NZ711245B2 - New Polypeptides having a Binding Affinity for the Neonatal Fc Receptor - Google Patents

Abstract

The present disclosure relates to a class of engineered polypeptides having a binding affinity for the neonatal Fc receptor (in the following referred to as FcRn), and provides an FcRn binding polypeptide comprising the sequence E X2 X3 X4 A X6 X7 EIRWLPNL X16 X17 X18 QR X21 AFI X25 X26 L X28 X29. The present disclosure also relates to the use of such an FcRn binding polypeptide as an agent for modifying pharmacokinetic and pharmacodynamic properties and as a therapeutic agent. he present disclosure also relates to the use of such an FcRn binding polypeptide as an agent for modifying pharmacokinetic and pharmacodynamic properties and as a therapeutic agent.

Description

NEW POLYPEPTIDES HAVING A BINDING AFFINITY FOR THE NEONATAL FC RECEPTOR Technical field of the invention The present disclosure relates to a class of engineered polypeptides having a binding affinity for the neonatal Fc receptor (in the following ed to as FcRn). The present disclosure also relates to the use of such an FcRn binding polypeptide as an agent for modifying pharmacokinetic and pharmacodynamic ties of a biomolecule, eg. a pharmaceutical, and as a therapeutic agent. 1O Background The neonatal Fc or (FcRn) is a heterodimeric protein consisting of a transmembrane MHC class |-|ike heavy chain (FcRn o-chain) and the 62— lobulin light chain, the latter also forming a part of MHC class I molecules (Simister and Mostov (1989) Nature 337:184—7; Burmeister etal. (1994) Nature 372:379—83).

FcRn is inantly located in endosomes and is able to bind to serum albumin and immunoglobulin G (lgG) at pH 3 6.5 and e them at pH 2 7.0 (reviewed in Roopenian (2007) Nat Rev Immunol 7:715—25).

FcRn carries out several distinct tasks in mammals (reviewed in Roopenian, supra). FcRn is involved in recycling of endocytosed lgG and serum albumin, thus avoiding their degradation in the lysosome, giving them longer half—life and higher availability in the blood than other serum proteins.

When lgG, serum albumin and other serum proteins are passively pinocytosed by cells in t with blood, the pH becomes gradually lower in the formed endosomes, which permits the binding of lgG and serum albumin to FcRn. The receptor is then, together with its bound ligand, transported via recycling endosomes back to the plasma membrane. After ing to the plasma membrane, the pH increases to above 7, at which point the bound ligand is released.

FcRn is also recognized for its ability to transport [96 over barriers such as the ta, the upper airway epithelium, the blood-brain barrier and the proximal small intestine. in mammals, the properties of FcRn are used to transcytose lgG from a mother to a fetus via the placenta, and to transcytose lgG from a mother’s milk to the blood stream of an infant in the proximal small intestine.

The expression pattern of FcRn differs between species. However, FcRn is widely expressed by cells in the blood brain barrier, upper airway epithelium, kidneys and vascular endothelia, and by antigen presenting cells as well as by other cells of hematopoietic origin in most species (reviewed in ian (2007), supra).

Antibodies and peptides with affinity towards FcRn (Liu et al. (2007) J lmmunol 179:2999-3011, Mezo et al. (2008) Proc Natl Acad Sci U S A 105:2337—42) and B2—microglobulin (Getman and Balthasar (2005) J Pharm Sci 94:718-29) have been developed with a view to inhibit the binding between endogenous lgG and FcRn. Another approach has been to mutate the Fc region of the lgG to get a higher affinity for FcRn va et al. (2006) lnt lmmunol 18:1759—69, Vaccaro etal. (2005) Nat Biotechnol 23:1283-8).

Fusion to the Fc domain or to albumin is a widely used strategy to increase the in vivo half-life of proteins. However, the large size of such fusion proteins adversly affects tissue penetration and reduces the specificity to the fusion partner (Valles ef al. (2011) J interferon Cytokine Res -184). On the other hand, mutations have been made in the Fc fragment of antibodies administered to non human primates to prolong half-life (Hinton et al. (2004) J Biol Chem 279:6213—6). However, this approach is only d in use to therapeutic antibodies, and cannot be olated to other therapeutic proteins unless the proteins in question are fused to Fc fragments, which also results in large size molecules. A number of chemical and recombinant methods have been devised to improve protein half—life, such as PEGylation and genetic fusions of the protein to the Fc domain of lgG or albumin (reviewed in enberger et al. (2009) Nat Biotechnol 21:1186-1190).

PEGylation of proteins has been ed to decrease their potency and 3O contribute to their immunoreactivity.

Fc—fusion proteins have also been used for oral and pulmonary delivery mediated by the FcRn (Low ef al., (2005) Human reproduction Jul;20(7):1805~ 13), r similar problems relating to tissue ation and reduced specificity remain, due to the size of the fusion molecules.

Hence, there is large need in the field for the continued ion of z les with high ty for FcRn. in ular, small binding molecules are needed that, when present as a fusion partner, do not adversely affect the properties of the molecules they are fused to and do not contribute to immunoreactivity.

Summary of the invention It is an object of the present disclosure to provide new FcRn binding agents for use in modifying pharmacokinetic and/or pharmacodynamic properties of a biomolecule, e.g. a pharmaceutical.

It is also an object of the t disclosure to provide new FcRn g agents for use as therapeutic agents in their own right, alone or as combination treatment.

It is an object of the present disclosure to provide a molecule allowing for efficient targeting of FcRn, while alleviating the above—mentioned and other drawbacks of current therapies.

These and other objects which are evident to the skilled person from the present sure are met by different aspects of the invention as d in the appended claims and as generally disclosed .

