Polypeptides binding to specific epitopes of neonatal Fc receptor

Abstract

The present invention relates to polypeptides capable of binding to specific epitopes on neonatal Fc receptor (FcRn). In particular, the present invention relates to novel and improved polypeptides comprising an immunoglobulin single variable domain (ISVD), such as a heavy chain single variable domain, capable of binding to a specific epitope on FcRn. The invention further relates to constructs, compounds, molecules or chemical entities comprising at least one of these ISSVDs that bind to a specific epitope on FcRn. The invention further relates to methods for producing such polypeptides and the use of such polypeptides for various applications including, but not limited to, extending the in vivo half-life of a therapeutic compound and / or preventing and / or treating a disease and / or disorder, such as, but not limited to, a proliferative disease, an inflammatory disease, an infectious disease or an autoimmune disease.

Description

1. Field of the Invention

[0001] The present invention relates to polypeptides capable of binding to specific epitopes on the neonatal Fc receptor (FcRn).

[0002] In particular, the present invention relates to novel and improved polypeptides comprising an immunoglobulin single variable domain (ISVD), such as a heavy chain single variable domain, capable of binding to a specific epitope on FcRn. The present invention further relates to constructs, compounds, molecules or chemical entities comprising at least one of these ISVDs that bind to specific epitopes on FcRn.

[0003] The present invention further relates to methods for generating such polypeptides and the use of such polypeptides for various applications, including but not limited to extending the in vivo half-life of therapeutic compounds and / or preventing and / or treating diseases and / or disorders, such as but not limited to proliferative diseases, inflammatory diseases, infectious diseases or autoimmune diseases. 2. Background Art

[0004] The neonatal Fc receptor (FcRn) is a unique beta (β)-2-microglobulin (β2m)-associated protein that is structurally related to the major histocompatibility class I (MHC-I) family, but which cannot present antigenic peptides to T cells. FcRn is widely expressed and is mainly expressed in parenchymal cells, endothelial cells and hematopoietic cells. FcRn is mainly localized intracellularly, but high surface levels have also been detected on immune cells such as monocytes or macrophages. FcRn has two known ligands (i.e., IgG and albumin), which together account for nearly 70% of the total serum proteins.

[0005] Although IgG subtypes are the basis of the immune response, albumin serves as a carrier protein in addition to being an important regulator of blood colloidal pressure. Despite these differences, IgG and albumin share a long serum half-life because of their interaction with FcRn, which spares them from intracellular degradation through a cellular recycling mechanism. This recycling mechanism occurs in a strictly pH-dependent manner, where binding to FcRn occurs after uptake in endosomal compartments at low pH (pH 5 - 6) and release at the cell surface at physiological pH (usually pH of about 7.4). Another function of FcRn is to transport IgG from the mother to the offspring, thus providing the naive and immature immune system of the newborn with the experience and protection formed in adult progenitor cells. This process is developmentally regulated as it occurs prenatally in rodents and humans via the inverted yolk sac or placenta, respectively, but uniquely persists at a significant level during early postnatal life in rodents due to high FcRn expression levels in the intestinal epithelium. This functional manifestation of FcRn and its ability to transcytose IgG are not limited to the newborn but persist throughout life and allow for the targeted delivery of IgG to sites where the presence of this type of antibody enhances immunity, a process widely used by IgG-based therapeutic agents. Finally, the function of FcRn is differentially determined by whether IgG is a single molecule and thus monomeric, or presented as an immune complex. In the latter case, FcRn has been shown to critically regulate the innate immune response as well as the processing and presentation of antigens contained within IgG immune complexes.

[0006] Due to the many different important physiological roles of FcRn, there is clearly a need for improved FcRn binders for therapeutic applications. 3. Summary of the Invention

[0007] The inventors have identified improved FcRn-binding polypeptides that have several advantages compared to FcRn binders described in the prior art.

[0008] One of the advantageous features of the FcRn-binding polypeptides according to the present invention is that these bind to a unique epitope on FcRn that is different from the epitopes bound by known natural FcRn ligands (i.e., serum albumin and IgG). Thus, the inventors have identified a new epitope present on FcRn and have developed polypeptides that can specifically bind to this epitope without interfering with the natural physiological functions of FcRn. Accordingly, the polypeptides of the present invention can be applied to a variety of prophylactic, diagnostic, and therapeutic applications where biological pathways mediated by or involving FcRn play an important role, while avoiding the occurrence of any potential side effects or at least limiting their severity.

[0009] In a first aspect, the present invention thus provides a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) that specifically binds to an epitope on FcRn, wherein the polypeptide is characterized in that the epitope comprises at least one of the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q and / or 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0010] In certain specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least one of the following combinations of amino acid residues:

[0011] a) 4H and 5L, and / or

[0012] b) 98L, 99G, 100P, 101D and 102N, and / or

[0013] c) 167L, 171R, 174L, 175E and 177K, and / or

[0014] d) 255Q, 256H, 257A, 259L, 260A and 262P,

[0015] The amino acid residues are numbered according to SEQ ID NO:1.

[0016] In certain further specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least one of the following combinations of amino acid residues:

[0017] a) 2E, 3S, 4H and 5L, and / or

[0018] b) 97E, 98L, 99G, 100P, 101D and 102N, and / or

[0019] c) 98L, 99G, 100P, 101D, 102N and 103T, and / or

[0020] d) 167L, 168E, 171R, 174L, 175E and 177K, and / or

[0021] e) 205P, 206P and 207E, and / or

[0022] f) 255Q, 256H, 257A, 259L, 260A, 261Q and 262P,

[0023] The amino acid residues are numbered according to SEQ ID NO:1.

[0024] In certain further specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least one of the following combinations of amino acid residues:

[0025] a) 1A, 2E, 3S, 4H and 5L, and / or

[0026] b) 164R, 167L, 168E, 171R, 174L, 175E and 177K, and / or

[0027] c) 204Y, 205P, 206P and 230E, and / or

[0028] d) 205P, 206P, 207E and 208L,

[0029] The amino acid residues are numbered according to SEQ ID NO:1.

[0030] According to certain embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 98L, 99G, 100P, 101D, 102N, 167L, 171R, 174L, 175E, 177K, 207E, 255Q, 256H, 257A, 259L, 260A and 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0031] According to further specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 167L, 168E, 171R, 174L, 175E, 177K, 205P, 206P, 207E, 255Q, 256H, 257A, 259L, 260A, 261Q and 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0032] According to still further specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q and 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0033] In certain embodiments, the FcRn-binding polypeptides of the invention bind to a novel epitope on FcRn as disclosed herein in a pH-dependent manner such that their binding affinity at acidic pH, particularly between 5.0 and 6.8, is at least three-fold higher than their binding affinity at neutral or physiological pH of about 7.4. In these certain embodiments, the polypeptides of the invention exhibit conditional specific binding to FcRn and thus have an extended in vivo serum half-life by exploiting the in vivo FcRn-mediated recycling mechanism. Accordingly, the polypeptides of the invention can be used to extend the in vivo half-life of therapeutic targets or therapeutic molecules of interest to which they are appropriately linked, bound, or fused.

[0034] In certain specific embodiments, the polypeptides of the invention have a molecular weight of at least 30 kDa, particularly between 30 kDa and 100 kDa. According to these specific embodiments, the compounds of the invention can be most effectively and optimally applied to a variety of applications, particularly for the extension of the in vivo half-life of therapeutic molecules.

[0035] According to certain specific embodiments, the polypeptides of the invention comprise at least one immunoglobulin single variable domain (ISVD) that specifically binds to an epitope on FcRn, characterized in that the epitope on FcRn to which the polypeptide binds is different from the epitope on FcRn bound by serum albumin.

[0036] According to certain specific embodiments, the polypeptides of the invention comprise at least one immunoglobulin single variable domain (ISVD) that specifically binds to an epitope on FcRn, characterized in that the epitope on FcRn to which the polypeptide binds is different from the epitope on FcRn bound by immunoglobulin G (IgG).

[0037] In certain specific embodiments, the invention provides FcRn-binding polypeptides comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein at least one ISVD is composed of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity-determining regions (CDR1 to CDR3, respectively), and characterized in that:

[0038] a) CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:8 [=S Y A M Y] and / or has an amino acid sequence having 4, 3, 2, or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO:8; and

[0039] b) CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:9 [= A I S S G G G S T D Y A D S V K G] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO:9; and

[0040] c) CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:10 [= D T L Y T S L T S Y S Y] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO:10.

[0041] In certain specific embodiments, the present invention provides an FcRn-binding polypeptide, the FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein at least one ISVD is composed of 4 framework regions (FR1 to FR4 respectively) and 3 complementary determining regions (CDR1 to CDR3 respectively), and is characterized in that:

[0042] a) CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:11 [= G F T F S S Y A M Y] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO:11; and

[0043] b) CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:12 [= A I S S G G G S T D] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO:12; and

[0044] c) CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:13 [= D T L Y T S L T S Y S Y] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO:13.

[0045] In a specific embodiment, the FcRn-binding polypeptide of the present invention is characterized in that the at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein has the sequence of SEQ ID NO:14 or SEQ ID NO:15, or a sequence having 90% to 95% sequence identity with the sequence of SEQ ID NO:14 or SEQ ID NO:15.

[0046] In certain embodiments, the polypeptide according to the present invention specifically binds to FcRn, such as but not limited to human FcRn or cynomolgus monkey FcRn, and its dissociation constant (K D ) is between 10 3 nM and 10 -2 nM or lower. Preferably, the K D is determined by Kinexa, BLI or SPR, such as by SPR.

[0047] In certain embodiments, the polypeptide according to the present invention specifically binds to FcRn, and its affinity (K A ) is between 10 -3 nM -1 and 10 2 nM -1 .

[0048] In certain embodiments, the polypeptide according to the present invention specifically binds to FcRn, and its association rate constant (k on ) is selected from at least about 10 2 M -1 s -1 , at least about 10 3 M -1 s -1 , at least about 10 4 M -1 s -1 , at least about 10 5 M -1 s -1 , at least about 10 6 M -1 s -1 , at least about 10 7 M -1 s -1 and at least about 10 8 M -1 s -1 , preferably as measured by surface plasmon resonance or BLI.

[0049] In certain embodiments, the polypeptide according to the present invention specifically binds to FcRn, and its dissociation rate constant (k off ) is selected from at most about 10 -1 s -1 , at most about 10 -2 s -1 , at most about 10 -3 s -1 , at most about 10 -4 s -1 , at most about 10 -5 s -1 and at most about 10-6 s -1 , preferably as measured by surface plasmon resonance or BLI.

[0050] In certain specific embodiments, the present invention provides a polypeptide, wherein at least one ISVD that specifically binds to FcRn is composed of 4 framework regions (FR1 to FR4 respectively) and 3 complementary determining regions (CDR1 to CDR3 respectively), and is characterized in that:

[0051] a) CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 8 [=S Y A M Y] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO: 8; and

[0052] b) CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO: 9 [=A I S S G G G S T D Y A D S V K G] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO: 9; and

[0053] c) CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO: 10 [=D T L Y T S L T S Y S Y] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO: 10.

[0054] In certain specific embodiments, the present invention provides a polypeptide, wherein at least one ISVD that specifically binds to FcRn is composed of 4 framework regions (FR1 to FR4 respectively) and 3 complementary determining regions (CDR1 to CDR3 respectively), and is characterized in that:

[0055] a) CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=G F T F S S Y A M Y] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO: 11; and

[0056] b) CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [=A I S S G G G S T D] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO: 12; and

[0057] c) The CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 13 [= D T L Y T S L T S Y SY] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO: 13.

[0058] In certain embodiments, the FcRn-binding polypeptide of the present invention is characterized in that at least one of the ISVDs that specifically binds to the epitope on FcRn as disclosed herein has the sequence of SEQ ID NO: 14 or SEQ ID NO: 15, or has 90% to 95% sequence identity with the sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0059] In certain specific embodiments, the improved FcRn-binding polypeptide of the present invention binds to FcRn in a pH-dependent manner.

[0060] In certain specific embodiments, the FcRn-binding polypeptide of the present invention binds to a new epitope on FcRn in a pH-dependent manner, such that the binding affinity at acidic pH, particularly at an acidic pH between 5.0 and 6.8, is at least three times higher than the binding affinity at a pH of about 7.4.

[0061] In a specific embodiment, at an acidic pH between 5.0 and 6.8, the polypeptide according to the present invention specifically binds to FcRn, and its affinity (K A ) is between 10 -3 nM -1 and 10 2 nM -1 . The affinity (K A ) of these polypeptides for FcRn at acidic pH, preferably at a pH between 5.0 and 6.8, is at least three times higher than the affinity (K A ) of the same polypeptide for FcRn at a neutral or physiological pH of about 7.4. In a further specific embodiment, at an acidic pH between 5.0 and 6.8, the polypeptide according to the present invention binds to FcRn with an affinity that is at least ten times higher than the affinity (K A ) of the same polypeptide for FcRn at a neutral or physiological pH of about 7.4. In yet a further specific embodiment, at an acidic pH between 5.0 and 6.8, the polypeptide according to the present invention binds to FcRn with an affinity that is at least fifty times higher, such as at least one hundred times higher, than the affinity (K A ) of the same polypeptide for FcRn at a neutral or physiological pH of about 7.4.

[0062] In certain specific embodiments, the present invention provides a polypeptide as described herein, the polypeptide being characterized in that at least one ISVD binds to FcRn at a neutral or physiological pH of about 7.4, with an affinity that is at least three-fold lower, such as at least ten-fold lower, such as at least fifty-fold lower, such as at least one hundred-fold lower than the affinity of the at least one ISVD for FcRn at an acidic pH between 5.0 and 6.8.

[0063] In a specific embodiment, at a physiological pH, such as at a pH of about 7.4, the at least one ISVD binds to FcRn with a K 4 value of less than 10 A L / mol.

[0064] In certain specific embodiments, the present invention provides a polypeptide as described herein, the polypeptide being characterized in that at a neutral or physiological pH, such as at a pH of about 7.4, the at least one ISVD does not detectably, selectively, or specifically bind to FcRn, or exhibits no binding or substantially no binding to FcRn.

[0065] In a specific embodiment, at an acidic pH between 5.0 and 6.8, the polypeptide according to the present invention specifically binds to FcRn, with a dissociation constant (K D ) between 10 3 nM and 10 -2 nM or lower. Preferably, the K D is determined by Kinexa, BLI, or SPR, for example, by SPR.

[0066] In a specific embodiment, at an acidic pH between 5.0 and 6.8, the polypeptide according to the present invention specifically binds to FcRn, with an association rate constant (k on ) selected from at least about 10 2 M -1 s -1 , at least about 10 3 M -1 s -1 , at least about 10 4 M -1 s -1 , at least about 10 5 M -1 s -1 , at least about 10 6 M -1 s -1 , at least about 10 7 M -1 s -1 and at least about 10 8 M -1 s -1, preferably as measured by surface plasmon resonance or BLI.

[0067] In certain embodiments, at an acidic pH between 5.0 and 6.8, the polypeptide according to the invention specifically binds to FcRn, and its dissociation rate constant (k off ) is selected from up to about 10 -1 s -1 , up to about 10 -2 s -1 , up to about 10 -3 s -1 , up to about 10 -4 s -1 , up to about 10 -5 s -1 , and up to about 10 -6 s -1 , preferably as measured by surface plasmon resonance or BLI.

[0068] The polypeptide of the invention binds to a specific, unique and novel epitope on FcRn, which is different from the epitopes bound by the natural ligands of FcRn, namely serum albumin and IgG.

[0069] In certain embodiments, the FcRn-binding polypeptide according to the invention preferably causes that when it binds to or otherwise associates with an FcRn molecule, the binding of the FcRn molecule to serum albumin and / or IgG is not (significantly) affected, reduced or inhibited. In this particular embodiment, in a cross-blocking assay (as described herein), when the FcRn-binding polypeptide binds to or otherwise associates with an FcRn molecule, the displacement of serum albumin is less than 40%, such as less than 30%, less than 20%, less than 10% or substantially no detectable displacement (e.g., in a competition assay based on ELISA or Alphascreen). In this particular embodiment, in a cross-blocking assay (as described herein), when the FcRn-binding polypeptide binds to or otherwise associates with an FcRn molecule, the displacement of IgG is less than 40%, such as less than 30%, less than 20%, less than 10% or substantially no detectable displacement (e.g., in a competition assay based on ELISA or Alphascreen).

[0070] In other specific embodiments, the present invention provides polypeptides, which are characterized in that they comprise at least one ISVD, and the at least one ISVD specifically binds to amino acid residues on FcRn that do not participate in the binding of FcRn to serum albumin and / or IgG. In this specific embodiment, the polypeptide comprises at least one ISVD, and the at least one ISVD specifically binds to an epitope on FcRn, and the epitope comprises amino acid residues that are not included in the epitope on FcRn bound by serum albumin and / or the epitope on FcRn bound by IgG. In this specific embodiment, the polypeptide comprises at least one ISVD, and the at least one ISVD specifically binds to amino acid residues on FcRn that are not bound by serum albumin and / or IgG.

[0071] According to a specific embodiment, the FcRn-binding polypeptide of the present invention also preferably competes with polypeptides having the amino acid sequences of SEQ ID NO: 14 and / or SEQ ID NO: 15 for binding to FcRn and / or "cross-blocks" (as defined herein) the binding of polypeptides having the amino acid sequences of SEQ ID NO: 14 and / or SEQ ID NO: 15 to FcRn.

[0072] In a specific embodiment, the FcRn-binding polypeptide of the present invention binds to substantially the same amino acid residues and / or epitopes on FcRn as those bound by SEQ ID NO: 14 and / or SEQ ID NO: 15, and even more preferably shares substantially the same amino acid interactions with FcRn as those of SEQ ID NO: 14 and / or SEQ ID NO: 15. For this purpose, according to a specific but non-limiting aspect, the FcRn-binding polypeptide according to the present invention preferably has the same CDRs as the sequences of SEQ ID NO: 14 and / or SEQ ID NO: 15, or, compared with the sequences of SEQ ID NO: 14 and / or SEQ ID NO: 15, preferably contains only such mutations (such as conservative amino acid substitutions) in its CDRs, and the mutations still allow it to have the same or substantially the same amino acid interactions with FcRn as those of SEQ ID NO: 14 and / or SEQ ID NO: 15.

[0073] In certain specific embodiments, the present invention provides the polypeptides as described herein, which are characterized in that the ISVD is a (single) domain antibody, V HH 、V HH 、humanized V HH or camelized V H 。

[0074] In certain embodiments, the present invention provides a polypeptide comprising:

[0075] at least one ISVD that specifically binds FcRn in a pH-dependent manner and at least one additional moiety, wherein the polypeptide is characterized by

[0076] a) the at least one ISVD specifically binds FcRn in a pH-dependent manner such that the binding affinity at a pH between 5.0 and 6.8 is at least three-fold higher than the binding affinity at a pH of about 7.4, and

[0077] b) the polypeptide has a molecular weight of at least 30 kDa.

[0078] In certain further specific embodiments, the polypeptide of the present invention has a molecular weight between about 30 kDa and 200 kDa, such as between about 30 kDa and 100 kDa.

[0079] In certain further specific embodiments, the at least one additional moiety is a protein moiety, such as a serum protein.

[0080] In certain further specific embodiments, the at least one additional moiety is a protein-binding moiety, such as a serum protein-binding moiety.

[0081] In certain further specific embodiments, the at least one additional moiety is a serum protein-binding moiety, such as a serum albumin-binding moiety.

[0082] In certain further specific embodiments, at least one serum albumin-binding moiety specifically binds to amino acid residues on serum albumin that do not participate in the binding of serum albumin to FcRn.

[0083] In certain further specific embodiments, the present invention provides a polypeptide as described herein, wherein the at least one serum albumin-binding moiety is at least one ISVD that specifically binds to serum albumin, such as a (single) domain antibody, V HH 、V HH 、humanized V HH 、or camelized V H 。

[0084] In certain further specific embodiments, at least one additional moiety is at least one ISVD that binds to serum albumin.

[0085] In certain additional specific embodiments, at least one additional moiety is at least one ISVD that binds to serum albumin and is substantially composed of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity-determining regions (CDR1 to CDR3 respectively), where CDR1 is SFGMS (SEQ ID NO:16), CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO:17) and CDR3 is GGSLSR (SEQ ID NO:18), and the CDRs are determined according to the Kabat definition; and / or where CDR1 is GFTFRSFGMS (SEQ ID NO:19), CDR2 is SISGSGSDTL (SEQ ID NO:20) and CDR3 is GGSLSR (SEQ ID NO:21), and the CDRs are determined according to the AbM definition (Kontermann et al., 2010).

[0086] In certain specific embodiments, at least one additional moiety is the Fc region of an immunoglobulin (Ig). Thus, in certain specific embodiments, the present invention provides at least one ISVD that specifically binds to FcRn in a pH-dependent manner, and at least one additional moiety, characterized in that:

[0087] a) The at least one ISVD specifically binds to FcRn in a pH-dependent manner such that the binding affinity at a pH between 5.0 and 6.8 is at least three times higher than the binding affinity at a pH of approximately 7.4,

[0088] b) The polypeptide has a molecular weight of at least 30 kDa, particularly between approximately 30 kDa and 100 kDa, and

[0089] c) The at least one additional moiety is the Fc region or Fc domain of an immunoglobulin (Ig).

[0090] In certain specific embodiments, the at least one additional moiety is a non-protein moiety, such as but not limited to a polyethylene glycol (PEG) moiety.

[0091] Thus, in certain specific embodiments, the present invention provides at least one ISVD that specifically binds to FcRn in a pH-dependent manner and at least one additional moiety, characterized in that:

[0092] a) The at least one ISVD specifically binds to FcRn in a pH-dependent manner such that the binding affinity at a pH between 5.0 and 6.5 is at least three times higher than the binding affinity at a pH of approximately 7.4,

[0093] b) The polypeptide has a molecular weight of at least 30 kDa, particularly between approximately 30 kDa and 100 kDa, and

[0094] c) The at least one additional moiety is a non-protein moiety, such as but not limited to a polyethylene glycol (PEG) moiety.

[0095] The polypeptide preferably further comprises one or more other groups, residues, moieties or binding units optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, moieties or binding units provide the polypeptide with an increased half-life compared to the corresponding polypeptide without the one or more other groups, residues, moieties or binding units.

[0096] In certain embodiments, the present invention provides a polypeptide as described herein, characterized in that the polypeptide further comprises a therapeutic moiety.

[0097] In certain embodiments, the present invention provides a polypeptide as described herein, characterized in that the polypeptide further comprises a therapeutic moiety, the therapeutic moiety comprising an ISVD such as a (single) domain antibody, V HH 、V HH 、humanized V HH or camelized V H 。

[0098] In another aspect, the present invention provides a nucleic acid or nucleic acid sequence encoding a polypeptide according to the present invention.

[0099] In another aspect, the present invention provides a vector comprising a nucleic acid or nucleic acid sequence according to the present invention.

[0100] In yet another aspect, the present invention provides a host cell transformed or transfected with a nucleic acid or nucleic acid sequence according to the present invention or with a vector according to the present invention.

[0101] In another aspect, the present invention provides a method or process for producing a polypeptide according to the present invention, the method comprising at least the following steps:

[0102] a. Expressing the nucleic acid sequence in a suitable (non-human) host cell or host organism or another suitable expression system; subsequently optionally:

[0103] b. Isolating and / or purifying the polypeptide according to the present invention.

[0104] In yet another aspect, the present invention provides a pharmaceutical composition comprising a polypeptide according to the present invention or a polypeptide produced by a process according to the present invention.

[0105] In another aspect, the present invention provides a polypeptide according to the present invention or a polypeptide produced by a process according to the present invention, for treating a subject in need thereof.

[0106] In another aspect, the present invention provides a method for delivering a prophylactic or therapeutic polypeptide to a specific location, tissue, or cell type in the body, the method comprising the step of administering to a subject a polypeptide of the present invention or a polypeptide produced by a process according to the present invention.

[0107] In another aspect, the present invention provides a polypeptide of the present invention or a polypeptide produced by a process according to the present invention, the polypeptide being for delivering a prophylactic or therapeutic polypeptide to a specific location, tissue, or cell type in the body.

[0108] In yet another aspect, the present invention provides a polypeptide of the present invention or a polypeptide produced by a process according to the present invention, the polypeptide being for use in therapy.

[0109] In yet another aspect, the present invention provides a polypeptide of the present invention or a polypeptide produced by a process according to the present invention, the polypeptide being for preventing, treating, or ameliorating a disease selected from the group consisting of proliferative diseases, inflammatory diseases, infectious diseases, and autoimmune diseases.

[0110] In another aspect, the present invention provides a method for preventing, treating, or ameliorating a disease selected from the group consisting of proliferative diseases, inflammatory diseases, infectious diseases, and autoimmune diseases, the method comprising at least the step of administering to a subject in need thereof a polypeptide of the present invention or a polypeptide produced by a method of the present invention.

[0111] In another aspect, the present invention provides a kit comprising a polypeptide of the present invention, a nucleic acid or nucleic acid sequence of the present invention, a vector of the present invention, or a host cell of the present invention.

[0112] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, which will be clear to the person skilled in the art. Reference is made, for example, to the standard manuals mentioned in paragraph a) on page 46 of WO 08 / 020079.

[0113] It must be noted that, as used herein, the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a reagent" includes one or more such different reagents, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that may modify or replace the methods described herein.

[0114] Unless otherwise specified, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to ascertain, using only routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0115] As used anywhere in this document, the term "and / or" includes the meanings of "and", "or", and "any other combination of all or any of the elements connected by said term".

[0116] As used in the context of the parameters or parameter ranges provided herein, the term "about" shall have the following meaning. Unless otherwise specified, when the term "about" is applied to a particular value or range, that value or range is interpreted as being as accurate as the method used to measure it. If no error range is specified in the application, the last significant digit of the numerical value indicates its accuracy. In the absence of other error ranges being given, the maximum range is determined by applying the rounding convention to the last significant digit. For example, for a pH value of about pH 2.7, the error range is 2.65 - 2.74. However, for the following parameters, specific ranges shall be used: Temperatures expressed in °C and without significant digits shall have an error range of ±1 °C (e.g., a temperature value of about 50 °C represents 50 °C ± 1 °C); times expressed in hours shall have an error range of 0.1 hour, regardless of significant digits (e.g., a time value of about 1.0 hour represents 1.0 hour ± 0.1 hour; a time value of about 0.5 hour represents 0.5 hour ± 0.1 hour).

[0117] In this application, any parameter indicated by the term "about" is also considered to be disclosed without the term "about". In other words, embodiments that mention a parameter value using the term "about" should also describe the embodiments themselves that relate to the numerical value of said parameter. For example, an embodiment specifying a pH value of "about pH 2.7" should also disclose the embodiment itself specifying a pH value of "pH 2.7"; an embodiment specifying a pH range of "between about pH 2.7 and about pH 2.1" should also describe the embodiment specifying a pH range of "between pH 2.7 and pH 2.1", etc.

[0118] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" or "comprising" shall be understood to mean including a stated integer or step or group of integers or steps but not excluding any other integer or step or group of integers or steps. When used herein, the term "comprise" may be replaced by the term "contain" or "include" or sometimes when used herein, by the term "have". 4. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 The sequence of human FcRn, wherein the amino acid residues identified as being involved (directly or indirectly) in binding to at least one polypeptide of the present invention are shown in bold and underlined.

[0120] Figure 2(A, B, C, and D) show the three-dimensional protein structure of the alpha chain of FcRn (also known as the Fc fragment of the IgG receptor and transporter (FcGRT)), where the epitope of the FcRn-binding polypeptide according to a particular embodiment of the invention is darkly shaded in (A), and the epitopes of the Fc domain of IgG and (C) serum albumin are lightly shaded. In (D), the combination shows the epitope of the FcRn-binding polypeptide according to a particular embodiment of the invention (darkly shaded in the center) and the epitope of the Fc domain of IgG (lightly shaded on the right) and the epitope of serum albumin (lightly shaded on the left and upper center).

[0121] Figure 3 (A, B, C, and D) show the three-dimensional protein structure of the heterodimeric FcRn (also known as the Fc fragment of the IgG receptor and transporter (FcGRT) and beta-2-microglobulin), where the epitope of the FcRn-binding polypeptide according to a particular embodiment of the invention is darkly shaded in (A), the epitopes of the Fc domain of IgG and (C) serum albumin are lightly shaded. In (D), the combination shows the epitope of the FcRn-binding polypeptide according to a particular embodiment of the invention (darkly shaded in the center) and the epitope of the Fc domain of IgG (lightly shaded on the right) and the epitope of serum albumin (lightly shaded on the left and upper center).

[0122] Figure 4 After intravenous bolus administration at 1.9 mg / kg in female Tg32 mice, the mean (+ / - SD, n = 2) serum concentration-time curves of different test bispecific ISVD constructs comprising an FcRn-binding ISVD and an albumin-binding ISVD (ALB23002) compared to a control bispecific ISVD construct comprising a control ISVD (IRR) and an albumin-binding ISVD (ALB23002).

