U.S. patent application number 09/990586 was filed with the patent office on 2003-06-12 for antibodies for inhibiting blood coagulation and methods of use thereof.
This patent application is currently assigned to Sunol Molecular Corporation. Invention is credited to Jiao, Jin-An, Luepschen, Lawrence, Nieves, Esperanza Liliana, Wong, Hing C..
Application Number | 20030109680 09/990586 |
Document ID | / |
Family ID | 25536300 |
Filed Date | 2003-06-12 |
United States Patent
Application |
20030109680 |
Kind Code |
A1 |
Wong, Hing C. ; et
al. |
June 12, 2003 |
Antibodies for inhibiting blood coagulation and methods of use
thereof
Abstract
The invention includes antibodies that provide superior
anti-coagulant activity by binding native human TF with high
affinity and specificity. Antibodies of the invention can
effectively inhibit blood coagulation in vivo. Antibodies of the
invention can bind native human TF, either alone or present in a
TF:FVIIa complex, effectively preventing factor X or FIX binding to
TF or that complex, and thereby reducing blood coagulation.
Preferred antibodies of the invention specifically bind a
conformational epitope predominant to native human TF, which
epitope provides an unexpectedly strong antibody binding site. Also
provided are humanized antibodies and fragments thereof that bind
to the TF.
Inventors: |
Wong, Hing C.; (Fort
Lauderdale, FL) ; Jiao, Jin-An; (Fort Lauderdale,
FL) ; Nieves, Esperanza Liliana; (Plantation, FL)
; Luepschen, Lawrence; (Miami, FL) |
Correspondence
Address: |
EDWARDS & ANGELL, LLP
P.O. BOX 9169
BOSTON
MA
02209
US
|
Assignee: |
Sunol Molecular Corporation
|
Family ID: |
25536300 |
Appl. No.: |
09/990586 |
Filed: |
November 21, 2001 |
Current U.S.
Class: |
530/388.15 ;
530/388.25 |
Current CPC
Class: |
C07K 16/467 20130101;
A61K 2039/505 20130101; A61P 7/02 20180101; C07K 16/36 20130101;
C07K 2317/24 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
530/388.15 ;
530/388.25 |
International
Class: |
C07K 016/44 |
Claims
What is claimed is:
1. A humanized antibody that binds specifically to human tissue
factor (TF) to form a complex, wherein factor X or factor IX
binding to the complex and the FX or FIX activation by TF:VIIa are
inhibited.
2. The humanized antibody of claim 1, wherein the antibody has a
dissociation constant (K.sub.d) for the TF of less than about 0.5
nM.
3. The humanized antibody of claim 1 or 2, wherein the antibody is
further characterized by increasing blood clotting time by at least
about 5 seconds as determined by a standard prothrombin (PT)
clotting assay at an antibody concentration of <15 nM.
4. The humanized antibody of claim 1 or 2, wherein the antibody has
a binding specificity for the TF about equal or greater than the
antibody obtained from cell line H36.D2.B7 deposited under ATCC
Accession No. HB-12255.
5. The humanized antibody of claim 1 or 2, wherein the antibody has
a binding affinity for the TF about equal to or greater than the
antibody obtained from cell line H36.D2.B7 deposited under ATCC
Accession No. HB-12255.
6. The humanized antibody of claim 1 or 2, wherein the antibody
comprises at least one fully murine complimentarily determining
region (CDR).
7. The humanized antibody of claim 1 or 6, wherein the antibody
comprises at least one fully human framework (FR) region.
8. The humanized antibody of claim 1, wherein the antibody has at
least about 90% amino acid sequence identity to a human
antibody.
9. The humanized antibody of claim 1, wherein the variable region
of the humanized antibody has at least about 70% amino acid
sequence identity to a human antibody variable region.
10. The humanized antibody of claim 1, wherein each of frameworks
(FRs) 1, 2, 3 and 4 has at least about 95% amino acid sequence
identity to the light chain FR sequences shown in FIG. 12A (SEQ ID
NO. ______).
11. The humanized antibody of claim 1, wherein the antibody
comprises a light chain constant region having at least about 95%
amino acid sequence identity to the sequence shown in FIG. 14A or
15A (SEQ ID NO. ______).
12. The humanized antibody of claim 1, wherein each of frameworks
(FRs) 1, 2, 3 and 4 has at least about 95% amino acid sequence
identity to the heavy chain sequences shown in FIG. 13A (SEQ ID NO.
______).
13. The humanized antibody of claim 12, wherein the antibody
further comprises a heavy chain constant region having at least
about 95% amino acid sequence identity to sequence shown in FIG.
14B or 15B (SEQ ID NO. ______).
14. The humanized antibody of claim 1, wherein the antibody has an
IgG1 (hOAT) or IgG4 (hFAT) isotype.
15. A human TF binding fragment of the humanized antibody of claim
1.
16. The human TF binding fragment of claim 15, wherein the fragment
is Fab, Fab', or F(ab).sub.2.
17. A humanized antibody comprising at least one murine
complementarity determining region (CDR), wherein the antibody
binds specifically to human tissue factor (TF) to form a complex,
and further wherein factor X or factor IX binding to TF or TF:FVIIa
and activation by TF:FVIIa thereto is inhibited.
18. The humanized antibody of claim 17, wherein all the CDR (light
and heavy chain) are murine.
19. The humanized antibody of claim 17, wherein the antibody
further comprises as least one human framework (FR) region.
20. The humanized antibody of claim 19, wherein the amino acid
sequences of all the FR (light and heavy chain) are human or within
2 amino acid substitutions of being human.
21. The humanized antibody of claim 17, wherein the first CDR
(CDR1) of the heavy chain hypervariable region is at least 95%
identical to the CDR1 amino acid sequence shown in FIG. 13B (SEQ ID
NO. ______).
22. The humanized antibody of claim 17, wherein the second CDR
(CDR2) of the heavy chain hypervariable region is at least 95%
identical to the CDR2 amino acid sequence shown in FIG. 13C (SEQ ID
NO. ______).
23. The humanized antibody of claim 17, wherein the third CDR
(CDR3) of the heavy chain hypervariable region is at least 95%
identical to the CDR3 amino acid sequence shown in FIG. 13D (SEQ ID
NO. ______).
24. The humanized antibody of claim 17, wherein the first CDR
(CDR1) of the light chain hypervariable region is at least 95%
identical to the CDR1 amino acid sequence shown in FIG. 12B (SEQ ID
NO. ______).
25. The humanized antibody of claim 17, wherein the second CDR
(CDR2) of the light chain hypervariable region is at least 95%
identical to the CDR2 amino acid sequence shown in FIG. 12C (SEQ ID
NO. ______).
26. The humanized antibody of claim 17, wherein the third CDR
(CDR3) of the light chain hypervariable region is at least 95%
identical to the CDR3 amino acid sequence shown in FIG. 12D (SEQ ID
NO. ______).
27. The humanized antibody of claim 19, wherein the first framework
(FR1) of the heavy chain hypervariable region is at least 95%
identical to the amino acid sequence shown in FIG. 13A (SEQ ID NO.
______).
28. The humanized antibody of claim 27, wherein the FR1 comprises
at least one of the following amino acid changes: E1 to Q; Q5 to V;
P9 to G; L11 to V; V12 to K; Q19 to R; and T24 to A.
29. The humanized antibody of claim 19, wherein the second
framework (FR2) of the heavy chain hypervariable region is at least
95% identical to the amino acid sequence shown in FIG. 13A (SEQ ID
NO. ______).
30. The humanized antibody of claim 29, wherein the FR2 comprises
at least one of the following amino acid changes: 41H to P; and 44S
to G.
31. The humanized antibody of claim 19, wherein the third framework
(FR3) of the heavy chain hypervariable region is at least 95%
identical to the amino acid sequence shown in FIG. 13A (SEQ ID NO.
______).
32. The humanized antibody of claim 31, wherein the FR3 comprises
at least one of the following amino acid changes: 76S to T; 77T to
S; 80F to Y; 82H to E; 84N to S; 87T to R; 89D to E; and 91S to
T.
33. The humanized antibody of claim 19, wherein the fourth
framework (FR4) of the heavy chain hypervariable region is at least
95% identical to the amino acid sequence shown in FIG. 13A (SEQ ID
No. ______).
34. The humanized antibody of claim 33, wherein the FR4 comprises
the following amino acid change: 113L to V.
35. The humanized antibody of claim 19, wherein the first framework
(FR1) of the light chain hypervariable region is at least about 95%
identical to the amino acid sequence shown in FIG. 12A (SEQ ID NO.
______).
36. The humanized antibody of claim 35, wherein the FR1 comprises
at least one of the following amino acid changes: 11QL to L; 15L to
V; 17E to D; and 18 to R.
37. The humanized antibody of claim 19, wherein the second
framework (FR2) of the light chain hypervariable region is at least
about 95% identical to the amino acid sequence shown in FIG. 12A
(SEQ ID NO. ______).
38. The humanized antibody of claim 37, wherein the FR2 has the
following amino acid changes: 37Q to L.
39. The humanized antibody of claim 19, wherein the third framework
(FR3) of the light chain hypervariable region is at least about 95%
identical to the amino acid sequence shown in FIG. 12A (SEQ ID NO.
______).
40. The humanized antibody of claim 39, wherein the FR3 has the
following amino acid changes: 70K to D, 74K to T, 80A to P, 84A to
V, and 85N to T.
41. The humanized antibody of claim 40, wherein the fourth
framework (FR4) of the light chain hypervariable region is at least
about 95% identical to the amino acid sequence shown in FIG. 12A
(SEQ ID NO. ______).
42. The humanized antibody of claim 41, wherein the FR4 comprises
the following amino acid changes: 100A to Q; and 106L to I.
43. A human TF binding fragment of the humanized antibody of claim
17.
44. The human TF binding fragment of claim 43, wherein the fragment
is Fab, Fab', or F(ab).sub.2.
45. A humanized antibody comprising at least one murine
complementarity determining region (CDR), wherein the antibody
binds specifically to human tissue factor (TF) to form a complex,
and further wherein factor X or factor IX binding to TF or TF:FVIIa
and activation by TF:FVIIa thereto is inhibited, the antibody
comprising on the heavy chain: a) a first CDR (CDR1) which is at
least 95% identical to CDR1 amino acid sequence shown in FIG. 13B
(SEQ ID NO. ______), b) a second CDR (CDR2) which is at least 95%
identical to the CDR2 amino acid sequence shown in FIG. 13C (SEQ ID
NO. ______), c) a third CDR (CDR3) which is at least 95% identical
to the CDR3 amino acid sequence shown in FIG. 13D (SEQ ID NO.
______), d) a first framework (FR1) which is at least 95% identical
to the amino acid sequence shown in FIG. 12A (SEQ ID NO. ______),
e) a second framework (FR2) which is at least 95% identical to the
amino acid sequence shown in FIG. 12A (SEQ ID NO. ______), f) a
third framework (FR3) which is at least 95% identical to the amino
acid sequence shown in FIG. 12A (SEQ ID NO. ______), and g) a
fourth framework (FR4) which is at least 95% identical to the amino
acid sequence shown in FIG. 12A (SEQ ID No. ______);
46. The antibody of claim 45 further comprising on the light chain,
h) a first CDR (CDR1) which is at least 95% identical to CDR1 amino
acid sequence shown in FIG. 12B (SEQ ID NO. ______), i) a second
CDR (CDR2) which is at least 95% identical to the CDR2 amino acid
sequence shown in FIG. 12C (SEQ ID NO. ______), j) a third CDR
(CDR3) which is at least 95% identical to the CDR3 amino acid
sequence shown in FIG. 12C (SEQ ID NO. ______), k) a first
framework (FR1) which is at least 95% identical to the amino acid
sequence shown in FIG. 12A (SEQ ID NO. ______), l) a second
framework (FR2) which is at least 95% identical to the amino acid
sequence shown in FIG. 12A (SEQ ID NO. ______), m) a third
framework (FR3) which is at least 95% identical to the amino acid
sequence shown in FIG. 12A (SEQ ID NO. ______), and n) a fourth
framework (FR4) which is at least 95% identical to the amino acid
sequence shown in FIG. 12A (SEQ ID NO. ______).
47. The antibody of claim 45 further comprising the light chain
constant sequence of FIG. 14A (SEQ ID No. ______) or FIG. 15A (SEQ
ID No. ______).sub.--
48. The antibody of claim 45 further comprising the heavy chain
constant region of FIG. 14B (SEQ ID No. ______) or FIG. 15B (SEQ ID
No. ______).
49. A human TF binding fragment of the humanized antibody of claim
45.
50. The human TF binding fragment of claim 45, wherein the fragment
is Fab, Fab', or F(ab).sub.2.
51. A humanized antibody comprising on the heavy chain: a) a first
CDR (CDR1) identical to the CDR1 amino acid sequence shown in FIG.
13B (SEQ ID NO. ______), b) a second CDR (CDR2) identical to the
CDR2 amino acid sequence shown in FIG. 13C (SEQ ID NO. ______), c)
a third CDR (CDR3) identical to the CDR3 amino acid sequence shown
in FIG. 13D (SEQ ID NO. ______), d) a first framework (FR1)
identical to the amino acid sequence shown in FIG. 13A (SEQ ID NO.
______), e) a second framework (FR2) identical to the amino acid
sequence shown in FIG. 13A (SEQ ID NO. ______), f) a third
framework (FR3) identical to the amino acid sequence shown in FIG.
13A (SEQ ID NO. ______); and g) a fourth framework (FR4) identical
to the amino acid sequence shown in FIG. 13A (SEQ ID No. ______);
and on the light chain: h) a first CDR (CDR1) identical to CDR1
amino acid sequence shown in FIG. 12B (SEQ ID NO. ______), i) a
second CDR (CDR2) identical to the CDR2 amino acid sequence shown
in FIG. 12C (SEQ ID NO. ______), j) a third CDR (CDR3) identical to
the CDR3 amino acid sequence shown in FIG. 12D (SEQ ID NO. ______),
k) a first framework (FR1) identical to the amino acid sequence
shown in FIG. 12A (SEQ ID NO. ______), l) a second framework (FR2)
identical to the amino acid sequence shown in FIG. 12A (SEQ ID NO.
______), m) a third framework (FR3) identical to the amino acid
sequence shown in FIG. 12A (SEQ ID NO. ______), and n) a fourth
framework (FR4) identical to the amino acid sequence shown in FIG.
12A (SEQ ID No. ______).
52. The antibody of claim 51 further comprising the light chain
constant sequence of FIG. 14A (SEQ ID No. ______) or FIG. 15A (SEQ
ID No. ______).
53. The antibody of claim 51 further comprising the heavy chain
constant sequence of FIGS. 14B (SEQ ID No. ______) or 15B (SEQ ID
No. ______).
54. The humanized antibody of claim 51, wherein the antibody has an
IgG1 or IgG4 isotype.
55. A human TF binding fragment of the humanized antibody of claim
4.
56. The human TF binding fragment of claim 55, wherein the fragment
is Fab, Fab', or F(ab).sub.2.
57. The humanized antibody of claim 1, wherein the antibody is a
monoclonal antibody.
58. A single-chain antibody comprising the hypervariable region of
the antibody of claim 1.
59. An isolated nucleic acid encoding at least one of the heavy or
light chain of the humanized antibody of claim 1.
60. A recombinant vector comprising the isolated nucleic acid of
claim 59.
61. A host cell comprising the recombinant vector of claim 60.
62. A composition comprising the humanized antibody of claim 1, and
at least one pharmaceutically acceptable carrier.
63. A method of inhibiting blood coagulation in a mammal, the
method comprising administering to the mammal an effective amount
of the humanized antibody of claim 1 or fragment thereof that binds
specifically to human tissue factor (TF) to form a complex, wherein
factor X or factor IX binding to TF or TF:FVIIa and activation by
TF:FVIIa thereto is inhibited, the method further comprising
forming a specific complex between the antibody and the TF to
inhibit the blood coagulation.
64. A method of inhibiting blood coagulation in a mammal, the
method comprising administering to the mammal, an effective amount
of the humanized antibody of claim 7 comprising at least or
fragment thereof, wherein the antibody or fragment binds
specifically to human tissue factor (TF) to form a complex, and
further wherein factor X or factor IX binding to TF or TF:FVIIa and
activation by TF:FVIIa thereto is inhibited, the method further
comprising forming a specific complex between the antibody and the
TF to inhibit the blood coagulation.
65. A method of inhibiting blood coagulation in a mammal, the
method comprising administering to the mammal, an effective amount
of a humanized antibody or fragment thereof wherein the antibody
binds specifically to human tissue factor (TF) to form a complex,
and further wherein factor X or factor IX binding to TF or TF:FVIIa
and activation by TF:FVIIa thereto is inhibited, the antibody or
fragment comprising on the heavy chain: a) a first CDR (CDR1) which
is at least 95% identical to CDR1 amino acid sequence shown in FIG.
13B (SEQ ID NO. ______), b) a second CDR (CDR2) which is at least
95% identical to the CDR2 amino acid sequence shown in FIG. 13C
(SEQ ID NO. ______), c) a third CDR (CDR3) which is at least 95%
identical to the CDR3 amino acid sequence shown in FIG. 13D (SEQ ID
NO. ______), d) a first framework (FR1) which is at least 95%
identical to the amino acid sequence shown in FIG. 13A (SEQ ID NO.
______), e) a second framework (FR2) which is at least 95%
identical to the amino acid sequence shown in FIG. 13A (SEQ ID NO.
______), f) a third framework (FR3) which is at least 95% identical
to the amino acid sequence shown in FIG. 13A (SEQ ID NO. ______),
g) a fourth framework (FR4) which is at least 95% identical to the
amino acid sequence shown in FIG. 13A (SEQ ID No. ______); and on
the light chain, h) a first CDR (CDR1) which is at least 95%
identical to CDR1 amino acid sequence shown in FIG. 12B (SEQ ID NO.
______), i) a second CDR (CDR2) which is at least 95% identical to
the CDR2 amino acid sequence shown in FIG. 12C (SEQ ID NO. ______),
j) a third CDR (CDR3) which is at least 95% identical to the CDR3
amino acid sequence shown in FIG. 12D (SEQ ID NO. ______), k) a
first framework (FR1) which is at least 95% identical to the amino
acid sequence shown in FIG. 12A (SEQ ID NO. ______), l) a second
framework (FR2) which is at least 95% identical to the amino acid
sequence shown in FIG. 12A (SEQ ID NO. ______), m) a third
framework (FR3) which is at least 95% identical to the amino acid
sequence shown in FIG. 12A (SEQ ID NO. ______), n) a fourth
framework (FR4) which is at least 95% identical to the amino acid
sequence shown in FIG. 12A (SEQ ID No. ______), o) a light chain
constant region which is at least 95% identical to the amino acid
sequence shown in FIG. 14A (SEQ ID No. ______) or FIG. 15A (SEQ ID
No. ______), and p) a heavy chain constant region which is at least
95% identical to the amino acid sequence shown in FIG. 14B (SEQ ID
No. ______) or FIG. 15B (SEQ ID No. ______).
66. A method of inhibiting blood coagulation in a mammal, the
method comprising administering to the mammal, an effective amount
of a humanized antibody or fragment thereof wherein the antibody
binds specifically to human tissue factor (TF) to form a complex,
and further wherein factor X or factor IX binding to TF or TF:FVIIa
and activation by TF:FVIIa thereto is inhibited, the antibody or
fragment comprising on the heavy chain: a) a first CDR (CDR1)
identical to CDR1 amino acid sequence shown in FIG. 13B (SEQ ID NO.
______), b) a second CDR (CDR2) identical to the CDR2 amino acid
sequence shown in FIG. 13C (SEQ ID NO. ______), c) a third CDR
(CDR3) identical to the CDR3 amino acid sequence shown in FIG. 13D
(SEQ ID NO. ______), d) a first framework (FR1) identical to the
amino acid sequence shown in FIG. 13A (SEQ ID NO. ______), e) a
second framework (FR2) identical to the amino acid sequence shown
in FIG. 13A (SEQ ID NO. ______), f) a third framework (FR3)
identical to the amino acid sequence shown in FIG. 13A (SEQ ID NO.
______), g) a fourth framework (FR4) identical to the amino acid
sequence shown in FIG. 13A (SEQ ID No. ______), and on the light
chain: h) a first CDR (CDR1) identical to CDR1 amino acid sequence
shown in FIG. 12B (SEQ ID NO. ______), i) a second CDR (CDR2)
identical to the CDR2 amino acid sequence shown in FIG. 12C (SEQ ID
NO. ______), j) a third CDR (CDR3) identical to the CDR3 amino acid
sequence shown in FIG. 12D (SEQ ID NO. ______), k) a first
framework (FR1) identical to the amino acid sequence shown in FIG.
12A (SEQ ID NO. ______), l) a second framework (FR2) identical to
the amino acid sequence shown in FIG. 12A (SEQ ID NO. ______), m) a
third framework (FR3) identical to the amino acid sequence shown in
FIG. 12A (SEQ ID NO. ______), n) a fourth framework (FR4) identical
to the amino acid sequence shown in FIG. 12A (SEQ ID NO. ______),
o) a light chain constant region which is identical to the amino
acid sequence shown in FIG. 14A (SEQ ID No. ______) or FIG. 15A
(SEQ ID No. ______), and p) a heavy chain constant region which is
identical to the amino acid sequence shown in FIG. 14B (SEQ ID No.
______) or FIG. 15B (SEQ ID No. ______).
67. A method of detecting tissue factor (TF) in a biological
sample, the method comprising contacting a biological sample with
the antibody of claim 1 under conditions conducive to forming a
complex and detecting the complex as being indicative of the TF in
the biological sample.
68. A method for producing the humanized antibody of claim 1,
wherein the method comprises providing a host cell transformed with
either 1) a first expression vector encoding the light chain of the
humanized antibody or fragment thereof and a second expression
vector encoding the heavy chain of the humanized antibody or
fragment thereof, or 2) a single expression vector encoding both
the light chain and the heavy chain of the humanized antibody or
fragment thereof, maintaining the host cell under growth conditions
in which each chain is expressed; and isolating the humanized
antibody or fragment thereof.
