U.S. patent application number 10/535873 was filed with the patent office on 2006-05-11 for antibodies binding to human magic roundabout (mr), polypeptides and uses thereof for inhibition angiogenesis.
Invention is credited to Roy Bicknell, Lorna Mary Dyet Stewart, Steven Suchting.
Application Number | 20060099143 10/535873 |
Document ID | / |
Family ID | 32328072 |
Filed Date | 2006-05-11 |
United States Patent
Application |
20060099143 |
Kind Code |
A1 |
Bicknell; Roy ; et
al. |
May 11, 2006 |
Antibodies binding to human magic roundabout (mr), polypeptides and
uses thereof for inhibition angiogenesis
Abstract
A method of inhibiting angiogenesis in an individual in need
thereof comprising administering an antibody that selectively binds
to the extracellular region of human magic roundabout (MR) to the
individual. An antibody that has the amino acid sequences i) to
iii), the amino acid sequences iv) to vi), or the amino acid
sequences i) to vi): i) S A S S S V S Y M Y ii) L T S N L A S iii)
Q Q W S S N P L T iv) D Y N L N v) V I N P N Y G T T S Y N Q K F K
G vi) G R D Y F G Y. A method of inhibiting angiogenesis in an
individual in need thereof comprising administering the
extracellular domain (residues 1-467) of MR, or a fragment thereof
that inhibits angiogenesis, to the individual. A method of
inhibiting endothelial cell migration and/or proliferation
comprising administering the extracellular domain of MR, or a
fragment thereof that inhibits endothelial cell migration and/or
proliferation.
Inventors: |
Bicknell; Roy; (Oxford,
GB) ; Suchting; Steven; (Oxford, GB) ;
Stewart; Lorna Mary Dyet; (London, GB) |
Correspondence
Address: |
JAECKLE FLEISCHMANN & MUGEL, LLP
190 Linden Oaks
ROCHESTER
NY
14625-2812
US
|
Family ID: |
32328072 |
Appl. No.: |
10/535873 |
Filed: |
November 20, 2003 |
PCT Filed: |
November 20, 2003 |
PCT NO: |
PCT/GB03/05059 |
371 Date: |
November 21, 2005 |
Current U.S.
Class: |
424/1.49 ;
424/155.1; 435/320.1; 435/344; 435/69.1; 530/388.8; 536/23.53 |
Current CPC
Class: |
A61P 27/02 20180101;
A61P 9/10 20180101; A61K 2039/53 20130101; A61P 43/00 20180101;
A61P 19/02 20180101; A61P 35/00 20180101; A61P 17/06 20180101; A61P
29/00 20180101; C07K 2317/76 20130101; A61P 9/00 20180101; A61K
2039/505 20130101; A61P 15/00 20180101; C07K 16/2803 20130101; A61P
3/04 20180101; C07K 16/18 20130101; A61K 31/70 20130101 |
Class at
Publication: |
424/001.49 ;
424/155.1; 530/388.8; 435/069.1; 435/320.1; 435/344;
536/023.53 |
International
Class: |
A61K 51/00 20060101
A61K051/00; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; A61K 39/395 20060101 A61K039/395; C07K 16/30 20060101
C07K016/30; C07K 16/46 20060101 C07K016/46; C12N 5/06 20060101
C12N005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 20, 2002 |
GB |
0227080.9 |
Sep 12, 2003 |
GB |
0321401.2 |
Claims
1. A method of inhibiting angiogenesis in an individual in need
thereof or in vitro comprising administering an antibody that
selectively binds to the extracellular region (residues 1-467, SEQ
ID NO: 3) of human magic roundabout (MR) to the individual or to
tissue or cells in vitro.
2. (canceled)
3. (canceled)
4. A method according to claim 1 wherein the antibody selectively
binds to the Ig region of MR (residues 46-209, SEQ ID NO: 4).
5. A method according to claim 4 wherein the antibody selectively
binds to the IgA region of MR (residues 46-116, SEQ ID NO: 5).
6. A method according to claim 4 wherein the antibody selectively
binds to the IgB region of MR (residues 151-209, SEQ ID NO: 6).
7. A method according to claim 1 wherein the antibody has at least
one light chain variable region incorporating the following CDRs:
TABLE-US-00035 CDR1: S A S S S V S Y M Y (SEQ ID NO: 9) CDR2: L T S
N L A S (SEQ ID NO: 10) CDR3: Q Q W S S N P L T. (SEQ ID NO:
11)
8. A method according to claim 7 wherein the antibody has at least
one light chain variable region comprising the amino acid sequence
Q I V L T Q S P A L M S A S P G E K V T M T C S A S S S V S Y M Y W
Y Q Q K P R S S P K P W I Y L T S N L A S G V P A R F S G S G S G T
S Y S L T I S S M E A E D A A T Y Y C Q Q W S S N P L T F G A G T K
L E L K (SEQ ID NO: 12).
9. A method according to claim 1 wherein the antibody has at least
one heavy chain variable region incorporating the following CDRs:
TABLE-US-00036 CDR1: D Y N L N (SEQ ID NO: 13) CDR2: V I N P N Y G
T T S Y N Q K (SEQ ID NO: 14) F K G CDR3: G R D Y F G Y. (SEQ ID
NO: 15)
10. A method according to claim 9 wherein the antibody has at least
one heavy chain variable region comprising the amino acid sequence
Q V K/Q L Q E S G P E L V K P G A S V K I S C K A S G Y S L T D Y N
L N W V K Q N K G K S L E W I G V I N P N Y G T T S Y N Q K F K G K
A T L T V D Q S S S T T Y M Q L N S L T S E D S A V Y Y C A R G R D
Y F G Y W G Q G T T V T V S S (SEQ ID NOs: 16-17).
11. A method according to claim 1 wherein the antibody has at least
one light chain variable region incorporating the following CDRs:
TABLE-US-00037 CDR1: S A S S S V S Y M Y (SEQ ID NO: 9) CDR2: L T S
N L A S (SEQ ID NO: 10) CDR3: Q Q W S S N P L T. (SEQ ID NO:
11)
and wherein the antibody has at least one heavy chain variable
region incorporating the following CDRs: TABLE-US-00038 CDR1: D Y N
L N (SEQ ID NO: 13) CDR2: V I N P N Y G T T S Y N Q K (SEQ ID NO:
14) F K G CDR3: G R D Y F G Y. (SEQ ID NO: 15)
12. A method of inhibiting angiogenesis in an individual in need
thereof or in vitro comprising administering a polynucleotide
encoding an antibody that selectively binds to the extracellular
region (residues 1-467, SEQ ID NO: 3) of human magic roundabout
(MR) to the individual or to tissue or cells in vitro.
13. (canceled)
14. (canceled)
15. An antibody that contains the amino acid sequences i) to iii),
the amino acid sequences iv) to vi), or preferably the amino acid
sequences i) to vi): TABLE-US-00039 i) S A S S S V S Y M Y (SEQ ID
NO: 9) ii) L T S N L A S (SEQ ID NO: 10) iii) Q Q W S S N P L T
(SEQ ID NO: 11) iv) D Y N L N (SEQ ID NO: 13) v) V I N P N Y G T T
S Y N Q K F (SEQ ID NO: 14) K G vi) G R D Y F G Y; (SEQ ID NO:
15)
or an antibody that selectively binds the Ig region of MR (residues
46-209, SEQ ID NO: 4) but does not selectively bind to the peptides
LLQPPARGHAHDGQALSTDL (SEQ ID NO: 28) or LSQSPGAVPQALVAWRA (SEQ ID
NO: 29).
16. An antibody according to claim 15 having at least one light
chain variable region incorporating the following CDRs:
TABLE-US-00040 CDR1: S A S S S V S Y M Y (SEQ ID NO: 9) CDR2: L T S
N L A S (SEQ ID NO: 10) CDR3: Q Q W S S N P L T. (SEQ ID NO:
11)
17. An antibody according to claim 16 having at least one light
chain variable region comprising the amino acid sequence Q I V L T
Q S P A L M S A S P G E K V T M T C S A S S S V S Y M Y W Y Q Q K P
R S S P K P W I Y L T S N L A S G V P A R F S G S G S G T S Y S L T
I S S M E A E D A A T Y Y C Q Q W S S N P L T F G A G T K L E L K
(SEQ ID NO: 12).
18. An antibody according to claim 15 having at least one heavy
chain variable region incorporating the following CDRs:
TABLE-US-00041 CDR1: D Y N L N (SEQ ID NO: 13) CDR2: V I N P N Y G
T T S Y N Q K (SEQ ID NO: 14) F K G CDR3: G R D Y F G Y. (SEQ ID
NO: 15)
19. An antibody according to claim 18 having at least one heavy
chain variable region comprising the amino acid sequence Q V K/Q L
Q E S G P E L V K P G A S V K I S C K A S G Y S L T D Y N L N W V K
Q N K G K S L E W I G V I N P N Y G T T S Y N Q K F K G K A T L T V
D Q S S S T T Y M Q L N S L T S E D S A V Y Y C A R G R D Y F G Y W
G Q G T T V T V S S (SEQ IDNOs: 16-17).
20. An antibody according to claim 15 having at least one light
chain variable region incorporating the following CDRs:
TABLE-US-00042 CDR1: S A S S S V S Y M Y (SEQ ID NO: 9) CDR2: L T S
N L A S (SEQ ID NO: 10) CDR3: Q Q W S S N P L T (SEQ ID NO: 11)
and the antibody having at least one heavy chain variable region
incorporating the following CDRs: TABLE-US-00043 CDR1: D Y N L N
(SEQ ID NO: 13) CDR2: V I N P N Y G T T S Y N Q K (SEQ ID NO: 14) F
K G CDR3: G R D Y F G Y. (SEQ ID NO: 15)
21. An antibody that selectively binds to the MR epitope bound by
an antibody according to claim 15 having at least one kappa light
chain variable region comprising the amino acid sequence 0 I V L T
Q S P A L M S A S P G E K V T M T C S A S S S V S Y M Y W Y O O K P
R S S P K P W I Y L T S N L A S G V P A R F S G S G S G T S Y S L T
I S S M E A E D A A T Y Y C Q Q W S S N P L T F G A G T K L E L K
(SEQ ID NO: 12) and at least one heavy chain variable region
comprising the amino acid sequence Q V K/Q L Q E S G P E L V K P G
A S V K I S C K A S G Y S L T D Y N L N W V K O N K G K S L E W I G
V I N P N Y G T T S Y N Q K F K G K A T L T V D Q S S S T T Y M O L
N S L T S E D S A V Y Y C A R G R D Y F G Y W G Q G T T V T V S S
(SEQ IDNOs: 16-17).
22. A polynucleotide encoding an antibody according to claim
15.
23. A polynucleotide according to claim 22 comprising one or more
of the nucleotide sequences: TABLE-US-00044 i) AGT GCC AGC TCA AGT
GTA AGT (SEQ ID NO: 18) TAC ATG TAC ii) TCT CAC ATC CAA CCT GGC TTC
T (SEQ ID NO: 19) iii) CAG CAG TGG AGT AGT AAC CCA (SEQ ID NO: 20)
CTC ACG iv) GAC TAC AAC CTG AAC (SEQ ID NO: 22) v) GTA ATT AAT CCA
AAC TAT GGT (SEQ ID NO: 23) ACT AGT TAC AAT CAG AAG TTC AAG GGC,
and vi) GGG AGG GAT TAC TTC GGC TAC. (SEQ ID NO: 24)
24. A polynucleotide according to claim 22 comprising the
nucleotide sequence CAA ATT GTT CTC ACC CAG TCT CCA GCA CTC ATG TCT
GCA TCT CCA GGG GAG AAG GTC ACC ATG ACC TGC AGT GCC AGC TCA AGT GTA
AGT TAC ATG TAC TGG TAC CAG CAG AAG CCA AGA TCC TCC CCC AAA CCC TGG
ATT TAT CTC ACA TCC AAC CTG GCT TCT GGA GTC CCT GCT CGC TTC AGT GGC
AGT GGG TCT GGG ACC TCT TAC TCT CTC ACA ATC AGC AGC ATG GAG GCT GAA
GAT GCT GCC ACT TAT TAC TGC CAG CAG TGG AGT AGT AAC CCA CTC ACG TTC
GGT GCT GGG ACC AAG CTG GAG CTG AAA (SEQ ID NO: 21).
25. A polynucleotide according to claim 22 comprising the
nucleotide sequence CAG GTC AAG (or A/CAA) CTG CAG GAG TCA GGA CCT
GAG CTG GTG AAG CCT GGC GCT TCA GTG AAG ATA TCC TGC AAG GCT TCT GGT
TAC TCA CTC ACT GAC TAC AAC CTG AAC TGG GTG AAG CAG AAC AAA GGA AAG
AGC CTT GAG TGG ATT GGA GTA ATT AAT CCA AAC TAT GGT ACT AGT TAC AAT
CAG AAG TTC AAG GGC AAG GCC ACA TTG ACT GTA GAC CAA TCT TCC AGC ACA
ACC TAC ATG CAG CTC AAC AGC CTG ACA TCT GAG GAC TCT GCA GTC TAT TAC
TGT GCA AGA GGG AGG GAT TAC TTC GGC TAC TGG GGC CAA GGG ACC ACG GTC
ACC GTC TCC TCA (SEQ ID NO: 25-27).
26. A polynucleotide according to claim 22 comprising the
nucleotide sequence CAA ATT GTT CTC ACC CAG TCT CCA GCA CTC ATG TCT
GCA TCT CCA GGG GAG AAG GTC ACC ATG ACC TGC AGT GCC AGC TCA AGT GTA
AGT TAC ATG TAC TGG TAC CAG CAG AAG CCA AGA TCC TCC CCC AAA CCC TGG
ATT TAT CTC ACA TCC AAC CTG GCT TCT GGA GTC CCT GCT CGC TTC AGT GGC
AGT GGG TCT GGG ACC TCT TAC TCT CTC ACA ATC AGC AGC ATG GAG GCT GAA
GAT GCT GCC ACT TAT TAC TGC CAG CAG TGG AGT AGT AAC CCA CTC ACG TTC
GGT GCT GGG ACC AAG CTG GAG CTG AAA (SEQ ID NO: 21) and the
nucleotide sequence CAG GTC AAG (or A/CAA) CTG CAG GAG TCA GGA CCT
GAG CTG GTG AAG CCT GGC GCT TCA GTG AAG ATA TCC TGC AAG GCT TCT GGT
TAC TCA CTC ACT GAC TAC AAC CTG AAC TGG GTG AAG CAG AAC AAA GGA AAG
AGC CTT GAG TGG ATT GGA GTA ATT AAT CCA AAC TAT GGT ACT AGT TAC AAT
CAG AAG TTC AAG GGC AAG GCC ACA TTG ACT GTA GAC CAA TCT TCC AGC ACA
ACC TAC ATG CAG CTC AAC AGC CTG ACA TCT GAG GAC TCT GCA GTC TAT TAC
TGT GCA AGA GGG AGG GAT TAC TTC GGC TAC TGG GGC CAA GGG ACC ACG GTC
ACC GTC TCC TCA (SEQ ID NO: 25-27) .
27. (canceled)
28. An antibody according to claim 15 that selectively binds the
IgA region of MR (residues 46-116, SEQ ID NO: 5) but does not
selectively bind to the peptide LLQPPARGHAHDGQALSTDL (SEQ ID NO:
28), or that selectively binds the IgB region of MR (residues
151-209, SEQ ID NO: 6) but does not selectively bind to the peptide
LSQSPGAVPQALVAWRA (SEQ ID NO: 29).
29. (canceled)
30. A compound comprising an antibody according to claim 15 and a
directly or indirectly cytotoxic moiety.
31. A compound according to claim 30 wherein the cytotoxic moiety
is selected from a directly cytotoxic chemotherapeutic agent, a
directly cytotoxic polypeptide, a moiety which is able to convert a
relatively non-toxic prodrug into a cytotoxic drug, a
radiosensitizer, a directly cytotoxic nucleic acid, a nucleic acid
molecule that encodes a directly or indirectly cytotoxic
polypeptide, a nucleic acid molecule that encodes a therapeutic
polypeptide, or a radioactive atom.
32. A compound according to claim 31 wherein the radioactive atom
is any one of phosphorus-32, iodine-125, iodine-131, indium-111,
rhenium-186, rhenium-188 or yttrium-90.
33. A compound according to claim 30 wherein the antibody and the
cytotoxic moiety are polypeptides which are fused.
34. A polynucleotide encoding a compound according to claim 33.
35. A compound comprising an antibody according to claim 15 and a
readily detectable moiety.
36. A compound according to claim 35 wherein the readily detectable
moiety comprises a suitable amount of any one of iodine-123,
iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, technitium-99m, gadolinium, manganese or iron.
37. A vector comprising the polynucleotide of claim 22.
38. A host cell comprising the polynucleotide of claim 22.
39. A stable host cell line producing an antibody resulting from
incorporation in the cell line of an exogenous polynucleotide
according to claim 22.
40. A pharmaceutical composition comprising an antibody according
to claim 15, or a polynucleotide encoding an antibody according to
claim 15, or a compound comprising an antibody according to claim
15 and a directly or indirectly cytotoxic moiety, or a compound
comprising an antibody according to claim 15 and a readily
detectable moiety, and a pharmaceutically acceptable carrier.
41. A pharmaceutical composition according to claim 40 suitable for
administration to a patient by injection.
42. (canceled)
43. (canceled)
44. A method of inhibiting angiogenesis in an individual in need
thereof or in vitro comprising administering a compound comprising
an antibody according to claim 15 and a directly or indirectly
cytotoxic moiety, or comprising administering a compound comprising
an antibody according to claim 15 and a readily detectable moiety,
to the individual or to tissue or cells in vitro.
45. (canceled)
46. A method of producing an antibody, the method comprising
expressing a polynucleotide according to claim 22.
