NZ613543B2 - Anticoagulant antidotes - Google Patents
Anticoagulant antidotes Download PDFInfo
- Publication number
- NZ613543B2 NZ613543B2 NZ613543A NZ61354312A NZ613543B2 NZ 613543 B2 NZ613543 B2 NZ 613543B2 NZ 613543 A NZ613543 A NZ 613543A NZ 61354312 A NZ61354312 A NZ 61354312A NZ 613543 B2 NZ613543 B2 NZ 613543B2
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- New Zealand
- Prior art keywords
- antibody
- dabigatran
- seq
- fab
- antibody molecule
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- AMCPECLBZPXAPB-UHFFFAOYSA-N propane-1,2,3-triol;sodium Chemical compound [Na].OCC(O)CO AMCPECLBZPXAPB-UHFFFAOYSA-N 0.000 description 1
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- KEAYESYHFKHZAL-UHFFFAOYSA-N sodium Chemical class [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
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- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- GRONZTPUWOOUFQ-UHFFFAOYSA-M sodium;methanol;hydroxide Chemical compound [OH-].[Na+].OC GRONZTPUWOOUFQ-UHFFFAOYSA-M 0.000 description 1
- 239000012265 solid product Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
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- 150000008163 sugars Chemical class 0.000 description 1
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- 238000004114 suspension culture Methods 0.000 description 1
- AOCSUUGBCMTKJH-UHFFFAOYSA-N tert-butyl N-(2-aminoethyl)carbamate Chemical compound CC(C)(C)OC(=O)NCCN AOCSUUGBCMTKJH-UHFFFAOYSA-N 0.000 description 1
- ZFQWJXFJJZUVPI-UHFFFAOYSA-N tert-butyl N-(4-aminobutyl)carbamate Chemical compound CC(C)(C)OC(=O)NCCCCN ZFQWJXFJJZUVPI-UHFFFAOYSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-PHYPRBDBSA-N α-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
Classifications
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39583—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials not provided for elsewhere, e.g. haptens, coenzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Abstract
Disclosed are antibodies comprising the CDRs GYTFTDYYMH, ETNPRNGGTTYNEKFKG, GTSGYDYFDY, RSSQSIVHSDGNIYLE, KVSYRFS and FQASHVPYT.
Description
ANTICOAGULANT ANTIDOTES
TECHNICAL FIELD
The t ion pertains to the field of medicine, in particular to the field of
anticoagulant therapy.
BACKGROUND ATION
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.
An important class of oral anticoagulants acts by antagonizing the effects of vitamin K, for
example the coumarins which include in. A second class of compounds t
coagulation indirectly via a cofactor such as antithrombin III or heparin cofactor II. This
includes l 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 parinux, rinux) inhibit only factor Xa via antithrombin
III. Heparinoids (danaparoid, xide, 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 irudin,
Iepirudin, desirudin), and the monovalent compounds argatroban and dabigatran.
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 ng events when they occur (Zikria and
, Current Opinion in Hematology 2009, 16(5): 347-356). One way to achieve this is
by neutralizing the activity of the agulant compound present in the patient after
administration.
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 hage
(Lauritzen, B. et al, Blood, 2005, 607A-608A.). Also reported are protamine fragments (US
Patent No. 6,624,141) and small synthetic peptides (US Patent No. 6,200,955) as heparin or low
molecular weight n tes; and thrombin muteins (US Patent No. 6,060,300) as
antidotes for thrombin inhibitor. Prothrombin intermediates and derivatives have been reported
as antidotes to n and synthetic thrombin inhibitors (US Patent 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 ent factor Xa inhibitors such as fondaparinux and
idraparinux (Bijsterveld, NR et al, Circulation, 2002, 106: 2550-2554; Bijsterveld, NR 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.
International patent application WO2011089183 discloses antibodies that can bind and
neutralize the activity of tran.
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
ing to a first aspect of the invention, there is provided an antibody molecule against
dabigatran comprising 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.
According to a second aspect of the ion, there is provided use of an antibody molecule
according to the first aspect of the ion for the manufacture of a medicament for the
therapy or tion of side effects of agulant therapy, and/or for reversal of an
overdosing of an agulant.
According to a third aspect of the invention, there is provided a method of manufacturing an
antibody molecule according to the first aspect of the invention, comprising
AH26(10039265_1):JIN
(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, wherein the host
cell is not within a human.
According to a fourth aspect of the invention, there is provided a kit comprising an antibody
according to the first aspect of the invention, or a pharmaceutical composition thereof.
According to a fifth aspect of the invention, there is provided a kit comprising:
(a) an antibody according to the first aspect of the invention, or a
pharmaceutical composition thereof;
(b) a ner; and
(c) a label.
According to a sixth aspect of the invention, there is provided a kit comprising an dy
according to the first aspect of the ion, and dabigatran, dabigatran etexilate, a prodrug of
dabigatran or a pharmaceutically acceptable salt thereof.
In a further aspect, the present invention s to an antibody molecule capable of neutralizing
the ty of an agulant.
In a further aspect, the antibody molecule has binding specificity for the anticoagulant.
In a further aspect, the anticoagulant is a direct thrombin inhibitor, a Factor Xa inhibitor, or a
n K antagonist.
AH26(10039265_1):JIN
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.
In another aspect, the present invention relates to an antibody molecule against
dabigatran, dabigatran exetilate, and/or an O-acylglucuronide of dabigatran.
In a further aspect, the present invention relates to an antibody molecule against
dabigatran, tran exetilate, and/or an glucuronide of dabigatran with reduced
immunogenicity in man.
In a further aspect, the present invention relates to an antibody molecule against
dabigatran, dabigatran ate, and/or an O-acylglucuronide of dabigatran with improved
physicochemical properties, in particular improved solubility in aqueous solvents.
In a further aspect, the present ion 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 ed production yields.
In a r aspect, the antibody le is a polyclonal antibody, a monoclonal dy,
a human antibody, a humanized antibody, a chimeric dy, a fragment of an antibody,
in ular a Fab, Fab’, or F(ab’)2 fragment, a single chain antibody, in particular a
single chain le nt (scFv), a domain antibody, a nanobody, a diabody, or a
DARPin.
In a further aspect, the present invention relates to an dy molecule as described
above for use in ne.
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 a further aspect, the side effect is a bleeding event.
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
dy molecule as described above to a patient in need thereof.
In another aspect, the present invention relates to a kit comprising an antibody molecule
as described, together with a ner and a label.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Increased time to clotting seen with increased concentrations of dabigatran
using the thrombin clotting time assay. The 200 nM concentration resulted in an d
elevation in ng time over baseline and was used in the first and second set of
experiments. The 500 nM tration (supratherapeutic) was used in the last set of
experiments.
Figure 2: Four different antibodies to dabigatran (A-D) all lized 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, ng was ged
to 51 seconds. Each antibody was added to plasma preincubated with 200 nM of
dabigatran and further ted 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.
Figure 3: The effect of sing trations 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 tran 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. on of a non specific rabbit
WO 30834
polyclonal antibody (square) had no effect on reversing the anticoagulant effect of
dabigatran.
Figure 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 old se). 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
1O completely reversed the in clotting time to baseline levels and resulted in complete
neutralization of the anticoagulant effect of even supratherapeutic concentrations of
dabigatran.
Figure 5: A mouse monoclonal antibody (Clone 22) reverses the anticoagulant effect of
dabigatran in human plasma and in human whole blood. lncreasing 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 dy dose
ently inhibited the tran mediated prolongation of clotting time.
Figure 6: A mouse Fab generated from the Clone 22 antibody es the
anticoagulant effect of dabigatran in human . 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
additon of 0.4 U/mL of thrombin and clotting time was ed. 100% inhibition was
defined as the complete block of the dabigatran mediated increase in clotting time. The
Fab dose dependently inhibited the tran induced prolongation in clotting time in
human plasma.
Figure 7: A mouse monoclonal dy (Clone 22) reverses the anticoagulant effect of
dabigatran ucuronide 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 additon of 0.4
U/mL of in and ng 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.
Figure 8: Selected chimeric antibodies t 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 additon of 0.4 U/mL of thrombin and
clotting time was measured. 100% inhibition was defined as the complete block of the
tran ed increase in clotting time. The antibodies dose dependently inhibited
the dabigatran induced prolongation in ng time in human plasma.
Figure 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.
Figure 10: Fab VH5c/Vk18 (SEQ ID NO: 99 and SEQ ID NO: 100) and k21 (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 bed above.
Figure 11: l structure of the Fab-Dabigatran complexes. A: Crystal structure of Fab
18/15 (W02011089183) 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
mation of dabigatran as seen in the crystal structure with VH5c/Vk18.
Figure 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).
Figure 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 sion constructs.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect, the t invention relates to an antibody molecule capable of
neutralizing the activity of an anticoagulant.
Antibodies (also known as immunoglobulins, abbreviated lg) are gamma globulin proteins
that can be found in blood or other bodily fluids of vertebrates, and are used by the
immune system to fy 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 ized by an dy 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 , 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).
Vlfithin the t 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 lnterest, National Institutes of Health, Bethesda (1983)).
The art has further developed antibodies and made them ile tools in medicine and
technology. Thus, in the context of the present invention the terms “antibody molecule” or
ody” (used mously herein) do not only include antibodies as they may be
found in , sing e.g. two light chains and two heavy chains, orjust two heavy
chains as in camelid species, but furthermore encompasses all molecules sing at
least one paratope with binding specificity to an antigen and structural similarity to a
variable domain of an immunoglobulin.
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’)2 fragment, a single chain
antibody, in ular a single chain variable nt , a Small r
lmmunopharmaceutical (SMIP), a domain antibody, a nanobody, a diabody.
Polyclonal dies 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.
Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are identical in
1O 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 ic antibody-producing B
cell with a myeloma (B cell cancer) cell (Kohler G, Milstein C. Continuous cultures of fused
cells secreting antibody of ined specificity. Nature 1975;256:495-7.) atively,
monoclonal antibodies may be produced by recombinant expression in host cells
(Norderhaug L, n T, Michaelsen TE, Sandlie |. (May 1997). "Versatile vectors for
transient and stable expression of recombinant antibody molecules in mammalian cells.".
J lmmunol Methods 204 (1): 77—87; see also below).
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
ic 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 dy sing 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 AF. “Chimeric antibodies”. Int Rev l. 1993;10(2—3):165-76; Riechmann L,
Clark M, Waldmann H, Winter G (1988). "Reshaping human antibodies for therapy".
Nature: 332:323.)
Furthermore, technologies have been ped for creating antibodies based on
sequences derived from the human genome, for example by phage display or using
enic animals (VVO 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, 7; Knappik
et al., J. Mol. Biol. 296: 57-86, 2000; 8. 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 lmmunol.
1995;13(1):65-93.; Briiggemann M, Taussig MJ. "Production of human antibody
oires in transgenic mice”. Curr Opin Biotechnol. 1997 Aug;8(4):455-8.). Such
antibodies are “human antibodies” in the context of the present invention.
Antibody molecules according to the present invention also e fragments of
immunoglobulins which retain antigen binding properties, like Fab, Fab’, or F(ab’)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 (VVO 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 cal antigen binding fragments, so-called Fab fragments, each with a single
n binding site, and a residual Fc fragment. Pepsin treatment yields an F(ab')2.
Methods of producing Fab molecules by recombinant expression in host cells are outlined
in more detail below.
A number of technologies have been developed for placing variable domains of
immunoglobulins, or molecules derived from such variable domains, in a different
lar t. Those should be also considered as “antibody les” in
accordance with the present invention. In general, these antibody molecules are r 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 s of the heavy and light chains of immunoglobulins,
linked er with a short , usually serine (S) or glycine (G) (WO 49; WO
91/17271; Huston et al; International Reviews of Immunology, Volume 10, 1993, 195 -
217). e domain antibodies” or ,,nanobodies” harbour an antigen-binding site in a
single lg-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
WO 30834
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 04, Holliger et al., Proc
Natl Acad Sci U S A. 1993 Jul 15;90(14):6444-8). Other examples for antibody-like
molecules are immunoglobulin super family antibodies (lgSF; Srinivasan and Roeske,
t Protein Pept. Sci. 2005, 6(2): 185-96). A ent concept leads to the so-called
Small Modular lmmunopharmaceutical (SMIP) which comprises a Fv domain linked to
single-chain hinge and effector domains devoid of the constant domain CH1
(WO 02/056910).
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 munoglobulin “antibody ”, sometimes called “scaffold
proteins”, may be based on the genes of protein A, the lipocalins, a fibronectin domain, an
ankyrin consensus repeat domain, and doxin (Skerra, Current Opinion in
Biotechnology 2007, 18(4): 295-304). A preferred embodiment in the context of the
present ion are designed ankyrin repeat proteins (DARPin’s; r et al., J Mol
Biol. 2008 Oct 24;382(5): 1211-27; Stumpp MT, Amstutz P. Curr Opin Drug Discov Devel.
2007 Mar;10(2):153-9).
The dy molecule may be fused (as a fusion protein) or othenNise 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 ular to g half-life of
such antibody molecules in the circulation, such as pegylation (WO 71; WO
98/48837; WO 2004081026), fusing or othenNise covalently attaching the antibody
molecule to another antibody molecule having affinity to a serum protein like albumin (VVO
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).
2012/055397
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 urally ted molecules.
ty is the interaction between a single n-binding site on an antibody molecule
and a single epitope. It is sed by the association constant KA = kass/kdiss, or the
dissociation constant KD = kdiss/kass .
In one aspect of the invention, the antibody binds to the agulant with an affinity, as
1O determined e.g. by e plasmon resonance analysis (Malmqvist M., "Surface plasmon
resonance for detection and measurement of antibody-antigen affinity and kinetics.", Curr
Opin lmmunol. 1993 2):282-6.), with a KD value ranging from 0.1 pM to 100 uM,
preferably 1 pM to 100 uM, preferably 1 pM to 1 uM. Antibody affinity can also be
measured using kinetic exclusion assay (KinExA) technology (Darling, R.J., and Brault P-
A., ic exclusion assay technology: Characterization of Molecular Interactions.”
ASSAY and Drug Development Technologies. 2004, Dec 2(6): 647-657).
The binding affinity of an antibody le may be enhanced by a process known as
ty 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 dies are therefore also embraced in the present invention.
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 ty, 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 ive to thrombin, such as the ecarin clotting time or the thrombin clotting time (H.
Bounameaux, Marbet GA, 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).
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 lmmunol 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).
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 tor, or a vitamin
K antagonist. Examples of vitamin K antagonists are the coumarins, which include
warfarin. es of indirect predominantly factor Xa tors are the n group of
1O substances acting through activation of antithrombin III including several low molecular
weight heparin ts (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 nds argatroban and
dabigatran.
Thus, in a r aspect, the agulant is dabigatran, argatroban, tran,
ximelagatran, hirudin, bivalirudin, lepirudin, desirudin, apixaban, edoxaban, otamixaban,
rivaroxaban, defibrotide, ramatroban, antithrombin III, or drotrecogin alpha.
A red anticoagulant in the context of the present invention is dabigatran (CA8
2119141, N-[2-(4-Amidinophenylaminomethyl)methyl-1H-benzimidazol
ylcarbonyl]-N-(2-pyridyl)-beta-alanine) having the chemical formula (II):
(EH3 NH2
0com N
HOW\0N
0 N / (II)
Dabigatran is known from WO 98/37075, which discloses compounds with a thrombininhibiting
effect and the effect of prolonging the thrombin time, under the name yl-
2-[N-(4-amidinophenyl)-aminomethyl]-benzimidazolyl-carboxylic acid-N-(2-pyridyl)-N-
(2-hydroxycarbonylethyl)-amide. See also Hauel et al. J Med Chem 2002, 45 (9): 1757—
Dabigatran is applied as a prodrug of formula (III):
C|3H3 NH
N AW
YN o 0 CH3
0 N
EtOW\ON
\ ( I” )
O N /
The compound of formula III (named dabigatran etexilate, CAS 2119159; ethyl 3-[(2-
{[4-(hexyloxycarbonylamino-imino-methyl)-phenylamino]-methyl}methyl-1H-
benzimidazolecarbonyl)-pyridinyl-amino]-propionate) is converted into the active
compound (II) after entering the body. A preferred polymorph of dabigatran etexilate is
dabigatran etexilate mesylate.
The main indications for dabigatran are the post-operative prevention of deep-vein
thrombosis, the ent of established deep vein thrombosis and the prevention of
strokes in patients with atrial fibrillation son et al., Lancet 2007, 370 (9591): 949—56;
an 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): 3).
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
acylglucuronide, in on to minor ysis to the aglycon, may undergo
ymatic acyl migration in aqueous on, resulting in the formation of the 2-O-, 3-
O-, and ylglucuronides. Experiments with the purified 1-O-acylglucuronide and its
isomeric rearrangement products revealed equipotent prolongation of the activated partial
thromboplastin time compared with dabigatran.
In another aspect of the invention, the dy molecule binds both to dabigatran and
dabigatran etexilate.
In another aspect of the ion, the dy molecule binds both to dabigatran and O-
1O acylglucuronides of dabigatran, in particular the 1-O-acylglucuronide of dabigatran.
In another aspect of the invention, the antibody molecule binds rmore to the 2-O-, 3-
O-, and 4-O-acylglucuronides of dabigatran.
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
acylglucuronide of dabigatran.
In the following, references to SEQ ID NOs. referto the sequences of Table 1 and the
sequence listing which is part of this ation, unless indicated othenNise.
In another aspect of the invention, the antibody molecule has binding specificity for
tran 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 ting 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.
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.
In r 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.
In another aspect of the invention, the antibody molecule has binding icity 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.
In another aspect of the invention, the antibody le 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.
In another aspect of the invention, the dy 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.
In another aspect of the invention, the antibody molecule has binding icity 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.
In another aspect of the ion, the antibody le 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.
In r aspect of the invention, the antibody molecule has binding specificity for
dabigatran and ses 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.
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.
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.
In another aspect of the invention, the antibody molecule has g 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.
In another aspect of the invention, the antibody molecule has binding icity 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
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domain with a CDR1 of SEQ ID NO: 64, a CDR2 of SEQ ID NO: 65, and a CDR3 of SEQ
ID NO: 69.
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.
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.
In another aspect of the invention, the antibody molecule comprises a heavy chain
le domain of SEQ ID NO: 74, and a light chain variable domain of SEQ ID No: 75.
In another aspect of the invention, the antibody molecule ses a heavy chain
variable domain of SEQ ID NO: 76, and a light chain variable domain of SEQ ID No: 77.
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.
In another aspect of the invention, the dy molecule comprises a heavy chain
variable domain of SEQ ID NO: 80, and a light chain variable domain of SEQ ID No: 81.
In r 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.
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.
In another aspect of the ion, 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.
In another aspect of the invention, the antibody molecule comprises a heavy chain
le domain of SEQ ID NO: 88, and a light chain variable domain of SEQ ID No: 89.
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.
In another aspect of the invention, the dy molecule comprises a heavy chain
variable domain of SEQ ID NO: 92, and a light chain le domain of SEQ ID No: 93.
In another aspect of the invention, the antibody le comprises a heavy chain
variable domain of SEQ ID NO: 92, and a light chain le domain of SEQ ID No: 94.
1O In another aspect of the invention, any one of the aforementioned light chain variable
s is fused to a constant domain of SEQ ID NO: 97.
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.
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.
In certain aspects, the invention concerns antibodies against dabigatran which have a
high solubility in aqeous media and a low tendency of aggregation.
