FVIII PEPTIDES FOR IMMUNE TOLERANCE INDUCTION AND IMMUNODIAGNOSTICS

Abstract

The present invention is related to peptides that can be used to reduce the immune response against FVIII or to induce tolerance to human FVIII in patients with, e.g., hemophilia A. Furthermore, the peptides can be used for immunodiagnostic purposes to detect FVIII-specific CD4.sup.+ T cells to monitor patients with hemophilia A during replacement therapy and during immune tolerance induction therapy.

Claims

1. A peptide consisting of the amino acid sequence:
(R.sup.1).sub.x—P—(R.sup.2).sub.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; R.sup.1 is an amino acid sequence consisting of from 1 to 80 amino acids; R.sup.2 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 peptide of claim 1, wherein x and y are both zero.

3. The peptide of claim 1, wherein x is one and y is zero.

4. The peptide of claim 1, wherein x is zero and y is one.

5. The peptide of claim 1, wherein x and y are both one.

6. The peptide of claim 1, wherein the peptide consists of from 9 to 100 amino acids.

7. The peptide of claim 6, wherein the peptide consists of from 9 to 50 amino acids.

8. The peptide of claim 6, wherein the peptide consists of from 9 to 25 amino acids.

9. A composition comprising a peptide according to claim 1.

10. The composition of claim 9, wherein the composition is formulated for pharmaceutical administration.

11. The composition of claim 9, wherein the composition further comprises a second polypeptide, the second polypeptide consisting of the amino acid sequence:
(R.sup.1).sub.x—P—(R.sup.2).sub.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; R.sup.1 is an amino acid sequence consisting of from 1 to 80 amino acids; R.sup.2 is an amino acid sequence consisting of from 1 to 80 amino acids; and each of x and y are independently zero or one.

12. 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 according to claim 1.

13. 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 claim 1; and b) expressing the peptide in the culture of cells.

14. A method of identifying a FVIII peptide-specific T cell, the method comprising: a) combining a plurality of CD4.sup.+ T cells with a peptide complexed with a MHC class II multimer, wherein the peptide is a FVIII peptide according to claim 1; and b) identifying at least one of the members of the plurality of CD4.sup.+ T cells that is specific for the peptide complexed with the MHC class II multimer.

15. The method of claim 14, wherein the MHC class II multimer is a MHC class II tetramer.

16. The method of claim 14, wherein the peptide or MHC class II multimer further comprises a detectable moiety.

17. The method of claim 14, further comprising isolating the at least one CD4.sup.+ T cells that is specific for the peptide.

18. The method of claim 17, wherein the CD4.sup.+ T cells is isolated using flow cytometry.

19. A fusion protein comprising: a Factor VIII peptide according to claim 1; and a second peptide.

20. The fusion protein of claim 19, wherein the second peptide is a reporter peptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

[0073] 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.

[0074] The present invention is based in-part on the discovery that several regions of FVIII, specifically FVIII.sup.102-122, FVIII.sup.246-266, and FVIII.sup.141-1424, 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.

[0075] Peptides of the present invention include peptides having at least a portion of the regions FVIII.sup.102-122, FVIII.sup.246-266, and FVIII.sup.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.sup.102-122, FVIII.sup.246-266, or FVIII.sup.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.sup.102-122, FVIII.sup.246-266, and FVIII.sup.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

[0076] 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.

[0077] 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.

[0078] 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.

[0079] “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.

[0080] 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.

[0081] 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).

[0082] 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.

[0083] 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

[0084] 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.

[0085] In a specific embodiment, the FVIII peptide has the sequence: (R.sup.1).sub.x—P—(R.sup.2).sub.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, R.sup.1 is an amino acid sequence consisting of from 1 to 40 amino acids, and R.sup.2 is an amino acid sequence consisting of from 1 to 40 amino acids.

[0086] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0087] 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.

[0088] Generally, the FVIII peptides of the present invention can include any sequence of amino acids present in the identified region of FVIII.sup.102-122, FVIII.sup.246-266, or FVIII.sup.1401-1424, or a modified variant that can, for example, have a retained function similar or identical to FVIII.sup.102-122, FVIII.sup.246-266, or FVIII.sup.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.sup.102-122, FVIII.sup.246-266, or FVIII.sup.1401-1424.

[0089] 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.sup.102-122, FVIII.sup.246-266, or FVIII.sup.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.sup.102-122, FVIII.sup.246-266, or FVIII.sup.1401-1424 region.

[0090] 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 (R.sup.1).sub.x and (R.sup.2).sub.y. In certain embodiments, R.sup.1 and R.sup.2 can range from 1 to about 80 amino acids in length. Alternatively, R.sup.1 and R.sup.2 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.

[0091] In one embodiment, the present invention provides a polypeptide having the sequence (R.sup.1).sub.x—P—(R.sup.2).sub.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, R.sup.1 is an amino acid sequence consisting of from 1 to 80 amino acids, and R.sup.2 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, R.sup.1 and R.sup.2 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-US-00001 TABLE 1 Regions of FVIII including T-cell epitopes Regions including Amino Acid Sequence T cell epitopes FVIII.sup.102-119 TVVITLKNMASHPVSLHA (SEQ ID NO: 10) FVIII.sup.246-266 AWPKMHTVNGYVNRSLPGLIG (SEQ ID NO: 68) FVIII.sup.474-494 GEVGDTLLIIFKNQASRPYNI (SEQ ID NO: 159) FVIII.sup.540-560 PTKSDPRCLTRYYSSFVNMER (SEQ ID NO: 250) FVIII.sup.1401-1424 QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO: 344) FVIII.sup.1785-1805 EVEDNIMVTFRNQASRPYSFY (SEQ ID NO: 477) FVIII.sup.2025-2045 LHAGMSTLFLVYSNKCQTPLG (SEQ ID NO: 568) FVIII.sup.2160-2180 NPPIIARYIRLHPTHYSIRST (SEQ ID NO: 659) FVIII.sup.102-122 TVVITLKNMASHPVSLHAVGV (SEQ ID NO: 740)

[0092] As described above, the FVIII peptides of the present invention can include any sequence of amino acids present in the identified region of FVIII.sup.1401-1424 or a modified variant that can, for example, have a retained function similar or identical to FVIII.sup.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.sup.474-494; SEQ ID NO: 159), PTKSDPRCLTRYYSSFVNMER (FVIII.sup.540-560; SEQ ID NO:250), EVEDNIMVTFRNQASRPYSFY (FVIII.sup.1785-1805; SEQ ID NO:477), LHAGMSTLFLVYSNKCQTPLG (FVIII.sup.2025-2045; SEQ ID NO:568), NPPIIARYIRLHPTHYSIRST (FVIII.sup.2160-2180; SEQ ID NO:659), TVVITLKNMASHPVSLHA (FVIII.sup.102-119; SEQ ID NO:10), AWPKMHTVNGYVNRSLPGLIG (FVIII.sup.246-266; SEQ ID NO:68), and TVVITLKNMASHPVSLHAVGV (FVIII.sup.102-122; SEQ ID NO:740).

[0093] 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.sup.474-494, FVIII.sup.540-560, FVIII.sup.1785-1805, FVIII.sup.2025-2045, FVIII.sup.2160-2180, FVIII.sup.102-119, FVIII.sup.246-266, or FVIII.sup.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.sup.474-494 FVIII.sup.540-560, FVIII.sup.1785-1805, FVIII.sup.2025-2045, FVIII.sup.2160-2180, FVIII.sup.102-119, FVIII.sup.246-266, or FVIII.sup.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.sup.474-494, FVIII.sup.540-560, FVIII.sup.1785-1805, FVIII.sup.2025-2045, FVIII.sup.2160-2180, FVIII.sup.102-119, FVIII.sup.246-266, or FVIII.sup.102-122 regions. The FVIII peptides disclosed here can be made using methods described above with respect to the FVIII peptides relating to FVIII.sup.1401-1424.

[0094] A. Factor VIII.sup.102-119 Peptides

[0095] In one embodiment, the present invention provides a polypeptide having the sequence (R.sup.1).sub.x—P—(R.sup.2).sub.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.sup.102-119 peptide having the sequence: TVVITLKNMASHPVSLHA (SEQ ID NO: 10), R.sup.1 is an amino acid sequence consisting of from 1 to 80 amino acids, and R.sup.2 is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one.

[0096] 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.sup.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.

[0097] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0098] In certain embodiments, R.sup.1 is an amino acid sequence consisting of from 1 to 40 amino acids, and R.sup.2 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-US-00002 TABLE 2 Exemplary FVIII.sup.102-119 Peptides Peptide Sequence SEQ ID NO: FVIII.sup.102-119-1 TVVITLKNM  1 FVIII.sup.102-119-2 TVVITLKNMA  2 FVIII.sup.102-119-3 TVVITLKNMAS  3 FVIII.sup.102-119-4 TVVITLKNMASH  4 FVIII.sup.102-119-5 TVVITLKNMASHP  5 FVIII.sup.102-119-6 TVVITLKNMASHPV  6 FVIII.sup.102-119-7 TVVITLKNMASHPVS  7 FVIII.sup.102-119-8 TVVITLKNMASHPVSL  8 FVIII.sup.102-119-9 TVVITLKNMASHPVSLH  9 FVIII.sup.102-119-10 TVVITLKNMASHPVSLHA 10 FVIII.sup.102-119-11 VVITLKNMA 11 FVIII.sup.102-119-12 VVITLKNMAS 12 FVIII.sup.102-119-13 VVITLKNMASH 13 FVIII.sup.102-119-14 VVITLKNMASHP 14 FVIII.sup.102-119-15 VVITLKNMASHPV 15 FVIII.sup.102-119-16 VVITLKNMASHPVS 16 FVIII.sup.102-119-17 VVITLKNMASHPVSL 17 FVIII.sup.102-119-18 VVITLKNMASHPVSLH 18 FVIII.sup.102-119-19 VVITLKNMASHPVSLHA 19 FVIII.sup.102-119-20 VITLKNMAS 20 FVIII.sup.102-119-21 VITLKNMASH 21 FVIII.sup.102-119-22 VITLKNMASHP 22 FVIII.sup.102-119-23 VITLKNMASHPV 23 FVIII.sup.102-119-24 VITLKNMASHPVS 24 FVIII.sup.102-119-25 VITLKNMASHPVSL 25 FVIII.sup.102-119-26 VITLKNMASHPVSLH 26 FVIII.sup.102-119-27 VITLKNMASHPVSLHA 27 FVIII.sup.102-119-28 ITLKNMASH 28 FVIII.sup.102-119-29 ITLKNMASHP 29 FVIII.sup.102-119-30 ITLKNMASHPV 30 FVIII.sup.102-119-31 ITLKNMASHPVS 31 FVIII.sup.102-119-32 ITLKNMASHPVSL 32 FVIII.sup.102-119-33 ITLKNMASHPVSLH 33 FVIII.sup.102-119-34 ITLKNMASHPVSLHA 34 FVIII.sup.102-119-35 TLKNMASHP 35 FVIII.sup.102-119-36 TLKNMASHPV 36 FVIII.sup.102-119-37 TLKNMASHPVS 37 FVIII.sup.102-119-38 TLKNMASHPVSL 38 FVIII.sup.102-119-39 TLKNMASHPVSLH 39 FVIII.sup.102-119-40 TLKNMASHPVSLHA 40 FVIII.sup.102-119-41 LKNMASHPV 41 FVIII.sup.102-119-42 LKNMASHPVS 42 FVIII.sup.102-119-43 LKNMASHPVSL 43 FVIII.sup.102-119-44 LKNMASHPVSLH 44 FVIII.sup.102-119-45 LKNMASHPVSLHA 45 FVIII.sup.102-119-46 KNMASHPVS 46 FVIII.sup.102-119-47 KNMASHPVSL 47 FVIII.sup.102-119-48 KNMASHPVSLH 48 FVIII.sup.102-119-49 KNMASHPVSLHA 49 FVIII.sup.102-119-50 NMASHPVSL 50 FVIII.sup.102-119-51 NMASHPVSLH 51 FVIII.sup.102-119-52 NMASHPVSLHA 52 FVIII.sup.102-119-53 MASHPVSLH 53 FVIII.sup.102-119-54 MASHPVSLHA 54 FVIII.sup.102-119-55 ASHPVSLHA 55

