solution o~ MBS (25 mg/ml in PBS) was
added to a stirring solution of ovalbumin (lO mg/ml in .
P~S) and the reaction mixture stirred at room :
temperature for 30 mins. The activated ovalbumin was ~;
then separated ~rom free MBS by gel filtration on a
,
Sephadex G-25 column equilibrated with PBS. Peptide was
dissolved in PBS and added to the activated ovalbumin so
that a final concentration of l mole of peptide for each
50 amino acids of carrier resulted. The pH was adjusted
to 7-7.5 and the reaction stirred for three hours at ..
:20 room temperature. The mixture was dialysed against
large~volumes of PBS overnight and stored at -20C ~or
:~not longer than 3 weeks.
To determine the peptide/carrier protein ratio a
procedure d~scribed by Habeeb can be u~ed (Habeeb,
A.F.S.A., Anal. Biochem., 14, 328 (l966)3. In this, the
free amino acids are determined by the trinitro~enzene
;~ sulphonic acid (TNBS) method using borate buffer, pH
9Ø ~The absorbence of the solution is read at 335~ nm
and the percentage of modified free amino acid groups i5
~ calculated. The free thiol assay as described by Ellman :
can also be used to assess the ef~iciency of conjuga~ion
(~nderson, W.L. and Wet1aufer, D.B., Anal. Biochem.~ 67.
493 (1975)~)-
:
Immunisatisn
C57 Black mice were pre-bled 2-3 days before the
. primary inoculatiQn, the sera separated and stored at
20C. This was the negative control mouse sera. Each
~0 mouse was immunised with 40 ~g of peptide in FCA and
rJ ~ rj r3
WO93/2121~ PCT/GB93~00808
- 25 -
subsequently with 20 ~g of peptide in FIA 2 to 3 weeks
later. The inoculation volume was O.l ml in each case
and all injec~ions were given subcutaneously. Animals
were bled immediately prior to the booster injection,
and then two weeks and four weeks later.
ELI5A
The ELISA protocol was as described in Example 2,
except that sera were diluted l/50 and l/lO0 in dilution
~uffer, and the antibody used was alkaline pho~phatase
conjugated goat anti-mouse IgG~
'
_ sult~
l. 2/24 (8%) animals responded to the peptide
according to formula Ia conjugated using glutaraldehyde
when compared to controls after the third bleeding.
Thus:~it is possible to raise antibodies against this
p~ptide when it has been non-specifically conjugated to
: a~carrier~ : :
2. ~ 14~24 (58%) of the mice had high levels of ~.
an*ibadies to the peptide after only one immunisation
2~5`~ with~the peptide accor~ing to~formula Ia conjugated to
:ova1bumin by the:specific linkage using MBS. This is a
strong:and specif~ic response. : ~ -
Tho results~show:that the peptide according to~
~ 30 ~formula Ia, when conjugated to ovalbumin using ~BS, is a
:~ : powerful i~munogen inducing very high levels o~ antibody
a~te~ a single immunisation and that antibodies c`an bel
:~aised even if the peptide is non-specifical~ly bound~to
~ ovalbumin.
: .
WO93/21218 2 ~ 3 3 - 26 - PCT/GB93/00808
SEQUENCE LISTING
Number of SequPnces ll
(l) Information for Se~ I.D. No: l
(i) Characteristation of sequence:
(A) Length: 13 Amino acids
(B) Type: ~mino acid
(D) Topology: Linear
(ii) Type of molecule: Peptide
(xi~ Description o~ sequence: Seq. I.~. No: l
Gln Gln ~is Leu Leu Gln Leu Thr Val Trp Gly Ile Lys
~: ~ 1 S 1~ .