In another aspect of the invention, the antibody molecule is a scFv le. 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)-(Iinker peptide)-(Iight chain variable
domain), or (light chain le )-(Iinker peptide)-( heavy chain variable domain).
Processes are known in the art which allow inant expression of nucleic acids
encoding st constructs in host cells (like E. coli, Pichia pastoris, or mammalian cell lines,
e.g. CHO or NSO), 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).
In particular, the scFv antibody les 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
ineering 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 x media components
1O such as soy peptone or yeast extract may be beneficial. The process for tation is
performed in a fed-batch mode. Conditions: Temperature 20 — 40 °C, pH 5.5 — 7.5, D0 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 g the respective
inducer into the fermenter over a prolonged time or a combination thereof. The tion
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.
The E. coli cell mass is resuspended in 4- to 8—fold amount of lysis buffer (phosphate or
Tris , pH 7-8.5). Cell lysis is preferably performed by high pressure homogenization
followed by ry of the pellet by centrifugation in bowl, tubular bowl or disc stack
fuges. 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-HCI or 8—10 mM Urea. After incubation for 30-60 minutes on is centrifuged
and supernatant containing the target protein red for uent refolding.
ing 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 NaCI, 1-3 M urea,
0.5-1 M ne, 2-6 mM of redox system such as e.g. / cystine or oxidized/reduced
glutathione, pH 9.5-10.5. After incubation for 24-72 h at 4°C refolding solution is optionally
filtrated using a 0.22 pm filter, diluted and pH adjusted to pH 0. Protein is separated
via cation ge chromatography in binding mode (e.g. arl p S-650M,
SP Sepharose FF or S HyperCelTM) at pH 7.0-8.5. Elution is performed by a linear
increasing NaCI 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 HyperCelTM) 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. r assays for characterization of the scFv include mass
ometry, RP-HPLC and SE-HPLC.
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 CH1 domain (a so-called Fd nt), and the light chain variable domain may be
fused to a CL domain.
In another aspect of the ion, the antibody molecule ses 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.
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.
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.
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.
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
NSO). Processes are known in the art which allow proper folding, ation, 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. lmmunol. 2007, 44: 1307-
1O 1315; s et al. J. togr. B. Analyt. l. Biomed. Life Sci. 2003;786:161-
176.).
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 ,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 LipofectamineTM and
PlusTM reagent rogen) according to the manufacturer’s instructions. After 48 hours,
the cells are subjected to selection in medium containing 200ug/mL of the antibiotic G418
and without hypoxanthine and ine to generate stably transfected cell populations.
These stable transfectants are subsequently ted 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
to 11 days to produce Fab protein material.
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 g densities of 3 X105—2 x 105 cells/mL respectively. Cells are
grown in shake flasks in Multitron HT tors (lnfors) at 5% C02, 37°C and .
For fed-batch experiments, cells are seeded at 3x105 cells/mL into shake flasks in Bl-
proprietary tion medium without antibiotics or MTX. The cultures are agitated at
120 rpm in 37°C and 5% C02 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-
etary feed solution is added every 24 hrs. Samples from the supernatant are taken
at different time points to dermine the Fab product concentration by ELISA. After 10 to 11
days, the cell culture fluid is harvested by fugation and transferred to the purification
labs.
The Fab molecule is purified from the supernatant of the fed-batch cultures by means of
tography 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
ties, the Fab is captured by cation exchange chromatography (CEX) exploiting the
pl of the molecule. Host cell ns and contaminants, e.g. DNA or viruses, are removed
by additional orthogonal purification steps.
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 terization of the Fab product include mass
spectrometry, isoelectric ng and size ion chromatography. Binding activity is
followed by BlAcore analysis.
Quantification of Fab or full-length lgG molecules in the supernatant of the cell cultures is
performed via ch enzyme linked immunosorbent assay (ELISA). The full-length
lgG can be detected using antibodies raised against Fc fragment (Jackson
lmmuno Research Laboratories) and human kappa light chain (peroxidase-conjugated,
Sigma). The Fab fragment is immobilized by goat polyclonal anti-Human lgG (H and L,
Novus) and detected by sheep polyclonal antibodies raised against human lgG
(peroxidase-conjugated, The Binding Site).
Fab molecules can also be generated from full-length antibody molecules by enzymatic
ge. The advantage of this approach is that platform processes for robust and
efficient fermentation and purification are applicable which are le for up-scaling
and high yields at the desired product quality. For purification affinity tography
using a recombinant Protein A resin can be used as primary capture step which usually
results in high purities.
For this purpose, the heavy chain ng Fab ces are fused to the Fc-region of a
human lgG 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 200ug/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 e medium. Once the cells have adapted, they are subjected to fed-
batch fermentations over 10 to 11 days to produce lgG protein material.
The lgG n is purified from the culture atant by using recombinant Protein A-
affinity chromatography. To obtain the desired neutralizing Fab fragment the full-length
lgG is then incubated in the presence of papain which cleaves the lgG within the hinge
region, thereby releasing two Fab fragments and the Fc-moiety.
The Fab molecule is isolated by affinity chromatography, e.g. Protein G or Protein L.
Alternatively, in case of low binding ties 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 onal
cation steps.
In another aspect of the ion, the antibody molecule is an amino acid sequence
t of an antibody molecule as described herein.
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, ed
that the final construct possesses the desired characteristics. The amino acid changes
also may alter post-translational ses of the humanized or variant antibody, such as
changing the number or position of glycosylation sites.
A useful method for identification of certain es 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 vely 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 ted at the target codon
1O or region and the expressed antibody variants are ed for the desired activity.
Amino acid sequence insertions include amino- and/or yl-terminal fusions ranging
in length from one residue to polypeptides containing a hundred or more residues, as well
as equence insertions of single or multiple amino acid residues. Examples of
al insertions include an antibody fused to an epitope tag. Other insertional variants
of the dy molecule include a fusion to the N- or C-terminus of the dy of an
enzyme or a polypeptide which increases the serum half-life of the antibody.
Another type of t 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 bed below in
reference to amino acid s, may be introduced and the products screened.
Original e Exemplary Substitutions Preferred 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
lie (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 (8) thr thr
Thr (T) ser ser
1O Trp (W) tyr; phe tyr
Tyr (Y) phe;trp; thr; ser phe
Val (V) leu; ile; met; phe ala; norleucine; leu
In protein chemistry, it is generally ed 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 hobicity 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 ties:
(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.
Non-conservative tutions will entail ging a member of one of these classes
for another class.
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 le, prevent aberrant crosslinking, or provide for
established points of conjugation to a cytotoxic or cytostatic compound. sely,
cysteine bond(s) may be added to the antibody to e its stability (particularly where
the antibody is an antibody fragment such as an Fv fragment).
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) ed 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.
y, 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 les as fusions to the gene
lll product of M13 packaged within each particle. The phage-displayed variants are then
1O 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 cial to e a l structure of
the antigen-antibody complex to identify t points n the antibody and human
Dabigatran. Such contact residues and neighboring residues are candidates for
substitution according to the ques 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.
Another type of amino acid variant of the antibody alters the original ylation 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.
In some embodiments, it may be desirable to modify the antibodies of the invention to add
glycosylations sites. Glycosylation of antibodies is typically either ed 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
ce of either of these tripeptide ces in a polypeptide creates a potential
glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-
alactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine
or threonine, although 5-hydroxyproline or oxylysine 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 ine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
Nucleic acid molecules encoding amino acid sequence ts 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 irected)
mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared t
or a riant version of an antibody molecule as described herein. As outlined above,
the antigen of the dy 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.
Immunization protocols for animals are well-known in the art. To achieve a proper immune
se, 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
ably small c molecules, which sometimes do not stimulate antibody
formation upon administration to an animal. It may ore be necessary to attach the
antigen to a macromolecule, as a hapten.
In a further aspect, the present invention relates to an antibody molecule as described
above for use in medicine.
In a further , the present invention relates to a pharmaceutical composition
comprising an antibody molecule as described before, and a pharmaceutical carrier.
To be used in therapy, the antibody molecule is included into pharmaceutical
itions appropriate to facilitate administration to s or humans. Typical
formulations of the antibody molecule can be prepared by mixing the dy molecule
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with physiologically acceptable carriers, excipients or stabilizers, in the form of lyophilized
or otherwise dried formulations or aqueous solutions or s 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 c acids and their salts; antioxidants including ascorbic acid
and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium de; 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
1O immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone or polyethylene glycol
(PEG); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, harides, oligosaccharides or polysaccharides and other
carbohydrates ing 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 xes); and/or ionic
or non-ionic surfactants such as TWEENTM (polysorbates), PLURONICST'V' or fatty acid
, fatty acid ethers or sugar esters. Also organic solvents can be contained in the
antibody formulation such as l or isopropanol. The excipients may also have a
e-modifying or absorption-modifying function.
In one aspect, the ceutical compositon 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.
The preferred mode of application is parenteral, by on or injection (intraveneous,
intramuscular, subcutaneous, intraperitoneal, intradermal), but other modes of application
such as by inhalation, ermal, intranasal, buccal, oral, may also be applicable.
In a further aspect, the present invention relates to an dy molecule as described
above for use in the therapy or prevention of side effects of anticoagulant therapy, in
ular bleeding events.
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
WO 30834
disease or disorder as described herein, in ular the side effects of anticoagulant
therapy.
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 agulant, in ular dabigatran
or dabigatran ate.
In a further aspect, the present invention s to an antibody molecule as described
above for use as an antidote of an anticoagulant, in particular dabigatran or dabigatran
1O exetilate.
In a further aspect, the present invention relates to a method of treatment or prevention of
side effects of anticoagulant therapy, comprising administering an ive amount of an
antibody molecule as described above to a patient in need f.
In a further aspect, the present invention relates to a method of treatment of an
overdosing event in agulant therapy, comprising administering an effective amount
of an antibody molecule as described above to a patient in need thereof.
