U.S. patent application number 14/710182 was filed with the patent office on 2015-08-27 for therapeutic factor viii antibodies.
This patent application is currently assigned to NOVO NORDISK A/S. The applicant listed for this patent is Jes T. Clausen, Ida Hilden, Heidi L. Holmberg, Kasper Lamberth, Henrik Oestergaard. Invention is credited to Jes T. Clausen, Ida Hilden, Heidi L. Holmberg, Kasper Lamberth, Henrik Oestergaard.
Application Number | 20150239984 14/710182 |
Document ID | / |
Family ID | 43587481 |
Filed Date | 2015-08-27 |
United States Patent
Application |
20150239984 |
Kind Code |
A1 |
Oestergaard; Henrik ; et
al. |
August 27, 2015 |
THERAPEUTIC FACTOR VIII ANTIBODIES
Abstract
The present invention relates to therapeutic FVIII antibodies.
In particular, the present invention relates to FVIII antibodies
having the ability to prolong the circulatory half life of FVIII.
The present invention furthermore relates to use of such antibodies
in treatment and prophylaxis of haemophilia A.
Inventors: |
Oestergaard; Henrik;
(Oelstykke, DK) ; Hilden; Ida; (Vanloese, DK)
; Holmberg; Heidi L.; (Oelsted, DK) ; Lamberth;
Kasper; (Stenloese, DK) ; Clausen; Jes T.;
(Hoeng, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Oestergaard; Henrik
Hilden; Ida
Holmberg; Heidi L.
Lamberth; Kasper
Clausen; Jes T. |
Oelstykke
Vanloese
Oelsted
Stenloese
Hoeng |
|
DK
DK
DK
DK
DK |
|
|
Assignee: |
NOVO NORDISK A/S
Bagsvaerd
DK
|
Family ID: |
43587481 |
Appl. No.: |
14/710182 |
Filed: |
May 12, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13822952 |
Jun 26, 2013 |
9062115 |
|
|
PCT/EP2011/065986 |
Sep 15, 2011 |
|
|
|
14710182 |
|
|
|
|
61386783 |
Sep 27, 2010 |
|
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Current U.S.
Class: |
424/145.1 ;
435/69.6; 530/387.9; 530/388.25 |
Current CPC
Class: |
A61K 39/3955 20130101;
C07K 2317/34 20130101; C07K 2317/70 20130101; C07K 2317/92
20130101; A61P 7/04 20180101; C07K 16/36 20130101; A61K 38/37
20130101; C07K 2317/56 20130101 |
International
Class: |
C07K 16/36 20060101
C07K016/36; A61K 38/37 20060101 A61K038/37; A61K 39/395 20060101
A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 22, 2010 |
EP |
10178292.8 |
Claims
1. A monoclonal Factor VIII antibody having the ability to bind to
activated human Factor VIII, wherein said antibody, upon binding to
activated Factor VIII, reduces dissociation of the A2 domain, and
wherein said antibody does not interfere with vWF binding.
2. The monoclonal antibody according to claim 1, wherein said
antibody does not accelerate thrombin activation.
3. The antibody according to claim 1, wherein said antibody binds
to the A2 domain.
4. The antibody according to claim 1, wherein said antibody binds
to the A3 domain.
5. The antibody according to claim 1, wherein said antibody binds
to an epitope identical with or overlapping with the peptide
fragment 407-428 (SEQ ID NO 15) and/or 591-602 (SEQ ID NO: 16).
6. The antibody according to claim 1, wherein said antibody binds
to an epitope identical with or overlapping with the peptide
fragment 1965-1976 (SEQ ID NO: 17).
7. The antibody according to claim 1, wherein the antibody competes
with binding to the 4F143 antibody.
8. The antibody according to claim 1, wherein said antibody
comprises one or more CDR sequences having at least 95% identity
with one or more of the CDR sequences selected from the list
consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
11, SEQ ID NO: 12, and SEQ ID NO: 13.
9. The antibody according to claim 5, wherein said antibody
comprises a VL sequence having at least 95% identity with the SEQ
ID NO: 9 and a VH sequence having at least 95% identity with SEQ ID
NO: 10.
10. A method for treating haemophilia A comprising administering an
antibody according to claim 1 to a subject in need thereof.
11. The method for treating haemophilia A of claim 10 comprising
administering the antibody in combination with a Factor VIII
molecule.
12. A pharmaceutical composition comprising an antibody according
to claim 1.
13. The pharmaceutical composition according to claim 12, further
comprising a Factor VIII molecule.
14. The pharmaceutical composition according to claim 12, wherein
said composition is for subcutaneous administration.
15. A method of making an antibody according to claim 1, wherein
said method comprises incubation of a host cell encoding such
antibody under conditions suitable for expressing said antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 13/822,952, filed Jun. 26, 2013 (Notice of Allowance Mailed
Mar. 4, 2015) which is a 35 U.S.C. .sctn.371 National Stage
application of International Application PCT/EP2011/065986 (WO
2012/038315), filed Sep. 15, 2011, which claimed priority of
European Patent Application 10178292.8, filed Sep. 22, 2010; this
application claims priority under 35 U.S.C. .sctn.119 of U.S.
Provisional Application 61/386,783; filed Sep. 27, 2010; the
contents of which are hereby incorporated by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 12, 2015, is named 8126US01_SeqListing_ST25.txt and is
30,613 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to treatment of haemophilia A.
In particular, the present invention relates to therapeutic Factor
VIII antibodies as well as use of Factor VIII antibodies for
treatment of haemophilia A.
BACKGROUND OF THE INVENTION
[0004] Haemophilia A is an inherited bleeding disorder caused by
deficiency or dysfunction of coagulation factor VIII (FVIII)
activity. The clinical manifestation is not on primary
haemostasis--formation of the blood clot occurs normally--but the
clot is unstable due to a lack of secondary thrombin formation.
[0005] Haemophilia A is currently treated by intravenously
injection of coagulation factor FVIII which is either isolated from
blood or produced recombinantly. Treatment can be either on-demand
or prophylactic. Recent published data support that prophylaxis has
significant advantages over on-demand treatment. These include
reduction in bleeding frequency and lower risk of developing
haemophilic arthropathy, both resulting in a better quality of life
for the patients. However, while prophylaxis enables a virtually
symptom-free life for the patients, it requires frequent injections
in a peripheral vein, typically three times a week, which is known
to be painful, difficult, and time consuming. In addition, repeated
venipuncture is not always possible in young children.
Consequently, a product supporting less frequent administration
and/or administration via a more convenient and safe route such as
e.g. subcutaneous administration, would to a greater extent enable
regular prophylactic treatment.
[0006] A FVIII antibody having the ability to enhance the
activation of wt FVIII is disclosed in US20090297503. This
antibody, however, is also shown to impair binding of wt FVIII to
vWF. Impairment of FVIII:vWF binding is generally believed to be
undesirable as the circulatory half life of Factor VIII is many
fold higher upon vWF binding.
[0007] There is thus a need in the art for therapies that support
infrequent administration and/or is capable of enhancing the
activity of endogenous FVIII, and consequently the procoagulant
response, in patients suffering from haemophilia A. Patients with
endogenous FVIII include haemophilia A patients suffering from the
mild to moderate form and a certain fraction of the severe
patients.
SUMMARY OF THE INVENTION
[0008] The present invention relates to a monoclonal Factor VIII
antibody having the ability to bind to activated human Factor VIII,
wherein said antibody, upon binding to activated Factor VIII,
reduces or inhibits dissociation of the A2 domain, and wherein said
antibody does not interfere with vWF binding. The present invention
furthermore relates to therapeutic use of such molecules.
[0009] Such antibodies may be useful for prolonging the lifetime of
the FVIIIaIFIXa complex resulting in more thrombin being generated
and consequently improved clot formation. Such antibodies are thus
suitable for treatment of patients suffering from haemophilia A and
not completely devoid of endogenous FVIII such as patients with
mild and moderate haemophilia A and a subset of patients having
severe haemophilia A. Optionally such antibodies may be used in
combination with Factor VIII replacement therapy.
DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows result from the functional chromogenic primary
screening assay. One medium control with no disassociation time
(Max) and a medium control with a 7.5-min dissociation time (Min)
defined the assay window. Several of the samples inhibited FVIII
activity evidenced by activities below Min in the assay.
Interestingly, a significant fraction of the samples were able to
stabilize FVIII to a greater extent than the control with no
dissociation time (Max).
[0011] FIG. 2 shows antibodies tested in five different
concentrations (0-50 nM) at four different dissociation timepoints
(0-25 minutes) with phospholipids added together with FVIII
(phospholipid dependent) or with phospholipids added together with
FIXa/FX-mix (phospholipid independent). The measured signal
(absorbance at 405 nm) is proportional to the remaining FVIIIa
activity after dissociation. Comparison of the two reaction
conditions demonstrate that the majority of antibodies are able to
stabilize FVIIIa in the presence of phospholipid whereas the degree
of FVIIIa stabilization in the absence of phospholipid during
FVIIIa decay is less. For most antibodies, maximal stabilization of
FVIIIa is observed even at the lowest antibody concentration. One
exception is 4F136 with less stabilization at the lowest antibody
concentrations indicating a relatively low affinity
interaction.
[0012] FIG. 3. Effect of 4F143 on the time-dependent spontaenous
decay of activated FVIII or FVIII S289L. FVIII at 0.3 nM was
rapidly activated with 40 nM thrombin for 30 sec at room
temperature and pH 7.4 followed by addition of hirudin to
inactivate thrombin. At the indicated time points, the decay
mixture was diluted into FIXa/phospholipid and the initial rate of
FX activation determined as milli absorbance units at 405 nm per
minute (mAU/min). (A) Binding of 4F143 does not stabilize FVIIIa
against spontaneous dissociation in the absence of phospholipid.
Reactions were performed in the absence of phospholipid and either
in the absence ( ) or the presence (.tangle-solidup.) of 20 nM
4F143 antibody during activation and subsequent decay of FVIIIa.
(B) Significant stabilization of FVIIIa by 4F143 in the presence of
phospholipid. Stabilization is not dependent on the pre-association
of FVIII and antibody before thrombin activation. Reactions were
performed in the presence of 10 .mu.M phospholipid during FVIII
activation and decay. Symbols indicate ( ) No antibody present,
(.tangle-solidup.) 20 nM 4F143 present during FVIIIa activation and
decay, and () 20 nM 4F143 present during FVIIIa decay. (C) 4F143 is
able to slow the rate of dissociation of FVIIIa S289L to
approximately the level observed for wt FVIIIa in the absence of
antibody. Reactions were performed in the presence of 10 .mu.M
phospholipid during activation and decay of FVIII or FVIII S289L; (
, .box-solid.) No antibody present, (.diamond-solid.) 20 nM 4F143
present during FVIII S289L activation and decay.
[0013] FIG. 4. Effect of antibodies on the binding of FVIII to
immobilized vWF in a solid-phase binding assay. (A) Titration of
vWF with 0.05 to 6.4 nM FVIII in the absence of antibody. Analysis
of data according to a one-site binding model gave an apparent
dissociation constant (K.sub.d) of 0.29.+-.0.01 nM, which agrees
well with published values (Vlot et al., 1995). Results are shown
as mean.+-.standard deviation (n=4). (B) The effect of antibody on
FVIII binding to vWF was investigated at a single FVIII
concentration (0.8 nM) giving half maximal binding to vWF in the
absence of antibody. Antibody concentrations ranged from 0 to 162
nM with the highest concentrations being more than one order of
magnitude above the measured K.sub.d for the FVIII-antibody
interaction (see Table 2). Bound FVIII was detected using an
anti-FVIII antibody recognizing a non-overlapping epitope. None of
the antibodies tested were found to affect the interaction of FVIII
with vWF. Results are shown as mean.+-.standard deviation
(n=2).
[0014] FIG. 5. Effect of antibody on the activation of FVIII by
thrombin. Activation of 100 nM FVIII by 1 nM thrombin was performed
at 37.degree. C. and in the absence ( , stipled line) or presence
of 100 nM ESH5 (.diamond-solid.), ESH8 (.largecircle.), moAb216
(.quadrature.), 4F143 (.tangle-solidup.), 4F50 (), or 4F140
(.box-solid.). At indicated time points further activation of FVIII
was quenched by addition of excess hirudin and the extent of FVIII
activation quantified by rpHPLC as the amount of free A1 subunit.
ESH5, ESH8, and moAb216 were all found to accelerate the activation
of FVIII, whereas no increased rate of activation was observed for
4F143, 4F50, and 4F140.
