U.S. patent application number 14/337357 was filed with the patent office on 2014-12-25 for anticoagulant antidotes.
This patent application is currently assigned to BOEHRINGER INGELHEIM INTERNATIONAL GMBH. The applicant listed for this patent is Keith CANADA, Robert COPENHAVER, Norbert HAUEL, Tobias LITZENBURGER, Christopher Ronald SARKO, Sanjaya SINGH, Joanne VAN RYN, Alisa K. WATERMAN. Invention is credited to Keith CANADA, Robert COPENHAVER, Norbert HAUEL, Tobias LITZENBURGER, Christopher Ronald SARKO, Sanjaya SINGH, Joanne VAN RYN, Alisa K. WATERMAN.
Application Number | 20140377265 14/337357 |
Document ID | / |
Family ID | 45876804 |
Filed Date | 2014-12-25 |
United States Patent
Application |
20140377265 |
Kind Code |
A1 |
VAN RYN; Joanne ; et
al. |
December 25, 2014 |
ANTICOAGULANT ANTIDOTES
Abstract
The present invention relates to antibody molecules against
anticoagulants, in particular dabigatran, and their use as
antidotes of such anticoagulants.
Inventors: |
VAN RYN; Joanne;
(Warthausen, DE) ; CANADA; Keith; (Southbury,
CT) ; COPENHAVER; Robert; (Portland, OR) ;
HAUEL; Norbert; (Schemmerhofen, DE) ; LITZENBURGER;
Tobias; (Mittelbiberach, DE) ; SARKO; Christopher
Ronald; (New Milford, CT) ; SINGH; Sanjaya;
(Sandy Hook, CT) ; WATERMAN; Alisa K.; (Weston,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VAN RYN; Joanne
CANADA; Keith
COPENHAVER; Robert
HAUEL; Norbert
LITZENBURGER; Tobias
SARKO; Christopher Ronald
SINGH; Sanjaya
WATERMAN; Alisa K. |
Warthausen
Southbury
Portland
Schemmerhofen
Mittelbiberach
New Milford
Sandy Hook
Weston |
CT
OR
CT
CT
CT |
DE
US
US
DE
DE
US
US
US |
|
|
Assignee: |
BOEHRINGER INGELHEIM INTERNATIONAL
GMBH
Ingelheim am Rhein
DE
|
Family ID: |
45876804 |
Appl. No.: |
14/337357 |
Filed: |
July 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13432296 |
Mar 28, 2012 |
8821871 |
|
|
14337357 |
|
|
|
|
61469207 |
Mar 30, 2011 |
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Current U.S.
Class: |
424/135.1 ;
424/133.1; 424/175.1; 435/69.6; 530/387.3; 530/389.8 |
Current CPC
Class: |
A61K 39/395 20130101;
C07K 2317/565 20130101; C07K 2299/00 20130101; C07K 2317/24
20130101; A61P 7/02 20180101; A61K 31/4439 20130101; A61P 39/00
20180101; C07K 2317/92 20130101; C07K 2317/14 20130101; C07K 16/44
20130101; A61K 39/39583 20130101; C07K 2317/55 20130101; C07K
2317/76 20130101; A61K 31/4439 20130101; A61P 7/04 20180101; A61K
2300/00 20130101; A61P 39/02 20180101; A61K 2039/505 20130101; C07K
2317/21 20130101; C07K 2317/56 20130101 |
Class at
Publication: |
424/135.1 ;
530/389.8; 530/387.3; 424/175.1; 424/133.1; 435/69.6 |
International
Class: |
C07K 16/44 20060101
C07K016/44; A61K 31/4439 20060101 A61K031/4439; A61K 39/395
20060101 A61K039/395 |
Claims
1. An antibody molecule against dabigatran comprising a heavy chain
variable domain with a CDR1 selected from the group consisting of
SEQ ID NO: 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, and 61, a CDR2
selected from the group consisting of SEQ ID NO: 2, 8, 14, 20, 26,
32, 38, 44, 50, 56, and 62, and a CDR3 selected from the group
consisting of SEQ ID NO: 3, 15, 21, 27, 33, 39, 45, 51, 57, and 63,
and a light chain variable domain with a CDR1 selected from the
group consisting of SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, 52,
and 58, a CDR2 selected from the group consisting of SEQ ID NO: 5,
11, 17, 23, 29, 35, 41, 47, 53, and 59, and a CDR3 selected from
the group consisting of SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48,
54, 60, and 66.
2. The antibody molecule of claim 1 comprising a heavy chain
variable domain with a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO:
2, and a CDR3 of SEQ ID NO: 3, and a light chain variable domain
with a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 5, and a CDR3 of
SEQ ID NO: 6.
3. The antibody molecule of claim 1 comprising a heavy chain
variable domain with a CDR1 of SEQ ID NO: 7, a CDR2 of SEQ ID NO:
8, and a CDR3 of SEQ ID NO: 9, and a light chain variable domain
with a CDR1 of SEQ ID NO: 10, a CDR2 of SEQ ID NO: 11, and a CDR3
of SEQ ID NO: 12.
4. The antibody molecule of claim 1 comprising a heavy chain
variable domain with a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO:
14, and a CDR3 of SEQ ID NO: 15, and a light chain variable domain
with a CDR1 of SEQ ID NO: 16, a CDR2 of SEQ ID NO: 17, and a CDR3
of SEQ ID NO: 18.
5. The antibody molecule of claim 1 comprising a heavy chain
variable domain with a CDR1 of SEQ ID NO: 19, a CDR2 of SEQ ID NO:
20, and a CDR3 of SEQ ID NO: 21, and a light chain variable domain
with a CDR1 of SEQ ID NO: 22, a CDR2 of SEQ ID NO: 23, and a CDR3
of SEQ ID NO: 24.
6. The antibody molecule of claim 1 comprising a heavy chain
variable domain with a CDR1 of SEQ ID NO: 25, a CDR2 of SEQ ID NO:
26, and a CDR3 of SEQ ID NO: 27, and a light chain variable domain
with a CDR1 of SEQ ID NO: 28, a CDR2 of SEQ ID NO: 29, and a CDR3
of SEQ ID NO: 30.
7. The antibody molecule of claim 1 comprising a heavy chain
variable domain with a CDR1 of SEQ ID NO: 31, a CDR2 of SEQ ID NO:
32, and a CDR3 of SEQ ID NO: 33, and a light chain variable domain
with a CDR1 of SEQ ID NO: 34, a CDR2 of SEQ ID NO: 35, and a CDR3
of SEQ ID NO: 36.
8. The antibody molecule of claim 1 comprising a heavy chain
variable domain with a CDR1 of SEQ ID NO: 37, a CDR2 of SEQ ID NO:
38, and a CDR3 of SEQ ID NO: 39, and a light chain variable domain
with a CDR1 of SEQ ID NO: 40, a CDR2 of SEQ ID NO: 41, and a CDR3
of SEQ ID NO: 42.
9. The antibody molecule of claim 1 comprising a heavy chain
variable domain with a CDR1 of SEQ ID NO: 43, a CDR2 of SEQ ID NO:
44, and a CDR3 of SEQ ID NO: 45, and a light chain variable domain
with a CDR1 of SEQ ID NO: 46, a CDR2 of SEQ ID NO: 47, and a CDR3
of SEQ ID NO: 48.
10. The antibody molecule of claim 1 comprising a heavy chain
variable domain with a CDR1 of SEQ ID NO: 49, a CDR2 of SEQ ID NO:
50, and a CDR3 of SEQ ID NO: 51, and a light chain variable domain
with a CDR1 of SEQ ID NO: 52, a CDR2 of SEQ ID NO: 53, and a CDR3
of SEQ ID NO: 54.
11. The antibody molecule of claim 1 comprising a heavy chain
variable domain with a CDR1 of SEQ ID NO: 55, a CDR2 of SEQ ID NO:
56, and a CDR3 of SEQ ID NO: 57, and a light chain variable domain
with a CDR1 of SEQ ID NO: 58, a CDR2 of SEQ ID NO: 59, and a CDR3
of SEQ ID NO: 60.
12. The antibody molecule of claim 1 comprising a heavy chain
variable domain with a CDR1 of SEQ ID NO: 61, a CDR2 of SEQ ID NO:
62, and a CDR3 of SEQ ID NO: 63, and a light chain variable domain
with a CDR1 of SEQ ID NO: 64, a CDR2 of SEQ ID NO: 65, and a CDR3
of SEQ ID NO: 66.
13. (canceled)
14. The antibody molecule of claim 1 comprising a heavy chain
variable domain of SEQ ID NO: 70, and a light chain variable domain
of SEQ ID No: 71.
15. The antibody molecule of claim 1 comprising a heavy chain
variable domain of SEQ ID NO: 72, and a light chain variable domain
of SEQ ID No: 73.
16. The antibody molecule of claim 1 comprising a heavy chain
variable domain of SEQ ID NO: 74, and a light chain variable domain
of SEQ ID No: 75.
17. The antibody molecule of claim 1 comprising a heavy chain
variable domain of SEQ ID NO: 76, and a light chain variable domain
of SEQ ID No: 77.
18. The antibody molecule of claim 1 comprising a heavy chain
variable domain of SEQ ID NO: 78, and a light chain variable domain
of SEQ ID No: 79.
19. The antibody molecule of claim 1 comprising a heavy chain
variable domain of SEQ ID NO: 80, and a light chain variable domain
of SEQ ID No: 81.
20. The antibody molecule of claim 1 comprising a heavy chain
variable domain of SEQ ID NO: 82, and a light chain variable domain
of SEQ ID No: 83.
21. The antibody molecule of claim 1 comprising a heavy chain
variable domain of SEQ ID NO: 84, and a light chain variable domain
of SEQ ID No: 85.
22. The antibody molecule of claim 1 comprising a heavy chain
variable domain of SEQ ID NO: 86, and a light chain variable domain
of SEQ ID No: 87.
23. The antibody molecule of claim 1 comprising a heavy chain
variable domain of SEQ ID NO: 88, and a light chain variable domain
of SEQ ID No: 89.
24. The antibody molecule of claim 1 comprising a heavy chain
variable domain of SEQ ID NO: 90, and a light chain variable domain
of SEQ ID No: 91.
25. (canceled)
26. (canceled)
27. The antibody molecule of claim 1, wherein the light chain
variable domain is fused to a constant domain of SEQ ID NO: 97.
28. The antibody molecule of claim 1, wherein the heavy chain
variable domain is fused to a constant domain of SEQ ID NO: 98.
29. The antibody molecule of claim 1 comprising a heavy chain of
SEQ ID NO: 95, and a light chain of SEQ ID No: 96.
30. (canceled)
31. (canceled)
32. The antibody molecule of claim 1, wherein the antibody is a
polyclonal antibody, a monoclonal antibody, a human antibody, a
humanized antibody, a chimeric antibody, a fragment of an antibody,
in particular a Fab, Fab', or F(ab').sub.2 fragment, a single chain
antibody, in particular a single chain variable fragment (scFv), a
Small Modular Immunopharmaceutical (SMIP), a domain antibody, a
nanobody, a diabody, or a Designed Ankyrin Repeat Protein
(DARPin).
33. The antibody molecule of claim 1 for use in medicine.
34. Antibody molecule of claim 1 for use in the therapy or
prevention of side effects of anticoagulant therapy, and/or for
reversal of an overdosing of an anticoagulant.
35. Antibody molecule of claim 34, wherein the side effect is a
bleeding event.
36. Method of treatment or prevention of side effects of
anticoagulant therapy, or of an overdosing event in anticoagulant
therapy, comprising administering an effective amount of an
antibody molecule of claim 1 or 32 to a patient in need
thereof.
37. Method of manufacturing an antibody molecule of claim 1 or 32,
comprising (a) providing a host cell comprising one or more nucleic
acids encoding said antibody molecule in functional association
with an expression control sequence, (b) cultivating said host
cell, and (c) recovering the antibody molecule from the cell
culture.
38. A kit comprising an antibody of claim 1 or 32, or a
pharmaceutical composition thereof.
39. A kit comprising: (a) an antibody of claim 1 or 32, or a
pharmaceutical composition thereof; (b) a container; and (c) a
label.
40. A kit comprising an antibody of claim 1 or 32, and dabigatran,
dabigatran etexilate, a prodrug of dabigatran or a pharmaceutically
acceptable salt thereof.
41. A method for neutralizing or partially neutralizing dabigatran
or 1-O-acylglucuronide of dabigatran in a patient being treated
with dabigatran, dabigatran etexilate, a prodrug of dabigatran or a
pharmaceutically acceptable salt thereof, comprising administering
an antibody of claim 1 or 32, or a pharmaceutical composition
thereof.
42. A method for neutralizing or partially neutralizing dabigatran
or 1-O-acylglucuronide of dabigatran in a patient comprising: (a)
confirming that a patient was being treated with dabigatran,
dabigatran etexilate, a prodrug of dabigatran or a pharmaceutically
acceptable salt thereof, and the amount that was taken by the
patient; (b) neutralizing dabigatran or 1-O-acylglucuronide with an
antibody of claim 1 or 32 prior to performing a clotting or
coagulation test or assay wherein dabigatran or the
1-O-acylglucuronide of dabigatran would interfere with the accurate
read out of the test or assay results; (c) performing the clotting
or coagulation test or assay on a sample taken from the patient to
determine the level of clot formation without dabigatran or
1-O-acylglucuronide of dabigatran present; and (d) adjusting an
amount of dabigatran, dabigatran etexilate, a prodrug of dabigatran
or a pharmaceutically acceptable salt thereof administered to the
patient in order to achieve the appropriate balance between clot
formation and degradation in a patient.
43. A method for reducing the concentration of dabigatran or
1-O-acylglucuronide of dabigatran in plasma of a patient being
treated with dabigatran, dabigatran etexilate, a prodrug of
dabigatran or a pharmaceutically acceptable salt thereof,
comprising the step of administering an antibody of claim 1 or 32
or pharmaceutical composition thereof that neutralizes the activity
of dabigatran or 1-O-acylglucuronide in the patient.
44. A method of reversal of the anticoagulant effect of dabigatran
or 1-O-acylglucuronide of dabigatran in a patient being treated
with dabigatran, dabigatran etexilate, a prodrug of dabigatran or a
pharmaceutically acceptable salt thereof, wherein the patient
either has major bleeding considered life-threatening or leading to
hemodynamic compromise, or wherein the patient requires emergency
medical procedures, comprising the step of administering an
antibody of claim 1 or 32 or pharmaceutical composition thereof
that neutralizes the activity of dabigatran or 1-O-acylglucuronide
in the patient.
45. A method for reversing or reducing the activity of dabigatran
or 1-O-acylglucuronide of dabigatran in a patient experiencing
bleeding or at risk for bleeding due to an impaired clotting
ability or trauma, comprising the steps of: (d) determining the
amount of dabigatran or 1-O-acylglucuronide of dabigatran present
in the patient; (e) administering an effective amount of an
antibody of claim 1 or 32 or pharmaceutical composition thereof to
reverse or reduce the activity of dabigatran or 1-O-acylglucuronide
of dabigatran determined in the patient; and (f) monitoring a
thrombin clotting time of the patient to ensure a reversal or
reduction in activity of dabigatran or 1-O-acylglucuronide of
dabigatran has been reached.
46. (canceled)
47. (canceled)
48. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention pertains to the field of medicine, in
particular to the field of anticoagulant therapy.
BACKGROUND INFORMATION
[0002] Anticoagulants are substances that prevent coagulation; that
is, they stop blood from clotting. Anticoagulants are widely used
in human therapy as a medication for thrombotic disorders, for
example primary and secondary prevention of deep vein thrombosis,
pulmonary embolism, myocardial infarctions and strokes in those who
are predisposed.
[0003] An important class of oral anticoagulants acts by
antagonizing the effects of vitamin K, for example the coumarins
which include warfarin. A second class of compounds inhibit
coagulation indirectly via a cofactor such as antithrombin III or
heparin cofactor II. This includes several low molecular weight
heparin products which catalyse the inhibition of predominantly
factor Xa (and to a lesser degree thrombin) via antithrombin III
(bemiparin, certoparin, dalteparin, enoxaparin, nadroparin,
parnaparin, reviparin, tinzaparin), Smaller chain oligosaccharides
(fondaparinux, idraparinux) inhibit only factor Xa via antithrombin
III. Heparinoids (danaparoid, sulodexide, dermatan sulfate) act via
both cofactors and inhibit both factor Xa and thrombin. A third
class represents the direct inhibitors of coagulation. Direct
factor Xa inhibitors include apixaban, edoxaban, otamixaban,
rivaroxaban, and direct thrombin inhibitors include the bivalent
hirudins (bivalirudin, lepirudin, desirudin), and the monovalent
compounds argatroban and dabigatran.
[0004] As blood clotting is a biological mechanism to stop
bleeding, a side effect of anticoagulant therapy may be unwanted
bleeding events. It is therefore desirable to provide an antidote
to be able to stop such anticoagulant-related bleeding events when
they occur (Zikria and Ansell, Current Opinion in Hematology 2009,
16(5): 347-356). One way to achieve this is by neutralizing the
activity of the anticoagulant compound present in the patient after
administration.
[0005] Currently available anticoagulant antidotes are protamine
(for neutralization of heparin) and vitamin K for neutralization of
vitamin K antagonists like warfarin. Fresh frozen plasma and
recombinant factor VIIa have also been used as non-specific
antidotes in patients under low molecular weight heparin treatment,
suffering from major trauma or severe hemorrhage (Lauritzen, B. et
al, Blood, 2005, 607A-608A.). Also reported are protamine fragments
(U.S. Pat. No. 6,624,141) and small synthetic peptides (U.S. Pat.
No. 6,200,955) as heparin or low molecular weight heparin
antidotes; and thrombin muteins (U.S. Pat. No. 6,060,300) as
antidotes for thrombin inhibitor. Prothrombin intermediates and
derivatives have been reported as antidotes to hirudin and
synthetic thrombin inhibitors (U.S. Pat. Nos. 5,817,309 and
6,086,871). For direct factor Xa inhibitors, inactive factor Xa
analogs have been proposed as antidotes (WO2009042962).
Furthermore, recombinant factor VIIa has been used to reverse the
effect of indirect antithrombin III dependent factor Xa inhibitors
such as fondaparinux and idraparinux (Bijsterveld, N R et al,
Circulation, 2002, 106: 2550-2554; Bijsterveld, N R et al, British
J. of Haematology, 2004 (124): 653-658). A review of methods of
anticoagulant reversal is provided in Schulman and Bijsterveld,
Transfusion Medicine Reviews 2007, 21(1): 37-48.
[0006] International patent application WO2011089183 discloses
antibodies that can bind and neutralize the activity of
dabigatran.
[0007] There is a need to provide improved antidotes for
anticoagulant therapy, and in particular to provide antidotes for
direct thrombin inhibitors like dabigatran for which no specific
antidotes have been disclosed so far.
BRIEF SUMMARY OF THE INVENTION
[0008] In one aspect, the present invention relates to an antibody
molecule capable of neutralizing the activity of an
anticoagulant.
[0009] In a further aspect, the antibody molecule has binding
specificity for the anticoagulant.
[0010] In a further aspect, the anticoagulant is a direct thrombin
inhibitor, a Factor Xa inhibitor, or a vitamin K antagonist.
[0011] In a further aspect, the anticoagulant is dabigatran,
argatroban, melagatran, ximelagatran, hirudin, bivalirudin,
lepirudin, desirudin, apixaban, otamixaban, edoxaban, rivaroxaban,
defibrotide, ramatroban, antithrombin III, or drotrecogin
alpha.
[0012] In another aspect, the present invention relates to an
antibody molecule against dabigatran, dabigatran exetilate, and/or
an O-acylglucuronide of dabigatran.
[0013] In a further aspect, the present invention relates to an
antibody molecule against dabigatran, dabigatran exetilate, and/or
an O-acylglucuronide of dabigatran with reduced immunogenicity in
man.
[0014] In a further aspect, the present invention relates to an
antibody molecule against dabigatran, dabigatran exetilate, and/or
an O-acylglucuronide of dabigatran with improved physicochemical
properties, in particular improved solubility in aqueous
solvents.
[0015] In a further aspect, the present invention relates to an
antibody molecule against dabigatran, dabigatran exetilate, and/or
an O-acylglucuronide of dabigatran with improved produceability in
host cells, in particular resulting in improved production
yields.
[0016] In a further aspect, the antibody molecule is a polyclonal
antibody, a monoclonal antibody, a human antibody, a humanized
antibody, a chimeric antibody, a fragment of an antibody, in
particular a Fab, Fab', or F(ab').sub.2 fragment, a single chain
antibody, in particular a single chain variable fragment (scFv), a
domain antibody, a nanobody, a diabody, or a DARPin.
[0017] In a further aspect, the present invention relates to an
antibody molecule as described above for use in medicine.
[0018] In a further aspect, the present invention relates to an
antibody molecule as described above for use in the therapy or
prevention of side effects of anticoagulant therapy.
[0019] In a further aspect, the side effect is a bleeding
event.
[0020] In a further aspect, the present invention relates to a
method of treatment or prevention of side effects of anticoagulant
therapy, comprising administering an effective amount of an
antibody molecule as described above to a patient in need
thereof.
[0021] In another aspect, the present invention relates to a kit
comprising an antibody molecule as described, together with a
container and a label.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1: Increased time to clotting seen with increased
concentrations of dabigatran using the thrombin clotting time
assay. The 200 nM concentration resulted in an .about.5-fold
elevation in clotting time over baseline and was used in the first
and second set of experiments. The 500 nM concentration
(supratherapeutic) was used in the last set of experiments.
[0023] FIG. 2: Four different antibodies to dabigatran (A-D) all
neutralized the prolonged clotting time of dabigatran in human
plasma. Baseline clotting in human plasma was 10.9 seconds, when
200 nM dabigatran was preincubated with plasma, clotting was
prolonged to 51 seconds. Each antibody was added to plasma
preincubated with 200 nM of dabigatran and further incubated for 5
min. The thrombin clotting time was then initiated by addition of
thrombin. Each antibody could reverse the clotting time of
dabigatran to different degrees. The most concentrated solution
resulted in the largest reversal of anticoagulant activity.
