Improved anti-albumin nanobodies and uses thereof

Abstract

Albumin-binding variants were selected via yeast display technology, and the variants showed improved binding to MSA or both HSA and MSA. All variants were subcloned into a pHEN expression plasmid and expressed in E. coli WK6 cells. The purified nanobodies were then analyzed for their ability to bind to immobilized MSA. Biotinylated nanobodies were then tested for their circulating half-life in vivo. The effect of these nanobodies anti-albumin linked to another nanobody, such as the single-domain antibody anti-D'D3, was then examined, as described in WO 2017 / 129630 and WO 2018 / 091621.

Description

[Technical Field]

[0001] The present invention relates to anti-albumin nanobodies and their use for therapy. [Background technology]

[0002] In certain diseases, the native or endogenous protein is defective or missing in patients, especially due to inherited genetic defects. In other diseases, the level of the native or endogenous protein is not sufficient to have normal function in patients compared to healthy people, and the level of the endogenous protein is lower in patients than in healthy subjects.

[0003] There are many ways to overcome these problems. In particular, the use of polypeptides, such as proteins, for therapeutic applications has expanded in recent years due to advanced knowledge of the molecular biological principles underlying many diseases and the availability of improved recombinant expression and delivery systems for human polypeptides. In the prior art, the short circulating half-life of polypeptide therapeutics has been addressed by covalently attaching polymers to the polypeptide. However, many problems have been observed with polymer attachment. For example, polymer attachment can lead to reduced drug activity. Furthermore, certain reagents used to couple polymers to proteins are insufficiently reactive, thus requiring long reaction times during which protein denaturation and / or inactivation can occur. Furthermore, incomplete or uneven attachment can lead to a mixed population of compounds with different properties.

[0004] However, there are few methods in the art to increase the half-life and levels of endogenous or exogenous proteins that are defective, missing, or insufficient to function properly (WO 2018 / 091621).

[0005] Single domain antibodies (sdAbs) or nanobodies or VHHs that target albumin are well known to those skilled in the art.By binding to both human albumin and mouse albumin, the anti-albumin nanobody (such as ALB8; SEQ ID NO: 1, described in WO2006 / 122787) described in the art can be used in preclinical mouse models before proceeding to primate and human studies.However, the binding of ALB8 to mouse albumin is relatively weak (the half-maximal binding of ALB8 to MSA requires 35 times higher sdAb concentration compared to the binding to HSA). Therefore, there remains a need for anti-albumin nanobodies that increase the half-life and levels of endogenous or exogenous proteins, thereby increasing the efficacy or reducing the amount and / or injection frequency of therapeutic proteins administered to patients, which would also reduce the cost of treatment. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2018 / 091621 [Patent Document 2] International Publication No. 2006 / 122787 Summary of the Invention [Means for solving the problem]

[0007] The present invention provides - a CDR1 having the sequence set forth as SEQ ID NO: 2, a CDR2 having the sequence set forth as SEQ ID NO: 3, and a CDR3 having the sequence set forth as SEQ ID NO: 4; - a CDR1 having the sequence set forth as SEQ ID NO: 6, a CDR2 having the sequence set forth as SEQ ID NO: 7, and a CDR3 having the sequence set forth as SEQ ID NO: 8; - a CDR1 having the sequence set forth as SEQ ID NO: 10, a CDR2 having the sequence set forth as SEQ ID NO: 11, and a CDR3 having the sequence set forth as SEQ ID NO: 12; - a CDR1 having the sequence set forth as SEQ ID NO: 14, a CDR2 having the sequence set forth as SEQ ID NO: 15, and a CDR3 having the sequence set forth as SEQ ID NO: 16, or - an isolated single domain antibody (sdAb) against albumin, comprising a CDR1 having the sequence set forth as SEQ ID NO: 18, a CDR2 having the sequence set forth as SEQ ID NO: 19, and a CDR3 having the sequence set forth as SEQ ID NO: 20.

[0008] In particular, the invention is defined by the claims. DETAILED DESCRIPTION OF THE INVENTION

[0009] Single domain antibodies (sdAb) or nanobodies that target albumin are well known to those skilled in the art.By binding to both human albumin and mouse albumin, the anti-albumin nanobody (such as ALB8; SEQ ID NO: 1, described in WO2006 / 122787) described in the art can be used in preclinical mouse models before proceeding to primate and human studies.However, the binding of ALB8 to mouse albumin is relatively weak (the half-maximal binding of ALB8 to MSA requires 35 times higher sdAb concentration compared to the binding to HSA).

[0010] SEQ ID NO: 1 [ka]

[0011] Therefore, we decided to carry out an affinity maturation process. Two libraries were generated. In the first library, each amino acid in CDR1, CDR2, and CDR3 was replaced with one of 19 possible other amino acids (except cysteine). In the second library, the combined CDR sequences contained one, two, or three mutations, resulting in 19 possible amino acid substitutions for each amino acid in the three CDRs.

[0012] The sequences were amplified by PCR and recombined into the yeast display plasmid pSTALK-Halo to generate libraries with a complexity of 703 yeast cells for the simple variant library and 3.62 million yeast cells for the simple, double, and triple variant libraries. Forty-eight random clones from each library were sequenced to verify the mutagenesis conditions. To facilitate cloning into the yeast expression system, the N-terminal portion of the framework sequence was modified from EVQLVESGGGLV (SEQ ID NO: 41) to QVQLQQSGGGFV (SEQ ID NO: 42) (the bolded amino acids were changed). This modified sequence was selectively used in the yeast display system.

[0013] Albumin-binding variants were selected via yeast display technology, and the variants showed improved binding to MSA. All variants were subcloned into the pHEN expression plasmid (here carrying the original EVQLVESGGGLV N-terminal sequence) and expressed in E. coli WK6 cells. The purified nanobodies were then analyzed for their ability to bind to immobilized MSA. Biotinylated nanobodies were then tested in vivo for their circulating half-life. They were then tested for the efficacy of these nanobodies linked to other nanobodies, such as the single-domain antibody anti-D'D3, as described in WO 2017 / 129630 and WO 2018 / 091621.

[0014] Mice with reduced VWF levels (von Willebrand disease type 1) received a single dose of a bispecific single-domain antibody. VWF and FVIII levels were followed over a 14-day period. These data show that there was a statistically significant increase in VWF and FVIII levels that was maintained for 10 days.

[0015] Single domain antibodies against albumin Thus, in a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: - a CDR1 having the sequence set forth as SEQ ID NO: 2, a CDR2 having the sequence set forth as SEQ ID NO: 3, and a CDR3 having the sequence set forth as SEQ ID NO: 4; - a CDR1 having the sequence set forth as SEQ ID NO: 6, a CDR2 having the sequence set forth as SEQ ID NO: 7, and a CDR3 having the sequence set forth as SEQ ID NO: 8; - a CDR1 having the sequence set forth as SEQ ID NO: 10, a CDR2 having the sequence set forth as SEQ ID NO: 11, and a CDR3 having the sequence set forth as SEQ ID NO: 12; - a CDR1 having the sequence set forth as SEQ ID NO: 14, a CDR2 having the sequence set forth as SEQ ID NO: 15, and a CDR3 having the sequence set forth as SEQ ID NO: 16, or - an isolated single domain antibody (sdAb) against albumin, comprising a CDR1 having the sequence set forth as SEQ ID NO: 18, a CDR2 having the sequence set forth as SEQ ID NO: 18, and a CDR3 having the sequence set forth as SEQ ID NO: 20.

[0016] In another embodiment, there is provided an isolated single domain antibody against albumin according to the present invention, wherein the single domain antibody - a CDR1 having at least 70% identity to the sequence set forth as SEQ ID NO: 2, a CDR2 having at least 70% identity to the sequence set forth as SEQ ID NO: 3, and a CDR3 having at least 70% identity to the sequence set forth as SEQ ID NO: 4; - a CDR1 having at least 70% identity with the sequence set forth as SEQ ID NO: 6, a CDR2 having at least 70% identity with the sequence set forth as SEQ ID NO: 7, and a CDR3 having at least 70% identity with the sequence set forth as SEQ ID NO: 8; - a CDR1 having at least 70% identity with the sequence set forth as SEQ ID NO: 10, a CDR2 having at least 70% identity with the sequence set forth as SEQ ID NO: 11, and a CDR3 having at least 70% identity with the sequence set forth as SEQ ID NO: 12; - a CDR1 having at least 70% identity with the sequence shown as SEQ ID NO: 14, a CDR2 having at least 70% identity with the sequence shown as SEQ ID NO: 15, and a CDR3 having at least 70% identity with the sequence shown as SEQ ID NO: 16, - comprising a CDR1 having at least 70% identity to the sequence set forth as SEQ ID NO: 18, a CDR2 having at least 70% identity to the sequence set forth as SEQ ID NO: 19, and a CDR3 having at least 70% identity to the sequence set forth as SEQ ID NO: 20.

[0017] In a further embodiment there is provided an isolated single domain antibody against albumin according to the present invention, wherein the single domain antibody comprises: - OptiAlb-03 (SEQ ID NO: 5), - OptiAlb-07 (SEQ ID NO: 9); - OptiAlb-09 (SEQ ID NO: 13); OptiAlb-11 (SEQ ID NO: 17) or - OptiAlb-12 (SEQ ID NO: 21).

[0018] As used herein, the term "single-domain antibody" (sdAb) has its general meaning in the art and refers to a single heavy-chain variable domain of an antibody of the type that can be found in camelid mammals that naturally lack light chains. Such single-domain antibodies are also called VHHs or "nanobodies." For a general description of (single) domain antibodies, see the prior art cited above, as well as EP 0,368,684, Ward et al. (Nature 1989 Oct 12; 341(6242):544-6), Holt et al., Trends Biotechnol, 2003, 21(11):484-490; and WO 06 / 030220 and WO 06 / 003388. The amino acid sequence and structure of a single-domain antibody can be considered to consist of four framework regions or FRs, referred to in the art and herein as framework region 1 or FR1. Each framework region, interrupted by three complementarity-determining regions or CDRs, is referred to in the art as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively, as "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4." A single domain antibody can thus be defined as an amino acid sequence having the following general structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3. In the context of the present invention, the amino acid residues of a single domain antibody are numbered according to the general numbering of a VH domain as given by the amino acid numbering system of the international ImMunoGeneTics information system (http: / / imgt.cines.fr / ).

[0019] As used herein, the term "amino acid sequence" has its general meaning: a sequence of amino acids that gives a protein its primary structure. According to the present invention, an amino acid sequence may be modified with one, two, or three conservative amino acid substitutions without significant loss of interactive binding ability. "Conservative amino acid substitution" means that an amino acid may be substituted with another amino acid having a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0020] According to the present invention, a first amino acid sequence having at least 70% identity to a second amino acid sequence means that the first sequence has 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%, or 99% identity to the second amino acid sequence. Amino acid sequence identity is typically determined using a suitable sequence alignment algorithm, such as BLAST P (Karlin and Altschul, 1990), and default parameters.

[0021] In the sense of the present invention, "identity" is calculated by comparing two aligned sequences within a comparison window. Sequence alignment allows the number of common positions (nucleotides or amino acids) for the two sequences within the comparison window to be determined. Therefore, the number of common positions is divided by the total number of positions within the comparison window and multiplied by 100 to obtain the identity percentage. The determination of the sequence identity percentage can be performed manually or by using well-known computer programs.

[0022] As used herein, the terms "purified" and "isolated," in reference to the sdAbs of the present invention, mean that the sdAb is present in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" means at least 75%, more preferably at least 85%, even more preferably at least 95%, more preferably at least 98% by weight of antibody compared to the total weight of macromolecules present.

[0023] As used herein, the term "nucleic acid molecule" has its general meaning in the art and refers to a DNA or RNA molecule.

[0024] As used herein, the term "albumin" refers to a transport protein that binds to and transports various ligands. Albumin is found in plasma and differs from other blood proteins in that it is not glycosylated. In certain embodiments, the albumin is human serum albumin (HSA) present in human blood. The naturally occurring human HSA gene has the nucleotide sequence set forth in GenBank accession number NM_000477, and the naturally occurring human HSA protein has the amino acid sequence set forth in GenBank accession number NP_000468, SEQ ID NO: 71. Mouse nucleotide and amino acid sequences have also been described (GenBank accession numbers NM_009654 and NP_033784).

[0025] SEQ ID NO: 71. Human HSA protein MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFH DNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCE KPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0026] The inventors have isolated five single domain antibodies (sdAbs) with the required properties and characterized and therefore determined the complementarity determining regions (CDRs) of the sdAbs (Table A).

[0027] [Table 1-1] [Table 1-2]

[0028] In a particular embodiment, the present invention relates to an isolated single domain antibody (sdAb OptiAlb-03) comprising a CDR1 having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to the sequence set forth as SEQ ID NO:2, a CDR2 having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to the sequence set forth as SEQ ID NO:3, and a CDR3 having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to the sequence set forth as SEQ ID NO:4.

[0029] Amino acid sequence identity is preferably determined using an appropriate sequence alignment algorithm, such as BLAST P (Karlin and Altschul, Proc. Natl. Acad. Science USA 87(6):2264-2268 (1990)), and default parameters.

[0030] In some embodiments, an isolated single domain antibody according to the invention comprises a CDR1 having the sequence set forth as SEQ ID NO:2, a CDR2 having the sequence set forth as SEQ ID NO:3, and a CDR3 having the sequence set forth as SEQ ID NO:4.

[0031] In some embodiments, the isolated single domain antibody according to the invention has the sequence set forth as SEQ ID NO:5.

[0032] In particular, the present invention relates to an isolated single domain antibody (sdAb OptiAlb-07) comprising a CDR1 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the sequence set forth as SEQ ID NO:6, a CDR2 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the sequence set forth as SEQ ID NO:7, and a CDR3 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the sequence set forth as SEQ ID NO:8.

[0033] Amino acid sequence identity is preferably determined using an appropriate sequence alignment algorithm, such as BLAST P (Karlin and Altschul, Proc. Natl. Acad. Science USA 87(6):2264-2268 (1990)), and default parameters.

