HK1246211A1

HK1246211A1 - Fviii peptides for immune tolerance induction and immunodiagnostics

Info

Publication number: HK1246211A1
Application number: HK18105977.6A
Authority: HK
Inventors: Katharina Nora Steinitz; Paula Maria Wilhelmina Van Helden; Birgit Maria Reipert; Hans-Peter Schwarz; Hartmut Ehrlich
Original assignee: Baxalta GmbH; Baxalta Incorporated
Priority date: 2010-10-27
Filing date: 2018-05-09
Publication date: 2018-09-07

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Patent Application Serial Number 61/407,402, filed on October 27, 2010 , U.S. Provisional Patent Application Serial Number 61/467,894 , filed on March 25, 2011, and U.S. Provisional Patent Application Serial Number 61/502,476 , filed on June 29, 2011, the disclosures of which are hereby incorporated by reference in their entireties for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT NOT APPLICABLE REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK NOT APPLICABLE BACKGROUND OF THE INVENTION

Factor VIII (FVIII) is a protein found in blood plasma that acts as a cofactor in the cascade of reactions leading to blood coagulation. Hemophilia A is caused by a reduction or deficiency of functional FVIII protein and is one of the most common bleeding disorders that affects about 1 in 5000-10000 men. Clinical symptoms in hemophilia are frequent muscle and joint bleeds, and trauma can even lead to life threatening situations. Currently, effective treatments for hemophilia include replacing the missing FVIII protein using intravenous application of recombinant or plasma derived FVIII products. Such preparations are generally administered either in response to a bleeding episode (on-demand therapy) or at frequent, regular intervals to prevent uncontrolled bleeding (prophylaxis). Unfortunately, the appearance of neutralizing anti-FVIII antibodies (FVIII inhibitors) is a major complication during replacement therapy with FVIII products. Approximately 25% of the patients receiving treatment develop this immunity to FVIII protein, thus making further control of bleeding very difficult.

The cause for this immune response to FVIII protein has not been fully elucidated, but the specifics of a patient's immune system can affect their response to therapy. Normally, the immune system develops a tolerance to certain antigens, e.g., "self" antigens. This feature is important because, otherwise, if a self antigen is recognized as a foreign antigen, autoimmune disease results. Hemophilia A patients, in particular, have a genetic defect in their FVIII gene, which causes the immune system to not recognize the administered FVIII protein as a "self" antigen. Thus, when FVIII protein is administered during coagulation factor replacement therapy, the patient's immune system recognizes the FVIII protein as a foreign antigen or an altered self protein and develops anti-FVIII antibodies accordingly.

The FVIII inhibitors, i.e., anti-FVIII antibodies are produced by plasma cells derived from FVIII specific B cells. B cells need the help of activated CD4⁺ T-cells to proliferate and differentiate into anti-FVIII antibody producing plasma cells. For example, FVIII protein is recognized by B and T lymphocytes in different ways. The induction of anti-FVIII antibodies is T helper cell dependent. B cells recognize whole protein epitopes via their specific B cell receptor. T-cells on the other hand, recognize proteins in the form of processed peptides complexed with an MHC class II molecule presented on the surface of an antigen presenting cell. Each CD4⁺ T-cell clone recognizes only one specific peptide-MHC complex. For presenting the peptides to the T-cells, MHC class II molecules have an open binding groove that allows peptides of various lengths to fit in and be presented on the surface of a cell. Moreover, the MHC class II protein contains four binding pockets that differ for the various haplotypes (Jones et al., Nature Rev. Immunol. 6:271-282 (2006)). Only specific amino acids fit into these binding pockets, and the minimal size of binding peptides is nine amino acids. Notably, different MHC class II haplotypes can present different peptides. Thus, it is likely that a patient's MHC class II haplotype influences the risk of developing anti-FVIII antibodies. Indeed, several studies have shown that there is a correlation of the human MHC class II haplotype HLA-DRB1*1501 with an increased risk for anti-FVIII antibody development (Pavlova et al., J. Thromb. Haemost. 7:2006-2015 (2009); Oldenburg et al., Thromb. Haemost. 77:238-242 (1997); Hay et al., Thromb. Haemost. 77:234-237 (1997)).

Certain approaches have been explored to address the challenges associated with treating hemophilia by administration of FVIII protein. For example, WO 03/087161 discloses modified FVIII proteins, in which the immune characteristics of the FVIII protein are modified by reducing or removing the number of potential T-cell epitopes present on the protein. A number of regions that include T-cell epitopes along the FVIII protein were identified, including, e.g., FVIII^2030-2044. According to the disclosure, removal of such regions could be used to provide functional FVIII protein that did not induce production of anti-FVIII antibodies. WO 09/071886 also discloses specific regions of FVIII protein that were predicted to give rise to HLA-DR2 binding peptides that are involved in a patient's immune response, such as, e.g., FVIII^475-495. FVIII^542-562, FVIII^1785-1805, and FVIII^2158-2178. The peptides were identified for possible use in inducing immune tolerance in a patient.

While there have been advances in identifying regions of FVIII protein involved in the immune response, there is still a need to identify other regions of FVIII protein that can be used for developing other therapeutic peptides and methodologies that can, for example, be used to treat patients having hemophilia A.

BRIEF SUMMARY OF THE INVENTION

The present invention is based on the identification of regions of FVIII protein related to the immune response against FVIII molecules. More specifically, a FVIII peptide including the region of FVIII protein can be used to induce tolerance to human FVIII in patients with, e.g., hemophilia A. Furthermore, the FVIII peptides can be used for immunodiagnostic purposes to monitor patients with hemophilia A during replacement therapy and during immune tolerance induction therapy.

In one aspect, the present invention provides a method of inducing an immune tolerance to FVIII in a subject in need thereof, the method comprising a step of: administering to the subject a therapeutically effective amount of a peptide having an amino acid sequence consisting of: (R¹)_x-P-(R²)_y, wherein: P is an amino acid sequence having at least 85% identity to at least nine consecutive amino acids of a sequence selected from SEQ ID NOS:10, 68, 344, and 740; R¹ is an amino acid sequence consisting of from 1 to 80 amino acids; R² is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one.

In one embodiment of the methods provided above, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:10.

In one embodiment of the methods provided above, P is an amino acid sequence identical to a sequence of at least nine consecutive amino acids of SEQ ID NO:10.

In one embodiment of the methods provided above, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:68.

In one embodiment of the methods provided above, P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:68.

In one embodiment of the methods provided above, P is an amino acid sequence identical to a sequence of at least nine consecutive amino acids of SEQ ID NO:68.

In one embodiment of the methods provided above, P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:344.

In one embodiment of the methods provided above, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:344.

In one embodiment of the methods provided above, P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:344.

In one embodiment of the methods provided above, P is an amino acid sequence identical to a sequence of at least nine consecutive amino acids of SEQ ID NO:344.

In one embodiment of the methods provided above, P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:740.

In one embodiment of the methods provided above, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:740.

In one embodiment of the methods provided above, P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:740.

In one embodiment of the methods provided above, P is an amino acid sequence identical to a sequence of at least nine consecutive amino acids of SEQ ID NO:740.

In one embodiment of the methods provided above, x and y are both zero.

In one embodiment of the methods provided above, x is one and y is zero.

In one embodiment of the methods provided above, x is zero and y is one.

In one embodiment of the methods provided above, x and y are both zero.

In one embodiment of the methods provided above, the peptide consists of from 9 to 100 amino acids.

In one embodiment of the methods provided above, the peptide consists of from 9 to 50 amino acids.

In one embodiment of the methods provided above, the peptide consists of from 9 to 25 amino acids.

In one embodiment of the methods provided above, administration of the pharmaceutical composition prevents development of anti-FVIII antibodies in the subject.

In one embodiment of the methods provided above, administration of the pharmaceutical composition reduces an amount anti-FVIII antibodies present in the subject.

In one aspect, the present invention provides a peptide consisting of the amino acid sequence: (R¹)_x-P-(R²)_y, wherein: P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a sequence selected from SEQ ID NOS:10, 68, 159, 250, 344, 477, 568, 659, and 740; R¹ is an amino acid sequence consisting of from 1 to 80 amino acids; R² is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one.

In one embodiment of the peptides provided above, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:10.

In one embodiment of the peptides provided above, P is an amino acid sequence identical to a sequence of at least nine consecutive amino acids of SEQ ID NO:10.

In one embodiment of the peptides provided above, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:68.

In one embodiment of the peptides provided above, P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:68.

In one embodiment of the peptides provided above, P is an amino acid sequence identical to a sequence of at least nine consecutive amino acids of SEQ ID NO:68.

In one embodiment of the peptides provided above, P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:344.

In one embodiment of the peptides provided above, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:344.

In one embodiment of the peptides provided above, P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:344.

In one embodiment of the peptides provided above, P is an amino acid sequence identical to a sequence of at least nine consecutive amino acids of SEQ ID NO:344.

In one embodiment of the peptides provided above, P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:740.

In one embodiment of the peptides provided above, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:740.

In one embodiment of the peptides provided above, P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:740.

In one embodiment of the peptides provided above, P is an amino acid sequence identical to a sequence of at least nine consecutive amino acids of SEQ ID NO:740.

In one embodiment of the peptides provided above, x and y are both zero.

In one embodiment of the peptides provided above, x is one and y is zero.

In one embodiment of the peptides provided above, x is zero and y is one.

In one embodiment of the peptides provided above, x and y are both zero.

In one embodiment of the peptides provided above, the peptide consists of from 9 to 100 amino acids.

In one embodiment of the peptides provided above, the peptide consists of from 9 to 50 amino acids.

In one embodiment of the peptides provided above, the peptide consists of from 9 to 25 amino acids.

In one aspect, the present invention provides a composition comprising a peptide as described herein.

In one embodiment of the compositions provided above, the composition is formulated for pharmaceutical administration.

In one embodiment of the compositions provided above, the composition further comprises a second polypeptide, the second polypeptide consisting of the amino acid sequence: (R¹)_x-P-(R²)_y, wherein: P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a sequence selected from SEQ ID NOS:10, 68, 159, 250, 477, 568, 659, and 740; R¹ is an amino acid sequence consisting of from 1 to 80 amino acids; R² is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one.

In one aspect, the present invention provides a method of making a FVIII peptide, the method comprising the steps of: a) providing a culture of cells comprising a polynucleotide that encodes a FVIII peptide according to any one of claims 24 to 41; and b) expressing the peptide in the culture of cells.

In one aspect, the present invention provides a method of identifying a FVIII peptide-specific T cell, the method comprising: a) combining a plurality of CD4+ T cells with a peptide complexed with a MHC class II multimer, wherein the peptide is a FVIII peptide according to any one of claims 24 to 41; and b) identifying at least one of the members of the plurality of CD4+ T cells that is specific for the peptide complexed with the MHC class II multimer.

In one embodiment of the methods provided above, the MHC class II multimer is a MHC class II tetramer.

In one embodiment of the methods provided above, the peptide or MHC class II multimer further comprises a detectable moiety.

In one embodiment of the methods provided above, the method further comprises isolating at least one CD4+ T cell that is specific for the peptide.

In one embodiment of the methods provided above, the CD4+ T cell is isolated using flow cytometry.

In one aspect, the present invention provides a fusion protein comprising a FVIII peptide as provided herein and a second peptide.

In one embodiment of the methods provided above, the second peptide is a reporter peptide.

In one embodiment of the methods provided above, the fusion protein is encoded by a nucleic acid.

In one embodiment of the methods provided above, the FVIII peptide is chemically linked to the second peptide.

In one aspect, the FVIII peptides provided herein are used to induce immune tolerance towards human FVIII for the prevention of FVIII inhibitor development.

In one aspect, the FVIII peptides provided herein are used to induce tolerance towards human FVIII for the treatment of patients with established FVIII inhibitors.

In one aspect, the FVIII peptides provided herein are used to generate reagents suitable for direct staining of FVIII specific T cells (e.g., MHC class II multimers or MHC class II tetramers) in immune monitoring of patients during replacement therapy or during immune tolerance induction therapy.

In one aspect, the FVIII peptides provided herein are used to identify antigen specific T cells. In one embodiment, these reagents can be used to track FVIII specific T cells in in vitro and in ex vivo settings. In another embodiment, these reagents can be used to isolate and further characterize FVIII specific T cells. In one embodiment, fluorescent activated cell sorting (FACS) or single cell PCR can be used for these purposes.

In one aspect, the FVIII peptides provided herein are used for immune monitoring of FVIII specific T cells during immune tolerance induction therapy.

In one aspect, the FVIII peptides provided herein are used for immune monitoring of FVIII specific T cells during FVIII treatment.

In one aspect, the FVIII peptides provided herein are used for immunodiagnostics of FVIII specific T cells during clinical development of new immune modulators for the prevention of FVIII inhibitors.

BRIEF DESCRIPTION OF THE DRAWINGS

Not applicable.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is related to Factor VIII (FVIII) peptides that can be used to induce tolerance to FVIII protein in, for example, patients with hemophilia A. Furthermore, the peptides can be used for immunodiagnostic purposes to monitor FVIII-specific T cells in patients with hemophilia A during replacement therapy and during immune tolerance induction therapy.

The present invention is based in-part on the discovery that several regions of FVIII, specifically FVIII^102-122, FVIII^246-161, and FVIII^1401-1424, are involved in the immune response mounted against FVIII protein during Factor VIII replacement therapy or connected with acquired hemophilia. The amino acid sequences of the regions identified are TVVITLKNMASHPVSLHAVGV (SEQ ID NO:740), AWPKMHTVNGYVNRSLPGLIG (SEQ ID NO:68), and QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344), respectively. It is believed that the present invention provides for the first time identification of these FVIII protein regions and their relationship to the immune response to FVIII protein.

Peptides of the present invention include peptides having at least a portion of the regions FVIII^102-122, FVIII^246-266, and FVIII^1401-1424 that complexes with a MHC class II molecule to produce a T cell epitope capable of being recognized by T cells involved in a patient's immune response. In some embodiments, the peptides include at least nine contiguous amino acids that correspond to nine contiguous amino acids in FVIII^102-122, FVIII^246-266, or FVIII^1401-1424. As described further below, the peptides provided herein also include peptides longer than nine amino acids in length as well as variants of the FVIII^102-122, FVIII^246-266, and FVIII^1401-1424 sequences. Such an identification of the peptides of the present invention can have implications in improving and advancing therapeutic strategies designed to treat diseases related to blood coagulation, such as hemophilia A.

II. Definitions

The term "Factor VIII protein" or "FVIII protein" refers to any FVIII molecule which has at least a portion of the B domain intact, and which exhibits biological activity that is associated with native human FVIII protein. The FVIII molecule can be full-length FVIII. The FVIII molecule may also be a conservatively modified variant of native FVIII. The FVIII protein can be derived from human plasma or be produced by recombinant engineering techniques. Additional characterization of FVIII protein can be, e.g., found at paragraphs [0042]-[0055] in US 2010/0168018 , which is incorporated by reference herein.

