Polypeptides that bind to specific epitopes of neonatal Fc receptors

Abstract

The present invention relates to polypeptides capable of binding to specific epitopes on the neonatal Fc receptor (FcRn). In particular, the present invention relates to new and improved polypeptides comprising immunoglobulin single variable domains (ISVDs), e.g., heavy chain single variable domains, capable of binding to specific epitopes on FcRn. The present invention further relates to constructs, compounds, molecules, or chemical entities comprising at least one of these ISVDs that bind to a specific epitope on FcRn. The present invention further relates to methods for producing such polypeptides and to the use of such polypeptides for a variety of 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.

Description

[Technical Field]

[0001] 1.Technical Field 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 new and improved polypeptides comprising immunoglobulin single variable domains (ISVDs), e.g., heavy chain single variable domains, capable of binding to specific epitopes on FcRn. The present invention further relates to constructs, compounds, molecules, or chemical entities comprising at least one of these ISVDs that bind to a specific epitope on FcRn.

[0003] The present invention further relates to methods for producing such polypeptides and to the use of such polypeptides for a variety of 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. [Background technology]

[0004] 2.Technical background Neonatal Fc receptors (FcRn) are characteristically beta (β)-2-microglobulin (β2m)-related proteins that are structurally related to the major histocompatibility class I (MHC-I) family but are unable to present antigenic peptides to T cells. FcRn is ubiquitously expressed, primarily on parenchymal, endothelial, and hematopoietic cells. While primarily localized intracellularly, high surface levels of FcRn have also been detected on immune cells such as monocytes and macrophages. FcRn has two known ligands, IgG and albumin, which together account for approximately 70% of all serum proteins.

[0005] While IgG subtypes are essential for immune responses, albumin functions as a carrier protein in addition to being a key regulator of colloid oncotic pressure. Despite these differences, IgG and albumin share a long serum half-life due to their interaction with FcRn, which rescues them from intracellular degradation through an intracellular recycling mechanism. This recycling mechanism occurs in a strictly pH-dependent manner, resulting in binding to FcRn followed by internalization into endosomal compartments at low pH (pH 5-6) and release to the cell surface at physiological pH (typically around pH 7.4). Another function of FcRn is the transport of IgG from mother to child, thereby providing the naive and immature immune system of newborns with the experience and protection developed in adult ancestors. This process is developmentally regulated, occurring prenatally in rodents and through the everted yolk sac or placenta in humans, respectively, but continues at significant levels intrinsically during early postnatal life in rodents due to the high expression of FcRn in the intestinal epithelium. This functional expression of FcRn and its ability to transcytose IgG persist beyond the neonate and throughout life, allowing for the targeted delivery of IgG to sites where the presence of this type of antibody enhances immunity. This process is widely exploited by IgG-based therapeutics. Finally, the function of FcRn is determined differently depending on whether IgG is present as a single molecule, i.e., a monomer, or as an immune complex. In the latter case, FcRn has been shown to process and present antigens contained within the IgG immune complex, as well as critically regulate innate immune responses. Summary of the Invention [Problem to be solved by the invention]

[0006] Because FcRn has many different important physiological roles, there is a clear need for improved FcRn binding agents for therapeutic use. [Means for solving the problem]

[0007] 3. Overview of this technology The present inventors have identified improved FcRn-binding polypeptides that have several advantages over FcRn-binding agents as described in the prior art.

[0008] One advantageous feature of the FcRn-binding polypeptides according to the present invention is that they bind to a unique epitope on FcRn, which is different from the epitopes bound by known natural FcRn ligands (i.e., serum albumin and IgG). Therefore, the present inventors have identified a novel epitope present on FcRn and developed a polypeptide capable of specifically binding to that epitope without interfering with the natural physiological role of FcRn. Thus, the polypeptides of the present invention can be applied to several preventive, diagnostic, and therapeutic applications in which 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 therefore provides a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) that specifically binds to an epitope on FcRn, 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0010] In certain particular embodiments, the polypeptides of the invention comprise epitopes comprising 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 characterized in that it includes at least one of Amino acid residues are numbered according to SEQ ID NO:1.

[0011] In certain further specific embodiments, the polypeptides of the invention comprise epitopes comprising the following combination 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 d) 167L, 168E, 171R, 174L, 175E and 177K, and / or e) 205P, 206P and 207E, and / or f) 255Q, 256H, 257A, 259L, 260A, 261Q, and 262P and Amino acid residues are numbered according to SEQ ID NO:1.

[0012] In certain still further specific embodiments, the polypeptides of the invention comprise epitopes comprising 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 characterized in that it includes at least one of Amino acid residues are numbered according to SEQ ID NO:1.

[0013] According to certain embodiments, the polypeptides of the invention are 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, wherein the amino acid residues are numbered according to SEQ ID NO:1.

[0014] According to a further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0015] According to yet a further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0016] In certain embodiments, the FcRn-binding polypeptides of the present invention bind to novel epitopes on FcRn as disclosed herein in a pH-dependent manner, such that the binding affinity at acidic pH, particularly at acidic pHs of 5.0 to 6.8, is at least three-fold higher than the binding affinity at neutral or physiological pH of about 7.4. In these specific embodiments, the polypeptides of the present invention exhibit conditional specific binding to FcRn and, as a result, have extended in vivo serum half-lives by utilizing the recycling mechanism mediated by FcRn in vivo. Thus, the polypeptides of the present 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, conjugated, or fused.

[0017] 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, which allows the compounds of the invention to be most effectively and optimally applied for some applications, particularly for extending the in vivo half-life of therapeutic molecules.

[0018] According to certain particular 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 to which serum albumin binds.

[0019] According to certain particular 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 to which immunoglobulin G (IgG) binds.

[0020] In certain specific embodiments, the present invention provides FcRn-binding polypeptides comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); a) CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 8 [=SYAMY] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 8; b) CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO: 9 [=AISSGGGSTDYADSVKG] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 9; c) CDR3 (according to Kabat) is characterized in that it has the amino acid sequence of SEQ ID NO: 10 [=DTLYTSLTSYSY] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 10.

[0021] In certain specific embodiments, the present invention provides FcRn-binding polypeptides comprising at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein, wherein the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); a) CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=GFTFSSYAMY] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 11; b) CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [=AISSGGGSTD] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 12; c) CDR3 (according to AbM) is characterized in that it has the amino acid sequence of SEQ ID NO: 13 [=DTLYTSLTSYSY] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 13.

[0022] In a specific embodiment, the FcRn-binding polypeptide of the present 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, or a sequence having 90%, e.g., 95%, sequence identity to the sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0023] In a particular embodiment, the polypeptide according to the invention is 3 nM~10 -2 The dissociation constant (K D ) and specifically binds to FcRn, such as, but not limited to, human FcRn or cynomolgus monkey FcRn. D is determined by Kinexa, BLI or SPR, for example determined by SPR.

[0024] In a particular embodiment, the polypeptide according to the invention is -3 nM -1 ~10 2 nM -1 Affinity (K A ) specifically binds to FcRn.

[0025] In a particular embodiment, the polypeptide according to the invention has a denaturing activity of at least about 10, preferably as measured by surface plasmon resonance or BLI. 2 M -1 s -1 , at least about 103 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 A binding rate constant (k on ) specifically binds to FcRn.

[0026] In a particular embodiment, the polypeptide according to the invention has a denaturing activity of up to about 10, preferably as measured by surface plasmon resonance or BLI. -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 Dissociation rate constant (k off ) specifically binds to FcRn.

[0027] In certain specific embodiments, the present invention provides a polypeptide in which at least one ISVD that specifically binds to FcRn is comprised of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), a) CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 8 [=SYAMY] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 8; b) CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO: 9 [=AISSGGGSTDYADSVKG] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 9; c) CDR3 (according to Kabat) is characterized in that it has the amino acid sequence of SEQ ID NO: 10 [=DTLYTSLTSYSY] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 10.

[0028] In certain specific embodiments, the present invention provides a polypeptide in which at least one ISVD that specifically binds to FcRn is comprised of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), a) CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=GFTFSSYAMY] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 11; b) CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [=AISSGGGSTD] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 12; c) CDR3 (according to AbM) is characterized in that it has the amino acid sequence of SEQ ID NO: 13 [=DTLYTSLTSYSY] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 13.

[0029] In a specific embodiment, the FcRn-binding polypeptide of the present 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, or has 90%, e.g., 95%, sequence identity with the sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0030] In certain specific embodiments, the improved FcRn-binding polypeptides of the present invention are such that they bind to FcRn in a pH-dependent manner.

[0031] In certain specific embodiments, the FcRn-binding polypeptides of the present invention bind to novel epitopes on FcRn in a pH-dependent manner, such that the binding affinity at acidic pH, particularly at an acidic pH of 5.0 to 6.8, is at least three-fold higher than the binding affinity at a pH of about 7.4.

[0032] In a particular embodiment, the polypeptide according to the present invention is soluble in 10% or more of the soluble fraction at an acidic pH of 5.0 to 6.8. -3 nM -1 ~10 2 nM -1 Affinity (K A ) specifically binds to FcRn. The affinity (K) of these polypeptides for FcRn at an acidic pH, preferably a pH of 5.0 to 6.8 A ) is the affinity (K ) of the same polypeptide for FcRn at neutral or physiological pH of about 7.4. A In a further specific embodiment, the affinity of a polypeptide according to the present invention for FcRn at an acidic pH of 5.0 to 6.8 is at least three times higher than that of the same polypeptide at a neutral or physiological pH of about 7.4 (K A In yet a further specific embodiment, the polypeptides according to the present invention bind to FcRn with an affinity (K) at an acidic pH of 5.0 to 6.8 that is at least 10 times higher than the affinity (K) of the same polypeptide for FcRn at a neutral or physiological pH of about 7.4. A ) binds to FcRn with at least 50-fold greater affinity, such as at least 100-fold greater affinity.

[0033] In certain particular embodiments, the invention provides a polypeptide as described herein, 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 3-fold, such as at least 10-fold, such as at least 50-fold, such as at least 100-fold lower than the affinity with which the polypeptide binds to FcRn at an acidic pH of 5.0 to 6.8.

[0034] In certain embodiments, at least one ISVD has a pH of 10 or less at physiological pH, e.g., a pH of about 7.4. 4 K lower than l / mol A It binds to FcRn at a value

[0035] In certain specific embodiments, the invention provides polypeptides as described herein, characterized in that at least one ISVD does not detectably, selectively, or specifically bind to FcRn at neutral or physiological pH, e.g., a pH of about 7.4, or exhibits no binding to FcRn, or does not essentially bind to FcRn.

[0036] In a particular embodiment, the polypeptide according to the present invention is soluble in 10% or more of the soluble fraction at an acidic pH of 5.0 to 6.8. 3 nM~10 -2 The dissociation constant (K D ) specifically binds to FcRn. D is determined by Kinexa, BLI or SPR, for example determined by SPR.

[0037] In a particular embodiment, at an acidic pH of 5.0 to 6.8, the polypeptide according to the present invention has a solubility of at least about 10, preferably as measured by surface plasmon resonance or BLI. 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 A binding rate constant (k on ) specifically binds to FcRn.

[0038] In a particular embodiment, at an acidic pH of 5.0 to 6.8, the polypeptide according to the present invention has a maximum of 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 Dissociation rate constant (k off ) specifically binds to FcRn.

[0039] The polypeptides of the present invention bind to a specific, unique and novel epitope on FcRn, which is distinct from the epitopes bound by the natural ligands of FcRn, namely serum albumin and IgG.

[0040] In a specific embodiment, the FcRn-binding polypeptides according to the present invention are preferably such that, when they bind to or otherwise associate with an FcRn molecule, they do not (significantly) affect, reduce, or inhibit the binding of the FcRn molecule to serum albumin and / or IgG. In this specific embodiment, in a cross-blocking assay (as described herein), when the FcRn-binding polypeptide binds to or otherwise associates with an FcRn molecule, there is less than 40%, e.g., less than 30%, less than 20%, or less than 10%, or essentially no displacement is detected (e.g., in an ELISA or Alphascreen-based competitive assay). In this specific embodiment, in a cross-blocking assay (as described herein), when the FcRn-binding polypeptide binds to or otherwise associates with an FcRn molecule, there is less than 40%, e.g., less than 30%, less than 20%, or less than 10%, or essentially no displacement is detected (e.g., in an ELISA or Alphascreen-based competitive assay).

[0041] In a further specific embodiment, the present invention provides a polypeptide comprising at least one ISVD that specifically binds to an amino acid residue on FcRn that is not involved in the binding of FcRn to serum albumin and / or IgG. In this specific embodiment, the polypeptide comprises at least one ISVD that specifically binds to an epitope on FcRn, wherein the ISVD comprises an amino acid residue that is not included in the epitope on FcRn to which serum albumin binds and / or the epitope on FcRn to which IgG binds. According to this specific embodiment, the polypeptide comprises at least one ISVD that specifically binds to an amino acid residue on FcRn that is not bound by serum albumin and / or IgG.

[0042] According to certain embodiments, the FcRn-binding polypeptides of the present invention are also preferably such that they compete for binding to FcRn with polypeptides comprising the amino acid sequence of SEQ ID NO: 14 and / or SEQ ID NO: 15 and / or that they "cross-block" (as defined herein) the binding to FcRn by polypeptides comprising the amino acid sequence of SEQ ID NO: 14 and / or SEQ ID NO: 15.

[0043] In a specific embodiment, the FcRn-binding polypeptides of the present invention are such that they bind to essentially 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 such that they share the interaction with FcRn of essentially the same amino acids as those of SEQ ID NO: 14 and / or SEQ ID NO: 15. To this end, according to a specific, but non-limiting aspect, the FcRn-binding polypeptides according to the present invention preferably have CDRs identical to those of the sequences of SEQ ID NO: 14 and / or SEQ ID NO: 15, or preferably comprise, compared to the sequences of SEQ ID NO: 14 and / or SEQ ID NO: 15, within their CDRs only those mutations (such as conservative amino acid substitutions) that still enable them to undergo interaction with FcRn of the same or essentially the same amino acids as those of SEQ ID NO: 14 and / or SEQ ID NO: 15.

[0044] In certain specific embodiments, the present invention provides a method for the production of antibodies in which the ISVD is a (single) domain antibody, Nanobody®, V HH , V HH , humanized V HH , or camelization V H The present invention provides a polypeptide as described herein, characterized in that:

[0045] In certain embodiments, the present invention provides a method for producing a pharmaceutical composition comprising: A polypeptide comprising at least one ISVD that specifically binds to FcRn in a pH-dependent manner and at least one further moiety, a) at least one ISVD specifically binds to FcRn in a pH-dependent manner such that the binding affinity at pH 5.0-6.8 is at least three times higher than the binding affinity at a pH of about 7.4; b) the polypeptide has a molecular weight of at least 30 kDa The present invention provides a polypeptide characterized by:

[0046] In certain further specific embodiments, the polypeptides of the invention have a molecular weight of between about 30 kDa and 200 kDa, for example between about 30 kDa and 100 kDa.

[0047] In certain further particular embodiments, the at least one further moiety is a protein moiety, such as a serum protein.

[0048] In certain further particular embodiments, at least one further moiety is a protein-binding moiety, such as a serum protein-binding moiety.

[0049] In certain further particular embodiments, at least one further moiety is a serum protein binding moiety, such as a serum albumin binding moiety.

[0050] In certain further specific embodiments, at least one serum albumin binding moiety specifically binds to an amino acid residue on serum albumin that is not involved in binding of serum albumin to FcRn.

[0051] In certain further particular embodiments, the present invention provides a method for the preparation of a serum albumin-binding molecule, wherein the at least one serum albumin binding moiety is at least one ISVD, such as a (single) domain antibody, Nanobody®, V HH , V HH , humanized V HH , or camelization V H The present invention provides a polypeptide as described herein, characterized in that:

[0052] In certain further specific embodiments, the at least one further moiety is at least one ISVD that binds to serum albumin.

[0053] In certain further particular embodiments, the at least one further moiety is at least one ISVD that binds to serum albumin and essentially consists of four framework regions (FR1 to FR4, respectively) and three 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), with the CDRs determined according to the Kabat definition; and / or an ISVD wherein CDR1 is GFTFRSFGMS (SEQ ID NO: 19), CDR2 is SISGSGSDTL (SEQ ID NO: 20), and CDR3 is GGSLSR (SEQ ID NO: 21), with the CDRs determined according to the AbM definition (Kontermann et al., 2010).

[0054] In certain embodiments, the at least one further moiety is an 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 further moiety, characterized in that: a) at least one ISVD specifically binds to FcRn in a pH-dependent manner such that the binding affinity at pH 5.0-6.8 is at least three times higher than the binding affinity at a pH of about 7.4; b) the polypeptide has a molecular weight of at least 30 kDa, in particular between about 30 kDa and 100 kDa; c) At least one further moiety is an Fc region or domain of an immunoglobulin (Ig).

[0055] In certain embodiments, at least one additional moiety is a non-protein moiety, such as, but not limited to, a polyethylene glycol (PEG) moiety.

[0056] 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 further moiety, characterized in that: a) at least one ISVD specifically binds to FcRn in a pH-dependent manner such that the binding affinity at pH 5.0-6.5 is at least three times higher than the binding affinity at a pH of about 7.4; b) the polypeptide has a molecular weight of at least 30 kDa, in particular between about 30 kDa and 100 kDa; c) At least one further moiety is a non-protein moiety, such as, but not limited to, a polyethylene glycol (PEG) moiety.

[0057] The polypeptide preferably further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptide linkers, which provide the polypeptide with an increased half-life compared to a corresponding polypeptide that does not comprise said one or more other groups, residues, moieties or binding units.

[0058] In certain embodiments, the invention provides a polypeptide as described herein, wherein the polypeptide further comprises a therapeutic moiety.

[0059] In a particular embodiment, the present invention provides a method for the preparation of a polypeptide comprising: a (single) domain antibody, a Nanobody®, a V HH , V HH , humanized V HH , or camelization V H The present invention provides a polypeptide as described herein, further comprising a therapeutic moiety comprising an ISVD such as

[0060] In a further aspect, the present invention provides a nucleic acid or nucleic acid sequence encoding a polypeptide according to the present invention.

[0061] In another aspect, the present invention provides a vector comprising a nucleic acid or nucleic acid sequence according to the present invention.

[0062] 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 invention, or with a vector according to the invention.

[0063] In a further aspect, the present invention provides a method or process for producing a polypeptide according to the present invention, said method comprising: a. expressing the nucleic acid sequence in a suitable (non-human) host cell or host organism, or in another suitable expression system; optionally followed by: b. At least comprising the step of isolating and / or purifying the polypeptide according to the present invention.

[0064] In a still further 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.

[0065] In a further aspect, the present invention provides a polypeptide of the present invention, or a polypeptide produced according to a process of the present invention, for use in treating a subject in need thereof.

[0066] In a further 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 administering to a subject a polypeptide of the invention, or a polypeptide produced by a process according to the invention.

[0067] In a further aspect, the invention provides a polypeptide of the invention, or a polypeptide produced according to a process of the invention, for use in delivering a prophylactic or therapeutic polypeptide to a specific location, tissue or cell type in the body.

[0068] In a still further aspect, the present invention provides a polypeptide of the invention, or a polypeptide produced according to the process of the invention, for use in therapy.

[0069] In a still further aspect, the present invention provides a polypeptide of the invention, or a polypeptide produced according to the process of the invention, for use in the prevention, treatment or amelioration of a disease selected from the group consisting of a proliferative disease, an inflammatory disease, an infectious disease and an autoimmune disease.

[0070] In another aspect, the present invention provides a method for preventing, treating, or ameliorating a disease selected from the group consisting of a proliferative disease, an inflammatory disease, an infectious disease, and an autoimmune disease, comprising at least the step of administering a polypeptide of the present invention, or a polypeptide produced by a method of the present invention, to a subject in need thereof.

[0071] In a further aspect, the present invention provides a kit comprising a polypeptide of the invention, a nucleic acid or nucleic acid sequence of the invention, a vector of the invention, or a host cell of the invention.

[0072] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, which will be apparent to those skilled in the art, see for example the standard handbooks referred to in WO 08 / 020079, page 46, paragraph a).

[0073] It should 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 of such different reagents, and reference to a "method" includes reference to equivalent steps and methods known to those skilled in the art that may be modified or substituted for the methods described herein.

[0074] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

[0075] The term "and / or" wherever used in this specification includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0076] The term "about" when used in the context of a parameter or range of parameters provided herein shall have the following meaning: Unless otherwise indicated, when the term "about" is applied to a particular value or range, that value or range shall be interpreted as being as accurate as the method used to measure it. Unless a tolerance is specified when applied, the last decimal place of the numerical value indicates its degree of precision. Unless another tolerance is given, the maximum tolerance is ascertained by applying rounding conventions to the last decimal place; for example, for a pH value of approximately pH 2.7, the tolerance is 2.65 to 2.74. However, for the following parameters, special tolerances shall apply: temperatures specified in °C without decimal places shall have a tolerance of ±1°C (e.g., a temperature value of approximately 50°C means 50°C ±1°C); durations stated in hours shall have a tolerance of 0.1 hours regardless of the decimal place (e.g., a time value of approximately 1.0 hour means 1.0 hour ±0.1 hour; a time value of approximately 0.5 hour means 0.5 hour ±0.1 hour).

[0077] In this application, any parameter designated with the term "about" is also contemplated as being disclosed without the term "about." In other words, an embodiment that refers to a parameter value using the term "about" is also intended to represent an embodiment directed to such a numerical value of said parameter. For example, an embodiment that specifies a pH as "about pH 2.7" is also intended to disclose an embodiment that specifies such a pH as "pH 2.7"; an embodiment that specifies a pH range as "about pH 2.7 to about pH 2.1" is also intended to represent an embodiment that specifies a pH range as "pH 2.7 to pH 2.1," etc.

[0078] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise," and variations such as "comprises" and "comprising," should be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be replaced with the terms "containing" or "including," or, as sometimes used herein, the term "having." [Brief explanation of the drawings]

[0079] 4. Brief description of the drawings [Figure 1] The sequence of human FcRn, with amino acid residues identified as being involved (directly or indirectly) in binding to at least one of the polypeptides of the invention shown in bold and underlined. [Figure 2A-D] 1 shows the three-dimensional protein structure of the α-chain of FcRn (also known as the Fc fragment of IgG receptor and transporter (FcGRT)), with (A) the epitope of an FcRn-binding polypeptide according to a specific embodiment of the invention darkly shaded, (B) the epitope of the Fc domain of IgG, and (C) the epitope of serum albumin lightly shaded. In (D), the epitope of an FcRn-binding polypeptide according to a specific embodiment of the invention (dark shaded in the center) is shown in combination with both the epitope of the Fc domain of IgG (light shaded on the right) and the epitope of serum albumin (light shaded on the left and in the upper center). [Figure 3A-D]1 shows the three-dimensional protein structure of the heterodimeric FcRn (also known as the Fc fragment of IgG receptor and transporter (FcGRT) and β2-microglobulin), with (A) the epitope of an FcRn-binding polypeptide according to a specific embodiment of the invention darkly shaded, (B) the epitope of the Fc domain of IgG, and (C) the epitope of serum albumin lightly shaded. In (D), the epitope of an FcRn-binding polypeptide according to a specific embodiment of the invention (dark shaded in the center) is shown in combination with the epitope of the Fc domain of IgG (light shaded on the right) and the epitope of serum albumin (light shaded on the left and in the upper center). [Figure 4] Mean (+ / -SD, n=2) serum concentration-time profiles of various test bispecific ISVD constructs containing FcRn-binding ISVD and albumin-binding ISVD (ALB23002) compared to control bispecific ISVD constructs containing control ISVD (IRR) and albumin-binding ISVD (ALB23002) following iv bolus administration at 1.9 mg / kg to female Tg32 mice. [Figure 5] Mean (+ / - SD, n=2) serum concentration-time profiles of test bispecific ISVD constructs comprising parental VHH T0263018B11 or its mutant variants and albumin-binding ISVD (ALB23002) compared to control bispecific ISVD constructs comprising control ISVD (IRR) and albumin-binding ISVD (ALB23002) following iv bolus administration at 1.9 mg / kg to female Tg32 mice. [Figure 6] Mean (+ / -SD, n=2) serum concentration-time profiles of multivalent ISVD test constructs, including an FcRn-binding ISVD, an albumin-binding ISVD, and additional ISVDs directed against potentially relevant therapeutic targets, following iv bolus administration to female Tg32 mice. [Figure 7] Mean (+ / -SD, n=2–3) serum concentration-time profiles of multivalent ISVD test constructs containing one or more FcRn-binding ISVDs and one or more control ISVDs after iv bolus administration at 4.5 mg / kg to female Tg32 mice. [Figure 8] Schematic diagram of the structural format of fusion polypeptide constructs, for example, as described in Example 14. The Fc domain in the constructs was an IgG4 FALA Fc framework sequence variant containing knobs-in-holes mutations as described herein. One of these constructs (i.e., TP049) contained additional amino acid differences or mutations in the Fc framework sequence (i.e., I253A, H310A, H435A) and is designated as IgG Fc(IHH). The FcRn Nanobody® VHH used was in each case the T0263018B11 sequence (FcRn ISVD 1 in the figure) as described herein. A Nanobody® VHH that does not bind to FcRn or any other putative target is designated as "Control ISVD" in the figure. The Nanobody® VHH sequences in these fusion proteins were fused to the N-terminus and / or C-terminus of the Fc chain via a linker (as described in detail herein), i.e., via an IgG1 hinge (e.g., SEQ ID NO: 126) and / or a GS linker (e.g., a 35GS linker, SEQ ID NO: 48), respectively, as described herein. [Figure 9] Surface plasmon resonance (SPR) sensorgrams on a Biacore 8K+ instrument using different coating densities of FcRn (up to 5000 RU). Nanobody® VHH-Fc proteins were characterized by measuring their affinity for human FcRn at pH 6.0 (A) and pH 7.4 (B). [Figure 10] Mean (+ / -SD, n=4-6) serum concentration-time profile of ISVD-Fc fusion protein containing FcRn-binding ISVD and the Fc region of IgG4 FALA after iv bolus administration to female Tg32 mice. DETAILED DESCRIPTION OF THE INVENTION

[0080] 5. Detailed Description The present inventors have identified a unique epitope on FcRn that is distinct from the epitopes of its natural ligands, such as serum albumin and IgG. Additionally, and more importantly, the present inventors have developed novel polypeptides that can specifically bind to that epitope on FcRn without interfering with the natural physiological role of FcRn. Polypeptides as 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 isoforms. Isoforms are alternative protein sequences that can be generated from the same gene by a single biological event or by a combination of biological events, such as the use of alternative promoters, alternative splicing, alternative initiation, and ribosomal frameshifting, all as known in the art.