Thus, in the first aspect of the disclosure, there is provided a neonatal Fc receptor (FcRn) binding polypeptide, comprising an FcRn binding motif, BM, which motif consists of the amino acid sequence EX2 X3 X4 AX6 X7 EIR 16 X17 X18 QR X21 AF|X25 X26LX28 X29 wherein, independently from each other, X2 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y; X3 is selected from A, D, E, F, G, H, l, K, L, M, N, Q, R, S, T, V, Wand X4 is selected from A, D, E, F, G, H, l, K, L, N, Q, R, S, T, V, W and Y; ' X6 is selected from A, D, E, F, G, H, l, K, L, N, Q, R, S, T, V, W and Y; X7 is selected from A, F, H, l, K, L, N, Q, R, S, T, V, W and Y; X16 is selected from N and T; X17 is selected from F, W and Y; X18 is selected from A, D, E and N; X21 is selected from A, S, V and W; X25 is selected from A, D, E, F, G, H, l, K, L, N, Q, R, S, T, V, W and Y; X26 is selected from K and S; X28 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y; X29 is selected from D and R.

The above definition of a class of ce related, FcRn binding polypeptides is based on a statistical analysis of a number of random ptide variants of a parent scaffold, that were selected for their interaction with FcRn in several different ion experiments. The identified FcRn binding motif, or “BM", corresponds to the target binding region of the parent scaffold, which region constitutes two alpha helices within a three— l bundle protein domain. In the parent ld, the varied amino acid residues of the two BM helices constitute a binding surface for interaction with the constant Fc part of antibodies, In the present disclosure, the random variation of binding surface es and subsequent selection of variants have replaced the Fc interaction capacity with a capacity for interaction with FcRn.

In one embodiment of said FcRn binding polypeptide, the BM consists of the amino acid ce EX2 X3 X4 AXs X7 EIR WLPNLTX17 X18 QR X21 AFlX25 KLX28 D wherein, independently from each other, X2 is selected from A, D, E, F, H, l, K, L, N, Q, R, S, T, V, Wand Y; X3 is selected from A, D, E, F, G, H, l, K, L, M, N, Q, R, S, T, V, W and X4 is selected from A, D, E, F, G, H, l, K, L, N, Q, R, S, T, V, W and Y; X8 is selected from A, D, E, F, G, H, l, K, L, N, Q, R, S, T, V, W and Y; X7 is selected from A, F, H, l, K, L, N, Q, R, S, T, V, W and Y; X17 is selected from F, W and Y; X18 is selected from A, D, E and N; X21 is selected from A, S, V and W; X25 is selected from A, D, E, F, G, H, l, K, L, N, Q, R, S, T, V, W and Y; X28 is selected from A, D, E, F, H, l, K, L, N, Q, R, S, T, V, W and Y.

In r embodiment of the first aspect of the disclosure, said neonatal Fc receptor (FcRn) binding ptide comprises an FcRn binding motif, BM, which motif consists of the amino acid sequence EX2 X3 X4 AXs X7 EIR 16X17 X18 QR X21 AFIX25 8 X29 wherein, independently from each other, 1O X2 is selected from A, D, E, F, H, l, K, L, N, Q, R, S, T, V, W and Y; X3 is selected from A, D, E, F, G, H, l, K, L, M, N, Q, R, S, T, V, Wand X4 is selected from A, D, E, F, G, H, l, K, L, N, Q, R, S, T, V, W and Y; X6 is selected from A, E, F, G, H, l, K, Q, R, S and V; X7 is selected from A, F, H, K, N, Q, R, S and V; X16 is selected from N and T; X17 is selected from F, W and Y; X18 is selected from A, D, E and N; X21 is selected from A, S, V and W; X25 is selected from D, E, G, H, l, K, L, N, Q, R, S, T, V, W and Y; X26 is selected from K and S; X23 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y; X29 is selected from D and R.

In another embodiment of the first aspect, there is provided an FcRn binding polypeptide, wherein, independently from each other, X2 is selected from A, D, E, F, H, l, K, L, N, Q, R, S, T, V, W and Y; X3 is selected from A, D, E, F, H, l, K, L, M, N, Q, R, S, T, V, W and Y; X4 is selected from A, D, E, F, H, l, K, L, N, Q, R, S, T, V, W and Y; X5 is selected from A, E, F, G, H, l, K, Q, R and 8; X7 is selected from A, F, H, K, N, Q, R, S and V; X16 is selected from N and T; X17 is selected from F and Y; X18 is D; X21 is V; X25 is selected from D, E, H, I, K, L, N, Q, R, S, T, V, W and Y; X26 is selected from K and S; X28 is selected from A, D, E, F, H, l, K, L, N, Q, R, S, T, V and W and.

X29 is selected from D and R. in another embodiment of the first aspect, the BM consists of an amino acid sequence selected from i) EX2 X8 X4 AXe HEIR WLPNLTX17 X18 QR X21 AFlX25 KLX28 D wherein, independently from each other, X2 is selected from A, D, E, F, H, l, K, L, N, Q, R, S, T, V, W and Y; X3 is selected from A, D, E, G, H, K, L, M, N, Q, R, S, T, V and Y; X4 is selected from A, D, E, F, G, l, K, L, N, Q, R, S, T, V and Y; X8 is selected from A, G, K, R, S and V; X17 is selected from F, W and Y; X18 is selected from A, D, E and N; X21 is selected from A, S, V and W; X25 is selected from D, G, H, K, L, N, R, V and W; X28 is selected from A, D, E, H, K, L, N, Q, R, S, T, W and Y; ii) an amino acid sequence which has at least 96 % ty to said sequence. in yet r embodiment of said , the BM in sequence i) consists of an amino acid sequence selected from EX2 X3 X4 AXe HElR WLPNLTX17 X18 QR X21 AFlX25 KLX28 D wherein, independently from each other, X2 is selected from A, D, E, F, N, Q, R, S and W; X3 is selected from D, E, G, H, K, M, N, Q, S and T; X4 is selected from A, D, E, G, N, Q, R, S, T, V and Y; X6 is selected from A, G, S and V; X17 is selected from F, W and Y; X18 is selected from A, D, E and N; X21 is selected from A, S, V and W; X25 is selected from D, G, H, K, L, N, R and V; and X28 is selected from A, E, H, L, N, Q, R, S, T, W and Y.