[0015] FIG. 6. HX monitored by mass spectrometry identifies regions
of FVIII involved in 4F143 and 4F41 binding. (A) Mass/charge
spectra corresponding to the peptide fragment aa 407-428 (SEQ ID NO
15), YKSQYLNNGPQRIGRKYKKVRF ([M+H]+=549.5128, z=5), (B) Mass/charge
spectra corresponding to the peptide fragment aa 591-602 (SEQ ID NO
16), IQRFLPNPAGVQ ([M+H]+=670.3730, z=2) both identified to be part
of the epitope of 4F143 binding to FVIII. (C) Mass/charge spectra
corresponding to the peptide fragment 1965-1976 (SEQ ID NO 17),
VRKKEEYKMALY (m/z=524.9335, z=3), identified to be part of the
epitope of 4F41 binding to FVIII. For all spectra the upper panels
show the non-deuterated controls, middle and lower panels show the
peptide after 30 s in-exchange with D20 in the absence or presence
of mAb, respectively.
[0016] FIG. 7. Hydrogen exchange time-plots of representative
peptides of FVIII in the presence of 4F143. Deuterium incorporation
(Da) of FVIII peptides is plotted against time on a logarithmic
scale in the absence (.box-solid.) or presence (.quadrature.) of
4F143. Peptides covering residues aa 392-403 and 429-436 represent
regions of FVIII that are unaffected by complex formation with
4F143. Peptides covering residues aa 407-428, and 415-428 represent
regions of FVIII that are part of the binding epitope of 4F143.
[0017] FIG. 8. Sequence coverage of HX analyzed peptides of FVIII
in the presence of 4F143. The primary sequence (using mature
numbering) is displayed above the HX analyzed peptides (shown as
horizontal bars) for both epitope regions identified, i.e., the
sequence (A) aa 407-428 and (B) aa 591-602. Peptides showing
similar exchange patterns both in the presence and absence of 4F143
are displayed in with no fills (.quadrature.) whereas peptides
showing reduced deuterium incorporation upon 4F143 binding are
filled in black (.box-solid.).
[0018] FIG. 9. Hydrogen exchange time-plots of representative
peptides of FVIII in the presence of 4F41. Deuterium incorporation
(Da) of FVIII peptides is plotted against time on a logarithmic
scale in the absence (.box-solid.) or presence (.DELTA.) of 4F41.
Peptides covering the residues aa 1963-1972, 1963-1974, and
1965-1976 represent regions of FVIII that are part of the binding
epitope of 4F41, changes in deuterium exchange rate are observed
for short incubation times, i.e., 10 s and 30s. The peptide
covering residues aa 1984-1988 represents regions of FVIII that are
unaffected by complex formation with 4F41.
[0019] FIG. 10. Sequence coverage of HX analyzed peptides of FVIII
in the presence and absence of 4F41. The primary sequence (using
mature numbering) is displayed above the HX analyzed peptides
(shown as horizontal bars). Peptides showing similar exchange
patterns both in the presence and absence of 4F41 are displayed in
with no fills (.quadrature.), and peptides showing reduced
deuterium incorporation at short incubation times, i.e., <100 s
upon 4F41 binding are filled in black (.box-solid.).
DESCRIPTION OF THE INVENTION
Definitions
[0020] Factor VIII antibody: Factor VIII antibodies according to
the present invention have the ability to bind to activated Factor
VIII and they furthermore preferably have the ability to bind to
Factor VIII both before and after thrombin activation. The
antibodies according to the invention may furthermore have the
ability to bind to modified Factor VIII variants such as e.g.
Factor VIII molecules conjugated with one or more side groups.
Antibodies according to the invention may furthermore have the
ability to bind to fusion proteins comprising Factor VIII and
optionally conjugated with side groups. Antibodies according to the
invention may furthermore have the ability to bind to Factor VIII
variants comprising amino acid deletions, substitutions and/or
additions such as those found in haemophilia A patients or e.g. B
domain truncated/deleted Factor VIII, Factor VIII with decreased
ability to bind to vWF, Factor VIII variants with modified ability
to bind to various molecules (such as e.g. LRP), optionally
conjugated with side groups and optionally being part of a fusion
protein. Antibodies according to the present invention may in other
words bind to any Factor VIII variant having Factor VIII activity.
Antibodies according to the present invention may typically have a
circulatory half life that is significantly longer compared to the
circulatory half life of wt FVIII. Antibodies according to the
invention can furthermore be administered e.g. subcutaneously which
is an administration form that is usually more convenient and easy
to use than IV administration.
[0021] The term "antibody" or "Factor VIII antibody", as used
herein, is intended to refer to immunoglobulin molecules and
fragments thereof that have the ability to specifically bind to
Factor VIII and/or FVIIIa. Full-length antibodies comprise four
polypeptide chains, two heavy (H) chains and two light (L) chains
interconnected by disulfide bonds. Each heavy chain is comprised of
a heavy chain variable region (abbreviated herein as HCVR or VH)
and a heavy chain constant region. The heavy chain constant region
is comprised of three domains, CH1, CH2 and CH3. Each light chain
is comprised of a light chain variable region (abbreviated herein
as LCVR or VL) and a light chain constant region. The light chain
constant region is comprised of one domain, CL. The VH and VL
regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Thus, within the definition
of an antibody according to the invention is also one or more
fragments of an antibody that retain the ability to specifically
bind to Factor VIII. It has been shown that the antigen-binding
function of an antibody can be performed by fragments of a
full-length antibody. Examples of binding fragments encompassed
within the term "antibody" include (i) a Fab fragment, a monovalent
fragment consisting of the VL, VH, CL and CH I domains; (ii) F(ab)2
and F(ab')2 fragments, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the VH and CH1 domains; (iv) a Fv
fragment consisting of the VL and VH domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546), which consists of a VH domain; and (vi) an isolated
complementarity determining region (CDR). Furthermore, although the
two domains of the Fv fragment, VL and VH, are coded for by
separate genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the VL and VH regions pair to form monovalent
molecules (known as single chain Fv (scFv); see e.g., Bird et al.
(1988) Science 242:423-426: and Huston et al. (1988) Proc. Natl.
Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also
intended to be encompassed within the term "Factor VIII antibody"
according to the present invention. Other forms of single chain
antibodies, such as diabodies are also encompassed in the term
"Factor VIII antibody". Diabodies are bivalent, bispecific
antibodies in which VH and VL domains are expressed on a single
polypeptide chain, but using a linker that is too short to allow
for pairing between the two domains on the same chain, thereby
forcing the domains to pair with complementary domains of another
chain and creating two antigen binding sites (see e.g., Holliger,
P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak,
R. J., et al. (1994) Structure 2:1121-1123).
[0022] The terms "human antibody", "human antibodies", as used
herein, means Factor VIII antibodies according to the invention
having variable and constant regions derived from human germline
immunoglobulin sequences. The human Factor VIII antibodies of the
invention may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in vivo), for example in the CDRs and in particular CDR3.
[0023] The term "humanized antibody" in this context refers to CDR
sequences from a Factor VIII antibody according to the invention
that have been grafted onto a human scaffold. Factor VIII
antibodies according to the present invention may thus be e.g.
human antibodies or humanized antibodies.
[0024] The term "epitope" as used herein means any antigenic
determinant on an antigen to which the antibody binds. Epitopic
determinants usually consist of chemically active surface groupings
of molecules such as amino acids or sugar side chains and usually
have spe-cific three dimensional structural characteristics, as
well as specific charge characteristics.
The terms "immunoreacts" or "immunoreacting", as used herein, means
any binding of an antibody to its epitope with a dissociation
constant Kd lower than 10-4 M. The terms "immunoreacts" or
"immunoreacting" are used where appropriate inter-changeably with
the term "specifically bind". Epitopes are often referred to as one
or more regions in the amino acid sequence, and/or individual amino
acid residues of the FVIII molecule that is/are covered by an
antibody upon FVIII:FVIII antibody binding. Antibodies binding to a
region that is overlapping e.g. with a subsection or a region of
"an epitope" are also regarded as antibodies binding to this
epitope as long as the antibody can be said to form non-covalent
interactions with or to cover at least one, preferably at least
two, more preferably at least three, more preferably at least four
and most preferably at least 5-10 of the amino acids within the
FVIII epitope.
[0025] The term "affinity", as used herein, means the strength of
the binding of an antibody to an epitope. The affinity of an
antibody is measured by the dissociation constant Kd, defined as
[Ab].times.[Ag]/[Ab-Ag] where [Ab-Ag] is the molar concentration of
the antibody-antigen complex, [Ab] is the molar concentration of
the unbound antibody and [Ag] is the molar concentration of the
unbound antigen. The affinity constant Ka is defined by 1/Kd.
Preferred methods for determining Mabs specificity and affinity by
competitive inhibition can be found in Harlow, et al., Antibodies:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1988), Colligan et al., eds., Current
Protocols in Immunology, Greene Publishing Assoc. and Wiley
Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol.
92:589-601 (1983), which references are entirely incorporated
herein by reference.
[0026] Co-administration: Factor VIII antibodies according to the
invention can be co-administered together with therapeutic Factor
VIII molecules that may be either derived froom blood or produced
using recombinant techniques. Co-administration may be performed by
IV administration of a pharmaceutical formulation comprising both
types of therapeutic proteins. Co-administration may also be
performed by IV administration of a pharmaceutical composition
comprising therapeutic Factor VIII molecules and IV or subcutaneous
administration of a composition comprising Factor VIII antibodies
according to the present invention. Co-administration can be done
either simultaneously or with an interval of from about one minute
to one month, one hour to one day, or one day to one week.
Administration of antibodies according to the present invention,
optionally in the form as co-administration with a FVIII molecule
or FVIII variant/derivative may be performed e.g. once every day,
once every week, once every second week, once every third week, or
once every month.
[0027] Factor VIII molecules: FVIII/Factor VIII is a large, complex
glycoprotein that primarily is produced by hepatocytes. Human FVIII
consists of 2351 amino acids, including signal peptide, and
contains several distinct domains, as defined by homology. There
are three A-domains, a unique B-domain, and two C-domains. The
domain order can be listed as NH2-A1-A2-B-A3-C1-C2-COOH. FVIII
circulates in plasma as two chains, separated at the B-A3 border.
The chains are connected by bivalent metal ion-bindings. The
A1-A2-B chain is termed the heavy chain (HC) while the A3-C1-C2 is
termed the light chain (LC).
[0028] FVIII circulates associated with von Willebrand Factor
(VWF). VWF is a large multimeric glycoprotein that serves as a
carrier for FVIII and is required for normal platelet adhesion to
components of the vessel wall. The plasma-half life of FVIII in
complex with VWF is approximately 12 hours.
[0029] FVIII is activated by thrombin or FXa by cleavages in the HC
and LC, which releases FVIIIa from VWF. This process produces a
heterotrimeric molecule consisting of A1 and A2 domains
non-covalently linked to the A3-C1-C2 light chain through ionic
interactions. The FVIIIa molecule is inherently unstable as a
consequence of spontaneous A2 subunit dissociation and concomitant
loss of cofactor activity (References: Fay (1991) JBC,
266:8957-8962; Lamphear (1992) JBC, 267:3725-3730; Fay (1992) JBC
267:13246-13250; Fay (1993) JBC 268:17861-17866; Fay (1996) JBC
271:6027-6032; Parker (2007) JBC 281:13922-13930; Parker (2007)
Biochemistry 46:9737-9742). Dissociation occurs with a half-life of
approximately 2 min and appears to be the predominant physiological
mechanism for down-regulation of the FVIIIa/FIXa complex (Fay P J
(2004) Blood Reviews, 18:1-15). FVIIIa can also be inactivated by
the anticoagulant serine protease, activated protein C (APC) which
cleaves FVIIIa at additional site in the heavy chain. However, the
physiological relevance of this pathway appears to be minor (Fay P
J (2004) Blood Reviews, 18:1-15).
[0030] "Native FVIII" is the full length human FVIII molecule as
shown in SEQ ID NO. 1 (amino acid 1-2332). The B-domain is spanning
amino acids 741-1648 in SEQ ID NO 1.