[0099] B. Factor VIII.sup.246-266 Peptides

[0100] In one embodiment, the present invention provides a polypeptide having the sequence (R.sup.1).sub.x—P—(R.sup.2).sub.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.sup.246-266 peptide having the sequence: AWPKMHTVNGYVNRSLPGLIG (SEQ ID NO:68), R.sup.1 is an amino acid sequence consisting of from 1 to 80 amino acids, and R.sup.2 is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one.

[0101] 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.sup.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.sup.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.

[0102] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0103] 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-US-00003 TABLE 3 Exemplary FVIII.sup.246-266 Peptides Peptide Sequence SEQ ID NO: FVIII.sup.246-266-1 AWPKMHTVN  56 FVIII.sup.246-266-2 AWPKMHTVNG  57 FVIII.sup.246-266-3 AWPKMHTVNGY  58 FVIII.sup.246-266-4 AWPKMHTVNGYV  59 FVIII.sup.246-266-5 AWPKMHTVNGYVN  60 FVIII.sup.246-266-6 AWPKMHTVNGYVNR  61 FVIII.sup.246-266-7 AWPKMHTVNGYVNRS  62 FVIII.sup.246-266-8 AWPKMHTVNGYVNRSL  63 FVIII.sup.246-266-9 AWPKMHTVNGYVNRSLP  64 FVIII.sup.246-266-10 AWPKMHTVNGYVNRSLPG  65 FVIII.sup.246-266-11 AWPKMHTVNGYVNRSLPGL  66 FVIII.sup.246-266-12 AWPKMHTVNGYVNRSLPGLI  67 FVIII.sup.246-266-13 AWPKMHTVNGYVNRSLPGLIG  68 FVIII.sup.246-266-14 WPKMHTVNG  69 FVIII.sup.246-266-15 WPKMHTVNGY  70 FVIII.sup.246-266-16 WPKMHTVNGYV  71 FVIII.sup.246-266-17 WPKMHTVNGYVN  72 FVIII.sup.246-266-18 WPKMHTVNGYVNR  73 FVIII.sup.246-266-19 WPKMHTVNGYVNRS  74 FVIII.sup.246-266-20 WPKMHTVNGYVNRSL  75 FVIII.sup.246-266-21 WPKMHTVNGYVNRSLP  76 FVIII.sup.246-266-22 WPKMHTVNGYVNRSLPG  77 FVIII.sup.246-266-23 WPKMHTVNGYVNRSLPGL  78 FVIII.sup.246-266-24 WPKMHTVNGYVNRSLPGLI  79 FVIII.sup.246-266-25 WPKMHTVNGYVNRSLPGLIG  80 FVIII.sup.246-266-26 PKMHTVNGY  81 FVIII.sup.246-266-27 PKMHTVNGYV  82 FVIII.sup.246-266-28 PKMHTVNGYVN  83 FVIII.sup.246-266-29 PKMHTVNGYVNR  84 FVIII.sup.246-266-30 PKMHTVNGYVNRS  85 FVIII.sup.246-266-31 PKMHTVNGYVNRSL  86 FVIII.sup.246-266-32 PKMHTVNGYVNRSLP  87 FVIII.sup.246-266-33 PKMHTVNGYVNRSLPG  88 FVIII.sup.246-266-34 PKMHTVNGYVNRSLPGL  89 FVIII.sup.246-266-35 PKMHTVNGYVNRSLPGLI  90 FVIII.sup.246-266-36 PKMHTVNGYVNRSLPGLIG  91 FVIII.sup.246-266-37 KMHTVNGYV  92 FVIII.sup.246-266-38 KMHTVNGYVN  93 FVIII.sup.246-266-39 KMHTVNGYVNR  94 FVIII.sup.246-266-40 KMHTVNGYVNRS  95 FVIII.sup.246-266-41 KMHTVNGYVNRSL  96 FVIII.sup.246-266-42 KMHTVNGYVNRSLP  97 FVIII.sup.246-266-43 KMHTVNGYVNRSLPG  98 FVIII.sup.246-266-44 KMHTVNGYVNRSLPGL  99 FVIII.sup.246-266-45 KMHTVNGYVNRSLPGLI 100 FVIII.sup.246-266-46 KMHTVNGYVNRSLPGLIG 101 FVIII.sup.246-266-47 MHTVNGYVN 102 FVIII.sup.246-266-48 MHTVNGYVNR 103 FVIII.sup.246-266-49 MHTVNGYVNRS 104 FVIII.sup.246-266-50 MHTVNGYVNRSL 105 FVIII.sup.246-266-51 MHTVNGYVNRSLP 106 FVIII.sup.246-266-52 MHTVNGYVNRSLPG 107 FVIII.sup.246-266-53 MHTVNGYVNRSLPGL 108 FVIII.sup.246-266-54 MHTVNGYVNRSLPGLI 109 FVIII.sup.246-266-55 MHTVNGYVNRSLPGLIG 110 FVIII.sup.246-266-56 HTVNGYVNR 111 FVIII.sup.246-266-57 HTVNGYVNRS 112 FVIII.sup.246-266-58 HTVNGYVNRSL 113 FVIII.sup.246-266-59 HTVNGYVNRSLP 114 FVIII.sup.246-266-60 HTVNGYVNRSLPG 115 FVIII.sup.246-266-61 HTVNGYVNRSLPGL 116 FVIII.sup.246-266-62 HTVNGYVNRSLPGLI 117 FVIII.sup.246-266-63 HTVNGYVNRSLPGLIG 118 FVIII.sup.246-266-64 TVNGYVNRS 119 FVIII.sup.246-266-65 TVNGYVNRSL 120 FVIII.sup.246-266-66 TVNGYVNRSLP 121 FVIII.sup.246-266-67 TVNGYVNRSLPG 122 FVIII.sup.246-266-68 TVNGYVNRSLPGL 123 FVIII.sup.246-266-69 TVNGYVNRSLPGLI 124 FVIII.sup.246-266-70 TVNGYVNRSLPGLIG 125 FVIII.sup.246-266-71 VNGYVNRSL 126 FVIII.sup.246-266-72 VNGYVNRSLP 127 FVIII.sup.246-266-73 VNGYVNRSLPG 128 FVIII.sup.246-266-74 VNGYVNRSLPGL 129 FVIII.sup.246-266-75 VNGYVNRSLPGLI 130 FVIII.sup.246-266-76 VNGYVNRSLPGLIG 131 FVIII.sup.246-266-77 NGYVNRSLP 132 FVIII.sup.246-266-78 NGYVNRSLPG 133 FVIII.sup.246-266-79 NGYVNRSLPGL 134 FVIII.sup.246-266-80 NGYVNRSLPGLI 135 FVIII.sup.246-266-81 NGYVNRSLPGLIG 136 FVIII.sup.246-266-82 GYVNRSLPG 137 FVIII.sup.246-266-83 GYVNRSLPGL 138 FVIII.sup.246-266-84 GYVNRSLPGLI 139 FVIII.sup.246-266-85 GYVNRSLPGLIG 140 FVIII.sup.246-266-86 YVNRSLPGL 141 FVIII.sup.246-266-87 YVNRSLPGLI 142 FVIII.sup.246-266-88 YVNRSLPGLIG 143 FVIII.sup.246-266-89 VNRSLPGLI 144 FVIII.sup.246-266-90 VNRSLPGLIG 145 FVIII.sup.246-266-91 NRSLPGLIG 146

[0104] C. Factor VIII.sup.474-494 Peptides

[0105] In one embodiment, the present invention provides a polypeptide having the sequence (R.sup.1).sub.x—P—(R.sup.2).sub.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.sup.474-494 peptide having the sequence: GEVGDTLLIIFKNQASRPYNI (SEQ ID NO: 159), R.sup.1 is an amino acid sequence consisting of from 1 to 80 amino acids, and R.sup.2 is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one.

[0106] 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.sup.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.sup.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.