As the skilled person will realize, the function of any polypeptide, including the FcRn binding capacity of the polypeptide of the present disclosure, is dependent on the tertiary structure of the polypeptide. It is therefore possible to make minor s to the sequence of amino acids in a polypeptide without ing the function thereof. Thus, the disclosure encompasses modified variants of the FcRn binding ptide, which are such that the FcRn binding characteristics are retained.

Therefore, as described above, also encompassed by the present disclosure is a FcRn binding polypeptide comprising an amino acid sequence with 96 % or r identity to a polypeptide as defined in i).

In some embodiments, such changes may be made in all positions of the ces of the FcRn binding polypeptide as disclosed herein. in other embodiments, such changes may be made only in the non-variable positions, also denoted as scaffold amino acid residues. In such cases, changes are not allowed in the variable positions, i.e. positions denoted with an “X” in sequence i). For example, it is possible that an amino acid residue belonging to a certain functional grouping of amino acid residues (e.g. hydrophobic, hydrophilic, polar etc) could be exchanged for another amino acid e from the same functional group.

The term "% ty”, as used throughout the specification, may for example be calculated as s. The query ce is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al. (1994) Nucleic Acids Research 22:4673—4680). A comparison is made over the window corresponding to the st of the aligned sequences. The shortest of the aligned sequences may in some instances be the target sequence. In other instances, the query sequence may constitute the st of the aligned sequences. The amino acid es at each position are compared, and the percentage of positions in the query sequence that have identical correspondences in the target sequence is reported as % identity.

Below follows a list of ments which further specify amino acid residue Xn, wherein n is an integer which denotes the position of said residue within the ptide described . To clarify, in cases where the BM comprised in the polypeptide may consist of either a given amino acid sequence or an amino acid ce with at least a given % identity to said given amino acid sequence, the Xn as used herein refers to an amino acid residue in said given amino acid sequence. For example, when applicable, Xn refers to an amino acid residue in sequence i) above. in one embodiment, X2 is selected from A, D, E, F, l, L, N, Q, R, S, T, V, W and Y.

In one embodiment, X2 is selected from A, D, F, l, L, N, Q, R, S, T, V, W and Y.

In one embodiment, X2 is selected from A, D, F, l, L, N, Q, R, S, V and W.

In one embodiment, X2 is selected from A, l, L, N, Q, R, S, T, V, W and in one embodiment, X2 is selected from A, l, L, N, Q, S, T, V and W. in one embodiment, X2 is selected from A, l, L, N, Q, V and W.

In one embodiment, X2 is selected from A, l, L, Q, V and W.

In one embodiment, X2 is selected from A, l, L and Q.

In one embodiment, X2 is selected from I, L and Q.

In one embodiment, X2 is selected from l and Q.

In one embodiment, X2 is l. in one embodiment, X2 is Q. in one embodiment, X3 is ed from A, D, E, G, H, K, L, M, N, Q, R, S, T, V and Y. in one embodiment, X3 is selected from A, D, E, H, K, L, M, N, Q, R, S, T, V and Y.

In one embodiment, X3 is selected from A, D, E, G, H, K, L, M, N, Q, R, S and T.

In one embodiment, X3 is ed from A, D, E, G, H, K, M, N, Q, S and T. in one embodiment, X3 is selected from A, D, E, G, H, M, N, Q, S and in one embodiment, X3 is selected from A, D, E, K, N, Q, S and T.

In one embodiment, X3 is selected from A, D, E, K, Q, and T.

In one embodiment, X3 is selected from A, D, E, Q and T.

In one embodiment, X3 is selected from D, E and T.

In one embodiment, X3 is selected from D and E.

In one embodiment, X3 is D.

In one embodiment, X3 is E.

In one embodiment, X4 is selected from A, D, E, F, G, I, K, L, N, Q, R, S, T, V and Y.

In one embodiment, X4 is ed from A, D, E, G, N, Q, R, S, T and V. ‘ In one embodiment, X4 is ed from A, D, E, F, I, K, L, N, Q, R, S, T and V.

In one embodiment, X4 is selected from A, D, E, I, K, N, Q, R, S and T.

In one embodiment, X4 is selected from A, D, E, I, K, Q, S and T.

In one embodiment, X4 is selected from A, D, I, K, Q and S.

In one embodiment, X4 is selected from A, D, E, K and S.

In one embodiment, X4 is selected from A, D, K and S.

In one embodiment, X4 is selected from A, D, E and K.

In one embodiment, X4 is selected from A, D and K.

In one embodiment, X4 is selected from A and D.

In one embodiment, X4 is selected from A and E.

In one embodiment, X4 is A.

In one embodiment, X4 is D.

In one embodiment, X4 is E.

In one embodiment, X5 is selected from A, G, K, Q, R, S and V.

In one ment, X6 is selected from A, G, K, R, S and V.

In one embodiment, X6 is selected from A, G, K, R and S.

In one embodiment, X6 is selected from A, G, K, S and V.

In one embodiment, X5 is selected from A, G, K and V.

In one embodiment, X6 is selected from A, G, K and S.

In one embodiment, X5 is selected from A, G and K.

In one ment, X6 is selected from A, G and V.

In one embodiment, X6 is selected from A and G.

In one embodiment, X6 is A.

In one embodiment, X5 is G.

In one embodiment, X7 is selected from A and H.

In one ment, X7 is H.

In one embodiment, X16 is T.

In one embodiment, X15 is N.

In one embodiment, X17 is selected from F and Y.

In one embodiment, X17 is F.