47. A method of combating a disease or condition selected from
tumours/cancer, psoriasis, atherosclerosis, menorrhagia,
endometriosis, arthritis (both inflammatory and rheumatoid),
macular degeneration, Paget's disease, retinopathy and its vascular
complications (including proliferative and of prematurity, and
diabetic retinopathy), benign vascular proliferations, fibroses,
obesity and inflammation in an individual in need thereof
comprising administering an antibody that selectively binds to the
extracellular region of MR, or administering the MR ectodomain, or
a fragment thereof that inhibits angiogenesis, to the
individual.
48. (canceled)
49. (canceled)
50. (canceled)
51. (canceled)
52. (canceled)
53. A method of combating a disease or condition involving
unwanted, undesirable or inappropriate endothelial cell migration
and/or proliferation in an individual, the method comprising
administering the MR ectodomain or a fragment thereof that inhibits
endothelial cell migration and/or proliferation, or a
polynucleotide encoding the MR ectodomain or a fragment thereof
that inhibits endothelial cell migration and/or proliferation, to
the individual.
54. (canceled)
55. A method according to claim 53 wherein the disease or condition
involving unwanted, undesirable or inappropriate endothelial cell
migration and/or proliferation is selected from tumours/cancer,
psoriasis, atherosclerosis, menorrhagia, endometriosis, arthritis
(both inflammatory and rheumatoid), macular degeneration, Paget's
disease, retinopathy and its vascular complications (including
proliferative and of prematurity, and diabetic retinopathy), benign
vascular proliferations, fibroses, obesity and inflammation.
56. An in vitro method of inhibiting endothelial cell migration
and/or proliferation comprising administering the MR ectodomain or
a fragment thereof that inhibits endothelial cell migration and/or
proliferation, a polynucleotide encoding the MR ectodomain or a
fragment thereof that inhibits endothelial cell migration and/or
proliferation, to tissue or cells in vitro.
57. (canceled)
58. (canceled)
59. (canceled)
60. A method of inhibiting angiogenesis in an individual in need
thereof or in vitro comprising administering the MR ectodomain, or
a fragment thereof that inhibits angiogenesis, or administering a
polynucleotide encoding the MR ectodomain, or a fragment thereof
that inhibits angiogenesis, to the individual or to tissue and
cells in vitro.
61. (canceled)
62. (canceled)
63. (canceled)
64. (canceled)
65. (canceled)
66. (canceled)
67. (canceled)
68. (canceled)
69. (canceled)
70. (canceled)
71. (canceled)
72. (canceled)
73. (canceled)
74. A method according to claim 12 wherein the antibody selectively
binds to the Ig region of MR (residues 46-209, SEQ ID NO: 4).
75. A method according to claim 74 wherein the antibody selectively
binds to the IgA region of MR (residues 46-116, SEQ ID NO: 5).
76. A method according to claim 74 wherein the antibody selectively
binds to the IgB region of MR (residues 151-209, SEQ ID NO: 6).
77. A vector comprising the polynucleotide of claim 34.
78. A host cell comprising the polynucleotide of claim 34.
79. A host cell comprising the vector of claim 37.
80. A host cell comprising the vector of claim 77.
81. A stable host cell line producing a compound resulting from
incorporation in the cell line of an exogenous polynucleotide
according to claim 34.
82. A stable host cell line producing an antibody resulting from
incorporation in the cell line of a vector according to claim
37.
83. A stable host cell line producing a compound resulting from
incorporation in the cell line of a vector according to claim
77.
84. A method of producing a compound, the method comprising
expressing a polynucleotide according to claim 34.
85. A method of producing an antibody, the method comprising
culturing a stable host cell line according to claim 39.
86. A method of producing a compound, the method comprising
culturing a stable host cell line according to claim 81.
87. A method of producing an antibody, the method comprising
culturing a stable host cell line according to claim 82.
88. A method of producing a compound, the method comprising
culturing a stable host cell line according to claim 83.
Description
[0001] The present invention relates to antibodies and
polypeptides, and in particular to ECSM4 antibodies and
polypeptides that inhibit angiogenesis and their use therefor.
[0002] Endothelial cells form a single cell layer that lines all
blood vessels and regulates exchanges between the blood stream and
the surrounding tissues. New blood vessels develop from the walls
of existing small vessels by the outgrowth of these endothelial
cells which have the capacity to form hollow capillary tubes even
when isolated in culture. In vivo, damaged tissues and some tumours
attract a blood supply by secreting factors that stimulate nearby
endothelial cells to construct new capillary sprouts. Tumours that
fail to attract a blood supply are severely limited in their
growth.
[0003] The process whereby new vessels originate as capillaries,
which sprout from existing small vessels, is called angiogenesis.
It can therefore be seen that angiogenesis plays a major role in
normal tissue development and repair and in the progression of some
pathological conditions.
[0004] Once the vascular system is fully developed, endothelial
cells of blood vessels normally remain quiescent with no new vessel
formation, with the exception of the formation of new blood vessels
in natural wound healing. However, a deregulation of blood vessel
growth and an abnormal increase in vessel density can occur in
diseases or conditions such as tumourigenesis, diabetic
retinopathy, psoriasis and inflammation. Therefore the ability to
inhibit inappropriate or undesirable angiogenesis may be useful in
the treatment of these diseases or conditions.
[0005] Human magic roundabout (MR; also known as endothelial
cell-specific molecule 4, ECSM4) has previously been shown to have
a highly endothelial-cell selective expression profile (Huminiecki
& Bicknell (2000), Genome Research, 10, 1796-1806; and WO
02/36771). MR expression in vivo was shown to be restricted to
sites of active angiogenesis, notably tumour vessels (Huminiecki et
al. (2002) Genomics, 79(4), 547-552).
[0006] Based on this information it was suggested in WO 02/36771
that compounds comprising a moiety that binds to MR, such as an
antibody, and a further functional moiety, may be useful for a
variety of medical purposes including imaging the vascular
epithelium; diagnosing or prognosing a condition involving the
vascular endothelium; assessing the efficacy of anti-angiogenic
therapies; detecting endothelial damage; detecting a tumour or
tumour neovasculature or cardiac disease or endometriosis or
atherosclerosis; treating a proliferative disease involving the
vascular endothelium such as cancer, psoriasis, diabetic
retinopathy, artherosclerosis or menorrhagia; introducing genetic
material into vascular endothelial cells; and modulating
angiogenesis.
[0007] For example, WO 02/36771 teaches a compound comprising a
moiety that binds to MR, such as an antibody, and a further moiety
such as an inhibitor of angiogenesis (page 27). WO 02/36771 also
teaches a compound comprising a moiety that binds to MR, such as an
antibody, and a further moiety such as a cytotoxic moiety that
destroys or slows or reverses the growth of the neovasculature
(page 35).
[0008] However, in each case, the moiety that binds to MR merely
directs the functional moiety to a desired endothelial location for
use. WO 02/36771 does not suggest that the moiety that binds to MR
is itself functional, let alone that it inhibits MR or can be used
to inhibit angiogenesis.
[0009] We have now shown that an antibody that selectively binds to
the extracellular region of MR results in inhibition of
angiogenesis.
[0010] On the paragraph spanning pages 71-72, WO 02/36771 states
that both antibodies which stimulate or activate MR and antibodies
which prevent stimulation and activation of MR could be used to
modulate angiogenesis. However, it does not suggest that an
antibody that selectively binds to the extracellular region of MR
can be used to inhibit angiogenesis. Moreover, to the best of the
inventors' knowledge, neither WO 02/36771 nor any other document
shows any evidence that an antibody that selectively binds to the
extracellular region of MR does, in fact, inhibit angiogenesis.
[0011] There is thus provided in accordance with a first aspect of
the invention a method of inhibiting angiogenesis in an individual
in need thereof comprising administering an antibody that
selectively binds to the extracellular region of human magic
roundabout (MR) to the individual.
[0012] By "inhibiting angiogenesis" we include the meaning of
reducing the rate or level of angiogenesis. The reduction can be a
low level reduction of about 10%, or about 20%, or about 30%, or
about 40% of the rate or level of angiogenesis. Preferably, the
reduction is a medium level reduction of about 50%, or about 60%,
or about 70%, or about 80% reduction of the rate or level of
angiogenesis. More preferably, the reduction is a high level
reduction of about 90%, or about 95%, or about 99%, or about 99.9%,
or about 99.99% of the rate or level of angiogenesis. Most
preferably, inhibition can also include the elimination of
angiogenesis or its reduction to an undetectable level.
[0013] Methods and assays for determining the rate or level of
angiogenesis, and hence for determining whether and to what extent
an antibody inhibits angiogenesis, are known in the art.
[0014] For example, U.S. Pat. No. 6,225,118 B1 to Grant et al,
incorporated herein by reference, describes a multicellular in
vitro assay for modelling the combined stages of angiogenesis
namely the proliferation, migration and differentiation stages of
cell development.
[0015] The AngioKit, Catalogue No. ZHA-1000, by TCS CellWorks Ltd,
Buckingham MK18 2LR, UK, is a suitable model of human angiogenesis
for analysing the angiogenic or anti-angiogenic properties of test
compounds.
[0016] The rate or level of angiogenesis can also be determined
using the aortic ring assay described in Example 2.
[0017] Preferably, the antibody also inhibits angiogenesis in vivo,
especially in mammals, and most preferably in humans
[0018] By "MR" we include the gene product of the human magic
roundabout gene (also known as ECSM4) and naturally occurring
variants thereof. The cDNA and amino acid sequence of MR are found
in Genbank Accession Nos. AF361473 and AAL31867, and are shown in
FIG. 1 (SEQ ID NOs: 1 and 2, respectively).
[0019] MR is a transmembrane protein and has been predicted to have
an extracellular region at residues 1-467 (SEQ ID NO: 3), a
transmembrane region at residues 468-490, and an intracellular
region at residues 491-1007 (Huminiecki et al., 2002). The
extracellular region of MR has an immunoglobulin (Ig) region at
residues 46-209 (SEQ ID NO: 4), which can be further subdefined
into an IgA domain at residues 46-116 (SEQ ID NO: 5), and an IgB
domain at residues 151-209 (SEQ ID NO: 6), and two fibronectin type
III domains at residues 252-335 (SEQ ID NO: 7) and 347-432 (SEQ ID
NO: 8). The MR amino acid residue numbering is that given in
AF361473, AAL31867 and in FIG. 1B.
[0020] By an antibody that "selectively binds" a specified domain
or region of MR, such as an Ig domain, we include the meaning that
the antibody binds the specific domain with a greater affinity than
for any other region of MR. Preferably, the antibody binds the
specified domain of MR with at least 2, or at least 5, or at least
10 or at least 50 times greater affinity than any other region of
MR. More preferably, the antibody binds the specific domain of MR
with at least 100, or at least 1,000, or at least 10,000 times
greater affinity than any other region of MR. Such binding may be
determined by methods well known in the art. Preferably, the
antibody selectively binds a particular epitope within MR and does
not bind other epitopes.
[0021] Preferably, when the antibody is administered to an
individual, the antibody binds MR at the specified domain with a
greater affinity than for any other molecule in the individual.
Preferably, the agent binds MR at the specific domain with at least
2, or at least 5, or at least 10 or at least 50 times greater
affinity than for any other molecule in the individual. More
preferably, the agent binds MR at the specific domain with at least
100, or at least 1,000, or at least 10,000 times greater affinity
than any other molecule in the individual.
[0022] Inhibition of angiogenesis may be useful in combating any
disease or condition involving unwanted, undesirable or
inappropriate angiogenesis. Such conditions include tumours/cancer,
psoriasis, atherosclerosis, menorrhagia, endometriosis, arthritis
(both inflammatory and rheumatoid), macular degeneration, Paget's
disease, retinopathy and its vascular complications (including
proliferative and of prematurity, and diabetic retinopathy), benign
vascular proliferations, fibroses, obesity and inflammation.
[0023] By cancer is included Kaposi's sarcoma, leukaemia, lymphoma,
myeloma, solid carcinomas (both primary and secondary (metastasis),
vascular tumours including haemangioma (both capillary and juvenile
(infantile)), haemangiomatosis and haemagioblastoma.
[0024] The tumours that may be treated by the methods of the
invention include any tumours which are associated with new blood
vessel production.
[0025] The term "tumour" is to be understood as referring to all
forms of neoplastic cell growth, including tumours of the lung,
liver, blood cells, skin, pancreas, stomach, colon, prostate,
uterus, breast, lymph glands and bladder. Solid tumours are
especially suitable. However, blood cancers, including leukaemias
and lymphomas are now also believed to involve new blood vessel
formation and may be treated by the methods of the invention.
[0026] The invention thus includes a method of combating a disease
or condition selected from tumours/cancer, psoriasis,
atherosclerosis, menorrhagia, endometriosis, arthritis (both
inflammatory and rheumatoid), macular degeneration, Paget's
disease, retinopathy and its vascular complications (including
proliferative and of prematurity, and diabetic retinopathy), benign
vascular proliferations, fibroses, obesity and inflammation in an
individual, the method comprising administering an antibody that
selectively binds to the extracellular region of MR to the
individual.
[0027] By "combating" we include the meaning that the method can be
used to alleviate symptoms of the disorder (ie the method is used
palliatively), or to treat the disorder, or to prevent the disorder
(ie the method is used prophylactically).
[0028] Thus, the invention comprises a method of treating a patient
who has a disease in which angiogenesis contributes to pathology,
the method comprising the step of administering to the patient an
antibody that selectively binds to the extracellular region of
MR.
[0029] Typically, the disease is associated with undesirable
neovasculature formation and the treatment reduces this to a useful
extent.
[0030] The therapy (treatment) may be on humans or animals.
Preferably, the methods of the inventions are used to treat
humans.
[0031] By "antibody" we include not only whole immunoglobulin
molecules but also fragments thereof such as Fab, F(ab')2, Fv and
other fragments thereof that retain the antigen-binding site.
Similarly the term "antibody" includes genetically engineered
derivatives of antibodies such as single chain Fv molecules (scFv)
and domain antibodies (dAbs). The term also includes antibody-like
molecules which may be produced using phage-display techniques or
other random selection techniques for molecules which bind to MR or
to specified regions of MR. Thus, the term antibody includes all
molecules which contain a structure, preferably a peptide
structure, which is part of the recognition site (ie the part of
the antibody that binds or combines with the epitope or antigen) of
a natural antibody.
[0032] The variable heavy (V.sub.H) and variable light (V.sub.L)
domains of the antibody are involved in antigen recognition, a fact
first recognised by early protease digestion experiments. Further
confirmation was found by "humanisation" of rodent antibodies.
Variable domains of rodent origin may be fused to constant domains
of human origin such that the resultant antibody retains the
antigenic specificity of the rodent parented antibody (Morrison et
al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).
[0033] That antigenic specificity is conferred by variable domains
and is independent of the constant domains is known from
experiments involving the bacterial expression of antibody
fragments, all containing one or more variable domains. These
molecules include Fab-like molecules (Better et al (1988) Science
240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038);
single-chain Fv (ScFv) molecules where the V.sub.H and V.sub.L
partner domains are linked via a flexible oligopeptide (Bird et al
(1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci.
USA 85, 5879) and single domain antibodies (dabs) comprising
isolated V domains (Ward et al (1989) Nature 341, 544). A general
review of the techniques involved in the synthesis of antibody
fragments which retain their specific binding sites is to be found
in Winter & Milstein (1991) Nature 349,293-299.
[0034] By "ScFv molecules" we mean molecules wherein the V.sub.H
and V.sub.L partner domains are linked via a flexible oligopeptide.
Engineered antibodies, such as ScFv antibodies, can be made using
the techniques and approaches described in J. Huston et al., (1988)
"Protein engineering of antibody binding sites: recovery of
specific activity in an anti-digoxin single chain Fv analogue
produced in E. coli", Proc. Natl. Acad. Sci. USA, 85, pp.
5879-5883, and in A. Pluckthun, (June 1991) "Antibody engineering;
Advances from use of E. coli expression systems", Bio/technology
vol 9, incorporated herein by reference.
[0035] The advantages of using antibody fragments, rather than
whole antibodies, are several-fold. The smaller size of the
fragments may lead to improved pharmacological properties, such as
better penetration to the target site. Effector functions of whole
antibodies, such as complement binding, are removed. Fab, Fv, ScFv
and dAb antibody fragments can all be expressed in and secreted
from E. coli, thus allowing the facile production of large amounts
of the fragments.
[0036] Whole antibodies, and F(ab').sub.2 fragments are "bivalent".
By "bivalent" we mean that the antibodies and F(ab').sub.2
fragments have two antigen combining sites. In contrast, Fab, Fv,
ScFv and dAb fragments are monovalent, having only one antigen
combining site.
[0037] Although the antibody may be a polyclonal antibody, it is
preferred if it is a monoclonal antibody. In some circumstance,
particularly if the antibody is going to be administered repeatedly
to a human patient, it is preferred if the monoclonal antibody is a
human monoclonal antibody or a humanised monoclonal antibody.
[0038] Suitable monoclonal antibodies which are reactive as
described herein may be prepared by known techniques, for example
those disclosed in "Monoclonal Antibodies; A manual of techniques",
H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies:
Techniques and Application", SGR Hurrell (CRC Press, 1982).
Polyclonal antibodies may be produced which are polyspecific or
monospecific. It is preferred that they are monospecific.
[0039] Chimaeric antibodies are discussed by Neuberger et al (1998,
8.sup.th International Biotechnology Symposium Part 2,
792-799).
[0040] It is preferred if the antibody is a humanised antibody.
Suitably prepared non-human antibodies can be "humanised" in known
ways, for example by inserting the CDR regions of mouse antibodies
into the framework of human antibodies. Humanised antibodies can be
made using the techniques and approaches described in M. Verhoeyen,
C. Milstein and G. Winter (1988) "Reshaping human antibodies:
Grafting an antilysozyme activity", Science, 239, 1534-1536, and in
C. Kettleborough et al., (1991) "Humanisation of a mouse monoclonal
antibody by CDR grafting; The importance of framework residues in
loop conformation", Protein Engineering, 14(7), 773-783,
incorporated herein by reference.