[0107] In certain embodiments, R.sup.1 is an amino acid sequence consisting of from 1 to 40 amino acids, and R.sup.2 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-US-00004 TABLE 4 Exemplary FVIII.sup.474-494 Peptides Peptide Sequence SEQ ID NO: FVIII.sup.474-494-1 GEVGDTLLI 147 FVIII.sup.474-494-2 GEVGDTLLII 148 FVIII.sup.474-494-3 GEVGDTLLIIF 149 FVIII.sup.474-494-4 GEVGDTLLIIFK 150 FVIII.sup.474-494-5 GEVGDTLLIIFKN 151 FVIII.sup.474-494-6 GEVGDTLLIIFKNQ 152 FVIII.sup.474-494-7 GEVGDTLLIIFKNQA 153 FVIII.sup.474-494-8 GEVGDTLLIIFKNQAS 154 FVIII.sup.474-494-9 GEVGDTLLIIFKNQASR 155 FVIII.sup.474-494-10 GEVGDTLLIIFKNQASRP 156 FVIII.sup.474-494-11 GEVGDTLLIIFKNQASRPY 157 FVIII.sup.474-494-12 GEVGDTLLIIFKNQASRPYN 158 FVIII.sup.474-494-13 GEVGDTLLIIFKNQASRPYNI 159 FVIII.sup.474-494-14 EVGDTLLII 160 FVIII.sup.474-494-15 EVGDTLLIIF 161 FVIII.sup.474-494-16 EVGDTLLIIFK 162 FVIII.sup.474-494-17 EVGDTLLIIFKN 163 FVIII.sup.474-494-18 EVGDTLLIIFKNQ 164 FVIII.sup.474-494-19 EVGDTLLIIFKNQA 165 FVIII.sup.474-494-20 EVGDTLLIIFKNQAS 166 FVIII.sup.474-494-21 EVGDTLLIIFKNQASR 167 FVIII.sup.474-494-22 EVGDTLLIIFKNQASRP 168 FVIII.sup.474-494-23 EVGDTLLIIFKNQASRPY 169 FVIII.sup.474-494-24 EVGDTLLIIFKNQASRPYN 170 FVIII.sup.474-494-25 EVGDTLLIIFKNQASRPYNI 171 FVIII.sup.474-494-26 VGDTLLIIF 172 FVIII.sup.474-494-27 VGDTLLIIFK 173 FVIII.sup.474-494-28 VGDTLLIIFKN 174 FVIII.sup.474-494-29 VGDTLLIIFKNQ 175 FVIII.sup.474-494-30 VGDTLLIIFKNQA 176 FVIII.sup.474-494-31 VGDTLLIIFKNQAS 177 FVIII.sup.474-494-32 VGDTLLIIFKNQASR 178 FVIII.sup.474-494-33 VGDTLLIIFKNQASRP 179 FVIII.sup.474-494-34 VGDTLLIIFKNQASRPY 180 FVIII.sup.474-494-35 VGDTLLIIFKNQASRPYN 181 FVIII.sup.474-494-36 VGDTLLIIFKNQASRPYNI 182 FVIII.sup.474-494-37 GDTLLIIFK 183 FVIII.sup.474-494-38 GDTLLIIFKN 184 FVIII.sup.474-494-39 GDTLLIIFKNQ 185 FVIII.sup.474-494-40 GDTLLIIFKNQA 186 FVIII.sup.474-494-41 GDTLLIIFKNQAS 187 FVIII.sup.474-494-42 GDTLLIIFKNQASR 188 FVIII.sup.474-494-43 GDTLLIIFKNQASRP 189 FVIII.sup.474-494-44 GDTLLIIFKNQASRPY 190 FVIII.sup.474-494-45 GDTLLIIFKNQASRPYN 191 FVIII.sup.474-494-46 GDTLLIIFKNQASRPYNI 192 FVIII.sup.474-494-47 DTLLIIFKN 193 FVIII.sup.474-494-48 DTLLIIFKNQ 194 FVIII.sup.474-494-49 DTLLIIFKNQA 195 FVIII.sup.474-494-50 DTLLIIFKNQAS 196 FVIII.sup.474-494-51 DTLLIIFKNQASR 197 FVIII.sup.474-494-52 DTLLIIFKNQASRP 198 FVIII.sup.474-494-53 DTLLIIFKNQASRPY 199 FVIII.sup.474-494-54 DTLLIIFKNQASRPYN 200 FVIII.sup.474-494-55 DTLLIIFKNQASRPYNI 201 FVIII.sup.474-494-56 TLLIIFKNQ 202 FVIII.sup.474-494-57 TLLIIFKNQA 203 FVIII.sup.474-494-58 TLLIIFKNQAS 204 FVIII.sup.474-494-59 TLLIIFKNQASR 205 FVIII.sup.474-494-60 TLLIIFKNQASRP 206 FVIII.sup.474-494-61 TLLIIFKNQASRPY 207 FVIII.sup.474-494-62 TLLIIFKNQASRPYN 208 FVIII.sup.474-494-63 TLLIIFKNQASRPYNI 209 FVIII.sup.474-494-64 LLIIFKNQA 210 FVIII.sup.474-494-65 LLIIFKNQAS 211 FVIII.sup.474-494-66 LLIIFKNQASR 212 FVIII.sup.474-494-67 LLIIFKNQASRP 213 FVIII.sup.474-494-68 LLIIFKNQASRPY 214 FVIII.sup.474-494-69 LLIIFKNQASRPYN 215 FVIII.sup.474-494-70 LLIIFKNQASRPYNI 216 FVIII.sup.474-494-71 LIIFKNQAS 217 FVIII.sup.474-494-72 LIIFKNQASR 218 FVIII.sup.474-494-73 LIIFKNQASRP 219 FVIII.sup.474-494-74 LIIFKNQASRPY 220 FVIII.sup.474-494-75 LIIFKNQASRPYN 221 FVIII.sup.474-494-76 LIIFKNQASRPYNI 222 FVIII.sup.474-494-77 IIFKNQASR 223 FVIII.sup.474-494-78 IIFKNQASRP 224 FVIII.sup.474-494-79 IIFKNQASRPY 225 FVIII.sup.474-494-80 IIFKNQASRPYN 226 FVIII.sup.474-494-81 IIFKNQASRPYNI 227 FVIII.sup.474-494-82 IFKNQASRP 228 FVIII.sup.474-494-83 IFKNQASRPY 229 FVIII.sup.474-494-84 IFKNQASRPYN 230 FVIII.sup.474-494-85 IFKNQASRPYNI 231 FVIII.sup.474-494-86 FKNQASRPY 232 FVIII.sup.474-494-87 FKNQASRPYN 233 FVIII.sup.474-494-88 FKNQASRPYNI 234 FVIII.sup.474-494-89 KNQASRPYN 235 FVIII.sup.474-494-90 KNQASRPYNI 236 FVIII.sup.474-494-91 NQASRPYNI 237

[0108] D. Factor VIII.sup.540-560 Peptides

[0109] In one embodiment, the present invention provides a polypeptide having the sequence (R.sup.1).sub.x—P—(R.sup.2).sub.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.sup.540-560 peptide having the sequence: PTKSDPRCLTRYYSSFVNMER (SEQ ID NO:250), R.sup.1 is an amino acid sequence consisting of from 1 to 80 amino acids, and R.sup.2 is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one.

[0110] 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.sup.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.sup.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.

[0111] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0112] 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-US-00005 TABLE 5 Exemplary FVIII.sup.540-560 Peptides Peptide Sequence SEQ ID NO: FVIII.sup.540-560-1 PTKSDPRCL 238 FVIII.sup.540-560-2 PTKSDPRCLT 239 FVIII.sup.540-560-3 PTKSDPRCLTR 240 FVIII.sup.540-560-4 PTKSDPRCLTRY 241 FVIII.sup.540-560-5 PTKSDPRCLTRYY 242 FVIII.sup.540-560-6 PTKSDPRCLTRYYS 243 FVIII.sup.540-560-7 PTKSDPRCLTRYYSS 244 FVIII.sup.540-560-8 PTKSDPRCLTRYYSSF 245 FVIII.sup.540-560-9 PTKSDPRCLTRYYSSFV 246 FVIII.sup.540-560-10 PTKSDPRCLTRYYSSFVN 247 FVIII.sup.540-560-11 PTKSDPRCLTRYYSSFVNM 248 FVIII.sup.540-560-12 PTKSDPRCLTRYYSSFVNME 249 FVIII.sup.540-560-13 PTKSDPRCLTRYYSSFVNMER 250 FVIII.sup.540-560-14 TKSDPRCLT 251 FVIII.sup.540-560-15 TKSDPRCLTR 252 FVIII.sup.540-560-16 TKSDPRCLTRY 253 FVIII.sup.540-560-17 TKSDPRCLTRYY 254 FVIII.sup.540-560-18 TKSDPRCLTRYYS 255 FVIII.sup.540-560-19 TKSDPRCLTRYYSS 256 FVIII.sup.540-560-20 TKSDPRCLTRYYSSF 257 FVIII.sup.540-560-21 TKSDPRCLTRYYSSFV 258 FVIII.sup.540-560-22 TKSDPRCLTRYYSSFVN 259 FVIII.sup.540-560-23 TKSDPRCLTRYYSSFVNM 260 FVIII.sup.540-560-24 TKSDPRCLTRYYSSFVNME 261 FVIII.sup.540-560-25 TKSDPRCLTRYYSSFVNMER 262 FVIII.sup.540-560-26 KSDPRCLTR 263 FVIII.sup.540-560-27 KSDPRCLTRY 264 FVIII.sup.540-560-28 KSDPRCLTRYY 265 FVIII.sup.540-560-29 KSDPRCLTRYYS 266 FVIII.sup.540-560-30 KSDPRCLTRYYSS 267 FVIII.sup.540-560-31 KSDPRCLTRYYSSF 268 FVIII.sup.540-560-32 KSDPRCLTRYYSSFV 269 FVIII.sup.540-560-33 KSDPRCLTRYYSSFVN 270 FVIII.sup.540-560-34 KSDPRCLTRYYSSFVNM 271 FVIII.sup.540-560-35 KSDPRCLTRYYSSFVNME 272 FVIII.sup.540-560-36 KSDPRCLTRYYSSFVNMER 273 FVIII.sup.540-560-37 SDPRCLTRY 274 FVIII.sup.540-560-38 SDPRCLTRYY 275 FVIII.sup.540-560-39 SDPRCLTRYYS 276 FVIII.sup.540-560-40 SDPRCLTRYYSS 277 FVIII.sup.540-560-41 SDPRCLTRYYSSF 278 FVIII.sup.540-560-42 SDPRCLTRYYSSFV 279 FVIII.sup.540-560-43 SDPRCLTRYYSSFVN 280 FVIII.sup.540-560-44 SDPRCLTRYYSSFVNM 281 FVIII.sup.540-560-45 SDPRCLTRYYSSFVNME 282 FVIII.sup.540-560-46 SDPRCLTRYYSSFVNMER 283 FVIII.sup.540-560-47 DPRCLTRYY 284 FVIII.sup.540-560-48 DPRCLTRYYS 285 FVIII.sup.540-560-49 DPRCLTRYYSS 286 FVIII.sup.540-560-50 DPRCLTRYYSSF 287 FVIII.sup.540-560-51 DPRCLTRYYSSFV 288 FVIII.sup.540-560-52 DPRCLTRYYSSFVN 289 FVIII.sup.540-560-53 DPRCLTRYYSSFVNM 290 FVIII.sup.540-560-54 DPRCLTRYYSSFVNME 291 FVIII.sup.540-560-55 DPRCLTRYYSSFVNMER 292 FVIII.sup.540-560-56 PRCLTRYYS 293 FVIII.sup.540-560-57 PRCLTRYYSS 294 FVIII.sup.540-560-58 PRCLTRYYSSF 295 FVIII.sup.540-560-59 PRCLTRYYSSFV 296 FVIII.sup.540-560-60 PRCLTRYYSSFVN 297 FVIII.sup.540-560-61 PRCLTRYYSSFVNM 298 FVIII.sup.540-560-62 PRCLTRYYSSFVNME 299 FVIII.sup.540-560-63 PRCLTRYYSSFVNMER 300 FVIII.sup.540-560-64 RCLTRYYSS 301 FVIII.sup.540-560-65 RCLTRYYSSF 302 FVIII.sup.540-560-66 RCLTRYYSSFV 303 FVIII.sup.540-560-67 RCLTRYYSSFVN 304 FVIII.sup.540-560-68 RCLTRYYSSFVNM 305 FVIII.sup.540-560-69 RCLTRYYSSFVNME 306 FVIII.sup.540-560-70 RCLTRYYSSFVNMER 307 FVIII.sup.540-560-71 CLTRYYSSF 308 FVIII.sup.540-560-72 CLTRYYSSFV 309 FVIII.sup.540-560-73 CLTRYYSSFVN 310 FVIII.sup.540-560-74 CLTRYYSSFVNM 311 FVIII.sup.540-560-75 CLTRYYSSFVNME 312 FVIII.sup.540-560-76 CLTRYYSSFVNMER 313 FVIII.sup.540-560-77 LTRYYSSFV 314 FVIII.sup.540-560-78 LTRYYSSFVN 315 FVIII.sup.540-560-79 LTRYYSSFVNM 316 FVIII.sup.540-560-80 LTRYYSSFVNME 317 FVIII.sup.540-560-81 LTRYYSSFVNMER 318 FVIII.sup.540-560-82 TRYYSSFVN 319 FVIII.sup.540-560-83 TRYYSSFVNM 320 FVIII.sup.540-560-84 TRYYSSFVNME 321 FVIII.sup.540-560-85 TRYYSSFVNMER 322 FVIII.sup.540-560-86 RYYSSFVNM 323 FVIII.sup.540-560-87 RYYSSFVNME 324 FVIII.sup.540-560-88 RYYSSFVNMER 325 FVIII.sup.540-560-89 YYSSFVNME 326 FVIII.sup.540-560-90 YYSSFVNMER 327 FVIII.sup.540-560-91 YSSFVNMER 328