[0123] Figure 5 After intravenous bolus administration at 1.9 mg / kg in female Tg32 mice, compared to a control bispecific ISVD construct comprising a control ISVD (IRR) and an albumin-binding ISVD (ALB23002), the test bispecific ISVD construct comprising the parental V HH T0263018B11 or its mutant variant and an albumin-binding ISVD (ALB23002) mean (+ / - SD, n = 2) serum concentration-time curve.

[0124] Figure 6Mean (+ / - SD, n = 2) serum concentration-time curves of a multivalent ISVD test construct comprising an FcRn-binding ISVD, an albumin-binding ISVD, and additional ISVDs directed to potentially relevant therapeutic targets after intravenous bolus administration in female Tg32 mice.

[0125] Figure 7 Mean (+ / - SD, n = 2 - 3) serum concentration-time curves of a multivalent ISVD test construct comprising one or more FcRn-binding ISVDs and one or more control ISVDs after intravenous bolus administration at 4.5 mg / kg in female Tg32 mice.

[0126] Figure 8 Schematic representation of the structural forms of the fusion polypeptide constructs as described, for example, in Example 14. The Fc domain in the constructs is an IgG4 FALA Fc backbone sequence variant with the knobs-into-holes mutations as described herein. One of these constructs (i.e., TP049) contains additional amino acid differences or variations (i.e., I253A, H310A, H435A) in the Fc backbone sequence, designated as IgG Fc (IHH). The FcRn VHH is in each case the T0263018B11 sequence (FcRn ISVD 1 in the figure) as described herein. The VHH that does not bind to FcRn or any other contemplated target is designated as "control ISVD" in the figure. In these fusion proteins, the VHH sequences are fused to the N- and / or C-terminus of the Fc chain via linkers (as described in detail herein), i.e., via the IgG1 hinge (e.g., SEQ ID NO: 126) and / or GS linker (e.g., 35GS linker, SEQ ID NO: 48), as described herein.

[0127] Figure 9 Surface plasmon resonance (SPR) sensorgrams using different coating densities of FcRn (up to 5000 RU) on a Biacore 8K+ instrument. Characterization by affinity determination of human FcRn at pH 6.0 (A) and pH 7.4 (B) of VHH-Fc proteins.

[0128] Figure 10 Mean (+ / - SD, n = 4 - 6) serum concentration-time curves of an ISVD-Fc fusion protein comprising an FcRn-binding ISVD and the Fc region of IgG4 FALA after intravenous bolus administration in female Tg32 mice. 5. Detailed Description

[0129] The present inventors have identified unique epitopes on FcRn that are distinct from the epitopes of the natural ligands of FcRn, such as serum albumin and IgG. Moreover, and even more importantly, the inventors have developed novel polypeptides that can specifically bind to the FcRn epitope without interfering with the natural physiological functions of FcRn. The polypeptides disclosed herein comprise at least one "immunoglobulin single variable domain" (ISVD) that binds to human FcRn (SEQ ID NO:1) or its (polymorphic) variants or isotypes. An isotype is an alternative protein sequence that can be generated from the same gene by a single biological event or by a combination of biological events, such as alternative promoter usage, alternative splicing, alternative initiation, and ribosomal frameshifting, all of which are known in the art.

[0130] Amino acid residues will be interchangeably indicated herein according to the standard three-letter or one-letter amino acid codes as mentioned in Table B-1 below.

[0131] Table B-1: Common Amino Acids

[0132]

[0133]

[0134] When an amino acid residue is indicated as "X" or "Xaa", it means that the amino acid residue is unspecified, unless the context requires a more restrictive interpretation. For example, if the description provides the amino acid sequence of a CDR in which one (or more) of the amino acid residues is indicated by "X", the description may further specify which amino acid residue(s) may be present at that particular position of the CDR.

[0135] The amino acids are those L-amino acids that are common in naturally occurring proteins and are listed in Table B-1. Those amino acid sequences containing D-amino acids are not intended to be covered by this definition. Any amino acid sequence containing post-translationally modified amino acids can be described as the initially translated amino acid sequence using the symbols shown in Table B-1 and the position of the modification; for example, hydroxylation or glycosylation, but these modifications should not be explicitly shown in the amino acid sequence. Any peptide or protein that can be represented as a sequence-modified connection, cross-link, and end-cap, non-peptide bond, etc. is covered by this definition. The terms "protein", "peptide", "protein / peptide", and "polypeptide" are used interchangeably throughout this disclosure and, for the purposes of this disclosure, have the same meaning. Each term refers to an organic compound consisting of a linear chain of two or more amino acids. The compound can have ten or more amino acids; twenty-five or more amino acids; fifty or more amino acids; one hundred or more amino acids, two hundred or more amino acids, and even three hundred or more amino acids. Those skilled in the art will understand that polypeptides generally contain fewer amino acids than proteins, although there is no well-recognized cut-off point for the number of amino acids that distinguishes polypeptides from proteins; polypeptides can be prepared by chemical synthesis or recombinant methods; and proteins are generally prepared in vitro or in vivo by recombinant methods known in the art.

[0136] When a nucleotide sequence or an amino acid sequence is said to "comprise" another nucleotide sequence or amino acid sequence, respectively, or to be "substantially composed of" another nucleotide sequence or amino acid sequence, this can mean that the latter nucleotide sequence or amino acid sequence has been incorporated into the first-mentioned nucleotide sequence or amino acid sequence, respectively, but more usually this generally means that the first-mentioned nucleotide sequence or amino acid sequence respectively contains within its sequence a segment of nucleotides or amino acid residues having the same nucleotide sequence or amino acid sequence as the latter sequence, regardless of how the first-mentioned sequence was actually produced or obtained (e.g., it may have been produced or obtained by any suitable method described herein). By way of non-limiting example, when an ISVD is said to comprise a CDR sequence, this may mean that the CDR sequence has been incorporated into the ISVD, but more usually this generally means that the ISVD contains within its sequence a segment of amino acid residues having the same amino acid sequence as the CDR sequence, regardless of how the ISVD was produced or obtained. It should also be noted that when the latter amino acid sequence has a particular biological or structural function, it preferably has a biological or structural function that is substantially the same, similar or equivalent in the first-mentioned amino acid sequence (in other words, the first-mentioned amino acid sequence preferably enables the latter sequence to perform a biological or structural function that is substantially the same, similar or equivalent). For example, when an ISVD is said to respectively comprise a CDR sequence or a framework sequence, the CDR sequence and the framework preferably are able to respectively act as a CDR sequence or a framework sequence in the ISVD. In addition, when a nucleotide sequence is said to comprise another nucleotide sequence, the first-mentioned nucleotide sequence preferably enables the amino acid sequence encoded by the latter nucleotide sequence to form part of the expression product (e.g., a polypeptide) when it is expressed as an expression product (in other words, the latter nucleotide sequence is in the same reading frame as the first-mentioned larger nucleotide sequence).

[0137] As used herein, the term "domain" generally refers to a globular region of an antibody chain, particularly of a heavy-chain antibody, or a polypeptide substantially composed of such a globular region. Generally, such a domain will contain peptide loops (e.g., 3 or 4 peptide loops), which are stabilized, for example, as a sheet or by disulfide bonds.

[0138] In the context of the technology of the present invention, "binding" to a particular target molecule has the ordinary meaning understood in the context of an antibody and its corresponding antigen in the art.

[0139] The epitope specifically targeted by the polypeptide of the present invention is an epitope on FcRn, which comprises at least one of the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q and / or 262P, and the amino acid residues are numbered according to the order or numbering of the amino acid residues in the amino acid sequence of SEQ ID NO:1.

[0140] The term "epitope" refers to an epitope on an antigen that is recognized by an antigen-binding molecule (such as an ISVD or a polypeptide comprising an ISVD), and more particularly by the antigen-binding site of said molecule. The terms "epitope" and "antigenic determinant" may also be used interchangeably herein. An antigen-binding molecule (such as an antibody, an ISVD, a polypeptide of the present invention, or generally an antigen-binding protein or polypeptide or a fragment thereof) that can (specifically) bind to a specific epitope, antigenic determinant, antigen or protein (or at least a part, fragment or epitope thereof), has an affinity for and / or is specific for a specific epitope, antigenic determinant, antigen or protein is referred to as "for" or "against" said epitope, antigenic determinant, antigen or protein.

[0141] In certain specific embodiments, the epitope as disclosed herein is a linear epitope, which comprises consecutive amino acid residues of a segment of the FcRn primary sequence.

[0142] In certain specific embodiments, the epitope as disclosed herein is a conformational epitope, which comprises at least two non-consecutive amino acid residues and / or segments of amino acid residues of the FcRn primary sequence that are close to each other in the FcRn tertiary structure and form a specific binding surface with the three-dimensional structure of the polypeptide of the present invention.

[0143] According to certain specific embodiments, the epitope on FcRn as disclosed herein, which is targeted by the polypeptide according to the present invention, is characterized in that it is different from the epitope on FcRn targeted by serum albumin (see Figure 2 and Figure 3 ).

[0144] According to certain specific embodiments, the epitope on FcRn as disclosed herein, which is targeted by the polypeptide according to the present invention, is characterized in that it is different from the epitope on FcRn targeted by immunoglobulin G (IgG) (see Figure 2 and Figure 3 ).

[0145] According to certain embodiments, the polypeptides of the invention are characterized in that the epitopes on the FcRn to which they bind comprise at least one of the following combinations of amino acid residues:

[0146] a) 4H and 5L, and / or

[0147] b) 98L, 99G, 100P, 101D and 102N, and / or

[0148] c) 167L, 171R, 174L, 175E and 177K, and / or

[0149] d) 255Q, 256H, 257A, 259L, 260A and 262P,

[0150] The amino acid residues are numbered according to the order or numbering of the amino acid residues in the amino acid sequence of SEQ ID NO:1.

[0151] According to certain further embodiments, the polypeptides of the invention are characterized in that the epitopes to which they bind comprise at least one of the following combinations of amino acid residues:

[0152] a) 2E, 3S, 4H and 5L, and / or

[0153] b) 97E, 98L, 99G, 100P, 101D and 102N, and / or

[0154] c) 98L, 99G, 100P, 101D, 102N and 103T, and / or

[0155] d) 167L, 168E, 171R, 174L, 175E and 177K, and / or

[0156] e) 205P, 206P and 207E, and / or

[0157] f) 255Q, 256H, 257A, 259L, 260A, 261Q and 262P,

[0158] The amino acid residues are numbered according to the order or numbering of the amino acid residues in the amino acid sequence of SEQ ID NO:1.

[0159] According to certain further embodiments, the polypeptides of the invention are characterized in that the epitopes to which they bind comprise at least one of the following combinations of amino acid residues:

[0160] a) 1A, 2E, 3S, 4H and 5L, and / or

[0161] b) 164R, 167L, 168E, 171R, 174L, 175E and 177K, and / or

[0162] c) 204Y, 205P, 206P and 230E, and / or

[0163] d) 205P, 206P, 207E and 208L,

[0164] The amino acid residues are numbered according to the order or numbering of the amino acid residues in the amino acid sequence of SEQ ID NO:1.

[0165] According to yet another embodiment, the polypeptides of the invention are characterized in that the epitopes they bind comprise at least the following amino acid residues: 4H, 5L, 98L, 99G, 100P, 101D, 102N, 167L, 171R, 174L, 175E, 177K, 207E, 255Q, 256H, 257A, 259L, 260A and 262P, and the amino acid residues are numbered according to the order or numbering of the amino acid residues in the amino acid sequence of SEQ ID NO:1.

[0166] According to a further specific embodiment, the polypeptides of the invention are characterized in that the epitopes they bind comprise at least the following amino acid residues: 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 167L, 168E, 171R, 174L, 175E, 177K, 205P, 206P, 207E, 255Q, 256H, 257A, 259L, 260A, 261Q and 262P, and the amino acid residues are numbered according to the order or numbering of the amino acid residues in the amino acid sequence of SEQ ID NO:1.

[0167] According to yet a further specific embodiment, the polypeptides of the invention are characterized in that the epitopes they bind comprise at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q and 262P, and the amino acid residues are numbered according to the order or numbering of the amino acid residues in the amino acid sequence of SEQ ID NO:1 (see Figure 1 、 Figure 2 and Figure 3 ).

[0168] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q and / or 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0169] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q and / or 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0170] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q and / or 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0171] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q and / or 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0172] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q and / or 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0173] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q and / or 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0174] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A and / or 261Q, and the amino acid residues are numbered according to SEQ ID NO:1.

[0175] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L and / or 260A, and the amino acid residues are numbered according to SEQ ID NO:1.

[0176] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A and / or 259L, and the amino acid residues are numbered according to SEQ ID NO:1.

[0177] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H and / or 257A, and the amino acid residues are numbered according to SEQ ID NO:1.

[0178] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q and / or 255Q, and the amino acid residues are numbered according to SEQ ID NO:1.

[0179] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L and / or 209Q, and the amino acid residues are numbered according to SEQ IDNO:1.

[0180] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A and / or 261Q, and the amino acid residues are numbered according to SEQ ID NO:1.

[0181] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A and / or 261Q, and the amino acid residues are numbered according to SEQ ID NO:1.

[0182] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L and / or 260A, and the amino acid residues are numbered according to SEQ ID NO:1.

[0183] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A and / or 261Q, and the amino acid residues are numbered according to SEQ ID NO:1.

[0184] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A and / or 259L, and the amino acid residues are numbered according to SEQ ID NO:1.

[0185] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A and / or 259L, and the amino acid residues are numbered according to SEQ ID NO:1.

[0186] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A and / or 259L, and the amino acid residues are numbered according to SEQ ID NO:1.

[0187] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A and / or 259L, and the amino acid residues are numbered according to SEQ ID NO:1.

[0188] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A and / or 261Q, and the amino acid residues are numbered according to SEQ ID NO:1.

[0189] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q and / or 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0190] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H and / or 257A, and the amino acid residues are numbered according to SEQ ID NO:1.

[0191] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H and / or 257A, and the amino acid residues are numbered according to SEQ ID NO:1.

[0192] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H and / or 257A, and the amino acid residues are numbered according to SEQ ID NO: 1.

[0193] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A and / or 261Q, and the amino acid residues are numbered according to SEQ ID NO: 1.

[0194] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q and / or 256H, and the amino acid residues are numbered according to SEQ ID NO: 1.

[0195] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q and / or 256H, and the amino acid residues are numbered according to SEQ ID NO: 1.

[0196] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A and / or 261Q, and the amino acid residues are numbered according to SEQ ID NO:1.

[0197] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A and / or 261Q, and the amino acid residues are numbered according to SEQ ID NO:1.

[0198] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q and / or 255Q, and the amino acid residues are numbered according to SEQ ID NO:1.

[0199] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 98L, 99G, 100P, 101D, 102N, 167L, 171R, 174L, 175E, 177K, 255Q, 256H, 257A, 259L, 260A and 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0200] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H and 5L.

[0201] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 98L, 99G, 100P, 101D and 102N.

[0202] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 167L, 171R, 174L, 175E, and 177K.

[0203] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 255Q, 256H, 257A, 259L, 260A, and 262P.

[0204] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 167L, 171R, 174L, 175E, and 177K.

[0205] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 98L, 99G, 100P, 101D, and 102N.

[0206] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 255Q, 256H, 257A, 259L, 260A, and 262P.

[0207] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 167L, 171R, 174L, 175E, 177K, 255Q, 256H, 257A, 259L, 260A, and 262P.

[0208] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 167L, 171R, 174L, 175E, 177K, 255Q, 256H, 257A, 259L, 260A, and 262P.

[0209] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 98L, 99G, 100P, 101D, 102N, 167L, 171R, 174L, 175E, 177K, 255Q, 256H, 257A, 259L, 260A, and 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0210] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 98L, 99G, 100P, 101D, 102N, 167L, 171R, 174L, 175E, and 177K, and the amino acid residues are numbered according to SEQ ID NO:1.

[0211] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 98L, 99G, 100P, 101D, 102N, 167L, 171R, 174L, 175E, and 177K, and the amino acid residues are numbered according to SEQ ID NO:1.

[0212] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 97E, 98L, 99G, 100P, 101D, 102N, 98L, 99G, 100P, 101D, 102N, 103T, 167L, 168E, 171R, 174L, 175E, 177K, 205P, 206P, 207E, 255Q, 256H, 257A, 259L, 260A, 261Q, and 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0213] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, and 5L.

[0214] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 97E, 98L, 99G, 100P, 101D, and 102N.

[0215] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 98L, 99G, 100P, 101D, 102N, and 103T.

[0216] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 167L, 168E, 171R, 174L, 175E, and 177K.

[0217] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 205P, 206P, and 207E.

[0218] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 255Q, 256H, 257A, 259L, 260A, 261Q, and 262P.

[0219] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 97E, 98L, 99G, 100P, 101D, and 102N.

[0220] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 167L, 168E, 171R, 174L, 175E, and 177K.

[0221] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 205P, 206P, and 207E.

[0222] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 98L, 99G, 100P, 101D, 102N, and 103T.

[0223] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 205P, 206P, 207E, 255Q, 256H, 257A, 259L, 260A, 261Q, and 262P.

[0224] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 98L, 99G, 100P, 101D, 102N, 103T, 167L, 168E, 171R, 174L, 175E, 177K, 205P, 206P, and 207E.

[0225] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 97E, 98L, 99G, 100P, 101D, 102N, 167L, 168E, 171R, 174L, 175E, 177K, 255Q, 256H, 257A, 259L, 260A, 261Q, and 262P.

[0226] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 205P, 206P, 207E, 255Q, 256H, 257A, 259L, 260A, 261Q, and 262P.

[0227] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 97E, 98L, 99G, 100P, 101D, 102N, 255Q, 256H, 257A, 259L, 260A, 261Q, and 262P.

[0228] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 255Q, 256H, 257A, 259L, 260A, 261Q, and 262P.

[0229] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 97E, 98L, 99G, 100P, 101D, 102N, 167L, 168E, 171R, 174L, 175E, 177K, 255Q, 256H, 257A, 259L, 260A, 261Q, and 262P.

[0230] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 167L, 168E, 171R, 174L, 175E, 177K, 205P, 206P, 207E, 255Q, 256H, 257A, 259L, 260A, 261Q, and 262P.

[0231] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 97E, 98L, 99G, 100P, 101D, 102N, 255Q, 256H, 257 a 、259L、260 a 、261Q and 262P.

[0232] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 97E, 98L, 99G, 100P, 101D, 102N, 98L, 99G, 100P, 101D, 102N, and 103T.

[0233] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 97E, 98L, 99G, 100P, 101D, 102N, 205P, 206P, and 207E.

[0234] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 97E, 98L, 99G, 100P, 101D, 102N, 167L, 168E, 171R, 174L, 175E and 177K.

[0235] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 167L, 168E, 171R, 174L, 175E, 177K, 205P, 206P and 207E.

[0236] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 167L, 168E, 171R, 174L, 175E, 177K, 98L, 99G, 100P, 101D, 102N and 103T.

[0237] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 167L, 168E, 171R, 174L, 175E, 177K, 255Q, 256H, 257A, 259L, 260A, 261Q and 262P.

[0238] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 205P, 206P, 207E, 255Q, 256H, 257A, 259L, 260A, 261Q and 262P.

[0239] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 97E, 98L, 99G, 100P, 101D, 102N, 98L, 99G, 100P, 101D, 102N, 103T, 167L, 168E, 171R, 174L, 175E, 177K, 205P, 206P, 207E, 255Q, 256H, 257A, 259L, 260A, 261Q and 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0240] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 97E, 98L, 99G, 100P, 101D, 102N, 98L, 99G, 100P, 101D, 102N, 103T, 167L, 168E, 171R, 174L, 175E, 177K, 205P, 206P and 207E, and the amino acid residues are numbered according to SEQ ID NO:1.

[0241] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 97E, 98L, 99G, 100P, 101D, 102N, 98L, 99G, 100P, 101D, 102N, 103T, 167L, 168E, 171R, 174L, 175E, 177K, 205P, 206P, and 207E, and the amino acid residues are numbered according to SEQ ID NO:1.

[0242] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 230E, 205P, 206P, 207E, 208L, and the amino acid residues are numbered according to SEQ ID NO:1.

[0243] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, and 5L.

[0244] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 164R, 167L, 168E, 171R, 174L, 175E, and 177K.

[0245] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 204Y, 205P, 206P, and 230E.

[0246] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 205P, 206P, 207E, and 208L.

[0247] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 164R, 167L, 168E, 171R, 174L, 175E, and 177K.

[0248] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 204Y, 205P, 206P, and 230E.

[0249] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 205P, 206P, 207E, and 208L.

[0250] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 204Y, 205P, 206P, 230E, 205P, 206P, 207E, and 208L.

[0251] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 204Y, 205P, 206P, 230E, 205P, 206P, 207E, and 208L.

[0252] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 230E, 205P, 206P, 207E, 208L, and the amino acid residues are numbered according to SEQ ID NO:1.

[0253] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, and 230E, and the amino acid residues are numbered according to SEQ ID NO:1.

[0254] According to other specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, and 230E, and the amino acid residues are numbered according to SEQ ID NO:1.

[0255] According to certain embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 5L, 98L, 99G, 100P, 101D, 102N, 167L, 171R, 174L, 175E, 177K, 207E, 255Q, 256H, 257A, 259L, 260A, and 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0256] According to certain embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 98L, 99G, 100P, 101D, 102N, 167L, 171R, 174L, 175E, 177K, 207E, 255Q, 256H, 257A, 259L and 260A, and the amino acid residues are numbered according to SEQ ID NO:1.

[0257] According to certain embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 98L, 99G, 100P, 101D, 102N, 167L, 171R, 174L, 175E, 177K, 207E, 255Q, 256H, 257A, 259L, 260A and 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0258] According to certain embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 98L, 99G, 100P, 101D, 102N, 167L, 171R, 174L, 175E, 177K, 207E, 255Q, 256H, 257A and 259L, and the amino acid residues are numbered according to SEQ ID NO:1.

[0259] According to certain embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 98L, 99G, 100P, 101D, 102N, 167L, 171R, 174L, 175E, 177K, 207E, 255Q, 256H, 257A and 259L, and the amino acid residues are numbered according to SEQ ID NO:1.

[0260] According to further specific embodiments, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 167L, 168E, 171R, 174L, 175E, 177K, 205P, 206P, 207E, 255Q, 256H, 257A, 259L, 260A, 261Q and 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0261] According to a further specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 167L, 168E, 171R, 174L, 175E, 177K, 205P, 206P, 207E, 255Q, 256H, 257A, 259L, 260A and 261Q, and the amino acid residues are numbered according to SEQ ID NO: 1.

[0262] According to yet another specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q and 262P, and the amino acid residues are numbered according to SEQ ID NO: 1.

[0263] According to yet another specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A and 261Q, and the amino acid residues are numbered according to SEQ ID NO: 1.

[0264] According to yet another specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A and 261Q, and the amino acid residues are numbered according to SEQ ID NO: 1.

[0265] According to yet another specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q and 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0266] According to yet another specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L and 260A, and the amino acid residues are numbered according to SEQ ID NO:1.

[0267] According to yet another specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L and 260A, and the amino acid residues are numbered according to SEQ ID NO:1.

[0268] According to yet another specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q and 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0269] According to yet another specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A and 259L, and the amino acid residues are numbered according to SEQ ID NO:1.

[0270] According to yet another specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A and 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0271] According to yet another specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A and / or 262P, and the amino acid residues are numbered according to SEQ ID NO:1.

[0272] According to yet another specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A and / or 261Q, and the amino acid residues are numbered according to SEQ ID NO:1.

[0273] According to yet another specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, and / or 257A, and the amino acid residues are numbered according to SEQ ID NO:1.

[0274] According to yet another specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, and / or 256H, and the amino acid residues are numbered according to SEQ ID NO:1.

[0275] According to yet another specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, and / or 255Q, and the amino acid residues are numbered according to SEQ ID NO:1.

[0276] According to yet another specific embodiment, the polypeptide of the present invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, and / or 209Q, and the amino acid residues are numbered according to SEQ ID NO:1.

[0277] The polypeptide according to the present invention specifically binds to the epitope on FcRn through at least one immunoglobulin single variable domain (ISVD) that interacts with the FcRn epitope disclosed herein.

[0278] 5.1 Immunoglobulin single variable domain

[0279] The term "immunoglobulin single variable domain" (ISVD), which can be used interchangeably with "single variable domain", defines an immunoglobulin molecule in which the antigen-binding site resides in and is formed by a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (such as monoclonal antibodies) or their fragments (such as Fab, Fab', F(ab') 2 , scFv, diabody), in which two immunoglobulin domains, particularly two variable domains, interact to form the antigen-binding site. Generally, in conventional immunoglobulins, the heavy chain variable domain (V H ) and the light chain variable domain (V L ) interact to form the antigen-binding site. In such cases, the complementarity-determining regions (CDRs) of both V H and V L will contribute to the antigen-binding site, i.e., a total of 6 CDRs will be involved in the formation of the antigen-binding site.

[0280] Given the above definitions, the antigen-binding domains of conventional 4-chain antibodies (such as IgG, IgM, IgA, IgD, or IgE molecules; known in the art) or Fab fragments, F(ab') 2 fragments, Fv fragments (such as disulfide-linked Fv or scFv fragments) or diabodies (all known in the art) derived from such conventional 4-chain antibodies will generally not be considered immunoglobulin single variable domains because in these cases, it is not one (single) immunoglobulin domain that binds to the corresponding epitope, but a pair (associated) of immunoglobulin domains (such as the light and heavy chain variable domains), i.e., the V H -V L pair that binds to the corresponding epitope, which together bind to the corresponding epitope.

[0281] In contrast, an immunoglobulin single variable domain is capable of specifically binding to an epitope without pairing with another immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single V H , a single V HH or a single V L domain.

[0282] Thus, the single variable domain can be a light chain variable domain sequence (e.g., a V L sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a V H sequence or a V HHa sequence) or a suitable fragment thereof; provided that it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain such that a single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0283] An immunoglobulin single variable domain (ISVD) can be, for example, a heavy chain ISVD such as V H 、V HH , including camelized V H or humanized V HH . Preferably, it is V HH , including camelized V H or humanized V HH . The heavy chain ISVD can be derived from a conventional four-chain antibody or a heavy chain antibody.

[0284] For example, the immunoglobulin single variable domain can be a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb) or a molecule (as defined herein and including, but not limited to, V HH ); other single variable domains, or any suitable fragment of any of them.

[0285] In particular, the immunoglobulin single variable domain can be an immunoglobulin single variable domain (such as V HH , including humanized V HH or camelized V H ) or a suitable fragment thereof. [Note: is a registered trademark of Ablynx N.V]

[0286] The "V HH domain" is also referred to as V HH , V HH antibody fragment and V HH antibody, the antigen-binding immunoglobulin variable domain originally described as a "heavy chain antibody" (i.e., "antibody without light chain"; Hamers-Casterman et al. Nature 363:446-448, 1993). The term "V HH domain" has been chosen to distinguish these variable domains from the heavy chain variable domains present in conventional four-chain antibodies (which are referred to herein as "V H domains") and from the light chain variable domains present in conventional four-chain antibodies (which are referred to herein as "V L domains"). For V HHFor further description, reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0287] Typically, the generation of immunoglobulins involves immunization of experimental animals, fusion of cells producing immunoglobulins to generate hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be generated by screening natural or synthetic libraries (e.g., by phage display).

[0288] Immunoglobulin sequences such as V HH ) have been widely described in various publications, among which WO 94 / 04678, Hamers-Casterman et al. 1993 and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74:277-302, 2001) can be cited as examples. In these methods, camelids are immunized with a target antigen to induce an immune response against the target antigen. The V HH library obtained from the immunization is further screened against the V HH that binds the target antigen.

[0289] In these cases, the generation of antibodies requires purified antigen for immunization and / or screening. The antigen can be purified from natural sources or during recombinant production.

[0290] Immunization and / or screening of immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0291] This technique can use immunoglobulin sequences from different sources, including mouse, rat, rabbit, donkey, human, and camelid immunoglobulin sequences. The technique also includes fully human, humanized, or chimeric sequences. For example, the present invention includes camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, such as the camelized dAb described by Ward et al. (see, for example, WO 94 / 04678 and Riechmann, Febs Lett., 339:285-290, 1994 and Prot. Eng., 9:531-537, 1996). In addition, the present invention also uses fused immunoglobulin sequences, such as to form multivalent and / or multispecific constructs (for those containing one or more V HHMultivalent and multispecific polypeptides of domains and their preparation, also referring to Conrath et al., J. Biol. Chem., Vol. 276, 10.7346 - 7350, 2001, and for example WO96 / 34103 and WO 99 / 23221), and immunoglobulin sequences comprising a label or other functional moiety (such as a toxin, label, radiochemical, etc.), which may be derived from the immunoglobulin sequences of the technology of the present invention.

[0292] "Humanized V HH " comprises an amino acid sequence corresponding to that of the naturally occurring V HH domain but which has been "humanized", i.e., by substituting one or more amino acid residues in the amino acid sequence of the said naturally occurring V HH sequence (and in particular the framework sequence) with one or more amino acid residues present at one or more corresponding positions in the V H domain of a conventional 4-chain antibody from humans (such as those shown above). This can be carried out in a manner known per se, which will be clear to the person skilled in the art, for example based on the further description herein and the prior art (such as WO 2008 / 020079). Similarly, it should be noted that such humanized V HH can be obtained in any suitable manner known per se and is thus not strictly limited to polypeptides obtained from polypeptides that have used a naturally occurring V HH domain as a starting material.