[0041] The antibodies may be human antibodies in the sense that
they have the amino acid sequence of human anti-MR antibodies but
they may be prepared using methods known in the art that do not
require immunisation of humans. For example, transgenic mice are
available which contain, in essence, human immunoglobulin genes
(see Vaughan et al (1998) Nature Biotechnol. 16, 535-539.
[0042] A second aspect of the invention provides use of an antibody
that selectively binds to the extracellular region of MR in the
preparation of a medicament for inhibiting angiogenesis.
[0043] The medicament may be useful in combating any disease or
condition involving unwanted or inappropriate angiogenesis. Such
conditions include those described above with reference to the
first aspect of the invention.
[0044] The invention thus includes the use of an antibody that
selectively binds to the extracellular region of MR in the
preparation of a medicament for combating a disease or condition
selected from tumours/cancer, especially solid tumours, psoriasis,
atherosclerosis, menorrhagia, endometriosis, arthritis (both
inflammatory and rheumatoid), macular degeneration, Paget's
disease, retinopathy and its vascular complications (including
proliferative and of prematurity, and diabetic retinopathy), benign
vascular proliferations, fibroses, obesity and inflammation.
[0045] A third aspect of the invention provides an in vitro method
of inhibiting angiogenesis comprising administering an antibody
that selectively binds to the extracellular region of MR to tissue
or cells in vitro. The cells may be established cell lines, or
cells that have been removed from an individual. The tissue or
cells are preferably mammalian tissue or cells, and most preferably
are human tissue or cells.
[0046] In an embodiment of each of the first three aspects of the
invention, the antibody selectively binds to the Ig region of MR.
The Ig region of MR is located at residues 46-209 of MR (FIG. 3,
SEQ ID NO: 4).
[0047] In one preferred embodiment, the antibody selectively binds
to the IgA domain of MR, which is located at residues 46-116 of MR
(FIG. 4A, SEQ ID NO: 5).
[0048] In an alternative preferred embodiment, the antibody
selectively binds to the IgB domain of MR, which is located at
residues 151-209 of MR (FIG. 4B, SEQ ID NO: 6).
[0049] In an embodiment of each of the first three aspects of the
invention, the antibody has at least one light chain variable
region incorporating the following CDRs: TABLE-US-00001 CDR1: S A S
S S V S Y M Y (SEQ ID NO: 9) CDR2: L T S N L A S (SEQ ID NO: 10)
CDR3: Q Q W S S N P L T (SEQ ID NO: 11)
[0050] In a more specific embodiment, the antibody may have at
least one light chain variable region comprising the amino acid
sequence: TABLE-US-00002 Q I V L T Q S P A L .M S A S P G E (SEQ ID
NO: 12) K V T M T C S A S S S V S Y M Y W Y Q Q K P R S S P K P W I
Y L T S N L A S G V P A R F S G S G S G T S Y S L T I S S M E A E D
A A T Y Y C Q Q W S S N P L T F G A G T K L E L K.
[0051] Preferably, the light chain is a kappa light chain.
[0052] In an embodiment of each of the first three aspects of the
invention, the antibody has at least one heavy chain variable
region incorporating the following CDRs: TABLE-US-00003 CDR1: D Y N
L N (SEQ ID NO: 13) CDR2: V I N P N Y G T T S Y N Q K (SEQ ID NO:
14) F K G CDR3: G R D Y F G Y (SEQ ID NO: 15)
[0053] In a more specific embodiment, the antibody may have at
least one heavy chain variable region comprising the amino acid
sequence: TABLE-US-00004 (SEQ ID NOs: 16 and 17) Q V K/Q L Q E S G
P E L V K P G A S V K I S C K A S G Y S L T D Y N L N W V K Q N K G
K S L E W I G V I N P N Y G T T S Y N Q K F K G K A T L T V D Q S S
S T T Y M Q L N S L T S E D S A V Y Y C A R G R D Y F G Y W G Q G T
T V T V S S,
where K/Q means that either K or Q is present at that position (K
is present in SEQ ID NO: 16, while Q is present in SEQ ID NO:
17).
[0054] In a yet more specific embodiment, the antibody has at least
one light chain variable region as defined above and at least one
heavy chain variable region as defined above.
[0055] A fourth aspect of the invention provides a method of
inhibiting angiogenesis in an individual in need thereof comprising
administering a polynucleotide encoding an antibody as defined
above to the individual.
[0056] A fifth aspect of the invention provides the use of a
polynucleotide encoding an antibody as defined above in the
preparation of a medicament for inhibiting angiogenesis.
[0057] A sixth aspect of the invention provides an in vitro method
of inhibiting angiogenesis comprising administering a
polynucleotide encoding an antibody as defined above to tissue or
cells in vitro.
[0058] A seventh aspect of the invention provides an antibody that
contains the amino acid sequences i) to iii), the amino acid
sequences iv) to vi), or preferably the amino acid sequences i) to
vi): TABLE-US-00005 i) S A S S S V S Y M Y (SEQ ID NO: 9) ii) L T S
N L A S (SEQ ID NO: 10) iii) Q Q W S S N P L T (SEQ ID NO: 11) iv)
D Y N L N (SEQ ID NO: 13) v) V I N P N Y G T T S Y N Q K F (SEQ ID
NO: 14) K G vi) G R D Y F G Y. (SEQ ID NO: 15)
[0059] While the CDRs that determine specificity of binding to an
antigen may be determined empirically, it has been demonstrated
that in a significant number of cases the IgH CDR3 is the most
important CDR region. The invention thus includes an antibody with
an Ig heavy chain CDR3 region having the amino acid sequence G R D
Y F G Y (SEQ ID NO: 15).
[0060] Preferably, the antibody selectively binds to the
extracellular region of MR (residues 1-467 of MR, FIG. 2B, SEQ ID
NO: 3), and the selective binding to MR is conferred by the
presence of these amino acid sequences. Preferably, the antibody
inhibits a function of MR. Such functions include the inhibition of
ligand binding, the interaction with other cell surface molecules
and the inhibition of activation of the receptor.
[0061] Preferably, the antibody has at least one light chain
variable region incorporating the following CDRs: TABLE-US-00006
CDR1: S A S S S V S Y M Y (SEQ ID NO: 9) CDR2: L T S N L A S (SEQ
ID NO: 10) CDR3: Q Q W S S N P L T (SEQ ID NO: 11)
[0062] More preferably, the antibody has at least one light chain
variable region comprising the amino acid sequence: TABLE-US-00007
Q I V L T Q S P A L M S A S P G E (SEQ ID NO: 12) K V T M T C S A S
S S V S Y M Y W Y Q Q K P R S S P K P W I Y L T S N L A S G V P A R
F S G S G S G T S Y S L T I S S M E A E D A A T Y Y C Q Q W S S N P
L T F G A G T K L E L K.
[0063] Preferably, the light chain is a kappa light chain.
[0064] Preferably, the antibody has at least one heavy chain
variable region incorporating the following CDRs: TABLE-US-00008
CDR1: D Y N L N (SEQ ID NO: 13) CDR2: V I N P N Y G T T S Y N Q K
(SEQ ID NO: 14) F K G CDR3: G R D Y F G Y (SEQ ID NO: 15)
[0065] More preferably, the antibody has at least one heavy chain
variable region comprising the amino acid sequence: TABLE-US-00009
(SEQ ID NOS: 16 and 17) Q V K/Q L Q E S G P E L V K P G A S V K I S
C K A S G Y S L T D Y N L N W V K Q N K G K S L E W I G V I N P N Y
G T T S Y N Q K F K G K A T L T V D Q S S S T T Y M Q L N S L T S E
D S A V Y Y C A R G R D Y F G Y W G Q G T T V T V S S.
[0066] Yet more preferably, the antibody has at least one light
chain variable region as defined above as defined above in the
seventh aspect of the invention and at least one heavy chain
variable region as defined above in the seventh aspect of the
invention.
[0067] Most preferably, the antibody has at least one light chain
variable region comprising the amino acid sequence: TABLE-US-00010
Q I V L T Q S P A L M S A S P G E (SEQ ID NO: 12) K V T M T C S A S
S S V S Y M Y W Y Q Q K P R S S P K P W I Y L T S N L A S G V P A R
F S G S G S G T S Y S L T I S S M E A E D A A T Y Y C Q Q W S S N P
L T F G A G T K L E L K
[0068] and at least one heavy chain variable region comprising the
amino acid sequence: TABLE-US-00011 (SEQ ID NOs: 16 and 17) Q V K/Q
L Q E S G P E L V K P G A S V K I S C K A S G Y S L T D Y N L N W V
K Q N K G K S L E W I G V I N P N Y G T T S Y N Q K F K G K A T L T
V D Q S S S T T Y M Q L N S L T S E D S A V Y Y C A R G R D Y F G Y
W G Q G T T V T V S S.
[0069] It is preferred if the antibody is a humanised antibody.
[0070] It is further preferred if the antibody is a humanised
antibody having the following CDRs: TABLE-US-00012 light chain
CDR1: S A S S S V S Y (SEQ ID NO: 9) M Y light chain CDR2: L T S N
L A S (SEQ ID NO: 10) light chain CDR3: Q Q W S S N P L (SEQ ID No:
11) T heavy chain CDR1: D Y N L N (SEQ ID NO: 13) heavy chain CDR2:
V I N P N Y G T (SEQ ID NO: 14) T S Y N Q K F K G heavy chain CDR3:
G R D Y F G Y (SEQ ID NO: 15)
[0071] An eighth aspect of the invention provides an antibody that
selectively binds to the MR epitope selectively bound by an
antibody having at least one light chain variable region comprising
the amino acid sequence: TABLE-US-00013 Q I V L T Q S P A L M S A S
P G E (SEQ ID NO: 12) K V T M T C S A S S S V S Y M Y W Y Q Q K P R
S S P K P W I Y L T S N L A S G V P A R F S G S G S G T S Y S L T I
S S M E A E D A A T Y Y C Q Q W S S N P L T F G A G T K L E L K
[0072] and at least one heavy chain variable region comprising the
amino acid sequence: TABLE-US-00014 (SEQ ID NOs: 16 and 17) Q V K/Q
L Q E S G P E L V K P G A S V K I S C K A S G Y S L T D Y N L N W V
K Q N K G K S L E W I G V I N P N Y G T T S Y N Q K F K G K A T L T
V D Q S S S T T Y M Q L N S L T S E D S A V Y Y C A R G R D Y F G Y
W G Q G T T V T V S S.
[0073] By an antibody that selectively binds to an MR epitope
selectively bound by another defined antibody, we include an
antibody that competes with the defined antibody. Such antibodies
can be determined, for example, using competitive binding assays,
preferably high throughput binding assays, as are well known to a
person of skill in the art. Suitable assays include a
cross-competition ELISA in which an extracellular fragment of MR is
incubated with the defined antibody and a test antibody, to
determine whether or not the test antibody competes with the
defined antibody for binding to the MR epitope.
[0074] A ninth aspect of the invention provides a polynucleotide
encoding an antibody as defined in the seventh or eighth aspects of
the invention.
[0075] In an embodiment, the polynucleotide comprises at least one
of the nucleotide sequences: TABLE-US-00015 i) AGT GCC AGC TCA AGT
GTA AGT (SEQ ID NO: 18) TAC ATG TAC ii) TCT CAC ATC CAA CCT GGC TTC
T (SEQ ID NO: 19) iii) CAG CAG TGG AGT AGT AAC CCA (SEQ ID NO: 20)
CTC ACG
[0076] Preferably, the polynucleotide comprises two or all three of
the nucleotide sequences i), ii) and iii).
[0077] Preferably, the polynucleotide comprises the nucleotide
sequence: TABLE-US-00016 CAA ATT GTT CTC ACC CAG TCT CCA (SEQ ID
NO: 21) GCA CTC ATG TCT GCA TCT CCA GGG GAG AAG GTC ACC ATG ACC TGC
AGT GCC AGC TCA AGT GTA AGT TAC ATG TAC TGG TAC CAG CAG AAG CCA AGA
TCC TCC CCC AAA CCC TGG ATT TAT CTC ACA TCC AAC CTG GCT TCT GGA GTC
CCT GCT CGC TTC AGT GGC AGT GGG TCT GGG ACC TCT TAC TCT CTC ACA ATC
AGC AGC ATG GAG GCT GAA GAT GCT GCC ACT TAT TAC TGC CAG CAG TGG AGT
AGT AAC CCA CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA.
[0078] In an alternative or additional embodiment, the
polynucleotide comprises at least one of the nucleotide sequences:
TABLE-US-00017 iv) GAC TAC AAC CTG AAC (SEQ ID NO: 22) v) GTA ATT
AAT CCA AAC TAT GGT ACT (SEQ ID NO: 23) AGT TAC AAT CAG AAG TTC AAG
GGC, and vi) GGG AGG GAT TAC TTC GGC TAC (SEQ ID NO: 24)
[0079] Preferably, the polynucleotide comprises two or all three of
the nucleotide sequences iv), v) and vi).
[0080] Preferably, the polynucleotide comprises the nucleotide
sequence: TABLE-US-00018 CAG GTC AAG(or A/CAA) CTG CAG GAG TCA GGA
CCT GAG CTG GTG AAG CCT GGC GCT TCA GTG AAG ATA TCC TGC AAG GCT TCT
GGT TAC TCA CTC ACT GAC TAC AAC CTG AAC TGG GTG AAG CAG AAC AAA GGA
AAG AGC CTT GAG TGG ATT GGA GTA ATT AAT CCA AAC TAT GGT ACT AGT TAC
AAT CAG AAG TTC AAG GGC AAG GCC ACA TTG ACT GTA GAC CAA TCT TCC AGC
ACA ACC TAC ATG CAG CTC AAC AGC CTG ACA TCT GAG GAC TCT GCA GTC TAT
TAC TGT GCA AGA GGG AGG GAT TAC TTC GGC TAC TGG GGC CAA GGG ACC ACG
GTC ACC GTC TCC TCA.
[0081] (The polynucleotide having AAG in the third codon is SEQ ID
NO: 25; the polynucleotide having AAA in the third codon is SEQ ID
NO: 26; and the polynucleotide having CAA in the third codon is SEQ
ID NO: 27).
[0082] Most preferably, the polynucleotide comprises at least one
nucleotide sequence: TABLE-US-00019 CAA ATT GTT CTC ACC CAG TCT CCA
(SEQ ID NO: 21) GCA CTC ATG TCT GCA TCT CCA GGG GAG AAG GTC ACC ATG
ACC TGC AGT GCC AGC TCA AGT GTA AGT TAC ATG TAC TGG TAC CAG CAG AAG
CCA AGA TCC TCC CCC AAA CCC TGG ATT TAT CTC ACA TCC AAC CTG GCT TCT
GGA GTC CCT GCT CGC TTC AGT GGC AGT GGG TCT GGG ACC TCT TAC TCT CTC
ACA ATC AGC AGC ATG GAG GCT GAA GAT GCT GCC ACT TAT TAC TGC CAG CAG
TGG AGT AGT AAC CCA CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG
AAA,
[0083] and at least one nucleotide sequence: TABLE-US-00020 (SEQ ID
NOs: 25-27) CAG GTC AAG(or A/CAA) CTG CAG GAG TCA GGA CCT GAG CTG
GTG AAG CCT GGC GCT TCA GTG AAG ATA TCC TGC AAG GCT TCT GGT TAC TCA
CTC ACT GAC TAC AAC CTG AAC TGG GTG AAG CAG AAC AAA GGA AAG AGC CTT
GAG TGG ATT GGA GTA ATT AAT CCA AAC TAT GGT ACT AGT TAC AAT CAG AAG
TTC AAG GGC AAG GCC ACA TTG ACT GTA GAC CAA TCT TCC AGC ACA ACC TAC
ATG CAG CTC AAC AGC CTG ACA TCT GAG GAC TCT GCA GTC TAT TAC TGT GCA
AGA GGG AGG GAT TAC TTC GGC TAC TGG GGC CAA GGG ACC ACG GTC ACC GTC
TCC TCA.
[0084] In an embodiment, the two coding regions may be on the same
polynucleotide, for example on a polynucleotide for expression of a
ScFv antibody.
[0085] A tenth aspect of the invention provides an antibody that
selectively binds the Ig region of MR (residues 46-209, SEQ ID NO:
4) but which does not selectively bind to the peptide
LLQPPARGHAHDGQALSTDL (residues 91-109 of MR, SEQ ID NO: 28) or to
the peptide LSQSPGAVPQALVAWRA (residues 165-181 of MR, SEQ ID NO:
29).
[0086] In an embodiment, the invention includes an antibody that
selectively binds the IgA region of MR (residues 46-116, SEQ ID NO:
5) but does not selectively bind to the peptide
LLQPPARGHAHDGQALSTDL (residues 91-109 of MR, SEQ ID NO: 28).
[0087] In an alternative embodiment, the invention includes an
antibody that selectively binds the IgB region of MR (residues
151-209, SEQ ID NO: 6) but does not selectively bind to the peptide
LSQSPGAVPQALVAWRA (residues 165-181 of MR, SEQ ID NO: 29).
[0088] An eleventh aspect of the invention provides a
polynucleotide that encodes an antibody as defined in the tenth
aspect of the invention.
[0089] A twelfth aspect of the invention provides a compound
comprising an antibody as defined above in the seventh, eighth and
tenth aspects of the invention, and a cytotoxic moiety.
[0090] The cytotoxic moiety is preferably directly or indirectly
toxic to cells in neovasculature or cells which are in close
proximity to and associated with neovasculature.
[0091] By "directly cytotoxic" we include the meaning that the
moiety is one which on its own is cytotoxic. By "indirectly
cytotoxic" we include the meaning that the moiety is one which,
although is not itself cytotoxic, can induce cytotoxicity, for
example by its action on a further molecule or by further action on
it.
