CN117157316A

CN117157316A - Modified plasma coagulation factor VIII and methods of use thereof

Info

Publication number: CN117157316A
Application number: CN202180096784.0A
Authority: CN
Inventors: 王启钊; 于道展
Original assignee: Ainuojian Gene Therapy Co ltd
Current assignee: Ainuojian Gene Therapy Co ltd
Priority date: 2021-03-30
Filing date: 2021-03-30
Publication date: 2023-12-01
Also published as: WO2022211791A1; JP2024511851A; EP4314041A1

Abstract

The present application describes modified hum1.An factor VIII polypeptides having enhanced factor VIII activity. In some embodiments, the modified human factor VIII polypeptide comprises one or more amino acid substitutions at positions a20, T21, F57, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332, R378, I610, and/or I661. Such polypeptides and viral vectors encoding such polypeptides may be used to treat FVIII deficiency, such as hemophilia a.

Description

Modified plasma coagulation factor VIII and methods of use thereof

Technical Field

The present application relates generally to medical treatment, and in particular to modified plasma factor VIII polypeptides and their use in the treatment of hemophilia a.

Background

Hemophilia a is an X-linked recessive disease caused by a deficiency of functional plasma coagulation factor VIII (hFVIII). In hemophilia a patients, blood does not clot properly, resulting in excessive bleeding at the time of injury. The bleeding phenotype is typically associated with residual factor (residual factor) activity: people with severe disease (factor activity <1% normal) have frequent spontaneous bleeding; people with moderate disease (factor activity 1% -5% normal) rarely bleed spontaneously, but bleed with minor trauma; people with mild disease (factor activity 5% -40% normal) bleed during interventional surgery or trauma.

Current treatment of severe hemophilia a (factor VIII activity < 1%) requires periodic intravenous infusion of either recombinant factor VIII (rFVIII) or plasma concentrated factor VIII. Individuals with moderate and mild hemophilia a can be treated as desired without periodic preventive regimens. Infusion therapy is expensive and introduces a risk of infectious disease. rFVIII therapies have proven to be expensive due to the expense of production, purification and formulation. rFVIII treatment still requires intravenous administration because of the limited bioavailability of other routes of administration. The cost and limited availability of rFVIII have hampered the widespread implementation of this therapeutic strategy.

Gene therapy provides an alternative to infusion therapy. However, current gene therapies require high doses of viral vectors, which increases the costs associated with treatment. However, difficulties in implementing gene therapy techniques include vector toxicity and insufficient levels of factor VIII expression.

Thus, engineering FVIII to increase clotting activity, as demonstrated by increased secretion, increased specific activity, or both, would significantly increase rFVIII production in cell culture production or transgenic animals and increase the success potential of gene therapy strategies for hemophilia a. Thus, there is a need for improved vectors and constructs that are capable of efficiently expressing sufficient amounts of hFVIII protein to increase FVIII production or reduce the required dose of viral vectors to tolerable levels.

Disclosure of Invention

One aspect of the application relates to a modified hFVIII polypeptide (mhFVIII) comprising one or more mutations compared to a wild-type hFVIII polypeptide or a reference polypeptide.

In some embodiments, the mhFVIII comprises one or more amino acid substitutions at positions a20, T21, F57, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332, R378, I610, and/or I661.

In some embodiments, the mhFVIII comprises one or more amino acid substitutions selected from the amino acid substitutions listed in table 1.

In some embodiments, the mhFVIII comprises one or more amino acid substitutions at a position selected from a20K, T21I, T3521V, F L, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S P, R378S, I610M and I661V. In some embodiments, the mhFVIII comprises a single amino acid substitution.

In some embodiments, the mhFVIII comprises an amino acid substitution in each of amino acids a20K and T21I.

In some embodiments, the mhFVIII comprises amino acid substitutions a20K and T21V.

In some embodiments, the mhFVIII comprises the amino acid substitutions T21I, L V and I80V.

In some embodiments, the mhFVIII comprises amino acid substitutions T21I, L69V, I, 80 and L178F.

In some embodiments, the mhFVIII comprises amino acid substitutions T21I, L69V, I V and I661V.

In some embodiments, the mhFVIII comprises amino acid substitutions T21I, L69V, I80, L178F, and I661V.

In some embodiments, the mhFVIII comprises the amino acid substitutions R199K, H212Q, I215V, R269K, I310V, L318F and S332P.

In some embodiments, the mhFVIII comprises amino acid substitutions T21I, L69V, I80V, L178F, H212Q, I215V, R269K, L F and I661V.

In some embodiments, the mhFVIII comprises amino acid substitutions a20K, T21V, L69V, I V, L178F, H212Q, I215V, R269K, L F and I661V.

In some embodiments, the mhFVIII comprises amino acid substitutions T21I, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S332P and I661V.

In some embodiments, the mhFVIII comprises amino acid substitutions a20K, T21V, L69V, I80V, L F, R199K, H212Q, I215V, R269K, I310V, L F, S332P and I661V.

In some embodiments, the mhFVIII consists of a single polypeptide comprising the A1, A2, A3, C1 and C2 domains of hFVIII.

In some embodiments, the mhFVIII consists of a single polypeptide comprising: (1) A1, A2, A3, C1 and C2 domains of hFVIII; and (2) a truncated B domain of hFVIII.

In some embodiments, the mhFVIII consists of a heavy chain polypeptide comprising the A1 and A2 domains of hFVIII and a light chain polypeptide comprising the A3, C1 and C2 domains of hFVIII. In some embodiments, the heavy chain polypeptide further comprises a truncated B domain of hFVIII and a light chain polypeptide comprising the A3, C1, and C2 domains of hFVIII.

In some embodiments, the mhFVIII comprises a heavy chain of human FVIII and a light chain of FVIII from a different species, e.g., a light chain of canine FVIII.

Another aspect of the application relates to an isolated polynucleotide encoding said mhFVIII of the application.

Another aspect of the application relates to an expression cassette comprising: the polynucleotides of the application; and regulatory sequences operably linked to the polynucleotide.

Another aspect of the application relates to an expression vector comprising said polynucleotide of the application. In some embodiments, the expression vector is a plasmid. In some embodiments, the expression vector is a viral vector. In some embodiments, the expression vector is an AAV vector.

Another aspect of the application relates to a host cell comprising said expression vector of the application.

Another aspect of the application relates to a pharmaceutical composition comprising said mhFVIII of the application and a pharmaceutically acceptable carrier.

Another aspect of the application relates to a pharmaceutical composition comprising the expression vector of the application and a pharmaceutically acceptable carrier.

Another aspect of the application relates to a method of treating a subject suffering from factor VIII deficiency. The method comprises the step of administering to the subject an effective amount of the mhFVIII, the expression vector or the host cell of the application.

Another aspect of the application relates to a recombinant AAV vector comprising nucleotides encoding mhFVIII, wherein the mhFVIII comprises one or more amino acid substitutions at a position selected from a20K, T I, T21 5621V, F L, L69V, I178F, R199K, H212Q, I215V, R269K, I310 318F, S332P, R S, I610M and I661V, and wherein the AAV vector is capable of expressing the mhFVIII in a host cell. In some embodiments, the mhFVIII comprises a truncated B domain of hFVIII.

Another aspect of the application relates to a method of expressing mhFVIII. The method comprises the following steps: (a) Introducing into a host cell an expression vector comprising: a polynucleotide comprising a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding the mhFVIII, wherein the mhFVIII comprises one or more amino acid substitutions at a position selected from the group consisting of a20K, T I, T21V, F L, L69V, I80 8238 178F, R K, H212Q, I215V, R269K, I310 318F, S332P, R378S, I610M and I661V; and a regulatory sequence operably linked to the polynucleotide; (b) Allowing the host cell to grow under conditions suitable for expression and secretion of the mhFVIII; (c) Harvesting a culture medium from the host cells, and (d) purifying the mhFVIII from the harvested culture medium.

Drawings

FIG. 1 shows an expression plasmid (pANG-CAG-hBDDF 8) encoding wild-type human FVIII with a deletion in the B domain (hBDDF 8). The hbdf 8 coding sequences (including heavy and light chains) are under the control of a CAG promoter for detecting the functional activity of various modified hbdf 8-proteins (also referred to as "mutant hFVIII" or "hFVIII mutants") according to the application.

FIG. 2 shows an alignment between the hBDDF8 heavy chain (hBDDF 8-HC) and the modified hBDDF8 heavy chain (qwBDDF 8-HC) containing 17 substitution mutations at the 16 amino acid positions for analysis of hFVIII mutant activity. Other constructs comprising various single, double and multiple substitutions thereof are further described in fig. 3 and example 1. Further analysis of the relative functional activity of these mutants is shown in FIGS. 4-9, described below.

FIG. 3 summarizes exemplary substitution mutants in hBDDF8-HC for analysis of hFVIII functional activity.

Figure 4 shows the relative functional activity of single amino acid substituted hFVIII mutants (i.e. modified hfdf 8 proteins) in HuH7 cells (compared to the functional activity of hFVIII (i.e. unmodified hfdf 8 protein)).

Figure 5 shows the relative functional activity of single amino acid substituted hFVIII mutants (i.e. modified hfdf 8 proteins) in HEK 293T cells (compared to the functional activity of hFVIII (i.e. unmodified hfdf 8 proteins)).

Figure 6 shows the relative functional activity of a particular single amino acid substitution mutation in amino acid 21 of the hFVIII heavy chain (i.e., the hbdf 8 protein modified at amino acid position 21) in HEK 293T cells (compared to the functional activity of hFVIII (i.e., the unmodified hbdf 8 protein).

Figure 7 shows the relative functional activity of the double amino acid substitutions (compared to each, as well as to unmodified hbdf 8 protein and single amino acid substitution mutant T21I) containing a combination of T21I and various substitutions in amino acid 20 of the hFVIII heavy chain (i.e., hbdf 8 protein modified at amino acid position 21) in HEK 293T cells.

FIG. 8 shows the functional activity of various hFVIII-HC mutants with single or multiple mutations (as indicated) in Huh7 cells 24 hours or 48 hours post-transfection (compared to the hBDDF8 cDNA in pANG-CAG-hBDDF 8).

FIG. 9 shows the functional activity of various hFVIII-HC mutants (including those with single and multiple mutations (as indicated)) in HEK 293T cells 24, 48 and 72 hours post-transfection (compared to that of hFVIII (i.e.unmodified hBDDF8 protein).

FIG. 10 shows the functional activity of various hFVIII-HC mutants (including those with single or multiple mutations (as indicated)) in CHO cells 24 hours or 48 hours post-transfection (compared to that of hFVIII (i.e.unmodified hBDDF8 protein).

FIG. 11A shows the domains of selected hFVIII mutants (and wild-type) and human/canine hybrid FVIII mutants. Fig. 11B shows the functional activity of the B-domain-free hFVIII mutant (and wild-type) and the human/canine hybrid FVIII mutant of fig. 11A in HEK 293T cells at 48 hours post-transfection (as compared to the functional activity of hFVIII (i.e., unmodified hfdf 8 protein).

FIG. 12 shows an expression plasmid (pANG-TTR-hBDDF 8) similar to the expression plasmid in FIG. 1, except that the CAG promoter is replaced by a TTR promoter.

FIG. 13 shows functional activity of various mhFVIII constructs and hBDDF8 from rAAV2 vectors expressed in Huh7 cells 72 hours post-transfection.

Detailed Description

I. Definition of the definition

Various terms relating to the biomolecules of the present application are used hereinabove and throughout the specification and claims.

The phrase "activity-enhanced FVIII (actFVIII or actF 8)" refers to a modified hFVIII (hF 8) that has been genetically engineered such that the encoded protein exhibits an increase in activity of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to unmodified wild-type hFVIII. The nucleotide sequence of unmodified wild-type hFVIII is set forth in SEQ ID NO. 1, comprising a nucleotide sequence encoding a signal peptide of 19 amino acids (MQIELSCFFLCLLRFCFS (SEQ ID NO: 2)). The amino acid sequence of unmodified wild-type hFVIII comprising a signal peptide is set forth in SEQ ID NO. 3. The amino acid sequence of unmodified wild-type hFVIII without signal peptide is set forth in SEQ ID NO. 4.

The term "hddf 8 protein", "hddf 8 polypeptide" or "hddf 8" refers to a wild-type hFVIII protein with a deletion in the B domain. In some embodiments, the deletion encompasses a majority of the B domain, including sequences responsive to multiple cuts within the wild-type B domain. Exemplary hBDDF8 polypeptides have the amino acid sequence shown in SEQ ID NO. 5 (with signal peptide) or SEQ ID NO. 6 (without signal peptide) comprising an hFVIII heavy chain (SEQ ID NO. 7), a truncated B domain (SEQ ID NO. 8) and a light chain (SEQ ID NO. 9).

The phrase "one or more" followed by a list of elements or species is intended to encompass any arrangement of elements or species in the list. Thus, for example, the phrase "one or more substitution mutations selected from A, B, C, D, E and F" can include any combination of substitution mutations comprising A, B, C, D, E and/or F.

As used herein, a range may be expressed as from one particular integer value to another particular integer value. When such a range is expressed, it should be understood that any and all integer values within the range define different embodiments in accordance with the application, and that the full range of embodiments is included within the range, and that the range further includes any and all subranges between any integer value pair within the initial range.

With respect to the nucleic acids of the present application, the term "isolated nucleic acid", when applied to DNA, refers to a DNA molecule that has been isolated from the naturally occurring genome of the organism from which it is derived (in the 5 'and 3' directions) from sequences immediately adjacent thereto. For example, an "isolated nucleic acid" may include a DNA or cDNA molecule inserted into a vector (e.g., a plasmid or viral vector) or integrated into the DNA of a prokaryote or eukaryote. The nucleic acid codons can be optimized to enhance expression in mammalian cells.

With respect to the RNA molecules of the present application, the term "isolated nucleic acid" refers primarily to RNA molecules encoded by the isolated DNA molecules as defined above. Alternatively, the term may refer to an RNA molecule that is sufficiently isolated from the RNA molecule to which it binds in its natural state (i.e., in a cell or tissue) to exist in a "substantially pure" form (the term "substantially pure" is defined below).

With respect to proteins, the term "isolated protein" or "isolated and purified protein" refers herein to a protein produced by expression of an isolated nucleic acid molecule of the application. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins that naturally bind, so as to exist in a "substantially pure" form.

The term "hFVIII polypeptide" refers to a full length human FVIII protein, a fragment of a human FVIII protein, a domain and a combination of domains of a human FVIII protein that substantially retains the biological function of hFVIII.

The term "mutant hFVIII polypeptide" or "modified hFVIII polypeptide" refers to a polypeptide that has one or more amino acid differences (e.g., one or more amino acid substitutions) from a reference hFVIII polypeptide. The reference hFVIII polypeptide may be a wild-type hFVIII protein with or without a signal peptide, a wild-type hFVIII protein with modifications, such as a wild-type hFVIII protein with a deletion in the B domain (e.g., hfdf 8) or a fragment of hFVIII, hFVIII without a B domain, a domain or combination of domains with or without further modifications of hFVIII. In some embodiments, a "reference hFVIII polypeptide" of a "modified hFVIII polypeptide" refers to a hFVIII polypeptide prior to modification. In some embodiments, the term "mutant hFVIII polypeptide" or "modified hFVIII polypeptide" refers to a hybrid FVIII polypeptide comprising a human FVIII heavy chain and FVIII light chains from different species (e.g., light chains from canine FVIII).

The term "hFVIII variant" as used herein refers to a "mutant hFVIII protein" or a "modified hFVIII protein" that substantially retains hFVIII biological function.

A "conservative amino acid substitution" is a substitution of an amino acid residue for a functionally similar residue. Examples of conservative substitutions include the substitution of nonpolar (hydrophobic) residues such as isoleucine, valine, leucine or methionine for one another; substitution of charged or polar (hydrophilic) residues with each other, such as between arginine and lysine, between glutamine and asparagine, or between threonine and serine; substitution of basic residues such as lysine or arginine with each other; substitution of acidic residues, such as aspartic acid or glutamic acid, with each other; substitution of aromatic residues such as phenylalanine, tyrosine or tryptophan with each other; or substitution of alanine or glycine. The mutant FVIII proteins of the application may comprise amino acids having one or more conservative substitutions relative to the reference protein and retaining some or all of the activity of the reference protein, as described herein.

The term "expression cassette" as used herein refers to a nucleic acid construct comprising nucleic acid elements sufficient to express a polynucleotide of interest. Typically, an expression cassette comprises a polynucleotide of interest operably linked to regulatory sequences (e.g., promoters and enhancers). In some embodiments, the expression cassette may comprise additional elements, such as introns, polyadenylation sites, woodchuck hepatitis virus post-transcriptional response elements (WPREs), secretion signal sequences, and/or other elements known to affect the level of the coding sequence expression.

The term "control sequences" refers to transcriptional control sequences of a gene, which may be found 5 'or 3' to the coding region, within the coding region, or within an intron. Examples of regulatory sequences include, but are not limited to, promoters and enhancers.

