A polynucleotide, comprising a contiguous nucleotide sequence coding for a human antibody with factor VIII specificity, or complementary to a nucleotide sequence coding for a human antibody for factor VIII specificity, or capable of selectively hybridizing under stringent conditions to such nucleotide sequence. Such polynucleotide may be used as a probe or primer for detection of factor VIII inhibitors, or be used for producing a recombinant polypeptide. A polypeptide, comprising a contiguous amino acid sequence corresponding to or mimicking a fragment or derivative of a human antibody with factor VIII specificity capable of specific binding to factor VIII. An antibody, comprising a recombinant human antibody with factor VIII specificity or an anti-idiotypic antibody directed against a human antibody with factor VIII specificity. Pharmaceutical compositions which contain such polypeptide or antibody.

Description of the Invention

SUMMARY OF THE INVENTION

This invention relates to methods for diagnosis and treatment using inhibitory antibodies directed against factor VIII. Methods are disclosed that show how to arrive at nucleotide and amino acid sequences that encode factor VIII specific antibodies. This invention discloses diagnostic tests that allow for detection of nucleotide and amino acid sequences that encode factor VIII specific antibodies within a heterogeneous mixture of antibody-encoding nucleotide or amino acid sequences. This invention further discloses how to use recombinant antibody fragments which bind specifically to factor VIII as novel therapeutic agents for the treatment of patients with factor VIII inhibitors.

The invention provides a polynucleotide in substantially isolated form, comprising a contiguous nucleotide sequence (a) coding for a human antibody with factor VIII specificity, or (b) complementary to a nucleotide sequence coding for a human antibody with factor VIII specificity, or (c) capable of selectively hybridizing under stringent conditions to nucleotide sequence (a) or (b).

Preferably, the contiguous nucleotide sequence is at least 8, preferably at least 10 nucleotides.

In a preferred embodiment, the invention provides a probe or primer which comprises a polynucleotide as defined herein, optionally further comprising a detectable label, such as a radioactive atom or group, an enzyme, a fluorescent or luminescent group, a dye or biotin.

The invention also provides an assay kit for detecting nucleic acid coding for a human antibody with factor VIII specificity, comprising a probe or primer as defined herein in a suitable container.

Furthermore, the invention provides a nucleic acid amplification and detection kit for detecting nucleic acid coding for a human antibody with factor VIII specificity, comprising a pair of primers as defined herein capable of priming the synthesis of cDNA, and optionally further comprising a probe as defined herein capable of selectively hybridizing to (the complement of) a region of the nucleic acid to be detected between and not including the sequences from which the primers are derived.

The invention provides a method for assaying a sample for the presence or absence of nucleic acid coding for a human antibody with factor VIII specificity, comprising contacting the sample with a probe as defined herein under conditions that allow the selective hybridization of said probe to the (complement of the) nucleic acid to be detected in the sample, and determined whether polynucleotide duplexes comprising said probe are formed.

The invention also provides a method for assaying a sample for the presence or absence of nucleic acid coding for a human antibody with factor VIII specificity, comprising subjecting nucleic acid present in the sample to a nucleic acid amplification process using a pair of primers as defined herein capable of priming the synthesis of cDNA, contacting the nucleic acid resulting from the amplification process with a probe as defined herein under conditions that allow the selective hybridization of said probe to the (complement of the) nucleic acid to be detected in the sample, and determining whether polynucleotide duplexes comprising said probe are formed.

Furthermore, the invention provides a method of producing a recombinant polypeptide, comprising providing a polynucleotide coding for said polypeptide, preparing a recombinant vector containing said polynucleotide operably linked to a control sequence capable of providing for the expression of the polynucleotide by a host cell, transforming a host cell with said recombinant vector, growing said host cell under conditions that provide for the expression of the polynucleotide and optionally isolating the thus produced polypeptide, wherein said polynucleotide codes for a human antibody with factor VIII specificity, or a fragment or derivative thereof capable of specific binding to factor VIII.

According to another aspect, the invention provides a polypeptide in substantially isolated form, comprising a contiguous amino acid sequence corresponding to or mimicking a fragment or derivative of a human antibody with factor VIII specificity capable of specific binding to factor VIII. In a preferred embodiment of the invention, the contiguous amino acid sequence is capable of reducing the activity of factor VIII inhibiting antibodies.

Preferably, the fragment is (part of) a variable region of the heavy chain or light chain of said antibody, and the derivative is preferably a single chain Fv fragment of said antibody.

The invention furthermore provides an antibody in substantially isolated form, comprising a recombinant human antibody with factor VIII specificity.