[0113] E. Factor VIII.sup.1401-1424 Peptides

[0114] In one embodiment, the present invention provides a polypeptide having the sequence (R.sup.1).sub.x—P—(R.sup.2).sub.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.sup.1401-1424 peptide having the sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344), R.sup.1 is an amino acid sequence consisting of from 1 to 80 amino acids, and R.sup.2 is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one.

[0115] 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.sup.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.sup.1401-1424 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.

[0116] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0117] 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-US-00006 TABLE 6 Exemplary FVIII.sup.1401-1424 Peptides SEQ Peptide Sequence ID NO: FVIII.sup.1401-1424-1 QANRSPLPI 329 FVIII.sup.1401-1424-2 QANRSPLPIA 330 FVIII.sup.1401-1424-3 QANRSPLPIAK 331 FVIII.sup.1401-1424-4 QANRSPLPIAKV 332 FVIII.sup.1401-1424-5 QANRSPLPIAKVS 333 FVIII.sup.1401-1424-6 QANRSPLPIAKVSS 334 FVIII.sup.1401-1424-7 QANRSPLPIAKVSSF 335 FVIII.sup.1401-1424-8 QANRSPLPIAKVSSFP 336 FVIII.sup.1401-1424-9 QANRSPLPIAKVSSFPS 337 FVIII.sup.1401-1424-10 QANRSPLPIAKVSSFPSI 338 FVIII.sup.1401-1424-11 QANRSPLPIAKVSSFPSIR 339 FVIII.sup.1401-1424-12 QANRSPLPIAKVSSFPSIRP 340 FVIII.sup.1401-1424-13 QANRSPLPIAKVSSFPSIRPI 341 FVIII.sup.1401-1424-14 QANRSPLPIAKVSSFPSIRPIY 342 FVIII.sup.1401-1424-15 QANRSPLPIAKVSSFPSIRPIYL 343 FVIII.sup.1401-1424-16 QANRSPLPIAKVSSFPSIRPIYLT 344 FVIII.sup.1401-1424-17 ANRSPLPIA 345 FVIII.sup.1401-1424-18 ANRSPLPIAK 346 FVIII.sup.1401-1424-19 ANRSPLPIAKV 347 FVIII.sup.1401-1424-20 ANRSPLPIAKVS 348 FVIII.sup.1401-1424-21 ANRSPLPIAKVSS 349 FVIII.sup.1401-1424-22 ANRSPLPIAKVSSF 350 FVIII.sup.1401-1424-23 ANRSPLPIAKVSSFP 351 FVIII.sup.1401-1424-24 ANRSPLPIAKVSSFPS 352 FVIII.sup.1401-1424-25 ANRSPLPIAKVSSFPSI 353 FVIII.sup.1401-1424-26 ANRSPLPIAKVSSFPSIR 354 FVIII.sup.1401-1424-27 ANRSPLPIAKVSSFPSIRP 355 FVIII.sup.1401-1424-28 ANRSPLPIAKVSSFPSIRPI 356 FVIII.sup.1401-1424-29 ANRSPLPIAKVSSFPSIRPIY 357 FVIII.sup.1401-1424-30 ANRSPLPIAKVSSFPSIRPIYL 358 FVIII.sup.1401-1424-31 ANRSPLPIAKVSSFPSIRPIYLT 359 FVIII.sup.1401-1424-32 NRSPLPIAK 360 FVIII.sup.1401-1424-33 NRSPLPIAKV 361 FVIII.sup.1401-1424-34 NRSPLPIAKVS 362 FVIII.sup.1401-1424-35 NRSPLPIAKVSS 363 FVIII.sup.1401-1424-36 NRSPLPIAKVSSF 364 FVIII.sup.1401-1424-37 NRSPLPIAKVSSFP 365 FVIII.sup.1401-1424-38 NRSPLPIAKVSSFPS 366 FVIII.sup.1401-1424-39 NRSPLPIAKVSSFPSI 367 FVIII.sup.1401-1424-40 NRSPLPIAKVSSFPSIR 368 FVIII.sup.1401-1424-41 NRSPLPIAKVSSFPSIRP 369 FVIII.sup.1401-1424-42 NRSPLPIAKVSSFPSIRPI 370 FVIII.sup.1401-1424-43 NRSPLPIAKVSSFPSIRPIY 371 FVIII.sup.1401-1424-44 NRSPLPIAKVSSFPSIRPIYL 372 FVIII.sup.1401-1424-45 NRSPLPIAKVSSFPSIRPIYLT 373 FVIII.sup.1401-1424-46 RSPLPIAKV 374 FVIII.sup.1401-1424-47 RSPLPIAKVS 375 FVIII.sup.1401-1424-48 RSPLPIAKVSS 376 FVIII.sup.1401-1424-49 RSPLPIAKVSSF 377 FVIII.sup.1401-1424-50 RSPLPIAKVSSFP 378 FVIII.sup.1401-1424-51 RSPLPIAKVSSFPS 379 FVIII.sup.1401-1424-52 RSPLPIAKVSSFPSI 380 FVIII.sup.1401-1424-53 RSPLPIAKVSSFPSIR 381 FVIII.sup.1401-1424-54 RSPLPIAKVSSFPSIRP 382 FVIII.sup.1401-1424-55 RSPLPIAKVSSFPSIRPI 383 FVIII.sup.1401-1424-56 RSPLPIAKVSSFPSIRPIY 384 FVIII.sup.1401-1424-57 RSPLPIAKVSSFPSIRPIYL 385 FVIII.sup.1401-1424-58 RSPLPIAKVSSFPSIRPIYLT 386 FVIII.sup.1401-1424-59 SPLPIAKVS 387 FVIII.sup.1401-1424-60 SPLPIAKVSS 388 FVIII.sup.1401-1424-61 SPLPIAKVSSF 389 FVIII.sup.1401-1424-62 SPLPIAKVSSFP 390 FVIII.sup.1401-1424-63 SPLPIAKVSSFPS 391 FVIII.sup.1401-1424-64 SPLPIAKVSSFPSI 392 FVIII.sup.1401-1424-65 SPLPIAKVSSFPSIR 393 FVIII.sup.1401-1424-66 SPLPIAKVSSFPSIRP 394 FVIII.sup.1401-1424-67 SPLPIAKVSSFPSIRPI 395 FVIII.sup.1401-1424-68 SPLPIAKVSSFPSIRPIY 396 FVIII.sup.1401-1424-69 SPLPIAKVSSFPSIRPIYL 397 FVIII.sup.1401-1424-70 SPLPIAKVSSFPSIRPIYLT 398 FVIII.sup.1401-1424-71 PLPIAKVSS 399 FVIII.sup.1401-1424-72 PLPIAKVSSF 400 FVIII.sup.1401-1424-73 PLPIAKVSSFP 401 FVIII.sup.1401-1424-74 PLPIAKVSSFPS 402 FVIII.sup.1401-1424-75 PLPIAKVSSFPSI 403 FVIII.sup.1401-1424-76 PLPIAKVSSFPSIR 404 FVIII.sup.1401-1424-77 PLPIAKVSSFPSIRP 405 FVIII.sup.1401-1424-78 PLPIAKVSSFPSIRPI 406 FVIII.sup.1401-1424-79 PLPIAKVSSFPSIRPIY 407 FVIII.sup.1401-1424-80 PLPIAKVSSFPSIRPIYL 408 FVIII.sup.1401-1424-81 PLPIAKVSSFPSIRPIYLT 409 FVIII.sup.1401-1424-82 LPIAKVSSF 410 FVIII.sup.1401-1424-83 LPIAKVSSFP 411 FVIII.sup.1401-1424-84 LPIAKVSSFPS 412 FVIII.sup.1401-1424-85 LPIAKVSSFPSI 413 FVIII.sup.1401-1424-86 LPIAKVSSFPSIR 414 FVIII.sup.1401-1424-87 LPIAKVSSFPSIRP 415 FVIII.sup.1401-1424-88 LPIAKVSSFPSIRPI 416 FVIII.sup.1401-1424-89 LPIAKVSSFPSIRPIY 417 FVIII.sup.1401-1424-90 LPIAKVSSFPSIRPIYL 418 FVIII.sup.1401-1424-91 LPIAKVSSFPSIRPIYLT 419 FVIII.sup.1401-1424-92 PIAKVSSFP 420 FVIII.sup.1401-1424-93 PIAKVSSFPS 421 FVIII.sup.1401-1424-94 PIAKVSSFPSI 422 FVIII.sup.1401-1424-95 PIAKVSSFPSIR 423 FVIII.sup.1401-1424-96 PIAKVSSFPSIRP 424 FVIII.sup.1401-1424-97 PIAKVSSFPSIRPI 425 FVIII.sup.1401-1424-98 PIAKVSSFPSIRPIY 426 FVIII.sup.1401-1424-99 PIAKVSSFPSIRPIYL 427 FVIII.sup.1401-1424-100 PIAKVSSFPSIRPIYLT 428 FVIII.sup.1401-1424-101 IAKVSSFPS 429 FVIII.sup.1401-1424-102 IAKVSSFPSI 430 FVIII.sup.1401-1424-103 IAKVSSFPSIR 431 FVIII.sup.1401-1424-104 IAKVSSFPSIRP 432 FVIII.sup.1401-1424-105 IAKVSSFPSIRPI 433 FVIII.sup.1401-1424-106 IAKVSSFPSIRPIY 434 FVIII.sup.1401-1424-107 IAKVSSFPSIRPIYL 435 FVIII.sup.1401-1424-108 IAKVSSFPSIRPIYLT 436 FVIII.sup.1401-1424-109 AKVSSFPSI 437 FVIII.sup.1401-1424-110 AKVSSFPSIR 438 FVIII.sup.1401-1424-111 AKVSSFPSIRP 439 FVIII.sup.1401-1424-112 AKVSSFPSIRPI 440 FVIII.sup.1401-1424-113 AKVSSFPSIRPIY 441 FVIII.sup.1401-1424-114 AKVSSFPSIRPIYL 442 FVIII.sup.1401-1424-115 AKVSSFPSIRPIYLT 443 FVIII.sup.1401-1424-116 KVSSFPSIR 444 FVIII.sup.1401-1424-117 KVSSFPSIRP 445 FVIII.sup.1401-1424-118 KVSSFPSIRPI 446 FVIII.sup.1401-1424-119 KVSSFPSIRPIY 447 FVIII.sup.1401-1424-120 KVSSFPSIRPIYL 448 FVIII.sup.1401-1424-121 KVSSFPSIRPIYLT 449 FVIII.sup.1401-1424-122 VSSFPSIRP 450 FVIII.sup.1401-1424-123 VSSFPSIRPI 451 FVIII.sup.1401-1424-124 VSSFPSIRPIY 452 FVIII.sup.1401-1424-125 VSSFPSIRPIYL 453 FVIII.sup.1401-1424-126 VSSFPSIRPIYLT 454 FVIII.sup.1401-1424-127 SSFPSIRPI 455 FVIII.sup.1401-1424-128 SSFPSIRPIY 456 FVIII.sup.1401-1424-129 SSFPSIRPIYL 457 FVIII.sup.1401-1424-130 SSFPSIRPIYLT 458 FVIII.sup.1401-1424-131 SFPSIRPIY 459 FVIII.sup.1401-1424-132 SFPSIRPIYL 460 FVIII.sup.1401-1424-133 SFPSIRPIYLT 461 FVIII.sup.1401-1424-134 FPSIRPIYL 462 FVIII.sup.1401-1424-135 FPSIRPIYLT 463 FVIII.sup.1401-1424-136 PSIRPIYLT 464