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 f, comprising the step of administering a reversal
agent that neutralizes the activity of dabigatran or 1-O-acylglucuronide in the patient.
In a further aspect, the present invention s to a reversal agent that neutralizes the
activity of dabigatran or 1-O-acylglucuronide for use in a patient being d 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.
In a further aspect, the present invention s 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 tran or 1-O-acylglucuronide in the
patient.
In a r aspect, the present invention s to a method of reversal of the
anticoagulant effect of dabigatran or 1-O-acylglucuronide of tran in a patient being
1O treated with tran, 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 ylglucuronide in the
patient.
In a preferred ment, the reversal agent is an antibody molecule against dabigatran
which is capable of lizing the anticoagulant ty of dabigatran, dabigatran
etexilate, and/or 1-O-acylglucuronide. In another preferred ment, the reversal
agent is an antibody molecule against dabigatran as bed herein.
Preferably, the concentration of dabigatran or 1-O-acylglucuronide of dabigatran in
plasma is greater than 0 nM but less than 1000 uM 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.
In a further , the concentration of dabigatran or 1-O-acylglucuronide of dabigatran
in plasma is greater than 0 nM but less than 1000 uM, 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.
In a further , the concentration of dabigatran or 1-O-acylglucuronide of dabigatran
in plasma is n 30 nM and 1000 uM, and wherein the reversal agent used to
neutralize the activity of dabigatran or 1-O-acylglucuronide is present in a ratio of between
nM and 1000 uM of dabigatran or 1-O-acylglucuronide of dabigatran to reversal agent.
In another aspect, the present invention s to a method for reversing or reducing the
activity of dabigatran or 1-O-acylglucuronide of dabigatran in a t experiencing
bleeding or at risk for bleeding due to an impaired clotting ability or trauma, sing the
steps of:
(a) determining the amount of dabigatran or 1-O-acylglucuronide of dabigatran
present in the patient;
1O (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
(c) monitoring a thrombin clotting time of the patient to ensure a reversal or ion
in activity of dabigatran or ylglucuronide of tran has been reached.
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 tran or 1
acylglucuronide of dabigatran in the patient.
The "therapeutically effective amount" of the dy 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.
Dabigatran, for example, may achieve a plasma tration 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
tran 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 stered; the time elapsed
since such administration, the nature of the antigen molecule, the condition of the patient,
2012/055397
and other factors. The skilled expert knows methods to establish doses which are both
eutically effective and safe.
In a further aspect, the present invention relates to an antibody molecule with binding
affinity to dabigatran and/or dabigatran etexilate. Preferably, the dy molecule binds
to the dabigatran and/or tran etexilate with an affinity, as determined e.g. by
surface plasmon resonance analysis (Malmqvist M., ce plasmon nce for
detection and measurement of antibody-antigen affinity and cs. "Curr Opin lmmunol.
1993 Apr;5(2):282—6.) or c exclusion assay (KinExA) technology (Darling, R.J., and
1O Brault P-A., “Kinetic exclusion assay technology: Characterization of Molecular
Interactions.” ASSAY and Drug Development Technologies. 2004, Dec 2(6): 647-657),
with a KD value ranging from 0.1 pM to 100 uM, preferably 1 pM to 100 uM, more
preferably 1 pM to 1 uM.
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 adsorbent assay (ELISA), like those described in the examples.
Thus, in a further aspect, the present invention relates to analytical and diagnostic kits
sing antibody molecules a bed , and to respective analytical and
diagnostic methods.
In a r aspect, the present invention relates to a method of manufacturing an antibody
molecule of any one of the preceding claims, comprising
(a) ing 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.
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
2012/055397
example, bottles, vials, and test tubes. The containers may be formed from a variety of
materials such as glass, metal, plastic or combinations f. The ner 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 ate, a prodrug of dabigatran or a pharmaceutically acceptable
salt thereof. The label on the container of the antibody indicates that the ceutical
composition is used for lizing or lly neutralizing dabigatran, dabigatran
etexilate, a prodrug of dabigatran or a ceutically acceptable salt thereof in vivo.
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.
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.
In r ment, 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 doage unit of the tran, tran etexilate,
prodrug of dabigatran or pharmaceutically able 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 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.
In an alternate embodiment, the kit comprises (1) a first pharmaceutical composition
comprising dabigatran, tran 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
admininstration 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.
The invention also provides a diagnostic method to neutralize or partially neutralize
dabigatran or 1-O-acylglucuronide of dabigatran in a t being d with dabigatran,
dabigatran etexilate, a prodrug of dabigatran or a pharmaceutically acceptable salt
thereof, sing administering any one of the antibodies described herein, a
ation thereof or a pharmaceutical composition thereof. Specifically, the ion
provides a method for neutralizing or partially neutralizing dabigatran or 1
acylglucuronide of dabigatran in a patient comprising the steps of (a) ming that a
patient was being d 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 ation 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 tran 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 tran or ylglucuronide 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 ogen levels, activated protein C resistance or related
tests.
EXAMPLES
I. PRODUCTION OF POLYCLONAL ABIGATRAN ANTIBODIES
For the tion 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).
For the screening, an enzyme horseradish peroxidase (HRP)-conjugate was produced
and an enzyme-immunosorbent assay (ELISA) developed.
1O Further purification of the polyclonal antibodies was performed by affinity chromatography
on protein A sepharose FF.
1. MATERIALS AND S
Test nd (dabigatran)
a—obigatran, zwitter ion
Structural formula:
25H25N703
olecular weight: 471.5 g/mol
1.1 HAPTEN USED FOR SYNTHESIS OF IMMUNOGEN AND TRACER
Haptem
Structural formula
ongand:
HZN/\/\/H\n/\/N\©o
C30H35N802 * HCI
molecular weight: 577.13 g/mol
Hapten2
Structural formula
ongand:
/\/N N
O N / X HCI
C27H31N902 * HCI
molecular : 550.07 g/mol
1.2 SYNTHESIS OF S
The haptens Hapten1 and Hapten2 were synthesized as follows:
Hagten‘l 2-[(4-Carbamimidoyl-phenylamino)-methyl]methyl-1H-benzoimidazole
carboxylic acid [2-(4-amino-butylcarbamoyl)-ethyl]-phenyl-amide
>—\ NH
N—<: :>—<
/\/\/ 2
H2N W
1a 3-[(4-Methylaminonitro-benzoyl)-phenyl-amino]-propionic acid methyl ester
O +-_O
,o N o’
H3C W
To a solution of 4-methylaminonitro-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
1O three hours the rection mixture was evaporated to dryness, the remaining solid triturated
with water and the solid product isolated through tion.
Yield: 99%
C18H19N305 (357.36)
TLC a gel; Dichloromethane/ethanol 19:1): Rf: 0.48
lb 3-[(3-Aminomethylamino-benzoyl)-phenyl-amino]-propionic acid methyl ester
,0 N
H30 \H/V
The nitro group of product 1a was d by hydrogenation at room temperature in
ethanol with Pd (10% on charcoal) as catalyst.
Yield: 99%
C18H21N303 (327.38)
TLC (silica gel; Dichloromethane/ethanol 9:1): Rf: 0.23
Mass spectrum (ESI): [M+H]+ = 328
1O 1c [2-(4-Cyano-phenylamino)-acetylamino]methylamino-benzoyl}-phenyl-
amino)-propionic acid methyl ester
H C/OWN
3 \
The t of lb (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.
Yield: 97%
C27H27N5O4 (485.54)
Mass spectrum (ESI): [M+H]+ = 486
fl 3-({2-[(4-Cyano-phenylamino)-methyl]—1-methyl-1H-benzoimidazolecarbonyl}-
phenyl-amino)-propionic acid methyl ester
A on 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 ing
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
1O on silica gel (eluent: dichloromethane/ethanol 1:1).
Yield: 58%
C27H25N503 (467.52)
TLC (silica gel; Dichloromethane/ethanol 9:1): Rf: 0.71
Mass spectrum (ESI): [M+H]+ = 468
1_e 3-({2-[(4-Cyano-phenylamino)-methyl]—1-methyl-1H-benzoimidazolecarbonyl}-
phenyl-amino)-propionic acid
To a on of the product of 1d (13.0 mmol) in 100mL methanol sodium hydroxide (20.0
mmol) was added. The mixture was d for 2.5 hours at 40°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°C.
Yield: 88%
C26H23N503 9)
TLC (silica gel; Dichloromethane/ethanol 9:1): Rf: 0.33
Mass spectrum (ESI): [M+H]+ = 454
1f {4-[3-({2-[(4-Cyano-phenylamino)-methyl]—1-methyl-1H-benzoimidazole
carbonyl}-phenyl-amino)-propionylamino]-butyl}-carbamic acid tert-butyl ester
/> \
N N
%%12;:TN
i430 0 \©
A solution of the product of 1e (5.23 mmol), 2-(1H-benzotriazoleyl)-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 s. Then (4-aminobutyl
)-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.
Yield: 92%
C35H41N7O4 5)
TLC (silica gel; romethane/ethanol 9:1): Rf: 0.51
lg 2-[(4-Carbamimidoyl-phenylamino)-methyl]methyl-1H-benzoimidazole
carboxylic acid [2-(4-amino-butylcarbamoyl)-ethyl]—phenyl-amide
H NH
o N/ N
H < > <
H NH
HZN/\/\/ WN 2
The product of 1f (4.81 mmol) was dissolved in a ted solution of HCI in ethanol (250
mL), the mixture stirred at room temperature over night and then evaporated to dryness at
°C. The remainig 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 ation 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
°C. The product was then dissolved in 30 mL water, the solution stirred with ca.2g
1O charcoal, filtered and evaporated to dryness.