The term "Factor VIII peptide" or "FVIII peptide" refers to the peptides described herein that include an amino acid sequence corresponding to a region of FVIII protein discovered to be important in an immune response against FVIII. A FVIII peptide includes at least nine amino acids that complex with a MHC class II protein for presentation to T cells involved in the immune response. Additional amino acids can be present on either end of the at least nine amino acid core of the peptide. In some embodiments, a FVIII peptide can include a sequence identical to the particular region of native human FVIII protein. In other embodiments, a FVIII peptide can be a conservatively modified variant of a region of FVIII protein. As described further herein, a FVIII peptide can be characterized by a certain percent identity, e.g., 85% identical, relative to the sequence of a region of native human FVIII protein.

The term "amino acid" refers to naturally occurring and non-natural amino acids, including amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Naturally occurring amino acids can include, e.g., D-and L-amino acids. The amino acids used herein can also include non-natural amino acids. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., any carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, or methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

"Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given peptide. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

As to amino acid sequences, one of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid or peptide sequence that alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M). See, e.g., Creighton, Proteins (1984).

The terms "identical" or percent "identity," in the context of two or more nucleic acids or peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection.

By "therapeutically effective amount or dose" or "sufficient amount or dose" herein is meant a dose that produces effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Augsburger & Hoag, Pharmaceutical Dosage Forms (vols. 1-3, 3rd Ed. 2008); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (3rd Ed., 2008); Pickar, Dosage Calculations (8th Ed., 2007); and Remington: The Science and Practice of Pharmacy, 21st Ed., 2005, Gennaro, Ed., Lippincott, Williams & Wilkins).

III. FVIII Peptides

The present invention relates to FVIII peptides that correspond to regions of FVIII protein involved in an immune response against FVIII. In one aspect, the present invention provides a FVIII peptide consisting of a consecutive sequence of nine amino acids that is at least 85 % identical to nine consecutive amino acids in one of the following amino acid sequences: AWPKMHTVNGYVNRSLPGLIG (SEQ ID NO:68); QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344); or TVVITLKNMASHPVSLHAVGV (SEQ ID NO:740), wherein the peptide consists of from 9 to 180 amino acids.

In a specific embodiment, the FVIII peptide has the sequence: (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a sequence selected from SEQ ID NOS:68, 344, and 740, R1 is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one. In one embodiment, R¹ is an amino acid sequence consisting of from 1 to 40 amino acids, and R² is an amino acid sequence consisting of from 1 to 40 amino acids.

In one embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, S0, S1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acids.

In one embodiment, the FVIII peptide consists of from 9 to 150 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 100 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 50 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 25 amino acids. In yet other embodiments, the FVIII peptide consists of from 9 to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 amino acids.

Generally, the FVIII peptides of the present invention can include any sequence of amino acids present in the identified region of FVIII^102-122, FVIII^246-266, or FVIII^1401-1424, or a modified variant that can, for example, have a retained function similar or identical to FVIII^102-122, FVIII^246-266, or FVIII^1401-1424. In particular, the FVIII peptides of the present invention include a sequence of amino acids that includes a T cell epitope. The FVIII peptides include a sequence of at least nine amino acids that can range in percent identity relative to the amino acid sequence AWPKMHTVNGYVNRSLPGLIG (SEQ ID NO:68); QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344); or TVVITLKNMASHPVSLHAVGV (SEQ ID NO:740). For example, a FVIII peptides can have nine amino acids that are identical or at least 50%, 60%, 70%, 80%, or 85% percent identical to any of nine consecutive amino acids in FVIII^102-122, FVIII^246-266, or FVIII^1401-1424.

In another group of embodiments, the FVIII peptides can have amino acid sequences greater than nine amino acids, in which the amino acid sequences include a region that can be identical or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent identical to the sequence of consecutive amino acids in FVIII^102-122, FVIII^246-266, or FVIII^1401-1424. One of ordinary skill in the art will appreciate that known mutagenesis techniques, such as alanine substitution, can be used to identify modified variants that retain the function of the FVIII^102-122, FVIII^246-266, or FVIII^1401-1424 region.

In addition, the FVIII peptides can further include additional sequences of amino acids on either end of the core sequence of the FVIII peptides discussed above. The additional sequences are designated (R¹)_x and (R²)_y. In certain embodiments, R¹ and R² can range from 1 to about 80 amino acids in length. Alternatively, R¹ and R² can range from 1 to about 40 amino acids in length. In certain embodiments, each of the subscripts x and y are independently zero or one. In some embodiments, both x and y can be zero. In other embodiments, x can be one and y can be zero. In yet other embodiments, x can be zero and y can be one. In another embodiment, both x and y are one. Additional amino acids on either end can be added for a variety of reasons, including increased stability of the peptides, improved binding to MHC class II molecules and/or T cells, as well as other aspects that will be appreciated by one of ordinary skill in the art.

In one embodiment, the present invention provides a polypeptide having the sequence (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a Factor VIII region identified in Table 1, R¹ is an amino acid sequence consisting of from 1 to 80 amino acids, and R² is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one. Alternatively, R¹ and R² can range from 1 to about 40 amino acids in length. In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of a Factor VIII region identified in Table 1. In another embodiment, P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of a Factor VIII region identified in Table 1. In some embodiments, both x and y can be zero. In other embodiments, x can be one and y can be zero. In other embodiments, x can be zero and y can be one. In yet another embodiment, both x and y can be one. In one embodiment, the FVIII peptide consists of from 9 to 150 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 100 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 50 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 25 amino acids.

Table 1. Regions of FVIII including T-cell epitopes

Regions including T cell epitopes	Amino Acid Sequence
TVVITLKNMASHPVSLHA (SEQ ID NO:10)
AWPKMHTVNGYVNRSLPGLIG (SEQ ID NO:68)
GEVGDTLLIIFKNQASRPYNI (SEQ ID NO:159)
PTKSDPRCLTRYYSSFVNMER (SEQ ID NO:250)
QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344)
EVEDNIMVTFRNQASRPYSFY (SEQ ID NO:477)
LHAGMSTLFLVYSNKCQTPLG (SEQ ID NO:568)
NPPIIARYIRLHPTHYSIRST (SEQ ID NO:659)
TVVITLKNMASHPVSLHAVGV (SEQ ID NO:740)

As described above, the FVIII peptides of the present invention can include any sequence of amino acids present in the identified region of FVIII^1401-1424 or a modified variant that can, for example, have a retained function similar or identical to FVIII^1401-1424. In certain embodiments, the peptides can cover the whole B-domain of human FVIII protein. The present invention also can include other FVIII peptides that include a peptide having a sequence of at least nine amino acids that can range in percent identity relative to any one of the following amino acid sequences: GEVGDTLLIIFKNQASRPYNI (FVIII^474-494; SEQ ID NO:159), PTKSDPRCLTRYYSSFVNMER (FVIII^540-560; SEQ ID NO:250), EVEDNIMVTFRNQASRPYSFY (FVIII^1785-1805; SEQ ID NO:477), LHAGMSTLFLVYSNKCQTPLG (FVIII^2025-2045; SEQ ID NO:568), NPPIIARYIRLHPTHYSIRST (FVIII^2160-2180; SEQ ID NO:659), TVVITLKNMASHPVSLHA (FVIII^102-119; SEQ ID NO:10), AWPKMHTVNGYVNRSLPGLIG (FVIII^246-266; SEQ ID NO:68), and TVVITLKNMASHPVSLHAVGV (FVIII^102-122; SEQ ID NO:740).

For example, the FVIII peptides having nine amino acids that are identical or at least 50%, 60%, 70%, 80%, or 85% percent identical to any of nine consecutive amino acids in FVIII^474-494, FVIII^540-560, FVIII^1785-1805, FVIII^2025-2045, FVIII^2160-2180, FVIII^102-119, FVIII^246-260, or FVIII^102-122. In another group of embodiments, the FVIII peptides can have amino acid sequences greater than nine amino acids, in which the amino acid sequences can be identical or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent identical to any of nine consecutive amino acids in FVIII^474-494 FVIII^540-560, FVIII^1785-1805, FVIII^2025-2045, FVIII^2160-2180, FVIII^102-119, FVIII^246-266, or FVIII^102-122. One of ordinary skill in the art will appreciate that known mutagenesis techniques, such as alanine substitution, can be used to identify modified variants that retain the function of the FVIII^474-494, FVII^540-560, FVIII^1785-1805, FVIII^2025-2045, FVIII^2160-2180, FVIII^102-119, FVIII^246-266 or FVIII^102-122 regions. The FVIII peptides disclosed here can be made using methods described above with respect to the FVIII peptides relating to FVIII^1401-1424.

A. Factor VIII^102-119 Peptides

In one embodiment, the present invention provides a polypeptide having the sequence (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^102-119 peptide having the sequence: TVVITLKNMASHPVSLHA (SEQ ID NO:10), R¹ is an amino acid sequence consisting of from 1 to 80 amino acids, and R² is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one.

In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^102-119 peptide having the sequence: TVVITLKNMASHPVSLHA (SEQ ID NO:10). In one embodiment, P is an amino acid sequence having at least 85% identity to a sequence selected from SEQ ID NOS: 1 to 55 (SEQ ID NO:10). In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOS:1 to 55. In one embodiment, P is an amino acid sequence selected from SEQ ID NOS:1 to 55. In some embodiments, both x and y can be zero. In other embodiments, x can be one and y can be zero. In other embodiments, x can be zero and y can be one. In yet another embodiment, both x and y can be one.

In one embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R¹ and R² arc seperately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acids.

In certain embodiments, R¹ is an amino acid sequence consisting of from 1 to 40 amino acids, and R² is an amino acid sequence consisting of from 1 to 40 amino acids. In one embodiment, the FVIII peptide consists of from 9 to 150 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 100 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 50 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 25 amino acids. In yet other embodiments, the FVIII peptide consists of from 9 to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 3R, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, S6, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, R6, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 amino acids.

Table 2. Exemplary FVIII^102-119 Peptides


TVVITLKNM	1
TVVITLKNMA	2
TVVITLKNMAS	3
TVVITLKNMASH	4
TVVITLKNMASHP	5
TVVITLKNMASHPV	6
TVVITLKNMASHPVS	7
TVVITLKNMASHPVSL	8
TVVITLKNMASHPVSLH	9
TVVITLKNMASHPVSLHA	10
VVITLKNMA	11
VVITLKNMAS	12
VVITLKNMASH	13
VVITLKNMASHP	14
VVITLKNMASHPV	15
VVITLKNMASHPVS	16
VVITLKNMASHPVSL	17
VVITLKNMASHPVSLH	18
VVITLKNMASHPVSLHA	19
VITLKNMAS	20
VITLKNMASH	21
VITLKNMASHP	22
VITLKNMASHPV	23
VITLKNMASHPVS	24
VITLKNMASHPVSL	25
VITLKNMASHPVSLH	26
VITLKNMASHPVSLHA	27
ITLKNMASH	28
ITLKNMASHP	29
ITLKNMASHPV	30
ITLKNMASHPVS	31
ITLKNMASHPVSL	32
ITLKNMASHPVSLH	33
ITLKNMASHPVSLHA	34
TLKNMASHP	35
TLKNMASHPV	36
TLKNMASHPVS	37
TLKNMASHPVSL	38
TLKNMASHPVSLH	39
TLKNMASHPVSLHA	40
LKNMASHPV	41
LKNMASHPVS	42
LKNMASHPVSL	43
LKNMASHPVSLH	44
LKNMASHPVSLHA	45
KNMASHPVS	46
KNMASHPVSL	47
KNMASHPVSLH	48
KNMASHPVSLHA	49
NMASHPVSL	50
NMASHPVSLH	51
NMASUPVSLHA	52
MASHPVSLH	53
MASHPVSLHA	54
ASHPVSLHA	55

B. Factor VIII^246-266 Peptides

In one embodiment, the present invention provides a polypeptide having the sequence (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^246-266 peptide having the sequence: AWPKMHTVNGYVNRSLPGLIG (SEQ ID NO:68), R¹ is an amino acid sequence consisting of from 1 to 80 amino acids, and R² is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one.

In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^246-266 peptide having the sequence: AWPKMHTVNGYVNRSLPGLIG (SEQ ID NO:68). In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^246-266 peptide having the sequence: AWPKMHTVNGYVNRSLPGLIG (SEQ ID NO:68). In one embodiment, P is an amino acid sequence having at least 85% identity to a sequence selected from SEQ ID NOS:56 to 146. In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOS:56 to 146. In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence selected from SEQ ID NOS:56 to 146. In one embodiment, P is an amino acid sequence selected from SEQ ID NOS:56 to 146. In some embodiments, both x and y can be zero. In other embodiments, x can be one and y can be zero. In other embodiments, x can be zero and y can be one. In yet another embodiment, both x and y can be one.

In one embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acids.

Table 3. Exemplary FVIII^246-266 Peptides


AWPKMHTVN	56
AWPKMHTVNG	57
AWPKMHTVNGY	58
AWPKMHTVNGYV	59
AWPKMHTVNGYVN	60
AWPKMHTVNGYVNR	61
AWPKMHTVNGYVNRS	62
AWPKMHTVNGYVNRSL	63
AWPKMHTVNGYVNRSLP	64
AWPKMHTVNGYVNRSLPG	65
AWPKMHTVNGYVNRSLPGL	66
AWPKMHTVNGYVNRSLPGLI	67
AWPKMHTVNGYVNRSLPGLIG	68
WPKMHTVNG	69
WPKMHTVNGY	70
WPKMHTVNGYV	71
WPKMHTVNGYVN	72
WPKMHTVNGYVNR	73
WPKMHTVNGYVNRS	74
WPKMHTVNGYVNRSL	75
WPKMHTVNGYVNRSLP	76
WPKMHTVNGYVNRSLPG	77
WPKMHTVNGYVNRSLPGL	78
WPKMHTVNGYVNRSLPGLI	79
WPKMHTVNGYVNRSLPGLIG	80
PKMHTVNGY	81
PKMHTVNGYV	82
PKMHTVNGYVN	83
PKMHTVNGYVNR	84
PKMHTVNGYVNRS	85
PKMHTVNGYVNRSL	86
PKMHTVNGYVNRSLP	87
PKMHTVNGYVNRSLPG	88
PKMHTVNGYVNRSLPGL	89
PKMHTVNGYVNRSLPGLI	90
PKMHTVNGYVNRSLPGLIG	91
KMHTVNGYV	92
KMHTVNGYVN	93
KMHTVNGYVNR	94
KMHTVNGYVNRS	95
KMHTVNGYVNRSL	96
KMHTVNGYVNRSLP	97
KMHTVNGYVNRSLPG	98
KMHTVNGYVNRSLPGL	99
KMHTVNGYVNRSLPGLI	100
KMHTVNGYVNRSLPGLIG	101
MHTVNGYVN	102
MHTVNGYVNR	103
MHTVNGYVNRS	104
MHTVNGYVNRSL	105
MHTVNGYVNRSLP	106
MHTVNGYVNRSLPG	107
MHTVNGYVNRSLPGL	108
MHTVNGYVNRSLPGLI	109
MHTVNGYVNRSLPGLIG	110
HTVNGYVNR	111
HTVNGYVNRS	112
HTVNGYVNRSL	113
UTVNCYVNRSLP	114
HTVNGYVNRSLPG	115
HTVNGYVNRSLPGL	116
HTVNGYVNRSLPGLI	117
HTVNGYVNRSLPGLIG	118
TVNGYVNRS	119
TVNGYVNRSL	120
TVNGYVNRSLP	121
TVNGYVNRSLPG	122
TVNGYVNRSLPGL	123
TVNGYVNRSLPGLI	124
TVNGYVNRSLPGLIG	125
VNGYVNRSL	126
VNGYVNRSLP	127
VNGYVNRSLPG	128
VNGYVNRSLPGL	129
VNGYVNRSLPGLI	130
VNGYVNRSLPGLIG	131
NGYVNRSLP	132
NGYVNRSLPG	133
NGYVNRSLPGL	134
NGYVNRSLPGLI	135
NGYVNRSLPGLIG	136
GYVNRSLPG	137
GYVNRSLPGL	138
GYVNRSLPGLI	139
GYVNRSLPGLIG	140
YVNRSLPGL	141
YVNRSLPGLI	142
YVNRSLPGLIG	143
VNRSLPGLI	144
VNRSLPGLIG	145
NRSLPGLIG	146

C. Factor VIII^474-494 Peptides

In one embodiment, the present invention provides a polypeptide having the sequence (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^474-494 peptide having the sequence: GEVGDTLLIIFKNQASRPYNI (SEQ ID NO:159), R¹ is an amino acid sequence consisting of from 1 to 80 amino acids, and R² is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one.