The term "promoter" as used herein refers to a nucleotide sequence capable of controlling expression of a coding sequence or functional RNA. Typically, the polynucleotide of interest is located 3' to the promoter sequence. In some embodiments, the promoter is entirely derived from a native gene. In some embodiments, the promoter consists of different elements derived from different naturally occurring promoters. In some embodiments, the promoter comprises a synthetic nucleotide sequence. It will be appreciated by those skilled in the art that different promoters will direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions, or the presence or absence of a drug or transcription co-factor. Ubiquitous, cell type-specific, tissue-specific, developmental stage-specific, and conditional promoters, such as drug-responsive promoters (e.g., tetracycline-responsive promoters), are well known to those skilled in the art. Examples of promoters include, but are not limited to, phosphoglycerate Kinase (PKG) promoter, CAG, NSE (neuron specific enolase), synapsin or NeuN promoter, SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad-MLP); herpes Simplex Virus (HSV) promoters, cytomegalovirus (CMV) promoters such as CMV immediate early promoter region (CMVIE), SFFV promoters, osteosarcoma virus (RSV) promoters, synthetic promoters, hybrid promoters, and the like. Promoters may be from humans, but also from other species, including from mice. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene promoter, may also be used herein. In some embodiments, the promoter is a heterologous promoter. In some embodiments, the promoter sequence consists of proximal and more distal upstream elements, and may comprise enhancer elements.

The term "heterologous promoter" as used herein refers to a promoter that is not found in nature operably linked to a given coding sequence.

The term "enhancer" refers to a nucleotide sequence that can stimulate the activity of a promoter, and can be an innate element (element) of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter.

The term "operatively linked" or "operatively linked" refers to the binding of two or more nucleic acid fragments to a single nucleic acid fragment, such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when the promoter is capable of affecting the expression of the coding sequence (e.g., the coding sequence is under the transcriptional control of the promoter). The coding sequence may be operably linked to the regulatory sequence in sense or antisense orientation.

As used herein, the term "secretion signal sequence," "signal peptide," or variant thereof refers to an amino acid sequence that has the function of enhancing (as described above) the secretion of an operably linked polypeptide from a cell as compared to the secretion level of the native polypeptide. As defined above, "enhanced" secretion refers to an increase in the relative proportion of polypeptide synthesized by a cell that is secreted from the cell; the absolute amount of secreted protein has not to be increased. In some embodiments, substantially all (i.e., at least 95%, 97%, 98%, 99% or more) of the polypeptide is secreted. However, as long as the secretion level is increased compared to the native polypeptide, it is not necessary that substantially all or even a substantial portion of the polypeptide is secreted. Typically, the secretion signal sequence is cleaved within the endoplasmic reticulum, and in some embodiments, the secretion signal sequence is cleaved prior to secretion. However, the secretion signal sequence does not have to be cleaved as long as the secretion of the polypeptide from the cell is enhanced and the polypeptide is functional. Thus, in some embodiments, the secretion signal sequence is partially or fully retained. The secretion signal sequence may be derived in whole or in part from the secretion signal of the secreted polypeptide (i.e., derived from a precursor) and/or may be wholly or partially synthetic. The length of the secretion signal sequence is not critical; typically, the known secretion signal sequence is about 10-15 to 50-60 amino acids in length. In addition, known secretion signals from secreted polypeptides may be altered or modified (e.g., by amino acid substitutions, deletions, truncations, or insertions) so long as the resulting secretion signal sequence serves to enhance secretion of the operably linked polypeptide. The secretion signal sequence of the invention may comprise, consist essentially of, or consist of a naturally occurring secretion signal or modification thereof (as described above). Many secreted proteins and sequences are known in the art that are secreted directly from cells. The secretion signal sequences of the invention may also be wholly or partially synthetic or artificial. Synthetic or artificial secretion signal peptides are known in the art, see, e.g., "Human secretory signal peptide description by hidden Markov model and generation of a strong artificial signal peptide for secreted protein expression" by Barash et al, "biochem. Biophys. Res. Comm 294:835-42 (2002); the disclosure of which is incorporated herein in its entirety. The term "operably linked" refers to the placement of regulatory sequences required for expression of a coding sequence in a DNA molecule in place relative to the coding sequence, thereby affecting the expression of the coding sequence. This same definition sometimes applies to the arrangement of coding sequences and transcriptional control elements (e.g., promoters, enhancers, and termination elements) in an expression vector. The definition also sometimes applies to the arrangement of the nucleic acid sequences of the first and second nucleic acid molecules, wherein a hybrid nucleic acid molecule is produced.

The term "substantially pure" refers to a formulation comprising at least 50-60% by weight of a compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the formulation contains at least 75% by weight, most preferably 90-99% by weight of the compound of interest. Purity is measured by methods suitable for the compound of interest (e.g., chromatography, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

The phrase "consisting essentially of … … when referring to a particular nucleotide sequence or amino acid sequence" refers to a sequence having the characteristics of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence itself and molecular modifications that do not affect the basic and novel properties of the sequence.

The term "oligonucleotide" as used herein refers to primers and probes of the present application and is defined as a nucleic acid molecule consisting of two or more, preferably more than three, ribonucleotides or deoxyribonucleotides. The exact size of the oligonucleotide will depend on various factors and the particular application in which the oligonucleotide is used. The term "probe" as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, whether R A or DNA, whether naturally occurring as in a purified restriction enzyme digest or synthetically produced, that is capable of annealing or specifically hybridizing to a nucleic acid having a sequence complementary to the probe. The probe may be single-stranded or double-stranded. The exact length of the probe depends on many factors, including temperature, source of the probe, and method of use. For example, for diagnostic applications, an oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides, depending on the complexity of the target sequence.

The term "percent identity" is used herein to refer to a comparison between nucleic acid or amino acid sequences. Nucleic acid and amino acid sequences are often compared using, for example, the computer program in the national library of medicine (National Library of Medicine) BLAST alignment program.

As used herein, a "corresponding" nucleic acid or amino acid or sequence is a nucleic acid or amino acid present at a site in a FVIII or mutant FVIII molecule or fragment thereof, which has the same structure and/or function as a site in a FVIII molecule of another species, although the number of nucleic acids or amino acids may not be the same. The sequence "corresponding" to another FVIII sequence corresponds substantially to such a sequence and hybridizes under stringent conditions to the human FVIIIDNA sequence designated SEQ ID NO. 1. Sequences "corresponding" to another FVIII sequence also include sequences that result in expression of FVIII or a procoagulant hybrid FVIII as claimed or fragments thereof and which will hybridize to a nucleic acid molecule comprising SEQ ID No. 1 but which are genetically redundant.

As used herein, a "unique" amino acid residue or sequence refers to an amino acid sequence or residue in a FVIII molecule of one species that differs from a homologous residue or sequence in a FVIII molecule of another species.

As used herein, "specific activity" refers to the activity that will correct coagulation defects in human factor VIII deficient plasma. In a standard assay, specific activity is measured in units of clotting activity per milligram of total FVIII protein, wherein the clotting time of human FVIII deficient plasma is compared to the clotting time of normal human plasma. One FVIII activity unit is activity present in one milliliter of normal human plasma. In the assay, the shorter the clot formation time, the greater the activity of FVIII assayed. In the human FVIII assay, heterozygous human/porcine FVIII has co-clotting activity. Such activity, as well as the activity of other heterozygous or heterozygous equivalent FVIII molecules or fragments thereof, may be less than, equal to or greater than the activity of plasma derived or recombinant human FVIII.

As used herein, the "subunits" of human or animal FVIII are the heavy and light chains of a protein. The heavy chain of FVIII comprises three domains, al, A2 and B. The light chain of FVIII also comprises three domains, A3, C1 and C2.

The terms "epitope," "antigenic site," and "antigenic determinant" as used herein are synonymous and are defined as the portion of human, animal, hybrid, or heterozygous equivalent FVIII or fragments thereof specifically recognized by an antibody. It may consist of any number of amino acid residues and may depend on the primary, secondary or tertiary structure of the protein. According to the present disclosure, a hybrid FVIII comprising at least one epitope, a hybrid FVIII equivalent or a fragment of one of them may be used as a reagent in the diagnostic assays described below. In some embodiments, the heterozygous or heterozygous equivalent FVIII or fragments thereof are not or less cross-reactive with all naturally occurring inhibitory FVIII antibodies compared to human or porcine FVIII.

As used herein, the term "immunogenic site" is defined as a region of human or animal FVIII, heterozygous or heterozygous equivalent FVIII or fragments thereof that specifically elicits the production of antibodies against FVIII, heterozygous equivalent or fragments in a human or animal as determined by conventional protocols (e.g., immunoassays as described herein, such as ELISA or Bethesda assays). It may consist of any number of amino acid residues, and it may depend on the primary, secondary or tertiary structure of the protein. In some embodiments, the heterozygous or heterozygous equivalent FVIII or fragment thereof is non-immunogenic or less immunogenic than human or porcine FVIII in an animal or human.

As used herein, "FVIII deficiency" refers to a lack of clotting activity caused by: (1) producing defective FVIII; (2) insufficient or no production of FVIII; or (3) partial or total inhibition of FVIII. Hemophilia a is a FVIII deficiency caused by a deficiency in the X-linked gene and by the deletion or deficiency of the FVIII protein encoded thereby.

Modified hFVIII polypeptide (mhFVIII)

One aspect of the application relates to a modified hFVIII polypeptide (mhFVIII) comprising one or more mutations compared to a wild-type hFVIII polypeptide or an unmodified reference polypeptide. In some embodiments, the mhFVIII when expressed in a host cell results in increased hFVIII activity in the host cell as compared to a wild type hFVIII or a reference protein (e.g., hfdf 8) expressed in the same type of host cell under the same conditions.

Human FVIII encodes a protein with 2351 amino acids (signal peptide with 19 amino acids and mature protein with 2332 amino acids). It is arranged with a series of structural "domains": NH (NH) ₂ -SP-A1-A1-A2-A2-B-A3-A3-Cl-C2-COOH. As used herein, a FVIII "domain" is defined by a contiguous sequence of amino acids, characterized by, for example, internal amino acid sequence identity and thrombin proteolytic cleavage sites with the structurally related domains. Furthermore, the term "no domain" or "lack of domain"It is understood to have at least 95% or 100% domain deletion. Unless otherwise indicated, the FVIII domain is defined by the following amino acid residues in hFVIII (as shown in SEQ ID NO: 3) arranged from amino terminus to carboxyl terminus: SP, amino acid residues 1-19; al domain, amino acid residues 20-354; a1 domain, amino acid residues 355-391, A2 domain, amino residues 392-728; a2 domain, amino acid residues 729-760, b domain, amino residues 761-l667; a3 domain, amino acid residues 1668-l708; a3 domain, amino acid residues 1709-2039; c1 domain, amino acid residues 2040-2192; and C2 domain, amino acid residues 2193-2351.

A1-a ₁ -A2-a ₂ -B (amino acid (aa) 20-1667) sequence or A1-a ₁ -A2-a ₂ The (aa 1-740) sequence is commonly referred to as hFVIII heavy chain. a, a ₃ The sequence A3-C1-C2 (aa l 668-2351) is commonly referred to as hFVIII light chain. FVIII is proteolytically activated by thrombin or factor Xa, which dissociates from von Willebrand factor to form FVIIIa with procoagulant function. The biological function of FVIIIa is to increase the catalytic efficiency of factor IXa on factor X activation by several orders of magnitude. Thrombin activated FVIIIa is A1-a of 160kDa ₁ /A2-a ₂ /a ₃ -A3-C1-C2 heterotrimers forming complexes with factor IXa and factor X on the surface of platelets or monocytes.

The cDNA sequence encoding wild type human FVIII has the nucleotide sequence as set forth in SEQ ID NO. 1. In SEQ ID NO. 1, the first 57 nucleotides of the FVIII open reading frame encode a signal peptide sequence (SEQ ID NO. 2) that is normally cleaved from the mature FVIII protein.

In some embodiments, the modified hFVIII polypeptides of the application comprise one or more amino acid substitutions in the region corresponding to amino acid residues 20-171 of the wild-type hFVIII amino acid sequence set forth in SEQ ID NO. 3. In some embodiments, the modified hFVIII polypeptides of the application comprise one or more substitutions at positions a20, T21, F57, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332, R378, I610, and I661.

Referring to the mutants or modifications described herein, the positional designation indicated by the single letter code of the amino acid followed by a number refers to the amino acid residue and its position in wild type hFVIII (SEQ ID NO: 3). For example, the designation "A20" refers to the amino acid residue alanine (A) at position 20 of the wild-type hFVIII sequence (SEQ ID NO: 3). Similarly, the substitution designations indicated by the first single letter code followed by a number for an amino acid and the second single letter code followed by an amino acid refer to the substitution of the original amino acid residue at the position indicated by the number in wild-type hFVIII (SEQ ID NO: 3) with the second amino acid. For example, the designation "A20K" refers to the substitution of the amino acid residue alanine (A) at position 20 of wild-type hFVIII (SEQ ID NO: 3) with the amino acid residue lysine (K). Amino acid position nomenclature and substitution nomenclature also applies to domains of hFVIII, hFVIII heavy and light chains, hFVIII fragments, polypeptides having a common sequence with hFVIII, and/or other hFVIII derived sequences, such as hfdf 8.

In some embodiments, the application provides mhFVIII comprising an amino acid substitution selected from one or more of amino acid residues a20, T21, F57, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332, R378, I610, and I661. The mhFVIII may comprise any combination of mutations encompassing these 16 amino acid positions. An exemplary mhFVIII for use in accordance with the application is depicted in figure 3 with single and multiple mutations identified in the box.

In some embodiments, the mhFVIII of the application comprises one or more amino acid substitutions selected from the group consisting of a20K, T21I, T V, F57L, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S P, R378S, I M and I661V.

In some embodiments, the mhFVIII of the application comprises an amino acid substitution at position T21. Preferred substitutions include T21I and T21V. In some embodiments, the mhFVIII further comprises one or more amino acid substitutions at a position selected from the group consisting of a20, F57, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332, R378, I610, and I661.

In some embodiments, mhVIII of the application comprises amino acid substitutions at positions a20 and T21. In some embodiments, the mhFVIII of the application comprises amino acid substitutions a20K and T21I (2M 1 mutant), or amino acid substitutions a20K and T21V (2M 2 mutant).

In some embodiments, mhFVIII of the application comprises amino acid substitutions at positions T21, L69 and I80. In some embodiments, mhFVIII of the application comprises amino acid substitutions T21I, L V and I80V (3M 1 mutant).

In some embodiments, mhFVIII of the application comprises amino acid substitutions at positions T21, L69, I80 and L178. In some embodiments, the mhFVIII of the application comprises the amino acid substitutions T21I, L69V, I80 and L178F (4M 1 mutant).

In some embodiments, mhFVIII of the application comprises amino acid substitutions at positions T21, L69, I80 and I661. In some embodiments, mhFVIII of the application comprises amino acid substitutions T21I, L69V, I V and I661V (4M 3 mutant).

In some embodiments, mhFVIII of the application comprises amino acid substitutions at positions T21, L69, I80, L178 and I661. In some embodiments, mhFVIII of the application comprises amino acid substitutions T21I, L69V, I80V, L178F and I661V (5M 4 mutant).

In some embodiments, mhFVIII of the application comprises amino acid substitutions at positions R199, H212, I215, R269, I310, L318, and S332. In some embodiments, the mhFVIII of the application comprises the amino acid substitutions R199K, H212Q, I215V, R269K, I310V, L F and S332P (7M 2 mutant).

In some embodiments, mhFVIII of the application comprises amino acid substitutions at positions T21, L69, I80, L178, H212, I215, R269, L318 and I661. In some embodiments, mhFVIII of the application comprises amino acid substitutions T21I, L69V, I80V, L178F, H212Q, I215V, R269K, L318F and I661V (9M 1 mutant).

In some embodiments, mhFVIII of the application comprises amino acid substitutions at positions a20, T21, L69, I80, L178, H212, I215, R269, L318 and I661. In some embodiments, the mhFVIII of the application comprises amino acid substitutions a20K, T21V, L V, I80V, L178F, H Q, I215V, R269K, L F and I661V (10M 1 mutant).

In some embodiments, mhFVIII of the application comprises amino acid substitutions at positions T21, L69, I80, L178, R199, H212, I215, R269, I310, L318, S322, and I661. In some embodiments, the mhFVIII of the application comprises the amino acid substitutions T21I, L69V, I V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S P and I661V (12M 1 mutant).

In some embodiments, mhFVIII of the application comprises amino acid substitutions at positions a20, T21, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332, and I661. In some embodiments, the mhFVIII of the application comprises amino acid substitutions a20K, T21V, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S P and I661V (13M 1 mutant).

In some embodiments, the amino acid positions corresponding to the amino acid substitutions described above may be substituted with other conservative substitutions. Table 1 provides a list of exemplary amino acid substitutions at amino acid positions a20, T21, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332, and I661.

Table 1. Exemplary amino substitutions.

In some embodiments, the mhFVIIIs described above comprises a deletion in the B domain ("no B domain"). Examples of hFVIII polypeptides comprising deletions in the B domain are described in U.S. patent No.6,800,461, U.S. patent No.6,780,614 u.s.s. and patent application publication No.2004/0197875, which are incorporated herein by reference.

In some embodiments, the mhFVIII of the application comprises a single chain polypeptide. In some embodiments, the mhFVIII of the application comprises a single chain hFVIII polypeptide having a truncated or deleted B domain. In some embodiments, mhFVIII of the application comprises a heterodimer of a Heavy Chain (HC) comprising an A1 domain and an A2 domain and a Light Chain (LC) comprising an A3 domain, a C1 domain and a C2 domain. In some embodiments, mhFVIII of the application comprises a Heavy Chain (HC) comprising an A1 domain, an A2 domain and a full length or truncated B domain and a heterodimer of a Light Chain (LC) comprising an A3 domain, a C1 domain and a C2 domain. In some embodiments, mhFVIII of the application comprises a heterotrimer of A1 domain-containing polypeptide, an A2 domain-containing polypeptide, and A3 domain, C1 domain, and C2 domain-containing polypeptide.