[0081] Amino acid residues are referred to herein interchangeably according to the standard three-letter or one-letter amino acid code, as referenced in Table B-1 below.

[0082] [Table 1]

[0083] When an amino acid residue is designated as "X" or "Xaa," this means that the amino acid residue is unspecified unless a more restrictive interpretation is required by the context. For example, when the amino acid sequence of a CDR is described and one (or more) of the amino acid residues is designated as "X," this description can further specify which amino acid residue is (may be) present at that particular position in the CDR.

[0084] The amino acids are L-amino acids commonly found in naturally occurring proteins and are listed in Table B-1. Those amino acid sequences containing D-amino acids are not intended to be encompassed by this definition. Any amino acid sequence containing post-translationally modified amino acids may be described as the originally translated amino acid sequence using the symbols shown in Table B-1, along with the modified positions, e.g., hydroxylation or glycosylation, but these modifications shall not be explicitly indicated in the amino acid sequence. Any peptide or protein that can be represented as a sequence modified by linkages, cross-links, and end caps, non-peptidyl bonds, etc., is encompassed by this definition. The terms "protein," "peptide," "protein / peptide," and "polypeptide" are used interchangeably throughout this disclosure, and each has the same meaning for purposes of this disclosure. Each term refers to an organic compound made from two or more amino acids in a linear chain. The compound may have 10 or more amino acids, 25 or more amino acids, 50 or more amino acids, 100 or more amino acids, 200 or more amino acids, or even 300 or more amino acids. Those skilled in the art will understand that, although polypeptides generally contain fewer amino acids than proteins, there is no art-recognized demarcation line in the number of amino acids that distinguishes a protein from a polypeptide; that polypeptides can be made by chemical synthesis or recombinant methods; and that proteins are generally made in vitro or in vivo by recombinant methods as known in the art.

[0085] When a nucleotide sequence or amino acid sequence is said to "comprise" or "consist essentially of" another nucleotide sequence or amino acid sequence, respectively, this may mean that the latter nucleotide sequence or amino acid sequence is incorporated into the first-mentioned nucleotide sequence or amino acid sequence, respectively, but more generally means that the first-mentioned nucleotide sequence or amino acid sequence contains within its sequence a stretch of nucleotides or amino acid residues, respectively, having the same nucleotide sequence or amino acid sequence as the latter sequence, regardless of how the first-mentioned sequence was actually generated or obtained (which may be by any suitable method described herein). By way of non-limiting example, when an ISVD is said to contain a CDR sequence, this may mean that the CDR sequence is incorporated into the ISVD, but more generally means that the ISVD contains within its sequence a stretch of amino acid residues comprising the same amino acid sequence as the CDR sequence, regardless of how the ISVD was generated or obtained. It should also be noted that, if the latter amino acid sequence has a particular biological or structural function, it preferably has essentially the same, similar, or equivalent biological or structural function as the first-mentioned amino acid sequence (in other words, the first-mentioned amino acid sequence is preferably such that the latter sequence is capable of performing essentially the same, similar, or equivalent biological or structural function). For example, when an ISVD is said to comprise a CDR sequence or a framework sequence, respectively, it is preferred that the CDR sequence and framework are capable of functioning as a CDR sequence or a framework sequence, respectively, within said ISVD. Also, when a nucleotide sequence is said to comprise another nucleotide sequence, it is preferred that the first-mentioned nucleotide sequence is such that, when it is expressed into an expression product (e.g., a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of said expression product (in other words, the latter nucleotide sequence is in the same reading frame as the first-mentioned larger nucleotide sequence).

[0086] The term "domain" as used herein generally refers to a globular region of an antibody chain, particularly a heavy chain antibody globular region, or to a polypeptide consisting essentially of such a globular region. Typically, such a domain comprises peptide loops (e.g., three or four peptide loops) stabilized, for example, as a sheet or by disulfide bonds.

[0087] In the context of the present technology, "binding" to a particular target molecule has its ordinary meaning in the art as understood in the context of antibodies and their corresponding antigens.

[0088] The epitopes against which the polypeptides of the present invention are specifically directed are epitopes on FcRn that contain 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, wherein 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.

[0089] The term "antigenic determinant" 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), more particularly by the antigen-binding site of said molecule. The terms "antigenic determinant" and "epitope" may also be used interchangeably herein. An antigen-binding molecule (such as an antibody, an ISVD, a polypeptide of the invention, or generally an antigen-binding protein or polypeptide or fragment thereof) that is able to (specifically) bind to, has affinity for, and / or has specificity for a particular antigenic determinant, epitope, antigen, or protein (or for at least a part, fragment, or epitope thereof) is said to be "against" or "directed against" said antigenic determinant, epitope, antigen, or protein.

[0090] In certain embodiments, an epitope as disclosed herein is a linear epitope comprising a stretch of consecutive amino acid residues of the primary sequence of FcRn.

[0091] In certain embodiments, an epitope as disclosed herein is a conformational epitope comprising at least two non-contiguous amino acid residues and / or a stretch of amino acid residues in the primary sequence of FcRn that are close to each other in the tertiary structure of FcRn and form a specific binding surface with the three-dimensional structure of the polypeptide of the invention.

[0092] According to certain specific embodiments, the epitopes on FcRn disclosed herein from which the polypeptides according to the present invention are derived are characterized by being different from the epitopes on FcRn from which serum albumin is derived (see Figures 2 and 3).

[0093] According to certain specific embodiments, the epitopes on FcRn disclosed herein from which the polypeptides according to the present invention are derived are characterized in that they are different from the epitopes on FcRn from which immunoglobulin G (IgG) is derived (see Figures 2 and 3).

[0094] According to certain embodiments, the polypeptides of the present invention bind to epitopes on FcRn that contain the following combination 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 the order or numbering of the amino acid residues in the amino acid sequence of SEQ ID NO:1.

[0095] According to certain further embodiments, the polypeptides of the invention are those in which the epitopes to which they bind comprise the following combination 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 d) 167L, 168E, 171R, 174L, 175E and 177K, and / or e) 205P, 206P and 207E, and / or f) 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.

[0096] According to certain further embodiments, the polypeptides of the invention are those in which the epitopes to which they bind comprise the following combination 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 the order or numbering of the amino acid residues in the amino acid sequence of SEQ ID NO:1.

[0097] According to still further embodiments, the polypeptides of the invention are characterized in that the epitopes to which 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, wherein 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.

[0098] According to a further particular embodiment, the polypeptides of the invention are characterized in that the epitopes to which 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, wherein 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.

[0099] According to yet further particular embodiments, the polypeptides of the invention are characterized in that the epitopes to which 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, wherein 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 Figures 1, 2 and 3).

[0100] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0101] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0102] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0103] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0104] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0105] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0106] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0107] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0108] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0109] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0110] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0111] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0112] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0113] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0114] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0115] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0116] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0117] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0118] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0119] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0120] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0121] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0122] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0123] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0124] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0125] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0126] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0127] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0128] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0129] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0130] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0131] According to another particular embodiment, the polypeptide of the 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, the amino acid residues being numbered according to SEQ ID NO: 1.

[0132] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 4H and 5L.

[0133] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 98L, 99G, 100P, 101D and 102N.

[0134] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 167L, 171R, 174L, 175E and 177K.

[0135] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 255Q, 256H, 257A, 259L, 260A and 262P.

[0136] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 167L, 171R, 174L, 175E and 177K.

[0137] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 98L, 99G, 100P, 101D and 102N.

[0138] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 4H, 5L, 255Q, 256H, 257A, 259L, 260A and 262P.

[0139] According to another particular embodiment, the polypeptide of the 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.

[0140] According to another particular embodiment, the polypeptide of the 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.

[0141] According to another particular embodiment, the polypeptide of the 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, the amino acid residues being numbered according to SEQ ID NO: 1.

[0142] According to another particular embodiment, the polypeptide of the 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, the amino acid residues being numbered according to SEQ ID NO: 1.

[0143] According to another particular embodiment, the polypeptide of the 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, the amino acid residues being numbered according to SEQ ID NO: 1.

[0144] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0145] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H and 5L.

[0146] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 97E, 98L, 99G, 100P, 101D and 102N.

[0147] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 98L, 99G, 100P, 101D, 102N and 103T.

[0148] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 167L, 168E, 171R, 174L, 175E and 177K.

[0149] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 205P, 206P and 207E.

[0150] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 255Q, 256H, 257A, 259L, 260A, 261Q, and 262P.

[0151] According to another particular embodiment, the polypeptide of the 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.

[0152] According to another particular embodiment, the polypeptide of the 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.

[0153] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 2E, 3S, 4H, 5L, 205P, 206P and 207E.

[0154] According to another particular embodiment, the polypeptide of the 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.

[0155] According to another particular embodiment, the polypeptide of the 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.

[0156] According to another particular embodiment, the polypeptide of the 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.

[0157] According to another particular embodiment, the polypeptide of the 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.

[0158] According to another particular embodiment, the polypeptide of the 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.

[0159] According to another particular embodiment, the polypeptide of the 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.

[0160] According to another particular embodiment, the polypeptide of the 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.

[0161] According to another particular embodiment, the polypeptide of the 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.

[0162] According to another particular embodiment, the polypeptide of the 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.

[0163] According to another particular embodiment, the polypeptide of the invention has an epitope located at the following amino acid residues: 97E, 98L, 99G, 100P, 101D, 102N, 255Q, 256H, 257 a , 259L, 260 a , 261Q, and 262P.

[0164] According to another particular embodiment, the polypeptide of the 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.

[0165] According to another particular embodiment, the polypeptide of the 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.

[0166] According to another particular embodiment, the polypeptide of the 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.

[0167] According to another particular embodiment, the polypeptide of the 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.

[0168] According to another particular embodiment, the polypeptide of the 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.

[0169] According to another particular embodiment, the polypeptide of the 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.

[0170] According to another particular embodiment, the polypeptide of the 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.

[0171] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0172] According to another particular embodiment, the polypeptide of the 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, the amino acid residues being numbered according to SEQ ID NO: 1.

[0173] According to another particular embodiment, the polypeptide of the 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, the amino acid residues being numbered according to SEQ ID NO: 1.

[0174] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0175] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 1A, 2E, 3S, 4H and 5L.

[0176] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 164R, 167L, 168E, 171R, 174L, 175E and 177K.

[0177] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 204Y, 205P, 206P and 230E.

[0178] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 205P, 206P, 207E and 208L.

[0179] According to another particular embodiment, the polypeptide of the 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.

[0180] According to another particular embodiment, the polypeptide of the 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.

[0181] According to another particular embodiment, the polypeptide of the 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.

[0182] According to another particular embodiment, the polypeptide of the invention is characterized in that the epitope comprises at least the following amino acid residues: 204Y, 205P, 206P, 230E, 205P, 206P, 207E and 208L.

[0183] According to another particular embodiment, the polypeptide of the 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.

[0184] According to another particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0185] According to another particular embodiment, the polypeptide of the 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, the amino acid residues being numbered according to SEQ ID NO: 1.

[0186] According to another particular embodiment, the polypeptide of the 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, the amino acid residues being numbered according to SEQ ID NO: 1.

[0187] According to certain embodiments, the polypeptides of the invention are 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, wherein the amino acid residues are numbered according to SEQ ID NO:1.

[0188] According to certain embodiments, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO:1.

[0189] According to certain embodiments, the polypeptides of the invention are 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, wherein the amino acid residues are numbered according to SEQ ID NO:1.

[0190] According to certain embodiments, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO:1.

[0191] According to certain embodiments, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO:1.

[0192] According to a further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0193] According to a further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0194] According to yet a further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0195] According to a still further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0196] According to a still further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0197] According to yet a further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0198] According to a still further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0199] According to a still further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0200] According to yet a further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0201] According to a still further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0202] According to yet a further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0203] According to yet further particular embodiments, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0204] According to yet a further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0205] According to yet a further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0206] According to yet a further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0207] According to yet a further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0208] According to yet a further particular embodiment, the polypeptide of the 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, wherein the amino acid residues are numbered according to SEQ ID NO: 1.

[0209] The polypeptides according to the present invention specifically bind to an FcRn epitope as disclosed herein via at least one immunoglobulin single variable domain (ISVD) that interacts with that epitope on FcRn.

[0210] 5.1 Immunoglobulin Single Variable Domains The term "immunoglobulin single variable domain" (ISVD) is used interchangeably with "single variable domain" and defines an immunoglobulin molecule in which the antigen-binding site is present on and formed by a single immunoglobulin domain. This term distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, dis-scFv), in which two immunoglobulin domains, in particular two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (V H ) and the light chain variable domain (V L ) interact to form the antigen-binding site. In this case, V H and V L Both complementarity-determining regions (CDRs) of the nucleotides contribute to the antigen-binding site, i.e., a total of six CDRs are involved in forming the antigen-binding site.

[0211] In view of the above definition, the antigen-binding domain of a conventional four-chain antibody (e.g., an IgG, IgM, IgA, IgD, or IgE molecule known in the art), or of a Fab fragment, a F(ab')2 fragment, an Fv fragment, such as a disulfide-linked Fv or scFv fragment, or a diabody derived from such a conventional four-chain antibody (all known in the art), is not normally considered to be an immunoglobulin single variable domain, because in these cases, binding to a corresponding epitope of an antigen is typically not by one (single) immunoglobulin domain, but by a pair of (associated) immunoglobulin domains, such as a light and heavy chain variable domain, i.e., the V of an immunoglobulin domain that together binds to an epitope of the corresponding antigen. H -V L This is because it occurs in pairs.

[0212] In contrast, an immunoglobulin single variable domain can specifically bind to an epitope of an antigen without being paired with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain consists of a single VH , a single V HH、 or a single V L Formed by domains.

[0213] Therefore, a single variable domain may be used with any light chain variable domain sequence (e.g., V), as long as 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 the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit). L -sequence) or a suitable fragment thereof, or a heavy chain variable domain sequence (e.g., V H -sequence or V HH sequence) or a suitable fragment thereof.

[0214] Immunoglobulin single variable domains (ISVDs) include, for example, camelized V H or humanized V HH Including V H , V HH Preferably, the heavy chain ISVD is a camelized V H or humanized V HH Contains V HH The heavy chain ISVD can be derived from a conventional four-chain antibody or a heavy chain antibody.

[0215] For example, an immunoglobulin single variable domain may be a single domain antibody (or a suitable amino acid sequence for use as a single domain antibody), a "dAb" or dAb (or a suitable amino acid sequence for use as a dAb), or (as defined herein, V HH The antibody may be a Nanobody® molecule (such as, but not limited to, a Nanobody® molecule), other single variable domain, or any suitable fragment of any one of these.

[0216] In particular, the immunoglobulin single variable domain is a Nanobody® immunoglobulin single variable domain (humanized V HH Or Camelization V H Contains V HHetc.) or a suitable fragment thereof [Note: Nanobody® is a registered trademark of Ablynx NV].

[0217] "V HH Domain" is V HH , V HH antibody fragments, and V HH Also known as antibodies, they were originally described as the antigen-binding immunoglobulin variable domains of "heavy chain antibodies" (i.e., "antibodies without light chains"; Hamers-Casterman et al., Nature 363:446-448, 1993). HH The term "variable domain" refers to these variable domains, as compared to the heavy chain variable domains (herein referred to as "V" domains) present in conventional four-chain antibodies. H domain) present in conventional four-chain antibodies and the light chain variable domain (referred to herein as "V L The domain name was chosen to distinguish it from the domains referred to as "domains." HH For a further description, see the review by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0218] Typically, producing immunoglobulins involves immunizing laboratory animals, fusing immunoglobulin-producing cells to create hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be produced by screening naive or synthetic libraries, e.g., by phage display.

[0219] Nanobody® V HHThe generation of immunoglobulin sequences such as V is widely described in various published documents, among which WO 94 / 04678, Hamers-Casterman et al. 1993 and Muyldermans et al., 2001 (Reviews in Molecular Biotechnology 74; 277-302, 2001). In these methods, camelids are immunized with a target antigen in order to induce an immune response against said antigen. The V obtained from said immunization is HH The repertoire of V HH are screened for binding to the target antigen.

[0220] In these instances, the generation of antibodies requires purified antigen for immunization and / or screening. The antigen can be purified from a natural source or during recombinant production.

[0221] Peptide fragments of such antigens can be used to immunize and / or screen for immunoglobulin sequences.

[0222] The present technology can use immunoglobulin sequences of different origins, including mouse, rat, rabbit, donkey, human, and camelid immunoglobulin sequences. The technology also encompasses fully human, humanized, or chimeric sequences. For example, the present invention encompasses camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, e.g., camelized dAbs, as described by Ward et al. (see, e.g., WO 94 / 04678 and Riechmann, Febs Lett., 339:285-290, 1994 and Prot. Eng., 9:531-537, 1996). Furthermore, the present invention also encompasses fused immunoglobulin sequences (one or more V domains), e.g., to form multivalent and / or multispecific constructs. HHFor multivalent and multispecific polypeptides containing domains and their preparation, see Conrath et al., J. Biol. Chem., Vol. 276, 10, 7346-7350, 2001, and also see, for example, WO 96 / 34103 and WO 99 / 23221), as well as immunoglobulin sequences of the present technology that include tags or other functional moieties derived from the immunoglobulin sequences, such as toxins, labels, radiochemicals, etc.

[0223] "Humanized V HH " is a naturally occurring V HH domain, but is "humanized," i.e., HH One or more amino acid residues in the amino acid sequence (and particularly in the framework sequences) of the sequence are replaced with V from a conventional human four-chain antibody. H The term "humanized" includes amino acid sequences that have been "humanized" by substituting one or more of the amino acid residues (e.g., as shown above) that occur at the corresponding positions in the domain. This can be done in a manner known per se and will be clear to the skilled artisan, for example, based on the further description herein and the prior art (e.g., WO 2008 / 020079). Again, such humanized V HH can be obtained by any suitable method known per se and therefore is not a naturally occurring V HH It should be noted that the invention is not strictly limited to polypeptides obtained using a polypeptide containing the domain as a starting material.

[0224] "Camelization V H " is a naturally occurring V H The naturally occurring V domains correspond to the amino acid sequence of the V domain but are "camelized", i.e., derived from traditional four-chain antibodies. H One or more amino acid residues in the amino acid sequence of the V domain of a heavy chain antibody HHThis includes amino acid sequences that have been "camelized" by substituting one or more of the amino acid residues occurring at the corresponding positions in the V domain. This can be done in a manner known per se and will be clear to the skilled artisan based on the further explanations herein and the prior art (e.g. WO 2008 / 020079). Such "camelizing" substitutions can be made by substituting one or more of the amino acid residues occurring at the corresponding positions in the V domain. H -V L Preferably, the amino acid residues are inserted at amino acid positions forming and / or present at the interface and / or at the so-called Camelidae hallmark residues, as defined herein (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996), supra). H V, which is used as a starting material or starting point for generating or designing H The sequence is V from mammals H sequence, more preferably human V H Array, e.g. V H 3 sequence. However, such camelized V H can be obtained by any suitable method known per se, and therefore can be obtained without using naturally occurring V as starting material. H It should be noted that the present invention is not strictly limited to polypeptides obtained using polypeptides containing the domain.

[0225] The preferred structure of an immunoglobulin single variable domain sequence can be viewed as consisting of four framework regions ("FR"), referred to in the art and herein as "framework region 1" ("FR1"), "framework region 2" ("FR2"), "framework region 3" ("FR3"), and "framework region 4" ("FR4"), respectively, which are interrupted by three "complementarity-determining regions" ("CDR"), referred to in the art and herein as "complementarity-determining region 1" ("CDR1"), "complementarity-determining region 2" ("CDR2"), and "complementarity-determining region 3" ("CDR3"), respectively.

[0226] As further described in paragraph q) of pages 58 and 59 of WO 08 / 020079 (incorporated herein by reference), the amino acid residues of immunoglobulin single variable domains are designated by the V sequence given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). H According to the general numbering of domains, the V from camelids in the article by Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2); 185-195; see e.g., Figure 2 of this publication) HH It can be numbered as it applies to the domain. H Domain and V HHIt should be noted that, as is well known in the art for domains, the total number of amino acid residues may vary from CDR to CDR and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to the Kabat numbering may be unoccupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This generally means that the Kabat numbering may or may not correspond to the actual numbering of amino acid residues in the actual sequence. H Domains and V HH The total number of amino acid residues in a domain will usually be in the range of 110 to 120, and often 112 to 115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described herein.

[0227] In this application, unless otherwise indicated, CDR sequences are determined according to AbM numbering as described in Kontermann and Duebel (Eds. 2010, Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, FR1 contains amino acid residues 1 to 25, CDR1 contains amino acid residues 26 to 35, FR2 contains amino acid residues 36 to 49, CDR2 contains amino acid residues 50 to 58, FR3 contains amino acid residues 59 to 94, CDR3 contains amino acid residues 95 to 102, and FR4 contains amino acid residues 103 to 113.

[0228] The CDR regions may be determined according to different methods: in the Kabat CDR determination, FR1 of an immunoglobulin single variable domain comprises amino acid residues 1 to 30, CDR1 of an immunoglobulin single variable domain comprises amino acid residues 31 to 35, FR2 of an immunoglobulin single variable domain comprises amino acid residues 36 to 49, CDR2 of an immunoglobulin single variable domain comprises amino acid residues 50 to 65, FR3 of an immunoglobulin single variable domain comprises amino acid residues 66 to 94, CDR3 of an immunoglobulin single variable domain comprises amino acid residues 95 to 102, and FR4 of an immunoglobulin single variable domain comprises amino acid residues 103 to 113.

[0229] In such immunoglobulin sequences, the framework regions may be any suitable framework sequence, and examples of suitable framework sequences will be clear to the skilled person on the basis of, for example, standard handbooks and the further disclosure and prior art referred to herein.

[0230] The framework sequences are preferably immunoglobulin framework sequences or (suitable combinations of) framework sequences derived from immunoglobulin framework sequences (e.g., by humanization or camelization). For example, the framework sequences may be those of a light chain variable domain (e.g., V L -sequence) and / or heavy chain variable domain (e.g., V H -sequence or V HH In one particularly preferred embodiment, the framework sequences are derived from V HH -framework sequences derived from a conventional V-sequence (which may optionally be partially or fully humanized), or a camelized V-sequence (as defined herein). H It can be either an array.

[0231] In particular, the framework sequences present in the ISVD sequences used in the present invention are those that are suitable for humanized VSVD sequences. HH or Camelization V HContains V HH and the like. Some preferred, but non-limiting examples of (suitable combinations of) such framework sequences will become apparent from the further disclosure herein.

[0232] Again, any suitable fragment (or combination of fragments) of the foregoing may also be used, such as a fragment comprising one or more CDR sequences adjacent to and / or linked through one or more framework sequences (e.g., in the same order as those CDR and framework sequences may be present in the full-sized immunoglobulin sequence from which the fragment is derived), as generally described herein for immunoglobulin sequences.

[0233] It should be noted, however, that the present invention is not limited with respect to the origin of the ISVD sequence (or the nucleotide sequence used to express it), nor with respect to the manner in which the ISVD sequence or nucleotide sequence is generated or obtained (or generated or obtained). Thus, the ISVD sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In a specific, but non-limiting embodiment, the ISVD sequence is a "humanized" (as defined herein) immunoglobulin sequence (e.g., a partially or fully humanized mouse or rabbit immunoglobulin sequence, particularly a partially or fully humanized V HHimmunoglobulin sequences), "camelized" immunoglobulin sequences (as defined herein), and immunoglobulin sequences obtained by techniques such as affinity maturation (e.g., starting 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 that are well known to those skilled in the art; or any suitable combination of any of the foregoing.