[0092] In one embodiment the cytotoxic moiety is a cytotoxic
chemotherapeutic agent. Cytotoxic chemotherapeutic agents are well
known in the art.
[0093] Cytotoxic chemotherapeutic agents, such as anticancer
agents, include: alkylating agents including nitrogen mustards such
as mechlorethamine (HN.sub.2), cyclophosphamide, ifosfamide,
melphalan (L-sarcolysin) and chlorambucil; ethylenimines and
methylmelamines such as hexamethylmelamine, thiotepa; alkyl
sulphonates such as busulfan; nitrosoureas such as carmustine
(BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin
(streptozotocin); and triazenes such as decarbazine (DTIC;
dimethyltriazenoimidazole-carboxamide); Antimetabolites including
folic acid analogues such as methotrexate (amethopterin);
pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU),
floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine
arabinoside); and purine analogues and related inhibitors such as
mercaptopurine (6-mercaptopurine; 6-MP), thioguanine
(6-thioguanine; TG) and pentostatin (2'-deoxycoformycin). Natural
Products including vinca alkaloids such as vinblastine (VLB) and
vincristine; epipodophyllotoxins such as etoposide and teniposide;
antibiotics such as dactinomycin (actinomycin D), daunorubicin
(daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin
(mithramycin) and mitomycin (mitomycin C); enzymes such as
L-asparaginase; and biological response modifiers such as
interferon alphenomes. Miscellaneous agents including platinum
coordination complexes such as cisplatin (cis-DDP) and carboplatin;
anthracenedione such as mitoxantrone and anthracycline; substituted
urea such as hydroxyurea; methyl hydrazine derivative such as
procarbazine (N-methylhydrazine, M1H); and adrenocortical
suppressant such as mitotane (o,p'-DDD) and aminoglutethimide;
taxol and analogues/derivatives; and hormone agonists/antagonists
such as flutamide and tamoxifen.
[0094] Various of these agents have previously been attached to
antibodies and other target site-delivery agents, and so compounds
of the invention comprising these agents may readily be made by the
person skilled in the art. For example, carbodiimide conjugation
(Bauminger & Wilchek (1980) Methods Enzymol. 70, 151-159;
incorporated herein by reference) may be used to conjugate a
variety of agents, including doxorubicin, to antibodies.
[0095] Carbodiimides comprise a group of compounds that have the
general formula R--N.dbd.C.dbd.N--R', where R and R' can be
aliphatic or aromatic, and are used for synthesis of peptide bonds.
The preparative procedure is simple, relatively fast, and is
carried out under mild conditions. Carbodiimide compounds attack
carboxylic groups to change them into reactive sites for free amino
groups.
[0096] The water soluble carbodiimide,
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is
particularly useful for conjugating a functional moiety to an
antibody and may be used to conjugate doxorubicin to tumor homing
peptides. The conjugation of doxorubicin and an antibody requires
the presence of an amino group, which is provided by doxorubicin,
and a carboxyl group, which is provided by the antibody such as an
antibody or peptide.
[0097] In addition to using carbodiimides for the direct formation
of peptide bonds, EDC also can be used to prepare active esters
such as N-hydroxysuccinimide (NHS) ester. The NHS ester, which
binds only to amino groups, then can be used to induce the
formation of an amide bond with the single amino group of the
doxorubicin. The use of EDC and NHS in combination is commonly used
for conjugation in order to increase yield of conjugate formation
(Bauminger & Wilchek, supra, 1980).
[0098] Other methods for conjugating a cytotoxic moiety to an
antibody can also be used. For example, sodium periodate oxidation
followed by reductive alkylation of appropriate reactants can be
used, as can glutaraldehyde cross-linking. However, it is
recognised that, regardless of which method of producing a compound
of the invention is selected, a determination must be made that the
antibody maintains its targeting ability and that the attached
moiety maintains its relevant function.
[0099] In a further embodiment of the invention, the cytotoxic
moiety is a cytotoxic peptide or polypeptide moiety by which we
include any moiety which leads to cell death. Cytotoxic peptide and
polypeptide moieties are well known in the art and include, for
example, ricin, abrin, Pseudomonas exotoxin, tissue factor and the
like. Methods for linking them to targeting moieties such as
antibodies are also known in the art. The use of ricin as a
cytotoxic agent is described in Burrows & Thorpe (1993) Proc.
Natl. Acad. Sci. USA 90, 8996-9000, incorporated herein by
reference, and the use of tissue factor, which leads to localised
blood clotting and infarction of a tumour, has been described by
Ran et al (1998) Cancer Res. 58, 4646-4653 and Huang et al (1997)
Science 275, 547-550. Tsai et al (1995) Dis. Colon Rectum 38,
1067-1074 describes the abrin A chain conjugated to a monoclonal
antibody and is incorporated herein by reference. Other ribosome
inactivating proteins are described as cytotoxic agents in WO
96/06641. Pseudomonas exotoxin may also be used as the cytotoxic
polypeptide moiety (see, for example, Aiello et al (1995) Proc.
Natl. Acad. Sci. USA 92, 10457-10461; incorporated herein by
reference).
[0100] Certain cytokines, such as TNF.alpha. and IL-2, may also be
useful as cytotoxic agents.
[0101] Certain radioactive atoms may also be cytotoxic if delivered
in sufficient doses. Thus, the cytotoxic moiety may comprise a
radioactive atom which, in use, delivers a sufficient quantity of
radioactivity to the target site so as to be cytotoxic. Suitable
radioactive atoms include phosphorus-32, iodine-125, iodine-131,
indium-111, rhenium-186, rhenium-188 or yttrium-90, or any other
isotope which emits enough energy to destroy neighbouring cells,
organelles or nucleic acid. Preferably, the isotopes and density of
radioactive atoms in the compound of the invention are such that a
dose of more than 4000 cGy (preferably at least 6000, 8000 or 10000
cGy) is delivered to the target site and, preferably, to the cells
at the target site and their organelles, particularly the
nucleus.
[0102] The radioactive atom may be attached to the antibody in
known ways. For example EDTA or another chelating agent may be
attached to the antibody and used to attach .sup.111In or .sup.90Y.
Tyrosine residues may be labelled with .sup.125I, or .sup.131I.
[0103] The cytotoxic moiety may be a suitable indirectly cytotoxic
polypeptide. In a particularly preferred embodiment, the indirectly
cytotoxic polypeptide is a polypeptide which has enzymatic activity
and can convert a relatively non-toxic prodrug into a cytotoxic
drug. When the targeting moiety is an antibody this type of system
is often referred to as ADEPT (Antibody-Directed Enzyme Prodrug
Therapy). The system requires that the targeting moiety locates the
enzymatic portion to the desired site in the body of the patient
(ie the site expressing MR, such as new vascular tissue associated
with a tumour) and after allowing time for the enzyme to localise
at the site, administering a prodrug which is a substrate for the
enzyme, the end product of the catalysis being a cytotoxic
compound. The object of the approach is to maximise the
concentration of drug at the desired site and to minimise the
concentration of drug in normal tissues (see Senter, P. D. et al
(1988) "Anti-tumor effects of antibody-alkaline phosphatase
conjugates in combination with etoposide phosphate" Proc. Natl.
Acad. Sci. USA 85, 4842-4846; Bagshawe (1987) Br. J. Cancer 56,
531-2; and Bagshawe, K. D. et al (1988) "A cytotoxic agent can be
generated selectively at cancer sites" Br. J. Cancer. 58,
700-703.)
[0104] The cytotoxic substance may be any existing anti-cancer drug
such as an alkylating agent; an agent which intercalates in DNA; an
agent which inhibits any key enzymes such as dihydrofolate
reductase, thymidine synthetase, ribonucleotide reductase,
nucleoside kinases or topoisomerase; or an agent which effects cell
death by interacting with any other cellular constituent. Etoposide
is an example of a topoisomerase inhibitor.
[0105] Reported prodrug systems include: a phenol mustard prodrug
activated by an E. coli .beta.-glucuronidase (Wang et al, 1992 and
Roffler et al, 1991); a doxorubicin prodrug activated by a human
.beta.-glucuronidase (Bosslet et al, 1994); further doxorubicin
prodrugs activated by coffee bean .alpha.-galactosidase (Azoulay et
al, 1995); daunorubicin prodrugs, activated by coffee bean
.alpha.-D-galactosidase (Gesson et al, 1994); a 5-fluorouridine
prodrug activated by an E. coli .beta.-D-galactosidase (Abraham et
al, 1994); and methotrexate prodrugs (eg methotrexate-alanine)
activated by carboxypeptidase A (Kuefner et al, 1990, Vitols et al,
1992 and Vitols et al, 1995). These and others are included in
Table 1. TABLE-US-00021 TABLE 1 Enzyme Prodrug Carboxypeptidase G2
Derivatives of L-glutamic acid and benzoic acid mustards, aniline
mustards, phenol mustards and phenylenediamine mustards;
fluorinated derivatives of these Alkaline phosphatase Etoposide
phosphate Mitomycin phosphate Beta-glucuronidase p-Hydroxyaniline
mustard-glucuronide Epirubicin-glucuronide Penicillin-V-amidase
Adriamycin-N phenoxyacetyl Penicillin-G-amidase N-(4'-hydroxyphenyl
acetyl) palytoxin Doxorubicin and melphalan Beta-lactamase Nitrogen
mustard-cephalosporin p-phenylenediamine; doxorubicin derivatives;
vinblastine derivative-cephalosporin, cephalosporin mustard; a
taxol derivative Beta-glucosidase Cyanophenylmethyl-beta-D-gluco-
pyranosiduronic acid Nitroreductase
5-(Azaridin-1-yl-)-2,4-dinitrobenzamide Cytosine deaminase
5-Fluorocytosine Carboxypeptidase A Methotrexate-alanine
[0106] (This table is adapted from Bagshawe (1995) Drug Dev. Res.
34, 220-230, from which full references for these various systems
may be obtained; the taxol derivative is described in Rodrigues, M.
L. et al (1995) Chemistry & Biology 2, 223).
[0107] Suitable enzymes for forming part of the enzymatic portion
of the invention include: exopeptidases, such as carboxypeptidases
G, G1 and G2 (for glutamylated mustard prodrugs), carboxypeptidases
A and B (for MTX-based prodrugs) and aminopeptidases (for
2-.alpha.-aminocyl MTC prodrugs); endopeptidases, such as eg
thrombolysin (for thrombin prodrugs); hydrolases, such as
phosphatases (eg alkaline phosphatase) or sulphatases (eg aryl
sulphatases) (for phosphylated or sulphated prodrugs); amidases,
such as penicillin amidases and arylacyl amidase; lactamases, such
as .beta.-lactamases; glycosidases, such as .beta.-glucuronidase
(for .beta.-glucuronomide anthracyclines), .alpha.-galactosidase
(for amygdalin) and .beta.-galactosidase (for .beta.-galactose
anthracycline); deaminases, such as cytosine deaminase (for 5FC);
kinases, such as urokinase and thymidine kinase (for gancyclovir);
reductases, such as nitroreductase (for CB1954 and analogues),
azoreductase (for azobenzene mustards) and DT-diaphorase (for
CB1954); oxidases, such as glucose oxidase (for glucose), xanthine
oxidase (for xanthine) and lactoperoxidase; DL-racemases, catalytic
antibodies and cyclodextrins.
[0108] The prodrug is relatively non-toxic compared to the
cytotoxic drug. Typically, it has less than 10% of the toxicity,
preferably less than 1% of the toxicity as measured in a suitable
in vitro cytotoxicity test.
[0109] It is likely that the moiety which is able to convert a
prodrug to a cytotoxic drug will be active in isolation from the
rest of the compound but it is necessary only for it to be active
when (a) it is in combination with the rest of the compound and (b)
the compound is attached to, adjacent to or internalised in target
cells.
[0110] When each moiety of the compound is a polypeptide, the two
portions may be linked together by any of the conventional ways of
cross-linking polypeptides, such as those generally described in
O'Sullivan et al (1979) Anal. Biochem. 100, 100-108. For example,
the anti-MR antibody may be enriched with thiol groups and the
further moiety reacted with a bifunctional agent capable of
reacting with those thiol groups, for example the
N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Amide and
thioether bonds, for example achieved with
m-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more
stable in vivo than disulphide bonds.
[0111] Alternatively, the compound may be produced as a fusion
compound by recombinant DNA techniques whereby a length of DNA
comprises respective regions encoding the two moieties of the
compound of the invention either adjacent one another or separated
by a region encoding a linker peptide which does not destroy the
desired properties of the compound. Conceivably, the two portions
of the compound may overlap wholly or partly.
[0112] The DNA is then expressed in a suitable host to produce a
polypeptide comprising the compound of the invention.
[0113] The cytotoxic moiety may be a radiosensitizer.
Radiosensitizers include fluoropyrimidines, thymidine analogues,
hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated
pyrimidines, 3-aminobenzamide, 3-aminobenzodiamide, etanixadole,
pimonidazole and misonidazole (see, for example, McGinn et al
(1996) J. Natl. Cancer ITst. 88, 1193-11203; Shewach & Lawrence
(1996) Invest. New Drugs 14, 257-263; Horsman (1995) Acta Oncol.
34, 571-587; Shenoy & Singh (1992) Clin. Invest. 10, 533-551;
Mitchell et al (1989) Int. J. Radiat. Biol. 56, 827-836; Iliakis
& Kurtzman (1989) Int. J. Radiat. Oncol. Biol. Phys. 16,
1235-1241; Brown (1989) Int. J. Radiat. Oncol. Biol. Phys. 16,
987-993; Brown (1985) Cancer 55, 2222-2228).
[0114] Also, delivery of genes into cells can radiosensitise them,
for example delivery of the p53 gene or cyclin D (Lang et al (1998)
J. Neurosurg. 89, 125-132; Coco Martin et al (1999) Cancer Res. 59,
1134-1140).
[0115] The further moiety may be one which becomes cytotoxic, or
releases a cytotoxic moiety, upon irradiation. For example, the
boron-10 isotope, when appropriately irradiated, releases .alpha.
particles which are cytotoxic (see for example, U.S. Pat. No.
4,348,376 to Goldenberg; Primus et al (1996) Bioconjug. Chem. 7,
532-535).
[0116] Similarly, the cytotoxic moiety may be one which is useful
in photodynamic therapy such as photofrin (see, for example,
Dougherty et al (1998) J. Natl. Cancer Inst. 90, 889-905).
[0117] The cytotoxic moiety may be a nucleic acid molecule which is
directly or indirectly cytotoxic. For example, the nucleic acid
molecule may be an antisense oligonucleotide which, upon
localisation at the target site is able to enter cells and lead to
their death. The oligonucleotide, therefore, may be one which
prevents expression of an essential gene, or one which leads to a
change in gene expression which causes apoptosis.
[0118] Examples of suitable oligonucleotides include those directed
at bcl-2 (Ziegler et al (1997) J. Natl. Cancer Inst. 89,
1027-1036), and DNA polymerase a and topoisomerase II.alpha. (Lee
et al (1996) Anticancer Res. 16, 1805-1811.
[0119] Peptide nucleic acids may be useful in place of conventional
nucleic acids (see Knudsen & Nielsen (1997) Anticancer Drugs 8,
113-118).
[0120] A thirteenth aspect of the invention provides a
polynucleotide encoding a compound as defined above in the twelfth
aspect of the invention, wherein the antibody and the cytotoxic
moiety are polypeptides which are fused.
[0121] A fourteenth aspect of the invention provides a compound
comprising an antibody as defined above in the seventh, eighth and
tenth aspects of the invention, and a readily detectable
moiety.
[0122] A compound comprising an anti-MR antibody as defined above
and a readily detectable moiety can be used, in combination with an
appropriate detection method, to detect the location of the
compound in the individual, and hence to identify the sites and
extent of angiogenesis in the individual, as well as inhibiting the
angiogenesis in the individual.
[0123] By a "readily detectable moiety" we include the meaning that
the moiety is one which, when located at the target site following
administration of the compound of the invention into a patient, may
be detected, typically non-invasively from outside the body and the
site of the target located. Thus, the compounds of this embodiment
of the invention are useful in imaging and diagnosis.
[0124] Typically, the readily detectable moiety is or comprises a
radioactive atom which is useful in imaging. Suitable radioactive
atoms include technetium-99m or iodine-123 for scintigraphic
studies. Other readily detectable moieties include, for example,
spin labels for magnetic resonance imaging (MRI) such as iodine-123
again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron. Clearly, the compound of
the invention must have sufficient of the appropriate atomic
isotopes in order for the molecule to be readily detectable.
[0125] The radio- or other labels may be incorporated in the
compound of the invention in known ways. For example, if the
antibody is a polypeptide it may be biosynthesised or may be
synthesised by chemical amino acid synthesis using suitable amino
acid precursors involving, for example, fluorine-19 in place of
hydrogen. Labels such as .sup.99mTc, .sup.123I, .sup.186Rh
.sup.188Rh and .sup.111In can, for example, be attached via
cysteine residues in the antibody. Yttrium-90 can be attached via a
lysine residue. The IODOGEN method (Fraker er al (1978) Biochein.
Biophys. Res. Comm. 80, 49-57) can be used to incorporate
iodine-123. Reference ("Monoclonal Antibodies in
Immunoscintigraphy", J-F Chatal, CRC Press, 1989) describes other
methods in detail.
[0126] A fifteenth aspect of the invention provides a vector
comprising a polynucleotide as defined above in the ninth, eleventh
and thirteenth aspects of the invention.
[0127] Typical prokaryotic vector plasmids are: pUC18, pUC19,
pBR322 and pBR329 available from Biorad Laboratories (Richmond,
Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5
available from Pharmacia (Piscataway, N.J., USA); pBS vectors,
Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A,
pNH46A available from Stratagene Cloning Systems (La Jolla, Calif.
