U.S. patent application number 13/749180 was filed with the patent office on 2013-08-08 for therapeutic uses of monoclonal antibodies to the angiotensin-ii type-1 receptor.
This patent application is currently assigned to Gavin Paul Vinson. The applicant listed for this patent is John Richard Puddefoot, Queen Mary & Westfield College, University of London, Gavin Paul Vinson. Invention is credited to Stewart Barker, John Richard Puddefoot, Gavin Paul Vinson.
Application Number | 20130202604 13/749180 |
Document ID | / |
Family ID | 9942753 |
Filed Date | 2013-08-08 |
United States Patent
Application |
20130202604 |
Kind Code |
A1 |
Vinson; Gavin Paul ; et
al. |
August 8, 2013 |
Therapeutic Uses of Monoclonal Antibodies to the Angiotensin-II
Type-1 Receptor
Abstract
The use of monoclonal antibodies to the angiotensin-II type-I
receptor is provided for the treatment of cancer and vascular
smooth muscle cell proliferation. Specifically, use is provided of
a monoclonal antibody or a fragment thereof to a peptide comprising
the N-terminal portion of the angiotensin-II type-1 receptor
defined by the sequence TABLE-US-00001 (SEQ ID NO: 1) MILNSSTEDG
IKRIQDDCPK AGRHNYIFVM IPTLYSIIFV VGIFG in the preparation of a
medicament for the treatment of cancer or in the preparation of a
medicament for the treatment of vascular smooth muscle (VSM) cell
proliferation.
Inventors: |
Vinson; Gavin Paul; (London,
GB) ; Puddefoot; John Richard; (London, GB) ;
Barker; Stewart; (London, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of London; Queen Mary & Westfield College,
Vinson; Gavin Paul
Puddefoot; John Richard |
London
London
London |
|
GB
GB
GB |
|
|
Assignee: |
Vinson; Gavin Paul
London
GB
Queen Mary & Westfield College, University of London
London
GB
Puddefoot; John Richard
London
GB
|
Family ID: |
9942753 |
Appl. No.: |
13/749180 |
Filed: |
January 24, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13087770 |
Apr 15, 2011 |
8383108 |
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13749180 |
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10525277 |
Feb 22, 2005 |
7951904 |
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PCT/GB2003/003758 |
Aug 21, 2003 |
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13087770 |
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Current U.S.
Class: |
424/135.1 ;
424/133.1; 424/139.1; 514/21.3; 530/324; 530/350; 530/387.3 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 7/06 20130101; A61P 37/04 20180101; A61P 35/00 20180101; A61K
39/00 20130101; C07K 16/2869 20130101; A61K 47/646 20170801; A61K
2039/505 20130101; A61K 39/3955 20130101; A61P 9/00 20180101; C07K
14/72 20130101; A61P 9/10 20180101 |
Class at
Publication: |
424/135.1 ;
530/387.3; 530/324; 530/350; 514/21.3; 424/139.1; 424/133.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 21, 2002 |
GB |
0219524.6 |
Claims
1. A single chain Fv (scFv) molecule that binds to a peptide
comprising the N-terminal portion of the angiotensin-II type-1
receptor.
2. The scFv according to claim 1 wherein the peptide comprising the
N-terminal portion of the angiotensin-II type-1 receptor comprises
Glu-Asp-Gly-Ile-Lys-Arg-Ile-Gln-Asp-Asp (SEQ ID NO:2), wherein,
independently, Glu can be replaced with Asp or Gln, Asp can be
replaced with Glu, Gly can be replaced with Ala, Ile can be
replaced with Ala, Lys can be replaced with Arg, Arg can be
replaced with Lys, and Gln can be replaced with Asn.
3. The scFv according to claim 1 wherein the peptide comprising the
N-terminal portion of the angiotensin-II type-1 receptor comprises
Glu-Asp-Gly-Ile-Lys-Arg-Ile-Gln-Asp-Asp (SEQ ID NO:2).
4. The scFv according to claim 1 wherein the peptide comprising the
N-terminal portion of the angiotensin-II type-1 receptor comprises
Met-Ile-Leu-Asn-Ser-Ser-Thr-Glu-Asp-Gly-Ile-Lys-Arg-Ile-Gln-Asp-Asp-Cys-P-
ro-Lys-Ala-Gly-Arg-His-Asn-Tyr-Ile-Phe-Val-Met-Ile-Pro-Thr-Leu-Tyr-Ser-Ile-
-Ile-Phe-Val-Val-Gly-Ile-Phe-Gly (SEQ ID NO:1).
5. The scFv according to claim 1 which is an scFv derived from
monoclonal antibody 6313/G2 produced by the hybridoma cell line
designated by European Collection of Animal Cell Cultures (ECACC)
accession number 93072117.
6. The scFv according to claim 1 which is humanised.
7. The scFv according to claim 5 which is humanised.
8. A composition comprising the scFv according to claim 1.
9. A composition comprising a peptide sequence comprising the
N-terminal portion of the angiotensin-II type-1 receptor defined by
the sequence_MILNSSTEDG IKRIQDDCPK AGRHNYIFVM IPTLYSIIFV VGIFG (SEQ
ID NO:1) or a fragment thereof comprising at least five amino acid
residues.
10. The composition according to claim 9, wherein the peptide is
conjugated to a carrier protein.
11. The composition according to claim 9 further comprising an
adjuvant.
12. The composition according to claim 9 wherein the peptide
comprises up to 45 amino acids, and comprises EDGIKRIQDD (SEQ ID
NO:2).
13. The composition according to claim 12, wherein the peptide is
conjugated to a carrier protein.
14. A method of treating cancer or a disease or condition
associated with vascular smooth muscle cell proliferation
comprising administering to a subject in need thereof a
therapeutically effective amount of a monoclonal antibody, or a
fragment thereof, that binds to a peptide; wherein the peptide
comprises an N-terminal portion of an angiotensin-II type-1
receptor comprising the sequence MILNSSTEDG IKRIQDDCPK AGRHNYIFVM
IPTLYSIIFV VGIFG (SEQ ID NO:1), a conservative mutant thereof, or
an active fragment thereof comprising at least five amino acid
residues.
15. The method according to claim 14 wherein the active fragment is
a hexapeptide, heptapeptide, octapeptide, nonapeptide, or
decapeptide.
16. The method according to claim 14 wherein the peptide comprises
the sequence EDGIKRIQDD (SEQ ID NO:2), a conservative mutant
thereof, or an active fragment thereof comprising at least five
amino acid residues.
17. The method according to claim 16 wherein the conservative
mutant comprises any one or more of the following amino acid
substitutions: position 1 is E, D or Q, position 2 is D or E,
position 3 is G or A, position 4 is I or A, position 5 is K or R,
position 6 is R or K, position 7 is I or A, position 8 is Q or N,
and position 9 and 10, independently, are each either D or E.