In some embodiments, the mhFVIII described above is derived from wild-type hBDDF8 comprising a deletion in the B domain with the native hFVIII signal peptide set forth in SEQ ID NO 5. The secreted form of mhFVIII does not comprise the signal peptide sequence shown in SEQ ID NO. 2.

In some embodiments, mhFVIII of the application results in 5% -100-fold, 10% -50-fold, 50% -25-fold, 2-100-fold, 2-80-fold, 2-60-fold, 2-40-fold, 2-20-fold, 2-10-fold, 2-5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 70-fold, at least 80-fold, or at least 100-fold improvement in FVIII activity when expressed in vitro (in vitro) or in vivo (in vivo). In vitro FVIII activity can be determined by analyzing tissue culture medium from mhFVIII expressing cells. In vivo FVIII activity can be determined by analyzing plasma collected from an individual receiving mhFVIII infusion or from an expression vector expressing mhFVIII.

In some embodiments, mhFVIII of the application as described above may be further modified to additionally include, delete or modify other FVIII sequences to confer other desired properties, such as reduced antigenicity, increased stability, increased circulatory half-life through binding to serum binding proteins, increased protein secretion, increased affinity for factor IXa and/or factor X, reduced affinity for von Willebrand factor, increased glycosylation, altered inactivating cleavage, altered at least one calcium binding site, and/or increased shelf life.

For example, in some embodiments, mhFVIII of the application may be modified to additionally include amino acid substitutions responsible for immunogenicity and/or antigenicity of human FVIII, as described in U.S. patent No., U.S. patent No.2003/0166536, e.g., R503 503 504 505 506 506 507 507 508 508 508 509 511 512 514 516 517 518 519 521 522 523 525 526 527 2218 2242 2246 2271F, or any combination thereof.

In other embodiments, mhFVIII of the application as described above may be modified to additionally include amino acid substitutions providing secretion enhancement as described in U.S. patent application No.2016/102133, such as I105V, Y124F, A127S, D134E, Q H, F148L, G151K, H153Q, M166T and L171P or any combination thereof.

In other embodiments, mhFVIII of the application as described above may be modified to additionally include amino acid substitutions that confer greater stability to activated FVIII by virtue of fused A2 and A3 domains. In particular, FVIII can be modified by substitution of the cysteine residues at positions 683 and 1845 (i.e., Y683C, T1845C) to yield mutant FVIII that forms a C683-Cl845 disulfide bond covalently linking the A2 and A3 domains.

In other embodiments, mhFVIII of the application as described above may be further modified to additionally include amino acid substitutions conferring altered inactivating cleavage sites. For example, a355 or a581 may be substituted for reducing sensitivity of the mutant FVIII to cleaving enzymes that normally inactivate wild type FVIII.

In other embodiments, the mhFVIIIs of the application as described above may be further modified to additionally include amino acid substitutions that confer enhanced affinity for factor IXa.

In other embodiments, mhFVIII of the application as described above may be further modified to additionally include amino acid substitutions that confer increased circulatory half-life. This can be accomplished by a variety of methods including, but not limited to, by reducing interactions with heparan sulfate (heparan sulfate).

In other embodiments, mhFVIII of the application as described above may be further modified to additionally comprise amino acid substitutions conferring a recognition sequence for glycosylation at asparagine residues. Such modifications can be used to evade detection by the presence of (existing) inhibitory antibodies (low antigenic FVIII) and by reducing the likelihood of producing inhibitory antibodies (low immunogenic FVIII). In one representative embodiment, the modified FVIII is mutated to incorporate a consensus amino acid sequence for N-linked glycosylation, e.g. N-X-S/T.

In other embodiments, mhFVIIIs of the application as described above may be further modified to additionally comprise a mutation to (i) delete von Willebrand factor binding site, (ii) add a mutation at a759, and/or (iii) add an amino acid sequence spacer between the A2 domain and the A3 domain, wherein the amino acid spacer is of sufficient length that upon activation, the procoagulant FVIII protein becomes a heterodimer.

In some embodiments, the mhFVIII of the application is a hybrid FVIII comprising a human FVIII heavy chain and a FVIII light chain from a different species, e.g., a light chain from canine FVIII. In some embodiments, the human FVIII heavy chain further comprises one or more amino acid substitutions described in the present application. In some embodiments, the hybrid FVIII comprises a truncated B domain. In some embodiments, the hybrid FVIII consists of a single polypeptide comprising (1) a wild type human FVIII heavy chain sequence or a modified human FVIII heavy chain sequence, and (B) a FVIII light chain sequence from a different species, e.g., a light chain from canine FVIII. In some embodiments, the modified human FVIII heavy chain sequence comprises one or more amino acid substitutions described in the present application. In some embodiments, the modified human FVIII heavy chain sequence comprises an hddf 8 sequence or a modified hddf 8 with one or more amino acid substitutions described in the present application.

III.mhFVIII encoding polynucleotides, expression cassettes and expression vectors

Another aspect of the application relates to an isolated polynucleotide encoding mhFVIII of the application, including all possible nucleic acids encoding substitutions and/or other mutations of the ranges (break) described herein. The isolated nucleic acid may be RNA or DNA.

In certain embodiments, the polynucleotide encodes a mhFVIII polypeptide that is codon optimized for expression in a variety of human, primate, or mammalian cells, such as HuH7, HEK293T, or CHO cells. Polynucleotides encoding mhFVIII of the application may be codon optimized to increase activity, stability or expression in a host cell without altering the encoded amino acid sequence.

Codons consist of a set of three nucleotides, either encoding a particular amino acid or resulting in translation termination (i.e., stop codon). The genetic code is redundant in that multiple codons specify the same amino acid, i.e., 61 codons in total encode 20 amino acids. Codon optimisation replaces codons present in the polynucleotide sequence with preferred codons encoding the same amino acid, e.g. preferred codons for mammalian expression. Thus, the amino acid sequence is not altered during this process. Codon optimisation may be performed using gene optimisation software. The codon-optimized nucleotide sequence is translated and aligned with the original protein sequence to ensure that the amino acid sequence is unchanged. Methods of codon optimization are known in the art and are described, for example, in U.S. application publication No.2008/0194511 and U.S. patent No.6,114,148.

In some embodiments, the mhFVIII protein is expressed as single chain B domain free mhFVIII. In some embodiments, the mhFVIII protein is expressed from one or more nucleic acids in the form of a double-stranded (DC) protein comprising the Heavy (HC) and Light (LC) chains of hFVIII. In some embodiments, the mhFVIII protein is expressed from one or more nucleic acids in the form of a heterotrimer of a polypeptide comprising an A1 domain, a polypeptide comprising an A2 domain, and a polypeptide comprising A3, C1, and C2 domains.

In some embodiments, the mhFVIII-encoding polynucleotides of the application include a coding sequence for expression of a wild-type hFVIII amino-terminal signal peptide (SEQ ID NO: 2) that is removed from the mature protein. In some embodiments, mhFVIIIs of the application are derived from an hBDDF8 protein having the amino acid sequence of SEQ ID NO:5 (with signal peptide) or SEQ ID NO:6 (without signal peptide). Because the signal peptide sequence can affect expression levels, the mhFVIII encoding polynucleotide can be engineered to express mhFVIII carrying any one of a variety of heterologous N-terminal signal peptides known in the art.

In some embodiments, a mhFVIII encoding polynucleotide of the application as described above may be modified to additionally include, delete or otherwise modify other FVIII sequences that confer other desired properties, such as reduced antigenicity, increased stability, increased circulation half-life through binding to serum binding proteins, increased protein secretion, increased affinity for factor IXa and/or factor X, reduced affinity for von Willebrand factor, increased glycosylation, altered inactivating cleavage, altered one or more calcium binding sites, and/or increased shelf life, as described above.

Another aspect of the application relates to an expression cassette for expressing mhFVIII as described herein. In some embodiments, the expression cassette comprises a nucleotide sequence encoding mhFVIII of the application and regulatory sequences operably linked to the nucleotide sequence. In some embodiments, the regulatory sequence comprises a promoter.

Another aspect of the application relates to an expression vector capable of expressing mhFVIII of the application in vitro and/or in vivo. In some embodiments, the expression vector is a non-viral vector, such as a plasmid. In some embodiments, the expression vector is a viral vector, such as an AAV vector or a lentiviral vector.

Expression vectors for expressing mhFVIII of the application typically comprise one or more regulatory sequences operably linked to the polynucleotide sequence to be expressed. It will be appreciated by those skilled in the art that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, and the like. The expression vectors of the application can be introduced into host cells to produce mhFVIII as described herein.

Suitable expression vectors for directing expression in mammalian cells typically include promoters and other transcriptional and translational control sequences known in the art. In certain embodiments, mammalian expression vectors are capable of preferentially directing expression of a polynucleotide in a particular cell type (e.g., using tissue-specific regulatory elements to express the polynucleotide). Tissue-specific regulatory elements are known in the art and may include, for example, hepatocyte-specific promoters and/or enhancers (e.g., albumin promoter, a-1 antitrypsin promoter, apoE enhancer). Alternatively, constitutive promoters active in almost any cell type (e.g., HCMV) may be used.

In certain preferred embodiments, the expression vector is a viral vector. Viral vectors typically have one or more viral genes removed and include a gene/promoter cassette inserted into the viral genome insertion site for insertion of exogenous transgenes, including the mutated FVIII genes described herein. The necessary functions of the removed genes may be provided by cell lines that have been engineered to trans-express the gene products of the early genes. Exemplary viral vectors include, but are not limited to, adeno-associated virus (AAV) vectors, retroviral vectors, including lentiviral vectors, adenoviral vectors, herpesviral vectors, and alphaviral vectors. Other viral vectors include viral vectors such as astrovirus, coronavirus, orthomyxovirus, papillomavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, togavirus (togavirus), and the like. The viral vector may comprise any suitable nucleic acid construct, for example a DNA or RNA construct, and may be single-stranded, double-stranded or multiple-stranded (multiplexed).

Once the DNA construct of the application is prepared, it can be integrated into a host cell. Thus, another aspect of the application relates to a method of preparing a recombinant cell comprising a mhFVIII nucleic acid. Basically, this requires introducing a DNA construct into a cell by transformation, transduction, electroporation, calcium phosphate precipitation, liposomes, etc., and selecting cells that have either bound the DNA extrachromosomally or have integrated the DNA into the host genome. In some embodiments, cells expressing the mhFVIII are transplanted into a subject for use in treating hemophilia.

In some embodiments, the mhFVIII protein is expressed from a viral vector for administration to hemophilia patients. In some embodiments, the viral vector is an AAV vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is an adenovirus vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a herpes viral vector. In some embodiments, methods are provided for administering one or more AAV vectors encoding mhFVIII.

Recombinant AAV and lentiviral vectors have found wide use in a variety of gene therapy applications. Their utility in such applications is largely due to the high efficiency of gene transfer in vivo achieved in various organ environments. AAV and lentiviral particles can be advantageously used as vectors for efficient gene delivery. Such viral particles have many desirable characteristics for such applications, including their propensity to dividing and non-dividing cells. Early clinical experience with these vectors also showed no persistent toxicity, little or no detectable immune response. AAV is known to infect a variety of cell types in vitro and in vivo through receptor-mediated endocytosis or transport. These vector systems have been tested in humans to target retinal epithelium, liver, skeletal muscle, airways, brain, joints and hematopoietic stem cells. Non-viral vectors based on plasmid DNA or micro loops (miniloops) may also be suitable gene transfer vectors for large genes encoding FVIII.

In some embodiments, the mhFVIII coding sequence is provided as a component of a viral vector packaged in a capsid. In some embodiments, AAV vectors are used for in vivo delivery of mhFVIII of the application. In this case, the AAV vector comprises at least one mhFVIII and associated expression control sequences for controlling expression of the mhFVIII sequence. Exemplary AAV vectors for expressing mhFVIII sequences may include promoter-enhancer regulatory regions for FVIII expression and cis acting ITRs (which function to facilitate replication and packaging of mhFVIII nucleic acid into AAV capsids and integration of mhFVIII nucleotides into the genome of target cells). Preferably, the rep and cap genes of the AAV vector are deleted and replaced with mhFVIII sequences and their associated expression control sequences. The mhFVIII sequence is typically inserted adjacent to one or both (i.e., flanking) AAV TR or TR elements sufficient for viral replication. Most preferably, only the necessary parts of the vector, such as ITR and LTR elements, respectively, are included. In some embodiments, two or more AAV vectors are used for in vivo delivery of mhFVIII of the application. In this case, each AAV vector is constructed as described above and carries a portion of the mhFVIII coding sequence (e.g., one vector carries the coding sequence of the mhFVIII heavy chain and the other vector carries the coding sequence of the mhFVIII light chain).

Regulatory sequences suitable for promoting tissue-specific expression of mutated hFVIII sequences in target cells are used for in vitro or in vivo expression of said mhFVIII. The introduction of a tissue-specific regulatory element in the expression construct of the application provides at least partial tissue tropism for the expression of mhFVIII or functional fragments thereof. For example, nucleic acid sequences encoding mutant FVIII under the control of the Cytomegalovirus (CMV) promoter or the CAG promoter can be used for skeletal muscle expression or hAAT-ApoE and others for liver-specific expression. Hematopoietic specific promoters in AAV and lentiviral vectors may also be used to drive expression of mhFVIII in vivo.

The viral capsid component of the packaged viral vector may be a parvoviral capsid. AAVCap and chimeric capsids are preferred. Examples of suitable parvoviral viral capsid components are capsid components from the parvoviridae family, such as autonomous parvovirus (autonomous parvovirus) or dependent virus (dependovirus). For example, the viral capsid may be an AAV capsid (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid; one skilled in the art will appreciate that there may be other variants that perform the same or similar function that have not been identified), or may include components from two or more AAV capsids. The complete complement (complement) of AAV Cap proteins includes VP1, VP2, and VP3. An ORF comprising a nucleotide sequence encoding an AAV VP capsid protein may comprise less than the complete complement AAV Cap protein, or may provide the complete complement of the AAV Cap protein.

One or more of the AAV-Cap proteins may be chimeric proteins, including the amino acid sequences AAV-Cap from two or more viruses, preferably two or more AAVs, as described in Rabinowitz et al, U.S. Pat. No.6,491,907. For example, a chimeric viral capsid can comprise an AAV1-Cap protein or subunit and at least one AAV2-Cap or subunit. Chimeric capsids may, for example, comprise AAV capsids having one or more B19-Cap subunits, e.g., AAV-Cap proteins or subunits may be substituted with B19-Cap proteins or subunits. For example, the Vp3 subunit of the AAV capsid may be replaced with the Vp2 subunit of B19.

Packaging cells can be cultured to produce the packaged viral vectors of the application. The packaging cell may include (1) a viral vector function, (2) a packaging function, and (3) a helper function. The viral vector functions typically include a portion of a parvoviral genome, such as an AAV genome, in which rep and cap are deleted and replaced with a mutated FVIII sequence and its associated expression control sequences, as described above.

In certain embodiments, the viral vector function may suitably be provided as a multiplex vector template, as described in U.S. patent publication No.2004/0029106 to Samulski et al. The multiplex vector is a dimeric self-complementary (sc) polynucleotide (typically DNA). For example, the DNA of the multiplex carrier may be selected so that a double stranded hairpin structure is formed due to base pairing within the strand. Both strands of the multiplex DNA vector may be packaged within the viral capsid. The multiplex vector provides a function comparable to that of a double stranded DNA viral vector and can alleviate the need for target cells to synthesize DNA complementary to the single stranded genome normally enveloped by the virus.

The TR (degradable and non-degradable) selected for the viral vector is preferably an AAV sequence (from any AAV serotype). Degradable AAV ITRs need not have wild-type TR sequences (e.g., wild-type sequences may be altered by insertions, deletions, truncations, or missense mutations) so long as TR mediates desired functions, such as viral packaging, integration, and/or proviral rescue, etc. TR may be a synthetic sequence that functions as an AAV inverted terminal repeat, such as the "double-D sequence" described in U.S. patent No.5,478,745 to Samulski et al. Typically, but not necessarily, multiple TRs are from the same parvovirus, e.g., both TR sequences are from AAV2.

The packaging function includes a capsid component. The capsid component is preferably derived from a parvoviral capsid, such as an AAV capsid or chimeric AAV capsid function. Examples of suitable parvoviral viral capsid components are capsid components from parvoviridae, such as autonomous parvoviruses or dependent viruses. For example, the capsid component may be selected from AAV capsids, such as AAV1-AAV12 and other novel capsids not yet identified, or from non-human primate sources. The capsid component may comprise components from two or more AAV capsids.

In certain embodiments, one or more VP capsid proteins may comprise a chimeric protein comprising amino acid sequences from two or more viruses, preferably two or more AAV. For example, the chimeric viral capsid can include a capsid region from an adeno-associated virus (AAV) and at least one capsid region from a B19 virus. The chimeric capsids may, for example, comprise an AAV capsid having one or more B19 capsid subunits, e.g., the AAV capsid subunits may be substituted with B19 capsid subunits. For example, the VP1, VP2, or VP3 subunit of the AAV capsid can be replaced by the VP1, VP2, or VP3 subunit of B19. As another example, the chimeric capsid may comprise an AAV type 2 capsid, wherein the VP1 subunit has been substituted with a VP1 subunit from an AAV type 1, 3, 4, 5 or 6 capsid, preferably a type 3, 4 or 5 capsid. Alternatively, the chimeric parvovirus has an AAV type 2 capsid wherein the VP2 subunit has been replaced with a VP2 subunit from an AAV type 1, 3, 4, 5 or 6 capsid, preferably a type 3, 4 or 5 capsid. Also preferred are chimeric parvoviruses in which the VP3 subunit from AAV type 1, 3, 4, 5 or 6 (more preferably type 3, 4 or 5) is replaced with the VP3 subunit of an AAV type 2 capsid. Alternatively, chimeric parvoviruses are preferred in which two AAV type 2 subunits are substituted with subunits from AAV of different serotypes (e.g., AAV types 1, 3, 4, 5, or 6). In an exemplary chimeric parvovirus according to this embodiment, the VP1 and VP2, or VP1 and VP3, or VP2 and VP3 subunits of an AAV type 2 capsid are substituted with corresponding subunits of an AAV of a different serotype (e.g., AAV type 1, 3, 4, 5, or 6). Also, in some other preferred embodiments, the chimeric parvovirus has an AAV type 1, 3, 4, 5 or 6 capsid (preferably type 2, 3 or 5 capsid), wherein one or both subunits have been replaced with subunits from AAV of a different serotype, as described above for AAV type 2.