TABLE-US-00001 SEQ ID NO 1: ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKS-
FPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASH-
PVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGG-
SHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHK-
FILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVY-
WHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQ-
TLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLT-
DSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLN-
NGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNI-
YPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSS-
FVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENI-
QRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTD-
FLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRG-
MTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPEN-
DIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDP-
SPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLD-
FKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGG-
PLSLSEENNDSKLLESGLMNSQESS-
WGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIEN
SPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEG-
PIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGP-
SPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLD-
NLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGA-
YAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISP-
NTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQI-
DYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSS-
FPSIRPIYLTRVLFQDNSSHLPAASYRK-
KDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLP
KTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIK-
WNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAF-
KKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRT-
TLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFI-
AAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEH-
LGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETK-
TYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIG-
PLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERN-
CRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNE-
NIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEM-
LPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAP-
KLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLD-
GKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLR-
MELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFAT-
WSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKE-
FLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRI-
HPQSWVHQIALRMEVLGCEAQDLY
[0031] "Factor VIII molecules" that are co-administrated along with
Factor VIII antibodies according to the present invention may be
Factor VIII isolated from blood plasma and/or recombinant Factor
VIII. Factor VIII molecules to be coadministrated together with the
Factor FVIII antibodies according to the invention may be e.g. B
domain truncated Factor FVIII molecules wherein e.g. the remaining
domains correspond closely to the sequence as set forth in amino
acid no 1-740 and 1649-2332 in SEQ ID NO. 1 (although there may
also e.g. be one or more alterations within the vWF binding region
between residues 1670-1684). B domain truncated molecules
co-administered with Factor VIII antibodies according to the
invention may differ slightly from the sequence set forth in SEQ ID
NO 1, meaning that the remaining domains (i.e. the three A-domains
and the two C-domains) may differ slightly e.g. 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10 amino acids, alternatively may differ about 1%, 2%,
3%, 4% or 5% from the amino acid sequence as set forth in SEQ ID NO
1 (amino acids 1-740 and 1649-2332) due to the fact that mutations
can be introduced in order to e.g. reduce vWF binding capacity.
Furthermore, it is plausible that amino acid modifications
(substitutions, deletions, etc.) are introduced other places in the
molecule in order to modify the binding capacity of Factor VIII
with various other components such as e.g. LRP, various receptors,
other coagulation factors, cell surfaces, introduction and/or
abolishment of glycosylation sites, etc.
[0032] Factor VIII molecules that are co-adminstered along with
Factor VIII antibodies according to the present invention have
Factor VIII activity, meaning the ability to function in the
coagulation cascade in a manner functionally similar or equivalent
to FVIII, induce the formation of FXa via interaction with FIXa on
an activated platelet, and support the formation of a blood clot.
The activity can be assessed in vitro by techniques well known in
the art such as e.g. clot analysis, endogenous thrombin potential
analysis, etc. Factor VIII molecules co-adminstered with Factor
VIII antibodies according to the invention have FVIII activity
being at least about 10%, at least 20%, at least 30%, at least 40%,
at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, and 100% or even more than 100% of that of native human
FVIII.
[0033] Reduction/Inhibition of dissociation of the A2 subunit
refers to the situation where the rate of dissociation of the A2
subunit from activated FVIII is reduced as compared to e.g. the
rate of dissociation of wtFVIII (optionally in the presence of a
control antibody) as measured in the presence of antibody and e.g
5, 10, 15, 20, or 25 minutes after activation of FVIII to FVIIIa.
Antibodies according to the present invention result in a reduction
of dissociation of the A2 domain of 10% or more, preferably 15% or
more, more preferably 20% or more, more preferably 25% or more,
more preferably 30% or more, more preferably 35% or more, more
preferably 40% or more, more preferably 45% or more, more
preferably 50% or more, more preferably 55% or more, more
preferably 60% or more, more preferably 65% or more, more
preferably 70% or more, more preferably 75% or more, more
preferably 80% or more, and most preferably 90% or more. Such
property can be determined in a functional decay assay as described
in Example 4 and elsewhere (Fay et al. (1996) JBC, 271:6027-6032;
Parker et al. (2006) JBC, 281:13922-13930). The assay measures the
cofactor activity of FVIIIa as a function of time following
activation of FVIII by thrombin. The cofactor activity of FVIIIa is
measured as its ability to stimulate the conversion of FX to FXa in
the presence of a suitable phospholipid surface and factor IXa.
[0034] B domain: The B-domain in Factor VIII spans amino acids
741-1648 in SEQ ID NO 1. The B-domain is cleaved at several
different sites, generating large heterogeneity in circulating
plasma FVIII molecules. The exact function of the heavily
glycosylated B-domain is unknown. What is known is that the domain
is dispensable for FVIII activity in the coagulation cascade. This
apparent lack of function is supported by the fact that B domain
deleted/truncated FVIII appears to have in vivo properties
identical to those seen for full length native FVIII.
[0035] B domain truncated/deleted Factor VIII molecule: Endogenous
full length FVIII is synthesized as a single-chain precursor
molecule. Prior to secretion, the precursor is cleaved into the
heavy chain and the light chain. Recombinant B domain-deleted FVIII
can be produced from two different strategies. Either the heavy
chain without the B-domain and the light chain are synthesized
individually as two different polypeptide chains (two-chain
strategy) or the B-domain deleted FVIII is synthesized as a single
precursor polypeptide chain (single-chain strategy) that is cleaved
into the heavy and light chains in the same way as the full-length
FVIII precursor.
[0036] In a B domain-deleted FVIII precursor polypeptide, the heavy
and light chain moieties are normally separated by a linker. To
minimize the risk of introducing immunogenic epitopes in the B
domain-deleted FVIII, the sequence of the linker is preferable
derived from the FVIII B-domain. As a minimum, the linker must
comprise a recognition site for the protease that cleaves the B
domain-deleted FVIII precursor polypeptide into the heavy and light
chain. In the B domain of full length FVIII, amino acid 1644-1648
constitutes this recognition site. The thrombin site leading to
removal of the linker on activation of B domain-deleted FVIII is
located in the heavy chain. Thus, the size and amino acid sequence
of the linker is unlikely to influence its removal from the
remaining FVIII molecule by thrombin activation.
Deletion/truncation of the B domain is an advantage for production
of FVIII. Nevertheless, parts of the B domain can be included in
the linker without reducing the productivity. The negative effect
of the B domain on productivity has not been attributed to any
specific size or sequence of the B domain.
[0037] B-domain truncated/deleted Factor VIII variants that can be
co-administered with Factor VIII antibodies according to the
invention may contain one or more O-glycosylation sites. However,
according to a preferred embodiment, the molecule comprises only
one O-linked oligosaccharides in the truncated B-domain--an example
thereof is the BDD-FVIII 40 KDa PEG (O-glycan) molecule disclosed
in FIG. 7 in WO09108806, wherein the amino acid sequence of the B
domain is: SFSQNSRHPSQNPPVLKRHQR (SEQ ID NO 2). An O-linked glycan
may be used for conjugating the Factor VIII molecule with various
side groups as described in the methods in e.g. WO0331464.
[0038] The Factor VIII molecule that is co-administered with Factor
VIII antibodies according to the invention comprises a number of
N-linked oligosaccharides and each of these may likewise
potentially serve as an anchor for attachment of a side group (as
disclosed in .g. WO0331464).
[0039] The length of the B domain in the wt FVIII molecule is about
907 amino acids. The length of the truncated B domain in Factor
VIII molecules co-administered with Factor VIII antibodies
according to the present invention may vary from about 10 to about
800 amino acids, such as e.g. from about 10 amino acids to about
700 acids, such as e.g. about 12-500 amino acids, 12-400 amino
acids, 12-300 amino acids, 12-200 amino acids, 15-100 amino acids,
15-75 amino acids, 15-50 amino acids, 15-45 amino acids, 20-45
amino acids, 20-40 amino acids, or 20-30 amino acids. The truncated
B-domain may comprise fragments of the heavy chain and/or the light
chain and/or an artificially introduced sequence that is not found
in the wt FVIII molecule. The terms "B-domain truncated" and
"B-domain deleted" may be used interchangeably herein.
[0040] Von Willebrandt Factor (vWF): vWF is a large
mono-/multimeric glycoprotein present in blood plasma and produced
constitutively in endothelium (in the Weibel-Palade bodies),
megakaryocytes (a-granules of platelets), and subendothelial
connective tissue. Its primary function is binding to other
proteins, particularly Factor VIII and it is important in platelet
adhesion to wound sites.
[0041] Factor VIII is bound to vWF while inactive in circulation;
Factor VIII degrades rapidly or is cleared when not bound to vWF.
Antibodies according to the present invention do not interfere with
vWF binding to FVIII or FVIII variants. Non-interference with vWF
binding is in connection with the present invention defined as a
reduced binding to vWF of preferably 0%, or alternatively less than
2%, or less than 5%, or less than 10%, or less than 15%, or less
than 20%, or less than 25%, or less than 30% at a concentration of
antibody ensuring FVIII saturation, such as an antibody
concentration 10-fold above the measured dissociation constant for
the antibody-FVIII interaction. An assay that can be used to
measure vWF:FVIII binding is disclosed e.g. in Example 5.
[0042] The term "reduced capacity to bind vWF" is herein meant to
encompass Factor VIII variants, that are co-administered with
Factor VIII antibodies according to the invention, wherein the
capacity to bind vWF is decreased by at least 50%, preferably by at
least 60%, more preferably by at least 70%, more preferably by at
least 80%, more preferably by at least 90%, and most preferably
about 100%. FVIII binding to vWF may be measured either by an
solid-phase assay or as direct binding to immobilized vWF using
surface plasmon resonance. The region in Factor VIII responsible
for binding to vWF is the region spanning residues 1670-1684 as
disclosed in EP0319315. It is envisaged that Factor VIII point
and/or deletion mutants involving this area will modify the ability
to bind to vWF. Examples of particularly preferred point mutations
according to the present invention include variants comprising one
or more of the following point mutations: Y1680F, Y1680R, Y1680N,
and E1682T, and Y1680C. If the FVIII variants co-administered along
with antibodies according to the invention, are modified with
relation to their capacity to bind to vWF, then such FVIII variants
are preferably protracted e.g. with a protracting group such as
e.g. PEG, fatty acid derivates, albumin, etc.
[0043] Suitable host cells for producing antibodies according to
the invention as well as recombinant factor VIII protein, that can
be co-administered with Factor VIII antibodies according to the
invention, are preferably of mammalian origin in order to ensure
that the molecule is properly processed during folding and
post-translational modification, eg. glycosylation and sulfatation.
In practicing the present invention, the cells are mammalian cells,
more preferably an established mammalian cell line, including,
without limitation, CHO (e.g., ATCC CCL 61), COS-1 (e.g., ATCC CRL
1650), baby hamster kidney (BHK), and HEK293 (e.g., ATCC CRL 1573;
Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines. A
preferred BHK cell line is the tk-ts13 BHK cell line (Waechter and
Baserga, Proc. Natl. Acad. Sci. USA 79:1106-1110, 1982),
hereinafter referred to as BHK 570 cells. The BHK 570 cell line is
available from the American Type Culture Collection, 12301 Parklawn
Dr., Rockville, Md. 20852, under ATCC accession number CRL 10314. A
tk- ts13 BHK cell line is also available from the ATCC under
accession number CRL 1632. A preferred CHO cell line is the CHO K1
cell line available from ATCC under accession number CCI61 as well
as cell lines CHO-DXB11 and CHO-DG44.
[0044] Other suitable cell lines include, without limitation, Rat
Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC
CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC
1469 (ATCC CCL 9.1); DUKX cells (CHO cell line) (Urlaub and Chasin,
Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980) (DUKX cells also
being referred to as DXB11 cells), and DG44 (CHO cell line) (Cell,
33: 405, 1983, and Somatic Cell and Molecular Genetics 12: 555,
1986). Also useful are 3T3 cells, Namalwa cells, myelomas and
fusions of myelomas with other cells. In some embodiments, the
cells may be mutant or recombinant cells, such as, e.g., cells that
express a qualitatively or quantitatively different spectrum of
enzymes that catalyze post-translational modification of proteins
(e.g., glycosylation enzymes such as glycosyl transferases and/or
glycosidases, or processing enzymes such as propeptides) than the
cell type from which they were derived. DUKX cells (CHO cell line)
are especially preferred.
[0045] Currently preferred cells are HEK293, COS, Chinese Hamster
Ovary (CHO) cells, Baby Hamster Kidney (BHK) and myeloma cells, in
particular Chinese Hamster Ovary (CHO) cells.
[0046] Modified circulatory half life: Administration of Factor
VIII antibodies according to the present invention alone or in
combination with therapeutic Factor VIII molecules may result in a
prolonged circulatory half life of endogenous Factor VIII or a
prolonged half life of endogenous Factor FVIII in combination with
therapeutic Factor VIII, or a prolonged half life of therapeutic
Factor VIII. Circulatory half life is preferably increased at least
10%, preferably at least 15%, preferably at least 20%, preferably
at least 25%, preferably at least 30%, preferably at least 35%,
preferably at least 40%, preferably at least 45%, preferably at
least 50%, preferably at least 55%, preferably at least 60%,
preferably at least 65%, preferably at least 70%, preferably at
least 75%, preferably at least 80%, preferably at least 85%,
preferably at least 90%, preferably at least 95%, preferably at
least 100%, more preferably at least 125%, more preferably at least
150%, more preferably at least 175%, more preferably at least 200%,
and most preferably at least 250% or 300%. Even more preferably,
such molecules have a circulatory half life that is increased at
least 400%, 500%, 600%, or even 700%.