The invention furthermore provides a pharmaceutical composition for the treatment of factor VIII inhibition in a human individual, comprising a polypeptide as defined herein or an antibody as defined herein, together with a pharmaceutically acceptable carrier. Optionally, the composition further contains factor VIII, or a substitute of factor VIII.

The invention also provides a method of treatment of factor VIII inhibition in a human individual comprising administering (an effective amount to reduce or prevent said factor VIII inhibition of) a polypeptide as defined herein or an antibody as defined herein, optionally together with factor VIII or a substitute of factor VIII.

DETAILED DESCRIPTION OF THE INVENTION

A number of investigators have addressed the epitope-specificity and mode of action of factor VIII inhibitory antibodies. Molecular cloning of the factor VIII cDNA revealed that factor VIII consists of a series of repeated domains which appear in the order A1-A2-B-A3-C1-C2. In plasma, factor VIII circulates as a heterodimer which consists of a heavy chain of variable length (90-220 kDa) and a light chain of 80 kDa. The factor VIII light chain consists of the domains A3-C1-C2 while the factor VIII heavy chain comprises the domains A1-A2-B. Heterogeneity of the factor VIII heavy chain is caused by limited proteolysis within the B-domain which contains several sites that are sensitive towards proteolytic cleavage. In plasma, factor VIII circulates in complex with von Willebrand factor, a large multimeric protein involved in the initial steps of platelet adhesion to a damaged vessel wall. Binding to von Willebrand factor protects factor VIII from proteolytic degradation. The physiological importance of this interaction is underscored by the low levels of factor VIII in plasma of patients that lack von Willebrand factor. Factor VIII is a precursor molecule which upon activation functions as a cofactor for factor IXa in the phospholipid and Ca.sup.2+-dependent conversion of factor X to factor Xa. Activation of factor VIII involves proteolytic cleavages in both the heavy and light chain of factor VIII. Thrombin is considered to be the physiological activator of factor VIII and cleaves at Arg.sup.372, Arg.sup.740 and Arg.sup.1689 of factor VIII. Thrombin activated factor VIII thus consists of a hetero-trimer of the separate A1 and A2-domains and the cleaved factor VIII light chain (A3-C1-C2). Cleavage at Arg.sup.1689 of the factor VIII light chain results in removal of amino-acid sequence Glu.sup.1649-Arg.sup.1689 which is essential for binding of factor VIII to von Willebrand factor. Sofar, three major binding sites for factor VIII inhibitors have been characterized (Scandella et al. 1994, Blood 86: 1811-1819; Healey et al. 1995, J. Biol. Chem. 270: 14505-14509; Fijnvandraat et al. 1998, Blood 91: 2347-2352).

Amino acid residues Val.sup.2248-Ser.sup.2312 in the C2-domain constitute a binding site for factor VIII inhibitors. The large size of this epitope suggests that a number of antibodies which bind to different amino acid regions in this area occur in plasma of patients with inhibitors of C2-specificity. The mechanisms of action of anti-C2 antibodies has been explored in considerable detail. Most of these antibodies interfere with binding of factor VIII to phospholipids. Furthermore, some of the antibodies with C2-specificity also inhibit the interaction of factor VIII with its carrier von Willebrand factor. A new mechanism for inhibition of factor VIII by a human alloantibody has been described recently (Saenko et al. 1996, J. Biol. Chem. 271: 27424-27431). A human alloantibody that binds only to the amino-terminal portion (Val.sup.2248-Gly.sup.2285) of the C2-epitope has been shown to inhibit the thrombin induced release of factor VIII from von Willebrand factor.

Amino acid residues Arg.sup.484-Ile.sup.508 in the A2-domain of factor VIII constitute a major epitope for factor VIII inhibitors. Studies on the mechanism of inhibition of anti-A2 antibodies have shown that anti-A2 antibodies interfere with conversion of factor X to Xa by the lipid bound factor VIIIa-factor IXa-complex (Lollar et al., 1995). The anti-A2-antibodies do not interfere with binding of factor X to the factor VIIIa-factor IXa complex but simply limit the conversion of factor X.

A third major epitope of factor VIII inhibitors has been found in the A3-domain of factor VIII. Binding of inhibitory antibodies was dependent on the presence of amino acids Gln.sup.1178-Met.sup.1823. Previous studies have shown that this site constitute a binding site for factor IXa and indeed antibodies binding to this site interfered with complex assembly of factor VIIIa and factor IXa (Fijnvandraat et al. 1998. Blood 91: 2347-2352). In a number of patients with an inhibitor, inhibitory antibodies directed against other epitopes have been observed. An early study has shown that inhibitory antibodies may recognize amino acid region Met.sup.336-Arg.sup.372 of factor VIII (Ware et al. 1998. Proc. Natl. Acad. Sci USA 85: 3165-3169). The mechanism of inhibition has not yet been explored but recently a binding site for factor X has been proposed in this part of the factor VIII molecule (Lapan, K. A. and Fay, P. J. 1997. J. Biol. Chem. 272: 2082-2088).