[0234] Likewise, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, for example a sequence isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), a nucleotide sequence isolated from a library (and in particular an expression library), a nucleotide sequence prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence prepared by PCR using overlapping primers, or a nucleotide sequence prepared using DNA synthesis techniques known per se.

[0235] As described above, the ISVD is the V of a Nanobody®. HH or a suitable fragment thereof. For a general description of ISVDs, reference is made to the prior art cited herein, as well as the further description below. However, in this regard, this description and prior art are primarily directed to the so-called "V H 3 Class ISVD (i.e., V such as DP-47, DP-51, or DP-29) HIt should be noted that the present invention describes ISVDs (ISVDs) with a high degree of sequence homology to three classes of human germline sequences. However, in its broadest sense, the present invention can generally be used with any type of ISVD, including, for example, so-called "V" ISVDs, as described in WO 2007 / 118670. H 4 class" (i.e., V such as DP-78) H It should be noted that ISVDs belonging to four classes of ISVDs with a high degree of sequence homology to human germline sequences are also used.

[0236] In general, ISVDs (especially (partially) humanized V HH Sequence and camelized V H V containing arrays HH An ISVD (sequence) may be characterized by the presence of one or more "hallmark residues" (again as further described herein) of one or more framework sequences (as described herein). Thus, in general, an ISVD has the (generic) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and one or more of the hallmark residues are as further defined herein.

[0237] In particular, ISVD has a (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and the immunoglobulin sequence may be an immunoglobulin sequence having the following structure: Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and framework sequences are as further defined herein.

[0238] More specifically, ISVD is a (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and the immunoglobulin sequence may be an immunoglobulin sequence having the following structure: Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively. One or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are selected from the hallmark residues set forth in Table A-0 below.

[0239] [Table 2]

[0240] [Table 3]

[0241] 5.2 Specificity The terms "specificity," "specifically binds," or "specific binding" refer to the number of different target molecules, such as antigens from the same organism, to which a particular binding unit, such as an ISVD, can bind with sufficiently high affinity (see below). "Specificity," "specifically binds," or "specific binding" are used interchangeably herein with "selectivity," "selectively binds," or "selective binding." Binding units, such as ISVDs, preferably bind specifically to their designated targets. The specificity / selectivity of a binding unit can be determined based on affinity. Affinity indicates the strength or stability of a molecular interaction. Affinity is generally determined by the K D The affinity is expressed in units of mol / l (or M). A It can also be expressed as K A is 1 / K D is equal to (mol / l) -1 (or M -1 Affinity is expressed in units of K. Affinity is a measure of the strength of binding between a moiety and a binding site on a target molecule. DThe smaller the value of , the stronger the binding strength between the target molecule and the targeting moiety. Typically, the binding units used in this technology (such as ISVD) are 10 -5 ~10 -12 mol / l or less, preferably 10 -7 ~10 -12 mol / l or less, more preferably 10 -8 ~10 -1 Dissociation constant (K in mol / l D ) (e.g., 10 5 ~10 12 mol / l or more, preferably 10 7 ~10 12 mol / l or more, more preferably 10 8 ~10 12 Binding constant (K in mol / l A ) and bind to their targets. 10 -4 Any K above mol / l D value (or 10 4 Any K less than l / mol A The K value) is generally considered to be indicative of nonspecific binding. The K value of a biological interaction, such as the binding of an immunoglobulin sequence to an antigen, is considered to be specific. D is typically 10 -5 mol / l (10,000 nM or 10 μM) to 10 -12 mol / l (0.001 nM or 1 pM) or less. Thus, specific / selective binding is measured using the same measurement method, e.g., SPR, with binding units (or polypeptides containing same) in the range of 10 -5 ~10 -12 K in mol / l or less D binds to FcRn at a value of 10 -4 K above mol / l DThis may mean binding to a related target at a specific value. Thus, the ISVD preferably exhibits at least half the binding affinity, and more preferably at least the same binding affinity, for human FcRn compared to an ISVD consisting of the amino acids of SEQ ID NO: 14 or 15, 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 a polypeptide comprising it) can also specifically bind to FcRn from cynomolgus monkeys. Specific binding of a binding unit to a designated target can be determined by any suitable method known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays, as well as various modifications thereof known per se in the art; as well as other techniques mentioned herein. The dissociation constant may be an actual dissociation constant or an apparent dissociation constant, as will be apparent to those skilled in the art. Methods for determining dissociation constants will be apparent to those skilled in the art and include, for example, the techniques mentioned below. -4 mol / l or 10 -3 mol / l (e.g., 10 -2 It will also be apparent that it may not be possible to measure the dissociation constant in mol / l. Optionally, and as will be apparent to those skilled in the art, the (actual or apparent) dissociation constant may be calculated in terms of the (actual or apparent) binding constant (K A ) based on the relationship [K D =1 / K A] can be calculated by the following formula: The affinity of a molecular interaction between two molecules can be measured by different techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (e.g., 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 real-time biospecific interactions to be analyzed by detecting changes in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions to obtain k on , k off measurement, hence K D (or K A) values are obtained. Surface plasmon resonance can be performed, for example, using the well-known BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson et al. (1993, Ann. Biol. Clin. 51;19-26), Jonsson et al. (1991 Biotechniques 11;620-627), Johnson et al. (1995, J. Mol. Recognit. 8;125-131), and Johnson et al. (1991, Anal. Biochem. 198;268-277). Another well-known biosensor technique 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 proteins immobilized on a biosensor chip (signal beam). Changes in the number of molecules bound to the biosensor chip 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 chip surface. Interactions can be measured in real time, allowing the determination of binding and dissociation rates, as well as affinity. BLI can be performed, for example, using the well-known Octet® system (ForteBio, a division of Pall Life Sciences, Menlo Park, USA). Alternatively, affinity can be measured by equilibrium exclusion binding (KinExA) (see, e.g., Drake et al., 2004, Anal. Biochem., 328;35-43) using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA).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 antibody / antigen complexes is passed through a column containing beads precoated with the antigen (or antibody), allowing free antibody (or antigen) to bind to the coated molecules. Detection of the captured antibody (or antigen) is achieved by a fluorescently labeled protein conjugated to the antibody (or antigen). The GYROLAB® Immunoassay System provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al., 2013, Bioanalysis 5;1765-74).

[0242] In a particular embodiment, the polypeptide according to the invention is 6 M -1 ~10 11 M -1 Affinity (K A ) specifically binds to the epitope on FcRn described above.

[0243] In a particular embodiment, the polypeptide according to the invention is -6 nM~10 -11 The dissociation constant (K D ) specifically binds to the epitope on FcRn described above. D is determined by Kinexa, BLI or SPR, for example determined by SPR.

[0244] In a particular embodiment, the polypeptide according to the invention has a denaturing activity of at least about 10, preferably as measured by surface plasmon resonance or BLI. 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 106 M -1 s -1 , at least about 10 7 M -1 s -1 , and at least about 10 8 M -1 s -1 A binding rate constant (k on ) specifically binds to the epitope on FcRn described above.

[0245] In a particular embodiment, the polypeptide according to the invention has a denaturing activity of up to about 10, preferably as measured by surface plasmon resonance or BLI. -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 Dissociation rate constant (k off ) specifically binds to the epitope on FcRn described above.

[0246] In certain embodiments, the FcRn-binding polypeptides of the present invention are such that they are cross-reactive between human FcRn and FcRn derived from at least one, preferably at least two, more preferably at least three, and up to essentially all of the following mammalian species: mouse, dog, rat, rabbit, guinea pig, pig, sheep, bovine, and cynomolgus monkey.

[0247] When an ISVD is said to exhibit "(improved) cross-reactivity in binding to human and non-human primate FcRn" compared to another ISVD, the ISVD is D or k offThis means that the ratio of binding activity to human FcRn to non-human primate FcRn (as expressed in units of ) is lower than the same ratio calculated for other ISVDs in the same assay. The favorable cross-reactivity of binding to human FcRn and non-human primate FcRn allows the toxicity of the FcRn-binding polypeptides of the present invention to be evaluated in preclinical studies conducted in non-human primates.

[0248] In a specific embodiment, the FcRn-binding polypeptides of the present invention are such that they are (at least) cross-reactive between human FcRn and cynomolgus monkey FcRn, and preferably also cross-reactive between either human FcRn and / or cynomolgus monkey FcRn, on the one hand, and at least one, preferably both, rat FcRn and porcine FcRn, on the other hand. For convenience, stretches of amino acids in the FcRn sequence that are assumed to be part of the putative epitope of the polypeptides of the present invention are highlighted. Without being limited to any particular mechanism or hypothesis, it is assumed that, since the polypeptides of the present invention are cross-reactive, they are (essentially) capable of binding to (one or more amino acid residues within) the corresponding stretches of amino acid residues present in the amino acid sequences of those mammalian FcRn proteins.

[0249] In general, an FcRn-binding polypeptide of the present invention can bind to human FcRn with an affinity of less than 500 nM, preferably less than 200 nM, and more preferably less than 10 nM (again, both measured using SPR); and can be considered to be cross-reactive between human FcRn and an FcRn derived from one of the other species mentioned above if it can bind to FcRn from those above-mentioned species with an affinity of less than 500 nM, preferably less than 200 nM, and more preferably less than 10 nM.

[0250] In a specific embodiment, 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 the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 2 [=X1X2X3MY], or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 5 [=X a X b X c FX e X1X2X3MY], wherein X a is A, D, E, F, G, H, K, L, M, N, Q, S, T, W, or Y; X b is F, I, L, M, V, or Y; X c is A, E, G, I, L, M, N, P, S, T, V, W, or Y; X e is D, E, G, K, M, N, P, Q, S or T, X1 is A, D, E, G, K, N, Q, S, T, or V; X2 is A, F, L, M, N, Q, S, T, V, or Y; and X3 is A, D, E, G, H, M, N, Q, S, T, or V; and, CDR2 (according to Kabat) is SEQ ID NO: 3 [=AIX3X4GGGX8X9X 10 YADSVKG] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 6 [=AIX3X4GGGX8X9X 10 ], wherein: X3 is A, D, E, G, H, P, Q, R, S, T, or V; X4 is A, D, E, G, K, M, N, P, Q, R, S, T, V, W, or Y; X8 is A, D, G, H, K, L, M, Q, S, T, or V; X9 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X 10 is A, D, E, H, S, T, V, or Y; and CDR3 (according to Kabat) is SEQ ID NO: 4 [=DX2X3X4TX6X7TX9YX 11 X 12 or the CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 7 [=DX2X3X4TX6X7TX9YX 11 X 12 ], wherein: X2 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; X3 is L or N, preferably L; X4 is F, L, W, or Y; X6 is A, D, E, K, L, M, Q, S, or W; X7 is L, M, or V; X9 is A, D, E, F, G, H, K, L, M, Q, R, S, T, V, W or Y; X 11 is A, E, L, M, Q, S, T, V, or W, and X 12 is T or Y; However, the following amino acids: X1 and X3 in SEQ ID NO: 2 and X6 in SEQ ID NO: 4 are not simultaneously E, D, and D, respectively; or X in SEQ ID NO:5 a , X1 and X3 and X6 of SEQ ID NO: 7 are not simultaneously T, E, D, and D, respectively; or X1 and X3 of SEQ ID NO: 2 and X9 of SEQ ID NO: 4 are not simultaneously E, D, and K, respectively; or X in SEQ ID NO:5 a , X1 and X3 and X9 of SEQ ID NO: 7 are not simultaneously T, E, D, and K, respectively; or X3 of SEQ ID NO: 2 and X9 of SEQ ID NO: 4 are not simultaneously D and K, respectively; or X in SEQ ID NO:5 a and X3 and X9 of SEQ ID NO: 7 are not simultaneously T, D and K, respectively; or X1 and X3 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously E, D, Q, D, and K, respectively; or X in SEQ ID NO:5 a , X1 and X3, X4 of SEQ ID NO: 6, and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, E, D, Q, D, and K, respectively; or X1 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously E, Q, D, and K, respectively; or X in SEQ ID NO:5 a and X1, X4 of SEQ ID NO: 6, and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, E, Q, D, and K, respectively; or X3 of SEQ ID NO: 2 and X6 of SEQ ID NO: 4 are simultaneously D and not D, respectively; or X in SEQ ID NO:5 a and X3 and X6 of SEQ ID NO: 7 are not simultaneously T, D, and D, respectively; or X1 and X3 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 of SEQ ID NO: 4 are not simultaneously E, D, Q, and D, respectively; or X in SEQ ID NO:5 a , X1 and X3, X4 of SEQ ID NO: 6, and X6 of SEQ ID NO: 7 are not simultaneously T, E, D, Q, and D, respectively; or X1 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously E, D, and K, respectively; or X3 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously D, Q, D, and K, respectively; or X1 and X3 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X9 of SEQ ID NO: 4 are not simultaneously E, D, Q, and K, respectively; or X in SEQ ID NO:5 a , X1 and X3, X4 of SEQ ID NO: 6, and X9 of SEQ ID NO: 7 are not simultaneously T, E, D, Q, and K, respectively; or X3 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously D, D, and K, respectively; or X3 of SEQ ID NO: 2 and X9 of SEQ ID NO: 4 are not simultaneously D and S, respectively; or X in SEQ ID NO:5 a and X3 and X9 of SEQ ID NO: 7 are not simultaneously T, D, and S, respectively; or X1 and X3 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously E, D, D, and S, respectively; or X6 and X9 in SEQ ID NO: 4 are not simultaneously D and K, respectively; or X in SEQ ID NO:5 a and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, D, and K, respectively; or X1 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously E, Q, D, and K, respectively; or X3 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously D, Q, D, and S, respectively; or X in SEQ ID NO:5 a and X3, X4 of SEQ ID NO: 6, and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, D, Q, D, and S, respectively; or X1 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously E, Q, D, and S, respectively; or X in SEQ ID NO:5 a and X1, X4 of SEQ ID NO: 6, and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, E, Q, D, and S, respectively; or X1 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously E, D, and K, respectively; or X in SEQ ID NO:5 a and X1 and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, E, D, and K, respectively; or X3 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously D, D, and K, respectively; or X in SEQ ID NO:5 a and X3 and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, D, D, and K, respectively; or X1 and X3 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously E, D, D, and K, respectively; or X3 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously D, D, and S, respectively; or X1 and X3 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously E, D, Q, D, and S, respectively; or X in SEQ ID NO:5 a , X1 and X3, X4 of SEQ ID NO: 6, and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, E, D, Q, D, and S, respectively; or X1 and X3 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously E, D, Q, D, and K, respectively; or X in SEQ ID NO:5 a , X1 and X3, and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, E, D, D, and S, respectively; or X3 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously D, D, and S, respectively; or X in SEQ ID NO:5 a and X3 and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, D, D, and S, respectively; or X1 and X3 in SEQ ID NO: 2 and X9 in SEQ ID NO: 4 are not simultaneously E, D, and S, respectively; or X4 of SEQ ID NO: 3 and X9 of SEQ ID NO: 4 are not simultaneously Q and S, respectively; or X in SEQ ID NO:5 a and X4 of SEQ ID NO: 6 and X9 of SEQ ID NO: 7 are not simultaneously T, Q, and S, respectively.

[0251] In a specific embodiment, 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 the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 2 [=X1X2X3MY], or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 5 [=X a X b X c FX e X1X2X3MY], wherein X a is G or T, X b is F, X c is T, X e is S, X1 is S, D, or E; X2 is Y, and X3 is A or D, and CDR2 (according to Kabat) is SEQ ID NO: 3 [=AIX3X4GGGX8X9X 10 YADSVKG] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 6 [=AIX3X4GGGX8X9X 10 ], wherein: X3 is S, X4 is Q or S; X8 is S, X9 is T, and X 10 is D, and CDR3 (according to Kabat) is SEQ ID NO: 4 [=DX2X3X4TX6X7TX9YX 11 X 12 or the CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 7 [=DX2X3X4TX6X7TX9YX 11 X 12 ], wherein: X2 is T, X3 is L, X4 is Y, X6 is D, E, or S; X7 is L, X9 is K, R, S, or W; X 11 is S, and X 12 is Y, However, the following amino acids: X1 and X3 in SEQ ID NO: 2 and X6 in SEQ ID NO: 4 are not simultaneously E, D, and D, respectively; or X in SEQ ID NO:5 a , X1 and X3 and X6 of SEQ ID NO: 7 are not simultaneously T, E, D, and D, respectively; or X1 and X3 of SEQ ID NO: 2 and X9 of SEQ ID NO: 4 are not simultaneously E, D, and K, respectively; or X in SEQ ID NO:5 a , X1 and X3 and X9 of SEQ ID NO: 7 are not simultaneously T, E, D, and K, respectively; or X3 of SEQ ID NO: 2 and X9 of SEQ ID NO: 4 are not simultaneously D and K, respectively; or X in SEQ ID NO:5 a and X3 and X9 of SEQ ID NO: 7 are not simultaneously T, D and K, respectively; or X1 and X3 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously E, D, Q, D, and K, respectively; or X in SEQ ID NO:5 a , X1 and X3, X4 of SEQ ID NO: 6, and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, E, D, Q, D, and K, respectively; or X1 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously E, Q, D, and K, respectively; or X in SEQ ID NO:5 a and X1, X4 of SEQ ID NO: 6, and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, E, Q, D, and K, respectively; or X3 of SEQ ID NO: 2 and X6 of SEQ ID NO: 4 are simultaneously D and not D, respectively; or X in SEQ ID NO:5 a and X3 and X6 of SEQ ID NO: 7 are not simultaneously T, D, and D, respectively; or X1 and X3 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 of SEQ ID NO: 4 are not simultaneously E, D, Q, and D, respectively; or X in SEQ ID NO:5 a , X1 and X3, X4 of SEQ ID NO: 6, and X6 of SEQ ID NO: 7 are not simultaneously T, E, D, Q, and D, respectively; or X1 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously E, D, and K, respectively; or X3 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously D, Q, D, and K, respectively; or X1 and X3 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X9 of SEQ ID NO: 4 are not simultaneously E, D, Q, and K, respectively; or X in SEQ ID NO:5 a , X1 and X3, X4 of SEQ ID NO: 6, and X9 of SEQ ID NO: 7 are not simultaneously T, E, D, Q, and K, respectively; or X3 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously D, D, and K, respectively; or X3 of SEQ ID NO: 2 and X9 of SEQ ID NO: 4 are not simultaneously D and S, respectively; or X in SEQ ID NO:5 a and X3 and X9 of SEQ ID NO: 7 are not simultaneously T, D, and S, respectively; or X1 and X3 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously E, D, D, and S, respectively; or X6 and X9 in SEQ ID NO: 4 are not simultaneously D and K, respectively; or X in SEQ ID NO:5 a and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, D, and K, respectively; or X1 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously E, Q, D, and K, respectively; or X3 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously D, Q, D, and S, respectively; or X in SEQ ID NO:5 aand X3, X4 of SEQ ID NO: 6, and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, D, Q, D, and S, respectively; or X1 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously E, Q, D, and S, respectively; or X in SEQ ID NO:5 a and X1, X4 of SEQ ID NO: 6, and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, E, Q, D, and S, respectively; or X1 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously E, D, and K, respectively; or X in SEQ ID NO:5 a and X1 and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, E, D, and K, respectively; or X3 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously D, D, and K, respectively; or X in SEQ ID NO:5 a and X3 and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, D, D, and K, respectively; or X1 and X3 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously E, D, D, and K, respectively; or X3 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously D, D, and S, respectively; or X1 and X3 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously E, D, Q, D, and S, respectively; or X in SEQ ID NO:5 a , X1 and X3, X4 of SEQ ID NO: 6, and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, E, D, Q, D, and S, respectively; or X1 and X3 of SEQ ID NO: 2, X4 of SEQ ID NO: 3, and X6 and X9 of SEQ ID NO: 4 are not simultaneously E, D, Q, D, and K, respectively; or X in SEQ ID NO:5 a , X1 and X3, and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, E, D, D, and S, respectively; or X3 in SEQ ID NO: 2 and X6 and X9 in SEQ ID NO: 4 are not simultaneously D, D, and S, respectively; or X in SEQ ID NO:5 a and X3 and X6 and X9 of SEQ ID NO: 7 are not simultaneously T, D, D, and S, respectively; or X1 and X3 in SEQ ID NO: 2 and X9 in SEQ ID NO: 4 are not simultaneously E, D, and S, respectively; or X4 of SEQ ID NO: 3 and X9 of SEQ ID NO: 4 are not simultaneously Q and S, respectively; or X in SEQ ID NO:5 a and X4 of SEQ ID NO: 6 and X9 of SEQ ID NO: 7 are not simultaneously T, Q, and S, respectively.

[0252] In a specific embodiment, 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 the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 2 [=X1X2X3MY], or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 5 [=X a X b X c FX e X1X2X3MY], wherein X a is A, D, E, F, G, H, K, L, M, N, Q, S, T, W, or Y; X b is F, I, L, M, V, or Y; X c is A, E, G, I, L, M, N, P, S, T, V, W, or Y; X e is D, E, G, K, M, N, P, Q, S or T, X1 is A, D, E, G, K, N, Q, S, T, or V; X2 is A, F, L, M, N, Q, S, T, V, or Y; and X3 is A, D, E, G, H, M, N, Q, S, T, or V; and CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO: 9 [=AISSGGGSTDYADSVKG] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [=AISSGGGSTD], and It is characterized in that the CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO: 10 [=DTLYTSLTSYSY] or the CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 13 [=DTLYTSLTSYSY].

[0253] In a specific embodiment, 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 the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 8 [=SYAMY] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=GFTFSSYAMY], and CDR2 (according to Kabat) is SEQ ID NO: 3 [=AIX3X4GGGX8X9X 10 YADSVKG] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 6 [=AIX3X4GGGX8X9X 10 ], wherein: X3 is A, D, E, G, H, P, Q, R, S, T, or V; X4 is A, D, E, G, K, M, N, P, Q, R, S, T, V, W, or Y; X8 is A, D, G, H, K, L, M, Q, S, T, or V; X9 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X 10 is A, D, E, H, S, T, V, or Y; and It is characterized in that the CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO: 10 [=DTLYTSLTSYSY] or the CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 13 [=DTLYTSLTSYSY].

[0254] In a specific embodiment, 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 the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 8 [=SYAMY] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=GFTFSSYAMY], and CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO: 9 [=AISSGGGSTDYADSVKG] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [=AISSGGGSTD], and CDR3 (according to Kabat) is SEQ ID NO: 4 [=DX2X3X4TX6X7TX9YX 11 X 12 or the CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 7 [=DX2X3X4TX6X7TX9YX 11 X 12 ], wherein: X2 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; X3 is L or N, preferably L; X4 is F, L, W, or Y; X6 is A, D, E, K, L, M, Q, S, or W; X7 is L, M, or V; X9 is A, D, E, F, G, H, K, L, M, Q, R, S, T, V, W or Y; X 11 is A, E, L, M, Q, S, T, V, or W, and X 12 is T or Y; However, the following amino acids, X6 and X9 of SEQ ID NO: 4, are not simultaneously D and K, respectively, and X6 and X9 of SEQ ID NO: 7 are not simultaneously D and K, respectively.

[0255] In a specific embodiment, 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 the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 2 [=X1X2X3MY], or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 5 [=X a X b X c FX e X1X2X3MY], wherein X a is G or T, X1 is S, D, or E; X2 is Y, and X3 is A or D, and CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO: 9 [=AISSGGGSTDYADSVKG] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [=AISSGGGSTD], and It is characterized in that the CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO: 10 [=DTLYTSLTSYSY] or the CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 13 [=DTLYTSLTSYSY].

[0256] In a specific embodiment, 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 the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 8 [=SYAMY] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=GFTFSSYAMY], and CDR2 (according to Kabat) is SEQ ID NO: 3 [=AIX3X4GGGX8X9X 10 YADSVKG] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 6 [=AIX3X4GGGX8X9X 10 ], wherein: X3 is S, X4 is Q or S; X8 is S, X9 is T, and X 10 is D, and It is characterized in that the CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO: 10 [=DTLYTSLTSYSY] or the CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 13 [=DTLYTSLTSYSY].

[0257] In a specific embodiment, 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 the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 8 [=SYAMY] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=GFTFSSYAMY], and CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO: 9 [=AISSGGGSTDYADSVKG] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [=AISSGGGSTD], and CDR3 (according to Kabat) is SEQ ID NO: 4 [=DX2X3X4TX6X7TX9YX 11 X 12 or the CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 7 [=DX2X3X4TX6X7TX9YX 11 X 12 ], wherein: X2 is T, X3 is L, X4 is Y, X6 is D, E, or S; X7 is L, X9 is K, R, S, or W; X 11 is S, and X 12 is Y, However, the following amino acids, X6 and X9 of SEQ ID NO: 4, are not simultaneously D and K, respectively, and X6 and X9 of SEQ ID NO: 7 are not simultaneously D and K, respectively.