69. A method for producing a humanized antibody, wherein the method
comprises: a) comparing the amino acid sequence of a light chain
framework from a rodent antibody against a collection of
corresponding human antibody framework sequences, b) selecting a
human framework sequence from the collection having the greatest
amino acid sequence identity to the corresponding rodent light
chain framework, c) mutagenizing a DNA segment encoding the rodent
light chain framework to encode a humanized light chain framework
having an amino acid sequence that is substantially identical to
the human framework sequence selected in step b), d) repeating
steps a) thru c) for each individual framework of the rodent light
chain to produce a plurality of DNA sequences in which each
sequence encodes a humanized light chain framework, wherein each of
the corresponding human framework sequences selected in step b) are
from the same or different human antibody, e) assembling into a
first vector encoding at least the light chain variable region of
the rodent antibody, the DNA sequences encoding the humanized
framework sequences produced in step d); and f) introducing the
assembled vector into a host under conditions sufficient to produce
the humanized antibody.
70. The method of claim 69, wherein the method further comprises:
g) comparing the amino acid sequence of a heavy chain framework
from the rodent antibody against a collection of corresponding
human antibody framework sequences, h) selecting a human framework
sequence from the collection having the greatest amino acid
sequence identity to the corresponding rodent heavy chain
framework, i) mutagenizing a DNA segment encoding the rodent heavy
chain framework to encode a humanized heavy chain framework having
an amino acid sequence that is substantially identical to the human
framework sequence selected in step h); and j) repeating steps g)
thru i) for each individual framework of the rodent heavy chain to
produce a plurality of DNA sequences in which each sequence encodes
a humanized heavy chain framework, wherein each of the
corresponding human framework sequences selected in step h) are
from the same or different human antibody.
71. The method of claim 70, wherein the method further comprises
assembling into a second vector encoding at least the heavy chain
variable region of the rodent antibody, the DNA sequences encoding
the humanized framework sequences produced in step j); and
introducing the assembled first and second vectors into a host
under conditions sufficient to produce the humanized antibody.
72. The method of claim 70, wherein the method further comprises
assembling into the first vector encoding at least the light chain
variable region of the rodent antibody, the DNA sequences encoding
the humanized framework sequences produced in step j); and
introducing the assembled first vector into a host under conditions
sufficient to produce the humanized antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Application U.S. Ser. No. ______ entitled Antibodies For Inhibiting
Blood Coagulation and Methods of Use Thereof by Jiao, J. et al. as
filed on Oct. 29, 2001 which application is related to U.S. patent
application Ser. No. 09/293,854 filed on Apr. 16, 1999 which
application is a divisional of U.S. Ser. No. 08/814,806 (now U.S.
Pat. No. 5,986,065). The disclosures of said U.S. Provisional
Application U.S. Ser. No. ______ as filed on Oct. 29, 2001, U.S.
patent application Ser. No. 09/293,854 and U.S. Pat. No. 5,986,065
are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to novel human tissue factor
antibodies and methods of using the antibodies to inhibit tissue
factor-related functions such as blood coagulation, angiogenesis,
tumor metastasis, and inflammation. In particular, the invention
relates to novel antibodies that can specifically bind native human
tissue factor with high affinity and prevent factor X or factor IX
binding and activation. The antibodies of the invention are useful
for a variety of applications, particularly for reducing blood
coagulation in vivo.
[0004] 2. Background
[0005] Blood clotting assists homeostasis by minimizing blood loss.
Generally, blood clotting requires vessel damage, platelet
aggregation, activation of coagulation factors and inhibition of
fibrinolysis. The coagulation factors act through a cascade that
relates the vessel damage to formation of a blood clot (see
generally L. Stryer, Biochemistry, 3rd Ed, W. H. Freeman Co., New
York; and A. G. Gilman et al., The Pharmacological Basis of
Therapeutics, 8th Edition, McGraw Hill Inc., New York, pp.
1311-1331).
[0006] There is general agreement that factor X (FX) activation to
factor Xa (FXa) (or factor IX activation to factor IXa) is a
critical step in the blood coagulation process. Generally, FX (or
FIX) is converted to FXa (or FIXa) by binding a catalytically
active complex that includes "tissue factor" (TF). TF is a
controllably-expressed cell membrane protein that binds factor
VII/Via to produce the catalytically active complex (TF:FVIIa). A
blood clot follows FXa-mediated (or FIXa) activation of
prothrombin. Blood clotting can be minimized by inactivation of TF
to non-native forms which cannot optimally produce the TF:FVIIa
complex. Excessive activation of the coagulation cascade through
formation of FXa (or FIXa) is believed to contribute to various
thromboses including restenosis.
[0007] Thrombosis may be associated with invasive medical
procedures such as cardiac surgery (e.g. angioplasty),
abdominothoracic surgery, arterial surgery, peripheral vascular
bypass grafts, deployment of an implementation (e.g., a stent or
catheter), or endarterectomy. Further, thrombosis may accompany
various thromboembolic disorders and coagulopathies such as stroke,
pulmonary embolism (e.g., atrial fibrillation with embolization),
coronary artery disease or acute coronary syndromes (e.g., unstable
angina or myocardial infarction), atherosclerosis or other
thrombo-occlusive disorders, deep vein thrombosis and disseminated
intravascular coagulation, respectively. Manipulation of body
fluids can also result in an undesirable thrombus, particularly in
blood transfusions or fluid sampling, as well as procedures
involving extracorporeal circulation (e.g., cardiopulmonary bypass
surgery) and renal dialysis.
[0008] Anti-coagulants are frequently used to alleviate or avoid
blood clots associated with thrombosis. Blood clotting often can be
minimized or eliminated by administering a suitable anti-coagulant
or mixture thereof, including one or more of a coumarin derivative
(e.g., warfarin, Coumadin or dicumarol) or a charged polymer (e.g.,
heparin, low molecular weight heparin, hirudin or hirulog) or
anti-platelet agents (e.g., ReoPro, Integrilin, Aggrestat, Plavix,
Ticlid or aspirin). See e.g., Gilman et al., supra, R. J. Beigering
et al., Ann. Hematol., 72:177 (1996); J. D. Willerson, Circulation,
94:866 (1996).
[0009] However, use of anti-coagulants is often associated with
side effects such as hemorrhaging, re-occlusion, "white-clot"
syndrome, irritation, birth defects, thrombocytopenia and hepatic
dysfunction. Long-term administration of anti-coagulants can
particularly increase risk of life-threatening illness (see e.g.,
Gilman et al., supra).
[0010] Certain antibodies with anti-platelet activity have also
been used to alleviate various thromboses. For example, ReoPro.TM.
is a therapeutic antibody fragment that is routinely administered
to alleviate various thromboembolic disorders such as those arising
from angioplasty, myocardial infarction, unstable angina and
coronary artery stenoses. Additionally, ReoPro.TM. can be used as a
prophylactic to reduce the risk of myocardial infarction and angina
(J. T. Willerson, Circulation, 94:866 (1996); M. L. Simmons et al.,
Circulation, 89:596 (1994)).
[0011] Certain anti-coagulant antibodies are also known.
Particularly, certain TF-binding antibodies have been reported to
inhibit blood coagulation, presumably by interfering with assembly
of a catalytically active TF:FVIIa complex (see e.g., Jeske et al.,
SEM in THROM. and HEMO, 22:213 (1996); Ragni et al., Circulation,
93:1913 (1996); European Patent No. 0 420 937 B1; W. Ruf et al.,
Throm. Haemosp., 66:529 (1991); M. M. Fiorie et al., Blood, 8:3127
(1992)).
[0012] However, current TF-binding antibodies exhibit significant
disadvantages which can minimize their suitably as anti-coagulants.
For example, current TF-binding antibodies do not exhibit
sufficient binding affinity for optimal anti-coagulant activity.
Accordingly, for many thrombotic conditions, to compensate for such
ineffective binding affinities, unacceptably high antibody levels
must be administered to minimize blood coagulation.
[0013] It would thus be desirable to have an anti-coagulant
antibody that binds native human TF with high affinity and
selectivity to thereby inhibit undesired blood coagulation and the
formation of blood clots. It would be further desirable to have
such an anti-coagulant antibody that prevents the binding of factor
X (or factor IX) to TF:FVIIa complex.
SUMMARY OF THE INVENTION
[0014] We have now discovered antibodies that provide superior
anti-coagulant activity by binding native human TF with high
affinity and specificity. Antibodies of the invention can
effectively inhibit blood coagulation in vivo. Antibodies of the
invention can bind native human TF, either alone or present in a
TF:FVIIa complex, effectively preventing factor X (or factor IX)
binding to TF or that complex, and thereby reducing blood
coagulation.
[0015] Preferred antibodies of the invention are monoclonal and
specifically bind a conformational epitope predominant to native
human TF, which epitope provides a site for the unexpectedly strong
antibody binding. Indeed, preferred antibodies of the invention
bind to native human TF at least about 5 times greater, more
typically at least about ten times greater than the binding
affinity exhibited by prior anti-coagulant antibodies.
Additionally, preferred antibodies of the invention are selective
for native human TF, and do not substantially bind non-native or
denatured TF. H36.D2.B7 (secreted by hybridoma ATCC HB-12255 and
often referred to as H36) is an especially preferred antibody of
the invention.
[0016] Preferred antibodies of the invention bind TF so that FX (or
FIX) does not effectively bind to the TF:FVIIa complex whereby FX
(or FIX) is not effectively converted to its activated form (FXa or
FIXa). Preferred antibodies of the invention can inhibit TF
function by effectively blocking FX (or FIX) binding or access to
TF molecules. See, for instance, the results of Example 3 which
follows.
[0017] Preferred antibodies of the invention also do not
significantly inhibit the interaction or binding between TF and
factor VIIa, or inhibit activity of a TF:FVIIa complex with respect
to materials other than FX and Factor IX. See, for instance, the
results of Example 4 which follows.
[0018] The invention also provides nucleic acids that encode
antibodies of the invention. Nucleic acid and amino acid sequences
(SEQ ID:NOS 1-4) of variable regions of H36.D2.B7 are set forth in
FIGS. 1A and 1B of the drawings.
[0019] In preferred aspects, the invention provides methods for
inhibiting blood coagulation and blood clot formation, and methods
for reducing human TF levels.
[0020] In general, antibodies of the invention will be useful to
modulate virtually any biological response mediated by FX (or FIX)
binding to TF or the TF:FVIIa complex, including blood coagulation
as discussed above, inflammation, tumor angiogenesis and
metastasis, and other disorders.
[0021] Antibodies of the invention are particularly useful to
alleviate various thromboses, particularly to prevent or inhibit
restenosis, or other thromboses following an invasive medical
procedure such as arterial or cardiac surgery (e.g., angioplasty).
Antibodies of the invention also can be employed to reduce or even
effectively eliminate thrombotic occlusion arising from activation
of blood coagulation in such non-surgical cardiovascular conditions
including but not limited to coronary artery disease, acute
coronary syndromes (e.g., unstable angina and myocardial
infarction) and atherosclerosis. Antibodies of the invention also
can be employed to reduce or even effectively eliminate blood
coagulation arising from use of medical implementation (e.g., a
catheter, stent or other medical device). Preferred antibodies of
the invention will be compatible with many anti-coagulant,
anti-platelet and thrombolytic compositions, thereby allowing
administration in a cocktail format to boost or prolong inhibition
of blood coagulation.
[0022] Antibodies of the invention also can be employed as an
anti-coagulant in extracorporeal circulation of a mammal,
particularly a human subject. In such methods, one or more
antibodies of the invention is administered to the mammal in an
amount sufficient to inhibit blood coagulation prior to or during
extracorporeal circulation such as may be occur with
cardiopulmonary bypass surgery, organ transplant surgery or other
prolonged surgeries.
[0023] Antibodies of the invention also can be used as a carrier
for drugs, particularly pharmaceuticals targeted for interaction
with a blood clot such as strepokinase, tissue plasminogen
activator (t-PA) or urokinase. Similarly, antibodies of the
invention can be used as a cytotoxic agent by conjugating a
suitable toxin to the antibody. Conjugates of antibodies of the
invention also can be used to reduce tissue factor levels in a
mammal, particularly a human, by administering to the mammal an
effective amount of an antibody of the invention which is
covalently linked to a cytotoxic agent or an effector molecule to
provide complement-fixing ability and antibody-dependent
cell-mediated cytotoxicity, whereby the antibody conjugate contacts
cells expressing tissue factor to thereby reduce tissue factor
levels in the mammal.
[0024] Antibodies of the invention also can be employed in in vivo
diagnostic methods including in vivo diagnostic imaging of native
human TF.
[0025] Antibodies of the invention also can be used in in vitro
assays to detect native TF in a biological sample including a body
fluid (e.g., plasma or serum) or tissue (e.g., a biopsy sample).
More particularly, various heterogeneous and homogeneous
immunoassays can be employed in a competitive or non-competitive
format to detect the presence and preferably an amount of native TF
in the biological sample.
[0026] Such assays of the invention are highly useful to determine
the presence or likelihood of a patient having a blood coagulation
or a blood clot. That is, blood coagulation is usually accompanied
by and the result of TF expression on cell surfaces such as
monocytes, macrophages, and endothelial cells lining the
vasculature. Thus, the detection of TF in a body fluid sample by an
assay of the invention will be indicative of blood coagulation.
[0027] Antibodies of the invention also can be used to prepare
substantially pure native TF, particularly native human TF, from a
biological sample. Antibodies of the invention also can be used for
detecting and purifying cells which express native TF.
[0028] Antibodies of the invention also can be employed as a
component of a diagnostic kit, e.g. for detecting and preferably
quantitating native TF in a biological sample.
[0029] The invention also provides humanized antibodies that bind
specifically to human tissue factor (TF) to form a complex. In a
preferred embodiment, blood factor X or factor IX binding to the
complex is significantly inhibited. Preferably, the humanized
antibody includes at least one murine complementarity determining
region (CDR), preferably one, two, three or four of such murine
CDRs. Further provided are TF binding fragments of such humanized
antibodies.
[0030] In another aspect, the invention provides methods of
inhibiting blood coagulation in a mammal that include administering
to the mammal an effective amount of the humanized antibody or
fragment thereof that binds specifically to human tissue factor
(TF) to form a complex. A preferred antibody for use in the method
significantly reduces factor X or factor IX binding to the complex.
Preferred methods further include forming a specific complex
between the antibody and the TF or TF:FVIIa complex to inhibit the
blood coagulation.
[0031] The invention also provides methods of inhibiting blood
coagulation in a mammal that include administering to the mammal,
an effective amount of a humanized antibody or fragment thereof
comprising at least one murine complementarity determining region
(CDR). A preferred humanized antibody for use with the method binds
specifically to human tissue factor (TF) to form a complex.
Preferably, factor X or factor IX binding to the complex is
significantly reduced. Preferred methods further include forming a
specific complex between the antibody and the TF to inhibit the
blood coagulation.
[0032] Other aspects of the invention are discussed infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIGS. 1A and 1B shows the nucleic acid (SEQ ID NOS:1 and 3)
and amino acid (SEQ ID NOS:2 and 4) sequences of light chain and
heavy chain variable regions of H36.D2.B7 with hypervariable
regions (CDRs or Complementarity Determining Regions) underlined
(single underline for nucleic acid sequences and double underline
for amino acid sequences).
[0034] FIG. 2 shows association (K.sub.a) and disassociation
(K.sub.d) constants of anti-tissue factor antibodies as determined
by ELISA or BIACore analysis.
[0035] FIG. 3 shows inhibition of TF:FVIIa complex mediated FX
activation by pre-incubation with anti-tissue factor
antibodies.
[0036] FIG. 4 shows inhibition of TF:FVIIa activity toward the
FVIIa-specific chromogenic substrate S-2288 by anti-tissue factor
antibodies.
[0037] FIG. 5 shows the capacity of the H36 antibody to increase
prothrombin time (PT) in a TF-initiated coagulation assay.
[0038] FIGS. 6A and 6B graphically show the relationship between
FXa formation and molar ratio of the H36 antibody and rHTF. FIG.
6A: H36 was pre-incubated with the TF:FVIIa complex prior to adding
FX. FIG. 6B: H36, TF:FVIIa and FX were added simultaneously.
[0039] FIG. 7 shows inhibition of TF:FVIIa activity by the H36
antibody in a J-82 cell activation assay.
[0040] FIGS. 8A and 8B are representations of dot blots showing
that the H36 antibody binds a conformational epitope on rhTF. Lane
1-native rHTF, Lane 2-native rhTF treated with 8M urea, Lane
3-native rHTF treated with 8M urea and 5 mM DTT. In FIG. 8A, the
blot was exposed for approximately 40 seconds, whereas in FIG. 8B,
the blot was exposed for 120 seconds.
[0041] FIGS. 9A-B are drawings showing human IgG1-cH36 HC variable
region cloning and expression vectors. HC cloning vector (9A) and
HC expression vector (9B).
[0042] FIGS. 9C-D are drawings showing human IgG4-cH36 HC variable
region cloning and expression vectors. HC cloning vector (9C) and
HC expression vector (9D).
[0043] FIGS. 10A-B are drawings showing cH36 LC variable region
cloning and expression vectors. LC cloning vector (10A) and LC
expression vector (10B).
[0044] FIG. 11 is a drawing showing a plasmid map of humanized
anti-TF IgG1 antibody expression vector (pSUN 34).
[0045] FIGS. 12A-D are drawings showing sequences of partially and
fully humanized light chain (LC) variable regions. Light chain CDR
sequences CDR sequences of cH36 are shown in FIGS. 12B-D. Sequence
named "LC-09" is representative of a fully humanized LC framework
region.
[0046] FIGS. 13A-D are sequences of partially and fully humanized
heavy chain (LC) variable regions. Heavy chain CDR sequences for
cH36 and HC-08 are shown in FIGS. 13B-D. Sequence named "HC-08" is
fully humanized HC framework region
[0047] FIGS. 14A-B are drawings showing humanized IgG one
anti-tissue factor antibody (hOAT (IgG1) constant regions.
[0048] FIGS. 15A-B are drawings showing humanized IgG four
anti-tissue factor antibody (hFAT) (IgG4) constant regions.
DETAILED DESCRIPTION OF THE INVENTION
[0049] As discussed above, preferred antibodies of the invention
exhibit substantial affinity for native human TF. In particular,
preferred antibodies of the invention exhibit an association
constant (K.sub.a, M.sup.-1) for native human TF of at least about
1.times.10.sup.8 as determined by surface plasmon analysis
(particularly, BIACore analysis in accordance with the procedures
of Example 1 which follows), more preferably at least about
5.times.10.sup.8 as determined by surface plasmon analysis, still
more preferably a K.sub.a (K.sub.a, M.sup.-1) for native human TF
of at least about 1.times.10.sup.10 as determined by surface
plasmon resonance analysis. Such substantial binding affinity of
antibodies of the invention contrast sharply from much lower
binding affinities of previously reported antibodies.
[0050] In this regard, a quite low of effective concentration of an
antibody of the invention can be employed, e.g. a relatively low
concentration of antibody can be employed to inhibit TF function as
desired (e.g. at least about 95, 98 or 99 percent inhibition) in an
in vitro assay such as described in Example 3 which follows.
[0051] The preferred antibodies are also highly specific for native
human TF, and preferably do not substantially bind with non-native
TF. Preferred antibodies do not substantially bind non-native TF or
other immunologically unrelated molecules as determined, e.g. by
standard dot blot assay (e.g. no or essentially no binding to
non-native TF visually detected by such dot blot assay). References
herein to "non-native TF" mean a naturally-occurring or recombinant
human TF that has been treated with a chaotropic agent so that the
TF is denatured. Typical chaotropic agents include a detergent
(e.g. SDS), urea combined with dithiothreotol or
.beta.-mercaptoethanol; guanidine hydrochloride and the like. The
H36, H36.D2 or H36.D2.B7 antibody does not substantially bind to
such non-native TF. See, for instance, the results of Example 8
which follows and is a dot blot assay.
[0052] As discussed above, preferred antibodies of the invention
also bind with TF so that FX (or FIX) does not effectively bind to
the TF:FVIIa complex whereby FX (or FIX) is not effectively
converted to its activated form (FXa or FIXa). Particularly
preferred antibodies of the invention will strongly inhibit FX
activation by a TF:FVIIa complex, e.g. an inhibition of at least
about 50%, more preferably at least about 80%, and even more
preferably at least about 90% or 95%, even at low TF concentrations
such as less than about 1.0 nM TF, or even less than about 0.20 nM
or 0.10 nM TF, as determined by a standard in vitro binding assay
such as that of Example 3 which follows and includes contacting FX
(or FIX) with a TF: FVIIa complex both in the presence (i.e.
experimental sample) and absence (i.e. control sample) of an
antibody of the invention and determining the percent difference of
conversion of FX to FXa (or FIX to FIXa) between the experimental
and control samples.
[0053] Antibodies of the invention are preferably substantially
pure when used in the disclosed methods and assays. References to
an antibody being "substantially pure" mean an antibody or protein
which has been separated from components which naturally accompany
it. For example, by using standard immunoaffinity or protein A
affinity purification techniques, an antibody of the invention can
be purified from a hybridoma culture by using native TF as an
antigen or protein A resin. Similarly, native TF can be obtained in
substantially pure form by using an antibody of the invention with
standard immunoaffinity purification techniques. Particularly, an
antibody or protein is substantially pure when at least 50% of the
total protein (weight % of total protein in a given sample) is an
antibody or protein of the invention. Preferably the antibody or
protein is at least 60 weight % of the total protein, more
preferably at least 75 weight %, even more preferably at least 90
weight %, and most preferably at least 98 weight % of the total
material. Purity can be readily assayed by known methods such as
SDS (PAGE) gel electrophoresis, column chromatography (e.g.,
affinity chromatography) or HPLC analysis.
[0054] The nucleic acid (SEQ ID NOS: 1 and 3) and amino acid (SEQ
ID NOS: 2 and 4) sequences of a preferred antibody of the invention
(H36.D2.B7) are shown in FIGS. 1A and 1B of the drawings. SEQ ID
NOS. 1 and 2 are the nucleic acid and amino acid respectively of
the light chain variable region, and SEQ ID NOS. 3 and 4 are the
nucleic acid and amino acid respectively of the heavy chain
variable region, with hypervariable regions (CDRs or
Complementarity Determining Regions) underlined in all of those
sequences.
[0055] Additional preferred antibodies of the invention will have
substantial amino acid? sequence identity to either one or both of
the light chain or heavy sequences shown in FIGS. 1A and 1B. More
particularly, preferred antibodies include those that have at least
about 70 percent homology (amino acid sequence identity) to SEQ ID
NOS. 2 and/or 4, more preferably about 80 percent or more homology
to SEQ ID NOS. 2 and/or 4, still more preferably about 85, 90 or 95
percent or more homology to SEQ ID NOS. 2 and/or 4.