The restricted epitope specificity of factor VIII inhibitors suggests that a limited number of dominant B-cell epitopes is involved in the immune response to factor VIII. Apparently, human anti-factor VIII antibodies synthesized by B-cell clones from a variety of patients are surprisingly similar with respect to epitope specificity. This suggests that the primary amino acid and nucleotide sequence of antibodies with factor VIII specificity is similar at the molecular level. Based on this it is desirable to define the presence and epitope specificity of anti-factor VIII antibodies by simply addressing the presence of nucleotide sequences that correspond to antibodies with factor VIII inhibiting capacity. Sofar, the primary sequences of anti-factor VIII antibodies have been poorly defined. Davies and co-workers have suggested an association between factor VIII inhibitors and use of VH gene segment DP73 (Davies et al. 1997. Thromb. Haemostas. supplement: 2352A). The nucleotide and primary amino acid sequence of these antibodies has not been disclosed and details with respect to the epitope specificity of these antibodies are lacking. Clearly, there is a need to define the primary amino acid and nucleotide sequence of factor VIII antibodies in more detail. Such sequence information can be used to design diagnostic tests which can be used to monitor the occurrence of B-cell clones that produce factor VIII inhibitors in patients with haemophilia A. These diagnostic tests can be extremely sensitive and give information on the epitope specificity of factor VIII inhibitors.

Studies directed at defining the epitope specificity and mode of action of these antibodies are limited by the heterogeneity of these antibodies in the plasma of these patients. Clearly, more stringent diagnostic criteria would be required to define the properties of factor VIII inhibitors in more detail.

A sudden increase in the frequency of inhibitor development in a group of previously treated patients has been associated with a particular pasteurized factor VIII concentrate manufactured in the Netherlands (Roosendaal et al. 1993. Blood 81: 2180-2186). These factor VIII inhibitors are directed against the factor VIII light chain and epitope mapping revealed that the majority of inhibitors reacted with epitopes in the A3-C1 and the C2-domain of factor VIII (Sawamoto et al. 1998. Thromb. Haemostas. 79: 62-68). Recently, a second pasteurized factor VIII concentrate has been implicated in the development of inhibitors in a group of previously treated patients. Also in this case the inhibitory antibodies were predominantly of factor VIII light chain specificity (Peerlinck et al. 1997. Thromb. Haemostas. 77: 80-86). It has been suggested that inhibitor development in these patients is due to small alterations in the factor VIII molecule which have been induced by the manufacturing process. This may indicate that the antibodies that developed in these patients have different properties compared to the factor VIII inhibitory antibodies that develop in other patients. Clearly, knowledge of nucleotide and amino acid sequence of factor VIII specific antibodies could provide additional information on the etiology of factor VIII inhibitor which is desirable for the characterization of the antibody response in patients who have received these factor VIII concentrates.