[0258] In a specific embodiment, 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 the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 8 [=SYAMY] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=GFTFSSYAMY], S at position 1 of SEQ ID NO: 8 or position 6 of SEQ ID NO: 11 is changed to A, D, E, G, K, N, Q, T, or V; and / or Y at position 2 of SEQ ID NO: 8 or position 7 of SEQ ID NO: 11 is changed to A, F, L, M, N, Q, S, T, or V; and / or A at position 3 of SEQ ID NO: 8 or position 8 of SEQ ID NO: 11 is changed to D, E, G, H, M, N, Q, S, T, or V; and CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO: 9 [=AISSGGGSTDYADSVKG] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [=AISSGGGSTD], and It is characterized in that the CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO: 10 [=DTLYTSLTSYSY] or the CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 13 [=DTLYTSLTSYSY].

[0259] In a specific embodiment, 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 the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 8 [=SYAMY] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=GFTFSSYAMY], and CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO: 9 [=AISSGGGSTDYADSVKG], or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [=AISSGGGSTD], S at position 3 of SEQ ID NO: 9 or SEQ ID NO: 12 is changed to A, D, E, G, H, P, Q, R, T, or V; and / or S at position 4 of SEQ ID NO: 9 or SEQ ID NO: 12 is changed to A, D, E, G, K, M, N, P, Q, R, T, V, W, or Y; and / or S at position 8 of SEQ ID NO: 9 or SEQ ID NO: 12 is changed to A, D, G, H, K, L, M, Q, T, or V; and / or T at position 9 of SEQ ID NO: 9 or position 9 of SEQ ID NO: 12 is changed to A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; and / or D at position 10 of SEQ ID NO: 9 or position 10 of SEQ ID NO: 12 is changed to A, E, H, S, T, V, or Y; and It is characterized in that the CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO: 10 [=DTLYTSLTSYSY] or the CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 13 [=DTLYTSLTSYSY].

[0260] In a specific embodiment, 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 the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 8 [=SYAMY] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=GFTFSSYAMY], and CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO: 9 [=AISSGGGSTDYADSVKG] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [=AISSGGGSTD], and CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO: 10 [=DTLYTSLTSYSY], or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 13 [=DTLYTSLTSYSY], T at position 2 of SEQ ID NO: 10 or position 2 of SEQ ID NO: 13 is changed to A, D, E, F, G, I, L, M, Q, S, V, W or Y, preferably A, E, F, G, I, L, M, Q, S, V, W or Y; and / or the L at position 3 of SEQ ID NO: 10 or position 3 of SEQ ID NO: 13 is changed to N, preferably the amino acid at position 3 of SEQ ID NO: 13 is L (i.e., unchanged); and / or Y at position 4 of SEQ ID NO: 10 or position 4 of SEQ ID NO: 13 is changed to F, L, or W, and / or S at position 6 of SEQ ID NO: 10 or SEQ ID NO: 13 is changed to A, D, E, K, L, M, Q, or W; and / or L at position 7 of SEQ ID NO: 10 or position 7 of SEQ ID NO: 13 is changed to M or V, and / or S at position 9 of SEQ ID NO: 10 or SEQ ID NO: 13 is changed to A, D, E, F, G, H, K, L, M, Q, R, T, V, W or Y; and / or S at position 11 of SEQ ID NO: 10 or SEQ ID NO: 13 is changed to A, E, L, M, Q, T, V, or W; and / or The Y at position 12 of SEQ ID NO: 10 or position 12 of SEQ ID NO: 13 is changed to T; However, the amino acids at positions 6 and 9 of SEQ ID NO: 10 or positions 6 and 9 of SEQ ID NO: 13 must not be simultaneously changed to D and K, respectively.

[0261] In a specific embodiment, 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 the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 8 [=SYAMY] or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=GFTFSSYAMY], S at position 1 of SEQ ID NO: 8 or position 6 of SEQ ID NO: 11 is changed to D or E, and / or A at position 3 of SEQ ID NO: 8 or position 8 of SEQ ID NO: 11 is changed to D, and CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO: 9 [=AISSGGGSTDYADSVKG], or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [=AISSGGGSTD], S at position 4 of SEQ ID NO: 9 or SEQ ID NO: 12 is changed to Q; and CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO: 10 [=DTLYTSLTSYSY], or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 13 [=DTLYTSLTSYSY], S at position 6 of SEQ ID NO: 10 or SEQ ID NO: 13 is changed to D or E, and / or S at position 9 of SEQ ID NO: 10 or SEQ ID NO: 13 is changed to K, R, or W; However, the amino acids at positions 6 and 9 of SEQ ID NO: 10 or positions 6 and 9 of SEQ ID NO: 13 must not be simultaneously changed to D and K, respectively.

[0262] In a specific embodiment, 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 the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 8 [=SYAMY], or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=GFTFSSYAMY], wherein S at position 1 of SEQ ID NO: 8 or position 6 of SEQ ID NO: 11 is changed to D or E, and / or A at position 3 of SEQ ID NO: 8 or position 8 of SEQ ID NO: 11 is changed to D; and CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO: 9 [=AISSGGGSTDYADSVKG] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [=AISSGGGSTD], and It is characterized in that the CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO: 10 [=DTLYTSLTSYSY] or the CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 13 [=DTLYTSLTSYSY].

[0263] In a specific embodiment, 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 the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 8 [=SYAMY], or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=GFTFSSYAMY], and CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO: 9 [=AISSGGGSTDYADSVKG], or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [=AISSGGGSTD], S at position 4 of SEQ ID NO: 9 or SEQ ID NO: 12 is changed to Q; and It is characterized in that the CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO: 10 [=DTLYTSLTSYSY] or the CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 13 [=DTLYTSLTSYSY].

[0264] In a specific embodiment, 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 the at least one ISVD consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively), wherein CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 8 [=SYAMY], or CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=GFTFSSYAMY]; and CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO: 9 [=AISSGGGSTDYADSVKG] or CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [=AISSGGGSTD], and CDR3 (according to Kabat) has the amino acid sequence of SEQ ID NO: 10 [=DTLYTSLTSYSY], or CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 13 [=DTLYTSLTSYSY], S at position 6 of SEQ ID NO: 10 or SEQ ID NO: 13 is changed to D or E, and / or S at position 9 of SEQ ID NO: 10 or SEQ ID NO: 13 is changed to K, R, or W; However, the amino acids at positions 6 and 9 of SEQ ID NO: 10 or positions 6 and 9 of SEQ ID NO: 13 must not be simultaneously changed to D and K, respectively.

[0265] In certain specific embodiments, the present invention provides a polypeptide in which at least one ISVD that specifically binds to FcRn is comprised of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), a) CDR1 (according to Kabat) has the amino acid sequence of SEQ ID NO: 8 [=SYAMY] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 8; b) CDR2 (according to Kabat) has the amino acid sequence of SEQ ID NO: 9 [=AISSGGGSTDYADSVKG] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 9; c) CDR3 (according to Kabat) is characterized in that it has the amino acid sequence of SEQ ID NO: 10 [=DTLYTSLTSYSY] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 10.

[0266] In certain specific embodiments, the present invention provides a polypeptide in which at least one ISVD that specifically binds to FcRn is comprised of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), a) CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=GFTFSSYAMY] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 11; b) CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [=AISSGGGSTD] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 12; c) CDR3 (according to AbM) is characterized in that it has the amino acid sequence of SEQ ID NO: 13 [=DTLYTSLTSYSY] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO: 13.

[0267] In a specific embodiment, the FcRn-binding polypeptide of the present 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.

[0268] In a specific embodiment, the FcRn-binding polypeptide of the present invention is characterized in that at least one ISVD that specifically binds to an epitope on FcRn as disclosed herein has at least 80%, for example 90%, or even 95% sequence identity with the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0269] In certain specific embodiments, the present invention provides a method for the production of antibodies in which the ISVD is a (single) domain antibody, Nanobody®, V HH , V HH , humanized V HH , or camelization V H The present invention provides a polypeptide as described herein, characterized in that:

[0270] In certain embodiments, a polypeptide as described herein comprising an ISVD having one or more CDRs that contain one, two, three, or four amino acid differences binds to FcRn with approximately the same affinity as binding by an amino acid sequence or polypeptide that contains a CDR that does not contain four, three, two, or one amino acid difference, wherein the affinity is measured by surface plasmon resonance.

[0271] When comparing two immunoglobulin single variable domains, the term "amino acid difference" refers to the insertion, deletion or substitution of a single amino acid residue at the position in the first sequence that is compared with the second sequence, and it will be understood that the two immunoglobulin single variable domains may contain one, two or more such amino acid differences.

[0272] For example, for purposes of comparing two or more immunoglobulin single variable domains or other amino acid sequences, such as the polypeptides of the invention, the percentage of "sequence identity" (also referred to herein as "amino acid identity") between a first and a second amino acid sequence may be calculated or determined, for example, by dividing the number of amino acid residues in a 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%, as described in paragraph f) on pages 49 and 50 of WO 08 / 020079 (herein incorporated by reference) (wherein each deletion, insertion, substitution or addition of amino acid residues in a second amino acid sequence compared to a first amino acid sequence is considered to be a difference at a single amino acid residue (position), i.e., an "amino acid difference" as defined herein); alternatively, the degree of sequence identity between two amino acid sequences may be calculated using known computer algorithms for aligning sequences, such as NCBI Blast v2.0 using standard settings. Several other techniques, computer algorithms and settings for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0967284, EP 1085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357768-A.

[0273] Generally, for purposes of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation methods outlined herein above, the amino acid sequence containing the greatest number of amino acid residues is considered the "first" amino acid sequence, and the other amino acid sequence is considered the "second" amino acid sequence.

[0274] In determining the degree of sequence identity between two immunoglobulin single variable domains, those skilled in the art may also take into account so-called "conservative" amino acid substitutions, which may generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which have 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, from WO 04 / 037999, GB-A-3 357 768, WO 98 / 49185, WO 00 / 46383, and WO 01 / 09300, and (preferred) types and / or combinations of such substitutions may be selected based on the appropriate teachings from WO 04 / 037999 and WO 98 / 49185, as well as from the further literature cited therein. Examples of conservative substitutions are described further below in this specification.

[0275] Any amino acid substitutions applied to the polypeptides described herein may also be based on the analysis of the frequency of amino acid changes between homologous proteins of different species developed by Schulz et al., 1978 (Principles of Protein Structure, Springer-Verlag), or on the analysis of structure formation potential developed by Chou and Fasman 1975 (Biochemistry 13;211) and 1978 (Adv. Enzymol. 47;45-149), or on the analysis of structure formation potential developed by Eisenberg et al., 1984 (Proc. Natl. Acad. Sci. USA 81;140-144), Kyte & Doolittle 1981 (J. Molec. Biol. 157;105-132), and Goldman et al. al., 1986 (Ann. Rev. Biophys. Chem. 15;321-353), all of which are incorporated herein by reference in their entirety. Information regarding the primary, secondary and tertiary structure of ISVDs is provided in the description herein and in the general background art cited above. For this purpose, V from llamas was also used. HH Crystal structures of domains have been shown, for example, by Desmyter et al. 1996 (Nature Structural Biology, 3; 803), Spinelli et al. 1996 (Natural Structural Biology 3; 752-757), and Decaniere et al., 1999 (Structure, 7; 361). H V in the domain H / V L Further information regarding some of the amino acid residues that form the interface, and potential camelizing substitutions at these positions, can be found in the prior art cited above. Immunoglobulin single variable domains and nucleic acid sequences are said to be "exactly the same" if they have 100% sequence identity (as defined herein) over their entire length.

[0276] Compared with the sequences of SEQ ID NO: 14 and / or 15, the FcRn-binding polypeptides of the present invention also preferably contain one or more humanized substitutions; and / or It contains (at least) one or more mutations (i.e., amino acid substitutions, deletions, or additions, particularly substitutions) that reduce binding by pre-existing antibodies; and may optionally contain one or more further mutations as described herein (e.g., to improve the chemical stability of the FcRn-binding polypeptide).

[0277] For suitable humanizing substitutions (and suitable combinations thereof), see, for example, 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), as well as Tables A-3 to A-8 in WO 08 / 020079 (which list possible humanizing substitutions). Some preferred, but non-limiting, examples of such humanizing substitutions are Q108L and A14P, or suitable combinations thereof. Such humanizing substitutions may be suitably combined with one or more other mutations (e.g., one or more mutations that reduce binding by a pre-existing antibody) as described herein.

[0278] For suitable mutations (and suitable combinations of such mutations) that may reduce binding by existing antibodies, see, e.g., WO 2012 / 175741 and WO 2015 / 173325, also, e.g., WO 2013 / 024059 and WO 2016 / 118733.

[0279] Modifications to the amino acid sequence of the polypeptides or ISVDs described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the polypeptide or ISVD. Amino acid sequence variants of the polypeptides or ISVDs described herein are prepared by introducing appropriate nucleotide changes into a nucleic acid encoding the polypeptide or ISVD, or by peptide synthesis.

[0280] Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the polypeptide or ISVD as described herein. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties. Amino acids can also be altered to alter 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). Preferably, substitutions are conservative substitutions as described herein. Additionally or alternatively, 1, 2, 3, 4, 5, or 6 amino acids may be inserted or deleted in each of the CDRs (depending, of course, 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 may be inserted or deleted in each of the FRs.

[0281] A useful method for identifying specific residues or regions of a polypeptide or ISVD as described herein that are preferred sites for mutagenesis is the so-called "alanine scanning mutagenesis" method, as described by Cunningham and Wells 1989 (Science, 244:1081-1085). In this method, a residue or 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. Further or other variants are then introduced at or for the substitution sites to refine those amino acid positions that are functionally sensitive to the substitution. Thus, while the site for introducing an amino acid sequence variant is predetermined, the nature of the mutation per se need not be. 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.

[0282] Preferably, amino acid sequence insertions comprise amino- and / or carboxyl-terminal fusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues to polypeptides containing 100 or more residues.

[0283] Another type of variant is an amino acid substitution variant. These variants preferably have (at least) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in the amino acid sequence, ISVD, or polypeptide replaced with different residues. The most important sites for substitution mutagenesis include the CDRs, particularly the hypervariable regions, although modifications of the FRs are also contemplated. For example, if the CDR sequence contains 6 amino acids, it is contemplated that 1, 2, or 3 of these amino acids will be substituted. Similarly, if the CDR sequence contains 15 amino acids, it is contemplated that 1, 2, 3, 4, 5, or 6 of these amino acids will be substituted.

[0284] Generally, when amino acids are substituted in one or more or all of the CDRs, it is preferred that the resulting "substituted" sequence be at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, and even particularly preferably 80% or even 90% identical to the "original" CDR sequence. This means that the length of the CDR will depend on how identical it is to the "substituted" sequence. For example, a CDR having five amino acids will preferably be 80% identical to its substituted sequence, since it has at least one substituted amino acid. Thus, the CDRs of an amino acid sequence, ISVD, or polypeptide may have different degrees of identity to their substituted sequences; for example, CDR1 may have 80%, while CDR3 may have 90%.

[0285] Preferred amino acid substitutions are conservative substitutions, preferably in which one amino acid residue within the following groups (a) to (e) is replaced with another amino acid residue within the same group: (a) small, aliphatic, non-polar or slightly 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, non-polar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp. More preferred conservative substitutions are: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu. However, the polypeptide retains its ability to specifically bind to an epitope on FcRn as described herein and exhibits an affinity (e.g., 10 or less) as described herein at an acidic pH (e.g., pH 5.0-6.8). -6 ~10 -11 K of M D ) and / or its CDRs have at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, and more particularly preferably 80% identity with the "original" CDR sequences, any substitutions (including non-conservative substitutions) are contemplated.

[0286] As described in the Examples section, only after rigorous screening and selection methods have the inventors been able to identify ISVDs that bind to FcRn in a pH-dependent manner. Thus, the present invention relates to polypeptides comprising at least one ISVD that binds to FcRn at acidic pH, selected from the group consisting of SEQ ID NOs: 14 and 15 (Table A-1).

[0287] Preferred CDR sequences are presented in Table A-1 as SEQ ID NOS: 8-13 and 104-105. Sequence analysis further revealed that there are many possible sequence variations in the CDRs (see Table A-1 as SEQ ID NOS: 2-7).

[0288] [Table 4]

[0289] [Table 5]

[0290] [Table 6]

[0291] The polypeptides of the present invention bind to specific, unique and novel epitopes on FcRn, which are distinct from the epitopes on FcRn to which the natural ligands of FcRn, i.e., serum albumin and IgG, bind (shown in Figures 2 and 3).

[0292] In a specific embodiment, the FcRn-binding polypeptides according to the present invention are such that, when they bind to or otherwise associate with an FcRn molecule, the binding of the FcRn molecule to serum albumin and / or IgG is not (significantly) affected, reduced or inhibited.

[0293] In a further specific embodiment, the present invention provides a polypeptide comprising 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 involvement of the at least one ISVD and the amino acid residues on FcRn in the binding to serum albumin and / or IgG can be determined, for example, by determining the interaction of the amino acid residues on FcRn with the amino acid residues on the at least one ISVD, the amino acid residues on serum albumin, and / or the amino acid residues on IgG, for example, by crystallographic studies. In this specific embodiment, the polypeptide comprises at least one ISVD that specifically binds to an epitope on FcRn, wherein the ISVD comprises amino acid residues that are not included in the epitope on FcRn to which serum albumin and / or IgG bind. According to this specific 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.

[0294] According to certain embodiments, the FcRn-binding polypeptides of the present invention are also preferably such that they compete for binding to FcRn with polypeptides comprising the amino acid sequences of SEQ ID NOs: 14 and / or 15 and / or such that they "cross-block" (as defined herein) the binding of polypeptides comprising the amino acid sequences of SEQ ID NOs: 14 and / or 15 to FcRn.

[0295] The terms "cross-blocking," "cross-blocked," and "cross-blocking" are used interchangeably herein and refer to the ability of an immunoglobulin single variable domain or polypeptide to interfere with the binding of a ligand to its target, e.g., the binding of a natural ligand to its receptor. 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 natural ligand, to its target, and therefore can be said to cross-block according to the invention, can be determined using competitive binding assays. One particularly suitable quantitative cross-blocking assay uses FACS or ELISA-based techniques or Alphascreen to measure the competition between labeled (e.g., His-tagged or biotinylated) immunoglobulin single variable domains or polypeptides according to the invention and other binding agents in terms of binding to their targets. FACS-, ELISA-, or Alphascreen-displacement-based assays suitable for determining whether a binding molecule cross-blocks or is capable of cross-blocking a polypeptide are well known. It will be understood that these assays can be used with any of the immunoglobulin single variable domains or other binding agents described herein. Thus, in general, a cross-blocking polypeptide according to the invention is one that binds to a target in the above cross-blocking assays such that, for example, in the assay and in the presence of a second polypeptide or in the presence of a natural ligand, the displacement recorded by an immunoglobulin single variable domain or polypeptide according to the invention is 60% to 100% (e.g. in an ELISA / Alphascreen-based competitive assay) or 80% to 100% (e.g. in a FACS-based competitive assay) of the maximum theoretical displacement by the potential cross-blocker being tested present in an amount of 0.01 mM or less (e.g. displacement by a cold (e.g. unlabeled) immunoglobulin single variable domain or polypeptide that needs to be cross-blocked).

[0296] In a specific embodiment, the FcRn-binding polypeptides of the present invention are such that they bind to essentially the same amino acid residues and / or epitopes on FcRn as the amino acid residues and / or epitopes bound by the polypeptides comprising the amino acid sequences of SEQ ID NOs: 14 and / or 15, and even more preferably, they share essentially the same amino acid interactions as the polypeptides comprising the amino acid sequences of SEQ ID NOs: 14 and / or 15. To this end, according to specific, but non-limiting aspects, the FcRn-binding polypeptides according to the present invention preferably have CDRs identical to the sequences of SEQ ID NOs: 14 and / or 15, or preferably contain, compared to the sequences of SEQ ID NOs: 14 and / or 15, within their CDRs only those mutations (such as conservative amino acid substitutions) that still enable them to undergo interaction with FcRn of the same or essentially the same amino acids as the polypeptides comprising the sequences of SEQ ID NOs: 14 and / or 15.

[0297] In certain embodiments, the FcRn-binding polypeptides of the present invention specifically bind to a unique epitope on FcRn as disclosed herein in a pH-dependent manner, such that the binding affinity at acidic pH, particularly at an acidic pH of 5.0 to 6.8, is at least three-fold higher than the binding affinity at a neutral or physiological pH of 7.4. In these specific embodiments, the polypeptides of the present invention exhibit conditional specific binding to FcRn and, as a result, have extended in vivo serum half-lives by utilizing the recycling mechanism mediated by FcRn in vivo. Thus, the polypeptides of the present 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, conjugated, or fused.

[0298] As used herein, a pH of 5.0 to 6.8 refers to an acidic physiological pH, and can be, for example, a pH value less than or greater than either 5.0 or 6.8, and / or any pH value 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 slightly greater or slightly less than these. As used herein, a pH of 7.4 refers to a neutral physiological pH, and can be, for example, a pH of 7.2 to 7.4 (or slightly greater or slightly less than these, as the case may be).

[0299] The pH-dependent interaction of FcRn-binding polypeptides with FcRn in certain specific embodiments of the present invention ensures that these polypeptides are optimally and rapidly trapped in the acidic environment of the endosome and efficiently released after recycling when they again encounter pH 7.4 (i.e., outside the endosome).

[0300] Thus, the inventors have been able to demonstrate that polypeptides according to certain embodiments of the present invention exhibit increased in vivo half-life by specifically binding to FcRn in a pH-dependent manner (as demonstrated by the Examples further described herein).

[0301] The half-life of a polypeptide according to the invention, or a fusion protein, construct or compound comprising same (and as further described herein), 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, for example due to degradation of the sequence or compound by natural mechanisms and / or clearance or sequestration of the sequence or compound. In particular, the half-life may be as defined in WO 2009 / 068627.

[0302] The in vivo half-life of a polypeptide according to the invention, or a fusion protein, construct or compound comprising the same (as further described herein), can be determined by any method known per se, such as pharmacokinetic analysis. Suitable techniques will be clear to those skilled in the art and may, for example, generally involve administering to a warm-blooded animal (i.e., a human or another suitable animal, such as a mouse, rabbit, rat, pig, dog or primate, such as a monkey of the genus Macaca (particularly Macaca fascicularis) and / or a rhesus monkey (Macaca mulatta) and a baboon (Papio ursinus)) an appropriate dose of an amino acid sequence, compound or polypeptide of the invention; collecting a blood sample or other sample from the animal; measuring the level or concentration of the amino acid sequence, compound or polypeptide of the invention in the blood sample; and calculating from the (plot of) the data thus obtained the time until 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. See, for example, the Experimental Section below, as well as standard handbooks, e.g., Kenneth, A. et al., Chemical Stability of Pharmaceuticals; A Handbook for Pharmacists and Peters et al., Pharmacokine analysis; A Practical Approach (1996). See also "Pharmacokinetics," M. Gibaldi & D. Perron, published by Marcel Dekker, 2nd Rev. edition (1982). Half-life can be expressed using parameters such as t½-α, t½-β, and area under the curve (AUC).See, for example, the Experimental Section below, as well as standard handbooks, such as Kenneth et al., 1996 (Chemical Stability of Pharmaceuticals; A Handbook for Pharmacists) and Peters et al., 1996 (Pharmacokinetic Analysis; A Practical Approach). See also Gibaldi & Perron 1982 (Pharmacokinetics, Dekker M, 2nd Rev. edition). As used herein, "increased half-life" refers to an increase in any one of these parameters, e.g., any two of these parameters, or essentially all three of these parameters. The term "increased half-life" or "increased half-life" particularly refers to an increase in t1 / 2-β, with or without t1 / 2-α and / or AUC, or both.

[0303] Thus, in certain embodiments, polypeptides according to the invention and comprising at least one ISVD that binds to a unique epitope on FcRn as according to the invention and disclosed herein, as well as fusion proteins, constructs and compounds comprising same (as further described herein), have an increased half-life compared to known FcRn-binding polypeptides, (fusion) proteins, constructs or compounds as described in the prior art.

[0304] In general, it is preferred that the polypeptides according to the present invention, and fusion proteins, constructs and compounds comprising same (as further described herein), have a half-life (measured either in humans or in suitable animals (e.g., mice or cynomolgus monkeys)) that is at least 1.5-fold, preferably at least 2-fold, for example at least 5-fold, for example at least 10-fold or more than 20-fold, or more, than the half-life of known FcRn-binding amino acid sequences and proteins of the prior art.