In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^474-494 peptide having the sequence: GEVGDTLLIIFKNQASRPYNI (SEQ ID NO:159). In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^474-494 peptide having the sequence: GEVGDTLLIIFKNQASRPYNI (SEQ ID NO:159). In one embodiment, P is an amino acid sequence having at least 85% identity to a sequence selected from SEQ ID NOS:147 to 237. In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOS:147 to 237. In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence selected from SEQ ID NOS: 147 to 237. In one embodiment, P is an amino acid sequence selected from SEQ ID NOS: 147 to 237. In some embodiments, both x and y can be zero. In other embodiments, x can be one and y can be zero. In other embodiments, x can be zero and y can be one. In yet another embodiment, both x and y can be one.

In certain embodiments, R¹ is an amino acid sequence consisting of from 1 to 40 amino acids, and R² is an amino acid sequence consisting of from 1 to 40 amino acids. In one embodiment, the FVIII peptide consists of from 9 to 150 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 100 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 50 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 25 amino acids. In yet other embodiments, the FVIII peptide consists of from 9 to 10, 11, 12, 13,14,15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 amino acids.

Table 4. Exemplary FVIII^474-494 Peptides


GEVGDTLL1	147
GEVGDTLLII	148
GEVGDTLLIIF	149
GEVGDTLLIIFK	150
GEVGDTLLIIFKN	151
GEVGDTLLIIFKNQ	152
GEVGDTLLIIFKNQA	153
GEVGDTLLIIFKNQAS	154
GEVGDTLLIIFKNQASR	155
GEVGDTLLIIFKNQASRP	156
GEVGDTLLIIFKNQASRPY	157
GEVGDTLLIIFKNQASRPYN	158
GEVGDTLLUFKNQASRPYNI	159
EVGDTLLII	160
EVGDTLLIIF	161
EVGDTLLDFK	162
EVGDTLLIIFKN	163
LVGDTLLIIFKNQ	164
EVGDTLLIIFKNQA	165
EVGDTLLIIFKNQAS	166
EVGDTLLIIFKNQASR	167
EVGDTLLIIFKNQASRP	168
EVGDTLLIIFKNQASRPY	169
EVGDTLLIIFKNQASRPYN	170
EVGDTLLIIFKNQASRPYNI	171
VGDTLLIIF	172
VGDTLLIIFK	173
VGDTLLIIFKN	174
VGDTLLIIFKNQ	175
VGDTLLIIFKNQA	176
VGDTLLIIFKNQAS	177
VGDTLLIIFKNQASR	178
VGDTLLIIFKNQASRP	179
VGDTLLIIFKNQASRPY	180
VGDTLLIIFKNQASRPYN	181
VGDTLLIIFKNQASRPYNI	182
GDTLLIIFK	183
GDTLLIIFKN	184
GDTLLIIFKNQ	185
GDTLLIIFKNQA	186
GDTLLIIFKNQAS	187
GDTLLIIFKNQASR	188
GDTLLIIFKNQASRP	189
GDTLLIIFKNQASRPY	190
GDTLLIIFKMQASRPYN	191
GDTLLIIFKNQASRPYNI	192
DTLLIIFKN	193
DTLLIIFKNQ	194
DTLLIIFKNQA	195
DTLLIIFKNQAS	196
DTLLIIFKNQASR	197
DTLLIIFKNQASRP	198
DTLLIIFKNQASRPY	199
DTLLIIFKNQASRPYN	200
DTLLIIFKNQASRPYNI	201
TLLIIFKNQ	202
TLLIIFKNQA	203
TLLIIFKNQAS	204
TLLIIFKNQASR	205
TLLIIFKNQASRP	206
TLLIIFKNQASRPY	207
TLLIIFKNQASRPYN	208
TLLIIFKNQASRPYNI	209
LLIIFKNQA	210
LLIIFKNQAS	211
LLIIFKNQASR	212
LLIIFKNQASRP	213
LLIIFKNQASRPY	214
LLIIFKNQASRPYN	215
LLIIFKNQASRPYNI	216
LIIFKNQAS	217
LIIFKNQASR	218
LIIFKNQASRP	219
LIIFKNQASRPY	220
LIIFKNQASRPYN	221
LIIFKNQASRPYNI	222
IIFKNQASR	223
IIFKNQASRP	224
IIFKNQASRPY	225
IIFKNQASRPYN	226
IIFKNQASRPYNI	227
IFKNQASRP	228
IFKNQASRPY	229
IFKNQASRPYN	230
IFKNQASRPYNI	231
FKNQASRPY	232
FKNQASRPYN	233
FKNQASRPYNI	234
KNQASRPYN	235
KNQASRPYNI	236
NQASRPYNI	237

D. Factor VIII^540-560 Peptides

In one embodiment, the present invention provides a polypeptide having the sequence (R^J)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^540-560 peptide having the sequence: PTKSDPRCLTRYYSSFVNMER (SEQ ID NO:250), R¹ is an amino acid sequence consisting of from 1 to 80 amino acids, and R² is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y arc independently zero or one.

In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^540-560 peptide having the sequence: PTKSDPRCLTRYYSSFVNMER (SEQ ID NO:250). In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^540-560 peptide having the sequence: PTKSDPRCLTRYYSSFVNMER (SEQ ID NO:250). In one embodiment, P is an amino acid sequence having at least 85% identity to a sequence selected from SEQ ID NOS:238 to 328. In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOS:238 to 328. In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence selected from SEQ ID NOS:238 to 328. In one embodiment, P is an amino acid sequence selected from SEQ ID NOS:238 to 328. In some embodiments, both x and y can be zero. In other embodiments, x can be one and y can be zero. In other embodiments, x can be zero and y can be one. In yet another embodiment, both x and y can be one.

In one embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, '?6, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, S5, 56, 57, 58, 59, 60, 61, 62, 63, 64, 6S, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acids.

In one embodiment, the FVIII peptide consists of from 9 to 150 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 100 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 50 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 25 amino acids. In yet other embodiments, the FVIII peptide consists of from 9 to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, ?3, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 1.55, 160, 165, 170, 175, or 180 amino acids.

Table 5. Exemplary FVIII^540-560 Peptides


PTKSDPRCL	238
PTKSDPRCLT	239
PTKSDPRCLTR	240
PTKSDPRCLTRY	241
PTKSDPRCLTRYY	242
PTKSDPRCLTRYYS	243
PTKSDPRCLTRYYSS	244
PTKSDPRCLTRYYSSF	245
PTKSDPRCLTRYYSSFV	246
PTKSDPRCLTRYYSSFVN	247
PTKSDPRCLTRYYSSFVNM	248
PTKSDPRCLTRYYSSFVNME	249
PTKSDPRCLTRYYSSFVNMER	250
TKSDPRCLT	251
TKSDPRCLTR	252
TKSDPRCLTRY	253
TKSDPRCLTRYY	254
TKSDPRCLTRYYS	255
TKSDPRCLTRYYSS	256
TKSDPRCLTRYYSSF	257
TKSDPRCLTRYYSSFV	258
TKSDPRCLTRYYSSFVN	259
TKSDPRCLTRYYSSFVNM	260
TKSDPRCLTRYYSSFVNME	261
TKSDPRCLTRYYSSFVNMER	262
KSDPRCLTR	263
KSDPRCLTRY	264
KSDPRCLTRYY	265
KSDPRCLTRYYS	266
KSDPRCLTRYYSS	267
KSDPRCLTRYYSSF	268
KSDPRCLTRYYSSFV	269
KSDPRCLTRYYSSFVN	270
KSDPRCLTRYYSSFVNM	271
KSDPRCLTRYYSSFVNME	272
KSDPRCLTRYYSSFVNMLR	273
SDPRCLTRY	274
SDPRCLTRYY	275
SDPRCLTRYYS	276
SDPRCLTRYYSS	277
SDPRCLTRYYSSF	278
SDPRCLTRYYSSFV	279
SDPRCLTRYYSSFVN	280
SDPRCLTRYYSSFVNM	281
SDPRCLTRYYSSFVNME	282
SDPRCLTRYYSSFVNMER	283
DPRCLTRYY	284
DPRCLTRYYS	285
DPRCLTRYYSS	286
DPRCLTRYYSSF	287
DPRCLTRYYSSFV	288
DPRCLTRYYSSFVN	289
DPRCLTRYYSSFVNM	290
DPRCLTRYYSSFVNME	291
DPRCLTRYYSSFVIVMER	292
PRCLTRYYS	293
PRCLTRYYSS	294
PRCLTRYYSSF	295
PRCLTRYYSSFV	296
PRCLTRYYSSFVN	297
PRCLTRYYSSFVNM	298
PRCLTRYYSSFVNME	299
PRCLTRYYSSFVNMER	300
RCLTRYYSS	301
RCLTRYYSSF	302
RCLTRYYSSFV	303
RCLTRYYSSFVN	304
RCLTRYYSSFVNM	305
RCLTRYYSSFVNME	306
RCLTRYYSSFVNMER	307
CLTRYYSSF	308
CLTRYYSSFV	309
CLTRYYSSFVN	310
CLTRYYSSFVNM	311
CLTRYYSSFVNME	312
CLTRYYSSFVNMER	313
LTRYYSSFV	314
LTRYYSSFVN	315
LTRYYSSFVNM	316
LTRYYSSFVNME	317
LTRYYSSFVNMER	318
TRYYSSFVN	319
TRYYSSFVNM	320
TRYYSSFVNME	321
TRYYSSFVNMER	322
RYYSSFVNM	323
RYYSSFVNME	324
RYYSSFVNMER	325
YYSSFVNME	326
YYSSFVNMER	327
YSSFVNMER	328

E. Factor VIII^1401-1424 Peptides

In one embodiment, the present invention provides a polypeptide having the sequence (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^1401-1424 peptide having the sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344), R¹ is an amino acid sequence consisting of from 1 to 80 amino acids, and R² is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one.

In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^1401-1424 peptide having the sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344). In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^1401-1124 peptide having the sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344). In one embodiment, P is an amino acid sequence having at least 85% identity to a sequence selected from SEQ ID NOS:329 to 464. In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOS:329 to 464. In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence selected from SEQ ID NOS:329 to 464. In one embodiment, P is an amino acid sequence selected from SEQ ID NOS:329 to 464. In some embodiments, both x and y can be zero. In other embodiments, x can be one and y can be zero. In other embodiments, x can be zero and y can be one. In yet another embodiment, both x and y can be one.

In one embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acids.

Table 6. Exemplary FVIII^1401-1424 Peptides


QANRSPLPI	329
QANRSPLPIA	330
QANRSPLPIAK	331
QANRSPLPIAKV	332
QANRSPLPIAKVS	333
QANRSPLPIAKVSS	334
QANRSPLPIAKVSSF	335
QANRSPLPIAKVSSFP	336
QANRSPLPIAKVSSFPS	337
QANRSPLPIAKVSSFPSI	338
QANRSPLPIAKVSSFPSIR	339
QANRSPLPIAKVSSFPSIRP	340
QANRSPLPIAKVSSFPSLRPI	341
QANRSPLPLAKVSSFPSIRPIY	342
QANRSPLPIAKVSSFPSIRPIYL	343
QAMRSPLPIAKVSSFPSIRPIYLT	344
ANRSPLPIA	345
ANRSPLPIAK	346
ANRSPLPIAKV	347
ANRSPLPIAKVS	348
ANRSPLPIAKVSS	349
ANRSPLPIAKVSSF	350
ANRSPLPIAKVSSFP	351
ANRSPLPIAKVSSFPS	352
ANRSPLPIAKVSSFPSI	353
ANRSPLPIAKVSSFPSIR	354
ANRSPLPIAKVSSFPSIRP	355
ANRSPLPIAKVSSFPSIRPI	356
ANRSPLPIAKVSSFPSIRPIY	357
ANRSPLPIAKVSSFPSIRPIYL	358
ANRSPLPIAKVSSFPSIRPIYLT	359
NRSPLPIAK	360
NRSPLPIAKV	361
NRSPLPIAKVS	362
NRSPLPIAKVSS	363
NRSPLPIAKVSSF	364
NRSPLPIAKVSSFP	365
NRSPLPIAKVSSFPS	366
NRSPLPIAKVSSFPSI	367
NRSPLPIAKVSSFPSIR	368
NRSPLPLAKVSSFPSIRP	369
NRSPLPIAKVSSFPSIRPI	370
NRSPLPIAKVSSFPSIRPIY	371
NRSPLPIAKVSSFPSIRPIYL	372
NRSPLPIAKVSSFPSIRPIYLT	373
RSPLPIAKV	374
RSPLPIAKVS	375
RSPLPIAKVSS	376
RSPLPIAKVSSF	377
RSPLPIAKVSSFP	378
RSPLPIAKVSSFPS	379
RSPLPIAKVSSFPSI	380
RSPLPIAKVSSFPSIR	381
RSPLPIAKVSSFPSIRP	382
RSPLPIAKVSSFPSIRPI	383
RSPLPIAKVSSFPSIRPIY	384
RSPLP1AKVSSFPSIRPIYL	385
RSPLPIAKVSSFPSIRPIYLT	386
SPLPIAKVS	387
SPLPIAKVSS	388
SPLPIAKVSSF	389
SPLPIAKVSSFP	390
SPLPIAKVSSFPS	391
SPLPIAKVSSFPSI	392
SPLPIAKVSSFPSIR	393
SPLPIAKVSSFPSIRP	394
SPLPIAKVSSFPSIRPI	395
SPLPIAKVSSFPSIRPIY	396
SPLPIAKVSSFPSIRPIYL	397
SPLPIAKVSSFPSIRPIYLT	398
PLPIAKVSS	399
PLPIAKVSSF	400
PLPIAKVSSFP	401
PLPIAKVSSFPS	402
PLPIAKVSSFPSI	403
PLPIAKVSSFPSIR	404
PLPIAKVSSFPSIRP	405
PLPIAKVSSFPSIRPI	406
PLPIAKVSSFPSIRPIY	407
PLPIAKVSSFPSIRPIYL	408
PLPIAKVSSFPSIRPIYLT	409
LPIAKVSSF	410
LPIAKVSSFP	411
LPIAKVSSFPS	412
LPIAKVSSFPSI	413
LPIAKVSSFPSIR	414
LPIAKVSSFPSIRP	415
LPIAKLVSSFPSIRPI	416
LPLAKVSSFPSIRPIY	417
LPIAKVSSFPSIRPIYL	418
LPIAKVSSFPSIRPIYLT	419
PIAKVSSFP	420
PIAKVSSFPS	421
PIAKVSSFPSI	422
PIAKVSSFPSIR	423
PIAKVSSFPSIRP	424
PLAKVSSFPSIRPI	425
PIAKVSSFPSIRPIY	426
PIAKVSSFPSIRPIYL	427
PIAKVSSFPSIRPIYLT	428
IAKVSSFPS	429
IAKVSSFPSI	430
IAKVSSFPSIR	431
IAKVSSFPSIRP	432
IAKVSSFPSIRPI	433
IAKVSSFPSIRPIY	434
IAKVSSFPSIRPIYL	435
IAKVSSFPSIRPIYLT	436
AKVSSFPSI	437
AKVSSFPSIR	438
AKVSSFPSIRP	439
AKVSSFPSIRPI	440
AKVSSFPSIRPIY	441
AKVSSFPSIRPIYL	442
AKVSSFPSIRPIYLT	443
KVSSFPSIR	444
KVSSFPSIRP	445
KVSSFPSIRPI	446
KVSSFPSIRPIY	447
KVSSFPSIRPIYL	448
KVSSFPS1RPIYLT	449
VSSFPSIRP	450
VSSFPSIRPI	451
VSSFPSIRPIY	452
VSSFPSIRPIYL	453
VSSFPSIRTIYLT	454
SSFPSIRPI	455
SSFPSIRPIY	456
SSFPSIRPIYL	457
SSFPSIRPIYLT	458
SFPSIRPIY	459
SFPSIRPIYL	460
SFPSIRPIYLT	461
FPSIRPIYL	462
FPSIRPIYLT	463
PSIRPIYLT	464

F. Factor VIII^1785-1805 Peptides

In one embodiment, the present invention provides a polypeptide having the sequence (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^1785-1805 peptide having the sequence: EVEDNIMVTFRNQASRPYSFY (SEQ ID NO:477), R¹ is an amino acid sequence consisting of from 1 to 80 amino acids, and R² is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one. In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^1785-1805 peptide having the sequence: EVEDNIMVTFRNQASRPYSFY (SEQ ID NO:477).