Until now, the primary nucleotide and amino acid sequence of anti-factor VIII antibodies has not been disclosed. This invention describes the nucleotide sequences that encode human antibodies with factor VIII-specificity. Based on the primary sequence of these antibodies, oligonucleotide primers are designed that allow for detection of B-cells that produce antibodies with affinity for factor VIII. Detection of factor VIII specific B-cells may be accomplished using both mRNA, cDNA or DNA which are derived from lymphocytes of patients. Genomic DNA, RNA and cDNA are prepared from lymphocytes by methods that are generally known in the art. Some methods for the detection of factor VIII specific B-cell clones are listed below. Other methods for the detection of nucleotide sequences of factor VIII specific antibodies, disclosed in this invention, are considered to fall within the scope of this invention. Selective amplification of heavy chain variable sequences (VH-genes) can be used to detect nucleotide sequences that encode antibodies that are part of the human antibody repertoire that can bind specifically to factor VIII. The variable part of the human heavy chain is assembled from the variable heavy chain regions (VH), the diversity regions (D) and the joining regions (J). Fusion of these three different gene segments is not a precise event and this so-called "junctional diversity", together with the process of nucleotide addition and deletion, results in the generation of the hypervariable complementary determining region 3 CDR3. The human light chain is assembled in a similar manner but lacks diversity region D. Additional sequence diversity of both heavy and light chain sequences is generated by somatic hypermutation and together with the mechanisms outlined above this ultimately results in the generation of high affinity antibodies. Knowledge on the nucleotide sequences that encode factor VIII-specific antibody allows for the detection of this specific antibody in the repertoire of patients who are at risk of developing factor VIII-specific antibodies (such as haemophilia A patients who are treated with factor VIII or patients with acquired haemophilia). Amplification may be performed with a combination of oligonucleotide primers directed against constant regions or variable regions of heavy and light chain of factor VIII-specific antibodies. Detection of factor VIII specific antibodies may be performed using one oligonucleotide primer derived from the variable parts of the nucleotide sequences encoding factor VIII antibodies and one oligonucleotide primer that is derived from the constant regions of factor VIII specific antibodies. Detection may also be performed using two oligonucleotide primers specific for variable parts of the nucleotide sequence that encodes an antibody that binds to factor VIII. The methods described herein also include the amplification of immunoglobulin genes using oligonucleotide primers that are directed against the constant regions of the immunoglobulin genes. Subsequent detection of nucleotide sequences of factor VIII specific antibodies can be performed using selective hybridization with (radiolabelled) oligonucleotide primers that are directed against the variable parts of the nucleotide sequence encoding factor VIII specific antibodies. From the above it follows that oligonucleotide primers are preferentially but not exclusively directed towards the constant and variable regions of factor VIII specific antibodies. In example 5, methods are disclosed that can be used to detect the presence of factor VIII specific antibodies in a mixture of nucleotide sequences. Combination of oligonucleotide primers derived from the nucleotide sequence of factor VIII specific antibodies can be used to directly assess the presence of factor VIII specific antibodies in the antibody-repertoire of patients. Alternatively, analysis by methods that include but are not limited to sequencing analysis, re-amplification of obtained fragments with more specific oligonucleotide primers, digestion with restriction enzymes and selective hybridization may be utilized to address the presence of factor VIII antibodies. Quantification of the amount of nucleotide sequences encoding factor VIII antibodies may be obtained by various methods that are generally known in the art and include but are not limited to the following. The amount of radioactivity incorporated into a PCR-fragment that encodes part of a factor VIII specific antibody can be determined. Furthermore, radioactively labelled oligonucleotide probes can be used to estimate the amount of a nucleotide sequence encoding a factor VIII specific antibody in a mixture of DNA fragments that code for part of a patients antibody repertoire. Quantitative PCR-amplification can be performed using for example dye-modified oligonucleotide primers which allow for direct monitoring of the amount of PCR-product generated during amplification.

Other methods that selectively detect and quantify specific nucleotide sequences that encode factor VIII specific antibodies may be devised by an average expert in the art. These methods are considered to fall within the scope of the present invention.

Examples 1-10 provide details on the identification and detection of nucleotide sequences that encode factor VIII specific antibodies in haemophilia patients. These examples teach how to arrive at the nucleotide sequence of factor VIII inhibitors and provide information on how to use this information for the detection of factor VIII specific antibodies.