In one embodiment, X18 is ed from A, D and E.

In one embodiment, X18 is selected from A and D.

In one embodiment, X18 is D.

In one embodiment, X21 is selected from V and W.

In one embodiment, X21 is V.

In one ment, X25 is selected from D, E, G, H, K, L, N, Q, R, V and W.

In one embodiment, X25 is selected from D, G, H, K, L, N, R, V and W.

In one embodiment, X25 is ed from D, G, H, K, L, N, R and V.

In one embodiment, X25 is selected from H, L, R, V and W.

In one embodiment, X25 Is selected from H, R, V and W.

In one embodiment, X25 is selected from H, R and V.

In one embodiment, X25 is ed from H, L and R.

In one embodiment, X25 is selected from H and R.

In one embodiment, X25 is selected from H and V.

In one embodiment, X25 is H.

In one embodiment, X25 is K.

In one embodiment, X25 is S.

In one embodiment, X28 is selected from A, D, E, H, K, L, N, Q, R, S, T, W and Y.

In one embodiment, X28 is ed from A, D, E, K, L, N, Q, R, S, T, W and Y.

In one embodiment, X28 is selected from A, D, E, L, R, S, T, W and Y.

In one embodiment, X28 is selected from A, D, K, L, N, Q, R, S, T and In one embodiment, X28 is selected from A, D and R.

In one embodiment, X28 is selected from A and R.

In one embodiment, X28 is selected from D and R.

In one embodiment, X28 is A.

In one embodiment, X28 is R.

In one embodiment, X29 is D.

In one embodiment, X29 is R.

In one embodiment, X5X7 is selected from AH and GH.

In one embodiment, X6X7 is AH.

In one embodiment, X6X7 is GH.

In one embodiment, X17X18 is selected from FD and YD.

In one embodiment, X17X18 is FD.

In a more ic embodiment defining a sub-class of the FcRn binding polypeptide, the sequence ls at least three of the six conditions I— I. X5 is selected from A, G, K and S, such as in particular A; 1O II. X7 is H; III. X17 is selected from F and Y, such as in particular F; IV. X18 is D; V. X21 is selected from V and W, such as in particular V; VI. X25 is selected from H and R, such as in particular H.

In some examples of an FcRn binding polypeptide according to the first aspect, said sequence fulfills at least four of the six conditions l—Vl. More specifically, the sequence may fulfill at least five of the six conditions I—Vl, such as all of the six ions l—VI.

As described in detail in the experimental section to follow, the selection of FcRn binding ptide variants has led to the identification of a number of dual FcRn g motif (BM) sequences. These sequences constitute dual embodiments according to this aspect. The sequences of individual FcRn binding motifs are presented in Figure 1 and as SEQ ID NO:1—353. Hence, in one embodiment of the FcRn binding polypeptide according to this aspect, the sequence is selected from the group consisting of SEQ ID NO:1—353. In one embodiment, the sequence is selected from the group consisting of SEQ ID NO:1—15, SEQ ID NO:17—14O and SEQ ID 3O NO:353. In one embodiment, the sequence is selected from the group consisting of SEQ ID NO:1-2 and SEQ ID NO:17—140. In one ment, the sequence is selected from the group consisting of SEQ ID NO:1—2, SEQ ID NO:17—92, SEQ ID NO:94-103, SEQ ID NO:105—125 and SEQ ID NO:127— 140. In one embodiment, the sequence is selected from the group consisting of SEQ ID NO:1—8, SEQ ID NO:13 SEQ ID NO:19-20, SEQ ID NO:23, SEQ ID N0228, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:75—77 and SEQ ID NO:353. In r embodiment, the ce is selected from the group consisting of SEQ ID N0:1, SEQ ID N0:23, SEQ ID N0228, SEQ ID N0z41, SEQ ID N0:44, SEQ ID N0265, SEQ ID NO:73 and SEQ ID NO:75—77. In yet another embodiment, the ce is selected from SEQ ID N0:1, SEQ ID N0:23, SEQ ID N0z44, SEQ ID N0:65, SEQ ID NO:75 and SEQ ID NO:77. In one embodiment, the ce Is selected from SEQ ID N021, SEQ ID N023 and SEQ ID NO:75. In one embodiment, the sequence is SEQ ID N021.

In some embodiments of the present disclosure, the BM as defined 1O above “forms part of” a three—helix bundle protein domain. This is understood to mean that the sequence of the BM is “inserted” into or “grafted” onto the sequence of the al three—helix bundle domain, such that the BM replaces a similar structural motif in the original domain. For example, without wishing to be bound by theory, the BM is thought to constitute two of the three helices of a three—helix bundle, and can ore replace such a two-helix motif within any three—helix bundle. As the skilled person will realize, the replacement of two helices of the three—helix bundle domain by the two BM helices has to be med so as not to affect the basic structure of the polypeptide. That is, the overall folding of the Ca backbone of the polypeptide according to this embodiment of the invention is substantially the same as that of the three—helix bundle protein domain of which it forms a part, e.g. having the same elements of ary structure in the same order etc.

Thus, a BM according to the disclosure “forms part” of a helix bundle domain if the ptide according to this embodiment of the aspect has the same fold as the original domain, implying that the basic structural properties are shared, those ties e.g. ing in similar CD spectra. The skilled person is aware of other parameters that are relevant.

In particular embodiments, the FcRn binding motif (BM) thus forms part of a three—helix bundle protein domain. For example, the BM may essentially constitute two alpha helices with an interconnecting loop, within said three- helix bundle protein domain. In particular embodiments, said three—helix bundle protein domain is selected from domains of bacterial receptor proteins.