[0034] In some embodiments, an isolated single domain antibody according to the invention comprises a CDR1 having the sequence set forth as SEQ ID NO:6, a CDR2 having the sequence set forth as SEQ ID NO:7, and a CDR3 having the sequence set forth as SEQ ID NO:8.

[0035] In some embodiments, the isolated single domain antibody according to the invention has the sequence set forth as SEQ ID NO:9.

[0036] In particular, the present invention relates to an isolated single domain antibody (sdAb OptiAlb-09) comprising a CDR1 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the sequence set forth as SEQ ID NO: 10, a CDR2 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the sequence set forth as SEQ ID NO: 11, and a CDR3 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the sequence set forth as SEQ ID NO: 12.

[0037] Amino acid sequence identity is preferably determined using an appropriate sequence alignment algorithm, such as BLAST P (Karlin and Altschul, Proc. Natl. Acad. Science USA 87(6):2264-2268 (1990)), and default parameters.

[0038] In some embodiments, an isolated single domain antibody according to the invention comprises a CDR1 having the sequence set forth as SEQ ID NO: 10, a CDR2 having the sequence set forth as SEQ ID NO: 11, and a CDR3 having the sequence set forth as SEQ ID NO: 12.

[0039] In some embodiments, the isolated single domain antibody according to the invention has the sequence set forth as SEQ ID NO:13.

[0040] In particular, the present invention relates to an isolated single domain antibody (sdAb OptiAlb-11) comprising a CDR1 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the sequence set forth as SEQ ID NO: 14, a CDR2 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the sequence set forth as SEQ ID NO: 15, and a CDR3 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the sequence set forth as SEQ ID NO: 16.

[0041] Amino acid sequence identity is preferably determined using an appropriate sequence alignment algorithm, such as BLAST P (Karlin and Altschul, Proc. Natl. Acad. Science USA 87(6):2264-2268 (1990)), and default parameters.

[0042] In some embodiments, an isolated single domain antibody according to the invention comprises a CDR1 having the sequence set forth as SEQ ID NO: 14, a CDR2 having the sequence set forth as SEQ ID NO: 15, and a CDR3 having the sequence set forth as SEQ ID NO: 16.

[0043] In some embodiments, the isolated single domain antibody according to the invention has the sequence set forth as SEQ ID NO:17.

[0044] In particular, the present invention relates to an isolated single domain antibody (sdAb OptiAlb-12) comprising a CDR1 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the sequence set forth as SEQ ID NO: 18, a CDR2 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the sequence set forth as SEQ ID NO: 19, and a CDR3 having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the sequence set forth as SEQ ID NO: 20.

[0045] Amino acid sequence identity is preferably determined using an appropriate sequence alignment algorithm, such as BLAST P (Karlin and Altschul, Proc. Natl. Acad. Science USA 87(6):2264-2268 (1990)), and default parameters.

[0046] In some embodiments, an isolated single domain antibody according to the invention comprises a CDR1 having the sequence set forth as SEQ ID NO: 18, a CDR2 having the sequence set forth as SEQ ID NO: 19, and a CDR3 having the sequence set forth as SEQ ID NO: 20.

[0047] In some embodiments, the isolated single domain antibody according to the invention has the sequence set forth as SEQ ID NO:21.

[0048] Furthermore, it should be noted that the above-mentioned single domain antibody anti-albumin cross-reacts with mouse albumin, which is the subject of preclinical and toxicological studies. It should be noted that the single domain antibodies of the present invention exhibit high affinity for both human and mouse albumin (see Example 19).

[0049] In some embodiments, the single domain antibody is a "humanized" single domain antibody. As used herein, the term "humanized" refers to a single domain antibody of the present invention in which the amino acid sequence corresponding to the amino acid sequence of a naturally occurring VHH domain has been "humanized," i.e., by replacing one or more amino acid residues (particularly amino acid residues in the framework sequences) in the amino acid sequence of said naturally occurring VHH sequence with one or more amino acid residues present at the corresponding positions in a VH domain from a conventional chain antibody of human origin. Methods for humanizing single domain antibodies are well known in the art. Typically, humanizing substitutions should be selected such that the resulting humanized single domain antibody still retains the favorable properties of the single domain antibody of the present invention. Those skilled in the art are able to determine and select suitable humanizing substitutions or suitable combinations of humanizing substitutions.

[0050] A further aspect of the present invention refers to cross-competing single domain antibodies that cross-compete with the single domain antibodies of the present invention for binding albumin. In some embodiments, the cross-competing single domain antibodies of the present invention cross-compete with the single domain antibodies of the present invention for binding albumin. - a CDR1 having the sequence set forth as SEQ ID NO: 2, a CDR2 having the sequence set forth as SEQ ID NO: 3, and a CDR3 having the sequence set forth as SEQ ID NO: 4; - a CDR1 having the sequence set forth as SEQ ID NO: 6, a CDR2 having the sequence set forth as SEQ ID NO: 7, and a CDR3 having the sequence set forth as SEQ ID NO: 8; - a CDR1 having the sequence set forth as SEQ ID NO: 10, a CDR2 having the sequence set forth as SEQ ID NO: 11, and a CDR3 having the sequence set forth as SEQ ID NO: 12; - a CDR1 having the sequence set forth as SEQ ID NO: 14, a CDR2 having the sequence set forth as SEQ ID NO: 15, and a CDR3 having the sequence set forth as SEQ ID NO: 16, or - cross-competes with a single domain antibody comprising a CDR1 having the sequence set forth as SEQ ID NO: 18, a CDR2 having the sequence set forth as SEQ ID NO: 18, and a CDR3 having the sequence set forth as SEQ ID NO: 20.

[0051] In some embodiments, a cross-competing single domain antibody of the invention cross-competes for binding albumin with a single domain antibody comprising or consisting of the sequence set forth as SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, or SEQ ID NO:21.

[0052] As used herein, the term "cross-competition" refers to single domain antibodies that share the ability to bind to a specific region of an antigen. In the present disclosure, a cross-competing single domain antibody has the ability to interfere with the binding of another single domain antibody to an antigen in a standard competitive binding assay. Such a single domain antibody may, according to non-limiting theory, bind to the same, related, or nearby (e.g., structurally similar or spatially proximal) epitope as the single domain antibody it competes with. Single domain antibody A reduces the binding of single domain antibody B by at least 60%, specifically at least 70%, more specifically at least 80%, compared to a positive control lacking one of the single domain antibodies, and vice versa. As those skilled in the art will appreciate, competition can be assessed in a variety of assay setups. One suitable assay involves the use of Biacore technology, which can measure the degree of interaction using surface plasmon resonance technology (e.g., by using a BIAcore 3000 instrument (Biacore, Uppsala, Sweden)). Another assay for measuring cross-competition uses an ELISA-based approach. Furthermore, a high-throughput process for "binning" antibodies based on their cross-competition is described in WO 2003 / 48731.

[0053] According to the present invention, the above-mentioned cross-competing antibodies retain the activity of a single antibody comprising or consisting of the sequence set forth as SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, or SEQ ID NO:21.

[0054] According to the present invention, the above-mentioned cross-competing antibodies exhibit high affinity for both human albumin and mouse albumin.

[0055] In some embodiments, the above-mentioned cross-competing antibodies and / or single domains of the invention bind to conformational epitopes comprising at least the following amino acid sequences: an amino acid sequence ranging from amino acid residue 491 to amino acid residue 499 of SEQ ID NO:71, an amino acid sequence ranging from amino acid residue 516 to amino acid residue 526 of SEQ ID NO:71, and an amino acid sequence ranging from amino acid residue 559 to amino acid residue 565 of SEQ ID NO:71.

[0056] Chimeric polypeptides of the present invention: To verify the effectiveness of the above-mentioned single-domain antibodies, the inventors generated bispecific single-domain antibody proteins by fusion of the single-domain antibody OptiAlb-12 with the single-domain antibody KB-VWF-013 (targeting the D'D3 domain of VWF, previously described in patent publications WO 2017 / 129630 and WO 2018 / 091621).

[0057] This variant was expressed, purified, and tested for its ability to cross-link VWF to albumin in vitro.

[0058] The bispecific single-domain antibody (designated KB-V13A12) was then used in in vivo studies. Mice with reduced VWF levels (von Willebrand disease type 1) received a single dose of the bispecific single-domain antibody. VWF and FVIII levels were monitored over a 14-day period.

[0059] These data show that there is a clear increase in VWF and FVIII levels that is maintained over 10 days.

[0060] Thus, in some embodiments, the chimeric polypeptides of the invention may also provide at least one further binding site for any desired protein, polypeptide, antigen, antigenic determinant or epitope, which may be targeted to the same protein, polypeptide, antigen, antigenic determinant or epitope to which the single domain antibody of the invention is retargeted, or which may be targeted to a different protein, polypeptide, antigen, antigenic determinant or epitope than the single domain antibody of the invention.

[0061] Typically, a chimeric polypeptide of the invention comprises a single domain antibody of the invention, which is fused at its N-terminus, its C-terminus, or both its N-terminus and its C-terminus to at least one further amino acid sequence, i.e., to provide a fusion protein. A chimeric polypeptide comprising only one single domain antibody according to the invention is referred to herein as a "monovalent" polypeptide. A polypeptide comprising, or consisting essentially of, two or more single domain antibodies according to the invention is referred to herein as a "multivalent" polypeptide.

[0062] In some embodiments, a chimeric polypeptide comprises at least one single domain antibody of the invention and at least one other binding unit (i.e., directed against another epitope, antigen, target, protein, or polypeptide), which is typically also a single domain antibody. Such polypeptides are referred to herein as "multispecific" polypeptides, as opposed to polypeptides comprising the same single domain antibody ("monospecific" polypeptides).

[0063] A "bispecific" polypeptide of the invention is a polypeptide comprising at least one single domain antibody against a first antigen (i.e., albumin) and at least one further binding site for a second antigen (i.e., different from albumin), whereas a "trispecific" polypeptide of the invention is a polypeptide comprising at least one single domain antibody against a first antigen (i.e., albumin), at least one further binding site for a second antigen (i.e., different from albumin), and at least one further binding site for a third antigen (i.e., different from both the first and second antigens), etc.

[0064] Thus, in a second aspect, the present invention relates to a chimeric polypeptide comprising a polypeptide and at least one single domain antibody directed against albumin.

[0065] As used herein, the term "protein" or "polypeptide" refers to a polymer of two or more natural or unnatural amino acids.

[0066] A "fusion" or "chimeric" protein or polypeptide comprises a first amino acid sequence linked to a second amino acid sequence that is not naturally linked in nature. Amino acid sequences that are normally present in separate proteins can be combined in a fusion polypeptide. Fusion proteins can be produced, for example, by chemical synthesis, or by creating and translating a polynucleotide that encodes the polypeptide regions in the desired relationship. "Fusion" or "chimeric" polypeptides and proteins comprise a combination of a first polypeptide chain, e.g., a single domain antibody against VWF, and a second polypeptide chain, e.g., a single domain antibody against albumin.

[0067] In a particular embodiment, an anti-albumin single domain antibody according to the invention is linked to another single domain antibody that recognizes an endogenous plasma protein such as VWF. Such anti-albumin single domain antibodies are called bispecific single domain antibodies.

[0068] Thus, in a particular embodiment, the present invention relates to a bispecific single domain antibody comprising an anti-albumin single domain antibody according to the invention and another single domain antibody which recognizes an endogenous plasma protein.

[0069] In certain embodiments, the bispecific single domain antibody according to the invention increases the circulating half-life of an endogenous plasma protein.

[0070] In certain embodiments, the bispecific single domain antibody according to the invention increases endogenous plasma levels.

[0071] In another embodiment, the anti-albumin single domain antibody according to the invention is linked to another single domain antibody that recognizes the polypeptide (eg VWF concentrate) that is injected into the subject.

[0072] In certain embodiments, the bispecific single domain antibody according to the invention increases the circulating half-life of the exogenous polypeptide.

[0073] In another embodiment, an anti-albumin single domain antibody according to the invention is combined with a single domain antibody targeting any polypeptide.

[0074] In certain embodiments, a bispecific single domain antibody according to the invention increases the circulating half-life of a single domain antibody targeting another polypeptide.

[0075] In another embodiment, the anti-albumin single domain antibody is fused to another polypeptide (which is not an sdAb, but a polypeptide such as VWF).

[0076] In certain embodiments, the bispecific single domain antibody according to the invention increases the half-life of the polypeptide.

[0077] As used herein, the term "half-life" refers to the biological half-life of a particular polypeptide in vivo.Half-life can be expressed by the time required for half of the amount administered to a subject to be removed from the circulatory system and / or other tissues of the animal.When the clearance curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic, with a rapid α phase and a longer β phase.

[0078] In one embodiment, the chimeric polypeptide comprises any polypeptide, particularly a therapeutic polypeptide, preferably having low blood levels or a short half-life, allowing for repeated administration to a patient in need thereof. Such therapeutic polypeptides include VWF, FVII, FVIII, FIX, antithrombin, fibrinogen, protein C, or protein S, complement proteins (particularly C2, C9, mannose-binding lectin (MBL), C1-inhibitor, factor H-related protein-3), serpins (particularly serpin A1, serpin A3, serpin A5, Serpin A6, serpin A7, serpin A8, serpin A10, serpin C1 (=antithrombin), serpin D1, serpin E1, serpin F2, serpin G1, serpin I1), fibrinolysis-related proteins (tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen), protein hormones, growth factors, interleukins, insulin, glucagon, osteogerin (OPG), angiopoietin-2 (ANGPT2) or furin, but are not limited to these.

[0079] In certain embodiments, the chimeric polypeptide comprises at least one isolated single domain antibody.

[0080] In certain embodiments, the chimeric polypeptide comprises a clotting factor (also called a blood clotting factor).