[0305] It is also preferred that the polypeptides according to the present invention, as well as fusion proteins, constructs and compounds comprising same (as further described herein), have a half-life (measured either in humans or in suitable animals such as mice or cynomolgus monkeys) that is increased by at least 30%, at least 50%, at least 75%, for example at least 100%, or that is increased by more than 200%, for example more than 300%, more than 400%, more than 500% or more, compared to the half-life of known FcRn-binding amino acid sequences and proteins of the prior art.

[0306] Additionally, polypeptides according to the present invention, which comprise at least one ISVD that binds a unique epitope on FcRn and are fused to another moiety, e.g., a therapeutic moiety, as well as fusion proteins, constructs and compounds comprising the same (as further described herein), have an increased half-life compared to the other moiety, e.g., the therapeutic moiety itself.

[0307] In general, it is preferred that the constructs or fusion proteins described herein have a half-life (as measured either in humans or in a suitable animal, such as a mouse or cynomolgus monkey) that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times that of the corresponding other moiety, e.g., the therapeutic moiety, itself.

[0308] As mentioned, in one embodiment, the FcRn binding agents of the present invention are domain antibodies, single domain antibodies, "dAbs," V HH or Nanobody® V HH (For example, V HH , humanized V HH Or Camelization V H , e.g., camelized human V H ) can be used to increase the half-life of immunoglobulin single variable domain(s).

[0309] Furthermore, the FcRn-binding polypeptides provided by the present invention, as well as fusion proteins, constructs and compounds comprising same (as further described herein), preferably have a half-life in humans (defined as t1 / 2β) of more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, e.g., more than 12 hours, e.g., about 1 day, 2 days, 1 week, 2 weeks, and up to the half-life of serum albumin or IgG in humans (estimated to be about 19 days), although the latter may be less critical.

[0310] FcRn-binding polypeptides according to different embodiments of the present invention are also preferably such that they are any of the following: greater than 6 hours, preferably greater than 12 hours, more preferably greater than 24 hours, even more preferably greater than 72 hours; for example, such that it has a serum half-life in humans (expressed as t1 / 2β) of about 1 week, 2 weeks, and up to the half-life of serum albumin or IgG in humans (estimated to be about 19 days); and / or such that when linked to a therapeutic moiety or entity it confers on the resulting polypeptide of the invention a serum half-life in humans (expressed as t1 / 2β) of greater than 6 hours, preferably greater than 12 hours, more preferably greater than 24 hours, and even more preferably greater than 72 hours; for example, such that it has a serum half-life in humans (expressed as t1 / 2β) of about 1 week, 2 weeks, and up to the half-life of serum albumin or IgG in humans (estimated to be about 19 days).

[0311] The half-life in a mammalian species other than humans mainly depends on the half-life of native serum albumin in that species, as well as the binding properties (e.g., affinity) of the FcRn-binding polypeptide of the present invention for FcRn derived from that mammalian species, among other factors. According to a preferred embodiment of the present invention, when the FcRn-binding polypeptide of the present invention cross-reacts (as defined herein) between human FcRn and FcRn derived from another mammalian species, the half-life of the FcRn-binding polypeptide of the present invention (and / or the compound of the present invention comprising said FcRn-binding polypeptide) measured in that species is preferably at least 5%, e.g., at least 10%, more preferably at least 25%, e.g., about 50%, and in some cases up to 100%, of the half-life of serum albumin in that species.

[0312] In a particular embodiment, the polypeptide according to the present invention is soluble in 10% or more of the soluble fraction at an acidic pH of 5.0 to 6.8. -3 nM -1 ~10 2 nM -1 Affinity (K A ) specifically binds to FcRn. The affinity (K) of these polypeptides for FcRn at an acidic pH, preferably a pH of 5.0 to 6.8 A ) is the affinity (K ) of the same polypeptide for FcRn at neutral or physiological pH of 7.4. A ) at least three times higher than

[0313] In a further specific embodiment, the affinity of the polypeptide according to the present invention for FcRn at an acidic pH of 5.0 to 6.8 (K ) is lower than that of the same polypeptide at a neutral or physiological pH of 7.4. A In yet a further specific embodiment, the polypeptide according to the present invention binds to FcRn with an affinity (K) at an acidic pH of 5.0 to 6.8 that is at least 10 times higher than the affinity (K) of the same polypeptide for FcRn at a neutral or physiological pH of 7.4. A ) binds to FcRn with at least 50-fold greater affinity, such as at least 100-fold greater affinity.

[0314] In certain particular embodiments, the 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 3-fold, such as at least 10-fold, such as at least 50-fold, such as at least 100-fold lower than the affinity with which it binds to FcRn at an acidic pH of 5.0 to 6.8.

[0315] In certain embodiments, at physiological pH, e.g., a pH of 7.4, at least one ISVD is 10 4 K lower than l / mol A It binds to FcRn at a value

[0316] In certain particular embodiments, the invention provides polypeptides as described herein, characterized in that at least one ISVD does not detectably, selectively, or specifically bind to FcRn at neutral or physiological pH, e.g., a pH of 7.4, or exhibits no binding to FcRn, or does not essentially bind to FcRn.

[0317] In a particular embodiment, the polypeptide according to the present invention is soluble in 10% or more of the soluble fraction at an acidic pH of 5.0 to 6.8. -6 M~10 -11 The dissociation constant (K D ) specifically binds to FcRn. D is determined by Kinexa, BLI or SPR, for example determined by SPR.

[0318] In a particular embodiment, the polypeptide according to the present invention is soluble in 10% or more of the soluble fraction at an acidic pH of 5.0 to 6.8. 3 nM~10 -2 The dissociation constant (K D ) specifically binds to FcRn at an acidic pH, preferably a pH of 5.0 to 6.8. D ) is the dissociation constant (K) of the same amino acid sequence and polypeptide for FcRn at neutral or physiological pH of about 7.4. DIn a further specific embodiment, the affinity of a polypeptide according to the present invention for FcRn at an acidic pH of 5.0 to 6.8 is at least three times better (i.e., lower) than the affinity (K) of the same polypeptide at a neutral or physiological pH of about 7.4. D In yet a further specific embodiment, the polypeptides according to the present invention bind to FcRn with an affinity (K) at an acidic pH of 5.0 to 6.8 that is at least 10 times higher / better than the same polypeptides at a neutral or physiological pH of about 7.4. D ) binds to FcRn with at least 50-fold higher, e.g., at least 100-fold higher / better affinity.

[0319] In certain particular embodiments, the 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 3-fold, such as at least 10-fold, such as at least 50-fold, such as at least 100-fold lower / poorer than the affinity with which it binds to FcRn at an acidic pH of 5.0 to 6.8.

[0320] In certain embodiments, at least one ISVD is 10 -4 K greater than mol / l D It binds to FcRn at a value

[0321] In certain particular embodiments, the invention provides polypeptides as described herein, characterized in that at least one ISVD does not detectably, selectively, or specifically bind to FcRn at neutral or physiological pH, e.g., a pH of 7.4, or exhibits no binding to FcRn, or does not essentially bind to FcRn.

[0322] Therefore, the present invention provides an average K D values, e.g., an average K below 900 nM Dand even more preferably an average K value of 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. D Preferably, the polypeptide further comprises at least one ISVD that binds to FcRn at a K value of D is determined by Kinexa, BLI or surface plasmon resonance (SPR), e.g., determined by SPR. Preferably, the average K D is measured by SPR of the recombinant protein.

[0323] The present invention also relates to a polypeptide as described herein comprising at least one ISVD that binds to FcRn at acidic pH with an EC50 value of between 1000 nM and 1 pM, such as with an average EC50 value of 1000 nM or less, even more preferably with 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.

[0324] In a particular embodiment, at an acidic pH of 5.0 to 6.8, the polypeptide according to the present invention has a solubility of at least about 10, preferably as measured by surface plasmon resonance or BLI. 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 6M -1 s -1 , at least about 10 7 M -1 s -1 , and at least about 10 8 M -1 s -1 A binding rate constant (k on ) specifically binds to FcRn.

[0325] In a particular embodiment, at an acidic pH of 5.0 to 6.8, the polypeptide according to the present invention has a maximum of 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 Dissociation rate constant (k off ) specifically binds to FcRn.

[0326] In a particular embodiment, the polypeptide according to the present invention has a maximum yield of 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 Dissociation rate constant (k off ) specifically binds to FcRn at an acidic pH, preferably a pH of 5.0 to 6.8. off) is the dissociation rate constant (K) of the same amino acid sequence and polypeptide for FcRn at neutral or physiological pH of 7.4. off In a further specific embodiment, the dissociation rate constant (K) of a polypeptide according to the present invention for FcRn at an acidic pH of 5.0 to 6.8 is at least three times lower than that of the same polypeptide at a neutral or physiological pH of 7.4. off ) with a dissociation rate constant (K off In yet a further specific embodiment, the polypeptide according to the present invention binds to FcRn at an acidic pH of 5.0 to 6.8 with a dissociation rate constant (K) for FcRn that is lower than that of the same polypeptide at neutral or physiological pH of 7.4. off ) at least 50-fold lower, e.g., at least 100-fold lower, dissociation rate constant (K off ) binds to FcRn.

[0327] In certain specific embodiments, the present invention provides a method for the preparation of FcRn-binding proteins comprising: at least one ISVD having a dissociation rate constant (K off ) that is at least 3-fold, such as at least 10-fold, such as at least 50-fold, such as at least 100-fold higher than the dissociation rate constant (K off ) and characterized in that it binds to FcRn at a neutral or physiological pH of 7.4.

[0328] In certain particular embodiments, the invention provides polypeptides as described herein, characterized in that at least one ISVD does not detectably, selectively, or specifically bind to FcRn at neutral or physiological pH, e.g., a pH of 7.4, or exhibits no binding to FcRn, or does not essentially bind to FcRn.

[0329] Thus, the present invention provides improved FcRn-binding agents that can be used for a variety of applications, including, but not limited to, extending the in vivo half-life of therapeutic compounds (existing or future). In certain embodiments, the polypeptides of the present invention have high affinity for a unique conformational epitope on FcRn that is distinct from the epitopes on FcRn bound by serum albumin and IgG.

[0330] In a specific embodiment, the polypeptides of the present invention have high affinity for a specific epitope on FcRn at an acidic pH of 5.0 to 6.8, while exhibiting reduced binding to FcRn or no detectable selective and / or specific binding at a physiological pH of 7.4 or at a neutral pH.

[0331] 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, which allows the compounds of the invention to be most effectively and optimally applied for some applications, particularly for extending the in vivo half-life of therapeutic molecules.

[0332] Thus, in certain embodiments, the present invention provides A polypeptide comprising at least one ISVD that specifically binds to FcRn in a pH-dependent manner and at least one further moiety, a) at least one ISVD specifically binds to FcRn in a pH-dependent manner such that the binding affinity at pH 5.0 to 6.8 is at least three times higher than the binding affinity at pH 7.4; b) the polypeptide has a molecular weight of at least 30 kDa The present invention provides a polypeptide characterized by:

[0333] In certain further particular embodiments, the polypeptide has a molecular weight of between about 30 kDa and 200 kDa, for example between about 30 kDa and 100 kDa.

[0334] 5.3 Multispecific Polypeptides Generally, a polypeptide according to the invention comprising or consisting essentially of a single building block, a single immunoglobulin single variable domain or a single Nanobody® ISVD is referred to herein as a "monovalent" protein or polypeptide, a "monovalent construct," a "monovalent building block," a "monovalent immunoglobulin single variable domain," a "monovalent Nanobody® ISVD" or a "monovalent Nanobody® VD," respectively. HH "It is called "

[0335] A polypeptide comprising or consisting essentially of two or more immunoglobulin single variable domains (e.g., at least two immunoglobulin single variable domains of the invention) is referred to herein 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.

[0336] At least one building block, ISVD, Nanobody® ISVD or Nanobody® V HH is directed against a first antigen (i.e., against a first target, such as FcRn), and comprises at least one building block, an ISVD, a Nanobody® ISVD, or a Nanobody® V HH or at least two building blocks, ISVD, Nanobody® ISVD or Nanobody® V, directed against a second antigen (i.e., against a second target different from the first target, e.g., serum albumin or a therapeutic target other than FcRn). HH The polypeptides of the invention comprising the building blocks ISVD, Nanobody® ISVD or Nanobody® V are also referred to as "multispecific" polypeptides of the invention, and the building blocks present in such polypeptides include ISVD, Nanobody® ISVD or Nanobody® V HHare also referred to herein as being "multivalent formats" or "multispecific formats." Thus, for example, a "bispecific" polypeptide of the invention comprises at least one building block, an ISVD, Nanobody® ISVD, or Nanobody® V, directed against a first target (e.g., FcRn). HH and at least one further building block, ISVD, Nanobody® ISVD or Nanobody® V, directed against a second target (i.e. directed against a second target different from said first target, e.g. serum albumin). HH and a "trispecific" polypeptide of the invention is a polypeptide comprising at least one building block, an ISVD, Nanobody® ISVD or Nanobody® V, directed against a first target (e.g., FcRn). HH and a second building block, ISVD, Nanobody® ISVD or Nanobody® V, directed against a second target different from the first target (e.g., serum albumin). HH and at least one further building block, ISVD, Nanobody® ISVD or Nanobody® V, directed against a third antigen (i.e., a therapeutic target other than both the first and second target, e.g., FcRn and serum albumin). HH As is apparent from the present specification, the present invention relates to a multispecific polypeptide of the present invention, which comprises a first building block, ISVD, Nanobody® ISVD or Nanobody® V, directed against a first target. HH , a second building block against a second target, ISVD, Nanobody® ISVD or Nanobody® V HH and any number of building blocks, ISVDs, Nanobody® ISVDs or Nanobody® Vs, directed against one or more targets, which may be the same or different from the first target and / or the second target, respectively.HH The present invention is not limited to bispecific polypeptides in that they may include at least:

[0337] The terms bispecific polypeptide, bispecific format, bispecific construct, bispecific Nanobody® construct, bispecific and bispecific ISVD construct are used interchangeably herein.

[0338] As is clear from the above and further description herein, the immunoglobulin single variable domains of the invention can be used to form the polypeptides of the invention as "building blocks", e.g., by suitably combining them with other groups, residues, moieties or binding units, to form compounds or constructs as described herein (such as, but not limited to, the bivalent / trivalent / tetravalent / multivalent and bi / tri / quadruplex / multispecific polypeptides of the invention as described herein), thereby combining one or more desired properties or biological functions in one molecule.

[0339] In a further particular embodiment, the present invention provides a polypeptide wherein said at least one ISVD and said at least one further binding moiety are linked to each other directly or via a linker.

[0340] The compounds, constructs or polypeptides of the invention can generally be prepared by a method comprising at least one step of suitably linking one or more immunoglobulin single variable domains of the invention to one or more further groups, residues, moieties or binding units, optionally via one or more suitable linkers, so as to provide the compounds, constructs or polypeptides of the invention. The polypeptides of the invention can also be prepared by a method which generally comprises at least the steps of providing a nucleic acid encoding the polypeptide of the invention, expressing said nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention. Such a method will be clear to the skilled person and can be carried out in a manner known per se, for example on the basis of the methods and techniques further described herein. The process of designing / selecting and / or preparing a compound, construct or polypeptide of the invention, starting from a polypeptide comprising at least one ISVD of the invention, is also referred to herein as "formatting" said polypeptide of the invention, and a polypeptide of the invention which is part of a compound, construct or polypeptide of the invention is said to be "formatted" into or to be "in the format" of said compound, construct or polypeptide of the invention. Examples of ways in which the polypeptides of the invention can be formatted, and examples of such formats, will be apparent to the skilled artisan based on the disclosure herein, and such formatted immunoglobulin single variable domains or polypeptides form further aspects of the invention.

[0341] For example, such further groups, residues, moieties or binding units may be one or more further immunoglobulins, such that the compound or construct is a (fusion) protein or (fusion) polypeptide. In a preferred but non-limiting aspect, said one or more other groups, residues, moieties or binding units are immunoglobulin single variable domains (ISVDs). Even more preferably, said one or more other groups, residues, moieties or binding units are 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 , Camelization V H , or Nanobody® V HH Alternatively, such groups, residues, moieties or binding units may, for example, be chemical groups, residues, moieties which may or may not be biologically and / or pharmacologically active in themselves. For example, and without limitation, such groups may be linked to one or more immunoglobulin single variable domains or polypeptides of the invention to provide "derivatives" of the ISVDs or polypeptides of the invention, as further described herein.

[0342] Also within the scope of the present invention are compounds or constructs comprising or consisting essentially of one or more derivatives as described herein, and optionally further comprising one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers. Preferably, said one or more other groups, residues, moieties or binding units are immunoglobulin single variable domains. In the compounds or constructs described above, the one or more immunoglobulin single variable domains of the invention and the one or more groups, residues, moieties or binding units may be linked to each other directly and / or via one or more suitable linkers or spacers. For example, if the one or more groups, residues, moieties or binding 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.

[0343] In some embodiments, a polypeptide comprises at least two or more immunoglobulin single variable domains disclosed herein. In some embodiments, a polypeptide consists essentially of two or more immunoglobulin single variable domains disclosed herein. A polypeptide "consisting essentially of" two or more immunoglobulin single variable domains is a polypeptide that, in addition to two or more immunoglobulin single variable domains disclosed herein, lacks any additional immunoglobulin single variable domains. For example, a polypeptide consisting essentially of two immunoglobulin single variable domains does not contain 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 contain additional functionality, such as a label, a toxin, one or more linkers, linking sequences, etc. These additional functionality include both amino acid-based and non-amino acid-based groups. In some embodiments, a polypeptide consists of one or more immunoglobulin single variable domains disclosed herein. It should be understood that the terms "polypeptide construct" and "polypeptide" can be used interchangeably herein (unless the context clearly dictates otherwise).

[0344] In some embodiments, the polypeptide comprises a multivalent or multispecific construct comprising an immunoglobulin single variable domain disclosed herein. In some embodiments, the polypeptide comprises one or more antibody-based 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 Nanobody® V HH is.

[0345] It will be understood that the order (arrangement) of building blocks, such as a first building block, a second building block, a third building block, etc., in a polypeptide can be selected according to the needs of the skilled artisan, as well as according to the relative affinity, which may depend on the position of these building blocks in the polypeptide. Whether or not a polypeptide includes a linker is a matter of design choice. However, some arrangements, with or without a linker, may result in preferred binding properties compared to other arrangements. For example, the order of the first and second building blocks in a polypeptide of the present invention can be (from N-terminus to C-terminus): (i) the first building block (e.g., the first Nanobody® V) HH a first ISVD (e.g., a second Nanobody® V)-[linker]-second building block (e.g., a second Nanobody® V) HH (ii) a second ISVD, such as a second Nanobody® V; or (iii) a second building block (e.g., a second Nanobody® V) HH a second ISVD such as a first Nanobody® V HH (wherein the linker is optional). All configurations are encompassed by the present invention.

[0346] A polypeptide according to the present invention comprising at least one ISVD and at least one other binding moiety is such that the at least one ISVD and the at least one other binding moiety are linked to each other directly or via a linker, such as a peptidic linker. The use of linkers to link two or more (poly)peptides is well known in the art. One frequently used class of peptide linkers is known as "Gly-Ser" or "GS" linkers. These are linkers consisting essentially of glycine (G) and serine (S) residues, usually containing a GGGGS (SEQ ID NO: 86) motif (e.g., a linker of the formula (Gly-Gly-Gly-Gly-Ser) n (where n can be 1, 2, 3, 4, 5, 6, 7, or more). Some frequently used examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 87), the 15GS linker (n=3), and the 35GS linker (n=7). See, e.g., Chen et al. Adv. Drug Deliv. Rev. 2013 Oct 15;65(10);1357-1369; and Klein et al., Protein Eng. Des. Sel. (2014) 27(10);325-330. In specific, but non-limiting, embodiments, the linker is selected from the group consisting of the 3A, 3GS, 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS, and 35GS (SEQ ID NOs: 37-48) linkers. In other particular embodiments, the linker may be an IgG hinge region, such as an IgG1 hinge region (SEQ ID NO: 50 or 126). Preferably, the linker may be the short hinge region of IgG1 (SEQ ID NO: 126) and / or a 35GS linker (SEQ ID NO: 48).

[0347] [Table 7]

[0348] In certain further particular embodiments, the at least one further moiety is a protein moiety, such as a further ISVD.

[0349] In certain further particular embodiments, the at least one further moiety is a protein moiety, such as a serum protein, including but not limited to serum albumin.

[0350] In certain further particular embodiments, the at least one further 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.

[0351] In certain further specific embodiments, at least one serum albumin binding moiety specifically binds to an amino acid residue on serum albumin that is not involved in binding of serum albumin to FcRn.

[0352] In certain further particular embodiments, the present invention provides a method for the production of antibodies in which at least one ISVD that specifically binds to serum albumin is a (single) domain antibody, V HH , Nanobody® V HH , humanized V HH , or camelization V H The present invention provides a polypeptide as described herein, characterized in that:

[0353] In certain further specific embodiments, the at least one further moiety is at least one ISVD that binds to serum albumin.

[0354] In certain further particular embodiments, the at least one further moiety is at least one ISVD that binds to serum albumin and essentially consists of four framework regions (FR1 to FR4, respectively) and three 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), the CDRs being determined according to the Kabat definition; and / or an ISVD wherein CDR1 is GFTFRSFGMS (SEQ ID NO: 19), CDR2 is SISGSGSDTL (SEQ ID NO: 20), and CDR3 is GGSLSR (SEQ ID NO: 21), the CDRs being determined according to the AbM definition (Kontermann et al., 2010).

[0355] WO 06 / 122787 describes a number of ISVDs that bind to (human) serum albumin. These ISVDs include the Nanobody® V, designated Alb-1 (SEQ ID NO: 52 in WO 06 / 122787). HH and humanized variants thereof, such as Alb-8 (SEQ ID NO: 62 in WO 06 / 122787). Additionally, WO 2012 / 175400 describes a further improved version of Alb-1, called Alb-23.

[0356] In a specific embodiment, the polypeptide of the invention comprises at least one ISVD that binds to an epitope on FcRn as disclosed herein, and further comprises an ISVD 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 a variant thereof, as set forth on pages 7-9 of WO 2012 / 175400. These include serum albumin binding moieties, as well as albumin binders described in WO 2012 / 175741, WO 2015 / 173325, WO 2017 / 080850, WO 2017 / 085172, WO 2018 / 104444, WO 2018 / 134235, and WO 2018 / 134234. Some preferred serum albumin binders are also listed in Table A-3.

[0357] [Table 8]

[0358] In certain embodiments of the invention, a polypeptide of the invention comprises at least one ISVD that binds to an epitope on FcRn as disclosed herein and further comprises a serum albumin-binding ISVD having the complete amino acid sequence of ALB23002 (SEQ ID NO: 35, see Table A-3).

[0359] Also in preferred embodiments, the amino acid sequence of the ISVD that binds human serum albumin may have greater than 90%, such as greater than 95% or greater than 99% sequence identity to SEQ ID NO: 35, and optionally the CDRs are as defined above as SEQ ID NOs: 16-18 (according to Kabat) or SEQ ID NOs: 19-21 (according to AbM). In particular, it is preferred that the ISVD that binds human serum albumin has the amino acid sequence of SEQ ID NO: 35.

[0360] If such an ISVD that binds to human serum albumin has two or one amino acid difference in at least one CDR compared to the corresponding reference CDR sequence (as defined above as SEQ ID NOs: 16-18 (according to Kabat) or SEQ ID NOs: 19-21 (according to AbM)), the ISVD preferably has at least half the binding affinity, preferably at least the same binding affinity, to human serum albumin as construct ALB23002, the binding affinity being measured using the same method, such as SPR.

[0361] When such an ISVD that binds to human serum albumin has a C-terminal position, it may exhibit a C-terminal alanine (A) or glycine (G) stretch and is preferably selected from SEQ ID NOs: 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 other than the C-terminal position (i.e., is not the C-terminal ISVD of the polypeptide of the invention) and is selected from SEQ ID NOs: 22, 23, 26, 28 and 35 (see Table A-3).

[0362] In certain embodiments, a polypeptide of the invention comprises at least a first immunoglobulin single variable domain (ISVD) and at least a second immunoglobulin single variable domain (ISVD), wherein said at least a first ISVD has high affinity / specifically binds to FcRn at acidic pH, and said at least a second ISVD has high affinity / specifically binds to serum albumin. Preferred sequences of polypeptides, polypeptide constructs, ISVD building blocks, and combinations of CDR and FR sequences according to certain embodiments of the invention are shown in Tables A-4 to A-8.

[0363] [Table 9]

[0364] [Table 10]

[0365] [Table 11]

[0366] [Table 12]

[0367] [Table 13]

[0368] In a particular embodiment, the present invention provides a method for the preparation of a polypeptide comprising: a (single) domain antibody, a Nanobody®, a V HH , V HH , humanized V HH , or camelization V H The present invention provides a polypeptide as described herein, further comprising a therapeutic moiety comprising:

[0369] In a specific embodiment, a polypeptide of the invention comprises at least a first, at least a second, and at least a third immunoglobulin single variable domain (ISVD), wherein said at least a first ISVD has high affinity for / specifically binds to FcRn at acidic pH, said at least a second ISVD has high affinity for / specifically binds to serum albumin, and said at least a third ISVD has high affinity for / specifically binds to a therapeutically relevant antigen other than FcRn and serum albumin.