[0118] F. Factor VIII.sup.1785-1805 Peptides

[0119] In one embodiment, the present invention provides a polypeptide having the sequence (R.sup.1).sub.x—P—(R.sup.2).sub.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.sup.1785-1805 peptide having the sequence: EVEDNIMVTFRNQASRPYSFY (SEQ ID NO:477), R.sup.1 is an amino acid sequence consisting of from 1 to 80 amino acids, and R.sup.2 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.sup.1785-1805 peptide having the sequence: EVEDNIMVTFRNQASRPYSFY (SEQ ID NO:477).

[0120] 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.sup.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.

[0121] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0122] 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-US-00007 TABLE 7 Exemplary FVIII.sup.1785-1805 Peptides Peptide Sequence SEQ ID NO: FVIII.sup.1785-1805-1 EVEDNIMVT 465 FVIII.sup.1785-1805-2 EVEDNIMVTF 466 FVIII.sup.1785-1805-3 EVEDNIMVTFR 467 FVIII.sup.1785-1805-4 EVEDNIMVTFRN 468 FVIII.sup.1785-1805-5 EVEDNIMVTFRNQ 469 FVIII.sup.1785-1805-6 EVEDNIMVTFRNQA 470 FVIII.sup.1785-1805-7 EVEDNIMVTFRNQAS 471 FVIII.sup.1785-1805-8 EVEDNIMVTFRNQASR 472 FVIII.sup.1785-1805-9 EVEDNIMVTFRNQASRP 473 FVIII.sup.1785-1805-10 EVEDNIMVTFRNQASRPY 474 FVIII.sup.1785-1805-11 EVEDNIMVTFRNQASRPYS 475 FVIII.sup.1785-1805-12 EVEDNIMVTFRNQASRPYSF 476 FVIII.sup.1785-1805-13 EVEDNIMVTFRNQASRPYSFY 477 FVIII.sup.1785-1805-14 VEDNIMVTF 478 FVIII.sup.1785-1805-15 VEDNIMVTFR 479 FVIII.sup.1785-1805-16 VEDNIMVTFRN 480 FVIII.sup.1785-1805-17 VEDNIMVTFRNQ 481 FVIII.sup.1785-1805-18 VEDNIMVTFRNQA 482 FVIII.sup.1785-1805-19 VEDNIMVTFRNQAS 483 FVIII.sup.1785-1805-20 VEDNIMVTFRNQASR 484 FVIII.sup.1785-1805-21 VEDNIMVTFRNQASRP 485 FVIII.sup.1785-1805-22 VEDNIMVTFRNQASRPY 486 FVIII.sup.1785-1805-23 VEDNIMVTFRNQASRPYS 487 FVIII.sup.1785-1805-24 VEDNIMVTFRNQASRPYSF 488 FVIII.sup.1785-1805-25 VEDNIMVTFRNQASRPYSFY 489 FVIII.sup.1785-1805-26 EDNIMVTFR 490 FVIII.sup.1785-1805-27 EDNIMVTFRN 491 FVIII.sup.1785-1805-28 EDNIMVTFRNQ 492 FVIII.sup.1785-1805-29 EDNIMVTFRNQA 493 FVIII.sup.1785-1805-30 EDNIMVTFRNQAS 494 FVIII.sup.1785-1805-31 EDNIMVTFRNQASR 495 FVIII.sup.1785-1805-32 EDNIMVTFRNQASRP 496 FVIII.sup.1785-1805-33 EDNIMVTFRNQASRPY 497 FVIII.sup.1785-1805-34 EDNIMVTFRNQASRPYS 498 FVIII.sup.1785-1805-35 EDNIMVTFRNQASRPYSF 499 FVIII.sup.1785-1805-36 EDNIMVTFRNQASRPYSFY 500 FVIII.sup.1785-1805-37 DNIMVTFRN 501 FVIII.sup.1785-1805-38 DNIMVTFRNQ 502 FVIII.sup.1785-1805-39 DNIMVTFRNQA 503 FVIII.sup.1785-1805-40 DNIMVTFRNQAS 504 FVIII.sup.1785-1805-41 DNIMVTFRNQASR 505 FVIII.sup.1785-1805-42 DNIMVTFRNQASRP 506 FVIII.sup.1785-1805-43 DNIMVTFRNQASRPY 507 FVIII.sup.1785-1805-44 DNIMVTFRNQASRPYS 508 FVIII.sup.1785-1805-45 DNIMVTFRNQASRPYSF 509 FVIII.sup.1785-1805-46 DNIMVTFRNQASRPYSFY 510 FVIII.sup.1785-1805-47 NIMVTFRNQ 511 FVIII.sup.1785-1805-48 NIMVTFRNQA 512 FVIII.sup.1785-1805-49 NIMVTFRNQAS 513 FVIII.sup.1785-1805-50 NIMVTFRNQASR 514 FVIII.sup.1785-1805-51 NIMVTFRNQASRP 515 FVIII.sup.1785-1805-52 NIMVTFRNQASRPY 516 FVIII.sup.1785-1805-53 NIMVTFRNQASRPYS 517 FVIII.sup.1785-1805-54 NIMVTFRNQASRPYSF 518 FVIII.sup.1785-1805-55 NIMVTFRNQASRPYSFY 519 FVIII.sup.1785-1805-56 IMVTFRNQA 520 FVIII.sup.1785-1805-57 IMVTFRNQAS 521 FVIII.sup.1785-1805-58 IMVTFRNQASR 522 FVIII.sup.1785-1805-59 IMVTFRNQASRP 523 FVIII.sup.1785-1805-60 IMVTFRNQASRPY 524 FVIII.sup.1785-1805-61 IMVTFRNQASRPYS 525 FVIII.sup.1785-1805-62 IMVTFRNQASRPYSF 526 FVIII.sup.1785-1805-63 IMVTFRNQASRPYSFY 527 FVIII.sup.1785-1805-64 MVTFRNQAS 528 FVIII.sup.1785-1805-65 MVTFRNQASR 529 FVIII.sup.1785-1805-66 MVTFRNQASRP 530 FVIII.sup.1785-1805-67 MVTFRNQASRPY 531 FVIII.sup.1785-1805-68 MVTFRNQASRPYS 532 FVIII.sup.1785-1805-69 MVTFRNQASRPYSF 533 FVIII.sup.1785-1805-70 MVTFRNQASRPYSFY 534 FVIII.sup.1785-1805-71 VTFRNQASR 535 FVIII.sup.1785-1805-72 VTFRNQASRP 536 FVIII.sup.1785-1805-73 VTFRNQASRPY 537 FVIII.sup.1785-1805-74 VTFRNQASRPYS 538 FVIII.sup.1785-1805-75 VTFRNQASRPYSF 539 FVIII.sup.1785-1805-76 VTFRNQASRPYSFY 540 FVIII.sup.1785-1805-77 TFRNQASRP 541 FVIII.sup.1785-1805-78 TFRNQASRPY 542 FVIII.sup.1785-1805-79 TFRNQASRPYS 543 FVIII.sup.1785-1805-80 TFRNQASRPYSF 544 FVIII.sup.1785-1805-81 TFRNQASRPYSFY 545 FVIII.sup.1785-1805-82 FRNQASRPY 546 FVIII.sup.1785-1805-83 FRNQASRPYS 547 FVIII.sup.1785-1805-84 FRNQASRPYSF 548 FVIII.sup.1785-1805-85 FRNQASRPYSFY 549 FVIII.sup.1785-1805-86 RNQASRPYS 550 FVIII.sup.1785-1805-87 RNQASRPYSF 551 FVIII.sup.1785-1805-88 RNQASRPYSFY 552 FVIII.sup.1785-1805-89 NQASRPYSF 553 FVIII.sup.1785-1805-90 NQASRPYSFY 554 FVIII.sup.1785-1805-91 QASRPYSFY 555

[0123] G. Factor VIII.sup.2025-2045 Peptides

[0124] In one embodiment, the present invention provides a polypeptide having the sequence (R.sup.1).sub.x—P—(R.sup.2).sub.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.sup.2025-2045 peptide having the sequence: LHAGMSTLFLVYSNKCQTPLG (SEQ ID NO:568), R.sup.1 is an amino acid sequence consisting of from 1 to 80 amino acids, and R.sup.2 is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one.

[0125] 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.sup.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.sup.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.