Packaged viral vectors typically include a mutant FVIII sequence and expression control sequences flanked by TR elements sufficient to result in packaging of the vector DNA and subsequent expression of the mutant FVIII sequence in transduced cells. The viral vector function may be provided to the cell, for example, as a component of a plasmid or amplicon. The viral vector function may exist extrachromosomally within the cell line and/or may be integrated into the chromosomal DNA of the cell.

Methods and cell lines for production of mhFVIII proteins

Another aspect of the application relates to a method of preparing mhFVIII of the application. This requires growing the host cell of the application under conditions in which the expression vector converts the host cell to express mhFVIII. The expressed mhFVIII is then isolated.

Another aspect of the application relates to a host cell comprising an isolated nucleic acid molecule encoding said mhFVIII of the application. The host cell may contain the isolated nucleic acid molecule as a DNA molecule in the form of an episomal plasmid, or it may be stably integrated into the genome of the host cell. Furthermore, the host cell may constitute an expression system for producing the mhFVIII protein. Suitable host cells may be, but are not limited to, animal cells (e.g., human HuH7 and HEK293 cells, chinese hamster ovary cells ("CHO"), baby hamster kidney cells ("BHK")), bacterial cells (e.g., escherichia coli), insect cells (e.g., sf9 cells), fungal cells, yeast cells (e.g., saccharomyces cerevisiae or schizosaccharomyces), plant cells (e.g., arabidopsis or tobacco cells), algal cells, and the like. Mammalian cells suitable for practicing the application additionally include COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281), CHO (ATCC No. CCL 61), heLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573), CHOP, huH7, HEK293 and NS-1 cells.

Another aspect of the application relates to a method of producing mhFVIII from cultured cells. In some embodiments, the method comprises the steps of: (a) Introducing into a host cell an expression vector comprising: a polynucleotide comprising a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding the mhFVIII, wherein the mhFVIII comprises at least one or more amino acid substitutions at a position selected from the group consisting of a20K, T I, T21V, F57L, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L F, S332P, R S, I610M and I661V; and a regulatory sequence operably linked to the polynucleotide; (b) Growing the host cell carrying the expression vector under conditions suitable for expression and secretion of the mhFVIII; and (c) harvesting the host cells and/or the medium of the host cells, and (d) purifying the mhFVIII from the harvested medium and/or host cells.

In one embodiment, the host cell is grown in vitro in a growth medium. Suitable growth media may include, but are not limited to, growth media containing von Willebrand factor (referred to herein as "VWF"). In this embodiment, the host cell may comprise a transgene encoding VWF, or VWF may be introduced as a supplement into the growth medium. VWF in the growth medium will allow the mhFVIII to achieve higher expression levels. Once the recombinant FVIII is secreted into the growth medium, it can be isolated from the growth medium using techniques known to those of ordinary skill in the relevant recombinant DNA and protein techniques, including those described herein. In another embodiment, the method of making the mhFVIII of the application further comprises disrupting the host cell prior to isolating the mhFVIII. In this embodiment, the mhFVIII is separated from cell debris.

The mhFVIII is preferably produced in substantially pure form. In a particular embodiment, the purity of the substantially pure recombinant FVIII is at least about 80%, more preferably at least 90%, most preferably at least 95%, 98%, 99% or 99.9%. Substantially pure recombinant FVIII can be obtained by conventional techniques well known in the art. Typically, substantially pure mhFVIII is secreted into the growth medium of the recombinant host cell. Alternatively, substantially pure mhFVIII is produced but not secreted into the growth medium. In this case, to isolate substantially pure mhFVIII, host cells carrying the recombinant plasmid are propagated, lysed by sonication, heating or chemical treatment, and the homogenate centrifuged to remove cell debris. The supernatant is then subjected to a sequential ammonium sulphate precipitation. The fraction containing substantially pure mhFVIII is gel filtered in a dextran or polyacrylamide column of appropriate size to isolate mhFVIII. If necessary, the protein fraction (containing substantially pure mhFVIII) may be further purified by high performance liquid chromatography ("HPLC").

V. therapeutic methods

Another aspect of the application relates to a method of treating a patient suffering from FVIII deficiency.

In some embodiments, the method comprises the step of administering to a patient in need thereof an effective amount of mhFVIII of the application. In some embodiments, the mhFVIII is administered intravenously in purified form.

In other embodiments, the method comprises the step of administering to a patient in need thereof an effective amount of an expression vector comprising a coding sequence for mhFVIII of the application, wherein the expression vector is capable of expressing the mhFVIII in the patient.

In other embodiments, the method comprises the step of administering to a patient in need thereof an effective amount of a cell comprising a coding sequence for mhFVIII of the application, wherein the cell is capable of expressing the mhFVIII in the patient after transplantation. In some embodiments, the cell is a dermal fibroblast. In some embodiments, the cell is an autologous cell. In some embodiments, the method comprises the step of introducing a mhFVIII encoding sequence into a population of target cells, wherein the target cells are isolated from a subject in need of such treatment; expressing the mhFVIII in the target cell, and infusing an effective amount of mhFVIII expressing cells into the subject.

In some embodiments, the FVIII deficiency is hemophilia a. In such cases, expression of mhFVIII of the application can enhance coagulation in patients that are otherwise susceptible to uncontrolled bleeding (e.g., intra-articular, intracranial, or gastrointestinal bleeding) due to FVIII deficiency, including hemophilia patients who have developed anti-human FVIII antibodies. The target cells of the vector are cells capable of expressing a polypeptide having FVIII activity, such as those of the mammalian liver system, endothelial cells, and other cells having appropriate cellular mechanisms to process the precursors to produce proteins having FVIII activity.

Administration of the mhFVIII protein or an expression vector encoding mhFVIII or a cell expressing mhFVIII to a FVIII deficient patient may functionally reestablish the coagulation cascade (coagulation cascade). The mhFVIII protein or expression vector encoding mhFVIII or cell expressing mhFVIII may be administered alone or in combination with other therapeutic agents in a pharmaceutically acceptable or biologically compatible composition.

In some embodiments, the method comprises administering intravenously a pharmaceutical composition comprising mhFVIII protein to a patient according to the same procedure as used for infusion of human or animal FVIII. Suitable effective amounts of mhFVIII may include, but are not limited to, from about 10 to about 500 units/kg of patient body weight.

The dosage of treatment of the mhFVIII-encoding expression vector or mhFVIII protein or mhFVIII-expressing cells will vary with the severity of FVIII deficiency. Typically, the dosage level is adjusted in frequency, duration, and units according to the severity and duration of each patient's bleeding episode. Thus, the expression vector encoding mhFVIII or the mhFVIII protein or cell expressing mhFVIII is contained in a pharmaceutically acceptable carrier, delivery vehicle or stabilizer in an amount sufficient to deliver a therapeutically effective amount of the protein to a patient to stop bleeding as measured by standard coagulation assays.

FVIII is classically defined as a substance present in normal plasma that corrects clotting defects in the plasma of hemophilia a individuals. The in vitro coagulation activity of purified and partially purified forms of FVIII was used to calculate the dose of mhFVIII infused in human patients and is a reliable indicator of recovery activity from patient plasma and correction of in vivo bleeding defects. No differences were reported between the in vitro standard assays of the novel FVIII molecules and their behavior in dog infusion models or human patients.

Typically, the desired plasma FVIII activity level in the patient is achieved by administration of said mhFVIII at 30-200% of normal levels. In one embodiment, the preferred dosage for intravenous administration of therapeutic mhFVIII is a dosage of about 5 to 500 units/kg body weight, particularly 10 to 100 units/kg body weight, further particularly 20 to 40 units/kg body weight; the frequency of intervals is about 8 to 24 hours (in severely affected hemophiliacs); and the duration of treatment in days is 1-10 days, or until the bleeding episode is resolved. Patients using inhibitors may require different amounts of mhFVIII than their previous forms of FVIII. For example, a patient may need less mhFVIII because it has higher specific activity than wild type VIII and its antibody reactivity is reduced. As with human or plasma-derived FVIII treatment, the amount of therapeutic mhFVIII infusion is determined by a single-stage FVIII clotting assay, and in selected cases, in vivo recovery is determined by measuring FVIII in the patient's plasma after infusion. It should be understood that for any particular subject, the particular dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the mhFVIII as claimed.

The treatment may take the form of a single intravenous administration of the mtFVIII, or periodic or continuous administration over a longer period of time, as desired. Alternatively, therapeutic mhFVIII may be administered subcutaneously or orally with liposomes at different time intervals in one or several doses.

Administration of the expression vector to a human subject or animal in need thereof may be by any means known in the art for administration of viral vectors. Exemplary modes of administration include rectal, transmucosal, topical, transdermal, inhaled, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intra-articular), and the like, as well as direct tissue or organ injection, or intrathecal, direct intramuscular, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection. Injectables can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in liquid prior to injection, or emulsion forms. Alternatively, the virus may be administered in a local rather than systemic manner, for example in a depot (depot) or sustained release formulation.

In certain preferred embodiments, the expression vector is administered intramuscularly, more preferably by intramuscular injection or topically. The vectors disclosed herein may be administered to the lungs of a subject by any suitable means, but are preferably administered by administering an aerosol suspension of respirable particles composed of the parvoviral vectors of the present invention (which are inhaled by the subject). The inhalable particles may be liquid or solid. Aerosols of liquid particles comprising the parvoviral vectors (e.g., AAV) of the invention may be produced by any suitable means, for example using a pressure-driven aerosol nebulizer or an ultrasonic nebulizer known to those skilled in the art (see, e.g., U.S. patent No.4,501,729).

The dose of the viral vector expressing mhFVIII will depend on the mode of administration, the disease or disorder to be treated, the condition of the individual subject, the particular viral vector and the gene to be delivered, and can be determined in a conventional manner. Exemplary dosages for achieving a therapeutic effect are at least about 10 ⁵ 、10 ⁶ 、10 ⁷ 、10 ⁸ 、10 ⁹ 、10 ¹⁰ 、10 ¹¹ 、10 ¹² 、10 ¹³ 、10 ¹⁴ 、10 ¹⁵ A transduction unit or more, preferably about 10 ⁸ -10 ¹³ Transduction units, more preferably 10 ¹² Viral titer of the transduction unit. Polynucleotides encoding mtFVIII of the applicationCan be administered as a component of a DNA molecule having regulatory elements suitable for expression in a target cell. The mtFVIII encoding polynucleotides of the application can be administered as a component of a viral plasmid or viral particle (e.g., an AAV particle). The viral particles may be administered as individual viral particles by direct in vivo delivery to the portal vasculature of a subject in need thereof, or as an ex vivo treatment (ex vivo treatment) comprising the use of the viral particles to transduce cells ex vivo and then introducing the transduced cells back into the subject in vivo (in vivo).

The polynucleotide encoding mtFVIII can be administered as a single-chain molecule containing both heavy and light chain portions, or split into two or more molecules (e.g., heavy and heavy chains) in multiple independent viral or non-viral vectors for delivery into a patient's host cells.

In some embodiments, the expression vector is a viral vector. Viral vectors that can be used in the present application include, but are not limited to, adeno-associated viral (AAV) vectors of various serotypes (e.g., AAV-1 to AAV-12 and pseudotyped vectors thereof), hybrid AAV vectors, retroviral vectors, including lentiviral vectors and pseudotyped lentiviral vectors (e.g., human Immunodeficiency Virus (HIV) and Feline Immunodeficiency Virus (FIV)); adenovirus vectors, herpes simplex virus vectors, vaccinia virus vectors, non-viral vectors, and other vectors. Furthermore, any of the viral vectors may be modified to include tissue specific promoters/enhancers and the like.

VI pharmaceutical composition

Another aspect of the application relates to a pharmaceutical composition comprising (1) a mhFVIII polypeptide of the application, an expression vector encoding mhFVIII or a cell expressing mhFVIII, and (2) a pharmaceutically acceptable carrier.

Exemplary pharmaceutically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline. Physiologically acceptable carriers include pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are those that are not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing an undesirable biological effect that exceeds the beneficial biological effect of the material. In some embodiments, the pharmaceutical composition is formulated for injection.

For injection, the carrier is typically a liquid. As injection medium, water is preferably used which contains additives commonly used for injection solutions, such as stabilizers, salts or saline and/or buffers. For other methods of administration, the carrier may be solid or liquid. For administration by inhalation, the carrier will be respirable and preferably in solid or liquid particulate form.

The application is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.

Examples

Example 1: construction, expression and characterization of factor VIII mutants

And (3) a template: plasmid pANG-CAG-hBDDF8 was used as a template to introduce multiple hFVIII mutations into the coding region of the hFVIII heavy chain. As shown in FIG. 1, pANG-CAG-hBDDF8 (SEQ ID NO: 15) contains the human factor VIII (hBDDF 8) cDNA under the control of the CAG promoter. Furthermore, pANG-CAG-hbdf 8 carries a deletion in the B domain, resulting in a functionally defective B domain. FIG. 2 identifies hFVIII mutations constructed in pANG-CAG-hBDDF 8. Figure 3 shows two hFVIII mutations analyzed in this study, including single amino acid substitutions and combinations thereof.

Single mutation: using a GIBSONMethod A single mutation corresponding to A20K, T21I, T V, F57L, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S P, R378S, I M and I661V of human factor VIII was introduced into pANG-CAG-hBDDF8 plasmid. The obtained plasmid comprises pANG-CAG-hBDDF8-A20K, pANG-CAG-hBDDF8, pANG-CAG-hBDDF8-T21I, pANG-CAG-hBDDF8-T21V, pANG-CAG-hBDDF8-F57L, pANG-CAG-hBDDF8-L69V, pANG-CAG-hBDDF8-I80V、pANG-CAG-hBDDF8-L178F、pANG-CAG-hBDDF8-R199K、pANG-CAG-hBDDF8-H212Q、pANG-CAG-hBDDF8-I215V、pANG-CAG-hBDDF8-R269K、pANG-CAG-hBDDF8-I310V 、 pANG-CAG-hBDDF8-L318F 、pANG-CAG-hBDDF8-S332P 、 pANG-CAG-hBDDF8-R378S 、pANG-CAG-hBDDF8-I610M、pANG-CAG-hBDDF8-I661V。

T21 mutant: an avrII restriction site was introduced in pANG-CAG-hBDDF8 by substituting P for T21. The resulting plasmid pANG-CAG-hBDDF8-T21P was used as a template to generate a multiple point mutation at amino acid position 21. pANG-CAG-hBDDF8-T21P was digested by AvrII and used as a template for mutant construction as described herein. 19 oligonucleotides of NNN corresponding to the T21 position were recombined into pANG-CAG-hBDDF8 by HIFI assembly. Mutant plasmids include pANG-CAG-hBDDF8-T21V, pANG-CAG-hBDDF8-T21I … … pANG-CAG-hBDDF8-T21G and the like. The last letter indicates the amino acid substitution at that particular position. Similar strategies may be used to generate other substitutions in accordance with the present invention.

A20 mutation with T21I: T21I and NNN correspond to alanine 20 of 19 oligonucleotides through HIFI assembly recombination into AvrII digested pANG-CAG-hBDDF 8-T21P. The obtained mutant plasmids include pANG-CAG-hBDDF8-T21V, pANG-CAG-hBDDF8-T21I … … pANG-CAG-hBDDF8-T21G and the like. Similar strategies can be used to generate other substitutions according to the invention, such as pANG-CAG-hBDDF8-A20K/T21V (2M 2).

Combination of mutations: hFVIII mutants with multiple HC mutations were constructed in pANG-CAG-hBDDF8 (as shown in FIG. 3). In one embodiment, DNA fragments encoding substitution mutations a20K, T21V, L69V, I V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S332P and I661V were chemically synthesized and used to replace the corresponding regions in pANG-CAG-hbdf 8. The resulting plasmid pANG-CAG-BDDF8-13M1 expresses the mutant factor VIII protein (13M 1) with the 13 mutations described above.

In another embodiment, DNA fragments encoding substitution mutations T21I, L69V, I V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S P and I661V were chemically synthesized and used to replace the corresponding region of pANG-CAG-hbdf 8. The resulting plasmid pANG-CAG-BDDF8-12M1 expresses the mutant factor VIII protein (12M 1) with the 12 mutations described above.

In another embodiment, DNA fragments encoding substitution mutations a20K, T21V, L69V, I V, L178F, H212Q, I215V, R269K, L F and I661V were chemically synthesized and used to replace the corresponding regions of pANG-CAG-hbdf 8. The resulting plasmid pANG-CAG-BDDF8-10M1 expresses the mutant factor VIII protein (10M 1) with the 10 mutations described above.

In another embodiment, DNA fragments encoding substitution mutations T21I, L69V, I80V, L178F, H212Q, I215V, R269K, L318F and I661V were chemically synthesized and used to replace the corresponding regions of pANG-CAG-hbdf 8. The resulting plasmid pANG-CAG-BDDF8-9M1 expresses the mutant factor VIII protein (9M 1) with the 9 mutations described above.