[0047] Side chain/side group: Factor VIII Side groups may comprise
a hydrophilic polymer such as e.g. a PEG molecule, molecules of a
mainly hydrophobic nature, such as e.g. fatty acids, molecules of
peptidic origin, etc. Side groups are usually conjugated to Factor
VIII via a linker. Such conjugated Factor VIII molecules may be
co-administered with Factor VIII antibodies according to the
invention.
[0048] Pharmaceutical composition: A pharmaceutical composition is
herein preferably meant to encompass compositions comprising Factor
VIIII antibodies according to the present invention optionally in
combination with Factor VIII molecules suitable for parenteral
administration, such as e.g. ready-to-use sterile aqueous
compositions or dry sterile compositions that can be reconstituted
in e.g. water or an aqueous buffer. The compositions according to
the invention may comprise various pharmaceutically acceptable
excipients, stabilizers, etc.
[0049] Additional ingredients in such compositions may include
wetting agents, emulsifiers, antioxidants, bulking agents, tonicity
modifiers, chelating agents, metal ions, oleaginous vehicles,
proteins (e.g., human serum albumin, gelatine or proteins) and a
zwitterion (e.g., an amino acid such as betaine, taurine, arginine,
glycine, lysine and histidine). Such additional ingredients, of
course, should not adversely affect the overall stability of the
pharmaceutical formulation of the present invention. Parenteral
administration may be performed by subcutaneous, intramuscular,
intraperitoneal or intravenous injection by means of a syringe,
optionally a pen-like syringe. Alternatively, parenteral
administration can be performed by means of an infusion pump. A
further option is a composition which may be a solution or
suspension for the administration of the FVIII antibody compound in
the form of a nasal or pulmonal spray. As a still further option,
the pharmaceutical compositions containing the FVIII compound of
the invention may also be adapted to transdermal administration,
e.g. by needle-free injection or from a patch, optionally an
iontophoretic patch, or transmucosal, e.g. buccal,
administration.
[0050] The term "treatment", as used herein, refers to the medical
therapy of any human or other animal subject in need thereof. Said
subject is expected to have undergone physical examination by a
medical practitioner, who has given a tentative or definitive
diagnosis which would indicate that the use of said specific
treatment is beneficial to the health of said human or other animal
subject. The timing and purpose of said treatment may vary from one
individual to another, according to the status quo of the subject's
health. Thus, said treatment may be prophylactic, palliative,
symptomatic and/or curative.
LIST OF EMBODIMENTS
[0051] The present invention includes the following non-limiting
embodiments:
Embodiment 1
[0052] A monoclonal Factor VIII antibody having the ability to bind
to activated human Factor VIII, wherein said antibody, upon binding
to activated Factor VIII, reduces dissociation of the A2 domain,
and wherein said antibody does not interfere with vWF binding.
Embodiment 2
[0053] An antibody according to embodiment 1, wherein the reduction
of A2 subunit association occurs in the absence or presence of a
phospholipid surface.
Embodiment 3
[0054] An antibody according to embodiment 1, wherein the reduction
of A2 subunit dissociation from the activated Factor VIII molecule
is improved in the presence of a phospholipid surface.
Embodiment 4
[0055] An antibody according to embodiment 2 or 3, wherein
administration of this antibody results in increased thrombin
activation in the presence of platelets.
Embodiment 5
[0056] A monoclonal antibody according to any one of embodiments
1-4, wherein said antibody does not accellerate thrombin
activation.
Embodiment 6
[0057] An antibody according to any one of embodiments 1-5, wherein
said antibody binds to the A2 domain.
Embodiment 7
[0058] An antibody according to any one of embodiments 1-5, wherein
said antibody binds to the A3 domain.
Embodiment 8
[0059] An antibody according to any one of embodiments 1-6,
wherein, wherein said antibody comprises one, two, three, four, or
five CDR sequences having at least 95% identity, more preferably at
least 96% identity, more preferably at least 97% identity with,
more preferably at least 98% identity with, more preferably at
least 99% identity with, or most preferably 100% identity with one,
two, three, four, or five of the CDR sequences selected from the
list consisting of: SEQ ID NO: 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID
NO 11, SEQ ID NO 12, and SEQ ID NO 13 or the list consisting of:
SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 21, SEQ ID NO
22, and SEQ ID NO 23.
Embodiment 9
[0060] An antibody according to embodiment 8, wherein the CDR
sequences of said antibody have at least 95% identity, preferably
at least 96% identity, preferably at least 97% identity, preferably
at least 98% identity, preferably at least 99% identity, and most
preferably 100% identity with the following CDR sequences: SEQ ID
NO: 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 11, SEQ ID NO 12, and
SEQ ID NO 13 or SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO
21, SEQ ID NO 22, and SEQ ID NO 23
Embodiment 10
[0061] An antibody according to any one of embodiments 8-9, wherein
said antibody comprises a VL sequence having at least 95% identity,
preferably at least 96%, preferably at least 97% identity,
preferably at least 98% identity, and most preferably 100% identity
with SEQ ID NO 10 or SEQ ID NO 20 and a VH sequence having at least
least 95% identity, preferably at least 96%, preferably at least
97% identity, preferably at least 98% identity, and most preferably
100% identity with SEQ ID NO 9 OR SEQ ID NO 15.
Embodiment 11
[0062] An antibody according to any one of embodiments 1-10,
wherein said antibody binds to an epitope identical with or
overlapping with the peptide fragment 407-428 (SEQ ID NO 15) and/or
591-602 (SEQ ID NO 16).
Embodiment 12
[0063] An antibody according to any one of embodiments 1-10,
wherein said antibody binds to an epitope identical with or
overlapping the peptide fragment 1965-1976 (SEQ ID NO 17).
Embodiment 13
[0064] An antibody according to any of embodiments 1-12, wherein
said antibody competes with binding to the 4F143 antibody.
Embodiment 14
[0065] A DNA molecule comprising a DNA sequence encoding an
antibody according to any one of embodiments 1-13. Optionally this
DNA molecule is embedded in an expression vector.
Embodiment 14A
[0066] A host cell comprising the DNA molecule according to
embodiment 14.
Embodiment 15
[0067] Use of an antibody according to any one of embodiments 1-13
as a medicament for treatment of haemophilia A, such as mild,
moderate, or severe haemophilia A.
Embodiment 16
[0068] A pharmaceutical composition comprising an antibody
according to any one of embodiments 1-13 and optionally a
pharmaceutically acceptable excipient.
Embodiment 17
[0069] A pharmaceutical composition comprising an antibody
according to any one of embodiments 1-13 and a Factor VIII molecule
and optionally a pharmaceutically acceptable excipient. The
pharmaceutical composition according to any one of embodiments 16
or 17 may be for subcutaneous administration.
Embodiment 18
[0070] A method of making an antibody according to any one of
embodiments 1-13, wherein said method comprises incubation of a
host cell comprising a DNA molecule encoding such antibody under
conditions suitable for expressing said antibody.
Embodiment 19
[0071] A method of treatment of a hemophiliac disease comprising
administering to a patient in need thereof a therapeutically
effective amount of a molecule according to any one of embodiments
1-13, optionally in combination with a Factor VIII molecule. The
molecule according to any one of embodiments 1-13 may be in the
form of a pharmaceutical composition according to embodiment 16 or
17.
EXAMPLES
[0072] Proteins--B-domain deleted factor VIII (FVIII) was prepared
recombinantly in chinese hamster ovary (CHO) cells as described
elsewhere (Thim et al., 2010). Recombinant hirudin (Rydel et al.,
1990) was cloned into pET-26b(+) (Novagen, San Diego, Calif.) and
purified following periplasmic expression in Escherichia coli via
the introduced LeuGln(His).sub.6-tag using standard nickel
nitrilo-triacetic acid (Ni-NTA) chromatography.
Example 1
[0073] Antibody Production--Monoclonal antibodies (mAbs) of the
present invention can be produced by a variety of techniques,
including conventional monoclonal methodology e.g., the standard
somatic cell hybridization technique of Kohler and Milstein (1975)
Nature 256:495. Although somatic cell hybridization procedures are
prefered, in principle, other techniques for production of
monoclonal antibody can be employed e.g., viral or oncogenic
transformation of B lymphocytes, phage display techniques using
libraries of human or other species (mouse, rabbit, rat, guinea
pig) antibody genes.
[0074] RBF, Balb/c, NMRICF1 or FVIII deficient mice were used for
immunizations and production of mouse monoclonal antibodies. As
antigen for immunization FVIII was used either pre-activated with
thrombin or on the pro-cofactor form. Injections were made
subcutaneously in the back of the mice. FVIII (20 pg) was mixed
with complete Freund's adjuvant for the first injection. In the
subsequent immunizations, incomplete Freund's adjuvant was used
with same concentration of the antigen. Ten days after the last
immunization, eye-blood from mice was screened, using ELISA, for
FVIII specific antibodies. Mice with positive serum titres were
boosted with 10 pg of the FVIII variant used for initial
immunization by intravenous injection and sacrificed after three
days. The spleens were removed aseptically and dispersed to a
single cell suspension. Fusion of spleen cells and myeloma cells
(FOX, X63, SP2/0) was done by the PEG-method or by
electrofusion.
[0075] Monoclonal antibodies were purified by means of protein A
affinity chromatography.
[0076] Detection of Stabilizing Anti-FVIIIa Antibodies in Hybridoma
Supernatants (Primary Screen)--The ability of anti-FVIIIa
antibodies to stabilize FVIIIa was evaluated in a functional
chromogenic primary screening assay as follows: 30 .mu.l of
anti-FVIII supernatants were transferred to 96-well Spectramax
microtiter plates followed by 20 .mu.l of 1.04 nM FVIII.
Subsequently, 20 .mu.l of 14 nM thrombin (Roche, Germany) were
added and incubated for 5 minutes at room temperature allowing
FVIII to be activated. After incubation thrombin was inactivated by
adding 20 .mu.l containing 50 ATU/ml hirudin and 162.5 .mu.M 25:75
PS:PC phospholipids (Rossix, Sweden). Activated FVIII was then
allowed to dissociate for 7.5 minutes at room temperature followed
by quantification of remaining FVIIIa activity. To this end a 40
.mu.l-mixture of 1.3 nM FIXa and 162.5 nM FX (Enzyme Research, USA)
was added and incubated for 5 minutes at room temperature followed
by addition of 100 .mu.l of the FXa substrate S-2765 at 920 .mu.M
(Chromogenix, Sweden). Following 5 min incubation at room
temperature 25 .mu.l 1 M citric acid (Merck, Germany), pH 3, were
added to stop the reaction. The absorbance at 405 nm was measured
on an Envision plate reader (PerkinElmer, USA) with absorbance at
620 nm used as reference wavelength. Three medium controls were
included in the assay: one with no dissociation time (max activity)
and two, with a 7.5-min dissociation time (minimum activity) and
three, with a 7.5-min dissociation time and with FVIII replaced by
buffer (background). The two first control samples defined the
assay window and the third control was subtracted from all
measurements. The data in FIG. 1 demonstrate the ability of the
anti-FVIII supernatants to stabilize FVIII against spontaneous
disassociation.
[0077] Detection of Stabilizing Anti-FVIIIa Antibodies in Hybridoma
Supernatants (Secondary Screen)--Anti-FVIIa supernatants from the
primary screen were rescreened in a secondary time course assay to
evaluate their effect on FVIIIa decay at several time points and at
two antibody concentrations. The assay was performed as follows: 15
or 30 .mu.l of anti-FVIII supernatant were transferred to 96-well
Spectramax microtiter plates followed by 20 .mu.l of 1.04 nM FVIII.
Thrombin (20 .mu.l of 14 nM; Roche, Germany) was added and
incubated for 5 min at room temperature allowing FVIII to be
activated. Following activation, thrombin was inactivated by adding
20 .mu.l containing 50 ATU/ml hirudin and 162.5 .mu.M 25:75 PS:PC
phospholipids (Rossix, Sweden). Activated FVIII was then allowed to
dissociate for 7.5, 15, and 25 min at room temperature. Remaining
FVIIIa activity was measured by the addition of a 40 .mu.l-mixture
of 1.3 nM FIXa and 162.5 nM FX (Enzyme Research, USA) and incubated
for 5 min at room temperature followed by addition of 100 .mu.l 920
.mu.M S-2765 chromogenic FXa substrate S-2765 (Chromogenix,
Sweden). Five minutes later 25 .mu.l 1 M citric acid (Merck,
Germany), pH 3, were added to stop the reaction. The absorbance at
405 nm was measured on an Envision plate reader (PerkinElmer, USA)
with absorbance at 620 nm used as reference wavelength. A medium
control was included in the assay to verify the dependence of FVIII
in the assay. The control had a dissociation time of 7.5 minutes
and buffer was added instead of FVIII.