18. The method according to claim 14 wherein the monoclonal
antibody is humanized.
19. The method according to claim 14 wherein the antibody fragment
is a Fab, F(ab').sub.2, Fv, or scFv fragment.
20. The method according to claim 14 wherein the monoclonal
antibody is 6313/G2 produced by the hybridoma cell line designated
by accession no. 93072117.
Description
FIELD
[0001] The present invention relates to therapeutic uses of
monoclonal antibodies to the angiotensin-II type-I receptor, in
particular in the treatment of cancer and vascular smooth muscle
cell proliferation.
BACKGROUND
[0002] Angiotensin-II plays a central role in mammalian electrolyte
homeostasis and blood pressure control (Peach Physiol. Rev 57
313-370 (1977); Vinson et al "The Adrenal Cortex", Prentice Hall,
Englefield Heights (1992)). Two main types of angiotensin-II
receptors, designated types 1 and 2 (AT1 and AT2), have been
recognised, but the majority of the well known actions of
angiotensin-II occur via the AT1 subtype (Herblin et al Am. J.
Hypertens. 4 299S-302S (1991); Ouali et al J. Steroid. Biochem.
Mol. Biol. 43 271-280 (1992)).
[0003] A monoclonal antibody 6313/G2 to the AT1 receptor subtype
(Barker et al J. Mol. Endocrinol. 11 241-245 (1993)) has been used
to study the distribution of the receptor (Vinson et al Mol. Med.
Today 1 35-38 (1995)). The monoclonal antibody has been suggested
for use as a therapeutic agent to control vaso-constriction, for
example in the treatment of hypertension or uterine
contractions.
[0004] The antibody has been used as a specific imaging agent in
various tissues, for example laryngeal cancer (Marsigliante et al
Cancer Letters 110 19-27 (1996)), kidney (Harrison-Bernard et al
Am. J. Physiol. 42 F170-F177 (1997); Cheng et al Am. J. Physiol. 43
F10-F17 (1998)), and brain (Yang et al J. Neuroscience 17 1660-1669
(1997)). The antibody has been shown to block angiotensin-II
induced AT1 receptor internalisation and PKC activation but
conversely promotes the calcium response (Kapas et al Biochem.
Biophys. Res. Comm. 204 1292-1298 (1994; Vinson et al J.
Endocrinol. 141 R5-R9 (1994)). The presence of AT1 and At2
receptors in breast tumours has been reported with local production
of angiotensin (Inwang et al Brit. J. Cancer 75 1279-1283 (1997);
Tahmasebi et al Eur. J. Cancer 34 1777-1782 (1998)).
[0005] Monoclonal antibody 6313/G2 is secreted by a hybridoma cell
line deposited on 21 Jul. 1993 with the European Collection of
Animal Cell Cultures (ECACC), Porton Down, United Kingdom, under
the Budapest Treaty, and designated by the accession no. 93072117.
The deposit was made by Dr Gavin P Vinson and Dr Stewart Barker,
Department of Biochemistry, Queen Mary & Westfield College,
Mile End Road, London E1 4NS. The depositor has authorised the
applicant to refer to the deposited material in the application and
has given his unreserved and irrevocable consent to the deposited
material being made available to the public in accordance with Rule
28(1)(d) of the European Patent Convention.
[0006] The hybridoma cell line produces an antibody that
specifically binds to amino acid residues 8 to 17 of the rat
vascular smooth muscle AT1 receptor, which sequence is also found
in the AT1 receptor of human and bovine cells. The epitope sequence
is as follows:
TABLE-US-00002 (SEQ ID NO: 2) EDGIKRIQDD
Or, alternatively expressed as,
TABLE-US-00003 (SEQ ID NO: 2)
NH.sub.2-Glu-Asp-Gly-Ile-Lys-Arg-Ile-Gln-Asp-Asp-COOH
SUMMARY
[0007] It has now been surprisingly found that monoclonal
antibodies to the peptide sequence comprising the N-terminal
sequence of the angiotensin-II type-1 receptor have additional
therapeutic uses in certain medical conditions where such uses were
not previously suggested or shown. Furthermore, these therapeutic
effects are seen in the ability of the monoclonal antibodies to
block the harmful actions of angiotensin-II in the medical
conditions concerned whilst preserving the beneficial actions of
the molecule. A functionally important role for the entire
N-terminal sequence has now been realised.
[0008] According to a first aspect of the invention, there is
provided the use of a monoclonal antibody or a fragment thereof to
a peptide comprising the N-terminal portion of the angiotensin-II
type-1 receptor defined by the sequence
TABLE-US-00004 (SEQ ID NO: 1) MILNSSTEDG IKRIQDDCPK AGRHNYIFVM
IPTLYSIIFV VGIFG
or a fragment thereof, in the preparation of a medicament for the
treatment of cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The invention will now be further described by way of
reference to the following Examples which are provided for the
purposes of illustration only and are not to be construed as being
limiting on the invention. Reference is made to a number of Figures
in which:
[0010] FIG. 1 shows the effects of antibody on PNT1a cell
proliferation.
[0011] FIG. 2 shows the effects of antibody on aortic smooth muscle
cell proliferation by assaying uptake of tritiated thymidine.
[0012] FIG. 3 shows the results of cell adhesion assay of MCF-7
cells on extracellular matrix protein.
[0013] FIG. 4 shows the results of chemoinvasion assay of MCF-7
cells on extracellular matrix protein.
[0014] FIG. 5 shows results of Western blots in assay of expression
of integrins alpha-3 and beta-1 in breast cancer cells.
[0015] FIG. 6 shows antibody 6313/G2 stimulation of calcium
responses in MCF-7 cells.
[0016] FIG. 7 shows antibody 6313/G2 stimulation of calcium
responses in RASMC.
[0017] FIG. 8 shows a schematic diagram of the actions of
angiotensin-II, the site of the monoclonal antibody activation and
the site of monoclonal antibody block.
[0018] FIG. 9 shows sequence homologies for the N-terminal
sequences of angiotensin-II type-1 receptor from different species,
where "X" denotes a missing residue, and "-" denotes an identical
residue (bovine AT1 is SEQ ID NO:66; ovine AT1 is SEQ ID NO:67;
rabbit AT1 is SEQ ID NO:68; rat AT1b is SEQ ID NO:69; guinea pig
AT1 is SEQ ID NO:70; rat AT1a and gerbil AT1 are both SEQ ID NO:71;
and mouse AT1a is SEQ ID NO:72).