Yield: 90%
CsoHssNBOZ (540.67)
TLC (reversed phase RP-8; ol/5% aqueous NaCl solution 9:1): R = 0.79
Mass spectrum (ESI): [M+H]+ = 541
‘ = 575/7
Hapten2 2-[(4-Carbamimidoyl-phenylamino)-methyl]methyl-1H-benzoimidazole
carboxylic acid [2-(2-amino-ethylcarbamoyl)-ethyl]-pyridinyl-amide
H H
HZN/\/ WN N 2
O N /
2a [(4-Cyano-phenylamino)-methyl]—1-methyl-1H-benzoimidazolecarbonyl}-
pyridinyl-amino)-propionic acid
CH3 3
/ /
O\ N N4©7:NH o
H3CVOWN \ \
‘ HOWN ‘
o N / o N /
To a on of sodium hydroxide (50.0 mmol) in 500 mL ethanol and 50 mL water was
added 3-({2-[(4-Cyano-phenylamino)-methyl]methyl-1H-benzoimidazolecarbonyl}-
pyridinyl-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.
1O Yield: 78%
N603 (454.48)
2b {2-[3-({2-[(4-Cyano-phenylamino)-methyl]methyl-1H-benzoimidazole
carbonyl}-pyridinyl-amino)-propionylamino]-ethyl}-carbamic acid tert-butyl ester
/> \
O N :N
H N
3C 0 U
A solution of the product of 2a (2.20 mmol), 2-(1H-benzotriazoleyl)—1,1,3,3-
tetramethyluronium luoroborate (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 ed through extraction with ethyl acetate and
purified by chromatography (silica gel; dichloromethane/methanol 15:1).
Yield: 61%
N804 (596.68)
Mass spectrum (ESI): [M+H]+ = 597
[M+H]' = 595
2c 2—[(4-Carbamimidoyl-phenylamino)-methyl]methyl-1H-benzoimidazole
carboxylic acid [2-(2-amino-ethylcarbamoyl)-ethyl]-pyridinyl-amide
H H4?H NH
HZN/\/ WN N 2
O N /
The product of 2b (1.34 mmol) was added to a saturated HCI solution in dry ethanol (30
1O mL). The solution was stirred at room temperature for 5 hours, then evaporated to dryness
at 30°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), ed and ated in order to separate the product
from nic salts.
Yield: 27%
C27H31N902 (513.61)
Mass spectrum (ESI): [M+Cl]' = 548/50
[M+HC|+C|]' = 584/6
[M+H]+ = 514
2. ALS
2.1 CHEMICALS FOR REAGENT SYNTHESIS
name specification upplier atalogue no.
1,4-Benzoquinone
Bovines Serum Albumin
(BSA)
1,1’-CarbonyI-di-(1,2,4-
Freund’s adjuvant (CFA)
Freund’s adjuvant (IFA)
Glycerine
Sodium perborate
2012/055397
2.2 CHEMICALS FOR ELISA
analytical grade Riedel-DeHaenf-3114
analytical grade Riedel-De Haen £0743
2. 3 S FOR ELISA
. .05 M NazI'IPO4 / KH2PO4
0.15 M NaCl, pH = 7.4
A weeks at approximately +4°C
buffer 2 s buffer 1, with 5 g/l BSA ssay buffer
stability. 10 days at approximately +4°C
s buffer 1,
ith 5 g/l BSA and 0.1 g/L thimerosal
A weeks at approximately +4°C
-phenylene diamine
.5 mmol/L sodium perborate
itric acid:
. months at approximately +4°C
ith perborate:
days at approximately +4°C
ash solution ater, 0.5 g/L Tween 20 late washing
stability: 10 days at ambient temperature
' .25 M H2804 rrests o-phenylene
oiamine colour
years at ambient temperature oevelopment
Water from an at Maxima-HPLC ultra pure water processing system was used to
prepare buffer solutions.
3. SYNTHESIS OF IMMUNOGENS
In order to stimulate the immune system of rabbits to produce onal antibodies
against dabigatran, three immunogens (lot. nos. GL256, GL258, and GL262,) were
synthesized by coupling the haptens HAPTEN1 and HAPTEN2 to the carrier n
bovine serum albumin (BSA) using 1,4-benzoquinone or 1,1’-carbonyl-di-(1,2,4-triazol) as
coupling reagent.
For the synthesis of GL256, 1,4-benzoquinone was used as a homobifunctional
compound with two ve 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 arbonyl-di-(1,2,4-triazol) as coupling
reagent with different input ratios of the hapten to the r protein.
3.1 SYNTHESIS OF GL256
To the solution of 0.75 uMol BSA in 8.5 mL 0.1 M KHZPO4-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. AftenNards 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).
2.5 mL (0.15 uMol) of the purified BSA-solution were added slowly under stirring to a
solution of the 525 uMol hapten HAPTEN1 dissolved in 2 mL 0.1 M NaHCOs,/Na2COS-
buffer 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.
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 d
hapten remained in the hapten-carrier conjugates.
The immunogen was stored frozen in aliquots at -20°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
A solution of 158 uMol HAPTEN2 in 6.3 mL N,N-dimethylformamide (DMF) was ed
at room temperature. 158 uMol 1,1’-carbonyl-di-(1,2,4-triazol) was added and incubated
first for 4 hours at 10°C and ards for 30 min at room temperature. The chemical
reaction was d with thin-layer chromatography and was about 20-25%.
Then 0.75 uMol BSA were dissolved in 2 mL 0.13 M NaHCOs and 1 mL N,N-
dimethylformamide (DMF) was added dropwise under stirring. The pH was adjusted to
approximately 8.3. AftenNards the hapten solution (6.3 mL) and 4 mL 0.13 M NaHCOs
were added dropwise to the BSA on 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.
After incubation at room temperature over night under stirring ions, 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.
The immunogen was stored frozen in aliquots at -20°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 t of immunogen in the final solution was
0.28 mg GL258 / mL.
3.3 SYNTHESIS OF GL262
A solution of 225 uMol HAPTEN2 in 8.75 mL N,N-dimethylformamide (DMF) was
ed at room ature. 225 uMol 1,1’-carbonyl-di-(1,2,4-triazol) was added and
incubated for 4 hours at 10°C. The chemical reaction was checked with thin-layer
chromatography and was about 20-25%.
Then 0.49 uMol BSA were dissolved in 2 mL 0.13 M NaHCOs and 1 mL N,N-
dimethylformamide (DMF) was added dropwise under stirring. The pH was adjusted to
approximately 8.2. AftenNards the hapten solution (8.75 mL) and 6 mL 0.13 M NaHCOs
1O 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.
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 ed in the hapten-carrier conjugates.
The immunogen was stored frozen in aliquots at -20°C. The degree of substitution of BSA
with hapten in the supernatant of the immunogen was about 1:32 as ined by UV
absorption spectrometry at 302 nm. The content of immunogen in the final on was
0.71 mg GL262 / mL
4. SYNTHESIS OF CONJUGATE
4.1 SYNTHESIS OF GL261
A on of 37.4 uMol HAPTEN2 in 1.5 mL N,N-dimethylformamide (DMF) was prepared
at room temperature. 37.5 uMol 1,1’-carbonyl-di-(1,2,4-triazol) was added and incubated
first for 4 hours at 10°C and afterwards for 30 min at room temperature. The chemical
reaction was checked with ayer chromatography and was about 20-25%.
WO 30834
Then 1.125 uMol enzyme horseradish peroxidase (HRP) were dissolved in 0.4 mL 0.13 M
NaHCOs and 0.267 mL N,N- dimethylformamide (DMF) was added dropwise under
stirring. The pH was adjusted to approximately 8.2. AftenNards 0.9 mL of the hapten
solution (22.5 uMol) and 0.57 mL 0.13 M NaHCOs 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.
After incubation at room temperature over night under stirring conditions, the HRP
conjugate was ted from organic solvents and the excess of hapten by gel
chromatography. The solution passed a sephadex G25 column brated with 0.1 M
phosphate buffer pH 7.0.
The final tration 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 . The tracer solution was labelled
as lot no. GL261 and stored in aliquots at -20°C.
The degree of substitution of HRP with hapten was 1:02 as determined by UV
spectroscopy at 302 nm.
The specific activity of the tracer was measured in BSA-blocked microtiter plates using 0-
phenylene-diamine (OPD) as substrate and native HRP as reference material. The
e of diluted HRP standards or the hapten-HRP conjugate and substrate solution
were ted 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.
Summary of tracer specifications:
HAPTEN2 - horseradish peroxidase
108 U/mg 611 U/ml
(substrate Guajacol and H202, 25°C)
at approximately -20°C
1:40000
. IMMUNIZATION AND PRODUCTION OF ANTIBODIES
.1 IMMUNIZATION OF RABBITS
Twelve female chinchilla rabbits, 3 months old, were immunized with an emulsion of 100
ug immunogen GL256, GL258 and GL262 in 0.5 mL 0.9 % NaCl solution and 0.5 mL of
complete Freund’s adjuvant (CFA). Several r immunizations ed in the next
month. For the third immunization 0.5 mL of lete Freund’s adjuvant (IFA) was
used. Each immunization was performed at four subcutaneous and four intramuscular
sites.
1O Group A — immunogen GL256
Rabbit 1 #50
Rabbit 2 #51
Rabbit 3 #52
Rabbit 4 #53
Group B — immunogen GL258
Rabbit 5 #54
Rabbit 6 #55
Rabbit 7 #56
Rabbit 8 #57
Group C — immunogen GL262
Rabbit 9 #46
Rabbit 10 #47
Rabbit 11 #48
Rabbit 12 #49
Immunization scheme
in CFA
Day 29 Second immunization with 100 ug immunogen / mL per
animal
in CFA
Day 57 Third immunization with 100 ug 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 ug immunogen / mL per
animal
in CFA
Day 91 Second bleeding (25 mL per animal)
Day 112 Fifth immunization with 100 ug immunogen /mL per animal in
Day 122 Assignment of the animal numbers was d
Third final ng (Exsanguination)*
*Rabbit no. 1-12 were exsanguinated completely 10 days after the fifth zation.