92037, USA).
[0128] A typical mammalian cell vector plasmid is pSVL available
from Pharmacia (Piscataway, N.J., USA). This vector uses the SV40
late promoter to drive expression of cloned genes, the highest
level of expression being found in T antigen-producing cells, such
as COS-1 cells. An example of an inducible mammalian expression
vector is pMSG, also available from Pharmacia (Piscataway, N.J.,
USA). This vector uses the glucocorticoid-inducible promoter of the
mouse mammary tumour virus long terminal repeat to drive expression
of the cloned gene.
[0129] Useful yeast plasmid vectors are pRS403-406 and pRS413-416
and are generally available from Stratagene Cloning Systems (La
Jolla, Calif. 92037, USA). Plasmids pRS403, pRS404, pRS405 and
pRS406 are Yeast Integrating plasmids (YIps) and incorporate the
yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids
pRS413-416 are Yeast Centromere plasmids (YCps).
[0130] Methods well known to those skilled in the art can be used
to construct expression vectors containing the coding sequence and,
for example appropriate transcriptional or translational controls.
One such method involves ligation via homopolymer tails.
Homopolymer polydA (or polydC) tails are added to exposed 3'-OH
groups on the DNA fragment to be cloned by terminal
deoxynucleotidyl transferases. The fragment is then capable of
annealing to the polydT (or polydG) tails added to the ends of a
linearised plasmid vector. Gaps left following annealing can be
filled by DNA polymerase and the free ends joined by DNA
ligase.
[0131] Another method involves ligation via cohesive ends.
Compatible cohesive ends can be generated on the DNA fragment and
vector by the action of suitable restriction enzymes. These ends
will rapidly anneal through complementary base pairing and
remaining nicks can be closed by the action of DNA ligase.
[0132] A further method uses synthetic molecules called linkers and
adaptors. DNA fragments with blunt ends are generated by
bacteriophage T4 DNA polymerase or E. coli DNA polymerase I which
remove protruding 3' termini and fill in recessed 3' ends.
Synthetic linkers, pieces of blunt-ended double-stranded DNA which
contain recognition sequences for defined restriction enzymes, can
be ligated to blunt-ended DNA fragments by T4 DNA ligase. They are
subsequently digested with appropriate restriction enzymes to
create cohesive ends and ligated to an expression vector with
compatible termini. Adaptors are also chemically synthesised DNA
fragments which contain one blunt end used for ligation but which
also possess one preformed cohesive end.
[0133] Synthetic linkers containing a variety of restriction
endonuclease sites are commercially available from a number of
sources including International Biotechnologies Inc, New Haven,
Conn., USA.
[0134] A desirable way to modify the DNA encoding the polypeptide
of the invention is to use the polymerase chain reaction as
disclosed by Saiki et al (1988) Science 239, 487-491. In this
method the DNA to be enzymatically amplified is flanked by two
specific oligonucleotide primers which themselves become
incorporated into the amplified DNA. The specific primers may
contain restriction endonuclease recognition sites which can be
used for cloning into expression vectors using methods known in the
art.
[0135] A sixteenth aspect of the invention provides a host cell
comprising a polynucleotide as defined in the ninth, eleventh and
thirteenth aspects of the invention, or a vector as defined in the
fifteenth aspect of the invention.
[0136] Many expression systems are known, including systems
employing: bacteria (eg. E. coli and Bacillus subtilis) transformed
with, for example, recombinant bacteriophage, plasmid or cosmid DNA
expression vectors; yeasts (eg. Saccharomyces cerevisiae)
transformed with, for example, yeast expression vectors; insect
cell systems transformed with, for example, viral expression
vectors (eg. baculovirus); plant cell systems transfected with, for
example viral or bacterial expression vectors; animal cell systems
transfected with, for example, adenovirus expression vectors.
[0137] The vectors can include a prokaryotic replicon, such as the
Col E1 ori, for propagation in a prokaryote, even if the vector is
to be used for expression in other, non-prokaryotic cell types. The
vectors can also include an appropriate promoter such as a
prokaryotic promoter capable of directing the expression
(transcription and translation) of the genes in a bacterial host
cell, such as E. coli, transformed therewith.
[0138] A promoter is an expression control element formed by a DNA
sequence that permits binding of RNA polymerase and transcription
to occur. Promoter sequences compatible with exemplary bacterial
hosts are typically provided in plasmid vectors containing
convenient restriction sites for insertion of a DNA segment of the
present invention.
[0139] The polynucleotide in a suitable host cell may be expressed
to produce the antibody or compound of the invention. Thus, the
polynucleotide may be used in accordance with known techniques,
appropriately modified in view of the teachings contained herein,
to construct an expression vector, which is then used to transform
an appropriate host cell for the expression and production of the
antibody or compound of the invention. Such techniques include
those disclosed in U.S. Pat. No. 4,440,859 issued 3 Apr. 1984 to
Rutter et al, U.S. Pat. No. 4,530,901 issued 23 Jul. 1985 to
Weissman, U.S. Pat. No. 4,582,800 issued 15 Apr. 1986 to Crowl,
U.S. Pat. No. 4,677,063 issued 30 Jun. 1987 to Mark et al, U.S.
Pat. No. 4,678,751 issued 7 Jul. 1987 to Goeddel, U.S. Pat. No.
4,704,362 issued 3 Nov. 1987 to Itakura et al, U.S. Pat. No.
4,710,463 issued 1 Dec. 1987 to Murray, U.S. Pat. No. 4,757,006
issued 12 Jul. 1988 to Toole, Jr. et al, U.S. Pat. No. 4,766,075
issued 23 Aug. 1988 to Goeddel et al and U.S. Pat. No. 4,810,648
issued 7 Mar. 1989 to Stalker, all of which are incorporated herein
by reference.
[0140] The polynucleotide may be joined to a wide variety of other
DNA sequences for introduction into an appropriate host. The
companion DNA will depend upon the nature of the host, the manner
of the introduction of the DNA into the host, and whether episomal
maintenance or integration is desired.
[0141] Generally, the polynucleotide is inserted into an expression
vector, such as a plasmid, in proper orientation and correct
reading frame for expression. If necessary, the DNA may be linked
to the appropriate transcriptional and translational regulatory
control nucleotide sequences recognised by the desired host,
although such controls are generally available in the expression
vector. Thus, the DNA insert may be operatively linked to an
appropriate promoter. Bacterial promoters include the E. coli lacI
and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the
phage .lamda. PR and PL promoters, the phoA promoter and the trp
promoter. Eukaryotic promoters include the CMV immediate early
promoter, the HSV thymidine kinase promoter, the early and late
SV40 promoters and the promoters of retroviral LTRs. Other suitable
promoters will be known to the skilled artisan. The expression
constructs will desirably also contain sites for transcription
initiation and termination, and in the transcribed region, a
ribosome binding site for translation. (Hastings et al,
International Patent No. WO 98/16643, published 23 Apr. 1998)
[0142] The vector is then introduced into the host through standard
techniques. Generally, not all of the hosts will be transformed by
the vector and it will therefore be necessary to select for
transformed host cells. One selection technique involves
incorporating into the expression vector a DNA sequence marker,
with any necessary control elements, that codes for a selectable
trait in the transformed cell. These markers include dihydrofolate
reductase, G418 or neomycin resistance for eukaryotic cell culture,
and tetracyclin, kanamycin or ampicillin resistance genes for
culturing in E. coli and other bacteria.
[0143] Alternatively, the gene for such selectable trait can be on
another vector, which is used to co-transform the desired host
cell.
[0144] Host cells that have been transformed by the recombinant DNA
of the invention are then cultured for a sufficient time and under
appropriate conditions known to those skilled in the art in view of
the teachings disclosed herein to permit the expression of the
polypeptide, which can then be recovered.
[0145] The antibody or compound can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium
sulphate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification.
[0146] An seventeenth aspect of the invention provides a stable
host cell line producing an antibody as defined in the seventh,
eighth or tenth aspects of the invention, or a compound as defined
in the twelfth aspect of the invention wherein the antibody and the
cytotoxic moiety are polypeptides which are fused, resulting from
incorporation in the cell line an exogenous polynucleotide as
defined in the ninth, eleventh and thirteenth aspects of the
invention, or a vector as defined in the fifteenth aspect of the
invention.
[0147] A eighteenth aspect of the invention provides a
pharmaceutical composition or formulation comprising an antibody as
defined in the seventh, eighth or tenth aspects of the invention,
or a polynucleotide as defined in the ninth, eleventh and
thirteenth aspects of the invention, or a compound as defined in
the twelfth or fourteenth aspects of the invention, and a
pharmaceutically acceptable carrier.
[0148] By "pharmaceutically acceptable" is included that the
formulation is sterile and pyrogen free. Suitable pharmaceutical
carriers are well known in the art of pharmacy.
[0149] The carrier(s) must be "acceptable" in the sense of being
compatible with the compound of the invention and not deleterious
to the recipients thereof. Typically, the carriers will be water or
saline which will be sterile and pyrogen free; however, other
acceptable carriers may be used.
[0150] In an embodiment, the pharmaceutical compositions or
formulations of the invention are for parenteral administration,
more particularly for intravenous administration.
[0151] In a preferred embodiment, the pharmaceutical composition is
suitable for intravenous administration to a patient, for example
by injection.
[0152] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents.
[0153] In an alternative preferred embodiment, the pharmaceutical
composition is suitable for topical administration to a
patient.
[0154] Preferably, the formulation is a unit dosage containing a
daily dose or unit, daily sub-dose or an appropriate fraction
thereof, of the active ingredient.
[0155] The antibody, polynucleotide or compound of the invention
will normally be administered orally or by any parenteral route, in
the form of a pharmaceutical formulation comprising the active
ingredient, optionally in the form of a non-toxic organic, or
inorganic, acid, or base, addition salt, in a pharmaceutically
acceptable dosage form. Depending upon the disorder and patient to
be treated, as well as the route of administration, the
compositions may be administered at varying doses.
[0156] In human therapy, the antibody, polynucleotide or compound
of the invention can be administered alone but will generally be
administered in admixture with a suitable pharmaceutical excipient,
diluent or carrier selected with regard to the intended route of
administration and standard pharmaceutical practice.
[0157] For example, the antibody, polynucleotide or compound of the
invention can be administered orally, buccally or sublingually in
the form of tablets, capsules, ovules, elixirs, solutions or
suspensions, which may contain flavouring or colouring agents, for
immediate-, delayed- or controlled-release applications. The
antibody, polynucleotide or compound of invention may also be
administered via intracavernosal injection.
[0158] Such tablets may contain excipients such as microcrystalline
cellulose, lactose, sodium citrate, calcium carbonate, dibasic
calcium phosphate and glycine, disintegrants such as starch
(preferably corn, potato or tapioca starch), sodium starch
glycollate, croscarmellose sodium and certain complex silicates,
and granulation binders such as polyvinylpyrrolidone,
hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC),
sucrose, gelatin and acacia. Additionally, lubricating agents such
as magnesium stearate, stearic acid, glyceryl behenate and talc may
be included.
[0159] Solid compositions of a similar type may also be employed as
fillers in gelatin capsules. Preferred excipients in this regard
include lactose, starch, a cellulose, milk sugar or high molecular
weight polyethylene glycols. For aqueous suspensions and/or
elixirs, the compounds of the invention may be combined with
various sweetening or flavouring agents, colouring matter or dyes,
with emulsifying and/or suspending agents and with diluents such as
water, ethanol, propylene glycol and glycerin, and combinations
thereof.
[0160] The antibody, polynucleotide or compound of the invention
can also be administered parenterally, for example, intravenously,
intra-arterially, intraperitoneally, intrathecally,
intraventricularly, intrasternally, intracranially, intramuscularly
or subcutaneously, or they may be administered by infusion
techniques. They are best used in the form of a sterile aqueous
solution which may contain other substances, for example, enough
salts or glucose to make the solution isotonic with blood. The
aqueous solutions should be suitably buffered (preferably to a pH
of from 3 to 9), if necessary. The preparation of suitable
parenteral formulations under sterile conditions is readily
accomplished by standard pharmaceutical techniques well-known to
those skilled in the art.
[0161] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents. The
formulations may be presented in unit-dose or multi-dose
containers, for example sealed ampoules and vials, and may be
stored in a freeze-dried (lyophilised) condition requiring only the
addition of the sterile liquid carrier, for example water for
injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets of the kind previously described.
[0162] For oral and parenteral administration to human patients,
the daily dosage level of the antibody, polynucleotide or compound
of the invention will usually be from 1 to 1000 mg per adult (i.e.
from about 0.015 to 15 mg/kg), administered in single or divided
doses.
[0163] Thus, for example, the tablets or capsules of the antibody,
polynucleotide or compound of the invention may contain from 1 mg
to 1000 mg of active agent for administration singly or two or more
at a time, as appropriate. The physician in any event will
determine the actual dosage which will be most suitable for any
individual patient and it will vary with the age, weight and
response of the particular patient. The above dosages are exemplary
of the average case. There can, of course, be individual instances
where higher or lower dosage ranges are merited and such are within
the scope of this invention.
[0164] The antibody, polynucleotide or compound of the invention
can also be administered intranasally or by inhalation and are
conveniently delivered in the form of a dry powder inhaler or an
aerosol spray presentation from a pressurised container, pump,
spray or nebuliser with the use of a suitable propellant, e.g.
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoro-ethane, a hydrofluoroalkane such as
1,1,1,2-tetrafluoroethane (HFA 134A, or
1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA), carbon dioxide or
other suitable gas. In the case of a pressurised aerosol, the
dosage unit may be determined by providing a valve to deliver a
metered amount. The pressurised container, pump, spray or nebuliser
may contain a solution or suspension of the active compound, e.g.
using a mixture of ethanol and the propellant as the solvent, which
may additionally contain a lubricant, e.g. sorbitan trioleate.
Capsules and cartridges (made, for example, from gelatin) for use
in an inhaler or insufflator may be formulated to contain a powder
mix of a compound of the invention and a suitable powder base such
as lactose or starch.
[0165] Aerosol or dry powder formulations are preferably arranged
so that each metered dose or "puff" contains at least 1 mg of an
antibody, polynucleotide or compound of the invention for delivery
to the patient. It will be appreciated that he overall daily dose
with an aerosol will vary from patient to patient, and may be
administered in a single dose or, more usually, in divided doses
throughout the day.
[0166] Alternatively, the antibody, polynucleotide or compound of
the invention can be administered in the form of a suppository or
pessary, or they may be applied topically in the form of a lotion,
solution, cream, ointment or dusting powder. The compounds of the
invention may also be transdermally administered, for example, by
the use of a skin patch. They may also be administered by the
ocular route, particularly for treating diseases of the eye.
[0167] For ophthalmic use, the antibody, polynucleotide or compound
of the invention can be formulated as micronised suspensions in
isotonic, pH adjusted, sterile saline, or, preferably, as solutions
in isotonic, pH adjusted, sterile saline, optionally in combination
with a preservative such as a benzylalkonium chloride.
Alternatively, they may be formulated in an ointment such as
petrolatum.
[0168] For application topically to the skin, the antibody,
polynucleotide or compound of the invention can be formulated as a
suitable ointment containing the active compound suspended or
dissolved in, for example, a mixture with one or more of the
following: mineral oil, liquid petrolatum, white petrolatum,
propylene glycol, polyoxyethylene polyoxypropylene compound,
emulsifying wax and water. Alternatively, they can be formulated as
a suitable lotion or cream, suspended or dissolved in, for example,
a mixture of one or more of the following: mineral oil, sorbitan
monostearate, a polyethylene glycol, liquid paraffin, polysorbate
60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl
alcohol and water.
[0169] Formulations suitable for topical administration in the
mouth include lozenges comprising the active ingredient in a
flavoured basis, usually sucrose and acacia or tragacanth;
pastilles comprising the active ingredient in an inert basis such
as gelatin and glycerin, or sucrose and acacia; and mouth-washes
comprising the active ingredient in a suitable liquid carrier.
[0170] For veterinary use, a compound of the invention is
administered as a suitably acceptable formulation in accordance
with normal veterinary practice and the veterinary surgeon will
determine the dosing regimen and route of administration which will
be most appropriate for a particular animal.
[0171] A nineteenth aspect of the invention provides an antibody as
defined in the seventh, eighth or tenth aspects of the invention,
or a polynucleotide as defined in the ninth, eleventh and
thirteenth aspects of the invention, or a compound as defined in
the twelfth or fourteenth aspects of the invention, for use in
medicine.
[0172] A twentieth aspect of the invention provides the use of an
antibody as defined in the seventh, eighth, or tenth aspects of the
invention, or a polynucleotide as defined in the ninth, eleventh
and thirteenth aspects of the invention, or a compound as defined
in the twelfth or fourteenth aspects of the invention, in the
preparation of a medicament for inhibiting angiogenesis.
[0173] Conditions which involve unwanted or undesirable
angiogenesis are described above.
[0174] A twenty-first aspect of the invention provides a method of
inhibiting angiogenesis in an individual in need thereof comprising
administering an antibody as defined in the seventh, eighth, or
tenth aspects of the invention, or a polynucleotide as defined in
the ninth, eleventh and thirteenth aspects of the invention, or a
compound as defined in the twelfth or fourteenth aspects of the
invention, to the individual.
[0175] In an embodiment, polypeptides, such as antibodies, may be
delivered using an injectable sustained-release drug delivery
system. These are designed specifically to reduce the frequency of
injections. An example of such a system is Nutropin Depot which
encapsulates recombinant human growth hormone (rhGH) in
biodegradable microspheres that, once injected, release rhGH slowly
over a sustained period.
[0176] The polypeptide can be administered by a surgically
implanted device that releases the drug directly to the required
site. For example, Vitrasert releases ganciclovir directly into the
eye to treat CMV retinitis. The direct application of this toxic
agent to the site of disease achieves effective therapy without the
drug's significant systemic side-effects.