[0024] FIG. 3: The effect of increasing concentrations of
polyclonal antibody (antibody D) added to human plasma that had
been preincubated with 200 nM dabigatran was measured. Baseline
clotting time was 11 seconds, addition of dabigatran prolonged
clotting to 63.7 seconds. The effect of increasing dilutions of
antibody on reversing the prolonged thrombin clotting time with
dabigatran was then tested. The lowest concentration reduced the
thrombin clotting time to 43.9 seconds. Higher concentrations
completely reduced the thrombin clotting time to baseline levels
and resulted in complete neutralization of the anticoagulant effect
of dabigatran. Addition of a non specific rabbit polyclonal
antibody (square) had no effect on reversing the anticoagulant
effect of dabigatran.
[0025] FIG. 4: The effect of increasing concentrations of
polyclonal antibody (antibody D) added to human plasma that had
been preincubated with 500 nM dabigatran was measured. Baseline
clotting time was 10.9 seconds, addition of this higher
concentration of dabigatran prolonged clotting to 111.7 seconds
(.about.10-fold increase). The effect of a 1:2 dilution of antibody
or stock solution reversed the prolonged thrombin clotting time
with dabigatran in a concentration dependent manner. The highest
concentration also completely reversed the thrombin clotting time
to baseline levels and resulted in complete neutralization of the
anticoagulant effect of even supratherapeutic concentrations of
dabigatran.
[0026] FIG. 5: A mouse monoclonal antibody (Clone 22) reverses the
anticoagulant effect of dabigatran in human plasma and in human
whole blood. Increasing concentrations of mouse antibody were added
to human plasma or whole blood that had been preincubated with 30
nM dabigatran. The assay was initiated by the addition of 1.5-2
U/mL of thrombin and clotting time was measured. 100% dabigatran
activity was defined as the difference in clotting time in the
presence and absence of compound. The antibody dose dependently
inhibited the dabigatran mediated prolongation of clotting
time.
[0027] FIG. 6: A mouse Fab generated from the Clone 22 antibody
reverses the anticoagulant effect of dabigatran in human plasma.
Increasing concentrations of mouse Fab were added to human plasma
that had been preincubated with 7 nM dabigatran. The intact
antibody was also tested as a positive control. The assay was
initiated by the addition of 0.4 U/mL of thrombin and clotting time
was measured. 100% inhibition was defined as the complete block of
the dabigatran mediated increase in clotting time. The Fab dose
dependently inhibited the dabigatran induced prolongation in
clotting time in human plasma.
[0028] FIG. 7: A mouse monoclonal antibody (Clone 22) reverses the
anticoagulant effect of dabigatran acylglucuronide in human plasma.
Increasing concentrations of mouse antibody were added to human
plasma that had been preincubated with 7 nM of dabigatran
acylglucuronide or dabigatran. The assay was initiated by the
addition of 0.4 U/mL of thrombin and clotting time was measured.
100% inhibition was defined as the complete block of the compound
mediated increase in clotting time. The antibody dose dependently
inhibited the dabigatran acylglucuronide induced prolongation in
clotting time in human plasma.
[0029] FIG. 8: Selected chimeric antibodies inhibit dabigatran
activity in the thrombin clotting time assay. Increasing
concentrations of antibody were added to human plasma that had been
preincubated with 7 nM dabigatran. The intact antibody was also
tested as a positive control. The assay was initiated by the
addition of 0.4 U/mL of thrombin and clotting time was measured.
100% inhibition was defined as the complete block of the dabigatran
mediated increase in clotting time. The antibodies dose dependently
inhibited the dabigatran induced prolongation in clotting time in
human plasma.
[0030] FIG. 9: Fab VH5c/Vk18 (SEQ ID NO: 99 and SEQ ID NO: 100) and
VH5c/Vk21 (SEQ ID NO: 99 and SEQ ID NO: 101) inhibit dabigatran
activity in the thrombin clotting time plasma assay. The assay was
performed as described above.
[0031] FIG. 10: Fab VH5c/Vk18 (SEQ ID NO: 99 and SEQ ID NO: 100)
and VH5c/Vk21 (SEQ ID NO: 99 and SEQ ID NO: 101) inhibit dabigatran
activity in the plasma and whole blood thrombin clotting time
assay. The assay was performed as described above.
[0032] FIG. 11: Crystal structure of the Fab-Dabigatran complexes.
A: Crystal structure of Fab 18/15 (WO2011089183) in complex with
dabigatran. B: Crystal structure of Fab VH5c/Vk18 (SEQ ID NO: 99
and SEQ ID NO: 100) in complex with dabigatran. C: Conformation of
dabigatran as seen in the crystal structure with Fab 18/15. D:
Extended conformation of dabigatran as seen in the crystal
structure with VH5c/Vk18.
[0033] FIG. 12: Spatial aggregation propensities (SAP) calculated
for (A) Fab 18/15 (B) Fab VH5c/Vk18 and (C) Fab VH5c/Vk21
comprising the CDRs (left panels) or the whole Fv region (right
panels).
[0034] FIG. 13: Titers of (A) Fab 18/15 (B) Fab VH5c/Vk18 and (C)
Fab VH5c/Vk21 from fed batch runs of CHO cells transfected with
corresponding Fab expression constructs.
DETAILED DESCRIPTION OF THE INVENTION
[0035] In one aspect, the present invention relates to an antibody
molecule capable of neutralizing the activity of an
anticoagulant.
[0036] Antibodies (also known as immunoglobulins, abbreviated Ig)
are gamma globulin proteins that can be found in blood or other
bodily fluids of vertebrates, and are used by the immune system to
identify and neutralize foreign objects, such as bacteria and
viruses. They are typically made of basic structural units--each
with two large heavy chains and two small light chains--to form,
for example, monomers with one unit, dimers with two units or
pentamers with five units. Antibodies can bind, by non-covalent
interaction, to other molecules or structures known as antigens.
This binding is specific in the sense that an antibody will only
bind to a specific structure with high affinity. The unique part of
the antigen recognized by an antibody is called an epitope, or
antigenic determinant. The part of the antibody binding to the
epitope is sometimes called paratope and resides in the so-called
variable domain, or variable region (Fv) of the antibody. The
variable domain comprises three so-called complementary-determining
region (CDR's) spaced apart by framework regions (FR's).
[0037] Within the context of this invention, reference to CDR's is
based on the definition of Chothia (Chothia and Lesk, J. Mol. Biol.
1987, 196: 901-917), together with Kabat (E. A. Kabat, T. T. Wu, H.
Bilofsky, M. Reid-Miller and H. Perry, Sequence of Proteins of
Immunological Interest, National Institutes of Health, Bethesda
(1983)).
[0038] The art has further developed antibodies and made them
versatile tools in medicine and technology. Thus, in the context of
the present invention the terms "antibody molecule" or "antibody"
(used synonymously herein) do not only include antibodies as they
may be found in nature, comprising e.g. two light chains and two
heavy chains, or just two heavy chains as in camelid species, but
furthermore encompasses all molecules comprising at least one
paratope with binding specificity to an antigen and structural
similarity to a variable domain of an immunoglobulin.
[0039] Thus, an antibody molecule according to the invention may be
a polyclonal antibody, a monoclonal antibody, a human antibody, a
humanized antibody, a chimeric antibody, a fragment of an antibody,
in particular a Fv, Fab, Fab', or F(ab').sub.2 fragment, a single
chain antibody, in particular a single chain variable fragment
(scFv), a Small Modular Immunopharmaceutical (SMIP), a domain
antibody, a nanobody, a diabody.
[0040] Polyclonal antibodies represent a collection of antibody
molecules with different amino acid sequences and may be obtained
from the blood of vertebrates after immunization with the antigen
by processes well-known in the art.
[0041] Monoclonal antibodies (mAb or moAb) are monospecific
antibodies that are identical in amino acid sequence. They may be
produced by hybridoma technology from a hybrid cell line (called
hybridoma) representing a clone of a fusion of a specific
antibody-producing B cell with a myeloma (B cell cancer) cell
(Kohler G, Milstein C. Continuous cultures of fused cells secreting
antibody of predefined specificity. Nature 1975; 256:495-7.).
Alternatively, monoclonal antibodies may be produced by recombinant
expression in host cells (Norderhaug L, Olafsen T, Michaelsen T E,
Sandlie I. (May 1997). "Versatile vectors for transient and stable
expression of recombinant antibody molecules in mammalian cells.".
J Immunol Methods 204 (1): 77-87; see also below).
[0042] For application in man, it is often desirable to reduce
immunogenicity of antibodies originally derived from other species,
like mouse. This can be done by construction of chimeric
antibodies, or by a process called "humanization". In this context,
a "chimeric antibody" is understood to be an antibody comprising a
sequence part (e.g. a variable domain) derived from one species
(e.g. mouse) fused to a sequence part (e.g. the constant domains)
derived from a different species (e.g. human). A "humanized
antibody" is an antibody comprising a variable domain originally
derived from a non-human species, wherein certain amino acids have
been mutated to resemble the overall sequence of that variable
domain more closely to a sequence of a human variable domain.
Methods of chimerisation and -humanization of antibodies are
well-known in the art (Billetta R, Lobuglio A F. "Chimeric
antibodies". Int Rev Immunol. 1993; 10(2-3):165-76; Riechmann L,
Clark M, Waldmann H, Winter G (1988). "Reshaping human antibodies
for therapy". Nature: 332:323.).
[0043] Furthermore, technologies have been developed for creating
antibodies based on sequences derived from the human genome, for
example by phage display or using transgenic animals (WO 90/05144;
D. Marks, H. R. Hoogenboom, T. P. Bonnert, J. McCafferty, A. D.
Griffiths and G. Winter (1991) "By-passing immunisation. Human
antibodies from V-gene libraries displayed on phage." J. Mol.
Biol., 222, 581-597; Knappik et al., J. Mol. Biol. 296: 57-86,
2000; S. Carmen and L. Jermutus, "Concepts in antibody phage
display". Briefings in Functional Genomics and Proteomics 2002
1(2):189-203; Lonberg N, Huszar D. "Human antibodies from
transgenic mice". Int Rev Immunol. 1995; 13(1):65-93.; Bruggemann
M, Taussig M J. "Production of human antibody repertoires in
transgenic mice". Curr Opin Biotechnol. 1997 August; 8(4):455-8.).
Such antibodies are "human antibodies" in the context of the
present invention.
[0044] Antibody molecules according to the present invention also
include fragments of immunoglobulins which retain antigen binding
properties, like Fab, Fab', or F(ab').sub.2 fragments. Such
fragments may be obtained by fragmentation of immunoglobulins e.g.
by proteolytic digestion, or by recombinant expression of such
fragments. For example, immunoglobulin digestion can be
accomplished by means of routine techniques, e.g. using papain or
pepsin (WO 94/29348), or endoproteinase Lys-C (Kleemann, et al,
Anal. Chem. 80, 2001-2009, 2008). Papain or Lys-C digestion of
antibodies typically produces two identical antigen binding
fragments, so-called Fab fragments, each with a single antigen
binding site, and a residual Fc fragment. Pepsin treatment yields
an F(ab').sub.2. Methods of producing Fab molecules by recombinant
expression in host cells are outlined in more detail below.
[0045] A number of technologies have been developed for placing
variable domains of immunoglobulins, or molecules derived from such
variable domains, in a different molecular context. Those should be
also considered as "antibody molecules" in accordance with the
present invention. In general, these antibody molecules are smaller
in size compared to immunoglobulins, and may comprise a single
amino acid chain or be composed of several amino acid chains. For
example, a single-chain variable fragment (scFv) is a fusion of the
variable regions of the heavy and light chains of immunoglobulins,
linked together with a short linker, usually serine (S) or glycine
(G) (WO 88/01649; WO 91/17271; Huston et al; International Reviews
of Immunology, Volume 10, 1993, 195-217). "Single domain
antibodies" or "nanobodies" harbour an antigen-binding site in a
single Ig-like domain (WO 94/04678; WO 03/050531, Ward et al.,
Nature. 1989 Oct. 12; 341(6242):544-6; Revets et al., Expert Opin
Biol Ther. 5(1):111-24, 2005). One or more single domain antibodies
with binding specificity for the same or a different antigen may be
linked together. Diabodies are bivalent antibody molecules
consisting of two amino acid chains comprising two variable domains
(WO 94/13804, Holliger et al., Proc Natl Acad Sci USA. 1993 Jul.
15; 90(14):6444-8). Other examples for antibody-like molecules are
immunoglobulin super family antibodies (IgSF; Srinivasan and
Roeske, Current Protein Pept. Sci. 2005, 6(2): 185-96). A different
concept leads to the so-called Small Modular Immunopharmaceutical
(SMIP) which comprises a Fv domain linked to single-chain hinge and
effector domains devoid of the constant domain CH1 (WO
02/056910).
[0046] In a further aspect, an antibody molecule of the invention
may even only have remote structural relatedness to an
immunoglobulin variable domain, or no such relation at all, as long
as it has a certain binding specificity and affinity comparable to
an immunoglobulin variable domain. Such non-immunoglobulin
"antibody mimics", sometimes called "scaffold proteins", may be
based on the genes of protein A, the lipocalins, a fibronectin
domain, an ankyrin consensus repeat domain, and thioredoxin
(Skerra, Current Opinion in Biotechnology 2007, 18(4): 295-304). A
preferred embodiment in the context of the present invention are
designed ankyrin repeat proteins (DARPin's; Steiner et al., J Mol
Biol. 2008 Oct. 24; 382(5): 1211-27; Stumpp M T, Amstutz P. Curr
Opin Drug Discov Devel. March; 10(2):153-9).
[0047] The antibody molecule may be fused (as a fusion protein) or
otherwise linked (by covalent or non-covalent bonds) to other
molecular entities having a desired impact on the properties of the
antibody molecule. For example, it may be desirable to improve
pharmacokinetic properties of antibody molecules, stability e.g. in
body fluids such as blood, in particular in the case of single
chain antibodies or domain antibodies. A number of technologies
have been developed in this regard, in particular to prolong
half-life of such antibody molecules in the circulation, such as
pegylation (WO 98/25971; WO 98/48837; WO 2004081026), fusing or
otherwise covalently attaching the antibody molecule to another
antibody molecule having affinity to a serum protein like albumin
(WO 2004041865; WO 2004003019), or expression of the antibody
molecule as fusion protein with all or part of a serum protein like
albumin or transferrin (WO 01/79258).
[0048] In a further aspect, the antibody molecule has binding
specificity for the anticoagulant. "Binding specificity" means that
the antibody molecule has a significantly higher binding affinity
to the anticoagulant than to structurally unrelated molecules.
[0049] Affinity is the interaction between a single antigen-binding
site on an antibody molecule and a single epitope. It is expressed
by the association constant K.sub.A=k.sub.ass/k.sub.diss, or the
dissociation constant K.sub.D=k.sub.diss/k.sub.ass.
[0050] In one aspect of the invention, the antibody binds to the
anticoagulant with an affinity, as determined e.g. by surface
plasmon resonance analysis (Malmqvist M., "Surface plasmon
resonance for detection and measurement of antibody-antigen
affinity and kinetics.", Curr Opin Immunol. 1993 April;
5(2):282-6.), with a K.sub.D value ranging from 0.1 pM to 100
.mu.M, preferably 1 pM to 100 .mu.M, preferably 1 pM to 1 .mu.M.
Antibody affinity can also be measured using kinetic exclusion
assay (KinExA) technology (Darling, R. J., and Brault P-A.,
"Kinetic exclusion assay technology: Characterization of Molecular
Interactions." ASSAY and Drug Development Technologies. 2004,
December 2(6): 647-657).
[0051] The binding affinity of an antibody molecule may be enhanced
by a process known as affinity maturation (Marks et al., 1992,
Biotechnology 10:779-783; Barbas, et al., 1994, Proc. Nat. Acad.
Sci, USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155).
Affinity matured antibodies are therefore also embraced in the
present invention.
[0052] In a further aspect of the invention, the antibody molecule
is capable of neutralizing the activity of the anticoagulant. That
is, upon binding to the antibody molecule, the anticoagulant is no
longer able to exert its anticoagulant activity, or exerts this
activity at a significantly decreased magnitude. Preferably, the
anticoagulant activity is decreased at least 2fold, 5fold, 10fold,
or 100fold upon antibody binding, as determined in an activity
assay which is appropriate for the anticoagulant at issue,
particularly a clotting assay that is sensitive to thrombin, such
as the ecarin clotting time or the thrombin clotting time (H.
Bounameaux, Marbet G A, Lammle B, et al. "Monitoring of heparin
treatment. Comparison of thrombin time, activated partial
thromboplastin time, and plasma heparin concentration, and analysis
of the behaviour of antithrombin III". American Journal of Clinical
Pathology 1980 74(1): 68-72).
[0053] For manufacturing the antibody molecules of the invention,
the skilled artisan may choose from a variety of methods well known
in the art (Norderhaug et al., J Immunol Methods 1997, 204 (1):
77-87; Kipriyanow and Le Gall, Molecular Biotechnology 26: 39-60,
2004; Shukla et al., 2007, J. Chromatography B, 848(1): 28-39).
[0054] Anticoagulants are well-known in the art, as outlined above.
In a further aspect of the invention, the anticoagulant is a direct
thrombin inhibitor, a Factor Xa inhibitor, or a vitamin K
antagonist. Examples of vitamin K antagonists are the coumarins,
which include warfarin. Examples of indirect predominantly factor
Xa inhibitors are the heparin group of substances acting through
activation of antithrombin III including several low molecular
weight heparin products (bemiparin, certoparin, dalteparin,
enoxaparin, nadroparin, parnaparin, reviparin, tinzaparin), certain
oligosaccharides (fondaparinux, idraparinux), heparinoids
(danaparoid, sulodexide, dermatan sulfate), and the direct factor
Xa inhibitors (apixaban, otamixaban, rivaroxaban). Examples of
thrombin inhibitors include the bivalent hirudins (bivalirudin,
lepirudin, desirudin), and the monovalent compounds argatroban and
dabigatran.
[0055] Thus, in a further aspect, the anticoagulant is dabigatran,
argatroban, melagatran, ximelagatran, hirudin, bivalirudin,
lepirudin, desirudin, apixaban, edoxaban, otamixaban, rivaroxaban,
defibrotide, ramatroban, antithrombin III, or drotrecogin
alpha.
[0056] A preferred anticoagulant in the context of the present
invention is dabigatran (CAS 211914-51-1,
N-[2-(4-Amidinophenylaminomethyl)-1-methyl-1H-benzimidazol-5-ylcarbonyl]--
N-(2-pyridyl)-beta-alanine) having the chemical formula (II):
##STR00001##
[0057] Dabigatran is known from WO 98/37075, which discloses
compounds with a thrombin-inhibiting effect and the effect of
prolonging the thrombin time, under the name
1-Methyl-2-[N-(4-amidinophenyl)-aminomethyl]-benzimidazol-5-yl-carboxylic
acid-N-(2-pyridyl)-N-(2-hydroxycarbonylethyl)-amide. See also Hauel
et al. J Med Chem 2002, 45 (9): 1757-66.
[0058] Dabigatran is applied as a prodrug of formula (III):
##STR00002##
[0059] The compound of formula III (named dabigatran etexilate, CAS
211915-06-9; ethyl
3-[(2-{[4-(hexyloxycarbonylamino-imino-methyl)-phenylamino]-methyl}-1-met-
hyl-1H-benzimidazole-5-carbonyl)-pyridin-2-yl-amino]propionate) is
converted into the active compound (II) after entering the body. A
preferred polymorph of dabigatran etexilate is dabigatran etexilate
mesylate.
[0060] The main indications for dabigatran are the post-operative
prevention of deep-vein thrombosis, the treatment of established
deep vein thrombosis and the prevention of strokes in patients with
atrial fibrillation (Eriksson et al., Lancet 2007, 370 (9591):
949-56; Schulman S et al, N Engl J Med 2009, 361 (24): 2342-52;
Connolly S et al., N Engl J Med 2009, 361 (12): 1139-51; Wallentin
et al., Lancet 2010, 376 (9745): 975-983).
[0061] In the human body, glucuronidation of the carboxylate moiety
is the major human metabolic pathway of dabigatran (Ebner et al.,
Drug Metab. Dispos. 2010, 38(9):1567-75). It results in the
formation of the 1-O-acylglucuronide (beta anomer). The
1-O-acylglucuronide, in addition to minor hydrolysis to the
aglycon, may undergo nonenzymatic acyl migration in aqueous
solution, resulting in the formation of the 2-O-, 3-O-, and
4-O-acylglucuronides. Experiments with the purified
1-O-acylglucuronide and its isomeric rearrangement products
revealed equipotent prolongation of the activated partial
thromboplastin time compared with dabigatran.
[0062] In another aspect of the invention, the antibody molecule
binds both to dabigatran and dabigatran etexilate.
[0063] In another aspect of the invention, the antibody molecule
binds both to dabigatran and O-acylglucuronides of dabigatran, in
particular the 1-O-acylglucuronide of dabigatran.
[0064] In another aspect of the invention, the antibody molecule
binds furthermore to the 2-O-, 3-O-, and 4-O-acylglucuronides of
dabigatran.
[0065] In another aspect of the invention, the antibody molecule is
capable of neutralizing the activity of dabigatran and
O-acylglucuronides of dabigatran, in particular the
1-O-acylglucuronide of dabigatran.
[0066] In the following, references to SEQ ID NOs. refer to the
sequences of Table 1 and the sequence listing which is part of this
application, unless indicated otherwise.
[0067] In another aspect of the invention, the antibody molecule
has binding specificity for dabigatran and comprises a heavy chain
variable domain with a CDR1 selected from the group consisting of
SEQ ID NO: 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, and 67, a CDR2
selected from the group consisting of SEQ ID NO: 2, 8, 14, 20, 26,
32, 38, 44, 50, 56, 62, and 68, and a CDR3 selected from the group
consisting of SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, and
63, and a light chain variable domain with a CDR1 selected from the
group consisting of SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, 52,
58, and 64, a CDR2 selected from the group consisting of SEQ ID NO:
5, 11, 17, 23, 29, 35, 41, 47, 53, 59, and 65, and a CDR3 selected
from the group consisting of SEQ ID NO: 6, 12, 18, 24, 30, 36, 42,
48, 54, 60, 66, and 69.
