Therapeutic Factor Viii Antibodies

Oestergaard; Henrik ;   et al.

Patent Application Summary

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 Number20150239984 14/710182
Document ID /
Family ID43587481
Filed Date2015-08-27

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

Application Number Filing Date Patent Number
13822952 Jun 26, 2013 9062115
PCT/EP2011/065986 Sep 15, 2011
14710182
61386783 Sep 27, 2010

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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