[0370] It will be understood that (as demonstrated in the Examples section) the ISVD that binds FcRn and the ISVD that binds serum albumin and / or the ISVD that binds a therapeutic target other than FcRn and albumin can be arranged in any order in the polypeptides of the invention. More particularly, in one embodiment, the ISVD that binds FcRn is located at the N-terminus and the ISVD that binds another antigen is located at the C-terminus. In another embodiment, the ISVD that binds the other antigen is located at the N-terminus and the ISVD that binds FcRn is located at the C-terminus.

[0371] The present invention further provides compounds or constructs, in particular proteins or polypeptides, comprising or consisting essentially of one or more ISVDs or polypeptides of the invention, and optionally further comprising one or more other groups, residues, moieties or binding units. As will be clear to the skilled artisan from the further disclosure herein, such further groups, residues, moieties, binding units or amino acid sequences may or may not provide additional functionality to the polypeptide of the invention (and / or the compound or construct in which it is found), and may or may not alter the properties of the polypeptide of the invention.

[0372] In certain embodiments, the at least one further moiety is an 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 further moiety, characterized in that: a) at least one ISVD specifically binds to FcRn in a pH-dependent manner such that the binding affinity at pH 5.0 to 6.8 is at least three times higher than the binding affinity at pH 7.4; b) the polypeptide has a molecular weight of at least 30 kDa, in particular between about 30 kDa and 100 kDa; c) At least one further moiety is an Fc region or domain of an immunoglobulin (Ig).

[0373] In certain embodiments, the invention provides a polypeptide as described herein, wherein at least one further moiety is an Fc region or Fc domain of immunoglobulin G (IgG). In further specific embodiments, the invention provides a polypeptide as described herein, wherein at least one further moiety is an Fc region or Fc domain of immunoglobulin G type 4 (IgG4).

[0374] In certain embodiments, the invention provides a polypeptide as described herein, wherein at least one further moiety is an Fc region or domain of immunoglobulin A (IgA).

[0375] In some particular embodiments, the Fc domain is a wild-type Fc domain of an immunoglobulin. A wild-type Fc domain has a K of greater than 500 nM, e.g., greater than 600 nM. D It can bind to FcRn at high levels.

[0376] In certain other specific embodiments, the Fc domain has a K of 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 of 5.0 to 6.8). D In certain other specific embodiments, the Fc domain may be a mutant Fc domain of an immunoglobulin that binds to FcRn at a pH between 5.0 and 6.8, or at a neutral or physiological pH, e.g., 7.4, or that does not detectably, selectively, or specifically bind to FcRn, or that exhibits no or essentially no binding to FcRn.

[0377] Suitable constructs and formats comprising at least one polypeptide according to the invention and at least one Fc domain will become clear from the further description herein. Suitable mutant variants of Fc domains and suitable formats of Fc domain constructs are well known in the art and are described in, inter alia, published patent applications EP 2654790, US 10239944, US 20120251531, US 9133274, WO 2014065945, WO 2015150447 and WO 2021016571.

[0378] Thus, as described above, the polypeptides according to the present invention further comprise an IgG Fc domain. The IgG Fc domain refers to the C-terminal non-antigen-binding region of the immunoglobulin G heavy chain, which comprises at least a portion of the constant region. In certain embodiments, the Fc domain may be a native Fc region, i.e., as occurs in a natural antibody, or a variant Fc region comprising one or more alterations, mutations, or variations compared to a native Fc domain. In certain embodiments, the IgG Fc domain may also be a fragment of a native Fc domain or a fragment of a variant Fc domain. In certain embodiments, the polypeptides as described herein comprise a native Fc domain of human IgG, for example, preferably a native Fc of human IgG4 (e.g., Uniprot sequence P01861, SEQ ID NO: 129). In other embodiments, the polypeptides according to the present invention comprise a variant Fc domain having altered binding properties for Fc ligands compared to the unmodified parent Fc molecule. For example, the polypeptides described herein can include an Fc region having one or more of amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 replaced with a different amino acid residue, such that the variant Fc region has altered affinity for an effector ligand, e.g., an Fc receptor or the C1 component of complement, as described in U.S. Pat. Nos. 5,624,821 and 5,648,260 (both by Winter et al.).

[0379] In certain embodiments, polypeptides of the invention comprise an Fc variant domain with reduced effector function, in particular the so-called "FALA" or "LALA" Fc variants in which residues 234 and 235 are substituted with 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 as described herein and the mutation S228P. These Fc variants are referred to as "pFALA."

[0380] In certain embodiments, polypeptides according to the invention comprise an Fc variant domain that exhibits improved binding to the FcRn receptor compared to a native Fc domain, including those that comprise substitutions at one or more of residues 259, 308, 428, and 434 of the Fc region. Other variants that enhance 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, and 434A (Shields et al., Journal of Biological Chemistry, 2001, 276(9):6591-6604).

[0381] In certain specific embodiments, a polypeptide according to the invention comprises an Fc variant domain in which methionine 428 has been substituted with a lysine and asparagine 434 has been substituted with a serine.

[0382] In certain embodiments, polypeptides according to the invention comprise an Fc variant domain that exhibits reduced or no binding to the FcRn receptor compared to a native Fc domain, including those that comprise a substitution at one or more of residues 253, 310, and 453 of the Fc region.

[0383] In a specific embodiment, a polypeptide according to the invention comprises an Fc variant domain in which isoleucine 428 is substituted with alanine, histidine 310 is substituted with alanine, and histidine 453 is substituted with alanine, optionally in combination with histidine 453 substituted with alanine.

[0384] In certain embodiments, at least one additional moiety is a non-protein moiety, such as, but not limited to, a polyethylene glycol (PEG) moiety.

[0385] 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 further moiety, characterized in that: a) at least one ISVD specifically binds to FcRn in a pH-dependent manner such that the binding affinity at pH 5.0-6.5 is at least three times higher than the binding affinity at pH 7.4; b) the polypeptide has a molecular weight of at least 30 kDa, in particular between about 30 kDa and 100 kDa; c) At least one further moiety is a non-protein moiety, such as, but not limited to, a polyethylene glycol (PEG) moiety.

[0386] In certain embodiments, the invention provides a polypeptide as described herein, wherein the polypeptide further comprises a therapeutic moiety.

[0387] In certain embodiments, at least one therapeutic moiety comprises or consists essentially of a therapeutic protein, polypeptide, compound, factor, or other entity. In preferred embodiments, the therapeutic moiety is directed against a desired antigen or target, is capable of binding to the desired antigen (particularly, is capable of specifically binding to the desired antigen), and / or is capable of interacting with the desired target. In another embodiment, at least one therapeutic moiety comprises or consists essentially of a therapeutic protein or polypeptide. In further embodiments, 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 a fragment of an immunoglobulin), e.g., an antibody or antibody fragment (including but not limited to an ScFv fragment), or another suitable protein scaffold, e.g., a protein A domain (such as Affibodies™), tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors, engineered ankyrin repeats, avimers and PDZ domains (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).

[0388] In yet another embodiment, 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.

[0389] In a preferred embodiment, at least one therapeutic moiety is a domain antibody, a single domain antibody, a "dAb" or a V HH (e.g., Nanobody® V HH , humanized V HH Or Camelization V H ) or IgNAR domain.

[0390] For example, but not by way of limitation, such polypeptides, compounds or constructs of the present invention may be - at least one FcRn-binding agent of the present invention and at least one ISVD (preferably Nanobody® V) against a therapeutic target; HH ) and; or - at least one FcRn-binding agent of the present invention, at least one serum albumin binder (as described herein), and at least one ISVD (preferably Nanobody® V) directed against a therapeutic target; HH ) and; or - at least one FcRn-binding agent of the present invention, at least one Fc region of an immunoglobulin (as described herein), and at least one ISVD (preferably a Nanobody® V) against a therapeutic target; HH ) and; or - at least one FcRn-binding agent of the present invention, at least one serum albumin-binding agent (as described herein), at least one Fc region of an immunoglobulin (as described herein), and at least one ISVD (preferably a Nanobody® V) directed against a therapeutic target; HH ) may be included.

[0391] These polypeptides, compounds or constructs of the invention may comprise two or more building blocks, ISVD or Nanobody® V HH , optionally one or more polypeptides, one or more other groups, drugs, agents, residues, moieties or binding units may be directly linked to each other (e.g. as described in WO 99 / 23221) and / or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof.

[0392] Suitable spacers or linkers for use in multivalent and multispecific polypeptides will be apparent to those skilled in the art and may generally be any linker or spacer used in the art for linking amino acid sequences. Preferably, said linker or spacer is suitable for use in the construction of proteins or polypeptides intended for pharmaceutical use.

[0393] For example, the linker may be a suitable amino acid sequence, particularly an amino acid sequence of 1 to 50, preferably 1 to 30, e.g., 1 to 10 amino acid residues. Preferred examples of such amino acid sequences include (gly)-(gly4ser)3 or (gly3ser2)3, as described in WO 99 / 42077. x ser y ) z Preferred linkers include gly-ser linkers, as well as the 30GS, 15GS, 9GS, and 7GS linkers described in the Ablynx applications referenced herein (see, e.g., WO 06 / 040153 and WO 06 / 122825), and hinge-like regions, such as those of naturally occurring heavy chain antibodies or analogous sequences (as described in WO 94 / 04678). Preferred linkers are shown in Table A-2.

[0394] Some other particularly preferred linkers are polyalanine (eg, AAA), and linkers GS30 (SEQ ID NO: 85 of WO 06 / 122825) and GS9 (SEQ ID NO: 84 of WO 06 / 122825).

[0395] Other suitable linkers generally include organic compounds or polymers, particularly those suitable for use with proteins for pharmaceutical applications, for example, poly(ethylene glycol) moieties have been used to link antibody domains, see e.g., WO 04 / 081026.

[0396] It is within the scope of the present invention that the length, degree of flexibility, and / or other properties of the linker used (although not critical, as is typically the case for linkers used with scFv fragments) may have some effect on the properties of the final polypeptide of the invention, including, but not limited to, affinity, specificity, or avidity for one or more FcRn or other antigens. Based on the disclosure herein, one skilled in the art will be able to determine, optionally after some limited routine experimentation, the optimal linker for use in a particular polypeptide of the invention.

[0397] For example, building blocks, ISVDs or Nanobody V directed against first and second targets. HH In the multivalent polypeptide of the invention comprising HH is preferably such that it is capable of binding to its cognate target, e.g., an antigenic determinant on the respective target. Again, based on the disclosure herein, one skilled in the art will be able, optionally after some limited routine experimentation, to determine the optimal linker for use in a particular polypeptide of the invention.

[0398] The linker used may also confer one or more other advantageous properties or functionalities to the polypeptide of the invention and / or (e.g., the ISVD, Nanobody V of the invention). HHIt is also within the scope of the present invention to provide one or more sites for forming derivatives (as described herein for derivatives of polypeptides) and / or for attaching functional groups. For example, a linker comprising one or more charged amino acid residues can provide improved hydrophilic properties, while a linker forming or comprising a small epitope or tag can be used for detection, identification, and / or purification purposes. Again, based on the disclosure herein, one of skill in the art will be able to determine the optimal linker for use with a particular polypeptide of the invention, optionally after some limited routine experimentation.

[0399] Finally, when more than one linker is used in a polypeptide of the invention, these linkers may be the same or different, and again, based on the disclosure herein, one skilled in the art will be able to determine the optimal linker for use in a particular polypeptide of the invention, optionally after some limited routine experimentation.

[0400] Typically, the polypeptides of the present invention are linear polypeptides for ease of expression and production, but the present invention is not limited thereto in its broadest sense. For example, the polypeptides of the present invention may be composed of three or more building blocks, ISVDs, or Nanobody Vs. HH When the building blocks, ISVDs or Nanobody V are included, they can be linked using a linker with three or more "arms", each "arm" being a building block, ISVD or Nanobody V, resulting in a "star-shaped" construct. HH Although generally less preferred, circular constructs can also be used.

[0401] 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 by any method 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 by any method known per se for the preparation of antibodies, in particular for the preparation of antibody fragments (including, but not limited to, (single) domain antibodies and ScFv fragments). Some preferred, but non-limiting, methods for preparing the polypeptides and constructs include the methods and techniques described herein.

[0402] Methods for producing the polypeptides, ISVDs, compounds and constructs of the present invention include: Expressing a nucleic acid encoding said ISVD, polypeptide or protein construct of the invention in a suitable host cell or host organism (herein also referred to as "host of the invention") or another suitable expression system, optionally followed by: The method may include a step of isolating and / or purifying the polypeptides, ISVDs, compounds and constructs of the present invention thus obtained.

[0403] In particular, such a method Cultivating and / or maintaining a host cell or host organism of the invention under conditions such that said 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 followed by: The method may include a step of isolating and / or purifying the polypeptide, ISVD, compound and / or construct of the present invention thus obtained.

[0404] 5.2 Nucleic Acid Sequences and Genetic Constructs Thus, the present invention also relates to nucleic acids or nucleotide sequences (also referred to as "nucleic acids of the invention" or "nucleotide sequences of the invention") that encode the ISVDs, polypeptides, compounds, (fusion) proteins or (multispecific) constructs of the invention. Nucleic acids of the invention may be in the form of single-stranded or double-stranded DNA or RNA, preferably in the form of double-stranded DNA. For example, nucleotide sequences of the invention may be genomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage specifically adapted for expression in the intended host cell or host organism).

[0405] According to one embodiment of the present invention, the nucleic acids of the present invention are in essentially isolated form, as defined herein. The nucleic acids of the present invention may also be in the form of, present in, and / or part of a vector, such as a plasmid, cosmid, or YAC, which may also be in essentially isolated form. A nucleic acid sequence is considered to be "essentially isolated" if it has been separated from at least one other component with which it is normally associated 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 trace component, e.g., compared to its natural biological source and / or the reaction or culture medium from which it was obtained. In particular, a nucleic acid sequence or amino acid sequence is considered "essentially isolated" if it has been purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold, and up to 1000-fold or more. A nucleic acid sequence in "essentially isolated form" is preferably essentially homogeneous, as determined using a suitable technique, e.g., a suitable chromatographic technique such as polyacrylamide gel electrophoresis.

[0406] For purposes of comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first nucleotide sequence and a second nucleotide sequence may be calculated by dividing the number of nucleotides in the first nucleotide sequence that are identical to nucleotides at corresponding positions in the second nucleotide sequence by the total number of nucleotides in the first nucleotide sequence, and multiplying by 100% (where each deletion, insertion, substitution, or addition of nucleotides in the second nucleotide sequence compared to the first nucleotide sequence is considered to be a difference at a single nucleotide (position)).

[0407] Alternatively, the degree of sequence identity between two or more nucleotide sequences may be calculated using a known computer algorithm for aligning sequences, 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 0967284, EP 1085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357768-A. Typically, for the purpose of determining the percentage of "sequence identity" between two nucleotide sequences according to the calculation method outlined hereinabove, the nucleotide sequence with the most nucleotides is considered to be the "first" nucleotide sequence, and the other nucleotide sequence is considered to be the "second" nucleotide sequence.

[0408] The nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information on the polypeptide or protein constructs of the invention presented herein, and / or isolated from suitable natural sources. It will also be clear to the skilled artisan that, to prepare the nucleic acids of the invention, several nucleotide sequences, e.g. at least one nucleotide sequence encoding an immunoglobulin single variable domain of the invention and, e.g., nucleic acids encoding one or more linkers, can also be linked together in a suitable manner.

[0409] Techniques for generating the nucleic acids of the invention will be clear to those skilled in the art and may include, 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 portions thereof), introducing mutations that result in the expression of truncated expression products; introducing one or more restriction sites (e.g., to create cassettes and / or regions that can be easily digested and / or ligated using suitable restriction enzymes), and / or introducing mutations by PCR reactions using one or more "mismatched" primers. These and other techniques will be clear to those skilled in the art, and reference is again made to standard handbooks such as Sambrook et al. and Ausubel et al., referred to herein, and to the Examples below.

[0410] The nucleic acids of the present invention may also be in the form of, present in, and / or part of a genetic construct, as will be apparent to those skilled in the art. Such genetic constructs generally comprise at least one nucleic acid of the present invention, optionally linked to one or more elements of genetic constructs known per se, such as, for example, one or more suitable regulatory elements (e.g., suitable promoters, enhancers, terminators, etc.) and the further elements of genetic constructs referred to herein. Such genetic constructs comprising at least one nucleic acid of the present invention are also referred to herein as "genetic constructs of the present invention."

[0411] The genetic constructs of the present invention may be DNA or RNA, preferably double-stranded DNA. The genetic constructs of the present invention may also be in a form suitable for transforming an intended host cell or host organism, for integration into the genomic DNA of the intended host cell, or for independent replication, maintenance, and / or inheritance in the intended host organism. For example, the genetic constructs of the present invention may be in the form of a vector, such as a plasmid, cosmid, YAC, viral vector, or transposon. In particular, the vector may be an expression vector, i.e., a vector capable of effecting expression in vitro and / or in vivo (e.g., in a suitable host cell, host organism, and / or expression system).

[0412] In a preferred but non-limiting embodiment, the genetic construct of the present invention comprises: a) at least one nucleic acid of the invention operably linked to: b) one or more regulatory elements, such as a promoter and optionally a suitable terminator; Also, optionally, c) one or more further elements of a genetic construct known per se, wherein the terms "regulatory element," "promoter," "terminator," and "operably linked" have their usual meaning in the art (as further described herein); said "further elements" present in a genetic construct may, for example, be 3'- or 5'-UTR sequences, leader sequences, selection markers, expression markers / reporter genes, and / or elements that may facilitate or increase the (efficiency of) transformation or integration. These and other suitable elements for such genetic constructs will be clear to the skilled artisan and may depend, for example, on the type of construct used; the intended host cell or host organism; the method for expressing the nucleotide sequence of the invention of interest (e.g., by constitutive, transient, or inducible expression); and / or the transformation technique used. For example, regulatory sequences, promoters, and terminators known per se for expressing and producing antibodies and antibody fragments (including, but not limited to, (single) domain antibodies and scFv fragments) may be used in an essentially similar manner.

[0413] Preferably, in a genetic construct of the present invention, the at least one nucleic acid of the present invention and the regulatory element, and optionally the one or more further elements, are "operably linked" to each other, which generally means that they are in a functional relationship with each other. For example, a promoter is considered to be "operably linked" to a coding sequence if the promoter is capable of initiating or otherwise controlling / regulating the transcription and / or expression of the coding sequence (the coding sequence should be understood to be "under the control of" the promoter). Generally, when two nucleotide sequences are operably linked, they are in the same orientation and usually also in the same reading frame. They are also usually essentially contiguous, although this may not be required.

[0414] 5.3 Hosts and Host Cells The nucleic acids of the invention and / or the genetic constructs of the invention can be used to transform a host cell or host organism, i.e. to express and / or produce the polypeptide or protein construct of the invention. Preferably, the host is a non-human host. Suitable hosts or host cells will be clear to the skilled artisan and can be, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell line, or any suitable fungal, prokaryotic or eukaryotic organism, e.g. Gram-negative strains such as Escherichia coli strains; Proteus strains, for example Proteus mirabilis strains; Pseudomonas strains, for example Pseudomonas fluorescens strains; and Bacillus strains, for example Bacillus subtilis strains or Bacillus brevis strains; Streptomyces strains, for example Streptomyces lividans strains; Staphylococcus strains, for example Staphylococcus carnosus strains; and Lactococcus strains, for example Lactococcus lactis bacterial strains, including, but not limited to, Gram-positive strains such as Lactis strains; Trichoderma species, such as Trichoderma reesei cells, Neurospora species, such as Neurospora crassa cells; Sordaria species, such as Sordaria macrospora cells; Aspergillus species, such as Aspergillus niger or Aspergillus sojae cells; or fungal cells, including, but not limited to, cells from other filamentous fungi; Saccharomyces species, such as Saccharomyces cerevisiae cells. cells of Schizosaccharomyces species, such as Schizosaccharomyces pombe;Pichia species, for example Pichia pastoris or Pichia methanolica cells; Hansenula species, for example Hansenula polymorpha cells; Kluyveromyces species, for example Kluyveromyces lactis cells; Arxula species, for example Arxula adeninivorans cells; Yarrowia species, for example Yarrowia lipolytica cells. amphibian cells or cell lines such as Xenopus oocytes; cells or cell lines of insect origin, such as cells / cell lines of Lepidoptera, including but not limited to cells / cell lines of Drosophila origin, such as Spodoptera SF9 and Sf21 cells or Schneider and Kc cells; plants or plant cells, for example tobacco plants; and / or mammalian cells or cell lines, such as cells or cell lines of human origin, cells or cell lines of mammalian origin, 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 expressing and producing antibodies and antibody fragments (including but not limited to (single) domain antibodies and scFv fragments), which will be clear to the skilled person. As well as the general background art cited herein above, see, 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);See also Joosten et al. 2003 (Microb. Cell Fact. 2;1); Joosten et al. 2005 (Appl. Microbiol. Biotechnol. 66;384-92); and further references cited therein.

[0415] With regard to expression of polypeptides, ISVDs, compounds or constructs in cells, they may be expressed as so-called "intracellular antibodies", as described, for example, in WO 94 / 02610, WO 95 / 22618 and U.S. Pat. No. 7,004,940; WO 03 / 014960; Cattaneo and Biocca 1997 (Intracellular Antibodies; Development and Applications. Landes and Springer-Verlag); and Kontermann 2004 (Methods 34; 163-170).

[0416] According to one preferred, but non-limiting embodiment of the invention, the polypeptide, ISVD, (fusion) protein or construct of the invention is produced in bacterial cells, in particular bacterial cells suitable for large-scale pharmaceutical production, such as cells of the above-mentioned strains.

[0417] According to another preferred, but non-limiting embodiment of the invention, the polypeptide, ISVD, (fusion) protein or construct of the invention is produced in yeast cells, in particular yeast cells suitable for large-scale pharmaceutical production, such as cells of the species mentioned above.

[0418] 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, in particular in human cells or cells of a human cell line, more in particular in human cells or cells of a human cell line suitable for large-scale drug production, such as the cell lines mentioned herein above.

[0419] Suitable techniques for transforming the hosts or host cells of the present invention will be apparent to those skilled in the art and will depend on the intended host cell / host organism and the genetic construct used - again, reference is made to the handbooks and patent applications mentioned above.

[0420] After transformation, a step may be carried out to detect and select those host cells or host organisms which have been successfully transformed with a nucleotide sequence / genetic construct of the invention. This step may for example be a selection step based on a selectable marker present in the genetic construct of the invention, or it may be a step which involves detecting a polypeptide of the invention, for example using a specific antibody.

[0421] Transformed host cells (which may be in the form of stable expression strains) or host organisms (which may be in the form of stable mutants or strains) are further aspects of the present invention.

[0422] Preferably, these host cells or host organisms are such that they express or are (e.g. under suitable conditions) (at least) capable of expressing) the ISVD, polypeptide, compound, (fusion) protein or construct of the invention (in the case of a host organism: in at least one cell, part, tissue or organ thereof). The invention also includes further generations, progeny and / or descendants of the host cells or host organisms of the invention, obtained for example by cell division or sexual or asexual reproduction.

[0423] Thus, in another aspect, the present invention relates to a host or host cell that expresses (or is capable of expressing under suitable conditions) an ISVD, polypeptide, (fusion) protein or construct of the invention; and / or comprises a nucleic acid encoding same. Some preferred, but non-limiting examples of such hosts or host cells may be as generally described in WO 04 / 041867, WO 04 / 041865 or WO 09 / 068627. For example, the ISVDs, polypeptides, (fusion) proteins and constructs of the invention may advantageously be expressed, produced or manufactured in a yeast strain, such as a Pichia pastoris strain. See also 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 same in Pichia and other hosts / host cells.

[0424] In order to bring about / obtain expression of the polypeptide, ISVD, (fusion) protein or construct of the invention, the transformed host cell or transformed host organism may generally be kept, maintained and / or cultured under conditions such that the (desired) ISVD, polypeptide, (fusion) protein or construct of the invention is expressed / produced. Suitable conditions will be clear to those skilled in the art and will usually depend not only on the host cell / host organism used but also on the regulatory elements controlling the expression of the (relevant) nucleotide sequence of the invention. Again, reference is made to the handbooks and patent applications mentioned above in the paragraph on the genetic constructs of the invention.

[0425] In general, suitable conditions may include the use of a suitable culture medium, the presence of a suitable food source and / or suitable nutrients, the use of a suitable temperature, and optionally the presence of a suitable inducer or compound (e.g., when the nucleotide sequence of the invention is under the control of an inducible promoter), all of which may be selected by one of skill in the art. Again, under such conditions, the ISVD, polypeptide, (fusion) protein or construct of the invention may be expressed in a constitutive manner, in a transient manner, or only when appropriately induced.