[0081] As used herein, the term "clotting factor" refers to a naturally or recombinantly produced molecule or analog thereof involved in the hemostatic process. In other words, it refers to a molecule with procoagulant activity, i.e., a molecule involved in the conversion of fibrinogen to an insoluble fibrin mesh that clots or clots blood, or a molecule with anticoagulant activity. Procoagulant factors include factor V, factor VII, factor VIII, factor IX, factor X, and prothrombin. Anticoagulant factors include protein C, protein S, protein Z, antithrombin, protease nexin-1, tissue factor pathway inhibitor, and protein Z-dependent protease inhibitor (ZPI). In a specific embodiment, the chimeric polypeptide according to the present invention is a clotting factor selected from the group consisting of FVII, FVIII, protein C, and protein S. The clotting factors of the present invention may also be variants of wild-type clotting factors. The term "variant" includes insertions, deletions, and substitutions, either conservative or non-conservative, which changes do not substantially alter the active site or domain that confers the biological activity of the respective clotting factor. Preferably, the clotting factor is selected from the group consisting of FVII, FVIII, and FX.

[0082] In a particular embodiment, said chimeric polypeptide, wherein said at least one further single domain antibody targets a coagulation factor selected from the group consisting of VWF, FVII, FVIII, antithrombin, fibrinogen, protein C, protein S, complement proteins (in particular C2, C9, mannose-binding lectin (MBL), C1-inhibitor, factor H-related protein-3), serpins (in particular serpin A1, serpin A3, serpin A5, serpin A6, serpin A7, serpin A8, serpin A10, serpin C1 (=antithrombin), serpin D1, serpin E1, serpin F2, serpin G1, serpin I1), fibrinolysis-related proteins (tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen), protein hormones or growth factors and interleukins.

[0083] In another embodiment, the chimeric polypeptide according to the invention is targeted to VWF by at least one other single domain antibody.

[0084] The term "VWF" has its common meaning in the art and refers to human von Willebrand factor (VWF), a glycemic protein involved in blood coagulation. VWF is a monomer composed of several homologous domains, each covering a different function: D1-D2-D'-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK. The naturally occurring human VWF protein has the amino acid sequence shown in GeneBank accession number NP_000543.2. The monomers are then arranged into dimers or multimers by cross-linking cysteine residues via disulfide bonds. Therefore, VWF multimers can be very large, consisting of more than 40 monomers, also referred to as high molecular weight (HMW) VWF multimers.

[0085] In some embodiments, the at least one other single domain antibody against VWF is as described in WO 2017 / 129630 and WO 2018 / 091621.

[0086] In another embodiment, the chimeric polypeptide according to the invention comprises at least one additional single domain directed against VWF: - a CDR1 having the sequence set forth as SEQ ID NO: 22, a CDR2 having the sequence set forth as SEQ ID NO: 23, and a CDR3 having the sequence set forth as SEQ ID NO: 24, or - comprising a CDR1 having the sequence set forth as SEQ ID NO: 26, a CDR2 having the sequence set forth as SEQ ID NO: 27, and a CDR3 having the sequence set forth as SEQ ID NO: 28. The sequences of the single domain antibodies against the D'D3 domain of VWF (KB-VWF-013 KB and KB-VWF-080) are shown in Table (B) below.

[0087] [Table 2]

[0088] In certain embodiments, the chimeric polypeptide according to the invention is a bispecific polypeptide.

[0089] 1. A chimeric polypeptide according to the present invention, wherein the bispecific polypeptide comprises a sequence consisting of, but not limited to, the following sequences: SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:47, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70.

[0090] In another embodiment, the chimeric polypeptide according to the invention comprises a single domain antibody anti-albumin and a single domain antibody anti-VWF.

[0091] In the context of the present invention, the chimeric polypeptide according to the invention is a bispecific polypeptide comprising a single domain antibody anti-albumin, and a single domain antibody anti-VWF having the following fusion sequence SEQ ID NO: 30 (KB-V13A12).

[0092] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-VWF has the following fusion sequence SEQ ID NO: 30 (KB-V13A12).

[0093] SEQ ID NO:30: [ka]

[0094] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-VWF has the following fusion sequence SEQ ID NO: 31 (KB-V13A12 / V12L).

[0095] SEQ ID NO:31: [ka]

[0096] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-VWF has the following fusion sequence SEQ ID NO: 32 (KB-V13A12 / E46V).

[0097] SEQ ID NO:32: [ka]

[0098] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-VWF has the following fusion sequence SEQ ID NO: 33 (KB-V13A12 / T78S).

[0099] SEQ ID NO:33: [ka]

[0100] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-VWF has the following fusion sequence SEQ ID NO: 34 (KB-V13A12 / V12L-E46V).

[0101] SEQ ID NO:34: [ka]

[0102] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-VWF has the following fusion sequence SEQ ID NO: 35 (KB-V13A12 / V12L-T78S).

[0103] SEQ ID NO:35: [ka]

[0104] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-VWF has the following fusion sequence SEQ ID NO: 36 (KB-V13A12 / E46V-T78S).

[0105] SEQ ID NO:36: [ka]

[0106] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-VWF has the following fusion sequence SEQ ID NO: 37 (KB-V13A12 / V12L-E46V-T78S).

[0107] SEQ ID NO:37: [ka]

[0108] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-VWF has the following fusion sequence SEQ ID NO: 43 (KB-V13A12 / V5L).

[0109] SEQ ID NO:43: [ka]

[0110] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-VWF has the following fusion sequence SEQ ID NO: 44 (KB-V13A12 / V5L-E46V).

[0111] SEQ ID NO:44: [ka]

[0112] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-VWF has the following fusion sequence SEQ ID NO: 45 (KB-V13A12 / V5L-T78S).

[0113] SEQ ID NO:45: [ka]

[0114] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-VWF has the following fusion sequence SEQ ID NO: 46 (KB-V13A12 / V5L-E46V-T78S).

[0115] SEQ ID NO:46: [ka]

[0116] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-VWF has the following fusion sequence SEQ ID NO: 47 (KB-V80A12).

[0117] SEQ ID NO:47: [ka]

[0118] In a specific embodiment, the chimeric polypeptide according to the invention comprises a single domain antibody anti-albumin and a single domain antibody anti-VWF, wherein the single domain antibody anti-VWF targets the CK domain of VWF. The sequence of the single domain antibody against the CK domain of VWF (KB-VWF-040) is shown in Table (C) below.

[0119] In a particular embodiment, the sequence of a single domain antibody against the CK domain of VWF (KB-VWF-040) is shown in Table (C) below.

[0120] [Table 3]

[0121] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-VWF has the following fusion sequence SEQ ID NO: 52 (KB-VWF-040).

[0122] SEQ ID NO:52 [ka]

[0123] In another embodiment, the chimeric polypeptide according to the invention is directed to at least one other single domain antibody targeted to protein S (PS).

[0124] As used herein, the term "protein S" (PS) refers to a natural anticoagulant that acts as a cofactor for activated protein C (APC) in the proteolytic inactivation of activated factor V (FVa) and factor VIII (FVIIIa), as well as tissue factor pathway inhibitor alpha (TFPIα) in the inhibition of activated factor X (FXa).

[0125] In another embodiment, the chimeric polypeptide according to the invention comprises a single domain antibody anti-albumin and a single domain antibody anti-protein S.

[0126] In some embodiments, the at least one other single domain antibody against VWF is as described in WO 2022 / 002880.

[0127] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-protein S has the following fusion sequence SEQ ID NO:38.

[0128] SEQ ID NO:38: [ka]

[0129] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and two single domain antibodies anti-protein S has the following fusion sequence SEQ ID NO:39.

[0130] SEQ ID NO:39: [ka]

[0131] In another embodiment is a chimeric polypeptide according to the invention, wherein at least one further single domain antibody targets protease nexin-1 (PN-1).

[0132] As used herein, the term "protease nexin-1" (PN-1), also known as SERPINE2, refers to a member of the family of serine protease inhibitors called serpins, which are important regulators of many biological events. PN-1 is a serpin that is barely detectable in plasma but is found in many organs and produced by most cell types, including monocytes, platelets, and vascular cells. PN-1 is a 45-50 kDa glycoprotein encoded by the SERPINE2 gene on human chromosome 2q33-q35. PN-1 is a 378-amino acid residue single chain with three cysteine residues that do not form disulfide bonds within the protein core of the molecule (Bouton et al., 2012 and McGrogan et al., 1988; Boulaftali et al., 2010).

[0133] In some embodiments, the at least one other single domain antibody against protease nexin-1 is as described in WO 2020 / 54619. In certain embodiments, single domain antibodies anti-albumin and single domain antibodies anti-protease

[0134] A chimeric polypeptide according to the invention comprising xin-1 has the following fusion sequence SEQ ID NO:40.

[0135] SEQ ID NO:40: [ka]

[0136] In another embodiment, the chimeric polypeptide according to the invention is directed to at least one other single domain antibody targeted to antithrombin.

[0137] As used herein, the term "antithrombin," also known as SERPIN C1, has its common meaning in the art and refers to a small glycoprotein that inactivates several enzymes of the coagulation system. Antithrombin activity is increased by the anticoagulant heparin, which enhances the binding of antithrombin to factors IIa and Xa.

[0138] In another embodiment, a chimeric polypeptide according to the invention comprises a single domain antibody anti-albumin and a single domain antibody anti-antithrombin.

[0139] In some embodiments, at least one additional single domain antibody against antithrombin (KB-AT-01) is shown in the table below (Table D).

[0140] [Table 4]

[0141] In a particular embodiment, at least one additional single domain against antithrombin is a chimeric polypeptide according to the present invention, comprising a CDR1 having the sequence set forth as SEQ ID NO: 53, a CDR2 having the sequence set forth as SEQ ID NO: 54, and a CDR3 having the sequence set forth as SEQ ID NO: 55.

[0142] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-antithrombin has the following fusion sequence SEQ ID NO: 57 (KB-AT01A12).

[0143] SEQ ID NO:57 [ka]

[0144] In another embodiment, the chimeric polypeptide according to the invention is directed to coagulation factor X by at least one further single domain antibody.

[0145] As used herein, the term "clotting factor X" or "factor X" has its common meaning in the art and refers to a secreted serine protease involved in the coagulation mechanism. It functions as the first enzyme in the coagulation cascade to form fibrin. Factor X normally circulates in plasma as an inactive molecule, but activation of factor X is involved in both the intrinsic and extrinsic coagulation pathways.

[0146] In another embodiment, a chimeric polypeptide according to the invention comprises a single domain antibody anti-albumin and a single domain antibody anti-factor X.

[0147] In some embodiments, at least one additional single domain antibody against Factor X (KB-FX-E3) is shown in the table below (Table E).

[0148] [Table 5]

[0149] In certain embodiments, the at least one additional single domain for Factor X is a chimeric polypeptide according to the invention, comprising a CDR1 having the sequence set forth as SEQ ID NO: 58, a CDR2 having the sequence set forth as SEQ ID NO: 59, and a CDR3 having the sequence set forth as SEQ ID NO: 60.

[0150] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-factor X has the following fusion sequence SEQ ID NO: 62 (KB-X3A12).

[0151] SEQ ID NO: 62 [ka]

[0152] In another embodiment, the chimeric polypeptide according to the invention is directed to at least one further single domain antibody targeted to coagulation factor IX.

[0153] As used herein, the term "blood coagulation factor IX" or "factor IX" has its common meaning in the art and refers to a blood coagulation factor, a serine protease zymogen. Upon activation, FIX is converted into an active serine protease, which, in the presence of Ca and membrane phospholipids, hydrolyzes one arginine-isoleucine bond of factor X to form activated factor X.

[0154] In another embodiment, the chimeric polypeptide according to the invention comprises a single domain antibody anti-albumin and a single domain antibody anti-factor IX.

[0155] In some embodiments, the at least one additional single domain antibody against Factor IX (KB-FIX-D9) is shown in the table below (Table F).

[0156] [Table 6]

[0157] In a specific embodiment, at least one additional single domain against Factor IX is a chimeric polypeptide according to the present invention comprising a CDR1 having the sequence set forth as SEQ ID NO: 63, a CDR2 having the sequence set forth as SEQ ID NO: 64, and a CDR3 having the sequence set forth as SEQ ID NO: 65.

[0158] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-factor X has the following fusion sequence SEQ ID NO: 67 (KB-F9D9A12).

[0159] SEQ ID NO: 67 [ka]

[0160] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-factor X has the following fusion sequence of SEQ ID NO: 68 (KB-F9D9A12 / LTRAE).

[0161] SEQ ID NO: 68 [ka]

[0162] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-factor X has the following fusion sequence of SEQ ID NO: 69 (KB-F9D9A12 / FTRVV).

[0163] SEQ ID NO: 69 [ka]

[0164] In a particular embodiment, a chimeric polypeptide according to the invention comprising a single domain antibody anti-albumin and a single domain antibody anti-factor X has the following fusion sequence SEQ ID NO: 70 (KB-F9D9A12 / LTRVV).

[0165] SEQ ID NO: 70 [ka]

[0166] In certain embodiments, the chimeric polypeptide exhibits increased blood levels of the protein of interest relative to albumin when administered to a subject, compared to a corresponding polypeptide that is not linked to the single domain antibody administered to the subject.

[0167] Typically, a chimeric polypeptide of the invention comprises at least one single domain antibody of the invention, which is fused at the N-terminus, the C-terminus, or both the N-terminus and the C-terminus, i.e. (eventually via at least one further amino acid sequence) to provide a fusion protein.

[0168] Alternatively, a chimeric polypeptide of the invention comprises at least one single domain antibody of the invention inserted into a therapeutic polypeptide.

[0169] As used herein, the term "inserted into" refers to the position of the single domain antibody to albumin in the chimeric polypeptide relative to the analogous position in a naturally occurring polypeptide, such as a mature human VWF polypeptide. This term refers to the properties of the chimeric polypeptide relative to the naturally occurring polypeptide and does not indicate, imply, or infer any method or process by which the chimeric polypeptide was made.