Non-limiting examples of such s are the five different three—helical domains of Protein A from Staphylococcus aureus, such as domain B, and derivatives thereof. In some embodiments, the three—helical bundle protein domain is a variant of protein Z, which is derived from domain B of staphylococcal Protein A. in embodiments where the FcRn binding polypeptide of the invention forms part of a three-helix bundle protein domain, the FcRn binding polypeptide may se an amino acid sequence selected from: iii) K—[BM—DPSQS XaXbLLXc EAKKL Q; wherein [BM] is an FcRn binding motif as defined herein, provided that X29 is D; ’10 Xa is selected from A and S; Xb is selected from N and E; Xc is selected from A, S and C; Xd is ed from E, N and S; Xe is ed from D, E and S; Xf is selected from A and S; iv) an amino acid sequence which has at least 93 % identity to a sequence defined by iii). in embodiments where the FcRn binding polypeptide of the invention forms part of a three-helix bundle protein , the FcRn binding polypeptide may comprise an amino acid sequence selected from: V) K-[BAfl—QPEQS XaXbLLXc EAKKL XdXeXfQ; wherein [BM] is an FcRn binding motif as defined herein, provided that X29 is R; Xa is selected from A and S; 3O Xb is selected from N and E; Xc is selected from A, S and C; Xd is selected from E, N and S; Xe is selected from D, E and S; Xr is selected from A and S; and vi) an amino acid sequence which has at least 93 % identity to a sequence defined by v).

As discussed above, polypeptides comprising minor changes as compared to the above amino acid sequences which do not largely affect the tertiary structure and the function thereof are also within the scope of the present disclosure. Thus, in some embodiments, sequence iv) or sequence vi) has at least 95 %, for example at least 97 % identity to a sequence defined by iii) and v), respectively.

In one embodiment, Xa in sequence iii) or v) is A. In an alternative embodiment, Xa in ce iii) or v) is S. in one embodiment, Xb in sequence iii) or v) is N. In an ative embodiment, Xb in sequence iii) or v) is E.

In one embodiment, Xc in sequence ill) or v) is A. in an alternative embodiment, X0 in sequence iii) or v) is S. in yet another alternative embodiment, Xc in sequence ill) or v) is C. in one embodiment, Xd in sequence iii) or v) is E. in one embodiment, Xd in sequence iii) or v) is N.

In one embodiment, Xd in sequence ill) or v) is S. in one embodiment, Xe in sequence iii) or v) is D.

In one embodiment, Xe in sequence iii) or v) is E.

In one embodiment, Xe in ce iii) or v) is S. in one embodiment, XdXe in sequence iii) or v) is selected from EE, ES, SE and SS. ln one ment, XdXe in sequence iii) or v) is ES. in one ment, XdXe in sequence iii) or v) is SE.

In one embodiment, Xf in sequence iii) or v) is A. in an alternative embodiment, Xf in sequence iii) or v) is S. 3O In one embodiment, in ce iii) or v), Xa is A; Xb is N; Xc is A and Xf is A. in one embodiment, in ce iii) or v), Xa is A; Xb is N; X0 is C and Xf is A.

In one embodiment, in sequence iii) or v), X3 is S; Xb is E; X0 is S and Xf is S.

In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is C and Xf is S.

In one embodiment, in sequence iii) or v), Xa is A; Xb is N; X0 is A; XdXe is ND and Xf is A.

In one embodiment, in sequence iii) or v), Xa is A; Xb is N; Xc is C; XdXe is ND and Xf is A.

In one embodiment, in ce iii) or v), Xa is S; Xb is E; Xc is S; XdXe is ND and Xf is S.

In one embodiment, in Sequence iii) or v), Xa is S; Xb is E; Xc is C; XdXe is ND and Xf is S.

In one embodiment, in sequence iii) or v), X3 is A; Xb is N; Xc is A; XdXe is SE and Xf is A.

In one embodiment, in sequence iii) or v), Xa is A; Xb is N; Xc is C; XdXe is SE and Xf is A.

In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is S; XdXe is SE and Xf is S.

In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is C; XdXe is SE and Xf is S.

In one embodiment, in sequence iii) or v), Xa is A; Xb is N; Xc is A; XdXe is ES and Xf is A.

In one embodiment, in sequence iii) or v), Xa is A; Xb is N; Xc is C; XdXe is ES and Xf is A.

In one embodiment, in ce iii) or v), Xa is S; Xb is E; Xc is S; XdXe is ES and Xf is S.

In one embodiment, in sequence iii) or v), Xa is S; Xb is E; Xc is C; XdXe is ES and Xr is S.

In yet a further embodiment, sequence iii) in the definition of FcRn binding polypeptides above is selected from the group consisting of SEQ ID NO:354—706. In one embodiment, sequence iii) is selected from the group consisting of SEQ ID NO:354—368, SEQ ID NO:370-493 and SEQ ID NO:706.

In one embodiment, sequence iii) is selected from the group consisting of SEQ ID NO:354—355 and SEQ ID NO:370—493. In one ment, sequence iii) is selected from the group consisting of SEQ ID —355, SEQ ID NO:370—445, SEQ ID NO:447-456, SEQ ID NO:458—478 and SEQ ID N02480— 493. In one embodiment, sequence iii) is selected from the group consisting of SEQ ID -361, SEQ ID NO:366, SEQ ID —373, SEQ ID NO:376, SEQ ID NO:381, SEQ ID N02394, SEQ ID NO:397, SEQ ID NO:418, SEQ ID NO:423, SEQ ID NO:426, SEQ ID NO:428—43O and SEQ ID NO:706.

In another embodiment, sequence iii) is selected from the group ting of SEQ ID NO:354, SEQ ID NO:376, SEQ ID NO:381, SEQ ID N02394, SEQ ID NO:397, SEQ ID NO:418, SEQ ID NO:426 and SEQ ID NO:428-430. In yet another embodiment, sequence iii) is selected from SEQ ID NO:354, SEQ ID NO:376, SEQ ID NO:397, SEQ ID NO:418, SEQ ID NO:428 and SEQ ID NO:430. In one embodiment, sequence iii) is selected from SEQ ID NO:354, SEQ ID NO:376 and SEQ ID NO:428. In one embodiment, sequence iii) is SEQ ID NO:354.