[0068] In another aspect of the invention, the antibody molecule
has binding specificity for dabigatran and comprises a heavy chain
variable domain with a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO:
2, and a CDR3 of SEQ ID NO: 3, and a light chain variable domain
with a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 5, and a CDR3 of
SEQ ID NO: 6.
[0069] In another aspect of the invention, the antibody molecule
has binding specificity for dabigatran and comprises a heavy chain
variable domain with a CDR1 of SEQ ID NO: 7, a CDR2 of SEQ ID NO:
8, and a CDR3 of SEQ ID NO: 9, and a light chain variable domain
with a CDR1 of SEQ ID NO: 10, a CDR2 of SEQ ID NO: 11, and a CDR3
of SEQ ID NO: 12.
[0070] In another aspect of the invention, the antibody molecule
has binding specificity for dabigatran and comprises a heavy chain
variable domain with a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO:
14, and a CDR3 of SEQ ID NO: 15, and a light chain variable domain
with a CDR1 of SEQ ID NO: 16, a CDR2 of SEQ ID NO: 17, and a CDR3
of SEQ ID NO: 18.
[0071] In another aspect of the invention, the antibody molecule
has binding specificity for dabigatran and comprises a heavy chain
variable domain with a CDR1 of SEQ ID NO: 19, a CDR2 of SEQ ID NO:
20, and a CDR3 of SEQ ID NO: 21, and a light chain variable domain
with a CDR1 of SEQ ID NO: 22, a CDR2 of SEQ ID NO: 23, and a CDR3
of SEQ ID NO: 24.
[0072] In another aspect of the invention, the antibody molecule
has binding specificity for dabigatran and comprises a heavy chain
variable domain with a CDR1 of SEQ ID NO: 25, a CDR2 of SEQ ID NO:
26, and a CDR3 of SEQ ID NO: 27, and a light chain variable domain
with a CDR1 of SEQ ID NO: 28, a CDR2 of SEQ ID NO: 29, and a CDR3
of SEQ ID NO: 30.
[0073] In another aspect of the invention, the antibody molecule
has binding specificity for dabigatran and comprises a heavy chain
variable domain with a CDR1 of SEQ ID NO: 31, a CDR2 of SEQ ID NO:
32, and a CDR3 of SEQ ID NO: 33, and a light chain variable domain
with a CDR1 of SEQ ID NO: 34, a CDR2 of SEQ ID NO: 35, and a CDR3
of SEQ ID NO: 36.
[0074] In another aspect of the invention, the antibody molecule
has binding specificity for dabigatran and comprises a heavy chain
variable domain with a CDR1 of SEQ ID NO: 37, a CDR2 of SEQ ID NO:
38, and a CDR3 of SEQ ID NO: 39, and a light chain variable domain
with a CDR1 of SEQ ID NO: 40, a CDR2 of SEQ ID NO: 41, and a CDR3
of SEQ ID NO: 42.
[0075] In another aspect of the invention, the antibody molecule
has binding specificity for dabigatran and comprises a heavy chain
variable domain with a CDR1 of SEQ ID NO: 43, a CDR2 of SEQ ID NO:
44, and a CDR3 of SEQ ID NO: 45, and a light chain variable domain
with a CDR1 of SEQ ID NO: 46, a CDR2 of SEQ ID NO: 47, and a CDR3
of SEQ ID NO: 48.
[0076] In another aspect of the invention, the antibody molecule
has binding specificity for dabigatran and comprises a heavy chain
variable domain with a CDR1 of SEQ ID NO: 49, a CDR2 of SEQ ID NO:
50, and a CDR3 of SEQ ID NO: 51, and a light chain variable domain
with a CDR1 of SEQ ID NO: 52, a CDR2 of SEQ ID NO: 53, and a CDR3
of SEQ ID NO: 54.
[0077] In another aspect of the invention, the antibody molecule
has binding specificity for dabigatran and comprises a heavy chain
variable domain with a CDR1 of SEQ ID NO: 55, a CDR2 of SEQ ID NO:
56, and a CDR3 of SEQ ID NO: 57, and a light chain variable domain
with a CDR1 of SEQ ID NO: 58, a CDR2 of SEQ ID NO: 59, and a CDR3
of SEQ ID NO: 60.
[0078] In another aspect of the invention, the antibody molecule
has binding specificity for dabigatran and comprises a heavy chain
variable domain with a CDR1 of SEQ ID NO: 61, a CDR2 of SEQ ID NO:
62, and a CDR3 of SEQ ID NO: 63, and a light chain variable domain
with a CDR1 of SEQ ID NO: 64, a CDR2 of SEQ ID NO: 65, and a CDR3
of SEQ ID NO: 66.
[0079] In another aspect of the invention, the antibody molecule
has binding specificity for dabigatran and comprises a heavy chain
variable domain with a CDR1 of SEQ ID NO: 67, a CDR2 of SEQ ID NO:
68, and a CDR3 of SEQ ID NO: 9, and a light chain variable domain
with a CDR1 of SEQ ID NO: 64, a CDR2 of SEQ ID NO: 65, and a CDR3
of SEQ ID NO: 69.
[0080] In another aspect of the invention, the antibody molecule
comprises a heavy chain variable domain of SEQ ID NO: 70, and a
light chain variable domain of SEQ ID No: 71.
[0081] In another aspect of the invention, the antibody molecule
comprises a heavy chain variable domain of SEQ ID NO: 72, and a
light chain variable domain of SEQ ID No: 73.
[0082] In another aspect of the invention, the antibody molecule
comprises a heavy chain variable domain of SEQ ID NO: 74, and a
light chain variable domain of SEQ ID No: 75.
[0083] In another aspect of the invention, the antibody molecule
comprises a heavy chain variable domain of SEQ ID NO: 76, and a
light chain variable domain of SEQ ID No: 77.
[0084] In another aspect of the invention, the antibody molecule
comprises a heavy chain variable domain of SEQ ID NO: 78, and a
light chain variable domain of SEQ ID No: 79.
[0085] In another aspect of the invention, the antibody molecule
comprises a heavy chain variable domain of SEQ ID NO: 80, and a
light chain variable domain of SEQ ID No: 81.
[0086] In another aspect of the invention, the antibody molecule
comprises a heavy chain variable domain of SEQ ID NO: 82, and a
light chain variable domain of SEQ ID No: 83.
[0087] In another aspect of the invention, the antibody molecule
comprises a heavy chain variable domain of SEQ ID NO: 84, and a
light chain variable domain of SEQ ID No: 85.
[0088] In another aspect of the invention, the antibody molecule
comprises a heavy chain variable domain of SEQ ID NO: 86, and a
light chain variable domain of SEQ ID No: 87.
[0089] In another aspect of the invention, the antibody molecule
comprises a heavy chain variable domain of SEQ ID NO: 88, and a
light chain variable domain of SEQ ID No: 89.
[0090] In another aspect of the invention, the antibody molecule
comprises a heavy chain variable domain of SEQ ID NO: 90, and a
light chain variable domain of SEQ ID No: 91.
[0091] In another aspect of the invention, the antibody molecule
comprises a heavy chain variable domain of SEQ ID NO: 92, and a
light chain variable domain of SEQ ID No: 93.
[0092] In another aspect of the invention, the antibody molecule
comprises a heavy chain variable domain of SEQ ID NO: 92, and a
light chain variable domain of SEQ ID No: 94.
[0093] In another aspect of the invention, any one of the
aforementioned light chain variable domains is fused to a constant
domain of SEQ ID NO: 97.
[0094] In another aspect of the invention, any one of the
aforementioned heavy chain variable domains is fused to a constant
domain of SEQ ID NO: 98.
[0095] In another aspect of the invention, the antibody molecule
comprises a heavy chain of SEQ ID NO: 95, and a light chain of SEQ
ID No: 96.
[0096] In certain aspects, the invention concerns antibodies
against dabigatran which have a high solubility in aqueous media
and a low tendency of aggregation.
[0097] In another aspect of the invention, the antibody molecule is
a scFv molecule. In this format, the variable domains disclosed
herein may be fused to each other with a suitable linker peptide.
The construct may comprise these elements in the order, from N
terminus to C terminus, (heavy chain variable domain)-(linker
peptide)-(light chain variable domain), or (light chain variable
domain)-(linker peptide)-(heavy chain variable domain).
[0098] Processes are known in the art which allow recombinant
expression of nucleic acids encoding sFv constructs in host cells
(like E. coli, Pichia pastoris, or mammalian cell lines, e.g. CHO
or NS0), yielding functional scFv molecules (see e.g. Rippmann et
al., Applied and Environmental Microbiology 1998, 64(12):
4862-4869; Yamawaki et al., J. Biosci. Bioeng. 2007, 104(5):
403-407; Sonoda et al., Protein Expr. Purif. 2010, 70(2):
248-253).
[0099] In particular, the scFv antibody molecules of the invention
can be produced as follows. The constructs can be expressed in
different E. coli strains like W3110, TG1, BL21, BL21 (DE3),
HMS174, HMS174 (DE3), MM294 under control of an inducible promoter.
This promoter can be chosen from lacUV5, tac, T7, trp, trc, T5,
araB. The cultivation media are preferably fully defined according
to Wilms et al., 2001 (Wilms et al., Biotechnology and
Bioengineering 2001, 73(2): 95-103), DeLisa et al., 1999 (DeLisa et
al., Biotechnology and Bioengineering 1999, 65(1): 54-64) or
equivalent. However, supplementation of the batch medium and/or
feed medium with amino acids such as isoleucine, leucine, lysine,
methionine, phenylalanine, threonine, tryptophan and valin or
complex media components such as soy peptone or yeast extract may
be beneficial. The process for fermentation is performed in a
fed-batch mode. Conditions: Temperature 20-40.degree. C., pH
5.5-7.5, DO is kept above 20%. After consumption of the initial
carbon source the culture is fed with the feed media stated above
(or equivalent). When a dry cell weight of 40 to 100 g/L is reached
in the fermenter the culture is induced with an appropriate inducer
corresponding to the used promoter system (e.g. IPTG, lactose,
arabinose). The induction can either be performed as a pulsed full
induction or as a partial induction by feeding the respective
inducer into the fermenter over a prolonged time or a combination
thereof. The production phase should last 4 hours at least. The
cells are recovered by centrifugation in bowl centrifuges, tubular
bowl centrifuges or disc stack centrifuges, the culture supernatant
is discarded.
[0100] The E. coli cell mass is resuspended in 4- to 8-fold amount
of lysis buffer (phosphate or Tris buffer, pH 7-8.5). Cell lysis is
preferably performed by high pressure homogenization followed by
recovery of the pellet by centrifugation in bowl, tubular bowl or
disc stack centrifuges. Pellet containing scFv inclusion bodies is
washed 2-3 times with 20 mM Tris, 150 mM NaCl, 5 mM EDTA, 2 M Urea,
0.5% Triton X-100, pH 8.0 followed by two wash steps using 20 mM
Tris, 150 mM NaCl, 5 mM EDTA, pH 8.0. scFv inclusion bodies are
finally recovered by centrifugation in bowl, tubular bowl or disc
stack centrifuges. Solubilisation of scFv inclusion bodies can be
performed in 100 mM Glycine/NaOH, 5 mM EDTA, 20 mM dithiothreitol,
pH 9.5-10.5 containing chaotropic agents such as 6 M Guanidine-HCl
or 8-10 mM Urea. After incubation for 30-60 minutes solution is
centrifuged and supernatant containing the target protein recovered
for subsequent refolding. Refolding is preferably performed in fed
batch mode by diluting the protein solution 1:10-1:50 in refolding
buffer to a final protein concentration of 0.1-0.5 mg/ml. Refolding
buffer can contain 50-100 mM Tris and/or 50-100 mM Glycine, 50-150
mM NaCl, 1-3 M urea, 0.5-1 M arginine, 2-6 mM of redox system such
as e.g. cytein/cystine or oxidized/reduced glutathione, pH
9.5-10.5. After incubation for 24-72 h at 4.degree. C. refolding
solution is optionally filtrated using a 0.22 .mu.m filter, diluted
and pH adjusted to pH 7.0-8.0. Protein is separated via cation
exchange chromatography in binding mode (e.g. Toyopearl GigaCap
S-650M, SP Sepharose FF or S HyperCel.TM.) at pH 7.0-8.5. Elution
is performed by a linear increasing NaCl gradient. Fractions
containing the target protein are pooled and subsequently separated
on anion exchange column in non-binding mode (e.g. Toyopearl
GigaCap Q-650M, Q-Sepharose FF, Q HyperCel.TM.) followed by a
cation exchange polishing step (eg. SP Sepharose HP). Fractions
containing the target protein with a purity level of minimally 90%
are pooled and formulated by diafiltration or size exclusion
chromatography in PBS. Identity and product quality of the produced
scFv molecule are analysed by reducing SDS-PAGE where the scFv can
be detected in one major band of approx. 26 kDa. Further assays for
characterization of the scFv include mass spectrometry, RP-HPLC and
SE-HPLC.
[0101] In another aspect of the invention, the antibody molecule is
a Fab molecule. In that format, the variable domains disclosed
above may each be fused to an immunoglobulin constant domain,
preferably of human origin. Thus, the heavy chain variable domain
may be fused to a CH.sub.1 domain (a so-called Fd fragment), and
the light chain variable domain may be fused to a CL domain.
[0102] In another aspect of the invention, the antibody molecule
comprises a heavy chain of SEQ ID NO: 99, and a light chain of SEQ
ID No: 100. Preferably, the antibody molecule is a Fab
molecule.
[0103] In another aspect of the invention, the antibody molecule
comprises a heavy chain of SEQ ID NO: 99, and a light chain of SEQ
ID No: 101. Preferably, the antibody molecule is a Fab
molecule.
[0104] In another aspect of the invention, the antibody molecule is
a Fab molecule which consists of a heavy chain of SEQ ID NO: 99,
and a light chain of SEQ ID No: 100.
[0105] In another aspect of the invention, the antibody molecule is
a Fab molecule which consists of a heavy chain of SEQ ID NO: 99,
and a light chain of SEQ ID No: 101.
[0106] Nucleic acids encoding Fab constructs may be used to express
such heavy and light chains in host cells, like E. coli, Pichia
pastoris, or mammalian cell lines (e.g. CHO, or NS0). Processes are
known in the art which allow proper folding, association, and
disulfide bonding of these chains into functional Fab molecules
comprising a Fd fragment and a light chain (Burtet et al., J.
Biochem. 2007, 142(6), 665-669; Ning et al., Biochem. Mol. Biol.
2005, 38: 204-299; Quintero-Hernandez et al., Mol. Immunol. 2007,
44: 1307-1315; Willems et al. J. Chromatogr. B. Analyt. Technol.
Biomed. Life Sci. 2003; 786:161-176.).
[0107] In particular, Fab molecules of the invention can be
produced in CHO cells as follows. CHO-DG44 cells (Urlaub, G., Kas,
E., Carothers, A. M., and Chasin, L. A. (1983). Deletion of the
diploid dihydrofolate reductase locus from cultured mammalian
cells. Cell 33, 405-412.) growing in suspension in serum-free
medium are transfected with expression constructs encoding heavy
and light chain of the Fab molecule using Lipofectamine.TM. and
PIus.TM. reagent (Invitrogen) according to the manufacturer's
instructions. After 48 hours, the cells are subjected to selection
in medium containing 200 .mu.g/mL of the antibiotic G418 and
without hypoxanthine and thymidine to generate stably transfected
cell populations. These stable transfectants are subsequently
subjected to gene amplification by adding methotrexate (MTX) in
increasing concentrations (up to 100 or 400 nM) into the culture
medium. Once the cells have adapted, they are subjected to
fed-batch fermentations over 10 to 11 days to produce Fab protein
material.
[0108] Suspension cultures of CHO-DG44 cells and stable
transfectants thereof are incubated in chemically defined,
serum-free cultivation media. Seed stock cultures are
sub-cultivated every 2-3 days with seeding densities of
3.times.10.sup.5-2.times.10.sup.5 cells/mL respectively. Cells are
grown in shake flasks in Multitron HT incubators (Infors) at 5%
CO.sub.2, 37.degree. C. and 120 rpm. For fed-batch experiments,
cells are seeded at 3.times.10.sup.5 cells/mL into shake flasks in
BI-proprietary production medium without antibiotics or MTX. The
cultures are agitated at 120 rpm in 37.degree. C. and 5% CO.sub.2
which is later reduced to 2% as cell numbers increase. Culture
parameters including cell count, viability, pH, glucose and lactate
concentrations are determined daily and pH is adjusted to pH 7.0
using carbonate as needed. BI-proprietary feed solution is added
every 24 hrs. Samples from the supernatant are taken at different
time points to determine the Fab product concentration by ELISA.
After 10 to 11 days, the cell culture fluid is harvested by
centrifugation and transferred to the purification labs.
[0109] The Fab molecule is purified from the supernatant of the
fed-batch cultures by means of chromatography and filtration. As
primary capture step affinity chromatography, e.g. Protein G or
Protein L, are applied. Alternatively, in case of low binding
affinities and capacities, the Fab is captured by cation exchange
chromatography (CEX) exploiting the pl of the molecule. Host cell
proteins and contaminants, e.g. DNA or viruses, are removed by
additional orthogonal purification steps.
[0110] Identity and product quality of the produced Fab molecule
are analysed by electrophoretic methods, e.g. SDS-PAGE, by which
Fab can be detected as one major band of approx. 50 kDa. Further
assays for characterization of the Fab product include mass
spectrometry, isoelectric focusing and size exclusion
chromatography. Binding activity is followed by BIAcore
analysis.
[0111] Quantification of Fab or full-length IgG molecules in the
supernatant of the cell cultures is performed via sandwich enzyme
linked immunosorbent assay (ELISA). The full-length IgG can be
detected using antibodies raised against human-Fc fragment (Jackson
Immuno Research Laboratories) and human kappa light chain
(peroxidase-conjugated, Sigma). The Fab fragment is immobilized by
goat polyclonal anti-Human IgG (H and L, Novus) and detected by
sheep polyclonal antibodies raised against human IgG
(peroxidase-conjugated, The Binding Site).
[0112] Fab molecules can also be generated from full-length
antibody molecules by enzymatic cleavage. The advantage of this
approach is that platform processes for robust and efficient
fermentation and purification are applicable which are amenable for
up-scaling and high yields at the desired product quality. For
purification affinity chromatography using a recombinant Protein A
resin can be used as primary capture step which usually results in
high purities.
[0113] For this purpose, the heavy chain encoding Fab sequences are
fused to the Fc-region of a human IgG antibody molecule. The
resulting expression constructs are then transfected into CHO-DG44
cells growing in suspension in serum-free medium using lipofection.
After 48 hours, the cells are subjected to selection in medium
containing 200 .mu.g/mL of the antibiotic G418 and without
hypoxanthine and thymidine to generate stably transfected cell
populations. These stable transfectants are subsequently subjected
to gene amplification by adding methotrexate (MTX) in increasing
concentrations (up to 100 or 400 nM) into the culture medium. Once
the cells have adapted, they are subjected to fed-batch
fermentations over 10 to 11 days to produce IgG protein
material.
[0114] The IgG protein is purified from the culture supernatant by
using recombinant Protein A-affinity chromatography. To obtain the
desired neutralizing Fab fragment the full-length IgG is then
incubated in the presence of papain which cleaves the IgG within
the hinge region, thereby releasing two Fab fragments and the
Fc-moiety.
[0115] The Fab molecule is isolated by affinity chromatography,
e.g. Protein G or Protein L. Alternatively, in case of low binding
affinities and capacities, the Fab is captured by cation exchange
chromatography (CEX) exploiting the pl of the molecule. Host cell
proteins and contaminants, e.g. Papain, DNA or viruses, are removed
by additional orthogonal purification steps.
[0116] In another aspect of the invention, the antibody molecule is
an amino acid sequence variant of an antibody molecule as described
herein.
[0117] Amino acid sequence variants of antibodies can be prepared
by introducing appropriate nucleotide changes into the antibody
DNA, or by peptide synthesis. Such variants include, for example,
deletions from, and/or insertions into and/or substitutions of,
residues within the amino acid sequences of the antibodies of the
examples herein. Any combination of deletions, insertions, and
substitutions is made to arrive at the final construct, provided
that the final construct possesses the desired characteristics. The
amino acid changes also may alter post-translational processes of
the humanized or variant antibody, such as changing the number or
position of glycosylation sites.
[0118] A useful method for identification of certain residues or
regions of the antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis," as described
by Cunningham and Wells (Science, 244:1081-1085 (1989)). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (typically alanine) to
affect the interaction of the amino acids with antigen. Those amino
acid locations demonstrating functional sensitivity to the
substitutions then are refined by introducing further or other
variants at, or for, the sites of substitution. Thus, while the
site for introducing an amino acid sequence variation is
predetermined, the nature of the mutation per se need not be
predetermined. For example, to analyze the performance of a
mutation at a given site, alanine scanning or random mutagenesis is
conducted at the target codon or region and the expressed antibody
variants are screened for the desired activity.
[0119] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody fused to an
epitope tag. Other insertional variants of the antibody molecule
include a fusion to the N- or C-terminus of the antibody of an
enzyme or a polypeptide which increases the serum half-life of the
antibody.
[0120] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antibody molecule removed and a different residue inserted in its
place. The sites of greatest interest for substitutional
mutagenesis include the hypervariable regions, but FR alterations
are also contemplated. Conservative substitutions are shown in the
Table below under the heading of "preferred substitutions". If such
substitutions result in a change in biological activity, then more
substantial changes, denominated "exemplary substitutions", or as
further described below in reference to amino acid classes, may be
introduced and the products screened.