In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^1785-1805 peptide having the sequence: EVEDNIMVTFRNQASRPYSFY (SEQ ID NO:477). In one embodiment, P is an amino acid sequence having at least 85% identity to a sequence selected from SEQ ID NOS:465 to 555. In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOS:465 to 555. In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence selected from SEQ ID NOS:465 to 555. In one embodiment, P is an amino acid sequence selected from SEQ ID NOS:465 to 555. In some embodiments, both x and y can be zero. In other embodiments, x can be one and y can be zero. In other embodiments, x can be zero and y can be one. In yet another embodiment, both x and y can be one.

In one embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 2, 3,4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 4R, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acids.

In one embodiment, the FVIII peptide consists of from 9 to 150 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 100 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 50 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 25 amino acids. In yet other embodiments, the FVIII peptide consists of from 9 to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 amino acids.

Table 7. Exemplary FVIII^1785-1805 Peptides


EVEDNIMVT	465
EVEDNIMVTF	466
EVEDNIMVTFR	467
EVEDNIMVTFRN	468
EVEDNIMVTFRNQ	469
EVEDNIMVTFRNQA	470
EVEDNIMVTFRNQAS	471
EVEDNIMVTFRNQASR	472
EVEDNIMVTFRNQASRP	473
EVEDNIMVTFRNQASRPY	474
EVEDNIMVTFRNQASRPYS	475
EVEDNIMVTFRNQASRPYSF	476
EVEDNIMVTFRNQASRPYSFY	477
VEDNIMVTF	478
VEDNIMVTFR	479
VEDNIMVTFRN	480
VEDNIMVTFRNQ	481
VEDMMVTFRNQA	482
VEDNIMVTFRNQAS	483
VEDNIMVTFRNQASR	484
VEDNIMVTFRNQASRP	485
VEDNIMVTFRNQASRPY	486
VEDNIMVTFRNQASRPYS	487
VEDNIMVTFRNQASRPYSF	488
VEDNIMVTFRNQASRPYSFY	489
EDNIMVTFR	490
EDNIMVTFRN	491
EDNIMVTFRNQ	492
EDNIMVTFRNQA	493
EDNIMVTTRNQAS	494
EDNIMVTFRNQASR	495
EDNIMVTFRNQASRP	496
EDNIMVTFRNQASRPY	497
EDNIMVTFRNQASRPYS	498
EDNIMVTFRNQASRPYSF	499
EDNIMVTFRNQASRPYSFY	500
DNIMVTFRN	501
DNIMVTFRNQ	502
DNIMVTFRNQA	503
DNIMVTFRNQAS	504
DNIMVTFRNQASR	505
DNIMVTFRNQASRP	506
DNIMVTFRNQASRPY	507
DNIMVTFRNQASRPYS	508
DNIMVTFRNQASRPYSF	509
DNIMVTFRNQASRPYSFY	510
NIMVTFRNQ	511
NIMVTFRNQA	512
NIMVTFRNQAS	513
NIMVTFRNQASR	514
NIMVTFRNQASRP	515
NIMVTFRNQASRPY	516
NIMVTFRNQASRPYS	517
NIMVTFRNQASFPYSF	518
NIMVTFRNQASRPYSFY	519
IMVTFRNQA	520
IMVTFRNQAS	521
IMVTFRNQASR	522
IMVTFRNQASRP	523
IMVTFRNQASRPY	524
IMVTFRNQASRPYS	525
IMVTFRNQASRPYSF	526
IMVTFRNQASRPYSFY	527
MVTFRNQAS	528
MVTFRNQASR	529
MVTFRNQASRP	530
MVTFRNQASRPY	531
MVTFRNQASRPYS	532
MVTFRNQASRPYSF	533
MVTFRNQASRPYSFY	534
VTFRNQASR	535
VTFRNQASRP	536
VTFRNQASRPY	537
VTFRNQASRPYS	538
VTFRNQASRPYSF	539
VTFRNQASRPYSFY	540
TFRNQASRP	541
TFRNQASRPY	542
TFRNQASRPYS	543
TFRNQASRPYSF	544
TFRNQASRPYSFY	545
FRNQASRPY	546
FRNQASRPYS	547
FRNQASRPYSF	548
FRNQASRPYSFY	549
RNQASRPYS	550
RNQASRPYSF	551
RNQASRPYSFY	552
NQASRPYSF	553
NQASRPYSFY	554
QASRPYSFY	555

G. Factor VIII^II15-2045 Peptides

In one embodiment, the present invention provides a polypeptide having the sequence (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^2025-2045 peptide having the sequence: LHAGMSTLFLVYSNKCQTPLG (SEQ ID NO: 568), R¹ is an amino acid sequence consisting of from 1 to 80 amino acids, and R² is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one.

In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^2025-2045 peptide having the sequence: LHAGMSTLFLVYSNKCQTPLG (SEQ ID NO:568). In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^2025-2045 peptide having the sequence: LHAGMSTLFLVYSNKCQTPLG (SEQ ID NO:568). In one embodiment, P is an amino acid sequence having at least 85% identity to a sequence selected from SEQ ID NOS:556 to 646. In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOS:556 to 646. In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence selected from SEQ ID NOS:556 to 646. In one embodiment, P is an amino acid sequence selected from SEQ ID NOS:556 to 646. In some embodiments, both x and y can be zero. In other embodiments, x can be one and y can be zero. In other embodiments, x can be zero and y can be one. In yet another embodiment, both x and y can be one.

In one embodiment, the FVIII peptide consists of from 9 to 150 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 100 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 50 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 25 amino acids. In yet other embodiments, the FVIII peptide consists of from 9 to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, S0, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, R5, R6, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 amino acids.

Table 8. Exemplary FVIII^2525-2045 Peptides


LHAGMSTLF	556
LHAGMSTLFL	557
LHAGMSTLFLV	558
LHAGMSTLFLVY	559
LHAGMSTLFLVYS	560
LHAGMSTLFLVYSN	561
LHAGMSTLFLVYSNK	562
LHAGMSTLFLVYSNKC	563
LHAGMSTLFLVYSNKCQ	564
LHAGMSTLFLVYSNKCQT	565
LHAGMSTLFLVYSNKCQTP	566
LHAGMSTLFLVYSNKCQTPL	567
LHAGMSTLFLVYSNKCQTPLG	568
HAGMSTLFL	569
HAGMSTLFLV	570
HAGMSTLFLVY	571
HAGMSTLFLVYS	572
HAGMSTLFLVYSN	573
HAGMSTLFLVYSNK	574
HAGMSTLFLVYSNKC	575
HAGMSTLFLVYSNKCQ	576
HAGMSTLFLVYSNKCQT	577
HAGMSTLFLVYSNKCQTP	578
HAGMSTLFLVYSNKCQTPL	579
HAGMSTLFLVYSNKCQTPLG	580
AGMSTLFLV	581
AGMSTLFLVY	582
AGMSTLFLVYS	583
AGMSTLFLVYSN	584
AGMSTLFLVYSNK	585
AGMSTLFLVYSNKC	586
AGMSTLFLVYSNKCQ	587
AGMSTLFLVYSNKCQT	588
AGMSTLFLVYSNKCQTP	589
AGMSTLFLVYSNKCQTPL	590
AGMSTLFLVYSNKCQTPLG	591
GMSTLFLVY	592
GMSTLFLVYS	593
GMSTLFLVYSN	594
GMSTLFLVYSNK	595
GMSTLFLVYSNKC	596
GMSTLFLVYSNKCQ	597
GMSTLFLVYSNKCQT	598
GMSTLFLVYSNKCQTP	599
GMSTLFLVYSNKCQTPL	600
GMSTLFLVYSNKCQTPLG	601
MSTLFLVYS	602
MSTLFLVYSN	603
MSTLFLVYSNK	604
MSTLFLVYSNKC	605
MSTLFLVYSNKCQ	606
MSTLFLVYSNKCQT	607
MSTLFLVYSNKCQTP	608
MSTLFLVYSNKCQTPL	609
MSTLFLVYSNKCQTPLG	610
STLFLVYSN	611
STLFLVYSNK	612
STLFLVYSNKC	613
STLFLVYSNKCQ	614
STLFLVYSNKCQT	615
STLFLVYSNKCQTP	616
STLFLVYSNKCQTPL	617
STLFLVYSNKCQTPLG	618
TLFLVYSNK	619
TLFLVYSNKC	620
TLFLVYSNKCQ	621
TLFLVYSNKCQT	622
TLFLVYSNKCQTP	623
TLFLVYSNKCQTPL	624
TLFLVYSNKCQTPLG	625
LFLVYSNKC	626
LFLVYSNKCQ	627
LFLVYSNKCQT	628
LFLVYSNKCQTP	629
LFLVYSNKCQTPL	630
LFLVYSNKCQTPLG	631
FLVYSNKCQ	632
FLVYSNKCQT	633
FLVYSNKCQTP	634
FLVYSNKCQTPL	635
FLVYSNKCQTPLG	636
LVYSNKCQT	637
LVYSNKCQTP	638
LVYSNKCQTPL	639
LVYSNKCQTPLG	640
VYSNKCQTP	641
VYSNKCQTPL	642
VYSNKCQTPLG	643
YSNKCQTPL	644
YSNKCQTPLG	645
SNKCQTPLG	646

H. Factor VIII^2160-2180 Peptides

In one embodiment, the present invention provides a polypeptide having the sequence (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^2160-2180 peptide having the sequence: NPPIIARYIRLHPTHYSIRST (SEQ ID NO:659), R¹ is an amino acid sequence consisting of from 1 to 80 amino acids, and R² is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one. In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^2160-2180 peptide having the sequence: NPPIIARYIRLHPTHYSIRST (SEQ ID NO:659). In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^2160-2180 peptide having the sequence: NPPIIARYIRLHPTHYSIRST (SEQ ID NO:659). In one embodiment, P is an amino acid sequence having at least 85% identity to a sequence selected from SEQ ID NOS:647 to 737. In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOS:647 to 737. In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence selected from SEQ ID NOS:647 to 737. In one embodiment, P is an amino acid sequence selected from SEQ ID NOS:647 to 737. In some embodiments, both x and y can be zero. In other embodiments, x can be one and y can be zero. In other embodiments, x can be zero and y can be one. In yet another embodiment, both x and y can be one.

Table 9. Exemplary FVIII^2160-2180 Peptides


NPPIIARYI	647
NPPIIARYIR	648
NPPIIARYFRL	649
NPPIIARYIRLH	650
NPPIIARYIRLHP	651
NPPIIARYIRLHPT	652
NPPIIARYIRLHPTH	653
NPPIIARYIRLHPTHY	654
NPPIIARYIRLHPTHYS	655
NPPIIARYIRLHPTHYSI	656
NPPIIARYIRLHPTHYSIR	657
NPPIIARYIRLHPTHYSIRS	658
NPPIIARYIRLHPTHYSIRST	659
PPIIARYIR	660
PPIIARYIRL	661
PPIIARYIRLH	662
PPIIARYIRLHP	663
PPIIARYIRLHPT	664
PPIIARYIRLHPTH	665
PPIIARYIRLHPTHY	666
PPIIARYIRLHPTHYS	667
PPIIARYIRLHPTHYSI	668
PPIIARYIRLHPTHYSIR	669
PPIIARYIRLHPTHYSIRS	670
PPIIARYIRLHPTHYSIRST	671
PIIARYIRL	672
PIIARYIRLH	673
PIIARYIRLHP	674
PIIARYIRLHPT	675
PIIARYIRLHPTH	676
PIIARYIRLHPTHY	677
PIIARYIRLHPTHYS	678
PIIARYIRLHPTHYSI	679
PIIARYIRLHPTHYSIR	680
PIIARYIRLHPTHYSIRS	681
PIIARYIRLHPTHYSIRST	682
IIARYIRLH	683
IIARYIRLHP	684
IIARYIRLHPT	685
IIARYIRLHPTH	686
IIARYIRLHPTHY	687
IIARYIRLHPTHYS	688
IIARYIRLHPTHYSI	689
IIARYIRLHPTHYSIR	690
IIARYIRLHPTHYSIRS	691
HARYIRLHPTHYSIRST	692
IARYIRLHP	693
IARYIRLHPT	694
IARYIRLHPTH	695
IARYIRLHPTHY	696
IARYIRLHPTHYS	697
IARYIRLHPTHYSI	698
IARYIRLHPTHYSIR	699
IARYIRLHPTHYSIRS	700
IARYIRLHPTHYSIRST	701
ARYIRLHPT	702
ARYIRLHPTH	703
ARYIRLHPTHY	704
ARYIRLHPTHYS	705
ARYIRLHPTHYSI	706
ARYIRLHPTHYSIR	707
ARYIRLHPTHYSIRS	708
ARYIRLHPTHYSIRST	709
RYIRLHPTH	710
RYIRLHPTHY	711
RYIRLHPTHYS	712
RYIRLHPTHYSI	713
RYIRLHPTHYSIR	714
RYIRLHPTHYSIRS	715
RYIRLHPTHYSIRST	716
YIRLHPTHY	717
YIRLHPTHYS	718
YIRLHPTHYSI	719
YIRLHPTHYSIR	720
YIRLHPTHYSIRS	721
YIRLHPTHYSIRST	722
IRLHPTHYS	723
IRLHPTHYSI	724
IRLHPTHYSIR	725
IRLHPTHYSIRS	726
IRLHPTHYSIRST	727
RLHPTHYSI	728
RLHPTHYSIR	729
RLHPTHYSIRS	730
RLHPTHYSIRST	731
LHPTHYSIR	732
LHPTHYSIRS	733
LHPTHYSIRST	734
HPTHYSIRS	735
HPTHYSIRST	736
PTHYSIRST	737

I. Factor VIII^102-122 Peptides

In one embodiment, the present invention provides a polypeptide having the sequence (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^102-122 peptide having the sequence: TVVITLKNMASHPVSLHAVGV (SEQ ID NO:740), R¹ is an amino acid sequence consisting of from 1 to 80 amino acids, and R² is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one.