[0293] "Camelized V H " comprises an amino acid sequence corresponding to that of the naturally occurring V H domain but which has been "camelized", i.e., by substituting one or more amino acid residues in the amino acid sequence of the naturally occurring V H domain of a conventional 4-chain antibody with one or more amino acid residues present at one or more corresponding positions in the V HH domain of a heavy chain antibody. This can be carried out in a manner known per se, which will be clear to the person skilled in the art, for example based on the further description herein and the prior art (such as WO 2008 / 020079). As defined herein, such "camelized" substitutions are preferably made at the V H -V L junction formation and / or at amino acid positions where they are present and / or inserted at so-called camelid signature residues as defined herein (see for example WO 94 / 04678 and Davies and Riechmann (1994 and 1996), ibid.). Preferably, the V H used as a starting material or starting point for generating or designing camelized VH The sequence is preferably a V from a mammal H sequence, more preferably a human V H sequence, such as a V H 3 sequence. However, it should be noted that such camelized V can be obtained in any suitable manner known per se H , and is thus not strictly limited to polypeptides obtained using polypeptides containing naturally occurring V H domains as starting materials.

[0294] The preferred structure of an immunoglobulin single variable domain sequence can be considered to be composed of four framework regions ("FR"), which are respectively referred to as "framework region 1" ("FR1"), "framework region 2" ("FR2"), "framework region 3" ("FR3") and "framework region 4" ("FR4") in the art and herein. The framework regions are interrupted by three complementarity determining regions ("CDR"), which are respectively referred to as "complementarity determining region 1" ("CDR1"), "complementarity determining region 2" ("CDR2") and "complementarity determining region 3" ("CDR3") in the art and herein.

[0295] As further described in paragraph q) on pages 58 and 59 of WO 08 / 020079 (incorporated herein by reference), the amino acid residues of an immunoglobulin single variable domain can be numbered according to the general numbering of the V H domain given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, publication number 91), as applied to the V Figure 2 domains from camelids in the article of Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2):185-195; see, for example, HH ) of this publication. It should be noted that, as is well known in the art for V H domains and for V HH domains, the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. V H domains and V HHThe total number of amino acid residues in the domain is usually in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0296] In the present application, unless otherwise specified, CDR sequences are determined according to the AbM numbering described in Kontermann and Dübel (Eds. 2010, Antibody Engineering, Volume 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33 - 51). According to this method, FR1 contains the amino acid residues at positions 1 - 25, CDR1 contains the amino acid residues at positions 26 - 35, FR2 contains the amino acids at positions 36 - 49, CDR2 contains the amino acid residues at positions 50 - 58, FR3 contains the amino acid residues at positions 59 - 94, CDR3 contains the amino acid residues at positions 95 - 102, and FR4 contains the amino acid residues at positions 103 - 113.

[0297] The determination of the CDR region can also be carried out according to different methods. In the CDR determination according to Kabat, FR1 of the immunoglobulin single variable domain contains the amino acid residues at positions 1 - 30, CDR1 of the immunoglobulin single variable domain contains the amino acid residues at positions 31 - 35, FR2 of the immunoglobulin single variable domain contains the amino acids at positions 36 - 49, CDR2 of the immunoglobulin single variable domain contains the amino acid residues at positions 50 - 65, FR3 of the immunoglobulin single variable domain contains the amino acid residues at positions 66 - 94, CDR3 of the immunoglobulin single variable domain contains the amino acid residues at positions 95 - 102, and FR4 of the immunoglobulin single variable domain contains the amino acid residues at positions 103 - 113.

[0298] In such immunoglobulin sequences, the framework sequence can be any suitable framework sequence, and examples of suitable framework sequences will be clear to the person skilled in the art based on standard manuals as well as additional disclosures and the prior art mentioned herein.

[0299] The framework sequence is preferably an immunoglobulin framework sequence or a framework sequence (suitable combination) derived from an immunoglobulin framework sequence (e.g., by humanization or camelization). For example, the framework sequence can be derived from the light chain variable domain (e.g., V L sequence) and / or the heavy chain variable domain (e.g., V H sequence or V HH sequence). In a particularly preferred aspect, the framework sequence is derived from V HHThe framework sequence of the sequence (wherein the framework sequence may optionally have been partially or fully humanized) or a conventional V sequence that has been camelized (as defined herein). H sequence (as defined herein).

[0300] In particular, the framework sequence present in the ISVD sequences used in the present invention may contain one or more signature residues (as defined herein), such that the ISVD sequences are molecules, such as V HH , including humanized V HH or camelized V H . Some preferred but non-limiting examples of such framework sequences (suitable combinations thereof) will become clear from the further disclosure herein.

[0301] In addition, as described generally herein for immunoglobulin sequences, suitable fragments (or combinations of fragments) of any of the foregoing may also be used, such as fragments containing one or more framework sequences flanked appropriately and / or one or more CDR sequences linked via one or more framework sequences (e.g., in the same order as these CDRs and framework sequences may occur in the full-size immunoglobulin sequence from which the fragment is derived).

[0302] However, it should be noted that the present invention is not limited with respect to the source of the ISVD sequence (or the nucleotide sequence used to represent the ISVD sequence), and with respect to the manner of generating or obtaining (or having generated or obtained) the ISVD sequence or nucleotide sequence. Thus, the ISVD sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In a particular but non-limiting aspect, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to "humanized" (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and particularly partially or fully humanized V HH sequences), "camelized" (as defined herein) immunoglobulin sequences, and immunoglobulin sequences that have been obtained by techniques such as affinity maturation (starting, for example, from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences known to those skilled in the art; or any suitable combination of any of the foregoing.

[0303] Similarly, a nucleotide sequence can be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and can be, for example, a sequence isolated by PCR from a suitable naturally occurring template (e.g., DNA or RNA isolated from cells), a nucleotide sequence that has been isolated from a library (and especially an expression library), a nucleotide sequence that has been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence that has been prepared by PCR using overlapping primers, or a nucleotide sequence that has been prepared using DNA synthesis techniques known per se.

[0304] As described above, the ISVD can be V HH or a suitable fragment thereof. For a general description of the ISVD, reference is made to the further description below, as well as the prior art cited herein. However, in this regard, it should be noted that this description and the prior art mainly describe the so-called "V H class 3" ISVDs (i.e., ISVDs that have a high sequence homology with human germline sequences of V H class 3 such as DP-47, DP-51 or DP-29). However, it should be noted that the present invention can generally use any type of ISVD in its broadest sense and, for example, also use ISVDs belonging to the so-called "V H class 4" (i.e., ISVDs that have a high sequence homology with human germline sequences of V H class 4 such as DP-78), as described, for example, in WO 2007 / 118670.

[0305] Generally, the ISVD (especially the V HH sequence, including (partial) humanized V HH sequences and camelized V H sequences) can be characterized by the presence of one or more "signature residues" (as described herein) in one or more framework sequences (also as further described herein). Thus, generally, the ISVD can be defined as an immunoglobulin sequence having the following (general) structure:

[0306] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0307] where FR1 to FR4 respectively refer to framework regions 1 to 4, and where CDR1 to CDR3 respectively refer to complementarity-determining regions 1 to 3, and where one or more of the signature residues are as further defined herein.

[0308] In particular, the ISVD can be an immunoglobulin sequence having the (general) structure

[0309] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0310] Wherein FR1 to FR4 respectively refer to framework regions 1 to 4, and wherein CDR1 to CDR3 respectively refer to complementary determining regions 1 to 3, and wherein the framework sequences are as further defined herein.

[0311] More particularly, the ISVD can be an immunoglobulin sequence having the (general) structure

[0312] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0313] Wherein FR1 to FR4 respectively refer to framework regions 1 to 4, and wherein CDR1 to CDR3 respectively refer to complementary determining regions 1 to 3, and wherein:

[0314] According to Kabat numbering, one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 are selected from the signature residues mentioned in Table A-0 below.

[0315] Table A-0: Signature residues in ISVD

[0316]

[0317]

[0318] 5.2 Specificity

[0319] The terms "specificity", "specifically bind" or "specific binding" refer to the number of different target molecules (such as antigens) from the same organism that a specific binding unit (such as an ISVD) can bind with a sufficiently high affinity (see below). "Specificity", "specifically bind" or "specific binding" are used interchangeably herein with "selectivity", "selectively bind" or "selective binding". A binding unit such as an ISVD preferably binds specifically to its designated target. The specificity / selectivity of a binding unit can be determined based on affinity. Affinity represents the strength or stability of a molecular interaction. Affinity is usually given by K D or the dissociation constant, expressed in units of mol / L (or M). Affinity can also be expressed as the association constant K A which is equal to 1 / K D and is expressed in units of (mol / L) -1 (or M -1 ). Affinity is a measure of the binding strength between a moiety and the binding site on the target molecule: K DThe lower the value, the stronger the binding strength between the target molecule and the targeting moiety. Generally, the binding unit (such as ISVD) used in the technology of the present invention will have a dissociation constant (K -5 from 10 -12 to 10 -7 mol / L or lower, and preferably from 10 -12 to 10 -8 mol / L or lower, and more preferably from 10 -1 to 10 D mol / L of dissociation constant (K 5 )(i.e., an association constant (K 12 from 10 7 to 10 12 L / mol or higher, and preferably from 10 8 to 10 12 L / mol or higher, and more preferably from 10 A )) binds to its target. It is generally considered that any K -4 value greater than 10 D mol / L (or any K 4 value less than 10 A L / mol) indicates non-specific binding. The K D of a biological interaction considered to be specific (such as the binding of an immunoglobulin sequence to an antigen) is typically in the range of 10 -5 mol / L (10000 nM or 10 μM) to 10 -12 mol / L (0.001 nM or 1 pM) or lower. Thus, specific / selective binding can mean that, using the same measurement method such as SPR, the binding unit (or the polypeptide containing it) binds to FcRn with a K -5 from 10 -12 to 10 D value, and binds to other molecules with a K -4 value greater than 10 DValues are combined with relevant targets. Thus, compared to an ISVD composed of the amino acids of SEQ ID NO: 14 or 15, the ISVD preferably exhibits at least half, more preferably at least the same, binding affinity for human FcRn, where the binding affinity is measured using the same method such as SPR. Specific binding to a particular target from a particular species does not exclude that the binding unit can also specifically bind to a similar target from a different species. For example, specific binding to human FcRn does not exclude that the binding unit (or the polypeptide comprising the binding unit) can also specifically bind to FcRn from cynomolgus monkeys. Specific binding of the binding unit to its designated target can be determined by any suitable means known per se (including, for example, Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays) and different variants thereof known per se in the art; as well as other techniques mentioned herein. The dissociation constant can be an actual or apparent dissociation constant, as will be clear to the person skilled in the art. The methods for determining the dissociation constant are clear to the person skilled in the art and include, for example, the techniques mentioned below. In this regard, it will also be clear that it may not be possible to measure dissociation constants greater than 10 -4 mol / L or 10 -3 mol / L (e.g., 10 -2 mol / L). Optionally, as will also be clear to the person skilled in the art, the (actual or apparent) dissociation constant can be calculated from the (actual or apparent) association constant (K A ) through the relationship [K D = 1 / K A . The affinity of the molecular interaction between two molecules can be measured via different techniques known per se (such as the well-known surface plasmon resonance (SPR) biosensor technology (see, for example, Ober et al., 2001, Intern. Immunology 13:30 1551 - 1559)). As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of real-time biospecific interactions by detecting changes in the protein concentration in a biosensor matrix, where one molecule is immobilized on a biosensor chip and the other molecule passes over the immobilized molecule under flow conditions, thereby generating k on , k off measurements and thus generating a K D (or K A ) value. For example, this can be done using the well-known Performed by the BIAcore International AB, GE Healthcare, Uppsala, Sweden and Piscataway, NJ system. For further description, see Jonsson et al. (1993, Ann. Biol. Clin. 51:19-26), Jonsson et al. (1991, Biotechniques 11:620-627), Johnsson et al. (1995, J. Mol. Recognit. 8:125-131) and Johnnson et al. (1991, Anal. Biochem. 198:268-277). Another well-known biosensor technology for determining the affinity of biomolecular interactions is Biolayer Interferometry (BLI) (see, e.g., Abdiche et al., 2008, Anal. Biochem. 377:209-217). As used herein, the term "Biolayer interferometry" or "BLI" refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor tip (signal beam). Changes in the number of molecules bound to the biosensor tip result in a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Since interactions can be measured in real time, association and dissociation rates as well as affinity can be determined. For example, BLI can use the well-known system (ForteBio, a division of Pall Life Sciences, Menlo Park, USA). Alternatively, the platform (Sapidyne Instruments Inc, Boise, USA) can be used to measure affinity in a Kinetic Exclusion Assay (KinExA) (see, e.g., Drake et al., 2004, Anal. Biochem., 328:35-43). As used herein, the term "KinExA" refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrium solution of an antibody / antigen complex is passed through a column with beads pre-coated with the antigen (or antibody), allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the thus-captured antibody (or antigen) is accomplished with a fluorescently labeled protein that binds the antibody (or antigen). Immunoassay systems provide a platform for automated bioanalysis and rapid sample turnover (Fraley et al., 2013, Bioanalysis 5:1765-74).

[0320] In certain embodiments, the polypeptide according to the invention specifically binds to the above epitope on FcRn, with an affinity (K A ) between 10 6 M -1 and 10 11 M -1 .

[0321] In certain embodiments, the polypeptide according to the invention specifically binds to the above epitope on FcRn, with a dissociation constant (K D ) between 10 -6 nM and 10 -11 M or lower. Preferably, the K D is determined by Kinexa, BLI or SPR, for example by SPR.

[0322] In certain embodiments, the polypeptide according to the invention specifically binds to the above epitope on FcRn, with an association rate constant (k on ) selected from at least about 10 2 M -1 s -1 , at least about 10 3 M -1 s -1 , at least about 10 4 M -1 s -1 , at least about 10 5 M -1 s -1 , at least about 10 6 M -1 s -1 , at least about 10 7 M -1 s -1 , and at least about 10 8 M -1 s -1 , preferably as measured by surface plasmon resonance or BLI.

[0323] In certain embodiments, the polypeptide according to the invention specifically binds to the above epitope on FcRn, with a dissociation rate constant (k off ) selected from at most about 10 -1 s -1 , at most about 10 -2 s -1 , at most about 10 -3 s -1 , at most about 10 -4 s -1 , at most about 10 -5 s -1 , and at most about 10 -6 s-1 , preferably as measured by surface plasmon resonance or BLI.

[0324] In certain embodiments, the FcRn-binding polypeptides of the invention are cross-reactive between human FcRn and FcRn from at least one, preferably at least two, more preferably at least three and up to substantially all of the following mammalian species: mouse, dog, rat, rabbit, guinea pig, pig, sheep, cow, and cynomolgus monkey.

[0325] When an ISVD is said to exhibit "(improved) cross-reactivity for binding to human FcRn and non-human primate FcRn" compared to another ISVD, it means that for the ISVD, the ratio of the binding activity to human FcRn and to non-human primate FcRn (expressed as K D or k off ) is lower than that same ratio calculated for other ISVDs in the same assay. Good cross-reactivity for binding to human FcRn and non-human primate FcRn allows the assessment of the toxicity of the FcRn-binding polypeptides according to the invention in pre-clinical studies conducted on non-human primates.

[0326] In certain embodiments, the FcRn-binding polypeptides of the invention are (at least) cross-reactive between human FcRn and cynomolgus monkey FcRn, and also preferably between human FcRn and / or cynomolgus monkey FcRn on the one hand and at least one, preferably two, of rat FcRn and pig FcRn on the other hand. For convenience, in the sequence of FcRn, amino acid segments that are considered to be part of the putative epitope of the polypeptides of the invention are highlighted. Without being limited to any particular mechanism or hypothesis, it is assumed that the polypeptides of the invention are (substantially) capable of binding to the corresponding amino acid residue segments (one or more amino acid residues therein) present in the amino acid sequences of those mammalian FcRn proteins, and the polypeptides of the invention are cross-reactive with said amino acid residue segments.

[0327] Generally, the FcRn-binding polypeptides of the invention can be considered to be cross-reactive between human FcRn and FcRn from one of the other species described above when they bind to human FcRn with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM; and bind to FcRn from those above-mentioned species with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM (both using SPR assays).

[0328] In certain embodiments, the present invention provides an FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein at least one ISVD is composed of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), and is characterized in that:

[0329] CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:2 [= X 1 X 2 X 3 M Y] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:5 [= X a X b X c F X e X 1 X 2 X 3 M Y], wherein

[0330] X a is A, D, E, F, G, H, K, L, M, N, Q, S, T, W or Y,

[0331] X b is F, I, L, M, V or Y,

[0332] X c is A, E, G, I, L, M, N, P, S, T, V, W or Y,

[0333] X e is D, E, G, K, M, N, P, Q, S or T,

[0334] X 1 is A, D, E, G, K, N, Q, S, T or V,

[0335] X 2 is A, F, L, M, N, Q, S, T, V or Y, and

[0336] X 3 is A, D, E, G, H, M, N, Q, S, T or V,

[0337] And

[0338] CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:3 [= A I X 3 X 4 G G G X 8 X 9 X 10Y A D S V K G] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:6 [= A I X 3 X 4 G G G X 8 X 9 X 10 , where

[0339] X 3 is A, D, E, G, H, P, Q, R, S, T or V,

[0340] X 4 is A, D, E, G, K, M, N, P, Q, R, S, T, V, W or Y,

[0341] X 8 is A, D, G, H, K, L, M, Q, S, T or V,

[0342] X 9 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y, and

[0343] X 10 is A, D, E, H, S, T, V or Y,

[0344] And

[0345] CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:4 [= D X 2 X 3 X 4 T X 6 X 7 T X 9 YX 11 X 12 or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:7 [= D X 2 X 3 X 4 T X 6 X 7 T X 9 YX 11 X 12 , where

[0346] X 2 is A, D, E, F, G, I, L, M, Q, S, T, V, W or Y, preferably A, E, F, G, I, L, M, Q, S, T, V, W or Y

[0347] X 3 is L or N, preferably L,

[0348] X 4 is F, L, W or Y,

[0349] X 6 is A, D, E, K, L, M, Q, S or W,

[0350] X 7 is L, M or V,

[0351] X 9 is A, D, E, F, G, H, K, L, M, Q, R, S, T, V, W or Y,

[0352] X 11 is A, E, L, M, Q, S, T, V or W, and

[0353] X 12 is T or Y,

[0354] provided that the amino acid

[0355] X of SEQ ID NO:2 1 and X 3 and X of SEQ ID NO:4 6 are not simultaneously E, D and D, respectively, or

[0356] X of SEQ ID NO:5 a , X 1 and X 3 and X of SEQ ID NO:7 6 are not simultaneously T, E, D and D, respectively, or

[0357] X of SEQ ID NO:2 1 and X 3 and X of SEQ ID NO:4 9 are not simultaneously E, D and K, respectively, or

[0358] X of SEQ ID NO:5 a , X 1 and X 3 and X of SEQ ID NO:7 9 are not simultaneously T, E, D and K, respectively, or

[0359] X of SEQ ID NO:2 3 and X of SEQ ID NO:4 9 are not simultaneously D and K, respectively, or

[0360] X of SEQ ID NO:5 a and X 3and X of SEQ ID NO:7 9 are not simultaneously T, D, and K, respectively, or

[0361] X of SEQ ID NO:2 1 and X 3 and X of SEQ ID NO:3 4 and X of SEQ ID NO:4 6 or X 9 are not simultaneously E, D, Q, D, and K, respectively, or

[0362] X of SEQ ID NO:5 a and X 1 and X 3 and X of SEQ ID NO:6 4 and X of SEQ ID NO:7 6 and X 9 are not simultaneously T, E, D, Q, D, and K, respectively, or

[0363] X of SEQ ID NO:2 1 and X of SEQ ID NO:3 4 and X of SEQ ID NO:4 6 and X 9 are not simultaneously E, Q, D, and K, respectively, or

[0364] X of SEQ ID NO:5 a and X 1 and X of SEQ ID NO:6 4 and X of SEQ ID NO:7 6 and X 9 are not simultaneously T, E, Q, D, and K, respectively, or

[0365] X of SEQ ID NO:2 3 and X of SEQ ID NO:4 6 are not simultaneously D and D, respectively, or

[0366] X of SEQ ID NO:5 a and X 3 and X of SEQ ID NO:7 6 are not simultaneously T, D, and D, respectively, or

[0367] X of SEQ ID NO:2 1 and X 3 and X of SEQ ID NO:3 4 and X of SEQ ID NO:4 6 are not simultaneously E, D, Q, and D, respectively, or

[0368] X of SEQ ID NO:5 a 、X 1 and X 3 、X of SEQ ID NO:6 4 and X of SEQ ID NO:7 6 are not simultaneously T, E, D, Q, and D, respectively, or

[0369] X of SEQ ID NO:2 1 and X of SEQ ID NO:4 6 and X 9 are not simultaneously E, D, and K, respectively, or

[0370] X of SEQ ID NO:2 3 、X of SEQ ID NO:3 4 and X of SEQ ID NO:4 6 and X 9 are not simultaneously D, Q, D, and K, respectively, or

[0371] X of SEQ ID NO:2 1 and X 3 、X of SEQ ID NO:3 4 and X of SEQ ID NO:4 9 are not simultaneously E, D, Q, and K, respectively, or

[0372] X of SEQ ID NO:5 a 、X 1 and X 3 、X of SEQ ID NO:6 4 and X of SEQ ID NO:7 9 are not simultaneously T, E, D, Q, and K, respectively, or

[0373] X of SEQ ID NO:2 3 and X of SEQ ID NO:4 6 and X 9 are not simultaneously D, D, and K, respectively, or

[0374] X of SEQ ID NO:2 3 and X of SEQ ID NO:4 9 are not simultaneously D and S, respectively, or

[0375] X of SEQ ID NO:5 a and X 3 and X of SEQ ID NO:7 9Not simultaneously T, D, and S, respectively, or

[0376] X of SEQ ID NO:2 1 and X 3 and X of SEQ ID NO:4 6 and X 9 Not simultaneously E, D, D, and S, respectively, or

[0377] X of SEQ ID NO:4 6 and X 9 Not simultaneously D and K, respectively, or

[0378] X of SEQ ID NO:5 a and X of SEQ ID NO:7 6 and X 9 Not simultaneously T, D, and K, respectively, or

[0379] X of SEQ ID NO:2 1 X of SEQ ID NO:3 4 and X of SEQ ID NO:4 6 and X 9 Not simultaneously E, Q, D, and K, respectively, or

[0380] X of SEQ ID NO:2 3 X of SEQ ID NO:3 4 and X of SEQ ID NO:4 6 and X 9 Not simultaneously D, Q, D, and S, respectively, or

[0381] X of SEQ ID NO:5 a and X 3 X of SEQ ID NO:6 4 and X of SEQ ID NO:7 6 and X 9 Not simultaneously T, D, Q, D, and S, respectively, or

[0382] X of SEQ ID NO:2 1 X of SEQ ID NO:3 4 X of SEQ ID NO:4 6 and X 9 Not simultaneously E, Q, D, and S, respectively, or

[0383] X of SEQ ID NO:5 a and X 1 X of SEQ ID NO:6 4, X of SEQ ID NO:7 6 and X 9 are not simultaneously T, E, Q, D, and S, respectively, or

[0384] X of SEQ ID NO:2 1 and X of SEQ ID NO:4 6 and X 9 are not simultaneously E, D, and K, respectively, or

[0385] X of SEQ ID NO:5 a and X 1 and X of SEQ ID NO:7 6 and X 9 are not simultaneously T, E, D, and K, respectively, or

[0386] X of SEQ ID NO:2 3 and X of SEQ ID NO:4 6 and X 9 are not simultaneously D, D, and K, respectively, or

[0387] X of SEQ ID NO:5 a and X 3 and X of SEQ ID NO:7 6 and X 9 are not simultaneously T, D, D, and K, respectively, or

[0388] X of SEQ ID NO:2 1 and X 3 and X of SEQ ID NO:4 6 and X 9 are not simultaneously E, D, D, and K, respectively, or

[0389] X of SEQ ID NO:2 3 and X of SEQ ID NO:4 6 and X 9 are not simultaneously D, D, and S, respectively, or

[0390] X of SEQ ID NO:2 1 and X 3 , X of SEQ ID NO:3 4 and X of SEQ ID NO:4 6 or X 9 are not simultaneously E, D, Q, D, and S, respectively, or

[0391] X of SEQ ID NO:5 a , X 1 and X 3, X of SEQ ID NO:6 4 and X of SEQ ID NO:7 6 and X 9 are not simultaneously T, E, D, Q, D, and S, respectively, or

[0392] X of SEQ ID NO:2 1 and X 3 , X of SEQ ID NO:3 4 and X of SEQ ID NO:4 6 or X 9 are not simultaneously E, D, Q, D, and K, respectively, or

[0393] X of SEQ ID NO:5 a , X 1 and X 3 and X of SEQ ID NO:7 6 and X 9 are not simultaneously T, E, D, D, and S, respectively, or

[0394] X of SEQ ID NO:2 3 , X of SEQ ID NO:4 6 and X 9 are not simultaneously D, D, and S, respectively, or

[0395] X of SEQ ID NO:5 a and X 3 , X of SEQ ID NO:7 6 and X 9 are not simultaneously T, D, D, and S, respectively, or

[0396] X of SEQ ID NO:2 1 and X 3 and X of SEQ ID NO:4 9 are not simultaneously E, D, and S, respectively, or

[0397] X of SEQ ID NO:3 4 and X of SEQ ID NO:4 9 are not simultaneously Q and S, respectively, or

[0398] X of SEQ ID NO:5 a , X of SEQ ID NO:6 4 and X of SEQ ID NO:7 9 are not simultaneously T, Q, and S, respectively.