DESCRIPTION OF EMBODIMENTS
[0019] In the above, and throughout this specification, the amino
acid residues are designated by the usual IUPAC single letter
nomenclature. The single letter designations may be correlated with
the classical three letter designations of amino acid residues as
follows:
TABLE-US-00005 A = Ala C = Cys D = Asp E = Glu F = Phe G = Gly H =
His I = Ile K = Lys L = Leu M = Met N = Asn P = Pro Q = Gln R = Arg
S = Ser T = Thr V = Val W = Trp Y = Tyr
[0020] As used herein, the term "peptide" includes oligopeptide or
polypeptide and these terms may be used interchangeably.
[0021] The peptide will be of at least the minimum size necessary
to confer antigenicity: usually it will be of at least six or seven
residues, but may be of any suitable length up to, for example, 20
amino acid residues. Preferably, it may be or nine or ten residues.
The best peptides may be expected to correspond to topographical
surface features of the natural angiotensin-II type-1 receptor
molecule, that is to say those features having some
three-dimensional feature protruding from or extending into the
ambient surface level of the receptor. Preferred peptides
correspond to the region 1 to 45, preferably residues 8 to 17.
[0022] Probably the most simple way of ensuring that at least part
of the molecule is antigenically equivalent to the peptide is for
that part of the molecule to comprise a sequence of amino acid
residues which is identical to or conformationally similar to the
peptide. However, any other way of producing antigenic equivalence
may be used: an example is to use an anti-idiotype antibody or
other (even non-proteinaceous) analogue.
[0023] The invention therefore encompasses the use of monoclonal
antibodies against short peptides (preferably of 10 amino acid
residues or fewer, but generally of at least 4 or 5 amino acid
residues, for example 6 to 8 residues, or 7 to 9 residues) sharing
structural homology with the angiotensin-II type-1 receptor.
[0024] In a preferred embodiment, the invention therefore
encompasses the use of a monoclonal antibody against a peptide
sequence comprising the amino acid sequence:
TABLE-US-00006 (SEQ ID NO: 2) EDGIKRIQDD
or an active fragment thereof and/or conservative mutant thereof.
This sequence is taken from rat angiotensin-II type-1 receptor
sequence, residues 8 to 17. Conservative substitutions in this
fragment would be D for E, E for D, A for G, L or I, R for K, K for
R, N for Q, or any combination of these.
[0025] The sequence EDGIKRIQDD (SEQ ID NO:2) is fully 100%
conserved between the species human, chimpanzee, murine (AT1b) and
(AT1b), bovine, canine, ovine, rabbit, and rat (AT1b). Variations
are seen in residue 8 for guinea pig which has Q, rat (AT1a) which
has D and gerbil which has D. The sequence homologies for these
species for the full region 1 to 45 of the N-terminal sequence are
shown in FIG. 9.
[0026] Accordingly, a preferred consensus sequence for the peptide
corresponding to residues 8 to 17 of the angiotensin-II type-1
receptor has the structure:
TABLE-US-00007 (SEQ ID NO: 2) EDGIKRIQDD
where the following residues may each independently be as follows:
residue 8 may be E, D or Q, residue 9 may be D or E, residue 10 may
be G or A, residue 11 may be I or A, residue 12 may be K or R,
residue 13 may be R or K, residue 14 may be I or A, residue 15 may
be Q or N, and residues 16 and 17 may each either be D or E.
[0027] The peptide will generally be antigenic and capable of
stimulating the production of antibodies which, when administered
can be used in the treatment of cancer.
[0028] As stated above, an active subfragment of the specified
sequence may be used as defined. Active subfragments may consist of
or include pentapeptides, including one or more of:
TABLE-US-00008 (SEQ ID NO: 3) TEDGI (SEQ ID NO: 4) EDGIK (SEQ ID
NO: 5) DGIKR (SEQ ID NO: 6) GIKRI (SEQ ID NO: 7) IKRIQ (SEQ ID NO:
8) KRIQD (SEQ ID NO: 9) RIQDD (SEQ ID NO: 10) IQDDC
[0029] Active subfragments may also consist of or include
hexapeptides, including one or more of:
TABLE-US-00009 (SEQ ID NO: 11) STEDGI (SEQ ID NO: 12) TEDGIK (SEQ
ID NO: 13) EDGIKR (SEQ ID NO: 14) DGIKRI (SEQ ID NO: 15) GIKRIQ
(SEQ ID NO: 16) IKRIQD (SEQ ID NO: 17) KRIQDD (SEQ ID NO: 18)
RIQDDC (SEQ ID NO: 19) IQDDCP
[0030] Active subfragments may alternatively consist of or include
heptapeptides, including one or more of:
TABLE-US-00010 (SEQ ID NO: 20) SSTEDGI (SEQ ID NO: 21) STEDGIK (SEQ
ID NO: 22) TEDGIKR (SEQ ID NO: 23) EDGIKRI (SEQ ID NO: 24) DGIKRIQ
(SEQ ID NO: 25) GIKRIQD (SEQ ID NO: 26) IKRIQDD (SEQ ID NO: 27)
KRIQDDC (SEQ ID NO: 28) RIQDDCP (SEQ ID NO: 29) IQDDCPK
[0031] Further, active subfragments may consist of or include
octapeptides, including:
TABLE-US-00011 (SEQ ID NO: 30) NSSTEDGI (SEQ ID NO: 31) SSTEDGIK
(SEQ ID NO: 32) STEDGIKR (SEQ ID NO: 33) TEDGIKRI (SEQ ID NO: 34)
EDGIKRIQ (SEQ ID NO: 35) DGIKRIQD (SEQ ID NO: 36) GIKRIQDD (SEQ ID
NO: 37) IKRIQDDC (SEQ ID NO: 38) KRIQDDCP (SEQ ID NO: 39) RIQDDCPK
(SEQ ID NO: 40) IQDDCPKA
[0032] Further, active subfragments may consist of or include
nonapeptides, including:
TABLE-US-00012 (SEQ ID NO: 41) LNSSTEDGI (SEQ ID NO: 42) NSSTEDGIK
(SEQ ID NO: 43) SSTEDGIKR (SEQ ID NO: 44) STEDGIKRI (SEQ ID NO: 45)
TEDGIKRIQ (SEQ ID NO: 46) EDGIKRIQD (SEQ ID NO: 47) DGIKRIQDD (SEQ
ID NO: 48) GIKRIQDDC (SEQ ID NO: 49) IKRIQDDCP (SEQ ID NO: 50)
KRIQDDCPK (SEQ ID NO: 51) RIQDDCPKA (SEQ ID NO: 52) IQDDCPKAG
[0033] Further, active subfragments may consist of or include
decapeptides, including:
TABLE-US-00013 (SEQ ID NO: 53) ILNSSTEDGI (SEQ ID NO: 54)
LNSSTEDGIK (SEQ ID NO: 55) NSSTEDGIKR (SEQ ID NO: 56) SSTEDGIKRI
(SEQ ID NO: 57) STEDGIKRIQ (SEQ ID NO: 58) TEDGIKRIQD (SEQ ID NO:
59) EDGIKRIQDD (SEQ ID NO: 60) DGIKRIQDDC (SEQ ID NO: 61)
GIKRIQDDCP (SEQ ID NO: 62) IKRIQDDCPK (SEQ ID NO: 63) KRIQDDCPKA
(SEQ ID NO: 64) RIQDDCPKAG (SEQ ID NO: 65) IQDDCPKAGR
[0034] Preferred fragments include those containing some, for
example at least four residues of the sequence EDGIKRIQDD (SEQ ID
NO:2).