In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^102-122 peptide having the sequence: TVVITLKNMASHPVSLHAVGV (SEQ ID NO:740). In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^102-122 peptide having the sequence: TVVITLKNMASHPVSLHAVGV (SEQ ID NO:740).

In the context of the present invention, FVIII^102-122 peptides also include FVIII^102-119 peptides. Accordingly, In one embodiment, P is an amino acid sequence having at least 85% identity to a sequence selected from SEQ ID NOS:1 to 55 and 738 to 773. In one embodiment, P is an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOS: 1 to 55 and 738 to 773. In one embodiment, P is an amino acid sequence having at least 95% identity to a sequence selected from SEQ ID NOS:1 to 55 and 738 to 773. In one embodiment, P is an amino acid sequence selected from SEQ ID NOS:1 to 55 and 738 to 773. In some embodiments, both x and y can be zero. In other embodiments, x can be one and y can be zero. In other embodiments, x can be zero and y can be one. In yet another embodiment, both x and y can be one.

In one embodiment, the FVIII peptide consists of from 9 to 150 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 100 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 50 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 25 amino acids. In yet other embodiments, the FVIII peptide consists of from 9 to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 amino acids.

Table 10. Exemplary FVIII^102-122 Peptides


TVVITLKNMASHPVSLHAV	738
TVVITLKNMASHPVSLHAVG	739
TVVITLKNMASHPVSLHAVGV	740
VVITLKNMASHPVSLHAV	741
VVITLKNMASHPVSLHAVG	742
VVITLKNMASHPVSLHAVGV	743
VITLKNMASHPVSLHAV	744
VITLKNMASHPVSLHAVG	745
VITLKNMASHPVSLHAVGV	746
ITLKNMASHPVSLHAV	747
ITLKNMASHPVSLHAVG	748
ITLKNMASHPVSLHAVGV	749
TLKNMASHPVSLHAV	750
TLKNMASHPVSLHAVG	751
TLKNMASHPVSLHAVGV	752
LKNMASHPVSLHAV	753
LKNMASHPVSLHAVG	754
LKNMASHPVSLHAVGV	755
KNMASHPVSLHAV	756
KNMASHPVSLHAVG	757
KNMASHPVSLHAVGV	758
NMASHVSLHAV	759
NMASHPVSLHAVG	760
NMASHPVSLHAVGV	761
MASHPVSLHAV	762
MASHPVSLHAVG	763
MASHPVSLHAVGV	764
ASHPVSLHAV	765
ASHPVSLHAVG	766
ASHPVSLHAVGV	767
SHPVSLHAV	768
SHPVSLHAVG	769
SHPVSLHAVGV	770
HPVSLHAVG	771
HPVSLHAVGV	772
PVSLHAVGV	773

IV. Methods of Producing FVIII Peptides

In another aspect, the present invention further relates to methods for producing FVIII peptides. In some embodiments, the FVIII peptides of the present invention can be produced using solid phase (e.g., Fmoc or t-Boc) or liquid phase synthesis techniques generally known in the art. See, e.g., Chan & White, Eds., Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Oxford University Press, 2000); Benoiton, Chemistry of Peptide Synthesis (CRC Press, 2005); Howl, Peptide Synthesis and Applications (Humana Press, 2010).

In one embodiment, the present invention includes a method of making a FVIII peptide, the method comprising: a) synthesizing a peptide using solid phase or liquid phase synthesis techniques, the FVIII peptide having the sequence: (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a sequence selected from SEQ ID NOS:68, 344, and 740, R1 is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one. In one embodiment, R¹ is an amino acid sequence consisting of from 1 to 40 amino acids, and R² is an amino acid sequence consisting of from 1 to 40 amino acids. In certain embodiments, the peptides can cover the whole B-domain of human FVIII protein.

In other embodiments, the peptides can be produced using recombinant techniques. In one embodiment, the present invention includes a method of making a FVIII peptide, the method comprising the steps of: a) providing a culture of cells comprising a vector that encodes a FVIII peptide, the FVIII peptide having the sequence: (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a sequence selected from SEQ ID NOS:68, 344, and 740, R¹ is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one. In one embodiment, R¹ is an amino acid sequence consisting of from 1 to 40 amino acids, and R² is an amino acid sequence consisting of from 1 to 40 amino acids. In certain embodiments, the peptides can cover the whole B-domain of human FVIII protein.

In one embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acids.

In one embodiment, the present invention provides a method for making a FVIII peptide, the method comprising the steps of: a) providing a culture of cells comprising a polynucleotide that encodes a FVIII peptide, the peptide having the sequence: (R¹)_x-P-(R²)_y , wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a sequence selected from SEQ ID NOS:10, 68, 159, 250, 344, 477, 568, 659, and 740, R1 is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one; and b) expressing the peptide in the culture of cells.

In one embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 20 amino acids, In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, or 80 amino acids.

In one embodiment of the methods for producing FVIII peptides, the FVIII peptide consists of from 9 to 150 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 100 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 50 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 25 amino acids. In yet other embodiments, the FVIII peptide consists of from 9 to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 amino acids.

The FVIII peptides of the present invention can be produced by expression in a suitable prokaryotic or eukaryotic host system. Examples of eukaryotic cells include, without limitation, mammalian cells, such as CHO, COS, HEK 293, BHK, SK-Hep, and HepG2; insect cells, for example SF9 cells, SF21 cells, S2 cells, and High Five cells; and yeast cells, for example Saccharomyces or Schizosaccharomyces cells. In one embodiment, the FVIII peptides can be expressed in bacterial cells, yeast cells, insect cells, avian cells, mammalian cells, and the like. In some embodiments, the peptides can be expressed in a human cell line, a hamster cell line, or a murine cell line. In one particular embodiment, the cell line is a CHO, BHK, or HEK cell line.

A wide variety of vectors can be used for the expression of the FVIII peptides and can be selected from eukaryotic and prokaryotic expression vectors. The vectors will include a nucleotide sequence necessary for expression of at least one of the FVIII peptides disclosed herein. Examples of vectors for prokaryotic expression include plasmids such as pRSET, pET, pBAD, etc., wherein the promoters used in prokaryotic expression vectors include lac, trc, trp, recA, araBAD, etc. Examples of vectors for eukaryotic expression include: (i) for expression in yeast, vectors such as pAO, pPIC, pYES, pMET, using promoters such as AOX1, GAP, GAL1, AUG1, etc; (ii) for expression in insect cells, vectors such as pMT, pAc5, pIB, pMIB, pBAC, etc., using promoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh, etc., and (iii) for expression in mammalian cells, vectors such as pSVL, pCMV, pRc/RSV, pcDNA3, pBPV, etc., and vectors derived from viral systems such as vaccinia virus, adeno-associated viruses, herpes viruses, retroviruses, etc., using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin.

In some embodiments of the present invention, the nucleic acid sequences for producing the FVIII peptides further include other sequences suitable for a controlled expression of a protein such as promoter sequences, enhancers, TATA boxes, transcription initiation sites, polylinkers, restriction sites, poly-A-sequences, protein processing sequences, selection markers, and the like which are generally known to a person of ordinary skill in the art.

The culture media used for the cells producing the FVIII peptides can be based on a suitable basal medium well known in the art, e.g., DMEM, Ham's F12, Medium 199, McCoy, or RPMI. The basal medium can include a number of ingredients, including amino acids, vitamins, organic and inorganic salts, and sources of carbohydrate. Each ingredient can be present in an amount that supports the cultivation of a cell, such amounts being generally known to a person skilled in the art. The medium can include auxiliary substances, such as buffer substances, e.g., sodium bicarbonate, antioxidants, stabilizers to counteract mechanical stress, or protease inhibitors. If necessary, a non-ionic surfactant such as copolymers and/or mixtures of polyethylene glycols and polypropylene glycols can be added.

In some embodiments, the culture medium is free of exogenously added protein. "Protein free" and related terms refers to protein that is from a source exogenous to or other than the cells in the culture, which naturally shed proteins during growth. In another embodiment, the culture medium is polypeptide free. In another embodiment, the culture medium is serum free. In another embodiment the culture medium is animal protein free. In another embodiment the culture medium is animal component free. In another embodiment, the culture medium contains protein, e.g., animal protein from serum such as fetal calf serum. In another embodiment, the culture has recombinant proteins exogenously added. In another embodiment, the proteins are from a certified pathogen free animal.

Methods of preparing animal protein-free and chemically defined culture mediums are known in the art, for example in US 2008/0009040 and US 2007/0212770 , which are both incorporated herein for all purposes. In one embodiment, the culture medium used in the methods described herein is animal protein-free or oligopeptide-free medium. In certain embodiments, the culture medium may be chemically defined. The term "chemically defined" as used herein shall mean, that the medium does not comprise any undefined supplements, such as, for example, extracts of animal components, organs, glands, plants, or yeast. Accordingly, each component of a chemically defined medium is accurately defined.

In certain embodiments, the methods of the present invention can include the use of a cell-culture system operated in, for example, batch-mode, semi-batch mode, fed-batch mode, or continuous mode. A batch culture can be a large scale cell culture in which a cell inoculum is cultured to a maximum density in a tank or fermenter, and harvested and processed as a batch. A fed-batch culture can be a batch culture which is supplied with either fresh nutrients (e.g., growth-limiting substrates) or additives (e.g., precursors to products). A continuous culture can be a suspension culture that is continuously supplied with nutrients by the inflow of fresh medium, wherein the culture volume is usually constant. Similarly, continuous fermentation can refer to a process in which cells or micro-organisms are maintained in culture in the exponential growth phase by the continuous addition of fresh medium that is exactly balanced by the removal of cell suspension from the bioreactor. Furthermore, the stirred-tank reactor system can be used for suspension, perfusion, chemostatic, and/or microcarrier cultures. Generally, the stirred-tank reactor system can be operated as any conventional stirred-tank reactor with any type of agitator such as a Rushton, hydrofoil, pitched blade, or marine.

In certain embodiments, the cell-culture methods of the invention can include the use of a microcarrier. In some embodiments, the cell-cultures of the embodiments can be performed in large bioreactors under conditions suitable for providing high volume-specific culture surface areas to achieve high cell densities and protein expression. One means for providing such growth conditions is to use microcarriers for cell-culture in stirred tank bioreactors. The concept of cell-growth on microcarriers was first described by van Wezel (van Wezel, A.L., Nature 216:64-5 (1967)) and allows for cell attachment on the surface of small solid particles suspended in the growth medium. These methods provide for high surface-to-volume ratios and thus allow for efficient nutrient utilization. Furthermore, for expression of secreted proteins in eukaryotic cell lines, the increased surface-to-volume ratio allows for higher levels of secretion and thus higher protein yields in the supernatant of the culture. Finally, these methods allow for the easy scale-up of eukaryotic expression cultures.

The cells expressing FVIII peptides can be bound to a spherical or a porous microcarrier during cell culture growth. The microcarrier can be a microcarrier selected from the group of microcarriers based on dextran, collagen, plastic, gelatine and cellulose and others. It is also possible to grow the cells to a biomass on spherical microcarriers and subculture the cells when they have reached final fermenter biomass and prior to production of the expressed protein on a porous microcarrier or vice versa. Suitable spherical microcarriers can include smooth surface microcarriers, such as Cytodex™ 1, Cytodex™ 2, and Cytodex™ 3 (GE Healthcare) and macroporous microcarriers such as Cytopore™ 1, Cytopore™ 2, Cytoline™ 1, and Cytoline™ 2 (GE Healthcare).

One of ordinary skill in the art will appreciate that the FVIII peptides produced by the synthetic and/or recombinant methods described above can include natural and/or non-natural amino acids, including amino acid analogs and/or amino acid mimetics.

V. Factor FVIII Peptide Compositions for Inducing Immune Tolerance

In another aspect, the FVIII peptides disclosed herein can be included in a pharmaceutical composition. In one embodiment, the present invention provides a pharmaceutical composition comprising a Factor FVIII^246-266 peptide, Factor VIII^1401-1424 peptide, or Factor VIII^102-122 peptide, as described herein.

In one embodiment, the pharmaceutical composition comprises a Factor VIII^246-266 peptide as described herein. In another embodiment, the pharmaceutical composition further comprises a FVIII^474-494 peptide, FVIII^540-560 peptide, FVIII^1785-1805 peptide, FVIII^2025-2045 peptide, FVIII^2100-2180 peptide, FVIII^102-119 peptide, FVIII^1401-1424 peptide, FVIII^102-122 peptide, or second FVIII^246-266 peptide, as described herein.

In another embodiment, the pharmaceutical composition comprises a Factor VIII^1401-1424 peptide as described herein. In another embodiment, the pharmaceutical composition further comprises a FVIII^474-494 peptide, FVIII^540-560 peptide, FVIII^1785-1805 peptide, FVIII^2025-2045 peptide, FVIII^2160-2180 peptide, FVIII^102-119 peptide, FVIII^246-266 peptide, FVIII^162-122 peptide, or second FVIII^1401-1424 peptide, as described herein.

In another embodiment, the pharmaceutical composition comprises a Factor VIII^102-122 peptide as described herein. In another embodiment, the pharmaceutical composition further comprises a FVIII^474-494 peptide, FVIII^540-560 peptide, FVIII^1785-1805 peptide, FVIII^2025-2045 peptide, FVIII^2160-2180 peptide, FVIII^102-119 peptide, FVIII^246-206 peptide, FVIII^1401-1424 peptide, or second FVIII^102-122 peptide, as described herein.

In a specific embodiment, the present invention provides a pharmaceutical composition comprising a peptide having the sequence: (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a sequence selected from SEQ ID NOS:68, 344, and 740, R1 is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one. In one embodiment, R¹ is an amino acid sequence consisting of from 1 to 40 amino acids, and R² is an amino acid sequence consisting of from 1 to 40 amino acids.

In a specific embodiment, the pharmaceutical composition further comprises a second polypeptide, the second polypeptide having the sequence: (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a sequence selected from SEQ ID NOS: 10, 68, 159, 250, 344, 477, 568, 659, and 740, R1 is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one. In one embodiment, R¹ is an amino acid sequence consisting of from 1 to 40 amino acids, and R² is an amino acid sequence consisting of from 1 to 40 amino acids.

In one embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, or 80 amino acids.