[0399] In certain embodiments, the present invention provides an FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein at least one ISVD is composed of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity-determining regions (CDR1 to CDR3, respectively), and is characterized in that:

[0400] CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:2 [= X 1 X 2 X 3 M Y] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:5 [= X a X b X c F X e X 1 X 2 X 3 M Y], wherein

[0401] X a is G or T,

[0402] X b is F,

[0403] X c is T,

[0404] X e is S,

[0405] X 1 is S, D or E,

[0406] X 2 is Y, and

[0407] X 3 is A or D,

[0408] And

[0409] CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:3 [= A I X 3 X 4 G G G X 8 X 9 X 10 Y A D S V K G] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:6 [= A I X 3 X 4 G G G X 8 X 9 X 10 , wherein

[0410] X 3 is S,

[0411] X 4 is Q or S,

[0412] X 8 is S,

[0413] X 9 is T, and

[0414] X 10 is D,

[0415] and

[0416] CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:4 [= D X 2 X 3 X 4 T X 6 X 7 T X 9 YX 11 X 12 or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:7 [= D X 2 X 3 X 4 T X 6 X 7 T X 9 YX 11 X 12 , where

[0417] X 2 is T,

[0418] X 3 is L,

[0419] X 4 is Y,

[0420] X 6 is D, E or S,

[0421] X 7 is L,

[0422] X 9 is K, R, S or W,

[0423] X 11 is S, and

[0424] X 12 is Y,

[0425] provided that the amino acid

[0426] X of SEQ ID NO:2 1 and X 3 and X of SEQ ID NO:4 6 are not simultaneously E, D, and D, respectively, or

[0427] X of SEQ ID NO:5 a 、X 1 and X 3 and X of SEQ ID NO:7 6 are not simultaneously T, E, D, and D, respectively, or

[0428] X of SEQ ID NO:2 1 and X 3 and X of SEQ ID NO:4 9 are not simultaneously E, D, and K, respectively, or

[0429] X of SEQ ID NO:5 a 、X 1 and X 3 and X of SEQ ID NO:7 9 are not simultaneously T, E, D, and K, respectively, or

[0430] X of SEQ ID NO:2 3 and X of SEQ ID NO:4 9 are not simultaneously D and K, respectively, or

[0431] X of SEQ ID NO:5 a and X 3 and X of SEQ ID NO:7 9 are not simultaneously T, D, and K, respectively, or

[0432] X of SEQ ID NO:2 1 and X 3 、X of SEQ ID NO:3 4 and X of SEQ ID NO:4 6 or X 9 are not simultaneously E, D, Q, D, and K, respectively, or

[0433] X of SEQ ID NO:5 a 、X 1 and X 3 、X of SEQ ID NO:6 4 and X of SEQ ID NO:7 6 and X 9 are not simultaneously T, E, D, Q, D, and K, respectively, or

[0434] X of SEQ ID NO:2 1 、X of SEQ ID NO:3 4 and X of SEQ ID NO:4 6 and X 9 are not E, Q, D and K at the same time, or

[0435] X of SEQ ID NO:5 a and X 1 、X of SEQ ID NO:6 4 and X of SEQ ID NO:7 6 and X 9 are not T, E, Q, D and K at the same time, or

[0436] X of SEQ ID NO:2 3 and X of SEQ ID NO:4 6 are not both D and D, or

[0437] X of SEQ ID NO:5 a and X 3 and X of SEQ ID NO:7 6 are not T, D, and D at the same time, or

[0438] X of SEQ ID NO:2 1 and X 3 、X of SEQ ID NO:3 4 and X of SEQ ID NO:4 6 are not E, D, Q and D at the same time, or

[0439] X of SEQ ID NO:5 a , X 1 and X 3 、X of SEQ ID NO:6 4 and X of SEQ ID NO:7 6 are not T, E, D, Q and D at the same time, or

[0440] X of SEQ ID NO:2 1 and X of SEQ ID NO:4 6 and X 9 are not E, D and K at the same time, or

[0441] X of SEQ ID NO:2 3 、X of SEQ ID NO:3 4 and X of SEQ ID NO:4 6 and X 9are not simultaneously D, Q, D, and K, respectively, or

[0442] X of SEQ ID NO:2 1 and X 3 and X of SEQ ID NO:3 4 and X of SEQ ID NO:4 9 are not simultaneously E, D, Q, and K, respectively, or

[0443] X of SEQ ID NO:5 a and X 1 and X 3 and X of SEQ ID NO:6 4 and X of SEQ ID NO:7 9 are not simultaneously T, E, D, Q, and K, respectively, or

[0444] X of SEQ ID NO:2 3 and X of SEQ ID NO:4 6 and X 9 are not simultaneously D, D, and K, respectively, or

[0445] X of SEQ ID NO:2 3 and X of SEQ ID NO:4 9 are not simultaneously D and S, respectively, or

[0446] X of SEQ ID NO:5 a and X 3 and X of SEQ ID NO:7 9 are not simultaneously T, D, and S, respectively, or

[0447] X of SEQ ID NO:2 1 and X 3 and X of SEQ ID NO:4 6 and X 9 are not simultaneously E, D, D, and S, respectively, or

[0448] X of SEQ ID NO:4 6 and X 9 are not simultaneously D and K, respectively, or

[0449] X of SEQ ID NO:5 a and X of SEQ ID NO:7 6 and X 9 are not simultaneously T, D, and K, respectively, or

[0450] X of SEQ ID NO:2 1 and X of SEQ ID NO:34 and X of SEQ ID NO:4 6 and X 9 are not simultaneously E, Q, D, and K, respectively, or

[0451] X of SEQ ID NO:2 3 、X of SEQ ID NO:3 4 and X of SEQ ID NO:4 6 and X 9 are not simultaneously D, Q, D, and S, respectively, or

[0452] X of SEQ ID NO:5 a and X 3 、X of SEQ ID NO:6 4 and X of SEQ ID NO:7 6 and X 9 are not simultaneously T, D, Q, D, and S, respectively, or

[0453] X of SEQ ID NO:2 1 、X of SEQ ID NO:3 4 、X of SEQ ID NO:4 6 and X 9 are not simultaneously E, Q, D, and S, respectively, or

[0454] X of SEQ ID NO:5 a and X 1 、X of SEQ ID NO:6 4 、X of SEQ ID NO:7 6 and X 9 are not simultaneously T, E, Q, D, and S, respectively, or

[0455] X of SEQ ID NO:2 1 and X of SEQ ID NO:4 6 and X 9 are not simultaneously E, D, and K, respectively, or

[0456] X of SEQ ID NO:5 a and X 1 and X of SEQ ID NO:7 6 and X 9 are not simultaneously T, E, D, and K, respectively, or

[0457] X of SEQ ID NO:2 3 and X of SEQ ID NO:4 6 and X 9 are not simultaneously D, D, and K, respectively, or

[0458] X of SEQ ID NO:5 a and X 3 and X of SEQ ID NO:7 6 and X 9 are not simultaneously T, D, D, and K, respectively, or

[0459] X of SEQ ID NO:2 1 and X 3 and X of SEQ ID NO:4 6 and X 9 are not simultaneously E, D, D, and K, respectively, or

[0460] X of SEQ ID NO:2 3 and X of SEQ ID NO:4 6 and X 9 are not simultaneously D, D, and S, respectively, or

[0461] X of SEQ ID NO:2 1 and X 3 and X of SEQ ID NO:3 4 and X of SEQ ID NO:4 6 or X 9 are not simultaneously E, D, Q, D, and S, respectively, or

[0462] X of SEQ ID NO:5 a and X 1 and X 3 and X of SEQ ID NO:6 4 and X of SEQ ID NO:7 6 and X 9 are not simultaneously T, E, D, Q, D, and S, respectively, or

[0463] X of SEQ ID NO:2 1 and X 3 and X of SEQ ID NO:3 4 and X of SEQ ID NO:4 6 or X 9 are not simultaneously E, D, Q, D, and K, respectively, or

[0464] X of SEQ ID NO:5 a and X 1 and X 3 and X of SEQ ID NO:7 6 and X 9 are not simultaneously T, E, D, D, and S, respectively, or

[0465] X of SEQ ID NO:2 3 、X of SEQ ID NO:4 6 and X 9 are not simultaneously D, D, and S, respectively, or

[0466] X of SEQ ID NO:5 a and X 3 、X of SEQ ID NO:7 6 and X 9 are not simultaneously T, D, D, and S, respectively, or

[0467] X of SEQ ID NO:2 1 and X 3 as well as X of SEQ ID NO:4 9 are not simultaneously E, D, and S, respectively, or

[0468] X of SEQ ID NO:3 4 as well as X of SEQ ID NO:4 9 are not simultaneously Q and S, respectively, or

[0469] X of SEQ ID NO:5 a 、X of SEQ ID NO:6 4 as well as X of SEQ ID NO:7 9 are not simultaneously T, Q, and S, respectively.

[0470] In certain embodiments, the present invention provides an FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein at least one ISVD is composed of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity-determining regions (CDR1 to CDR3, respectively), and is characterized in that:

[0471] CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:2 [= X 1 X 2 X 3 M Y] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:5 [= X a X b X c F X e X 1 X 2 X 3 M Y], wherein

[0472] X ais A, D, E, F, G, H, K, L, M, N, Q, S, T, W or Y,

[0473] X b is F, I, L, M, V or Y,

[0474] X c is A, E, G, I, L, M, N, P, S, T, V, W or Y,

[0475] X e is D, E, G, K, M, N, P, Q, S or T,

[0476] X 1 is A, D, E, G, K, N, Q, S, T or V,

[0477] X 2 is A, F, L, M, N, Q, S, T, V or Y, and

[0478] X 3 is A, D, E, G, H, M, N, Q, S, T or V,

[0479] and

[0480] CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:9 [=A I S S G G G S T D Y A D S V K G] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:12 [=A I S S G G G S T D],

[0481] and

[0482] CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:10 [=D T L Y T S L T S Y S Y] or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:13 [=D T L Y T S L T S Y S Y].

[0483] In certain embodiments, the present invention provides an FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein at least one ISVD is composed of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity-determining regions (CDR1 to CDR3 respectively), and is characterized in that:

[0484] CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:8 [= S Y A M Y] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:11 [= G F T F S S Y A M Y]

[0485] And

[0486] CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:3 [= A I X 3 X 4 G G G X 8 X 9 X 10 Y A D S V K G] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:6 [= A I X 3 X 4 G G G X 8 X 9 X 10 , where

[0487] X 3 is A, D, E, G, H, P, Q, R, S, T or V,

[0488] X 4 is A, D, E, G, K, M, N, P, Q, R, S, T, V, W or Y,

[0489] X 8 is A, D, G, H, K, L, M, Q, S, T or V,

[0490] X 9 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y, and

[0491] X 10 is A, D, E, H, S, T, V or Y,

[0492] And

[0493] CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:10 [= D T L Y T S L T S Y SY] or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:13 [= D T L Y T S L T S Y S Y].

[0494] In certain embodiments, the present invention provides an FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein at least one ISVD is composed of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), and is characterized in that:

[0495] CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:8 [=S Y A M Y] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:11 [=G F T F S S Y A M Y]

[0496] And

[0497] CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:9 [=A I S S G G G S T D Y AD S V K G] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:12 [=A I S S G G G S T D],

[0498] And

[0499] CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:4 [=D X 2 X 3 X 4 T X 6 X 7 T X 9 YX 11 X 12 or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:7 [=D X 2 X 3 X 4 T X 6 X 7 T X 9 YX 11 X 12 , wherein

[0500] X 2 is A, D, E, F, G, I, L, M, Q, S, T, V, W or Y, preferably A, E, F, G, I, L, M, Q, S, T, V, W or Y

[0501] X 3 is L or N, preferably L,

[0502] X 4 is F, L, W or Y,

[0503] X 6 is A, D, E, K, L, M, Q, S, or W,

[0504] X 7 is L, M, or V,

[0505] X 9 is A, D, E, F, G, H, K, L, M, Q, R, S, T, V, W, or Y,

[0506] X 11 is A, E, L, M, Q, S, T, V, or W, and

[0507] X 12 is T or Y,

[0508] provided that the X of amino acid SEQ ID NO:4 6 and the X 9 are not simultaneously D and K, respectively, or the X of SEQ ID NO:7 6 and the X 9 are not simultaneously D and K, respectively.

[0509] In certain embodiments, the present invention provides an FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein at least one ISVD is composed of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity-determining regions (CDR1 to CDR3, respectively), and is characterized in that:

[0510] CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:2 [= X 1 X 2 X 3 M Y] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:5 [= X a X b X c F X e X 1 X 2 X 3 M Y], wherein

[0511] X a is G or T,

[0512] X 1 is S, D, or E,

[0513] X 2 is Y, and

[0514] X 3 is A or D,

[0515] and

[0516] CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:9 [=A I S S G G G S T D Y A D S V K G] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:12 [=A I S S G G G S T D],

[0517] and

[0518] CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:10 [=D T L Y T S L T S Y S Y] or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:13 [=D T L Y T S L T S Y S Y].

[0519] In certain embodiments, the present invention provides an FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein at least one ISVD is composed of 4 framework regions (FR1 to FR4 respectively) and 3 complementary determining regions (CDR1 to CDR3 respectively), and is characterized in that:

[0520] CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:8 [=S Y A M Y] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:11 [=G F T F S S Y A M Y]

[0521] and

[0522] CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:3 [=A I X 3 X 4 G G G X 8 X 9 X 10 Y A D S V K G] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:6 [=A I X 3 X 4 G G G X 8 X 9 X 10 , where

[0523] X 3 is S,

[0524] X 4 is Q or S,

[0525] X 8 is S,

[0526] X 9 is T, and

[0527] X 10 is D,

[0528] and

[0529] CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:10 [=D T L Y T S L T S Y SY] or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:13 [=D T L Y T S L T S Y S Y].

[0530] In certain embodiments, the present invention provides an FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein at least one ISVD is composed of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity-determining regions (CDR1 to CDR3 respectively), and is characterized in that:

[0531] CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:8 [=S Y A M Y] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:11 [=G F T F S S Y A M Y]

[0532] and

[0533] CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:9 [=A I S S G G G S T D Y AD S V K G] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:12 [=A I S S G G G S T D],

[0534] and

[0535] CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:4 [=D X 2 X 3 X 4 T X 6 X 7 T X 9 YX 11 X 12 or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:7 [=D X2 X 3 X 4 T X 6 X 7 T X 9 Y X 11 X 12 , wherein

[0536] X 2 is T,

[0537] X 3 is L,

[0538] X 4 is Y,

[0539] X 6 is D, E, or S,

[0540] X 7 is L,

[0541] X 9 is K, R, S, or W,

[0542] X 11 is S, and

[0543] X 12 is Y,

[0544] provided that the X of amino acid SEQ ID NO:4 6 and X 9 are not both D and K, respectively, or the X of SEQ ID NO:7 6 and X 9 are not both D and K, respectively.

[0545] In certain embodiments, the present invention provides an FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein at least one ISVD is composed of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity-determining regions (CDR1 to CDR3, respectively), and is characterized in that:

[0546] CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:8 [=S Y A M Y] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:11 [=G F T F S S Y A M Y], wherein

[0547] the S at position 1 of SEQ ID NO:8 or position 6 of SEQ ID NO:11 has been changed to A, D, E, G, K, N, Q, T, or V, and / or

[0548] Y at position 2 of SEQ ID NO:8 or position 7 of SEQ ID NO:11 has been changed to A, F, L, M, N, Q, S, T or V, and / or

[0549] A at position 3 of SEQ ID NO:8 or position 8 of SEQ ID NO:11 has been changed to D, E, G, H, M, N, Q, S, T or V, and

[0550] CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:9 [=A I S S G G G S T D Y A D S V K G] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:12 [=A I S S G G G S T D], and

[0551] CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:10 [=D T L Y T S L T S Y S Y] or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:13 [=D T L Y T S L T S Y S Y].

[0552] In certain embodiments, the present invention provides an FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein at least one ISVD is composed of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity-determining regions (CDR1 to CDR3 respectively), and is characterized in that:

[0553] CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:8 [=S Y A M Y] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:11 [=G F T F S S Y A M Y]

[0554] and

[0555] CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:9 [=A I S S G G G S T D Y A D S V K G] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:12 [=A I S S G G G S T D], wherein

[0556] S at position 3 of SEQ ID NO:9 or position 3 of SEQ ID NO:12 has been changed to A, D, E, G, H, P, Q, R, T or V, and / or

[0557] The S at position 4 of SEQ ID NO:9 or position 4 of SEQ ID NO:12 has been changed to A, D, E, G, K, M, N, P, Q, R, T, V, W, or Y, and / or

[0558] The S at position 8 of SEQ ID NO:9 or position 8 of SEQ ID NO:12 has been changed to A, D, G, H, K, L, M, Q, T, or V, and / or

[0559] The T at position 9 of SEQ ID NO:9 or position 9 of SEQ ID NO:12 has been changed to A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y, and / or

[0560] The D at position 10 of SEQ ID NO:9 or position 10 of SEQ ID NO:12 has been changed to A, E, H, S, T, V, or Y, and

[0561] CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:10 [= D T L Y T S L T S Y SY] or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:13 [= D T L Y T S L T S Y S Y].

[0562] In certain embodiments, the present invention provides an FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein at least one ISVD is composed of 4 framework regions (FR1 to FR4 respectively) and 3 complementary determining regions (CDR1 to CDR3 respectively), and is characterized in that:

[0563] CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:8 [= S Y A M Y] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:11 [= G F T F S S Y A M Y]

[0564] And

[0565] CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:9 [= A I S S G G G S T D Y AD S V K G] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:12 [= A I S S G G G S T D]

[0566] And

[0567] The CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:10 [=D T L Y T S L T S Y SY] or the CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:13 [=D T L Y T S L T S Y S Y], wherein

[0568] the T at position 2 of SEQ ID NO:10 or position 2 of SEQ ID NO:13 has been changed to A, D, E, F, G, I, L, M, Q, S, V, W or Y, preferably to A, E, F, G, I, L, M, Q, S, V, W or Y, and / or

[0569] the L at position 3 of SEQ ID NO:10 or position 3 of SEQ ID NO:13 has been changed to N, preferably wherein the amino acid at position 3 of SEQ ID NO:13 is L (i.e., it has not changed), and / or

[0570] the Y at position 4 of SEQ ID NO:10 or position 4 of SEQ ID NO:13 has been changed to F, L or W, and / or

[0571] the S at position 6 of SEQ ID NO:10 or position 6 of SEQ ID NO:13 has been changed to A, D, E, K, L, M, Q or W, and / or

[0572] the L at position 7 of SEQ ID NO:10 or position 7 of SEQ ID NO:13 has been changed to M or V, and / or

[0573] the S at position 9 of SEQ ID NO:10 or position 9 of SEQ ID NO:13 has been changed to A, D, E, F, G, H, K, L, M, Q, R, T, V, W or Y, and / or

[0574] the S at position 11 of SEQ ID NO:10 or position 11 of SEQ ID NO:13 has been changed to A, E, L, M, Q, T, V or W, and / or

[0575] the Y at position 12 of SEQ ID NO:10 or position 12 of SEQ ID NO:13 has been changed to T,

[0576] provided that the amino acids at positions 6 and 9 of SEQ ID NO:10 or positions 6 and 9 of SEQ ID NO:13 are not simultaneously changed to D and K respectively.

[0577] In certain embodiments, the present invention provides an FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein at least one ISVD is composed of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity-determining regions (CDR1 to CDR3, respectively), and is characterized in that:

[0578] CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:8 [=S Y A M Y] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:11 [=G F T F S S Y A M Y], wherein

[0579] the S at position 1 of SEQ ID NO:8 or position 6 of SEQ ID NO:11 has been changed to D or E, and / or

[0580] the A at position 3 of SEQ ID NO:8 or position 8 of SEQ ID NO:11 has been changed to D, and

[0581] CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:9 [=A I S S G G G S T D Y AD S V K G] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:12 [=A I S S G G G S T D], wherein

[0582] the S at position 4 of SEQ ID NO:9 or position 4 of SEQ ID NO:12 has been changed to Q, and

[0583] CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:10 [=D T L Y T S L T S Y SY] or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:13 [=D T L Y T S L T S Y S Y], wherein

[0584] the S at position 6 of SEQ ID NO:10 or position 6 of SEQ ID NO:13 has been changed to D or E, and / or

[0585] the S at position 9 of SEQ ID NO:10 or position 9 of SEQ ID NO:13 has been changed to K, R or W,

[0586] provided that the amino acids at positions 6 and 9 of SEQ ID NO:10 or positions 6 and 9 of SEQ ID NO:13 are not simultaneously changed to D and K, respectively.

[0587] In certain embodiments, the present invention provides an FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn disclosed herein, wherein the at least one ISVD is composed of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity-determining regions (CDR1 to CDR3, respectively), and is characterized in that: CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:8 [=S Y A M Y] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:11 [=G F T F S S Y A M Y], wherein

[0588] the S at position 1 of SEQ ID NO:8 or position 6 of SEQ ID NO:11 has been changed to D or E, and / or

[0589] the A at position 3 of SEQ ID NO:8 or position 8 of SEQ ID NO:11 has been changed to D, and

[0590] CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:9 [=A I S S G G G S T D Y A D S V K G] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:12 [=A I S S G G G S T D], and

[0591] CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:10 [=D T L Y T S L T S Y S Y] or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:13 [=D T L Y T S L T S Y S Y].

[0592] In certain embodiments, the present invention provides an FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn disclosed herein, wherein the at least one ISVD is composed of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity-determining regions (CDR1 to CDR3, respectively), and is characterized in that: CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:8 [=S Y A M Y] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:11 [=G F T F S S Y A M Y], and

[0593] CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:9 [=A I S S G G G S T D Y A D S V K G] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:12 [=A I S S G G G S T D], wherein

[0594] the S at position 4 of SEQ ID NO:9 or position 4 of SEQ ID NO:12 has been changed to Q, and

[0595] CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:10 [=D T L Y T S L T S Y S Y] or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:13 [=D T L Y T S L T S Y S Y].

[0596] In certain embodiments, the present invention provides an FcRn-binding polypeptide, the FcRn-binding polypeptide comprising at least one ISVD that specifically binds to an epitope on FcRn disclosed herein, wherein at least one ISVD is composed of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity-determining regions (CDR1 to CDR3 respectively), and is characterized in that: CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:8 [=S Y A M Y] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:11 [=G F T F S S Y A M Y],

[0597] and

[0598] CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:9 [=A I S S G G G S T D Y A D S V K G] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO:12 [=A I S S G G G S T D], and

[0599] CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:10 [=D T L Y T S L T S Y S Y] or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO:13 [=D T L Y T S L T S Y S Y], wherein

[0600] the S at position 6 of SEQ ID NO:10 or position 6 of SEQ ID NO:13 has been changed to D or E, and / or

[0601] The S at position 9 of SEQ ID NO:10 or at position 9 of SEQ ID NO:13 has been changed to K, R or W,

[0602] provided that the amino acids at positions 6 and 9 of SEQ ID NO:10 or at positions 6 and 9 of SEQ ID NO:13 do not simultaneously change to D and K, respectively.

[0603] In certain specific embodiments, the present invention provides a polypeptide, wherein at least one ISVD that specifically binds to FcRn is composed of 4 framework regions (FR1 to FR4, respectively) and 3 complementary determining regions (CDR1 to CDR3, respectively), and is characterized in that:

[0604] a) CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO:8 [=S Y A M Y] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO:8; and

[0605] b) CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO:9 [=A I S S G G G S T D Y A D S V K G] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO:9; and

[0606] c) CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO:10 [=D T L Y T S L T S Y S Y] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO:10.

[0607] In certain specific embodiments, the present invention provides a polypeptide, wherein at least one ISVD that specifically binds to FcRn is composed of 4 framework regions (FR1 to FR4, respectively) and 3 complementary determining regions (CDR1 to CDR3, respectively), and is characterized in that:

[0608] a) CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO:11 [=G F T F S S Y A M Y] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO:11; and

[0609] b) The CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [= A I S S G G G S T D] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO: 12; and

[0610] c) The CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 13 [= D T L Y T S L T S Y SY] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO: 13.

[0611] In certain embodiments, the FcRn-binding polypeptide of the invention is characterized in that at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein has the sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0612] In certain embodiments, the FcRn-binding polypeptide of the invention is characterized in that at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein has at least 80%, such as 90%, or even 95% sequence identity with the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0613] In certain specific embodiments, the invention provides a polypeptide as described herein, which is characterized in that the ISVD is a (single) domain antibody, V HH 、V HH 、humanized V HH or camelized V H .

[0614] In certain embodiments, compared to the binding of a polypeptide having an amino acid sequence or polypeptide of a CDR having no 4, 3, 2 or 1 amino acid difference, a polypeptide comprising an ISVD having one or more CDRs having 1, 2, 3 or 4 amino acid differences as described herein binds to FcRn with approximately the same affinity, which is measured by surface plasmon resonance.

[0615] When comparing two immunoglobulin single variable domains, the term "amino acid difference" refers to an insertion, deletion or substitution of a single amino acid residue at a position in the first sequence compared to the second sequence; it is understood that two immunoglobulin single variable domains can contain one, two or more such amino acid differences.

[0616] For the purpose of comparing two or more immunoglobulin single variable domains or other amino acid sequences (such as the polypeptides of the present invention, etc.), the percentage of "sequence identity" (also referred to herein as "amino acid identity") between a first amino acid sequence and a second amino acid sequence can be calculated or determined as described in paragraph f) on pages 49 and 50 of WO 08 / 020079 (incorporated herein by reference), for example, by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], where each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence compared to the first amino acid sequence is considered a difference at a single amino acid residue (position), i.e., is considered an "amino acid difference" as defined herein; alternatively, the degree of sequence identity between two amino acid sequences can be calculated using a computer algorithm with standard settings for sequence alignment that is known in the art (such as NCBI Blast v2.0). For example, some other techniques, computer algorithms, and settings for determining the degree of sequence identity are described in WO 04 / 037999, EP 0 967 284, EP 1 085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2 357 768-A.

[0617] Generally, for determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the largest number of amino acid residues is taken as the "first" amino acid sequence, and the other amino acid sequence is taken as the "second" amino acid sequence.

[0618] In addition, when determining the degree of sequence identity between two immunoglobulin single variable domains, those skilled in the art can consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue with a similar chemical structure and has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example, in WO 04 / 037999, GB-A-3 357 768, WO 98 / 49185, WO 00 / 46383, and WO 01 / 09300; and the (preferred) types and / or combinations of such substitutions can be selected based on the relevant teachings of WO 04 / 037999 and WO 98 / 49185 and additional references cited therein. Examples of conservative substitutions are further described below.

[0619] Any amino acid substitution applied to the polypeptides described herein can also be based on the analysis of the frequency of amino acid variation between homologous proteins of different species developed by Schulz et al., 1978 (Principles of Protein Structure, Springer-Verlag), the analysis of structure-forming potential developed by Chou and Fasman in 1975 (Biochemistry 13:211) and 1978 (Adv. Enzymol. 47:45-149), and the analysis of protein hydrophobicity patterns developed by Eisenberg et al., 1984 (Proc. Natl. Acad. Sci. USA 81:140-144), Kyte and Doolittle 1981 (J Molec. Biol. 157:105-132), and Goldman et al., 1986 (Ann. Rev. Biophys. Chem. 15:321-353), all of which are incorporated herein by reference in their entirety. Information on the primary, secondary, and tertiary structures of ISVD is given in this specification and in the general background art cited above. In addition, for this purpose, the crystal structure of the V HH domain from camelids is given, for example, by Desmyter et al. 1996 (Nature Structural Biology, 3:803), Spinelli et al. 1996 (Natural Structural Biology 3:752-757), and Decanniere et al., 1999 (Structure, 7:361). Further information on some of the amino acid residues that form the V H / V H interface in a conventional V L domain and potential camelization substitutions at these positions can be found in the prior art cited above. An immunoglobulin single variable domain and a nucleic acid sequence are said to be "identical" if they have 100% sequence identity over their entire length (as defined herein).

[0620] Compared to the sequences of SEQ ID NO:14 and / or 15, the FcRn-binding polypeptides of the present invention preferably further contain (at least): one or more humanized substitutions;

[0621] and / or

[0622] one or more mutations (i.e., amino acid substitutions, deletions, or additions, particularly substitutions) that reduce the binding of pre-existing antibodies; and may optionally contain one or more additional mutations described herein (e.g., to improve the chemical stability of the FcRn-binding polypeptide).

[0623] For suitable humanizing substitutions (and suitable combinations thereof), for example, refer to WO 09 / 138519 (or the prior art cited in WO 09 / 138519) and WO 08 / 020079 (or the prior art cited in WO 08 / 020079), and also to Tables A-3 to A-8 in WO 08 / 020079 (which are lists showing possible humanizing substitutions). Some preferred but non-limiting examples of such humanizing substitutions are Q108L and A14P or suitable combinations thereof. Such humanizing substitutions can also be appropriately combined with one or more other mutations as described herein (such as one or more mutations that reduce the binding of pre-existing antibodies).

[0624] Regarding suitable mutations (and suitable combinations of such mutations) that can reduce the binding of pre-existing antibodies, for example, refer to WO 2012 / 175741 and WO 2015 / 173325, and also refer to, for example, WO 2013 / 024059 and WO 2016 / 118733.

[0625] Amino acid sequence modifications of the polypeptides or ISVDs described herein are contemplated. For example, it is desirable to improve the binding affinity and / or other biological properties of the polypeptides or ISVDs. Amino acid sequence variants of the polypeptides or ISVDs described herein are prepared by introducing appropriate nucleotide changes into the nucleic acids encoding the polypeptides or ISVDs or by peptide synthesis.

[0626] Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the polypeptides or ISVDs described herein. Any combination of deletions, insertions, and substitutions is made to obtain the final construct, provided that the final construct has the desired characteristics. Amino acid changes can also alter the post-translational processes of the binding molecule, such as changing the number or location of glycosylation sites. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids can be substituted in the CDRs, while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids can be substituted in the framework regions (FRs). The substitutions are preferably conservative substitutions as described herein. Additionally or alternatively, 1, 2, 3, 4, 5, or 6 amino acids can be inserted or deleted in each CDR (of course, depending on their length), while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids can be inserted or deleted in each FR.

[0627] A method for identifying certain residues or regions as preferred mutagenesis positions for polypeptides or ISVDs as described herein is referred to as "alanine scanning mutagenesis", as described by Cunningham and Wells 1989 (Science 244: 1081-1085). Here, residues or groups of target residues within the binding molecule are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the epitope. Then, the amino acid positions that exhibit functional sensitivity to the substitution are refined by introducing more or other variants at or around the substitution site. Thus, although the sites for introducing amino acid sequence variation are predetermined, the nature of the mutations themselves need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is performed at the target codon or region, and the expressed binding molecule variants are screened for the desired activity.

[0628] Preferably, amino acid sequence insertions include amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues to polypeptides containing 100 or more residues.

[0629] Another type of variant is an amino acid substitution variant. These variants have preferably (at least) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues substituted with different residues in the amino acid sequence, ISVD, or polypeptide. The sites of most interest for performing substitution mutagenesis include the CDRs, particularly the hypervariable regions, but FR alterations are also considered. For example, if a CDR sequence contains 6 amino acids, it is contemplated that one, two, or three of these amino acids are substituted. Similarly, if a CDR sequence contains 15 amino acids, it is contemplated that one, two, three, four, five, or six of these amino acids are substituted.

[0630] Generally, if amino acids are substituted in one or more or all of the CDRs, it is preferred that the resulting "substituted" sequence has at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, more particularly preferably 80%, or even more than 90% identity to the "original" CDR sequence. This means it depends on the length of the CDR, i.e., the degree to which the CDR is the same as the "substituted" sequence. For example, a CDR with 5 amino acids preferably has 80% identity to its substituted sequence such that at least one amino acid is substituted. Thus, the CDRs of an amino acid sequence, ISVD, or polypeptide can have different degrees of identity to their substituted sequences. For example, CDR1 may have 80% while CDR3 may have 90%.