[0056] Preferred antibodies of the invention will have high amino
acid sequence identity to hypervariable regions (shown with double
underlining in FIGS. 1A and 1B) of SEQ ID NOS. 2 and 4). Especially
preferred antibodies of the invention will have one, two or three
hypervariable regions of a light chain variable region that have
high sequence identity (at least 90% or 95% amino acid sequence
identity) to or be the same as one, two or three of the
corresponding hypervariable regions of the light chain variable
region of H36.D2.B7 (those hypervariable regions shown with
underlining in FIG. 1A and are the following: 1) LASQTID (SEQ ID
NO:5); 2) AATNLAD (SEQ ID NO:6); and 3) QQVYSSPFT (SEQ ID
NO:7)).
[0057] Especially preferred antibodies of the invention also will
have one, two or three hypervariable regions of a heavy chain
variable region that have high sequence identity (at least 90% or
95% amino acid sequence identity) to or be the same as one, two or
three of the corresponding hypervariable regions of the heavy chain
variable region of H36.D2.B7 (those hypervariable regions shown
with underlining in FIG. 1B and are the following: 1) TDYNVY (SEQ
ID NO:8); 2) YIDPYNGITIYDQNFKG (SEQ ID NO:9); and 3) DVTTALDF (SEQ
ID NO:10).
[0058] Nucleic acids of the invention preferably are of a length
sufficient (preferably at least about 100, 200 or 250 base pairs)
to bind to the sequence of SEQ ID NO: 1 and/or SEQ ID NO:3 under
the following moderately stringent conditions (referred to herein
as "normal stringency" conditions): use of a hybridization buffer
comprising 20% formamide in 0.8M saline/0.08M sodium citrate (SSC)
buffer at a temperature of 37.degree. C. and remaining bound when
subject to washing once with that SSC buffer at 37.degree. C.
[0059] More preferably, nucleic acids of the invention (preferably
at least about 100, 200 or 250 base pairs) will bind to the
sequence of SEQ ID NO:1 and/or SEQ ID NO:3 under the following
highly stringent conditions (referred to herein as "high
stringency" conditions): use of a hybridization buffer comprising
20% formamide in 0.9M saline/0.09M sodium citrate (SSC) buffer at a
temperature of 42.degree. C. and remaining bound when subject to
washing twice with that SSC buffer at 42.degree. C.
[0060] Nucleic acids of the invention preferably comprise at least
20 base pairs, more preferably at least about 50 base pairs, and
still more preferably a nucleic acid of the invention comprises at
least about 100, 200, 250 or 300 base pairs.
[0061] Generally preferred nucleic acids of the invention will
express an antibody of the invention that exhibits the preferred
binding affinities and other properties as disclosed herein.
[0062] Preferred nucleic acids of the invention also will have
substantial sequence identity to either one or both of the light
chain or heavy sequences shown in FIGS. 1A and 1B. More
particularly, preferred nucleic acids will comprise a sequence that
has at least about 70 percent homology (nucleotide sequence
identity) to SEQ ID NOS. 1 and/or 3, more preferably about 80
percent or more homology to SEQ ID NOS. 1 and/or 3, still more
preferably about 85, 90 or 95 percent or more homology to SEQ ID
NOS. 1 and/or 3.
[0063] Particularly preferred nucleic acid sequences of the
invention will have high sequence identity to hypervariable regions
(shown with underlining in FIGS. 1A and 1B) of SEQ ID NOS. 1 and
3). Especially preferred nucleic acids include those that code for
an antibody light chain variable region and have one, two or three
sequences that code for hypervariable regions and have high
sequence identity (at least 90% or 95% nucleotide sequence
identity) to or be the same as one, two or three of the sequences
coding for corresponding hypervariable regions of H36.D2.B7 (those
hypervariable regions shown with underlining in FIG. 1A and are the
following: 1) CTGGCAAGTCAGACCATTGAT (SEQ ID NO:11); 2) GCTGCCACC
AACTTGGCAGAT (SEQ ID NO: 12); and 3) CAACAAGTTTACAGTTCT CCATTCACGT
(SEQ ID NO: 13)).
[0064] Especially preferred nucleic acids also code for an antibody
heavy chain variable region and have one, two or three sequences
that code for hypervariable regions and have high sequence identity
(at least 90% or 95% sequence identity) to or be the same as one,
two or three of the sequences coding for corresponding
hypervariable regions of H36.D2.B7 (those hypervariable regions
shown with underlining in FIG. 1B and are the following: 1)
ACTGACTACAACGTGTAC (SEQ ID NO: 14); 2) TATATTGAT
CCTTACAATGGTATTACTATCTACGACCAGAACTTCAAGGGC (SEQ ID NO: 15); and 3)
GATGTGACTACGGCCCTTGACTTC (SEQ ID NO: 16)).
[0065] Nucleic acids of the invention are isolated, usually
constitutes at least about 0.5%, preferably at least about 2%, and
more preferably at least about 5% by weight of total nucleic acid
present in a given fraction. A partially pure nucleic acid
constitutes at least about 10%, preferably at least about 30%, and
more preferably at least about 60% by weight of total nucleic acid
present in a given fraction. A pure nucleic acid constitutes at
least about 80%, preferably at least about 90%, and more preferably
at least about 95% by weight of total nucleic acid present in a
given fraction.
[0066] Antibodies of the invention can be prepared by techniques
generally known in the art, and are typically generated to a
purified sample of native TF, typically native human TF, preferably
purified recombinant human tissue factor (rhTF). Truncated
recombinant human tissue factor or "rhTF" (composed of 243 amino
acids and lacking the cytoplasmic domain) is particularly preferred
to generate antibodies of the invention. The antibodies also can be
generated from an immunogenic peptide that comprises one or more
epitopes of native TF that are not exhibited by non-native TF.
References herein to "native TF" include such TF samples, including
such rhTF. As discussed above, monoclonal antibodies are generally
preferred, although polyclonal antibodies also can be employed.
[0067] More particularly, antibodies can be prepared by immunizing
a mammal with a purified sample of native human TF, or an
immunogenic peptide as discussed above, alone or complexed with a
carrier. Suitable mammals include typical laboratory animals such
as sheep, goats, rabbits, guinea pigs, rats and mice. Rats and
mice, especially mice, are preferred for obtaining monoclonal
antibodies. The antigen can be administered to the mammal by any of
a number of suitable routes such as subcutaneous, intraperitoneal,
intravenous, intramuscular or intracutaneous injection. The optimal
immunizing interval, immunizing dose, etc. can vary within
relatively wide ranges and can be determined empirically based on
this disclosure. Typical procedures involve injection of the
antigen several times over a number of months. Antibodies are
collected from serum of the immunized animal by standard techniques
and screened to find antibodies specific for native human TF.
Monoclonal antibodies can be produced in cells which produce
antibodies and those cells used to generate monoclonal antibodies
by using standard fusion techniques for forming hybridoma cells.
See G. Kohler, et al., Nature, 256:456 (1975). Typically this
involves fusing an antibody-producing cell with an immortal cell
line such as a myeloma cell to produce the hybrid cell.
Alternatively, monoclonal antibodies can be produced from cells by
the method of Huse, et al., Science, 256:1275 (1989).
[0068] One suitable protocol provides for intraperitoneal
immunization of a mouse with a composition comprising purified rhTF
complex conducted over a period of about two to seven months.
Spleen cells then can be removed from the immunized mouse. Serum
from the immunized mouse is assayed for titers of antibodies
specific for rhTF prior to excision of spleen cells. The excised
mouse spleen cells are then fused to an appropriate homogenic or
heterogenic (preferably homogenic) lymphoid cell line having a
marker such as hypoxanthine-guanine phosphoribosyltransfera- se
deficiency (HGPRT.sup.-) or thymidine kinase deficiency (TK.sup.-).
Preferably a myeloma cell is employed as the lymphoid cell line.
Myeloma cells and spleen cells are mixed together, e.g. at a ratio
of about 1 to 4 myeloma cells to spleen cells. The cells can be
fused by the polyethylene glycol (PEG) method. See G. Kohler, et
al., Nature, supra. The thus cloned hybridoma is grown in a culture
medium, e.g. RPMI-1640. See G. E. More, et al., Journal of American
Medical Association, 199:549 (1967). Hybridomas, grown after the
fusion procedure, are screened such as by radioimmunoassay or
enzyme immunoassay for secretion of antibodies that bind
specifically to the purified rhTF, e.g. antibodies are selected
that bind to the purified rhTF, but not to non-native TF.
Preferably an ELISA is employed for the screen. Hybridomas that
show positive results upon such screening can be expanded and
cloned by limiting dilution method. Further screens are preferably
performed to select antibodies that can bind to rhTF in solution as
well as in a human fluid sample. The isolated antibodies can be
further purified by any suitable immunological technique including
affinity chromatography. A hybridoma culture producing the
particular preferred H36.D2.B7 antibody has been deposited pursuant
to the Budapest Treaty with the American Type Culture Collection
(ATCC) at 12301 Parklawn Drive, Rockville, Md., 10852. The
hybridoma culture was deposited with the ATCC on Jan. 8, 1997 and
was assigned Accession Number ATCC HB-12255.
[0069] For human therapeutic applications, it may be desirable to
produce chimeric antibody derivatives, e.g. antibody molecules that
combine a non-human animal variable region and a human constant
region, to thereby render the antibodies less immunogenic in a
human subject than the corresponding non-chimeric antibody. A
variety of types of such chimeric antibodies can be prepared,
including e.g. by producing human variable region chimeras, in
which parts of the variable regions, especially conserved regions
of the antigen-binding domain, are of human origin and only the
hypervariable regions are of non-human origin. See also discussions
of humanized chimeric antibodies and methods of producing same in
S. L. Morrison, Science, 229:1202-1207 (1985); Oi et al.,
BioTechniques, 4:214 (1986); Teng et al., Proc. Natl. Acad. Sci.
U.S.A., 80:7308-7312 (1983); Kozbor et al., Immunology Today,
4:7279 (9183); Olsson et al., Meth. Enzymol., 9:3-16 (1982).
Additionally, transgenic mice can be employed. For example,
transgenic mice carrying human antibody repertoires have been
created which can be immunized with native human TF. Splenocytes
from such immunized transgenic mice can then be used to create
hybridomas that secrete human monoclonal antibodies that
specifically react with native human TF as described above. See N.
Lonberg et al., Nature, 368:856-859 (1994); L. L. Green et al.,
Nature Genet., 7:13-21 (1994); S. L. Morrison, Proc. Natl. Acad.
Sci. U.S.A., 81:6851-6855 (1994).
[0070] Nucleic acids which code for the antibodies of the invention
also can be prepared by polymerase chain reaction (see primers
disclosed in Example 1 which follows). See generally, Sambrook et
al., Molecular Cloning (2d ed. 1989). Such nucleic acids also can
be synthesized by known methods, e.g. the phosphate triester method
(see Oligonucleotide Synthesis, IRL Press (M. J. Gait, ed., 1984)),
or by using a commercially available automated oligonucleotide
synthesizer. Such a prepared nucleic acid of the invention can be
employed to express an antibody of the invention by known
techniques. For example, a nucleic acid coding for an antibody of
the invention can be incorporated into a suitable vector by known
methods such as by use of restriction enzymes to make cuts in the
vector for insertion of the construct followed by ligation. The
vector containing the inserted nucleic acid sequence, suitably
operably linked to a promoter sequence, is then introduced into
host cells for expression. See, generally, Sambrook et al., supra.
Selection of suitable vectors can be made empirically based on
factors relating to the cloning protocol. For example, the vector
should be compatible with, and have the proper replicon for the
host cell that is employed. Further, the vector must be able to
accommodate the inserted nucleic acid sequence. Suitable host cells
will include a wide variety of eukaryotic or prokaryotic cells such
as E. coli and the like.
[0071] The molecular weight of the antibodies of the invention will
vary depending on several factors such as the intended use and
whether the antibody includes a conjugated or recombinantly fused
toxin, pharmaceutical, or detectable label or the like. Also the
molecular weight will vary depending on nature and extent of
post-translational modifications if any (such as glycosylation) to
the antibody. The modifications are a function of the host used for
expression with E. coli producing non-glycosylated antibodies and
mammalian cells producing glycosylated antibodies. In general, an
antibody of the invention will have a molecular weight of between
approximately 20 to 150 kDa. Such molecular weights can be readily
are determined by molecular sizing methods such as SDS-PAGE gel
electrophoresis followed by protein staining or Western blot
analysis.
[0072] "Antibody of the invention" or other similar term refers to
whole immunoglobulin as well as immunologically active fragments
which bind native TF. The immunoglobulins and immunologically
active fragments thereof include an antibody-binding site (i.e.,
epitope capable of being specifically bound by an antibody
recognizing native human TF capable of specifically binding native
human TF). Exemplary antibody fragments include, for example, Fab,
F(v), Fab', F(ab').sub.2 fragments, "half molecules" derived by
reducing the disulfide bonds of immunoglobulins, single chain
immunoglobulins, or other suitable antigen binding fragments (see
e.g., Bird et al., Science, pp. 242-424 (1988); Huston et al.,
PNAS, (USA), 85:5879 (1988); Webber et al., Mol. Immunol., 32:249
(1995)). The antibody or immunologically active fragment thereof
may be of animal (e.g., a rodent such as a mouse or a rat), or
chimeric form (see Morrison et al., PNAS, 81:6851 (1984); Jones et
al., Nature, pp. 321, 522 (1986)). Single chain antibodies of the
invention can be preferred.
[0073] Similarly, a "nucleic acid of the invention" refers to a
nucleotide sequence which can be expressed to provide an antibody
of the invention as such term is specified to mean immediately
above.
[0074] As discussed above, antibodies of the invention can be
administered to a mammal, preferably a primate such as a human, to
prevent or reduce thrombotic occlusive disorders attributable to
TF-mediated activation of coagulation, typically in a composition
including one or more pharmaceutically acceptable non-toxic
carriers such as sterile water or saline, glycols such as
polyethylene glycol, oils of vegetable origin, and the like. In
particular, biocompatible, biodegradable lactide polymer, lactide
glycolide copolymer or polyoxyethylene, polyoxypropylene copolymers
may be useful excipients to control the release of the
antibody-containing compositions described herein. Other
potentially useful administration systems include ethylene vinyl
acetate copolymer particles, osmotic pumps, and implantable
infusion systems and liposomes. Generally, an anti-coagulant
composition of the invention will be in the form of a solution or
suspension (or a lyophilized form that can be reconstituted to a
solution or suspension), and will preferably include approximately
0.01% to 10% (w/w) of the antibody of the present invention,
preferably approximately 0.01% to 5% (w/w) of the antibody. The
antibody can be administered as a sole active ingredient in the
composition, or as a cocktail including one or more other
anti-coagulant (e.g., heparin, hirudin or hirulog, coumadin,
warfarin), anti-platelet (e.g., aspirin, Plavix, Ticlid, ReoPro,
Integrilin or Aggrestat), or thrombolytic agents (e.g., tissue
plasminogen activator, strepokinase and urokinase). Additionally,
antibodies of the invention can be administered prior to, or after
administration of one or more suitable anti-coagulant,
anti-platelet or thrombolytic agents to boost or prolong desired
anti-coagulation activity.
[0075] As also discussed above, antibodies of the invention can be
employed to reduce potential blood coagulation arising from use of
medical implementation, e.g. an indwelling device such as a
catheter, stent, etc. In one preferred method, the implementation
can be treated with an antibody of the invention (e.g., as a 1
mg/ml saline solution) prior to contact with a body fluid.
Alternatively, or in addition, an antibody of the invention can be
combined with the body fluid in an amount sufficient to minimize
blood clotting.
[0076] Therapeutic anti-coagulant compositions according to the
invention are suitable for use in parenteral or intravenous
administration, particularly in the form of liquid solutions. Such
compositions may be conveniently administered in unit dose and may
be prepared in accordance with methods known in the pharmaceutical
art. See Remington's Pharmaceutical Sciences, (Mack Publishing Co.,
Easton Pa., (1980)). By the term "unit dose" is meant a therapeutic
composition of the present invention employed in a physically
discrete unit suitable as unitary dosages for a primate such as a
human, each unit containing a pre-determined quantity of active
material calculated to produce the desired therapeutic effect in
association with the required diluent or carrier. The unit dose
will depend on a variety of factors including the type and severity
of thrombosis to be treated, capacity of the subject's blood
coagulation system to utilize the antibody, and degree of
inhibition or neutralization of FX (or FIX) activation desired.
Precise amounts of the antibody to be administered typically will
be guided by judgment of the practitioner, however, the unit dose
will generally depend on the route of administration and be in the
range of 10 ng/kg body weight to 50 mg/kg body weight per day, more
typically in the range of 100 ng/kg body weight to about 10 mg/kg
body weight per day. Suitable regimens for initial administration
in booster shots are also variable but are typified by an initial
administration followed by repeated doses at one or more hour
intervals by a subsequent injection or other administration.
Alternatively, continuous or intermittent intravenous infusions may
be made sufficient to maintain concentrations of at least from
about 10 nanomolar to 10 micromolar of the antibody in the
blood.
[0077] In some instances, it may be desirable to modify the
antibody of the present invention to impart a desirable biological,
chemical or physical property thereto. More particularly, it may be
useful to conjugate (i.e. covalently link) the antibody to a
pharmaceutical agent, e.g. a fibrinolytic drug such as t-PA,
streptokinase, or urokinase to provide fibrinolytic activity or to
a targeting agent such as a fibrin-binding domain. Such linkage can
be accomplished by several methods including use of a linking
molecule such as a heterobifunctional protein cross-linking agent,
e.g. SPDP, carbodimide, or the like, or by recombinant methods.
[0078] In addition to pharmaceuticals such as a fibrinolytic agent,
an antibody of the invention can be conjugated to a toxin of e.g.
plant or bacterial origin such as diphtheria toxin (i.e., DT),
shiga toxin, abrin, cholera toxin, ricin, saporin, pseudomonas
exotoxin (PE), pokeweed antiviral protein, or gelonin. Biologically
active fragments of such toxins are well known in the art and
include, e.g., DT A chain and ricin A chain. The toxin can also be
an agent active at cell surfaces such as phospholipases (e.g.,
phospholipase C). As another example, the toxin can be a
chemotherapeutic drug such as, e.g., vendesine, vincristine,
vinblastin, methotrexate, adriamycin, doxirubicin, bleomycin, or
cisplatin, or, the toxin can be a radionuclide such as, e.g.,
iodine-131, yttrium-90, rhenium-188 or bismuth-212 (see generally,
Moskaug et al., J. Biol. Chem., 264:15709 (1989); I. Pastan et al.,
Cell, 47:641 (1986); Pastan et al., Recombinant Toxins as Novel
Therapeutic Agents, Ann. Rev. Biochem., 61:331 (1992); Chimeric
Toxins Olsnes and Phil, Pharmac. Ther., 25:355 (1982); published
PCT Application No. WO 94/29350; published PCT Application No. WO
94/04689; and U.S. Pat. No. 5,620,939). Also, as discussed above,
in addition to a toxin, an antibody of the invention can be
conjugated to an effector molecule (e.g. IgG1 or IgG3) to provide
complement-fixing ability and antibody-dependent cell-mediated
cytoxicity upon administration to a mammal.
[0079] Such an antibody-cytotoxin or effector molecule conjugate
can be administered in a therapeutically effective amount to a
mammal, preferably a primate such as a human, where the mammal is
known to have or is suspected of having tumor cells, immune system
cells, or endothelia capable of expressing TF. Exemplary of such
tumor cells, immune system cells and endothelia include
malignancies of the breast and lung, monocytes and vascular
endothelia.
[0080] Antibodies of the invention also can be conjugated to a
variety of other pharmaceutical agents in addition to those
described above such as, e.g., drugs, enzymes, hormones, chelating
agents capable of binding a radionuclide, as well as other proteins
and polypeptides useful for diagnosis or treatment of disease. For
diagnostic purposes, the antibody of the present invention can be
used either detectably labeled or unlabeled. For example, a wide
variety of labels may be suitably employed to detectably-label the
antibody, such as radionuclides, fluors, enzymes, enzyme
substrates, enzyme cofactors, enzyme inhibitors, ligands such as,
e.g., haptens, and the like.
[0081] Diagnostic methods are also provided including in vivo
diagnostic imaging [see, e.g., A. K. Abbas, Cellular and Molecular
Immunology, pg. 328 (W. B. Saunders Co. 1991)]. For most in vivo
imaging applications, an antibody of the invention can be
detectably-labeled with, e.g., .sup.125I, .sup.32P, .sup.99Tc, or
other detectable tag, and subsequently administered to a mammal,
particularly a human, for a pre-determined amount of time
sufficient to allow the antibody to contact a desired target. The
subject is then scanned by known procedures such as scintigraphic
camera analysis to detect binding of the antibody. The analysis
could aid in the diagnosis and treatment of a number of thromboses
such as those specifically disclosed herein. The method is
particularly useful when employed in conjunction with cardiac
surgery, particularly angioplasty, or other surgical procedure
where undesired formation of a blood clot can occur, to visualize
the development or movement of a blood clot.
[0082] Antibodies of the invention also can be used to prepare
substantially pure (e.g., at least about 90% pure, preferably at
least about 96 or 97% pure) native TF, particularly native human TF
from a biological sample. For example, native TF can be obtained as
previously described (see e.g., L. V. M. Rao et al., Thrombosis
Res., 56:109 (1989)) and purified by admixing the solution with a
solid support comprising the antibody to form a coupling reaction
admixture. Exemplary solid supports include a wall of a plate such
as a microtiter plate, as well as supports including or consisting
of polystyrene, polyvinylchloride, a cross-linked dextran such as
Sephadex.TM. (Pharmacia Fine Chemicals), agarose, polystyrene beads
(Abbott Laboratories), polyvinyl chloride, polystyrene,
polyacrylmide in cross-linked form, nitrocellulose or nylon and the
like. The TF can then be isolated from the solid support in
substantially pure form in accordance with standard immunological
techniques. See generally Harlow and Lane supra and Ausubel et al.
supra).