In one embodiment, the second FVIII peptide consists of from 9 to 150 amino acids. In another embodiment, the second FVIII peptide consists of from 9 to 100 amino acids. In another embodiment, the second FVIII peptide consists of from 9 to 50 amino acids. In another embodiment, the second VIII peptide consists of from 9 to 25 amino acids. In yet other embodiments, the second FVIII peptide consists of from 9 to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 amino acids.

A. Administration

To administer compositions to a human or test animal, in one aspect, the compositions can include one or more pharmaceutically acceptable carriers. The phrases "pharmaceutically" or "pharmacologically" acceptable refer to molecular entities and compositions that are stable, inhibit protein or peptide degradation such as aggregation and cleavage products, and in addition do not produce allergic, or other adverse reactions when administered using routes well-known in the art, as described below. "Pharmaceutically acceptable carriers" include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.

The pharmaceutical compositions can be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and or surgical implantation at a particular site is contemplated as well. Generally, compositions are essentially free of pyrogens, as well as other impurities that could be harmful to the recipient.

Dosages and frequency of administration will depend upon various factors generally appreciated by those of skill in the art, including, e.g., the severity of a patient's hemophilia and/or whether immune tolerance is more effectively induced using larger or smaller doses. Typical daily doses may range from about 0.01 to 100 mg/kg. Doses in the range of 0.07-700 mg FVIII peptide per week may be effective and well tolerated, although even higher weekly doses may be appropriate and/or well tolerated. The principal determining factor in defining the appropriate dose is the amount of a particular FVIII peptide necessary to be therapeutically effective in a particular context. Repeated administrations may be required in order to achieve longer lasting immune tolerance. Single or multiple administrations of the compositions can be carried out with the dose levels and pattern being selected by the treating physician.

In one aspect, compositions of the invention can be administered by bolus. As another example, a FVIII peptide can be administered as a one-time dose. Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient. The frequency of dosing depends on the route of administration. The optimal pharmaceutical composition is determined by one skilled in the art depending upon the route of administration and desired dosage. See e.g., Remington: The Science and Practice of Pharmacy (Remington the Science and Practice of Pharmacy), 21st Ed. (2005, Lippincott Williams & Wilkins) the disclosure of which is hereby incorporated by reference. Such compositions influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agents. Depending on the route of administration, a suitable dose is calculated according to body weight, body surface area or organ size. Appropriate dosages may be ascertained through use of established assays for determining blood level dosages in conjunction with appropriate dose-response data. The final dosage regimen is determined by the attending physician, considering various factors which modify the action of drugs, e.g. the drug's specific activity, the severity of the damage and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors.

In some embodiments, the compositions comprising a FVIII peptide disclosed herein are lyophilized prior to administration. Lyophilization is carried out using techniques common in the art and should be optimized for the composition being developed, as described, e.g., in Tang et al., Pharm Res. 21:191-200, (2004) and Chang et al., Pharm Res. 13:243-9 (1996). Methods of preparing pharmaceutical compositions can include one or more of the following steps: adding a stabilizing agent to the mixture prior to lyophilizing, adding at least one agent selected from a bulking agent, an osmolarity regulating agent, and a surfactant to the mixture prior to lyophilization. A lyophilized formulation is, in one aspect, at least comprised of one or more of a buffer, a bulking agent, and a stabilizer. In this aspect, the utility of a surfactant is evaluated and selected in cases where aggregation during the lyophilization step or during reconstitution becomes an issue. An appropriate buffering agent is included to maintain the formulation within stable zones of pH during lyophilization.

The standard reconstitution practice for lyophilized material is to add back a volume of pure water or sterile water for injection (WFI) (typically equivalent to the volume removed during lyophilization), although dilute solutions of antibacterial agents are sometimes used in the production of pharmaceuticals for parenteral administration. Accordingly, methods are provided for preparation of reconstituted FVIII peptide compositions comprising the step of adding a diluent to a lyophilized FVIII peptide compositions.

In some embodiments, the lyophilized material may be reconstituted as an aqueous solution. A variety of aqueous carriers, e.g., sterile water for injection, water with preservatives for multi dose use, or water with appropriate amounts of surfactants (for example, an aqueous suspension that contains the active compound in admixture with excipients suitable for the manufacture of aqueous suspensions). In various aspects, such excipients are suspending agents, for example and without limitation, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents are a naturally-occurring phosphatide, for example and without limitation, lecithin, or condensation products of an alkylene oxide with fatty acids, for example and without limitation, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example and without limitation, heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example and without limitation, polyethylene sorbitan monooleate. In various aspects, the aqueous suspensions also contain one or more preservatives, for example and without limitation, ethyl, or n-propyl, p-hydroxybenzoate.

VI. Methods of Treatment

The present invention further relates to methods of treating a patient having a disease associated with the FVIII protein, such as hemophilia A or acquired hemophilia. Such methods can include administration of at least one of the FVIII peptides disclosed herein. In particular, the pharmaceutical compositions including at least one of the FVIII peptides can be administered to induce immune tolerance to FVIII protein in a patient.

In some embodiments, the methods for inducing an immune tolerance to FVIII can include preventing FVIII inhibitor development after administration of FVIII. The term "preventing" refers to allowing no substantially detectable immune response to FVIII. For example, a patient prior to administration of FVIII protein may not have any detectable anti-FVIII antibodies. However, after administration therapy with FVIII protein the level of detectable anti-FVIII antibodies can increase if a FVIII peptide is not administered to induce immune tolerance. The administration of the FVIII peptides disclosed herein can induce immune tolerance, thereby treating a patient having hemophilia.

In other embodiments, the methods for inducing an immune tolerance to FVIII protein can include treating patients already having established FVIII inhibitors. In these embodiments, administration of the FVIII peptide can reduce or eliminate the presence of anti-FVIII antibodies. The term "reduce" means a partial reduction in an immune response to FVIII protein. In certain embodiments, reducing the immune response can include a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in the immune response as compared to the level of the immune response in a patient prior to administration of the FVIII peptide. For example, the percentage reduction can be analyzed by measuring the amount of anti-FVIII antibodies present in the blood prior to and after administration of the FVIII peptide, using standard methods for determining the amount of FVIII antibodies present. In other embodiments, reduction of the immune response can include measuring reduced levels of CD4⁺ T cells specific for FVIII or FVIII specific B cells secreting FVIII antibodies, or a combination of all three, the T cells, B cells, and the anti-FVIII antibodies. Immune cells, such as T cells and B specific for FVIII can be isolated using methods generally known in the art.

In one aspect, the present invention includes a method of inducing immune tolerance to FVIII in a subject, the method comprising a step of administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a FVIII peptide as described herein. In a specific embodiment, the FVIII peptide is a Factor VIII^246-266 peptide, Factor VIII^1401-1424 peptide, or Factor VIII^102-122 peptide, as described herein.

In one embodiment, the method comprises a step of administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a Factor VIII^246-266 peptide as described herein. In another embodiment, the pharmaceutical composition further comprises a FVIII^474-494 peptide, FVIII^540-560 peptide, FVIII^1785-1805 peptide, FVIII^2025-2045 peptide, FVIII^2160-2180 peptide, FVIII^102-119 peptide, FVIII^1401-1424 peptide, FVIII^102-122 peptide, or second FVIII^246-266 peptide, as described herein.

In another embodiment, the method comprises a step of administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a Factor VIII^1401-1424 peptide as described herein. In another embodiment, the pharmaceutical composition further comprises a FVIII^474-494 peptide, FVIII^540-560 peptide, FVIII^1785-1805 peptide, FVIII^2025-2045 peptide, FVIII^2160-2180 peptide, FVIII^102-119 peptide, FVIII^246-266 peptide, FVIII^102-122 peptide, or second FVIII^1401-1424 peptide, as described herein.

In another embodiment, the method comprises a step of administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a Factor VIII^102-122 peptide as described herein. In another embodiment, the pharmaceutical composition further comprises a FVIII^474-494 peptide, FVIII^540-560 peptide, FVIII^1785-1805 peptide, FVIII^2025-2045 peptide, FVIII^2160-2180 peptide, FVIII^102.119 peptide, FVIII^246-266 peptide, FVIII^1401-1424 peptide, or second FVIII^102-122 peptide, as described herein.

In one embodiment, the present invention provides a method for inducing an immune tolerance to a FVIII protein, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a peptide having the sequence: (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a sequence selected from SEQ ID NOS:68, 344, and 740, R1 is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from I to 80 amino acids; and each of x and y are independently zero or one; thereby inducing an immune tolerance to FVIII protein in the subject. In certain embodiments, R¹ is an amino acid sequence consisting of from 1 to 40 amino acids, and R² is an amino acid sequence consisting of from 1 to 40 amino acids.

In one embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R¹ and R¹ are seperately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 6, 7, 8, 59, 60, 61, 62, 63, 64, 5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acids.

In one embodiment, the FVIII peptide consists of from 9 to 150 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 100 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 50 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 25 amino acids. In yet other embodiments, the FVIII peptide consists offrom 9 to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 amino acids.

The methods of inducing immune tolerance can further include combination therapies in which several peptides can be administered to induce immune tolerance. In one embodiment, the method of inducing immune tolerance further comprises administering a therapeutically effective amount of at least a second peptide having the sequence: (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a sequence selected from SEQ ID NOS:10, 68, 159, 250, 344, 477, 568, 659, and 740, R¹ is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one; thereby inducing an immune tolerance to FVIII protein in the subject. In certain embodiments, R¹ is an amino acid sequence consisting of from 1 to 40 amino acids, and R² is an amino acid sequence consisting of from 1 to 40 amino acids. In a particular embodiment, the second peptide consists of from 9 to 80 amino acids. In another particular embodiment, any additional amino acids in the second peptide are natural amino acids. In another particular embodiment, the second peptide consists of from 9 to 40 amino acids in length. In a specific embodiment, the second peptide consists of from 9 to 80 amino acids in length and any additional amino acids in the second peptide are natural amino acids.

In one embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, S6, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acids.

In one embodiment, the second FVIII peptide consists of from 9 to 150 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 100 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 50 amino acids. In another embodiment, the FVIII peptide consists of from 9 to 25 amino acids. In yet other embodiments, the FVIII peptide consists of from 9 to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, l 10, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 amino acids.

In a specific embodiment of method for inducing an immune tolerance, wherein the administered pharmaceutical composition comprises a peptide where P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:68, 344, or 740, the composition further comprises a second polypeptide, the second polypeptide having the sequence: (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a sequence selected from SEQ ID NOS:10, 68, 159, 250, 344, 477, 568, 659, and 740, R1 is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one.

In one embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R² and R² are seperately or both amino acid sequences consisting of from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acids.

In one aspect, the present invention provides the use of a FVIII peptide as described herein for the manufacture of a medicament for the treatment of an immune response generated against FVIII replacement therapy. In a specific embodiment, the FVIII peptide is a FVIII^1401-1424 peptide. In a related aspect, the present invention provides the use of a FVIII peptide as described herein for the manufacture of a medicament for the prevention of an immune response generated against FVIII replacement therapy. In a specific embodiment, the FVIII peptide is a FVIII^1401-1424 peptide.

In one aspect, the present invention provides a FVIII peptide for use as a medicament. In a specific embodiment, the invention provides a polypeptide having the sequence (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^1401-1424 peptide having the sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344), R¹ is an amino acid sequence consisting of from 1 to 80 amino acids, and R² is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one for use as a medicament.

In one aspect, the present invention provides a FVIII peptide for the treatment of an immune response generated against FVIII replacement therapy. In a specific embodiment, the invention provides a polypeptide having the sequence (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^1401-1424 peptide having the sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344), R¹ is an amino acid sequence consisting of from 1 to 80 amino acids, and R² is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one for the treatment of an immune response generated against FVIII replacement therapy.

In one aspect, the present invention provides a FVIII peptide for the prevention of an immune response generated against FVIII replacement therapy. In a specific embodiment, the invention provides a polypeptide having the sequence (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a Factor VIII^1401-1424 peptide having the sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344), R¹ is an amino acid sequence consisting of from 1 to 80 amino acids, and R² is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one for the prevention of an immune response generated against FVIII replacement therapy.

VII. Immunodiagnostics

In one aspect, the present invention provides a method for monitoring FVIII replacement therapy or FVIII immune tolerance induction therapy in a subject in need thereof by identifying the presence or level of a FVIII inhibitory antibody or CD4+ T cell that is specific for FVIII in a biological sample taken from the subject.

In one embodiment, the method comprises a method for monitoring FVIII replacement therapy in a subject in need thereof, the method comprising: contacting a biological sample from the subject with a FVIII^246-266 peptide, FVIII^1401-1424 peptide, or FVIII^102-122 peptide, as described herein; and detecting a complex formed between the FVIII peptide and a FVIII inhibitory antibody present in the sample. In one embodiment, the method comprises determining the level of FVIII inhibitory antibody in the sample. In yet another embodiment, the method comprises determining the level of a FVIII inhibitory antibody in at least two samples taken from the subject at different times, and comparing the levels of FVIII inhibitory antibody between the two samples, wherein an increase in the level of antibody over time is indicative of the formation of an immune response against FVIII administered to the subject during the course of the FVIII replacement therapy.

In another embodiment, the method comprises a method for monitoring FVIII immune tolerance induction therapy in a subject in need thereof, the method comprising: contacting a biological sample from the subject with a FVIII^246-266 peptide, FVIII^1401-1424 peptide, or FVIII^102-122 peptide, as described herein; and detecting a complex formed between the FVIII peptide and a FVIII inhibitory antibody present in the sample. In one embodiment, the method comprises determining the level of FVIII inhibitory antibody in the sample. In yet another embodiment, the method comprises determining the level of a FVIII inhibitory antibody in at least two samples taken from the subject at different times, and comparing the levels of FVIII inhibitory antibody between the two samples, wherein an decrease in the level of antibody over time is indicative of the formation of immune tolerance to FVIII protein in the subject.

In one embodiment, the method comprises a method for monitoring FVIII replacement therapy in a subject in need thereof, the method comprising: contacting a biological sample from the subject with a FVIII^246-266 peptide, FVIII^1401-1424 peptide, or FVIII^102-122 peptide, as described herein; and detecting a complex formed between the FVIII peptide and a CD4+ T cell specific for FVIII present in the sample. In one embodiment, the method comprises determining the level of CD4+ T cell specific for FVIII in the sample. In yet another embodiment, the method comprises determining the level of a CD4⁺ T cell specific for FVIII in at least two samples taken from the subject at different times, and comparing the levels of CD4+ T cell specific for FVIII between the two samples, wherein an increase in the level of antibody over time is indicative of the formation of an immune response against FVIII administered to the subject during the course of the FVIII replacement therapy. In a specific embodiment, the FVIII peptide is complexed with a MHC class II multimer.

In another embodiment, the method comprises a method for monitoring FVIII immune tolerance induction therapy in a subject in need thereof, the method comprising: contacting a biological sample from the subject with a FVIII^246-266 peptide, FVIII^1401-1424 peptide, or FVIII^102-122 peptide, as described herein; and detecting a complex formed between the FVIII peptide and a CD4+ T cell specific for FVIII present in the sample. In one embodiment, the method comprises determining the level of CD4+ T cell specific for FVIII in the sample. In yet another embodiment, the method comprises determining the level of a CD4+ T cell specific for FVIII in at least two samples taken from the subject at different times, and comparing the levels of CD4+ T cell specific for FVIII between the two samples, wherein an decrease in the level of antibody over time is indicative of the formation of immune tolerance to FVIII protein in the subject. In a specific embodiment, the FVIII peptide is complexed with a MHC class II multimer.