Also, in a further embodiment, there is ed an FcRn binding polypeptide as defined above, which comprises an amino acid sequence ed from: vii) YAK—[BM]-DPSQS SELLXC EAKKL NDSQA P; wherein [BM] is an FcRn binding motif as defined above and Xc is ed from A, S and C; and viii) an amino acid sequence which has at least 94 % identity to a sequence defined by vii).

Alternatively, there is provided an FcRn binding polypeptide as defined above, which comprises an amino acid sequence selected from: ix) FNK-[BM1—DPSQS ANLLXc EAKKL NDAQA P; wherein [BM] is an FcRn binding motif as defined above and Xc is selected from A and C; and x) an amino acid ce which has at least 94 % identity to a sequence defined by ix).

As discussed above, polypeptides comprising minor changes as compared to the above amino acid sequences that do not largely affect the tertiary structure and the function f are also within the scope of the present disclosure. Thus, in some embodiments, the FcRn g polypeptide as d above may comprise a sequence which is at least 96 %, such as at least 98 % identical to a sequence defined by vii) or ix).

In some embodiments, the FcRn binding motif may form part of a polypeptide comprising an amino acid sequence selected from ADNNFNK-[BM]—DPSQSANLLSEAKKLNESQAPK; ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; ADNKFNK-[BW—DPSVSKEILAEAKKLNDAQAPK; ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK; [BM]—DPSQSANVLGEAQKLNDSQAPK; VDNKFNK—[BM]-DPSQSANLLAEAKKLNDAQAPK; AEAKYAK—[BM]—DPSESSELLSEAKKLNKSQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLNDAQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLNDSQAPK; AEAKYAK—[BM]—DPSQSSELLSEAKKLNDSQAPK; AEAKYAK—[BM1—DPSQSSELLSEAKKLSESQAPK AEAKYAK—[BM]-DPSQSSELLSEAKKLESSQAPK VDAKYAK—[BM1—DPSQSSELLSEAKKLNDSQAPK; VDAKYAK—[BM]-DPSQSSELLSEAKKLSESQAPK; VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK; VDAKYAK—[BM1-DPSQSSELLAEAKKLNKAQAPK; and AEAKYAK—[BM]-DPSQSSELLAEAKKLNKAQAPK; wherein [BM] is an FcRn g motif as d above. in one embodiment, the FcRn binding polypeptide comprises an amino acid sequence selected from: xi) AEAKYAK-[BMJ-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is an FcRn binding motif as defined above; and xii) an amino acid sequence which has at least 94 % identity to the sequence defined in xi).

In one embodiment, sequence xi) is selected from the group consisting of SEQ lD NO:1060—1062. in one embodiment, the FcRn binding polypeptide comprises an amino acid sequence selected from: xiii) VDAKYAK—[BM]—DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is an FcRn g motif as defined above; and xiv) an amino acid sequence which has at least 94 % identity to the sequence defined in xiii).

Sequence xiii) in such a polypeptide may for example be selected from the group consisting of SEQ ID NO:707—1059. in one embodiment, sequence xiii) is selected from the group consisting of SEQ lD NO:707-721, SEQ lD N01723-846 and SEQ lD NO:1059. In one embodiment, ce xiii) is selected from the group ting of SEQ ID N01707—7O8 and SEQ lD -846. in one embodiment, sequence xiii) is selected from the group consisting of SEQ ID NO:707—708, SEQ ID —798, SEQ ID NO:800— 809, SEQ ID NO:811-831 and SEQ ID N02833—846. In one embodiment, ce xiii) is ed from the group consisting of SEQ ID NO:707—714, SEQ ID NO:719, SEQ ID NO:725-726, SEQ ID NO:729, SEQ ID NO:734, SEQ ID NO:747, SEQ ID NO:750, SEQ ID NO:771, SEQ ID NO:776, SEQ ID NO:779, SEQ ID NO:781—783 and SEQ ID NO:1059. In another embodiment, sequence xiii) is selected from the group consisting of SEQ ID NO:707, SEQ ID NO:729, SEQ ID NO:734, SEQ ID NO:747, SEQ ID NO:750, SEQ ID NO:771, SEQ ID NO:779 and SEQ ID NO:781—783. In yet r 1O embodiment, sequence xiii) is selected from SEQ ID NO:707, SEQ ID NO:729, SEQ ID NO:750, SEQ ID NO:771, SEQ ID NO:781 and SEQ ID NO:783. In one embodiment, sequence xiii) is selected from SEQ ID , SEQ ID NO:729 and SEQ ID NO:781. In one embodiment, sequence xiii) is SEQ ID NO:707.

Again, polypeptides comprising minor changes as compared to the above amino acid sequences which do not largely affect the ry structure and the function thereof are also within the scope of the present disclosure.

Thus, in some embodiments, the FcRn binding polypeptide as defined above may comprise a sequence which is at least 96 %, such as at least 98 % identical to a sequence defined by xi) or xiii).

The terms “FcRn binding” and ng affinity for FcRn” as used in this specification refer to a property of a polypeptide which may be tested for example by the use of surface plasmon resonance (SPR) technology or ELISA.

For example as described in the examples below, FcRn binding ty may be tested in an experiment in which FcRn, or a correctly folded fragment thereof, is immobilized on a sensor chip of the instrument, and the sample ning the polypeptide to be tested is passed over the chip. Alternatively, the polypeptide to be tested is immobilized on a sensor chip of the instrument, and a sample containing FcRn, or a correctly folded fragment f, is passed over the chip. The skilled person may then interpret the results ed by such experiments to establish at least a qualitative measure of the binding affinity of the polypeptide for FcRn. If a quantitative measure is desired, for example to determine a KD value for the interaction, e plasmon resonance methods may also be used. Binding values may for example be defined in a Biacore (GE Healthcare) or ProteOn XPR 36 (Bio— Rad) instrument. FcRn is suitably immobilized on a sensor chip of the instrument, and samples of the polypeptide whose affinity is to be determined are prepared by serial dilution and injected in random order. Ko values may then be calculated from the results using for example the 1:1 Langmuir binding model of the BlAevaluation 4.1 software, or other suitable re, provided by the instrument manufacturer.