[0083] As also discussed above, antibodies of the invention can be
employed to detect native human TF in a biological sample,
particularly native TF associated with a blood clot. Exemplary
biological samples include blood plasma, serum, saliva, urine,
stool, vaginal secretions, bile, lymph, ocular humors,
cerebrospinal fluid, cell culture media, and tissue, particularly
vascular tissues such as cardiac tissue. Samples may be suitably
obtained from a mammal suffering from or suspected of suffering
from a thrombosis, preferably restenosis, associated with, e.g., an
invasive medical procedure such as percutanous transluminal
coronary intervention, cardiopulmonary bypass surgery,
endarterectomy, peripheral vascular bypass grafts, reconstructive
or plastic surgery, joint replacement; a heart ailment such as
myocardial infarction, cardiomyopathy, valvular heart disease,
stable angina, unstable angina, or artrial fibrillation associated
with embolization; a coagulopathy including disseminated
intravascular coagulation, deep vein thrombosis, deployment of an
implementation such as a stent or catheter; shock (e.g., septic
shock syndrome), vascular trauma, liver disease, hemorrhagic
stroke, heat stroke, malignancies (e.g., pancreatic, ovarian, or
small lung cell carcinoma), lupus, eclampsia, perivascular
occlusive disease, and renal disease.
[0084] For such assays, an antibody of the invention can be
detectably-labeled with a suitable atom or molecule e.g.,
radioactive iodine, tritium, biotin, or reagent capable of
generating a detectable product such as an anti-iodiotypic antibody
attached to an enzyme such as P-galactosidase or horseradish
peroxidase, or a fluorescent tag (e.g., fluorescein or rhodamine)
in accordance with known methods. After contacting the biological
sample with the detectably-labeled antibody, any unreacted antibody
can be separated from the biological sample, the label (or product)
is detected by conventional immunological methods including
antibody capture assay, antibody sandwich assay, RIA, ELISA,
immunoprecipitation, immunoabsorption and the like (see Harlow and
Lane, supra; Ausubel et al. supra). Any label (or product) in
excess of that detected in a suitable control sample is indicative
of the presence of native TF, more particularly a blood clot, in
the biological sample. For example, antibodies of the invention can
be detectably labeled to detect, and preferably quantitate, native
TF in accordance with standard immunological techniques such as
antibody capture assay, ELISA, antibody sandwich assay, RIA,
immunoprecipitation, immunoabsorption and the like. In some cases,
particularly when a tissue is used, the immunological technique may
include tissue fixation with a reagent known to substantially
maintain protein conformation (e.g., dilute formaldehyde). See
generally, Ausubel et al., Current Protocols in Molecular Biology,
John Wiley & Sons, New York, (1989); Harlow and Lane in
Antibodies: A Laboratory Manual, CSH Publications, NY (1988).
[0085] Antibodies of the invention also can be used for detecting
and purifying cells which express native TF, including fibroblasts,
brain cells, immune cells, (e.g., monocytes), epithelia, as well as
certain malignant cells. Preferred methods of detecting and
purifying the cells include conventional immunological methods
(e.g., flow cytometry methods such as FACS, and immunopanning).
Substantially pure populations of cells expressing native TF are
useful in clinical and research settings, e.g., to establish such
cells as cultured cells for screening TF-binding antibodies.
[0086] The invention also provides test and diagnostic kits for
detection of native TF, particularly native human TF, in a test
sample, especially a body fluid such as blood, plasma, etc., or
tissue as discussed above. A preferred kit includes a detectably
labeled antibody of the invention. The diagnostic kit can be used
in any acceptable immunological format such as an ELISA format to
detect the presence or quantity of native TF in the biological
sample.
[0087] As discussed, the invention also features humanized
antibodies that specifically bind to human tissue factor to form a
binding complex. The tissue factor may be naturally-occurring or
recombinant (rHTF). Preferably, factor X or factor IX binding to
the complex is inhibited. In a preferred invention embodiment, the
humanized antibody has an affinity constant (Kd) for the hTF of
less than about 1 nM, preferably less than about 0.5 nM, more
preferably between from about 0.01 nM to about 0.4 nM. See Example
11, below for more information about determining affinity constants
for the humanized antibodies. By the phrase "specific binding" is
meant that the humanized antibodies form a detectable binding
complex with the TF and no other antigen as determined by standard
immunological techniques such as RIA, Western blot or ELISA.
[0088] Additional humanized antibodies of the invention are further
characterized by capacity to increase blood clotting time by at
least about 5 seconds as determined by a standard prothrombin (PT)
clotting assay. In preferred embodiments, the amount of humanized
antibody will be between from about 5 nM to about 75 nM, more
preferably about 10 nM to about 50 nM, in the assay. See Example 11
below (describing how to perform the standard PT clotting assay
with the humanized antibodies), for instance.
[0089] Additionally preferred humanized antibodies in accord with
the invention have a binding specificity for tissue factor,
preferably human TF, that is about equal or greater than the
antibody obtained from H36.D2.B7 deposited under ATCC Accession No.
HB-12255. Also preferred are humanized antibodies which have a
binding affinity for the TF about equal to or greater than the
antibody obtained from H36.D2.B7 deposited under ATCC Accession No.
HB-12255. Methods for determining binding specificity and affinity
are known in the field and include the specific assays described
below.
[0090] Further humanized antibodies in accord with the invention
include at least one murine complimentarity determining region
(CDR). As will be appreciated, immunoglobin light and heavy chain
share certain structural similarities eg., each includes a
framework of four regions (FR1-4) whose sequences are relatively
conserved. Each of FR1-4 (FR1, FR2, FR3, FR4) are covalently
connected by three CDRs i.e., CDR1, CDR2, CDR3. There is general
recognition that the four FRs largely adopt a beta-sheet
configuration and the interconnected CDRs form loops connecting,
and in some instances, forming part of the beta-sheet structure.
Most CDRs are held close to adjoining FRs, and with a corresponding
CDR from the opposite light or heavy chain, help form the antigen
binding site. A wide range of CDRs and FRs have been disclosed. See
eg., Kabat et al. in Sequences of Proteins of Immunological
Interest US Dept. of Health and Human Services, US Government
Printing Office (1987).
[0091] See also EP-A-0239400 and U.S. Pat. No. 5,985,279
(describing methods of making altered antibodies in which CDRs are
derived from different species than the FR).
[0092] By the phrase "humanized" is meant an immunoglobin that
includes a human framework region and one or more CDRs from a
non-human source, usually rodent such as a rat or mouse
immunoglobin. The non-human immunoglobin providing the CDRs is
called a "donor" and the human immunoglobin called the "acceptor".
Constant regions need not be present, as in, for example, certain
TF binding fragments of such immunoglobins. Preferred constant
regions, if present, are substantially identical to human
immunoglobin constant regions i.e., at least about 90% identical
with regard to the amino acid sequence, preferably at least about
95% identical or greater. Accordingly, nearly all parts of the
humanized immunoglobin, with the possible exception of the CDRs are
substantially identical to corresponding parts of
naturally-occurring human immunoglobin sequences.
[0093] By the phrase "humanized antibody" is meant an antibody that
includes a humanized light chain and a humanized heavy chain
immunoglobin. Methods for making and using such antibodies have
already been discussed above. See S.L. Morrison, supra; Oi et al.,
supra; Teng et al., supra; Kozbor et al., supra; Olsson et
al.,supra; and other references cited previously.
[0094] For example, an illustrative humanized antibody includes: 1)
light and heavy chain frameworks (FRs) that are each at least about
90% identical in amino acid sequence, preferably at least 95%
identical to corresponding human FRs, 2) at least one CDR from a
mouse, preferably all the CDRs from the mouse, 3) and an
immunoglobin constant region that is at least about 90% identical,
preferably at least 95% identical to a corresponding human
immunoglobin constant region. It will be appreciated that the donor
antibody has been "humanized" by the process of "humanization"
because the resultant humanized antibody is expected to bind to the
same antigen as the donor antibody that provides the CDRs.
[0095] It will be further appreciated that the humanized antibodies
provided herein may have one or more additional conservative amino
acid substitutions which can be contiguous or non-contiguous as
needed. For example, such substitutions will typically have
substantially little or no effect on antigen binding or other
immunoglobin functions. By the phrase "conservative substitution"
including plural forms is meant combinations of: glyala; valileleu;
aspglu; asngln; serthr, lysarg; and phetyr.
[0096] Additional humanized antibodies feature a variable region
that is at least 70% identical in amino acid sequence (eg., about
73% to 75% identical), to the corresponding variable region of one
or more native human immunoglobin sequences. Further humanized
antibodies in accord with the invention have at least 90% identity
over the entire antibody to one or more human antibodies.
[0097] More specific humanized antibodies of the invention are
those in each of frameworks (FRs) 1, 2, 3 and 4 has at least about
90% amino acid sequence identity, preferably at least about 95% or
greater identity to the light chain FR sequences shown in FIG. 12A
(SEQ ID NO. ______). Preferably, the sequence shown as "LC-09" in
FIG. 12A. Further preferred are those humanized antibodies that
include a light chain constant region having at least about 90%
amino acid sequence identity, preferably at least about 95%
sequence identity or greater to the sequence shown in FIG. 14A or
15A (SEQ ID NO. ______).
[0098] Further specific humanized antibodies are those in which
each of frameworks (FRs) 1, 2, 3 and 4 has at least about 90% amino
acid sequence identity, preferably about 95% identity or greater to
the heavy chain sequences shown in FIG. 13A (SEQ ID NO. ______).
Preferably, the sequence shown as "HC-08" in FIG. 13A. Additional
humanized antibodies have a heavy chain constant region with at
least about 90% amino acid sequence identity, preferably at least
about 95% identity or greater, to sequence shown in FIG. 14B or 15B
(SEQ ID NO. ______).
[0099] In certain embodiments, the humanized antibody will have an
IgG1 (hOAT) or IgG4 (HFAT) isotype. See Example 9.
[0100] Also provided by the present invention are functional
fragments of the humanized antibodies disclosed herein. Preferred
fragments specifically bind TF with an affinity constant (Kd) of
less than about 1 nM, preferably less than about 0.5 nM, more
preferably between from about 0.01 nM to about 0.4 nM. Specifically
preferred are antigen binding Fab, Fab', and F(ab).sub.2
fragments.
[0101] As discussed, the invention features humanized antibodies
that include at least one murine complementarity determining region
(CDR), eg., CDR1, CDR2, CDR3. In a preferred embodiment, the
antibodies bind specifically to human tissue factor (TF) to form a
complex. Typically, the factor X or factor IX binding to TF or
TF:VIIa and activation by TF:FVIIa thereto is inhibited. As
mentioned above, preferred CDRs (light and heavy chain) are from a
rodent source, typically the mouse.
[0102] In one embodiment of the humanized antibodies of the
invention, the antibodies further include at least one human
framework (FR) region. Preferably, all the FR regions (light and
heavy chain) are human.
[0103] In a more particular embodiment, the first CDR (CDR1) of the
heavy chain hypervariable region is at least 90% identical to the
CDR1 amino acid sequence shown in FIG. 13B (SEQ ID NO. ______),
preferably at least about 95% identical or greater to that
sequence. Typically, the second CDR (CDR2) of the heavy chain
hypervariable region is at least 90% identical to the CDR2 amino
acid sequence shown in FIG. 13C (SEQ ID NO. ______), preferably at
least about 95% identical or greater. Preferably also, the third
CDR (CDR3) of the heavy chain hypervariable region is at least 90%
identical to the CDR3 sequence shown in FIG. 13D (SEQ ID NO.
______), more preferably about 95% identical or greater to that
sequence.
[0104] Identity between two nucleic acid sequences can be
determined by inspection and/or use of conventional computer
software such as BLAST and FASTA.
[0105] In another invention embodiment, the first CDR (CDR1) of the
light chain hypervariable region is at least 90% identical to the
CDR1 amino acid sequence shown in FIG. 12B (SEQ ID NO. ______),
preferably at least about 95% identical or greater. Typically, the
second CDR (CDR2) of the light chain hypervariable region is at
least 90% identical to the CDR2 amino acid sequence shown in FIG.
12C (SEQ ID NO. ______), preferably about 95% identical or greater.
Preferably, the third CDR (CDR3) of the light chain hypervariable
region is at least 90% identical to the CDR3 amino acid sequence
shown in FIG. 12D (SEQ ID NO. ______), more preferably about 95%
identical or greater to that sequence.
[0106] Additional humanized antibodies of the invention include a
first framework (FR1) of the heavy chain hypervariable region which
FR1 is at least 90% identical to the amino acid sequence shown in
FIG. 13A (SEQ ID NO. ______) as "FR1 HC-08", preferably about 95%
identical or greater to that sequence. In one embodiment, the FR1
comprises at least one of the following amino acid changes: E1 to
Q; Q5 to V; P9 to G; L11 to V; V12 to K; Q19 to R; and T24 to A.
Preferably, the FR1 includes two, three, four, five, or six of
those changes with all of those amino acid changes being preferred
for many applications.
[0107] Further humanized antibodies of the invention include a
second framework (FR2) of the heavy chain hypervariable region
which FR2 is at least 90% identical to the sequence shown in FIG.
13A (SEQ ID NO. ______) as "FR2 HC-08", preferably about 95%
identical or greater to that sequence. In one embodiment, the FR2
at least one of the following amino acid changes: 41H to P; and 44S
to G. A preferred FR2 includes both of those amino acid
changes.
[0108] The invention also features humanized antibodies in which a
third framework (FR3) of the heavy chain hypervariable region is at
least 90% identical to the sequence shown in FIG. 13A (SEQ ID NO.
______) as "FR3 HC-08", preferably about 95% identical or greater
to that sequence. In one embodiment, the FR3 includes at least one
of the following amino acid changes: 76S to T; 77T to S; 80F to Y;
82H to E; 84N to S; 87T to R; 89D to E; and 91S to T. A preferred
FR3 includes two, three, four, five or six of those amino acid
changes with all seven of those amino acid changes being generally
preferred.
[0109] Also featured are humanized antibodies in which the fourth
framework (FR4) of the heavy chain hypervariable region is at least
90% identical to the amino acid sequence shown in FIG. 13A (SEQ ID
No. ______) as "FR4 HC-08", preferably at least about 95% identical
or greater to that sequence. Preferably, the FR4 includes the
following amino acid change: 113L to V.
[0110] Additional humanized antibodies in accord with the invention
feature a first framework (FR1) of the light chain hypervariable
region which is at least about 90% identical to the amino acid
sequence shown in FIG. 12A (SEQ ID NO. ______) as "FR1 LC-09",
preferably at least about 95% identical or greater to that
sequence. In one embodiment, the FR1 comprises at least one of the
following amino acid changes: 11Q to L; 15L to V; 17E to D; and 18
to R. A preferred FR1 includes two or three of such amino acid
changes with all four amino acid changes being generally
preferred.
[0111] The present invention also features humanized antibodies in
which a second framework (FR2) of the light chain hypervariable
region is at least about 90% identical to the amino acid sequence
shown in FIG. 12A (SEQ ID NO. ______) as "FR2 LC-09", preferably at
least about 95% identical or greater to that sequence. A preferred
FR2 has the following amino acid change: 37Q to L.
[0112] Also encompassed by the invention are humanized antibodies
in which a third framework (FR3) of the light chain hypervariable
region is at least about 90% identical to the amino acid sequence
shown in FIG. 12A (SEQ ID NO. ______) as "FR3 LC-09", preferably at
least about 95% identical or greater to that sequence. In one
embodiment, the FR3 has at least one of the following amino acid
changes: 70K to D, 74K to T, 80A to P, 84A to V, and 85N to T.
Preferably, the FR3 has two, three, or four of such amino acid
changes with all five of the changes being generally preferred.
[0113] Additional humanized antibodies of the invention include a
fourth framework (FR4) of the light chain hypervariable region
which FR4 is at least about 90% identical to the sequence shown in
FIG. 12A (SEQ ID NO. ______) as "FR4 LC-09", preferably at least
about 95% identical or greater to that sequence. In one embodiment,
the FR4 includes at least one and preferably all of the following
amino acid changes: 100A to Q; and 106L to I.
[0114] The invention also features a human TF binding fragment of
the foregoing humanized antibodies. Examples of such fragments
include Fab, Fab', and F(ab).sub.2.
[0115] In a particular embodiment, the invention features a
humanized antibody that includes at least one rodent
complementarity determining region (CDR), usually mouse.
Preferably, that antibody binds specifically to human tissue factor
(TF) to form a complex in which factor X or factor IX binding to TF
or TF/VIIa and activation by TF/VIIa thereto is inhibited. Also
preferably, the humanized antibody includes, on the heavy chain, at
least one of and more preferably all of the following
components:
[0116] a) a first CDR (CDR1) which is at least 95% identical to
CDR1 amino acid sequence shown in FIG. 13B (SEQ ID NO. ______),
[0117] b) a second CDR (CDR2) which is at least 95% identical to
the CDR2 amino acid sequence shown in FIG. 13C (SEQ ID NO.
______),
[0118] c) a third CDR (CDR3) which is at least 95% identical to the
CDR3 amino acid sequence shown in FIG. 13D (SEQ ID NO. ______),
[0119] d) a first framework (FR1) which is at least 95% identical
to the amino acid sequence shown in FIG. 13A (SEQ ID NO. ______) as
"FR1 HC-08",
[0120] e) a second framework (FR2) which is at least 95% identical
to the amino acid sequence shown in FIG. 13A (SEQ ID NO. ______) as
"FR2 HC-08",
[0121] f) a third framework (FR3) which is at least 95% identical
to the amino acid sequence shown in FIG. 13A (SEQ ID NO. ______) as
"FR3 HC-08", and
[0122] g) a fourth framework (FR4) which is at least 95% identical
to the amino acid sequence shown in FIG. 13A (SEQ ID NO. ______) as
"FR4 HC-08".
[0123] In a particular embodiment, the humanized antibody also
includes, on the light chain, at least one of and preferably all of
the following components:
[0124] h) a first CDR (CDR1) which is at least 95% identical to
CDR1 amino acid sequence shown in FIG. 12B (SEQ ID NO. ______),
[0125] i) a second CDR (CDR2) which is at least 95% identical to
the CDR2 amino acid sequence shown in FIG. 12C (SEQ ID NO.
______),
[0126] j) a third CDR (CDR3) which is at least 95% identical to the
CDR3 amino acid sequence shown in FIG. 12C (SEQ ID NO. ______),
[0127] k) a first framework (FR1) which is at least 95% identical
to the amino acid sequence shown in FIG. 12A (SEQ ID NO. ______) as
"FR1 LC-09",
[0128] l) a second framework (FR2) which is at least 95% identical
to the amino acid sequence shown in FIG. 12A (SEQ ID NO. ______) as
"FR2 LC-09",
[0129] m) a third framework (FR3) which is at least 95% identical
to the amino acid sequence shown in FIG. 12A (SEQ ID NO. ______) as
"FR3 LC-09", and
[0130] n) a fourth framework (FR4) which is at least 95% identical
to the amino acid sequence shown in FIG. 12A (SEQ ID No. ______) as
"FR4 LC-09". Preferably, the humanized antibody further includes
the light chain constant sequence of FIG. 14A (SEQ ID No. ______)
or FIG. 15A (SEQ ID No. ______). Also preferably, the antibody
includes the heavy chain constant region of FIG. 14B (SEQ ID No.
______) or FIG. 15B (SEQ ID No. ______).
[0131] The invention also features a humanized antibody that
includes, on the heavy chain, at least one of and preferably all of
the following components:
[0132] a) a first CDR (CDR1) identical to the CDR1 amino acid
sequence shown in FIG. 13B (SEQ ID NO. ______),
[0133] b) a second CDR (CDR2) identical to the CDR2 amino acid
sequence shown in FIG. 13C (SEQ ID NO. ______),
[0134] c) a third CDR (CDR3) identical to the CDR3 amino acid
sequence shown in FIG. 13D (SEQ ID NO. ______),
[0135] d) a first framework (FR1) identical to the amino acid
sequence shown in FIG. 13A (SEQ ID NO. ______) as "FR1 HC-08",
[0136] e) a second framework (FR2) identical to the amino acid
sequence shown in FIG. 13A (SEQ ID NO. ______) as "FR2 HC-08",
[0137] f) a third framework (FR3) identical to the amino acid
sequence shown in FIG. 13A (SEQ ID NO.) as "FR3 HC-08"; and
[0138] g) a fourth framework (FR4) identical to the amino acid
sequence shown in FIG. 13A (SEQ ID No. ______) as "FR4 HC-08".
[0139] In one embodiment, the humanized antibody further includes,
on the light chain, at least one of and preferably all of the
following components:
[0140] h) a first CDR (CDR1) identical to CDR1 amino acid sequence
shown in FIG. 12B (SEQ ID NO. ______),
[0141] i) a second CDR (CDR2) identical to the CDR2 amino acid
sequence shown in FIG. 12C (SEQ ID NO. ______),
[0142] j) a third CDR (CDR3) identical to the CDR3 amino acid
sequence shown in FIG. 12D (SEQ ID NO. ______),
[0143] k) a first framework (FR1) identical to the amino acid
sequence shown in FIG. 12A (SEQ ID NO. ______) as "FR1 LC-09",
[0144] l) a second framework (FR2) identical to the amino acid
sequence shown in FIG. 12A (SEQ ID NO. ______) as "FR2 LC-09",
[0145] m) a third framework (FR3) identical to the amino acid
sequence shown in FIG. 12A (SEQ ID NO. ______) as "FR3 LC-09",
and
[0146] n) a fourth framework (FR4) identical to the amino acid
sequence shown in FIG. 12A (SEQ ID No. ______) as "FR4 LC-09".
Preferably, the humanized antibody further includes the light chain
constant sequence of FIG. 14A (SEQ ID No. ______) or FIG. 15A (SEQ
ID No. ______). Also preferably, the antibody includes the heavy
chain constant region of FIG. 14B (SEQ ID No. ______) or FIG. 15B
(SEQ ID No. ______).
[0147] The humanized antibodies of the present invention may exist
in a variety of suitable forms in addition to whole antibodies;
including, for example, Fv, Fab, and F(ab').sub.2 as well as
bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J.
Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al.,
Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et
al., Science 242, 423-426 (1988), which are incorporated herein by
reference). (See, Hood et al., Immunology, Benjamin, N.Y., 2.sup.nd
ed. (1984), Harlow and Lane, Antibodies. A Laboratory Manual, Cold
Spring Harbor Laboratory (1988) and Hunkapiller and Hood, Nature,
323, 15-16 (1986), which are incorporated herein by
reference.).