TABLE-US-00001 Preferred Original Residue Exemplary Substitutions
Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys
Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C)
ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) ala
ala His (H) arg; asn; gln; lys; arg Ile (I) leu; val; met; ala;
phe; norleucine leu Leu (L) ile; norleucine; val; met; ala; phe ile
Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) tyr;
leu; val; ile; ala; tyr Pro (P) ala ala Ser (S) thr thr Thr (T) ser
ser Trp (W) tyr; phe tyr Tyr (Y) phe; trp; thr; ser phe Val (V)
leu; ile; met; phe ala; norleucine; leu
[0121] In protein chemistry, it is generally accepted that the
biological properties of the antibody can be accomplished by
selecting substitutions that differ significantly in their effect
on maintaining (a) the structure of the polypeptide backbone in the
area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. Naturally
occurring residues are divided into groups based on common
side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral
hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn,
gin, his, lys, arg; (5) residues that influence chain orientation:
gly, pro; and (6) aromatic: trp, tyr, phe.
[0122] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0123] Any cysteine residue not involved in maintaining the proper
conformation of the humanized or variant antibody also may be
substituted, generally with serine, to improve the oxidative
stability of the molecule, prevent aberrant crosslinking, or
provide for established points of conjugation to a cytotoxic or
cytostatic compound. Conversely, cysteine bond(s) may be added to
the antibody to improve its stability (particularly where the
antibody is an antibody fragment such as an Fv fragment).
[0124] A type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g., a
humanized or human antibody). Generally, the resulting variant(s)
selected for further development will have improved biological
properties relative to the parent antibody from which they are
generated. A convenient way for generating such substitutional
variants is affinity maturation using phage display. Briefly,
several hypervariable region sites (e.g., 6-7 sites) are mutated to
generate all possible amino substitutions at each site. The
antibody variants thus generated are displayed in a monovalent
fashion from filamentous phage particles as fusions to the gene III
product of M13 packaged within each particle. The phage-displayed
variants are then screened for their biological activity (e.g.,
binding affinity). In order to identify candidate hypervariable
region sites for modification, alanine scanning mutagenesis can be
performed to identify hypervariable region residues contributing
significantly to antigen binding. Alternatively, or in addition, it
may be beneficial to analyze a crystal structure of the
antigen-antibody complex to identify contact points between the
antibody and human Dabigatran. Such contact residues and
neighboring residues are candidates for substitution according to
the techniques elaborated herein. Once such variants are generated,
the panel of variants is subjected to screening as described herein
and antibodies with superior properties in one or more relevant
assays may be selected for further development.
[0125] Another type of amino acid variant of the antibody alters
the original glycosylation pattern of the antibody. By "altering"
is meant deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody.
[0126] In some embodiments, it may be desirable to modify the
antibodies of the invention to add glycosylations sites.
Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used. Thus, in order to glycosylate
a given protein, e.g., an antibody, the amino acid sequence of the
protein is engineered to contain one or more of the above-described
tripeptide sequences (for N-linked glycosylation sites). The
alteration may also be made by the addition of, or substitution by,
one or more serine or threonine residues to the sequence of the
original antibody (for O-linked glycosylation sites).
[0127] Nucleic acid molecules encoding amino acid sequence variants
of the antibody are prepared by a variety of methods known in the
art. These methods include, but are not limited to, isolation from
a natural source (in the case of naturally occurring amino acid
sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of an antibody molecule as described herein. As outlined above, the
antigen of the antibody molecule of the invention is an
anticoagulant. The antigen is used to generate the antibody
molecule, either by immunization of an animal, or by selecting
antibody sequences from sequence libraries, as with phage display
methods.
[0128] Immunization protocols for animals are well-known in the
art. To achieve a proper immune response, it may be necessary to
combine the antigen with an adjuvant, like aluminium phosphate,
aluminium hydroxide, squalene, or Freund's complete/incomplete
adjuvant. The antigens in the context of the present invention,
like dabigatran, are mostly comparably small organic molecules,
which sometimes do not stimulate antibody formation upon
administration to an animal. It may therefore be necessary to
attach the antigen to a macromolecule, as a hapten.
[0129] In a further aspect, the present invention relates to an
antibody molecule as described above for use in medicine.
[0130] In a further aspect, the present invention relates to a
pharmaceutical composition comprising an antibody molecule as
described before, and a pharmaceutical carrier.
[0131] To be used in therapy, the antibody molecule is included
into pharmaceutical compositions appropriate to facilitate
administration to animals or humans. Typical formulations of the
antibody molecule can be prepared by mixing the antibody molecule
with physiologically acceptable carriers, excipients or
stabilizers, in the form of lyophilized or otherwise dried
formulations or aqueous solutions or aqueous or non-aqueous
suspensions. Carriers, excipients, modifiers or stabilizers are
nontoxic at the dosages and concentrations employed. They include
buffer systems such as phosphate, citrate, acetate and other
anorganic or organic acids and their salts; antioxidants including
ascorbic acid and methionine; preservatives such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone or polyethylene
glycol (PEG); amino acids such as glycine, glutamine, asparagine,
histidine, arginine, or lysine; monosaccharides, disaccharides,
oligosaccharides or polysaccharides and other carbohydrates
including glucose, mannose, sucrose, trehalose, dextrins or
dextrans; chelating agents such as EDTA; sugar alcohols such as,
mannitol or sorbitol; salt-forming counter-ions such as sodium;
metal complexes (e.g., Zn-protein complexes); and/or ionic or
non-ionic surfactants such as TWEEN.TM. (polysorbates),
PLURONICS.TM. or fatty acid esters, fatty acid ethers or sugar
esters. Also organic solvents can be contained in the antibody
formulation such as ethanol or isopropanol. The excipients may also
have a release-modifying or absorption-modifying function.
[0132] In one aspect, the pharmaceutical composition comprises the
antibody molecule in an aqueous, buffered solution at a
concentration of 10-20 mg/ml, or a lyophilisate made from such a
solution.
[0133] The preferred mode of application is parenteral, by infusion
or injection (intravenous, intramuscular, subcutaneous,
intraperitoneal, intradermal), but other modes of application such
as by inhalation, transdermal, intranasal, buccal, oral, may also
be applicable.
[0134] In a further aspect, the present invention relates to an
antibody molecule as described above for use in the therapy or
prevention of side effects of anticoagulant therapy, in particular
bleeding events.
[0135] In a further aspect, the present invention relates to the
use of an antibody molecule as described herein for the manufacture
of a medicament for the treatment or prevention of a disease or
disorder as described herein, in particular the side effects of
anticoagulant therapy.
[0136] In a further aspect, the present invention relates to an
antibody molecule as described above for use in the reversal of an
overdosing of an anticoagulant, in particular dabigatran or
dabigatran exetilate.
[0137] In a further aspect, the present invention relates to an
antibody molecule as described above for use as an antidote of an
anticoagulant, in particular dabigatran or dabigatran
exetilate.
[0138] In a further aspect, the present invention relates to a
method of treatment or prevention of side effects of anticoagulant
therapy, comprising administering an effective amount of an
antibody molecule as described above to a patient in need
thereof.
[0139] In a further aspect, the present invention relates to a
method of treatment of an overdosing event in anticoagulant
therapy, comprising administering an effective amount of an
antibody molecule as described above to a patient in need
thereof.
[0140] In a further aspect, the present invention relates to a
method for reducing the concentration of dabigatran or
1-O-acylglucuronide of dabigatran in plasma of a patient being
treated with dabigatran, dabigatran etexilate, a prodrug of
dabigatran or a pharmaceutically acceptable salt thereof,
comprising the step of administering a reversal agent that
neutralizes the activity of dabigatran or 1-O-acylglucuronide in
the patient.
[0141] In a further aspect, the present invention relates to a
reversal agent that neutralizes the activity of dabigatran or
1-O-acylglucuronide for use in a patient being treated with
dabigatran, dabigatran etexilate, a prodrug of dabigatran or a
pharmaceutically acceptable salt thereof, wherein the patient
either has major bleeding considered life-threatening or leading to
hemodynamic compromise, or wherein the patient requires emergency
medical procedures.
[0142] In a further aspect, the present invention relates to a
method for reducing the concentration of dabigatran or
1-O-acylglucuronide of dabigatran in plasma of a patient being
treated with dabigatran, dabigatran etexilate, a prodrug of
dabigatran or a pharmaceutically acceptable salt thereof, wherein
the patient either has major bleeding considered life-threatening
or leading to hemodynamic compromise, or wherein the patient
requires emergency medical procedures, comprising the step of
administering a reversal agent that neutralizes the activity of
dabigatran or 1-O-acylglucuronide in the patient.
[0143] In a further aspect, the present invention relates to a
method of reversal of the anticoagulant effect of dabigatran or
1-O-acylglucuronide of dabigatran in a patient being treated with
dabigatran, dabigatran etexilate, a prodrug of dabigatran or a
pharmaceutically acceptable salt thereof, wherein the patient
either has major bleeding considered life-threatening or leading to
hemodynamic compromise, or wherein the patient requires emergency
medical procedures, comprising the step of administering a reversal
agent that neutralizes the activity of dabigatran or
1-O-acylglucuronide in the patient.
[0144] In a preferred embodiment, the reversal agent is an antibody
molecule against dabigatran which is capable of neutralizing the
anticoagulant activity of dabigatran, dabigatran etexilate, and/or
1-O-acylglucuronide. In another preferred embodiment, the reversal
agent is an antibody molecule against dabigatran as described
herein.
[0145] Preferably, the concentration of dabigatran or
1-O-acylglucuronide of dabigatran in plasma is greater than 0 nM
but less than 1000 .mu.M and wherein the reversal agent used to
neutralize the activity of dabigatran or 1-O-acylglucuronide is
present in a stoichiometric amount of dabigatran or
1-O-acylglucuronide of dabigatran to reversal agent.
[0146] In a further aspect, the concentration of dabigatran or
1-O-acylglucuronide of dabigatran in plasma is greater than 0 nM
but less than 1000 .mu.M, and wherein the reversal agent used to
neutralize the activity of dabigatran or 1-O-acylglucuronide is
present in a molar ratio of between 1:1 and 1:100 of dabigatran or
1-O-acylglucuronide of dabigatran to reversal agent.
[0147] In a further aspect, the concentration of dabigatran or
1-O-acylglucuronide of dabigatran in plasma is between 30 nM and
1000 .mu.M, and wherein the reversal agent used to neutralize the
activity of dabigatran or 1-O-acylglucuronide is present in a ratio
of between 30 nM and 1000 .mu.M of dabigatran or
1-O-acylglucuronide of dabigatran to reversal agent.
[0148] In another aspect, the present invention relates to a method
for reversing or reducing the activity of dabigatran or
1-O-acylglucuronide of dabigatran in a patient experiencing
bleeding or at risk for bleeding due to an impaired clotting
ability or trauma, comprising the steps of: [0149] (a) determining
the amount of dabigatran or 1-O-acylglucuronide of dabigatran
present in the patient; [0150] (b) administering an effective
amount of an agent to reverse or reduce the activity of dabigatran
or 1-O-acylglucuronide of dabigatran determined in the patient; and
[0151] (c) monitoring a thrombin clotting time of the patient to
ensure a reversal or reduction in activity of dabigatran or
1-O-acylglucuronide of dabigatran has been reached.
[0152] In a preferred aspect, the reversal of activity of
dabigatran or 1-O-acylglucuronide of dabigatran is 100%. In a
further preferred aspect, the reduction of activity of dabigatran
or 1-O-acylglucuronide of dabigatran is between 10 and 99% of
dabigatran or 1-O-acylglucuronide of dabigatran in the patient.
[0153] The "therapeutically effective amount" of the antibody to be
administered is the minimum amount necessary to prevent,
ameliorate, or treat the side effects of anticoagulant therapy, in
particular the minimum amount which is effective to stop bleeding.
This can be achieved with stoichiometric amounts of antibody
molecule.
[0154] Dabigatran, for example, may achieve a plasma concentration
in the magnitude of 200 nM when given at the recommended dose. When
a monovalent antibody molecule with a molecular weight of ca. 50 kD
is used, neutralization may be achieved for example at a dose of
about 1 mg/kg, when given intravenously as a bolus. In another
embodiment, the dose of a Fab molecule applied to a human patient
may be 50-1000 mg per application, for example 100, 200, 500, 750,
or 1000 mg. Depending on the situation, e.g. when dabigatran has
been overdosed in a patient, it may be adequate to apply an even
higher dose, e.g. 1250, 1500, 1750 or 2000 mg per application. The
appropriate dose may be different, depending on the type and dose
of anticoagulant administered; the time elapsed since such
administration, the nature of the antigen molecule, the condition
of the patient, and other factors. The skilled expert knows methods
to establish doses which are both therapeutically effective and
safe.
[0155] In a further aspect, the present invention relates to an
antibody molecule with binding affinity to dabigatran and/or
dabigatran etexilate. Preferably, the antibody molecule binds to
the dabigatran and/or dabigatran etexilate with an affinity, as
determined e.g. by surface plasmon resonance analysis (Malmqvist
M., "Surface plasmon resonance for detection and measurement of
antibody-antigen affinity and kinetics. "Curr Opin Immunol. April;
5(2):282-6.) or kinetic exclusion assay (KinExA) technology
(Darling, R. J., and Brault P-A., "Kinetic exclusion assay
technology: Characterization of Molecular Interactions." ASSAY and
Drug Development Technologies. 2004, December 2(6): 647-657), with
a K.sub.D value ranging from 0.1 pM to 100 .mu.M, preferably 1 pM
to 100 .mu.M, more preferably 1 pM to 1 .mu.M.
[0156] The antibody molecules of the invention can also be used for
analytical and diagnostic procedures, for example to determine
antigen concentration in samples such as plasma, serum, or other
body fluids. For example, the antigen molecules may be used in an
enzyme-linked immunoadsorbent assay (ELISA), like those described
in the examples. Thus, in a further aspect, the present invention
relates to analytical and diagnostic kits comprising antibody
molecules a described herein, and to respective analytical and
diagnostic methods.
[0157] In a further aspect, the present invention relates to a
method of manufacturing an antibody molecule of any one of the
preceding claims, comprising [0158] (a) providing a host cell
comprising one or more nucleic acids encoding said antibody
molecule in functional association with an expression control
sequence, [0159] (b) cultivating said host cell, and [0160] (c)
recovering the antibody molecule from the cell culture.
[0161] The invention further provides an article of manufacture and
kit containing materials useful for neutralization of oral
anticoagulants, particularly direct thrombin inhibitors. The
article of manufacture comprises a container with a label. Suitable
containers include, for example, bottles, vials, and test tubes.
The containers may be formed from a variety of materials such as
glass, metal, plastic or combinations thereof. The container holds
a pharmaceutical composition comprising the antibody described
herein or dabigatran, dabigatran etexilate, a prodrug of dabigatran
or a pharmaceutically acceptable salt thereof. The active agent in
the pharmaceutical composition is the particular antibody or
dabigatran, dabigatran etexilate, a prodrug of dabigatran or a
pharmaceutically acceptable salt thereof. The label on the
container of the antibody indicates that the pharmaceutical
composition is used for neutralizing or partially neutralizing
dabigatran, dabigatran etexilate, a prodrug of dabigatran or a
pharmaceutically acceptable salt thereof in vivo.
[0162] The kit of the invention comprises one or more of the
containers described above. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, syringes, and package inserts
with instructions for use.
[0163] In one embodiment of the invention, the kit comprises an
antibody of any one the antibodies described herein or a
pharmaceutical composition thereof. For example, the kit may
comprise (1) any one the antibodies described herein or a
pharmaceutical composition thereof, (2) a container and (3) a
label.
[0164] In another embodiment, the kit comprises an antibody of any
one the antibodies described herein or a pharmaceutical composition
thereof, and dabigatran, dabigatran etexilate, a prodrug of
dabigatran or a pharmaceutically acceptable salt thereof. The form
of dabigatran, dabigatran etexilate, a prodrug of dabigatran or a
pharmaceutically acceptable salt thereof may be in the form of a
solid, liquid or gel. In a preferred embodiment, the
pharmaceutically acceptable salt of dabigatran etexilate is a
mesylate salt. In yet another preferred embodiment, the strength
per dosage unit of the dabigatran, dabigatran etexilate, prodrug of
dabigatran or pharmaceutically acceptable salt thereof is between
about 50 mg and about 400 mg, about 75 mg and about 300 mg, about
75 mg and 150 mg, or about 110 mg and about 150 mg, given
once-a-day (QD) or twice-a-day (BID). For example, the kit may
comprise (1) any one the antibodies described herein or a
pharmaceutical composition thereof, (2) a pharmaceutical
composition of dabigatran, dabigatran etexilate, a prodrug of
dabigatran or a pharmaceutically acceptable salt thereof, (3) a
container and (4) a label.
[0165] In an alternate embodiment, the kit comprises (1) a first
pharmaceutical composition comprising dabigatran, dabigatran
etexilate, a prodrug of dabigatran or a pharmaceutically acceptable
salt thereof, (2) a second pharmaceutical composition comprising
any one the antibodies described herein or combination thereof, (3)
instructions for separate administration of said first and second
pharmaceutical compositions to a patient, wherein said first and
second pharmaceutical compositions are contained in separate
containers and said second pharmaceutical composition is
administered to a patient requiring neutralization or partial
neutralization of dabigatran or 1-O-acylglucuronide of
dabigatran.
[0166] The invention also provides a diagnostic method to
neutralize or partially neutralize dabigatran or
1-O-acylglucuronide of dabigatran in a patient being treated with
dabigatran, dabigatran etexilate, a prodrug of dabigatran or a
pharmaceutically acceptable salt thereof, comprising administering
any one of the antibodies described herein, a combination thereof
or a pharmaceutical composition thereof. Specifically, the
invention provides a method for neutralizing or partially
neutralizing dabigatran or 1-O-acylglucuronide of dabigatran in a
patient comprising the steps of (a) confirming that a patient was
being treated with dabigatran, dabigatran etexilate, a prodrug of
dabigatran or a pharmaceutically acceptable salt thereof, and the
amount that was taken by the patient; (b) neutralizing dabigatran
or 1-O-acylglucuronide with any one of the antibodies described
herein or combination thereof prior to performing a clotting or
coagulation test or assay wherein dabigatran or the
1-O-acylglucuronide of dabigatran would interfere with the accurate
read out of the test or assay results; (c) performing the clotting
or coagulation test or assay on a sample taken from the patient to
determine the level of clot formation without dabigatran or
1-O-acylglucuronide of dabigatran present; and (d) adjusting an
amount of dabigatran, dabigatran etexilate, a prodrug of dabigatran
or a pharmaceutically acceptable salt thereof administered to the
patient in order to achieve the appropriate balance between clot
formation and degradation in a patient. The molar ratio of antibody
to dabigatran or 1-O-acylglucuronide of dabigatran is in the molar
ratio of between 0.1 and 100, preferably between 0.1 and 10. The
accurate read out of the test or assay result may be an accurate
read out of fibrinogen levels, activated protein C resistance or
related tests.
EXAMPLES
I. Production of Polyclonal Anti-Dabigatran Antibodies
[0167] For the production of polyclonal anti-dabigatran antibodies,
3 different immunogens were produced with two different haptens and
different molar input ratios of the hapten and the carrier protein
(BSA).
[0168] For the screening, an enzyme horseradish peroxidase
(HRP)-conjugate was produced and an enzyme-immunosorbent assay
(ELISA) developed.
[0169] Further purification of the polyclonal antibodies was
performed by affinity chromatography on protein A sepharose FF.
1. Materials and Methods
Test Compound (Dabigatran)
TABLE-US-00002 [0170] Code: dabigatran, zwitter ion Structural
formula: ##STR00003## C.sub.25H.sub.25N.sub.7O.sub.3 molecular
weight: 471.5 g/mol
1.1 Hapten Used for Synthesis of Immunogen and Tracer
TABLE-US-00003 [0171] Code: Hapten1 Structural formula of ligand:
##STR00004## C.sub.30H.sub.36N.sub.8O.sub.2 * HCl molecular weight:
577.13 g/mol
TABLE-US-00004 Code: Hapten2 Structural formula of ligand:
##STR00005## C.sub.27H.sub.31N.sub.9O.sub.2 * HCl molecular weight:
550.07 g/mol
1.2 Synthesis of Haptens
[0172] The haptens Hapten1 and Hapten2 were synthesized as
follows:
Hapten1
2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidazol-
e-5-carboxylic acid
[2-(4-amino-butylcarbamoyl)-ethyl]phenyl-amide
##STR00006##
[0173] 1a 3-[(4-Methylamino-3-nitro-benzoyl)-phenyl-amino]propionic
acid methyl ester
##STR00007##
[0175] To a solution of 4-methylamino-3-nitro-benzoic acid chloride
(23.3 mmol) and 3-phenyl-amino-propionic acid methyl ester (23.3
mmol) in 80 mL dry tetrahydrofuran (THF) triethylamine (50.2 mmol)
was added dropwise under stirring at room temperature. After three
hours the reaction mixture was evaporated to dryness, the remaining
solid triturated with water and the solid product isolated through
filtration.
[0176] Yield: 99%
[0177] C.sub.18H.sub.19N.sub.3O.sub.5 (357.36)
[0178] TLC (silica gel; Dichloromethane/ethanol 19:1):
R.sub.f=0.48
1b 3[(3-Amino-4-methylamino-benzoyl)-phenyl-amino]-propionic acid
methyl ester
##STR00008##
[0180] The nitro group of product 1a was reduced by hydrogenation
at room temperature in ethanol with Pd (10% on charcoal) as
catalyst.
[0181] Yield: 99%
[0182] C.sub.18H.sub.21N.sub.3O.sub.3 (327.38)
[0183] TLC (silica gel; Dichloromethane/ethanol 9:1):
R.sub.f=0.23
[0184] Mass spectrum (ESI): [M+H].sup.+=328
1c
3-({3-[2-(4-Cyano-phenylamino)-acetylamino]-4-methylamino-benzoyl}-phen-
yl-amino)-propionic acid methyl ester
##STR00009##
[0186] The product of 1b (23.2 mmol) and N-(4-cyano-phenyl)-glycine
(23.2 mmol) were coupled with CDI (23.2 mmol) in dry THF at room
temperature. After completion of the reaction the mixture was
evaporated to dryness and the crude product was used without
further purification.