[0170] Importantly, to improve exposure of the single domain antibody in the context of the fusion protein, an amino acid linker can be placed at the N- or C-terminus of each single domain antibody sequence. Examples of linkers for use in the context of the present invention are (Gly3-Ser)4, (Gly3-Ser), Ser-Gly, or (Ala-Ala-Ala).

[0171] As used herein, the term "insertion site" refers to a position within a polypeptide, such as a VWF polypeptide, immediately upstream of a position at which a heterologous moiety may be inserted. An "insertion site" is designated by a number, which is the number of the amino acid in the polypeptide to which the insertion site corresponds, which is immediately N-terminal to the insertion site.

[0172] According to the present invention, a polypeptide comprising only one single domain antibody is referred to herein as a "monovalent" polypeptide. A polypeptide comprising, or consisting essentially of, two or more single domain antibodies according to the present invention is referred to herein as a "multivalent" polypeptide.

[0173] A chimeric polypeptide according to the present invention comprises at least one single domain antibody of the present invention, said single domain antibody being fused to the N-terminus, C-terminus, both N-terminus and C-terminus of a therapeutic polypeptide, or inserted within the sequence of a therapeutic polypeptide.

[0174] In some embodiments, the polypeptide comprises a single domain antibody of the present invention linked to an immunoglobulin domain. For example, the polypeptide comprises a single domain antibody of the present invention linked to an Fc portion (such as human Fc). The Fc portion may be useful for increasing the half-life and even production of the single domain antibody of the present invention. For example, the Fc portion can bind to serum proteins, thus extending the half-life of the single domain antibody.

[0175] In a particular embodiment is a chimeric polypeptide according to the invention, said polypeptide comprising at least one single domain antibody against a first antigen and at least one further binding site against a second antigen.

[0176] New single domain antibodies against the CK domain of VWF (KB-VWF-040), against coagulation factor X (KB-FX-E3), or again against factor IX, may be of interest for the prevention or treatment of bleeding disorders.

[0177] Thus, in another aspect, the present invention relates to an isolated single domain antibody (sdAb) against the CK domain of VWF, comprising a CDR1 having the sequence set forth as SEQ ID NO: 48, a CDR2 having the sequence set forth as SEQ ID NO: 49, and a CDR3 having the sequence set forth as SEQ ID NO: 50. In another embodiment, an isolated single domain antibody against the CK domain of VWF according to the present invention, said single domain antibody having at least 70% identity to the sequence set forth as SEQ ID NO: 51. In another embodiment, an isolated single domain antibody against the CK domain of VWF according to the present invention, said single domain antibody having or comprising the sequence set forth as SEQ ID NO: 51.

[0178] Thus, in another aspect, the present invention relates to an isolated single domain antibody (sdAb) against Factor X, comprising a CDR1 having the sequence set forth as SEQ ID NO: 58, a CDR2 having the sequence set forth as SEQ ID NO: 59, and a CDR3 having the sequence set forth as SEQ ID NO: 60. In another embodiment, an isolated single domain antibody against Factor X according to the present invention, wherein said single domain antibody has at least 70% identity to the sequence set forth as SEQ ID NO: 61. In another embodiment, an isolated single domain antibody against Factor X according to the present invention, wherein said single domain antibody has or comprises the sequence set forth as SEQ ID NO: 61.

[0179] Thus, in another aspect, the present invention relates to an isolated single domain antibody (sdAb) against Factor IX, comprising a CDR1 having the sequence set forth as SEQ ID NO: 63, a CDR2 having the sequence set forth as SEQ ID NO: 64, and a CDR3 having the sequence set forth as SEQ ID NO: 65. In another embodiment, an isolated single domain antibody against Factor IX according to the invention, wherein said single domain antibody has at least 70% identity to the sequence set forth as SEQ ID NO: 66. In another embodiment, an isolated single domain antibody against Factor X according to the invention, wherein said single domain antibody has or comprises the sequence set forth as SEQ ID NO: 66.

[0180] The present invention also relates to an isolated single domain antibody (sdAb) against the CK domain of VWF according to the present invention, an isolated single domain antibody (sdAb) against coagulation factor X according to the present invention or an isolated single domain antibody (sdAb) against coagulation factor IX according to the present invention for use in therapy, in particular for the prevention or treatment of bleeding disorders.

[0181] In accordance with the present invention, the single domain antibodies and polypeptides of the invention may be produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and making proteins are well known to those skilled in the art.

[0182] The single domain antibodies and polypeptides of the present invention can be synthesized in solution or on a solid support according to conventional techniques. Various automated synthesizers are commercially available and can be used according to known protocols described in Stewart and Young; Tam et al., 1983; Merrifield, 1986, and Barany and Merrifield, Gross and Meienhofer, 1979. The single domain antibodies and polypeptides of the present invention can also be synthesized by solid-phase techniques using an exemplary peptide synthesizer such as the Model 433A from Applied Biosystems Inc. The purity of any given protein produced by automated peptide synthesis or by recombinant methods can be determined using reverse-phase HPLC analysis. The chemical authenticity of each peptide can be established by any method known to those skilled in the art.

[0183] As an alternative to automated peptide synthesis, recombinant DNA techniques may be employed in which a nucleotide sequence encoding a selected polypeptide is inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression as described herein below. Recombinant methods are particularly preferred for producing longer polypeptides.

[0184] A variety of expression vector / host systems may be utilized to contain and express peptide or protein coding sequences. These include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors, yeast transformed with yeast expression vectors (Giga-Hama et al., 1999), viral expression vectors (e.g., baculovirus; Ghosh et al., 2002), viral expression vectors (e.g., california mosaic virus, CaMV, tobacco mosaic virus, TMV) or bacterial expression vectors (e.g., Ti or pBR322 plasmids; e.g., Babe et al., 2000), or animal cell systems. Those skilled in the art are aware of various techniques for optimizing mammalian expression of proteins; see, e.g., Kaufman, 2000; Colosimo et al., 2000. Mammalian cells useful for recombinant protein production include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, and 293 cells. Exemplary protocols for recombinant expression of peptide substrates or fusion polypeptides in bacteria, yeast, and other invertebrates are known to those of skill in the art and are briefly described below. Mammalian host systems for recombinant protein expression are also well known to those of skill in the art. Host cell lines may be selected for their particular ability to process expressed proteins or produce specific post-translational modifications useful for providing protein activity. Such polypeptide modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing, which cleaves the "prepro" form of a protein, may also be important for correct insertion, folding, and / or function. Different host cells, such as CHO, HeLa, MDCK, 293, WI38, etc., have specific cellular and characteristic mechanisms for such post-translational activities and can be selected to ensure the correct modification and processing of the introduced foreign protein.

[0185] Recombinant production of the single domain antibodies and polypeptides of the present invention requires the use of vectors containing polynucleotide molecules encoding the single domain antibodies and polypeptides of the present invention. Methods for preparing such vectors and for producing host cells transformed with such vectors are well known to those skilled in the art. Polynucleotide molecules used in such endeavors may be ligated to vectors that generally contain selectable markers and origins of replication for propagation in a host. These elements of expression constructs are well known to those skilled in the art. Generally, expression vectors contain DNA encoding a given protein operably linked to appropriate transcriptional or translational control sequences, such as those derived from mammalian, microbial, viral, or insect genes. Examples of control sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences for controlling transcription and translation.

[0186] The terms "expression vector," "expression construct," or "expression cassette" are used interchangeably throughout this specification and are meant to include any type of genetic construct containing a nucleic acid encoding a gene product in which part or all of the nucleic acid coding sequence can be transcribed.

[0187] The selection of a suitable expression vector for expressing a peptide or polypeptide of the present invention will, of course, depend on the particular host cell used, and will be appreciated by those skilled in the art.

[0188] Expression requires providing the vector with appropriate signals, such as enhancers / promoters from both viral and mammalian sources, that can be used to drive the expression of a nucleic acid of interest in a host cell. Typically, the nucleic acid to be expressed is under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the cellular or introduced synthetic machinery required to initiate the specific transcription of a gene. A nucleotide sequence is operably linked when the regulatory sequence is functionally related to the DNA encoding the protein of interest (e.g., a single-domain antibody). Thus, a promoter nucleotide sequence is operably linked to a given DNA sequence if it directs the transcription of the sequence.

[0189] In a particular embodiment, the present invention relates to nucleic acid molecules encoding the single domain antibodies of the invention and / or the chimeric polypeptides of the invention.

[0190] In a particular embodiment, the present invention relates to a vector comprising a nucleic acid of the present invention.

[0191] In a particular embodiment, the present invention relates to host cells transfected, infected or transformed with the nucleic acids of the invention and / or the vectors of the invention.

[0192] Chimeric polypeptide / albumin complexes according to the present invention In another aspect, the present invention relates to a chimeric polypeptide / albumin complex, wherein the chimeric polypeptide is the chimeric polypeptide of the present invention and an albumin polypeptide as described above.

[0193] In another embodiment, the chimeric polypeptide is a chimeric polypeptide / albumin complex according to the invention comprising another single domain antibody that recognizes an endogenous plasma protein, such as VWF. In a specific embodiment, the chimeric polypeptide / albumin complex according to the invention increases the circulating half-life of the endogenous plasma protein.

[0194] In certain embodiments, the chimeric polypeptide / albumin complexes according to the present invention increase endogenous plasma levels.

[0195] In another embodiment, a chimeric polypeptide / albumin complex according to the invention, wherein the chimeric polypeptide comprises a single domain antibody that recognizes a polypeptide (eg, VWF concentrate) that is injected into a subject.

[0196] In certain embodiments, a chimeric polypeptide / albumin complex according to the present invention increases the circulating half-life of the exogenous polypeptide.

[0197] In another embodiment, the chimeric polypeptide / albumin complex according to the invention, wherein the chimeric polypeptide comprises a single domain antibody targeting any polypeptide.

[0198] In certain embodiments, a chimeric polypeptide / albumin complex according to the invention increases the circulating half-life of a single domain antibody that targets another polypeptide.

[0199] In another embodiment, the chimeric polypeptide is a chimeric polypeptide / albumin complex according to the invention that comprises another polypeptide (which is not an sdAb, but is a polypeptide such as VWF, for example).

[0200] In certain embodiments, a chimeric polypeptide / albumin complex according to the present invention increases the half-life of the polypeptide.

[0201] In a further embodiment, the single domain antibody against at least one other target is PEGylated (such as rVWF (PEGrVWF)).

[0202] Polyethylene glycol (PEG) is widely used as a drug carrier due to its high biocompatibility and ease of modification. Conjugation to various drugs, proteins, and liposomes has been shown to improve retention time and reduce toxicity. PEG can be conjugated to active agents via hydroxyl groups at the chain ends and through other chemical methods, but PEG itself is limited to a maximum of two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials that would retain the biocompatibility properties of PEG but add the advantage of multiple attachment points per molecule (providing greater drug loading) and could be synthetically engineered to suit a variety of applications.

[0203] Treatment Methods and Uses The single domain antibody anti-albumin and / or chimeric polypeptides described above are suitable for use in therapeutic methods.

[0204] In a third aspect, the present invention relates to an isolated single domain antibody (sdAb) against albumin for use as a medicament.

[0205] In another aspect, the present invention relates to a chimeric polypeptide comprising a polypeptide and at least one single domain antibody of the invention for use as a medicament.

[0206] In yet another aspect, the present invention relates to a chimeric polypeptide / albumin complex of the invention for use as a medicament.

[0207] According to the present invention, a single domain antibody of the present invention, a chimeric polypeptide of the present invention, or a chimeric polypeptide / albumin complex of the present invention is administered in a therapeutically effective amount to a subject in need thereof.

[0208] In a particular embodiment is an isolated single domain antibody (sdAb) against albumin according to the invention or a chimeric polypeptide according to the invention for use in increasing the circulating half-life of an endogenous plasma protein, an exogenous polypeptide, another polypeptide or a single domain antibody against a polypeptide.

[0209] In a particular embodiment, the anti-albumin single domain antibody, the chimeric polypeptide, or the chimeric polypeptide / albumin complex according to the invention is for use in increasing the circulating half-life of an endogenous plasma protein.

[0210] In a particular embodiment, the anti-albumin single domain antibody, the chimeric polypeptide, or the chimeric polypeptide / albumin complex according to the invention is for use in increasing the circulating half-life of an exogenous polypeptide.

[0211] In a particular embodiment is an anti-albumin single domain antibody, chimeric polypeptide, or chimeric polypeptide / albumin complex according to the invention for use in increasing the circulating half-life of a single domain antibody targeting another polypeptide.

[0212] In a particular embodiment is an anti-albumin single domain antibody, a chimeric polypeptide, or a chimeric polypeptide / albumin complex according to the invention for use in increasing the half-life of a polypeptide.

[0213] In particular embodiments is an isolated single domain antibody against albumin according to the invention, a chimeric polypeptide comprising a polypeptide according to the invention and at least one single domain antibody against albumin, or a chimeric polypeptide / albumin complex according to the invention for use in a method for preventing or treating a bleeding disorder.

[0214] In another embodiment, the present invention relates to a method for preventing or treating a bleeding disorder in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an anti-albumin single domain antibody according to the present invention, a chimeric polypeptide according to the present invention, or a chimeric polypeptide / albumin complex according to the present invention.

[0215] In certain embodiments, the method according to the present invention, wherein the bleeding disorder is selected from the group consisting of, but not limited to, von Willebrand disease, hemophilia A or hemophilia B, protein C deficiency, protein S deficiency, antithrombin deficiency, factor XI deficiency, C1-esterase inhibitor deficiency, insulin deficiency, alpha-1-antitrypsin deficiency, complement C2 deficiency, or sickle cell disease.

[0216] In particular embodiments is an isolated single domain antibody against albumin according to the invention, a chimeric polypeptide comprising a polypeptide according to the invention and at least one single domain antibody against albumin, or a chimeric polypeptide / albumin complex according to the invention for use in a method for preventing or treating von Willebrand disease.