As will be appreciated by one of ordinary skill in the art, immune monitoring can be used, for example, to facilitate treatment of patients with hemophilia. For example, immune monitoring can be used to identify whether administration of the peptides and/or compositions of the present invention is preventing or reducing an immune response to a FVIII product. Dosage amounts and/or dosage intervals can be optimized by immune monitoring. In some embodiments, administration dosages can be tailored specifically based on results from immune monitoring of prevention or reduction of anti-FVIII antibodies. In addition, dosing intervals as well as dosage amounts can be determined for a particular patient or group of patients.

A. Methods of Identifying FVIII-Specific T Cells

In another aspect, the present invention includes methods of identifying antigen-specific T cells, more specifically T cells that are specific for FVIII protein and the FVIII peptides described herein. Such methods can, for example, be used for immunodiagnostics, such as immune monitoring of a patient. In one embodiment, the present invention includes a method of identifying FVIII peptide-specific T cells, the method comprising a) combining a plurality of CD4⁺ T cells with a FVIII peptide complexed with a MHC class II multimer, the FVIII peptide having the sequence: (R¹)_x-P-(R²)_y, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a sequence selected from SEQ ID NOS:68, 344, and 740, R1 is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one; and b) identifying at least one of the members of the plurality of CD4⁺ T cells that is specific for the peptide complexed with the MHC class II multimer. In some embodiments, R¹ is an amino acid sequence consisting of from 1 to 40 amino acids, and R² is an amino acid sequence consisting of from 1 to 40 amino acids.

In certain embodiments, the FVIII peptides disclosed herein can be used to generate reagents suitable for direct staining of FVIII specific T cells. For example, the MHC class II multimers that present the FVIII peptides of the present invention can include a variety of forms, such as a MHC class II tetramer. These MHC class II molecules can be further modified to include a diagnostic agent. Alternatively, the FVIII peptides that complex with the MHC class II multimers can include a diagnostic agent. The diagnostic agents (i.e., a detectable moiety) used in the present invention can include those generally known in the art for immune monitoring. For example, FVIII-specific T cells can be identified and/or isolated based on detection of a diagnostic agent associated with a FVIII peptide described herein that is presented by an MHC class II tetramer. Suitable diagnostic agents can include a fluorescent agent, a chemiluminescent agent, a radioactive agent, a contrast agent, and the like. Suitable fluorescence agents include those typically used in flow cytometry and can include but are not limited to fluorescein isothiocyanate, R-Phycoerythrin, Texas Red, Cy3, Cy5, Cy5.5, Cy7, and derivatives thereof.

In certain embodiments, the FVIII peptide can be used to re-stimulate CD4⁺ FVIII-specific T cells in vitro. In these embodiments, the re-stimulation of the T cells could be monitored by detection of proliferation, secretion of cytokines or chemokines, or the up- or down-regulation of certain activation markers that are known to those skilled in the art.

In some embodiments, detection of the diagnostic agent can be used to identify and/or isolate T cells specific for the FVIII peptides disclosed herein. For example, the reagents above (e.g., peptide, MHC class II tetramer, and diagnostic agent) can be used to track FVIII-specific T cells in vitro or ex vivo. In certain embodiments, the T cells can be further isolated and characterized using various techniques generally known in the art, such as flow cytometry, e.g., fluorescence activated cell sorting (FACS), and/or PCR, e.g., single cell PCR.

To carry out immune monitoring analyses, T cells that bind the FVIII peptide-MHC class II multimer complex include CD4⁺ T cells and can be isolated from a patient using a variety of methods generally known in the art. For example, T cells can be isolated and purified from a patient's blood, organs or other tissue. Isolation and identification of the FVIII specific T cells can be used for a variety of immunodiagnostic applications. In certain embodiments, the FVIII peptides or associated reagents can be used for immune monitoring of FVIII-specific T cells during clinical development of a new FVIII product. In other embodiments, the FVIII peptides can be used for immune monitoring of FVIII-specific T cells during immune tolerance induction therapy. In yet other embodiments, the FVIII peptides can be used for immune monitoring of FVIII-specific T cells during FVIII treatment.

FVIII. Kits of the Invention

The present invention also provides kits to facilitate and/or standardize use of compositions provided by the present invention, as well as facilitate the methods of the present invention. Materials and reagents to carry out these various methods can be provided in kits to facilitate execution of the methods. As used herein, the term "kit" is used in reference to a combination of articles that facilitate a process, assay, analysis or manipulation.

Kits can contain chemical reagents (e.g., FVIII peptides or polynucleotides encoding FVIII peptides) as well as other components. In addition, kits of the present invention can also include, for example but are not limited to, apparatus and reagents for sample collection and / or purification, apparatus and reagents for product collection and/or purification, reagents for bacterial cell transformation, reagents for eukaryotic cell transfection, previously transformed or transfected host cells, sample tubes, holders, trays, racks, dishes, plates, instructions to the kit user, solutions, buffers or other chemical reagents, suitable samples to be used for standardization, normalization, and / or control samples. Kits of the present invention can also be packaged for convenient storage and safe shipping, for example, in a box having a lid.

In some embodiments, for example, kits of the present invention can provide a FVIII peptide of the invention, a polynucleotide vector (e.g., a plasmid) encoding a FVIII peptide of the invention, bacterial cell strains suitable for propagating the vector, and reagents for purification of expressed fusion proteins. Alternatively, a kit of the present invention can provide the reagents necessary to conduct mutagenesis of a FVIII peptide in order to generate a conservatively modified variant of the FVIII peptide.

A kit can contain one or more compositions of the invention, for example, one or a plurality of FVIII peptides or one or a plurality of polynucleotides that encode the FVIII peptides. Alternatively, a kit can contain reagents (e.g., peptide, MHC class II tetramer, and diagnostic agent) for carrying out immune monitoring of a patient.

A kit of the invention also can contain one or a plurality of recombinant nucleic acid molecules, which encode the FVIII peptides, which can be the same or different, and can further include, for example, an operatively linked second polynucleotide containing or encoding a restriction endonuclease recognition site or a recombinase recognition site, or any polypeptide of interest. In addition, the kit can contain instructions for using the components of the kit, particularly the compositions of the invention that are contained in the kit.

IX. Specific Embodiments

In one embodiment, the present invention provides a FVIII peptide consisting of a consecutive sequence of nine amino acids that is at least 85 % identical to nine consecutive amino acids in the following amino acid sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344), and the peptide has the formula: (R1)x-peptide-(R2)y, wherein R1 is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from 1 to 80 amino acids; and each of the subscripts x and y are independently zero or one.

In one embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R¹ and R² are seperately or both amino acid sequences consisting of from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 11, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, or 80 amino acids.

In a specific embodiment of the peptides described above, x and y are both zero.

In a specific embodiment of the peptides described above, x is one and y is zero.

In a specific embodiment of the peptides described above, x is zero and y is one.

In a specific embodiment of the peptides described above, x and y are both one.

In a specific embodiment of the peptides described above, the consecutive sequence of nine amino acids is identical to nine consecutive amino acids in the amino acid sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344).

In one embodiment, the present invention provides a pharmaceutical composition comprising a FVIII peptide consisting of a consecutive sequence of nine amino acids that is at least 85 % identical to nine consecutive amino acids in the following amino acid sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344), and the peptide has the formula: (R1)x-peptide-(R2)y, wherein R1 is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from 1 to 80 amino acids; and each of the subscripts x and y are independently zero or one.

In a specific embodiment of the compositions described above, x and y are both zero.

In a specif embodiment of the compositions described above, x is one and y is zero.

In a specific embodiment of the compositions described above, x is zero and y is one.

In a specif embodiment of the compositions described above, x and y are both one.

In a specific embodiment of the compositions described above, the composition further comprises at least one peptide consisting of a consecutive sequence of nine amino acids that is at least 85 % identical to nine consecutive amino acids in an amino acid sequence independently selected from the group consisting of GEVGDTLLIIFKNQASRPYNI (SEQ ID NO:159), PTKSDPRCLTRYYSSFVNMER (SEQ ID NO:250), EVEDNIMVTFRNQASRPYSFY (SEQ ID NO:477), LHAGMSTLFLVYSNKCQTPLG (SEQ ID NO:568), NPPIIARYIRLHPTHYSIRST (SEQ ID NO:659), TVVITLKNMASHPVSLHA (SEQ ID NO:10), AWPKMHTVNGYVNRSLPGLIG (SEQ ID NO:68), and TVVITLKNMASHPVSLHAVGV (SEQ ID NO:740), wherein the at least one peptide is a maximum of 80 amino acids in length and wherein any additional amino acids in the at least one peptide are natural amino acids.

In one embodiment, the present invention provides a method of inducing an immune tolerance to FVIII in a subject, the method comprising a step of administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a FVIII peptide consisting of a consecutive sequence of nine amino acids that is at least 85 % identical to nine consecutive amino acids in the following amino acid sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO: 344), and the peptide has the formula: (R1)x-peptide-(R2)y, wherein R1 is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from 1 to 80 amino acids; each of the subscripts x and y are independently zero or one; and thereby inducing an immune tolerance to FVIII protein in the subject.

In a specific embodiment of the methods described above, the pharmaceutical composition further comprises at least one peptide consisting of a consecutive sequence of nine amino acids that is at least 85 % identical to nine consecutive amino acids in an amino acid sequence independently selected from the group consisting of GEVGDTLLIIFKNQASRPYNI (SEQ ID NO:159), PTKSDPRCLTRYYSSFVNMER (SEQ ID NO:250), EVEDNIMVTFRNQASRPYSFY (SEQ ID NO:477), LHAGMSTLFLVYSNKCQTPLG (SEQ ID NO:568), NPPIIARYIRLHPTHYSIRST (SEQ ID NO:659), TVVITLKNMASHPVSLHA (SEQ ID NO:10), AWPKMHTVNGYVNRSLPGLIG (SEQ ID NO:68), and TVVITLKNMASHPVSLHAVGV (SEQ ID NO:740), wherein the at least one peptide is a maximum of 80 amino acids in length and wherein any additional amino acids in the at least one peptide are natural amino acids.

In a specific embodiment of the methods described above, administration of the pharmaceutical composition prevents development anti-FVIII antibodies in the subject.

In a specific embodiment of the methods described above, administration of the pharmaceutical composition reduces an amount anti-FVIII antibodies present in the subject.

In a specific embodiment of the methods described above, x and y are both zero.

In a specific embodiment of the methods described above, x is one and y is zero.

In a specific embodiment of the methods described above, x is zero and y is one.

In a specific embodiment of the methods described above, x and y are both one.

In one embodiment, the present invention provides a method of making a FVIII peptide, the method comprising the steps of: a) providing a culture of cells comprising a vector that encodes a FVIII peptide consisting of a consecutive sequence of nine amino acids that is at least 85 % identical to nine consecutive amino acids in the following amino acid sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO: 344), and the peptide has the formula: (R1)x-peptide-(R2)y wherein R1 is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from 1 to 80 amino acids; each of the subscripts x and y are independently zero or one; and b) expressing the peptide in the culture of cells.

In a specific embodiment of the methods described above, x and y are both zero.

In a specific embodiment of the methods described above, x and y are both one.

In one embodiment, the present invention provides a method of making a FVIII peptide, the method comprising: a) synthesizing a peptide using solid phase or liquid phase synthesis techniques, the peptide consisting of a consecutive sequence of nine amino acids that is at least 85 % identical to nine consecutive amino acids in the following amino acid sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO: 344), and the peptide has the formula: (R1)x-pcptide-(R2)y wherein R1 is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from 1 to 80 amino acids; and each of the subscripts x and y are independently zero or one.

In a specific embodiment of the methods described above, x and y are both zero.

In a specific embodiment of the methods described above, x and y are both one.

In one embodiment, the present invention provides a method of identifying FVIII peptide-specific T cells, the method comprising: a) combining a plurality of CD4+ T cells with a FVIII peptide complexed with a MHC class II multimer, the FVIII peptide consisting of a consecutive sequence of nine amino acids that is at least 85 % identical to nine consecutive amino acids in the following amino acid sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO: 344), and the peptide has the formula: (R1)x-peptide-(R2)y, wherein R1 is an amino acid sequence consisting of from 1 to 80 amino acids; R2 is an amino acid sequence consisting of from 1 to 80 amino acids; each of the subscripts x and y are independently zero or one; and b) identifying at least one of the members of the plurality of CD4+ T cells that is specific for the peptide complexed with the MHC class II multimer.

In a specific embodiment of the methods described above, the MHC class 11 multimer is a MHC class II tetramer.

In a specific embodiment of the methods described above, the peptide or MHC class 11 multimer further comprises a diagnostic agent.

In a specific embodiment of the methods described above, the diagnostic agent identifies the at least one member of the plurality of CD4+ T cells that is specific for the peptide.

In a specific embodiment of the methods described above, the method further comprises isolating the at least one member of the plurality of CD4+ T cells that is specific for the peptide based on detection of the diagnostic agent.

In a specific embodiment of the methods described above, the at least one member of the plurality of CD4+ T cells is isolated with flow cytometry.

In a specific embodiment of the methods described above, x and y are both zero.

In a specific embodiment of the methods described above, x and y are both one.

The present invention will now be further illustrated in the following examples, without being limited thereto.

X. EXAMPLES Example 1

To better mimic the human MHC class II molecule for identifying FVIII peptides, a mouse model was developed for hemophilia A with a chimeric MHC class II molecule carrying a human HLA-DRB1*1501 specific binding site. This mouse was backcrossed to a mouse carrying a complete knock out of all murine MHC class II genes (Reipert et al., J. Thromb. Haemost. 7 Suppl. 1:92-97 (2009)). In this new transgenic mouse model, all CD4⁺ T cell responses are driven by the human MHC class II molecule. This mouse model was used to identify FVIII peptides presented by HLA-DRB1*1501 that drive anti-FVIII immune responses in these mice.

Materials and Methods

FVIII: Recombinant human FVIII (rFVIII) was produced as an albumin free bulk product (Baxter Neuchatel) and clinical sucrose formulated FVIII product (Advate, Baxter, Westlake Village, CA).

Hemophilic HLA-DRB15 E17 mice: HLA-DRB1*1501^+/- E17^-/- mice as described in Reipert et al., J. Thromb. Haemost. 7 Suppl. 1:92-97 (2009). Mice were all male and aged 8 to 12 weeks at the beginning of the experiment.

Immunization with human recombinant FVIII: HLA-DRB1*1501^+/- E17^-/- mice received between 4 and 8 intravenous or subcutaneous doses of 0.2 µg or 1 µg human rFVIII at weekly intervals. rFVIII was diluted in the original formulation buffer or Dulbecco phosphate buffered saline containing calcium and magnesium (DPBS; Sigma Aldrich, St. Louis, Missouri, USA).