[0426] It will also be clear to the skilled artisan that the polypeptides, ISVDs, (fusion) proteins or constructs of the invention may be (initially) produced in immature form (as mentioned above) and then may undergo post-translational modifications depending on the host cell / host organism used. The ISVDs, polypeptides, (fusion) proteins or constructs of the invention may also be glycosylated, again depending on the host cell / host organism used.

[0427] The polypeptide, ISVD, (fusion) protein or construct of the invention may then be isolated from the host cell / host organism and / or from the medium in which said host cell or host organism has been cultured using protein isolation and / or purification techniques known per se, such as (preparative) chromatographic and / or electrophoretic techniques, differential precipitation techniques, affinity techniques (e.g. using specific, cleavable amino acid sequences fused to the polypeptide or construct of the invention) and / or using preparative immunological techniques (i.e. using antibodies directed against the amino acid sequence to be isolated).

[0428] A polypeptide or protein is considered to be "essentially isolated (in) form" when it has been separated from at least one other component with which it is normally associated in said source or medium, such as another protein / polypeptide, another biological component or macromolecule, or at least one contaminant, impurity, or trace component, e.g., compared to its natural biological source and / or the reaction or culture medium from which it was obtained. In particular, a polypeptide or protein is considered to be "essentially isolated" when it has been 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 "essentially isolated form" is preferably essentially homogeneous, as determined using a suitable technique, e.g., a suitable chromatographic technique such as polyacrylamide gel electrophoresis.

[0429] 5.4 Pharmaceutical Compositions, Vaccines, and Treatment and / or Prevention Methods The present invention also relates to pharmaceutical compositions comprising the polypeptides, ISVD compounds, or constructs (multispecific polypeptides) of the invention. Accordingly, the present invention also provides uses of the polypeptides, ISVD compounds, or constructs (multispecific polypeptides) and / or pharmaceutical compositions of the invention. Accordingly, the present invention provides the polypeptides, ISVD compounds, or constructs (multispecific polypeptides) and / or pharmaceutical compositions of the invention for use in medicine. The present invention also relates to the use of the polypeptides, ISVD compounds, or constructs (multispecific polypeptides) and / or pharmaceutical compositions of the invention for therapeutic and / or prophylactic use (i.e., in therapeutic and / or prophylactic treatment methods). Accordingly, the present invention provides the polypeptides, ISVD compounds, or constructs (multispecific polypeptides) and / or pharmaceutical compositions of the invention for use in therapeutic and / or prophylactic treatment methods (i.e., for therapeutic and / or prophylactic use).

[0430] 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 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 via 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 used. The clinician will be able to select a suitable route of administration and a suitable pharmaceutical formulation or composition to be used for such administration depending on the disease or disorder to be prevented or treated and other factors familiar to the clinician.

[0431] As used herein, the term "therapeutic agent" refers to any agent that can be used in the prevention (prophylaxis), treatment and / or management of a disease or disorder, such as a hyperproliferative cell disorder, e.g., cancer, or one or more symptoms thereof. In certain embodiments, the term "therapeutic agent" refers to a multispecific polypeptide of the invention. Preferably, a therapeutic agent is an agent that is, has been, or is currently known to be useful in the treatment, prevention and / or management of a disease or disorder, or one or more symptoms thereof.

[0432] As used herein, a "therapeutically effective amount," in this context, refers to an amount of therapy alone 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 an amount of therapy sufficient to cure, ameliorate, stabilize, or control a disease and / or disorder, or one or more symptoms thereof. In another aspect, a therapeutically effective amount refers to an amount of therapy sufficient to reduce the symptoms of a disease and / or disorder. In another aspect, a therapeutically effective amount refers to an amount of therapy sufficient to postpone or minimize the spread of a disease and / or disorder. When used in reference to the amount of a multispecific polypeptide of the invention, the term can encompass an amount that improves the overall effect of a therapy, reduces or avoids undesirable effects, or enhances its therapeutic effect or synergistic effect with another therapy. In one embodiment, a therapeutically effective amount of a therapy reduces or avoids an undesirable effect, or enhances its therapeutic effect or a 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% compared to a control (e.g., a negative control such as phosphate buffered saline) in an assay known in the art or described herein.

[0433] As used herein, the term "therapy" refers to any protocol, method, and / or agent that can be used to treat, prevent (prophylactic), and / or manage a disease and / or disorder, or one or more symptoms thereof. In certain embodiments, the terms "therapies" and "therapy" refer to biologic, supportive, and / or other therapies known to those of skill in the art, such as health care professionals, that are useful in the treatment, prevention, and / or management of a disease and / or disorder, or one or more symptoms thereof.

[0434] As used herein, the terms "treat," "treatment," and "treating," in the context of administering a therapy to a subject, refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or disorder and / or the amelioration of one or more symptoms thereof, 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 relate to therapeutic and / or prophylactic (preventative) treatment. The term "prophylactic treatment" refers to a therapy for reducing susceptibility to a certain clinical condition. Accordingly, the terms "treat," "treatment," and "treating," and their cognates, refer to obtaining a desired pharmacological or physiological effect that extends to any treatment of a condition, disease, or disorder in a mammal, including a human. The effect may be prophylactic, in that the condition, disease, or disorder or its symptoms are completely or partially prevented, and / or may be therapeutic, in that the condition, disease, or disorder and / or adverse effects resulting from the condition, disease, or disorder are partially or completely cured. That is, "treatment" includes (1) preventing the occurrence or recurrence of a condition, disease, or disorder in a subject; (2) inhibiting the condition, disease, or disorder, e.g., arresting its onset; (3) halting or terminating the condition, disease, or disorder, or at least the symptoms associated therewith, such that the host is no longer afflicted with the condition, disease, or disorder, or its symptoms, e.g., causing regression of the condition, disease, or disorder, or its symptoms; or (4) alleviating, reducing, or ameliorating the condition, disease, or disorder, or the symptoms associated therewith (where ameliorating is used broadly to refer to at least reducing the magnitude of a parameter such as inflammation, pain, or immune deficiency).

[0435] The polypeptides, ISVDs, compounds or constructs of the invention and / or compositions comprising them are administered according to a treatment regimen suitable for preventing and / or treating the disease and / or disorder to be prevented or treated. A clinician will generally be 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 their symptoms, the particular polypeptide, ISVD, compound or construct of the invention to be used, the particular route of administration and pharmaceutical formulation or composition to be used, the patient's age, sex, weight, diet, general condition, and similar factors familiar to clinicians.

[0436] Generally, a treatment regimen will involve administering one or more polypeptides, ISVDs, compounds or constructs of the invention, or one or more compositions comprising them, in one or more pharmaceutically effective amounts or doses. The particular amount or dose to be administered can again be determined by the clinician based on the factors cited above.

[0437] Typically, the above methods will use a single polypeptide, ISVD, compound or construct of the invention, however, it is within the scope of the invention to use a combination of two or more polypeptides, ISVDs, compounds and / or constructs of the invention.

[0438] The polypeptides, ISVDs, compounds or constructs of the invention may also be used in combination with one or more additional pharmaceutically active compounds or ingredients, i.e., as a combined treatment regimen that may or may not provide a synergistic effect. Again, the clinician can select such additional compounds or ingredients, and the appropriate combined treatment regimen, based on the factors cited above and their professional judgment.

[0439] In particular, the polypeptides, ISVDs, compounds or constructs of the invention may be used in combination with other pharmaceutically active compounds or ingredients that are or may be used in the prevention and / or treatment of the diseases and / or disorders cited herein, which may or may not result in a synergistic effect. Examples of such compounds and ingredients, as well as routes, methods and pharmaceutical formulations or compositions for their administration, will be apparent to the clinician.

[0440] When two or more substances or components are to be used as part of a combined treatment regimen, they can be administered by the same route of administration or by different routes of administration, at substantially the same time or at different times (e.g., essentially simultaneously, sequentially, or according to an alternating regimen). When substances or components are to be administered simultaneously by the same route of administration, they may be administered in different pharmaceutical formulations or compositions, or as part of a combined pharmaceutical formulation or composition, as will be apparent to those skilled in the art.

[0441] In one aspect, the disclosure provides methods for administering immunoglobulin single variable domains and polypeptide constructs thereof, including one or more immunoglobulin single variable domains, polypeptides, compounds and / or constructs. In some embodiments, the immunoglobulin single variable domains, polypeptides, compounds and / or constructs are administered as a pharmaceutical composition. The pharmaceutical composition includes the immunoglobulin single variable domains and polypeptide constructs thereof in addition to a pharmaceutically acceptable carrier.

[0442] Because the compounds or polypeptides of the present invention have an improved half-life, they are preferably administered into the blood circulation. Thus, they can be administered by any suitable method that allows the compounds or polypeptides of the present invention to enter the blood circulation, such as intravenously, by injection or infusion, or any other suitable method, including oral administration, subcutaneous administration, intramuscular administration, administration through the skin, intranasal administration, administration via the lungs, etc. Suitable administration methods and routes will again be clear to those skilled in the art from the teachings of published patent applications, for example, of Ablynx NV, such as, for example, WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164, or WO 2009 / 068627.

[0443] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable risk / benefit ratio.

[0444] 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, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0445] Methods of preparing these formulations or compositions include the step of bringing into association the immunoglobulin single variable domain or polypeptide construct with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the immunoglobulin single variable domain or polypeptide construct with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0446] The present invention further provides the polypeptides, ISVD compounds or constructs (multispecific polypeptides) and / or pharmaceutical compositions of the invention as vaccines or as immunogenic compositions. Thus, the present invention further provides vaccines comprising the polypeptides, ISVD compounds or constructs (multispecific polypeptides) and / or pharmaceutical compositions of the invention.

[0447] As used herein, the term "vaccine" refers to a substance or composition that builds or improves 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. Vaccines can be prophylactic or therapeutic. In one aspect, a vaccine of the invention is a prophylactic vaccine.

[0448] Vaccines of the present invention may include an “adjuvant.” The term “adjuvant,” as used herein, refers to a substance that, when added to an immunogenic agent, non-specifically enhances or potentiates the immune response to the agent in a recipient host exposed to the mixture.

[0449] The Figures, Sequence Listing and Experimental Section / Examples are provided merely to further illustrate the present invention and should not be construed or understood as limiting the scope of the present invention and / or the appended claims in any way, unless expressly indicated otherwise herein.

[0450] The embodiments described and discussed herein are intended merely to teach those skilled in the art the best way known to the inventors to make and use the invention. As will be appreciated by those skilled in the art in light of the above teachings, modifications and variations of the above-described embodiments of the invention can be made without departing from the invention. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.

[0451] The present invention will now be further described by the following non-limiting preferred embodiments, examples and figures.

[0452] The contents of all references cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications), particularly with respect to the teachings referred to herein above, are hereby expressly incorporated by reference. [Example]

[0453] 6. Working Example 6.1 Example 1: Selection of FcRn-specific ISVDs by phage display FcRn-binding ISVD clones were identified from a synthetic library. Sequence diversity (both in length and amino acid composition) was introduced into the CDRs of the ISVD sequences in the synthetic ISVD library. The DNA library was cloned into the pAX319 vector. pAX319 is an expression vector derived from the commercially available plasmid vector pUC119. This vector encodes a C-terminal FLAG3 tag and a His6 tag in frame with the ISVD protein-coding sequence. pAX319 enabled the production of phage particles displaying individual ISVD proteins as fusion proteins with the pill protein. Phages were prepared according to standard protocols (see, e.g., the prior art and the applications filed by Ablynx NV cited herein), filter-sterilized, and stored at 4°C for further use. Between selection rounds, libraries were mined by alternating between recombinant biotinylated human FcRn / b2M (hFcRn; Immunitrack ITF01 or ACRO FCM-H82W4) and cynomolgus monkey FcRn / b2M (cFcRn; Immunitrack ITF05 or ACRO FCM-C82W5). Library phage particles were incubated with 0.05 nM to 50 nM biotinylated hFcRn or cFcRn for 2 h in the presence of 1 μM human b2M (Sino Biologicals 11976-H08H) and with or without 5 μM human serum albumin (HSA; Albumin Bioscience 9803), followed by capture on streptavidin- or neutravidin-coated magnetic beads or neutravidin-coated plates. Unbound phage were washed away with CPA buffer, pH 5.5, or PBS buffer, pH 6.0 (supplemented with 0.05% Tween-20 when beads were used), and bound phage were eluted by adding trypsin (1 mg / ml in PBS), CPA buffer, pH 7.4, or PBS buffer, pH 7.4, for 5 or 15 minutes. The protease activity of trypsin was immediately neutralized by adding the protease inhibitor AEBSF to a final concentration of 0.8 mM. As a control, selection without antigen was also performed in parallel.The eluted phages were used to infect exponentially growing E. coli (E. coli) TG-1 cells to rescue the phages. Phages prepared from the selected output were used as input for subsequent selection rounds. Clones obtained from different selection conditions were individually sorted into 96-well master plates for screening. Periplasmic extracts were prepared according to standard protocols (see, e.g., WO 03 / 035694, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, and other prior art and applications filed by Ablynx NV cited herein).

[0454] 6.2 Example 2: Screening and Sequence Analysis 6.2.1 Example 2.1. Binding to Human FcRn / b2M in ELISA 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% Tween 20 and 0.1% casein). High-binding 384-well Spectraplates (Perkin Elmer) were coated with 2 μg / mL neutravidin overnight at 4°C and blocked the next day for at least 1 h at room temperature in blocking buffer (PBS + 1% casein). Biotinylated FcRn / b2M (20 nM; human:ACRO(FCM-H82W4)) was captured for 1 h at room temperature (RT). Periplasmic extracts containing the Flag3-His6 tagged ISVD were diluted 1:10 or 1:50 in assay buffer supplemented with 0.05% Tween 20 + 0.1% casein and incubated on the coated wells for 1–2 h at room temperature. Unbound ISVDs were washed away, and bound ISVDs were detected using mouse anti-FLAG-HRP (Sigma; catalog no. A8592), which catalyzes the conversion of the chromogenic substrate esTMB (SDT Reagents). Absorbance at 450 / 620 nm was read on a Tecan Infinite M1000 or Clariostar-BMG (data not shown). ISVD clones that showed binding to human FcRn at acidic pH were sequenced by Sanger sequencing according to published procedures (Pardon et al., 2014).

[0455] 6.2.2 Example 2.2. Dissociation Rate Measurement by Surface Plasmon Resonance (SPR) After sequencing, unique sequences were relocated and new periplasmic extracts were generated. FLAG3-His6-tagged ISVD proteins were purified by IMAC using 200 μL of Phytip (Phynexus).

[0456] 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 pH 5.5, pH 6.0, or pH 7.4 + 150 mM NaCl + 0.005% Tw20) and injected over the ligand surface (association: 120 s at 45 μL / min, dissociation: 600 s–3600 s at 45 μL / min). Experiments were performed at 25°C. ISVD clones that showed no detectable dissociation rate at pH 7.4 or at least a 10-fold difference in dissociation rate between pH 7.4 and pH 5.5 or pH 6.0 were selected for purification. The dissociation rates obtained for a selection of clones are shown in Table 1.

[0457] [Table 14]

[0458] [Table 15]

[0459] 6.3 Example 3: Production, purification and characterization of ISVD k at pH 7.4 d (1 -s ) at pH 5.5 or 6.0 compared to d (1 -sISVDs with pH-dependent binding were screened for a >10-fold difference in dissociation rate (including retaining ISVDs that exhibit specific binding at pH 5.5 or 6.0 but lack a detectable dissociation rate at pH 7.4). These ISVDs were then expressed and purified as tag-free proteins. Finally, a limited number of ISVDs were further selected based on their optimal pH-dependent FcRn binding properties (maximum dissociation rate difference between pH 5.5 or 6.0 and 7.4 and highest affinity at pH 5.5 or 6.0), combined with their ability to be optimally expressed and produced at medium to large scales. The selected ISVDs were characterized by measuring their affinity for human and cynomolgus monkey FcRn at pH 6.0 and pH 7.4. Additionally, these binders were characterized by HSA competition FACS and grouped based on their potential FcRn epitopes (data not shown), confirming their binding to cell-expressed FcRn at acidic pH.

[0460] 6.3.1 Example 3.1 Generation of ISVD Expression Constructs and Protein Expression The ISVD-containing DNA fragments obtained by PCR using specific combinations of forward FR1 and reverse FR4 primers, each with a unique restriction site, were digested with the appropriate restriction enzymes and ligated into the corresponding 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 no. 60502 or custom-made, respectively) or TOP10 (ThermoFisher Scientific, catalog no. C404052 or C4081201, respectively) cells, which were grown under the appropriate antibiotic selection pressure (kanamycin or zeocin). Resistant clones were confirmed by Sanger sequencing of plasmid DNA (LGC Genomics).

[0461] Monovalent ISVDs were expressed in E. coli TG1 cells (Lucigen, Cat. No. 60502). See also 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 same in E. coli and other hosts / host cells.

[0462] The multivalent ISVD construct was expressed in P. pastoris. This yeast expression vector contains the AOX1 promoter and terminator, a zeocin resistance gene, and coding sequences for the Saccharomyces cerevisiae alpha mating factor signal peptide. The ISVD building blocks were combined with GS linkers and cloned into the expression vector by Golden Gate cloning (Engler C, Marillonnet S. Golden Gate cloning. Methods Mol Biol. 2014;1116:119-31). This expression vector contains two BpiI restriction sites for cloning PCR-amplified monovalent ISVDs with GS linkers contained in one or more vectors. All of these elements are flanked by BpiI sites. The use of unique nucleotide overhangs at each position of the cloning cassette allows for seamless ligation in a predetermined order. After confirmation by Sanger sequencing, the plasmid DNA from E. coli TOP10 was linearized and transformed by electroporation into an in-house prepared highly competent P. pastoris strain NRRLY-11430 (ATCC 76273).

[0463] 6.3.2 Example 3.2 Purification of ISVD His6-tagged ISVDs were purified by methods known in the art, and untagged ISVDs were purified by Protein A chromatography as known in the art, and concentrations were determined by measuring OD280 / OD340. Quality control was performed by SDS-PAGE and mass spectrometry.

[0464] 6.3.3 Example 3.3 Affinity Measurements by Surface Plasmon Resonance (SPR) of Monovalent and Bivalent ISVDs Selected ISVDs were characterized by measuring their affinity for human FcRn at pH 6.0 and pH 7.4.

[0465] The affinity of the monovalent purified untagged ISVD for human FcRn was measured on a ProteOn XPR36 (BioRad Laboratories, Inc.), MASS-2 (Sierra Sensors, GmbH), or SPR-32 (Bruker Corporation) instrument. Similarly, the affinity of bivalent ISVD constructs composed of two of the same monovalent building blocks of the purified untagged ISVD for human FcRn was measured on a ProteOn XPR36 (BioRad Laboratories, Inc.), MASS-2 (Sierra Sensors, GmbH), or SPR-32 (Bruker Corporation) instrument. To measure affinity, 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-immobilized with neutravidin; MASS-2). ISVDs were injected at six different concentrations (1–4000 nM), and binding was allowed for 120 s at 45 μl / min, followed by dissociation at 45 μl / min for 600–3600 s. Because of the lower binding at pH 7.4, affinity was measured using higher concentrations of ISVD at pH 7.4 compared to pH 5.5 or 6.0. Evaluation of the sensorgrams was based on a 1:1 Langmuir dissociation model, fitting the binding and dissociation rates simultaneously. The affinities of selected ISVDs for human FcRn at pH 6.0 and pH 7.4 are shown in Table 2.

[0466] [Table 16]

[0467] 6.4 Example 4: Genetic Engineering of T0263018B11 ELISAs were performed in PBS at pH 6.0 (diluted periplasmic extract 1:1000) and pH 7.4 (diluted periplasmic extract 1:10) as described in Example 2.1. After screening and sequencing, 206 unique clones (containing the FLAG3-His6 tag) were purified by phytip to generate periplasmic extracts, and the dissociation rates of these clones at human and cynomolgus FcRn at pH 6.0 and pH 7.4 were measured as described in Example 2.2 (see Table 3). Sixteen substitutions with a greater fold difference in kd at pH 6.0 compared to kd at pH 7.4 compared to the parent T0263018B11 were selected for subcloning and purification as tag-free ISVD proteins (as described in Examples 3.1 and 3.2) and characterized by affinity measurements (see Table 4).

[0468] [Table 17]

[0469] [Table 18]

[0470] [Table 19]

[0471] [Table 20]

[0472] [Table 21]

[0473] [Table 22]

[0474] [Table 23]

[0475] [Table 24]

[0476] [Table 25]

[0477] Based on the affinity data, a series of tag-free ISVD combinatorial variants was generated containing all possible combinations of the variants G26T, S31E, A33D, S52aQ, S100D, W100cS, and W100cK, including the parent amino acid at all six positions. In addition, several additional single variants at the same Kabat positions, namely, S31D, S100E, and W100cR, were examined. Periplasmic extracts containing these tag-free ISVD variants were generated, and dissociation rates at pH 6.0 were measured (see Table 5). Nineteen variants were selected for purification and further characterization by affinity measurements at pH 6.0 and pH 7.4 (see Table 6) and TSA (not shown). Based on the affinity data, five variants of FcRn ISVD T0263018B11 (including single variants and combinatorial variants) were selected for formatting and in vivo characterization (see Examples 8 and 10).

[0478] [Table 26]

[0479] [Table 27]

[0480] [Table 28]

[0481] [Table 29]

[0482] [Table 30]

[0483] 6.5 Example 5: Affinity Measurement by Surface Plasmon Resonance (SPR) of Pentavalent ISVDs Certain resulting mutational variants of the T0263018B11 ISVD building block were selected for further formatting by linking with one or more reference ISVD building blocks ("REF," which do not bind FcRn) to generate pentavalent ISVD formats. The affinity of the purified pentavalent ISVD constructs for human and cynomolgus monkey FcRn was measured under identical conditions using an assay similar to that described for the monovalent ISVD in Example 3. The affinities are shown in Table 7.

[0484] [Table 31]

[0485] 6.6 Example 6: Structural Interaction of FcRn-Binding ISVD T0263018B11 with FcRn The structure of the interaction between the FcRn-binding ISVD T0263018B11 and human FcRn(24-297;N125Q) / b2M(21-119) was confirmed by co-crystallization of the complex. The human FcRn(24-297;N125Q) / b2M(21-119) protein was produced in mammalian cells and contained the N125Q mutation to prevent N-glycosylation of the protein.

[0486] 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. The cryoprotectant was 18% PEG 80000, 200 mM NaCl, 50 mM TRIS (pH 8.0), 25% ethylene glycol at 19°C before freezing. Data sets were collected from both crystals on the EIGRR-X 6M detector (Dectris Ltd.) at beamline Proxima 1 from the synchrotron SOLEIL. The crystals belonged to the same space group, C2, and diffracted at 1.64 Å. Data were processed using autoproc (Vonrhein, C., Flensburg, C., Keller, P., Sharff, A., Smart, O., Paciorek, W., Womack, T. & Bricogne, G. (2011). autoPROC toolbox. Acta Cryst. D67, 293-302). Data processing and analysis were based on XDS (Kabsch, W. XDS. Acta Cryst. D66, 125-132 (2010)) and Aimless (PREvans and GN Murshudov, "How good are my data and what is the resolution?" Acta Cryst. D69, 1204-1214 (2013)). A model of FcRn(24-297;N125Q) / b2M(21-119) was obtained using the pdb structure 4N0U as a reference. A model of the FcRn-binding ISVD, T0263018B11, was constructed in Maestro (Schrodinger Release 2021-4; Maestro, Schroedinger, LLC, New York, NY, 2021). Molecular replacement was performed using Phaser (Coy et al., J. Appl. Cryst. (2007) 40, 658-674) in the CCP4 suite (Winn et al., Acta Cryst D67 (2011), 235-242).The structure was refined at 1.64 Å using cycles in Buster (Buster-TNT2.11.5, Global Phasing Ltd), followed by manual correction in COOT (Emsley, P. & Cowtan, K. (2004). Acta Cryst. D60, 2126-2132.) to a final Rfree of 24.3% and Rfactor of 20.7%.

[0487] Rsym and Rmerge - their formulas are as follows:

number

[0488] R and Rfree:

number

number

[0489] The residues involved in the epitope-paratope were determined using the software suite Schrodinger Release 2021-4; Maestro, Schroedinger, LLC, New York, NY, 2021. The FcRn epitope bound by T0263018B11 was compared with the epitopes bound by its natural ligands HSA and IgG, as found in PDB (Protein Database) ID: 4N0U, and the molecular surface representations are shown in Figures 2 and 3 (PyMOL 2.3.0).

[0490] 6.7 Example 7: Generation of Multivalent ISVD Constructs Containing Serum Albumin Binding ISVD ALB23002 and Affinity Measurement by Surface Plasmon Resonance (SPR) Based on their affinity for FcRn at pH 6.0 and pH 7.4, several ISVDs were selected for formatting with the HSA-binding ISVD building block, ALB23002 (SEQ ID NO: 35). The FcRn-binding ISVD building block was linked to ALB23002 by a 35GS linker (SEQ ID NO: 48), adding a C-terminal alanine. In certain formats, additional targeting ISVD building blocks (X, Y) were also added to represent potential future therapeutic leads. These formats were expressed in P. pastoris and purified as described in Examples 3.1 and 3.2.