This invention discloses the nucleotide and primary amino acid sequences of factor VIII specific antibodies. Factor VIII inhibitors are commonly directed against three major epitopes on factor VIII within the A2-A3 and C2-domain of factor VIII. In Example 4 the nucleotide and amino acid sequence of anti-C2 antibodies is disclosed. In examples 8 and 9, the nucleotide and amino acid sequence of anti-A2 and anti-A3-C1 antibodies is disclosed. This invention teaches how to arrive at the nucleotide and amino acid sequence of factor VIII specific antibodies and the methods disclosed in this invention can be used to derive the nucleotide and amino acid sequence of anti-factor VIII antibodies with specificity for other domains of factor VIII which are a target for factor VIII inhibitors. Anti-factor VIII antibodies encoded by the nucleotide sequences disclosed here, can be used for the development of therapeutic agents that are capable of limiting the biological activity of factor VIII inhibitors. These therapeutic agents preferentially contain, but are not limited to: 1. The antibody fragments (or agents based on the nucleotide or amino acid sequence) can be used for the generation of anti-idiotypic antibodies. Antibody fragments can be either administered together with factor VIII or administered alone. Also, peptides or related agents which are based on the primary amino acid sequence of the variable parts of factor VIII specific antibodies can be used to induce the formation of anti-idiotypic antibodies direct against factor VIII inhibitors. Anti-idiotypic antibodies can also be obtained by screening large (semi-synthetic) libraries that encode a wide variety of recombinant antibodies. The preparation of anti-idiotypic antibodies can also take place in animals that include but are not limited to mouse by injection of recombinant antibody disclosed in this invention. Anti-idiotypic antibodies can subsequently be obtained by methods that are known to those skilled in the art. An anti-idiotypic response to factor VIII specific antibodies may also be obtained by injection of DNA encoding part of the nucleotide sequences of factor VIII specific antibodies which can be obtained by the methods outlined in this invention. Immunization by injection of DNA is considered to be only modestly immunogenic and other agents are needed to obtain a sufficiently high immune response. Co-injection of plasmid DNA encoding IL-2, GM-CSF and tetanus toxoid has been used to enhance the immune response to injected DNA (Spellerberg et al. 1997. J. Immunol. 15:1885-1892). Similar methods can be applied to enhance the immune response towards DNA fragments encoding factor VIII specific antibodies. 2. The recombinant antibody fragments described in this invention can be used as a therapeutic for treatment of patients with an inhibitor. Examples 7 and 10 disclose that recombinant antibody fragments (termed scFv's) which bind specifically to the C2-domain, interfere with binding of inhibitory antibodies to factor VIII. These scFv's can be used for treatment of patients with inhibitory antibodies directed against the C2-domain. This invention discloses how to arrive at recombinant antibody fragments that bind specifically to the A2, A3-C1 and C2-domain of factor VIII. Using the methods outlined in this invention additional antibody fragments directed against these and other regions on factor VIII may be obtained. These regions include but are not limited to Arg.sup.484-Ile.sup.508 in the A2-domain, Gln.sup.1778-Met.sup.1823 in the A3-domain and Val.sup.2248-Ser.sup.2312 in the C2-domain. Recombinant antibodies directed against multiple epitopes preferentially will be part of a pharmaceutical preparation since most patients have inhibitory antibodies directed against multiple epitopes on factor VIII. The inhibitor neutralizing activity of the recombinant antibody fragments described in this invention may be modified by the introduction of point mutations in the constant and variable parts of these fragments. Furthermore, the recombinant antibody fragments described in this invention may be cloned into vectors which allow for expression of these fragments for example as Fab-fragment. Other vectors for expression of antibodies and antibody fragments are available to an average expert in the field (see for example: "Antibody Engineering; A Practical Approach" edited by Mc Cafferty et al. 1996. Oxford University Press). Methods to increase the affinity of recombinant antibodies or antibody fragments are readily available and can be used to modulate the biological activities of the recombinant antibody fragments described in this invention. Peptides and peptide-related agents which have been designed based on the amino acid sequence of the recombinant antibodies described in this invention (for example peptides derived from the amino acid sequence of CDR3) can be used to interfere with the activity of factor VIII inhibitors.

In summary, this invention provides an improved method to define factor VIII inhibitory antibodies. The methods described result in definition of the nucleotide sequence of factor VIII inhibitors and teaches how to utilize this information for the development of improved methods for the detection of factor VIII inhibitors. This invention also discloses pharmaceutical preparations, derived from the nucleotide and/or primary amino acid sequence of factor VIII specific antibodies, that can be used in the treatment of haemophilia A patients with inhibitory antibodies directed against factor VIII. The dose of the therapeutic agents to be administered to a patient, obviously depends on the affinity of the therapeutic agent for factor VIII. The affinity of the recombinant antibody fragments described in this invention can vary considerably as is disclosed in Examples 6 and 7. The dose to be administered also depends on bodyweight of the patient, the titre of the factor VIII inhibitor and the biological activities of the different components of the therapeutic agent used. The dose to be administered can be estimated according to methods that are disclosed in Examples 7 and 10. These methods may be complemented with dose finding studies which involve administration of the therapeutic agents in animal models and healthy individuals. In general, the dose administered will vary between 10 .mu.g-5 g/kg and more preferably between 100 .mu.g-1 g/kg of bodyweight per day.

The therapeutic agent may be administered in combination with factor VIII (or a substitute of factor VIII). The dose of factor VIII administered together with the therapeutic agent may vary between 0.1 and 2000 Units per kg and more preferably between 1 and 200 Units per kg of body weight per day.
 

Claim 1 of 13 Claims

1. An isolated antibody or fragment thereof capable of specific binding to factor VIII and comprising a heavy chain variable region of a human antibody with factor VIII specificity and a light chain variable region of a human antibody, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ. ID. NO: 23, SEQ. ID. NO: 25, SEQ. ID. NO: 32, SEQ. ID. NO: 34, SEQ. ID. NO: 36, SEQ. ID. NO: 38, SEQ. ID. NO: 49, and SEQ. ID. NO: 51.

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