[0126] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0127] 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-US-00008 TABLE 8 Exemplary FVIII.sup.2025-2045 Peptides Peptide Sequence SEQ ID NO: FVIII.sup.2025-2045-1 LHAGMSTLF 556 FVIII.sup.2025-2045-2 LHAGMSTLFL 557 FVIII.sup.2025-2045-3 LHAGMSTLFLV 558 FVIII.sup.2025-2045-4 LHAGMSTLFLVY 559 FVIII.sup.2025-2045-5 LHAGMSTLFLVYS 560 FVIII.sup.2025-2045-6 LHAGMSTLFLVYSN 561 FVIII.sup.2025-2045-7 LHAGMSTLFLVYSNK 562 FVIII.sup.2025-2045-8 LHAGMSTLFLVYSNKC 563 FVIII.sup.2025-2045-9 LHAGMSTLFLVYSNKCQ 564 FVIII.sup.2025-2045-10 LHAGMSTLFLVYSNKCQT 565 FVIII.sup.2025-2045-11 LHAGMSTLFLVYSNKCQTP 566 FVIII.sup.2025-2045-12 LHAGMSTLFLVYSNKCQTPL 567 FVIII.sup.2025-2045-13 LHAGMSTLFLVYSNKCQTPLG 568 FVIII.sup.2025-2045-14 HAGMSTLFL 569 FVIII.sup.2025-2045-15 HAGMSTLFLV 570 FVIII.sup.2025-2045-16 HAGMSTLFLVY 571 FVIII.sup.2025-2045-17 HAGMSTLFLVYS 572 FVIII.sup.2025-2045-18 HAGMSTLFLVYSN 573 FVIII.sup.2025-2045-19 HAGMSTLFLVYSNK 574 FVIII.sup.2025-2045-20 HAGMSTLFLVYSNKC 575 FVIII.sup.2025-2045-21 HAGMSTLFLVYSNKCQ 576 FVIII.sup.2025-2045-22 HAGMSTLFLVYSNKCQT 577 FVIII.sup.2025-2045-23 HAGMSTLFLVYSNKCQTP 578 FVIII.sup.2025-2045-24 HAGMSTLFLVYSNKCQTPL 579 FVIII.sup.2025-2045-25 HAGMSTLFLVYSNKCQTPLG 580 FVIII.sup.2025-2045-26 AGMSTLFLV 581 FVIII.sup.2025-2045-27 AGMSTLFLVY 582 FVIII.sup.2025-2045-28 AGMSTLFLVYS 583 FVIII.sup.2025-2045-29 AGMSTLFLVYSN 584 FVIII.sup.2025-2045-30 AGMSTLFLVYSNK 585 FVIII.sup.2025-2045-31 AGMSTLFLVYSNKC 586 FVIII.sup.2025-2045-32 AGMSTLFLVYSNKCQ 587 FVIII.sup.2025-2045-33 AGMSTLFLVYSNKCQT 588 FVIII.sup.2025-2045-34 AGMSTLFLVYSNKCQTP 589 FVIII.sup.2025-2045-35 AGMSTLFLVYSNKCQTPL 590 FVIII.sup.2025-2045-36 AGMSTLFLVYSNKCQTPLG 591 FVIII.sup.2025-2045-37 GMSTLFLVY 592 FVIII.sup.2025-2045-38 GMSTLFLVYS 593 FVIII.sup.2025-2045-39 GMSTLFLVYSN 594 FVIII.sup.2025-2045-40 GMSTLFLVYSNK 595 FVIII.sup.2025-2045-41 GMSTLFLVYSNKC 596 FVIII.sup.2025-2045-42 GMSTLFLVYSNKCQ 597 FVIII.sup.2025-2045-43 GMSTLFLVYSNKCQT 598 FVIII.sup.2025-2045-44 GMSTLFLVYSNKCQTP 599 FVIII.sup.2025-2045-45 GMSTLFLVYSNKCQTPL 600 FVIII.sup.2025-2045-46 GMSTLFLVYSNKCQTPLG 601 FVIII.sup.2025-2045-47 MSTLFLVYS 602 FVIII.sup.2025-2045-48 MSTLFLVYSN 603 FVIII.sup.2025-2045-49 MSTLFLVYSNK 604 FVIII.sup.2025-2045-50 MSTLFLVYSNKC 605 FVIII.sup.2025-2045-51 MSTLFLVYSNKCQ 606 FVIII.sup.2025-2045-52 MSTLFLVYSNKCQT 607 FVIII.sup.2025-2045-53 MSTLFLVYSNKCQTP 608 FVIII.sup.2025-2045-54 MSTLFLVYSNKCQTPL 609 FVIII.sup.2025-2045-55 MSTLFLVYSNKCQTPLG 610 FVIII.sup.2025-2045-56 STLFLVYSN 611 FVIII.sup.2025-2045-57 STLFLVYSNK 612 FVIII.sup.2025-2045-58 STLFLVYSNKC 613 FVIII.sup.2025-2045-59 STLFLVYSNKCQ 614 FVIII.sup.2025-2045-60 STLFLVYSNKCQT 615 FVIII.sup.2025-2045-61 STLFLVYSNKCQTP 616 FVIII.sup.2025-2045-62 STLFLVYSNKCQTPL 617 FVIII.sup.2025-2045-63 STLFLVYSNKCQTPLG 618 FVIII.sup.2025-2045-64 TLFLVYSNK 619 FVIII.sup.2025-2045-65 TLFLVYSNKC 620 FVIII.sup.2025-2045-66 TLFLVYSNKCQ 621 FVIII.sup.2025-2045-67 TLFLVYSNKCQT 622 FVIII.sup.2025-2045-68 TLFLVYSNKCQTP 623 FVIII.sup.2025-2045-69 TLFLVYSNKCQTPL 624 FVIII.sup.2025-2045-70 TLFLVYSNKCQTPLG 625 FVIII.sup.2025-2045-71 LFLVYSNKC 626 FVIII.sup.2025-2045-72 LFLVYSNKCQ 627 FVIII.sup.2025-2045-73 LFLVYSNKCQT 628 FVIII.sup.2025-2045-74 LFLVYSNKCQTP 629 FVIII.sup.2025-2045-75 LFLVYSNKCQTPL 630 FVIII.sup.2025-2045-76 LFLVYSNKCQTPLG 631 FVIII.sup.2025-2045-77 FLVYSNKCQ 632 FVIII.sup.2025-2045-78 FLVYSNKCQT 633 FVIII.sup.2025-2045-79 FLVYSNKCQTP 634 FVIII.sup.2025-2045-80 FLVYSNKCQTPL 635 FVIII.sup.2025-2045-81 FLVYSNKCQTPLG 636 FVIII.sup.2025-2045-82 LVYSNKCQT 637 FVIII.sup.2025-2045-83 LVYSNKCQTP 638 FVIII.sup.2025-2045-84 LVYSNKCQTPL 639 FVIII.sup.2025-2045-85 LVYSNKCQTPLG 640 FVIII.sup.2025-2045-86 VYSNKCQTP 641 FVIII.sup.2025-2045-87 VYSNKCQTPL 642 FVIII.sup.2025-2045-88 VYSNKCQTPLG 643 FVIII.sup.2025-2045-89 YSNKCQTPL 644 FVIII.sup.2025-2045-90 YSNKCQTPLG 645 FVIII.sup.2025-2045-91 SNKCQTPLG 646

[0128] H. Factor VIII.sup.2160-2180 Peptides

[0129] In one embodiment, the present invention provides a polypeptide having the sequence (R.sup.1).sub.x—P—(R.sup.2).sub.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.sup.2160-2180 peptide having the sequence: NPPIIARYIRLHPTHYSIRST (SEQ ID NO:659), R.sup.1 is an amino acid sequence consisting of from 1 to 80 amino acids, and R.sup.2 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.sup.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.sup.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.

[0130] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0131] 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-US-00009 TABLE 9 Exemplary FVIII.sup.2160-2180 Peptides Peptide Sequence SEQ ID NO: FVIII.sup.2160-2180-1 NPPIIARYI 647 FVIII.sup.2160-2180-2 NPPIIARYIR 648 FVIII.sup.2160-2180-3 NPPIIARYIRL 649 FVIII.sup.2160-2180-4 NPPIIARYIRLH 650 FVIII.sup.2160-2180-5 NPPIIARYIRLHP 651 FVIII.sup.2160-2180-6 NPPIIARYIRLHPT 652 FVIII.sup.2160-2180-7 NPPIIARYIRLHPTH 653 FVIII.sup.2160-2180-8 NPPIIARYIRLHPTHY 654 FVIII.sup.2160-2180-9 NPPIIARYIRLHPTHYS 655 FVIII.sup.2160-2180-10 NPPIIARYIRLHPTHYSI 656 FVIII.sup.2160-2180-11 NPPIIARYIRLHPTHYSIR 657 FVIII.sup.2160-2180-12 NPPIIARYIRLHPTHYSIRS 658 FVIII.sup.2160-2180-13 NPPIIARYIRLHPTHYSIRST 659 FVIII.sup.2160-2180-14 PPIIARYIR 660 FVIII.sup.2160-2180-15 PPIIARYIRL 661 FVIII.sup.2160-2180-16 PPIIARYIRLH 662 FVIII.sup.2160-2180-17 PPIIARYIRLHP 663 FVIII.sup.2160-2180-18 PPIIARYIRLHPT 664 FVIII.sup.2160-2180-19 PPIIARYIRLHPTH 665 FVIII.sup.2160-2180-20 PPIIARYIRLHPTHY 666 FVIII.sup.2160-2180-21 PPIIARYIRLHPTHYS 667 FVIII.sup.2160-2180-22 PPIIARYIRLHPTHYSI 668 FVIII.sup.2160-2180-23 PPIIARYIRLHPTHYSIR 669 FVIII.sup.2160-2180-24 PPIIARYIRLHPTHYSIRS 670 FVIII.sup.2160-2180-25 PPIIARYIRLHPTHYSIRST 671 FVIII.sup.2160-2180-26 PIIARYIRL 672 FVIII.sup.2160-2180-27 PIIARYIRLH 673 FVIII.sup.2160-2180-28 PIIARYIRLHP 674 FVIII.sup.2160-2180-29 PIIARYIRLHPT 675 FVIII.sup.2160-2180-30 PIIARYIRLHPTH 676 FVIII.sup.2160-2180-31 PIIARYIRLHPTHY 677 FVIII.sup.2160-2180-32 PIIARYIRLHPTHYS 678 FVIII.sup.2160-2180-33 PIIARYIRLHPTHYSI 679 FVIII.sup.2160-2180-34 PIIARYIRLHPTHYSIR 680 FVIII.sup.2160-2180-35 PIIARYIRLHPTHYSIRS 681 FVIII.sup.2160-2180-36 PIIARYIRLHPTHYSIRST 682 FVIII.sup.2160-2180-37 IIARYIRLH 683 FVIII.sup.2160-2180-38 IIARYIRLHP 684 FVIII.sup.2160-2180-39 IIARYIRLHPT 685 FVIII.sup.2160-2180-40 IIARYIRLHPTH 686 FVIII.sup.2160-2180-41 IIARYIRLHPTHY 687 FVIII.sup.2160-2180-42 IIARYIRLHPTHYS 688 FVIII.sup.2160-2180-43 IIARYIRLHPTHYSI 689 FVIII.sup.2160-2180-44 IIARYIRLHPTHYSIR 690 FVIII.sup.2160-2180-45 IIARYIRLHPTHYSIRS 691 FVIII.sup.2160-2180-46 IIARYIRLHPTHYSIRST 692 FVIII.sup.2160-2180-47 IARYIRLHP 693 FVIII.sup.2160-2180-48 IARYIRLHPT 694 FVIII.sup.2160-2180-49 IARYIRLHPTH 695 FVIII.sup.2160-2180-50 IARYIRLHPTHY 696 FVIII.sup.2160-2180-51 IARYIRLHPTHYS 697 FVIII.sup.2160-2180-52 IARYIRLHPTHYSI 698 FVIII.sup.2160-2180-53 IARYIRLHPTHYSIR 699 FVIII.sup.2160-2180-54 IARYIRLHPTHYSIRS 700 FVIII.sup.2160-2180-55 IARYIRLHPTHYSIRST 701 FVIII.sup.2160-2180-56 ARYIRLHPT 702 FVIII.sup.2160-2180-57 ARYIRLHPTH 703 FVIII.sup.2160-2180-58 ARYIRLHPTHY 704 FVIII.sup.2160-2180-59 ARYIRLHPTHYS 705 FVIII.sup.2160-2180-60 ARYIRLHPTHYSI 706 FVIII.sup.2160-2180-61 ARYIRLHPTHYSIR 707 FVIII.sup.2160-2180-62 ARYIRLHPTHYSIRS 708 FVIII.sup.2160-2180-63 ARYIRLHPTHYSIRST 709 FVIII.sup.2160-2180-64 RYIRLHPTH 710 FVIII.sup.2160-2180-65 RYIRLHPTHY 711 FVIII.sup.2160-2180-66 RYIRLHPTHYS 712 FVIII.sup.2160-2180-67 RYIRLHPTHYSI 713 FVIII.sup.2160-2180-68 RYIRLHPTHYSIR 714 FVIII.sup.2160-2180-69 RYIRLHPTHYSIRS 715 FVIII.sup.2160-2180-70 RYIRLHPTHYSIRST 716 FVIII.sup.2160-2180-71 YIRLHPTHY 717 FVIII.sup.2160-2180-72 YIRLHPTHYS 718 FVIII.sup.2160-2180-73 YIRLHPTHYSI 719 FVIII.sup.2160-2180-74 YIRLHPTHYSIR 720 FVIII.sup.2160-2180-75 YIRLHPTHYSIRS 721 FVIII.sup.2160-2180-76 YIRLHPTHYSIRST 722 FVIII.sup.2160-2180-77 IRLHPTHYS 723 FVIII.sup.2160-2180-78 IRLHPTHYSI 724 FVIII.sup.2160-2180-79 IRLHPTHYSIR 725 FVIII.sup.2160-2180-80 IRLHPTHYSIRS 726 FVIII.sup.2160-2180-81 IRLHPTHYSIRST 727 FVIII.sup.2160-2180-82 RLHPTHYSI 728 FVIII.sup.2160-2180-83 RLHPTHYSIR 729 FVIII.sup.2160-2180-84 RLHPTHYSIRS 730 FVIII.sup.2160-2180-85 RLHPTHYSIRST 731 FVIII.sup.2160-2180-86 LHPTHYSIR 732 FVIII.sup.2160-2180-87 LHPTHYSIRS 733 FVIII.sup.2160-2180-88 LHPTHYSIRST 734 FVIII.sup.2160-2180-89 HPTHYSIRS 735 FVIII.sup.2160-2180-90 HPTHYSIRST 736 FVIII.sup.2160-2180-91 PTHYSIRST 737