[0491] The affinity of purified multivalent ISVD constructs for human and cynomolgus monkey FcRn was measured using 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-immobilized with neutravidin). ISVD constructs were injected at six different concentrations (1–4000 nM), and binding was allowed for 120 s at 45 μl / min, followed by dissociation at 45 μl / min for 600–3600 s. Due to the lower binding at pH 7.4, affinity was measured using higher concentrations of ISVD constructs at pH 7.4 compared to pH 6.0. Sensorgram evaluation was based on a 1:1 Langmuir dissociation model, fitting the binding and dissociation rates simultaneously. The affinities are shown in Tables 8 and 9.

[0492] [Table 32]

[0493] [Table 33]

[0494] The affinity of purified multivalent ISVD constructs for human and mouse serum albumin (HSA and MSA, respectively) was measured using a ProteOn XPR36 (BioRad Laboratories, Inc.) instrument. HSA / MSA (HSA: Sigma-Aldrich-Sigma, catalog no. A8763; MSA: Albumin Bioscience, catalog no. 2601) was immobilized on a ProteOn GLC sensor chip (BioRad Laboratories, 1765011). ISVD constructs were injected at six different concentrations (1–500 nM) and allowed to bind for 120 s at 45 μl / min, followed by dissociation at 45 μl / min for 600–3600 s. Sensorgram evaluation was based on a 1:1 Langmuir dissociation model, fitting the binding and dissociation rates simultaneously. The affinities are shown in Tables 10 and 11.

[0495] [Table 34]

[0496] [Table 35]

[0497] 6.8 Example 8: Serum PK Assay Development and Optimization To assess the half-life of the pH-dependent FcRn-binding ISVDs genetically fused to either the serum albumin-binding ISVD (ALB23002, SEQ ID NO: 35) alone or in combination with additional target-binding ISVDs (X, Y) or a control ISVD (REF), pharmacokinetic studies were initiated in TG32 (B6.Cg-Fcgrttm1Dcr Tg(FCGRT) 32Dcr / Dcr) mice. Specific and sensitive ligand-binding assays were developed to measure the concentrations of all constructs in mouse serum.

[0498] Streptavidin-coated MSD GOLD 96-well SMALLSPOT® plates (Meso Scale Discovery) were blocked with Superblock T20™ (Thermo Scientific) for 30 minutes at room temperature. Plates were then washed and incubated with 1.0 μg / mL of biotinylated generic mAbs directed against the frameworks of different ISVD building blocks at 600 rpm for 1 hour at room temperature. Calibrators and QCs were prepared in pooled mouse serum. After washing the plates, calibrators, QCs, and samples were added to the plates at an MRD of 20 in PBS 0.1% casein and incubated at 600 rpm for 1 hour at room temperature. After washing, plates were incubated with 2.0 μg / mL of sulfo-labeled mAbs directed against specific ISVD building blocks, depending on the format being evaluated, at 600 rpm for 1 hour at room temperature. After washing the plate, 2x MSD Read buffer (Meso Scale Discovery) was added and the plate was read on a Sector Imager Quickplex SQ 120 (Meso Scale Discovery).

[0499] Interference in the hIgG assay was assessed in an in vivo experiment using a mixture of human IgG (hIVIG; Privigen®) to mimic competition for endogenous IgG.

[0500] 6.9 Example 9: Pharmacokinetics of Bispecific ISVD Constructs in Transgenic Mice Six to eight Tg32 mice (B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / DcrJ) were intravenously injected with 1.9 mg / kg of the bispecific ISVD construct into the tail. The bispecific ISVD construct consists of two genetically fused ISVD domains: one N-terminal negative control (IRR) ISVD or FcRn-targeting ISVD (FcRn), and one C-terminal albumin-targeting ISVD (Alb23002).

[0501] Blood was collected at different time points (two mice per time point) and serum was prepared. Serum samples were analyzed for the presence of the bispecific ISVD construct by ELISA as described in Example 8. The results are shown in Figure 4.

[0502] Half-life values were obtained by extrapolating endosomal FcRn affinity in vivo with a mechanistic model and are reported in Table 12.

[0503] From these results, we can conclude that the half-life of bispecific ISVDs containing both FcRn- and albumin-binding ISVDs can be significantly improved compared to constructs containing only the albumin-binding ISVD. A maximum half-life of 8.2 days was observed for the FcRn-Alb23002 bispecific ISVD (T026301170 containing the FcRn-binding ISVD T0263018B11) compared to 2.4 days for the control (IRR-Alb23002).

[0504] [Table 36]

[0505] 6.10 Example 10: Pharmacokinetics in Mice of Bispecific ISVD Constructs Containing Mutational Variants of T0263018B11 Two to eight Tg32 mice (B6.Cg-Fcgrttm1Dcr Tg(FCGRT) 32Dcr / DcrJ) were intravenously injected in the tail with 1.9 mg / kg of bispecific ISVD constructs. The bispecific ISVD constructs consisted of two genetically fused ISVD domains, one N-terminally either a negative control (IRR) ISVD or an FcRn-targeting ISVD (FcRn), and the C-terminally an albumin-targeting ISVD (ALB23002). The FcRn-targeting ISVD was either the parent sequence or a mutated variant with reduced affinity at neutral pH (see Example 4).

[0506] Blood was collected at different time points (two mice per time point) and serum was prepared. Serum samples were analyzed for the presence of the bispecific ISVD construct by ELISA as described in Example 8. The results are shown in Figure 5. Half-life values were obtained by estimating in vivo endosomal FcRn affinity with a mechanistic PBPK model and are reported in Table 13.

[0507] From these results, it can be concluded that the bispecific ISVD construct containing the mutated variant of T0263018B11 and the albumin-binding ISVD exhibited a significantly improved half-life compared to the control (IRR-Alb23002-IRR0122) ISVD.

[0508] For all mutation variants evaluated, half-life was improved by at least 2.2-fold and up to 3.7-fold compared to the control (IRR-Alb).

[0509] [Table 37]

[0510] 6.11 Example 11: Pharmacokinetics in Mice of Multivalent ISVD Constructs Comprising an FcRn-Binding ISVD (FcRn), a Serum Albumin-Binding ISVD (ALB23002), and Additional Target-Binding ISVD Building Blocks (X, Y) To mimic relevant competition with hIgG, Tg32 mice (B6.Cg-Fcgrttm1Dcr Tg(FCGRT) 32Dcr / DcrJ) were pretreated with a mixture of purified hIgG (hIVIG; Privigen®). Privigen® was administered intravenously once weekly, with the first administration occurring two days before the start of the PK study. A total of three 250 mg / kg injections of Privigen® were administered to achieve physiologically relevant hIgG serum concentrations throughout the study (data not shown). All groups received Privigen® treatment, and an additional group that did not receive Privigen® was included. This group allowed us to assess the impact on PK of hIgG that might be expected when competing for the same epitope on the FcRn-binding ISVD and IgG Fc.

[0511] Two days after the first Privigen® dose, four to six Tg32 mice (B6.Cg-Fcgrttm1Dcr Tg(FCGRT) 32Dcr / DcrJ) were intravenously injected in the tail with equimolar amounts of multivalent ISVD constructs consisting of three to four genetically fused ISVD domains (the format and building blocks used for these constructs are described in Example 7 above). Each construct contained two ISVD domains directed against different potentially therapeutically relevant targets (X, Y) fused to either 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 containing three non-targeted ISVDs (IRR) and one albumin-binding ISVD (Alb23002) was administered (IRR00164).

[0512] Blood was collected at different time points (two mice per time point) and serum was prepared. Serum samples were analyzed for the presence of multivalent ISVD constructs by ELISA as described in Example 7.

[0513] The results are shown in Figure 6. Half-life values were obtained by estimating in vivo endosomal FcRn affinity with a mechanistic PBPK model and are reported in Table 14.

[0514] In multivalent ISVD constructs containing ISVD domains directed against potentially relevant therapeutic targets, the combination of an FcRn-binding ISVD and an albumin-binding ISVD significantly improved half-life compared to a control ISVD construct containing only the albumin-binding ISVD. The length of the linker between the FcRn-binding ISVD and the albumin-binding ISVD, as well as the position of the FcRn-binding ISVD, did not affect the pharmacokinetic properties (half-life) of the constructs. In addition, half-life was unaffected by the presence of relevant levels of hIgG, suggesting that the FcRn-binding ISVD binds to a different epitope relative to that bound by IgG Fc. For all multivalent ISVD constructs evaluated, half-life was improved by 2-fold compared to the control ISVD format.

[0515] [Table 38]

[0516] 6.12 Example 12: Pharmacokinetics of Multivalent FcRn-Binding ISVD Constructs Containing pH-Modified Variants of T0263018B11 Six to nine Tg32 mice (B6.Cg-Fcgrttm1Dcr Tg(FCGRT) 32Dcr / DcrJ) were intravenously injected in the tail with 4.5 mg / kg of multivalent ISVD constructs. The multivalent ISVD constructs consisted of five genetically fused ISVD domains, including one or two FcRn-targeting ISVDs (FcRn) and a negative control (IRR) ISVD, or a control construct (IRR00245) containing only the negative control (IRR) ISVD. The FcRn-targeting ISVDs were either the parent sequence or mutated variants with reduced affinity at physiological pH (see Example 4).

[0517] Blood was collected at different time points (2-3 mice per time point) and serum was prepared. Serum samples were analyzed for the presence of the ISVD construct by ELISA as described in Example 8. The results are shown in Figure 7. Half-life values were obtained by estimating in vivo endosomal FcRn affinity with a mechanistic PBPK model and are reported in Table 15.

[0518] Multivalent ISVD constructs containing T0263018B11 or mutant variants of T0263018B11 showed improved half-life compared to the control (IRR00245) ISVD construct.

[0519] [Table 39]

[0520] 6.13 Example 13: Composition of Polypeptide Constructs of the Invention 6.13.1 Fc Domains in Polypeptides According to Certain Embodiments of the Invention The polypeptides according to the invention (multispecific polypeptides as described above) may further comprise an IgG Fc domain as described herein. The IgG Fc domain refers to the C-terminal non-antigen binding region of the immunoglobulin G heavy chain, which comprises at least a portion of the constant region. In certain embodiments, the Fc domain may be a native Fc region, i.e., as occurs in a natural antibody, or may be a variant Fc region comprising one or more alterations, mutations, or variations compared to a native Fc domain. In certain embodiments, the IgG Fc domain may also be a fragment of a native Fc domain or a fragment of a variant Fc domain.

[0521] 6.13.1.1 Native (i.e., wild-type) Fc domain of immunoglobulin G (IgG) In certain embodiments, a polypeptide as described herein comprises a native Fc domain of human IgG, e.g., preferably the native Fc of human IgG4 (e.g., Uniprot sequence P01861, SEQ ID NO: 129). Polypeptides comprising at least one such native Fc domain are produced and tested for beneficial PK properties.

[0522] 6.13.1.2 Variant Fc Domains with Reduced Effector Function In certain specific embodiments, polypeptides according to the invention comprise a variant Fc domain that has altered binding properties for an Fc ligand compared to an unmodified parent Fc molecule. For example, the polypeptides described herein may comprise an Fc region having one or more of amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 replaced with a different amino acid residue, such that the variant Fc region has altered affinity for an effector ligand, such as an Fc receptor or the C1 component of complement, as described in U.S. Patent Nos. 5,624,821 and 5,648,260 (both by Winter et al.).

[0523] In certain embodiments, polypeptides of the invention comprise an Fc variant domain with reduced effector function, in particular the so-called "FALA" or "LALA" Fc variants in which residues 234 and 235 are substituted with 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 as described herein and the mutation S228P. These Fc variants are referred to as "pFALA."

[0524] Polypeptides comprising at least one Fc domain with the above mutations were produced and tested for beneficial PK properties as described in Example 16 below.

[0525] 6.13.1.3 Variant Fc Domains of IgG with Improved Binding Affinity to the FcRn Receptor In certain embodiments, polypeptides according to the invention comprise an Fc variant domain that exhibits improved binding to the FcRn receptor compared to a native Fc domain, including those that comprise substitutions at one or more of residues 259, 308, 428, and 434 of the Fc region. Other variants that enhance 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, and 434A (Shields et al., Journal of Biological Chemistry, 2001, 276(9):6591-6604).

[0526] In certain specific embodiments, a polypeptide according to the invention comprises an Fc variant domain in which methionine 428 has been substituted with a lysine and asparagine 434 has been substituted with a serine.

[0527] Polypeptides containing at least one Fc domain with the above mutations were produced and tested for beneficial PK properties.

[0528] 6.13.1.4 Variant Fc Domains of IgG with Reduced or No Binding to the FcRn Receptor In certain embodiments, polypeptides according to the invention comprise an Fc variant domain that exhibits reduced or no binding to the FcRn receptor compared to a native Fc domain, including those that comprise a substitution at one or more of residues 253, 310, and 453 of the Fc region.

[0529] In a specific embodiment, a polypeptide according to the invention comprises an Fc variant domain in which isoleucine 428 is substituted with alanine, histidine 310 is substituted with alanine, and histidine 453 is substituted with alanine, optionally in combination with histidine 453 substituted with alanine.

[0530] Polypeptides containing at least one Fc domain are produced and tested for beneficial PK properties.

[0531] 6.14 Example 14: Generation and Expression of Fusion Protein Constructs Comprising FcRn-Binding ISVDs and IgG4 Fc Domains Asymmetric fusion proteins of FcRn-binding Nanobody® VHH (ISVD) linked to the Fc domain of IgG4 were generated using knobs-into-holes technology, which is commonly known in the art (e.g., as described in the patent publication WO 1996 / 27011 by Genentech and the scientific publications by Ridgway, JB et al., "'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization," Protein engineering 9, 7 (1996); 617-21 and Merchant et al., "An efficient route to human bispecific IgG," Nature Biotechnology 16, (1998); 677-681).

[0532] DNA fragments of the FcRn-binding Nanobody® VHH and / or control Nanobody® VHH and IgG4 Fc domains, obtained by PCR using specific combinations of forward and reverse primers each containing a specific BpiI restriction site, were cloned into appropriate expression vectors by Golden Gate cloning (Engler C, Marillonnet S. Golden Gate cloning. Methods Mol Biol. 2014;1116:119-31). After confirmation by Sanger sequencing, the plasmid DNA was then transfected into CHOEBNALT85 cells (QMCF Technology) for protein production. Nanobody® VHH-Fc fusion proteins were purified from the cell supernatant by a Protein A capture step followed by ion exchange and / or size exclusion chromatography purification steps.

[0533] The Fc domain in the constructs was the IgG4 pFALA Fc framework sequence variant with knobs-in-holes mutations as described herein (the so-called Fc variant with FALA mutations and mutation S228P), whereas the FcRn Nanobody® VHH used was in each case the T0263018B11 sequence as described herein. The Nanobody® VHH sequences in these fusion proteins were fused to the N- and / or C-terminus of the Fc chain, i.e., via the IgG1 hinge, via a linker (as described in detail herein) (i.e., SEQ ID NO: 126 and / or a GS linker, i.e., 35GS, see SEQ ID NO: 48, respectively (see Figure 8)). One of these constructs (i.e., TP049) contains additional amino acid differences or mutations in the Fc framework sequence (i.e., I253A, H310A, H435A) and is shown in Figure 8 as IgG Fc(IHH). This Fc sequence variant was generated to test constructs that do not exhibit binding to FcRn via its Fc domain and is further referred to herein as a non-binding Fc variant. As a control, a Nanobody® VHH-Fc fusion protein was generated that contained the same composition as the test construct, except that the Nanobody® VHH that binds FcRn was replaced with a Nanobody® VHH that does not bind FcRn or any other putative target (i.e., a variant of the IgG4 pFALA Fc backbone sequence linked to two Nanobody® VHHs that do not bind FcRn; see e.g., constructs TP003 and TP008 in Figure 8).

[0534] 6.15 Example 15: FcRn Binding Study of Nanobody® VHH(ISVD)-Fc Fusion Polypeptide Construct (IgG4 pFALA) Typically, a series of Nanobody® VHH(ISVD)-Fc fusion proteins consisting of an IgG4 Fc domain linked to (i) a Nanobody® VHH(SVD) that specifically binds to FcRn, and (ii) a Nanobody® VHH(ISVD) that does not bind to FcRn or any other putative target but is comprised solely in a polypeptide construct, were generated to result in a size (i.e., molecular weight) similar to the corresponding test constructs (TP003, TP008, TP048, TP049 and TP050), as described above.

[0535] Nanobody® VHH-Fc proteins were characterized by measuring their affinity for human FcRn at pH 6.0 and pH 7.4 on a Biacore 8K+ instrument. To measure affinity, approximately 80 RU of biotinylated human FcRn was captured on a Series S Sensor Chip SA. Nanobody® VHH-Fc fusion proteins were injected at nine different concentrations (0.5-1500 nM) and allowed to bind for 120 seconds at 30 μl / min, followed by dissociation for 600 seconds. Evaluation of the sensorgrams was based on Bivalent Analyte fits. The affinities for human FcRn at pH 6.0 and pH 7.4 are shown in Tables 16 and 17. All constructs showed specific binding to FcRn at pH 6.0 and pH 7.4.

[0536] [Table 40]

[0537] [Table 41]

[0538] Dissociation rate analyses were performed on a Biacore 8K+ instrument at pH 6.0 and pH 7.4 using different coating densities of FcRn (approximately 80, approximately 500, approximately 1500, approximately 3000, and approximately 5000 RU). For Fc-fusion constructs containing the FcRn-binding Nanobody® VHH T0263018B11 and intact Fc (constructs TP048 and TP050), the dissociation rate (kd1) slowed as the density of FcRn on the surface increased (up to 5000 RU, see Figure 9), suggesting an avidity effect due to simultaneous binding of the Nanobody® VHH T0263018B11 and the Fc domain to FcRn. This slow dissociation rate was not detected for TP049, which contains a non-binding Fc-variant domain (Tables 18 and 19).

[0539] [Table 42]

[0540] [Table 43]

[0541] 6.16 Example 16: Serum PK Assay Development and Optimization 6.16.1 Example 16.1 Assay for detecting ISVD-IgG4 Fc fusion polypeptides in mouse serum A specific and sensitive ligand-binding assay was developed in-house to detect Nanobody® VHH(ISVD)-IgG4 Fc fusion polypeptides. Streptavidin-coated MSD GOLD 96-well SMALLSPOT® plates (Meso Scale Discovery) were blocked with Superblock T20™ (Thermo Scientific) for 30 minutes at room temperature. Plates were then washed and incubated with 2.0 μg / mL of biotinylated generic mAb directed against the framework of the ISVD moiety used in each construct at 600 rpm for 1 hour at room temperature. Calibrators and QCs were prepared in pooled mouse serum. After washing the plates, calibrators, QCs, and samples were added at an MRD of 20–100 (depending on the construct) in PBS 0.1% casein and incubated at 600 rpm for 1 hour at room temperature. After washing, plates were incubated with 2.0 μg / mL of sulfo-labeled mAB directed against specific ISVD moieties, depending on the format being evaluated, for 1 hour at room temperature at 600 rpm. After washing the plates, 2× MSD Read buffer (Meso Scale Discovery) was added and the plates were read on a Sector Imager Quickplex SQ 120 (Meso Scale Discovery). Interference with the hIgG assay was assessed in an in vivo experiment using a mixture of human IgGs (hIVIG; Privigen®) to mimic endogenous IgG competition.

[0542] 6.16.2 Example 16.2. Pharmacokinetics in Mice with ISVD-IgG4 Fc Fusion Four to six Tg32 mice (B6.Cg-Fcgrttm1Dcr Tg(FCGRT) 32Dcr / DcrJ) were intravenously injected in the tail with 5 mg / kg of ISVD-Fc fusion. The ISVD-Fc constructs consisted of the same IgG Fc, except for TP049 (IHH), which had the mutations I253A, H310A, and H435A to abolish FcRn binding. The Fc constructs were genetically fused to either two ISVD domains: two non-targeting ISVDs (negative control; TP003) or one non-targeting ISVD and one FcRn-targeting ISVD (TP048, TP049, and TP050; see Figure 8 and Example 14).

[0543] Blood was collected at different time points (two mice per time point) and serum was prepared. Serum samples were analyzed for the presence of Nanobody® VHH-Fc fusions by ligand binding assay as described in Example 15. The results are shown in Figure 10.

[0544] PK parameters were obtained from non-compartmental analysis in Phoenix WinNonlin® (version 8.2.2.227. Certara) using the Plasma Data Module. Half-life values are reported in Table 20.

[0545] From these results, we conclude that fusing an FcRn-binding ISVD to an IgG Fc domain at either the N- or C-terminus significantly extends the half-life of the Fc domain. Constructs containing the FcRn ISVD (TP048 and TP050, respectively) exhibited half-lives of 174–185 h compared with 134 h for the control construct (TP003). An antibody (IHH) containing the I253A, H310A, and H435A mutations in the Fc domain exhibited dramatically reduced binding...

Claims

1. 1. A polypeptide comprising at least one immunoglobulin single variable domain (ISVD) that specifically binds to an epitope on FcRn, wherein 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, wherein the amino acid residues are numbered according to SEQ ID NO:

1.

2. The epitope comprises a combination of the following 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 polypeptide of claim 1, wherein the amino acid residues are numbered according to SEQ ID NO:

1.

3. The epitope comprises a combination of the following 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 3. The polypeptide of claim 1, wherein the amino acid residues are numbered according to SEQ ID NO:

1.

4. The epitope comprises a combination of the following 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 polypeptide of any one of claims 1 to 3, wherein the amino acid residues are numbered according to SEQ ID NO:

1.

5. 5. The polypeptide of claim 1, wherein 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, wherein the amino acid residues are numbered according to SEQ ID NO:

1.

6. 6. The polypeptide of any one of claims 1 to 5, wherein 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, wherein the amino acid residues are numbered according to SEQ ID NO:

1.

7. 7. The polypeptide of any one of claims 1 to 6, wherein 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, wherein the amino acid residues are numbered according to SEQ ID NO:

1.

8. 8. The polypeptide of claim 1, 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 pH 5.0 to 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, characterized in that the at least one ISVD that specifically binds to the epitope on FcRn specifically binds to an amino acid residue on FcRn that is not involved in binding of FcRn to serum albumin and / or that is not involved in binding of FcRn to IgG.

10. the at least one ISVD that specifically binds to the epitope on FcRn consists of four framework regions (FR1 to FR4, respectively) and three complementarity-determining regions (CDR1 to CDR3, respectively); a) CDR1 (according to AbM) has the amino acid sequence of SEQ ID NO: 11 [=GFTFSSYAMY] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with the sequence of SEQ ID NO: 11; b) CDR2 (according to AbM) has the amino acid sequence of SEQ ID NO: 12 [= AISSGGGSTD] and / or has an amino acid sequence which has 4, 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with the sequence of SEQ ID NO: 12; c) CDR3 (according to AbM) has the amino acid sequence of SEQ ID NO: 13 [=DTLYTSLTSYSY] and / or has an amino acid sequence that has 4, 3, 2 or only 1 "amino acid difference" (as defined herein) with the sequence of SEQ ID NO:

13. A polypeptide according to any one of claims 1 to 9.

11. 11. The polypeptide of 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, characterized in that the polypeptide further comprises at least one ISVD that specifically binds to (human) serum albumin.

13. the at least one ISVD that specifically binds to serum albumin consists of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively); 13. The polypeptide of claim 12, wherein CDR1 is SFGMS (SEQ ID NO: 16), CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO: 17), and CDR3 is GGSLSR (SEQ ID NO: 18), with the CDRs being 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), with the CDRs being determined according to the AbM definition.

14. The polypeptide according to any one of claims 1 to 13, characterized in that the polypeptide further comprises an Fc region of an immunoglobulin (Ig).

15. The polypeptide according to any one of claims 1 to 14, characterized in that the polypeptide further comprises at least one ISVD that specifically binds to a therapeutic target.

16. Use of a polypeptide according to any one of claims 1 to 15 for increasing the in vivo half-life of a therapeutic or diagnostic compound.

17. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 15 for the treatment of a disease or disorder.

18. A method for producing a polypeptide according to any one of claims 1 to 15, comprising: a. expressing in a suitable host cell or host organism or in another suitable expression system a nucleic acid sequence encoding the nucleic acid sequence of any one of claims 1 to 15; optionally followed by: b. Isolating and / or purifying the polypeptide according to any one of claims 1 to 15. The method includes at least the following.

19. A nucleic acid sequence encoding a polypeptide according to any one of claims 1 to 15.

20. 20. A non-human host or host cell comprising a nucleic acid sequence according to claim 19 or a vector that expresses a nucleic acid sequence according to claim 19.

21. A polypeptide according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 17 for use in therapy.

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