[0035] It should be noted that combinations of more than one of the
above sequences may be used.
[0036] Peptides and other molecules used to prepare monoclonal
antibodies for use in accordance with the invention may be rendered
antigenic, or presented, in a variety of ways.
[0037] For preference, an antigenic region (such as a peptide
fragment or sub-fragment) in a molecule in accordance with the
invention will contain the amino acid sequence of choice linked to
a carrier peptide or protein. It is generally preferred to have a
plurality, for example 5 to 10, copies of a peptide sequence (for
example one or more of the above sequences) linked to the carrier.
The carrier can for convenience be a generally large protein, which
is inert in material respects, and which is derived from a
different species or genus from that associated with the natural
growth hormone. Examples of carriers include albumins such as human
serum albumin, bovine serum albumin and ovalbumin (although not so
many peptides will probably be able to be carried in this last
case). Alternatively, keyhole limpet haemocyanin can be used. The
carrier will generally preferably come from a different species
from that on which the fragment is based.
[0038] It is not essential that peptide sequences as described
above be linked to albumins: they may be linked to other
macromolecules, such as .beta.-galactosidase, especially of
bacterial origin.
[0039] The invention encompasses the use of monoclonal antibodies
to molecules being peptides or having peptide regions which share
substantial (e.g. greater than 30%, 50% or even 70%, suitably, 80%,
85%, 90% or 95%) sequence homology with the above peptides.
Similarly, conservative amino acid substitutions may not decrease
the immunogenicity or antigenicity of peptides. Thus antigenically
similar homologues will elicit antibody which binds to
angiotensin-II type-1 receptors in the same region as the above
peptides define. It is well known that the use of homologues can be
a means of circumventing "self" tolerance. Thus the use of the
corresponding sequences from other species may be advantageous in
this invention.
[0040] It is alternatively possible for monoclonal antibodies to be
prepared against molecules which are or which comprise peptides to
be or to include polymers of sequences as described above.
Appropriate sequences can be polymerised either by cross-linking of
two cysteine residues to form disulphide bonds or by using external
chemical coupling agents (such as carbodiimide, glutaraldehyde or
other dialdehydes or di- (or poly-) functional carboxylic acids. As
a further alternative, recombinant DNA techniques could be used to
produce a peptide polymer.
[0041] It should be noted that the chemical coupling (which could
for example take place through the agency of lysine residues) and
disulphide bond formation are not limited to when the coupling
residues are at the end of the sequence: internal residues could
also be appropriate. Coupling residues, for example cysteine
residues, may be added as desired.
[0042] It may be found that it is not necessary to couple any of
the sequences described above with external peptides. They may be
antigenic on their own. In such a case, it may be advisable to
select particular adjuvants such as DEAE dextran and Merck
7426.
[0043] The monoclonal antibodies for used according to the present
invention can be prepared by immunising inbred mice by the standard
technique of Kohler & Milstein (Nature 256 495-497 (1975)). A
peptide corresponding to the epitope sequences described above can
be synthesised by any convenient chemical or biological route which
is then conjugated to bovine serum albumin (BSA), or another
suitable molecule, and then used to immunise the mice. Following a
booster injection of the peptide-BSA conjugate, the spleens of the
mice are removed, and the spleenocytes combined with mouse myeloma
cells. Mixed myeloma-lymphocyte hybrids can then be selected by
growth in hypoxanthine, thymidine and aminopterin in an appropriate
cell culture medium to inhibit proliferation of non-fused myeloma
cells and myeloma hybrids.
[0044] The hybridoma cells can be screened by ELISA for reactivity
against the epitope used of the angiotensin-II type-1 receptor by
adaptations of the technique described in Engvall et al Immunochem.
8 871 (1991). Alternatively, the antibody capture technique
described in Beckmann et al J. Immunol. 144 4212 (1990) may be
used. Positive hybridoma cells can be injected intraperitoneally
into syngeneic Balb/c mice to produce ascites containing high
concentrations of monoclonal antibodies raised against the epitope
used from the angiotensin-II type-1 receptor N-terminal sequence
described above. Alternatively, hybridoma cells can be grown in
vitro in flasks or roller bottles by various techniques. Monoclonal
antibodies produced in mouse ascites can be purified by ammonium
sulphate precipitation, followed by gel exclusion chromatography.
Alternatively, affinity chromatography based upon binding of
antibody to protein A or protein G can be used, as can affinity
chromatography based upon binding to the epitope used to generate
the monoclonal antibody. The monoclonal antibody 6313/G2 was
prepared as described in Barker et al J. Mol. Endocrinol. 11
241-245 (1993). Uses of the antibody in the treatment of
hypertension and in controlling uterine contractions were described
in WO-A-9509186. However, there was no suggestion of any broader
utility in other potential therapeutic areas.
[0045] The angiotensin-II type-1 receptor of the rat is described
in Murphy et al Nature 351 233-236 (1992) and the extracellular
domain identified as containing at least residues 8 to 17 is
represented by the amino acid sequence
TABLE-US-00014 (SEQ ID NO: 2) EDGIKRIQDD
The epitopes from the N-terminal sequence residues 1 to 45,
preferably 8 to 17, of the angiotensin-II type-1 receptor described
above may be varied modified by amino acid substitution, and/or
insertion, and/or deletion such that the overall shape and/or
conformation of the epitope is still antigenic.
[0046] In preferred embodiments of the invention, the monoclonal
antibody is 6313/G2. Monoclonal antibody 6313/G2 is secreted by a
hybridoma cell line deposited on 21 Jul. 1993 with the European
Collection of Animal Cell Cultures (ECACC), Porton Down, United
Kingdom, under the Budapest Treaty, and designated by the accession
no. 93072117. The hybridoma cell line can suitably be cultured
under standard conditions.
[0047] In uses of the present invention, the treatment of cancer
can include, but is not limited to, inhibition of metastasis,
inhibition of binding to matrix proteins of tumour cells,
inhibition of invasion by tumour cells and inhibition of tumour
cell proliferation. Examples of cancer tumours that may be
susceptible to such treatment include, but are not limited to
breast cancer and prostate cancer.