[0631] Preferred amino acid substitutions are conservative substitutions. Such conservative substitutions preferably refer to substitutions in which one amino acid in the following groups (a)-(e) is replaced by another amino acid residue in the same group: (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp. Further preferred conservative substitutions are as follows: Ala is substituted with Gly or with Ser; Arg is substituted with Lys; Asn is substituted with Gln or with His; Asp is substituted with Glu; Cys is substituted with Ser; Gln is substituted with Asn; Glu is substituted with Asp; Gly is substituted with Ala or with Pro; His is substituted with Asn or with Gln; Ile is substituted with Leu or with Val; Leu is substituted with Ile or with Val; Lys is substituted with Arg, with Gln or with Glu; Met is substituted with Leu, with Tyr or with Ile; Phe is substituted with Met, with Leu or with Tyr; Ser is substituted with Thr; Thr is substituted with Ser; Trp is substituted with Tyr; Tyr is substituted with Trp; and / or Phe is substituted with Val, with Ile or with Leu. However, any substitutions (including non-conservative substitutions) are contemplated as long as the polypeptide retains its ability to specifically bind to an epitope on FcRn as described herein, at an acidic pH (e.g., pH 5.0 to 6.8) with an affinity as described herein (e.g., at 10 -6 with 10 -11 K between M D ) has the ability to specifically bind to FcRn and / or its CDR has at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, and more particularly preferably 80% identity with the "original" CDR sequence.

[0632] As described in the Examples section, only after a rigorous screening and selection process the inventors were able to identify ISVDs that bind to FcRn in a pH-dependent manner. Thus, the present invention relates to a polypeptide comprising at least one ISVD that binds to FcRn at acidic pH, said ISVD being selected from the group consisting of SEQ ID NO: 14 and 15 (Table A-1).

[0633] Preferred CDR sequences are shown in Table A-1 as SEQ ID NOs 8 to 13 and 104 to 105. Sequence analysis further indicated that there are many possible sequence variations in the CDRs (see SEQ ID NOs: 2 to 7 in Table A-1).

[0634] Table A-1: Sequence IDs of Preferred ISVD and CDR Sequences

[0635]

[0636]

[0637]

[0638] The polypeptides of the present invention bind to specific, unique and novel epitopes on FcRn, which are different from the epitopes on FcRn to which the natural ligands of FcRn, namely serum albumin and IgG, bind (as shown in Figure 2 and Figure 3 ).

[0639] In certain embodiments, the FcRn-binding polypeptides according to the present invention are such that when they bind to or otherwise associate with FcRn molecules, the binding of FcRn molecules to serum albumin and / or IgG is not (significantly) affected, reduced or inhibited.

[0640] In other certain embodiments, the present invention provides polypeptides characterized in that they comprise at least one ISVD that specifically binds to amino acid residues on FcRn that are not involved in the binding of FcRn to serum albumin and / or IgG. The amino acid residues on FcRn involved in binding at least one ISVD, serum albumin and / or IgG can be determined, for example, by measuring the interaction of the amino acid residues on FcRn with the amino acid residues on at least one ISVD, with the amino acid residues on serum albumin and / or with the amino acid residues on IgG, for example, by crystallographic studies. In this particular embodiment, the polypeptide comprises at least one ISVD that specifically binds to an epitope on FcRn that comprises amino acid residues not included in the epitope on FcRn to which serum albumin binds and / or in the epitope on FcRn to which IgG binds. In this particular embodiment, the polypeptide comprises at least one ISVD that specifically binds to amino acid residues on FcRn that are not bound by serum albumin and / or IgG.

[0641] According to certain embodiments, the FcRn-binding polypeptides of the present invention also preferably compete with polypeptides having the amino acid sequences of SEQ ID NO: 14 and / or 15 for binding to FcRn and / or "cross-block" (as defined herein) the binding of polypeptides having the amino acid sequences of SEQ ID NO: 14 and / or 15 to FcRn.

[0642] The terms "cross-block," "cross-blocked," and "cross-blocking" are used interchangeably herein to refer to the ability of an immunoglobulin single variable domain or polypeptide to interfere with the binding of a ligand to its target, such as a native ligand to one or more of its receptors. A competitive binding assay can be used to determine the extent to which an immunoglobulin single variable domain or polypeptide of the invention is able to interfere with the binding of another compound, such as a native ligand, to its target, and thus whether it can be said to cross-block according to the invention. A particularly suitable quantitative cross-blocking assay uses a FACS- or ELISA-based method or Alphascreen to measure the competition between a labeled (e.g., His-tagged or biotinylated) immunoglobulin single variable domain or polypeptide of the invention and other binding agents for binding to the target. Suitable FACS-, ELISA-, or Alphascreen-based displacement assays for determining whether a binding molecule cross-blocks or is able to cross-block a polypeptide are well known. It should be understood that these assays can be used with any immunoglobulin single variable domain or other binding agent described herein. Thus, generally, a cross-blocking polypeptide according to the invention is, for example, a polypeptide that binds to the target in the above-described cross-blocking assay such that the recorded displacement of the immunoglobulin single variable domain or polypeptide of the invention in the presence of a second polypeptide or in the presence of a native ligand during the assay is between 60% and 100% (e.g., in an ELISA / Alphascreen-based competition assay) or between 80% and 100% (e.g., in a FACS-based competition assay) of the maximum theoretical displacement of the potential cross-blocking agent being tested present in an amount of 0.01 mM or less (e.g., the displacement of a cold (e.g., unlabeled) immunoglobulin single variable domain or polypeptide that needs to be cross-blocked).

[0643] In certain embodiments, the FcRn-binding polypeptide of the invention binds to amino acid residues and / or epitopes on FcRn that are substantially the same as the amino acid residues and / or epitopes to which a polypeptide having the amino acid sequence of SEQ ID NO: 14 and / or 15 binds, and even more preferably such that it has substantially the same amino acid interactions as a polypeptide having the amino acid sequence of SEQ ID NO: 14 and / or 15. To this end, according to certain but non-limiting aspects, the FcRn-binding polypeptide of the invention preferably has CDRs that are identical to the sequences of SEQ ID NO: 14 and / or 15, or, compared to the sequences of SEQ ID NO: 14 and / or 15, preferably contains only such mutations (such as conservative amino acid substitutions) in its CDRs that still allow it to make the same or substantially the same amino acid interactions with FcRn as a polypeptide having the sequences of SEQ ID NO: 14 and / or 15.

[0644] In certain embodiments, the FcRn-binding polypeptides of the invention specifically bind to a unique epitope on FcRn as disclosed herein in a pH-dependent manner such that their binding affinity at acidic pH, particularly between 5.0 and 6.8, is at least three-fold higher than their binding affinity at neutral or physiological pH of 7.4. In these particular embodiments, the polypeptides of the invention exhibit conditional specific binding to FcRn and thus have an extended in vivo serum half-life by exploiting the in vivo FcRn-mediated recycling mechanism. Accordingly, the polypeptides of the invention can be used to extend the in vivo half-life of a therapeutic target or therapeutic molecule of interest to which they are appropriately linked, bound, or fused.

[0645] As used herein, a pH between 5.0 and 6.8 refers to an acidic physiological pH value, e.g., it can be a pH value less than or greater than 5.0 or 6.8 and / or it can be any pH value within the range between 5.0 and 6.8, such as 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, or 6.7 or a pH value slightly greater or slightly less. As used herein, pH 7.4 refers to neutral physiological pH, e.g., it can be a pH between 7.2 and 7.4 (and possibly slightly greater or slightly less).

[0646] In certain particular embodiments of the invention, the pH-dependent interaction of the FcRn-binding polypeptides with FcRn ensures that these polypeptides are optimally and rapidly captured in the acidic environment of endosomes and are efficiently released after recycling when re-exposed to pH 7.4 (i.e., outside the endosome).

[0647] In this way, the inventors have been able to demonstrate that polypeptides according to one aspect of the invention exhibit an increased in vivo half-life by specifically binding to FcRn in a pH-dependent manner (as demonstrated by the examples further described herein).

[0648] The half-life of a polypeptide or a fusion protein, construct, or compound comprising it (and further described herein) according to the invention can generally be defined as the time it takes for the serum concentration of the amino acid sequence, compound, or polypeptide to decrease by 50% in vivo, e.g., due to degradation of the sequence or compound and / or clearance or sequestration of the sequence or compound by natural mechanisms. In particular, the half-life can be defined as in WO 2009 / 068627.

[0649] The in vivo half-life of a polypeptide according to the invention or a fusion protein, construct or compound (as further described herein) can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and can typically involve, for example, the following steps: appropriately administering a suitable dose of the amino acid sequence, compound or polypeptide of the invention to a warm-blooded animal (i.e., a human or another suitable mammal, such as a mouse, rabbit, rat, pig, dog or primate, for example a monkey from the genus Macaca (e.g., and in particular, cynomologus monkey (Macaca fascicularis) and / or rhesus monkey (Macaca mulatta)) and baboon (Papio ursinus)); collecting a blood sample or other sample from the animal; determining the level or concentration of the amino acid sequence, compound or polypeptide of the invention in the blood sample; and calculating, based on the data obtained therefrom (plotting), the time at which the level or concentration of the amino acid sequence, compound or polypeptide of the invention has decreased by 50% compared to the initial level at the time of administration. For example, reference is made to the experimental section below, and standard manuals such as Kenneth, A et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinete analysis: A Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi and D Perron, published by Marcel Dekker, 2nd revised edition (1982). The half-life can be expressed using parameters such as t1 / 2-α, t1 / 2-β and area under the curve (AUC). For example, reference is made to the experimental section below, and standard manuals such as Kenneth et al., 1996 (Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists) and Peters et al., 1996 (Pharmacokinetic Analysis: A Practical Approach). Reference is also made to Gibaldi and Perron 1982 (Pharmacokinetics, Dekker M, 2nd revised edition). In the present specification, "increase in half-life" means an increase in any one of these parameters, for example any two of these parameters, or substantially all three of these parameters.The term "increased half-life" or "increased half-life" particularly refers to an increase in t1 / 2-β, regardless of whether t1 / 2-α and / or AUC or both increase.

[0650] Thus, in certain embodiments, compared to known FcRn-binding polypeptides, (fusion) proteins, constructs or compounds described in the prior art, the polypeptides according to the invention comprising at least one ISVD that binds to a unique epitope on FcRn as disclosed herein and fusion proteins, constructs and compounds comprising the same (as further described herein) will have an increased half-life.

[0651] Generally, the polypeptides according to the invention and fusion proteins, constructs and compounds comprising the same (as further described herein) preferably have a half-life that is at least 1.5-fold, preferably at least 2-fold, such as at least 5-fold, such as at least 10-fold or more than 20-fold or greater (as measured in humans or suitable animals such as mice or cynomolgus monkeys) of the half-life of FcRn-binding amino acid sequences and proteins known in the prior art.

[0652] In addition, the polypeptides according to the invention and fusion proteins, constructs and compounds comprising the same (as further described herein) preferably have a half-life that is increased by at least 30%, at least 50%, at least 75%, such as at least 100% or increased by more than 200%, such as more than 300%, more than 400%, more than 500% or greater compared to the half-life of FcRn-binding amino acid sequences and proteins known in the prior art (as measured in humans or suitable animals such as mice or cynomolgus monkeys).

[0653] In addition, the polypeptides according to the invention and fusion proteins, constructs and compounds comprising the same (as further described herein) that comprise at least one ISVD that binds to a unique epitope on FcRn and is fused to another moiety (such as one or more therapeutic moieties) have an increased half-life compared to said another moiety (such as one or more therapeutic moieties themselves).

[0654] Generally, the constructs or fusion proteins described herein preferably have a half-life that is at least 1.5-fold, preferably at least 2-fold, such as at least 5-fold, such as at least 10-fold or more than 20-fold the half-life of the corresponding other moiety (such as the therapeutic moiety itself) (as measured in humans or suitable animals such as mice or cynomolgus monkeys).

[0655] As described, in one aspect, the FcRn binders of the invention can be used to increase (one or more) immunoglobulin single variable domains such as domain antibodies, single domain antibodies, "dAbs", V HH or V HH (such as V HH, humanized V HH or camelized V H such as camelized human V H ) half-life.

[0656] In addition, the FcRn-binding polypeptides provided by the present invention, as well as fusion proteins, constructs, and compounds comprising the same (as further described herein), preferably have the following half-life (defined as t1 / 2β) in humans: greater than 1 hour, preferably greater than 2 hours, more preferably greater than 6 hours, such as greater than 12 hours, for example, about one day, two days, one week, two weeks, and up to the half-life of human serum albumin or IgG (estimated to be about 19 days), although the latter may be less important.

[0657] The FcRn-binding polypeptides according to different embodiments of the present invention also preferably result in:

[0658] They have the following serum half-life (expressed as t1 / 2β) in humans: greater than 6 hours, preferably greater than 12 hours, more preferably greater than 24 hours, even more preferably greater than 72 hours; for example, about one week, two weeks, and up to the half-life of human serum albumin or IgG (estimated to be about 19 days); and / or result in: when they are linked to a therapeutic moiety or entity, they confer the following serum half-life (expressed as t1 / 2β) in humans to the resulting polypeptides of the present invention: greater than 6 hours, preferably greater than 12 hours, more preferably greater than 24 hours, even more preferably greater than 72 hours; for example, about one week, two weeks, and up to the half-life of human serum albumin or IgG (estimated to be about 19 days).

[0659] Among other factors, the half-life in mammalian species other than humans mainly depends on the binding characteristics (such as affinity) of the FcRn-binding polypeptides of the present invention for FcRn from the mammalian species and the half-life of naive serum albumin in the species. According to a preferred embodiment of the present invention, when the FcRn-binding polypeptides of the present invention have cross-reactivity (as defined herein) between human FcRn and FcRn from another mammalian species, then the half-life of the FcRn-binding polypeptides of the present invention (and / or the compounds of the present invention comprising the FcRn-binding polypeptides) measured in the species is preferably at least 5% of the half-life of serum albumin in the species, for example at least 10%, more preferably at least 25%, for example about 50%, and possibly up to 100%.

[0660] In a specific embodiment, at an acidic pH between 5.0 and 6.8, the polypeptides according to the present invention specifically bind FcRn, and their affinity (K A ) is between 10 -3 nM -1 and 10 2 nM -1between. These polypeptides have an affinity (K A ) for FcRn at acidic pH, preferably at a pH between 5.0 and 6.8, that is at least three-fold higher than the affinity (K A ) of the same polypeptide for FcRn at neutral or physiological pH of 7.4.

[0661] In other specific embodiments, at an acidic pH between 5.0 and 6.8, the polypeptide according to the invention binds to FcRn with an affinity that is at least ten-fold higher than the affinity (K A ) of the same polypeptide for FcRn at neutral or physiological pH of 7.4. In still other specific embodiments, at an acidic pH between 5.0 and 6.8, the polypeptide according to the invention binds to FcRn with an affinity that is at least fifty-fold higher than the affinity (K A ) of the same polypeptide for FcRn at neutral or physiological pH of 7.4, such as at least one hundred-fold higher.

[0662] In certain specific embodiments, the invention provides a polypeptide as described herein, characterized in that at least one ISVD binds to FcRn at neutral or physiological pH of 7.4 with an affinity that is at least three-fold lower, such as at least ten-fold lower, such as at least fifty-fold lower, such as at least one hundred-fold lower, than the affinity of the at least one ISVD for FcRn at acidic pH between 5.0 and 6.8.

[0663] In a specific embodiment, at physiological pH, such as at a pH of 7.4, the at least one ISVD binds to FcRn with a K 4 value of less than 10 A L / mol.

[0664] In certain specific embodiments, the invention provides a polypeptide as described herein, characterized in that at neutral or physiological pH, such as at a pH of 7.4, the at least one ISVD does not bind to FcRn detectably, selectively or specifically, or exhibits no binding or substantially no binding to FcRn.

[0665] In a specific embodiment, at an acidic pH between 5.0 and 6.8, the polypeptide according to the invention binds specifically to FcRn with a dissociation constant (K D ) between 10 -6 M and 10 -11 M or lower. Preferably, the K D is determined by Kinexa, BLI or SPR, such as by SPR.

[0666] In a specific embodiment, at an acidic pH between 5.0 and 6.8, the polypeptide according to the invention binds specifically to FcRn with a dissociation constant (K D)between 10 3 nM and 10 -2 nM or lower. These amino acid sequences and polypeptides have a dissociation constant (K D ) for FcRn at acidic pH, preferably at a pH between 5.0 and 6.8, that is at least three times better (i.e., lower value) than the dissociation constant (K D ) for the same amino acid sequence and polypeptide for FcRn at a neutral or physiological pH of about 7.4. In a further specific embodiment, at an acidic pH between 5.0 and 6.8, the polypeptide according to the invention binds to FcRn with an affinity that is at least ten times higher / better than the affinity (K D ) of the same polypeptide for FcRn at a neutral or physiological pH of about 7.4. In yet a further specific embodiment, at an acidic pH between 5.0 and 6.8, the polypeptide according to the invention binds to FcRn with an affinity that is at least fifty times higher / better than the affinity (K D ) of the same polypeptide for FcRn at a neutral or physiological pH of about 7.4, such as at least 100 times.

[0667] In certain specific embodiments, the present invention provides a polypeptide as described herein, characterized in that at least one ISVD binds to FcRn at a neutral or physiological pH of 7.4 with an affinity that is at least three times lower / worse than the affinity of the at least one ISVD for FcRn at an acidic pH between 5.0 and 6.8, such as at least ten times, such as at least fifty times, such as at least one hundred times.

[0668] In a specific embodiment, at physiological pH, such as at a pH of 7.4, at least one ISVD binds to FcRn with a K -4 value greater than 10 D mol / L.

[0669] In certain specific embodiments, the present invention provides a polypeptide as described herein, characterized in that at neutral or physiological pH, such as at a pH of 7.4, the at least one ISVD does not detectably, selectively or specifically bind to FcRn, or exhibits no binding or substantially no binding to FcRn.

[0670] Thus, the present invention relates to a polypeptide as described herein, which polypeptide comprises at least one ISVD that binds to FcRn and has an average K D value between 1000 nM and 10 pM, such as an average K D value of 900 nM or less, and even more preferably an average K DThe value is 800 nM or less, such as less than 700, 600, 500, 400, 300, 200, 100, 50 nM or even less, such as less than 40, 30, 20, 10, 5, 1 nM, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 pM or even less, such as less than 10 pM. Preferably, the K D is determined by Kinexa, BLI or surface plasmon resonance (SPR), for example by SPR. Preferably, the average K is measured by performing SPR on the recombinant protein D .

[0671] The present invention also relates to the polypeptides described herein, which polypeptides comprise at least one ISVD that binds to FcRn at acidic pH, and has an EC50 value between 1000 nM and 1 pM, such as an average EC50 value of 1000 nM or less, even more preferably an average EC50 value of 900 nM or less, such as less than 800, 700, 600, 500, 400, 300, 200, 100, 50 nM or even less, such as less than 40, 30, 20, 10, 5 or 1 nM or even less, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 pM or even less, such as less than 4 pM.

[0672] In certain embodiments, at an acidic pH between 5.0 and 6.8, the polypeptides according to the invention specifically bind to FcRn, and the binding rate constant (k on ) is selected from at least about 10 2 M -1 s -1 , at least about 10 3 M -1 s -1 , at least about 10 4 M -1 s -1 , at least about 10 5 M -1 s -1 , at least about 10 6 M -1 s -1 , at least about 10 7 M -1 s -1 and at least about 10 8 M -1 s -1 , preferably as measured by surface plasmon resonance or BLI.

[0673] In certain embodiments, at acidic pH between 5.0 and 6.8, the polypeptide according to the invention specifically binds to FcRn, and its dissociation rate constant (k off ) is selected from up to about 10 -1 s -1 、 up to about 10 -2 s -1 、 up to about 10 -3 s -1 、 up to about 10 -4 s -1 、 up to about 10 -5 s -1 and up to about 10 -6 s -1 , preferably as measured by surface plasmon resonance or BLI.

[0674] In certain embodiments, at acidic pH between 5.0 and 6.8, the polypeptide according to the invention specifically binds to FcRn, and its dissociation rate constant (k off ) is selected from up to about 10 -1 s -1 、 up to about 10 -2 s -1 、 up to about 10 -3 s -1 、 up to about 10 -4 s -1 、 up to about 10 -5 s -1 and up to about 10 -6 s -1 . The dissociation rate constant (k off ) of these amino acid sequences and polypeptides for FcRn at acidic pH, preferably at pH between 5.0 and 6.8, is at least three times lower than the dissociation rate constant (k off ) of the same amino acid sequences and polypeptides for FcRn at neutral or physiological pH of 7.4. In further certain embodiments, at acidic pH between 5.0 and 6.8, the dissociation rate constant (k off ) of the polypeptide according to the invention binding to FcRn is at least ten times lower than the dissociation rate constant (k off ) of the same polypeptide for FcRn at neutral or physiological pH of 7.4. In yet further certain embodiments, at acidic pH between 5.0 and 6.8, the dissociation rate constant (k off ) of the polypeptide according to the invention binding to FcRn is at least fifty times lower than the dissociation rate constant (k off ) of the same polypeptide for FcRn at neutral or physiological pH of 7.4, such as at least 100 times lower.

[0675] In certain specific embodiments, the present invention provides the polypeptides described herein, which are characterized in that the at least one ISVD binds to FcRn at a neutral or physiological pH of 7.4, and its dissociation rate constant (k off ) is at least 3 times higher, such as at least 10 times, such as at least 50 times, such as at least 100 times, than the dissociation rate constant (k off ) of the at least one ISVD binding to FcRn at an acidic pH between 5.0 and 6.8.

[0676] In certain specific embodiments, the present invention provides the polypeptides described herein, which are characterized in that at a neutral or physiological pH, such as at a pH of 7.4, the at least one ISVD does not detectably, selectively or specifically bind to FcRn, or exhibits no binding or substantially no binding to FcRn.

[0677] Accordingly, the present invention provides improved FcRn binders that can be used in various applications, including but not limited to extending the in vivo half-life of (existing or future) therapeutic compounds. In certain embodiments, the polypeptides of the present invention have a high affinity for a unique conformational epitope on FcRn, which is different from the epitopes on FcRn bound by serum albumin and IgG.

[0678] In certain embodiments, the polypeptides of the present invention have a high affinity for a specific epitope on FcRn at an acidic pH between 5.0 and 6.8, while showing reduced binding to FcRn or no detectable, selective and / or specific binding to FcRn at a physiological or neutral pH of 7.4.

[0679] In certain specific embodiments, the polypeptides of the present invention have a molecular weight of at least 30 kDa, particularly a molecular weight between 30 kDa and 100 kDa. According to these specific embodiments, the compounds of the present invention can be most effectively and optimally applied to a variety of applications, particularly for extending the in vivo half-life of therapeutic molecules.

[0680] Accordingly, in certain embodiments, the present invention provides polypeptides that comprise:

[0681] at least one ISVD that specifically binds to FcRn in a pH-dependent manner and at least one additional moiety, and the polypeptide is characterized in that

[0682] a) the at least one ISVD specifically binds to FcRn in a pH-dependent manner such that the binding affinity at a pH between 5.0 and 6.8 is at least three times higher than the binding affinity at a pH of 7.4, and

[0683] b) the polypeptide has a molecular weight of at least 30 kDa.

[0684] In certain additional specific embodiments, the polypeptide has a molecular weight between about 30 kDa and 200 kDa, such as between about 30 kDa and 100 kDa.

[0685] 5.3 Multispecific polypeptides

[0686] Generally, a polypeptide according to the invention comprising a single building block, a single immunoglobulin single variable domain or a single ISVD or consisting essentially thereof is herein referred to as a "monovalent" protein or polypeptide, "monovalent construct", "monovalent building block", "monovalent immunoglobulin single variable domain", "monovalent ISVD" or "monovalent V HH ".

[0687] A polypeptide comprising two or more immunoglobulin single variable domains (such as at least two immunoglobulin single variable domains of the invention) or consisting essentially thereof is herein referred to as a "multivalent" polypeptide, (fusion) protein, compound or "multivalent construct". Some non-limiting examples of such multivalent constructs will become clear from the further description herein.

[0688] The polypeptides of the invention contain at least two building blocks, ISVDs, ISVDs or Vs HH , wherein at least one building block, ISVD, ISVD or V HH is directed against a first antigen (i.e., against a first target, such as FcRn) and at least one building block, ISVD, ISVD or V HH is directed against a second antigen (i.e., against a second target different from the first target, such as serum albumin or a therapeutic target other than FcRn), will also be referred to as a "multispecific" polypeptide of the invention, and the building blocks, ISVDs, ISVDs or Vs HH present in such polypeptides will also be referred to herein as "multivalent forms" or "multispecific forms". Thus, for example, a "bispecific" polypeptide of the invention is a polypeptide that comprises at least one building block, ISVD, ISVD or V HH directed against a first target (such as FcRn) and at least one additional building block, ISVD, ISVD or V HH ,wherein the "trispecific" polypeptide of the present invention is a polypeptide that comprises at least one building block directed against a first target (e.g., FcRn), ISVD, ISVD or V HH a second building block directed against a second target different from the first target (e.g., serum albumin), ISVD, ISVD or V HH and at least one additional building block directed against a third antigen (i.e., different from the first and second targets, e.g., a therapeutic target other than FcRn and serum albumin), etc., ISVD, ISVD or V HH . It is clear from the specification that the present invention is not limited to bispecific polypeptides, and the multispecific polypeptides of the present invention may comprise at least a first building block directed against a first target, ISVD, ISVD or V HH a second building block directed against a second target, ISVD, ISVD or V HH and any number of building blocks directed against one or more targets, ISVD, ISVD or V HH , and these targets may be the same as or different from the first target and / or the second target, respectively.

[0689] The terms bispecific polypeptide, bispecific form, bispecific construct, bispecific construct, bispecific and bispecific ISVD construct are used interchangeably herein.

[0690] It will be clear from the foregoing and further description herein that the immunoglobulin single variable domain of the present invention can be used as a "building block" to form the polypeptides of the present invention, e.g., by appropriately combining them with other groups, residues, moieties or binding units to form compounds or constructs as described herein (e.g., but not limited to the divalent / trivalent / tetravalent / multivalent and bispecific / trispecific / tetraspecific / multispecific polypeptides of the present invention), which combine one or more desired properties or biological functions within a single molecule.

[0691] In a further specific embodiment, the present invention provides a polypeptide wherein the at least one ISVD and the at least one additional binding moiety are directly linked to each other or linked via a linker.

[0692] The compounds, constructs or polypeptides of the present invention can generally be prepared by a method that includes at least one step: appropriately linking one or more immunoglobulin single variable domains of the present invention to one or more other groups, residues, moieties or binding units, optionally via one or more suitable linkers, thereby providing the compounds, constructs or polypeptides of the present invention. The polypeptides of the present invention can also be prepared by a method that generally includes at least the following steps: providing a nucleic acid encoding the polypeptide of the present invention, expressing the nucleic acid in a suitable manner and recovering the expressed polypeptide of the present invention. Such methods can be carried out in a manner known per se, which will be clear to the person skilled in the art, for example based on the methods and techniques further described herein. Starting from a polypeptide comprising at least one ISVD of the present invention, the process of designing / selecting and / or preparing the compounds, constructs or polypeptides of the present invention is also referred to herein as "formatting" the polypeptide of the present invention; while the polypeptides of the present invention that form part of the compounds, constructs or polypeptides of the present invention are referred to as being "formatted" or "in the form of" the compounds, constructs or polypeptides of the present invention. Examples of the ways in which the polypeptides of the present invention can be formatted and examples of such forms will be clear to the person skilled in the art based on the disclosure herein; such formatted immunoglobulin single variable domains or polypeptides form another aspect of the present invention.

[0693] For example, such additional groups, residues, moieties or binding units can be one or more additional immunoglobulins, such that the compound or construct is a (fusion) protein or (fusion) polypeptide. In a preferred but non-limiting aspect, the one or more other groups, residues, moieties or binding units are immunoglobulin single variable domains (ISVDs). Even more preferably, the one or more other groups, residues, moieties or binding units are selected from domain antibodies, immunoglobulin single variable domains suitable for use as domain antibodies, single domain antibodies, immunoglobulin single variable domains (ISVDs) suitable for use as single domain antibodies, "dAbs", immunoglobulin single variable domains suitable for use as dAbs, V HH , humanized V HH , camelized V H or V HH . Alternatively, such groups, residues, moieties or binding units can be, for example, chemical groups, residues, moieties, which may or may not have biological and / or pharmacological activity per se. For example but not limited to, such groups can be linked to one or more immunoglobulin single variable domains or polypeptides of the present invention, thereby providing a "derivative" of the ISVD or polypeptide of the present invention, as further described herein.

[0694] Also within the scope of the present invention are compounds or constructs that comprise or consist essentially of one or more of the derivatives described herein and optionally further comprise one or more other groups, residues, moieties or linking units, optionally linked by one or more linkers. Preferably, the one or more other groups, residues, moieties or linking units are immunoglobulin single variable domains. In the above compounds or constructs, the one or more immunoglobulin single variable domains of the present invention and the one or more groups, residues, moieties or linking units may be directly linked to each other and / or linked via one or more suitable linkers or spacers. For example, when the one or more groups, residues, moieties or linking units are immunoglobulin single variable domains, the linker may also be an immunoglobulin single variable domain, such that the resulting compound or construct is a fusion protein or fusion polypeptide.