Exsanguination was performed via a carotid artery under anesthesia with xylazin
(Rompun®, Bayer, Leverkusen, Germany) and ketamine hydrochloride (Ketavet®, Parke-
Davis, Freiburg, Germany).
.2 ANALYSIS OF RABBIT SERA
Serum was prepared by centrifugation of the coagulated rabbit blood. A protein fraction
was obtained by um sulphate precipitation and ing through a Sephadex G25
column.
The individual protein fractions from the rabbit sera were screened for anti-dabigatran titer
by a standard ELISA ure.
Screening-ELISA:
CUIIIIIIIIIIIIIIIiIIiim 'U Procedure
protein fractions from each bleeding were adsorbed overnight at ambient
emperature onto microtiter plates (100 uL/well; 1, 2 or 4 ug/mL) in buffer 1.
ash microplates 4 times, 450 uL each
block with 250 uL buffer 3 for at least 1 hour
ash microplates 4 times, 450 uL each
add to each well of microtiter plate in triplicate:
50 uL buffer 2
50 uL calibration standards in buffer 2
uL tran-horseradish peroxidase (H RP) conjugate GL 261 (tracer)
(1/40000)
seal microplates with adhesive foil, complete sample distribution for all microplates
incubate for 4 h on a shaker at ambient temperature
E ash microplates 4 times, 450 uL each
F add to each well of iter plate 100 uL o-phenylene diamine HCI, 2.7 mg/mL
(one 30 mg tablet in 11 mL buffer 4)
incubate for 30 min in the dark at ambient temperature
add to each well of iter plate 100 uL H2804 (2.25 M)
shake for 5 minutes
read absorbance; test-wavelength: 490 nm, reference-wavelength: 650 nm
.3 DETECTION OF ABIGATRAN ANTIBODIES IN RABBIT SERA
Last three columns: values are for dabigatran
W0 2012/130834”1 2012/055397
biaeding 2
aoafing
can:
_ahnl
Finat ng
inununogane caaflng
cons
j almi
-4.4!. Q
-«g .3
-.2. N
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).
For the screening of the final bleeding 3, the cement of the tracer from the antibody
binding site with low concentrations of analyte (dabigatran) was used as main decision
criteria, e of the missing ation about the immunogen used. Therefore rabbits
no. 2, 3 and 5 were used for the further purification.
.4 PURIFICATION OF POLYCLONAL ANTIBODIES
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 um sulphate. The precipitate was
fuged for 30 min at 10°C at 4500 U/min, separated from the solution and re-
dissolved in Tris buffer. This procedure was repeated. Further cation was performed
by affinity chromatography on protein A ose 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
lgG were combined. Protein concentration was determined by UV spectroscopy at
280 nm.
Summary of antibody specifications:
HAPTEN2-BSA (lot no. GL258)
no. 5 (#54) serum (bleeding no. 2)
1.85 mg/mL
t approximately -20°C
HAPTEN1-BSA (GL256) or
HAPTEN2-BSA (lot no. GL258) or
HAPTEN2-BSA (lot no. GL262)
no. 2 serum collected (final bleeding)
t approximately -20°C
HAPTEN1-BSA (GL256) or
HAPTEN2—BSA (lot no. GL258) or
HAPTEN2—BSA (lot no. GL262)
no. 3 serum (final bleeding)
'.96 mg/mL
t approximately -20°C
HAPTEN1-BSA (GL256) or
HAPTEN2—BSA (lot no. GL258) or
HAPTEN2—BSA (lot no. GL262)
no. 5 serum (final bleeding)
.72 mg/mL
t approximately -20°C
ll. Neutralization of dabigatran
Two series of experiments were performed to show the effect of the antibodies against
dabigatran agulant activity in vitro. The four polyclonal antibodies were received in
1O the laboratory and r tested in human plasma. This was tested in the functional
assay, the thrombin clotting time.
Assay description:
Briefly human plasma is ed 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°C on the day of the
assay.
The thrombin clotting time is performed as follows. First thrombin is diluted to
manufacturer’s specification (3 lU/mL in) in the buffer ed (Dade Behring Test
kit) and prewarmed to 37°C. It is used within 2 hrs of being prepared. All assays were
performed on a commercially available CL4 clotting machine (Behnk Electronics,
Norderstadt, y). Fifty uL of plasma is pipetted into provided cuvettes with a
magnetic stirrer and allowed to stir for 2 min in the well preheated to 37°C in the CL4
machine. At this point 100 uL of the thrombin solution is added and the time required for
the plasma sample to clot is ed automatically by the CL4. Dabigatran is
preincubated for 5 min in plasma in the provided cuvettes, before adding thrombin and
starting the measurement. lf antibody is also tested (up 50 uL of stock solution), there is a
further 5 minute incubation at 37°C before beginning clotting (Le. 10 min total incubation
with tran, 5 min total incubation with antibody and then clotting is initiated with
1O throm bin).
Initially a dabigatran standard curve was performed by adding increasing concentrations
of dabigatran to human plasma and ing the time to clotting after addition of
thrombin (Figure 1). There was a concentration-dependent increase in the in
clotting time with increasing concentrations of dabigatran.
For the first set of neutralization experiments, a clinically relevant concentration of 200 nM
of tran was added to all plasma samples for neutralization. All 4 antibody
preparations were able to shorten the time to clotting in plasma containing dabigatran
(Figure 2). The extent of neutralization was related to the tration 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
ty in a separate set of experiments. It can be seen in Figure 3, there was a
concentration dependent inhibition of dabigatran-induced anticoagulant activity with
increasing concentrations of antibody. In addition when a ecific 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.
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 agulant activity of the t concentration of dabigatran (500 nM) in
the standard curve in Figure 1 was also tested. Figure 4 illustrates that antibody D could
also inhibit high concentrations of dabigatran.
III. PRODUCTION AND CHARACTERIZATION OF MONOCLONAL
ANTI-DABIGATRAN ANTIBODIES
1. Production of monoclonal anti-dabigatran antibodies and Fabs
1O Mice were zed with Hapten1 (see Example 1.1) conjugated to carrier proteins such
as hemocyanin and immunoglobulin and omas were ted according to
standard procedures. Monoclonal antibodies purified from the culture atants bound
to tran-protein conjugates and this binding could be competed with tran in
solution with half-maximal inhibition at concentrations in the range of 1 to 10 nM. Fabs
were generated by papain ge of the monoclonal antibodies with subsequent
elimination of the Fc domain via Protein A.
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 lgG constant regions were generated and protein was expressed in HEK293
cells and purified.
In order to reduce potential immunogenicity, sequences of mouse monoclonal antibody
clones 35E6 and 27A9 were humanized by standard s 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.
2. Characterization of monoclonal abigatran antibodies and Fabs
The sequences of the variable domains of 9 monoclonal antibody clones DBGZZ (clone
22), 35E6, 45B9, 48E1, 49F8, 6A7F1, 2F1E5, 3B4E7, 1F6G8, 2D2E3, and 27A9 are
depicted in Table 1. SEQ ID N03 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 K21 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
1O 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). K18
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).
In Table 1, the s “CDR” denote a complementarity determining , “VH” denotes
the variable region of a heavy chain, “VK” denotes the le 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 DBGZZ”
denotes the first CDR (CDR1) of the variable domain of the heavy chain of clone DBGZZ,
and “DBGZZVH” denotes the variable region of the heavy chain of clone DBGZZ.