[0177] Electroporation therapy (EPT) systems can also be employed
for the administration of polypeptides. A device which delivers a
pulsed electric field to cells increases the permeability of the
cell membranes to the drug, resulting in a significant enhancement
of intracellular drug delivery.
[0178] Polypeptides can also be delivered by electroincorporation
(EI). EI occurs when small particles of up to 30 microns in
diameter on the surface of the skin experience electrical pulses
identical or similar to those used in electroporation. In EI, these
particles are driven through the stratum corneum and into deeper
layers of the skin. The particles can be loaded or coated with
drugs or genes or can simply act as "bullets" that generate pores
in the skin through which the drugs can enter.
[0179] An alternative method of polypeptide delivery is the ReGel
injectable system that is thermo-sensitive. Below body temperature,
ReGel is an injectable liquid while at body temperature it
immediately forms a gel reservoir that slowly erodes and dissolves
into known, safe, biodegradable polymers. The active drug is
delivered over time as the biopolymers dissolve.
[0180] Polypeptide pharmaceuticals can also be delivered orally.
The process employs a natural process for oral uptake of vitamin
B.sub.12 in the body to co-deliver proteins and peptides. By riding
the vitamin B.sub.12 uptake system, the protein or peptide can move
through the intestinal wall. Complexes are synthesised between
vitamin B.sub.12 analogues and the drug that retain both
significant affinity for intrinsic factor (IF) in the vitamin
B.sub.12 portion of the complex and significant bioactivity of the
drug portion of the complex.
[0181] Polynucleotides may be administered by any effective method,
for example, parenterally (eg intravenously, subcutaneously,
intramuscularly) or by oral, nasal or other means which permit the
oligonucleotides to access and circulate in the patient's
bloodstream. Polynucleotides administered systemically preferably
are given in addition to locally administered polynucleotides, but
also have utility in the absence of local administration. A dosage
in the range of from about 0.1 to about 10 grams per administration
to an adult human generally will be effective for this purpose.
[0182] The polynucleotide may be administered as a suitable genetic
construct as is described below and delivered to the patient where
it is expressed. Typically, the polynucleotide in the genetic
construct is operatively linked to a promoter which can express the
antibody or compound in the cell.
[0183] Although genetic constructs for delivery of polynucleotides
can be DNA or RNA it is preferred if it is DNA.
[0184] Preferably, the genetic construct is adapted for delivery to
a human cell.
[0185] Means and methods of introducing a genetic construct into a
cell in an animal body are known in the art. For example, the
constructs of the invention may be introduced into cells by any
convenient method, for example methods involving retroviruses, so
that the construct is inserted into the genome of the cell. For
example, in Kuriyama et al (1991) Cell Struc. and Func. 16, 503-510
purified retroviruses are administered. Retroviral DNA constructs
comprising a polynucleotide as described above may be made using
methods well known in the art. To produce active retrovirus from
such a construct it is usual to use an ecotropic psi2 packaging
cell line grown in Dulbecco's modified Eagle's medium (DMEM)
containing 10% foetal calf serum (FCS). Transfection of the cell
line is conveniently by calcium phosphate co-precipitation, and
stable transformants are selected by addition of G418 to a final
concentration of 1 mg/ml (assuming the retroviral construct
contains a neo.sup.R gene). Independent colonies are isolated and
expanded and the culture supernatant removed, filtered through a
0.45 .mu.m pore-size filter and stored at -70.degree. C. For the
introduction of the retrovirus into the tumour cells, it is
convenient to inject directly retroviral supernatant to which 10
.mu.g/ml Polybrene has been added. For tumours exceeding 10 mm in
diameter it is appropriate to inject between 0.1 ml and 1 ml of
retroviral supernatant; preferably 0.5 ml.
[0186] Alternatively, as described in Culver et al (1992) Science
256, 1550-1552, cells which produce retroviruses are injected. The
retrovirus-producing cells so introduced are engineered to actively
produce retroviral vector particles so that continuous productions
of the vector occurred within the tumour mass in situ. Thus,
proliferating epidermmal cells can be successfully transduced in
vivo if mixed with retroviral vector-producing cells.
[0187] Targeted retroviruses are also available for use in the
invention; for example, sequences conferring specific binding
affinities may be engineered into pre-existing viral env genes (see
Miller & Vile (1995) Faseb J 9, 190-199 for a review of this
and other targeted vectors for gene therapy).
[0188] Other methods involve simple delivery of the construct into
the cell for expression therein either for a limited time or,
following integration into the genome, for a longer time. An
example of the latter approach includes liposomes (Nassander et al
(1992) Cancer Res. 52, 646-653).
[0189] For the preparation of immuno-liposomes MPB-PE
(N-[4-(p-maleimidophenyl)butyryl]-phosphatidylethanolamine) is
synthesised according to the method of Martin & Papahadjopoulos
(1982) J. Biol. Chem. 257, 286-288. MPB-PE is incorporated into the
liposomal bilayers to allow a covalent coupling of the antibody, or
fragment thereof, to the liposomal surface. The liposome is
conveniently loaded with the DNA or other genetic construct of the
invention for delivery to the target cells, for example, by forming
the said liposomes in a solution of the DNA or other genetic
construct, followed by sequential extrusion through polycarbonate
membrane filters with 0.6 .mu.m and 0.2 .mu.m pore size under
nitrogen pressures up to 0.8 MPa. After extrusion, entrapped DNA
construct is separated from free DNA construct by
ultracentrifugation at 80 000.times.g for 45 min. Freshly prepared
MPB-PE-liposomes in deoxygenated buffer are mixed with freshly
prepared antibody (or fragment thereof) and the coupling reactions
are carried out in a nitrogen atmosphere at 4.degree. C. under
constant end over end rotation overnight. The immunoliposomes are
separated from unconjugated antibodies by ultracentrifugation at 80
000.times.g for 45 min. Immunoliposomes may be injected
intraperitoneally or directly into a tumour.
[0190] Other methods of delivery include adenoviruses carrying
external DNA via an antibody-polylysine bridge (see Curiel Prog.
Med. Virol. 40, 1-18) and transferrin-polycation conjugates as
carriers (Wagner et al (1990) Proc. Natl. Acad. Sci. USA 87,
3410-3414). In the first of these methods a polycation-antibody
complex is formed with the DNA construct or other genetic construct
of the invention, wherein the antibody is specific for either
wild-type adenovirus or a variant adenovirus in which a new epitope
has been introduced which binds the antibody. The polycation moiety
binds the DNA via electrostatic interactions with the phosphate
backbone. The adenovirus, because it contains unaltered fibre and
penton proteins, is internalised into the cell and carries into the
cell with it the DNA construct of the invention. It is preferred if
the polycation is polylysine.
[0191] The polynucleotide may also be delivered by adenovirus
wherein it is present within the adenovirus particle, for example,
as described below.
[0192] In an alternative method, a high-efficiency nucleic acid
delivery system that uses receptor-mediated endocytosis to carry
DNA macromolecules into cells is employed. This is accomplished by
conjugating the iron-transport protein transferrin to polycations
that bind nucleic acids. Human transferrin, or the chicken
homologue conalbumin, or combinations thereof is covalently linked
to the small DNA-binding protein protamine or to polylysines of
various sizes through a disulfide linkage. These modified
transferrin molecules maintain their ability to bind their cognate
receptor and to mediate efficient iron transport into the cell. The
transferrin-polycation molecules form electrophoretically stable
complexes with DNA constructs or other genetic constructs of the
invention independent of nucleic acid size (from short
oligonucleotides to DNA of 21 kilobase pairs). When complexes of
transferrin-polycation and the DNA constructs or other genetic
constructs of the invention are supplied to the tumour cells, a
high level of expression from the construct in the cells is
expected.
[0193] High-efficiency receptor-mediated delivery of the DNA
constructs or other genetic constructs of the invention using the
endosome-disruption activity of defective or chemically inactivated
adenovirus particles produced by the methods of Cotten et al (1992)
Proc. Natl. Acad. Sci. USA 89, 6094-6098 may also be used. This
approach appears to rely on the fact that adenoviruses are adapted
to allow release of their DNA from an endosome without passage
through the lysosome, and in the presence of, for example
transferrin linked to the DNA construct or other genetic construct
of the invention, the construct is taken up by the cell by the same
route as the adenovirus particle.
[0194] This approach has the advantages that there is no need to
use complex retroviral constructs; there is no permanent
modification of the genome as occurs with retroviral infection; and
the targeted expression system is coupled with a targeted delivery
system, thus reducing toxicity to other cell types.
[0195] It will be appreciated that "naked DNA" and DNA complexed
with cationic and neutral lipids may also be useful in introducing
the DNA of the invention into cells of the individual to be
treated. Non-viral approaches to gene therapy are described in
Ledley (1995) Human Gene Therapy 6, 1129-1144.
[0196] Alternative targeted delivery systems are also known such as
the modified adenovirus system described in WO 94/10323 wherein,
typically, the DNA is carried within the adenovirus, or
adenovirus-like, particle. Michael et al (1995) Gene Theiapy 2,
660-668 describes modification of adenovirus to add a
cell-selective moiety into a fibre protein. Mutant adenoviruses
which replicate selectively in p53-deficient human tumour cells,
such as those described in Bischoff et al (1996) Science 274,
373-376 are also useful for delivering the genetic construct of the
invention to a cell. Thus, it will be appreciated that a further
aspect of the invention provides a virus or virus-like particle
comprising a genetic construct of the invention. Other suitable
viruses, viral vectors or virus-like particles include lentivirus
and lentiviral vectors, HSV, adeno-assisted virus (AAV) and
AAV-based vectors, vaccinia and parvovirus.
[0197] The genetic constructs of the invention can be prepared
using methods well known in the art.
[0198] A twenty-second aspect of the invention provides an in vitro
method of inhibiting angiogenesis comprising administering an
antibody as defined in the seventh, eighth, or tenth aspects of the
invention, or a polynucleotide as defined in the ninth, eleventh
and thirteenth aspects of the invention, or a compound as defined
in the twelfth or fourteenth aspects of the invention, to tissue or
cells in vitro.
[0199] A twenty-third aspect of the invention provides a method of
producing an antibody as defined in the seventh, eighth or tenth
aspects of the invention, or a compound as defined in the twelfth
aspect of the invention wherein the antibody and the cytotoxic
moiety are polypeptides which are fused, the method comprising
expressing a polynucleotide as defined in the ninth, eleventh and
thirteenth aspects of the invention, or culturing a stable host
cell line as defined in the seventeenth aspect of the
invention.
[0200] We have also shown that the extracellular fragment of MR
(residues 1-467, FIG. 2B, SEQ ID NO: 3, also known as the MR
ectodomain) inhibits migration of endothelial cells, including
bFGF- and VEGF-induced migration. Interestingly, the MR ectodomain
does not appear to affect endothelial cell attachment (data not
shown). The MR ectodomain, and fragments of it that show inhibitory
activity in the HUVEC migration assay, would be predicted to be
therapeutically useful in conditions in which unwanted, undesirable
or inappropriate endothelial cell migration contributes to the
pathology.
[0201] We have also shown that the MR ectodomain inhibits
proliferation of endothelial cells. The MR ectodomain, and
fragments of it that show anti-proliferative activity in an assay
such as that described in Example 5, would be predicted to be
therapeutically useful in conditions in which unwanted, undesirable
or inappropriate endothelial cell proliferation contributes to the
pathology.
[0202] A twenty-fourth aspect of the invention provides the MR
ectodomain, or a fragment thereof that inhibits endothelial cell
migration and/or proliferation.
[0203] It is appreciated that, in an embodiment, "a fragment of the
MR ectodomain that inhibits endothelial cell migration and/or
proliferation" may inhibit endothelial cell migration and not
endothelial cell proliferation, or may inhibit endothelial cell
proliferation and not endothelial cell migration. In an alternative
embodiment, "a fragment of the MR ectodomain that inhibits
endothelial cell migration and/or proliferation" may inhibit both
endothelial cell migration and endothelial cell proliferation. In
this embodiment, the fragment of the MR ectodomain does not
necessarily inhibit both endothelial cell migration and/or
proliferation to the same extent.
[0204] By "inhibiting endothelial cell migration and/or
proliferation" we include the meaning of reducing the rate or level
of endothelial cell migration and/or proliferation. The reduction
can be a low level reduction of about 10%, or about 20%, or about
30%, or about 40% of the rate or level of endothelial cell
migration and/or proliferation. Preferably, the reduction is a
medium level reduction of about 50%, or about 60%, or about 70%, or
about 80% reduction of the rate or level of endothelial cell
migration and/or proliferation. More preferably, the reduction is a
high level reduction of about 90%, or about 95%, or about 99%, or
about 99.9%, or about 99.99% of the rate or level of endothelial
cell migration and/or proliferation. Most preferably, inhibition
can also include the elimination of endothelial cell migration
and/or proliferation, or its reduction to an undetectable
level.
[0205] Methods and assays for determining the rate or level of
endothelial cell migration, and hence for determining whether and
to what extent any particular fragment of the MR ectodomain
inhibits endothelial cell migration, are known in the art and
include the HUVEC assay described in Example 4. Similarly, methods
and assays for determining the rate or level of endothelial cell
proliferation, and hence for determining whether and to what extent
any particular fragment of the MR ectodomain inhibits endothelial
cell proliferation, are well known in the art and include the HUVEC
assay described in Example 5.
[0206] By "a fragment of the MR ectodomain that inhibits
endothelial cell migration and/or proliferation" we include the MR
ectodomain that has been truncated or deleted, or a polypeptide
comprising at least 450 contiguous amino acid residues of the MR
ectodomain, which is sufficient to inhibit endothelial cell
migration and/or proliferation. More preferably, a fragment of the
ectodomain which is sufficient to inhibit endothelial cell
migration and/or proliferation comprises at least 400, or at least
350, or at least 300, or at least 250, or at least 200, or at least
150, or at least 100, or at least 90, or at least 80, or at least
70, or at least 60, or at least 50, or at least 40, or at least 30,
or at least 20, or at least 15, or at least 10 contiguous amino
acid residues of the MR ectodomain. It is more particularly
preferred if the fragment of the ectodomain which is sufficient to
inhibit endothelial cell migration and/or proliferation comprises
at least 60 contiguous amino acid residues of the MR ectodomain.
The inhibition of endothelial cell migration and/or proliferation
can be tested, for example, using the HUVEC assays as described in
Examples 4 and 5.
[0207] In an embodiment, the fragment of the ectodomain which is
sufficient to inhibit endothelial cell migration and/or
proliferation consists of or comprises the Ig region of MR
(residues 46-209, SEQ ID NO: 4).
[0208] In another embodiment, the fragment of the ectodomain which
is sufficient to inhibit endothelial cell migration and/or
proliferation consists of or comprises the IgA domain of MR
(residues 46-116, SEQ ID NO: 5) or the IgB domain of MR (residues
151-209, SEQ ID NO: 6).
[0209] We have shown that the MR ectodomain does not appear to
inhibit endothelial cell attachment (data not shown) and,
preferably, the fragment of the ectodomain which is sufficient to
inhibit endothelial cell migration and/or proliferation does not
inhibit endothelial cell attachment.
[0210] Thus the MR ectodomain, or fragment thereof which inhibits
endothelial cell migration, can be used to inhibit endothelial cell
migration and/or proliferation without inhibiting endothelial cell
attachment.
[0211] A twenty-fifth aspect of the invention provides the MR
ectodomain, or a fragment thereof that inhibits endothelial cell
migration and/or proliferation, for use in medicine.
[0212] A twenty-sixth aspect of the invention provides a method of
combating any disease or condition involving unwanted, undesirable
or inappropriate endothelial cell migration and/or proliferation in
an individual, the method comprising administering the MR
ectodomain, or a fragment thereof that inhibits endothelial cell
migration and/or proliferation, to the individual.
[0213] Thus, the invention includes a method of treating a patient
who has a disease or condition in which endothelial cell migration
and/or proliferation contributes to the pathology, the method
comprising the step of administering to the patient the MR
ectodomain, or a fragment thereof that inhibits endothelial cell
migration and/or proliferation.
[0214] A twenty-seventh aspect of the invention provides the use of
the MR ectodomain, or a fragment thereof that inhibits endothelial
cell migration and/or proliferation, in the preparation of a
medicament for combating any disease or condition involving
unwanted, undesirable or inappropriate endothelial cell migration
and/or proliferation.
[0215] It has been shown that adipose tissue mass can be regulated
by its vasculature (Rupnick, M. A. et al (2002) PNAS USA 99(16):
10730-10735). Furthermore, leptin, a known regulator of appetite
and metabolism, is also known to modulate both migration of
endothelial cells (Goetze, S. et al (2002) Hypertension 40(5):
748-754) and angiogenesis (Sierra-Honigmann, M. R. et al (1998)
Science 281: 1683). Hence inhibition of migration of endothelial
cells may reduce adipose tissue mass and be useful in treating
obesity.
[0216] Diseases or conditions involving unwanted, undesirable or
inappropriate endothelial cell migration and/or proliferation
include tumours/cancer, psoriasis, atherosclerosis, menorrhagia,
endometriosis, arthritis (both inflammatory and rheumatoid),
macular degeneration, Paget's disease, retinopathy and its vascular
complications (including proliferative and of prematurity, and
diabetic retinopathy), benign vascular proliferations, fibroses,
obesity and inflammation.
[0217] The invention thus includes a method of combating a disease
or condition selected from tumours/cancer, psoriasis,
atherosclerosis, menorrhagia, endometriosis, arthritis (both
inflammatory and rheumatoid), macular degeneration, Paget's
disease, retinopathy and its vascular complications (including
proliferative and of prematurity, and diabetic retinopathy), benign
vascular proliferations, fibroses, obesity and inflammation in an
individual, the method comprising administering the MR ectodomain,
or a fragment thereof that inhibits endothelial cell migration
and/or proliferation, to the individual.