In another embodiment, DNA fragments encoding the substitution mutations R199K, H212Q, I V, R269K, I310V, L F and S332P were chemically synthesized and used to replace the corresponding regions of pANG-CAG-hbdf 8. The resulting plasmid pANG-CAG-BDDF8-7M2 expresses the mutant factor VIII protein (7M 2) with the 7 mutations described above.

In another embodiment, DNA fragments encoding the substitution mutations T21I, L69V, I80V, L178F and I661V were chemically synthesized and used to replace the corresponding regions of pANG-CAG-hBDDF 8. The resulting plasmid pANG-CAG-BDDF8-5M4 expresses the mutant factor VIII protein (5M 4) with the 5 mutations described above.

In another embodiment, DNA fragments encoding the substitution mutations T21V, L69V, I V and I661V were chemically synthesized and used to replace the corresponding region of pANG-CAG-hbdf 8. The resulting plasmid pANG-CAG-BDDF8-4M3 expresses the mutant factor VIII protein (4M 3) with the 4 mutations described above.

In another embodiment, DNA fragments encoding the substitution mutations T21I, L69V, I V and L178F were chemically synthesized and used to replace the corresponding regions of pANG-CAG-hbdf 8. The resulting plasmid pANG-CAG-BDDF8-4M1 expresses the mutant factor VIII protein (4M 1) with the 4 mutations described above.

In another embodiment, DNA fragments encoding the substitution mutations T21I, L V and I80V were chemically synthesized and used to replace the corresponding regions of pANG-CAG-hbdf 8. The resulting plasmid pANG-CAG-BDDF8-3M1 expresses the mutant factor VIII protein (3M 1) with the 3 mutations described above.

To test the functional activity of the mutant constructs, HEK 293T, huH and CHO cells were cultured in DMEM containing 10% fetal bovine serum, penicillin (100U/ml) and streptomycin (100 μg/ml) at 37 ℃ in a humid environment provided with 5% carbon dioxide. HEK 293T, huH and CHO cells were transfected with mutants or wild type expression constructs in pANG-CAG-hDDF 8. Following transfection, cells were maintained in RPMI-1640 medium containing 2% inactivated fetal bovine serum. Cell culture media was collected at various times (24 hours, 48 hours, 72 hours) after transfection. Secreted FVIII activity was assayed using an Activated Partial Thromboplastin Time (APTT) assay. Normal human plasma was used as a standard.

Representative results of these assays are shown in FIGS. 4-10, 11B and 13. Briefly, FIGS. 4-6 show that functional activity of specific single amino acid substitution mutants was increased in HuH7 cells (FIG. 4) and HEK 293T cells (FIGS. 5, 6) compared to wild-type hFVIII at 48 hours post-transfection. Figure 6 shows that T21I and T21V mutants significantly improved FVIII activity in HEK 293T cells at 48 hours post-transfection. FIG. 7 shows that double mutants carrying A20K (A20K/T21I and A20K/T22V) further increased FVIII activity in HEK 293T cells at 48 hours post-transfection relative to T21 and T21V monosubstituted counterparts. Figures 8-10 show that the combination of multiple mutations significantly increased FVIII functional activity compared to wild type, single and double substitution mutants in HuH7, HEK 293T and CHO cells, respectively, 24 hours and 48 hours after transfection.

Example 2: construction, expression and functional Activity of heterozygous human/canine factor VIII mutants

To evaluate the functional activity of mutant hVIII of the application compared to mutant hybrid human/canine FVIII consisting of mutant hVIII heavy chain (hHC) and canine FVIII light chain (cLC), a series of B-domain free FVIII constructs were prepared and expressed in HEK 293T cells, as shown in fig. 11A and 11B.

Briefly, a mutant FVIII comprising a mutant human FVIII heavy chain and a canine FVIII light chain was constructed. Briefly, pANG-CAG-hBDDF8 was digested with CspCI and XhoI. A DNA fragment encoding a canine light chain (cLC) was chemically synthesized and used to replace the corresponding human light chain (hLC) region in pANG-CAG-hBDDF8 by the Gibson assembly method. The resulting plasmid pANG CAG-hHC cLC expressed the B-domain-free hybrid human/canine factor VIII polypeptide consisting of hHC and cLC (i.e., hHC-cLC). SEQ ID NO. 10 shows the amino acid sequence of the hHC-cLC protein with the native hFVIII signal peptide. SEQ ID NO. 11 shows the cDNA sequence encoding the protein in SEQ ID NO. 10. SEQ ID NO. 12 shows the amino acid sequence of the hHC-cLC protein without hFVIII signal peptide. SEQ ID NO. 13 shows the amino acid sequence of the truncated B domain in the hHC-cLC protein, while SEQ ID NO. 14 shows the amino acid sequence of the canine FVIII light chain (cLC).

Similar strategies were used to generate pANG-CAG-qwHC-2M1-cLC (2M 1 mutant) and pANG-CAG-qwHCC-9M1-cLC (9M 1 mutant) plasmids as shown in FIG. 11A (and abbreviated). Secreted FVIII activity was assayed using an Activated Partial Thromboplastin Time (APTT) assay. FIG. 11B shows the functional activity of various human/canine hybrid FVIII mutants in HEK 293T cells compared to the functional activity of hFVIII (i.e.unmodified hBDDF8 protein) 48 hours after transfection. As shown in fig. 11B, substitution of hLC with crc in this system resulted in increased FVIII activity compared to hfviii expressed from the parental plasmid hfdf 8.

In another aspect, the functional activity of a rAAV vector expressing mutant hdviii of the application is compared to a rAAV vector expressing the parental hddf 8. In this case, a series of rAAV expressing the hddf 8 protein and the modified hddf 8 protein were constructed and produced. The resulting rAAVs were used to infect Huh7 cells and analyzed for hVIII activity in the infected cells.

In this case, a first series of rAAV expressing mutant hVIII of the application was constructed in which the CAG promoter in hfdf 8 was replaced by the human TTR promoter. Briefly, pANG-CAG-hBDDF8 was digested with SnaBI and MluI. DNA fragments encoding the TTR promoter and intron were chemically synthesized and used to replace the CAG promoter region in pANG-CAG-hBDDF8 by the Gibson assembly method.

As shown in FIG. 12, the resulting plasmid pANG-TTR-hBDDF8 (SEQ ID NO: 18) expresses the hBDDF8 protein under the control of the TTR promoter. Similar strategies were used to generate pANG-TTR-qwBDDF8-2M1, pANG-TTR-qwBDDF8-2M2, pANG-TTR-qwBDDF8-9M1, pANG-TTR-qwBDDF8-10M1, pANG-TTR-qwBDDF8-12M1, pANG-TTR-qwBDDF8-13M1 plasmids.

AAV2 capsids were used to package pANG-TTR-hBDDF8 and its modified variant plasmids to produce rAAV therefrom. Briefly, pAAV Rep & Cap (serotype 2), pAd-cofactor (pAd-helper) and transgenic plasmids were co-transfected into HEK 293T cells cultured in a roller bottle (roller bottle) at a 1:1:1 ratio. rAAVs from transfected cell culture media were harvested 72 hours after transfection and purified by two rounds of CsCl gradient ultracentrifuge. Each rAAV was collected and extensively exchanged with PBS containing 5% D-sorbitol.

HuH7 cells were subjected to DMEM (Invitrogen, carlsbad, calif.) containing 10% FBS, penicillin (100U/ml) and streptomycin (100. Mu.g/ml) at 37℃in the presence of 5% CO ₂ Is grown in a humid environment. For each transduction experiment, 50,000 viable cells were seeded in 24-well plates 24 hours prior to transduction. rAAV was added directly to each plate well at 100,000 vg/cell. Secreted FVIII activity was analyzed at 72 hours post-transfection using an Activated Partial Thromboplastin Time (APTT) assay.

As shown in fig. 13, modified hbdf 8 exhibited increased FVIII activity when expressed by rAAV vector in Huh7 cells as compared to unmodified hbdf 8.

The above examples demonstrate that the mutant factor VIII products of the application exhibit increased functional activity compared to wild-type factor VIII. Thus, use of the mutants described herein can reduce production costs and required FVIII expression levels (relative to existing constructs). They may also allow for lower carrier doses to be administered by providing a higher active FVIII product.

The above description is intended to teach a person of ordinary skill in the art how to practice the application. This description is not intended to detail all such obvious modifications and alterations that will become apparent to the skilled worker upon reading the description. However, it is intended that all such obvious modifications and variations be included within the scope of the present application, which is defined by the following claims. The claims are intended to cover the claimed elements and steps in any order that is effective to achieve their intended objects unless the context clearly indicates to the contrary.

Sequence listing

<110> Ai Nuojian Gene therapy Co., ltd

Wang Qi

In the road

<120> modified plasma factor VIII and methods of use thereof

<130> 23US0670AF-CN

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 7056

<212> DNA

<213> Chile person

<220>

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<223> human factor VIII

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catcacagtg gggacatggt atttacccct gagtcaggcc tccaattaag attaaatgag 2640

aaactgggga caactgcagc aacagagttg aagaaacttg atttcaaagt ttctagtaca 2700

tcaaataatc tgatttcaac aattccatca gacaatttgg cagcaggtac tgataataca 2760

agttccttag gacccccaag tatgccagtt cattatgata gtcaattaga taccactcta 2820

tttggcaaaa agtcatctcc ccttactgag tctggtggac ctctgagctt gagtgaagaa 2880

aataatgatt caaagttgtt agaatcaggt ttaatgaata gccaagaaag ttcatgggga 2940

aaaaatgtat cgtcaacaga gagtggtagg ttatttaaag ggaaaagagc tcatggacct 3000

gctttgttga ctaaagataa tgccttattc aaagttagca tctctttgtt aaagacaaac 3060

aaaacttcca ataattcagc aactaataga aagactcaca ttgatggccc atcattatta 3120

attgagaata gtccatcagt ctggcaaaat atattagaaa gtgacactga gtttaaaaaa 3180

gtgacacctt tgattcatga cagaatgctt atggacaaaa atgctacagc tttgaggcta 3240

aatcatatgt caaataaaac tacttcatca aaaaacatgg aaatggtcca acagaaaaaa 3300

gagggcccca ttccaccaga tgcacaaaat ccagatatgt cgttctttaa gatgctattc 3360

ttgccagaat cagcaaggtg gatacaaagg actcatggaa agaactctct gaactctggg 3420

caaggcccca gtccaaagca attagtatcc ttaggaccag aaaaatctgt ggaaggtcag 3480

aatttcttgt ctgagaaaaa caaagtggta gtaggaaagg gtgaatttac aaaggacgta 3540

ggactcaaag agatggtttt tccaagcagc agaaacctat ttcttactaa cttggataat 3600

ttacatgaaa ataatacaca caatcaagaa aaaaaaattc aggaagaaat agaaaagaag 3660

gaaacattaa tccaagagaa tgtagttttg cctcagatac atacagtgac tggcactaag 3720

aatttcatga agaacctttt cttactgagc actaggcaaa atgtagaagg ttcatatgac 3780

ggggcatatg ctccagtact tcaagatttt aggtcattaa atgattcaac aaatagaaca 3840

aagaaacaca cagctcattt ctcaaaaaaa ggggaggaag aaaacttgga aggcttggga 3900

aatcaaacca agcaaattgt agagaaatat gcatgcacca caaggatatc tcctaataca 3960

agccagcaga attttgtcac gcaacgtagt aagagagctt tgaaacaatt cagactccca 4020

ctagaagaaa cagaacttga aaaaaggata attgtggatg acacctcaac ccagtggtcc 4080

aaaaacatga aacatttgac cccgagcacc ctcacacaga tagactacaa tgagaaggag 4140

aaaggggcca ttactcagtc tcccttatca gattgcctta cgaggagtca tagcatccct 4200

caagcaaata gatctccatt acccattgca aaggtatcat catttccatc tattagacct 4260

atatatctga ccagggtcct attccaagac aactcttctc atcttccagc agcatcttat 4320

agaaagaaag attctggggt ccaagaaagc agtcatttct tacaaggagc caaaaaaaat 4380

aacctttctt tagccattct aaccttggag atgactggtg atcaaagaga ggttggctcc 4440

ctggggacaa gtgccacaaa ttcagtcaca tacaagaaag ttgagaacac tgttctcccg 4500

aaaccagact tgcccaaaac atctggcaaa gttgaattgc ttccaaaagt tcacatttat 4560

cagaaggacc tattccctac ggaaactagc aatgggtctc ctggccatct ggatctcgtg 4620

gaagggagcc ttcttcaggg aacagaggga gcgattaagt ggaatgaagc aaacagacct 4680

ggaaaagttc cctttctgag agtagcaaca gaaagctctg caaagactcc ctccaagcta 4740

ttggatcctc ttgcttggga taaccactat ggtactcaga taccaaaaga agagtggaaa 4800

tcccaagaga agtcaccaga aaaaacagct tttaagaaaa aggataccat tttgtccctg 4860

aacgcttgtg aaagcaatca tgcaatagca gcaataaatg agggacaaaa taagcccgaa 4920

atagaagtca cctgggcaaa gcaaggtagg actgaaaggc tgtgctctca aaacccacca 4980

gtcttgaaac gccatcaacg ggaaataact cgtactactc ttcagtcaga tcaagaggaa 5040

attgactatg atgataccat atcagttgaa atgaagaagg aagattttga catttatgat 5100

gaggatgaaa atcagagccc ccgcagcttt caaaagaaaa cacgacacta ttttattgct 5160

gcagtggaga ggctctggga ttatgggatg agtagctccc cacatgttct aagaaacagg 5220

gctcagagtg gcagtgtccc tcagttcaag aaagttgttt tccaggaatt tactgatggc 5280

tcctttactc agcccttata ccgtggagaa ctaaatgaac atttgggact cctggggcca 5340

tatataagag cagaagttga agataatatc atggtaactt tcagaaatca ggcctctcgt 5400

ccctattcct tctattctag ccttatttct tatgaggaag atcagaggca aggagcagaa 5460

cctagaaaaa actttgtcaa gcctaatgaa accaaaactt acttttggaa agtgcaacat 5520

catatggcac ccactaaaga tgagtttgac tgcaaagcct gggcttattt ctctgatgtt 5580

gacctggaaa aagatgtgca ctcaggcctg attggacccc ttctggtctg ccacactaac 5640

acactgaacc ctgctcatgg gagacaagtg acagtacagg aatttgctct gtttttcacc 5700

atctttgatg agaccaaaag ctggtacttc actgaaaata tggaaagaaa ctgcagggct 5760

ccctgcaata tccagatgga agatcccact tttaaagaga attatcgctt ccatgcaatc 5820

aatggctaca taatggatac actacctggc ttagtaatgg ctcaggatca aaggattcga 5880

tggtatctgc tcagcatggg cagcaatgaa aacatccatt ctattcattt cagtggacat 5940

gtgttcactg tacgaaaaaa agaggagtat aaaatggcac tgtacaatct ctatccaggt 6000

gtttttgaga cagtggaaat gttaccatcc aaagctggaa tttggcgggt ggaatgcctt 6060

attggcgagc atctacatgc tgggatgagc acactttttc tggtgtacag caataagtgt 6120

cagactcccc tgggaatggc ttctggacac attagagatt ttcagattac agcttcagga 6180

caatatggac agtgggcccc aaagctggcc agacttcatt attccggatc aatcaatgcc 6240

tggagcacca aggagccctt ttcttggatc aaggtggatc tgttggcacc aatgattatt 6300

cacggcatca agacccaggg tgcccgtcag aagttctcca gcctctacat ctctcagttt 6360

atcatcatgt atagtcttga tgggaagaag tggcagactt atcgaggaaa ttccactgga 6420

accttaatgg tcttctttgg caatgtggat tcatctggga taaaacacaa tatttttaac 6480

cctccaatta ttgctcgata catccgtttg cacccaactc attatagcat tcgcagcact 6540

cttcgcatgg agttgatggg ctgtgattta aatagttgca gcatgccatt gggaatggag 6600

agtaaagcaa tatcagatgc acagattact gcttcatcct actttaccaa tatgtttgcc 6660

acctggtctc cttcaaaagc tcgacttcac ctccaaggga ggagtaatgc ctggagacct 6720

caggtgaata atccaaaaga gtggctgcaa gtggacttcc agaagacaat gaaagtcaca 6780

ggagtaacta ctcagggagt aaaatctctg cttaccagca tgtatgtgaa ggagttcctc 6840

atctccagca gtcaagatgg ccatcagtgg actctctttt ttcagaatgg caaagtaaag 6900

gtttttcagg gaaatcaaga ctccttcaca cctgtggtga actctctaga cccaccgtta 6960

ctgactcgct accttcgaat tcacccccag agttgggtgc accagattgc cctgaggatg 7020

gaggttctgg gctgcgaggc acaggacctc tactga 7056

<210> 2

<211> 19

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<223> human factor VIII Signal peptide

<400> 2

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser

<210> 3

<211> 2351

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<223> human factor VIII with Signal peptide