[0187] Yield: 97%
[0188] C.sub.27H.sub.27N.sub.5O.sub.4 (485.54)
[0189] Mass spectrum (ESI): [M+H].sup.+=486
1d
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-carbo-
nyl}-phenyl-amino)-propionic acid methyl ester
##STR00010##
[0191] A solution of the product of 1c (22.6 mmol) in 100 mL
concentrated acetic acid was heated to reflux for one hour. The
solution was then evaporated to dryness, the remaining solid
triturated with water and under stirring the pH was adjusted to
about 8-9. The crude product was isolated through extraction with
ethyl acetate and purified by chromatography on silica gel (eluent:
dichloromethane/ethanol 1:1).
[0192] Yield: 58%
[0193] C.sub.27H.sub.25N.sub.5O.sub.3 (467.52)
[0194] TLC (silica gel; Dichloromethane/ethanol 9:1):
R.sub.f=0.71
[0195] Mass spectrum (ESI): [M+H].sup.+=468
1e
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-carbo-
nyl}-phenyl-amino)-propionic acid
##STR00011##
[0197] To a solution of the product of 1d (13.0 mmol) in 100 mL
methanol sodium hydroxide (20.0 mmol) was added. The mixture was
stirred for 2.5 hours at 40.degree. C. and then evaporated to
dryness. The remaining solid was stirred with 100 mL water and the
pH was adjusted to about 6 with concentrated acetic acid. The
precipitated product was isolated by filtration, washed with water
and dried at 60.degree. C.
[0198] Yield: 88%
[0199] C.sub.26H.sub.23N.sub.5O.sub.3 (453.49)
[0200] TLC (silica gel; Dichloromethane/ethanol 9:1):
R.sub.f=0.33
[0201] Mass spectrum (ESI): [M+H].sup.+=454
1f
{4-[3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-c-
arbonyl}-phenyl-amino)-propionylamino]butyl}-carbamic acid
tert-butyl ester
##STR00012##
[0203] A solution of the product of 1e (5.23 mmol),
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU, 5.23 mmol) and N-methyl-morpholin (5.23
mmol) in 20 mL DMF was stirred at room temperature for 30 minutes.
Then (4-amino-butyl)-carbamic acid tert-butyl ester (5.23 mmol) was
added and the mixture stirred at room temperature for another 24
hours. The mixture was then diluted with water (100 mL) and the
product was isolated through extraction with ethyl acetate.
[0204] Yield: 92%
[0205] C.sub.35H.sub.41N.sub.7O.sub.4 (623.75)
[0206] TLC (silica gel; Dichloromethane/ethanol 9:1):
R.sub.f=0.51
1g
2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-c-
arboxylic acid [2-(4-amino-butylcarbamoyl)-ethyl]phenyl-amide
##STR00013##
[0208] The product of 1f (4.81 mmol) was dissolved in a saturated
solution of HCl in ethanol (250 mL), the mixture stirred at room
temperature over night and then evaporated to dryness at 30.degree.
C. The remaining raw material was dissolved in 200 mL dry ethanol,
then ammonium carbonate (48.1 mmol) was added and the mixture
stirred at room temperature over night. After evaporation of the
solvent the remaining raw material was triturated with ca. 5 mL
ethanol, the undissolved material separated by filtration and the
solvent evaporated at 30.degree. C. The product was then dissolved
in 30 mL water, the solution stirred with ca. 2 g charcoal,
filtered and evaporated to dryness.
[0209] Yield: 90%
[0210] C.sub.30H.sub.36N.sub.8O.sub.2 (540.67)
[0211] TLC (reversed phase RP-8; methanol/5% aqueous NaCl solution
9:1): R.sub.f=0.79
[0212] Mass spectrum (ESI): [M+H].sup.+=541 [0213]
[M+Cl].sup.-=575/7
Hapten2
2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidazol-
e-5-carboxylic acid
[2-(2-amino-ethylcarbamoyl)-ethyl]pyridin-2-yl-amide
##STR00014##
[0214] 2a
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole--
5-carbonyl}-pyridin-2-yl-amino)-propionic acid
##STR00015##
[0216] To a solution of sodium hydroxide (50.0 mmol) in 500 mL
ethanol and 50 mL water was added
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-carbony-
l}-pyridin-2-yl-amino)-propionic acid ethyl ester (41.4 mmol). The
mixture was stirred at room temperature for three hours, then ca.
350 mL ethanol were distilled off, ca. 100 mL water was added and
the pH was adjusted to 6. Then diethylether (50 mL) was added and
the mixture stirred over night. The product was isolated by
filtration and used without further purification.
[0217] Yield: 78%
[0218] C.sub.25H.sub.22N.sub.6O.sub.3 (454.48)
2b
{2-[3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-c-
arbonyl}-pyridin-2-yl-amino)-propionylamino]ethyl}-carbamic acid
tert-butyl ester
##STR00016##
[0220] A solution of the product of 2a (2.20 mmol),
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU, 2.20 mmol) and N-methyl-morpholin (2.20
mmol) in dry tetrahydrofuran (100 mL) was stirred at room
temperature for 15 minutes. Then (2-amino-ethyl)-carbamic acid
tert-butyl ester (2.20 mmol) was added and the mixture stirred at
room temperature for another 24 hours. The mixture was then diluted
with 40 mL water, the product was isolated through extraction with
ethyl acetate and purified by chromatography (silica gel;
dichloromethane/methanol 15:1).
[0221] Yield: 61%
[0222] C.sub.32H.sub.36N.sub.8O.sub.4 (596.68)
[0223] Mass spectrum (ESI): [M+H].sup.+=597 [0224]
[M+H].sup.-=595
2c
2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-c-
arboxylic acid
[2-(2-amino-ethylcarbamoyl)-ethyl]pyridin-2-yl-amide
##STR00017##
[0226] The product of 2b (1.34 mmol) was added to a saturated HCl
solution in dry ethanol (30 mL). The solution was stirred at room
temperature for 5 hours, then evaporated to dryness at 30.degree.
C. Ethanol (30 mL) and ammonium carbonate (13.0 mmol) were added
and the mixture stirred at room temperature over night. The solvent
was then evaporated, the residual material was triturated 5 times
with ca. 4 mL of a mixture of dichloromethane/methanol (30:1),
filtered and evaporated in order to separate the product from
inorganic salts.
[0227] Yield: 27%
[0228] C.sub.27H.sub.31N.sub.9O.sub.2 (513.61)
[0229] Mass spectrum (ESI): [M+Cl].sup.-=548/50 [0230]
[M+HCl+Cl].sup.-=584/6 [0231] [M+H].sup.+=514
2. Chemicals
TABLE-US-00005 [0232] 2.1 CHEMICALS FOR REAGENT SYNTHESIS catalogue
name specification supplier no. 1,4-Benzoquinone Fluka 12309
Bovines Serum Albumin Serva 11920 (BSA) 1,1'-Carbonyl-di-(1,2,4-
Fluka 21861 triazol) Citric acid analytical grade Riedel-De Haen
33114 N,N-dimethylformamide for synthesis Merck 822275 (DMF)
Ethanol analytical grade Baker 8006 Freund's adjuvant (CFA)
Complete Sigma F-5881 Freund's adjuvant (IFA) Incomplete Sigma
F-5506 Glycerine Pure Merck 104093 horseradish peroxidase 25000 U/
Boehringer 108090 HRP 100 mg Mannheim H.sub.2SO.sub.4 analytical
grade Riedel-De Haen 30743 KH.sub.2PO.sub.4 analytical grade Merck
4873 NaHCO.sub.3 analytical grade Merck 106329 Na.sub.2CO.sub.3
analytical grade Merck 106392 (NH.sub.4).sub.2SO.sub.4 analytical
grade Merck 101217 o-phenylene diamine 30 mg tablet Sigma P8412
Sodium perborate Pure Riedel-De Haen 11621 Thymol Pure Merck
8167
TABLE-US-00006 2.2 CHEMICALS FOR ELISA Name Specification supplier
catalogue no. Citric acid analytical grade Riedel-De Haen 33114
H.sub.2SO.sub.4 analytical grade Riedel-De Haen 30743
KH.sub.2PO.sub.4 analytical grade Merck 4873
Na.sub.2HPO.sub.4.cndot.2H.sub.2O analytical grade Merck 6580 NaCl
analytical grade Merck 6404 NaOH analytical grade Merck 6498
o-phenylene diamine 30 mg tablet Sigma P8412 Sodium perborate Pure
Riedel-De Haen 11621 Tween 20 Pure Serva 37470
TABLE-US-00007 2.3 BUFFERS FOR ELISA Name Ingredients use buffer 1
0.05M Na.sub.2HPO.sub.4/KH.sub.2PO.sub.4 coating 0.15M NaCl, pH =
7.4 stability: 4 weeks at approximately +4.degree. C. buffer 2 as
buffer 1, with 5 g/l BSA assay buffer stability: 10 days at
approximately +4.degree. C. buffer 3 as buffer 1, microplate with 5
g/l BSA and 0.1 g/L thimerosal blocking; storage stability: 4 weeks
at approximately +4.degree. C. buffer 4 0.1M citric acid, adjusted
to pH 5.0 substrate buffer with NaOH, for o-phenylene 6.5 mmol/L
sodium perborate diamine stability: citric acid: 6 months at
approximately +4.degree. C. with perborate: 10 days at
approximately +4.degree. C. wash water, 0.5 g/L Tween 20 microplate
solution washing stability: 10 days at ambient temperature stop
reagent 2.25M H.sub.2SO.sub.4 arrests o-phenylene diamine colour
stability: 5 years at ambient temperature development
[0233] Water from an Elgastat Maxima-HPLC ultra pure water
processing system was used to prepare buffer solutions.
3. Synthesis of Immunogens
[0234] In order to stimulate the immune system of rabbits to
produce polyclonal antibodies against dabigatran, three immunogens
(lot. nos. GL256, GL258, and GL262) were synthesized by coupling
the haptens HAPTEN1 and HAPTEN2 to the carrier protein bovine serum
albumin (BSA) using 1,4-benzoquinone or
1,1'-carbonyl-di-(1,2,4-triazol) as coupling reagent.
[0235] For the synthesis of GL256, 1,4-benzoquinone was used as a
homobifunctional compound with two reactive sites. First it reacts
at an acidic pH with amino groups at only one of the two sites and
at an alkaline pH at the other site with minimal polymerization.
GL258 and GL262 were synthesized using
1,1'-carbonyl-di-(1,2,4-triazol) as coupling reagent with different
input ratios of the hapten to the carrier protein.
3.1 Synthesis of GL256
[0236] To the solution of 0.75 .mu.Mol BSA in 8.5 mL 0.1 M
KH.sub.2PO.sub.4-buffer (pH=4.5), 0.416 mMol 1,4-benzoquinone (in
1.5 mL ethanol) was added and incubated for 1.5 h in the dark at
room temperature. Afterwards the solution passed a sephadex G25
column equilibrated in 0.15 M NaCl to eliminate the excess of
1,4-benzoquinone (final volume 12.5 mL).
[0237] 2.5 mL (0.15 .mu.Mol) of the purified BSA-solution were
added slowly under stirring to a solution of the 525 .mu.Mol hapten
HAPTEN1 dissolved in 2 mL 0.1 M
NaHCO.sub.31/Na.sub.2CO.sub.3-buffer (pH=8.5). During addition of
the BSA solution the pH was adjusted to approximately 8.0. The
molar input ratio of the hapten and the carrier protein was
3500:1.
[0238] After incubation at room temperature over night the
immunogen was dialysed 6 times against 1 litre of aqua. dest.
Thin-layer chromatography showed that no spots of unbound hapten
remained in the hapten-carrier conjugates.
[0239] The immunogen was stored frozen in aliquots at -20.degree.
C. The degree of substitution of BSA with hapten in the supernatant
of the immunogen was about 1:18 as determined by UV absorption
spectrometry at 302 nm. The content of immunogen in the final
solution was 0.75 mg GL256/mL
3.2 Synthesis of GL258
[0240] A solution of 158 .mu.Mol HAPTEN2 in 6.3 mL
N,N-dimethylformamide (DMF) was prepared at room temperature. 158
.mu.Mol 1,1'-carbonyl-di-(1,2,4-triazol) was added and incubated
first for 4 hours at 10.degree. C. and afterwards for 30 min at
room temperature. The chemical reaction was checked with thin-layer
chromatography and was about 20-25%. Then 0.75 .mu.Mol BSA were
dissolved in 2 mL 0.13 M NaHCO.sub.3 and 1 mL N,N-dimethylformamide
(DMF) was added dropwise under stirring. The pH was adjusted to
approximately 8.3. Afterwards the hapten solution (6.3 mL) and 4 mL
0.13 M NaHCO.sub.3 were added dropwise to the BSA solution under
stirring and the pH was adjusted to 8.4. The molar input ratio of
the hapten and the carrier protein was 210:1 for the immunogen
GL258.
[0241] After incubation at room temperature over night under
stirring conditions, the immunogen was dialysed 6 times against 1
litre of aqua. dest. Thin-layer chromatography showed that no spots
of unbound hapten remained in the hapten-carrier conjugates.
[0242] The immunogen was stored frozen in aliquots at -20.degree.
C. The degree of substitution of BSA with hapten in the supernatant
of the immunogen was about 1:5 as determined by UV absorption
spectrometry at 302 nm. The content of immunogen in the final
solution was 0.28 mg GL258/mL.
3.3 Synthesis of GL262
[0243] A solution of 225 .mu.Mol HAPTEN2 in 8.75 mL
N,N-dimethylformamide (DMF) was prepared at room temperature. 225
.mu.Mol 1,1'-carbonyl-di-(1,2,4-triazol) was added and incubated
for 4 hours at 10.degree. C. The chemical reaction was checked with
thin-layer chromatography and was about 20-25%.
[0244] Then 0.49 .mu.Mol BSA were dissolved in 2 mL 0.13 M
NaHCO.sub.3 and 1 mL N,N-dimethylformamide (DMF) was added dropwise
under stirring. The pH was adjusted to approximately 8.2.
Afterwards the hapten solution (8.75 mL) and 6 mL 0.13 M
NaHCO.sub.3 were added dropwise to the BSA solution under stirring
and the pH was adjusted to 8.3. The molar input ratio of the hapten
and the carrier protein was 460:1 for the immunogen GL262.
[0245] After incubation at room temperature over night under
stirring conditions, the immunogen was dialysed 6 times against 1
litre of aqua. dest. Thin-layer chromatography showed that no spots
of unbound hapten remained in the hapten-carrier conjugates.
[0246] The immunogen was stored frozen in aliquots at -20.degree.
C. The degree of substitution of BSA with hapten in the supernatant
of the immunogen was about 1:32 as determined by UV absorption
spectrometry at 302 nm. The content of immunogen in the final
solution was 0.71 mg GL262/mL
4. Synthesis of Conjugate
4.1 Synthesis of GL261
[0247] A solution of 37.4 .mu.Mol HAPTEN2 in 1.5 mL
N,N-dimethylformamide (DMF) was prepared at room temperature. 37.5
.mu.Mol 1,1'-carbonyl-di-(1,2,4-triazol) was added and incubated
first for 4 hours at 10.degree. C. and afterwards for 30 min at
room temperature. The chemical reaction was checked with thin-layer
chromatography and was about 20-25%.
[0248] Then 1.125 .mu.Mol enzyme horseradish peroxidase (HRP) were
dissolved in 0.4 mL 0.13 M NaHCO.sub.3 and 0.267 mL
N,N-dimethylformamide (DMF) was added dropwise under stirring. The
pH was adjusted to approximately 8.2. Afterwards 0.9 mL of the
hapten solution (22.5 .mu.Mol) and 0.57 mL 0.13 M NaHCO.sub.3 were
added dropwise to the HRP solution under stirring and the pH was
adjusted to 8.4. The molar input ratio of the hapten and the HRP
was 20:1 for the HRP conjugate GL261.
[0249] After incubation at room temperature over night under
stirring conditions, the HRP conjugate was separated from organic
solvents and the excess of hapten by gel chromatography. The
solution passed a sephadex G25 column equilibrated with 0.1 M
phosphate buffer pH 7.0.
[0250] The final concentration of hapten-HRP conjugate (tracer,
5.64 mg/mL) was spiked with BSA yielding a concentration of about
10 mg/mL, an equal volume of glycerine to prevent freezing and a
thymol crystal to prevent bacterial growth. The tracer solution was
labelled as lot no. GL261 and stored in aliquots at -20.degree.
C.
[0251] The degree of substitution of HRP with hapten was 1:0.2 as
determined by UV spectroscopy at 302 nm.
[0252] The specific activity of the tracer was measured in
BSA-blocked microtiter plates using o-phenylene-diamine (OPD) as
substrate and native HRP as reference material. The mixture of
diluted HRP standards or the hapten-HRP conjugate and substrate
solution were incubated for 30 min in the dark, stopped with
sulphuric acid and absorption measured at 490 nm. The remaining
activity was 94% of the native HRP and the specific activity of the
conjugate formulation in glycerine was 611 U/mL.
[0253] Summary of Tracer Specifications:
TABLE-US-00008 type: HAPTEN2 - horseradish peroxidase (lot no. GL
261) protein content: 5.64 mg/mL specific activity: 108 U/mg 611
U/ml (substrate Guajacol and H.sub.2O.sub.2, 25.degree. C.)
storage: at approximately -20.degree. C. working dilution:
1:40000
5. Immunization and Production of Antibodies
5.1 Immunization of Rabbits
[0254] Twelve female chinchilla rabbits, 3 months old, were
immunized with an emulsion of 100 .mu.g immunogen GL256, GL258 and
GL262 in 0.5 mL 0.9% NaCl solution and 0.5 mL of complete Freund's
adjuvant (CFA). Several booster immunizations followed in the next
month. For the third immunization 0.5 mL of incomplete Freund's
adjuvant (IFA) was used. Each immunization was performed at four
subcutaneous and four intramuscular sites.
Group A--immunogen GL256
Rabbit 1 #50
Rabbit 2 #51
Rabbit 3 #52
Rabbit 4 #53
[0255] Group B--immunogen GL258
Rabbit 5 #54
Rabbit 6 #55
Rabbit 7 #56
Rabbit 8 #57
[0256] Group C--immunogen GL262
Rabbit 9 #46
Rabbit 10 #47
Rabbit 11 #48
Rabbit 12 #49
TABLE-US-00009 [0257] Immunization scheme Day 1 First immunization
with 100 .mu.g immunogen/mL per animal in CFA Day 29 Second
immunization with 100 .mu.g immunogen/mL per animal in CFA Day 57
Third immunization with 100 .mu.g immunogen/mL per animal in IFA
the rabbit's state of the healthy might change for the worse by the
use of immunogens GL256 and GL258 rabbit 7 #56 was not treated Day
67 First bleeding (2 mL per animal) Day 81 Fourth immunization with
100 .mu.g immunogen/mL per animal in CFA Day 91 Second bleeding (25
mL per animal) Day 112 Fifth immunization with 100 .mu.g
immunogen/mL per animal in CFA Day 122 Assignment of the animal
numbers was mislaid Third final bleeding (Exsanguination)* *Rabbit
no. 1-12 were exsanguinated completely 10 days after the fifth
immunization. Exsanguination was performed via a carotid artery
under anesthesia with xylazin (Rompun .RTM., Bayer, Leverkusen,
Germany) and ketamine hydrochloride (Ketavet .RTM., Parke-Davis,
Freiburg, Germany).
5.2 Analysis of Rabbit Sera
[0258] Serum was prepared by centrifugation of the coagulated
rabbit blood. A protein fraction was obtained by ammonium sulphate
precipitation and desalting through a Sephadex G25 column.
[0259] The individual protein fractions from the rabbit sera were
screened for anti-dabigatran titer by a standard ELISA
procedure.
Screening-ELISA:
TABLE-US-00010 [0260] Step Procedure A protein fractions from each
bleeding were adsorbed overnight at ambient temperature onto
microtiter plates (100 .mu.L/well; 1, 2 or 4 .mu.g/mL) in buffer 1.
wash microplates 4 times, 450 .mu.L each block with 250 .mu.L
buffer 3 for at least 1 hour B wash microplates 4 times, 450 .mu.L
each C add to each well of microtiter plate in triplicate: +50
.mu.L buffer 2 +50 .mu.L calibration standards in buffer 2 +25
.mu.L dabigatran-horseradish peroxidase (HRP) conjugate GL 261
(tracer) ( 1/40000) D seal microplates with adhesive foil, complete
sample distribution for all microplates incubate for 4 h on a
shaker at ambient temperature E wash microplates 4 times, 450 .mu.L
each F add to each well of microtiter plate 100 .mu.L o-phenylene
diamine HCl, 2.7 mg/mL (one 30 mg tablet in 11 mL buffer 4)
incubate for 30 min in the dark at ambient temperature G add to
each well of microtiter plate 100 .mu.L H.sub.2SO.sub.4 (2.25M)
shake for 5 minutes H read absorbance; test-wavelength: 490 nm,
reference-wavelength: 650 nm
5.3 Detection of Anti-Dabigatran Antibodies in Rabbit Sera
[0261] Last three columns: values are for dabigatran
TABLE-US-00011 bleeding 2 coating conc conc. rabbit immunogene
[.mu.g/ml] [Mol] [Ext] [%] 1 #50 GL256 2 0 1.812 100% 2.E-12 1.574
87% 2.E-11 0.461 25% 2.E-10 0.059 3% 2 #51 GL256 1 0 2.193 100%
2.E-12 2.086 95% 2.E-11 1.515 69% 2.E-10 0.207 9% 3 #52 GL256 2 0
1.513 100% 2.E-12 1.419 94% 2.E-11 0.728 48% 2.E-10 0.107 7% 4 #53
GL256 2 0 1.474 100% 2.E-12 1.388 94% 2.E-11 0.848 58% 2.E-10 0.142
10% 5 #54 GL258 1 0 2.114 100% 2.E-12 1.892 89% 2.E-11 0.646 31%
2.E-10 0.159 8% 6 #55 GL258 1 0 1.295 100% 2.E-12 0.937 72% 2.E-11
0.265 20% 2.E-10 0.140 11% 7 #56 GL258 2 0 1.611 100% 2.E-12 1.372
85% 2.E-11 0.424 26% 2.E-10 0.145 9% 8 #46 GL258 1 0 1.640 100%
2.E-12 1.290 79% 2.E-11 0.425 26% 2.E-10 0.196 12% 9 #47 GL262 2 0
1.854 100% 2.E-12 1.534 83% 2.E-11 0.530 29% 2.E-10 0.254 14% 10
#48 GL262 2 0 1.458 100% 2.E-12 1.142 78% 2.E-11 0.300 21% 2.E-10
0.131 9% 11 #49 GL262 4 0 1.646 100% 2.E-12 1.393 85% 2.E-11 0.460
28% 2.E-10 0.257 16% 12 #50 GL262 2 0 1.605 100% 2.E-12 1.400 87%
2.E-11 0.389 24% 2.E-10 0.109 7%
TABLE-US-00012 Final bleeding coating conc conc. rabbit immunogene
[.mu.g/ml] [Mol] [Ext] [%] 1 ? 1 0 1.589 100% 2.E-12 1.442 91%
2.E-11 0.491 31% 2.E-10 0.130 8% 2 ? 1 0 1.375 100% 2.E-12 1.041
76% 2.E-11 0.293 21% 2.E-10 0.101 7% 3 ? 1 0 1.400 100% 2.E-12
1.081 77% 2.E-11 0.288 21% 2.E-10 0.097 7% 4 ? 1 0 1.183 100%
2.E-12 0.882 75% 2.E-11 0.396 33% 2.E-10 0.183 15% 5 ? 1 0 1.335
100% 2.E-12 1.066 80% 2.E-11 0.183 14% 2.E-10 0.057 4% 6 ? 1 0
1.214 100% 2.E-12 0.976 80% 2.E-11 0.250 21% 2.E-10 0.123 10% 7 ? 2
0 1.822 100% 2.E-12 1.702 93% 2.E-11 0.661 36% 2.E-10 0.189 10% 8 ?