[0217] In a particular embodiment is an isolated single domain antibody against albumin according to the present invention, a chimeric polypeptide comprising a polypeptide according to the present invention and at least one single domain antibody against albumin, or a chimeric polypeptide / albumin complex according to the present invention for use in a method for preventing or treating a subject with low levels of VWF.

[0218] In a particular embodiment is an anti-albumin single domain antibody according to the invention, a chimeric polypeptide according to the invention, or a chimeric polypeptide / albumin complex according to the invention for use in increasing blood levels of a therapeutic protein.

[0219] In a particular embodiment, the anti-albumin single domain antibody, chimeric polypeptide or chimeric polypeptide / albumin complex according to the invention is for use to increase blood levels of a coagulation factor selected from the group consisting of, but not limited to, VWF, FVII, FVIII, antithrombin, fibrinogen, protein C, protein S, complement proteins (in particular C2, C9, mannose-binding lectin (MBL), C1-inhibitor, factor H-related protein-3), serpins (in particular serpin A1, serpin A3, serpin A5, serpin A6, serpin A7, serpin A8, serpin A10, serpin C1 (=antithrombin), serpin D1, serpin E1, serpin F2, serpin G1, serpin I1), fibrinolysis-related proteins (tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen), protein hormones or growth factors and interleukins.

[0220] In certain embodiments, the chimeric polypeptides described above (such as KB-V13A12) are for increasing the level of VWF in a subject in need thereof.

[0221] In a particular embodiment, the chimeric polypeptide described above (such as KB-V13A12) for use in a method of treating von Willebrand disease type 1.

[0222] For example, an anti-albumin single domain antibody according to the invention, a chimeric polypeptide according to the invention, or a chimeric polypeptide / albumin complex according to the invention for use in a method for preventing and / or treating bleeding disorders.

[0223] Bleeding disorders that can be treated by administration of a single domain antibody according to the invention, a chimeric polypeptide according to the invention or a chimeric polypeptide / albumin complex of the invention include, but are not limited to, hemophilia, deficiencies or structural abnormalities of VWF, FVII, FVIII, antithrombin, fibrinogen, protein C, protein S, complement proteins (in particular C2, C9, mannose-binding lectin (MBL), C1-inhibitor, factor H-related protein-3), serpins (in particular serpin A1, serpin A3, serpin A5, serpin A6, serpin A7, serpin A8, serpin A10, serpin C1 (=antithrombin), serpin D1, serpin E1, serpin F2, serpin G1, serpin I1), fibrinolysis-related proteins (tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen), protein hormones or growth factors and interleukins.

[0224] In certain embodiments, the bleeding disorder that may be treated by administration of a single domain antibody, chimeric polypeptide or chimeric polypeptide / albumin complex according to the invention is selected from the group consisting of, but is not limited to: von Willebrand's disease, hemophilia A or hemophilia B, protein C deficiency, protein S deficiency, antithrombin deficiency, factor XI deficiency, C1-esterase inhibitor deficiency, insulin deficiency, alpha-1-antitrypsin deficiency, complement C2 deficiency, or sickle cell disease.

[0225] In certain embodiments, the bleeding disorder that may be treated by administration of a single domain antibody, chimeric polypeptide, or chimeric polypeptide / albumin complex according to the invention is von Willebrand's disease, hemophilia A, or hemophilia B.

[0226] In a particular embodiment, the bleeding disorder that may be treated by administration of a single domain antibody, chimeric polypeptide, or chimeric polypeptide / albumin complex according to the invention is von Willebrand disease type 1.

[0227] In a particular embodiment, the present invention relates to a method for extending or increasing the half-life of a therapeutic single domain antibody against a coagulation factor comprising the step of adding at least one sdAb against albumin to the therapeutic single domain antibody.

[0228] As used herein, the term "treating" or "treatment" refers to both preventative or prophylactic treatment and curative or disease-modifying treatment, including treatment of subjects at risk of or suspected of having a disease, as well as treatment of subjects suffering from or diagnosed with a disease or medical condition, including suppression of clinical recurrence. Treatment can be administered to a subject who has a medical disorder or who may eventually become disabled to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurrent disorder, or to prolong the subject's survival beyond that expected in the absence of such treatment. "Therapeutic regimen" refers to a pattern of disease treatment, e.g., an administration pattern used during treatment. The therapeutic regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to the subject during the initial period of the treatment regimen. The induction regimen may (partially or entirely) adopt a "loading regimen", which may include administering a higher dose of drug than the doctor uses during the maintenance regimen, administering drug more frequently than the doctor uses during the maintenance regimen, or both.The phrase "maintenance regimen" or "maintenance period" refers to the therapeutic regimen (or part of the therapeutic regimen) used to maintain the subject during the treatment of disease, for example, to keep the subject in remission for a long period of time (several months or years).The maintenance regimen may adopt continuous therapy (for example, administering drug at regular intervals, such as weekly, monthly, yearly, etc.) or intermittent therapy (for example, discontinued treatment, intermittent treatment, treatment at the time of relapse, or treatment at the time of achieving certain predetermined criteria (for example, pain, disease symptoms, etc.)).

[0229] As used herein, the term "administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance to a subject when the substance is present outside the body (e.g., an anti-albumin single domain antibody or chimeric polypeptide according to the present invention), such as by oral, mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery, and / or any other method of physical delivery described herein or known in the art. When a disease or symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease or symptom thereof. When a disease or symptom thereof is being prevented, administration of the substance typically occurs before the onset of the disease or symptom thereof. In certain embodiments, an anti-albumin single domain antibody or chimeric polypeptide according to the present invention is administered orally.

[0230] By "therapeutically effective amount" is meant a sufficient quantity of a polypeptide (or a nucleic acid encoding the polypeptide) to prevent use in a method of treating acute exacerbations of chronic obstructive pulmonary disease at a reasonable benefit / risk ratio applicable to any medical treatment. Naturally, the total daily dosage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate of the particular compound used; the duration of treatment; drugs used in combination with or concomitantly with the particular polypeptide used; and similar factors well known in the medical field. For example, it is well known in the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of a product can vary over a wide range, from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient for symptomatic administration to the patient being treated. The medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg. An effective amount of the agent is usually supplied at a dosage level of from 0.0002 mg / kg to about 20 mg / kg of body weight per day, particularly from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0231] Pharmaceutical Composition In a fourth aspect, the present invention relates to a pharmaceutical composition comprising a single domain antibody to albumin, a chimeric polypeptide, a chimeric polypeptide / albumin complex as described herein, and a pharmaceutically acceptable carrier.

[0232] In a particular embodiment is a pharmaceutical composition according to the invention for use in the prevention or treatment of the aforementioned bleeding disorders.

[0233] In certain embodiments, pharmaceutical compositions according to the invention may comprise any additional agent used in the prevention or treatment of bleeding disorders.

[0234] In a particular embodiment, the pharmaceutical composition according to the invention is for use in increasing the circulating half-life of an endogenous plasma protein.

[0235] In certain embodiments, a pharmaceutical composition according to the invention for use in increasing the circulating half-life of an exogenous polypeptide.

[0236] In a particular embodiment is a pharmaceutical composition according to the invention for use in increasing the circulating half-life of a single domain antibody that targets another polypeptide.

[0237] In a particular embodiment is a pharmaceutical composition according to the invention for use in increasing the half-life of a polypeptide.

[0238] In one embodiment, the aforementioned additional active agents may be contained in the same composition or may be administered separately.

[0239] In another embodiment, the pharmaceutical composition of the present invention relates to a combined preparation for simultaneous, separate or sequential use in the prevention and treatment of bleeding disorders.

[0240] The single domain antibodies and polypeptides of the invention (or the nucleic acids encoding them) may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices such as biodegradable polymers, to form pharmaceutical compositions. As used herein, the terms "pharmaceutical" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to mammals, particularly humans, as appropriate. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary.

[0241] In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical, or rectal administration, the active principle can be administered to animals and humans in unit dosage forms, either alone or in combination with another active principle, in admixture with a conventional pharmaceutical support. Suitable unit dosage forms include oral route forms such as tablets, gel capsules, powders, granules, and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intrathecal, and intranasal dosage forms, and rectal dosage forms. Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable formulation. These may be, in particular, isotonic, sterile, saline (monosodium phosphate or disodium phosphate, sodium, potassium, calcium, or magnesium chloride, and the like, or a mixture of such salts), or a dry composition, particularly a lyophilized composition, that allows the constitution of an injection solution upon addition of sterile water or saline, depending on the case.

[0242] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, formulations containing sesame oil, peanut oil, or aqueous propylene glycol, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid, so long as easy injectability exists. It must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. Solutions containing the compounds of the present invention as free bases or pharmacologically acceptable salts can be prepared in water, appropriately mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0243] The polypeptide (or the nucleic acid encoding it) can be formulated into a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids such as hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0244] Sterile injectable solution is prepared by incorporating active polypeptide in the required amount in suitable solvent, and optionally with some other components listed above, and then sterilized by filtration.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains basic dispersion medium and other necessary components listed above.For the preparation of sterile powder for sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces powder of active ingredient and any additional desired component from the solution that has been previously sterilized and filtered.

[0245] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, although drug release capsules and the like can also be used.

[0246] For parenteral administration in aqueous solution, for example, the solution should be appropriately buffered if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, suitable sterile aqueous media will be known to those skilled in the art in light of the present disclosure. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection fluid or injected at the proposed injection site. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any case, determine the appropriate dose for the individual subject.

[0247] The single domain antibody or chimeric polypeptide (or nucleic acid encoding same) according to the invention may be formulated into a therapeutic mixture to contain about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0, or even about 10 milligrams per dose. Multiple administrations are also possible. The invention is further illustrated by the following figures and examples.

[0248] Finally, the present invention also provides a kit comprising at least one single domain antibody or chimeric polypeptide of the present invention. A kit containing an anti-albumin single domain antibody or chimeric polypeptide of the present invention for use in a method of treatment.

[0249] The present invention is further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the present invention. [Brief explanation of the drawings]