[0132] I. Factor VIII.sup.102-122 Peptides

[0133] In one embodiment, the present invention provides a polypeptide having the sequence (R.sup.1).sub.x—P—(R.sup.2).sub.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.sup.102-122 peptide having the sequence: TVVITLKNMASHPVSLHAVGV (SEQ ID NO:740), R.sup.1 is an amino acid sequence consisting of from 1 to 80 amino acids, and R.sup.2 is an amino acid sequence consisting of from 1 to 80 amino acids, wherein each of x and y are independently zero or one.

[0134] 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.sup.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.sup.102-122 peptide having the sequence:

TABLE-US-00010 (SEQ ID NO: 740) TVVITLKNMASHPVSLHAVGV.

[0135] In the context of the present invention, FVIII.sup.102-122 peptides also include FVIII.sup.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.

[0136] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0137] 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-US-00011 TABLE 10 Exemplary FVIII.sup.102-122 Peptides Peptide Sequence SEQ ID NO: FVIII.sup.102-122-738 TVVITLKNMASHPVSLHAV 738 FVIII.sup.102-122-739 TVVITLKNMASHPVSLHAVG 739 FVIII.sup.102-122-740 TVVITLKNMASHPVSLHAVGV 740 FVIII.sup.102-122-741 VVITLKNMASHPVSLHAV 741 FVIII.sup.102-122-742 VVITLKNMASHPVSLHAVG 742 FVIII.sup.102-122-743 VVITLKNMASHPVSLHAVGV 743 FVIII.sup.102-122-744 VITLKNMASHPVSLHAV 744 FVIII.sup.102-122-745 VITLKNMASHPVSLHAVG 745 FVIII.sup.102-122-746 VITLKNMASHPVSLHAVGV 746 FVIII.sup.102-122-747 ITLKNMASHPVSLHAV 747 FVIII.sup.102-122-748 ITLKNMASHPVSLHAVG 748 FVIII.sup.102-122-749 ITLKNMASHPVSLHAVGV 749 FVIII.sup.102-122-750 TLKNMASHPVSLHAV 750 FVIII.sup.102-122-751 TLKNMASHPVSLHAVG 751 FVIII.sup.102-122-752 TLKNMASHPVSLHAVGV 752 FVIII.sup.102-122-753 LKNMASHPVSLHAV 753 FVIII.sup.102-122-754 LKNMASHPVSLHAVG 754 FVIII.sup.102-122-755 LKNMASHPVSLHAVGV 755 FVIII.sup.102-122-756 KNMASHPVSLHAV 756 FVIII.sup.102-122-757 KNMASHPVSLHAVG 757 FVIII.sup.102-122-758 KNMASHPVSLHAVGV 758 FVIII.sup.102-122-759 NMASHPVSLHAV 759 FVIII.sup.102-122-760 NMASHPVSLHAVG 760 FVIII.sup.102-122-761 NMASHPVSLHAVGV 761 FVIII.sup.102-122-762 MASHPVSLHAV 762 FVIII.sup.102-122-763 MASHPVSLHAVG 763 FVIII.sup.102-122-764 MASHPVSLHAVGV 764 FVIII.sup.102-122-765 ASHPVSLHAV 765 FVIII.sup.102-122-766 ASHPVSLHAVG 766 FVIII.sup.102-122-767 ASHPVSLHAVGV 767 FVIII.sup.102-122-768 SHPVSLHAV 768 FVIII.sup.102-122-769 SHPVSLHAVG 769 FVIII.sup.102-122-770 SHPVSLHAVGV 770 FVIII.sup.102-122-771 HPVSLHAVG 771 FVIII.sup.102-122-772 HPVSLHAVGV 772 FVIII.sup.102-122-773 PVSLHAVGV 773

IV. Methods of Producing FVIII Peptides

[0138] 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).

[0139] 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: (R.sup.1).sub.x—P—(R.sup.2).sub.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, R.sup.1 is an amino acid sequence consisting of from 1 to 40 amino acids, and R.sup.2 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.

[0140] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0141] 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: (R.sup.1).sub.x—P—(R.sup.2).sub.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, R.sup.1 is an amino acid sequence consisting of from 1 to 40 amino acids, and R.sup.2 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.

[0142] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0143] 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: (R.sup.1).sub.x—P—(R.sup.2).sub.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.

[0144] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0145] 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.

[0146] 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.

[0147] 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 (3-actin.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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).

[0155] 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

[0156] 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 VIII.sup.246-266 peptide, Factor VIII.sup.1401-1424 peptide, or Factor VIII.sup.102-122 peptide, as described herein.

[0157] In one embodiment, the pharmaceutical composition comprises a Factor VIII.sup.246-266 peptide as described herein. In another embodiment, the pharmaceutical composition further comprises a FVIII.sup.474-494 peptide, FVIII.sup.540-560 peptide, FVIII.sup.1785-1805 peptide, FVIII.sup.2025-2045 peptide, FVIII.sup.2160-2180 peptide, FVIII.sup.102-119 peptide, FVIII.sup.1401-1424 peptide, FVIII.sup.102-122 peptide, or second FVIII.sup.246-266 peptide, as described herein.

[0158] In another embodiment, the pharmaceutical composition comprises a Factor VIII.sup.1401-1424 peptide as described herein. In another embodiment, the pharmaceutical composition further comprises a FVIII.sup.474-494 peptide, FVIII.sup.540-560 peptide, FVIII.sup.1785-1805 peptide, FVIII.sup.2025-2045 peptide, FVIII.sup.2160-2180 peptide, FVIII.sup.102-119 peptide, FVIII.sup.246-266 peptide, FVIII.sup.102-122 peptide, or second FVIII.sup.1401-1424 peptide, as described herein.

[0159] In another embodiment, the pharmaceutical composition comprises a Factor VIII.sup.102-122 peptide as described herein. In another embodiment, the pharmaceutical composition further comprises a FVIII.sup.474-494 peptide, FVIII.sup.540-560 peptide, FVIII.sup.1785-1805 peptide, FVIII.sup.2025-2045 peptide, FVIII.sup.2160-2180 peptide, FVIII.sup.102-119 peptide, FVIII.sup.246-266 peptide, FVIII.sup.1401-1424 peptide, or second FVIII.sup.102-122 peptide, as described herein.

[0160] In a specific embodiment, the present invention provides a pharmaceutical composition comprising a peptide having the sequence: (R.sup.1).sub.x—P—(R.sup.2).sub.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, R.sup.1 is an amino acid sequence consisting of from 1 to 40 amino acids, and R.sup.2 is an amino acid sequence consisting of from 1 to 40 amino acids.

[0161] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0162] 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.

[0163] In a specific embodiment, the pharmaceutical composition further comprises a second polypeptide, the second polypeptide having the sequence: (R.sup.1).sub.x—P—(R.sup.2).sub.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, R.sup.1 is an amino acid sequence consisting of from 1 to 40 amino acids, and R.sup.2 is an amino acid sequence consisting of from 1 to 40 amino acids.

[0164] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0165] 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 FVIII 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.

[0166] A. Administration

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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, heptadecaethyleneoxycetanol, 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

[0174] 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.

[0175] 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.

[0176] 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.sup.+ 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.

[0177] 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.sup.246-266 peptide, Factor VIII.sup.1401-1424 peptide, or Factor VIII.sup.102-122 peptide, as described herein.

[0178] 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.sup.246-266 peptide as described herein. In another embodiment, the pharmaceutical composition further comprises a FVIII.sup.474-494 peptide, FVIII.sup.540-560 peptide, FVIII.sup.1785-1805 peptide, FVIII.sup.2025-2045 peptide, FVIII.sup.2160-2180 peptide, FVIII.sup.102-119 peptide, FVIII.sup.1401-1424 peptide, FVIII.sup.102-122 peptide, or second FVIII.sup.246-266 peptide, as described herein.