2 0 1.234 100% 2.E-12 1.085 88% 2.E-11 0.671 54% 2.E-10 0.147 12% 9
? 1 0 1.911 100% 2.E-12 1.862 97% 2.E-11 0.980 51% 2.E-10 0.292 15%
10 ? 1 0 1.933 100% 2.E-12 1.891 98% 2.E-11 1.055 55% 2.E-10 0.076
4% 11 ? 1 0 1.874 100% 2.E-12 1.817 97% 2.E-11 1.539 82% 2.E-10
0.181 10% 12 ? 2 0 1.599 100% 2.E-12 1.425 89% 2.E-11 0.475 30%
2.E-10 0.050 3%
[0262] After screening of the protein fractions of all rabbits from
bleeding 2, it was obvious that rabbit no. 5 (#54) had the highest
titre of anti-dabigatran antibodies with the preferred hapten
HAPTEN2. Furthermore, it was possible to displace the tracer from
the antibody binding sites with only low concentrations of analyte
(dabigatran).
[0263] For the screening of the final bleeding 3, the displacement
of the tracer from the antibody binding site with low
concentrations of analyte (dabigatran) was used as main decision
criteria, because of the missing information about the immunogen
used. Therefore rabbits no. 2, 3 and 5 were used for the further
purification.
5.4 Purification of Polyclonal Antibodies
[0264] The anti-serum of rabbit no. 5 (#54) bleeding no. 2 and
rabbits no. 2, 3 and 5 bleeding no. 3 (final bleeding) was
precipitated with ammonium sulphate. The precipitate was
centrifuged for 30 min at 10.degree. C. at 4500 U/min, separated
from the solution and re-dissolved in Tris buffer. This procedure
was repeated. Further purification was performed by affinity
chromatography on protein A sepharose FF. The column buffer was
0.01 M Tris pH=7.5 and 0.1 M glycine pH=3.0 was used for elution.
Fractions containing the rabbit IgG were combined. Protein
concentration was determined by UV spectroscopy at 280 nm.
[0265] Summary of Antibody Specifications:
TABLE-US-00013 immunogen: HAPTEN2-BSA (lot no. GL258) rabbit: no. 5
(#54) serum (bleeding no. 2) protein content: 1.85 mg/mL storage:
at approximately -20.degree. C.
TABLE-US-00014 immunogen: HAPTEN1-BSA (GL256) or HAPTEN2-BSA (lot
no. GL258) or HAPTEN2-BSA (lot no. GL262) rabbit: no. 2 serum
collected (final bleeding) protein content: 3.9 mg/mL storage: at
approximately -20.degree. C.
TABLE-US-00015 immunogen: HAPTEN1-BSA (GL256) or HAPTEN2-BSA (lot
no. GL258) or HAPTEN2-BSA (lot no. GL262) rabbit: no. 3 serum
(final bleeding) protein content: 9.96 mg/mL storage: at
approximately -20.degree. C.
TABLE-US-00016 immunogen: HAPTEN1-BSA (GL256) or HAPTEN2-BSA (lot
no. GL258) or HAPTEN2-BSA (lot no. GL262) rabbit: no. 5 serum
(final bleeding) protein content: 5.72 mg/mL storage: at
approximately -20.degree. C.
II. Neutralization of Dabigatran
[0266] Two series of experiments were performed to show the effect
of the antibodies against dabigatran anticoagulant activity in
vitro. The four polyclonal antibodies were received in the
laboratory and further tested in human plasma. This was tested in
the functional assay, the thrombin clotting time.
Assay Description:
[0267] Briefly human plasma is obtained by taking whole blood into
3.13% sodium citrate. This is then centrifuged to obtain platelet
free plasma and transferred to a separate tube and frozen until
required on the day of the assay. Plasma is thawed at 37.degree. C.
on the day of the assay.
[0268] The thrombin clotting time is performed as follows. First
thrombin is diluted to manufacturer's specification (3 IU/mL
thrombin) in the buffer provided (Dade Behring Test kit) and
prewarmed to 37.degree. C. It is used within 2 hrs of being
prepared. All assays were performed on a commercially available CL4
clotting machine (Behnk Electronics, Norderstadt, Germany). Fifty
.mu.L of plasma is pipetted into provided cuvettes with a magnetic
stirrer and allowed to stir for 2 min in the well preheated to
37.degree. C. in the CL4 machine. At this point 100 .mu.L of the
thrombin solution is added and the time required for the plasma
sample to clot is recorded automatically by the CL4. Dabigatran is
preincubated for 5 min in plasma in the provided cuvettes, before
adding thrombin and starting the measurement. If antibody is also
tested (up 50 .mu.L of stock solution), there is a further 5 minute
incubation at 37.degree. C. before beginning clotting (i.e. 10 min
total incubation with dabigatran, 5 min total incubation with
antibody and then clotting is initiated with thrombin).
[0269] Initially a dabigatran standard curve was performed by
adding increasing concentrations of dabigatran to human plasma and
measuring the time to clotting after addition of thrombin (FIG. 1).
There was a concentration-dependent increase in the thrombin
clotting time with increasing concentrations of dabigatran.
[0270] For the first set of neutralization experiments, a
clinically relevant concentration of 200 nM of dabigatran was added
to all plasma samples for neutralization. All 4 antibody
preparations were able to shorten the time to clotting in plasma
containing dabigatran (FIG. 2). The extent of neutralization was
related to the concentration of protein in each antibody
preparation. The antibody solution with the highest concentration
(D) was then serially diluted and tested for the ability to
neutralize 200 nM dabigatran anticoagulant activity in a separate
set of experiments. It can be seen in FIG. 3, there was a
concentration dependent inhibition of dabigatran-induced
anticoagulant activity with increasing concentrations of antibody.
In addition when a non-specific rabbit polyclonal antibody (blue
square) was added to plasma containing dabigatran, it had no
ability to neutralise the anticoagulant activity. The concentration
dependency and the lack of neutralization of a non specific
antibody indicate the reversal of anticoagulation by the antibody
is specific for dabigatran.
[0271] However, these concentrations of dabigatran are clinically
relevant, and bleeding or overdoses will probably occur with higher
concentrations. Thus the ability of an antibody to inhibit the
anticoagulant activity of the highest concentration of dabigatran
(500 nM) in the standard curve in FIG. 1 was also tested. FIG. 4
illustrates that antibody D could also inhibit high concentrations
of dabigatran.
III. Production and Characterization of Monoclonal Anti-Dabigatran
Antibodies
[0272] 1. Production of Monoclonal Anti-Dabigatran Antibodies and
Fabs
[0273] Mice were immunized with Hapten1 (see Example 1.1)
conjugated to carrier proteins such as hemocyanin and
immunoglobulin and hybridomas were generated according to standard
procedures. Monoclonal antibodies purified from the culture
supernatants bound to dabigatran-protein conjugates and this
binding could be competed with dabigatran in solution with
half-maximal inhibition at concentrations in the range of 1 to 10
nM. Fabs were generated by papain cleavage of the monoclonal
antibodies with subsequent elimination of the Fc domain via Protein
A.
[0274] The variable regions from the heavy and light chains of the
mouse antibodies were cloned and sequenced using standard methods.
The sequences were confirmed by protein analysis by mass
spectrometry and N-terminal sequencing of the antibodies. DNA
constructs encoding chimeric antibodies comprising the specific
mouse variable regions and human IgG constant regions were
generated and protein was expressed in HEK293 cells and
purified.
[0275] In order to reduce potential immunogenicity, sequences of
mouse monoclonal antibody clones 35E6 and 27A9 were humanized by
standard methods described above. Humanized Fabs were produced by
transient transfection in mammalian cells (e.g. HEK293; CHO cells)
and purified by affinity chromatography with benzamidine sepharose
followed by size exclusion chromatography.
[0276] 2. Characterization of Monoclonal Anti-Dabigatran Antibodies
and Fabs
[0277] The sequences of the variable domains of 9 monoclonal
antibody clones DBG22 (clone 22), 35E6, 45B9, 48E1, 49F8, 6A7F1,
2F1E5, 3B4E7, 1F6G8, 2D2E3, and 27A9 are depicted in Table 1. SEQ
ID NO's 67, 68, 69, 92, 93, 94, 99, 100 and 101 represent optimized
and/or humanized sequences. The Fab compound VH5C/VK18 comprises
HCVH5C (SEQ ID NO: 99) as heavy chain, and LCVK18 (SEQ ID NO: 100)
as light chain. The Fab compound VH5C/VK21 comprises HCVH5C (SEQ ID
NO: 99) as heavy chain, and LCVK21 (SEQ ID NO: 101) as light chain.
Thus, both VH5C/VK18 and VH5C/VK21 comprise a heavy chain variable
domain with a CDR1 of SEQ ID NO: 67, a CDR2 of SEQ ID NO: 68, and a
CDR3 of SEQ ID NO: 9, and a light chain variable domain with a CDR1
of SEQ ID NO: 64, a CDR2 of SEQ ID NO: 65, and a CDR3 of SEQ ID NO:
69. Both Fabs share a variable region of the heavy chain of SEQ ID
NO: 92 (VH5C). VH5C/VK18 comprises a variable region of the light
chain of SEQ ID NO: 93 (VK18), and VH5C/VK21 comprises a variable
region of the light chain of SEQ ID NO: 94 (VK21).
[0278] In Table 1, the letters "CDR" denote a complementarity
determining region, "VH" denotes the variable region of a heavy
chain, "VK" denotes the variable region of a kappa light chain,
"CL" denotes the constant region of a light chain, and "CH" denotes
the constant region of a heavy chain, "LC" denotes the light chain
of an antibody molecule, and "HC" denotes the heavy chain of an
antibody molecule. For example, "VHCDR1 DBG22" denotes the first
CDR (CDR1) of the variable domain of the heavy chain of clone
DBG22, and "DBG22VH" denotes the variable region of the heavy chain
of clone DBG22.
TABLE-US-00017 TABLE 1 SEQ ID NO Designation Sequence 1 VHCDR1
GFSLTSYIVD DBG22 2 VHCDR2 VIWAGGSTNYNSALRS DBG22 3 VHCDR3
AAYYSYYNYDGFAY DBG22 4 VKCDR1 KSSQSLLYTNGKTYLY DBG22 5 VKCDR2
LVSKLDS DBG22 6 VKCDR3 LQSTHFPHT DBG22 7 VHCDR1 GYTFTNYWMH 35E6 8
VHCDR2 ETNPRNGGTNYNEKFKR 35E6 9 VHCDR3 GTSGYDYFDY 35E6 10 VKCDR1
RSSQTIVHSNGNTYLE 35E6 11 VKCDR2 KVSNRFS 35E6 12 VKCDR3 FQASHFPYT
35E6 13 VHCDR1 GVSLFTYDVD 45B9 14 VHCDR2 VMWSGGTTNYNSALKS 45B9 15
VHCDR3 DRWSPGGFAY 45B9 16 VKCDR1 QSSQSLLYTNGKTYLH 45B9 17 VKCDR2
LVSKLDS 45B9 18 VKCDR3 LQSTHFPHT 45B9 19 VHCDR1 GFSLTSYDVD 48E1 20
VHCDR2 VIWAGGSTNYNSALKS 48E1 21 VHCDR3 DRWSPGGFAY 48E1 22 VKCDR1
KSSQSLLYTNGKTYLI 48E1 23 VKCDR2 LVSKLDS 48E1 24 VKCDR3 LQTTHFPHT
48E1 25 VHCDR1 GFSLSTYGVD 49F8 26 VHCDR2 LIWAGGSTTYNSAFKS 49F8 27
VHCDR3 ERSGDSPFGY 49F8 28 VKCDR1 KSSQSLLYTNGKTYLN 49F8 29 VKCDR2
LVSKLDS 49F8 30 VKCDR3 LQNSHFPHT 49F8 31 VHCDR1 GFTFSTYGMS 6A7F1 32
VHCDR2 SVTRGGNTYYPDSM 6A7F1 33 VHCDR3 DYSGWYFDV 6A7F1 34 VKCDR1
RSSQSIVHSNGDTFLE 6A7F1 35 VKCDR2 KVSNRFS 6A7F1 36 VKCDR3 FQGSRIPYT
6A7F1 37 VHCDR1 GFTLTNYGMN 2F1E5 38 VHCDR2 WINTYTGEPTYADDFKG 2F1E5
39 VHCDR3 SAGTDYFDY 2F1E5 40 VKCDR1 RASESVDSYGNSFMH 2F1E5 41 VKCDR2
LASNLES 2F1E5 42 VKCDR3 QQNNEDPWT 2F1E5 43 VHCDR1 GYTFTYYTIH 3B4E7
44 VHCDR2 YINPASSYTNYIQKFKD 3B4E7 45 VHCDR3 GANWDYFDY 3B4E7 46
VKCDR1 RSSQNIIQSNGNTYLE 3B4E7 47 VKCDR2 KVSNRFS 3B4E7 48 VKCDR3
FQGSHVPYT 3B4E7 49 VHCDR1 GYTFTSYTIH 1F6G8 50 VHCDR2
YINPSSGYTYYIQNFKD 1F6G8 51 VHCDR3 GANWDYFDY 1F6G8 52 VKCDR1
RSSQNIVQTNGNTYLE 1F6G8 53 VKCDR2 KVSSRFS 1F6G8 54 VKCDR3 FQGSHVPFT
1F6G8 55 VHCDR1 GYTFTHSGMN 2D2E3 56 VHCDR2 WINTNTGEPTYAEEFNGR 2D2E3
57 VHCDR3 SWWTDYFDY 2D2E3 58 VKCDR1 RSSQSIVHSNGNTYLE 2D2F8 59
VKCDR2 KVSNRFS 2D2E3 60 VKCDR3 FQGSHFPYT 2D2E3 61 VHCDR1 GYTFTNCYMH
27A9 62 VHCDR2 ETNPRNGGTNYNEKFKR 27A9 63 VHCDR3 GTSGYEYFDY 27A9 64
VKCDR1 RSSQSIVHSDGNIYLE 27A9 65 VKCDR2 KVSYRFS 27A9 66 VKCDR3
FQGSHVPYT 27A9 67 VHCDR1 5C GYTFTDYYMH 68 VHCDR2 5C
ETNPRNGGTTYNEKFKG 69 VKCDR3 18 FQASHVPYT 70 DBG22VH QVQLEQSGPG
LVAPSQRLSI TCTVSGFSLT SYIVDWVRQS PGKGLEWLGV IWAGGSTNYN SALRSRLSIT
KSNSKSQVFL QMNSLQTDDT AIYYCASAAY YSYYNYDGFA YWGQGTLVTV SA 71
DBG22VK DVVMTQTPLT LSVTIGQPAS ISCKSSQSLL YTNGKTYLYW LLQRPGQSPK
RLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGI YYCLQSTHFP HTFGGGTKLE
IK 72 35E6VH QVQLQQPGAE LVKPGASVKL SCKTSGYTFT NYWMHWVRQR PGQGLEWIGE
TNPRNGGTNY NEKFKRKATL TVDKSSNTAY MQLSSLTFGD SAVYYCTIGT SGYDYFDYWG
QGTTLTVSS 73 35E6VK DVLMTQTPLS LPVSLGDQAS ISCRSSQTIV HSNGNTYLEW
YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTGFTLKI SRVEAEDLGV YFCFQASHFP
YTFGGGTKLE IK 74 45B9VH QVQLKQSGPG LVAPSQSLSI TCTVSGVSLF TYDVDWVRQS
PGKDLEWLGV MWSGGTTNYN SALKSRLNIM KDSSKSQVFL KMSGLQTDDT GIYYCATDRW
SPGGFAYWGQ GTLVTVSA 75 45B9VK DVVMTQTPLT LSVLIGQPAS ISCQSSQSLL
YTNGKTYLHW LLQRPGQSPK RLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV
YYCLQSTHFP HTFGGGTKLE IR 76 48E1VH QVQLKQSGPG LVAPSQSLSI TCTVSGFSLT
SYDVDWVRQS PGKGLEWLGV IWAGGSTNYN SALKSRLIIS KDNSKNQVFL RMNSLQTDDT
AMYYCASDRW SPGGFAYWGQ GTLVTVSA 77 48E1VK DVVMTQTPLT LSVTIGQPAS
ISCKSSQSLL YTNGKTYLIW LLQRPGQSPK RLIHLVSKLD SGVPDRFSGS GSGTDFTLKI
SRVEAEDLGV FYCLQTTHFP HTFGGGTKLE IR 78 49F8VH QVQLKQSGPG LVAPSQSLSI
TCTVSGFSLS TYGVDWVRQS PKKGLEWLGL IWAGGSTTYN SAFKSRLSIS KDNSKSQVFL
KMNSLQTDDT AMYYCASERS GDSPFGYWGQ GTLVTVSA 79 49F8VK DVVMTQSPLI
LSVTIGQPAS ISCKSSQSLL YTNGKTYLNW LLQRPGQSPE RLIHLVSKLD SGVPDRFSGS
GSGTDFTLKI SRVEAEDLGV YYCLQNSHFP HTFGSGTKLE IK
80 6A7F1VH EVKLVESGGD LVRPGGSLKL SCAASGFTFS TYGMSWVRQS PEKRLEWVAS
VTRGGNTYYP DSMRGRFTIS RDNVGNILYL HLRSLRSEDT AIYFCARDYS GWYFDVWGAG
TTVTVSS 81 6A7F1VK DVLMTQIPLS LPVSLGDQAS ISCRSSQSIV HSNGDTFLEW
YLQKSGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSRIP
YTFGGGTKLE IK 82 3B4E7VH QVQLQQSGAE LARPGASVKM SCKASGYTFT
YYTIHWVKQR PGQGLEWIGY INPASSYTNY IQKFKDRATL TADKSSSTAY MQLSSLTSED
SAVFYCARGA NWDYFDYWGQ GTTLTVSS 83 3B4E7VK DVLMTQTPLS LPVSLGDQAS
ISCRSSQNII QSNGNTYLEW YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI
SRVEAEDLGV YYCFQGSHVP YTFGGGTNLE IK 84 2F1E5VH QIQLVQSGPE
LKKPGETVKI SCKSSGFTLT NYGMNWVKQV PGKGLRWMGW INTYTGEPTY ADDFKGRFAF
SLETSARTAY LQINNLKNED AATYFCARSA GTDYFDYWGQ GTTLTVSS 85 2F1E5VK
NFVLTQSPAS LAVSLGQRAT ISCRASESVD SYGNSFMHWC QQKPGQPPKL LIYLASNLES
GVPARFSGSG SRTDFTLTID PVEADDAATY YCQQNNEDPW TFGGGTKLEI K 86 1F6G8VH
QIQLVQSGPE LKKPGETVKI SCKSSGFTLT NYGMNWVKQV PGKGLRWMGW INTYTGEPTY
ADDFKGRFAF SLETSARTAY LQINNLKNED AATYFCARSA GTDYFDYWGQ GTTLTVSS 87
1F6G8VK DVLMTQTPLS LPVSLGDQAS ISCRSSQNIV QTNGNTYLEW YLQKPGQSPN
LLIYKVSSRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP FTFGGGTKLE
IK 88 2D2E3VH QAQIHLVQSG PELKKPGETV KISCKASGYT FTHSGMNWMK
QTPGKDLKWM GWINTNTGEP TYAEEFNGRF AFSLEASANT AYLQINNLKN EDTATYFCAR
SWWTDYFDYW GQGTTLTVSS 89 2D2E3VK DVLMTQTPLS LPVSLGDQTS ISCRSSQSIV
HSNGNTYLEW YLQKPGQSPE LLIYKVSNRF SGVPDRISGS GSGTDFTLKI SRVEAEDLGV
YYCFQGSHFP YTFGGGTKLE IT 90 27A9VH QVQLQQPGAE LVKPGASVKL SCKASGYTFT
NCYMHWVKQR PGQGLEWIGE TNPRNGGTNY NEKFKRKATL TVNKYSSTAY MQLSSLTSED
SAVYYCTIGT SGYEYFDYWG QGTTLTVSS 91 27A9VK NILMTQTPLS LPVSLGDQAS
ISCRSSQSIV HSDGNIYLEW YLQKPGQSPK VLIYKVSYRF SGVPDRFSGS GSGTYFTLKI
SRVEAEDLGV YFCFQGSHVP YTFGGGTKLE IK 92 VH5C QVQLVQSGAE VKKPGASVKV
SCKASGYTFT DYYMHWVRQA PGQGLEWMGE TNPRNGGTTY NEKFKGKATM TRDTSTSTAY
MELSSLRSED TAVYYCTIGT SGYDYFDYWG QGTLVTVSS 93 VK18 DIVMTQTPLS
LSVTPGQPAS ISCRSSQSIV HSDGNIYLEW YLQKPGQSPK LLIYKVSYRF SGVPDRFSGS
GSGTDFTLKI SRVEAEDVGV YYCFQASHVP YTFGQGTKLE IK 94 VK21 DIVMTQTPLS
LSVTPGQPAS ISCRSSQSIV HSDGNIYLEW YLQKPGQSPK LLIYKVSYRF SGVPDRFSGS
GSGTGFTLKI SRVEAEDVGV YYCFQASHVP YTFGGGTKLE IK 95 Clone 22
QVQLEQSGPG LVAPSQRLSI TCTVSGFSLT SYIVDWVRQS chimeric HC PGKGLEWLGV
IWAGGSTNYN SALRSRLSIT KSNSKSQVFL QMNSLQTDDT AIYYCASAAY YSYYNYDGFA
YWGQGTLVTV SAASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS
GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKRV EPKSCDKTHT
CPPCPAPEAA GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH
NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE
PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF
LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK 96 Clone 22
DVVMTQTPLT LSVTIGQPAS ISCKSSQSLL YTNGKTYLYW chimeric LC LLQRPGQSPK
RLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGI YYCLQSTHFP HTFGGGTKLE
IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE
QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 97 hCL Domain
RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD
SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC 98 hCH Domain
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS
GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPEAAGG
PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN
STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE
MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW
QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 99 HCVH5C QVQLVQSGAE VKKPGASVKV
SCKASGYTFT DYYMHWVRQA PGQGLEWMGE TNPRNGGTTY NEKFKGKATM TRDTSTSTAY
MELSSLRSED TAVYYCTIGT SGYDYFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG
TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY
ICNVNHKPSN TKVDKKVEPK SC 100 LCVK18 DIVMTQTPLS LSVTPGQPAS
ISCRSSQSIV HSDGNIYLEW YLQKPGQSPK LLIYKVSYRF SGVPDRFSGS GSGTDFTLKI
SRVEAEDVGV YYCFQASHVP YTFGQGTKLE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL
LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE
VTHQGLSSPV TKSFNRGEC 101 LCVK21 DIVMTQTPLS LSVTPGQPAS ISCRSSQSIV
HSDGNIYLEW YLQKPGQSPK LLIYKVSYRF SGVPDRFSGS GSGTGFTLKI SRVEAEDVGV
YYCFQASHVP YTFGGGTKLE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK
VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV
TKSFNRGEC
[0279] The mouse monoclonal antibody clone 22 was tested for its
ability to neutralize dabigatran anticoagulant activity in human
plasma in the thrombin clotting time assay outlined in Example II.