[0148] By the phrase "chimeric antibody" or related phrase
including plural forms is meant antibodies whose light and heavy
chain genes have been constructed, typically by genetic
engineering, from immunoglobulin gene segments belonging to
different species. For example, the variable (V) segments of the
genes from a mouse monoclonal antibody may be joined to human
constant (C) segments, such as .gamma..sub.1 .gamma..sub.3. A
typical therapeutic chimeric antibody is thus a hybrid protein
consisting of the V or antigen-binding domain from a mouse antibody
and the C or effector domain from a human antibody, although other
mammalian species may be used. A specifically preferred chimeric
antibody is the cH36 mouse-human chimera disclosed herein.
[0149] The humanized antibodies of the present invention can be
polyclonal or monoclonal, as needed, and may have an IgG1 or IgG4
isotype.
[0150] The humanized antibodies disclosed herein can be produced by
one or a combination of strategies including those already
referenced above. See eg., S. L. Morrison, supra; Oi et al., supra;
Teng et al., supra; Kozbor et al., supra; Olsson et al.,supra; and
other references cited previously.
[0151] In one approach, four general steps were employed to
humanize the antibodies. First, the amino acid sequences of the
mouse antibody light and heavy chains were obtained from the cH36
mouse-human chimeric antibody. Second, the cH36 antibody was
humanized by determining which human antibody framework region gave
the "best fit" i.e., most closely resembled the corresponding mouse
framework amino acid sequence. Third, relevant light and heavy
chain FR sequences were humanized, and fourth, transfection and
expression of isolated nucleic acid(s) that encode the humanized
light or heavy chain (or humanized light and heavy chain e.g., see
the mega vectors described below).
[0152] In some instances, a limited number of framework amino acids
of a humanized immunoglobin were chosen to be the same as the amino
acids at those positions in the donor rather than in the acceptor.
One advantage of this technique is to enhance affinity of the
antibody that includes the humanized immunoglobin chain. See also
U.S. Pat. Nos. 5,985,279; 5,693,762; and EP-A0239400 (disclosing
general methods for making humanized antibodies).
[0153] More particularly, the "best fit" approach was applied to
humanizing the chimeric anti-tissue factor antibody cH36 is
specifically preferred. In this approach, the murine light and
heavy chain variable sequences shown in FIGS. 1A and 1B (SEQ ID
NOS: 2 and 4) were used to search ("compare") all available protein
databases for those human antibody variable domain sequences that
are most homologous to the murine variable domain. See e.g., Kabat
et al., supra. A number of readily available computer programs can
be used to perform this step such as BLAST, FASTA and related
programs. Frameworks 1, 2, 3, and 4 of the light and heavy chain
were of special interest since these sites are almost universally
understood to hold the CDRs in proper orientation for antigen
binding. Output stemming from the search was typically a list of
sequences most homologous to the query mouse sequences, the percent
homology to each sequence, and an alignment of each human sequence
to the corresponding murine sequence. The analysis was generally
performed on the light and heavy chains independently.
[0154] According to the "best fit" approach, the number of
mismatched amino acids was minimized between the query mouse
framework sequence and the corresponding human framework sequence
in the database. In most cases, suitable human framework regions
were selected based on the following identity criteria. On the
light chain, the amino acid sequence of the murine FR1 was at least
about 80% identical to the corresponding human FR1; the murine FR1
was at least about 90% identical to the corresponding human FR2,
the murine FR3 was at least about 90% identical to the human FR3;
and the murine FR4 was at least about 75% identical to the
corresponding human FR4. And on the heavy chain, the amino acid
sequence of the murine FR1 was chosen to be at least about 80%
identical to the corresponding human FR1; the murine FR2 was at
least about 85% identical to the human FR2; the murine FR3 was
chosen to be at least about 70% identical to the corresponding
human FR3; and the murine FR4 was at least about 90% identical to
the corresponding human FR4. Typically, conservative amino acid
substitutions were favored when evaluating similar candidate human
framework sequences. It was found that when such factors were
considered the resulting human frameworks served as a good
reference point for humanization of the chimeric cH36 antibody.
[0155] Also preferably, according to the "best fit" approach all of
the human frameworks on the light and heavy chain were derived from
the same human antibody clone where possible.
[0156] Once a decision on a desired human framework was made,
recombinant polymerase chain reaction (PCR) techniques were used to
make desired amino acid substitutions in both the light and heavy
chains. Typically, oligonucleotides were made and used to
mutagenize mouse variable domain frameworks to contain desired
residues. Oligonucleotides having a variety of lengths were
employed. See WO 92107075 for general disclosure relating to
recombinant PCR and related methods.
[0157] In general, regular PCR was used for cloning, to introduce
cloning or diagnostic endonuclease sites, and to change amino acid
residues located at the ends of the variable regions. PCR-based
mutagenesis was used to change multiple amino acid residues at a
time, especially when these residues were in the middle of the
variable regions. Site directed mutagenesis was used to introduce
one or two amino acid substitutions at a time. After each step, the
partially humanized clones were sequenced and some of these
variable regions were later cloned into expression vectors. More
specific methods for performing these manipulations are described
in the Examples section.
[0158] After performing the foregoing "tbest fit" approach to
humanizing the chimeric cH36 antibody, mutagenized nucleic acids
encoding framework and/or CDR were linked to an appropriate DNA
encoding a light or heavy chain constant region. Such constructs
were then cloned into an expression vector, and transfected into
host cells, preferably mammalian cells. These steps were achieved
by using recombinant and cell culture techniques known in the
field. Accordingly, a humanized antibody of the invention can be
prepared by the following general method:
[0159] (a) preparing a first expression vector including a replicon
appropriate for the expression host and a suitable promoter
operably linked to a DNA sequence which encodes at least a variable
domain of an Ig heavy or light chain, the variable domain
comprising humanized framework regions 1-4 made according to the
"best fit" approach and murine CDRs 1-3 from the cH36 antibody,
[0160] (b) preparing a second replicable expression vector
including a suitable promoter operably linked to a DNA sequence
which encodes at least the variable domain of a complementary Ig
light or heavy chain respectively, that variable domain comprising
complementary humanized framework regions 1-4 made according to the
foregoing "best fit" approach and murine CDRs 1-3 from the cH36
antibody;
[0161] (c) transfecting a cell line with the first or both prepared
vectors; and
[0162] (d) culturing said transfected cell line to produce said
altered antibody.
[0163] Preferably the DNA sequence in steps (a) and (b) encode
suitable constant domains from the human antibody chain. Suitable
isotypes include IgG1 and IgG4, for example.
[0164] Alternatively, a suitable humanized antibody of the
invention can be prepared by making a single replicable "mega"
vector that includes an appropriate promoter operably linked to a
DNA sequence which encodes at least a variable domain of an Ig
heavy or light chain, the variable domain comprising humanized
framework regions 1-4 made according to the "best fit" approach and
murine CDRs 1-3 from the cH36 antibody. Preferably, the mega vector
will further include a suitable promoter operably linked to a DNA
sequence which encodes at least the variable domain of a
complementary Ig light or heavy chain respectively, that variable
domain comprising complementary humanized framework regions 1-4
made according to the foregoing "best fit" approach and murine CDRs
1-3 from the cH36 antibody. Use of the mega vector will often be
appropriate in invention embodiments in which humanized antibody
expression from a single vector is needed.
[0165] Other methods are well-suited for making the humanized
antibodies and fragments of this invention. In one embodiment, the
method includes at least one and preferably all of the following
steps:
[0166] a) comparing the amino acid sequence of a light chain
framework from a rodent antibody against a collection of
corresponding human antibody framework sequences, preferably a
mouse antibody,
[0167] b) selecting a human framework sequence from the collection
having the greatest amino acid sequence identity (i.e., at least
about 70% sequence identity) to the corresponding rodent light
chain framework,
[0168] c) mutagenizing a DNA segment encoding the rodent light
chain framework to encode a humanized light chain framework having
an amino acid sequence that is substantially identical (i.e., at
least about 95% identical) to the human framework sequence selected
in step b),
[0169] d) repeating steps a) thru c) for each individual framework
of the rodent light chain to produce a plurality of DNA sequences
in which each sequence encodes a humanized light chain framework in
which each of the corresponding human framework sequences selected
in step b) is preferably from the same or different human
antibody,
[0170] e) assembling into a first vector encoding at least the
light chain variable region of the rodent antibody, the DNA
sequences encoding the humanized framework sequences produced in
step d); and
[0171] f) introducing the assembled vector into a suitable host
under conditions sufficient to produce the humanized antibody.
Preferred light chain framework sequences for use with the method
include those specific mouse and humanized light chain frameworks
disclosed herein.
[0172] In one embodiment, the foregoing method for making the
humanized antibody further includes at least one and preferably all
of the following steps:
[0173] g) comparing the amino acid sequence of a heavy chain
framework from the rodent antibody against a collection of
corresponding human antibody framework sequences,
[0174] h) selecting a human framework sequence from the collection
having the greatest amino acid sequence identity (i.e., at least
about 70% sequence identity) to the corresponding rodent heavy
chain framework,
[0175] i) mutagenizing a DNA segment encoding the rodent heavy
chain framework to encode a humanized heavy chain framework having
an amino acid sequence that is substantially identical (i.e. at
least about 95% identical) to the human framework sequence selected
in step h); and
[0176] j) repeating steps g) thru i) for each individual framework
of the rodent heavy chain to produce a plurality of DNA sequences
in which each sequence encodes a humanized heavy chain framework.
Preferably, each of the corresponding human framework sequences
selected in step h) are from the same or different human antibody.
Preferred heavy chain framework sequences for use with the method
include those specific mouse and humanized heavy chain frameworks
disclosed herein.
[0177] More particular methods for making the humanized antibody
include assembling into a second vector encoding at least the heavy
chain variable region of the rodent antibody, the DNA sequences
encoding the humanized framework sequences produced in step j); and
introducing the assembled first and second vectors into the host
under conditions sufficient to produce the humanized antibody.
[0178] As discussed, it will often be preferable to express the
humanized antibodies of this invention from a single vector which
can sometimes be a "mega" vector. In one embodiment, the method
includes assembling into the first vector encoding at least the
heavy chain variable region and the light chain variable region of
the rodent antibody, the DNA sequences encoding the humanized
framework sequences produced in step j); and introducing the
further assembled first vector into the host under conditions
sufficient to produce the humanized antibody.
[0179] By the words "assembling" or "assembled" is meant use of
standard recombinant techniques to introduce subject DNA sequences
encoding the humanized frameworks into the vectors. Such assembly
can be performed by one or combination of approaches including, but
not limited to, introducing iterative changes to a single framework
sequence, cutting and pasting fragments together (via use of
restriction endonucleases and ligase), or by synthetic DNA
synthesis techniques. See generally Harlow and Lane supra and
Ausubel et al. supra.
[0180] The foregoing methods for making humanized antibodies can be
practiced with nearly any acceptable mutagenesis technique. In
particular, one or both of steps c) and i), above, can employ site
directed mutagenesis or standard PCR methods to replace desired
rodent amino acids in the framework with appropriate human amino
acids. Typically, the sequence of the modified (humanized)
framework corresponds to the selected human framework sequence from
the database.
[0181] The humanized antibody can be prepared using any suitable
recombinant expression system such as those disclosed in S. L.
Morrison, supra; Oi et al., supra; Teng et al., supra; Kozbor et
al., supra; Olsson et al.,supra; and other references cited
previously.
[0182] For example, suitable nucleic acids of the invention encode
at least one of the heavy or light chain of the humanized
antibodies or fragments thereof disclosed herein. Typically, the
nucleic acid is a recombinant DNA vector that includes the isolated
nucleic acid. The DNA vector will typically further include an
expression control polynucleotide sequence operably linked to the
humanized immunoglobulin coding sequences, including
naturally-associated or heterologous promoter regions. Preferably,
the expression control sequences will be eukaryotic promoter
systems in vectors capable of transforming or transfecting
eukaryotic host cells, but control sequences for prokaryotic hosts
may also be used. Once the vector has been incorporated into the
appropriate host, the host is maintained under conditions suitable
for high level expression of the nucleotide sequences, and, as
desired, the collection and purification of the light chains, heavy
chains, light/heavy chain dimers or intact antibodies, binding
fragments or other immunoglobulin forms may follow.
[0183] The nucleic acid sequences of the present invention capable
of ultimately expressing the desired humanized antibodies can be
formed from a variety of different polynucleotides (genomic or
cDNA, RNA, synthetic oligonucleotides, etc.) and components (e.g.,
V, J, D, and C regions), as well as by a variety of different
techniques. Joining appropriate genomic and synthetic sequences is
presently the most common method of production, but cDNA sequences
may also be utilized. See eg., S. L. Morrison, supra; Oi et al.,
supra; Teng et al., supra; Kozbor et al., supra; Olsson et al.
,supra; European Patent Publication No. 0239400 and Riechmann, L.
et al., Nature, 332, 323-327 (1988); and references cited
therein.
[0184] In one embodiment, suitable DNA expression vectors include
one or more selection markers, e.g., tetracycline, ampicillin, or
neomycin, to permit detection of those cells transformed with the
desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362, which is
incorporated herein by reference). E. coli is one prokaryotic host
useful particularly for cloning the polynucleotides of the present
invention. Other microbial hosts suitable for use include but are
not limited to bacilli, such as Bacillus subtilus, and other
enterobacteriacea, such as Salmonella, Serratia, various
Pseudomonas species and other microbes such as actinomycetes (e.g.,
Streptomyces species), yeast (e.g., Saccharomyces species) or fungi
(e.g., Aspergillus species). In these prokaryotic hosts, one can
also make expression vectors, which will typically contain
expression control sequences compatible with the host cell (e.g.,
promoters and an origin of replication). In addition, any number of
a variety of well-known promoters will be present, such as the
lactose promoter system, a tryptophan (trp) promoter system, a
beta-lactamase promoter system, or a promoter system from phage
lambda. The promoters will typically control expression, optionally
with an operator sequence, and have ribosome binding site sequences
and the like, for initiating and completing transcription and
translation. Other microbes, such as yeast, may also be used for
expression. Saccharomyces is a preferred host, with suitable
vectors having expression control sequences, such as promoters,
including 3-phosphoglycerate kinase or other glycolytic enzymes,
and an origin of replication, termination sequences and the like as
desired. Plants (e.g., Arabidopsis, Nicotinia, etc.) and plant cell
culture may also be used to express and produce the antibodies of
the present invention
[0185] In addition to forgoing microorganism-based systems,
mammalian tissue cell culture may also be used to express and
produce the polypeptides of the present invention (see, Winnacker,
From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987), which is
incorporated herein by reference).
[0186] In many instances, eukaryotic cells will be generally
preferred, typically CHO cell lines, various COS cell lines, NS0
cells, BK cells, HeLa cells, preferably myeloma cell lines, etc.,
or transformed B-cells of hybridomas. Expression vectors for these
cells can include expression control sequences, such as an origin
of replication, a promoter, and enhancer (Queen et al., Immunol.
Rev. 89, 46-68 (1986)), and necessary processing information sites,
such as ribosome binding sites, RNA splice sites, polyadenylation
sites, and transcriptional terminator sequences. Preferred
expression control sequences are promoters derived from
immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus,
cytomegalovirus and the like.
[0187] Preferred DNA vectors for practicing the invention include
the following operatively linked sequences: an antibiotic
resistance marker e.g., ampicillin resistance, F1 origin, and heavy
chain (HC) or light chain (LC) variable region. That variable
region can be inserted into an appropriate HC expression that
includes operatively linked in sequence: the HC variable region,
human IgG1 or IgG4 constant region, first poly A site, SV40
promoter, antibiotic resistance marker such as neomycin resistance,
second poly A site, cytomegelovirus (CMV) promoter/enhancer, and
suitable leader sequence.
[0188] Additionally preferred DNA vectors include the LC variable
region operatively linked to a rodent kappa intron (e.g., mouse)
which intron is operatively linked to a suitable human kappa
constant region; and antibiotic resistance marker such a neomycin
resistance.
[0189] As discussed, it will often be highly useful to express
humanized antibodies of the present invention from a single nucleic
acid. A preferred DNA vector is sometime referred to herein as a
"mega" vector and includes operatively linked in sequence the
following components: SV40 promoter, antibiotic resistance marker
such as neomycin, first poly A site, first CMV promoter/enhancer,
LC variable region, rodent kappa intron (e.g., mouse), human kappa
exon, second poly A site, second CMV promoter/enhancer, HC variable
sequence, and human IgG1 or IgG4 heavy chain constant region. A
specific example of such a mega vector is the humanized anti-TF
IgG1 antibody expression vector described below in Example ______.
See also FIG. 11.
[0190] The following three nucleic acid vectors pSUN36 (humanized
anti-TF antibody Ig G1-HC expression vector), pSUN37 (humanized
anti-TF antibody Ig G4-HC expression vector), and pSUN38 (humanized
anti-TF antibody LC expression vector) have been deposited pursuant
to the Budapest Treaty with the American Type Culture Collection
(ATCC) at 10801 University Boulevard, Manassas Va. 20110-2209. The
vectors were assigned the following Accession Numbers: PTA-3727
(pSUN36); PTA-3728 (pSUN37); and PTA-3729 (pSUN38).
[0191] A variety of suitable host cells can be used to produce the
humanized antibodies or fragments disclosed herein. In one
embodiment the method includes providing a host cell transfected
with either 1) a first expression vector encoding the light chain
of the humanized antibody or fragment thereof and a second
expression vector encoding the heavy chain of the humanized
antibody or fragment thereof, or 2) a single expression vector
encoding both the light chain and the heavy chain of the humanized
antibody or fragment thereof, maintaining the host cell under
growth conditions in which each chain is expressed; and isolating
the humanized antibody or fragment thereof.
[0192] For example, the cell line that is transfected to produce
the humanized antibody can be Chinese Hamster Ovary (CHO) cell
line, BK cell line or NSO cell line. Further acceptable cell lines
include recognized immortalized mammalian cell lines, preferably of
lymphoid origin, such as a myeloma, hybridoma, trioma or quadroma
cell lines. The cell line may also comprise a normal lymphoid cell,
such as a B-cell, which has been immortalized by transformation
with a virus, such as the Epstein-Barr virus. Methods for using CHO
cells for expression of a variety of proteins have been reported.
See e.g., Urlaub et al., Proc. Natl. Acad. Sci. U.S.A., 77
4216-4220 (1980)) and WO 87/04462. NSO cells, as described below in
the Examples section, are also preferred.
[0193] Although the cell line used to produce the humanized
antibody is preferably a mammalian cell line, any other suitable
cell, such as a bacterial cells, plant cells, insect cells or yeast
cells, may alternatively be used. In particular, it is envisaged
that E. coli-derived bacterial strains could be used.
[0194] Once expressed from an appropriate cell source, the whole
antibodies, their dimers, individual light and heavy chains, or
other immunoglobulin forms of the present invention such as
functional humanized antibody fragments can be recovered and
purified according to standard procedures. Such procedures include,
but are not limited to, ammonium sulfate precipitation, affinity
columns, column chromatography, gel electrophoresis and the like
(See, generally, Scopes, R., Protein Purification, Springer-Verlag,
N.Y. (1982)). Substantially pure humanized antibodies of the
invention and fragments thereof feature at least about 90 to 95%
homogeneity with about 98 to 99% or more homogeneity being
generally preferred for most pharmaceutical uses. Once purified,
partially or to homogeneity as desired, a humanized antibody may
then be used therapeutically or in developing and performing assay
procedures, immunofluorescent stainings, and the like (See,
generally, Immunological Methods, Vols. I and II, Lefkovits and
Pernis, eds., Academic Press, New York, N.Y. (1979 and 1981)).
[0195] A preferred method of purifying the present humanized
antibodies involves conventional affinity and ion exchange
chromatography, preferably using recombinant Protein A Sepharose
(to which human Ig G Fc has recognized high affinity). Antibody
containing fractions are collected and subjected to further ion
exchange chromatography, preferably using Q Sepharose. Antibody
containing protein peaks are pooled and dialyzed against an
appropriate solution or buffer, for instance, PBS.
[0196] Humanized antibodies and fragments thereof according to the
invention can be tested for function by one or a combination of
standard methods. Preferred tests assay for inhibition of TF
function. A preferred method is what is sometimes referred to
herein as a "standard prothrombin time" assay or related phrase.
The standard prothrombin time (PT) assay typically involves at
least one and preferably all of the following steps:
[0197] a) combining TF and factor VIIa to form a binding
complex,
[0198] b) contacting the binding complex with factor X (or factor
IX) under conditions conducive to forming factor Xa (or factor
IXa),
[0199] c) contacting the factor Xa with prothrombin to produce
thrombin, preferably in the presence of factor Va and lipids.
[0200] A preferred source of the TF for conducting the standard PT
assay is commercially available as Innovin. A preferred source for
the blood factors is a human plasma preparation called Ci-Trol
Coagulation Control.
[0201] The humanized antibodies and fragments thereof provided
herein can be readily tested in the assay. An aliquot of the
purified antibody or fragment, preferably about 200 nM to about
2000 nM, is added to the method, preferably before step a) although
addition at other points in the assay may be preferred for some
applications. Typically, the humanized antibody or fragment is
added to the Ci-Trol Coagulation Control followed by addition of
the TF.
[0202] Highly preferred humanized antibodies and fragments thereof
including whole IgG, Fab, Fab', F(ab).sub.2, and single chain
antibodies (comprising the antigen binding variable regions of the
humanized antibodies) will increase blood clotting time by at least
about 5 seconds when present in the standard assay at a
concentration of at least about 1 nM to about 20 nM, preferably
about 5 nM to about 15 nM, more preferably about 10 nm in the
assay. A typical control is a standard PT assay performed without
adding any antibody of fragment. Additionally preferred antibodies
and fragments of the invention achieve at least about 90%
inhibition of TF-dependent coagulation, preferably at least about
95% inhibition or greater when compared to the control. A specific
example of the standard PT assay is described in Examples 5 and
______.
[0203] Although a range of therapeutic anti-coagulant compositions
of the invention have been described above, other compositions that
include the humanized antibodies and fragments thereof are
contemplated. For example, such antibodies and fragments may be
used as the sole therapeutic agent or in combination with one or
more other humanized antibodies or fragments to achieve a desired
outcome. Such antibodies and fragments may also be used in
combination with other antibodies, particularly human monoclonal
antibodies reactive with other markers on cells responsible for the
disease.