Table 1
VHCDR1 GFSLTSY:
DBGZZ
VHCDRZ V..WAGGSTNYNSALRS
DBGZZ
VHCDR3 AAYYSYYNYDGFAY
DBGZZ
—VKCDR1
VV()2012/130834
DBG22
VKCDRZ LVSKLDS
DBG22
LQSTHFPHT
DBG22
GYTFTWYWMH
35E6
ETNPRWGGTNYN
35E6
GTSGYDYFDY
35E6
RSSQTIVHSNGNTYLI
35E6
35E6
2 VKCDR3 FQASiFPYT
35E6
13 VHCDR1 GVSLFTYDVD
4589
4 VHCDR2 VMWSGGTTNYNSALKS
4589
DRWSPGGFAY
16 VKCDR1 QSSQSLLYTNGKTYLH
4589
4589
4589
9 YDVD
48E1
0 :WAGGSTNYNSALKS
48E1
1 DRWSPGGFAY
48E1
22 {SSQSLLYTNGKTYLI
48E1
23 VKCDRZ LVSKLDS
48E1
24 LQTTHFPHT
48E1
GFSLSTYGVD
49F8
26 LIWAGGSTTYNSAFKS
49F8
27 PFGY
49F8
28 {SSQSLLYTNGKTYLN
49F8
29 S
49F8
LQNSHFPHT
49F8
31 GFTFSTYGMS
6A7F1
32 SVTRGGNTYYP:
6A7F1
33 DYSGWYFDV
6A7F1
34 VKCDR1
6A7F1
VKCDR2 {VSNRFS
6A7F1
36 FQGSQIPYT
9 SAGTDYF:
40 RASESVDSYGNS FMH
4 LASWLES
42 EDPWT
43 VHCDR1 GYTFTYYT:
3B4E7
44 VHCDRZ YINPASSYTNY:
3B4E7
:--GANWDYFDY3B4E7 RSSQW QSNGNTYLH
3B4E7
3B4E7
3B4E7
1FSG8
YINPSSGYTYYI
1FSG8
1FSG8
RSSQWIVQTNGNTYLH
1FSG8
VKCDR2 KVSSQFS
VV()2012/130834
1F6G8
54 VKCDR3 FQGSHVPFT
1F6G8
55 VHCDR1 GYTFTHSGMN
2D2E3
56 VHCDR2
2D2E3
57 VHCDR3 SWWTDYFDY
2D2E3
58 VKCDR1 RSSQS:ViSNGNTYL
2D2F8
59 VKCDR2 KVSNQFS
2D2E3
“-2D2E3 FQGSiFPYT
GYTFTWCYMH
27A9
ETNPRWGGTNYN
27A9
GTSGYIYFDY
27A9
RSSQS:VHS
27A9
KVSYQFS
27A9
“-27A9 FQGSiVPYT
VHCDR1 5C GYTFTDYYMH
PQWGGTTYN
E.—FQASiVPYT
SGPG LVAPSQRLS: TCTVSGFSLT SYIVDWVRQS
PGKGL?WLGV "WAGGSTNYN SALRSRLSIT KSNS<SQVFL
TDDT AIYYCASAAY YSYYNYDGFA YWGQGTLVTV
DVVWTQTPLT LSVTHGQPAS "SCKSSQSLL {TYLYW
QSP< RLIYLVSKLD SGVPDRFSGS DFTLK:
SRVfiAfiDVG YYCLQSTHFP HTFGGGTKLt
QVQLQQPGAI LVKPGASVKL SCKTSGYTFT NYWMiWVRQR
W G1 TNPRNGGTNY <ATL TVDKSSNTAY
MQLSSLTFG SAVYYCTIGT SGYDYFDYWG QGTTLTVSS
35E6VK i LPVSLGDQAS iSCQSSQTTV HSWGNTYL
<PGQSP< LLIYKVSWRF SGVPDQFSGS GSGTGFTL I
fiAfiDLGV YFCFQASiFP YTFGGGTKLfi <
45BQVH QVQLKQSGPG LVAPSQSLST TCTVSGVSL TYDVDWVRQS
PGKDLEWLGV MWSGGTTWYN SALKSRLNIW KDSS<SQVFL
{MSGLQTDDT GLYYCATDRW YWGQ GTLVTVSA
45BQVK DVVMTQTPLT "SCQSSQSLL {TYLHW
LLQRPGQSPK SGVPDRFSGS DFTLK:
VV()2012/130834
U) W<1 . ZDLGV YYCLQST HTFGGGTKL¥
76 48E1VH SGPG LVAPSQSLS: TCTVSGFSLT DWVRQS
PGKGL?WLGV "WAGGSTWYN SALKSRL S {NQVFL
RMNSLQTDDT AMYYCASDRW SPGGFAYWGQ
Iiilllllllllllll48E1VK DVVWTQTPLT LSVTHGQPAS "SCKSSQSLL
QSPK RLIHLVS<LD SGVPDRFSGS
SQVfiAiDLGV FYCLQTTiFP HTFGGGTKLt
\l O) 49F8VH SLS: TCTVSGFSLS DWVRQS
"WAGGSTTYW SAFKSQLSIS , {SQVFL
{MNSLQTDDT AMYYCASERS GDSPFGYWGQ GTLVTVSA
\lO 49F8VK DVVVTQSPL" LSVT GQPAS "SCKSSQSLL {TYLNW
LLQ' fl RT. HTIVS {TD SGVPDRFSGS DFTLK:
SQVZ .DLGV YYCLQNSiFP {TFGSGTKLt
6A7F1VH LVRPGGSLKL SCAASGFTFS TYGMSWVRQS
VTQGGNTYYP DSMQGRFTIS QDNVGNILYL
A YS GWYFDVWGAG TTVTVSS
O) _\ K IPLS LPVSLGDQAS :SCQSSQSIV {SNGDTFL
YLQ<SGQSP LLIYKVSWRF SGVPDRFSGS GSGTDFTL "
SRVfiAfiDLGV SQIP YTFGGGTKLt K
O)N 3B4E7VH QVQLQQSGAI LARPGASVKM SC<ASGYTFT YYT:{WVKQR
PGQGLfiW GY NBASSYTNY IQ<FKDRATL TADKSSSTAY
MQLSSLTS: SAVFYCAQGA NWDYFDYWGQ SS
Iiilllllllllllll3B4E7VK DVLWTQTPLS LPVSLGDQAS :SCRSSQN
<PGQSP< LLIYKVSWRF SGVPDQFSGS
fiAlDLGV YYCFQGSiVP YTFGGGTNLt
O)A 2F1E5VH 1 LKKPGfiTVK {SSGFTLT NYGMWWVKQV
:NTYTGEPTY iDFKGQFAF SLETSARTAY
AATYFCA’ GTDYFDYWGQ GTTLTVSS
0) 0| K LAVSLGQRAT :SCRASESVD
{PGQPP T."YT.AS\TT.'T. GVPARFSGSG
7 DDAATY QDPW TFGGGT<LZ
1F6G8VH 1 LKKPGfiTV< NYGMNWVKQV
:NTYTGEPTY SLETSARTAY
AATYFCAQSA GTTLTVSS
O) 1F6G8VK LPVSLGDQAS
LLIYKVSSRF
YYCFQGSiVP
O) 2D2E3VH fiLKKPGfiTV {ASGYT NWWK
WINTNTGEP ZfiENGRE AESLfiASAWT
HDTATYFCAR DYFDYW
2D2E3VK LPVSLGDQTS iSCQSSQS:
1TJ.YKVSNRF SGVPDRLSGS
YYCFQGSHFP YTFGGGTKLfi
27A9VH QVQLQQPGAI LVKPGASVKL SCKASGYTFT NCYMiWVKQR
PGQGLfiW G1 TNPRNGGTNY NEKFKR<ATL TVNKYSSTAY
MQLSSLTSE SAVYYCTIGT SGYEYFDYWG QGTTLTVSS
91 27A9VK NI-LWTQTPLG LPVGLGDQAG LGCRGGQGLV HGDGN YLZ
QGP< VL__YKVGYRF GGVPDRFGGG GGGTYFTL
L'ALDTIGV YFCFQGGHVP YTFGGGTKL. K
QVQLVQSGA VKKPGASVKV SCKASGYTF VRQA
PGQGLL'WMGZ TNPRNGGTTY NLKFKG<ATW TRDTGTGTAY
QG.I TAVYYCTLGT DYWG QGTLVTVGG
93 IL QPAG IGCQGGQGI
LL__YKVGYRF GGVPDRFGGG
1 .I YYCFQAGHVP YTFGQGTKLL
94 IL LGVTPGQPAG _GCRGGQGIV I YLZ
VGYRF GGVPDRFGGG GGGTGFTL
YYCFQAGLVP YTFGGGTKLL K
95 Cbne22 I LVAPGQRLG_I TCTVGGFGLT GYLVDWVRQG
CmmeflCHC I. WAGGGTVYN GALRGRLG..T KGVG<GQVFL
T I AGAAY YGYYNYDGFA YWGQGTLVTV
GAAGT<GPGV G<GT GGGTAALGCL VKDYFPLPVT
VGWNGGALTG GVHTFPAVLQ GGGLYGLGGV GLGT
QTY__CVVVHK PGNTKVD<QV LP<GCDKTHT CPPCPAPLAA
GGPGVFLFPP KPKDTLMLGR TPLVTCVVVD VGHL'DPL'VKE'
VWYVDGVLVH NAKTKPQLLQ YNGTYQVVGV LTVLLQDWLV
{CKVGN KALPAP L<T G<A<GQPRIL PQVYTLPPGQ
PYPG D AVLWL'GNG QPILNVY<TTP
I I LYGKLTVD<G QWQQGVVFGC GVWHLALHNL
{GLGLGP GK
Cbne22 DVVWTQTPLT LGVT._GQPAG LGCKGGQGLL YTVG<TYLYW
dflmeflcLC LLQQPGQGPK RLI.YLVG<LD GGVPDRFGGG GGGTDFTLK
GRVLALDVG GTLFP HTFGGGT<LL {QTVAAPGV
P__ PPPGDLQL KGGTAGVVCL LNNFYPRLA< VQWKVDNALQ
GGNGQLGVTL QDG<DGTYGL GGTLTLG<AD YL<HKVYACI
VTHQGLGGPV T<GFVQGLC
am RTVAAPGVh__ PPPGDLQLKG GTAGVVCLLV
WKVDNALQGG \GQLGVTLQD GKDGTYGLGG
LVT VT GFVRGLC
hCFIDomam {GPGVFP LAPGG<GTGG GTAALGCLVK DYFPLPVTVG
WNGGALTGGV TFPAVLQGG GLYGLGGVVT VPGGGLGTQT
YLCVVVHKPG \TKVD<QVILP {GCDKTLTCP PCPAPLAAGG
PPKP {DTLMLGRTP ILVTCVVVDVG {LDPL'VKE'VW
DGVLVHNA {TKPQL'QYN GTYQVVGVLT VLLQDWLVG<
{CKVGNKA LPAP L<T <A<GQPQLPQ VYTLPPGQLL
{VQVGLTC YPGI AVLWLGVGQP LNVY<TTPPV
DGDGGFFLY GKLTVD<GI QQGNVFGCGV WHLALHNLYT
<GLGLGPG<
HCVH5C QVQLVQGGAI VKKPGAGV GCKAGGYTFT DYYWHWVQQA
PGQGLLWMG TNPRNGGTTY NLKFKGKATM TRDTGTGTAY
MILTGGTIRGLD TAVYYCTLGT GGYDYFDYWG QGTLVTVGGA
GTKGPGVFPL APGGKGTGGG TAALGCLVKD YFPLPVTVGW
WSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY
__CNVNHKPSN TKVDKKVLPK SC
100 LCVK18 D._VWTQTPLS LSVTPGQPAS ISCRSSQS_V HSDGW YLLW
YLQ<PGQSPK LL._YKVSYRF SGVPDRFSGS GSGTDFTL<
LDVGV YYCFQASHVP YTFGQGT<LL RTVAAPSV
h'._b'PPSDLQL KSGTASVVCL LNNFYPRILA< V DNALQ
SGNSQL'SVTL QDSKDSTYSL SSTLTLS<AD YI<HKVYACIL
VTHQGLSSPV T<SFWRGILC
101 D.VWTQTPLS LSVTPGQPAS QSIV I YLLW
YLQ<PGQSPK LL__Y<VSYRF SGVPDRFSGS {I
SRVLALDVGV YYCFQASHVP YTFGGGT . SV
h'._b'PPSDLQL KSGTASVVCL LNNFYPR DNALQ
SGNSQLSVTL QDSKDSTYSL S I I<HKVYACL
VTHQGLSSPV T<SFWRGLC
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 (Figure 5).