Alternatively, as described in the examples below, FcRn binding affinity may be tested in an experiment in which samples of the polypeptide are 1O captured on dy coated ELISA plates, and biotinylated FcRn is added ed by streptavidin conjugated HRP. TMB substrate is added and the absorbance at 450 nm is measured using a multi-well plate reader, such as Victor3 (Perkin Elmer). The skilled person may then interpret the results obtained by such experiments to ish at least a qualitative measure of the binding affinity of the polypeptide for FcRn. If a quantitative measure is desired, for example to determine the K0 value (the half maximal effective concentration) for the ction, ELISA may also be used. The response of the ptides against a on series of biotinylated FcRn are ed using ELISA as described above. The d person may then interpret the results obtained by such ments and KD values may be calculated from the results using for example GraphPad Prism 5 and non—linear regression. in one embodiment, there is provided an FcRn binding polypeptide, which is capable of binding to FcRn at pH 6.0 such that the K0 value of the interaction is at most 1 x 10“6 M, such as at most 1 x 10‘7 M, such as at most 1 x10'8 M, such as at most 1 x10“9 M, such as at most 1 x10"10 M. An FcRn binding polypeptide according to this embodiment would bind, or remain bound, to FcRn in acidic pH conditions, such as pH 6.0, for example in a lysosome. if such a polypeptide were to enter an increasingly acidic intracellular environment, it would be recycled to the plasma membrane h its interaction with FcRn, and thus avoid degradation. in one embodiment, the KB value of the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is higher than the KB value of said interaction at pH 6.0. Thus, the FcRn binding polypeptide would bind to FcRn with higher affinity at pH 6.0 than at pH 7.4. in one ment, the KB value of said interaction at pH 7.4 is at least 2 times higher, such as at least 5 times higher, such as at least 10 times higher, such as at least 50 times higher, such as at least 100 times higher than the KB value of said interaction at pH 6.0.

In one embodiment, the KO value of the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is at least 1 x 10‘8 M, such as at least 1 x10'7 M, such as at least 1 x 10'6 M, such as at least 1 x 10‘5 M. In some embodiments, the only criterion for the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is that any FcRn binding polypeptide which has bound to FcRn during more acidic conditions is released more rapidly from FcRn when the pH value increases. 1O in an alternative ment, there is provided an FcRn binding polypeptide, for which the KD of said interaction at pH 7.4 is the same as or lower than the KB of said interaction at pH 6.0. An FcRn binding polypeptide according to this embodiment would bind or remain bound to FcRn in acidic pH ions (i.e. would have an off—rate at pH 6.0 which is iently slow to avoid release), for example in the lysosome, as well as in neutral or slightly basic pH conditions, for example on the plasma membrane. In a more specific embodiment, the K0 value of said interaction at pH 7.4 is at least 2 times lower, such as at least 5 times lower, such as at least 10 times lower, such as at least 50 times lower, such as at least 100 times lower than the KD value of said interaction at pH 6.0. in another embodiment, there is provided an FcRn binding polypeptide, which is capable of binding to FcRn at pH 7.4 such that the KD value of the interaction is at most 1 x 10'6 M, such as at most 1 x 10‘7 M, such as at most 1 x10“8 M, such as at most 1 x 10~9 M, such as at most 1 x 10'10 M. An FcRn binding polypeptide according to this embodiment would bind or remain bound for an extended time to FcRn in neutral or slightly basic pH conditions, such as pH 7.4, for example on the plasma membrane. The term “remain bound” should be understood to mean an interaction having a slow off—rate at given conditions. 3O in general, the skilled person knows that the KB value of an interaction is d as the ratio between the off—rate (koff) and the e (kon). Thus, a high KD value may be due to either a high koff, a low kon or both, and conversely, a low Kn value may be due to either a low koff, a high kon or both.

The skilled person will understand that various modifications and/or ons can be made to an FcRn binding polypeptide according to any aspect disclosed herein in order to tailor the polypeptide to a specific application without departing from the scope of the present disclosure.

For example, in one embodiment there is provided an FcRn binding polypeptide as described herein, which polypeptide has been extended by one or more amino acids at the C terminal and/or N terminal end. Such a polypeptide should be understood as a polypeptide having one or more additional amino acid residues at the very first and/or the very last position in the polypeptide chain. Thus, an FcRn binding polypeptide may comprise any suitable number of additional amino acid residues, for example at least one 1O additional amino acid e. Each additional amino acid residue may dually or collectively be added in order to, for example, improve production, purification, ization in vivo or in vitro, coupling, or ion of the ptide. Such additional amino acid es may comprise one or more amino acid residues added for the purpose of chemical coupling. One example of this is the addition of a cysteine residue. Such additional amino acid es may also provide a "tag” for purification or ion of the polypeptide, such as a His6 tag or a "myc" (c—myc) tag or a ”FLAG” tag for interaction with antibodies specific to the tag or immobilized metal affinity chromatography (lMAC) in the case of the hexahistidine tag.

The further amino acids as sed above may be coupled to the FcRn binding polypeptide by means of chemical conjugation (using known organic chemistry methods) or by any other means, such as expression of the FcRn binding polypeptide as a fusion protein orjoined in any otherfashion, either directly or via a linker, for example an amino acid linker.