[0048] In a second aspect of the invention, there is provided the
use of a monoclonal antibody to a peptide comprising the N-terminal
portion of the angiotensin-II type-1 receptor defined by the
sequence
TABLE-US-00015 (SEQ ID NO: 1) MILNSSTEDG IKRIQDDCPK AGRHNYIFVM
IPTLYSIIFV VGIFG
or a fragment thereof, in the preparation of a medicament for the
treatment of vascular smooth muscle (VSM) cell proliferation.
[0049] Treatment of vascular smooth muscle cell proliferation may
include the treatment of atherosclerosis, a complex disease
condition that shows an association with VSM cell
proliferation.
[0050] The first aspect of the invention therefore also extends to
a method for the treatment of cancer comprising administration to a
subject in need thereof a therapeutic amount of a monoclonal
antibody or a fragment thereof to a peptide comprising the
N-terminal portion of the angiotensin-II type-1 receptor or a
fragment thereof as defined by the amino acid sequences described
above.
[0051] The second aspect of the invention therefore also extends to
a method for the treatment of vascular smooth muscle cell
proliferation comprising administration to a subject in need
thereof a therapeutic amount of a monoclonal antibody or a fragment
thereof to a peptide comprising the N-terminal portion of the
angiotensin-II type-1 receptor or a fragment thereof as defined by
the amino acid sequence described above.
[0052] The antibodies used in accordance with the present invention
may be formulated for intravenous injection using appropriate
pharmaceutically acceptable adjuvants and/or diluents. Injection
may be intravenous, intramuscular, intraperitoneal, including
sub-cutaneous injection. Other modes of administration are not
excluded, such as for example orally via liposomes, enteric coated
capsules and the like.
[0053] Suitably, the antibodies used in accordance with the present
invention may be humanised antibodies as described in U.S. Pat. No.
4,816,567 and WO 94/10332; or microbodies as described in WO
94/09817; or transgenic antibodies as described in GB-A-2272440.
Such synthetic constructs include chimaeric molecules. Thus, for
example, uses of humanised (or primatised) antibodies or
derivatives thereof are within the scope of the present invention.
An example of a humanised antibody is an antibody having human
framework regions, but rodent hypervariable regions.
[0054] In addition to whole antibodies, the present invention
includes uses of derivatives of the monoclonal antibodies defined
above which are capable of binding to the epitope selected from the
N-terminal region of the angiotensin-II type-1 receptor described
above. Thus the present invention also includes uses of antibody
fragments. Examples of antibody fragments are given by Dougall et
al Tibtech 12 372-379 (1994).
[0055] Antibody fragments include, for example, Fab, F(ab').sub.2
and Fv fragments (Roitt et al "Immunology", Second edition (1989),
Churchill Livingstone, London). Fv fragments can be modified to
produce a synthetic construct known as a single chain Fv (scFv)
molecule. This includes a peptide linker covalently joining V.sub.h
and V.sub.l regions which contribute to the stability of the
molecule.
[0056] Other synthetic constructs include CDR peptides. These are
synthetic peptides comprising antigen binding determinants Peptide
mimetics may also be used. These molecules are usually
conformationally restricted organic rings which mimic the structure
of a CDR loop and which include antigen-interactive side chains.
Uses of such molecules able to bind to the desired epitope are
therefore also within the scope of the present invention.
[0057] In a third aspect of the invention, there is provided the
use of a peptide sequence comprising the N-terminal portion of the
angiotensin-II type-1 receptor defined by the sequence
TABLE-US-00016 (SEQ ID NO: 1) MILNSSTEDG IKRIQDDCPK AGRHNYIFVM
IPTLYSIIFV VGIFG
or a fragment thereof, in the preparation of a medicament for the
treatment of cancer.
[0058] In a fourth aspect of the invention there is provided a
vaccine composition comprising a peptide sequence comprising the
N-terminal portion of the angiotensin-II type-1 receptor defined by
the sequence
TABLE-US-00017 (SEQ ID NO: 1) MILNSSTEDG IKRIQDDCPK AGRHNYIFVM
IPTLYSIIFV VGIFG
or a fragment thereof. The vaccine composition may comprise a
polypeptide of the above sequence or an antigenic fragment as
defined above, optionally conjugated to a carrier protein. Means
for rendering such proteins or peptides antigenic are defined above
in relation to the earlier aspects of the invention. For example,
an albumin protein, such as human serum albumin, bovine serum
albumin and ovalbumin. Alternatively, keyhole limpet protein (also
sometimes referred to as keyhole limpet haemocyanin) can be used.
The carrier will generally be different come from a different
species from that on which the fragment is based. Other adjuvants
may also be present in the vaccine composition, for example a
saponin adjuvant, e.g. a Quillaja saponin or a derivative
thereof.
[0059] In a particularly preferred embodiment there is provided a
method for the inhibition of cancer cell growth, adhesion or
invasion comprising:
(1) formulating a monoclonal antibody or a fragment thereof to a
peptide comprising the N-terminal portion of the angiotensin-II
type-1 receptor as defined above, optionally conjugated to a
carrier peptide or protein in an appropriate pharmaceutically
acceptable adjuvant and/or diluent, such as for intravenous
injection (2) optionally further formulating the monoclonal
antibody preparation of (1) as a liposomal or enteric coated
capsule formulation (3) administration of the formulation of (2) or
(3) to a population of cancer cells in vitro or a subject suffering
from cancer. Alternatively, this embodiment may also comprise step
(1) and (2) only.
[0060] Such embodiments extend to the use of such formulations in
the preparation of medicaments for the treatment of cancer, for
example, prostate cancer, breast cancer (including breast cancer
cell adhesion or invasion).
[0061] In another preferred embodiment there is provided a method
for the inhibition of vascular smooth muscle cell proliferation in
which steps (1) to (3) described above are repeated mutatis
mutandis.
[0062] Preferred features for the second and subsequent aspects of
the invention are as for the first aspect mutatis mutandis.
[0063] The invention will now be further described by way of
reference to the following
[0064] Examples which are provided for the purposes of illustration
only and are not to be construed as being limiting on the
invention.
Example 1
Antibody 6313/G2 Inhibits Cell Proliferation in Prostate PNT1A
Cells
[0065] The tetrazolium salt, 3-(4,5
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is
widely used as an indictor for cellular oxidative metabolic
activity. On reduction MTT forms an intensely coloured formazan
product, which can be measured colorimetricly and thus is often
used for the quantitative assessment of cellular viability and
proliferation.