[0218] The invention also includes the use of the MR ectodomain, or
a fragment thereof that inhibits endothelial cell migration and/or
proliferation, in the preparation of a medicament for combating a
disease or condition selected from tumours/cancer, psoriasis,
atherosclerosis, menorrhagia, endometriosis, arthritis (both
inflammatory and rheumatoid), macular degeneration, Paget's
disease, retinopathy and its vascular complications (including
proliferative and of prematurity, and diabetic retinopathy), benign
vascular proliferations, fibroses, obesity and inflammation in an
individual.
[0219] A yet further aspect of the invention provides an in vitro
method of inhibiting endothelial cell migration and/or
proliferation comprising administering the MR ectodomain, or a
fragment thereof that inhibits endothelial cell migration and/or
proliferation, to tissue or cells in vitro. The cells may be
established cell lines, or cells that have been removed from an
individual. The tissue or cells are preferably mammalian tissue or
cells, and most preferably are human tissue or cells.
[0220] Furthermore, it is appreciated that administration of
nucleic acid encoding the extracellular fragment of MR or active
fragments thereof, might also be therapeutically useful.
[0221] A further aspect of the invention provides a polynucleotide
encoding the MR ectodomain, or a fragment thereof that inhibits
endothelial cell migration and/or proliferation, for use in
medicine.
[0222] A further aspect of the invention thus provides a method of
combating any disease or condition involving unwanted, undesirable
or inappropriate endothelial cell migration and/or proliferation in
an individual comprising administering a polynucleotide encoding
the MR ectodomain, or a fragment thereof that inhibits endothelial
cell migration and/or proliferation, to the individual.
[0223] A still further aspect of the invention provides the use of
a polynucleotide encoding the MR ectodomain, or a fragment thereof
that inhibits endothelial cell migration and/or proliferation, in
the preparation of a medicament for combating any disease or
condition involving unwanted, undesirable or inappropriate
endothelial cell migration and/or proliferation.
[0224] A yet further aspect of the invention provides an in vitro
method of inhibiting endothelial cell migration and/or
proliferation comprising administering a polynucleotide encoding
the MR ectodomain, or a fragment thereof that inhibits endothelial
cell migration and/or proliferation, to tissue or cells in vitro.
The cells may be established cell lines, or cells that have been
removed from an individual. The tissue or cells are preferably
mammalian tissue or cells, and most preferably are human tissue or
cells.
[0225] An additional aspect of the invention provides a vector
comprising a polynucleotide that encodes the MR ectodomain, or a
fragment thereof that inhibits endothelial cell migration and/or
proliferation. The invention also includes a host cell comprising a
polynucleotide that encodes the MR ectodomain, or a fragment
thereof that inhibits endothelial cell migration and/or
proliferation, or a vector comprising such a polynucleotide.
[0226] A further aspect of the invention provides a pharmaceutical
composition comprising the MR ectodomain, or a fragment thereof
that inhibits endothelial cell migration and/or proliferation, or a
polynucleotide encoding the MR ectodomain or the fragment thereof,
and a pharmaceutically acceptable carrier.
[0227] Preferably, the pharmaceutical composition is suitable for
intravenous administration to a patient.
[0228] The preferences for pharmaceutical formulations, routes of
administration, vectors, cell lines and so on, are the same in the
aspects of the invention directed to inhibiting endothelial cell
migration and/or proliferation using the MR ectodomain or a
fragment thereof as the preferences described above for the aspects
of the invention directed to anti-MR antibodies.
[0229] We have also shown that the MR ectodomain is sufficient to
inhibit formation of vessel sprouts in vitro in the aortic ring
assay, and in vivo in the sponge angiogenesis assay, and would be
predicted to be therapeutically useful in the inhibition of
angiogenesis. Furthermore, fragments (for example ones made
recombinantly or by de novo peptide synthesis) of the extracellular
region of MR that show inhibitory activity in the rat aortic ring
assay or the sponge angiogenesis assay would also be predicted to
be useful in the inhibition of angiogenesis.
[0230] A further aspect of the invention thus provides a method of
inhibiting angiogenesis in an individual in need thereof comprising
administering the MR ectodomain, or a fragment thereof that
inhibits angiogenesis, to the individual.
[0231] By "a fragment of the MR ectodomain that inhibits
angiogenesis" we include the MR ectodomain that has been truncated
or deleted, or a polypeptide comprising at least 450 contiguous
amino acid residues of the MR ectodomain, which is sufficient to
inhibit angiogenesis. More preferably, a fragment of the ectodomain
which is sufficient to inhibit angiogenesis comprises at least 400,
or at least 350, or at least 300, or at least 250, or at least 200,
or at least 150, or at least 100, or at least 90, or at least 80,
or at least 70, or at least 60, or at least 50, or at least 40, or
at least 30, or at least 20, or at least 15, or at least 10
contiguous amino acid residues of the MR ectodomain. It is more
particularly preferred if the fragment of the ectodomain which is
sufficient to inhibit angiogenesis comprises at least 60 contiguous
amino acid residues of the MR ectodomain. The inhibition of
angiogenesis can be tested, for example, using the aortic ring
assay as described in Example 2 or the sponge angiogenesis assay as
described in Example 3.
[0232] In an embodiment, the fragment of the ectodomain which is
sufficient to inhibit angiogenesis consists of or comprises the Ig
region of MR (residues 46-209, SEQ ID NO: 4).
[0233] In another embodiment, the fragment of the ectodomain which
is sufficient to inhibit angiogenesis consists of or comprises the
IgA domain of MR (residues 46-116, SEQ ID NO: 5) or the IgB domain
of MR (residues 151-209, SEQ ID NO: 6).
[0234] Another aspect of the invention provides the MR ectodomain,
or a fragment thereof that inhibits angiogenesis.
[0235] A further aspect of the invention provides the MR
ectodomain, or a fragment thereof that inhibits angiogenesis, for
use in medicine.
[0236] A still further aspect of the invention provides the use of
the MR ectodomain, or a fragment thereof that inhibits
angiogenesis, in the preparation of a medicament for inhibiting
angiogenesis.
[0237] A yet further aspect of the invention provides an in vitro
method of inhibiting angiogenesis comprising administering the MR
ectodomain, or a fragment thereof that inhibits angiogenesis, to
tissue or cells in vitro.
[0238] Furthermore, it is appreciated that administration of
nucleic acid encoding the extracellular fragment of MR or active
fragments thereof, would also be a useful mode of anti-angiogenesis
therapy.
[0239] A further aspect of the invention thus provides a method of
inhibiting angiogenesis in an individual in need thereof comprising
administering a polynucleotide encoding the MR ectodomain, or a
fragment thereof that inhibits angiogenesis.
[0240] A further aspect of the invention provides a polynucleotide
encoding the MR ectodomain, or a fragment thereof that inhibits
angiogenesis, for use in medicine.
[0241] A still further aspect of the invention provides the use of
a polynucleotide encoding the MR ectodomain, or a fragment thereof
that inhibits angiogenesis, in the preparation of a medicament for
inhibiting angiogenesis.
[0242] A yet further aspect of the invention provides an in vitro
method of inhibiting angiogenesis comprising administering a
polynucleotide encoding the MR ectodomain, or a fragment thereof
that inhibits angiogenesis, to tissue or cells in vitro.
[0243] An additional aspect of the invention provides a vector
comprising a polynucleotide that encodes the MR ectodomain, or a
fragment thereof that inhibits angiogenesis.
[0244] Another aspect of the invention provides a host cell
comprising a polynucleotide that encodes the MR ectodomain, or a
fragment thereof that inhibits angiogenesis, or a vector comprising
such a polynucleotide.
[0245] A further aspect of the invention provides a pharmaceutical
composition comprising the MR ectodomain, or a fragment thereof
that inhibits angiogenesis, or a polynucleotide encoding the MR
ectodomain or the fragment thereof, and a pharmaceutically
acceptable carrier.
[0246] Preferably, the pharmaceutical composition is suitable for
intravenous administration to a patient.
[0247] The preferences for diseases or conditions to be combated,
pharmaceutical formulations, routes of administration, vectors,
cell lines and so on, are the same in the aspects of the invention
directed to the MR ectodomain, or a fragment thereof that inhibits
angiogenesis, as the preferences described above for the aspects of
the invention directed to anti-MR antibodies.
[0248] All of the documents referred to herein are incorporated
herein, in their entirety, by reference.
[0249] The listing or discussion of a prior-published document in
this specification should not necessarily be taken as an
acknowledgement that the document is part of the state of the art
or is common general knowledge
[0250] The invention will now be described in more detail by
reference to the following Examples and Figures.
[0251] FIG. 1A shows the DNA sequence of the insert used to
generate plasmid N1 (SEQ ID NO: 1). FIG. 1B shows the amino acid
sequence encoded by the insert (SEQ ID NO: 2). This sequence is the
full length MR amino acid sequence.
[0252] FIG. 2A shows the DNA sequence of the insert used to
generate plasmid NH10 (SEQ ID NO: 30). FIG. 2B shows the amino acid
sequence encoded by the insert used for generating plasmid NH10.
This sequence is designated as the MR ectodomain and is the amino
acid sequence of the entire extracellular fragment of MR (residues
1-467, SEQ ID NO: 3).
[0253] FIG. 3 shows the amino acid sequence of the Ig region of MR
(residues 46-209, SEQ ID NO: 4).
[0254] FIG. 4A shows the amino acid sequence of the IgA domain of
MR (residues 46-116, SEQ ID NO: 5). FIG. 4B shows the amino acid
sequence of the IgB domain of MR (residues 151-209, SEQ ID NO:
5).
[0255] FIG. 5 shows a pictorial representation of the structure of
MR.
[0256] FIG. 6A is a graph and a table showing the effect of the
antibody (MR-7) and the soluble extracellular domain of MR (MR
Ecto) on formation of new vessels in the aortic ring assay.
[0257] FIG. 6B is a graph and a table showing the effect of the
soluble extracellular domain of MR (MR ectodomain, MR Ecto) on
formation of new vessels in the aortic ring assay with a human IgG
control.
[0258] FIG. 7 shows sprout formation from rat aorta in the presence
of antibody or soluble MR extracellular domain. Rat aorta sections
were cultured for 5 days in the presence of media alone (A), or in
media containing 100 .mu.g/ml MR-7 antibody (B), or 15 .mu.g/ml
soluble MR extracellular domain (residues 1-467) (C). Four pictures
are shown for each treatment group and are representative is of
sprouting levels from duplicate aortas.
[0259] FIG. 8 is a graph and a table showing the effect of the
soluble extracellular domain of MR (MR ectodomain) on formation of
new blood vessels in the sponge angiogenesis assay.
[0260] FIG. 9A is a graph and a table showing that the vascular
endothelial growth factor (VEGF) induced migration of primary human
endothelial cells is inhibited by the MR extracellular domain.
[0261] FIG. 9B is a graph and a table showing that the basic
fibroblast growth factor (bFGF) induced migration of primary human
endothelial cells is inhibited by the MR extracellular domain.
[0262] FIG. 10 is a graph and a table showing that the MR
ectodomain (Robo4-Fc) inhibits proliferation of primary human
endothelial cells.
EXAMPLE 1
Preparation of Antibodies
[0263] The cDNA constructs that were used were as described in
Table 2: TABLE-US-00022 TABLE 2 Full length MR cDNA MR
ectodomain-Fc MR IgA + B-Fc MR IgA-Fc
[0264] "Fc" refers to the Fc region of the pIG vector. It is human
IgG constant domains hinge, CH1, CH2, within the ends of the vector
(multiple cloning site and splice acceptor region included). The
nucleotide sequence of the vector is: TABLE-US-00023 (SEQ ID NO:
31) AAGCTTGATATCGAATTCTGCAGCCCGGGGGATCCGGAGGGAGGG
TGTCTGCTGGAAGCAGGCTCAGCGCTCCTGCCTGGACGCATGCCGG
CTATGCAGCCCCAGTCCAGGGCAGCAAGGCAGGCCCCGTCTGCCTC
TTCACCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGAGAGGG
TCTTCTGGCTTTTTCCCCAGGCTCTGGGCAGGCACAGGCTAGGTGC
CCCTAACCCAGGCCCTGCACACAAAGGGGCAGGTGCTGGGCTCAG
ACCTGCCAAGAGCCATATCCGGGAGGACCCTGGCCCTGACGTAAGC
CCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCGGACACCTTC
TCTCCTCCCAGATTCCAGTAACTCCCAATCTTCTCTCTGCAGAGCCC
AAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGGTAAGC
CAGCCCAGGCCTCGCCCTCCAGCTCAAGGCGGGACAGGTGCCCTA
GAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGTGCTGACACG
TCCACCTCCATCTCTTCCTCAGCACCTCAACTCCTGGGGGGACCGTC
AGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC
CGGACCCCTGAGGTCACATGGGTGGTGGTGGACGTGAGCCACGAA
GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG
CATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG
TACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA
ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAG
CCCCCATCGAGAAAACCATGTCCAAAGCCAAAGGTGGGACCCGTG
GGGTGCGAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCT
GCCCTGAGAGTGACCGCTGTACCAACCTCTGTCCTACAGGGCAGCC
CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGCCATGACCTG
ACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATC
CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA
ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTT
CTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA
GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC
CACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGC
GACGGCCGGCAAGCCCCGCTCCCCGGGCTCTCGCGGTCGCACGACC
ATGCTTGGCACGTACCCCCTGTACATACTTCCCGGGCGCCCAGCAT
GGAAATAAAGCACCCAGCGCTGCCCTGGGCCCCTGCGAGACTGTG
ATGGTTCTTTCCACGGGTCACCCCGAGTCTGAGGCCTGAGTGGCAT
GAGGGAGGCAGCGGCCGCGACTCTAG.
[0265] Generation of plasmid vectors N1 and NH10 for generation of
anti-MR antibodies using genetic immunisation was carried out as
follows. The plasmid vectors N1 and NHI0, encoding N1 (membrane
bound) and NH10 (soluble), were generated as follows:
[0266] N1 was generated by removal of the full length MR
pBluescript KS+ by a NotI digest. The product was cleaned from a
gel and ligated into pcDNA3 (which had been digested with
Not1).
[0267] NH10 was generated by amplifying the extracellular domain of
MR using primers which incorporated a 5' HinDIII site and a 3' NotI
site. This was ligated into a pcDNA3 vector which had been digested
with the same enzymes.
[0268] Genetic immunisation of mice to generate anti-MR antibodies
was carried out as follows:
[0269] The constructs N1 and NH10 were used to immunise mice from
three different genetic background according to the method reported
in (Boyle, J. S., A. Silva, et al (1997) "DNA immunization:
induction of higher avidity antibody and effect of route on T cell
cytotoxicity." Proc Natl Acad Sci USA 94(26): 14626-31). The mice
were immunised with an intramuscular injection of 100 .mu.g of
endotoxin free plasmid once every two weeks. Following the
immunisation with the DNA constructs, the mice were each injected
with 200 .mu.l of purified MR ectodomain intravenously as a final
boost before harvesting the spleen for generating the hybridomas.
Three different genetic strains of mice were tested for their
ability to generate suitable immune response to genetic
immunisation, as shown in Table 3. TABLE-US-00024 TABLE 3
Immunisation Immunisation Group with N1 with NH10 Group B - Balb/c
mice B1-B5 B6-B10 Group C - C57B1 mice C1-C5 C6-C10 Group M - MFI
outbred mice M1-M5 M6-M10
[0270] The schedule for immunisation is shown in Table 4.
TABLE-US-00025 TABLE 4 Day Pre Bleed -2 1.sup.st Immunisation I/M 0
2.sup.nd Immunisation I/M 14 3.sup.rd Immunisation I/M 28 Test
Bleed 35 I/V boost with cells or protein 200 .mu.l of 129 .mu.g/ml
59 purified ECSM4 protein I/V in PBSa Sacrifice the mice and remove
spleens for fusion. 63
[0271] During the course of immunisation, the test bleed were
assayed for anti-MR antibodies using the following capture ELISA.
This ELISA is a robust and flexible assay which can be used to
measure level of fusion protein in supernatant or presence/level of
antibody to the fusion protein in hybridoma supernatant. It is very
sensitive, detecting human IgG in the range 0.001 to 0.5 .mu.g/ml.
It has a low background, typically in the region of OD405=0.07. In
comparison, neat hybridoma supernatants in this system (MR-pIG)
give positive results of OD405>1.0 (and in some cases
>2.0).
Summary of ELISA Method
[0272] Coat plate with 5 .mu.g/ml goat anti-human IgG Fc-specific;
block with 1% BSA in PBS; add fusion protein supernatant or human
IgG control; add detection antibody or goat anti human alkaline
phosphatase conjugate; add secondary conjugate if unconjugated
detection antibody used; add pNPP substrate; stop with 3M NaOH
after 20-30 min.
Detailed Protocol for Capture ELISA for pIG Fusion Proteins
(1) Coat plate with 2 to 5 .mu.g/ml goat-anti-human IgG
(Fc-specific) purified unconjugated antibody diluted in PBS, e.g.
Sigma I-2136. 50 .mu.l/well, tap plate gently to ensure even
coverage of base of wells.
(2) Incubate overnight at +4.degree. C. Plates can be stored like
this for at least a week, as long as they are kept in a humidified
container to prevent drying out.
(3) Wash 3.times. with PBS-Tween 20 (0.04% Tween 20) by flooding
plate and tapping dry on tissue paper each time.
(4) Block with 1% BSA in PBS, 200 .mu.l/well. Incubate at room
temperature for 1-2 hours, or overnight at +4.degree. C. Plates can
be stored blocked at +4.degree. C., as for point (2) above.
(5) Repeat wash step (step 3).
[0273] (6) Coat plate with the fusion protein supernatant e.g.