Cell preparation: Spleens were obtained 3 to 7 days after the last immunization with rFVIII. Spleen cells were minced and passed through a 70 µm cell strainer (Becton Dickinson, Franklin Lakes, NJ). Single cells were collected in culture medium: RPMI 1640 medium (Gibco, Invitrogen, Life Technologies, Carlsbad, CA) supplemented with 10% preselected fetal calf serum (FCS; Hyclone, Logan, UT), 2 mM L-glutamine, 100 U/mL penicillin/streptomycin (both from Gibco), and 5x10^-5 M mercaptoethanol (Sigma-Aldrich). Erythrocytes were lysed using hypotonic buffer (pH 7.2) composed of 0.15 M ammonium chloride, 10 mM potassium bicarbonate (both from Merck, Darmstadt, Germany) and 0.1 mM ethylene-diaminetetraacetic acid (Sigma-Aldrich). Cells were washed and counted using a Coulter Counter Z1. Generation of T-cell hybridomas for identifying FVIII peptides

In vitro re-stimulation of spleen cells with human rFVIII: Spleen cells were re-stimulated in the presence of 20 µg/ml human FVIII in culture medium at a concentration of 1.5x10⁶ cells/ml for 3 or 10 days. The culture medium for the 10 day cultures was renewed after 6 days.

Fusion of mouse T cells with BW cells: In vitro re-stimulated spleen cell cultures and BW cells (α-β-) were washed twice with serum free culture medium and then combined at a ratio of 1:3 to 1:10 (T cells : BW cells). The BW cell line was derived from a mouse AKR/J T cell lymphoma. These cells had no T cell receptors on their surface (α-β-) and therefore any T cell receptor after fusion with mouse spleen cells is derived from the fusion partner. After a third washing step, the supernatant was removed. Fusion conditions were achieved by the addition of 1 ml polyethyleneglycol (PEG; 50% HybiMax, Sigma-Aldrich) within 45 seconds. After another 45 seconds of incubation, subsequently 50 ml serum free medium were added to prevent the toxic effect of PEG. Cells were centrifuged at 1300 rpm for 5 minutes without a break to form a very firm pellet. The supernatant was discarded and 50 ml new serum free medium were added very slowly aiming not to dislocate the pellet. The tube was inverted slowly until the cells were re-suspended and centrifuged as before. This was done twice to remove the remaining PEG. The last washing step was done with culture medium. Cells were then diluted and cultured in 96 well plates. The culture medium was changed for selection medium (HAT medium supplement, Sigma Aldrich) after 48 hours and growing clones were selected. Selection medium was kept for 2 weeks, afterwards the medium was subsequently changed back to normal culture medium.

Peptide specificity of FVIII-specific T cell hybridomas: T cell hybridomas were tested for their antigen specificity. For this purpose, 1x10⁵ cells were co-cultured with antigen presenting cells. We used either 5x10⁴ Mgar cells (expressing HLA-DRB1*1501) or 1x10⁵ whole spleen cells derived from naive HLA-DRB1*1501 - E17 mice. Cells were incubated with 10 µg/ml human rFVIII or with 1 µg/ml peptide/peptide pools for 24 hours at 37°C, 5%CO₂. The supernatants were collected and IL-2 release into the culture supernatant was measured using an IL-2 ELISA (BioLegend, San Diego, CA) or IL-2 Bio-Plex (Bio-Rad Laboratories, Hercules, CA) according to the manufacturers protocol. IL-2 release ≥ 20 pg/ml in the presence but not absence of FVIII (or peptides) was considered positive, or alternatively a 10 fold increase in IL-2 release in the presence of FVIII compared to the absence of FVIII was considered positive.

Subcloning of T cell hybridomas: To assure that each clone represents only one type of T cell, hybridoma all clones were sub-cloned. Hybridoma clones were diluted to a limiting dilution of 0.3 cells/well and co-cultured with 200 feeder cells /well. Feeder cells were produced by Mitomycin C treatment of the fusion partner cells, BW cells. 2x10⁸ BW cells were treated with 0.1 mg Mitomycin C from Streptomyces caespitosus (Sigma Aldrich) for 10 minutes at room temperature and 25 minutes at 37°C, 5% CO₂ in the incubator. Five growing subclones per clone were selected and tested for their FVIII specificity.

FVIII peptide pools used to specify specificities of T cell hybridomas: FVIII peptide pools were produced using the SPOT synthesis method as described by Ay et al. (Biopolymers 88:64-75 (2007)). Briefly, 15 mer peptides were synthesized on two identical cellulose membranes. Membranes were cut into vertical and horizontal stripes. Peptides were released from the membrane stripes and used as peptide pools in specificity tests as described above. Peptides were dissolved in DMSO (Hybrimax, Sigma Aldrich) and further diluted with PBS.

Results

181 FVIII specific hybridoma clones were produced. These clones were screened against a peptide library spanning the whole human FVIII. 15 mer peptides offset by three amino acids were used. Using this approach, six different FVIII regions that contained peptides bound to HLA-DRB1*1501 were identified. We found two peptide domains within the A1 domain, two peptides within the A2 domain, one within the B domain, two within the A3 domain and one peptide domain within the C1 domain of human FVIII. FVIII peptide^1401-1424 has not been described before (Table 11). Peptides FVIII^474-494, FVIII^545-559, FVIII^1788-1802 and FVIII^2161-2175 were already identified in WO 09/071886 , which used computer prediction programs followed by the T cell hybridoma technology. Peptide FVIII^2030-2044 was disclosed in WO 03/087161 . Peptide FVIII^2161-2180 was already published by Jacquemin et al., Blood 101 (4):1351-8 (2003).

Table 11. Regions of FVIII including T-cell epitopes

Regions including T cell epitopes	Amino Acid Sequence
	TVVITLKNMASHPVSLHAVGV (SEQ ID NO:740)
	TVVITLKNMASHPVSLHAVGV (SEQ ID NO:740)	AWPKMHTVNGYVNRSLPGLIG (SEQ ID NO:68)
	GEVGDTLLIIFKNQASRPYNI (SEQ ID NO:159)
	GEVGDTLLIIFKNQASRPYNI (SEQ ID NO:159)		PTKSDPRCLTRYYSSFVNMER (SEQ ID NO:250)
QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO.344)	A peptide of the present invention		PTKSDPRCLTRYYSSFVNMER (SEQ ID NO:250)
	EVEDNIMVTFRNQASRPYSFY (SEQ ID NO:477)
	EVEDNIMVTFRNQASRPYSFY (SEQ ID NO:477)	LHAGMSTLFLVYSNKCQTPLG (SEQ ID NO:568)
	NPPIIARYIRLHPTHYSIRST (SEQ ID NO:659)

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

The invention is also exemplified by the following preferred embodiments:

1. A method of inducing an immune tolerance to FVIII in a subject in need thereof, the method comprising a step of:
- administering to the subject a therapeutically effective amount of a peptide having an amino acid sequence consisting of: (R¹)_x-P-(R²)_y, wherein:
  - P is an amino acid sequence having at least 85% identity to at least nine consecutive amino acids of SEQ ID NO:344;
  - R¹ is an amino acid sequence consisting of from 1 to 80 amino acids;
  - R² is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one.
2. The method of item 1, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:344.
3. The method of item 1, wherein P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:344.
4. The method of item 1, wherein P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:344.
5. The method of item 1, wherein P is an amino acid sequence identical to a sequence of at least nine consecutive amino acids of SEQ ID NO:344.
6. The method according to any one of items 1 to 5, wherein x and y are both zero.
7. The method according to any one of items 1 to 5, wherein x is one and y is zero.
8. The method according to any one of items 1 to 5, wherein x is zero and y is one.
9. The method according to any one of items 1 to 5, wherein x and y are both one.
10. The method according to any one of items 1 to 5, wherein the peptide consists of from 9 to 100 amino acids.
11. The method of item 10, wherein the peptide consists of from 9 to 50 amino acids.
12. The method of item 10, wherein the peptide consists of from 9 to 25 amino acids.
13. The method according to any one of items 1 to 12, wherein the method further comprises a step of:
- administering to the subject a therapeutically effective amount of a second peptide having an amino acid sequence consisting of: (R¹)_x-P-(R²)_y, wherein:
  - P is an amino acid sequence having at least 85% identity to at least nine consecutive amino acids of a sequence selected from SEQ ID NOS: 10, 68, 159, 250, 344, 477, 568, 659, and 740;
  - R¹ is an amino acid sequence consisting of from 1 to 80 amino acids;
  - R² is an amino acid sequence consisting of from 1 to 80 amino acids; and
  - each of x and y are independently zero or one.
14. The method according to any one of items 1 to 13, wherein administration of the pharmaceutical composition prevents development of anti-FVIII antibodies in the subject.
15. The method according to any one of items 1 to 13, wherein administration of the pharmaceutical composition reduces an amount anti-FVIII antibodies present in the subject.
16. A peptide consisting of the amino acid sequence: (R¹)_x-P-(R²)_y, wherein:
- P is an amino acid sequence having at least 85% identity to at least nine consecutive amino acids of SEQ ID NO:344;
- R¹ is an amino acid sequence consisting of from 1 to 80 amino acids;
- R² is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one.
17. The peptide of item 16, wherein P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:344.
18. The peptide of item 16, wherein P is an amino acid sequence having at least 90% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:344.
19. The peptide of item 16, wherein P is an amino acid sequence having at least 95% identity to a sequence of at least nine consecutive amino acids of SEQ ID NO:344.
20. The peptide of item 16, wherein P is an amino acid sequence identical to a sequence of at least nine consecutive amino acids of (SEQ ID NO:344.
21. The peptide according to any one of items 16 to 20, wherein x and y are both zero.
22. The peptide according to any one of items 16 to 20, wherein x is one and y is zero.
23. The peptide according to any one of items 16 to 20, wherein x is zero and y is one.
24. The peptide according to any one of items 16 to 20, wherein x and y are both one.
25. The peptide according to any one of items 16 to 20, wherein the peptide consists of from 9 to 100 amino acids.
26. The peptide of item 25, wherein the peptide consists of from 9 to 50 amino acids.
27. The peptide of item 25, wherein the peptide consists of from 9 to 25 amino acids.
28. A composition comprising a peptide according to any one of items 16 to 27.
29. The composition of item 28, wherein the composition is formulated for pharmaceutical administration.
30. The composition of item 28 or 29, wherein the composition further comprises a second polypeptide, the second polypeptide consisting of the amino acid sequence: (R¹)_x-P-(R²)_y, wherein:
- P is an amino acid sequence having at least 85% identity to at least nine consecutive amino acids of a sequence selected from SEQ ID NOS:10, 68, 159,250,344, 477, 568, 659, and 740;
- R¹ is an amino acid sequence consisting of from 1 to 80 amino acids;
- R² is an amino acid sequence consisting of from 1 to 80 amino acids; and
- each of x and y are independently zero or one.
31. A method of making a FVIII peptide, the method comprising the steps of:
1. a) providing a culture of cells comprising a polynucleotide that encodes a FVIII peptide according to any one of items 16 to 27; and
2. b) expressing the peptide in the culture of cells.
32. A method of identifying a FVIII peptide-specific T cell, the method comprising:
1. a) combining a plurality of CD4⁺ T cells with a peptide complexed with a MHC class II multimer, wherein the peptide is a FVIII peptide according to any one of items 16 to 27; and
2. b) identifying at least one of the members of the plurality of CD4⁺ T cells that is specific for the peptide complexed with the MHC class II multimer.
33. The method of item 32, wherein the MHC class II multimer is a MHC class II tetramer.
34. The method of item 32 or 33, wherein the peptide or MHC class II multimer further comprises a detectable moiety.
35. The method according to any one of items 32 to 34, further comprising isolating the at least one CD4⁺ T cell that is specific for the peptide.
36. The method of item 35, wherein the CD4⁺ T cell is isolated using flow cytometry.
37. A fusion protein comprising:
- a Factor VIII peptide according to any one of items 16 to 27; and
- a second peptide.
38. The fusion protein of item 37, wherein the second peptide is a reporter peptide.
39. The fusion protein of item 37 or 38, wherein the fusion protein is encoded by a nucleic acid.
40. The fusion protein of item 37 or 38, wherein the FVIII peptide is chemically linked to the second peptide.

Claims

A peptide having the sequence: (R¹)_xP-(R²)_y,
wherein P is an amino acid sequence having at least 85% identity to the sequence of SEQ ID NO:68;

R¹ is an amino acid sequence consisting of from 1 to 80 amino acids;

R² is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one.
The peptide of claim 1, wherein P is an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:68, and wherein P is preferably an amino acid sequence having at least 95% identity to the sequence of SEQ ID NO:68.
The peptide of claim 1, wherein P is an amino acid sequence identical to the sequence of SEQ ID NO:68.
The peptide according to any one of claims 1 to 3, wherein x and y are both zero, or wherein x is one and y is zero, or wherein x is zero and y is one, or wherein x and y are both one.
The peptide according to any one of claims 1 to 4, wherein the peptide consists of from 21 to 100 amino acids, or wherein the peptide consists of from 21 to 50 amino acids, or wherein the peptide consists of from 21 to 25 amino acids.
A composition comprising a peptide according to any one of claims 1-5.
The composition of claim 6, wherein the composition is formulated for pharmaceutical administration.
The composition of claim 6 or 7, wherein the composition further comprises a second polypeptide, the second polypeptide consisting of the amino acid sequence: (R¹)_x-P-(R²)_y, wherein:
P is an amino acid sequence having at least 85% identity to a sequence of at least nine consecutive amino acids of a sequence selected from SEQ ID NOS:10, 68, 159, 250, 477, 568, 659, and 740;

R¹ is an amino acid sequence consisting of from 1 to 80 amino acids;

R² is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one.
A peptide according to any one of claims 1 to 5, for use in a method of inducing an immune tolerance to FVIII in a subject in need thereof.
The peptide for use according to of claim 9, wherein the use further comprises:
use of a therapeutically effective amount of a second peptide having the amino acid sequence: (R¹)_x,-P-(R²)_y,

wherein P is an amino acid sequence having at least 85% identity to at least nine consecutive amino acids of a sequence selected from SEQ ID NOS:10, 68, 159, 250, 344, 477, 568, 659, and 740;

R¹ is an amino acid sequence consisting of from 1 to 80 amino acids;

R² is an amino acid sequence consisting of from 1 to 80 amino acids; and

each of x and y are independently zero or one.
A method of identifying FVIII peptide-specific T cells, the method comprising:
a) combining a plurality of isolated or hybridoma CD4⁺ T cells with a peptide complexed with a MHC class II multimer, wherein the peptide is a FVIII peptide according to any one of claims 1 to 5; and

b) identifying at least one of the members of the plurality of CD4⁺ T cells that is specific for the peptide complexed with the MHC class II multimer.
The method of claim 11, wherein the MHC class II multimer is a MHC class II tetramer.
The method of claim 11 or 12, wherein the peptide or MHC class II multimer further comprises a detectable moiety.
The method according to any one of claims 11 to 13, further comprising isolating the at least one CD4⁺ T cell that is specific for the peptide.
The method of claim 14, wherein the CD4⁺ T cell is isolated using flow cytometry.
A fusion protein comprising:
a Factor VIII peptide according to any one of claims 1 to 5; and a second peptide.
The fusion protein of claim 16, wherein the second peptide is a reporter peptide.
The fusion protein of claims 16 or 17, wherein the fusion protein is encoded by a nucleic acid.
The fusion protein of claim 16 or 17, wherein the FVIII peptide is chemically linked to the second peptide.
A method for making a FVIII peptide, the method comprising the steps of:
a) Providing a culture of cells comprising a polynucleotide that encodes a FVIII peptide according to any one of claims 1 to 5; and

b) Expressing the peptide in the culture of cells.