[0204] A wide spectrum of important uses for the present antibodies
and fragments have been described above e.g., use to detect native
TF in a biological sample, use to detect and purify cells
expressing TF, and use to prevent or treat medical conditions such
as undesired blood coagulation in a human patient. In practice, the
humanized antibodies can be used as separately administered
compositions given in conjunction with other anti-clotting agents
including aspirin, coumadin, heparin, hirudin, or hirulog. Also
envisioned is co-administration with anti-platelet (e.g., ReoPro,
Integrilin, Aggrestat, Plavix, and/or Ticlid) and/or thrombolytic
agents (e.g., tissue plasminogen activator, strepokinase and
urokinase).
[0205] In embodiments in which the therapeutic anti-coagulant
compositions described herein include one or more humanized
antibodies or fragments, that composition may include a solution of
the antibody or a cocktail thereof dissolved in an acceptable
carrier, preferably an aqueous carrier. A variety of aqueous
carriers have already been referenced such as water, buffered
water, 0.4% saline, 0.3% glycine and the like. These solutions are
preferably sterile and generally free of particulate matter. These
compositions may be sterilized by conventional, well known
sterilization techniques. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, toxicity adjustment agents and the like, for
example sodium acetate, sodium chloride, potassium chloride,
calcium chloride, sodium lactate, etc. The concentration of
antibody in these formulations can vary widely, for example from
less than about 0.5%, usually at or at least about 1% to as much as
15 or 20% by weight and will be selected primarily based on fluid
volumes, viscosities, etc., in accordance with the particular mode
of administration selected. See generally, Remington's
Pharmaceutical Sciences, supra.
[0206] If desired, the therapeutic anti-coagulant compositions
described herein can be lyophilized for storage and reconstituted
in a suitable carrier prior to use. This technique has been shown
to be effective with conventional immune globulins. Any suitable
lyophilization and reconstitution techniques can be employed. It
will be appreciated by those skilled in the art that lyophilization
and reconstitution can lead to varying degrees of antibody activity
loss (e.g., with conventional immune globulins, IgM antibodies tend
to have greater activity loss than IgG antibodies) and that use
levels may have to be adjusted to compensate.
[0207] For some prophylactic applications, it will be helpful to
administer the therapeutic anti-coagulant compositions to a patient
not already in a detectable disease state to enhance the patient's
resistance to the disease. Such an amount is defined to be a
"prophylactically effective dose". In this use, the precise amounts
again depend upon the patient's state of health and general level
of immunity, but generally range from 0.1 to 25 mg per dose,
especially 0.5 to 2.5 mg per patient. A preferred prophylactic use
is for the prevention of undesired blood clotting following a
planned invasive medical procedure.
[0208] As discussed, the invention also features kits that include
subject antibodies or fragments thereof. In one embodiment, the
humanized antibodies or fragments thereof can be supplied for use
against or in the detection of TF antigen. Thus, for instance, one
or more humanized antibodies, fragments thereof, or single chain
antibodies may be provided, usually in a lyophilized form in a
container. Such antibodies, fragments, or single chain antibodies,
which may be conjugated to a previously mentioned label or toxin,
or unconjugated, are included in the kits with buffers, such as
Tris, phosphate, carbonate, etc., stabilizers, biocides, inert
proteins, e.g., serum albumin, or the like. Generally, these
materials will be present in less than about 5% by weight based on
the amount of active antibody, and usually present in total amount
of at least about 0.001% wt. based again on the antibody
concentration. Frequently, it will be desirable to include an inert
extender or excipient to dilute the active ingredients, where the
excipient may be present in from about 1 to 99% wt. of the total
composition. Where a second antibody capable of binding to the
chimeric antibody is employed in an assay, this will usually be
present in a separate vial. The second antibody is typically
conjugated to a label and formulated in an analogous manner with
the antibody formulations described above. The kit will generally
also include a set of instructions for use.
[0209] As discussed, the invention also provides a variety of
methods of inhibiting blood coagulation in a mammal, preferably a
primate such as a human patient.
[0210] For example, in one embodiment, the methods include
administering to the mammal a therapeutically effective amount of
at least one of, preferably one, two or three of the humanized
antibodies provided herein or a fragment thereof that binds
specifically to human tissue factor (TF) to form a complex.
Typically, factor X or factor IX binding to TF or TF:FVIIa and
activation by TF:FVIIa thereto is inhibited. In most embodiments,
the methods further include forming a specific complex between the
antibody and the TF to inhibit the blood coagulation.
[0211] Also provided are methods of inhibiting blood coagulation in
a mammal that include administering to the mammal, a
therapeutically effective amount of the humanized antibodies
disclosed herein or a fragment thereof. Typical antibodies and
fragments bind specifically to human tissue factor (TF) to form a
complex, and further wherein factor X or factor IX binding to TF or
TF:FVIIa and activation by TF:FVIIa thereto is inhibited. In most
embodiments, the methods further include forming a specific complex
between the antibody and the TF to inhibit the blood
coagulation.
[0212] In a more specific example, the invention provides methods
of inhibiting blood coagulation in a mammal that include
administering to the mammal, a therapeutically effective amount of
a humanized antibody or fragment thereof disclosed herein.
Typically, the antibody binds specifically to human tissue factor
(TF) to form a complex, and further wherein factor X or factor IX
binding to TF or TF:FVIIa and activation by TF:FVIIa thereto is
inhibited. Preferably, the humanized antibody or fragment includes,
on the heavy chain, at least one of and preferably all of the
following components:
[0213] a) a first CDR (CDR1) which is at least 95% identical to
CDR1 amino acid sequence shown in FIG. 13B (SEQ ID NO. ______),
[0214] b) a second CDR (CDR2) which is at least 95% identical to
the CDR2 amino acid sequence shown in FIG. 13C (SEQ ID NO.
______),
[0215] c) a third CDR (CDR3) which is at least 95% identical to the
CDR3 amino acid sequence shown in FIG. 13D (SEQ ID NO. ______),
[0216] d) a first framework (FR1) which is at least 95% identical
to the amino acid sequence shown in FIG. 13A (SEQ ID NO. ______) as
"FR1 HC-08",
[0217] e) a second framework (FR2) which is at least 95% identical
to the amino acid sequence shown in FIG. 13A (SEQ ID NO. ______) as
"FR2 HC-08",
[0218] f) a third framework (FR3) which is at least 95% identical
to the amino acid sequence shown in FIG. 13A (SEQ ID NO. ______) as
"FR3 HC-08",
[0219] g) a fourth framework (FR4) which is at least 95% identical
to the amino acid sequence shown in FIG. 13A (SEQ ID No. ______) as
"FR4 HC-08".
[0220] In a more specific invention embodiment, the humanized
antibody includes, on the light chain, at least one of, and
preferably all of the following components:
[0221] h) a first CDR (CDR1) which is at least 95% identical to
CDR1 amino acid sequence shown in FIG. 12B (SEQ ID NO. ______),
[0222] i) a second CDR (CDR2) which is at least 95% identical to
the CDR2 amino acid sequence shown in FIG. 12C (SEQ ID NO.
______),
[0223] j) a third CDR (CDR3) which is at least 95% identical to the
CDR3 amino acid sequence shown in FIG. 12D (SEQ ID NO. ______),
[0224] k) a first framework (FR1) which is at least 95% identical
to the amino acid sequence shown in FIG. 12A (SEQ ID NO. ______) as
"FR1 LC-09",
[0225] l) a second framework (FR2) which is at least 95% identical
to the amino acid sequence shown in FIG. 12A (SEQ ID NO. ______) as
"FR2 LC-09",
[0226] m) a third framework (FR3) which is at least 95% identical
to the amino acid sequence shown in FIG. 12A (SEQ ID NO. ______) as
"FR3 LC-09",
[0227] n) a fourth framework (FR4) which is at least 95% identical
to the amino acid sequence shown in FIG. 12A (SEQ ID No. ______) as
"FR4 LC-09",
[0228] o) a light chain constant region which is at least 95%
identical to the amino acid sequence shown in FIG. 14A (SEQ ID No.
______) or FIG. 15A (SEQ ID No. ______); and
[0229] p) a heavy chain constant region which is at least 95%
identical to the amino acid sequence shown in FIG. 14B (SEQ ID No.
______) or FIG. 15B (SEQ ID No. ______).
[0230] In a more specific embodiment of the foregoing method, the
humanized antibody or fragment thereof includes, on the heavy
chain, at least one of and preferably all of the following
components:
[0231] a) a first CDR (CDR1) identical to CDR1 amino acid sequence
shown in FIG. 13B (SEQ ID NO. ______),
[0232] b) a second CDR (CDR2) identical to the CDR2 amino acid
sequence shown in FIG. 13C (SEQ ID NO. ______),
[0233] c) a third CDR (CDR3) identical to the CDR3 amino acid
sequence shown in FIG. 13D (SEQ ID NO. ______),
[0234] d) a first framework (FR1) identical to the amino acid
sequence shown in FIG. 13A (SEQ ID NO. ______) as "FR1 HC-08",
[0235] e) a second framework (FR2) identical to the amino acid
sequence shown in FIG. 13A (SEQ ID NO. X as "FR2 HC-08",
[0236] f) a third framework (FR3) identical to the amino acid
sequence shown in FIG. 13A (SEQ ID NO. ______) as "FR3 HC-08",
[0237] g) a fourth framework (FR4) identical to the amino acid
sequence shown in FIG. 13A (SEQ ID NO. ______) as "FR HC-08";
[0238] and on the light chain:
[0239] h) a first CDR (CDR1) identical to CDR1 amino acid sequence
shown in FIG. 12B (SEQ ID NO. ______),
[0240] i) a second CDR (CDR2) identical to the CDR2 amino acid
sequence shown in FIG. 12C (SEQ ID NO. ______),
[0241] j) a third CDR (CDR3) identical to the CDR3 amino acid
sequence shown in FIG. 12D (SEQ ID NO. ______),
[0242] k) a first framework (FR1) identical to the amino acid
sequence shown in FIG. 12A (SEQ ID NO. ______) as "FR1 LC-09",
[0243] l) a second framework (FR2) identical to the amino acid
sequence shown in FIG. 12A (SEQ ID NO. ______) as "FR2 LC-09",
[0244] m) a third framework (FR3) identical to the amino acid
sequence shown in FIG. 12A (SEQ ID NO. ______) as "FR3 LC-09",
[0245] n) a fourth framework (FR4) identical to the amino acid
sequence shown in FIG. 12A (SEQ ID No. ______) as "FR4 LC-09",
[0246] o) a light chain constant region which is identical to the
amino acid sequence shown in FIG. 14A (SEQ ID No. ______) or FIG.
15A (SEQ ID No. ______), and
[0247] p) a heavy chain constant region which is identical to the
amino acid sequence shown in FIG. 14B (SEQ ID No. ______) or FIG.
15B (SEQ ID No. ______).
[0248] The invention also provides for a variety of methods of
detecting tissue factor (TF) in a biological sample. In one
embodiment, the method includes contacting a biological sample with
the humanized antibodies or fragments thereof disclosed herein
under conditions conducive to forming a complex and detecting the
complex as being indicative of the TF in the biological sample.
[0249] All documents mentioned herein are fully incorporated by
reference in their entirety.
[0250] The following non-limiting examples are illustrative of the
invention. In the following examples and elsewhere the antibodies
H36 and H36.D2 are referred to. Those antibodies are the same
antibody as H36.D2.B7, but H36 is derived from the mother clone,
and H36.D2 is obtained from the primary clone, whereas H36.D2.B7 is
obtained from the secondary clone. No differences have been
observed between those three clones with respect to ability to
inhibit TF or other physical properties. In general usage, H36 is
often used to indicate anti-TF antibody produced by any of these
clones or related cell lines producing the antibody.
EXAMPLE 1
Preparation and Cloning of Anti-rhTF Monoclonal Antibodies
Monoclonal antibodies against rhTF were prepared as follows.
[0251] A. Immunization and Boosts
[0252] Five female BALB/c mice were immunized with 10 .mu.g each of
lipidated, purified rhTF. The mice were initially sensitized
intraperitoneally using Hunter's Titermax adjuvant. Three final
boosts were administered in 0.85% NaCl. Boosts were 2, 5.5, and 6.5
months post initial sensitization. All boosts were given
intraperitoneally, except the first which was subcutaneous. The
final boost was given 3 days pre-fusion and 20 .mu.g was
administered.
[0253] B. Fusion of Mouse Spleen Lymphocytes with Mouse Myeloma
Cells
[0254] Lymphocytes from the spleen of one rhTF immunized BALB/c
mouse was fused to X63-Ag8.653 mouse myeloma cells using PEG 1500.
Following exposure to the PEG, the cells were incubated for one
hour in heat inactivated fetal bovine serum at 37.degree. C. The
fused cells were then resuspended in RPMI 1640 and incubated
overnight at 37.degree. C. with 10% CO.sub.2. The cells were plated
the next day using RPMI 1640 and supplemented with macrophage
culture supernatant.
[0255] C. ELISA Development
[0256] Plates for the ELISA assay were coated with 100 microliters
of recombinant tissue factor (0.25 .mu.g/ml) in a carbonate-based
buffer. All steps were performed at room temperature. Plates were
blocked with BSA, washed, and then the test samples and controls
were added. Antigen/antibody binding was detected by incubating the
plate with goat anti-mouse HRP conjugate (Jackson ImmunoResearch
Laboratories) and then using an ABTS peroxidase substrate system
(Kirkegaard and Perry Laboratories). Absorbance was read on an
automatic plate reader at a wavelength of 405 nm.
[0257] D. Stabilization of rhTF Hybridoma Cell Lines
[0258] Two weeks after fusion, screening of hybridoma colonies by
specific rhTF ELISA was started. Screening for new colonies
continued for three weeks. The positive clones were tested every
one to two weeks for continued antibody production until fifteen
stable clones were frozen down.
[0259] E. Primary and Secondary Cloning
[0260] Limiting dilution cloning was performed on each of the
positive stable hybridomas to obtain primary clones. The cells were
thawed, grown in culture for a short period of time, and then
diluted from 10 cells/well to 0.1 cells/well. Primary clones were
tested by anti-rhTF ELISA and five to six positive clones were
expanded and frozen.
[0261] Secondary clone of anti-rhTF antibody, H36.D2.B7, was
obtained from primary clone, H36.D2, prepared and stored in liquid
nitrogen as described above. Four different dilutions, 5
cells/well, 2 cells/well, 1 cell/well, 0.5 cells/well of the
primary clone were prepared in 96-wells microtiter plates to start
the secondary cloning. Cells were diluted in IMDM tissue culture
media containing the following additives: 20% fetal bovine serum
(FBS), 2 mM L-glutamine, 100 units/ml of penicillin, 100 .mu.g/ml
of streptomycin, 1% GMS-S, 0.075% NaHCO.sub.3. To determine clones
that secrete anti-rhTF antibody, supernatants from five individual
wells of the 0.2 cells/well microtiter plate were withdrawn after
two weeks of growth and tested for the presence of anti-rhTF
antibody by ELISA assays as described above. All five clones showed
positive results in the ELISA assay, with H36.D2.B7 being the best
antibody producer. All five clones were adapted and expanded in
RPMI media containing the following additive: 10% FBS, 2 mM
L-glutamine, 100 units/ml of penicillin, 100 .mu.g/ml of
streptomycin, 1% GMS-S, 0.075% NaHCO.sub.3, and 0.013 mg/ml of
oxalaacetic acid. H36.D2.B7 was purified by Protein A affinity
chromatography from the supernatant of cell culture and was tested
for its ability to inhibit TF:VIIa in a FX activation assay. The
results indicated that H36.D2.B7 had the same inhibition as H36.D2
antibody. All cells were stored in liquid nitrogen.
[0262] F. Isolation of Total RNA from H36.D2.B7
[0263] 269 .mu.g of total RNA was isolated from 2.7.times.10.sup.5
H36.D2.B7 hybridoma cells. The isolation of total RNA was performed
as described in the RNeasy Midi Kits protocol from Qiagen. The RNA
sample was stored in water at -20.degree. C. until needed.
[0264] G. cDNA Synthesis and Cloning of Variable Regions of
H36.D2.B7 Gene
[0265] To obtain the first strand of cDNA, a reaction mixture
containing 5 .mu.g of total RNA isolated as above, back primers
JS300 (all primers are identified below) for the heavy chain (HC)
and OKA 57 for the light chain (LC), RNase inhibitor, dNTP's, DTT,
and superscript II reverse transcriptase, was prepared and
incubated at 42.degree. C. for 1 hour. The reaction tube is then
incubated at 65.degree. C. for 15 minutes to stop the
transcription. After cooling down, five units of RNase H were then
added and the reaction was allowed to incubate at 37.degree. C. for
20 minutes. The cDNA sample was stored at -70.degree. C. until
needed.
[0266] PCR (polymerase chain reaction) was conducted separately to
clone the variable regions of both HC and LC of anti-rhTF,
H36.D2.B7 from the cDNA made as above (nucleic acid and amino acid
sequences of those HC and LC variable regions set forth in FIGS. 1A
and 1B). Three rounds of PCR were conducted. Round 1: PCR was run
for 35 cycles at 96.degree. C., 53.degree. C. and 72.degree. C.
using front primer JS002 and back primer JS300 for HC. For LC front
primer JS009 and back primer OKA 57 were used and PCR was rune for
35 cycles at 96.degree. C., 63.degree. C. and 72.degree. C. Round
2: PCR of both HC and LC was rune the same as in Round 1 with the
exception that pMC-18 was used for HC front primer and pMC-15 for
LC front primer. Round 3: PCR was run for 30 cycles at 96.degree.
C., 60-65.degree. C. and 72.degree. C. using H36HCF and H36HCR
primers for HC. For LC, PCR was run for 30 cycles at 96.degree. C.,
58.degree. C. and 72.degree. C. using H36LCF and H36LCR
primers.
[0267] The following primers were used for cloning H36.D2.B7
variable regions of HC and LC.
1 OKA 57: 5'-GCACCTCCAGATGTTAACTGCTC-3' (SEQ ID NO:17) JS300:
5'-GAARTAVCCCTTGACCAGGC-3' (SEQ ID NO:18) JS009:
5'-GGAGGCGGCGGTTCTGACATTGTGMTGWCMCARTC-3' (SEQ ID NO:19) JS002:
5'-ATTTCAGGCCCAGCCGGCCATGGCCGARGTYCA- RCTKCARCARYC-3' (SEQ ID
NO:20) pMC-15: 5'-CCCGGGCCACCATGKCCCCWRCTCAGYTYCTKG-3' (SEQ ID
NO:21) pMC-18: 5'-CCCGGGCCACCATGGRATGSAGCTGKGTMATSCTC-3' (SEQ ID
NO:22) H36HCF: 5'-ATATACTCGCGACAGCTACAGGTGTCCACTCCGAGATCCAG- CTGCA
(SEQ ID NO:23) GCAGTC-3' H36HCR:
5'-GACCTGAATTCTAAGGAGACTGTGAGAGTGG-3' (SEQ ID NO:24) H36LCF:
5'-TTAATTGATATCCAGATGACCCAGTCTCC-3' (SEQ ID NO:25) H36LCR:
TAATCGTTCGAAAAGTGTACTTACGTTTCAGCTCCAGCTTGGTCC (SEQ ID NO:26)
[0268] wherein in the above SEQ ID NOS: 17 through 26: K is G or T;
M is A or C; R is A or G; S is C or G; V is A, C or G; W is A or T;
Y is C or T.
EXAMPLE 2
Binding Activity of Antibodies of the Invention
[0269] Antibodies of the invention as prepared in Example 1 above
were employed. The rhTF molecule was expressed in E. coli and
purified by immunoaffinlty chromatography in accordance with
standard methods (see Harlow and Lane, supra, Ausubel et al.
supra). Antibody association (Ka) and dissociation (Kd) constants
were determined by ELISA and surface plasmon resonance (i.e.,
BIACore) assays (see e.g., Harlow and Lane, supra; Ausubel et al.
supra; Altschuh et al., Biochem., 31:6298 (1992); and the BIAcore
method disclosed by Pharmacia Biosensor). For BIACore assays, rhTF
was immobilized on a biosensor chip in accordance with the
manufacturer's instructions. Constants for each antibody were
determined at four antibody concentrations (0.125 nM, 0.25 nM, 0.5
nM, and 1 nM).
[0270] Protein concentrations were determined by standard assay
(M.M. Bradford, Anal. Biochem., 72:248 (1976)) using Bovine Serum
Albumin as a standard and a commercially available dye reagent
(Bio-Rad).
[0271] FIG. 2 shows association and disassociation constants for
each anti-TF antibody. Antibody H36 exhibited the highest
association rate (K.sub.a=3.1.times.10.sup.10 M.sup.-1) and the
lowest disassociation rate (K.sub.d=3.2.times.10.sup.-11 M) of any
of the anti-TF antibodies tested.
EXAMPLE 3
FXa-Specific Substrate Assay
[0272] In general, the experiments described herein were conducted
using rhTF lipidated with phosphatidycholine (0.07 mg/ml) and
phosphatidylserine (0.03 mg/ml) at a 70/30 w/w ratio in 50 mM
Tris-HCl, pH 7.5, 0.1% bovine serum albumin (BSA) for 30 minutes at
37.degree. C. A stock solution of preformed TF:FVIIa complex was
made by incubating 5 nM of the lipidated rhTF and 5 nM of FVIIa for
30 minutes at 37.degree. C. The TF:FVIIa complex was aliquoted and
stored at -70.degree. C. until needed. Purified human factors VII,
Vlla, and FX were obtained from Enyzme Research Laboratories, Inc.
The following buffer was used for all FXa and FVIIa assays: 25 mM
Hepes-NaOH, 5 mM CaCl.sub.2, 150 mM NaCl, 0.1% BSA, pH 7.5.