[0066] PNT1a, a prostate epithelial cell line were seeded into 96
well culture plate at a concentration of 1000 cells per well. Cells
were grown in the presence of RPM 11640 medium supplemented with 2
mM L-glutamine, 1% non-essential amino acids, 2% penicillin and
streptomycin, and 1 mM sodium pyruvate, 10% fetal calf serum, for
two days and subsequently rendered quiescent by incubation in RPMI
1640 serum free medium (200 .mu.l/well) for 24 hr. Stimulants were
then added at appropriate concentration and incubated for 24 hr and
96 hr. Four hours prior to end of incubation, 20 .mu.l of filtered
(0.2 .mu.m pore size) 5 mg/ml solution (in RPMI media) MTT was
added to each well and the incubation continued at 37.degree. C. At
the appropriate time point, 200 .mu.l of DMSO, followed by 25 .mu.l
of Sorensen's glycine buffer (0.1M glycine, 0.1M NaCl adjusted to
pH 10.5 with 1M NaOH) was added to each well, mixed thoroughly.
After 5 minutes, the absorbance was read at 545 nm.
[0067] Results are shown in FIG. 1 in which purified antibody (ab)
was added to PNT1a cells in culture. Concentrations of antibody
were (1:1600) 100 nmol/l, (1:160) 1 .mu.mol/l, (1:16) 10 .mu.mol/l.
Inhibition of proliferation was significant (P<0.05 or better)
at all concentrations of antibody used.
Example 2
Antibody 6313/G2 Inhibits Cell Proliferation in Vascular Smooth
Muscle Cells
[0068] ASMCs were isolated from rat thoracic and abdominal artery
(RASMC) and bovine aorta (BASMC) by the media explant method and
cultured over several passages. Segments of both abdominal and
thoracic aortas were obtained from rats by careful dissection from
killed rats. Segments of aorta were obtained from calves under
anaesthesia. The segments of aorta were placed in a depression
slide containing tissue culture medium, after which the adventitia
and the outer portion of each segment was carefully removed under a
dissecting microscope. The remaining inner portion of the tissue
and the intima were removed to a separate dissecting dish and
washed several times with fresh culture medium. At this point each
segment was cut into approximately 1 mm squares and placed on 25
cm.sup.2 tissue culture flask. The flasks were loosely capped and
placed in a humidified CO.sub.2 incubator. After two hours, 4 ml of
RPMI-1640 culture medium supplemented with 100 units/ml of
penicillin, 100 .mu.g/ml streptomycin, 4 .mu.mol/L L-glutamine and
20% FBS was carefully added to the flasks without dislodging the
tissue. Samples were fed with fresh medium after one week. The
cells from the explants were relatively confluent within a period
of approximately 2 weeks. They were then rinsed with PBS, and
subsequently trypsinized with a solution of 0.125% trypsin and
0.02% EDTA in PBS for 1-2 minutes at 37.degree. C. The resulting
suspension of cells was pipetted into 75 cm.sup.2 tissue culture
flasks containing 10 ml culture medium and incubated as above.
Experiments were performed with cells from passages 3-5.
[0069] A suspension of RASMC (10.sup.5 cells/ml) were prepared on
the first day of the experiment using RPMI-1640 supplemented with
20% FBS. One ml of this cell suspension was distributed to each
well of a 24-well multiwell dish. The medium was replaced 24 h
after the subculture with RPMI-1640 medium. The quiescent
(serum-derived) or serum-replete cells were incubated with the
appropriate experimental media for 48 hours 4 wells per group.
.sup.3H-methylthymidine (0.1 mCi/ml) 10 .mu.l was added to each
well (1 ml medium/well). 24 hours after the addition of radioactive
thymidine, media were aspirated and the cultured cells were rinsed
3 times with cold PBS. Cells were then dissolved in 0.5 ml of 0.1 N
NaOH and a 0.3 ml aliquot was mixed with 3.5 ml of scintillation
fluid and, after standing overnight at room temperature, tritium
content was assayed in a liquid scintillation counter.
[0070] Results are shown in FIG. 2 in which proliferation was
stimulated by 10 nmol/l angiotensin-II and inhibited by 6313/G2 (10
.mu.mol/L, **P<0.01).
Example 3
Antibody 6313/G2 Inhibits Cell Proliferation in Breast Cancer
Cells
[0071] MCF-7 cells (obtained from American Type Culture Collection
(ATCC) Manassas, Va. 20108, USA) were plated out in 24 well dishes
at a density of 5000 cells per well and grown for 24 hours in
Eagles's Minimum Essential Medium (MEM) containing 5% fetal bovine
serum (FBS). Cells were then incubated for a further 24 hours in
serum free medium. Following this, cells were grown in either serum
free medium alone (control wells) or serum free medium with
addition to experimental wells of angiotensin II alone (1-10 nM),
or with antibody 6313/G2. Each treatment was performed in
quadruplicate. Cells were then cultured for 24 hours. After 20
hours tritiated thymidine was added to each well (Amersham
Pharmacia Biotech, Amersham, UK, 50 .mu.Ci/ml, sp. activity 5
Ci/mmol) and cells were cultured for a further 4 hours. At the end
of this period the medium was aspirated and the cultured cells were
rinsed three times with ice-cold buffer solution (50 mM Tris-HCl,
pH 7.4). Cells were then dissolved in 1 ml 0.1N NaOH and 0.5 ml of
this solution was mixed with 3.5 ml of scintillation cocktail
(toluene scintillator, Packard Bioscience B.V. Groningen,
Netherlands) and tritium content was assayed.
Example 4
Antibody 6313 Inhibits Breast Cancer Cell Adhesion
[0072] A cell adhesion assay of MCF-7 cells on extracellular matrix
protein was carried out to investigate. Cell culture (flat
bottomed) 96 well plates were coated with graded amounts of
purified human matrix protein, Collagen type IV (50 .mu.g/well).
They were left overnight in a laminar flow cabinet to evaporate, at
room temperature.
[0073] MCF-7 cells (obtained from American Type Culture Collection
(ATCC) Manassas, Va. 20108, USA) were treated with 6163/G2 antibody
for 48 hours. Controls were untreated. Prior to use, each well was
treated with BSA (200 .mu.g/ml) to eliminate non-specific
binding.
[0074] 500 MCF-7 cells in cell culture medium (DMEM) were added to
each well and incubated at 37.degree. C. in a 5% CO.sub.2
environment for 1 hour. Wells were then washed 3 times with
serum-free DMEM and stained with Diff-quick fix (7 seconds), Diff
quick I (7 seconds) and Diff quick II (10 seconds) and washed once
with water.
[0075] Wells were then viewed under a microscope, and the numbers
of adhering cells counted. Antibody 6313/G2 significantly reduced
cell adhesion (P<0.05). Results are shown in FIG. 3 in which the
number of MCF-7 cells adhered to collagen (50 .mu.g/well) against
the treatments with and without antibody 6313.
Example 5
Antibody 6313 Inhibits Breast Cancer Cell Invasion
[0076] A chemoinvasion assay of MCF-7 cells on extracellular matrix
protein was carried out to investigate.8 .mu.m filter inserts were
coated with purified human collagen type IV matrix protein and left
overnight in a laminar flow cabinet to dry at room temperature.