[0179] 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.sup.1401-1424 peptide as described herein. In another embodiment, the pharmaceutical composition further comprises a FVIII.sup.474-494 peptide, FVIII.sup.540-560 peptide, FVIII.sup.1785-1805 peptide, FVIII.sup.2025-2045 peptide, FVIII.sup.2160-2180 peptide, FVIII.sup.102-119 peptide, FVIII.sup.246-266 peptide, FVIII.sup.102-122 peptide, or second FVIII.sup.1401-1424 peptide, as described herein.

[0180] 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.sup.102-122 peptide as described herein. In another embodiment, the pharmaceutical composition further comprises a FVIII.sup.474-494 peptide, FVIII.sup.540-560 peptide, FVIII.sup.1785-1805 peptide, FVIII.sup.2025-2045 peptide, FVIII.sup.2160-2180 peptide, FVIII.sup.102-119 peptide, FVIII.sup.246-266 peptide, FVIII.sup.1401-1424 peptide, or second FVIII.sup.102-122 peptide, as described herein.

[0181] 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: (R.sup.1).sub.x—P—(R.sup.2).sub.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; thereby inducing an immune tolerance to FVIII protein in the subject. In certain embodiments, R.sup.1 is an amino acid sequence consisting of from 1 to 40 amino acids, and R.sup.2 is an amino acid sequence consisting of from 1 to 40 amino acids.

[0182] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0183] 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.

[0184] 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: (R.sup.1).sub.x—P—(R.sup.2).sub.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, R.sup.1 is an amino acid sequence consisting of from 1 to 40 amino acids, and R.sup.2 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.

[0185] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0186] 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, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 amino acids.

[0187] 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: (R.sup.1).sub.x—P—(R.sup.2).sub.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.

[0188] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0189] 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.sup.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.sup.1401-1424 peptide.

[0190] 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 (R.sup.1).sub.x—P—(R.sup.2).sub.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.sup.1401-1424 peptide having the sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344), R.sup.1 is an amino acid sequence consisting of from 1 to 80 amino acids, and R.sup.2 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.

[0191] 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 (R.sup.1).sub.x—P—(R.sup.2).sub.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.sup.1401-1424 peptide having the sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344), R.sup.1 is an amino acid sequence consisting of from 1 to 80 amino acids, and R.sup.2 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.

[0192] 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 (R.sup.1).sub.x—P—(R.sup.2).sub.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.sup.1401-1424 peptide having the sequence: QANRSPLPIAKVSSFPSIRPIYLT (SEQ ID NO:344), R.sup.1 is an amino acid sequence consisting of from 1 to 80 amino acids, and R.sup.2 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

[0193] 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.

[0194] 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.sup.246-266 peptide, FVIII.sup.1401-1424 peptide, or FVIII.sup.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.

[0195] 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.sup.246-266 peptide, FVIII.sup.1401-1424 peptide, or FVIII.sup.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.

[0196] 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.sup.246-266 peptide, FVIII.sup.1401-1424 peptide, or FVIII.sup.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.

[0197] 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.sup.246-266 peptide, FVIII.sup.1401-1424 peptide, or FVIII.sup.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.

[0198] 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.

[0199] A. Methods of Identifying FVIII-Specific T Cells

[0200] 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.sup.+ T cells with a FVIII peptide complexed with a MHC class II multimer, the FVIII peptide having the sequence: (R.sup.1).sub.x—P—(R.sup.2).sub.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.sup.+ T cells that is specific for the peptide complexed with the MHC class II multimer. In some embodiments, R.sup.1 is an amino acid sequence consisting of from 1 to 40 amino acids, and R.sup.2 is an amino acid sequence consisting of from 1 to 40 amino acids.

[0201] 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.

[0202] In certain embodiments, the FVIII peptide can be used to re-stimulate CD4.sup.+ 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.

[0203] 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.

[0204] To carry out immune monitoring analyses, T cells that bind the FVIII peptide-MHC class II multimer complex include CD4.sup.+ 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.

VIII. Kits of the Invention

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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

[0210] 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.

[0211] In one embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 80 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 70 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 60 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 50 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 40 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 30 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 20 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 10 amino acids. In another embodiment, R.sup.1 and R.sup.2 are separately or both amino acid sequences consisting of from 1 to 5 amino acids. In yet other embodiment, R.sup.1 and R.sup.2 are separately 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.

[0212] In a specific embodiment of the peptides described above, x and y are both zero.

[0213] In a specific embodiment of the peptides described above, x is one and y is zero.

[0214] In a specific embodiment of the peptides described above, x is zero and y is one.

[0215] In a specific embodiment of the peptides described above, x and y are both one.

[0216] 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:

TABLE-US-00012 (SEQ ID NO: 344) QANRSPLPIAKVSSFPSIRPIYLT.

[0217] 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.

[0218] In a specific embodiment of the compositions described above, x and y are both zero.

[0219] In a specific embodiment of the compositions described above, x is one and y is zero.

[0220] In a specific embodiment of the compositions described above, x is zero and y is one.

[0221] In a specific embodiment of the compositions described above, x and y are both one.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] In a specific embodiment of the methods described above, administration of the pharmaceutical composition prevents development anti-FVIII antibodies in the subject.

[0226] In a specific embodiment of the methods described above, administration of the pharmaceutical composition reduces an amount anti-FVIII antibodies present in the subject.

[0227] In a specific embodiment of the methods described above, x and y are both zero.

[0228] In a specific embodiment of the methods described above, x is one and y is zero.

[0229] In a specific embodiment of the methods described above, x is zero and y is one.

[0230] In a specific embodiment of the methods described above, x and y are both one.

[0231] 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.

[0232] In a specific embodiment of the methods described above, x and y are both zero.

[0233] In a specific embodiment of the methods described above, x is one and y is zero.

[0234] In a specific embodiment of the methods described above, x is zero and y is one.

[0235] In a specific embodiment of the methods described above, x and y are both one.

[0236] 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-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.

[0237] In a specific embodiment of the methods described above, x and y are both zero.

[0238] In a specific embodiment of the methods described above, x is one and y is zero.

[0239] In a specific embodiment of the methods described above, x is zero and y is one.

[0240] In a specific embodiment of the methods described above, x and y are both one.

[0241] 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.

[0242] In a specific embodiment of the methods described above, the MHC class II multimer is a MHC class II tetramer.

[0243] In a specific embodiment of the methods described above, the peptide or MHC class II multimer further comprises a diagnostic agent.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] In a specific embodiment of the methods described above, x and y are both zero.

[0248] In a specific embodiment of the methods described above, x is one and y is zero.

[0249] In a specific embodiment of the methods described above, x is zero and y is one.

[0250] In a specific embodiment of the methods described above, x and y are both one.

[0251] The present invention will now be further illustrated in the following examples, without being limited thereto.

X. Examples

Example 1

[0252] 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.sup.+ 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

[0253] 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, Calif.).

[0254] Hemophilic HLA-DRB15 E17 mice: HLA-DRB1*1501.sup.+/− E17.sup.−/− 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.

[0255] Immunization with human recombinant FVIII: HLA-DRB1*1501.sup.+/− E17.sup.−/− 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, Mo., USA).

[0256] 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, N.J.). Single cells were collected in culture medium: RPMI 1640 medium (Gibco, Invitrogen, Life Technologies, Carlsbad, Calif.) supplemented with 10% preselected fetal calf serum (FCS; Hyclone, Logan, Utah), 2 mM L-glutamine, 100 U/mL penicillin/streptomycin (both from Gibco), and 5×10.sup.−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 Z 1.

Generation of T-Cell Hybridomas for Identifying FVIII Peptides

[0257] 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 1.5×10.sup.6 cells/ml for 3 or 10 days. The culture medium for the 10 day cultures was renewed after 6 days.

[0258] 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.

[0259] Peptide specificity of FVIII-specific T cell hybridomas: T cell hybridomas were tested for their antigen specificity. For this purpose, 1×10.sup.5 cells were co-cultured with antigen presenting cells. We used either 5×10.sup.4 Mgar cells (expressing HLA-DRB1*1501) or 1×10.sup.5 whole spleen cells derived from naïve 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% CO.sub.2. The supernatants were collected and IL-2 release into the culture supernatant was measured using an IL-2 ELISA (BioLegend, San Diego, Calif.) or IL-2 Bio-Plex (Bio-Rad Laboratories, Hercules, Calif.) 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.

[0260] 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. 2×10.sup.8 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% CO.sub.2 in the incubator. Five growing subclones per clone were selected and tested for their FVIII specificity.

[0261] 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

[0262] 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.sup.1401-1424 has not been described before (Table 11). Peptides FVIII.sup.474-494, FVIII.sup.545-559, FVIII.sup.1788-1802 and FVIII.sup.2161-2175 were already identified in WO 09/071886, which used computer prediction programs followed by the T cell hybridoma technology. Peptide FVIII.sup.2030-2044 was disclosed in WO 03/087161. Peptide FVIII.sup.2161-2180 was already published by Jacquemin et al., Blood 101(4):1351-8 (2003).

TABLE-US-00013 TABLE 11 Regions of FVIII including T-cell epitopes Regions including T cell epitopes Amino Acid Sequence Disclosures FVIII.sup.102-122 TVVITLKNMASHPVSLHAVGV FVIII.sup.107-121 disclosed in WO (SEQ ID NO: 740) 2003/087161 FVIII.sup.100-108 disclosed in WO/2009/095646 FVIII.sup.246-266 AWPKMHTVNGYVNRSLPGLIG FVIII.sup.253-268 disclosed in (SEQ ID NO: 68) WO/2009/095646 FVIII.sup.474-494 GEVGDTLLIIFKNQASRPYNI FVIII.sup.475-495 Disclosed in WO (SEQ ID NO: 159) 2009/071886 FVIII.sup.477-495 disclosed in WO/2009/095646 FVIII.sup.540-560 PTKSDPRCLTRYYSSFVNMER FVIII.sup.542-562 Disclosed in WO (SEQ ID NO: 250) 2009/071886 FVIII.sup.545-569 disclosed in WO/2009/095646 FVIII.sup.1401-1424 QANRSPLPIAKVSSFPSIRPIYLT A peptide of the present (SEQ ID NO: 344) invention FVIII.sup.1785-1805 EVEDNIMVTFRNQASRPYSFY FVIII.sup.1785-1805 Disclosed in WO (SEQ ID NO: 477) 2009/071886 FVIII.sup.1787-1805 disclosed in WO/2009/095646 FVIII.sup.2025-2045 LHAGMSTLFLVYSNKCQTPLG FVIII.sup.2030-2044 Disclosed in WO (SEQ ID NO: 568) 2003/087161 FVIII.sup.2160-2180 NPPIIARYIRLHPTHYSIRST FVIII.sup.2158-2178 Disclosed in WO (SEQ ID NO: 659) 2009/071886 and FVIII.sup.2161-2180 Jacquemin et al., supra. FVIII.sup.2164-2183 Disclosed in WO 2003/087161 FVIII.sup.2164-2188 disclosed in WO/2009/095646

[0263] 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.