<400> 3

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser

20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg

35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val

50 55 60

Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile

65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln

85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser

100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser

115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp

130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu

145 150 155 160

Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser

165 170 175

Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile

180 185 190

Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr

195 200 205

Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly

210 215 220

Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp

225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr

245 250 255

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val

260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile

275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser

290 295 300

Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met

305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His

325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro

340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp

355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser

370 375 380

Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr

385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro

405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn

420 425 430

Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met

435 440 445

Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu

450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu

465 470 475 480

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro

485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys

500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe

515 520 525

Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp

530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg

545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu

565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val

580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu

595 600 605

Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp

610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val

625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp

645 650 655

Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe

660 665 670

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr

675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro

690 695 700

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly

705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp

725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys

740 745 750

Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro

755 760 765

Ser Thr Arg Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp

770 775 780

Ile Glu Lys Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys

785 790 795 800

Ile Gln Asn Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser

805 810 815

Pro Thr Pro His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr

820 825 830

Glu Thr Phe Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn

835 840 845

Ser Leu Ser Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly

850 855 860

Asp Met Val Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu

865 870 875 880

Lys Leu Gly Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys

885 890 895

Val Ser Ser Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn

900 905 910

Leu Ala Ala Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met

915 920 925

Pro Val His Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys

930 935 940

Ser Ser Pro Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu

945 950 955 960

Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu

965 970 975

Ser Ser Trp Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe

980 985 990

Lys Gly Lys Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala

995 1000 1005

Leu Phe Lys Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser

1010 1015 1020

Asn Asn Ser Ala Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser

1025 1030 1035

Leu Leu Ile Glu Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu

1040 1045 1050

Ser Asp Thr Glu Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg

1055 1060 1065

Met Leu Met Asp Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met

1070 1075 1080

Ser Asn Lys Thr Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln

1085 1090 1095

Lys Lys Glu Gly Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met

1100 1105 1110

Ser Phe Phe Lys Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile

1115 1120 1125

Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro

1130 1135 1140

Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu

1145 1150 1155

Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys

1160 1165 1170

Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro

1175 1180 1185

Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu

1190 1195 1200

Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu

1205 1210 1215

Lys Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile

1220 1225 1230

His Thr Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu

1235 1240 1245

Leu Ser Thr Arg Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr

1250 1255 1260

Ala Pro Val Leu Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn

1265 1270 1275

Arg Thr Lys Lys His Thr Ala His Phe Ser Lys Lys Gly Glu Glu

1280 1285 1290

Glu Asn Leu Glu Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu

1295 1300 1305

Lys Tyr Ala Cys Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln

1310 1315 1320

Asn Phe Val Thr Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg

1325 1330 1335

Leu Pro Leu Glu Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp

1340 1345 1350

Asp Thr Ser Thr Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro

1355 1360 1365

Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala

1370 1375 1380

Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser

1385 1390 1395

Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser

1400 1405 1410

Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe

1415 1420 1425

Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys

1430 1435 1440

Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys

1445 1450 1455

Lys Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly

1460 1465 1470

Asp Gln Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser

1475 1480 1485

Val Thr Tyr Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp

1490 1495 1500

Leu Pro Lys Thr Ser Gly Lys Val Glu Leu Leu Pro Lys Val His

1505 1510 1515

Ile Tyr Gln Lys Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser

1520 1525 1530

Pro Gly His Leu Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr

1535 1540 1545

Glu Gly Ala Ile Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val

1550 1555 1560

Pro Phe Leu Arg Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser

1565 1570 1575

Lys Leu Leu Asp Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln

1580 1585 1590

Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys

1595 1600 1605

Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys

1610 1615 1620

Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys

1625 1630 1635

Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg

1640 1645 1650

Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu

1655 1660 1665

Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr

1670 1675 1680

Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile

1685 1690 1695

Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys

1700 1705 1710

Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr

1715 1720 1725

Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser

1730 1735 1740

Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr

1745 1750 1755

Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu

1760 1765 1770

His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp

1775 1780 1785

Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser

1790 1795 1800

Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly

1805 1810 1815

Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr

1820 1825 1830

Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu

1835 1840 1845

Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu

1850 1855 1860

Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His

1865 1870 1875

Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln

1880 1885 1890

Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp

1895 1900 1905

Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn

1910 1915 1920

Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His

1925 1930 1935

Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met

1940 1945 1950

Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser

1955 1960 1965

Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr

1970 1975 1980

Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr

1985 1990 1995

Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly

2000 2005 2010

Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly

2015 2020 2025

Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro

2030 2035 2040

Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala

2045 2050 2055

Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His

2060 2065 2070

Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser

2075 2080 2085

Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile

2090 2095 2100

Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser

2105 2110 2115

Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr

2120 2125 2130

Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn

2135 2140 2145

Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile

2150 2155 2160

Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg

2165 2170 2175

Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys

2180 2185 2190

Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln

2195 2200 2205

Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser

2210 2215 2220

Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp

2225 2230 2235

Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe

2240 2245 2250

Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys

2255 2260 2265

Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser

2270 2275 2280

Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys

2285 2290 2295

Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val

2300 2305 2310

Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His

2315 2320 2325

Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu

2330 2335 2340

Gly Cys Glu Ala Gln Asp Leu Tyr

2345 2350

<210> 4

<211> 2332

<212> PRT

<213> Chile person

<220>

<221> misc_feature

<223> Signal peptide-free human factor VIII

<400> 4

Ala 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

<210> 5

<211> 1457

<212> PRT

<213> Artificial work

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> human factor VIII without B domain with Signal peptide (hBDDF 8)

<400> 5

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser

20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg

35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val

50 55 60

Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile

65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln

85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser

100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser

115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp

130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu

145 150 155 160

Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser

165 170 175

Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile

180 185 190

Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr

195 200 205

Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly

210 215 220

Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp

225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr

245 250 255

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val

260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile

275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser

290 295 300

Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met

305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His

325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro

340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp

355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser

370 375 380

Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr

385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro

405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn

420 425 430

Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met

435 440 445

Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu

450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu

465 470 475 480

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro

485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys

500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe

515 520 525

Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp

530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg

545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu

565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val

580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu

595 600 605

Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp

610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val

625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp

645 650 655

Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe

660 665 670

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr

675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro

690 695 700

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly

705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp

725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys

740 745 750

Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu

755 760 765

Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln

770 775 780

Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu

785 790 795 800

Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe

805 810 815

Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp

820 825 830

Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln

835 840 845

Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr

850 855 860

Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His

865 870 875 880

Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile

885 890 895

Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser

900 905 910

Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg

915 920 925

Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val

930 935 940

Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp

945 950 955 960

Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu

965 970 975

Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His

980 985 990

Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe

995 1000 1005

Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn

1010 1015 1020

Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys

1025 1030 1035

Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr

1040 1045 1050

Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr

1055 1060 1065

Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe

1070 1075 1080

Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met

1085 1090 1095

Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met

1100 1105 1110

Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly

1115 1120 1125

Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser

1130 1135 1140

Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg

1145 1150 1155

Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro

1160 1165 1170

Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser

1175 1180 1185

Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro

1190 1195 1200

Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe

1205 1210 1215

Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp

1220 1225 1230

Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu

1235 1240 1245

Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn

1250 1255 1260

Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro

1265 1270 1275

Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly

1280 1285 1290

Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys

1295 1300 1305

Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn

1310 1315 1320

Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln

1325 1330 1335

Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu

1340 1345 1350

Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val

1355 1360 1365

Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys

1370 1375 1380

Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu

1385 1390 1395

Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp

1400 1405 1410

Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr

1415 1420 1425

Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala

1430 1435 1440

Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr

1445 1450 1455

<210> 6

<211> 1438

<212> PRT

<213> Artificial work

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> B-domain-free human factor VIII without native Signal peptide (hBDDF 8)

<400> 6

Ala 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 Pro Pro Val Leu Lys Arg His

740 745 750

Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile

755 760 765

Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp

770 775 780

Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys

785 790 795 800

Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly

805 810 815

Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser

820 825 830

Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser

835 840 845

Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu

850 855 860

Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr

865 870 875 880

Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile

885 890 895

Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe

900 905 910

Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His

915 920 925

Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe

930 935 940

Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro

945 950 955 960

Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln

965 970 975

Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr

980 985 990

Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro

995 1000 1005

Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg

1010 1015 1020

Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu

1025 1030 1035

Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met

1040 1045 1050

Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val

1055 1060 1065

Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn

1070 1075 1080

Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys

1085 1090 1095

Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His

1100 1105 1110

Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln

1115 1120 1125

Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile

1130 1135 1140

Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg

1145 1150 1155

Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro

1160 1165 1170

Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His

1175 1180 1185

Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr

1190 1195 1200

Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp

1205 1210 1215

Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe

1220 1225 1230

Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro

1235 1240 1245

Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser

1250 1255 1260

Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn

1265 1270 1275

Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp

1280 1285 1290

Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr

1295 1300 1305

Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn

1310 1315 1320

Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val

1325 1330 1335

Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly

1340 1345 1350

Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile

1355 1360 1365

Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn

1370 1375 1380

Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro

1385 1390 1395

Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg

1400 1405 1410

Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu

1415 1420 1425

Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr

1430 1435

<210> 7

<211> 745

<212> PRT

<213> human factor VIII heavy chain in hBDDF8 (without Signal peptide)

<400> 7

Ala 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

740 745

<210> 8

<211> 13

<212> PRT

<213> truncated B Domain in hBDDF8

<400> 8

Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln

1 5 10

<210> 9

<211> 693

<212> PRT

<213> human FVIII light chain in hBDDF8

<400> 9

Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu

1 5 10 15

Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu

20 25 30

Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser

35 40 45

Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val

50 55 60

Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg

65 70 75 80

Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe

85 90 95

Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu

100 105 110

Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val

115 120 125

Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr

130 135 140

Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly

145 150 155 160

Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr

165 170 175

Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp

180 185 190

Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val

195 200 205

His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu

210 215 220

Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe

225 230 235 240

Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met

245 250 255

Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr

260 265 270

Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp

275 280 285

Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr

290 295 300

Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser

305 310 315 320

Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu

325 330 335

Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser

340 345 350

Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His

355 360 365

Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr

370 375 380

Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala

385 390 395 400

Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr

405 410 415

Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile

420 425 430

Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln

435 440 445

Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile

450 455 460

Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser

465 470 475 480

Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile

485 490 495

Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu

500 505 510

His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met

515 520 525

Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys

530 535 540

Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met

545 550 555 560

Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg

565 570 575

Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln

580 585 590

Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly

595 600 605

Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser

610 615 620

Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys

625 630 635 640

Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn

645 650 655

Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln

660 665 670

Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu

675 680 685

Ala Gln Asp Leu Tyr

690

<210> 10

<211> 1457

<212> PRT

<213> hHC-cLC having Signal peptide

<400> 10

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser

20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg

35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val

50 55 60

Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile

65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln

85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser

100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser

115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp

130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu

145 150 155 160

Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser

165 170 175

Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile

180 185 190

Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr

195 200 205

Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly

210 215 220

Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp

225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr

245 250 255

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val

260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile

275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser

290 295 300

Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met

305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His

325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro

340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp

355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser

370 375 380

Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr

385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro

405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn

420 425 430

Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met

435 440 445

Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu

450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu

465 470 475 480

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro

485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys

500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe

515 520 525

Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp

530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg

545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu

565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val

580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu

595 600 605

Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp

610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val

625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp

645 650 655

Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe

660 665 670

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr

675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro

690 695 700

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly

705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp

725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys

740 745 750

Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Ser

755 760 765

Lys His His Gln Arg Glu Ile Thr Val Thr Thr Leu Gln Pro Glu Glu

770 775 780

Asp Lys Phe Glu Tyr Asp Asp Thr Phe Ser Ile Glu Met Lys Arg Glu

785 790 795 800

Asp Phe Asp Ile Tyr Gly Asp Tyr Glu Asn Gln Gly Leu Arg Ser Phe

805 810 815

Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp

820 825 830

Asp Tyr Gly Met Ser Arg Ser Pro His Ile Leu Arg Asn Arg Ala Gln

835 840 845

Ser Gly Asp Val Gln Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr

850 855 860

Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His

865 870 875 880

Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile

885 890 895

Val Val Thr Phe Lys Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser

900 905 910

Ser Leu Ile Ser Tyr Asp Glu Asp Glu Gly Gln Gly Ala Glu Pro Arg

915 920 925

Arg Lys Phe Val Asn Pro Asn Glu Thr Lys Ile Tyr Phe Trp Lys Val

930 935 940

Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp

945 950 955 960

Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu

965 970 975

Ile Gly Pro Leu Leu Ile Cys Arg Ser Asn Thr Leu Asn Pro Ala His

980 985 990

Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Val Phe Thr Ile Phe

995 1000 1005

Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Leu Glu Arg Asn

1010 1015 1020

Cys Arg Ala Pro Cys Asn Val Gln Lys Glu Asp Pro Thr Leu Lys

1025 1030 1035

Glu Asn Phe Arg Phe His Ala Ile Asn Gly Tyr Val Lys Asp Thr

1040 1045 1050

Leu Pro Gly Leu Val Met Ala Gln Asp Gln Lys Val Arg Trp Tyr

1055 1060 1065

Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe

1070 1075 1080

Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met

1085 1090 1095

Ala Val Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met

1100 1105 1110

Leu Pro Ser Gln Val Gly Ile Trp Arg Ile Glu Cys Leu Ile Gly

1115 1120 1125

Glu His Leu Gln Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser

1130 1135 1140

Lys Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg

1145 1150 1155

Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro

1160 1165 1170

Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser

1175 1180 1185

Thr Lys Asp Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro

1190 1195 1200

Met Ile Ile His Gly Ile Met Thr Gln Gly Ala Arg Gln Lys Phe

1205 1210 1215

Ser Ser Leu Tyr Val Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp

1220 1225 1230

Gly Asn Lys Trp His Ser Tyr Arg Gly Asn Ser Thr Gly Thr Leu

1235 1240 1245

Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn

1250 1255 1260

Ile Phe Asn Pro Pro Ile Ile Ala Gln Tyr Ile Arg Leu His Pro

1265 1270 1275

Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Leu Gly

1280 1285 1290

Cys Asp Phe Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys

1295 1300 1305

Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Leu Ser Ser

1310 1315 1320

Met Leu Ala Thr Trp Ser Pro Ser Gln Ala Arg Leu His Leu Gln

1325 1330 1335

Gly Arg Thr Asn Ala Trp Arg Pro Gln Ala Asn Asn Pro Lys Glu

1340 1345 1350

Trp Leu Gln Val Asp Phe Arg Lys Thr Met Lys Val Thr Gly Ile

1355 1360 1365

Thr Thr Gln Gly Val Lys Ser Leu Leu Ile Ser Met Tyr Val Lys

1370 1375 1380

Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Asn Trp Thr Leu

1385 1390 1395

Phe Leu Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Arg Asp

1400 1405 1410

Ser Ser Thr Pro Val Arg Asn Ala Leu Glu Pro Pro Leu Val Ala

1415 1420 1425

Arg Tyr Val Arg Leu His Pro Gln Ser Trp Ala His His Ile Ala

1430 1435 1440

Leu Arg Leu Glu Val Leu Gly Cys Asp Thr Gln Gln Pro Ala

1445 1450 1455

<210> 11

<211> 4374

<212> DNA

<213> hHC-cLC cDNA

<400> 11

atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60

accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120

ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180

acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240

gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300

gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360

ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420

gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480

aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540

gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600

gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660

tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720

gctgcatctg ctcgtgcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780

ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840

accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900

cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960

gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020

gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080

gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140

gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200

tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260

cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320

aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380

attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440

ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500

gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560

ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620

actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680

gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740

agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800

aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860

cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920

tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980

attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040

atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100

atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160

atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220

agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280

ttctcccaga atccaccagt ctcaaaacac catcaaaggg aaataaccgt tactactctt 2340

cagccagagg aagacaaatt tgagtatgat gacaccttct caattgaaat gaagagagaa 2400

gattttgaca tctacggcga ctatgaaaat cagggcctcc gcagctttca aaagaaaaca 2460

cgacactatt tcattgctgc agtggagcgt ctctgggatt atgggatgag tagatctccc 2520

catatactaa gaaacagggc tcaaagtggg gatgtccagc agttcaagaa ggtggttttc 2580

caggaattta ctgatggatc ctttactcag cccttatacc gtggagaact gaatgaacac 2640

ttgggactct tggggccata tataagagca gaagttgaag acaatatcgt ggtaactttc 2700

aaaaaccagg cctctcgtcc ctactccttc tattctagtc ttatttctta tgacgaagat 2760

gagggacaag gagcagaacc tagaagaaag tttgtcaacc ctaatgaaac caaaatttac 2820

ttttggaaag tgcagcatca tatggcaccc actaaagatg agtttgactg caaagcctgg 2880

gcttattttt ctgatgttga tttggagaaa gatgtgcact caggcttgat tggacccctt 2940

ctgatctgcc gcagtaacac actgaaccct gctcatggga gacaagtgac agtgcaggag 3000

tttgccctgg ttttcactat attcgatgag actaagagct ggtacttcac tgaaaacctg 3060

gaaaggaact gtagagctcc ctgcaatgtc cagaaggagg accctactct aaaagaaaac 3120

ttccgcttcc atgcaatcaa cggctatgtg aaggatacac tccctggctt agtaatggct 3180

caggatcaaa aggttcgatg gtatctgctc agcatgggca gcaacgaaaa cattcattcc 3240

attcacttca gtggacatgt gttcactgta cggaaaaaag aggaatataa aatggcagtc 3300

tacaacctct atccaggtgt ttttgagact gtggaaatgc taccatccca agttggaatc 3360

tggcggatag aatgccttat cggcgagcac ctgcaagccg ggatgagcac tctgtttctg 3420

gtgtacagca agaagtgtca gactccactg gggatggctt ccggacacat tagagatttt 3480

cagattacag cttcaggaca atatggacag tgggccccaa agctggccag acttcattat 3540

tccggatcaa tcaatgcctg gagcaccaag gatccctttt cctggatcaa ggtggatctc 3600

ttggcaccga tgattattca cggcatcatg acccaggggg cccgccagaa gttctccagc 3660

ctctacgtgt ctcagtttat catcatgtac agtctggatg gcaacaagtg gcacagttac 3720

cgagggaatt ccacggggac cttaatggtc ttctttggca acgtggattc atctgggatc 3780

aaacacaata tttttaaccc tccgattatt gctcagtaca tccgtttgca cccaacccat 3840

tacagcatcc gcagcactct tcgcatggag ctcttgggct gtgacttcaa cagttgcagc 3900

atgccgctgg ggatggagag taaagcaata tcagatgctc agatcactgc ctcgtcctac 3960

ctaagcagta tgcttgccac ttggtctcct tcccaagccc ggctgcacct gcagggcagg 4020

actaatgcct ggagacctca ggcaaataac ccaaaagagt ggctgcaagt ggacttccgg 4080

aagaccatga aagtcacagg aataaccacc cagggggtga aatctctcct catcagcatg 4140

tatgtgaagg agttcctcat ctccagtagt caagatggcc ataactggac tctgtttctt 4200

cagaatggca aagtcaaggt cttccaggga aaccgggact cctccacgcc tgtgcggaac 4260

gctctcgaac ccccgctggt ggctcgctac gtgcgcctgc acccgcagag ctgggcgcac 4320

cacatcgccc tgaggctgga ggtcctgggc tgcgacaccc agcagcccgc ctga 4374

<210> 12

<211> 1438

<212> PRT

<213> hHC-cLC without Signal peptide

<400> 12

Ala 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 Pro Pro Val Ser Lys His His