MCF-7 cells were treated with 6163/G2 antibody (hybridoma
supernatant) for 48 hours. Control cells were untreated.
[0077] Prior to use, BSA (100 .mu.g/ml) was added to each well for
1 hour. DMEM preconditioned by incubation with 3T3 fibroblast cells
was used as the chemoattractant. The coated inserts were placed in
each well to form an upper and a lower chamber. 10,000 MCF-7 cells
were added into the upper chamber with the addition of serum free
DMEM. Conditioned 3T3 cell medium was placed in the lower
compartment. Plates were covered and incubated at 37.degree. C. in
a humidified 5% CO.sub.2 environment for 24 hours.
[0078] After incubation, the cells remaining on the upper surface
of the filter were completely removed and the cells that had
traversed the collagen and attached to the lower surface of the
filter were stained with Diff-Quik and counted. Results are shown
in FIG. 4 in which the number of MCF-7 cells invaded through
collagen (50 .mu.u/well) against treatments with and without
antibody 6313. Antibody 6313 significantly inhibited cell invasion
(P>0.01).
Example 6
Effect of Antibody 6313 on Integrin Expression in Breast Cancer
Cells
[0079] The effect of antibody 6313/G2 on integrin expression was
investigated. The results show that antibody 6313 significantly
reduces integrins alpha 3 and beta 1 expression in breast cancer
cells.
[0080] MCF-7 cell lines was treated for 48 hours with antibody
6313/G2, controls were untreated. Cell membrane fractions were
prepared and fractionated non-reduced 8% SDS-PAGE gel.
[0081] Proteins were then transferred to a nitrocellulose membrane
overnight, 30V at 4.degree. C. Primary and secondary antibodies for
the integrins .alpha.3 and .beta.1 were used to detect these
components on the nitrocellulose membranes using established
methods for Western blotting. Luminescent bands were developed by
incubating the membrane in enhanced chemiluminescence (ECL) western
blotting detection reagents for 1 minute by hyper film ECL
exposure. Results are shown in FIG. 5 in which, C=control,
A=antibody tested, other lanes (M) are molecular weight
markers.
Example 7
Effect of Antibody 6313 on Calcium Responses in MCF-7 Cells and in
RASMC
[0082] The effect of antibody 6313 on calcium responses in MCF-7
cells and in RASMC was investigated. Antibody 6313 was found to
stimulate the calcium response in both.
[0083] For calcium ion ([Ca.sup.2+]) measurement, the cells were
loaded with 1 .mu.M fura-2 for 30 minutes in medium-modified
Krebs-Ringer bicarbonate solution (3.6 mM K.sup.+, 1.2 mM
Ca.sup.2+, 0.5 mM Mg.sup.2+, 5 mM Hepes and 20 mM HCO.sup.-) at
37.degree. C. For simultaneous measurements of measuring the
fluorescence of fura-2, the cells plated on coverslips were mounted
on the stage of an inverted microscope (Zeiss) in a modified
Krebs-Ringer bicarbonate solution. The excitation wavelengths were
340 nm and 380 nm, and emission was detected at 510 nm. Calcium ion
concentration ([Ca.sup.2+]) was calculated from the ration of
fluorescence intensities at excitation wavelengths of 340 nm and
380 nm.
[0084] Results are shown in FIG. 6 in MCF-7 cells and in RASMC. The
vertical arrow indicates the point of application if antibody
6313/G2. The increased ration of fluorescence intensities is
proportional to the intracellular calcium ion concentration.
Sequence CWU 1
1
72145PRTHomo sapiens 1Met Ile Leu Asn Ser Ser Thr Glu Asp Gly Ile
Lys Arg Ile Gln Asp 1 5 10 15 Asp Cys Pro Lys Ala Gly Arg His Asn
Tyr Ile Phe Val Met Ile Pro 20 25 30 Thr Leu Tyr Ser Ile Ile Phe
Val Val Gly Ile Phe Gly 35 40 45 210PRTHomo sapiens 2Glu Asp Gly
Ile Lys Arg Ile Gln Asp Asp 1 5 10 35PRTHomo sapiens 3Thr Glu Asp
Gly Ile 1 5 45PRTHomo sapiens 4Glu Asp Gly Ile Lys 1 5 55PRTHomo
sapiens 5Asp Gly Ile Lys Arg 1 5 65PRTHomo sapiens 6Gly Ile Lys Arg
Ile 1 5 75PRTHomo sapiens 7Ile Lys Arg Ile Gln 1 5 85PRTHomo
sapiens 8Lys Arg Ile Gln Asp 1 5 95PRTHomo sapiens 9Arg Ile Gln Asp
Asp 1 5 105PRTHomo sapiens 10Ile Gln Asp Asp Cys 1 5 116PRTHomo
sapiens 11Ser Thr Glu Asp Gly Ile 1 5 126PRTHomo sapiens 12Thr Glu
Asp Gly Ile Lys 1 5 136PRTHomo sapiens 13Glu Asp Gly Ile Lys Arg 1
5 146PRTHomo sapiens 14Asp Gly Ile Lys Arg Ile 1 5 156PRTHomo
sapiens 15Gly Ile Lys Arg Ile Gln 1 5 166PRTHomo sapiens 16Ile Lys
Arg Ile Gln Asp 1 5 176PRTHomo sapiens 17Lys Arg Ile Gln Asp Asp 1
5 186PRTHomo sapiens 18Arg Ile Gln Asp Asp Cys 1 5 196PRTHomo