[0273] Monoclonal antibodies were screened for capacity to block
TF:VIIa-mediated activation of FX to FXa. The FX activation was
determined in two discontinuous steps. In the first step (FX
activation), FX conversion to FXa was assayed in the presence of
Ca.sup.+2. In the second step (FXa activity assay), FX activation
was quenched by EDTA and the formation of FXa was determined using
a FXa-specific chromogenic substrate (S-2222). The S-2222 and
S-2288 (see below) chromogens were obtained from Chromogenix
(distributed by Pharmacia Hepar Inc.). FX activation was conducted
in 1.5 ml microfuge tubes by incubating the reaction with 0.08 nM
TF:VIIa, either pre-incubated with an anti-rhTF antibody or a
buffer control. The reaction was subsequently incubated for 30
minutes at 37.degree. C., then 30 mM FX was added followed by an
additional incubation for 10 minutes at 37.degree. C. FXa activity
was determined in 96-well microtiter plates. Twenty microliters of
sample was withdrawn from step one and admixed with an equal volume
of EDTA (500 mM) in each well, followed by addition of 0.144 ml of
buffer and 0.016 ml of 5 mM S-2222 substrate. The reaction was
allowed to incubate for an additional 15-30 minutes at 37.degree.
C. Reactions were then quenched with 0.05 ml of 50% acetic acid,
after which, absorbance at 405 nm was recorded for each reaction.
The inhibition of TF:FVIIa activity was calculated from OD.sub.405.
values in the experimental (plus antibody) and control (no
antibody) samples. In some experiments, an anti-hTF antibody,
TF:FVIIa, and FX were each added simultaneously to detect binding
competition. FIG. 3 shows that the H36.D2 MAb (in bold) inhibited
TF:FVIIa activity toward FX to a significantly greater extent (95%)
than other anti-rHTF Mabs tested.
EXAMPLE 4
FVIIa-Specific Substrate Assay
[0274] Monoclonal antibodies were further screened by an FVIIa
specific assay. In this assay, 5 nM lipidated rhTF was first
incubated with buffer (control) or 50 nM antibody (experimental) in
a 96-well microtiter plate for 30 minutes at 37.degree. C., then
admixed with 5 nM purified human FVIIa (V.sub.T=0.192 ml), followed
by 30 minutes incubation at 37.degree. C. Eight microliters of a 20
mM stock solution of the FVIIa specific substrate S-2288 was then
added to each well (final concentration, 0.8 mM). Subsequently, the
reaction was incubated for one hour at 37.degree. C. Absorbance at
405 nm was then measured after quenching with 0.06 ml of 50% acetic
acid. Percent inhibition of TF:FVIIa activity was calculated from
OD.sub.405nm values from the experimental and control samples.
[0275] FIG. 4 shows the H36 antibody did not significantly block
TF:FVIIa activity toward the S-2288 substrate when the antibody was
either pre-incubated with TF (prior to FVIIa addition) or added to
TF pre-incubated with FVIIa (prior to adding the antibody). This
indicates that H36 does not interfere with the interaction
(binding) between TF and FVIIa, and that H36 also does not inhibit
TF:FVIIa activity toward a peptide substrate.
EXAMPLE 5
Prothrombin Time (PT) Assay
[0276] Calcified blood plasma will clot within a few seconds after
addition of thromplastin (TF); a phenomenon called the "prothrombin
time" (PT). A prolonged PT is typically a useful indicator of
anti-coagulation activity (see e.g., Gilman et al. supra).
[0277] The H36.D2 antibody was investigated for capacity to affect
PT according to standard methods using commercially available human
plasma (Ci-Trol Control, Level I obtained from Baxter Diagnostics
Inc.). Clot reactions were initiated by addition of lipidated rhTF
in the presence of Ca.sup.+2. Clot time was monitored by an
automated coagulation timer (MLA Electra 800). PT assays were
initiated by injecting 0.2 ml of lipidated rhTF (in a buffer of 50
mM Tris-HCl, pH 7.5, containing 0.1% BSA, 14.6 mM CaCl.sub.2, 0.07
mg/ml of phosphatidylcholine, and 0.03 mg/ml of phosphatidylserine)
into plastic twin-well cuvettes. The cuvettes each contained 0.1 ml
of the plasma preincubated with either 0.01 ml of buffer (control
sample) or antibody (experimental sample) for 1-2 minutes. The
inhibition of TF-mediated coagulation by the H36.D2 antibody was
calculated using a TF standard curve in which the log [TF] was
plotted against log clot time.
[0278] FIG. 5 shows the H36.D2 antibody substantially inhibits
TF-initiated coagulation in human plasma. The H36.D2 antibody
increased PT times significantly, showing that the antibody is an
effective inhibitor of TF-initiated coagulation (up to
approximately 99% inhibition).
EXAMPLE 6
FX and H36.D2 Antibody Compete For Binding to the TF:FVIIa
Complex
[0279] Competition experinents were conducted between TF:FVIIa, FX
and the H36.D2 antibody. FIG. 6A illustrates the results of an
experiment in which a preformed TF:FVIIa complex (0.08 nM) was
pre-incubated at 37.degree. C. for 30 minutes in buffer including
0.02 nM, 0.04 nM, 0.08 nM and 0.16 nM of the H36.D2 monoclonal
antibody, respectively. FX (30 nM) was then added to the TF:FVIIa
and H36.D2 antibody mixture and the mixture allowed to incubate for
an additional 10 minutes at 37.degree. C. FX activation was
quenched with EDTA as described previously. The FXa produced
thereby was determined by the FXa-specific assay described in
Example 3, above.
[0280] FIG. 6B shows the results of an experiment conducted along
the lines just-described, except that the H36.D2 antibody,
pre-formed TF:FVIIa, and FX were added simultaneously to start the
FX activation assay.
[0281] The data set forth in FIGS. 6A and 6B show that the H36.D2
antibody and FX compete for binding to the pre-formed TF:FVIIa
complex.
EXAMPLE 7
Inhibition of TF Activity in Cell Culture
[0282] J-82 is a human bladder carcinoma cell line (available from
the ATCC) which abundantly expresses native human TF as a cell
surface protein. To see if the H36.D2 antibody could prevent FX
from binding to native TF displayed on the cell surface, a J-82 FX
activation assay was conducted in microtiter plates in the presence
of FVII (see D. S. Fair et al., J. Biol. Chem., 262:11692 (1987)).
To each well, 2.times.10.sup.5 cells was added and incubated with
either 50 ng FVII, buffer (control sample) or the anti-TF antibody
(experimental sample) for 2 hours at 37.degree. C. Afterwards, each
well was gently washed with buffer and 0.3 ml of FX (0.05 mg/ml)
was added to each well for 30 minutes at room temperature. In some
cases, the antibody was added at the same time as FX to detect
binding competition for the native TF. Thereafter, 0.05 ml aliquots
were removed and added to new wells in a 96-well microtiter plate
containing 0.025 ml of 100 mM EDTA. FXa activity was determined by
the FXa-specific assay as described in Example 3, above. Inhibition
of TF activity on the surface of the J-82 cells was calculated from
the OD.sub.405 nm in the absence (control sample) and presence of
antibody (experimental sample).
[0283] FIG. 7 shows that the H36.D2 antibody bound native TF
expressed on J-82 cell membranes and inhibited TF-mediated
activation of FX. These results indicate that the antibody competes
with FX for binding to native TF displayed on the cell surface.
Taken with the data of Example 8, below, the results also show that
the H36.D2 antibody can bind a conformational epitope on native TF
in a cell membrane.
EXAMPLE 8
Specific Binding of the H36.D2 Antibody to Native rhTF
[0284] Evaluation of H36.D2 binding to native and non-native rhTF
was performed by a simplified dot blot assay. Specifically, rhTF
was diluted to 30 .mu.g/ml in each of the following three buffers:
10 mM Tris-HCl, pH 8.0; 10 mM Tris-HCl, pH 8.0 and 8 M urea; and 10
mM Tris-HCl, pH 8.0, 8 M urea and 5 mM dithiothreitol. Incubation
in the Tris buffer maintains rhTF in native form, whereas treatment
with 8M urea and 5 nM dithiothreitol produces non-native
(denatured) rhTF. Each sample was incubated for 24 hours at room
temperature. After the incubation, a Millipore Immobilon (7.times.7
cm section) membrane was pre-wetted with methanol, followed by 25
mM Tris, pH 10.4, including 20% methanol. After the membranes were
air-dried, approximately 0.5 .mu.1,1 .mu.l, and 2 .mu.l of each
sample (30 .mu.g/ml) was applied to the membrane and air-dried.
After blocking the membrane by PBS containing 5% (w/v) skim milk
and 5% (v/v) NP-40, the membrane was probed with H36.D2 antibody,
followed by incubation with a goat anti-mouse IgG peroxidase
conjugate (obtained from Jackson ImmunoResearch Laboratories,
Inc.). After incubation with ECL Western Blotting reagents in
accordance with the manufacturer's instructions (Amersham), the
membrane was wrapped with plastic film (Saran Wrap) and exposed to
X-ray film for various times.
[0285] FIG. 8A shows that the H36.D2 monoclonal antibody binds a
conformational epitope on native TF in the presence of Tris buffer
or Tris buffer with 8M urea (lanes 1 and 2). The autoradiogram was
exposed for 40 seconds. However, when the native TF was denatured
with 8M urea and 5 mM DTT, H36.D2 binding was significantly reduced
or eliminated (lane 3). FIG. 8B shows an over-exposed autoradiogram
showing residual binding of the H36.D2 antibody to non-native
(i.e., denatured) rhTF. The over-exposure was for approximately 120
seconds. Treatment with 8M urea alone probably resulted in only
partial denaturation of the native rhTF since the two disulfide
bonds in TF are not reduced. It is also possible that the partially
denatured TF may refold back to native confirmation during later
blotting process when urea is removed. These results also clearly
distinguish preferred antibodies of the invention which do not bind
denatured TF from previously reported antibodies which do not
selectively bind to a conformational epitope and bind to denatured
TF (see U.S. Pat. No. 5,437,864 where in FIG. 18 Western Blot
analysis shows binding to TF denatured by SDS).
EXAMPLE 9
Humanization of Anti-Tissue Factor Antibody
[0286] The previous examples describe how to make and use a
particular murine antibody called H36.D2 (sometimes also called H36
as discussed above). The present example shows how to make and use
a humanized version of that antibody. A humanized H36 antibody has
a variety of uses including helping to minimize potential for human
anti-mouse antibody (HAMA) immunological responses. These and other
undesired responses pose problems for use of the H36 antibody in
human therapeutic applications.
[0287] A. Preparation of chimeric anti-tissue factor antibody
(cH36)
[0288] The H36 antibody described previously is an IgG2a murine
antibody. H36 was first converted to a mouse-human chimeric
antibody for clinical development. To do this, the heavy and light
chain genes for H36 were cloned (see U.S. Pat. No. 5,986,065). The
heavy chain variable region was fused to a human IgG4 constant (Fc)
domain and the light chain variable region was fused to a human
kappa light chain constant domain. The resulting IgG4K chimeric
antibody was designated Sunol-cH36. For multiple uses of H36 or
cH36 in patients with chronic diseases, a fully humanized cH36 is
preferred so that it will decease or eliminate any human anti-mouse
antibody immunological response. The humanization of cH36 is
described below.
[0289] B. Humanization of cH36 antibody
[0290] Humanization of the chimeric anti-tissue factor antibody
cH36 was achieved by using a "best-fit" method. This method takes
full advantage of the fact that a great number of human IgGs with
known amino acid sequences are available in the public database.
The individual frameworks of the mouse heavy and light variable
regions in cH36 are compared with their corresponding human
frameworks in the Kabat database (see http://immuno.bme.nwu.edu).
The following criteria were used to select the desired human IgG
frameworks for humanization: (1) The number of mismatched amino
acids was kept as low as possible. (2) Amino acids inside the
"vernier" zone (amino acids in this zone may adjust CDR structure
and fine-tune the fit to antigen, see Foote, J. and Winter, G., J.
of Mol. Bio. 224, (2) 487-499 [1992]) were left unchanged. (3)
Conservative amino acid substitutions were favored when evaluating
similar candidates. The matching program used for this comparison
can be found in Kabat's home page at immuno.bme.nwu.edu (Johnson G,
Wu T. "Kabat database and its application: Future directions."
Nucleic Acids Res. (2001) 29:205-206). The program finds and aligns
regions of homologies between the mouse sequences and human
sequences in the Kabat's database. By using this unique best-fit
method, it is anticipated that the humanized LC or HC variable
region of the target IgG may have all the four FRs derived from as
few as one human IgG molecule or to as many as four different human
IgG molecules.
[0291] (i). Selection of Human IgG Kappa Light Chain Variable
Region Frameworks
[0292] The amino acid sequence in each of the frameworks of cH36 LC
was compared with the amino acid sequence in the corresponding FR
in human IgG kappa light chain variable region in Kabat Database.
The best-fit FR was selected based on the three creteria described
above.
[0293] The amino acid sequence of human IgG kappa light chain
variable region with a Kabat Database ID No. 005191 was selected
for humanization of cH36 LC FR1. The amino acid sequence of human
IgG kappa light chain variable region with a Kabat Database ID No.
019308 was selected for humanization of cH36 LC FR2. The following
mutations were made in cH36 LC FR1 to match the amino acid sequence
of a human IgG kappa light chain variable region With a Kabat
Database ID No. 005191:Q11.fwdarw.L, L15.fwdarw.V, E17.fwdarw.D,
S18.fwdarw.R. One mutation Q37.fwdarw.L was made cH36 LC FR2 to
match the amino acid sequence of a human IgG kappa light chain
variable region with a Kabat Database ID No. 019308 (see Table 1A
for sequence information).
[0294] The amino acid sequence of a human IgG kappa light chain
variable region with a Kabat Database ID No. 038233 was selected
for humanization of cH36 LC FR3. The amino acid sequence of a human
IgG kappa light chain variable region with a Kabat Database ID No.
004733 was selected for humanization of cH36 LC FR4. The following
mutations were made in cH36 LC FR3 to match the amino acid sequence
of a human IgG kappa light chain variable region with a Kabat
Database ID No. 038233: K70.fwdarw.D, K74.fwdarw.T, A80.fwdarw.P,
V84.fwdarw.A, N85.fwdarw.T. Two mutations A100.fwdarw.Q and
L106.fwdarw.I were made cH36 LC FR4 to match the amino acid
sequence of a human IgG kappa light chain variable region with a
Kabat Database ID No. 0 004733 (see Table 1B for sequence
information).
[0295] (ii). Selection of Human IgG Heavy Chain Variable Region
Frameworks
[0296] The amino acid sequence in each of the frameworks of cH36 HC
was compared with the amino acid sequence in the corresponding FR
in human IgG heavy chain variable region in Kabat Database. The
best-fit FR was selected based on the three criteria described
above.
[0297] The amino acid sequence of a human IgG heavy chain variable
region with a Kabat Database ID No. 000042 was selected for
humanization of cH36 HC FR1. The amino acid sequence of a human IgG
heavy chain variable region with a Kabat Database ID No. 023960 was
selected for humanization of cH36 HC FR2. The following mutations
were made in cH36 HC FR1 to match the amino acid sequence of a
human IgG heavy chain variable region with a Kabat Database ID No.
000042: E1.fwdarw.Q, Q5.fwdarw.V, P9.fwdarw.G, L11.fwdarw.V,
V12.fwdarw.K, Q19.fwdarw.R, T24.fwdarw.A. Two mutations
H41.fwdarw.P and S44.fwdarw.G were made cH36 HC FR2 to match the
amino acid sequence of a human IgG heavy chain variable region with
a Kabat Database ID No. 023960 (see Table 2A for sequence
information).
[0298] The amino acid sequence of a human IgG heavy chain variable
region with a Kabat Database ID No. 037010 was selected for
humanization of cH36 HC FR3. The amino acid sequence of a human IgG
heavy chain variable region with a Kabat Database ID No. 000049 was
selected for humanization of cH36 HC FR4. The following mutations
were made in cH36 HC FR3 to match the amino acid sequence of a
human IgG heavy chain variable region with a Kabat Database ID No.
037010: S76.fwdarw.T, T77.fwdarw.S, F80.fwdarw.Y, H82.fwdarw.E,
N84.fwdarw.S, T87.fwdarw.R, D89.fwdarw.E, S91.fwdarw.T. One
mutations L113.fwdarw.V was made cH36 HC FR2 to match the amino
acid sequence of a human IgG heavy chain variable region with a
Kabat Database ID No. 000049 (see Table 2B for sequence
information).
2TABLE 1A TABLE 1A and 1B Comparison of cH36 and Human Light Chain
(LC) FR Sequences FR1 (23 AA) FR2 (15 AA) Names 1 10 20 35 48
DIQMTQSPASQSASLGESVTITC WYQQKPGKSPQLLIY cH36-LC
DIQMTQSPASLSASVGDRVTITC WYLQKPGKSPQLLIY Human LC
[0299]
3TABLE 1B FR3 (32 AA) FR4 (10 AA) Names 57 60 70 80 86 98 107
GVPSRFSGSGSGTKFSFKISSLQAEDFVNYYC FGAGTKLELK cH36- LC
GVPSRFSGSGSCTDFSFTISSLQPEDFATYYC FGQGTKLEIK Human- LC
[0300] Table 2A and 2B: Comparison of cH36 and Human Heavy Chain
(HC) FR Sequences
4TABLE 2A FR1 (30 AA) FR2 (14 AA) Names 1 10 20 29 36 44
EIQLQQSGPELVKPGASVQVSCKTSGYSFT WVRQSHGKSLE cH36-HC WIG
QIQLVQSGGEVKIKPGASVRVSCKASGYSFT WVRQSPGKGLE Human-HC WIG
[0301]
5TABLE 2B FR3 (32 AA) FR4 (11 AA) Names 67 75 85 95 107 117
KATLTVDKSSTTAFMHLNSLTSDDSAVYFCAR WGQGTTLTVSS cH36- HC
KATLTVDKSTSTAYMZLSSLRSEDTAVYFCAR WGQGTTVTVSS Human- HC
[0302] Once the decisions on the desired human frameworks were
made, the following three techniques were used to achieve the
desired amino acid substitutions in both the light and heavy
chains: (1) Regular PCR was used for cloning, to introduce cloning
or diagnostic endonuclease sites, and to change amino acid residues
located at the ends of the variable regions. (2) PCR-based
mutagenesis was used to change multiple amino acid residues at a
time, especially when these residues were in the middle of the
variable regions. (3) Site directed mutagenesis was used to
introduce one or two amino acid substitutions at a time. Site
directed mutagenesis was done following the protocol described in
Stratagene's "QuickChange Site-Directed Mutagenesis Kit" (Catalog
#200518).
[0303] After each step, the partially humanized clones were
sequenced and some of these variable regions were later cloned into
expression vectors. The plasmid tKMC180 was used to express LC
mutants, and pJRS 355 or pLAM 356 vector was used to express HC
mutants as IgG1 or IgG4, respectively. Some of these clones were
then combined and expressed transiently in COS cells to determine
the expression levels by ELISA.
[0304] The final fully humanized forms of the anti-TF heavy and
light variable regions were cloned into what is sometimes referred
to herein as a "mega vector" and transfected into CHO and NSO cells
for IgG expression. Stable cell lines were then used to produce
amounts of humanized anti-TF sufficient for analysis. The resulting
humanized versions are 100% human in origin (when the CDR sequences
are not considered). The humanized IgG4 kappa version is designated
HFAT (humanized IgG Four Anti-Tissue Factor antibody) and the IgG1
kappa version is designated hOAT (humanized IgG One Anti-Tissue
Factor antibody). These fully humanized versions of cH36 are
intended for treating chronic indications, such as thrombosis,
cancer and inflammatory diseases.
[0305] C. Humanization of Anti-TF Antibody Heavy Chain
[0306] 1. PCR amplification and cloning into pGem T-easy of anti-TF
mAb cH36 heavy chain (HC) variable region were performed using
plasmid pJAIgG4TF.A8 (an expression vector for chimeric H36) as
template and primers TFHC1s2 and TFHC1as2. Primer TFHC1s2
introduced a BsiWl site upstream of the initiation codon and also
an amino acid change E1 to Q in framework (FR) 1. Primer TFHC1as
introduced an amino acid change L113 to V in FR4. This step
resulted in the construct HC01.
[0307] 2. PCR-based mutagenesis using the previous construct (HC01)
and the following four primers generated construct HC02. Upstream
PCR used primers TFHC1s2 and TFHC7as. Downstream PCR used primers
TFHC7s and TFHC1as2. Overlap PCR using upstream and downstream PCR
products as templates and with primers TFHC1s2 and TFHC1as2 yielded
HC02. The use of primers TFHC7s and TFHC7as introduced two amino
acid changes in FR2: H41 to P and S44 to G.
[0308] 3. PCR-based mutagenesis using HC02 as template and the
following four primers generated construct HC03. Upstream PCR used
primers TFHC1s2 and TFHC5as2. Downstream PCR used primers TFHC5s
and TFHC1as2. PCR using upstream and downstream PCR products as
templates and with primers TFHC1s2 and TFHC1as2 yielded HC03. The
use of primers TFHC5s and TFHC5 as2 introduced three amino acid
changes in FR3: T87 to R, D89 to E, and S91 to T. A Bgl II site was
also introduced at position. 87.
[0309] 4. PCR amplification was performed using primers TFHC2s and
TFHC3 as and HC03 in pgem as template. TFHC2s sits upstream of the
cloning site in pgem. TFHC3 as sits in framework 3 and introduces
two amino acid changes in FR3: H82 to E and N84 to S. The resulting
PCR band was cloned into pgem and then the proper size insert was
digested with BsiW1 and Bgl II. Cloning of this fragment into HC03
yields HC04.
[0310] 5. PCR-based mutagenesis using HC04 as template and the
following primers resulted in HC05. Upstream PCR used primers
TFHC1s2 and TFHC6as. Downstream PCR used primers TFHC6s and
TFHC1as2. Mutagenic PCR using upstream and downstream PCR products
as templates and with primers TFHC1s2 and TFHC1as2 yielded HC05.
This step introduced the following amino acid changes in FR3: S76
to T, T77 to S, and F80 to Y.
[0311] 6. PCR-based mutagenesis using HC05 as template and the
following four primers generated HC06. Upstream PCR used primers
TFHC2s and TFHC2 as2. Downstream PCR used primers TFHC3s2 and
TFHC1as2. Amplification using TFHC2 as2 introduced an amino acid
change in FR1: P9 to G. Primer TFHC3s2 changes Q19 to R and T24 to
A. PCR using upstream and downstream PCR products as template and
with primers TFHC1s2 and TFHC1as2 yielded HC.sub.06.
[0312] 7. A point mutation from 1 to M in position 2 of FR1 was
spontaneously introduced during construction of HC06. PCR
amplification using HC06 as template and TFHC1s3 and TFHC1as2 as
primers, corrected this erroneous substitution and also introduced
an amino acid. change in FR1: Q5 to V. The resulting construct was
HC07.