740 745 750

Gln Arg Glu Ile Thr Val Thr Thr Leu Gln Pro Glu Glu Asp Lys Phe

755 760 765

Glu Tyr Asp Asp Thr Phe Ser Ile Glu Met Lys Arg Glu Asp Phe Asp

770 775 780

Ile Tyr Gly Asp Tyr Glu Asn Gln Gly Leu Arg Ser Phe Gln Lys Lys

785 790 795 800

Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly

805 810 815

Met Ser Arg Ser Pro His Ile Leu Arg Asn Arg Ala Gln Ser Gly Asp

820 825 830

Val Gln Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser

835 840 845

Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu

850 855 860

Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Val Val Thr

865 870 875 880

Phe Lys Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile

885 890 895

Ser Tyr Asp Glu Asp Glu Gly Gln Gly Ala Glu Pro Arg Arg Lys Phe

900 905 910

Val Asn Pro Asn Glu Thr Lys Ile Tyr Phe Trp Lys Val Gln His His

915 920 925

Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe

930 935 940

Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro

945 950 955 960

Leu Leu Ile Cys Arg Ser Asn Thr Leu Asn Pro Ala His Gly Arg Gln

965 970 975

Val Thr Val Gln Glu Phe Ala Leu Val Phe Thr Ile Phe Asp Glu Thr

980 985 990

Lys Ser Trp Tyr Phe Thr Glu Asn Leu Glu Arg Asn Cys Arg Ala Pro

995 1000 1005

Cys Asn Val Gln Lys Glu Asp Pro Thr Leu Lys Glu Asn Phe Arg

1010 1015 1020

Phe His Ala Ile Asn Gly Tyr Val Lys Asp Thr Leu Pro Gly Leu

1025 1030 1035

Val Met Ala Gln Asp Gln Lys Val Arg Trp Tyr Leu Leu Ser Met

1040 1045 1050

Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val

1055 1060 1065

Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Val Tyr Asn

1070 1075 1080

Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Gln

1085 1090 1095

Val Gly Ile Trp Arg Ile Glu Cys Leu Ile Gly Glu His Leu Gln

1100 1105 1110

Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Lys Lys Cys Gln

1115 1120 1125

Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile

1130 1135 1140

Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg

1145 1150 1155

Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Asp Pro

1160 1165 1170

Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His

1175 1180 1185

Gly Ile Met Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr

1190 1195 1200

Val Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Asn Lys Trp

1205 1210 1215

His Ser Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe

1220 1225 1230

Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro

1235 1240 1245

Pro Ile Ile Ala Gln Tyr Ile Arg Leu His Pro Thr His Tyr Ser

1250 1255 1260

Ile Arg Ser Thr Leu Arg Met Glu Leu Leu Gly Cys Asp Phe Asn

1265 1270 1275

Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp

1280 1285 1290

Ala Gln Ile Thr Ala Ser Ser Tyr Leu Ser Ser Met Leu Ala Thr

1295 1300 1305

Trp Ser Pro Ser Gln Ala Arg Leu His Leu Gln Gly Arg Thr Asn

1310 1315 1320

Ala Trp Arg Pro Gln Ala Asn Asn Pro Lys Glu Trp Leu Gln Val

1325 1330 1335

Asp Phe Arg Lys Thr Met Lys Val Thr Gly Ile Thr Thr Gln Gly

1340 1345 1350

Val Lys Ser Leu Leu Ile Ser Met Tyr Val Lys Glu Phe Leu Ile

1355 1360 1365

Ser Ser Ser Gln Asp Gly His Asn Trp Thr Leu Phe Leu Gln Asn

1370 1375 1380

Gly Lys Val Lys Val Phe Gln Gly Asn Arg Asp Ser Ser Thr Pro

1385 1390 1395

Val Arg Asn Ala Leu Glu Pro Pro Leu Val Ala Arg Tyr Val Arg

1400 1405 1410

Leu His Pro Gln Ser Trp Ala His His Ile Ala Leu Arg Leu Glu

1415 1420 1425

Val Leu Gly Cys Asp Thr Gln Gln Pro Ala

1430 1435

<210> 13

<211> 13

<212> PRT

<213> hHC-cLC truncated B Domain

<400> 13

Ser Phe Ser Gln Asn Pro Pro Val Ser Lys His His Gln

1 5 10

<210> 14

<211> 693

<212> PRT

Canine FVIII light chain in <213> hHC-cLC

<400> 14

Pro Pro Val Ser Lys His His Gln Arg Glu Ile Thr Val Thr Thr Leu

1 5 10 15

Gln Pro Glu Glu Asp Lys Phe Glu Tyr Asp Asp Thr Phe Ser Ile Glu

20 25 30

Met Lys Arg Glu Asp Phe Asp Ile Tyr Gly Asp Tyr Glu Asn Gln Gly

35 40 45

Leu Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val

50 55 60

Glu Arg Leu Trp Asp Tyr Gly Met Ser Arg Ser Pro His Ile Leu Arg

65 70 75 80

Asn Arg Ala Gln Ser Gly Asp Val Gln Gln Phe Lys Lys Val Val Phe

85 90 95

Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu

100 105 110

Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val

115 120 125

Glu Asp Asn Ile Val Val Thr Phe Lys Asn Gln Ala Ser Arg Pro Tyr

130 135 140

Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Asp Glu Asp Glu Gly Gln Gly

145 150 155 160

Ala Glu Pro Arg Arg Lys Phe Val Asn Pro Asn Glu Thr Lys Ile Tyr

165 170 175

Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp

180 185 190

Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val

195 200 205

His Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Arg Ser Asn Thr Leu

210 215 220

Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Val

225 230 235 240

Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Leu

245 250 255

Glu Arg Asn Cys Arg Ala Pro Cys Asn Val Gln Lys Glu Asp Pro Thr

260 265 270

Leu Lys Glu Asn Phe Arg Phe His Ala Ile Asn Gly Tyr Val Lys Asp

275 280 285

Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln Lys Val Arg Trp Tyr

290 295 300

Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser

305 310 315 320

Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Val

325 330 335

Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser

340 345 350

Gln Val Gly Ile Trp Arg Ile Glu Cys Leu Ile Gly Glu His Leu Gln

355 360 365

Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser Lys Lys Cys Gln Thr

370 375 380

Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala

385 390 395 400

Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr

405 410 415

Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Asp Pro Phe Ser Trp Ile

420 425 430

Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Met Thr Gln

435 440 445

Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Val Ser Gln Phe Ile Ile

450 455 460

Met Tyr Ser Leu Asp Gly Asn Lys Trp His Ser Tyr Arg Gly Asn Ser

465 470 475 480

Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile

485 490 495

Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Gln Tyr Ile Arg Leu

500 505 510

His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Leu

515 520 525

Gly Cys Asp Phe Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys

530 535 540

Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Leu Ser Ser Met

545 550 555 560

Leu Ala Thr Trp Ser Pro Ser Gln Ala Arg Leu His Leu Gln Gly Arg

565 570 575

Thr Asn Ala Trp Arg Pro Gln Ala Asn Asn Pro Lys Glu Trp Leu Gln

580 585 590

Val Asp Phe Arg Lys Thr Met Lys Val Thr Gly Ile Thr Thr Gln Gly

595 600 605

Val Lys Ser Leu Leu Ile Ser Met Tyr Val Lys Glu Phe Leu Ile Ser

610 615 620

Ser Ser Gln Asp Gly His Asn Trp Thr Leu Phe Leu Gln Asn Gly Lys

625 630 635 640

Val Lys Val Phe Gln Gly Asn Arg Asp Ser Ser Thr Pro Val Arg Asn

645 650 655

Ala Leu Glu Pro Pro Leu Val Ala Arg Tyr Val Arg Leu His Pro Gln

660 665 670

Ser Trp Ala His His Ile Ala Leu Arg Leu Glu Val Leu Gly Cys Asp

675 680 685

Thr Gln Gln Pro Ala

690

<210> 15

<211> 9618

<212> DNA

<213> pANG-CAG-hBDDF8

<400> 15

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtgccaac tccatcacta 120

ggggttcctt gtagttaatg attaacccgc catgctactt atttacgtag ccatgctcta 180

ggtaccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 240

ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 300

tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 360

tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 420

cgctattacc atgtcgaggc cacgttctgc ttcactctcc ccatctcccc cccctcccca 480

cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg 540

gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag 600

gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc 660

ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagcaagc tctagccgcg 720

cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg 780

cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc 840

ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg 900

ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg 960

ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg 1020

gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct 1080

acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgat 1140

ccacactttt tctttttctc cacaggtatc gattccacca tgcaaataga gctctccacc 1200

tgcttctttc tgtgcctttt gcgattctgc tttagtgcca ccagaagata ctacctgggt 1260

gcagtggaac tgtcatggga ctatatgcaa agtgatctcg gtgagctgcc tgtggacgca 1320

agatttcctc ctagagtgcc aaaatctttt ccattcaaca cctcagtcgt gtacaaaaag 1380

actctgtttg tagaattcac ggatcacctt ttcaacatcg ctaagccaag gccaccctgg 1440

atgggtctgc taggtcctac catccaggct gaggtttatg atacagtggt cattacactt 1500

aagaacatgg cttcccatcc tgtcagtctt catgctgttg gtgtatccta ctggaaagct 1560

tctgagggag ctgaatatga tgatcagacc agtcaaaggg agaaagaaga tgataaagtc 1620

ttccctggtg gaagccatac atatgtctgg caggtcctga aagagaatgg tccaatggcc 1680

tctgacccac tgtgccttac ctactcatat ctttctcatg tggacctggt aaaagacttg 1740

aattcaggcc tcattggagc cctactagta tgtagagaag ggagtctggc caaggaaaag 1800

acacagacct tgcacaaatt tatactactt tttgctgtat ttgatgaagg gaaaagttgg 1860

cactcagaaa caaagaactc cttgatgcag gatagggatg ctgcatctgc tcgggcctgg 1920

cctaaaatgc acacagtcaa tggttatgta aacaggtctc tgccaggtct gattggatgc 1980

cacaggaaat cagtctattg gcatgtgatt ggaatgggca ccactcctga agtgcactca 2040

atattcctcg aaggtcacac atttcttgtg aggaaccatc gccaggcgtc cttggaaatc 2100

tcgccaataa ctttccttac tgctcaaaca ctcttgatgg accttggaca gtttctactg 2160

ttttgtcata tctcttccca ccaacatgat ggcatggaag cttatgtcaa agtagacagc 2220

tgtccagagg aaccccaact acgaatgaaa aataatgaag aagcggaaga ctatgatgat 2280

gatcttactg attctgaaat ggatgtggtc aggtttgatg atgacaactc tccttccttt 2340

atccaaattc gctcagttgc caagaagcat cctaaaactt gggtacatta cattgctgct 2400

gaagaggagg actgggacta tgctccctta gtcctcgccc ccgatgacag aagttataaa 2460

agtcaatatt tgaacaatgg ccctcagcgg attggtagga agtacaaaaa agtccgattt 2520

atggcataca cagatgaaac ctttaagact cgtgaagcta ttcagcatga atcaggaatc 2580

ttgggacctt tactttatgg ggaagttgga gacacactgt tgattatatt taagaatcaa 2640

gcaagcagac catataacat ctaccctcac ggaatcactg atgtccgtcc tttgtattca 2700

aggagattac caaaaggtgt aaaacatttg aaggattttc caattctgcc aggagaaata 2760

ttcaaatata aatggacagt gactgtagaa gatgggccaa ctaaatcaga tcctcggtgc 2820

ctgacccgct attactctag tttcgttaat atggagagag atctagcttc aggactcatt 2880

ggccctctcc tcatctgcta caaagaatct gtagatcaaa gaggaaacca gataatgtca 2940

gacaagagga atgtcatcct gttttctgta tttgatgaga accgaagctg gtacctcaca 3000

gagaatatac aacgctttct ccccaatcca gctggagtgc agcttgagga tccagagttc 3060

caagcctcca acatcatgca cagcatcaat ggctatgttt ttgatagttt gcagttgtca 3120

gtttgtttgc atgaggtggc atactggtac attctaagca ttggagcaca gactgacttc 3180

ctttctgtct tcttctctgg atataccttc aaacacaaaa tggtctatga agacacactc 3240

accctattcc cattctcagg agaaactgtc ttcatgtcga tggaaaaccc aggtctatgg 3300

attctggggt gccacaactc agactttcgg aacagaggca tgaccgcctt actgaaggtt 3360

tctagttgtg acaagaacac tggtgattat tacgaggaca gttatgaaga tatttcagca 3420

tacttgctga gtaaaaacaa tgccattgaa ccaagaagct tctcccagaa tccaccagtc 3480

ttgaaacgcc atcaacgcga aataactcgt actactcttc agtcagatca agaggaaatt 3540

gactatgatg ataccatatc agttgaaatg aagaaggaag attttgacat ttatgatgag 3600

gatgaaaatc agagcccccg cagctttcaa aagaaaacac gacactattt tattgctgca 3660

gtggagaggc tctgggatta tgggatgagt agctccccac atgttctaag aaacagggct 3720

cagagtggca gtgtccctca gttcaagaaa gttgttttcc aggaatttac tgatggctcc 3780

tttactcagc ccttataccg tggagaacta aatgaacatt tgggactcct ggggccatat 3840

ataagagcag aagttgaaga taatatcatg gtaactttca gaaatcaggc ctctcgtccc 3900

tattccttct attctagcct tatttcttat gaggaagatc agaggcaagg agcagaacct 3960

agaaaaaact ttgtcaagcc taatgaaacc aaaacttact tttggaaagt gcaacatcat 4020

atggcaccca ctaaagatga gtttgactgc aaagcctggg cttatttctc tgatgttgac 4080

ctggaaaaag atgtgcactc aggcctgatt ggaccccttc tggtctgcca cactaacaca 4140

ctgaaccctg ctcatgggag acaagtgaca gtacaggaat ttgctctgtt tttcaccatc 4200

tttgatgaga ccaaaagctg gtacttcact gaaaatatgg aaagaaactg cagggctccc 4260

tgcaatatcc agatggaaga tcccactttt aaagagaatt atcgcttcca tgcaatcaat 4320

ggctacataa tggatacact acctggctta gtaatggctc aggatcaaag gattcgatgg 4380

tatctgctca gcatgggcag caatgaaaac atccattcta ttcatttcag tggacatgtg 4440

ttcactgtac gaaaaaaaga ggagtataaa atggcactgt acaatctcta tccaggtgtt 4500

tttgagacag tggaaatgtt accatccaaa gctggaattt ggcgggtgga atgccttatt 4560

ggcgagcatc tacatgctgg gatgagcaca ctttttctgg tgtacagcaa taagtgtcag 4620

actcccctgg gaatggcttc tggacacatt agagattttc agattacagc ttcaggacaa 4680

tatggacagt gggccccaaa gctggccaga cttcattatt ccggatcaat caatgcctgg 4740

agcaccaagg agcccttttc ttggatcaag gtggatctgt tggcaccaat gattattcac 4800

ggcatcaaga cccagggtgc ccgtcagaag ttctccagcc tctacatctc tcagtttatc 4860

atcatgtata gtcttgatgg gaagaagtgg cagacttatc gaggaaattc cactggaacc 4920

ttaatggtct tctttggcaa tgtggattca tctgggataa aacacaatat ttttaaccct 4980

ccaattattg ctcgatacat ccgtttgcac ccaactcatt atagcattcg cagcactctt 5040

cgcatggagt tgatgggctg tgatttaaat agttgcagca tgccattggg aatggagagt 5100

aaagcaatat cagatgcaca gattactgct tcatcctact ttaccaatat gtttgccacc 5160

tggtctcctt caaaagctcg acttcacctc caagggagga gtaatgcctg gagacctcag 5220

gtgaataatc caaaagagtg gctgcaagtg gacttccaga agacaatgaa agtcacagga 5280

gtaactactc agggagtaaa atctctgctt accagcatgt atgtgaagga gttcctcatc 5340

tccagcagtc aagatggcca tcagtggact ctcttttttc agaatggcaa agtaaaggtt 5400

tttcagggaa atcaagactc cttcacacct gtggtgaact ctctagaccc accgttactg 5460

actcgctacc ttcgaattca cccccagagt tgggtgcacc agattgccct gaggatggag 5520

gttctgggct gcgaggcaca ggacctctac tgactcgaga ataaaagatc agagctctag 5580

agatctgtgt gttggttttt tgtgtgcggc cgggatctga ggaaccccta gtgatggagt 5640

tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc 5700

gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg 5760

ccaacccccc cccccccccc cctgcaggcg attctcttgt ttgctccaga ctctcaggca 5820

atgacctgat agcctttgta gagacctctc aaaaatagct accctctccg gcatgaattt 5880

atcagctaga acggttgaat atcatattga tggtgatttg actgtctccg gcctttctca 5940

cccgtttgaa tctttaccta cacattactc aggcattgca tttaaaatat atgagggttc 6000

taaaaatttt tatccttgcg ttgaaataaa ggcttctccc gcaaaagtat tacagggtca 6060

taatgttttt ggtacaaccg atttagcttt atgctctgag gctttattgc ttaattttgc 6120

taattctttg ccttgcctgt atgatttatt ggatgttgga attcctgatg cggtattttc 6180

tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct 6240

ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 6300

gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 6360

tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 6420

gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 6480

ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 6540

atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 6600

tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 6660

tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 6720

gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 6780

aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 6840

gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 6900

ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 6960

gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 7020

gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 7080

atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 7140

ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 7200

cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 7260

cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 7320

gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 7380

cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 7440

cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 7500

taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 7560

ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 7620

aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 7680

caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 7740

taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 7800

gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 7860

cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 7920

taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 7980

agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 8040

ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 8100

gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 8160

acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 8220

acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 8280

tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 8340

ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 8400

agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagaattc 8460

ccatcatcaa taatatacct tattttggat tgaagccaat atgataatga gggggtggag 8520

tttgtgacgt ggcgcggggc gtgggaacgg ggcgggtgac gtagtagtct ctagaggtcc 8580

ccagcgacct tgacgggcat ctgcccggca tttctgacag ctttgtgaac tgggtggccg 8640

agaaggaatg ggagttgccg ccagattctg acatggatct gaatctgatt gagcaggcac 8700

ccctgaccgt ggccgagaag ctgcatcgct ggcgtaatag cgaagaggcc cgcaccgatc 8760

gcccttccca acagttgcgc agcctgaatg gcgaatggcg attccgttgc aatggctggc 8820

ggtaatattg ttctggatat taccagcaag gccgatagtt tgagttcttc tactcaggca 8880

agtgatgtta ttactaatca aagaagtatt gcgacaacgg ttaatttgcg tgatggacag 8940

actcttttac tcggtggcct cactgattat aaaaacactt ctcaggattc tggcgtaccg 9000

ttcctgtcta aaatcccttt aatcggcctc ctgtttagct cccgctctga ttctaacgag 9060

gaaagcacgt tatacgtgct cgtcaaagca accatagtac gcgccctgta gcggcgcatt 9120

aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 9180

gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggcg aacgtggcga 9240

gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 9300

cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt 9360

cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac 9420

gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt 9480

cccagtcacg acgttgtaaa acgacggcca gtgaattagg ttaattaagg cacacccgcc 9540

gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg 9600

gaagggcgat cggtgcgg 9618

<210> 16

<211> 791

<212> PRT

<213> hBDDF8 sequence in FIG. 2

<400> 16

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser

20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg

35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val

50 55 60

Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile

65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln

85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser

100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser

115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp

130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu

145 150 155 160

Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser

165 170 175

Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile

180 185 190

Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr

195 200 205

Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly

210 215 220

Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp

225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr

245 250 255

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val

260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile

275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser

290 295 300

Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met

305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His

325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro

340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp

355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser

370 375 380

Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr

385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro

405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn

420 425 430

Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met

435 440 445

Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu

450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu

465 470 475 480

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro

485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys

500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe

515 520 525

Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp

530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg

545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu

565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val

580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu

595 600 605

Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp

610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val

625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp

645 650 655

Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe

660 665 670

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr

675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro

690 695 700

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly

705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp

725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys

740 745 750

Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu

755 760 765

Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln

770 775 780

Glu Glu Ile Asp Tyr Asp Asp

785 790

<210> 17

<211> 791

<212> PRT

<213> substituted hBDDF8 sequence in FIG. 2

<400> 17

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser Lys Val Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser

20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg

35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Leu Pro Phe Asn Thr Ser Val Val

50 55 60

Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp His Leu Phe Asn Val

65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln

85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser

100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser

115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp

130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu

145 150 155 160

Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser

165 170 175

Tyr Phe Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile

180 185 190

Gly Ala Leu Leu Val Cys Lys Glu Gly Ser Leu Ala Lys Glu Lys Thr

195 200 205

Gln Thr Leu Gln Lys Phe Val Leu Leu Phe Ala Val Phe Asp Glu Gly

210 215 220

Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp

225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr

245 250 255

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser Val

260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile

275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser

290 295 300

Leu Glu Ile Ser Pro Val Thr Phe Leu Thr Ala Gln Thr Phe Leu Met

305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Pro Ser His Gln His

325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro

340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp

355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Ser Phe Asp Asp Asp Asn Ser

370 375 380

Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr

385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro

405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn

420 425 430

Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met

435 440 445

Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu

450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu

465 470 475 480

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro

485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys

500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe

515 520 525

Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp

530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg

545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu

565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val

580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu

595 600 605

Asn Met Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp

610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val

625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp

645 650 655

Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe

660 665 670

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr

675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro

690 695 700

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly

705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp

725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys

740 745 750

Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu

755 760 765

Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln

770 775 780

Glu Glu Ile Asp Tyr Asp Asp

785 790

<210> 18

<211> 8106

<212> DNA

<213> pANG-TTR-hBDDF8

<400> 18

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct acgcgtgtct gtctgcacat ttcgtagagc gagtgttccg atactctaat 180

ctccctaggc aaggttcata tttgtgtagg ttacttattc tccttttgtt gactaagtca 240

ataatcagaa tcagcaggtt tggagtcagc ttggcaggga tcagcagcct gggttggaag 300

gagggggtat aaaagcccct tcaccaggag aagccgtcac acagatccac aagctcctgc 360

tagcaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct 420

tgcgtgcctt gaattactga cactgacatc cactttttct ttttctccac aggtatcgat 480

gccaccatgc aaatagagct ctccacctgc ttctttctgt gccttttgcg attctgcttt 540

agtgccacca gaagatacta cctgggtgca gtggaactgt catgggacta tatgcaaagt 600

gatctcggtg agctgcctgt ggacgcaaga tttcctccta gagtgccaaa atcttttcca 660

ttcaacacct cagtcgtgta caaaaagact ctgtttgtag aattcacgga tcaccttttc 720

aacatcgcta agccaaggcc accctggatg ggtctgctag gtcctaccat ccaggctgag 780

gtttatgata cagtggtcat tacacttaag aacatggctt cccatcctgt cagtcttcat 840

gctgttggtg tatcctactg gaaagcttct gagggagctg aatatgatga tcagaccagt 900

caaagggaga aagaagatga taaagtcttc cctggtggaa gccatacata tgtctggcag 960

gtcctgaaag agaatggtcc aatggcctct gacccactgt gccttaccta ctcatatctt 1020

tctcatgtgg acctggtaaa agacttgaat tcaggcctca ttggagccct actagtatgt 1080

agagaaggga gtctggccaa ggaaaagaca cagaccttgc acaaatttat actacttttt 1140

gctgtatttg atgaagggaa aagttggcac tcagaaacaa agaactcctt gatgcaggat 1200

agggatgctg catctgctcg ggcctggcct aaaatgcaca cagtcaatgg ttatgtaaac 1260

aggtctctgc caggtctgat tggatgccac aggaaatcag tctattggca tgtgattgga 1320

atgggcacca ctcctgaagt gcactcaata ttcctcgaag gtcacacatt tcttgtgagg 1380

aaccatcgcc aggcgtcctt ggaaatctcg ccaataactt tccttactgc tcaaacactc 1440

ttgatggacc ttggacagtt tctactgttt tgtcatatct cttcccacca acatgatggc 1500

atggaagctt atgtcaaagt agacagctgt ccagaggaac cccaactacg aatgaaaaat 1560

aatgaagaag cggaagacta tgatgatgat cttactgatt ctgaaatgga tgtggtcagg 1620

tttgatgatg acaactctcc ttcctttatc caaattcgct cagttgccaa gaagcatcct 1680

aaaacttggg tacattacat tgctgctgaa gaggaggact gggactatgc tcccttagtc 1740

ctcgcccccg atgacagaag ttataaaagt caatatttga acaatggccc tcagcggatt 1800

ggtaggaagt acaaaaaagt ccgatttatg gcatacacag atgaaacctt taagactcgt 1860

gaagctattc agcatgaatc aggaatcttg ggacctttac tttatgggga agttggagac 1920

acactgttga ttatatttaa gaatcaagca agcagaccat ataacatcta ccctcacgga 1980

atcactgatg tccgtccttt gtattcaagg agattaccaa aaggtgtaaa acatttgaag 2040

gattttccaa ttctgccagg agaaatattc aaatataaat ggacagtgac tgtagaagat 2100

gggccaacta aatcagatcc tcggtgcctg acccgctatt actctagttt cgttaatatg 2160

gagagagatc tagcttcagg actcattggc cctctcctca tctgctacaa agaatctgta 2220

gatcaaagag gaaaccagat aatgtcagac aagaggaatg tcatcctgtt ttctgtattt 2280

gatgagaacc gaagctggta cctcacagag aatatacaac gctttctccc caatccagct 2340

ggagtgcagc ttgaggatcc agagttccaa gcctccaaca tcatgcacag catcaatggc 2400

tatgtttttg atagtttgca gttgtcagtt tgtttgcatg aggtggcata ctggtacatt 2460

ctaagcattg gagcacagac tgacttcctt tctgtcttct tctctggata taccttcaaa 2520

cacaaaatgg tctatgaaga cacactcacc ctattcccat tctcaggaga aactgtcttc 2580

atgtcgatgg aaaacccagg tctatggatt ctggggtgcc acaactcaga ctttcggaac 2640

agaggcatga ccgccttact gaaggtttct agttgtgaca agaacactgg tgattattac 2700

gaggacagtt atgaagatat ttcagcatac ttgctgagta aaaacaatgc cattgaacca 2760

agaagcttct cccagaatcc accagtcttg aaacgccatc aacgcgaaat aactcgtact 2820

actcttcagt cagatcaaga ggaaattgac tatgatgata ccatatcagt tgaaatgaag 2880

aaggaagatt ttgacattta tgatgaggat gaaaatcaga gcccccgcag ctttcaaaag 2940

aaaacacgac actattttat tgctgcagtg gagaggctct gggattatgg gatgagtagc 3000

tccccacatg ttctaagaaa cagggctcag agtggcagtg tccctcagtt caagaaagtt 3060

gttttccagg aatttactga tggctccttt actcagccct tataccgtgg agaactaaat 3120

gaacatttgg gactcctggg gccatatata agagcagaag ttgaagataa tatcatggta 3180

actttcagaa atcaggcctc tcgtccctat tccttctatt ctagccttat ttcttatgag 3240

gaagatcaga ggcaaggagc agaacctaga aaaaactttg tcaagcctaa tgaaaccaaa 3300

acttactttt ggaaagtgca acatcatatg gcacccacta aagatgagtt tgactgcaaa 3360

gcctgggctt atttctctga tgttgacctg gaaaaagatg tgcactcagg cctgattgga 3420

ccccttctgg tctgccacac taacacactg aaccctgctc atgggagaca agtgacagta 3480

caggaatttg ctctgttttt caccatcttt gatgagacca aaagctggta cttcactgaa 3540

aatatggaaa gaaactgcag ggctccctgc aatatccaga tggaagatcc cacttttaaa 3600

gagaattatc gcttccatgc aatcaatggc tacataatgg atacactacc tggcttagta 3660

atggctcagg atcaaaggat tcgatggtat ctgctcagca tgggcagcaa tgaaaacatc 3720

cattctattc atttcagtgg acatgtgttc actgtacgaa aaaaagagga gtataaaatg 3780

gcactgtaca atctctatcc aggtgttttt gagacagtgg aaatgttacc atccaaagct 3840

ggaatttggc gggtggaatg ccttattggc gagcatctac atgctgggat gagcacactt 3900

tttctggtgt acagcaataa gtgtcagact cccctgggaa tggcttctgg acacattaga 3960

gattttcaga ttacagcttc aggacaatat ggacagtggg ccccaaagct ggccagactt 4020

cattattccg gatcaatcaa tgcctggagc accaaggagc ccttttcttg gatcaaggtg 4080

gatctgttgg caccaatgat tattcacggc atcaagaccc agggtgcccg tcagaagttc 4140

tccagcctct acatctctca gtttatcatc atgtatagtc ttgatgggaa gaagtggcag 4200

acttatcgag gaaattccac tggaacctta atggtcttct ttggcaatgt ggattcatct 4260

gggataaaac acaatatttt taaccctcca attattgctc gatacatccg tttgcaccca 4320

actcattata gcattcgcag cactcttcgc atggagttga tgggctgtga tttaaatagt 4380

tgcagcatgc cattgggaat ggagagtaaa gcaatatcag atgcacagat tactgcttca 4440

tcctacttta ccaatatgtt tgccacctgg tctccttcaa aagctcgact tcacctccaa 4500

gggaggagta atgcctggag acctcaggtg aataatccaa aagagtggct gcaagtggac 4560

ttccagaaga caatgaaagt cacaggagta actactcagg gagtaaaatc tctgcttacc 4620

agcatgtatg tgaaggagtt cctcatctcc agcagtcaag atggccatca gtggactctc 4680

ttttttcaga atggcaaagt aaaggttttt cagggaaatc aagactcctt cacacctgtg 4740

gtgaactctc tagacccacc gttactgact cgctaccttc gaattcaccc ccagagttgg 4800

gtgcaccaga ttgccctgag gatggaggtt ctgggctgcg aggcacagga cctctactga 4860

ctcgagaata aaagatcaga gctctagaga tctgtgtgtt ggttttttgt gtgcggccgg 4920

gatctgagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 4980

ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga 5040

gcgagcgagc gcgcagagag ggagtggcca accccccccc ccccccccct gcaggcgatt 5100

ctcttgtttg ctccagactc tcaggcaatg acctgatagc ctttgtagag acctctcaaa 5160

aatagctacc ctctccggca tgaatttatc agctagaacg gttgaatatc atattgatgg 5220

tgatttgact gtctccggcc tttctcaccc gtttgaatct ttacctacac attactcagg 5280

cattgcattt aaaatatatg agggttctaa aaatttttat ccttgcgttg aaataaaggc 5340

ttctcccgca aaagtattac agggtcataa tgtttttggt acaaccgatt tagctttatg 5400

ctctgaggct ttattgctta attttgctaa ttctttgcct tgcctgtatg atttattgga 5460

tgttggaatt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 5520

tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc 5580

cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 5640

aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 5700

gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa 5760

tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 5820

tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 5880

ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 5940

ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 6000

aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 6060

gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 6120

ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 6180

gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 6240

cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 6300

cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 6360

acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 6420

caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 6480

taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 6540

ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 6600

aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 6660

agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 6720

atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 6780

tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 6840

tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 6900

gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 6960

taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 7020

aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 7080

ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 7140

catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 7200

ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 7260

ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 7320

agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 7380

taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 7440

atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 7500

cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 7560

ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 7620

accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 7680

gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc 7740

gttggccgat tcattaatgc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc 7800

aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg 7860

tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 7920

agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc 7980

ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc 8040

ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa 8100

aaagct 8106

Claims

1. An isolated modified human factor VIII (mhFVIII) comprising one or more amino acid substitutions at a position selected from the group consisting of a20, T21, F57, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332, R378, I610 and I661.

2. The isolated mhFVIII according to claim 1, wherein the mhFVIII comprises one or more amino acid substitutions selected from the amino acid substitutions listed in table 1.

3. The isolated mhFVIII according to claim 1 or 2, comprising one or more amino acid substitutions selected from the group consisting of a20K, T21I, T V, F3557L, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L F, S P, R378S, I M and I661V.

4. A mhFVIII according to any one of claims 1-3, comprising the following amino acid substitutions:

(1) A20K and T21I, or

(2) A20K and T21V, or

(3) T21I, L V and I80V, or

(4) T21I, L69V, I and L178F, or

(5) T21I, L69V, I V and I661V, or

(6) T21I, L69V, I, L178F and I661V, or

(7) R199K, H212Q, I V, R269K, I310V, L318F and S332P, or

(8) T21I, L69V, I80V, L178F, H212Q, I215V, R269K, L318F and I661V, or

(9) a20K, T21I, L V, I80V, L178F, H212Q, I215V, R269K, L318F and I661V, or

(10) T21I, L69V, I V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S P and I661V, or

(11) a20K, T21V, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S332P and I661V.

5. The mhFVIII according to any one of claims 1-4, wherein the mhFVIII consists of a single polypeptide.

6. The mhFVIII according to any one of claims 1-5, wherein the mhFVIII comprises a truncated B domain.

7. The mhFVIII according to any one of claims 1-6, wherein the mhFVIII comprises (1) wild type hFVIII or the A1 and A2 domains of mhFVIII, and (2) the A3, C1 and C2 domains of FVIII from a non-human species.

8. The mhFVIII according to claim 7, wherein the A3, C1 and C2 domains are from canine factor VIII.

9. An isolated polynucleotide encoding mhFVIII according to any one of claims 1-8, optionally comprising regulatory sequences operably linked to the polynucleotide.

10. An expression vector comprising the polynucleotide of claim 9.

11. The expression vector of claim 10, wherein the expression vector is a viral vector.

12. The expression vector of claim 11, wherein the viral vector is an AAV vector.

13. A host cell comprising the expression vector of claim 10.

14. A pharmaceutical composition comprising:

(1) The isolated mhFVIII of any one of claims 1-8, the expression vector of any one of claims 10-12, or the host cell of claim 13; and

(2) A pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14 for use in medicine.