[0695] In some embodiments, the polypeptide comprises at least two or more immunoglobulin single variable domains disclosed herein. In some embodiments, the polypeptide consists essentially of two or more immunoglobulin single variable domains disclosed herein. A polypeptide that "consists essentially of two or more immunoglobulin single variable domains" is a polypeptide that does not have additional immunoglobulin single variable domains other than the two or more immunoglobulin single variable domains disclosed herein. For example, a polypeptide consisting essentially of two immunoglobulin single variable domains does not include any additional immunoglobulin single variable domains. However, it should be understood that a polypeptide consisting essentially of two or more immunoglobulin single variable domains may include additional functions, such as labels, toxins, one or more linkers, binding sequences, etc. These additional functions include amino acid-based and non-amino acid-based groups. In some embodiments, the polypeptide is composed of one or more immunoglobulin single variable domains disclosed herein. It should be understood that the terms "polypeptide construct" and "polypeptide" may be used interchangeably herein (unless the context clearly dictates otherwise).

[0696] In some embodiments, the polypeptide includes a multivalent or multispecific construct comprising an immunoglobulin single variable domain disclosed herein. In some embodiments, the polypeptide comprises one or more antibody-based scaffolds and / or non-antibody-based scaffolds disclosed herein. In some embodiments, the polypeptide comprises a serum binding protein moiety. In some embodiments, the serum binding protein moiety is an immunoglobulin single variable domain. In some embodiments, the immunoglobulin single variable domain is V HH 。

[0697] It should be understood that the order (orientation) of the building blocks on the polypeptide, e.g., the first building block, the second building block, the third building block, etc., can be selected according to the needs of those skilled in the art and may depend on the relative affinities of these building blocks at their positions in the polypeptide. Whether the polypeptide contains a linker is a matter of design choice. However, some orientations with or without a linker may provide preferred binding properties compared to other orientations. For example, the order of the first and second building blocks in the polypeptides of the present invention can be (from the N-terminus to the C-terminus): (i) the first building block (e.g., the first ISVD, e.g., the first V HH ) - [linker] - the second building block (e.g., the second ISVD, e.g., the second V HH );or (ii) the second building block (e.g., the second ISVD, e.g., the second V HH ) - [linker] - the first building block (e.g., the first ISVD, e.g., the first V HH );(wherein the linker is optional). The present invention encompasses all orientations.

[0698] The polypeptides according to the present invention comprising at least one ISVD and at least one other binding moiety enable at least one ISVD and at least one other binding moiety to be directly linked to each other or linked via a linker (e.g., a peptide linker). Linking two or more (poly)peptides using a linker is well known in the art. A commonly used class of peptide linkers is referred to as "Gly-Ser" or "GS" linkers. These are linkers substantially composed of glycine (G) and serine (S) residues and typically contain one or more repeats of a peptide motif such as the GGGGS (SEQ ID NO:86) motif (e.g., the display (Gly-Gly-Gly-Gly-Ser) n, where n can be 1, 2, 3, 4, 5, 6, 7 or greater). Some common examples of such GS linkers are 9GS linker (GGGGSGGGS, SEQ ID NO: 87), 15GS linker (n = 3), and 35GS linker (n = 7). See, for example, Chen et al., Adv. Drug Deliv. Rev. October 15, 2013; 65(10):1357–1369; and Klein et al., Protein Eng. Des. Sel. (2014) 27(10):325-330. In certain specific but non-limiting embodiments, the linker is selected from linkers of 3A, 3GS, 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, and 35GS (SEQ ID NOs: 37 to 48). In other specific embodiments, the linker can be an IgG hinge region, such as the IgG1 hinge region (SEQ ID NO: 50 or 126). Preferably, the linker can be the IgG1 short hinge region (SEQ ID NO: 126) and / or the 35GS linker (SEQ ID NO: 48).

[0699] Table A-2: Linker Sequences ("ID" refers to SEQ ID NO as used herein)

[0700]

[0701]

[0702] In certain additional specific embodiments, at least one additional moiety is a protein moiety, such as an additional ISVD.

[0703] In certain additional specific embodiments, at least one additional moiety is a protein moiety, such as a serum protein, particularly but not limited to serum albumin.

[0704] In certain additional specific embodiments, at least one additional moiety is a protein-binding moiety, such as a serum protein-binding moiety, such as a serum albumin-binding moiety, particularly a serum protein-binding ISVD, more particularly a serum albumin-binding ISVD.

[0705] In certain additional specific embodiments, at least one serum albumin-binding moiety specifically binds to amino acid residues on serum albumin that are not involved in the binding of serum albumin to FcRn.

[0706] In certain additional specific embodiments, the present invention provides the polypeptide described herein, characterized in that at least one ISVD that specifically binds to serum albumin is a (single) domain antibody, V HH , V HH , humanized V HH , or camelized V H .

[0707] In certain further specific embodiments, at least one additional moiety is at least one ISVD that binds to serum albumin.

[0708] In certain further specific embodiments, at least one additional moiety is at least one ISVD that binds to serum albumin, which is substantially composed of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity-determining regions (CDR1 to CDR3 respectively), wherein CDR1 is SFGMS (SEQ ID NO:16), CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO:17) and CDR3 is GGSLSR (SEQ ID NO:18), and the CDRs are determined according to the Kabat definition; and / or wherein CDR1 is GFTFRSFGMS (SEQ ID NO:19), CDR2 is SISGSGSDTL (SEQ ID NO:20) and CDR3 is GGSLSR (SEQ ID NO:21), and the CDRs are determined according to the AbM definition (Kontermann et al., 2010).

[0709] International application WO 06 / 122787 describes a number of ISVDs that bind to (human) serum albumin. These ISVDs include one called Alb-1 V HH (SEQ ID NO:52 in WO 06 / 122787) and its humanized variants, such as Alb-8 (SEQ ID NO:62 in WO 06 / 122787). In addition, WO 2012 / 175400 describes a further improved form of Alb-1, called Alb-23.

[0710] In certain embodiments, the polypeptides of the invention comprise at least one ISVD disclosed herein that binds to an epitope on FcRn and further comprise a serum albumin binding moiety selected from: Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10, and Alb-23, preferably Alb-8 or Alb-23 or variants thereof, as shown on pages 7-9 of WO 2012 / 175400, and albumin binders as described in WO 2012 / 175741, WO 2015 / 173325, WO 2017 / 080850, WO 2017 / 085172, WO 2018 / 104444, WO 2018 / 134235, WO 2018 / 134234. Table A-3 also shows some preferred serum albumin binders.

[0711] Table A-3: ISVD Sequences That Bind Serum Albumin ("ID" refers to SEQ ID NO as used herein)

[0712]

[0713]

[0714] In certain embodiments of the invention, the polypeptides of the invention comprise at least one ISVD disclosed herein that binds to an epitope on FcRn and further comprise a serum albumin binding ISVD having the full amino acid sequence of ALB23002 (SEQ ID NO: 35, see Table A-3).

[0715] Also in preferred embodiments, the amino acid sequence of the ISVD that binds to human serum albumin can have greater than 90%, such as greater than 95% or greater than 99% sequence identity with SEQ ID NO: 35, wherein optionally the CDRs are defined as SEQ ID NOs: 16 to 18 (according to Kabat) or SEQ ID NOs: 19 to 21 (according to AbM) as defined above. In particular, the ISVD that binds to human serum albumin preferably has the amino acid sequence of SEQ ID NO: 35.

[0716] When such an ISVD that binds to human serum albumin has 2 or 1 amino acid differences in at least one CDR relative to the corresponding reference CDR sequence (as defined above as SEQ ID NOs: 16 to 18 (according to Kabat) or SEQ ID NOs: 19 to 21 (according to AbM)), the ISVD preferably has at least half the binding affinity for human serum albumin, preferably at least the same binding affinity, compared to the construct ALB23002, wherein the binding affinity is measured using the same method (such as SPR).

[0717] When the ISVD that binds to human serum albumin is at the C-terminal position, it can exhibit a C-terminal alanine (A) or glycine (G) extension and is preferably selected from SEQ ID NO: 24, 25, 27, 29, 30, 31, 32, 33, 34, and 36 (see Table A-3). In a preferred embodiment, the ISVD that binds to human serum albumin has another position different from the C-terminal position (i.e., not the C-terminal ISVD of the polypeptide of the present invention) and is selected from SEQ ID NO: 22, 23, 26, 28, and 35 (see Table A-3).

[0718] In a particular embodiment, the polypeptide of the present invention comprises at least a first and at least a second immunoglobulin single variable domain (ISVD), wherein the at least first ISVD has a high affinity / specific binding to FcRn at acidic pH, and the at least second ISVD has a high affinity / specific binding to serum albumin. The preferred sequences of the polypeptides, polypeptide constructs, ISVD building blocks, and combinations of CDR and FR sequences according to particular embodiments of the present invention are shown in Tables A-4 to A-8.

[0719] Table A-4: Configurations of multivalent ISVD constructs according to particular embodiments of the present invention

[0720]

[0721] Table A-5: Amino acid sequences of different monovalent VHH building blocks used in multispecific polypeptides according to the present invention ("ID" refers to SEQ ID NO. as used herein)

[0722]

[0723] Table A-6: Sequences of CDRs and frameworks according to AbM numbering ("ID" refers to the given SEQ ID NO.)

[0724]

[0725] Table A-7: Sequences of CDRs and frameworks according to Kabat numbering ("ID" refers to the given SEQ ID NO.)

[0726]

[0727]

[0728] Table A-8: Amino acid sequences of selected multivalent polypeptides ("ID" refers to the given SEQ ID NO.)

[0729]

[0730] In certain embodiments, the invention provides the polypeptides described herein, characterized in that the polypeptides further comprise a therapeutic moiety, which comprises a (single) domain antibody, V HH 、V HH 、humanized V HH or camelized V H 。

[0731] In certain embodiments, the polypeptides of the invention comprise at least a first, at least a second and at least a third immunoglobulin single variable domain (ISVD), wherein the at least first ISVD has high affinity / specific binding to FcRn at acidic pH; the at least second ISVD has high affinity / specific binding to serum albumin, and the at least third ISVD has high affinity / specific binding to a therapeutically relevant antigen other than FcRn and serum albumin.

[0732] It should be appreciated (as also demonstrated in the Examples section) that the ISVDs that bind to FcRn and the ISVDs that bind to serum albumin and / or the ISVDs that bind to therapeutic targets other than FcRn and albumin can be positioned in the polypeptides of the invention in any order. More specifically, in one embodiment, the ISVD that binds to FcRn is positioned at the N-terminus, while the ISVD that binds to another antigen is positioned at the C-terminus. In another embodiment, the ISVD that binds to other antigens is positioned at the N-terminus, while the ISVD that binds to FcRn is positioned at the C-terminus.

[0733] The invention also provides compounds or constructs, particularly proteins or polypeptides, that comprise one or more ISVDs or polypeptides of the invention or consist essentially thereof, and optionally further comprise one or more other groups, residues, moieties or binding units. As will be clear to those skilled in the art from the further disclosure herein, such other groups, residues, moieties, binding units or amino acid sequences may or may not provide additional functionality to the polypeptides of the invention (and / or the compounds or constructs in which it is present), and may or may not alter the properties of the polypeptides of the invention.

[0734] In certain embodiments, at least one additional moiety is the Fc region of an immunoglobulin (Ig). Thus, in certain specific embodiments, the invention provides at least one ISVD that specifically binds to FcRn in a pH-dependent manner, and at least one additional moiety, characterized in that:

[0735] a) The at least one ISVD specifically binds to FcRn in a pH-dependent manner such that the binding affinity at a pH between 5.0 and 6.8 is at least three-fold higher than the binding affinity at a pH of 7.4,

[0736] b) The polypeptide has a molecular weight of at least 30 kDa, particularly between about 30 kDa and 100 kDa, and

[0737] c) The at least one additional moiety is the Fc region or Fc domain of an immunoglobulin (Ig).

[0738] In certain embodiments, the invention provides a polypeptide as described herein, wherein the at least one additional moiety is the Fc region or Fc domain of immunoglobulin G (IgG). In certain embodiments, the invention provides a polypeptide as described herein, wherein the at least one additional moiety is the Fc region or Fc domain of immunoglobulin G type 4 (IgG4).

[0739] In certain embodiments, the invention provides a polypeptide as described herein, wherein the at least one additional moiety is the Fc region or Fc domain of immunoglobulin A (IgA).

[0740] In some particular embodiments, the Fc domain is the wild-type Fc domain of an immunoglobulin. The wild-type Fc domain can bind FcRn with a K D value greater than 500 nM, e.g., greater than 600 nM.

[0741] In certain other particular embodiments, the Fc domain can be a mutant Fc domain of an immunoglobulin that binds FcRn with a K D value less than about 550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125 or 100 nM (e.g., at a pH between 5.0 and 6.8). In certain other particular embodiments, the Fc domain can be a mutant Fc domain that does not detectably, selectively or specifically bind FcRn at a pH between 5.0 and 6.8 or at neutral or physiological pH, e.g., at a pH of 7.4, or exhibits no binding or substantially no binding to FcRn.

[0742] Suitable constructs and forms comprising at least one polypeptide according to the invention and at least one Fc domain will become apparent from the further description herein. Mutant variants of suitable Fc domains and suitable forms of Fc domain constructs are well known in the art and are described in published patent applications EP 2654790, US10239944, US20120251531, US 9133274, WO 2014065945, WO 2015150447 and WO 2021016571, among others.

[0743] Thus, as described above, the polypeptide according to the invention also comprises the Fc domain of IgG. The Fc domain of IgG refers to the C-terminal non-antigen-binding region of the immunoglobulin G heavy chain, which contains at least a portion of the constant region. In certain embodiments, the Fc domain can be a native Fc region, i.e., it exists in a native antibody, or it can be a variant Fc region that contains one or more alterations, mutations or variations compared to the native Fc domain. In certain embodiments, the Fc domain of IgG can also be a fragment of the native Fc domain or a fragment of a variant Fc domain. In some embodiments, the polypeptides described herein comprise the native Fc domain of human IgG, such as preferably the native Fc of human IgG4 (e.g., Uniprot sequence P01861, SEQ ID NO:129). In other embodiments, the polypeptide according to the invention comprises a variant Fc domain that has altered binding properties to Fc ligands relative to the unmodified parental Fc molecule. For example, the polypeptides described herein can comprise an Fc region in which one or more of the amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 are replaced by different amino acid residues such that the variant Fc region has altered affinity for effector ligands (e.g., Fc receptors or the C1 component of complement), as described in U.S. Patent Nos. 5,624,821 and 5,648,260 to Winter et al.

[0744] In certain embodiments, the polypeptide of the invention comprises an Fc variant domain with reduced effector function, particularly the so-called "FALA" or "LALA" Fc mutants in which residues 234 and 235 are replaced by alanine. Additional optional mutations include substitution of arginine residue 409 with lysine and deletion of lysine residue 447. In other embodiments, the Fc variant domain comprises the so-called "FALA" mutation and the S228P mutation described herein. These Fc variants are referred to as "pFALA".

[0745] In certain embodiments, the polypeptide according to the invention comprises an Fc variant domain which shows improved binding to the FcRn receptor as compared to the native Fc domain. Such Fc variants include those having substitutions at one or more of the Fc region residues 259, 308, 428 and 434. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8):6213 - 6216, Hinton et al., 2006, Journal of Immunology 176:346 - 356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Chemistry, 2001, 276(9):6591 - 6604).

[0746] In certain specific embodiments, the polypeptide according to the invention comprises an Fc variant domain in which methionine 428 is replaced by lysine and asparagine 434 is replaced by serine.

[0747] In certain embodiments, the polypeptide according to the invention comprises an Fc variant domain which shows reduced or no binding to the FcRn receptor as compared to the native Fc domain. Such Fc variants include those having substitutions at one or more of the Fc region residues 253, 310 and 453.

[0748] In certain embodiments, the polypeptide according to the invention comprises an Fc variant domain in which isoleucine 428 is replaced by alanine, histidine 310 is replaced by alanine, histidine 453 is replaced by alanine, optionally in combination with histidine 453 being replaced by alanine.

[0749] In certain embodiments, the at least one additional moiety is a non - protein moiety such as, but not limited to, a polyethylene glycol (PEG) moiety.

[0750] Thus, in certain specific embodiments, the invention provides at least one ISVD that specifically binds FcRn in a pH - dependent manner, and at least one additional moiety, characterized in that:

[0751] a) the at least one ISVD specifically binds FcRn in a pH - dependent manner such that the binding affinity at a pH between 5.0 and 6.5 is at least three - fold higher than the binding affinity at a pH of 7.4, and

[0752] b) The polypeptide has a molecular weight of at least 30 kDa, particularly between about 30 kDa and 100 kDa,

[0753] c) The at least one additional moiety is a non - protein moiety, such as but not limited to a polyethylene glycol (PEG) moiety.

[0754] In certain embodiments, the present invention provides a polypeptide as described herein, characterized in that the polypeptide further comprises a therapeutic moiety.

[0755] In certain embodiments, at least one therapeutic moiety comprises or consists essentially of a therapeutic protein, polypeptide, compound, factor or other entity. In a preferred embodiment, the therapeutic moiety is directed against a desired antigen or target, capable of binding to the desired antigen (particularly capable of specifically binding to the desired antigen), and / or capable of interacting with the desired target. In another embodiment, the at least one therapeutic moiety comprises or consists essentially of a therapeutic protein or polypeptide. In a further embodiment, the at least one therapeutic moiety comprises or consists essentially of a binding domain or binding unit, such as an immunoglobulin or immunoglobulin sequence (including but not limited to fragments of immunoglobulins), such as an antibody or antibody fragment (including but not limited to ScFv fragments), or another suitable protein scaffold, such as a protein A domain (such as Affibodies TM ), amylastatin, fibronectin, lipocalin, CTLA - 4, T - cell receptor, designed ankyrin repeats, avimer and PDZ domain (Binz et al., Nat. Biotech 2005, Vol. 23: 1257), and DNA - or RNA - based binding moieties, including but not limited to DNA or RNA aptamers (Ulrich et al., Comb Chem High Throughput Screen 2006 9(8): 619 - 32).

[0756] In yet another aspect, at least one therapeutic moiety comprises or consists essentially of an antibody variable domain, such as a heavy - chain variable domain or a light - chain variable domain.

[0757] In a preferred aspect, at least one therapeutic moiety comprises or consists essentially of at least one immunoglobulin single - variable domain, such as a domain antibody, single - domain antibody, "dAb" or V HH (e.g. V HH , humanized V HH or camelized V H ) or IgNAR domain.

[0758] For example but not limited to, such polypeptides, compounds or constructs of the present invention may comprise:

[0759] - At least one FcRn binder of the present invention and at least one ISVD against a therapeutic target (preferably V HH ); or

[0760] - At least one FcRn binder of the present invention and at least one serum albumin binder (as described herein) and at least one ISVD against a therapeutic target (preferably V HH ); or

[0761] - At least one FcRn binder of the present invention and at least one Fc region of an immunoglobulin (as described herein) and at least one ISVD against a therapeutic target (preferably V HH ); or

[0762] - At least one FcRn binder of the present invention and at least one serum albumin binder (as described herein) and at least one Fc region of an immunoglobulin (as described herein) and at least one ISVD against a therapeutic target (preferably V HH ).

[0763] In these polypeptides, compounds or constructs of the present invention, two or more building blocks, ISVDs or V HH and optionally one or more polypeptides, one or more other groups, drugs, medicaments, residues, moieties or binding units can be directly linked to each other (e.g. as described in WO 99 / 23221) and / or can be linked to each other via one or more suitable spacers or linkers, or any combination thereof.

[0764] Suitable spacers or linkers for multivalent and multispecific polypeptides will be apparent to the person skilled in the art and can generally be any linker or spacer used in the art for linking amino acid sequences. Preferably, the linker or spacer is suitable for constructing proteins or polypeptides intended for medical use.

[0765] For example, the linker can be a suitable amino acid sequence and in particular an amino acid sequence having between 1 and 50, preferably between 1 and 30, such as between 1 and 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, such as the type (gly x ser y ) z , such as (e.g. (gly 4 ser) 3 or (gly 3 ser 2) 3 , as described in WO 99 / 42077, and the 30GS, 15GS, 9GS and 7GS linkers described in the applications described by Ablynx herein (see, for example, WO06 / 040153 and WO 06 / 122825), and hinge-like regions, such as the hinge region of a naturally occurring heavy chain antibody or a similar sequence (as described in WO 94 / 04678). Preferred linkers are shown in Table A-2.

[0766] Some other particularly preferred linkers are polyalanine (such as AAA), and linker GS30 (SEQ ID NO:85 in WO 06 / 122825) and GS9 (SEQ ID NO:84 in WO 06 / 122825).

[0767] Other suitable linkers generally include organic compounds or polymers, especially those suitable for use in proteins for pharmaceutical use. For example, poly(ethylene glycol) moieties are used to link antibody domains, see, for example, WO 04 / 081026.

[0768] Encompassed within the scope of the present invention is that the length, degree of flexibility and / or other properties of one or more linkers used (but not critically, as it is commonly used in linkers for use in ScFv fragments) may have certain effects on the properties of the final polypeptide of the present invention, said properties of the final polypeptide including but not limited to affinity, specificity or avidity for FcRn or one or more other antigens. Based on the disclosure herein, the person skilled in the art will be able to optionally determine one or more optimal linkers for use in a specific polypeptide of the present invention after some limited routine experiments.

[0769] For example, in a multivalent polypeptide of the present invention comprising building blocks, ISVD or Nanobody V HH directed against a first and a second target, the length and flexibility of the linker preferably allow each building block, ISVD or Nanobody V HH of the present invention present in said polypeptide to bind its cognate target (such as an epitope on each target). Similarly, based on the disclosure herein, the person skilled in the art will be able to optionally determine one or more optimal linkers for use in a specific polypeptide of the present invention after some limited routine experiments.

[0770] Also within the scope of the present invention is: one or more linkers used confer one or more other advantageous properties or functions on the polypeptide of the present invention, and / or provide one or more sites for forming derivatives and / or for attaching functional groups (e.g., as described herein for the ISVD, Nanobody V HH(as described for derivatives of polypeptides). For example, linkers containing one or more charged amino acid residues can provide improved hydrophilic properties, while linkers that form or contain small epitopes or tags can be used for detection, identification, and / or purification purposes. Similarly, based on the disclosure herein, those skilled in the art will be able to optionally determine the optimal linker for a specific polypeptide of the present invention after some limited routine experiments.

[0771] Finally, when two or more linkers are used in the polypeptides of the present invention, these linkers can be the same or different. Similarly, based on the disclosure herein, those skilled in the art will be able to optionally determine the optimal linker for a specific polypeptide of the present invention after some limited routine experiments.

[0772] Generally, for ease of expression and production, the polypeptides of the present invention will be linear polypeptides. However, the present invention is not limited thereto in its broadest sense. For example, when the polypeptides of the present invention contain three or more building blocks, ISVDs, or Nanobody Vs HH they can be linked by using a linker having three or more "arms", each "arm" being linked to a building block, ISVD, or Nanobody V HH to provide a "star" construct. Cyclic ring constructs can also be used, although they are generally less preferred.

[0773] The present invention also relates to methods for preparing the polypeptides, ISVDs, compounds, and constructs described herein. The polypeptides, ISVDs, compounds, and constructs of the present invention can be prepared in a manner known per se, as will be clear to those skilled in the art from the further description herein. For example, the polypeptides, ISVDs, compounds, and constructs of the present invention can be prepared in any manner known per se for preparing antibodies, particularly for preparing antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments). Some preferred but non-limiting methods for preparing polypeptides and constructs include the methods and techniques described herein.

[0774] Methods for producing the polypeptides, ISVDs, compounds, and constructs of the present invention may include the following steps:

[0775] The nucleic acid encoding the ISVD, polypeptide, or protein construct described herein is expressed in a suitable host cell or host organism (also referred to herein as "the host of the present invention") or in another suitable expression system, optionally followed by:

[0776] Isolating and / or purifying the polypeptides, ISVDs, compounds, and constructs of the present invention thus obtained.

[0777] In particular, such methods may include the following steps:

[0778] Culturing and / or maintaining a host cell or host organism of the invention under conditions such that the host cell or host organism of the invention expresses and / or produces at least one polypeptide, ISVD, compound, and / or construct of the invention; optionally then

[0779] Isolating and / or purifying the polypeptide, ISVD, compound, and / or construct of the invention thus obtained.

[0780] 5.2 Nucleic acid sequences and gene constructs

[0781] Accordingly, the invention also relates to a nucleic acid or nucleotide sequence (also referred to as "nucleic acid of the invention" or "nucleotide sequence of the invention") encoding an ISVD, polypeptide, compound, (fusion) protein, or (multispecific) construct of the invention. The nucleic acid of the invention can be in the form of single-stranded or double-stranded DNA or RNA, and is preferably in the form of double-stranded DNA. For example, the nucleotide sequence of the invention can be genomic DNA, cDNA, or synthetic DNA (such as DNA whose codon usage has been specifically adjusted for expression in the intended host cell or host organism).

[0782] According to one embodiment of the invention, the nucleic acid of the invention is in substantially isolated form, as defined herein. The nucleic acid of the invention can also be in the form of a vector, present in a vector and / or as part of a vector, such as a plasmid, cosmid, or YAC, which can also be in substantially isolated form. A nucleic acid sequence is considered to be "(in) substantially isolated (form)" - for example, compared to its natural biological source and / or the reaction medium or culture medium from which it is obtained - when it is separated from at least one other component that is normally associated with it in said source or medium, such as another nucleic acid, another protein / polypeptide, another biological component or macromolecule, or at least one contaminant, impurity, or minor component. In particular, a nucleic acid sequence or amino acid sequence is considered to be "substantially isolated" when it is purified at least 2-fold, especially at least 10-fold, more especially at least 100-fold, and up to 1000-fold or more. The nucleic acid sequence in "substantially isolated form" is preferably substantially homogeneous, as determined using suitable techniques (such as suitable chromatography techniques, such as polyacrylamide gel electrophoresis).

[0783] For the purpose of comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated as follows: [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions in the second nucleotide sequence] / [the total number of nucleotides in the first nucleotide sequence]) × [100%], where each deletion, insertion, substitution, or addition of a nucleotide in the second nucleotide sequence compared to the first nucleotide sequence is considered a difference at a single nucleotide (position).

[0784] Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0, using standard settings. Some other techniques, computer algorithms and settings for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2 357 768-A. Generally, in order to determine the percentage of "sequence identity" between two nucleotide sequences according to the above calculation method, the nucleotide sequence with the largest number of nucleotides will be taken as the "first" nucleotide sequence and the other nucleotide sequence will be taken as the "second" nucleotide sequence.

[0785] The nucleic acids of the present invention can be prepared or obtained in a manner known per se based on the information given herein regarding the polypeptides or protein constructs of the present invention, and / or can be isolated from suitable natural sources. Additionally, it will be clear to the person skilled in the art that, in order to prepare the nucleic acids of the present invention, several nucleotide sequences (such as at least one nucleotide sequence encoding an immunoglobulin single variable domain of the present invention) and, for example, nucleic acids encoding one or more linkers can also be ligated together in a suitable manner.

[0786] The techniques for generating the nucleic acids of the present invention will be clear to the person skilled in the art and can include, for example, but are not limited to automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and / or synthetic sequences (or two or more parts thereof), introducing mutations that result in the expression of truncated expression products; introducing one or more restriction sites (for example, to generate cassettes and / or regions that are easily digestible and / or ligatable using suitable restriction enzymes), and / or introducing mutations by PCR reactions using one or more "mismatch" primers. These and other techniques will be clear to the person skilled in the art and reference is again made to the standard manuals mentioned in the text (such as Sambrook et al. and Ausubel et al.) and the examples below.

[0787] The nucleic acids of the present invention can also be in the form of, present in and / or part of a gene construct, as will be clear to the person skilled in the art. Such a gene construct generally contains at least one nucleic acid of the present invention, which is optionally ligated to one or more components of a gene construct known per se, such as one or more suitable regulatory components (such as one or more suitable promoters, enhancers, terminators, etc.) and additional components of the gene construct referred to herein. Such a gene construct containing at least one nucleic acid of the present invention is also referred to herein as "the gene construct of the present invention".

[0788] The gene construct of the present invention can be DNA or RNA, and is preferably double-stranded DNA. The gene construct of the present invention can also be in a form suitable for transforming the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell, or in a form suitable for independent replication, maintenance, and / or inheritance in the intended host organism. For example, the gene construct of the present invention can be in the form of a vector, such as a plasmid, cosmid, YAC, viral vector, or transposon. In particular, the vector can be an expression vector, i.e., a vector capable of providing in vitro and / or in vivo expression (e.g., in a suitable host cell, host organism, and / or expression system).

[0789] In a preferred but non-limiting embodiment, the gene construct of the present invention comprises

[0790] a) at least one nucleic acid of the present invention; operably linked to

[0791] b) one or more regulatory components, such as a promoter and optionally a suitable terminator;

[0792] and also optionally

[0793] c) one or more additional components of gene constructs known per se; wherein the terms "regulatory component", "promoter", "terminator", and "operably linked" have their usual meanings in the art (as further described herein); and wherein the said "additional components" present in the gene construct can be, for example, 3'- or 5'-UTR sequences, leader sequences, selection markers, expression markers / reporter genes, and / or components that may facilitate or increase transformation or integration (efficiency). These and other components suitable for such gene constructs will be clear to those skilled in the art and may depend, for example, on the type of construct used; the intended host cell or host organism; the mode of expression of the nucleotide sequence of the present invention of interest (e.g., by constitutive, transient, or inducible expression); and / or the transformation technique to be used. For example, regulatory sequences, promoters, and terminators known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments) can be used in a substantially similar manner.