[0250] [Figure 1] Binding of ALB8 to human and mouse serum albumin. Various concentrations of purified ALB8 were incubated with immobilized human serum albumin (HSA) or mouse serum albumin (MSA). Bound ALB8 was probed using a polyclonal peroxidase-labeled anti-cMyc antibody and detected via the hydrolysis of 3,3',5,5'-tetramethylbenzidine. [Figure 2A] Binding of mutated ALB8 variants to mouse serum albumin. Various concentrations of ALB8 and its variants were incubated with immobilized mouse serum albumin (MSA). Bound nanobodies were probed using a polyclonal peroxidase-labeled anti-cMyc antibody and detected via the hydrolysis of 3,3',5,5'-tetramethylbenzidine. [Figure 2B] Binding of mutated ALB8 variants to mouse serum albumin. Various concentrations of ALB8 and its variants were incubated with immobilized mouse serum albumin (MSA). Bound nanobodies were probed using a polyclonal peroxidase-labeled anti-cMyc antibody and detected via the hydrolysis of 3,3',5,5'-tetramethylbenzidine. [Figure 2C] Binding of mutated ALB8 variants to mouse serum albumin. Various concentrations of ALB8 and its variants were incubated with immobilized mouse serum albumin (MSA). Bound nanobodies were probed using a polyclonal peroxidase-labeled anti-cMyc antibody and detected via the hydrolysis of 3,3',5,5'-tetramethylbenzidine. [Figure 3A] Circulating survival of ALB8 and OptiAlb variants in mice. Factor VIII-deficient mice were administered biotinylated ALB8 or OptiAlb variants (2.5 mg / kg) intravenously, and blood was collected at 5 minutes, 4 hours, 24 hours, or 96 hours. Residual plasma levels of each biotinylated single domain antibody were determined. Plotted are residual single domain antibody levels versus time at t = 5 minutes (arbitrarily set to 100%). [Figure 3B]Circulating survival of ALB8 and OptiAlb variants in mice. Factor VIII-deficient mice were administered biotinylated ALB8 or OptiAlb variants (2.5 mg / kg) intravenously, and blood was collected at 5 minutes, 4 hours, 24 hours, or 96 hours. Residual plasma levels of each biotinylated single domain antibody were determined. Plotted are residual single domain antibody levels versus time at t = 5 minutes (arbitrarily set to 100%). [Figure 3C] Circulating survival of ALB8 and OptiAlb variants in mice. Factor VIII-deficient mice were administered biotinylated ALB8 or OptiAlb variants (2.5 mg / kg) intravenously, and blood was collected at 5 minutes, 4 hours, 24 hours, or 96 hours. Residual plasma levels of each biotinylated single domain antibody were determined. Plotted are residual single domain antibody levels versus time at t = 5 minutes (arbitrarily set to 100%). [Figure 4] Binding of OptiAlb-12 and ALB8 to human serum albumin. Various concentrations of ALB8 and its OptiAlb-12 were incubated with immobilized human serum albumin (HSA). Bound nanobodies were probed using a polyclonal peroxidase-labeled anti-cMyc antibody and detected via the hydrolysis of 3,3',5,5'-tetramethylbenzidine. [Figure 5] KB-V13A12 increases VWF and FVIII plasma levels. 129Sv mice expressing human VWF and human GpIbalpha were administered a single dose of KB-V13A12 (100 micrograms / mouse) via subcutaneous injection. Plasma levels of VWF and FVIII were determined before injection and on days 1, 3, 7, 10, and 14 after injection. A sustained, statistically significant increase in plasma levels of VWF and FVIII was observed over a 10-day period. [Figure 6]Correction of hemostasis after injection with KB-V13A12. KB-V13A12 was administered subcutaneously to VWD type 1 mice. Three days after injection, the distal tip of the tail was amputated from anesthetized mice. Blood loss was monitored for 30 minutes. WT and untreated VWD type 1 mice served as controls. Untreated VWD type 1 mice had significantly increased blood loss compared to WT mice, whereas KB-V13A12-treated VWD type 1 mice had similar blood loss to WT mice. [Figure 7] In vivo survival of von Willebrand factor in the presence of KB-V13A12. Purified recombinant VWF was administered intravenously to VWF-deficient mice (0.5 mg / kg) in the absence or presence of a 10-fold molar excess of KB-V13A12. 24 hours after injection, blood was collected and plasma was analyzed for the presence of residual VWF antigen. VWF concentrations in mice administered VWF alone were 6.6-fold lower compared to mice administered VWF in the presence of KB-V13A12. Plotted are the residual VWF levels (percentage of injected dose) for each individual mouse included in the study. Clearly, KB-V13A12 protects VWF from rapid clearance from the circulation. [Figure 8] KB-V80A12 differentially increases VWF and FVIII plasma levels. 129Sv mice expressing human VWF and human GpIbalpha were administered a single dose of KB-V80A12 (100 micrograms / mouse) via subcutaneous injection. Plasma levels of VWF and FVIII were determined before injection and on days 3, 6, 10, and 14 after injection. A sustained, statistically significant increase in plasma levels of FVIII over 10 days was observed, whereas a slight increase in VWF antigen levels was detected. [Figure 9]Epitope OptiAlb-12. Representation of OptiAlb-12 binding to albumin residues Thr49-Lys499, Glu516-Phe526, and His559-Lys56512. The three-dimensional structure of OptiAlb-12 was modeled using the VH structure available in crystal structure 7CJ2 (https: / / www.rcsb.org / structure / 7CJ2), and the albumin structure was taken from crystal structure 1AO6 (https: / / www.rcsb.org / structure / 1ao6). Figure prepared using PyMol. [Figure 10] Simultaneous binding of KB-V13A12 to VWF and albumin. Wells coated with human or mouse albumin were incubated with KB-V13A12 and then with various concentrations of human VWF. Bound VWF was probed using a peroxidase-labeled polyclonal anti-VWF antibody and detected via the hydrolysis of 3,3',5,5'-tetramethylbenzidine. Plotted is the response (OD450) to VWF concentration. [Figure 11] Binding of humanized KB-V13A12 variants to VWF. Varying concentrations of KB-V13A12, KB-V13A12 / T78S, KB-V13A12 / E46V-T78S, KB-V13A12 / V5L-E46V, KB-V13A12 / V5L-T78S, and KB-V13A12 / V5L-E46V-T78S were incubated with immobilized VWF. Bound nanobodies were probed using a polyclonal peroxidase-labeled anti-cMyc antibody and detected via hydrolysis of 3,3',5,5'-tetramethylbenzidine. Responses (OD450) versus single-domain antibody concentration are plotted. [Figure 12]Binding of KB-V13A12 to albumin and VWF at neutral and low pH. Human albumin-coated wells were incubated with KB-V13A12 followed by various concentrations of human VWF. After sequential incubation with 42 mM citric acid / 58 mM NaHPO / 150 mM NaCl (pH 5.6) or 9.2 mM citric acid / 90.9 mM NaHPO / 150 mM NaCl (pH 7.4) for 20 min at 37°C, bound VWF was probed using a peroxidase-labeled polyclonal anti-VWF antibody and detected via hydrolysis of 3,3',5,5'-tetramethylbenzidine. Plotted is the response (OD450) to VWF concentration. [Figure 13] Cross-linking of human albumin and antithrombin using KB-AT01A12. Wells coated with human or mouse albumin were incubated with KB-AT01A12 followed by various concentrations of human antithrombin. Bound antithrombin was probed using a peroxidase-labeled polyclonal anti-antithrombin antibody and detected via the hydrolysis of 3,3',5,5'-tetramethylbenzidine. Plotted is the response (OD450) versus antithrombin concentration. [Figure 14] KB-FXE3A12 simultaneously binds to HSA and factor X. Wells coated with human or mouse albumin were incubated with KB-FXE3A12 and then with various concentrations of human factor X. Bound factor X was probed using a peroxidase-labeled polyclonal anti-factor X antibody and detected via the hydrolysis of 3,3',5,5'-tetramethylbenzidine. Plotted is the response (OD450) to antithrombin concentration. [Figure 15]KB-F9D9A12 simultaneously binds to HSA and factor IX. Human albumin-coated wells were incubated with KB-F9D9A12 followed by various concentrations of human factor IX. Bound factor IX was probed using a peroxidase-labeled polyclonal anti-factor IX antibody and detected via the hydrolysis of 3,3',5,5'-tetramethylbenzidine. The plot shows the response (OD450) to antithrombin concentration. [Example]

[0251] Example 1 Expression and purification of ALB8. The nucleotide sequence encoding ALB8 was cloned into the pHEN6-plasmid, containing the PelB signal peptide to target the peripheral compartment as well as a C-terminal histidine tag and a cMyc tag. The plasmid was used to transform E. coli WK6 bacteria. E. coli WK6 clones expressing ALB8 were seeded, and a single clone was used to inoculate 3 ml of Luria-Bertani medium (LB) supplemented with ampicillin. This mixture was incubated at 37°C with agitation for 2.5 hours. This preculture was then added to 250 ml of pre-warmed Terrific Broth (TB) containing 0.1% glucose and 0.1 mg / ml ampicillin, and the bacteria were grown at 37°C with agitation until the optical density (OD) at 600 nm reached 1.0-1.3. The bacteria were collected via centrifugation, and the bacterial pellet was resuspended in 10 ml of TES buffer (0.2 M Tris (pH 8.0), 0.65 mM EDTA, 0.5 M sucrose). After incubation at 4°C for 1 hour, 20 ml of 4x diluted TES buffer was added, and the suspension was incubated at 4°C for 1 hour. The suspension was then centrifuged, and the supernatant was collected in a 50 ml Falcon tube. After another centrifugation step, the supernatant was filtered through a 0.22 micron filter.

[0252] The filtered protein was then purified via immobilized metal affinity chromatography using a 1 ml HiTrap-Talon column (GE Healthcare) according to the manufacturer's instructions. The eluted protein fractions were analyzed via SDS-Page. Purified ALB8 migrated essentially as a single band (data not shown). Fractions 2 and 3 were pooled and dialyzed against PBS. The protein concentration of the pooled fractions was 7.1 mg / ml, and the volume was 2 ml.

[0253] Example 2 Binding of ALB8 to human serum albumin and mouse serum albumin MaxiSorb microtiter plates were coated with human serum albumin (HSA) or mouse serum albumin (MSA) in carbonate buffer (pH 9.8) at 10 micrograms / ml overnight at 4°C. After washing with PBS / 0.1% Tween®-20, the wells were blocked by incubating with PBS / 1% bovine serum albumin (BSA) at 37°C for 1 hour. Serial dilutions of ALB8 were prepared in PBS / 0.1% Tween®-20 (0–10 micrograms / ml), and the preparations were incubated in the HSA- or MSA-coated wells at 37°C for 1 hour. After washing three times with PBS / 0.1% Tween®-20, bound ALB8 was probed using a polyclonal peroxidase-labeled anti-cMyc antibody (1:5,000 dilution) and detected via hydrolysis of 3,3',5,5'-tetramethylbenzidine. The response at OD450nm versus sdAb concentration is shown in Figure 1. The data were fitted using a model describing one-site binding (GraphPad Prism) to calculate half-maximal binding. Half-maximal binding of ALB8 to HSA was achieved at 0.08 micrograms / ml, whereas binding of ALB8 to MSA was achieved at a 35-fold higher concentration (2.86 micrograms / ml).

[0254] Example 3 Generation of randomly mutated ALB8-variants Two libraries were constructed by designing primers overlapping each of the three CDRs. In the first library, each amino acid in CDR1, CDR2, and CDR3 was replaced with one of 19 possible other amino acids (except cysteine). In the second library, the combined CDR sequences contained one, two, or three mutations, resulting in 19 possible amino acid substitutions for each amino acid in the three CDRs. The libraries were cloned into the pSTALK-Halo plasmid. These libraries were then transformed into yeast and subjected to yeast display analysis. 703 unique yeast clones were isolated from the first library, and 3.62 million yeast clones were obtained from the second library. 48 random clones from each library were sequenced to verify the diversity of the libraries.

[0255] Example 4 Isolation of yeast clones via cell sorting Surface expression of mutant variants was induced via incubation with galactose. One million yeast cells of the simple variant library were first incubated overnight at 4°C with Halo-Alexa660 ligand (for detection of yeast expressing sdAb on their surface) and 100 nM Alexa488-labeled MSA in PBS / 2% bovine albumin. The cells were then washed in PBS (pH 5.6) for 1 hour at 30°C before undergoing cell sorting. Fifty thousand (50,000) yeast cells positive for both Halo-Alexa660 and MSA-Alexa488 were sorted using a FAC ARIA III (Becton Dickinson). The sorted cells were amplified, and then one million cells were incubated overnight at 4°C with Halo-Alexa660 and 10 nM MSA-Alexa488. After washing with PBS (pH 5.6), a second round of sorting was performed, sorting 5,000 yeast cells. This second procedure was repeated once, allowing sorting of 15,000 clones positive for both Halo-Alexa660 and 10 nM MSA-Alexa488. Ninety-six clones were randomly picked and analyzed for their sequences, revealing the isolation of 11 distinct mutant clones.

[0256] A similar approach was used for the second library: 11 million yeast cells were incubated with Halo-Alexa660 and 100 nM MSA-Alexa488 in PBS / 2% bovine albumin overnight at 4°C. The cells were then washed in PBS (pH 5.6) at 30°C for 1 hour before cell sorting. Five hundred thousand yeast cells positive for both Halo-Alexa660 and MSA-Alexa488 were selected using a FAC ARIA III (Becton Dickinson). The selected cells were amplified, and then five million cells were incubated with Halo-Alexa660 and 10 nM MSA-Alexa488 overnight at 4°C. After washing in pH 5.6 buffer, a second round of sorting was performed. Three thousand clones were selected and amplified for a third round. In this third round, 1 million yeast cells were incubated with 10 nM MSA-Alexa4488 and Halo-Alexa660 ligand as described above, and another 1 million yeast cells were incubated with 1 nM MSA-Alexa4488 and Halo-Alexa660 ligand. Twenty-five thousand clones were selected from the 10 nM MSA-Alexa488 incubation and five thousand clones were selected from the 1 nM MSA-Alexa488 condition. Ninety-six clones were selected from each condition for sequence analysis, identifying 18 distinct mutant clones from the 10 nM MSA-Alexa488 condition and 17 clones from the 1 nM MSA-Alexa488 condition.

[0257] A total of 11 plus 18 plus 17 = 46 mutant clones were identified, and 14 unique sequences were obtained.

[0258] Example 5 Binding of Mutated ALB8 Variants to Mouse Serum Albumin The 14 mutated ALB8 variants (designated OptiAlb-01 to OptiAlb-14) were cloned into the pHEN6-plasmid and produced and purified as described in Example 1. The concentrations of the nanobodies were as follows:

[0259] [Table 7]

[0260] MaxiSorb microtiter plates were coated overnight at 4°C with mouse serum albumin (2 micrograms / ml) in carbonate buffer (pH 9.8). After washing with PBS / 0.1% Tween®-20, the wells were blocked by incubation with PBS / 1% bovine serum albumin (BSA) at 37°C for 1 hour. Serial dilutions of ALB8 and OptiAlb variants were prepared in PBS / 0.1% Tween®-20 (0–5 micrograms / ml), and the preparations were incubated in the MSA-coated wells at 37°C for 1 hour. After washing three times with PBS / 0.1% Tween®-20, bound proteins were probed using a polyclonal peroxidase-labeled anti-cMyc antibody (1:5,000 dilution) and detected via hydrolysis of 3,3',5,5'-tetramethylbenzidine. The response (OD450nm) versus single domain antibody concentration was then plotted (OptiAlb-01 to OptiAlb-05, Figure 2A; OptiAlb-06 to OptiAlb-10, Figure 2B; OptiAlb-11 to OptiAlb-14, Figure 2C; ALB8 shown in each panel of Figure 2).

[0261] As shown in Figure 2, all mutant ALB8s, except for OptiAlb-02, showed improved binding to MSA.

[0262] Example 6 Circulating survival of ALB8 and OptiAlb variants in mice ALB8 and OptiAlb variants were biotinylated using EZ-link NHS-PEG4-biotin (ThermoFisher) according to the manufacturer's instructions. The final protein concentrations were as follows:

[0263] [Table 8]

[0264] FVIII-deficient mice (bred on a C57B6 background) received biotinylated ALB8 or biotinylated OptiAlb variants at a dose of 2.5 mg / kg via intravenous injection into the retro-orbital sinus. Blood was collected 5 min, 4 h, 24 h, and 96 h after injection. Plasma samples were prepared by centrifugation (1,500 g for 20 min at room temperature) and analyzed for the presence of biotinylated single domain antibodies.

[0265] Protein concentration was determined as follows: 96-well MaxiSorp microtiter plates were coated overnight at 4°C with 50 microliters of a 5 microgram / ml streptavidin solution in carbonate buffer (pH 9.8). After washing with PBS / 0.1% Tween®-20, the wells were blocked with PBS / 1% bovine serum albumin (BSA) for 1 hour at 37°C. The wells were then washed with PBS / 0.1% Tween®-20. Serial dilutions (1 / 500 to 1 / 8000) of plasma samples were prepared in PBS / 0.1% Tween®-20. A serial dilution of purified biotinylated ALB8 was used as a reference. The samples were incubated with immobilized streptavidin for 1 hour at 37°C. The wells were then washed with PBS / 0.1% Tween®-20. Bound biotinylated nanobodies were probed using a polyclonal peroxidase-labeled anti-cMyc antibody and detected via the hydrolysis of 3,3′,5,5′-tetramethylbenzidine.