The antibody also effectively inhibited dabigatran function in human whole blood. A Fab
ted from this antibody blocked dabigatran ty in human plasma demonstrating
that monovalent antigen binding domains can neutralize compound agulant activity.
(Figure 6).
The major metabolic y 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,
tran acylglucuronides were ed from the urine of rhesus monkeys treated with
dabigatran and evalulated in the thrombin clotting time assay. The antibody dose
dependently reversed the dabigatran acylglucuronide-mediated prolongation of thrombin
dependent clotting in human plasma with r potency to that seen with dabigatran
(Figure 7). Thus the dy is effective in blocking the anticoagulant activity of
dabigatran metabolites found in humans.
The affinities of the Fab and the mouse-human chimeric antibodies comprising the
le domains of clone 22 were determined using Kinexa logy. A constant
concentration of Fab or ic antibody was incubated with various concentrations of
tran until equilibrium was reached. After this incubation the concentration of free
antibody was ined by ing the antibody on Neutravidin beads coupled with a
Biotin-conjugated dabigatran analog. The captured Fab was detected with an anti-Mouse
lgG (Fab specific) F(ab')2 fragment d with FITC. The captured chimeric antibodies
were detected with an anti-human lgG conjugated with Cy5. The dissociation constants
were calculated using a 1:1 g model. The results from these experiments are
summarized in the table below.
1O Affinity of anti-dabigatran antibodies
Antibody Apparent Kd
Clone 22 Fab 48 pM
Clone 22 Chimeric Ab 34 pM
Both the Fab and the chimeric antibodies bind dabigatran with high affinity.
Thrombin clotting time assay
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°C on the day of the
assay.
The thrombin clotting time is performed as follows. First in is diluted to
manufacturer’s ication (3 lU/mL thrombin) in the buffer provided (Dade Behring Test
kit) and prewarmed to 37°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 uL of plasma is pipetted into ed cuvettes with a
magnetic stirrer and allowed to stir for 2 min in the well preheated to 37°C in the CL4
machine. At this point 100 uL of the thrombin solution is added and the time required for
the plasma sample to clot is ed automatically by the CL4. Dabigatran is
preincubated for 5 min in plasma in the provided cuvettes, before adding thrombin and
starting the measurement. lf antibody is also tested (up 50 uL of stock solution), there is a
further 5 minute incubation at 37°C before beginning clotting (Le. 10 min total incubation
with tran, 5 min total incubation with antibody and then clotting is initiated with
thrombin).
Activity of chimeric antibodies and humanized Fabs in the thrombin time assay is shown in
Figures 8 -10, respectively.
Affinity determinations (Kinexa Method)
The ties of Fab and mouse-human chimeric antibodies were ined using
KinExA® logy. 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 dy on
Neutravidin beads coupled with a Biotin-conjugated dabigatran analog. The captured Fab
was detected with an anti-human lgG (Fab specific) F(ab')2 fragment labeled with FITC.
The captured chimeric antibodies were detected with an anti-human lgG conjugated with
Cy5. The dissociation constants (KD) were calculated using a 1:1 binding model.
To measure rate constants (kon and koff) with the ® 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 g 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, kon is measured ly and the off-
rate koff is calculated as koff = KD x kon.
Table: KD values of chimeric antibodies determined using KinExA® technology.
Chimeric Ab KD (pM)
Table: KD , kon and koff of humanized Fabs VH5C/VK18 and VH5C/VK21
H5C/VK18 133 pM 9.38e+005/Ms 1.25e-004 /s
H5C/VK21 147 pM 1.377e+006/Ms .029-004 /s
Fab-dabigatran complex ion and crystallization
The Fabs were concentrated to 10 mg/ml, mixed with a 2 molar excess of dabigatran and
incubated for 1 h at 4 °C. x and crystallization on were mixed 1:1. The
complex crystallizes in 25 % PEG 1500, 0.1 M SPG buffer (pH7).
Data collection and structure ination
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, 0., Paciorek, W., Womack, T. &
Bricogne, G. (2011). Data sing and analysis with the autoPROC toolbox. Acta
Cryst. D67, 293-302.).
Fab VH5C/VK21:Dabigatran ls grew in space group P212121 with unit cell
dimensions a=59.97 A, b=78.39 A, C: 87,67 A and diffract to 2.2 A 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 AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. J. Appl.
Cryst. (2007). 40, 658-674.) using a homologous Fab structure D 1C1 E) as the
starting search model. Analysis of the electron density map showed clear electron y
for dabigatran. The te 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-Biologica/
Crystallography 60: 2126-2132 Part 12 Sp. lss. 1 DEC 2004. Bricogne G., Blanc E.,
2012/055397
Brandl M., Flensburg C., Keller P., Paciorek W., Roversi P, Sharff A., Smart O.S.,
Vonrhein C., Womack TO. (2011). BUSTER version 2.11.2. Cambridge, United Kingdom:
Global Phasing Ltd).
Fab VH5C/VK18:Dabigatran crystals grew in space group P21 and P212121, tively.
Crystals with space group P21 showed unit cell dimensions of a=51.81 A, b=128.92 A, c=
60.26 A and ct to 1.9 A resolution. Crystals with space group P212121 showed unit
cell dimensions of a=48.20 A, b=59.74 A, c= 127.69 A and diffract to 2.2 A resolution.
Both complex structures were solved by molecular replacement with the program phaser
1O using the structure of Fab VH5C/VK21 as the starting search model. Analysis of the
electron density maps showed clear on 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)
The spatial aggregation sities (SAP) for each atom and each residue was
calculated as described in (1) with the exception that e 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 n data bank (PDB), yielding a mean (HPV and uch) and standard
deviation values (on and 0ch ) for both properties.
Z-scores for the Fv SAP and CDR SAP for the antibodies where then ated according
Z-score(Fv SAP) = ( Fv SAP - uF\,)/oFV and
Z-score(CDR SAP) = ( CDR SAP - uCDR)/OCDR.
Results (Figure 11):
Humanized Fab 18/15:
Z-score(Fv SAP) = 1.06
Z-score(CDR SAP) = 1.00
Humanized Fab VH5C/VK18:
Z-score(Fv SAP) = -0.61
Z-score(CDR SAP) = -0.84
Humanized Fab VH5C/VK21:
Z-score(Fv SAP) = -0.61
e(CDR SAP) = -0.78
Fab 18/15 (see W02011089183) has more solvent-exposed hydrophobic surface than the
average of known antibodies in the n data bank.
Surprisingly, both VH5C/VK18 (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 ive Z-
scores). This means that these compounds have an increased solubility in s media
and a lower tendency for ation, making them more suitable for stable drug
formulations with high antibody concentrations.
(1) Chennamsetty et. al., Proc Natl Acad Sci; 2009, 106(29), pg 11937-11942
(2) Cowan and Whittaker, Pept Res; 1990, 3(2), pg 75-80
Expression of Fab in CHO cells
Fabs were produced by transient transfection into CHO DG44 cells and subsequent
selection and generation of stable cell pools. Figure 13 shows the titers of fed batch runs
with Fab 18/15 (see W02011089183), 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.
I
Claims (12)
1. An antibody molecule against dabigatran comprising 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.
2. The antibody molecule of claim 1 comprising a heavy chain of SEQ ID NO: 99, and a light chain of SEQ ID No: 100.
3. The antibody le of claim 1 comprising a heavy chain of SEQ ID NO: 99, and a light chain of SEQ ID No: 101.
4. The antibody molecule of claim 1 which is a monoclonal antibody, a human antibody, a humanized dy, a chimeric antibody, a fragment of an dy, or a single chain antibody,.
5. The antibody molecule of claim 4, wherein said fragment of an antibody is a Fab, Fab’, or F(ab’)2 fragment.
6. The antibody molecule of claim 4, wherein the single chain antibody is a single chain variable fragment (scFv).
7. Use of an antibody molecule of any one of the preceding claims 1 to 6 for the manufacture of a medicament for the therapy or prevention of side effects of agulant therapy, and/or for reversal of an overdosing of an anticoagulant.
8. The use of claim 7, wherein the side effect is a bleeding event.
9. A method of manufacturing an antibody molecule of any one of claims 1 to 6, sing (a) providing a host cell comprising one or more nucleic acids encoding said antibody le in functional association with an expression control sequence, (b) cultivating said host cell, and AH26(10039265_1):JIN (c) recovering the dy molecule from the cell culture, wherein the host cell is not within a human.
10. A kit comprising an antibody of any one of claims 1 to 6, or a pharmaceutical composition thereof.
11. A kit comprising: (a) an antibody of any one of claims 1 to 6, or a ceutical composition thereof; (b) a ner; and (c) a label.
12. A kit comprising an antibody of any one of claims 1 to 6, and dabigatran, dabigatran etexilate, a prodrug of dabigatran or a pharmaceutically acceptable salt thereof. Boehringer Ingelheim International GmbH By the Attorneys for the Applicant SPRUSON & FERGUSON Per: AH26(10039265_1):JIN
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US201161469207P | 2011-03-30 | 2011-03-30 | |
US61/469,207 | 2011-03-30 | ||
PCT/EP2012/055397 WO2012130834A1 (en) | 2011-03-30 | 2012-03-27 | Anticoagulant antidotes |
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NZ613543B2 true NZ613543B2 (en) | 2015-11-03 |
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