The antibody completely reversed the dabigatran-mediated
prolongation of thrombin dependent clotting in human plasma in a
dose dependent manner (FIG. 5). The antibody also effectively
inhibited dabigatran function in human whole blood. A Fab generated
from this antibody blocked dabigatran activity in human plasma
demonstrating that monovalent antigen binding domains can
neutralize compound anticoagulant activity. (FIG. 6).
[0280] The major metabolic pathway of dabigatran in humans is
through the glucuronidation of the carboxylate moiety. Dabigatran
acylglucuronides have been shown to be pharmacologically active
(Ebner et al., Drug Metab. Dispos. 2010, 38(9):1567-75). To test
whether the mouse monoclonal antibody clone 22 could neutralize
these metabolites, dabigatran acylglucuronides were purified from
the urine of rhesus monkeys treated with dabigatran and evaluated
in the thrombin clotting time assay. The antibody dose dependently
reversed the dabigatran acylglucuronide-mediated prolongation of
thrombin dependent clotting in human plasma with similar potency to
that seen with dabigatran (FIG. 7). Thus the antibody is effective
in blocking the anticoagulant activity of dabigatran metabolites
found in humans.
[0281] The affinities of the Fab and the mouse-human chimeric
antibodies comprising the variable domains of clone 22 were
determined using Kinexa technology. A constant concentration of Fab
or chimeric antibody was incubated with various concentrations of
dabigatran until equilibrium was reached. After this incubation the
concentration of free antibody was determined by capturing the
antibody on Neutravidin beads coupled with a Biotin-conjugated
dabigatran analog. The captured Fab was detected with an anti-Mouse
IgG (Fab specific) F(ab')2 fragment labeled with FITC. The captured
chimeric antibodies were detected with an anti-human IgG conjugated
with Cy5. The dissociation constants were calculated using a 1:1
binding model. The results from these experiments are summarized in
the table below.
Affinity of Anti-Dabigatran Antibodies
TABLE-US-00018 [0282] Antibody Apparent K.sub.d Clone 22 Fab 48 pM
Clone 22 Chimeric Ab 34 pM
[0283] Both the Fab and the chimeric antibodies bind dabigatran
with high affinity.
Thrombin Clotting Time Assay
[0284] Briefly human plasma is obtained by taking whole blood into
3.13% sodium citrate. This is then centrifuged to obtain platelet
free plasma and transferred to a separate tube and frozen until
required on the day of the assay. Plasma is thawed at 37.degree. C.
on the day of the assay.
[0285] The thrombin clotting time is performed as follows. First
thrombin is diluted to manufacturer's specification (3 IU/mL
thrombin) in the buffer provided (Dade Behring Test kit) and
prewarmed to 37.degree. C. It is used within 2 hrs of being
prepared. All assays were performed on a commercially available CL4
clotting machine (Behnk Electronics, Norderstadt, Germany). Fifty
.mu.L of plasma is pipetted into provided cuvettes with a magnetic
stirrer and allowed to stir for 2 min in the well preheated to
37.degree. C. in the CL4 machine. At this point 100 .mu.L of the
thrombin solution is added and the time required for the plasma
sample to clot is recorded automatically by the CL4. Dabigatran is
preincubated for 5 min in plasma in the provided cuvettes, before
adding thrombin and starting the measurement. If antibody is also
tested (up 50 .mu.L of stock solution), there is a further 5 minute
incubation at 37.degree. C. before beginning clotting (i.e. 10 min
total incubation with dabigatran, 5 min total incubation with
antibody and then clotting is initiated with thrombin).
[0286] Activity of chimeric antibodies and humanized Fabs in the
thrombin time assay is shown in FIGS. 8-10, respectively.
Affinity Determinations (Kinexa Method)
[0287] The affinities of Fab and mouse-human chimeric antibodies
were determined using KinExA.RTM. technology. A constant
concentration of Fab or chimeric antibody was incubated with
various concentrations of dabigatran until equilibrium was reached.
After this incubation the concentration of free antibody was
determined by capturing the antibody on Neutravidin beads coupled
with a Biotin-conjugated dabigatran analog. The captured Fab was
detected with an anti-human IgG (Fab specific) F(ab')2 fragment
labeled with FITC. The captured chimeric antibodies were detected
with an anti-human IgG conjugated with Cy5. The dissociation
constants (K.sub.D) were calculated using a 1:1 binding model.
[0288] To measure rate constants (k.sub.on and k.sub.off) with the
KinExA.RTM. instrument, the Kinetics Direct method was used. In
this method, the binding partners are mixed in solution, and the
concentration of free active binding sites is probed over time as
active binding sites are depleted due to the formation of
complexes. Data points are collected at specified time intervals
and the signals are analyzed. In this way, k.sub.on is measured
directly and the off-rate k.sub.off is calculated as
k.sub.off=K.sub.D.times.k.sub.on.
TABLE-US-00019 TABLE K.sub.D values of chimeric antibodies
determined using KinExA .RTM. technology. Chimeric Ab K.sub.D (pM)
45B6 545 48E1 281 35E6 52 49F8 40 27A9 120
TABLE-US-00020 TABLE K.sub.D values, k.sub.on and k.sub.off of
humanized Fabs VH5C/VK18 and VH5C/VK21 Fab K.sub.D k.sub.on
k.sub.off (calculated) VH5C/VK18 133 pM 9.38e+005/Ms 1.25e-004/s
VH5C/VK21 147 pM 1.377e+006/Ms 2.02e-004/s
Fab-Dabigatran Complex Formation and Crystallization
[0289] The Fabs were concentrated to 10 mg/ml, mixed with a 2 molar
excess of dabigatran and incubated for 1 h at 4.degree. C. Complex
and crystallization solution were mixed 1:1. The complex
crystallizes in 25% PEG 1500, 0.1 M SPG buffer (pH7).
Data Collection and Structure Determination
[0290] Datasets for all crystals were collected on the Swiss light
Source beamline PXI-X06SA of the Paul Scherrer Institut. All
datasets were processed with the autoPROC package (Vonrhein, C.,
Flensburg, C., Keller, P., Sharff, A., Smart, O., Paciorek, W.,
Womack, T. & Bricogne, G. (2011). Data processing and analysis
with the autoPROC toolbox. Acta Cryst. D67, 293-302.).
[0291] Fab VH5C/VK21: Dabigatran crystals grew in space group
P212121 with unit cell dimensions a=59.97 .ANG., b=78.39 .ANG.,
c=87.67 .ANG. and diffract to 2.2 .ANG. resolution. The complex
structure was solved by molecular replacement with the program
phaser (Collaborative Computational Project, number 4. 1994. "The
CCP4 Suite: Programs for Protein Crystallography". Acta Cryst. D50,
760-763. Phaser crystallographic software. McCoy A J,
Grosse-Kunstleve R W, Adams P D, Winn M D, Storoni L C, Read R J.
J. Appl. Cryst. (2007). 40, 658-674.) using a homologous Fab
structure (PDB-ID 1C1E) as the starting search model. Analysis of
the electron density map showed clear electron density for
dabigatran. The complete structure was improved with multiple
rounds of model building with Coot and refinement with autoBUSTER
(Coot: model-building tools for molecular graphics" Emsley P,
Cowtan K Acta Crystallographica Section D-Biological
Crystallography 60: 2126-2132 Part 12 Sp. Iss. 1 DEC 2004. Bricogne
G., Blanc E., Brandi M., Flensburg C., Keller P., Paciorek W.,
Roversi P, Sharff A., Smart O. S., Vonrhein C., Womack T. O.
(2011). BUSTER version 2.11.2. Cambridge, United Kingdom: Global
Phasing Ltd).
[0292] Fab VH5C/VK18: Dabigatran crystals grew in space group P21
and P212121, respectively. Crystals with space group P21 showed
unit cell dimensions of a=51.81 .ANG., b=128.92 .ANG., c=60.26
.ANG. and diffract to 1.9 .ANG. resolution. Crystals with space
group P212121 showed unit cell dimensions of a=48.20 .ANG., b=59.74
.ANG., c=127.69 .ANG. and diffract to 2.2 .ANG. resolution. Both
complex structures were solved by molecular replacement with the
program phaser using the structure of Fab VH5C/VK21 as the starting
search model. Analysis of the electron density maps showed clear
electron density for dabigatran. The complete structures were
improved with multiple rounds of model building with Coot and
refinement with autoBUSTER.
In Silico Analysis of Spatial Aggregation Propensity (SAP)
[0293] The spatial aggregation propensities (SAP) for each atom and
each residue was calculated as described in (1) with the exception
that residue hydrophobicity parameters where taken from (2). The Fv
SAP is calculated as the sum over all positive residue SAP values
in the variable domains of the antibody. The CDR SAP is calculated
as the sum over all positive residue SAP values in the
complementary determining regions of the antibody. Fv SAP and CDR
SAP have been calculated for 850 different antibody structures from
the protein data bank (PDB), yielding a mean (.mu..sub.Fv, and
.mu..sub.CDR) and standard deviation values (.sigma..sub.Fv and
.sigma..sub.CDR) for both properties.
Z-scores for the Fv SAP and CDR SAP for the antibodies where then
calculated according to
Z-score(Fv SAP)=(Fv SAP-.mu..sub.Fv)/.sigma..sub.Fv and
Z-score(CDR SAP)=(CDR SAP-.mu..sub.CDR-.sigma..sub.CDR.
Results (FIG. 11):
Humanized Fab 18/15:
[0294] Z-score(Fv SAP)=1.06 [0295] Z-score(CDR SAP)=1.00
Humanized Fab VH5C/VK18:
[0295] [0296] Z-score(Fv SAP)=-0.61 [0297] Z-score(CDR
SAP)=-0.84
Humanized Fab VH5C/VK21:
[0297] [0298] Z-score(Fv SAP)=-0.61 [0299] Z-score (CDR
SAP)=-0.78
[0300] Fab 18/15 (see WO2011089183) has more solvent-exposed
hydrophobic surface than the average of known antibodies in the
protein data bank.
[0301] Surprisingly, both VH5CNK18 (SEQ ID NO: 99/SEQ ID NO: 100)
and VH5C/VK21 comprises SEQ ID NO: 99/SEQ ID NO: 101) have less
solvent-exposed hydrophobic surface than the average of known
antibodies in the protein data bank (negative Z-scores). This means
that these compounds have an increased solubility in aqueous media
and a lower tendency for aggregation, making them more suitable for
stable drug formulations with high antibody concentrations. [0302]
(1) Chemamsetty et. al., Proc Natl Acad Sci; 2009, 106(29), pg
11937-11942 [0303] (2) Cowan and Whittaker, Pept Res; 1990, 3(2),
pg 75-80
Expression of Fab in CHO Cells
[0304] Fabs were produced by transient transfection into CHO DG44
cells and subsequent selection and generation of stable cell pools.
FIG. 13 shows the titers of fed batch runs with Fab 18/15 (see
WO2011089183), Fab VH5c/Vk18 and Fab VH5c/Vk21. Surprisingly, Fabs
VH5c/Vk18 and VH5c/Vk21 show 5-10 fold higher titers as compared to
Fab 18/15.
Sequence CWU 1
1
101110PRTMus musculusSOURCE1..10/mol_type="protein" /note="VHCDR1
DBG22" /organism="Mus musculus" 1Gly Phe Ser Leu Thr Ser Tyr Ile
Val Asp 1 5 10216PRTMus musculusSOURCE1..16/mol_type="protein"
/note="VHCDR2 DBG22" /organism="Mus musculus" 2Val Ile Trp Ala Gly
Gly Ser Thr Asn Tyr Asn Ser Ala Leu Arg Ser 1 5 10 15 314PRTMus
musculusSOURCE1..14/mol_type="protein" /note="VHCDR3 DBG22"
/organism="Mus musculus" 3Ala Ala Tyr Tyr Ser Tyr Tyr Asn Tyr Asp
Gly Phe Ala Tyr 1 5 10 416PRTMus
musculusSOURCE1..16/mol_type="protein" /note="VKCDR1 DBG22"
/organism="Mus musculus" 4Lys Ser Ser Gln Ser Leu Leu Tyr Thr Asn
Gly Lys Thr Tyr Leu Tyr 1 5 10 15 57PRTMus
musculusSOURCE1..7/mol_type="protein" /note="VKCDR2 DBG22"
/organism="Mus musculus" 5Leu Val Ser Lys Leu Asp Ser 1 5 69PRTMus
musculusSOURCE1..9/mol_type="protein" /note="VKCDR3 DBG22"
/organism="Mus musculus" 6Leu Gln Ser Thr His Phe Pro His Thr 1 5
710PRTMus musculusSOURCE1..10/mol_type="protein" /note="VHCDR1
35E6" /organism="Mus musculus" 7Gly Tyr Thr Phe Thr Asn Tyr Trp Met
His 1 5 10817PRTMus musculusSOURCE1..17/mol_type="protein"
/note="VHCDR2 35E6" /organism="Mus musculus" 8Glu Thr Asn Pro Arg
Asn Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Arg 910PRTMus
musculusSOURCE1..10/mol_type="protein" /note="VHCDR3 35E6"
/organism="Mus musculus" 9Gly Thr Ser Gly Tyr Asp Tyr Phe Asp Tyr 1
5 101016PRTMus musculusSOURCE1..16/mol_type="protein" /note="VKCDR1
35E6" /organism="Mus musculus" 10Arg Ser Ser Gln Thr Ile Val His
Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 117PRTMus
musculusSOURCE1..7/mol_type="protein" /note="VKCDR2 35E6"
/organism="Mus musculus" 11Lys Val Ser Asn Arg Phe Ser 1 5
129PRTMus musculusSOURCE1..9/mol_type="protein" /note="VKCDR2 35E6"
/organism="Mus musculus" 12Phe Gln Ala Ser His Phe Pro Tyr Thr 1 5
1310PRTMus musculusSOURCE1..10/mol_type="protein" /note="VHCDR1
45B9" /organism="Mus musculus" 13Gly Val Ser Leu Phe Thr Tyr Asp
Val Asp 1 5 101416PRTMus musculusSOURCE1..16/mol_type="protein"
/note="VHCDR2 45B9" /organism="Mus musculus" 14Val Met Trp Ser Gly
Gly Thr Thr Asn Tyr Asn Ser Ala Leu Lys Ser 1 5 10 15 1510PRTMus
musculusSOURCE1..10/mol_type="protein" /note="VHCDR3 45B9"
/organism="Mus musculus" 15Asp Arg Trp Ser Pro Gly Gly Phe Ala Tyr
1 5 101616PRTMus musculusSOURCE1..16/mol_type="protein"
/note="VKCDR1 45B9" /organism="Mus musculus" 16Gln Ser Ser Gln Ser
Leu Leu Tyr Thr Asn Gly Lys Thr Tyr Leu His 1 5 10 15 177PRTMus
musculusSOURCE1..7/mol_type="protein" /note="VKCDR2 45B9"
/organism="Mus musculus" 17Leu Val Ser Lys Leu Asp Ser 1 5
189PRTMus musculusSOURCE1..9/mol_type="protein" /note="VKCDR3 45B9"
/organism="Mus musculus" 18Leu Gln Ser Thr His Phe Pro His Thr 1 5
1910PRTMus musculusSOURCE1..10/mol_type="protein" /note="VHCDR1
48E1" /organism="Mus musculus" 19Gly Phe Ser Leu Thr Ser Tyr Asp
Val Asp 1 5 102016PRTMus musculusSOURCE1..16/mol_type="protein"
/note="VHCDR2 48E1" /organism="Mus musculus" 20Val Ile Trp Ala Gly
Gly Ser Thr Asn Tyr Asn Ser Ala Leu Lys Ser 1 5 10 15 2110PRTMus
musculusSOURCE1..10/mol_type="protein" /note="VHCDR3 48E1"
/organism="Mus musculus" 21Asp Arg Trp Ser Pro Gly Gly Phe Ala Tyr
1 5 102216PRTMus musculusSOURCE1..16/mol_type="protein"
/note="VKCDR1 48E1" /organism="Mus musculus" 22Lys Ser Ser Gln Ser
Leu Leu Tyr Thr Asn Gly Lys Thr Tyr Leu Ile 1 5 10 15 237PRTMus
musculusSOURCE1..7/mol_type="protein" /note="VKCDR2 48E1"
/organism="Mus musculus" 23Leu Val Ser Lys Leu Asp Ser 1 5
249PRTMus musculusSOURCE1..9/mol_type="protein" /note="VKCDR3 48E1"
/organism="Mus musculus" 24Leu Gln Thr Thr His Phe Pro His Thr 1 5
2510PRTMus musculusSOURCE1..10/mol_type="protein" /note="VHCDR1
49F8" /organism="Mus musculus" 25Gly Phe Ser Leu Ser Thr Tyr Gly
Val Asp 1 5 102616PRTMus musculusSOURCE1..16/mol_type="protein"
/note="VHCDR2 49F8" /organism="Mus musculus" 26Leu Ile Trp Ala Gly
Gly Ser Thr Thr Tyr Asn Ser Ala Phe Lys Ser 1 5 10 15 2710PRTMus
musculusSOURCE1..10/mol_type="protein" /note="VHCDR3 49F8"
/organism="Mus musculus" 27Glu Arg Ser Gly Asp Ser Pro Phe Gly Tyr
1 5 102816PRTMus musculusSOURCE1..16/mol_type="protein"
/note="VKCDR1 49F8" /organism="Mus musculus" 28Lys Ser Ser Gln Ser
Leu Leu Tyr Thr Asn Gly Lys Thr Tyr Leu Asn 1 5 10 15 297PRTMus
musculusSOURCE1..7/mol_type="protein" /note="VKCDR2 49F8"
/organism="Mus musculus" 29Leu Val Ser Lys Leu Asp Ser 1 5
309PRTMus musculusSOURCE1..9/mol_type="protein" /note="VKCDR3 49F8"
/organism="Mus musculus" 30Leu Gln Asn Ser His Phe Pro His Thr 1 5
3110PRTMus musculusSOURCE1..10/mol_type="protein" /note="VHCDR1
6A7F1" /organism="Mus musculus" 31Gly Phe Thr Phe Ser Thr Tyr Gly
Met Ser 1 5 103214PRTMus musculusSOURCE1..14/mol_type="protein"
/note="VHCDR2 6A7F1" /organism="Mus musculus" 32Ser Val Thr Arg Gly
Gly Asn Thr Tyr Tyr Pro Asp Ser Met 1 5 10 339PRTMus
musculusSOURCE1..9/mol_type="protein" /note="VHCDR3 6A7F1"
/organism="Mus musculus" 33Asp Tyr Ser Gly Trp Tyr Phe Asp Val 1 5
3416PRTMus musculusSOURCE1..16/mol_type="protein" /note="VKCDR1
6A7F1" /organism="Mus musculus" 34Arg Ser Ser Gln Ser Ile Val His
Ser Asn Gly Asp Thr Phe Leu Glu 1 5 10 15 357PRTMus
musculusSOURCE1..7/mol_type="protein" /note="VKCDR2 6A7F1"
/organism="Mus musculus" 35Lys Val Ser Asn Arg Phe Ser 1 5
369PRTMus musculusSOURCE1..9/mol_type="protein" /note="VKCDR3
6A7F1" /organism="Mus musculus" 36Phe Gln Gly Ser Arg Ile Pro Tyr
Thr 1 5 3710PRTMus musculusSOURCE1..10/mol_type="protein"
/note="VHCDR1 2F1E5" /organism="Mus musculus" 37Gly Phe Thr Leu Thr
Asn Tyr Gly Met Asn 1 5 103817PRTMus
musculusSOURCE1..17/mol_type="protein" /note="VHCDR2 2F1E5"
/organism="Mus musculus" 38Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr
Tyr Ala Asp Asp Phe Lys 1 5 10 15 Gly 399PRTMus
musculusSOURCE1..9/mol_type="protein" /note="VHCDR3 2F1E5"
/organism="Mus musculus" 39Ser Ala Gly Thr Asp Tyr Phe Asp Tyr 1 5
4015PRTMus musculusSOURCE1..15/mol_type="protein" /note="VKCDR1
2F1E5" /organism="Mus musculus" 40Arg Ala Ser Glu Ser Val Asp Ser
Tyr Gly Asn Ser Phe Met His 1 5 10 15417PRTMus
musculusSOURCE1..7/mol_type="protein" /note="VKCDR2 2F1E5"
/organism="Mus musculus" 41Leu Ala Ser Asn Leu Glu Ser 1 5
429PRTMus musculusSOURCE1..9/mol_type="protein" /note="VKCDR3
2F1E5" /organism="Mus musculus" 42Gln Gln Asn Asn Glu Asp Pro Trp
Thr 1 5 4310PRTMus musculusSOURCE1..10/mol_type="protein"
/note="VHCDR1 3B4E7" /organism="Mus musculus" 43Gly Tyr Thr Phe Thr
Tyr Tyr Thr Ile His 1 5 104417PRTMus
musculusSOURCE1..17/mol_type="protein" /note="VHCDR2 3B4E7"
/organism="Mus musculus" 44Tyr Ile Asn Pro Ala Ser Ser Tyr Thr Asn
Tyr Ile Gln Lys Phe Lys 1 5 10 15 Asp 459PRTMus
musculusSOURCE1..9/mol_type="protein" /note="VHCDR3 3B4E7"
/organism="Mus musculus" 45Gly Ala Asn Trp Asp Tyr Phe Asp Tyr 1 5
4616PRTMus musculusSOURCE1..16/mol_type="protein" /note="VKCDR1
3B4E7" /organism="Mus musculus" 46Arg Ser Ser Gln Asn Ile Ile Gln
Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 477PRTMus
musculusSOURCE1..7/mol_type="protein" /note="VKCDR2 3B4E7"
/organism="Mus musculus" 47Lys Val Ser Asn Arg Phe Ser 1 5
489PRTMus musculusSOURCE1..9/mol_type="protein" /note="VKCDR3
3B4E7" /organism="Mus musculus" 48Phe Gln Gly Ser His Val Pro Tyr
Thr 1 5 4910PRTMus musculusSOURCE1..10/mol_type="protein"
/note="VHCDR1 1F6G8" /organism="Mus musculus" 49Gly Tyr Thr Phe Thr
Ser Tyr Thr Ile His 1 5 105017PRTMus
musculusSOURCE1..17/mol_type="protein" /note="VHCDR2 1F6G8"
/organism="Mus musculus" 50Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Tyr
Tyr Ile Gln Asn Phe Lys 1 5 10 15 Asp 519PRTMus
musculusSOURCE1..9/mol_type="protein" /note="VHCDR3 1F6G8"
/organism="Mus musculus" 51Gly Ala Asn Trp Asp Tyr Phe Asp Tyr 1 5
5216PRTMus musculusSOURCE1..16/mol_type="protein" /note="VKCDR1
1F6G8" /organism="Mus musculus" 52Arg Ser Ser Gln Asn Ile Val Gln
Thr Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 537PRTMus
musculusSOURCE1..7/mol_type="protein" /note="VKCDR2 1F6G8"
/organism="Mus musculus" 53Lys Val Ser Ser Arg Phe Ser 1 5
549PRTMus musculusSOURCE1..9/mol_type="protein" /note="VKCDR3
1F6G8" /organism="Mus musculus" 54Phe Gln Gly Ser His Val Pro Phe
Thr 1 5 5510PRTMus musculusSOURCE1..10/mol_type="protein"
/note="VHCDR1 2D2E3" /organism="Mus musculus" 55Gly Tyr Thr Phe Thr
His Ser Gly Met Asn 1 5 105618PRTMus
musculusSOURCE1..18/mol_type="protein" /note="VHCDR2 2D2E3"
/organism="Mus musculus" 56Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr
Tyr Ala Glu Glu Phe Asn 1 5 10 15 Gly Arg 579PRTMus
musculusSOURCE1..9/mol_type="protein" /note="VHCDR3 2D2E3"
/organism="Mus musculus" 57Ser Trp Trp Thr Asp Tyr Phe Asp Tyr 1 5
5816PRTMus musculusSOURCE1..16/mol_type="protein" /note="VKCDR1