sapiens 19Ile Gln Asp Asp Cys Pro 1 5 207PRTHomo sapiens 20Ser Ser
Thr Glu Asp Gly Ile 1 5 217PRTHomo sapiens 21Ser Thr Glu Asp Gly
Ile Lys 1 5 227PRTHomo sapiens 22Thr Glu Asp Gly Ile Lys Arg 1 5
237PRTHomo sapiens 23Glu Asp Gly Ile Lys Arg Ile 1 5 247PRTHomo
sapiens 24Asp Gly Ile Lys Arg Ile Gln 1 5 257PRTHomo sapiens 25Gly
Ile Lys Arg Ile Gln Asp 1 5 267PRTHomo sapiens 26Ile Lys Arg Ile
Gln Asp Asp 1 5 277PRTHomo sapiens 27Lys Arg Ile Gln Asp Asp Cys 1
5 287PRTHomo sapiens 28Arg Ile Gln Asp Asp Cys Pro 1 5 297PRTHomo
sapiens 29Ile Gln Asp Asp Cys Pro Lys 1 5 308PRTHomo sapiens 30Asn
Ser Ser Thr Glu Asp Gly Ile 1 5 318PRTHomo sapiens 31Ser Ser Thr
Glu Asp Gly Ile Lys 1 5 328PRTHomo sapiens 32Ser Thr Glu Asp Gly
Ile Lys Arg 1 5 338PRTHomo sapiens 33Thr Glu Asp Gly Ile Lys Arg
Ile 1 5 348PRTHomo sapiens 34Glu Asp Gly Ile Lys Arg Ile Gln 1 5
358PRTHomo sapiens 35Asp Gly Ile Lys Arg Ile Gln Asp 1 5 368PRTHomo
sapiens 36Gly Ile Lys Arg Ile Gln Asp Asp 1 5 378PRTHomo sapiens
37Ile Lys Arg Ile Gln Asp Asp Cys 1 5 388PRTHomo sapiens 38Lys Arg
Ile Gln Asp Asp Cys Pro 1 5 398PRTHomo sapiens 39Arg Ile Gln Asp
Asp Cys Pro Lys 1 5 408PRTHomo sapiens 40Ile Gln Asp Asp Cys Pro
Lys Ala 1 5 419PRTHomo sapiens 41Leu Asn Ser Ser Thr Glu Asp Gly
Ile 1 5 429PRTHomo sapiens 42Asn Ser Ser Thr Glu Asp Gly Ile Lys 1
5 439PRTHomo sapiens 43Ser Ser Thr Glu Asp Gly Ile Lys Arg 1 5
449PRTHomo sapiens 44Ser Thr Glu Asp Gly Ile Lys Arg Ile 1 5
459PRTHomo sapiens 45Thr Glu Asp Gly Ile Lys Arg Ile Gln 1 5
469PRTHomo sapiens 46Glu Asp Gly Ile Lys Arg Ile Gln Asp 1 5
479PRTHomo sapiens 47Asp Gly Ile Lys Arg Ile Gln Asp Asp 1 5
489PRTHomo sapiens 48Gly Ile Lys Arg Ile Gln Asp Asp Cys 1 5
499PRTHomo sapiens 49Ile Lys Arg Ile Gln Asp Asp Cys Pro 1 5
509PRTHomo sapiens 50Lys Arg Ile Gln Asp Asp Cys Pro Lys 1 5
519PRTHomo sapiens 51Arg Ile Gln Asp Asp Cys Pro Lys Ala 1 5
529PRTHomo sapiens 52Ile Gln Asp Asp Cys Pro Lys Ala Gly 1 5
5310PRTHomo sapiens 53Ile Leu Asn Ser Ser Thr Glu Asp Gly Ile 1 5
10 5410PRTHomo sapiens 54Leu Asn Ser Ser Thr Glu Asp Gly Ile Lys 1
5 10 5510PRTHomo sapiens 55Asn Ser Ser Thr Glu Asp Gly Ile Lys Arg
1 5 10 5610PRTHomo sapiens 56Ser Ser Thr Glu Asp Gly Ile Lys Arg
Ile 1 5 10 5710PRTHomo sapiens 57Ser Thr Glu Asp Gly Ile Lys Arg
Ile Gln 1 5 10 5810PRTHomo sapiens 58Thr Glu Asp Gly Ile Lys Arg
Ile Gln Asp 1 5 10 5910PRTHomo sapiens 59Glu Asp Gly Ile Lys Arg
Ile Gln Asp Asp 1 5 10 6010PRTHomo sapiens 60Asp Gly Ile Lys Arg
Ile Gln Asp Asp Cys 1 5 10 6110PRTHomo sapiens 61Gly Ile Lys Arg
Ile Gln Asp Asp Cys Pro 1 5 10 6210PRTHomo sapiens 62Ile Lys Arg
Ile Gln Asp Asp Cys Pro Lys 1 5 10 6310PRTHomo sapiens 63Lys Arg
Ile Gln Asp Asp Cys Pro Lys Ala 1 5 10 6410PRTHomo sapiens 64Arg
Ile Gln Asp Asp Cys Pro Lys Ala Gly 1 5 10 6510PRTHomo sapiens
65Ile Gln Asp Asp Cys Pro Lys Ala Gly Arg 1 5 10 6645PRTBovineAT1
66Met Ile Leu Asn Ser Ser Thr Glu Asp Gly Ile Lys Arg Ile Gln Asp 1
5 10 15 Asp Cys Pro Lys Ala Gly Arg His Asn Tyr Ile Phe Ile Met Ile
Pro 20 25 30 Thr Leu Tyr Ser Ile Ile Phe Val Val Gly Ile Phe Gly 35
40 45 6745PRTOvineAT1 67Met Ile Leu Asn Ser Ser Thr Glu Asp Gly Ile
Lys Arg Ile Gln Asp 1 5 10 15 Asp Cys Pro Lys Ala Gly Arg His Asn
Tyr Ile Phe Ile Met Ile Pro 20 25 30 Thr Leu Tyr Ser Ile Ile Phe
Val Val Gly Leu Phe Gly 35 40 45 6845PRTOryctolagus cuniculusAT1
68Met Met Leu Asn Ser Ser Thr Glu Asp Gly Ile Lys Arg Ile Gln Asp 1
5 10 15 Asp Cys Pro Lys Ala Gly Arg His Asn Tyr Ile Phe Val Met Ile
Pro 20 25 30 Thr Leu Tyr Ser Ile Ile Phe Val Val Gly Ile Phe Gly 35
40 45 6945PRTRatAT1b 69Met Ile Leu Asn Ser Ser Thr Glu Asp Gly Ile
Lys Arg Ile Gln Asp 1 5 10 15 Asp Cys Pro Lys Ala Gly Arg His Asn
Tyr Ile Phe Val Met Ile Pro 20 25 30 Thr Leu Tyr Ser Ile Ile Phe
Met Val Gly Ile Phe Gly 35 40 45 7044PRTCavia porcellusAT1 70Met
Ile Leu Asn Ser Ser Thr Gln Asp Gly Ile Lys Arg Ile Gln Asp 1 5 10
15 Asp Cys Pro Lys Gly Arg His Ser Tyr Ile Phe Val Met Ile Pro Thr
20 25 30 Leu Tyr Ser Ile Ile Phe Val Val Gly Ile Phe Gly 35 40
7143PRTArtificial SequenceRat AT1a and gerbil AT1 71Met Leu Asn Ser
Ser Asp Asp Gly Ile Lys Arg Ile Gln Asp Asp Cys 1 5 10 15 Pro Lys
Ala Gly Arg His Ser Tyr Ile Phe Val Met Ile Pro Thr Leu 20 25 30
Tyr Ser Ile Ile Phe Val Val Gly Ile Phe Gly 35 40 7242PRTMus
musculusAT1a 72Met Leu Asn Ser Ser Glu Asp Gly Ile Lys Arg Ile Gln
Asp Asp Cys 1 5 10 15 Pro Ser Gly Arg His Ser Tyr Ile Phe Val Met
Ile Pro Thr Leu Tyr 20 25 30 Ser Ile Met Phe Val Val Gly Ile Phe
Gly 35 40
* * * * *