[0794] Preferably, in the gene construct of the present invention, the at least one nucleic acid of the present invention and the regulatory component, and optionally the one or more additional components are "operably linked" to each other, which generally means that they are in a functional relationship with each other. For example, if a promoter is capable of initiating or otherwise controlling / regulating the transcription and / or expression of a coding sequence, the promoter is considered to be "operably linked" to the coding sequence (wherein the coding sequence should be understood to be "under the control" of the promoter). Generally, when two nucleotide sequences are operably linked, they will be in the same orientation and generally also in the same reading frame. They are generally also substantially contiguous, but this may not be necessary either.

[0795] 5.3 Hosts and host cells

[0796] The nucleic acid of the present invention and / or the gene construct of the present invention can be used to transform a host cell or a host organism, i.e., for expressing and / or producing the polypeptide or protein construct of the present invention. The host is preferably a non-human host. Suitable hosts or host cells will be apparent to those skilled in the art and can be, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism, such as:

[0797] Bacterial strains, including but not limited to Gram-negative strains such as Escherichia coli; Proteus, such as Proteus mirabilis; Pseudomonas, such as Pseudomonas fluorescens strains; and Gram-positive strains such as Bacillus, such as Bacillus subtilis or Bacillus brevis; Streptomyces, such as Streptomyces lividans; Staphylococcus, such as Staphylococcus carnosus; and Lactococcus, such as Lactococcus lactis strains; Fungal cells, including but not limited to cells from Trichoderma species, such as Trichoderma reesei; Neurospora, such as Neurospora crassa; Sordaria, such as Sordaria macrospora; Aspergillus, such as Aspergillus niger or Aspergillus sojae; or cells from other filamentous fungi; Yeast cells, including but not limited to cells from Saccharomyces species, such as Saccharomyces cerevisiae; Schizosaccharomyces, such as Schizosaccharomyces pombe; Pichia, such as Pichia pastoris or Pichia methanolica; Hansenula, such as Hansenula polymorpha; Kluyveromyces, such as Kluyveromyces lactis; Arxula, such as Arxula adeninivorans;Cells of the genus Yarrowia, such as cells of Yarrowia lipolytica; amphibian cells or cell lines, such as Xenopus oocytes; insect-derived cells or cell lines, such as cells / cell lines derived from Lepidoptera, including but not limited to Spodoptera SF9 and Sf21 cells or cells / cell lines derived from Drosophila, such as Schneider and Kc cells; plant or plant cells, such as tobacco plants; and / or mammalian cells or cell lines, such as cells or cell lines derived from humans, cells or cell lines from mammals, including but not limited to CHO cells, BHK cells (e.g., BHK-21 cells) and human cells or cell lines, such as HeLa, COS (e.g., COS-7) and PER.C6 cells; and all other hosts or host cells known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments), which will be clear to the person skilled in the art. Reference is also made to the general background art cited above, and for example WO 94 / 29457; WO 96 / 34103; WO 99 / 42077; Frenken et al. 1998 (Res. Immunol. 149:589-99); Riechmann and Muyldermans 1999 (J. Immunol. Met. 231:25-38); van der Linden 2000 (J. Biotechnol. 80:261-70); Joosten et al. 2003 (Microb. Cell Fact. 2:1); Joosten et al. 2005 (Appl. Microbiol. Biotechnol. 66:384-92); and further reference data cited herein.;

[0798] For the expression of polypeptides, ISVDs, compounds or constructs in cells, they can also be expressed as so-called "endosomes", as described in, for example, WO 94 / 02610, WO 95 / 22618 and US 7004940; WO 03 / 014960; Cattaneo and Biocca 1997 (Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag); and Kontermann 2004 (Methods 34:163-170).

[0799] According to a preferred but non-limiting embodiment of the present invention, the polypeptide, ISVD, (fusion) protein or construct of the present invention is produced in bacterial cells, particularly bacterial cells suitable for large-scale pharmaceutical production, such as cells of the above-mentioned strains.

[0800] According to another preferred but non-limiting embodiment of the present invention, the polypeptide, ISVD, (fusion) protein or construct of the present invention is produced in yeast cells, particularly yeast cells suitable for large-scale pharmaceutical production, such as cells of the above-mentioned species.

[0801] According to yet another preferred but non-limiting embodiment of the present invention, the polypeptide, ISVD, (fusion) protein or construct of the present invention is produced in mammalian cells, particularly in cells of human origin or human cell lines, and more particularly in cells of human cells or human cell strains (such as the cell strains mentioned above) suitable for large-scale pharmaceutical production.

[0802] Suitable techniques for transforming the host or host cell of the present invention will be apparent to the person skilled in the art and may depend on the intended host cell / host organism and the gene construct to be used. Reference is made again to the above-mentioned manuals and patent applications.

[0803] After transformation, steps may be carried out to detect and select those host cells or host organisms that have been successfully transformed with the nucleotide sequence / gene construct of the present invention. This may be, for example, a selection step based on a selectable marker present in the gene construct of the present invention or a step involving, for example, the detection of the polypeptide of the present invention using specific antibodies.

[0804] The transformed host cells (which may be in the form of a stable cell line) or host organisms (which may be in the form of a stable mutant line or strain) form a further aspect of the present invention.

[0805] Preferably, these host cells or host organisms are such that they express or are (at least) capable of expressing (e.g., under suitable conditions) the ISVD, polypeptide, compound, (fusion) protein or construct of the present invention (and in the case of a host organism: in at least one of its cells, parts, tissues or organs). The present invention also includes further generations, progeny and / or offspring of the host cells or host organisms of the present invention, for example obtained by cell division or by sexual or asexual reproduction.

[0806] Accordingly, in another aspect, the present invention relates to a host or host cell that expresses (or is capable of expressing, where appropriate) the ISVD, polypeptide, (fusion) protein or construct of the present invention; and / or a host or host cell containing a nucleic acid encoding the same. Some preferred but non-limiting examples of such hosts or host cells can generally be as described in WO 04 / 041867, WO04 / 041865 or WO 09 / 068627. For example, the ISVD, polypeptide, (fusion) protein and construct of the present invention can be advantageously expressed, produced or manufactured in yeast strains (such as strains of Pichia pastoris). Reference is also made to WO 04 / 25591, WO 10 / 125187, WO 11 / 003622 and WO 12 / 056000, which also describe the expression / production of immunoglobulin single variable domains and polypeptides comprising said immunoglobulin single variable domains in Pichia pastoris and other hosts / host cells.

[0807] To effect expression of the polypeptide, ISVD, (fusion) protein or construct of the present invention, the transformed host cell or transformed host organism can generally be maintained, sustained and / or cultured under conditions that allow expression / production of the (desired) ISVD, polypeptide, (fusion) protein or construct of the present invention. Suitable conditions will be apparent to the person skilled in the art and will generally depend on the host cell / host organism used, as well as the regulatory components controlling the expression of the (relevant) nucleotide sequence of the present invention. Reference is also made to the manuals and patent applications mentioned in the paragraph above regarding the gene constructs of the present invention.

[0808] Generally, suitable conditions can include using a suitable culture medium, the presence of a suitable food source and / or suitable nutrients, using a suitable temperature, and optionally the presence of a suitable inducer or compound (e.g., when the nucleotide sequence of the present invention is under the control of an inducible promoter); all of which can be selected by the person skilled in the art. Similarly, under such conditions, the ISVD, polypeptide, (fusion) protein or construct of the present invention can be expressed constitutively, transiently or only upon appropriate induction.

[0809] It will also be clear to the person skilled in the art that the polypeptide, ISVD, (fusion) protein or construct of the present invention can be produced (as described above) in an immature form and can then be post-translationally modified depending on the host cell / host organism used. In addition, the ISVD, polypeptide, (fusion) protein or construct of the present invention can be glycosylated, again depending on the host cell / host organism used.

[0810] The polypeptides, ISVDs, (fusion) proteins or constructs of the invention can then be isolated from the host cell / host organism and / or from the culture medium in which the host cell or host organism has been grown, using protein separation and / or purification techniques known per se, such as (preparative) chromatography and / or electrophoresis techniques, differential precipitation techniques, affinity techniques (e.g., using specific, cleavable amino acid sequences fused to the polypeptides or constructs of the invention) and / or preparative immunological techniques (i.e., using antibodies directed against the amino acid sequences to be isolated).

[0811] A polypeptide or protein is considered to be “(in) substantially isolated (form)” - e.g., as compared to its natural biological source and / or the reaction medium or culture medium from which it is obtained - when it is separated from at least one other component that is normally associated with it in said source or medium, such as another protein / polypeptide, another biological component or macromolecule or at least one contaminant, impurity or minor component. In particular, a polypeptide or protein is considered to be “substantially isolated” when it is purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold and up to 1000-fold or more. A polypeptide or protein in “substantially isolated form” is preferably substantially homogeneous, as determined using suitable techniques (e.g., suitable chromatographic techniques, such as polyacrylamide gel electrophoresis).

[0812] 5.4 Pharmaceutical Compositions, Vaccines and Therapeutic and / or Preventive Methods

[0813] The invention also relates to pharmaceutical compositions comprising the polypeptides, ISVD compounds or constructs (multispecific polypeptides) of the invention. Accordingly, the invention also provides for the use of the polypeptides, ISVD compounds or constructs (multispecific polypeptides) of the invention and / or the pharmaceutical compositions in medicine (as a drug). Accordingly, the invention provides polypeptides, ISVD compounds or constructs (multispecific polypeptides) and / or pharmaceutical compositions for use in medicine (as a drug). The invention also relates to the use of the polypeptides, ISVD compounds or constructs (multispecific polypeptides) of the invention and / or the pharmaceutical compositions for therapeutic and / or prophylactic applications (i.e., in methods of therapeutic and / or prophylactic treatment). Accordingly, the invention provides polypeptides, ISVD compounds or constructs (multispecific polypeptides) and / or pharmaceutical compositions of the invention for use in methods of therapeutic and / or prophylactic treatment (i.e., for therapeutic and / or prophylactic applications).

[0814] In the above methods, the polypeptides, ISVDs, compounds or constructs of the invention and / or compositions comprising them can be administered in any suitable manner, depending on the particular pharmaceutical formulation or composition to be used. Thus, the polypeptides, ISVDs, compounds or constructs of the invention and / or compositions comprising them can be administered, for example, orally, intraperitoneally, intravenously, subcutaneously, intramuscularly or by any other route of administration that bypasses the gastrointestinal tract, intranasally, transdermally, topically, by suppository, by inhalation, again depending on the particular pharmaceutical formulation or composition to be used. The clinician will be able to select the appropriate route of administration and the appropriate pharmaceutical formulation or composition for such administration, depending on the disease or disorder to be prevented or treated and other factors known to the clinician.

[0815] As used herein, the term "therapeutic agent" refers to any agent that can be used to prevent, treat and / or manage a disease or disorder, such as a hyperproliferative cell disorder, such as cancer or one or more symptoms thereof. In certain embodiments, the term "therapeutic agent" refers to the multispecific polypeptides of the invention. Preferably, the therapeutic agent is an agent known to be useful for, or that has been or is being used for, the treatment, prevention and / or management of a disease or disorder or one or more symptoms thereof.

[0816] As used herein, a "therapeutically effective amount" in this context refers to the amount of a single therapy or in combination with other therapies that provides a therapeutic benefit in the treatment and / or management of a disease and / or disorder. In one aspect, a therapeutically effective amount refers to a therapeutic amount sufficient to cure, modify, stabilize or control a disease and / or disorder or one or more symptoms thereof. In another aspect, a therapeutically effective amount refers to a therapeutic amount sufficient to alleviate the symptoms of a disease and / or disorder. In another aspect, a therapeutically effective amount refers to a therapeutic amount sufficient to delay or minimize the spread of a disease and / or disorder. In combination with the amount of the multispecific polypeptides of the invention, the term can include an amount that improves overall treatment, reduces or avoids unwanted effects, or enhances the therapeutic effect or synergistic effect with another therapy. In one embodiment, in assays known in the art or described herein, a therapeutically effective amount of a therapy reduces or avoids unwanted effects relative to a control (e.g., a negative control such as phosphate buffered saline), or increases the therapeutic effect or synergistic effect with another therapy by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%.

[0817] As used herein, the term "therapy" refers to any protocol, method, and / or agent that can be used to treat, prevent, and / or manage a disease and / or disorder or its symptoms. In certain embodiments, the term "therapy" ("therapies" and "therapy") refers to biotherapies, supportive therapies, and / or other therapies that can be used to treat, prevent, and / or manage a disease and / or disorder or one or more symptoms thereof known to those of skill in the art, such as medical personnel.

[0818] As used herein, in the context of administering one or more therapies to a subject, the terms "treat", "treatment", and "treating" refer to reducing or ameliorating the progression, severity, and / or duration of a disease or disorder, and / or ameliorating one or more symptoms resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents). In the context of the present invention, the terms "treat", "treatment", and "treating" can refer to therapeutic and / or prophylactic treatment. The term "prophylactic treatment" refers to a therapy that reduces susceptibility to a clinical condition. Thus, the terms "treat", "treatment", and "treating" and their equivalent terms refer to achieving the desired pharmacological or physiological effect, encompassing any treatment of a pathological condition, disease, or disorder in a mammal, including a human. The action can be prophylactic in terms of completely or partially preventing the pathological condition, disease, or disorder or its symptoms and / or therapeutic in terms of partially or completely curing the pathological condition, disease, or disorder and / or the adverse effects attributable to the pathological condition, disease, or disorder. That is, "treatment" includes (1) preventing the occurrence or recurrence of a pathological condition, disease, or disorder in a subject, (2) inhibiting a pathological condition, disease, or disorder, e.g., arresting its development, (3) stopping or terminating a pathological condition, disease, or disorder or at least the symptoms associated therewith such that the host is no longer afflicted with the pathological condition, disease, or disorder or its symptoms, e.g., causing regression of the pathological condition, disease, or disorder or its symptoms, or (4) alleviating, mitigating, or improving a pathological condition, disease, or disorder or the symptoms associated therewith, where improvement broadly refers to at least a reduction in the magnitude of a parameter, such as inflammation, pain, or immunodeficiency.

[0819] The polypeptide, ISVD, compound or construct of the present invention, and / or a composition comprising the same, is administered according to a treatment regimen suitable for preventing and / or treating the disease and / or disorder to be prevented or treated. A clinician is generally able to determine a suitable treatment regimen, depending on factors such as the stage of the disease and / or disorder to be treated, the severity of the disease and / or disorder to be treated and / or the severity of its symptoms, the specific polypeptide, ISVD, compound or construct of the present invention to be used, the specific route of administration and the pharmaceutical formulation or composition to be used, the age, sex, weight, diet, general condition of the patient, and similar factors known to the clinician.

[0820] Typically, the treatment regimen will include administering one or more polypeptides, ISVDs, compounds or constructs of the present invention, or one or more compositions comprising the same, in one or more pharmaceutically effective amounts or doses. The one or more specific amounts or doses to be administered can be determined by the clinician, again based on the above factors.

[0821] Typically, in the above methods, a single polypeptide, ISVD, compound or construct of the present invention will be used. However, it is within the scope of the present invention to use two or more polypeptides, ISVDs, compounds and / or constructs of the present invention in combination.

[0822] The polypeptide, ISVD, compound or construct of the present invention can also be used in combination with one or more additional pharmaceutically active compounds or principles, i.e., as part of a combination treatment regimen, which may or may not result in a synergistic effect. Again, based on the above factors and expert judgment, the clinician will be able to select such additional compounds or principles, as well as a suitable combination treatment regimen.

[0823] In particular, the polypeptide, ISVD, compound or construct of the present invention can be used in combination with other pharmaceutically active compounds or principles used for or useful for preventing and / or treating the diseases and / or disorders cited herein, with the result that a synergistic effect may or may not be obtained. Examples of such compounds and principles, as well as the routes, methods and pharmaceutical formulations or compositions for administering them, will be clear to the clinician.

[0824] When two or more substances or principles are used as part of a combination treatment regimen, they can be administered substantially simultaneously or at different times (e.g., substantially simultaneously, sequentially or according to an alternating pattern) by the same route of administration or by different routes of administration. When the substances or principles are administered simultaneously by the same route of administration, they can be administered as different pharmaceutical formulations or compositions, or as part of a combined pharmaceutical formulation or composition, as will be clear to the person skilled in the art.

[0825] In one aspect, the present disclosure provides methods for administering an immunoglobulin single variable domain and polypeptide constructs thereof comprising one or more immunoglobulin single variable domains, polypeptides, compounds, and / or constructs. In some embodiments, the immunoglobulin single variable domain, polypeptide, compound, and / or construct is administered as a pharmaceutical composition. In addition to the immunoglobulin single variable domain and its polypeptide constructs, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0826] Due to the increased half-life of the compounds or polypeptides of the present invention, it is preferred to administer them into the circulation. Thus, they can be administered in any suitable manner that allows the compounds or polypeptides of the present invention to enter the circulation, such as intravenously, by injection or infusion, or in any other suitable manner (including oral administration, subcutaneous administration, intramuscular administration, transdermal administration, intranasal administration, pulmonary administration, etc.). Suitable methods and routes of administration will be clear to those skilled in the art, again for example also from the teachings of the published patent applications of Ablynx N.V., such as WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164, or WO 2009 / 068627.

[0827] The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and that have a reasonable benefit / risk ratio commensurate therewith.

[0828] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, which participates in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0829] Methods for preparing these formulations or compositions include the step of associating the immunoglobulin single variable domain or polypeptide construct with a carrier and optionally one or more accessory ingredients. Generally, the formulations are prepared by uniformly and intimately bringing the immunoglobulin single variable domain or polypeptide construct into association with a liquid carrier or a subdivided solid carrier or both, and then, if necessary, shaping the product.

[0830] The present invention also provides a polypeptide, an ISVD compound or a construct (multispecific polypeptide) and / or a pharmaceutical composition as a vaccine or as an immunogenic composition. Accordingly, the present invention also provides a vaccine comprising the polypeptide, ISVD compound or construct (multispecific polypeptide) and / or pharmaceutical composition of the present invention.

[0831] As used herein, the term "vaccine" refers to a substance or composition that establishes or enhances immunity to a particular disease by inducing an adaptive immune response, including immunological memory, i.e., a substance or composition that stimulates the production of protective antibodies or a protective T cell response when administered to a subject in an effective amount. A vaccine can be prophylactic or therapeutic. In one aspect, the vaccine of the present invention is a prophylactic vaccine.

[0832] The vaccine of the present invention may comprise an "adjuvant". As used herein, the term "adjuvant" refers to a substance that nonspecifically enhances or potentiates the immune response of a recipient host to an immunogenic agent when added to the immunogenic agent and after exposure to the mixture.

[0833] The accompanying drawings, sequence listing and experimental section / examples are only for further illustration of the present invention and should not be construed or understood as limiting the scope of the present invention and / or the scope of the appended patent application in any way, unless expressly stated otherwise herein.

[0834] The embodiments described and discussed in this specification are only intended to teach those skilled in the art the best mode known to the inventors for making and using the present invention. Modifications and variations of the above embodiments of the present invention are possible without departing from the present invention, as will be understood by those skilled in the art from the above teachings. Accordingly, it is to be understood that the present invention may be practiced in a manner different from that specifically described within the scope of the patent application and its equivalents.

[0835] The present invention will now be further described by the following non-limiting preferred aspects, examples and drawings.

[0836] The entire contents of all references cited throughout this application (including literature references, issued patent applications, published patent applications and co-pending patent applications) are hereby expressly incorporated by reference herein, particularly for the teachings cited above.

[0837] 6. Examples

[0838] 6.1 Example 1: Selection of FcRn-Specific ISVD by Phage Display

[0839] Identification of FcRn-binding ISVD clones from a synthetic library. In the synthetic ISVD library, sequence diversity (length and amino acid composition) was introduced into the CDRs of the ISVD sequences. The DNA library was cloned into the pAX319 vector. pAX319 is an expression vector derived from the commercially available plasmid vector pUC119. In-frame with the ISVD protein-coding sequence, the vector encodes a C-terminal FLAG3 tag and His 6 tag. pAX319 allows the generation of phage particles displaying individual ISVD proteins as fusion proteins with the pIII protein. Phages were prepared according to standard protocols (see, for example, the prior art and applications filed by Ablynx N.V. cited herein) and stored at 4 °C after filter sterilization for further use. Recombinant biotinylated human FcRn / b2M (hFcRn; Immunitrack ITF01 or ACRO FCM-H82W4) and cynomolgus monkey FcRn / b2M (cFcRn; Immunitrack ITF05 or ACRO FCM-C82W5) were used to mine the library, alternating between selection rounds. Library phage particles were incubated with 0.05 nM - 50 nM biotinylated hFcRn or cFcRn in the presence of 1 μM human b2M (Sino Biologicals 11976-H08H) and in the presence or absence of 5 μM human serum albumin (HSA; Albumin Bioscience 9803) for 2 h, followed by capture on streptavidin- or neutravidin-coated magnetic beads or neutravidin-coated plates. Unbound phages were washed away with CPA pH 5.5 or PBS pH 6.0 buffer (supplemented with 0.05% Tween-20 when using beads), and bound phages were eluted by adding trypsin (1 mg / ml, in PBS), CPA pH 7.4 or PBS pH 7.4 buffer for 5 or 15 min. Trypsin activity was immediately neutralized by adding AEBSF protease inhibitor at a final concentration of 0.8 mM. As a control, selection in the absence of antigen was performed in parallel. The eluted phages were used to infect exponentially growing Escherichia coli TG-1 cells for phage rescue. Phages prepared from the selected output were used as input for subsequent selection rounds. Clones from different selection conditions were individually picked into a 96-well master plate for screening. Periplasmic extracts were prepared according to standard protocols (see, for example, WO 03 / 035694, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551 and other prior art and applications filed by Ablynx N.V. cited herein).

[0840] 6.2 Example 2: Screening and Sequence Analysis

[0841] 6.2.1 Example 2.1. Binding to human FcRn / b2M in ELISA

[0842] Binding to human FcRn / b2M in ELISA was performed in CPA pH 5.5, PBS pH 6.0 or PBS pH 7.4 (supplemented with 0.05% Tween20 and 0.1% casein). For this, high-binding 384-well Spectraplate (PerkinElmer) was coated overnight at 4 °C with 2 μg / mL neutrophilin and blocked the next day at RT in blocking buffer (PBS + 1% casein) for at least one hour. Biotinylated FcRn / b2M (20 nM; human: ACRO (FCM-H82W4)) was captured at room temperature (RT) for one hour. The periplasmic extract of ISVD with Flag 3 -His 6 tag was diluted 1:10 or 1:50 in assay buffer supplemented with 0.05% Tween20 + 0.1% casein and incubated on the coated wells at RT for one to two hours. Unbound ISVD was washed away, and the bound ISVD was detected using mouse anti-FLAG-HRP (Sigma; catalog number A8592) to catalyze the conversion of chromogenic substrate esTMB (SDT Reagents). Absorbance at 450 / 620 nm was read on a Tecan Infinite M1000 or Clariostar-BMG (data not shown). The ISVD clones showing binding to FcRn at acidic pH were sequenced using Sanger sequencing according to a generally known procedure (Pardon et al., 2014).

[0843] 6.2.2 Example 2.2. Determination of dissociation rate by surface plasmon resonance (SPR)

[0844] After sequencing, the unique sequences were rearranged and new periplasmic extracts were generated. The FLAG 3 -His 6 tagged ISVD protein was purified by IMAC using 200 μL Phytips (Phynexus).

[0845] All kinetic measurements were performed on a ProteOn XPR36 (BioRad Laboratories, Inc.), MASS-2 (Sierra Sensors, GmbH), or SPR-32 (Bruker Corporation). For dissociation rate measurements, biotinylated hFcRn / hb2M or cFcRn / cb2M was captured on a ProteOn NLC sensor (BioRad Laboratories, 1765021) or MASS-2 biotin tag capture (Bruker Corporation, 1862620) chip. Phytip-purified periplasmic extracts were diluted 1:10 in running buffer (CPA + 150 mM NaCl + 0.005% Tw20, pH 5.5, pH 6.0, or pH 7.4) and injected over the ligand surface (association: 45 μL / min, 120 s; dissociation: 45 μL / min, 600 s - 3600 s). Experiments were conducted at 25 °C. ISVD clones that did not show a detectable dissociation rate at pH 7.4 or had a dissociation rate difference of at least 10-fold between pH 7.4 and pH 5.5 or pH 6.0 were selected for purification. Dissociation rates obtained for the selected clones are shown in Table 1.

[0846] Table 1. Dissociation Rates of ISVD Clones for Human and Cynomolgus Monkey FcRn at pH 5.5 and pH 7.4

[0847]

[0848] Blank cell: dissociation rate not detected; Italic and Underline : indicative value

[0849] 6.3 Example 3: ISVD Generation, Purification, and Characterization

[0850] By screening k d (1 -s)ISVDs with pH-dependent binding obtained with a difference of more than 10-fold at pH 7.4 relative to pH 5.5 or 6.0 (also including ISVDs that retain specific binding at pH 5.5 or 6.0 but no dissociation rate is detected at pH 7.4) are used for tag-free protein expression and purification. A limited number of ISVDs were ultimately further selected based on their optimal pH-dependent FcRn binding properties (the largest dissociation rate difference between pH 5.5 or 6.0 and 7.4 and the highest affinity at pH 5.5 or 6.0) and their ability to be optimally expressed and produced at medium to large scales. The selection of ISVDs was characterized by affinity measurements for human and cynomolgus monkey FcRn at pH 6.0 and pH 7.4. In addition, these binders were characterized in HSA-competition FACS to group them based on their possible FcRn epitopes (data not shown) and to confirm binding to cell-expressed FcRn at acidic pH.

[0851] 6.3.1 Example 3.1 Generation of ISVD Expression Constructs and Protein Expression

[0852] The ISVD-containing DNA fragments obtained by PCR with specific combinations of forward FR1 and reverse FR4 primers each carrying unique restriction sites were digested with appropriate restriction enzymes and ligated into the matching cloning cassette of the ISVD expression vector (described below). The ligation mixture was then used to transform electrocompetent or chemically competent Escherichia coli TG1 (Lucigen, catalog number 60502 or custom) or TOP10 (ThermoFisher Scientific, catalog numbers C404052 or C4081201 respectively) cells, which were grown under appropriate antibiotic selection pressure (kanamycin or bleomycin). Resistant clones were verified by Sanger sequencing of plasmid DNA (LGC Genomics).

[0853] Monovalent ISVDs were expressed in Escherichia coli TG1 cells (Lucigen, catalog number 60502). Reference is also made to WO 04 / 25591, WO 10 / 125187, WO 11 / 003622 and WO 12 / 056000, which also describe the expression / production of immunoglobulin single variable domains and polypeptides comprising said immunoglobulin single variable domains in Escherichia coli and other hosts / host cells.

[0854] Multivalent ISVD constructs are expressed in Pichia pastoris. The yeast expression vector contains the AOX1 promoter and terminator, the G418 resistance gene, and the coding information for the Saccharomyces cerevisiae α-mating factor signal peptide. The ISVD building blocks are combined with GS linkers and cloned into the expression vector via Golden Gate cloning (Engler C, Marillonnet S. Golden Gate cloning. Methods Mol Biol. 2014;1116:119-31). The expression vector contains two BpiI restriction sites, which are used for cloning the PCR-amplified monovalent ISVDs contained in one or more vectors and the GS linkers. All these components are flanked by BpiI sites. The use of unique nucleotide overhangs at each position in the cloning cassette allows seamless ligation in a predefined order. After Sanger sequencing confirmation, the plasmid DNA derived from E. coli TOP10 is linearized and transformed into the internally prepared highly competent Pichia pastoris strain NRRL Y-11430 (ATCC 76273) by electroporation.

[0855] 6.3.2 Example 3.2 ISVD Purification

[0856] The His 6 -tagged ISVDs are purified by methods known in the art, and the tag-free ISVDs are purified by protein A chromatography known in the art, and the concentration is determined by OD280 / OD340 measurement. Quality control is performed by SDS-PAGE and mass spectrometry.

[0857] 6.3.3 Example 3.3 Affinity Determination of Monovalent and Bivalent ISVDs by Surface Plasmon Resonance (SPR)

[0858] The selected ISVDs are characterized by affinity determination for human FcRn at pH 6.0 and pH 7.4.