2D2F8" /organism="Mus musculus" 58Arg Ser Ser Gln Ser Ile Val His
Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 597PRTMus
musculusSOURCE1..7/mol_type="protein" /note="VKCDR2 2D2E3"
/organism="Mus musculus" 59Lys Val Ser Asn Arg Phe Ser 1 5
609PRTMus musculusSOURCE1..9/mol_type="protein" /note="VKCDR3
2D2E3" /organism="Mus musculus" 60Phe Gln Gly Ser His Phe Pro Tyr
Thr 1 5 6110PRTMus musculusSOURCE1..10/mol_type="protein"
/note="VHCDR1 27A9" /organism="Mus musculus" 61Gly Tyr Thr Phe Thr
Asn Cys Tyr Met His 1 5 106217PRTMus
musculusSOURCE1..17/mol_type="protein" /note="VHCDR2 27A9"
/organism="Mus musculus" 62Glu Thr Asn Pro Arg Asn Gly Gly Thr Asn
Tyr Asn Glu Lys Phe Lys 1 5 10 15 Arg 6310PRTMus
musculusSOURCE1..10/mol_type="protein" /note="VHCDR3 27A9"
/organism="Mus musculus" 63Gly Thr Ser Gly Tyr Glu Tyr Phe Asp Tyr
1 5 106416PRTMus musculusSOURCE1..16/mol_type="protein"
/note="VKCDR1 27A9" /organism="Mus musculus" 64Arg Ser Ser Gln Ser
Ile Val His Ser Asp Gly Asn Ile Tyr Leu Glu 1 5 10 15 657PRTMus
musculusSOURCE1..7/mol_type="protein" /note="VKCDR2 27A9"
/organism="Mus musculus" 65Lys Val Ser Tyr Arg Phe Ser 1 5
669PRTMus musculusSOURCE1..9/mol_type="protein" /note="VKCDR3 27A9"
/organism="Mus musculus" 66Phe Gln Gly Ser His Val Pro Tyr Thr 1 5
6710PRTMus musculusSOURCE1..10/mol_type="protein" /note="VHCDR1 5C"
/organism="Mus musculus" 67Gly Tyr Thr Phe Thr Asp Tyr Tyr Met His
1 5 106817PRTMus musculusSOURCE1..17/mol_type="protein"
/note="VHCDR2 5C" /organism="Mus musculus" 68Glu Thr Asn Pro Arg
Asn Gly Gly Thr Thr Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly 699PRTMus
musculusSOURCE1..9/mol_type="protein" /note="VKCDR3 18"
/organism="Mus musculus" 69Phe Gln Ala Ser His Val Pro Tyr Thr 1 5
70122PRTMus musculusSOURCE1..122/mol_type="protein" /note="DBG22VH"
/organism="Mus musculus" 70Gln Val Gln Leu Glu Gln Ser Gly Pro Gly
Leu Val Ala Pro Ser Gln 1 5 10 15 Arg Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Ile Val Asp Trp Val Arg
Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp
Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Arg 50 55 60 Ser Arg
Leu Ser Ile Thr Lys Ser Asn Ser Lys Ser Gln Val Phe Leu 65 70 75
80Gln Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95 Ser Ala Ala Tyr Tyr Ser Tyr Tyr Asn Tyr Asp Gly Phe Ala
Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 120
71112PRTMus musculusSOURCE1..112/mol_type="protein" /note="DBG22VK"
/organism="Mus musculus" 71Asp Val Val Met Thr Gln Thr Pro Leu Thr
Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys
Ser Ser Gln Ser Leu Leu Tyr Thr 20 25 30 Asn Gly Lys Thr Tyr Leu
Tyr Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu
Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75
80Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Leu Gln Ser
85 90 95 Thr His Phe Pro His Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 105 110 72119PRTMus
musculusSOURCE1..119/mol_type="protein" /note="35E6VH"
/organism="Mus musculus" 72Gln Val Gln Leu Gln Gln Pro Gly Ala Glu
Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Thr
Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met His Trp Val Arg
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Thr Asn
Pro Arg Asn Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Arg
Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75
80Met Gln Leu Ser Ser Leu Thr Phe Gly Asp Ser Ala Val Tyr Tyr Cys
85 90 95 Thr Ile Gly Thr Ser Gly Tyr Asp Tyr Phe Asp Tyr Trp Gly
Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 73112PRTMus
musculusSOURCE1..112/mol_type="protein" /note="35E6VK"
/organism="Mus musculus" 73Asp Val Leu Met Thr Gln Thr Pro Leu Ser
Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg
Ser Ser Gln Thr Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu
Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu
Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Gly Phe Thr Leu Lys Ile 65 70 75
80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Phe Gln Ala
85 90
95 Ser His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110 74118PRTMus musculusSOURCE1..118/mol_type="protein"
/note="45B9VH" /organism="Mus musculus" 74Gln Val Gln Leu Lys Gln
Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile
Thr Cys Thr Val Ser Gly Val Ser Leu Phe Thr Tyr 20 25 30 Asp Val
Asp Trp Val Arg Gln Ser Pro Gly Lys Asp Leu Glu Trp Leu 35 40 45
Gly Val Met Trp Ser Gly Gly Thr Thr Asn Tyr Asn Ser Ala Leu Lys 50
55 60 Ser Arg Leu Asn Ile Met Lys Asp Ser Ser Lys Ser Gln Val Phe
Leu 65 70 75 80Lys Met Ser Gly Leu Gln Thr Asp Asp Thr Gly Ile Tyr
Tyr Cys Ala 85 90 95 Thr Asp Arg Trp Ser Pro Gly Gly Phe Ala Tyr
Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115
75112PRTMus musculusSOURCE1..112/mol_type="protein" /note="45B9VK"
/organism="Mus musculus" 75Asp Val Val Met Thr Gln Thr Pro Leu Thr
Leu Ser Val Leu Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Gln
Ser Ser Gln Ser Leu Leu Tyr Thr 20 25 30 Asn Gly Lys Thr Tyr Leu
His Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu
Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75
80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Ser
85 90 95 Thr His Phe Pro His Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Arg 100 105 110 76118PRTMus
musculusSOURCE1..118/mol_type="protein" /note="48E1VH"
/organism="Mus musculus" 76Gln Val Gln Leu Lys Gln Ser Gly Pro Gly
Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Asp Val Asp Trp Val Arg
Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp
Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg
Leu Ile Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Phe Leu 65 70 75
80Arg Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95 Ser Asp Arg Trp Ser Pro Gly Gly Phe Ala Tyr Trp Gly Gln
Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 77112PRTMus
musculusSOURCE1..112/mol_type="protein" /note="48E1VK"
/organism="Mus musculus" 77Asp Val Val Met Thr Gln Thr Pro Leu Thr
Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys
Ser Ser Gln Ser Leu Leu Tyr Thr 20 25 30 Asn Gly Lys Thr Tyr Leu
Ile Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu
Ile His Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75
80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Phe Tyr Cys Leu Gln Thr
85 90 95 Thr His Phe Pro His Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Arg 100 105 110 78118PRTMus
musculusSOURCE1..118/mol_type="protein" /note="49F8VH"
/organism="Mus musculus" 78Gln Val Gln Leu Lys Gln Ser Gly Pro Gly
Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Ser Thr Tyr 20 25 30 Gly Val Asp Trp Val Arg
Gln Ser Pro Lys Lys Gly Leu Glu Trp Leu 35 40 45 Gly Leu Ile Trp
Ala Gly Gly Ser Thr Thr Tyr Asn Ser Ala Phe Lys 50 55 60 Ser Arg
Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75
80Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95 Ser Glu Arg Ser Gly Asp Ser Pro Phe Gly Tyr Trp Gly Gln
Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 79112PRTMus
musculusSOURCE1..112/mol_type="protein" /note="49F8VK"
/organism="Mus musculus" 79Asp Val Val Met Thr Gln Ser Pro Leu Ile
Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys
Ser Ser Gln Ser Leu Leu Tyr Thr 20 25 30 Asn Gly Lys Thr Tyr Leu
Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Glu Arg Leu
Ile His Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75
80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Asn
85 90 95 Ser His Phe Pro His Thr Phe Gly Ser Gly Thr Lys Leu Glu
Ile Lys 100 105 110 80117PRTMus
musculusSOURCE1..117/mol_type="protein" /note="6A7F1VH"
/organism="Mus musculus" 80Glu Val Lys Leu Val Glu Ser Gly Gly Asp
Leu Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met Ser Trp Val Arg
Gln Ser Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Val Thr
Arg Gly Gly Asn Thr Tyr Tyr Pro Asp Ser Met Arg 50 55 60 Gly Arg
Phe Thr Ile Ser Arg Asp Asn Val Gly Asn Ile Leu Tyr Leu 65 70 75
80His Leu Arg Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Phe Cys Ala
85 90 95 Arg Asp Tyr Ser Gly Trp Tyr Phe Asp Val Trp Gly Ala Gly
Thr Thr 100 105 110 Val Thr Val Ser Ser 115 81112PRTMus
musculusSOURCE1..112/mol_type="protein" /note="6A7F1VK"
/organism="Mus musculus" 81Asp Val Leu Met Thr Gln Ile Pro Leu Ser
Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asp Thr Phe Leu
Glu Trp Tyr Leu Gln Lys Ser Gly Gln Ser 35 40 45 Pro Lys Leu Leu
Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75
80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95 Ser Arg Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 105 110 82118PRTMus
musculusSOURCE1..118/mol_type="protein" /note="3B4E7VH"
/organism="Mus musculus" 82Gln Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Tyr Tyr 20 25 30 Thr Ile His Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn
Pro Ala Ser Ser Tyr Thr Asn Tyr Ile Gln Lys Phe 50 55 60 Lys Asp
Arg Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75
80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys
85 90 95 Ala Arg Gly Ala Asn Trp Asp Tyr Phe Asp Tyr Trp Gly Gln
Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 83112PRTMus
musculusSOURCE1..112/mol_type="protein" /note="3B4E7VK"
/organism="Mus musculus" 83Asp Val Leu Met Thr Gln Thr Pro Leu Ser
Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg
Ser Ser Gln Asn Ile Ile Gln Ser 20 25 30 Asn Gly Asn Thr Tyr Leu
Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu
Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75
80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Asn Leu Glu
Ile Lys 100 105 110 84118PRTMus
musculusSOURCE1..118/mol_type="protein" /note="2F1E5VH"
/organism="Mus musculus" 84Gln Ile Gln Leu Val Gln Ser Gly Pro Glu
Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ser
Ser Gly Phe Thr Leu Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys
Gln Val Pro Gly Lys Gly Leu Arg Trp Met 35 40 45 Gly Trp Ile Asn
Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly
Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Arg Thr Ala Tyr 65 70 75
80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Ala Ala Thr Tyr Phe Cys
85 90 95 Ala Arg Ser Ala Gly Thr Asp Tyr Phe Asp Tyr Trp Gly Gln
Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 85111PRTMus
musculusSOURCE1..111/mol_type="protein" /note="2F1E5VK"
/organism="Mus musculus" 85Asn Phe Val Leu Thr Gln Ser Pro Ala Ser
Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg
Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30 Gly Asn Ser Phe Met His
Trp Cys Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile
Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe
Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp 65 70 75
80Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn
85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 105 110 86118PRTMus musculusSOURCE1..118/mol_type="protein"
/note="1F6G8VH" /organism="Mus musculus" 86Gln Ile Gln Leu Val Gln
Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile
Ser Cys Lys Ser Ser Gly Phe Thr Leu Thr Asn Tyr 20 25 30 Gly Met
Asn Trp Val Lys Gln Val Pro Gly Lys Gly Leu Arg Trp Met 35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50
55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Arg Thr Ala
Tyr 65 70 75 80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Ala Ala Thr
Tyr Phe Cys 85 90 95 Ala Arg Ser Ala Gly Thr Asp Tyr Phe Asp Tyr
Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115
87112PRTMus musculusSOURCE1..112/mol_type="protein" /note="1F6G8VK"
/organism="Mus musculus" 87Asp Val Leu Met Thr Gln Thr Pro Leu Ser
Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg
Ser Ser Gln Asn Ile Val Gln Thr 20 25 30 Asn Gly Asn Thr Tyr Leu
Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Asn Leu Leu
Ile Tyr Lys Val Ser Ser Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75
80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95 Ser His Val Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 105 110 88120PRTMus
musculusSOURCE1..120/mol_type="protein" /note="2D2E3VH"
/organism="Mus musculus" 88Gln Ala Gln Ile His Leu Val Gln Ser Gly
Pro Glu Leu Lys Lys Pro 1 5 10 15 Gly Glu Thr Val Lys Ile Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 His Ser Gly Met Asn Trp
Met Lys Gln Thr Pro Gly Lys Asp Leu Lys 35 40 45 Trp Met Gly Trp
Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu 50 55 60 Glu Phe
Asn Gly Arg Phe Ala Phe Ser Leu Glu Ala Ser Ala Asn Thr 65 70 75
80Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr
85 90 95 Phe Cys Ala Arg Ser Trp Trp Thr Asp Tyr Phe Asp Tyr Trp
Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115
12089112PRTMus musculusSOURCE1..112/mol_type="protein"
/note="2D2E3VK" /organism="Mus musculus" 89Asp Val Leu Met Thr Gln
Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Thr Ser
Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly
Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45
Pro Glu Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50
55 60 Asp Arg Ile Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile 65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys
Phe Gln Gly 85 90 95 Ser His Phe Pro Tyr Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Thr 100 105 110 90119PRTMus
musculusSOURCE1..119/mol_type="protein" /note="27A9VH"
/organism="Mus musculus" 90Gln Val Gln Leu Gln Gln Pro Gly Ala Glu
Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asn Cys 20 25 30 Tyr Met His Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Thr Asn
Pro Arg Asn Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Arg
Lys Ala Thr Leu Thr Val Asn Lys Tyr Ser Ser Thr Ala Tyr 65 70 75
80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95 Thr Ile Gly Thr Ser Gly Tyr Glu Tyr Phe Asp Tyr Trp Gly
Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 91112PRTMus
musculusSOURCE1..112/mol_type="protein" /note="27A9VK"
/organism="Mus musculus" 91Asn Ile Leu Met Thr Gln Thr Pro Leu Ser
Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asp Gly Asn Ile Tyr Leu
Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Val Leu
Ile Tyr Lys Val Ser Tyr Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Tyr Phe Thr Leu Lys Ile 65 70
75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Phe Gln
Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 105 110 92119PRTArtificial
SequenceSOURCE1..119/mol_type="protein" /note="VH5C"
/organism="Artificial Sequence" 92Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu
Thr Asn Pro Arg Asn Gly Gly Thr Thr Tyr Asn Glu Lys Phe 50 55 60
Lys Gly Lys Ala Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Thr Ile Gly Thr Ser Gly Tyr Asp Tyr Phe Asp Tyr Trp
Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
93112PRTArtificial SequenceSOURCE1..112/mol_type="protein"
/note="VK18" /organism="Artificial Sequence" 93Asp Ile Val Met Thr
Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asp
Gly Asn Ile Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Tyr Arg Phe Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Phe Gln Ala 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 110 94112PRTArtificial
SequenceSOURCE1..112/mol_type="protein" /note="VK21"
/organism="Artificial Sequence" 94Asp Ile Val Met Thr Gln Thr Pro
Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asp Gly Asn Ile
Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys
Leu Leu Ile Tyr Lys Val Ser Tyr Arg Phe Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Gly Phe Thr Leu Lys Ile 65
70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln
Ala 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 105 110 95452PRTArtificial
SequenceSOURCE1..452/mol_type="protein" /note="Clone 22 chimeric
HC" /organism="Artificial Sequence" 95Gln Val Gln Leu Glu Gln Ser
Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Arg Leu Ser Ile Thr
Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Ile Val Asp
Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly
Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Arg 50 55
60 Ser Arg Leu Ser Ile Thr Lys Ser Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80Gln Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr
Cys Ala 85 90 95 Ser Ala Ala Tyr Tyr Ser Tyr Tyr Asn Tyr Asp Gly
Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ala
Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro
Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Ala Ala 225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310
315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435
440 445 Ser Pro Gly Lys 450 96219PRTArtificial
SequenceSOURCE1..219/mol_type="protein" /note="Clone 22 chimeric
LC" /organism="Artificial Sequence" 96Asp Val Val Met Thr Gln Thr
Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile
Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr 20 25 30 Asn Gly Lys
Thr Tyr Leu Tyr Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro
Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Leu
Gln Ser 85 90 95 Thr His Phe Pro His Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185
190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
97107PRTHomo sapiensSOURCE1..107/mol_type="protein" /note="hCL
Domain" /organism="Homo sapiens" 97Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65
70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105
98330PRTHomo sapiensSOURCE1..330/mol_type="protein" /note="hCH
Domain" /organism="Homo sapiens" 98Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65
70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195
200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu 225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33099222PRTArtificial SequenceSOURCE1..222/mol_type="protein"
/note="HCVH5C" /organism="Artificial Sequence" 99Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Glu Thr Asn Pro Arg Asn Gly Gly Thr Thr Tyr Asn Glu Lys
Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr 65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Thr Ser Gly Tyr Asp Tyr
Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220 100219PRTArtificial
SequenceSOURCE1..219/mol_type="protein" /note="LCVK18"
/organism="Artificial Sequence" 100Asp Ile Val Met Thr Gln Thr Pro
Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asp Gly Asn Ile
Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys
Leu Leu Ile Tyr Lys Val Ser Tyr Arg Phe Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65
70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln
Ala 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195
200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
101219PRTArtificial SequenceSOURCE1..219/mol_type="protein"
/note="LCVK21" /organism="Artificial Sequence" 101Asp Ile Val Met
Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30
Asp Gly Asn Ile Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Tyr Arg Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Gly Phe Thr
Leu Lys Ile 65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Phe Gln Ala 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155
160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
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