MR-ecto-pIG. Supernatant containing 0.5 to 1.0 .mu.g/ml fusion
protein gives a very strong result, so there is no need to purify
or concentrate. Incubate 1 hour at room temperature. Human IgG can
be used instead of supernatant as a positive control for detecting
the Fc domain and titrated for quantifying the amount of NABA-pIG
fusion protein present in the supernatant.
(7) Repeat wash step (step 3)
[0274] (8) Detect the pIG domain using goat anti-human IgG-alkaline
phosphatase conjugate, 1/5000 dilution in PBS (positive control),
or mouse serum from test bleed, various dilutions in PBS (1/10 to
1/1000). Incubate 1 hour at room temperature. This is followed by
an additional step of a secondary conjugate (anti-mouse-alkaline
phosphatase). Incubate for a further 1 hour at room
temperature.
(9) Repeat wash step (step 3). (Also between use of detection
antibody and secondary conjugate, if alternative method used)
(10) Measure colour change using Sigma pNPP substrate made up from
tablets, 50 .mu.l/well. Incubate for 20-30 min. in the dark then
stop the reaction by the addition of 50 .mu.l/well 3M NaOH. Read
the colour change at 405 nm.
[0275] Anti-MR antibodies were generated as follows: The spleens
harvested from the above mentioned immunised mice were fused to NSO
cells. The resulting hybridomas were tested for their ability
generate antibodies that recognise MR using ELISA. Of the
antibodies identified, one was chosen for further studies -MR7.
The MR7 Antibody was Characterised as Follows:
[0276] MR7 was tested for its ability to recognise various domains
of MR using ELISA. It was found that MR7 recognises the MR IgA
domain. The DNA sequence encoding the Complementary Determining
Regions (CDRs) of MR7 was determined by PCR amplification and
standard sequencing techniques using the primers shown below.
Primers
[0277] A mixture of eleven 5' primers (listed in Table 5) was used
to amplify the kappa chain CDRs. TABLE-US-00026 TABLE 5 Primer
Sequence MKV1 ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCTG (SEQ ID NO:
32) MKV2 ACTAGTCGACATGGAGWCAGACACACTCCTGYTATGGGT (SEQ ID NO: 33)
MKV3 ACTAGTCGACATGAGTGTGGCTCACTCAGGTCCTGGSGTTG (SEQ ID NO: 34) MKV4
ACTAGTCGACATGAGGRCCCCTGCTCAGWTTYTTGGMWTCT TG (SEQ ID NO: 35) MKV5
ACTAGTCGACATGGATTTWCAGGTGCAGATTWTCAGCTTC (SEQ ID NO: 36) MKV6
ACTAGTCGACATGAGGTKCCYTGYTCAGYTYCTGRGG (SEQ ID NO: 37) MKV7
ACTAGTCGACATGGGCWTCAAGATGGAGTCACAKWYYCWG G (SEQ ID NO: 38) MKV8
ACTAGTCGACATGTGGGGAYCTTKTTYAMMTTTTTCAATTG (SEQ ID NO: 39) MKV9
ACTAGTCGACATGGTRTCCWCASCTCAGTTCCTTG (SEQ ID NO: 40) MKV10
ACTAGTCGACATGTATATATGTTTGTTGTCTATTTCT (SEQ ID NO: 41) MKV11
ACTAGTCGACATGGAAGCCCCATGCTCAGCTTCTCTTCC (SEQ ID NO: 42)
[0278] The primer sequence which amplified the MR7 kappa chain 5'
end was MK5
[0279] The sequence of the 3' primer was GTTTGATCTAGAGCTTGGTCCC
(SEQ ID NO: 43) which amplifies from after CDR3 and adds an XbaI
restriction site on the end of the product if you need to clone the
product. The same product was also produced when the 5' mix was
used with the 3' constant region TABLE-US-00027 primer
TTGGAGGGCGTTATCCACCT. (SEQ ID NO: 44)
Heavy Chain Primers
[0280] The 5' primer was: TABLE-US-00028
ATCGGATCCAGGTSMARCTGCAGSAGTCWGG, (SEQ ID NO: 45)
[0281] and the 3' primer was: TABLE-US-00029 (SEQ ID NO: 46)
CTCGAATTCTGAGGAGACGGTGACCGTGGTCCCTTGGCCCC.
[0282] The redundancy code for these primers is shown in Table 6.
TABLE-US-00030 TABLE 6 IUB ambiguity code Nucleotides Code A + C M
aMino A + G R puRine A + T W Weak C + G S Strong C + T Y pYrimidine
G + T K Keto A + G + C V not T A + C + T H not G A + G + T D not C
C + G + T B not A A + G + C + T N aNy
MR7 Sequence
[0283] The amino acid sequence of the light and heavy V regions of
MR7 antibody is given below. The CDRs are underlined.
[0284] MR7 Kappa V Region: TABLE-US-00031 (SEQ ID NO: 12) Q I V L T
Q S P A L M S A S P G E K V T M T C S A S S S V S Y M Y W Y Q Q K P
R S S P K P W I Y L T S N L A S G V P A R F S G S G S G T S Y S L T
I S S M E A E D A A T Y Y C Q Q W S S N P L T F G A G T K L E L
K.
[0285] MR7 Heavy V Region: TABLE-US-00032 (SEQ ID NOs: 16-17) Q V
K/Q L Q E S G P E L V K P G A S V K I S C K A S G Y S L T D Y N L N
W V K Q N K G K S L E W I G V I N P N Y G T T S Y N Q K F K G K A T
L T V D Q S S S T T Y M Q L N S L T S E D S A V Y Y C A R G R D Y F
G Y W G Q G T T V T V S S,
[0286] The nucleotide sequence encoding the light and heavy V
regions of MR7 antibody. The CDRs are underlined.
[0287] MR7 Kappa V Region: TABLE-US-00033 (SEQ ID NO: 21)
CAA,ATT,GTT,CTC,ACC,CAG,TCT,CCA,GCA,CTC,ATG,TCT,
GCA,TCT,CCA,GGG,GAG,AAG,GTC,ACC,ATG,ACC,TGC,
AGT,GCC,AGC,TCA,AGT,GTA,AGT,TAC,ATG,TAC,TGG,TAC,
CAG,CAG,AAG,CCA,AGA,TCC,TCC,CCC,AAA,CCC,TGG,ATT,
TAT,CTC,ACA,TCC,AAC,CTG,GCT,TCT,GGA,GTC,CCT,GCT,
CGC,TTC,AGT,GGC,AGT,GGG,TCT,GGG,ACC,TCT,TAC,TCT,
CTC,ACA,ATC,AGC,AGC,ATG,GAG,GCT,GAA,GAT,GCT,GCC, ACT,TAT,TAC,TGC,
CAG,CAG,TGG,AGT,AGT,AAC,CCA,CTC,ACG,TTC,GGT,GCT,
GGG,ACC,AAG,CTG,GAG,CTG,AAA.
[0288] MR7 Heavy V Region: TABLE-US-00034 (SEQ ID NOs: 25-27)
CAG,GTC,AAG(orA/CAA),CTG,CAG,GAG,TCA,GGA,CCT,GAG,
CTG,GTG,AAG,CCT,GGC,GCT,TCA,GTG,AAG,ATA,TCC,TGC,
AAG,GCT,TCT,GGT,TAC,TCA,CTC,ACT,
GAC,TAC,AAC,CTG,AAC,TGG,GTG,AAG,CAG,AAC,AAA,GGA,
AAG,AGC,CTT,GAG,TGG,ATT,GGA,GTA,ATT,AAT,CCA,
AAC,TAT,GGT,ACT,AGT,TAC,AAT,CAG,AAG,TTC,AAG,GGC,
AAG,GCC,ACA,TTG,ACT,GTA,GAC,CAA,TCT,TCC,AGC,ACA,
ACC,TAC,ATG,CAG,CTC,AAC,AGC,CTG,ACA,TCT,GAG,GAC,
TCT,GCA,GTC,TAT,TAC,TGT,GCA,AGA,
GGG,AGG,GAT,TAC,TTC,GGC,TAC,TGG,GGC,CAA,GGG,ACC,
ACG,GTC,ACC,GTC,TCC,TCA.
EXAMPLE 2
The Antibody MR7 and the MR Ectodomain (Extracellular Fragment of
MR Residues 1-467) Inhibit Formation of Vessel Sprouts in the
Aortic Ring Assay
Summary
[0289] The role of MR in angiogenesis was investigated using the
rat aortic ring assay. Segments of rat aorta were embedded in
Matrigel and treated with either antibody MR7 or purified MR
ectodomain protein. The sprouting vessels were allowed to develop
over five day before scoring by three independent observers. The
averaged scores over 20-25 separate experiments are shown in FIGS.
6A and 6B. Inter-scorer reliability was assessed using the method
of Landis and Koch. The weighted kappa values calculated were 0.96
for MR7 and 0.93 for MR ectodomain. These kappa values show that
there was a high degree of consistency between independent
scorers.
Methods
[0290] Aortas were harvested from 200 g-300 g rats (6-8 weeks old)
and immediately placed in MCDB 131 media. Connective tissue was
removed and aortas cut into 1 mm-1.5 mm rings. 48-well plates were
coated with 110 .mu.l of Matrigel (BD Biosciences) diluted 1:1 with
PBS and allowed to gel at 37.degree. C. for 30 min. The rings were
placed in the wells and sealed in place with an overlay of 40 .mu.l
of Matrigel. Antibodies (100 .mu.g/ml) or MR ectodomain (soluble
robo4 extracellular domain) (15 .mu.g/ml) were added to wells in a
final volume of 250 .mu.l of MCDB 131 media containing 20% foetal
bovine serum and 50 .mu.g/ml endothelial cell growth supplement.
Media was changed after two days and aortas analysed and
photographed after five days.
Results
[0291] Representative photomicrographs of segments of the aortic
rings are shown in FIG. 7 A-C. As the figure shows, treatment of
the aortic rings with either MR7 or MR ectodomain resulted in
significant decrease in sprouting of vessels from the aortic
segment.
Statistical Analysis of Aortic Ring Assay
[0292] Aortic rings were scored according to the vessel growth on a
scale of 0 (low) to 4 (high) as follows: 0=no growth, 1=few
vessels, 2=intermediate vessels, 3=many vessels but sporadic
sprouting centres around the ring and 4=many vessels sprouting from
all regions of the ring.
[0293] All experiments were scored blind by three independent
researchers. Inter-scorer reliability was assessed using the method
of Landis and Koch (Biometrics, 1977, 33(1), 159-174). Weighted
kappa was calculated in order to establish the inter-rater
reliability of the examiners. Weighted kappa is given by: k w = p o
.function. ( w ) - p e .function. ( w ) 1 - p e .function. ( w )
##EQU1## where Po(w) and Pe(w) are the weighted observed and
expected agreement calculated with the formulas: p o .function. ( w
) = 1 n .times. i = 1 g .times. .times. j = 1 g .times. .times. w
ij .times. f if p e .function. ( w ) = 1 n 2 .times. i = 1 g
.times. .times. j = 1 g .times. .times. w ij .times. r i .times. c
j ##EQU2##
[0294] The i represents the category for one examiner and j
represent the category for the second examiner. The r.sub.i
represents the grand total of cases in category i for one examiner,
and c.sub.j represents the grand total of cases in category j for
the other examiner.
[0295] The difference between the categories considered to be 1,
therefore with the introduction of the new categories for the
description of the additional transition categories the difference
to one category to another was considered to be 0.5 points. The
total number of categories therefore g=9. The number of cases is
n=80.
[0296] The weight wij for the observed frequency fij of cases which
were in category i by one examiner and in category j for the second
examiner is calculated as: w ij = 1 - i - j g - 1 ##EQU3##
[0297] The strength of the agreement was regarded as poor if the
Kappa statistics was <0.00, slight for values 0.00-0.20, fair
for 0.21-0.40, moderate for 0.41-0.60, substantial for 0.61-0.80
and high for 0.81-1.00.
[0298] The data was then collated together and analysed by ANOVA
and the Kruskall Wallis method. The resulting p values show a
highly significant difference between the control group and those
treated with either MR7 or the ectodomain.
EXAMPLE 3
The MR Ectodomain (Extracellular Fragment of MR Residues 1-467)
Inhibits Formation of Vessel Sprouts In Vivo
[0299] The ability of the MR ectodomain to inhibit angiogenesis in
vivo was tested using a sponge angiogenesis assay (Hori Y. et al
(1996) "Differential effects of angiostatic steroids and
dexamethasone on angiogenesis and cytokine levels in rat sponge
implants" Br. J. Pharmacol. 118(7): 1584-1591) performed on female
C57 black mice. All mice received a subcutaneous sterile polyether
sponge (type 611-9) disc (15.times.5.times.5 mm) under the dorsal
skin at day 0. Test reagents were injected through the skin
directly into the sponges every second day for 21 days (100 .mu.l
injection volume). Groups of 2 mice received either PBS control; 10
ng/ml basic fibroblast growth factor (bFGF); or 10 ng/ml bFGF+100
.mu.g/ml MR ectodomain. Animals were scarified on day 21 and
sponges were removed, fixed in 3.7% paraformaldeyde and paraffin
embedded. 5 micron sections were stained with haematoxylin and
eosin and digital photos taken using a Zeiss Axioskop 2 plus
microscope with an Axiocam digital camera at 20.times.
magnification. The number of vessels invading the sponges were
counted as a measure of angiogenesis.
[0300] There were clear differences between the sponges from the
mice who were injected with bFGF alone (controls) and those who
were injected with both bFGF and the MR ectodomain. The differences
were:
[0301] a) significantly fewer vessel numbers in the MR ectodomain
treated sponges compared to control (FIG. 8; p=0.0014 using the
Student t-test);
[0302] b) an absence of very large vessels from the MR ectodomain
treated sponges; and
[0303] c) much lower fibroblast cell density in the MR ectodomain
treated sponges.
EXAMPLE 4
The MR Ectodomain (Extracellular Fragment of MR Residues 1-467)
Inhibits Migration of Primary Human Vascular Endothelial Cells
[0304] A primary human vascular endothelial cell (HUVEC) migration
assay was performed using the BD BioCoat.TM. Angiogenesis System
for Endothelial Cell Migration which is available as Catalog No.
354143 from BD Biosciences, Bedford, Mass., USA. Instructions for
using this kit can be found at
http://www.bdbiosciences.com/discovery_labware/Products/drug_discovery/in-
sert_systems/angiogenesis_system/pdf/Endothelial_Cell_Migration_Instruct
.pdf. This system uses a 24-multiwell insert system and consists of
a BD Falcon FluoroBlok PET membrane with 3 micron pore size coated
uniformly on the top side with fibronectin. Quantitation of cell
migration is achieved by post-labelling of cells with the
fluorescent dye Calcein AM and measuring the fluorescence of
migrating cells in a fluorescence plate reader. The FluoroBlok
membrane effectively blocks the passage of light from 490-700 nm at
>99% efficiency meaning labelled cells that have not migrated
are blocked from detection.
[0305] The upper chamber was seeded with 50,000 HUVEC/well in MCDB
131 medium supplemented with 1% heat-inactivated foetal calf serum
(FCS). The bottom chambers were loaded with or without bFGF (5
ng/ml), VEGF (10 ng/ml) and MR ectodomain (100 .mu.g/ml) in 750
.mu.l of MCDB 131+1% FCS. After 22 hr incubation at 37.degree. C.
the insert membranes were stained with 4 .mu.g/ml Calcein AM
(Molecular Probes) in Hanks Balanced Salt Solution (HBSS) for 90
min. Fluorescence on the bottom side of the membrane was measured
at excitation/emission wavelengths of 485/530 nm. Images were taken
using a Zeiss Axiovert 135 microscope with an Axiocam digital
camera at 10.times. magnification.
[0306] Both bFGF and VEGF are known to stimulate migration of
endothelial cells (Cross & Claesson-Welsh, 2001 Trends
Pharmacol Sci. 22(4): 201-207). As shown in FIGS. 9A and 9B, the MR
ectodomain was shown to significantly inhibit migration of HUVEC
cells induced by either bFGF or VEGF.
EXAMPLE 5
The MR Ectodomain (Extracellular Fragment of MR Residues 1-467)
Inhibits Endothelial Cell Proliferation
[0307] 5.times.10.sup.4 primary human vascular endothelial cells
(HUVEC) were seeded per well of a 6-well plate in 1.5 ml full
growth media containing treatment (6.25, 12.5, 25, 50 or 100
.mu.g/ml of the MR ectodomain (Robo4-Fc) or 100 .mu.g/ml human
IgG), or no treatment as a control. After four days incubation at
37.degree. C., the cells were washed in PBS and detached from the
wells by addition of 1 ml trypsin solution. After all the cells had
detached, 400 .mu.l of the cell suspension was transferred to 19.6
ml Isoton buffer (Beckman Coulter), and the number of cells in each
sample was determined in a Coulter Particle Count and Size Analyser
(Beckman Coulter). The experiment was carried out in triplicate,
and replicated three times.
[0308] As shown in FIG. 10, incubation in the presence of 12.5
.mu.g/ml MR ectodomain decreased proliferation of the HUVEC cells
to about 75% of the control levels, and higher concentrations of MR
ectodomain had an increasingly strong anti-proliferative
effect.
EXAMPLE 6
Treatment of a Patient Exhibiting Undesirable Angiogenesis by
Administering an Antibody that Specifically Binds to the
Extracellular Region of MR
[0309] A patient exhibiting undesirable angiogenesis is treated
with intravenous infusions of saline solutions of a pharmaceutical
composition comprising an antibody that specifically binds to the
extracellular region of MR. The infusions are administered weekly
for a time of 3 to 6 months.
EXAMPLE 7
Treatment of a Patient Exhibiting Undesirable Angiogenesis by
Administering the Extracellular Region of MR
[0310] A patient exhibiting undesirable angiogenesis is treated
with intravenous infusions of saline solutions of a pharmaceutical
composition comprising the MR ectodomain. The infusions are
administered weekly, typically for 3 to 6 months.
* * * * *
References