[0313] 8. Construct HC08 was made by PCR-based mutagenesis using
HC07 as template and the following primers. TFHC2s and TFHC2 as3
were used for the upstream product. The downstream product was
previously amplified using TFHC1s3 and TFHC1as2 (see step 7). The
use of primer TFHC2 as3 introduced two amino acid changes in FR1:
L11 to V and V12 to K. A spontaneous point mutation resulted in a F
to L change at position 64 in CDR2. Further screening and
sequencing yielded construct HC08R1, which has the correct sequence
of F at position 64 in CDR2.
[0314] 9. Two constructs, HC11 and HC12, were generated by
site-directed mutagenesis from HC07. Two complementary primers
TFHC8sP and TFHC8asP were used along with HC07 as template to
produce HC11 which contains three amino acid changes in FR1: G9 P,
Li 1 to V, and V12 to K. Then, HCil was methylated and column
purified for the next round of site directed mutagenesis. PCR using
HC11 as a template and the complementary primers TFHC9sL and
TFHCOasL generated HC12 which has a mutation from V11 to L in
FR1.
[0315] 10. Construct HC09 was derived from HC12 by performing PCR
using HC12 as a template and the complementary primers TFHC10sK and
TFHC10asK. HC09 contains an amino acid change: K12 to V in FR1.
[0316] 11. Construct HC10 was made from HC09. PCR using HC09 as a
template and the complementary primers LV-1 and LV-2 resulted in
the generation of HC10, which contains a mutation from L11 to V in
FR1.
[0317] After each mutation step, the partially humanized or fully
humanized clones were sequenced and some of these variable regions
were later cloned into expression vectors. pJRS 355 or pLAM 356
vector was used to express HC mutants fused to Fc of human IgG1 or
IgG4.
[0318] FIG. 13A summarizes steps 1-11 and shows incremental amino
acid changes introduced into FR1-4. Except HC08, all other heavy
chain mutants and cH36 contain F at position 64 in CDR2. HC08 has a
mutation from F to L at position 64. FIGS. 13B-D show the heavy
chain CDR sequences.
[0319] Primers Used for Heavy Chain Humanization
6 TFHC1s2 5' TTTCGTACGTCTTGTCCCAGATCCAGCTGCAGCAGTC 3' TFHC1as2 5'
AGCGAATTCTGAGGAGACTGTGACAGTGGTGCCTTGGCCCCAG 3' TFHC7s 5'
GTGAGGCAGAGCCCTGGAAAGGGCCTTGAGTGGATTGG 3' TFHC7as 5'
CCAATCCACTCAAGGCCCTTTCCAGGGCTCTGCCTCAC 3' TFHC5s 5'
GCATCTCAACAGCCTGAGATCTGAAGACACTGCAGTTTATTTCTGTG 3' TFHC5as2 5'
CTGCAGTGTCTTCAGATCTCAGGCTGTTGAGATGCATGAAGGC 3' TFHC3as 5'
GTCTTCAGATCTCAGGCTGCTGAGCTCCATGAAGGCTGTGGTG 3' TFHC2s 5'
TACGACTCACTATAGGGCGAATTGG 3' TFHC6s 5'
CTGTTGACAAGTCTACCAGCACAGCCTACATGGAGCTCAGCAG 3' TFHC6as 5'
CTGCTGAGCTCCATGTAGGCTGTGCTGGTAGACTTGTCAACAG 3' TFHC2as2 5'
GCACTGAAGCCCCAGGCTTCACCAGCTCACCTCCAGACTGCTGCAGC 3' TFHC3s2 5'
CTGGGGCTTCAGTGCGGGTATCCTGCAAGGCTTCTGGTTACTCATTCAC 3' TFHC1s3 5'
TCGTACGTCTTGTCCCAGATCCAGCTGGTGCAGTCTGGAGGTGAGC 3' TFHC2as3 5'
GCACTGAAGCCCCAGGCTTCTTCACCTCACCTCCAGACTGCACC 3' TFHC9sL 5'
GCAGTCTGGACCTGAGCTGAAGAAGCCTGGGG 3' TFHC9asL 5'
CCCCAGGCTTCTTCAGCTCAGGTCCAGACTGC 3' TFHC8sP 5'
GCTGGTGCAGTCTGGACCTGAGGTGAAGAAGCC 3' TFHC8asP 5'
GGCTTCTTCACCTCAGGTCCAGACTGCACCAGC3' TFHC10sK 5'
GCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTC 3' TFHC10asK 5'
GAAGCCCCAGGCTTCACCAGCTCAGGTCCAGACTGC 3' LV-1 5'
CAGTCTGGACCTGAGGTGGTGAAGCCTGGG 3' LV-2 5'
CCCAGGCTTCACCACCTCAGGTCCAGACTG 3'
[0320] D. Humanization of Anti-TF Antibody Light Chain
[0321] 1. PCR amplification was performed using plasmid
pJAIgG4TF.A8 (an expression vector for chimeric H36) as template
and primers TFLC1s2.1 and TFLC1as2. This step introduced a cloning
site, AgeI, upstream of the coding region. It also introduced the
L106I mutation in FR4. This step yielded the construct LC03.
[0322] 2. Site-directed mutagenesis was performed using
complementary primers TFLC5s and TFLC5as and LC03 as template. This
step introduced the mutation Q37L in FR2 and added a PstI site for
diagnostic purposes. This new construct is named LC04.
[0323] 3. PCR amplification was performed using LC04 as template
and primers TFHC2s and TFLC2as1. This step generated Fragment A
that will be used in step 6. This step introduced Q11L and L15V
mutations in FR1.
[0324] 4. PCR amplification was performed using LC04 as template
and primers TFLC1s2.1 and TFLC1asR. This introduced the KpnI site
at the end of LC variable region. Cloning of this PCR fragment into
pGEM yields pGEM04K that will be used in step 6.
[0325] 5. PCR amplification was performed using LC04 as template
and primers TFLC2s and TFLC4as. This step generated Fragment C that
will be used in step 6. Three mutations E17D, S18R in FR1 and A100Q
in FR4 were introduced in this step.
[0326] 6. PCR-based mutagenesis using Fragment A and Fragment C as
templates and primers TFHC2s and TFLC4 as yielded Fragment D.
Cloning of Fragment D into pGEM04K yielded the construct LC05.
[0327] 7. PCR amplification was performed using pGEM04K as template
and primers TFLC1s2.1 and TFLC4 as. This step generated Fragment H,
which is then cloned into pGEM04K. This introduced the A100Q
mutation in FR4 and the construct is named LC06.
[0328] 8. PCR amplification was performed using LC06 as template
and primers TFLC1s2.1 and TFLC3 as. This step generated Fragment I
that will be used in step 10. This introduced the K70D and the K74T
mutations in FR3.
[0329] 9. PCR amplification was performed using LC06 as template
and primers TFLC3s2 and TFLC4 as. This step generated Fragment F
that will be used in step 10. This introduced the A80P mutation in
FR3.
[0330] 10. PCR using Fragment I and Fragment F as templates and
primers TFLC1s2.1 and TFLC4 as yielded Fragment J. Cloning of
Fragment J into pGEM yielded the construct LC07.
[0331] 11. Site-directed mutagenesis was conduced using
complementary primers TFLC08sds and TFLC08sdsa and LC07 as
template. This step introduced the mutations V84A and N85T in FR3.
This construct is named LC08.
[0332] 12. The Agel to EcoO109I fragment from LC05 containing the
mutations Q11L, L15V, E17D, S18R and Q37L is cloned into LC08. This
yielded the construct LC09.
[0333] 13. Site-directed mutagenesis was conduced using LC09 as
template and the complementary primers LC105 and LC103. This step
introduced the T85N mutation in FR3 and yielded the construct
LC10.
[0334] 14. Site-directed mutagenesis was conducted using LC10 as
template and the complementary primers LC115 and LC113. This step
introduced the D70K mutation in FR3. This yielded the construct
LC11.
[0335] 15. Site-directed mutagenesis was conducted using LC11 as
template and the complementary primers LC125a and LC123a. This step
introduced the K42Q mutation in FR2. This yielded the construct
LC12.
[0336] After each mutation step, the partially humanized or fully
humanized LC clones were sequenced and some of these variable
regions were later cloned into expression vector tKMC180.
[0337] FIG. 12A summarizes steps 1-15 and shows incremental amino
acid changes introduced into FR1-4 of the light chain. FIGS. 12B-D
show the light chain CDR sequences.
[0338] Oligonucleotide Primers Used for Light Chain
Humanization
7 TFLC1as2: 5' TTCGAAAAGTGTACTTACGTTTGATCTCCAGCTTGGTCCCAG 3'
TFLC1s2.1: 5' ACCGGTGATATCCAGATGACCCAGTCTCC 3' TFLC5s: 5'
GGTTAGCATGGTATCTGCAGAAACCAGGG 3' TFLC5as: 5'
CCCTGGTTTCTGCAGATACCATGCTAACC 3' TFHC2s: 5'
TACGACTCACTATAGGGCGAATTGG 3' TFLC2as1: 5'
CCACAGATGCAGACAGGGAGGCAGGAGACTG 3' TFLC1asR: 5'
TTCGAAAAGTGTACTTACGTTTGATCTCCAGCTTGGTACCAGCACCGAACG 3' TFLC2s: 5'
CCTGTCTGCATCTGTGGGAGATAGGGTCACCATCACATGC 3' TFLC4as: 5'
GATCTCCAGCTTGGTACCCTGACCGAACGTGAATGG 3' TFLC3as: 5'
GTAGGCTGCTGATCGTGAAAGAAAAGTCTGTGCCAGATCC 3' TFLC3s2: 5'
CACGATCAGCAGCCTACAGCCTGAAGATTTTGTAAATTATTACTGTC 3' TFLC08sds: 5'
GCAGCCTACAGCCTGAAGATTTTGCAACTTATTACTGTCAACAAG 3' TFLC08sdsa: 5'
CTTGTTGACAGTAATAAGTTGCAAAATCTTCAGGCTGTAGGCTGC 3' LC105: 5'
CAGCAGCCTACAGCCTGAAGATTTTGCAAATTATTACTGTCAAC 3' LC103: 5'
GTTGACAGTAATAATTTGCAAAATCTTCAGGCTGTAGGCTGCTG 3' LC115: 5'
CAGTGGATCTGGCACAAAGTTTTCTTTCACGATCAGCAGC 3' LC113: 5'
GCTGCTGATCGTGAAAGAAAACTTTGTGCCAGATCCACTG 3' LC125a: 5'
CTGCAGAAACCAGGGCAATCTCCTCAGCTCCTG 3' LC123a: 5'
CAGGAGCTGAGGAGATTGCCCTGGTTTCTGCAG 3'
[0339] FIG. 14 shows hOAT (humanized cH36-IgG1) constant region
sequences of the light (FIG. 14A) and heavy chain (FIG. 14B). FIG.
15 shows hFAT (humanized cH36-IgG4) constant region sequences of
the light (FIG. 15A) and heavy chain (FIG. 15B). In each figure,
the last amino acid residue of the framework 4 (FR4) variable
region is connected to the first amino acid residue of the constant
region for hOAT and HFAT.
EXAMPLE 10
Expression and Purification of Humanized anti-TF Antibodies
[0340] The partially humanized or fully humanized LC and HC clones
were cloned into expression vectors. The plasmid tKMC180 (see FIGS.
10A-B) was used to express LC mutants fused to human kappa chain,
and pJRS 355 (see FIGS. 9A-B) or pLAM 356 (see FIGS. 9C-D) vector
was used to express HC mutants fused to Fc of human IgG1 or IgG4.
Some combinations of the HC and LC clones were then co-transfected
into COS cells. The transiently expressed IgGs in COS cells were
assayed for the whole IgG production and binding to TF by
ELISA.
[0341] The final fully-humanized forms of the anti-TF heavy and
light variable regions (combination of HC08 and LC09) were cloned
into Sunol's Mega expression vector (pSUN34, see FIG. 11) and
transfected into CHO and NSO cells for IgG expression. Stably
transfected cell lines producing the IgG4.kappa. or IgG1.kappa.
humanized anti-TF antibody were cloned. The selected stable cell
lines were then used to produce amounts of humanized anti-TF
sufficient for analysis. The resulting humanized versions are
approximately 95% human in origin (the CDR sequences are not
considered). The humanized IgG4 kappa version is designated hFAT
(humanized IgG Four Anti-Tissue Factor antibody) and the IgG1 kappa
version is designated hOAT (humanized IgG One Anti-Tissue Factor
antibody). These fully humanized versions of cH36 are intended for
treating chronic indications, such as cancer and inflammatory
diseases.
[0342] One of the NSO cell lines (OAT-NSO-P10A7) that expresses
hOAT (combination of HC08 and LC09) was thawed and extended in 10
mL of IMDM medium supplemented with 10% FBS in a 15 mL tube and
centrifuged. The cell pellet was resuspended in 10 mL of fresh
media and passed to a T25 flask and incubated at 37.degree. C. in
5% CO.sub.2. In order to prepare a sufficient number of cells to
inoculate a hollow fiber bioreactor, the cells were expanded to
obtain a total of 6.times.10.sup.8 cells. A bioreactor was set up
as per manufacturer's instruction manual. The harvested cells were
pelleted and resuspended in 60 mL of IMDM containing 35% FBS and
injected into the extracapillary space of the bioreactor.
Concentrations of glucose and lactate were monitored daily and the
harvest material was centrifuged and pooled. The harvested material
was tested for anti-TF antibody concentrations by ELISA assay. The
pooled sample containing anti-TF antibody (hOAT) were then purified
and analyzed as described below.
[0343] A. rProtein A Sepharose Fast Flow Chromatography
[0344] Recombinant humanized anti-TF monoclonal antibody consists
of two light and two heavy chains. Heavy chain is a fusion of mouse
variable region (unaltered or humanized as described above) and
human IgG1 or IgG4 Fc domain, while light chain contains mouse
variable region (unaltered or humanized as described above) and
human .kappa. domain. It is well established that human IgG Fc
region has high affinity for Protein A or recombinant Protein A
(rProtein A).
[0345] Harvest pools containing humanized anti-TF antibody (hOAT)
were adjusted to pH 8.0.+-.0.1 by adding 0.08 ml of 1 M Tris-HCl,
pH 8.0 per ml of sample. Then the sample is filtered through low
protein-binding 0.22 micron filters (e.g., Nalgene sterile
disposable tissue culture filter units with polyethersulfone
membrane from Nalge Nunc International, Cat. No. 167-0020).
Following sample application, rProtein A column (from Pharmacia) is
washed with 5 bed volumes of 20 mM Tris-HCl, pH 8.0 to remove
unbound materials such as media proteins. Since the harvest medium
contains high content of bovine serum, a stepwise pH gradient wash
was used to remove bovine IgG from the column. The stepwise pH
gradient was achieved by increasing the relative percentage of
Buffer B (100 mM acetic acid) in Buffer A (100 mM sodium acetate).
A typical pH stepwise wash employed 20%, 40%, and 60% Buffer B.
Elute the column with 100% Buffer B and collect fractions based on
A.sub.280. The pooled fractions were adjusted to pH 8.5 with
addition of 1 M Tris base.
[0346] B. Q Sepharose Fast Flow Chromatography
[0347] Anion ion exchange chromatography is very effective in
separating proteins according to their charges. The eluted and
pH-adjusted sample from rProtein A column was diluted with two
volumes of water, and the pH is checked and adjusted to 8.5. The
sample was then loaded to a 5 ml (1.6.times.2.5 cm) Q Sepharose
Fast Flow equilibrated with 20 mM Tris-HCl, pH 8.5 and the column
washed with (1) 5 bed volumes of 20 mM Tris-HCl, pH 8.5; and (2) 4
bed volumes of 20 mM Tris-HCl, pH 8.5 containing 100 mM NaCl. The
IgG protein was then eluted with bed volumes of 20 mM Tris-HCl, pH
8.5 containing 500 mM NaCl. The protein peaks were pooled and
buffer-exchanged into PBS using ultrafiltration device.
[0348] Using the same transfection, cell culture, and purification
methods, HFAT was also produced and purified.
EXAMPLE 11
Properties of Humanized Anti-TF Antibodies
[0349] A. Inhibition of TF Function by Humanized Anti-TF
Antibody
[0350] One of the key properties of anti-TF antibodies is its
ability to inhibit tissue factor-initiated blood coagulation. The
purified hOAT and hFAT were measured for their ability to inhibit
TF activity in a standard PT assay. PT assay is widely used to
measure tissue factor-dependent blood clotting times. The principal
of this assay is that tissue factor (TF) forms complex with factor
Vlla in plasma. This complex then activates factor X to FXa; FXa
then converts prothrombin to thrombin in the presence of factor Va
and phospholipids. Thrombin eventually leads to formation of a
blood clot. In standard PT assays, lipidated TF is added to plasma
to initiate blood coagulation and the clotting is recorded by an
Organon Teknika Coag-A-Mate Coagulation Analyzer or equivalent.
[0351] The anti-TF antibody, H36, inhibits human TF activity by a
unique mechanism. It binds to TF (free or in complex with factor
VIIa) in such a way that factor X and IX binding to TF:Vlla complex
is prohibited, thus FX and FIX activation by TF:VIIa is blocked
(see U.S. Pat. No. 5,986,065). In PT tests, the prolongation of
clotting times anti-TF antibody added into human plasma is a clear
indication that this TF-dependent coagulation is inhibited. The
clotting time is related to the amount of TF activity. A TF
standard curve is generated by measuring PT clotting times of
serially diluted TF. From the data of TF standard curve, the
inhibition of TF activity by anti-TF antibody is determined.
[0352] Reagents: Innovin (Cat No 68100-392) and Ci-Trol Coagulation
Control, Level I (Cat No 68100-336) are obtained from VWR.
Lipidated recombinant human TF was produced as described in Example
3.
[0353] Method: PT test is performed at 37 C using a Coagulation
Analyzer. PT reaction is initiated by adding 0.2 ml of lipidated
recombinant human tissue factor (e.g., Innovin) into 0.1 ml of
human plasma (Ci-Trol Control Level I) containing 0.01 ml buffer
(50 mM Tris-HCl, pH 7.5, 0.1% BSA) or anti-TF antibody.
[0354] 1. Add purified water to a vial of Innovin according to
manufacturer's instruction. Warm the reagent to 37.degree. C. The
reagent is stable for a few days if stored at 4-8.degree. C.
[0355] 2. Add 1 ml purified water to each vial of Ci-Trol. Mix to
solubilize. If more one vials are used, combine them into one
container (e.g., a 10 ml test tube). 1 ml Ci-Trol can run 5 assays
(each assay uses 2.times.0.1 ml=0.2 ml). Ci-Trol can be stored on
ice and last for a few hours.
[0356] 3. From anti-TF antibody stock, make a series of anti-TF
antibody solutions (200 nM to 1600 nM) with 50 mM Tris-HCl, pH 7.5,
0.1% BSA
[0357] 4. Add 10 .mu.l of 50 mM Tris-HCl, pH 7.5, 0.1% BSA or 10
.mu.l of diluted anti-TF to each well of the twin-well cuvette that
contains 0.1 ml of Ci-Trol. Use a pipette with 0.1 ml tip to mix
each well. Make sure no air bubbles are in the well. Following
mixing anti-TF (or buffer) with plasma (Ci-Trol), measure clotting
times within 10 min by adding 0.2 ml of Innovin to the plasma.
[0358] 5. For TF standard curve, first dilute Innovin (100% TF) to
20%, 10%, 5%and 2.5% with 50 mM Tris-HCl, pH 7.5, 0.1% BSA. Then PT
assays were performed as in Step 4 but using diluted Innovin
samples.
[0359] Table 3 is the summary of the effect of cH36, hOAT, and hFAT
on PT clotting times. Compared to the data in Table 4, cH36, hFAT,
and hOAT showed very potent inhibition of TF function. At a protein
concentration of above 12.9 nM, all antibodies achieved about 95%
inhibition. The results in Table 3 also indicate that humanization
of anti-TF, cH36, by the method described above did not have any
significant effect on cH36 inhibitory activity since both hFAT and
hOAT showed very similar ability to inhibit TF-dependent blood
coagulation as seen for cH36.
8TABLE 3 Effect on Prothrombin Times by Chimeric (cH36) and
Humanized) Anti-TF Antibodies (hFAT and hOAT) .sup.# Anti-TF
Antibody Concentrations (nM) PT Time (in seconds) in PT Assays cH36
hOAT hFAT 0 12.2 12.2 12.2 6.45 14.9 nd nd 9.7 17.8 16.5 nd 12.9
19.8 18.9 20.5 25.8 40 33.7 41.7 51.6 101.3 82.1 94.8 .sup.# All
assays used the same 100% TF activity (concentration) sample as in
Table 4.
[0360]
9TABLE 4 Clotting Times and Relative Tissue Factor Activities
(Concentrations) Relative TF Activities (Concentrations) PT
Clotting Times (Seconds) 100% (neat) 11.90 20% 13.225 10% 14.675 5%
16.700 2.5% 20.000
[0361] B. Determination of Affinity Constants
[0362] The affinity of humanized anti-TF antibody for TF was
determined by surface plasmon resonance (BIAcore from Pharmacia
Biosensor) with recombinant human tissue factor covalently
immobilized on a CM5 sensor chip. The affinity constants were the
average data calculated from four anti-TF monoclonal antibody
concentrations (0.125 nM, 0.25 nM, 0.5 nM, and 1 nM) by the BIAcore
computer software. The results in Table 5 indicate that
humanization of anti-TF, cH36, by the method described above did
not have any significant effect on cH36 affinity for TF since both
cH36 and HFAT have similar affinity for TF.
10TABLE 5 Apparent Affinity and Dissociation Constants of Anti-TF
Antibodies Anti-TF Antibody Apparent K.sub.a (M.sup.-1) Apparent
K.sub.d (M) H36 1.56 .times. 10.sup.10 6.4 .times. 10.sup.-11 cH36
7.94 .times. 10.sup.9 1.26 .times. 10.sup.-10 hFAT 2.99 .times.
10.sup.9 3.35 .times. 10.sup.-10
[0363] The invention has been described in detail with reference to
preferred embodiments thereof. However, it will be appreciated that
those skilled in the art, upon consideration of the disclosure, may
make modification and improvements within the spirit and scope of
the invention.
* * * * *
References