:~ '
: (2) Information for Seq. I.D. No: 2 :~
(;.) Characteristation of sequence: ;
: (A3 ;Length: 9 Amino acids
(B) Typ~e: Amino acid
(D); Topology: Linear
(ii) Type of molecule: Peptide
(xi) Description of sequence: Seq. I.D. Mo:
~ , ~
Leu~Gln Leu ~hr Val Trp Gly Ile Lys
(3)~ Information;for:5eq. I.D. No: 3
i) ~Characterlstation of sequence~
(A) Length: 13 Amino acids
(B) Type: Amino acid
(Dj ~opolo~y:! Li~ear
Type of molecule: Peptide
(xi) ~Description of sequence: Seq. I.D. No: 3
:-Gln Gln G}u ~et Leu Arg Leu Thr Val Trp Gly Thr ~ys
, ~ ~. :::
- , - -
WO93121218 ~1 ~ Y 0 3 ~ PCT/GB93/00~08
- 27 -
~4) Information for Seq. I.D. No: 4
(i) Characteristation of sequence:
(A~ Length: 9 Amino acids ':
~B) Type: Amino acid
(D) Topology: Linear
,";",
(ii) Type of molecule: Peptide .
~xi) Description of sequence: Seq. I.D. No: 4
Le~ Arg Leu Thr Val Trp Gly Thr Lys
l 5 ~:
(5) In~ormatio~ for Se~. I.D. No: 5
,
(i) Characteristation of sequence: ~ ~:
~A) Length: S Amino acids .
. (B) Type: A~ino acid
~: (D) Topology: Linear
, ~ , .
Type of molecule: Peptide
(xi~ Description~of~sequence: Seq.~ I.D. Mo: 5
Gly Gly Gly Gly Gly ;~
(6)~ Information~for Seq. I.~D. No::6; :
ij Characteristation o~ sequence~
::(A)~:Leng~h: 6 Am~no acids : ;:
f) Type: Amin~ acid
:: (D~ ~Topolo~y: Linear
ii) Type of ~molecule Peptide :
: (xi~ Description of sequence: Seq. IoD~ No: 6
Gly Pro~Gly Pro~Gly~Pro
(7)~ Information-for:Seq. I.D. Nof 7
(i) Characteristation of sequencP
(A) Length: 7 Amino acids ~ ~
' (Bf) Type: Amino acid ~:
W093/21218 ~7t r ~ û 3 3 PCT/GB93/00~08 ~
~ 28 - ' ;
(D) Topology: Linear
(ii) Type of molecule: Peptide
(xi) Description of sequence: Seq. I.D. ~o: 7
Gly Ser Ala Gly Ser Gly Ala
l 5
(8) Information for Seq. I.D. No: 8
(i) Characteristation of sequence:
(A) Length: 12 Amino acids
: (B) Type: Amino acid
(D) Topology: Linear
(ii) Type of molec~le: Peptide .
(xi) Description of sequence: Se~ D. No: 8
~ 1eu Gln Leu Thr Val Trp Gly Ile Lys Gly Cys Ala
,~ 1 5 1
(9) Information for Seq. I.D. No: 9
~: ~ (i) Chara~teristation of sequence:
(Aj Length: 15 ~mino acids
(B) Type: ~mino acid
(D) Topology: Linear:
(ii) Type of molecu}e: Peptide
(Xi) Description of sequence: Seq. I.D. No: 9 :
:: : ~ : :
Gln Gln G1u Met Leu;Arg Leu Thr Val Trp Gly Thr Lys Gly Cys
l 5 l~ 15
; ~ :
: : (lO) I~formation for Seq. I.D. No: lO ~ æ
: (i) Charactéristation of sequence:
~: (A) Length: ll ~mino acids
(B) Type: ~mino acid
) Topology: Linear
- (ii) Type of mol~cule: Peptide
xi3 Description of sequence: Seq. I.D~ No: lO
~:
,
WO93/21218 ~ 3 3 ~ PCr/~B93/00808
- 2~ -
Leu Arg Leu Thr Val Trp Gly Thr Lys Gly Cys
5 lO ~;
(11) Information for Seq. I.D. No~
(i) Characteristation of sequence;
(A) Length: 15 ~mino acids
( B ~ Type: Amino ac id
(D~ Topology: Linear
(ii) Type o:E molecule: Peptide
(xi) Description of sequence: Seq. ~.D. ~lo~
Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly I~e Lys Gly Cys
' `
":
~-;'"`,
,"~
- ~,
:~ ',``
~: ~" `.'"'
'~
', ~,, ~ , , , ~.
: - ~
~.