[0078] Characterization of Purified Anti-FVIII mAbs in Functional
Chromogenic Screening Assay--Purified anti-FVIIIa antibodies were
tested in a time course assay at different concentrations and in
the presence or absence of phospholipid to evaluate their effect on
the kinetics FVIIIa decay as well as dependence on the presence of
phospholipid and antibody concentration. The assay was performed as
follows: 30 .mu.l of purified anti-FVIII antibody were transferred
to 96-well Spectramax microtiter plates followed by 20 .mu.l of
1.04 nM FVIII (phospholipid independent) or alternatively 20 .mu.l
containing 1.04 nM FVIII and 162.5 .mu.M 25:75 PS:PC phospholipids
(Rossix, Sweden)(phospholipid dependent). Thrombin (20 .mu.l of 14
nM; Roche, Germany) was added and incubated for 5 min at room
temperature allowing FVIII to be activated. After the incubation
time thrombin was inactivated by adding 20 .mu.l 50 ATU/ml hirudin.
Activated FVIII was then allowed to dissociate for 7.5, 15, 25
minutes at room temperature. Remaining FVIIIa activity was measured
by the addition a 40-.mu.l mixture of 1.3 nM FIXa and 162.5 nM FX
(Enzyme Research, USA)(phospholipid dependent) or alternative a
40-.mu.l mixture of 1.3 nM FIXa, 162.5 nM FX (Enzyme Research, USA)
and 81.25 .mu.M 25:75 PS:PC phospholipids (Rossix,
Sweden)(phospholipid in dependent) and incubated for 5 min at room
temperature followed by addition of 100 .mu.l 920 .mu.M S-2765
chromogenic FXa substrate (Chromogenix, Sweden). After 5 min at
room temperature 25 .mu.l 1 M citric acid (Merck, Germany), pH 3,
were added to stop the reaction. The absorbance at 405 nm was
measured on an Envision plate reader (Perkin Elmer, USA) with
absorbance at 620 nm used as reference wavelength. A medium control
was included in the assay to verify the dependence of FVIII in the
assay. The control had a dissociation time of 7.5 minutes and
buffer was added instead of FVIII. The data in FIG. 2 demonstrate
the ability of the anti-FVIII supernatants to stabilize FVIII
against spontaneous disassociation over incubation times of 7.5,
15, and 25 minutes and that all the observed stabilization effects
are FVIII dependent.
Example 2
[0079] Epitope Binning of Antibodies--Antibodies were assigned to
epitope bins by performing competition binding to FVIII using a
tandem blocking assay (Abdiche et al., 2009) on a Biacore 3000
instrument (GE Healtcare, Uppsala, Sweden). The assay consisted of
three steps encompassing oriented capture of FVIII on the chip by
virtue of a immobilized non-interfering antibody (4F30) recognizing
the C2-domain followed by consecutive binding of primary and
secondary antibodies each at 200 nM to ensure saturation of FVIII.
Overlapping epitopes were observed as an inability of secondary
antibodies to bind following primary antibody binding and used to
group antibodies into epitope bins.
[0080] FVIII capture antibody (4F30) at 50 .mu.g/ml in 10 mM
acetate buffer, pH 5.0 was immobilized in flow cells 1 and 2 of a
CM5 chip using standard NHS/EDC coupling chemistry as described by
the manufacturer (GE Healthcare, Uppsala, Sweden). The final
coupling level was 10 kRU. Subsequent binding experiments were
performed at 25.degree. C. and a flow rate of 5 p/min in running
buffer (10 mM HEPES, 150 mM NaCl, 5 mM CaCl.sub.2, 0.005% Tween 20,
pH 7.4) using flow cell 1 for online reference subtraction. FVIII
was captured at a level of 400 RU by injecting 4 nM across flow
cell 2 for 2 min. This was followed by a 3-min exposure to 200 nM
primary antibody inject across both flow cells and finally an
identical injection of 200 nM secondary antibody. Regeneration was
performed at the end of each binding experiment by a 2-min pulse of
10 mM glycine, pH 2.0. The entire process was repeated for all
pairwise permutations of the antibodies listed in Table 1 except
for 4F136 which could not be used as primary antibody due to
insufficient FVIII affinity.
[0081] Based on these cross-competition studies, antibodies could
be grouped in two epitope bins denoted class 1 and class 2. Members
belonging to class 1 were 4F143, 4F50, 4F140, and 4F136, while
class 2 was represented by 4F11, 4F41, and 4F17 (Table 1). No
competition between antibodies across the two classes were
observed, whereas members within each class were mutually exclusive
with respect to FVIII binding indicating partially or completely
overlapping epitopes.
TABLE-US-00002 TABLE 1 Pairwise blocking results for antibodies
binding to immobilized FVIII in a Biacore 3000 instrument.
Following capture of FVIII to the chip using an immobilized
non-interfering antibody recognizing the C2- domain primary and
secondary antibodies were bound consecutively at 200 nM each to
ensure FVIII saturation. Antibodies fall in two epitope bins
denoted class 1 and class 2. No competition between antibodies
across the two classes were observed, whereas members within each
class were mutually exclusive with respect to FVIII binding
indicating partially or completely overlapping epitopes. Secondary
antibody Class 1 Class 2 4F143 4F50 4F140 4F136 4F11 4F41 4F17 Pri-
4F143 C C C C N N N mary 4F50 C C C C N N N anti- 4F140 C C C C N N
N body 4F136 -- -- -- -- -- -- -- 4F11 N N N N C C C 4F41 N N N N C
C C 4F17 N N N N C C C Abbreviations: `C`, competition i.e. no
binding of the secondary antibody; `N`, no competition, i.e.
binding of the secondary antibody; `--`, not tested due to poor
affinity.
Example 3
[0082] Affinity for FVIII--The kinetics of FVIII binding to
antibody was determined by surface plasmon resonance using a
Biacore 3000 instrument. Each antibody was captured to a level of
70-110 RU in flow cell 2 of a CM5 chip coated with rabbit
anti-mouse IgG antibody (GE Healthcare, Uppsala, Sweden) as
described by the manufacturer. Kinetic analysis was performed at
25.degree. C. at a flow rate of 30 .mu.l/min in running buffer
using flow cell 1 as reference. Serial two-fold dilutions of FVIII
from 0 to 40 nM were analyzed. Following 3-min equilibration of the
flow cells in running buffer, 150 .mu.l FVIII were injected. The
dissociation phase lasted 9 min and regeneration was performed with
a 3-min pulse of 10 mM glycine, pH 1.7. The obtained
reference-subtracted sensorgrams fitted well to a 1:1 Langmuir
binding model which allowed for estimation of association
(k.sub.on) and dissociation (k.sub.off) rate constants and the
equilibrium dissociation constant (K.sub.d=k.sub.off/k.sub.on)
using BIAevaluation 4.1 software (GE Healthcare, Uppsala,
Sweden).
TABLE-US-00003 TABLE 2 Surface plasmon resonance analysis of the
kinetics of FVIII binding to select antibodies. The listed
antibodies were captured by immobilized rabbit anti-mouse IgG
antibody and binding to FVIII were tested in concentrations ranging
from 0 to 40 nM. Binding curves fitted well to a 1:1 Langmuir
binding isotherm which provided estimates of association (k.sub.on)
and dissociation (k.sub.off) rate constants. The dissociation
constrant K.sub.d was calculated as k.sub.off/k.sub.on. Standard
errors obtained from the fits are shown. k.sub.on k.sub.off K.sub.d
(.times.10.sup.5 M.sup.-1s.sup.-1) (.times.10.sup.-4 s.sup.-1) (nM)
4F143 2.44 .+-. 0.04 8.49 .+-. 0.02 3.5 4F50 1.90 .+-. 0.06 10.60
.+-. 0.04 5.6 4F140 2.41 .+-. 0.19 8.07 .+-. 0.93 3.3
Example 4
[0083] Stabilization of FVIIIa--The effect of antibody on the
spontaneous dissociation of FVIIIa was measured in a functional
decay assay essentially as described elsewhere (Fay et al., 1996;
Parker et al., 2006). Activation of FVIII (0.36 nM) in a volume of
200 .mu.l was performed by combining with 20 .mu.l human
alpha-thrombin (American Diagnostica, Stamford, Conn., USA) to a
final thrombin concentration of 40 nM. Following 30 sec of
activation, 20 .mu.l recombinant hirudin (300 nM) were added to
inhibit thrombin and generated FVIIIa was allowed to decay for
defined periods. Residual FVIIIa was quantified by measuring its
ability to support conversion of FX into FXa. FVIIIa decay mixture
(20 .mu.l) was transferred to 60 .mu.l plasma-derived FIXa
(American Diagnostica) containing 25:75 PS:PC phospholipid vesicles
(Haematologic Technologies Inc., Essex Junction, VT, USA) to
assemble Xase complexes and following 15 sec incubation 20 .mu.l
human plasma-derived FX (Enzyme Research Laboratories, South Bend,
Ind., USA) were added. Final concentrations during FX activation
were 10 nM (FIXa), 25 .mu.M (phospholipid), and 150 nM (FX),
respectively. FX activation was allowed to proceed for 30 sec
before the reaction was terminted by dilution into an equal volume
of quench buffer (20 mM HEPES, 150 mM NaCl, 200 mM EDTA, 10 mM
Triton X-100, pH 7.4) and generated FXa was measured in the
presence of 0.4 mM S-2765 chromogenic substrate by measuring the
increase in absorbance at 405 nm over time (Chromogenix,
Instrumentation Laboratory Company, Bedford, Mass., USA). All
experiments were performed at room temperature in 20 mM HEPES, 150
mM NaCl, 5 mM CaCl.sub.2, 5 mg/ml BSA, pH 7.4 buffer in 96-well
plates (Nunc, Denmark) and with shaking to ensure rapid mixing.
Where indicated 10 .mu.M phospholipid and/or 20 nM antibody were
added together with FVIII or hirudin, or FVIII was replaced with
the variant FVIII S289L which has been shown to spontaneously
dissociate approximately 4-fold faster than wt FVIII upon
activation (Pipe et al., 2001).
[0084] Results from these experiments (FIG. 3) demonstrate that
4F143 and the other class 1 antibodies slow the rate of spontaneous
FVIIIa dissociation by a mechanism that is strictly dependent on
the presence of a phospholipid surface. Pre-association of FVIII
and antibody before thrombin activation is not required for
stabilization. Partial to complete rescue of the FVIII S289L
variant is observed. Similar rates of FX activation at the first
time point in the presence or absence of antibody indicate that the
antibody does not affect the rate of FX conversion to FXa under the
chosen experimental conditions.
Example 5
[0085] Effect of Antibody on the Interaction of FVIII with vWF--The
effect of antibody on the binding of FVIII to von Willebrand factor
(vWF) was studied by a solid-phase competition assay in which wells
coated with vWF were exposed to FVIII at different added antibody
concentrations (Layet et al., 1992; Ganz et al., 1991; Vlot et al.,
1995). Nunc MaxiSorp microtiterplate wells (Nunc, Denmark) were
coated with 1 .mu.g/ml of vWF (FVIII-free vWF from American
Diagnostica) in 20 mM Imidazole, 150 mM NaCl, 10 mM CaCl2, pH 7.3
overnight at 4.degree. C. and then blocked for 1 hour with the same
buffer supplemented with 10 mg/ml bovine serum albumin and 0.02%
(v/v) Tween 80 (blocking buffer). Coated wells were incubated for 1
hour at room temperature with 100 .mu.l FVIII diluted in blocking
buffer to concentrations ranging from 0.05 to 6.4 nM in the
presence of 0-162 nM antibody; the highest concentrations
significantly exceeding the measured K.sub.d (see Table 2) for the
FVIII-antibody interaction. After repeated washing with blocking
buffer, 3.33 nM biotinylated monoclonal anti-FVIII antibody 1F5
recognizing the 720-740 region was added in a volume of 100 .mu.l
blocking buffer and allowed to incubate for 15 min. Wells were
washed and peroxidase-conjugated streptavidin (xx) was added in 100
.mu.l blocking buffer and allowed to bind to residing biotin for 15
min. After repeated washing with blocking buffer, bound FVIII was
quantified as the amount of TMB (100 .mu.l TMB Plus, KEM-EN-TEC
Diagnostics, Denmark) processed by perioxidase. The reaction was
stopped after 5 minutes by the addition of an equal volume of 2 M
phosphoric acid and the amount of product formed was quantified by
absorbance at 450 nm in a SpectraMax plate reader.
[0086] As shown in FIG. 4 none of the antibodies tested (4F143,
4F140 or 4F50) affected the interaction of FVIII with vWF even at
concentrations ensuring essentially complete saturation of FVIII
with antibody.
Example 6
[0087] Effect of Antibody on the Activation of FVIII by
Thrombin--Conversion of FVIII to the activated cofactor occurs by
limited proteolysis at three sites in the heavy and light chain
catalyzed by thrombin or factor Xa, and with the former most likely
representing the physiologic activator (Pieters et al., 1989).