[0266] For each single-domain antibody, the residual plasma concentration was calculated and normalized to the plasma concentration 5 minutes after injection. These data were then plotted against time after injection (see Figure 3). Table 2 summarizes the relative plasma levels at 96 hours (as a percentage of the level at t = 5 minutes). Additionally, the table summarizes the ratio of the relative plasma level of each OptiAlb variant to ALB8. Five OptiAlb variants showed at least 1.8-fold higher plasma levels at 96 hours compared to ALB8: OptiAlb-03 (2.3-fold), OptiAlb-07 (2.4-fold), OptiAlb-09 (2.1-fold), OptiAlb-11 (1.8-fold), and OptiAlb-12 (2.4-fold). OptiAlb-03 has SEQ ID NO: 5, OptiAlb-07 has SEQ ID NO: 9, OptiAlb-09 has SEQ ID NO: 13, OptiAlb-11 has SEQ ID NO: 17 and OptiAlb-12 has SEQ ID NO: 21.

[0267] [Table 9]

[0268] Example 7 Binding of OptiAlb-12 and ALB8 to human serum albumin OptiAlb-12 and ALB8 were compared for binding to immobilized human serum albumin (HSA). MaxiSorb microtiter plates were coated with HSA (5 micrograms / ml) in carbonate buffer (pH 9.8) overnight at 4°C. After washing the wells with PBS / 0.1% Tween®-20, the wells were blocked by incubation with PBS / 1% bovine serum albumin (BSA) for 1 hour at 37°C. Serial dilutions of ALB8 and OptiAlb-12 were prepared in PBS / 0.1% Tween®-20 (0-10 micrograms / ml), and the preparations were incubated in the HSA-coated wells for 1 hour at 37°C. After washing three times with PBS / 0.1% Tween®-20, bound proteins were probed using a polyclonal peroxidase-conjugated anti-cMyc antibody (1:5,000 dilution) and detected via the hydrolysis of 3,3',5,5'-tetramethylbenzidine. Both ALB8 and OptiAlb-12 showed dose-dependent binding to immobilized HSA (Figure 4). Data were fitted using a model describing one-site binding (GraphPad Prism) to calculate half-maximal binding. Half-maximal binding of ALB8 to HSA was achieved at 0.083 micrograms / ml (95% confidence interval 0.073-0.094). Half-maximal binding of OptiAlb-12 to HSA was achieved at 0.038 micrograms / ml (95% confidence interval 0.034-0.044), a 2.2-fold lower concentration compared to ALB8 (p<0.0001).

[0269] Thus, OptiAlb-12 (identified by SEQ ID NO: 21) binds to both human and mouse serum albumin more efficiently than ALB8 and exhibits longer circulatory survival in mice.

[0270] Example 8 KB-V13A12 increases VWF and FVIII plasma levels A construct encoding KB-VWF-013 fused to OptiAlb-12 (SEQ ID NO: 30) was cloned into pHEN6-plasmid to include a C-terminal histidine and cMyc tag. The bispecific single domain antibody was expressed and purified as described in Example 1. The purified protein had a concentration of 5.96 mg / ml and is designated KB-V13A12.

[0271] Control immunosorbent assays showed that KB-V13A12 could simultaneously bind to both albumin and VWF.

[0272] The purified protein was then used for in vivo testing. Transgenic 129Sv mice expressing human von Willebrand factor (VWF) and the human glycoprotein Ivarfa were used in this study. VWF levels in these mice are 15 ± 4% of those in normal human plasma, and factor VIII (FVIII) activity levels are 44 ± 8% of those in normal human plasma. Therefore, this mouse model represents a model of von Willebrand disease type 1.

[0273] These mice received 100 micrograms of KB-V13A12 via subcutaneous injection. Blood samples were collected 24 hours before injection, and the plasma levels of VWF and FVIII from these samples were defined as t=0. Additional blood samples were collected on days 1, 3, 7, 10, and 14. Plasma samples were prepared and analyzed for levels of VWF antigen and FVIII activity.

[0274] The relative changes in plasma levels of VWF and FVIII are shown in Figures 5A and 5B, respectively. Plasma levels of VWF increased 1.9 ± 0.4-fold on day 1 (n = 9, p < 0.0001 compared to t = 0) and 2.1 ± 0.6-fold on day 10 (n = 5, p < 0.0001 compared to t = 0). On day 14, plasma levels were similar to those at t = 0 (1.3 ± 0.3-fold increase, p = 0.526).

[0275] Regarding FVIII activity, plasma levels increased 1.8±0.3-fold on day 1 (n=9, p<0.0001 compared to t=0) and 1.5±0.2-fold on day 10 (n=5, p=0.038 compared to t=0). On day 14, plasma levels were similar to those at t=0 (1.2±0.2-fold increase, p=0.537).

[0276] This indicates that KB-V13A12 efficiently increases endogenous plasma levels of VWF and FVIII after a single subcutaneous injection for at least 10 days in this mouse model for von Willebrand disease type 1.

[0277] Example 9 Correction of Hemostasis After Injection with KB-V13A12 To test whether the increased VWF and FVIII levels after injection with KB-V13A12 were functionally active, a tail clip bleeding model was applied. This experiment included three groups of mice (both male and female, 8-12 weeks old). Wild-type 129Sv mice were used as controls (WT; n=13), untreated transgenic 129Sv mice expressing human von Willebrand factor (VWF) and the human glycoprotein ivarufa (VWD type 1; n=26), and transgenic 129Sv mice expressing human von Willebrand factor (VWF) and the human glycoprotein ivarufa (VWD type 1+KB-V13A12; n=9) administered 100 µg of KB-V13A12 via subcutaneous injection to establish a bleeding tendency in these mice.

[0278] Three days after the KB-V13A12 injection, the distal 3 mm of the tail tip of each of the three groups of ketamine / xylazine-anesthetized mice was amputated. The amputated tail was immersed in a 50 ml tube filled with warm saline immediately after amputation. Blood was collected at 37°C for 30 minutes. After 30 minutes, the blood and saline mixture was centrifuged at 1,500 g. The red blood cell pellet was then dissolved in HO, and the amount of hemoglobin was obtained by reading the absorbance at 416 nm. The volume of blood lost in each sample was calculated from a standard curve, which was obtained by dissolving defined volumes of mouse blood (20 microliters, 40 microliters, 60 microliters, 80 microliters, and 100 microliters) in HO and extracting hemoglobin as described above. Blood loss for each individual mouse is shown in Figure 6.

[0279] Blood loss was significantly increased in VWD type 1 mice compared with WT mice (102 ± 123 microliters vs. 19 ± 37 microliters in VWD type 1 and WT mice, respectively, p = 0.0251). In contrast, blood loss in VWD type 1 + KB-V13A12 mice was similar to that in WT mice (47 ± 49 microliters, p = 0.711). Thus, KB-V13A12 treatment results in improved hemostasis in VWD type 1 mice.

[0280] Example 10 In vivo survival of recombinant von Willebrand factor in the absence or presence of KB-V13A12 Purified recombinant von Willebrand factor (VWF; 0.1 mg / ml) was incubated in PBS at room temperature for 30 minutes in the absence or presence of KB-V13A12 (0.12 mg / ml, 10-fold molar excess). VWF-deficient mice were then anesthetized with isoflurane, and VWF-containing solutions were intravenously infused via the retroorbital sinus at a dose of 0.5 mg VWF / kg body weight. At different time points (3 minutes and 24 hours), blood samples were obtained from isoflurane-anesthetized mice via retroorbital puncture, and plasma was prepared by centrifugation (1,500 g for 20 minutes at room temperature). Three mice received VWF alone, and four mice received VWF in the presence of KB-V13A12. Residual plasma concentrations of VWF were determined using an in-house ELISA measuring VWF using a polyclonal rabbit anti-VWF antibody (Dakocytomation, Glostrup, Denmark) as the capture agent and a peroxidase-labeled polyclonal rabbit anti-VWF antibody (Dakocytomation, Glostrup, Denmark) as the probe agent. Recovery 3 min after injection was similar for VWF alone and the VWF / KB-V13A12 combination (67 ± 42% and 86 ± 23% of the injected dose, respectively). In contrast, VWF levels were statistically significantly 6.6-fold lower in mice administered VWF alone (0.16 ± 0.02% of the injected dose) compared to VWF-deficient mice administered VWF in the presence of KB-V13A12 (1.06 ± 0.5% of the injected dose, p = 0.0323 when analyzed by an unpaired t-test with Welch's correction, Figure 7). This indicates that associating VWF with KB-V13A12 prolongs the survival of VWF in the circulation.

[0281] Example 11KB-V80A12 Differentially Increases VWF and FVIII Plasma Levels A construct encoding KB-VWF-080 fused to OptiAlb-12 (SEQ ID NO: 47) was cloned into pHEN6-plasmid to include a C-terminal histidine and cMyc tag. The bispecific single domain antibody was expressed and purified as described in Example 1. The purified protein was at a concentration of 5.2 milligrams / ml and is designated KB-V80A122.

[0282] Control immunosorbent assays showed that KB-V80A12 could simultaneously bind to both albumin and VWF.

[0283] The purified protein was then used for in vivo testing. Transgenic 129Sv mice expressing human von Willebrand factor (VWF) and the human glycoprotein Ivarfa were used in this study. VWF levels in these mice are 15 ± 4% of those in normal human plasma, and factor VIII (FVIII) activity levels are 44 ± 8% of those in normal human plasma. Therefore, this mouse model represents a model of von Willebrand disease type 1.

[0284] These mice received 100 micrograms of KB-V80A12 via subcutaneous injection. A blood sample was collected 24 hours before injection, and the plasma levels of VWF and FVIII from this sample were defined as t=0. Additional blood samples were collected on days 3, 6, 10, and 14. Plasma samples were prepared and analyzed for levels of VWF antigen and FVIII activity.

[0285] The relative changes in plasma levels of VWF and FVIII, respectively, are shown in Figure 8. Shown is the relative increase in VWF antigen or FVII activity at t = 0 relative to 1 day after injection. Plasma levels of VWF increased 1.3 ± 0.1-fold on day 3 (not significant compared to T = 0), 1.4 ± 0.2-fold on day 6 (p = 0.04 compared to T = 0), 1.4 ± 0.1-fold on day 10 (p = 0.05 compared to T = 0), and 1.2 ± 0.2-fold on day 14 (not significant compared to T = 0). Plasma levels of FVIII increased 2.2±0.2-fold on day 3 (p<0.0001 compared to T=0), 2.0±0.1-fold on day 6 (p=0.0001 compared to T=0), 1.7±0.3-fold on day 10 (p=0.0022 compared to T=0), and 1.6±0.2-fold on day 14 (p=0.0065 compared to T=0).

[0286] This indicates that KB-V80A12 efficiently increases endogenous plasma levels of FVIII, accompanied by a small increase in VWF levels after a single subcutaneous injection for at least 10 days in this mouse model for von Willebrand disease type 1.

[0287] Example 12 Epitope OptiAlb-12 The three-dimensional structure of OptiAlb-12 was modeled using the VH structure available in crystal structure 7CJ2 (https: / / www.rcsb.org / structure / 7CJ2), and the albumin structure was obtained from crystal structure 1AO6 (https: / / www.rcsb.org / structure / 1ao6). The OptiAlb-12 structure was docked onto the albumin structure using MAbYope (Bourquard et al. J Immunol 2018 201:3096-3105), as identified by OptiAlb-12. The top 30 structures all clustered at the same epitope within the DIII epitope of albumin, involving albumin residues: Thr491-Lys499, Glu516-Phe526, and His559-Lys565. The complex between human albumin and OptiAlb-12 is visualized in Figure 9. Residues were numbered according to the Uniprot sequence (PO2768).

[0288] Example 13: Simultaneous binding of KB-V13A12 to VWF and albumin Microtiter wells were coated with human serum albumin (HSA) or mouse serum albumin (MSA), both at 6 micrograms / ml, overnight at 4°C. The wells were emptied and incubated with PBS / 1% bovine serum albumin for 1 hour at 37°C. After washing four times with PBS / 0.1% Tween®-20, the wells were incubated with KB-V13A12 (200 nM) for 1 hour at 37°C. Control wells were incubated with PBS / 0.1% Tween®-20 for the same period. After washing four times with PBS / 0.1% Tween®-20, the wells were incubated with serial dilutions of purified VWF (0-6 micrograms / ml) for 1 hour at 37°C. After washing four times with PBS / 0.1% Tween®-20, bound VWF was probed with a rabbit polyclonal horseradish peroxidase-labeled anti-VWF antibody (DAKO, reference PO226) for 1 h at 37°C. After washing six times with PBS / 0.1% Tween®-20, the wells were incubated with 3,3',5,5'-tetramethylbenzidine for 5 min with gentle shaking. Hydrolysis was stopped by adding 1 M H2SO4, and the absorbance at 450 nm (OD450) was measured.

[0289] Analysis of the data revealed dose-dependent binding of VWF in the presence but not absence of KB-V13A12 (Figure 10). Binding was also absent in uncoated wells. Taken together, these data indicate that KB-V13A12 simultaneously binds to HSA and VWF, as well as MSA and VWF.

[0290] Example 14: Binding of humanized KB-V13A12 variants to VWF Microtiter wells were coated with human VWF (6 micrograms / ml) overnight at 4°C. The wells were emptied and incubated with PBS / 1% bovine serum albumin for 1 hour at 37°C. After washing four times with PBS / 0.1% Tween®-20, the wells were incubated with various concentrations of KB-V13A12 (SEQ ID NO: 30), KB-V13A12 / T78S (SEQ ID NO: 33), KB-V13A12 / E46V-T78S (SEQ ID NO: 36), KB-V13A12 / V5L-E46V (SEQ ID NO: 44), KB-V13A12 / V5L-T78S (SEQ ID NO: 45), KB-V13A12 / V5L-E46V-T After incubation with 78S (SEQ ID NO: 46) (0-100 nM) in PBS / 0.5% BSA / 0.1% Tween®-20 solution and washing four times with PBS / 0.1% Tween®-20, bound nanobodies were probed using a polyclonal peroxidase-labeled anti-cMyc antibody and detected by hydrolysis of 3,3',5,5'-tetramethylbenzidine for 5 min with gentle shaking. Hydrolysis was stopped by the addition of 1 M H2SO4, and the optical density at 450 nm (OD450) was measured.