[0859] The affinity of monovalent purified tag-free ISVD for human FcRn was determined on a ProteOn XPR36 (BioRad Laboratories, Inc.), MASS-2 (Sierra Sensors, GmbH), or SPR-32 (Bruker Corporation) instrument. In the same manner, the affinity of a bivalent ISVD construct containing two identical purified tag-free ISVD monovalent building blocks for human FcRn was determined on a ProteOn XPR36 (BioRad Laboratories, Inc.), MASS-2 (Sierra Sensors, GmbH), or SPR-32 (Bruker Corporation) instrument. For affinity measurements, approximately 1000 - 3000 RU (hFcRn / hb2M; ACRO Biosystems FCM-H82W4) was captured on a ProteOn NLC sensor (ProteOn) or a biotin-tag capture chip or a high-capacity amine chip (pre-fixed with neutrophilin; MASS-2). ISVD was injected at 6 different concentrations (between 1 and 4000 nM) and allowed to associate for 120 s at 45 μL / min and dissociate for 600 s - 3600 s at 45 μL / min. Since binding was lower at pH 7.4, higher ISVD concentrations were used to determine the affinity at pH 7.4 compared to pH 5.5 or 6.0. Evaluation of the sensorgrams was based on simultaneous fitting of the association and dissociation rates to a 1:1 Langmuir dissociation model. For some selected ISVDs, the affinity for human FcRn at pH 6.0 and pH 7.4 is shown in Table 2.

[0860] Table 2. Affinity of Monovalent and Bivalent ISVD for Human FcRn at pH 6.0 and pH 7.4

[0861]

[0862] 6.4 Example 4: Engineering of T0263018B11

[0863] As described in Example 2.1, ELISA was performed in PBS at pH 6.0 (diluting the periplasmic extract 1:1000) and pH 7.4 (diluting the periplasmic extract 1:10). After screening and sequencing, the phytip-purified periplasmic extract of 206 unique clones (containing the FLAG3-His6 tag) was generated, and the dissociation rates of these clones for human and cynomolgus monkey FcRn at pH 6.0 and pH 7.4 were determined as described in Example 2.2 (see Table 3). Sixteen substitutions with a greater fold difference in kd at pH 7.4 relative to pH 6.0 compared to the parental T0263018B11 were selected for subcloning and purification into tagless ISVD protein (as described in Examples 3.1 and 3.2) and characterized by affinity assays (see Table 4).

[0864] Table 3. Dissociation rates of T0263018B11 single-substitution variants for human and cynomolgus monkey FcRn at pH 6.0 and pH 7.4

[0865]

[0866]

[0867]

[0868]

[0869]

[0870]

[0871] ND: Not determined

[0872] Table 4. Affinities (K D values) of T0263018B11 single-substitution variants for human and cynomolgus monkey FcRn at pH 6.0 and pH 7.4

[0873]

[0874]

[0875] ND: Not determined

[0876] NA: Not available

[0877] Based on the avidity data, a set of untagged ISVD combinatorial variants was generated that included all possible combinations of the mutations G26T, S31E, A33D, S52aQ, S100D, W100cS, and W100cK, including the parental amino acids at all six positions. In addition, some additional single variants at the same Kabat positions were explored, namely S31D, S100E, and W100cR. Periplasmic extracts containing these untagged ISVD variants were generated and the dissociation rates were determined at pH 6.0 (see Table 5). Nineteen variants were selected for purification and further characterized by avidity assays at pH 6.0 and pH 7.4 (see Table 6) and TSA (not shown). Based on the avidity data, five variants (including single and combinatorial variants) of FcRn ISVD T0263018B11 were selected for formatting and in vivo characterization (see Examples 8 and 10).

[0878] Table 5. Analysis of the dissociation rates of T0263018B11 combinatorial variants for human FcRn at pH 6.0

[0879]

[0880]

[0881]

[0882] Table 6. Avidity of T0263018B11 combinatorial variants for human and cynomolgus monkey FcRn at pH 6.0 and pH 7.4

[0883]

[0884]

[0885] Blank cells: Avidity may not have been determined due to low binding; Italic and Underline : Indicative value; NA: Not available.

[0886] 6.5 Example 5: Affinity Determination of Pentavalent ISVD by Surface Plasmon Resonance (SPR)

[0887] Certain obtained mutant variants of the T0263018B11 ISVD building block were selected for further formatting by ligation to one or more reference ISVD building blocks ("REF", which do not bind FcRn) to generate pentavalent ISVD forms. The avidity of the purified pentavalent ISVD constructs for human and cynomolgus monkey FcRn was determined by a similar assay and under the same conditions as described for the monovalent ISVD in Example 3. The avidity is shown in Table 7.

[0888] Table 7. Affinity of pentavalent ISVD forms for human and cynomolgus FcRn at pH 6.0 and pH 7.4

[0889]

[0890]

[0891] 6.6 Example 6: Structural Interaction of FcRn-Binding ISVD T0263018B11 with FcRn

[0892] The structure of the interaction of the FcRn-binding ISVD T0263018B11 with human FcRn(24 - 297; N125Q) / β2M(21 - 119) was determined by co-crystallization of the complex. The human FcRn(24 - 297; N125Q) / β2M(21 - 119) protein was produced in mammalian cells and contained the N125Q mutation to avoid N-glycosylation of the protein.

[0893] The complex was concentrated to 9.5 mg / ml in 10 mM histidine, 150 mM NaCl pH 6. Crystallization was performed using the sitting-drop method. Containing 18% PEG80000, 200 mM NaCl, 50 mM TRIS pH 8.0 (19 °C), 25% ethylene glycol as cryoprotectant, and then frozen. Datasets were collected from two crystals at the beamline Proxima1 of the synchrotron SOLEIL on an EIGRR-X 6M detector (Dectris Ltd.). The crystals belong to the same space group C2 and diffract to The data was processed using autoproc (Vonrhein, C., Flensburg, C., Keller, P., Sharff, A., Smart, O., Paciorek, W., Womack, T., and Bricogne, G. (2011)). Data processing and analysis were performed using the autoPROC toolbox (Acta Cryst. D67, 293 - 302.), which relies on XDS (Kabsch, W. XDS. Acta Cryst. D66, 125 - 132 (2010)) and Aimless (P. R. Evans and G. N. Murshudov, 'How good are my data and what is the resolution?' Acta Cryst. D69, 1204 - 1214 (2013)). The model of FcRn(24 - 297; N125Q) / b2M(21 - 119) was obtained using the pdb structure 4N0U as a reference. The model of ISVDT0263018B11 in complex with FcRn was constructed in Maestro (Schrodinger Release 2021 - 4: Maestro, LLC, New York, NY, 2021). Molecular replacement was carried out using Phaser (Coy et al., J. Appl. Cryst. (2007) 40, 658 - 674) of the CCP4 suite (Winn et al., Acta Cryst D67 (2011), 235 - 242). Cycles using Buster (Buster - TNT 2.11.5, Global Phasing Ltd) were used to refine the structure, and then manual correction was performed in COOT (Emsley, P. and Cowtan, K. (2004). Acta Cryst. D60, 2126 - 2132.) to give a final Rfree of 24.3% and an Rfactor of 20.7%.

[0894] Rsym and Rmerge - the formula for both is:

[0895]

[0896] where is the mean of the unique reflection observations related to symmetry (or Friedel).

[0897] R and Rfree:

[0898]

[0899] where and They must be scaled proportionally to each other. R and Rfree differ in the calculation of their reflection sets (https: / / strucbio.biologie.uni-konstanz.de / ccp4wiki / index.php?title=R-factors).

[0900] The residues involved in the epitope-complementarity determining regions were identified using a suite of software: Schrodinger Release 2021-4: Maestro, LLC, New York, NY, 2021. The FcRn epitope bound by T0263018B11 was compared to the epitopes bound by the native ligands HSA and IgG present in PDB (Protein Data Bank) ID: 4N0U, and the molecular surface representations were as Figure 2 and Figure 3 shown (PyMOL 2.3.0).

[0901] 6.7 Example 7: Generation and Affinity Determination by Surface Plasmon Resonance (SPR) of Multivalent ISVD Constructs Containing ISVD ALB23002 That Binds Serum Albumin

[0902] Based on the FcRn affinity at pH 6.0 and pH 7.4, multiple ISVDs were selected for formatting with the HSA-binding ISVD building block ALB23002 (SEQ ID NO: 35). The FcRn-binding ISVD building blocks were linked to ALB23002 via a 35GS linker (SEQ ID NO: 48), and a C-terminal alanine was added. In some forms, additional target-binding ISVD building blocks (X, Y) were also added to represent potential future therapeutic leads. These forms were expressed in Pichia pastoris and purified as described in Examples 3.1 and 3.2.

[0903] The affinity of the purified multivalent ISVD constructs for human and cynomolgus monkey FcRn was determined on a MASS-2 (Sierra Sensors, Gmbh) instrument. hFcRn / hb2M and cFcRn / cb2M were captured on a biotin-tag capture chip or a high-capacity amine chip (pre-fixed with neutrophilin). The ISVD constructs were injected at 6 different concentrations (between 1 and 4000 nM) and allowed to associate for 120 s at 45 μL / min and dissociate for 600 s - 3600 s at 45 μL / min. Due to the lower binding at pH 7.4, higher ISVD construct concentrations were used to determine the affinity at pH 7.4 compared to pH 6.0. The evaluation of the sensorgrams was based on simultaneous fitting of the binding and dissociation rates to a 1:1 Langmuir dissociation model. The affinities are shown in Tables 8 and 9.

[0904] Table 8. Affinity of constructs containing FcRn-binding ISVD (FcRn) and serum albumin-binding ISVD (ALB23002) for human and cynomolgus monkey FcRn at pH 6.0 and pH 7.4

[0905]

[0906] NA: Not available; Italic and Underline : Indicative value

[0907] Table 9. Affinity of constructs containing FcRn-binding ISVD (FcRn), serum albumin-binding ISVD (ALB23002), and ISVD building blocks binding additional targets (X, Y) for human and cynomolgus monkey FcRn at pH 6.0 and pH 7.4

[0908]

[0909]

[0910] NA: Not available

[0911] The affinity of purified multivalent ISVD constructs for human and mouse serum albumin (HSA and MSA, respectively) was determined on a ProteOn XPR36 (BioRad Laboratories, Inc.) instrument. HSA / MSA (HSA: Sigma-Aldrich–Sigma, catalog number A8763; MSA: Albumin Bioscience, catalog number 2601) was immobilized on a ProteOn GLC sensor chip (Biorad Laboratories, 1765011). The ISVD constructs were injected at 6 different concentrations (between 1 and 500 nM) and allowed to associate for 120 s at 45 μL / min and dissociate for 600 s - 3600 s at 45 μL / min. Evaluation of the sensograms was based on simultaneous fitting of the association and dissociation rates to a 1:1 Langmuir dissociation model. The affinities are shown in Tables 10 and 11.

[0912] Table 10. Affinity of constructs containing FcRn-binding ISVD (FcRn) and serum albumin-binding ISVD (ALB23002) for human and mouse serum albumin at pH 6.0 and pH 7.4

[0913]

[0914] Italic and Underline : Indicative value

[0915] Table 11. Affinity and dissociation rates of constructs containing FcRn-binding ISVD (FcRn), serum albumin-binding ISVD (ALB23002), and ISVD building blocks (X, Y) binding to additional targets for human and mouse serum albumin at pH 6.0 and pH 7.4

[0916]

[0917]

[0918] 6.8 Example 8: Development and Optimization of Serum PK Assays

[0919] Pharmacokinetic experiments were initiated in TG32 (B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / Dcr) mice to evaluate the half-life of pH-dependent FcRn-binding ISVDs either alone as serum albumin-binding ISVD (ALB23002, SEQ ID NO:35) or in combination with ISVDs (X, Y) binding to additional targets or gene-fused to a control ISVD (REF). A specific and sensitive ligand-binding assay was developed to measure the concentration of all constructs in mouse serum.

[0920] Streptavidin-coated MSD GOLD 96-well plates (Meso Scale Discovery) were blocked with Superblock T20 TM (Thermo Scientific) for 30 minutes at RT. The plates were then washed and incubated with 1.0 μg / mL biotinylated universal mAb against the framework of different ISVD building blocks for 1 hour at RT at 600 rpm. Calibrators and QCs were prepared in pooled mouse serum. After washing the plates, the calibrators, QCs, and samples were applied to the plates at 20 MRD in PBS 0.1% casein and incubated for 1 hour at RT and 600 rpm. After washing, the plates were incubated with 2.0 μg / mL sulfo-tagged mAb against the specific ISVD building block, depending on the form being evaluated, for 1 hour at RT at 600 rpm. After washing the plates, 2x MSD read buffer (Meso Scale Discovery) was added, and the plates were read on a Sector Imager Quickplex SQ 120 (Meso scale Discovery).

[0921] For in vivo experiments using a mixture of human IgG (hIVIG; ) to mimic endogenous IgG competition, hIgG assay interference was evaluated.

[0922] 6.9 Example 9:Pharmacokinetics of Bispecific ISVD Constructs in Transgenic Mice

[0923] 1.9 mg / kg of the bispecific ISVD construct was injected into the tail vein of six to eight Tg32 mice (B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / DcrJ). The bispecific ISVD construct consists of two gene-fused ISVD domains, with a negative control (IRR) ISVD or an FcRn-targeting ISVD (FcRn) at the N-terminus and an albumin-targeting ISVD (Alb23002) at the C-terminus.

[0924] Blood was collected at different time points (two mice per time point) and serum was prepared. As described in Example 8, serum samples were analyzed by ELISA for the presence of the bispecific ISVD construct. The results are shown in Figure 4 in.

[0925] The half-life values were obtained by estimating the in vivo endosomal FcRn affinity in a mechanistic model and are reported in Table 12.

[0926] We can conclude from the results that the half-life of the bispecific ISVD containing both an FcRn-binding and an albumin-binding ISVD can be significantly improved compared to constructs containing only an albumin-binding ISVD. The half-life of the FcRn-Alb23002 bispecific ISVD was up to 8.2 days (for T026301170, which contains the FcRn-binding ISVD T0263018B11), while that of the control group (IRR-Alb23002) was 2.4 days.

[0927] Table 12. Calculated half-lives (days) of bispecific ISVD constructs (anti-FcRn-Alb23002 form)

[0928]

[0929] 6.10 Example 10: Pharmacokinetics in Mice of Bispecific ISVD Constructs Containing Mutant Variants of T0263018B11 Pharmacokinetics

[0930] 1.9 mg / kg of the bispecific ISVD construct was injected into the tail vein of two to eight Tg32 mice (B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / DcrJ). The bispecific ISVD construct consists of two gene-fused ISVD domains, with a negative control (IRR) ISVD or an FcRn-targeting ISVD (FcRn) at the N-terminus and an albumin-targeting ISVD (ALB23002) at the C-terminus. The FcRn-targeting ISVD is the parental sequence or a mutant variant with reduced affinity at neutral pH (see Example 4).

[0931] Blood was collected at different time points (2 mice per time point) and serum was prepared. As described in Example 8, serum samples were analyzed by ELISA for the presence of the bispecific ISVD construct. The results are shown in Figure 5 . The half-life values were obtained by estimating the in vivo endosomal FcRn affinity in a mechanistic PBPK model and are reported in Table 13.

[0932] We can conclude from the results that the half-life was significantly improved in the bispecific ISVD constructs containing the mutant variant of T0263018B11 and the albumin-binding ISVD, compared to the control (IRR-Alb23002-IRR0122) ISVD.

[0933] For all mutant variants evaluated, the half-life was increased by at least 2.2-fold and up to 3.7-fold compared to the control (IRR-Alb).

[0934] Table 13. Calculated half-lives of bispecific ISVD constructs (mutant variants in the form of anti-FcRn T0263018B11-Alb23002 or T0263018B11-Alb23002)

[0935]

[0936] 6.11 Example 11: Pharmacokinetics in Mice of Multivalent ISVD Constructs Containing FcRn-Binding ISVD (FcRn), Serum Albumin-Binding ISVD (ALB23002) and ISVD Building Blocks (X, Y) That Bind Additional Targets

[0937] To simulate the relevant competition with hIgG, Tg32 mice (B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / DcrJ) were pre-loaded with a mixture of purified hIgG (hIVIG; ). It was injected intravenously once a week, with the first administration 2 days before the start of the PK study. A total of 3 injections of 250 mg / kg of were administered, generating a physiologically relevant hIgG serum concentration during the study (data not shown). All groups received treatment, and another group did not receive This group allowed the evaluation of the effect of hIgG on PK, which was expected in the case where the FcRn-binding ISVD and IgG Fc compete for the same epitope.

[0938] At the first Two days after administration, equimolar amounts of multivalent ISVD constructs composed of 3 to 4 gene-fused ISVD domains (the forms and building blocks used in these constructs were described in Example 7 above) were injected into the tail veins of 4 to 6 Tg32 mice (B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / DcrJ). Each construct contained 2 ISVD domains (X, Y) targeting different potentially therapeutically relevant targets, which were fused to a single albumin-binding ISVD (Alb23002) (trivalent) or a combination of an FcRn-binding ISVD (FcRn) and an albumin-binding ISVD (Alb23002) (tetravalent). In addition, a tetravalent control ISVD (IRR00164) containing 3 non-targeting ISVDs (IRR) and one albumin-binding ISVD (Alb23002) was also administered.

[0939] Blood was collected at different time points (2 mice per time point) and serum was prepared. As described in Example 7, serum samples were analyzed by ELISA for the presence of multivalent ISVD constructs.

[0940] The results are shown in Figure 6 Half-life values were obtained by estimating endosomal FcRn affinity in vivo in a mechanistic PBPK model and are reported in Table 14.

[0941] Compared to control ISVD constructs containing only albumin-binding ISVDs, the combination of an FcRn-binding ISVD and an albumin-binding ISVD in multivalent ISVD constructs containing ISVD domains targeting potentially relevant therapeutic targets significantly improved the half-life. The position of the FcRn-binding ISVD and the linker length between the FcRn-binding ISVD and the albumin-binding ISVD did not affect the pharmacokinetic properties (half-life) of the constructs. In addition, the half-life was not affected by the presence of relevant levels of hIgG, indicating that the FcRn-binding ISVD binds to a different epitope compared to the epitope bound by IgG Fc. For all multivalent ISVD constructs evaluated, the half-life was increased by 2-fold compared to the control ISVD form.

[0942] Table 14. Forms and calculated half-lives of multivalent ISVD constructs evaluated in Tg32 mice

[0943]

[0944] * T02601257 was evaluated in the presence and absence of

[0945] $T026301366 contains a pH-engineered variant of the VHH 18B11 that binds to FcRn

[0946] 6.12 Example 12: Pharmacokinetics of Multivalent FcRn-Binding ISVD Constructs Containing pH-Engineered Variants of T0263018B11 Constructs

[0947] Intravenously inject 4.5 mg / kg of a multivalent ISVD construct into the tail vein of six to nine Tg32 mice (B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / DcrJ). The multivalent ISVD construct consists of a 5-gene fusion ISVD domain that contains one or two ISVDs targeting FcRn (FcRn) and a negative control (IRR) ISVD, or a control construct that contains only the negative control (IRR) ISVD (IRR00245). The ISVD targeting FcRn is the parental sequence or a mutant variant with reduced affinity at physiological pH (see Example 4).

[0948] Blood is collected at different time points (2-3 mice per time point) and serum is prepared. Serum samples are analyzed by ELISA for the presence of the ISVD construct as described in Example 8. The results are shown in Figure 7 . The half-life values are obtained by estimating the in vivo endosomal FcRn affinity in a mechanistic PBPK model and are reported in Table 15.

[0949] Compared to the control (IRR00245) ISVD construct, the multivalent ISVD constructs containing T0263018B11 or the T0263018B11 mutant variant showed improved half-lives.

[0950] Table 15. Forms and calculated half-lives of multivalent ISVD constructs evaluated in Tg32 mice

[0951]

[0952] *Higher uncertainty due to poor curve fitting

[0953] 6.13 Example 13: Composition of the Polypeptide Constructs of the Invention

[0954] 6.13.1 The Fc domain in the polypeptide according to a specific embodiment of the present invention

[0955] The polypeptides according to the invention (the multispecific polypeptides as described above) may also comprise an Fc domain of IgG, as described herein. The Fc domain of IgG refers to the C-terminal non-antigen-binding region of the immunoglobulin G heavy chain, which contains at least a portion of the constant region. In certain embodiments, the Fc domain may be a native Fc region, i.e., it exists in a native antibody, or it may be a variant Fc region that contains one or more alterations, mutations, or variations compared to the native Fc domain. In certain embodiments, the Fc domain of IgG may also be a fragment of the native Fc domain or a fragment of a variant Fc domain.

[0956] 6.13.1.1. Native (i.e., wild-type) Fc domain of immunoglobulin G (IgG)

[0957] In certain embodiments, the polypeptides as described herein comprise the native Fc domain of human IgG, such as preferably the native Fc of human IgG4 (e.g., Uniprot sequence P01861, SEQ ID NO: 129). Polypeptides containing at least one such native Fc domain are produced and tested for their beneficial PK properties.

[0958] 6.13.1.2. Variant Fc domains with reduced effector function

[0959] In certain specific embodiments, the polypeptides according to the invention comprise variant Fc domains that have altered binding properties to Fc ligands relative to the unmodified parental Fc molecule. For example, the polypeptides described herein may comprise an Fc region in which one or more of the amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 are replaced with different amino acid residues such that the variant Fc region has altered affinity for effector ligands (e.g., Fc receptors or the C1 component of complement), as described in U.S. Patent Nos. 5,624,821 and 5,648,260 to Winter et al.

[0960] In certain embodiments, the polypeptides of the invention comprise an Fc variant domain with reduced effector function, particularly the so-called "FALA" or "LALA" Fc mutants in which residues 234 and 235 are replaced with alanine. Additional optional mutations include replacement of arginine residue 409 with lysine and deletion of lysine residue 447. In other embodiments, the Fc variant domain comprises the so-called "FALA" mutation and the S228P mutation described herein. These Fc variants are referred to as "pFALA".

[0961] Polypeptides containing at least one Fc domain with the mutations described above are produced and tested for their beneficial PK properties, as described in Example 16 below.

[0962] 6.13.1.3. Variant Fc Domains of IgG with Improved Binding Affinity to the FcRn Receptor

[0963] In certain embodiments, the polypeptide according to the invention comprises an Fc variant domain which shows improved binding to the FcRn receptor as compared to the native Fc domain. Such Fc variants include those having substitutions at one or more of the Fc region residues 259, 308, 428 and 434. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8):6213 - 6216, Hinton et al., 2006, Journal of Immunology 176:346 - 356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Chemistry, 2001, 276(9):6591 - 6604).

[0964] In certain specific embodiments, the polypeptide according to the invention comprises an Fc variant domain in which methionine 428 is replaced by lysine and asparagine 434 is replaced by serine.

[0965] Polypeptides are produced that contain at least one Fc domain having the above mutations and their beneficial PK properties are tested.

[0966] 6.13.1.4. Variant Fc Domains of IgG with Reduced or No Binding to the FcRn Receptor

[0967] In certain embodiments, the polypeptide according to the invention comprises an Fc variant domain which shows reduced or no binding to the FcRn receptor as compared to the native Fc domain. Such Fc variants include those having substitutions at one or more ...

Claims

1. A polypeptide comprising at least one immunoglobulin single variable domain (ISVD) that specifically binds to an epitope on FcRn, wherein the polypeptide is characterized in that the epitope comprises at least one of the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q, and / or 262P, and the amino acid residues are numbered according to SEQ ID NO:

1.

2. The polypeptide according to claim 1, wherein the polypeptide is characterized in that the epitope comprises at least one of the following combinations of amino acid residues: a) 4H and 5L, and / or b) 98L, 99G, 100P, 101D, and 102N, and / or c) 167L, 171R, 174L, 175E, and 177K, and / or d) 255Q, 256H, 257A, 259L, 260A, and 262P, and the amino acid residues are numbered according to SEQ ID NO:

1.

3. The polypeptide according to any one of claims 1 or 2, wherein the polypeptide is characterized in that the epitope comprises at least one of the following combinations of amino acid residues: a) 2E, 3S, 4H, and 5L, and / or b) 97E, 98L, 99G, 100P, 101D, and 102N, and / or c) 98L, 99G, 100P, 101D, 102N, and 103T, and / or e) 167L, 168E, 171R, 174L, 175E, and 177K, and / or d) 205P, 206P, and 207E, and / or e) 255Q, 256H, 257A, 259L, 260A, 261Q, and 262P, and the amino acid residues are numbered according to SEQ ID NO:

1.

4. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide is characterized in that the epitope comprises at least one of the following combinations of amino acid residues: a) 1A, 2E, 3S, 4H, and 5L, and / or b) 164R, 167L, 168E, 171R, 174L, 175E, and 177K, and / or c) 204Y, 205P, 206P, and 230E, and / or d) 205P, 206P, 207E, and 208L, and the amino acid residues are numbered according to SEQ ID NO:

1.

5. The polypeptide according to any one of claims 1 to 4, wherein the epitope of the polypeptide comprises at least the following amino acid residues: 4H, 5L, 98L, 99G, 100P, 101D, 102N, 167L, 171R, 174L, 175E, 177K, 207E, 255Q, 256H, 257A, 259L, 260A, and 262P, and the amino acid residues are numbered according to SEQ ID NO:

1.

6. The polypeptide according to any one of claims 1 to 5, wherein the epitope of the polypeptide comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 167L, 168E, 171R, 174L, 175E, 177K, 205P, 206P, 207E, 255Q, 256H, 257A, 259L, 260A, 261Q, and 262P, and the amino acid residues are numbered according to SEQ ID NO:

1.

7. The polypeptide according to any one of claims 1 to 6, wherein the epitope of the polypeptide comprises at least the following amino acid residues: 1A, 2E, 3S, 4H, 5L, 32P, 97E, 98L, 99G, 100P, 101D, 102N, 103T, 164R, 167L, 168E, 171R, 174L, 175E, 177K, 204Y, 205P, 206P, 207E, 208L, 209Q, 255Q, 256H, 257A, 259L, 260A, 261Q, and 262P, and the amino acid residues are numbered according to SEQ ID NO:

1.

8. The polypeptide according to any one of claims 1 to 7, wherein the at least one immunoglobulin single variable domain (ISVD) specifically binds to the epitope on FcRn in a pH-dependent manner such that the binding affinity at a pH between 5.0 and 6.8 is at least three times higher than the binding affinity at a pH of 7.

4.

9. The polypeptide according to any one of claims 1 to 8, wherein the at least one ISVD that specifically binds to the epitope on FcRn specifically binds to the amino acid residues on FcRn that do not participate in the binding of FcRn to serum albumin and / or do not participate in the binding of FcRn to IgG.

10. The polypeptide according to any one of claims 1 to 9, wherein the at least one ISVD that specifically binds to the epitope on FcRn is composed of 4 framework regions (FR1 to FR4) and 3 complementary determining regions (CDR1 to CDR3), and is characterized in that: a) The CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [= G F T F S S YAM Y] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO: 11; and b) The CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [= AI S S G G GS T D] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO: 12; and c) The CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 13 [= D T L Y T S LT S Y S Y] and / or has an amino acid sequence having 4, 3, 2 or only 1 "amino acid difference" (as defined herein) from the sequence of SEQ ID NO:

13.

11. The polypeptide according to any one of claims 1 to 10, wherein the at least one ISVD that specifically binds to the epitope on FcRn has the sequence of SEQ ID NO: 14 or SEQ ID NO:

15.

12. The polypeptide according to any one of claims 1 to 11, wherein the polypeptide further comprises at least one ISVD that specifically binds to (human) serum albumin.

13. The polypeptide according to claim 12, wherein the at least one ISVD that specifically binds to serum albumin is composed of 4 framework regions (FR1 to FR4 respectively) and 3 complementary determining regions (CDR1 to CDR3 respectively), and is characterized in that: CDR1 is SFGMS (SEQ ID NO: 16), CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO: 17) and CDR3 is GGSLSR (SEQ ID NO: 18), and the CDR is determined according to the Kabat definition; and / or wherein CDR1 is GFTFRSFGMS (SEQ ID NO: 19), CDR2 is SISGSGSDTL (SEQ ID NO: 20) and CDR3 is GGSLSR (SEQ ID NO: 21), and the CDR is determined according to the AbM definition.

14. The polypeptide according to any one of claims 1 to 13, wherein the polypeptide further comprises the Fc region of an immunoglobulin (Ig).

15. The polypeptide according to any one of claims 1 to 14, wherein the polypeptide further comprises at least one ISVD that specifically binds to a therapeutic target.

16. Use of the polypeptide according to any one of claims 1 to 15 for prolonging the in vivo half-life of a therapeutic or diagnostic compound.

17. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 15, wherein the pharmaceutical composition is for treating a disease or disorder.

18. A method for producing a polypeptide as claimed in any one of claims 1 to 15, said method comprising at least the following steps: a. expressing a nucleic acid sequence encoding a polypeptide as claimed in any one of claims 1 to 15 in a suitable host cell or host organism or in another suitable expression system; subsequently optionally: b. isolating and / or purifying a polypeptide as claimed in any one of claims 1 to 15.

19. A nucleic acid sequence encoding a polypeptide as claimed in any one of claims 1 to 15.

20. A non-human host or host cell comprising a vector, said vector comprising the nucleic acid sequence as claimed in claim 19 or expressing the nucleic acid sequence as claimed in claim 19.

21. A polypeptide as claimed in any one of claims 1 to 14 or a pharmaceutical composition as claimed in claim 17, said polypeptide or said pharmaceutical composition for use in therapy.

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