Cleavage at R1689 in the light chain liberates the acidic a3 region
and causes the dissociation of FVIII from vWF. Cleavage of the
heavy chain occurs in the interdomainal regions at the A2-B
junction (R740) and the A1-A2 junction (Arg372), respectively.
Proteolysis at the latter site is essential for FVIII to gain
co-factor activity and can be monitored by the generation of the
50-kDa A1 subunit (Fay, 2004; Nogami et al., 2005). Recently an
anti-FVIII antibody was described that accelerated the proteolytic
activation of FVIII (Takeyama et al., 2010)(US 20090297503). In
addition we find that the well-known monoclonal anti-FVIII
antibodies ESH5 and ESH8 originally described by (Griffin et al.,
1986) and available from American Diagnostica Inc. (Stamford,
Conn., USA) also accelerate FVIII activation by thrombin. To
determine the effect of antibodies from the present invention on
the kinetics of FVIII activation, a proteolytic assay was used that
monitors A1 subunit generation by reversed-phase HPLC. This
particular assay was chosen in favor of a traditional functional
assays quantifying FVIIIa activity as a function of time to avoid
any confounding effects arising from the antibody-mediated
stabilization of FVIIIa against spontaneous decay.
[0088] Activation of FVIII (100 nM) by 1 nM thrombin (Haematologic
Technologies, Essex Junction, VT, USA) was performed in 20 mM
HEPES, 150 mM NaCl, 5 mM CaCl2, 0.01% (v/v) Tween 80, pH 7.4 buffer
at 37.degree. C. At defined intervals activation was quenched by
addition of 200 nM hirudin. Quenched samples were cooled on ice and
then analyzed by rpHPLC to quantify the amount of generated light
chain. Time-course studies demonstrated that the addition of
hirudin effectively prevented further activation of FVIII.
[0089] The FVIII light chain was quantified by injection of 10-20
.mu.l onto a Vydac C.sub.18 column (3.2.times.250 mm, 5 .mu.m, 300
.ANG.) in 34% solvent B. Mobile phases consisted of water
containing 0.09% (v/v) trifluoroacetic acid (solvent A) and
acetonitril containing 0.09% (v/v) trifluoroacetic acid (solvent
B). Separation was achieved by a linear gradient from 34 to 65%
solvent B over 15 min at a flow rate of 1 ml/min. The column was
maintained at 40.degree. C. and eluting FVIIIa subunits were
detected and quantified by fluorescence with excitation at 280 nm
and emission at 340 nm. Peak areas were converted to molar
concentrations based on a standard curve generated by injection of
defined amounts of FVIIIa prepared by thrombin activation. The peak
representing the A1 subunit was identified from the elution times
of the isolated FVIIIa subunits prepared according to published
procedures (Lapan and Fay, 1997).
[0090] As demonstrated in FIG. 5, ESH5 and ESH8 (American
Diagnostica Inc, Stamford, Conn., USA) were found to accelerate the
activation of FVIII by thrombin. Similarly, moAb216 were found to
accelerate FVIII activation in agreement with published studies
(Takeyama et al., 2010)(US 20090297503), whereas no acceleration
was observed for 4F143, 4F50, and 4F140.
Example 7
[0091] Effect of antibody on thrombin generation in haemophilia A
plasma--Washed platelets were prepared as described (Lisman et al.,
2005) and added to haemophilia A plasma (George King Bio-Medical
Inc) to a final density of 150,000 platelets/.mu.l. Eighty .mu.l of
the platelet-containing plasma was mixed with 5 .mu.l relipidated
tissue factor (Innovin, Dade, final dilution 1:50000 corresponding
to approx 0.12 pM tissue factor) in microtiter wells and preheated
10 min at 37.degree. C. in a Flouroskan Ascent plate reader (Thermo
Electron Corporation). Wild type FVIII or variants (2.7; 0.9, 0.3;
0.1; 0.033; 0.011; 0.0037 and 0.0012 nM final concentration) or
wild type FVIII co-formulated with 50 nM 4F143 antibody was added
in 15 .mu.l. Fluorogenic substrate (Z-Gly-GlyArg-AMC, Bachem, final
concentration 417 nM) mixed with CaCl.sub.2 (final concentration
16.7 mM) was added in 20 .mu.l before measuring fluorescence
(excitation at 390 nm and emission at 460 nm) continuously for one
hour. The fluorescence signal was corrected for
.alpha.2-macroglobulinbound thrombin activity and converted to
thrombin concentration by use of a calibrator and Thrombinoscope
software (Synapse BV) as described (Hemker et al., 2003). The
maximal level of thrombin activity (Table 3) obtained with 0.011 nM
FVIII was measured by the Thrombinoscope software. The the maximal
rate of thrombin generation was calculated from the parameters
obtained from the Thrombinoscope software, as follows: Maximal rate
of thrombin generation=maximal level of thrombin activity/(time to
peak thrombin activity--lagtime). Both parameters of thrombin
generation show that the antibody 4F143 enhanced the thrombin
generation of 0.1 nM FVIII.
TABLE-US-00004 TABLE 3 Parameters of thrombin generation obtained
by 0.01 nM FVIII with or without 4F143 added. Data for the
destabilized FVIII S289L variants are included. The data shows mean
.+-. standard error of the mean (SEM) of 5 individual experiments.
Both parameters demonstrate increased thrombin generation when
FVIII is combined with 4F143. Rate of Maximal level of thrombin
generation thrombin generation fold- fold- nM/min increase* nM
increase* FVIII 1.2 .+-. 0.4 1 29.7 .+-. 7.0 1 FVIII + 4F143 2.0
.+-. 0.9 1.8 50.3 .+-. 15.7 1.7 FVIII S289L 0.5 .+-. 0.1 0.39 18.0
.+-. 4.7 0.61 *compared to FVIII
Example 8
[0092] Epitope Mapping by HX-MS of FVIIIa Stabilizing mAbs on
FVIII--The HX-MS technology exploits that hydrogen exchange (HX) of
a protein can readily be followed by mass spectrometry (MS). By
replacing the aqueous solvent containing hydrogen with aqueous
solvent containing deuterium, incorporation of a deuterium atom at
a given site in a protein will give rise to an increase in mass of
1 Da. This mass increase can be monitored as a function of time by
mass spectrometry in quenched samples of the exchange reaction. The
deuterium labelling information can be sub-localized to regions in
the protein by pepsin digestion under quench conditions and
following the mass increase of the resulting peptides.
[0093] One use of HX-MS is to probe for sites involved in molecular
interactions by identifying regions of reduced hydrogen exchange
upon protein-protein complex formation. Usually, binding interfaces
will be revealed by marked reductions in hydrogen exchange due to
steric exclusion of solvent. Protein-protein complex formation may
be detected by HX-MS simply by measuring the total amount of
deuterium incorporated in either protein members in the presence
and absence of the respective binding partner as a function of
time. The HX-MS technique uses the native components, i.e. protein
and antibody or Fab fragment, and is performed in solution. Thus
HX-MS provides the possibility for mimicking the in vivo conditions
(for a recent review on the HX-MS technology, see Wales and Engen,
Mass Spectrom. Rev. 25, 158 (2006)).
[0094] Instrumentation and data recording--All proteins were buffer
exchanged into 20 mM Imidazole, 10 mM CaCl.sub.2, 150 mM NaCl,
adjusted to pH 7.3 before experiments. The HX experiments were
automated by a Leap robot (H/D-x PAL; Leap Technologies Inc.)
operated by the LeapShell software (Leap Technologies Inc.), which
performed initiation of the deuterium exchange reaction, reaction
time control, quench reaction, injection onto the UPLC system and
digestion time control. The Leap robot was equipped with two
temperature controlled stacks maintained at 20.degree. C. for
buffer storage and HX reactions and maintained at 2.degree. C. for
storage of protein and quench solution, respectively. The Leap
robot furthermore contained a cooled Trio VS unit (Leap
Technologies Inc.) holding the pepsin-, pre- and analytical
columns, and the LC tubing and switching valves at 1.degree. C. The
switching valves have been upgraded from HPLC to Microbore UHPLC
switch valves (Cheminert, VICI AG). For the inline pepsin
digestion, 100 .mu.L quenched sample containing 0.15 pmol FVIII was
loaded and passed over a Poroszyme.RTM. Immobilized Pepsin
Cartridge (2.1.times.30 mm (Applied Biosystems)) using a isocratic
flow rate of 200 .mu.L/min (0.1% formic acid:CH.sub.3OH 95:5). The
resulting peptides were trapped and desalted on a VanGuard
pre-column BEH C18 1.7 .mu.m (2.1.times.5 mm (Waters Inc.)).
Subsequently, the valves were switched to place the pre-column
inline with the analytical column, UPLC-BEH C18 1.7 .mu.m
(2.1.times.100 mm (Waters Inc.)), and the peptides separated using
a 9 min gradient of 15-40% B delivered at 150 .mu.L/min from an
AQUITY UPLC system (Waters Inc.). The mobile phases consisted of A:
0.1% formic acid in water and B: 0.1% formic acid in CH.sub.3CN.
The ESI MS data, and the elevated energy (MS.sup.E) experiments
were acquired in positive ion mode using a Q-Tof Premier MS (Waters
Inc.). Leucine-enkephalin was used as the lock mass ([M+H].sup.+
ion at m/z 556.2771) and data was collected in continuum mode.
[0095] Data Analysis--Peptic peptides were identified in separate
experiments using MSE methods (Waters Inc.). MSE data were
processed using BiopharmaLynx 1.2 (version 017). HX-MS raw data
files were subjected to continuous lockmass-correction. Data
analysis, i.e., centroid determination of deuterated peptides and
plotting of in-exchange curves, was performed using HX-Express
((Version Beta); Weis et al., J. Am. Soc. Mass Spectrom. 17, 1700
(2006)).
[0096] Epitope Mapping of 4F143 and 4F41--Amide hydrogen/deuterium
exchange (HX) was initiated by preparation of FVIII solutions in a
concentration of 30 .mu.M in the absence or presence of either
4F143 or 4F41 into the corresponding deuterated buffer, i.e., 20 mM
Imidazole, 10 mM CaCl2, 150 mM NaCl, prepared in D2O, 98% D2O
final, pH 7.3 (uncorrected value)). All HX reactions were carried
out at 20.degree. C. and contained 3 .mu.M FVIII in the absence or
presence of excess FVIII mAbs (4.5 uM) to ensure saturation of
FVIII with antibody. At appropriate time intervals ranging from 10
sec to 2 hours 46 min 40 s (10.000 s), aliquots of the HX reaction
were quenched by an equal volume of ice-cold quenching buffer 1.35M
TCEP (Tris(2-Carboxyethyl)-Phosphine Hydrochloride
(Calbiochem.RTM., EMD Chemicals inc.))) resulting in a final pH of
2.6 (uncorrected value). An example of raw data identifying the
4F143 epitope is shown in FIG. 6A.
[0097] 4F143 Epitope--The deuterium incorporation rate (HX
time-course) of 412 peptides, covering 82% of the primary sequence
of FVIII, were monitored in the presence and absence of 4F143 at 8
time points, i.e., 10 s, 30 s, 100 s, 300 s, 1.000 s, 3,000 s, and
10,000 s (FIG. 6A, FIG. 7, FIG. 8).
[0098] The observed exchange pattern in the presence or absence of
4F143 may be divided into two groups: One group of peptides display
an exchange pattern that is unaffected by the binding of 4F143
(FIGS. 7 (aa 392-403 and 429-436)), which comprises 98.2% of the
peptides. In contrast, another group of FVIII peptic peptides show
protection from exchange upon complex formation with 4F143 (FIG.
7), which includes 1.7% of the peptic peptides. For example at 30 s
exchange with D2O, approximately 1 amide is protected from exchange
in the region aa 407-428 upon 4F413 binding (FIG. 6A, FIG. 7). Two
regions were found to display protection upon 4F143 binding, one
region includes 5 peptic peptides covering the residues aa 407-428,
414-428, 415-428, 416-428 and 406-431, and an additional region
includes 2 peptic peptide covering the residues aa 591-602 and
593-597. The two epitope regions are both found within the A2
subdomain of FVIII.
[0099] Comparison of the relative amounts of exchange protection by
overlapping peptides enabled to narrow the affected regions of
FVIII upon complex formation with 4F143 to be found within the
sequence aa 407-428 and 591-602 (using mature numbering).
[0100] The relative exchange protection rate was determined for the
peptic petides included in the two epitopes regions by comparing HX
results of free FVIII vs FVIII in complex formation with 4F143.
[0101] For the epitope region within the sequence aa 407-428 the
relative exchange protection identifled for the peptides covering
residues aa 414-428, 415-428, 416-428 was found to be at a
comparable level and approximately 50% of the relative level
determined for the peptides covering residue aa 406-431,
407-428.