[0291] Data analysis revealed that all humanized variants were similar to KB-V13A12 in binding to VWF (Figure 11).

[0292] Example 15: Binding of KB-V13A12 to albumin and VWF at neutral and low pH Microtiter wells were coated with 6 micrograms / ml human serum albumin (HSA) overnight at 4°C. The wells were emptied and incubated with PBS / 1% bovine serum albumin for 1 hour at 37°C. After washing four times with PBS / 0.1% Tween®-20, the wells were incubated with KB-V13A12 (100 nM) for 1 hour at 37°C. Control wells were incubated with PBS / 0.1% Tween®-20 for the same period. After washing four times with PBS / 0.1% Tween®-20, the wells were incubated with serial dilutions of purified VWF from 0 to 6 micrograms / ml for 1 hour at 37°C. After washing four times with PBS / 0.1% Tween®-20, the wells were incubated in 42 mM citric acid / 58 mM NaHPO / 150 mM NaCl (pH 5.6) or 9.2 mM citric acid / 90.9 mM NaHPO / 150 mM NaCl (pH 7.4) for 20 min at 37°C. This step was repeated three times for 20 min. Bound VWF was then probed with a rabbit polyclonal horseradish peroxidase-conjugated anti-VWF antibody (DAKO, ref. PO226) for 1 h at 37°C. After washing six times with PBS / 0.1% Tween®-20, the wells were incubated with 3,3',5,5'-tetramethylbenzidine for 5 min with gentle shaking. Hydrolysis was stopped by adding 1 M HSO, and the absorbance at 450 nm (OD450) was measured.

[0293] Data analysis revealed that binding to albumin and VWF was similar at pH 7.4 and 5.6 (Figure 12).

[0294] Example 16 Cross-linking of human albumin and antithrombin using KB-AT01A12 A construct encoding KB-AT-01 fused to OptiAlb-12 (SEQ ID NO: 57) was cloned into pHEN6-plasmid to include a C-terminal histidine and cMyc tag. The bispecific single domain antibody was expressed and purified as described in Example 1. The purified protein was at a concentration of 1 mg / ml and is designated KB-AT01A12. Microtiter wells were coated with 6 micrograms / ml human serum albumin (HSA) overnight at 4°C. The wells were emptied and incubated with PBS / 1% bovine serum albumin for 1 hour at 37°C. After washing four times with PBS / 0.1% Tween®-20, the wells were incubated with KB-AT01A12 (100 nM) for 1 hour at 37°C. Control wells were incubated with PBS / 0.1% Tween®-20 for the same period. After washing four times with PBS / 0.1% Tween®-20, the wells were incubated with serial dilutions of purified antithrombin (0-6 micrograms / ml) for 1 hour at 37°C. After washing four times with PBS / 0.1% Tween®-20, the wells were incubated in 42 mM citric acid / 58 mM NaHPO / 150 mM NaCl (pH 5.6) or 9.2 mM citric acid / 90.9 mM NaHPO / 150 mM NaCl (pH 7.4) at 37°C for 20 minutes. This step was repeated three times for 20 minutes. Bound antithrombin was then probed with a rabbit polyclonal horseradish peroxidase-conjugated anti-antithrombin antibody (US Biologicals) for 1 hour at 37°C. After washing six times with PBS / 0.1% Tween®-20, the wells were incubated with 3,3',5,5'-tetramethylbenzidine for 5 minutes with gentle shaking. Hydrolysis was stopped by the addition of 1 M HSO, and the absorbance at 450 nm (OD450) was measured. Data analysis revealed that binding to albumin and antithrombin was similar at pH 7.4 and 5.6 (Figure 13).

[0295] Example 17: A construct encoding KB-FX-E3 fused to OptiAlb-12 (SEQ ID NO: 62) was cloned into pHEN6-plasmid. The bispecific single domain antibody was expressed and purified as described in Example 1. The purified protein was at a concentration of 0.6 mg / ml and is designated as KB-FXE3A12.

[0296] The purified protein was then used to test its ability to cross-link antithrombin to albumin.

[0297] Microtiter wells were coated with 6 micrograms / ml human serum albumin (HSA) overnight at 4°C. The wells were emptied and incubated with PBS / 0.1% Tween®-20 / 1% bovine serum albumin for 1 hour at 37°C. After washing four times with PBS / 0.1% Tween®-20, the wells were incubated with KB-FXE3A12 (100 nM) in PBS / 0.05% Tween®-20 / 0.5% BSA for 1 hour at 37°C. After washing four times with PBS / 0.1% Tween®-20, the wells were incubated with serial dilutions of purified Factor X (0-6 micrograms / ml) in PBS / 0.05% Tween®-20 / 0.5% BSA for 1 hour at 37°C. After washing four times with PBS / 0.1% Tween®-20, bound Factor X was probed with a rabbit polyclonal horseradish peroxidase-conjugated anti-Factor X antibody in PBS / 0.05% Tween®-20 / 0.5% BSA for 1 hour at 37°C. After washing six times with PBS / 0.1% Tween®-20, the wells were incubated with 3,3',5,5'-tetramethylbenzidine for 5 minutes with gentle shaking. Hydrolysis was stopped by the addition of 1 M H2SO4, and the optical density at 450 nm (OD450) was measured.

[0298] Analysis of the data revealed dose-dependent binding of factor X in the presence of KB-FXE3A12 (Figure 14). Binding was also absent in uncoated wells. Taken together, these data indicate that KB-FXE3A122 binds simultaneously to HSA and factor X.

[0299] Example 18: A construct encoding KB-F9-D9 fused to OptiAlb-12 (SEQ ID NO: 67) was cloned into pHEN6-plasmid to include a C-terminal histidine and cMyc tag. The bispecific single domain antibody was expressed and purified as described in Example 1. The purified protein was at a concentration of 2.7 mg / ml and is designated KB-F9D9A12. Microtiter wells were coated with 6 micrograms / ml human serum albumin (HSA) overnight at 4° C. The wells were emptied and incubated with PBS / 1% bovine serum albumin for 1 hour at 37° C. After washing four times with 20 mM Hepes / 0.15 M NaCl / 2.5 mM CaCl2 / 1% bovine serum albumin, the wells were incubated with KB-F9D9A12 (100 nM) for 1 hour at 37° C. in 20 mM Hepes / 0.15 M NaCl / 2.5 mM CaCl2 / 0.5% BSA / 0.05% Tween®-20. After washing four times with 20 mM Hepes / 0.15 M NaCl / 2.5 mM CaCl2 / 0.1% Tween®-20, the wells were incubated with serial dilutions of purified factor IX (0 to 12 micrograms / ml) in 20 mM Hepes / 0.15 M NaCl / 2.5 mM CaCl2 / 0.5% BSA / 0.05% Tween®-20 for 1 hour at 37°C. After washing four times with 20 mM Hepes / 0.15 M NaCl / 2.5 mM CaCl / 0.1% Tween®-20, bound Factor IX was probed with a rabbit polyclonal horseradish peroxidase-conjugated anti-Factor IX antibody in 20 mM Hepes / 0.15 M NaCl / 2.5 mM CaCl / 0.5% BSA / 0.05% Tween®-20 for 1 hour at 37°C. After washing six times with 20 mM Hepes / 0.15 M NaCl / 2.5 mM CaCl / 0.1% Tween®-20, the wells were incubated with 3,3',5,5'-tetramethylbenzidine for 5 minutes with gentle shaking. Hydrolysis was stopped by the addition of 1 M H2SO4, and the optical density at 450 nm (OD450) was measured.

[0300] Analysis of the data revealed dose-dependent binding of Factor IX in the presence of KB-F9D9A12 (FIG. 15). Binding was also absent in uncoated wells. Taken together, these data indicate that KB-F9D9A12 binds HSA and Factor IX simultaneously.

[0301] Example 19: Affinity of OptiAlb-12 for human and mouse albumin Real-time binding studies were performed using an openSPR instrument. OptiAlb-12 (SEQ ID NO: 21) was immobilized using EDC / NHS-amino coupling (1500-2000 RU), and unoccupied sites were blocked via subsequent incubation with an unrelated single domain antibody and ethanolamine (1 M). A control channel was blocked using an unrelated single domain antibody and ethanolamine in the absence of OptiAlb-12. Albumin binding to the OptiAlb-12 channel was corrected for binding to the control channel (<3% of binding to the OptiAlb-12-coated channel). SPR analysis was performed in PBS / 0.1% The experiments were performed in Tween®-20. Various concentrations of HSA or MSA (0-1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM) were perfused across both the control and OptiAlb-12-coated channels at a flow rate of 40 microliters / min at 25°C. Association was for 2 min, and dissociation was for 10 min. The channels were regenerated using 10 mM glycine-HCl, pH 1.5 (10 s at 200 microliters / min).

[0302] The sensorgrams of two independent experiments (Experiment 1 and Experiment 2) were analyzed using TraceDrawer software to calculate the affinity constants, which are summarized in Table 4.

[0303] [Table 10]

[0304] References: Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are incorporated by reference into this disclosure.

Claims

1. An isolated single-domain antibody (sdAb) against albumin, - CDR1 having the sequence described as SEQ ID NO: 2, CDR2 having the sequence described as SEQ ID NO: 3, and CDR3 having the sequence described as SEQ ID NO: 4; - CDR1 having the sequence described as sequence number 6, CDR2 having the sequence described as sequence number 7, and CDR3 having the sequence described as sequence number 8; - CDR1 having the sequence described as sequence number 10, CDR2 having the sequence described as sequence number 11, and CDR3 having the sequence described as sequence number 12; - CDR1 having the sequence described as sequence number 14, CDR2 having the sequence described as sequence number 15, and CDR3 having the sequence described as sequence number 16; or - An isolated single-domain antibody against albumin, comprising CDR1 having the sequence described as SEQ ID NO: 18, CDR2 having the sequence described as SEQ ID NO: 19, and CDR3 having the sequence described as SEQ ID NO:

20.

2. The aforementioned single-domain antibody - CDR1 having at least 70% identity with the sequence described as Sequence ID No. 2, CDR2 having at least 70% identity with the sequence described as Sequence ID No. 3, and CDR3 having at least 70% identity with the sequence described as Sequence ID No. 4; - CDR1 having at least 70% identity with the sequence described as Sequence ID No. 6, CDR2 having at least 70% identity with the sequence described as Sequence ID No. 7, and CDR3 having at least 70% identity with the sequence described as Sequence ID No. 8; - CDR1 having at least 70% identity with the sequence shown as Sequence ID No. 10, CDR2 having at least 70% identity with the sequence described as Sequence ID No. 11, and CDR3 having at least 70% identity with the sequence described as Sequence ID No. 12; - CDR1 having at least 70% identity with the sequence shown as SEQ ID NO: 14, CDR2 having at least 70% identity with the sequence shown as SEQ ID NO: 15, and CDR3 having at least 70% identity with the sequence shown as SEQ ID NO: 16; or - An isolated single-domain antibody against albumin according to claim 1, comprising CDR1 having at least 70% identity with the sequence described as SEQ ID NO: 18, CDR2 having at least 70% identity with the sequence described as SEQ ID NO: 19, and CDR3 having at least 70% identity with the sequence described as SEQ ID NO:

20.

3. The aforementioned single-domain antibody - OptiAlb-03 (SEQ ID NO: 5), - OptiAlb-07 (SEQ ID NO: 9); - OptiAlb-09 (SEQ ID NO: 13); - OptiAlb-11 (SEQ ID NO: 17) or - An isolated single-domain antibody against albumin according to claim 1, which is OptiAlb-12 (SEQ ID NO: 21).

4. A chimeric polypeptide comprising a polypeptide and at least one single-domain antibody against albumin as described in claim 1.

5. The chimeric polypeptide according to claim 4, wherein the polypeptide is a coagulation factor selected from, but not limited to, the group consisting of VWF, FVII, FVII, antithrombin, fibrinogen, protein C, protein S, complement proteins (especially C2, C9, mannose-binding lectin (MBL), C1 inhibitor, factor H-related protein-3), serpines (especially serpine A1, serpine A3, serpine A5, serpine A6, serpine A7, serpine A8, serpine A10, serpine C1 (=antithrombin), serpine D1, serpine E1, serpine F2, serpine G1, serpine I1), fibrinolysis-related proteins (tissue plasminogen activator, urokinase-type plasminogen activator, plasminogen), protein hormones, growth factors, interleukins, insulin, glucagon, osteoprotegerin (OPG), angiopoietin-2 (ANGPT2), or furin.

6. The chimeric polypeptide according to claim 5, wherein the single-domain antibody is fused to the N-terminus, the C-terminus, both the N-terminus and the C-terminus of the therapeutic polypeptide, or inserted into the sequence of the therapeutic polypeptide.

7. The chimeric polypeptide according to claim 4, wherein the chimeric polypeptide comprises two, three, four, or five single-domain antibodies against albumin.

8. The chimeric polypeptide according to claim 4, wherein the chimeric polypeptide has an sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 43, 44, 45, 46, 47, 52, 57, 62, 67, 68, 69, and 70.

9. A nucleic acid molecule encoding a single-domain antibody as described in claim 1.

10. A vector comprising the nucleic acid described in claim 9.

11. A pharmaceutical composition for preventing or treating hemorrhagic disorders in subjects requiring prevention or treatment of hemorrhagic disorders, comprising an isolated single-domain antibody (sdAb) against albumin as described in claim 1.

12. The pharmaceutical composition according to claim 11, wherein the hemorrhagic disorder is a hemorrhagic disorder selected from the group consisting of von Willebrand disease, hemophilia A or hemophilia B, protein C deficiency, protein S deficiency, antithrombin deficiency, factor XI deficiency, C1-esterase inhibitor deficiency, insulin deficiency, alpha-1-antitrypsin deficiency, complement C2 deficiency, or sickle cell disease.

13. A nucleic acid molecule encoding the chimeric polypeptide described in Claim 4.

14. A vector comprising the nucleic acid described in claim 13.

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