[0102] For the epitope region within the sequence aa 591-602 the
relative exchange protection identified for the peptide covering
residues aa 593-597 was found to be approximately 40% of the
relative proction level determined for the peptide covering
residues aa 591-602.
[0103] The two epitope regions covering the sequence aa 407-428 and
591-602 are found to be in structural close proximity when docking
onto the published crystal structure of FVIII Ngo, Jacky Chi Ki;
Huang, Mingdong; Roth, David A.; Furie, Barbara C.; Furie, Bruce.
Crystal Structure of Human Factor VIII: Implications for the
Formation of the Factor IXa-Factor Villa Complex. Structure
(Cambridge, Mass., United States) (2008), 16(4), 597-606.
[0104] 4F41 Epitope--The HX time-course of 412 peptides, covering
82% of the primary sequence of FVIII, were monitored in the
presence and absence 4F41 at 8 time points, i.e., 10 s, 30 s, 100
s, 300 s, 1.000 s, 3.000 s, and 10.000 s (FIG. 6B, FIG. 9, FIG.
10).
[0105] The observed exchange pattern in the presence or absence of
4F41 may be divided into two groups; one group of peptides displays
an exchange pattern that is unaffected by the binding of 4F41 (FIG.
9), which comprises 99.3% of the peptides; a second group shows
protection from exchange upon complex formation with 4F41 (FIG. 9),
which includes 0.7% of the peptic peptides.
[0106] The study of overlapping peptic peptides enabled the
sublocalization of the identified epitope region to be confined
within the sequence aa 1965-1970 (using mature numbering), which is
found in domain A3 of FVIII. Three peptides were identified to show
a significant lowered deuterium incorporation level detectable for
short incubation times, i.e., 10 s and 30 s. This clearly indicates
them to be situated within the epitope. These peptides covered the
sequence aa 1963-1972, 1963-1974, 1965-1976, respectively.
Example 9
[0107] Cloning and Sequencing of Mouse Anti-FVIII 4F143 and 4F50
Monoclonal Antibodies--This example describes cloning and
sequencing of the murine heavy chain and light chain sequences of
anti-FVIII antibody 4F143. Total RNA was extracted from hybridoma
cells using the RNeasy-Mini Kit from Qiagen and used as template
for cDNA synthesis. cDNA was synthesized in a 5'-RACE reaction
using the SMARTer.TM. RACE cDNA amplification kit from Clontech.
Subsequent target amplification of HC and LC sequences was
performed by PCR using Phusion Hot Start polymerase (Finnzymes) and
the universal primer mix (UPM) included in the SMARTer.TM. RACE kit
as forward primer. A reverse primer with the following sequence was
used for HC (VH domain) amplification:
TABLE-US-00005 (SEQ ID NO: 3) 5'-CCCTTGACCAGGCATCCCAG-3'
[0108] A reverse primer with the following sequence was used for LC
amplification:
TABLE-US-00006 (SEQ ID NO: 4)
5'-GCTCTAGACTAACACTCATTCCTGTTGAAGCTCTTG-3'
[0109] PCR products were separated by gel electrophoresis,
extracted using the GFX PCR DNA & Gel Band Purification Kit
from GE Healthcare Bio-Sciences and cloned for sequencing using a
Zero Blunt TOPO PCR Cloning Kit and chemically competent TOP10 E.
coli (Invitrogen). Colony PCR was performed on selected colonies
using an AmpliTaq Gold Master Mix from Applied Biosystems and
M13uni/M13rev primers. Colony PCR clean-up was performed using the
ExoSAP-IT enzyme mix (USB). Sequencing was performed at MWG
Biotech, Martinsried Germany using M13uni(-21)/M13rev(-29)
sequencing primers. Sequences were analyzed and annotated using the
VectorNTI program. All kits and reagents were used according to the
manufacturer's instructions.
Anti-FVIII 4F143
[0110] A single unique murine kappa type LC and a single unique
murine HC, subclass IgG1 was identified. Nucleic acid and amino
acid sequences are listed below, the leader peptide sequences are
not included.
[0111] Anti-FVIIIa 4F143 VH amino acid sequence (SEQ ID NO: 5)
(signal peptide sequence omitted, CDR1 (SEQ ID NO 6), CDR2 (SEQ ID
NO: 7), and CDR3 (SEQ ID NO: 8), respectively, are underlined):
TABLE-US-00007 1 QIQFVQSGPE LKKPGETVKI SCKASGYTFT NYGMNWVKQA
PGKGLKWMGW 51 INSYTGEPTY ADDFKGRFAF SLETSASTAY LQINNLKNED
TATYFCARGA 101 SYAMDYWGQG TSVTVSS
[0112] Anti-FVIIIa 4F143 VH nucleic acid sequence (SEQ ID NO: 9)
(signal peptide sequence omitted):
TABLE-US-00008
5'-CAGATCCAGTTCGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAA-
GATCTCCTGCAAGGCTTCTGGTTATACCTTCACAAACTATGGAATGAACTGGGTGAA-
GCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACTCCTACACTGGA-
GAGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAG-
CACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAA-
GAGGGGCTTCTTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA
[0113] Anti-FVIIIa 4F143 VL amino acid sequence (SEQ ID NO: 10)
(signal peptide sequence omitted, CDR1 (SEQ ID NO: 11), CDR2 (SEQ
ID NO 12), and CDR3 (SEQ ID NO: 13), respectively, are
underlined):
TABLE-US-00009 1 DVQITQSPSY LAASPGETIT INCRASKSIS KYLAWYQEKP
VKTNKLLIYS 51 GSTLQSGIPS RFSGSGSGTD FTLTISSLEP EDFAMYYCQQ
HYEYPLTFGA 101 GTKLELKR
[0114] Anti-FVIIIa 4F143 VL nucleic acid sequence (signal peptide
sequence omitted) (SEQ ID NO: 14):
TABLE-US-00010
5'-GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAAC-
CATTACTATTAATTGCAGGGCAAGTAAGAGCATTAGCAAATATTTAGCCTGGTATCAAGA-
GAAACCTGTGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACTTTGCAATCTG-
GAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAG-
TAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAACAGCATTATGAA-
TACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG
[0115] anti-FVIII 4F50
[0116] A single unique murine kappa type LC and a single unique
murine HC, subclass IgG1 was identified. Nucleic acid and amino
acid sequences are listed below, the leader peptide sequences are
not included.
[0117] Anti-FVIIIa 4F50 VH amino acid sequence (SEQ ID NO: 15)
(signal peptide sequence omitted, CDR1 (SEQ ID NO 16), CDR2 (SEQ ID
NO: 17), and CDR3 (SEQ ID NO: 18), respectively, are
underlined):
TABLE-US-00011 1 QIQFVQSGPE LKKPGETVKI SCKASGYTFT NYGMNWVKQA
PGKGLKWMGW 51 INSYTGEPTY ADDFKGRFDF SLETSASTAY LQINNLKNED
TATYFCARGA 101 SYAMDHWGQG TSVTVSS
[0118] Anti-FVIIIa 4F50 VH nucleotide sequence (SEQ ID NO: 19)
(signal peptide sequence omitted)
TABLE-US-00012
5'-CAGATCCAGTTCGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAA-
GATCTCCTGCAAGGCTTCTGGTTATACCTTCACAAACTATGGAATGAACTGGGTGAA-
GCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACTCCTACACTGGA-
GAGCCAACATATGCTGATGACTTCAAGGGACGGTTTGACTTCTCTTTGGAAACCTCTGCCAG-
CACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAA-
GAGGGGCTTCTTATGCTATGGACCACTGGGGTCAAGGAACCTCTGTCACCGTCTCCTCA
[0119] Anti-FVIIIa 4F50 VL amino acid sequence (SEQ ID NO: 20)
(signal peptide sequence omitted, CDR1 (SEQ ID NO: 21), CDR2 (SEQ
ID NO 22), and CDR3 (SEQ ID NO: 23), respectively, are
underlined):
TABLE-US-00013 1 DVQITQSPSY LAASPGETIS INCRASKSIS KYLAWYQEKP
VKTNKLLIYS 51 GSTLQSGIPS RFSGSGSGTD FTLTISSLEP EDFAMYYCQQ
HYEYPLTFGA 101 GTKLELKR
[0120] Anti-FVIIIa 4F50 VL nucleotide sequence (SEQ ID NO: 24)
(signal peptide sequence omitted)
TABLE-US-00014
5'-GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAACCATTAG-
TATTAATTGCAGGGCAAGTAAGAGCATTAGCAAATATTTAGCCTGGTATCAAGAGAAAC-
CTGTGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACTTTGCAATCTG-
GAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAG-
TAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAACAGCATTATGAA-
TACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG
Sequence CWU 1
1
2412332PRThomo sapiens 1Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu
Leu Ser Trp Asp Tyr 1 5 10 15 Met Gln Ser Asp Leu Gly Glu Leu Pro
Val Asp Ala Arg Phe Pro Pro 20 25 30 Arg Val Pro Lys Ser Phe Pro
Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45 Thr Leu Phe Val Glu
Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60 Arg Pro Pro
Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val 65 70 75 80 Tyr
Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90
95 Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala
100 105 110 Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp
Lys Val 115 120 125 Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val
Leu Lys Glu Asn 130 135 140 Gly Pro Met Ala Ser Asp Pro Leu Cys Leu
Thr Tyr Ser Tyr Leu Ser 145 150 155 160 His Val Asp Leu Val Lys Asp
Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175 Leu Val Cys Arg Glu
Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190 His Lys Phe
Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205 His
Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215
220 Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg
225 230 235 240 Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val
Tyr Trp His 245 250 255 Val Ile Gly Met Gly Thr Thr Pro Glu Val His
Ser Ile Phe Leu Glu 260 265 270 Gly His Thr Phe Leu Val Arg Asn His
Arg Gln Ala Ser Leu Glu Ile 275 280 285 Ser Pro Ile Thr Phe Leu Thr
Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300 Gln Phe Leu Leu Phe
Cys His Ile Ser Ser His Gln His Asp Gly Met 305 310 315 320 Glu Ala
Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335
Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340
345 350 Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser
Phe 355 360 365 Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr
Trp Val His 370 375 380 Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr
Ala Pro Leu Val Leu 385 390 395 400 Ala Pro Asp Asp Arg Ser Tyr Lys
Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415 Gln Arg Ile Gly Arg Lys
Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430 Asp Glu Thr Phe
Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445 Leu Gly
Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460
Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile 465
470 475 480 Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly
Val Lys 485 490 495 His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile
Phe Lys Tyr Lys 500 505 510 Trp Thr Val Thr Val Glu Asp Gly Pro Thr
Lys Ser Asp Pro Arg Cys 515 520 525 Leu Thr Arg Tyr Tyr Ser Ser Phe
Val Asn Met Glu Arg Asp Leu Ala 530 535 540 Ser Gly Leu Ile Gly Pro
Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp 545 550 555 560 Gln Arg Gly
Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575 Ser
Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585
590 Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe
595 600 605 Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe
Asp Ser 610 615 620 Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr
Trp Tyr Ile Leu 625 630 635 640 Ser Ile Gly Ala Gln Thr Asp Phe Leu
Ser Val Phe Phe Ser Gly Tyr 645 650 655 Thr Phe Lys His Lys Met Val
Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670 Phe Ser Gly Glu Thr
Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685 Ile Leu Gly
Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700 Leu
Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 705 710
715 720 Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn
Ala 725 730 735 Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro
Ser Thr Arg 740 745 750 Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu
Asn Asp Ile Glu Lys 755 760 765 Thr Asp Pro Trp Phe Ala His Arg Thr
Pro Met Pro Lys Ile Gln Asn 770 775 780 Val Ser Ser Ser Asp Leu Leu
Met Leu Leu Arg Gln Ser Pro Thr Pro 785 790 795 800 His Gly Leu Ser
Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe 805 810 815 Ser Asp
Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser 820 825 830
Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val 835
840 845 Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu
Gly 850 855 860 Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys
Val Ser Ser 865 870 875 880 Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro
Ser Asp Asn Leu Ala Ala 885 890 895 Gly Thr Asp Asn Thr Ser Ser Leu
Gly Pro Pro Ser Met Pro Val His 900 905 910 Tyr Asp Ser Gln Leu Asp
Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915 920 925 Leu Thr Glu Ser
Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp 930 935 940 Ser Lys
Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp 945 950 955
960 Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys
965 970 975 Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu
Phe Lys 980 985 990 Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser
Asn Asn Ser Ala 995 1000 1005 Thr Asn Arg Lys Thr His Ile Asp Gly
Pro Ser Leu Leu Ile Glu 1010 1015 1020 Asn Ser Pro Ser Val Trp Gln
Asn Ile Leu Glu Ser Asp Thr Glu 1025 1030 1035 Phe Lys Lys Val Thr
Pro Leu Ile His Asp Arg Met Leu Met Asp 1040 1045 1050 Lys Asn Ala
Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr 1055 1060 1065 Thr
Ser Ser Lys Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly 1070 1075
1080 Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe Lys
1085 1090 1095 Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile Gln Arg
Thr His 1100 1105 1110 Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro
Ser Pro Lys Gln 1115 1120 1125 Leu Val Ser Leu Gly Pro Glu Lys Ser
Val Glu Gly Gln Asn Phe 1130 1135 1140 Leu Ser Glu Lys Asn Lys Val
Val Val Gly Lys Gly Glu Phe Thr 1145 1150 1155 Lys Asp Val Gly Leu
Lys Glu Met Val Phe Pro Ser Ser Arg Asn 1160 1165 1170 Leu Phe Leu
Thr Asn Leu Asp Asn Leu His Glu Asn Asn Thr His 1175 1180 1185 Asn
Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr 1190 1195
1200 Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr Val Thr
1205 1210 1215 Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser
Thr Arg 1220 1225 1230 Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr
Ala Pro Val Leu 1235 1240 1245 Gln Asp Phe Arg Ser Leu Asn Asp Ser
Thr Asn Arg Thr Lys Lys 1250 1255 1260 His Thr Ala His Phe Ser Lys
Lys Gly Glu Glu Glu Asn Leu Glu 1265 1270 1275 Gly Leu Gly Asn Gln
Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys 1280 1285 1290 Thr Thr Arg
Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr 1295 1300 1305 Gln
Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu 1310 1315
1320 Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp Asp Thr Ser Thr
1325 1330 1335 Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr
Leu Thr 1340 1345 1350 Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala
Ile Thr Gln Ser 1355 1360 1365 Pro Leu Ser Asp Cys Leu Thr Arg Ser
His Ser Ile Pro Gln Ala 1370 1375 1380 Asn Arg Ser Pro Leu Pro Ile
Ala Lys Val Ser Ser Phe Pro Ser 1385 1390 1395 Ile Arg Pro Ile Tyr
Leu Thr Arg Val Leu Phe Gln Asp Asn Ser 1400 1405 1410 Ser His Leu
Pro Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly Val 1415 1420 1425 Gln
Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu 1430 1435
1440 Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln Arg Glu
1445 1450 1455 Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr
Tyr Lys 1460 1465 1470 Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp
Leu Pro Lys Thr 1475 1480 1485 Ser Gly Lys Val Glu Leu Leu Pro Lys
Val His Ile Tyr Gln Lys 1490 1495 1500 Asp Leu Phe Pro Thr Glu Thr
Ser Asn Gly Ser Pro Gly His Leu 1505 1510 1515 Asp Leu Val Glu Gly
Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile 1520 1525 1530 Lys Trp Asn
Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg 1535 1540 1545 Val
Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp 1550 1555
1560 Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys Glu
1565 1570 1575 Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala
Phe Lys 1580 1585 1590 Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys
Glu Ser Asn His 1595 1600 1605 Ala Ile Ala Ala Ile Asn Glu Gly Gln
Asn Lys Pro Glu Ile Glu 1610 1615 1620 Val Thr Trp Ala Lys Gln Gly
Arg Thr Glu Arg Leu Cys Ser Gln 1625 1630 1635 Asn Pro Pro Val Leu
Lys Arg His Gln Arg Glu Ile Thr Arg Thr 1640 1645 1650 Thr Leu Gln
Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile 1655 1660 1665 Ser
Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp 1670 1675
1680 Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr
1685 1690 1695 Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met
Ser Ser 1700 1705 1710 Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser
Gly Ser Val Pro 1715 1720 1725 Gln Phe Lys Lys Val Val Phe Gln Glu
Phe Thr Asp Gly Ser Phe 1730 1735 1740 Thr Gln Pro Leu Tyr Arg Gly
Glu Leu Asn Glu His Leu Gly Leu 1745 1750 1755 Leu Gly Pro Tyr Ile
Arg Ala Glu Val Glu Asp Asn Ile Met Val 1760 1765 1770 Thr Phe Arg
Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser 1775 1780 1785 Leu
Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 1790 1795
1800 Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys
1805 1810 1815 Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp
Cys Lys 1820 1825 1830 Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu
Lys Asp Val His 1835 1840 1845 Ser Gly Leu Ile Gly Pro Leu Leu Val
Cys His Thr Asn Thr Leu 1850 1855 1860 Asn Pro Ala His Gly Arg Gln
Val Thr Val Gln Glu Phe Ala Leu 1865 1870 1875 Phe Phe Thr Ile Phe
Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu 1880 1885 1890 Asn Met Glu
Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu 1895 1900 1905 Asp
Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly 1910 1915
1920 Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln
1925 1930 1935 Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu
Asn Ile 1940 1945 1950 His Ser Ile His Phe Ser Gly His Val Phe Thr
Val Arg Lys Lys 1955 1960 1965 Glu Glu Tyr Lys Met Ala Leu Tyr Asn
Leu Tyr Pro Gly Val Phe 1970 1975 1980 Glu Thr Val Glu Met Leu Pro
Ser Lys Ala Gly Ile Trp Arg Val 1985 1990 1995 Glu Cys Leu Ile Gly
Glu His Leu His Ala Gly Met Ser Thr Leu 2000 2005 2010 Phe Leu Val
Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala 2015 2020 2025 Ser
Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr 2030 2035
2040 Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser
2045 2050 2055 Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile
Lys Val 2060 2065 2070 Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile
Lys Thr Gln Gly 2075 2080 2085 Ala Arg Gln Lys Phe Ser Ser Leu Tyr
Ile Ser Gln Phe Ile Ile 2090 2095 2100 Met Tyr Ser Leu Asp Gly Lys
Lys Trp Gln Thr Tyr Arg Gly Asn 2105 2110 2115 Ser Thr Gly Thr Leu
Met Val Phe Phe Gly Asn Val Asp Ser Ser 2120 2125 2130 Gly Ile Lys
His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr 2135 2140 2145 Ile
Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg 2150 2155
2160 Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu
2165 2170 2175 Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr
Ala Ser 2180 2185 2190 Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser
Pro Ser Lys Ala 2195 2200 2205 Arg Leu His Leu Gln Gly Arg Ser Asn
Ala Trp Arg Pro Gln Val 2210 2215 2220 Asn Asn Pro Lys Glu Trp Leu
Gln Val Asp Phe Gln Lys Thr Met 2225 2230 2235 Lys Val Thr Gly Val
Thr Thr
Gln Gly Val Lys Ser Leu Leu Thr 2240 2245 2250 Ser Met Tyr Val Lys
Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly 2255 2260 2265 His Gln Trp
Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe 2270 2275 2280 Gln
Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp 2285 2290
2295 Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp
2300 2305 2310 Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys
Glu Ala 2315 2320 2325 Gln Asp Leu Tyr 2330 221PRTARTIFICIALAmino
acid sequence of truncated FVIII B domain in "N8" 2Ser Phe Ser Gln
Asn Ser Arg His Pro Ser Gln Asn Pro Pro Val Leu 1 5 10 15 Lys Arg
His Gln Arg 20 320DNAartificialprimer 3cccttgacca ggcatcccag
20436DNAartificialPrimer 4gctctagact aacactcatt cctgttgaag ctcttg
365117PRTartificial4F143 VH amino acid sequence 5Gln Ile Gln Phe
Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30
Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35
40 45 Gly Trp Ile Asn Ser Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp
Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr
Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Gly Ala Ser Tyr Ala Met Asp
Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115
65PRTartificial4F143 CDR1 amino acid sequence 6Asn Tyr Gly Met Asn
1 5 717PRTartificial4F143 CDR2 amino acid sequence 7Trp Ile Asn Ser
Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15 Gly
88PRTartificial4F143 CDR3 amino acid sequence 8Gly Ala Ser Tyr Ala
Met Asp Tyr 1 5 9351DNAartificial4F143 VH DNA sequence 9cagatccagt
tcgtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60tcctgcaagg
cttctggtta taccttcaca aactatggaa tgaactgggt gaagcaggct
120ccaggaaagg gtttaaagtg gatgggctgg ataaactcct acactggaga
gccaacatat 180gctgatgact tcaagggacg gtttgccttc tctttggaaa
cctctgccag cactgcctat 240ttgcagatca acaacctcaa aaatgaggac
acggctacat atttctgtgc aagaggggct 300tcttatgcta tggactactg
gggtcaagga acctcagtca ccgtctcctc a 35110108PRTartificial4F143 VL
amino acid sequence 10Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu
Ala Ala Ser Pro Gly 1 5 10 15 Glu Thr Ile Thr Ile Asn Cys Arg Ala
Ser Lys Ser Ile Ser Lys Tyr 20 25 30 Leu Ala Trp Tyr Gln Glu Lys
Pro Val Lys Thr Asn Lys Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr
Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu
Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Tyr Glu Tyr Pro Leu 85 90
95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105
1111PRTartificial4F143 CDR1 amino acid sequence 11Arg Ala Ser Lys
Ser Ile Ser Lys Tyr Leu Ala 1 5 10 127PRTartificial4F143 CDR2 amino
acid sequence 12Ser Gly Ser Thr Leu Gln Ser 1 5
139PRTartificial4F143 CDR3 13Gln Gln His Tyr Glu Tyr Pro Leu Thr 1
5 14324DNAartificial4F143 VL DNA sequence 14gatgtccaga taacccagtc
tccatcttat cttgctgcat ctcctggaga aaccattact 60attaattgca gggcaagtaa
gagcattagc aaatatttag cctggtatca agagaaacct 120gtgaaaacta
ataagcttct tatctactct ggatccactt tgcaatctgg aattccatca
180aggttcagtg gcagtggatc tggaacagat ttcactctca ccatcagtag
cctggagcct 240gaagattttg caatgtatta ctgtcaacag cattatgaat
acccgctcac gttcggtgct 300gggaccaagc tggagctgaa acgg
32415117PRTartificial4F50 VH amino acid sequence 15Gln Ile Gln Phe
Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30
Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35
40 45 Gly Trp Ile Asn Ser Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp
Phe 50 55 60 Lys Gly Arg Phe Asp Phe Ser Leu Glu Thr Ser Ala Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr
Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Gly Ala Ser Tyr Ala Met Asp
His Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115
165PRTartificial4F50 VH CDR1 amino acid sequence 16Asn Tyr Gly Met
Asn 1 5 1716PRTartificial4F50 VH CDR2 amino acid sequence 17Ile Asn
Ser Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys Gly 1 5 10 15
188PRTartificial4F50 VH CDR3 18Gly Ala Ser Tyr Ala Met Asp His 1 5
19351DNAartificial4F50 VH DNA sequence 19cagatccagt tcgtgcagtc
tggacctgag ctgaagaagc ctggagagac agtcaagatc 60tcctgcaagg cttctggtta
taccttcaca aactatggaa tgaactgggt gaagcaggct 120ccaggaaagg
gtttaaagtg gatgggctgg ataaactcct acactggaga gccaacatat
180gctgatgact tcaagggacg gtttgacttc tctttggaaa cctctgccag
cactgcctat 240ttgcagatca acaacctcaa aaatgaggac acggctacat
atttctgtgc aagaggggct 300tcttatgcta tggaccactg gggtcaagga
acctctgtca ccgtctcctc a 35120108PRTartificial4F50 VL amino acid
sequence 20Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser
Pro Gly 1 5 10 15 Glu Thr Ile Ser Ile Asn Cys Arg Ala Ser Lys Ser
Ile Ser Lys Tyr 20 25 30 Leu Ala Trp Tyr Gln Glu Lys Pro Val Lys
Thr Asn Lys Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr Leu Gln Ser
Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala
Met Tyr Tyr Cys Gln Gln His Tyr Glu Tyr Pro Leu 85 90 95 Thr Phe
Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105
2111PRTartificial4F50 VL CDR1 21Arg Ala Ser Lys Ser Ile Ser Lys Tyr
Leu Ala 1 5 10 229PRTartificial4F50 VL CDR2 amino acid sequence
22Gln Gln His Tyr Glu Tyr Pro Leu Thr 1 5 239PRTartificial4F50 VL
CDR3 23Gln Gln His Tyr Glu Tyr Pro Leu Thr 1 5
24324DNAartificial4F50 VL CDR3 amino acid sequence 24gatgtccaga
taacccagtc tccatcttat cttgctgcat ctcctggaga aaccattagt 60attaattgca
gggcaagtaa gagcattagc aaatatttag cctggtatca agagaaacct
120gtgaaaacta ataagcttct tatctactct ggatccactt tgcaatctgg
aattccatca 180aggttcagtg gcagtggatc tggaacagat ttcactctca
ccatcagtag cctggagcct 240gaagattttg caatgtatta ctgtcaacag
cattatgaat acccgctcac gttcggtgct 300gggaccaagc tggagctgaa acgg
324
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