The further amino acids as discussed above may for example comprise one or more polypeptide domain(s). A further polypeptide domain may provide the FcRn g polypeptide with r function, such as for example another binding on, or an enzymatic function, or a toxic function or a fluorescent signaling function, or combinations thereof.

A further polypeptide domain may moreover provide r FcRn binding moiety with the same FcRn binding function. Thus, in a further embodiment, there is provided an FcRn binding ptide in a multimeric form. Said multimer is understood to comprise at least two FcRn binding polypeptides as disclosed herein as monomer units, the amino acid sequences of which may be the same or different. Multimeric forms of the polypeptides may comprise a suitable number of domains, each having an FcRn binding motif, and each forming a monomer within the multimer. These s may have the same amino acid sequence, but alternatively, they may have different amino acid sequences. In other words, the FcRn binding polypeptide of the invention may form homo— or heteromultimers, for example homo— or heterodimers. In one embodiment, there is provided an FcRn binding polypeptide, wherein said monomeric units are covalently coupled together. In another embodiment, said FcRn g polypeptide monomer units are expressed as a fusion protein. ln one ment, there is provided an FcRn binding polypeptide in dimeric form.

Additionally, ”heterogenic" fusion polypeptides or proteins, or conjugates, in which an FcRn binding polypeptide described herein, or multimer thereof, constitutes a first , or first moiety, and the second and further moieties have other functions than g FcRn, are also contemplated and fall within the ambit of the present disclosure. The second and further moiety/moieties of the fusion polypeptide or conjugate in such a n suitably have a desired biological activity.

Thus, in a second aspect of the present disclosure, there is provided a fusion protein or a ate, sing a first moiety consisting of an FcRn binding polypeptide according to the first , and a second moiety consisting of a polypeptide having a desired biological activity. In another embodiment, said fusion protein or conjugate may additionally comprise further moieties, comprising desired biological activities that can be either the same or different from the biological activity of the second moiety.

Such heterogenic fusion polypeptides could also be used to create heteromultimeric complexes of higher order. One example is a heterodimeric x of two fusion polypeptides, each comprising an FcRn binding polypeptide according to the present disclosure in fusion with another moiety.

Such a complex could for example form a heterodimer in vivo or in vitro and be held together by non—covalent and/or covalent ctions. A specific example of such a complex is a Fab fragment, in which both the light chain and heavy chain are produced in fusion with one FcRn binding polypeptide each, and which may include an domain disulphide bond. Many biologically relevant, heterodimeric complexes known to the skilled person may be constructed using FcRn binding fusion proteins as monomer units.

In one embodiment of said fusion protein or conjugate, the total size of the molecule is below the threshold for efficient renal clearance upon stration to a ian subject.

In another embodiment of said fusion protein or conjugate, the total size of the le is above the threshold for efficient renal clearance upon administration to a mammalian subject. in one embodiment, there is provided a fusion protein or conjugate, wherein the in vivo half—life of said fusion protein or conjugate is longer than the in vivo half—life of the polypeptide having the desired biological activity per Non—limiting examples of a desired biological ty comprise a 1O therapeutic activity, a binding activity, and an enzymatic activity.

In one embodiment, said desired biological activity is a binding activity to a selected target.

One example of such a binding activity is a binding activity, which increases the in vivo half—life of a fusion n or conjugate. This fusion protein or conjugate may comprise at least one further moiety. in one particular embodiment, said target is albumin, binding to which increases the in vivo half—life of said fusion protein or conjugate. In one embodiment, said albumin binding activity is provided by an albumin binding domain (ABD) of streptococcal n G or a derivative thereof. For example, said fusion protein or conjugate, sing at least one further moiety, may comprise [FcRn g polypeptide moiety] — [albumin binding moiety] — [moiety with affinity for selected target]. It is to be understood that the three es in this example may be arranged in any order from the N— to the C—terminal of the polypeptide.

In one embodiment, when a complex between a target and the fusion protein or conjugate as described herein is formed (or maintained) at acidic pH, such as pH 6.0, the target is d from elimination by lysosomal ation. Thus, target half—life is ed. Half-life extension implies that the elimination rate of a target is lower when interacting with said fusion protein or conjugate than the elimination rate of the target molecule in the absence of said fusion protein or conjugate. Furthermore, it is ble in this embodiment that the g of target by the fusion protein or conjugate should not interfere ntially with the on of the target.

On the other hand, when a complex between the target and the fusion protein or conjugate as described herein is not maintained or not formed at acidic pH, the target is directed to the subcellular lysosomes where it is degraded. in one embodiment, there is provided a fusion protein or conjugate, wherein the rate of elimination of a selected, undesirable target from the subject is increased. increased elimination of an rable target implies increased elimination rate of the target from the body of the multicellular organism, as compared to a “normal” elimination rate of the target molecule per se, i.e. without previous interaction with the fusion protein or conjugate. in another embodiment, binding of a selected undesirable target could inactivate the on of the , thereby blocking its biological activity in situations where this is desirable. Such biological activity may for example be activation or blocking of ors or an enzymatic or otherwise toxic or undesirable activity. Such undesirable target may be an endogenous hormone, enzyme, cytokine, chemokine or a target having some other biological ty. By using an inactivating target binding, the biological activity is blocked until the target is delivered for degradation and released at a low pH value, and the target binding fusion protein is recycled to circulation.

This recycling of the target binding fusion protein (via its FcRn binding moiety) s it to yze" the removal of more than one molecule of the selected undesirable target.

Undesirabie targets may for example be foreign proteins and compounds, or lly expressed proteins that display elevated levels in plasma following a medical condition and where a therapeutic effect may be achieved by elimination of said n. The red target is not necessarily evenly distributed in the plasma but may be concentrated in certain regions, for e around a tumor or at sites of inflammation. miting es of targets are targets selected from the group consisting of allergens, amyloids, dies, auto-antigens, blood clotting factors, hormones, tumor cells, drug molecules, cytokines, chemokines, proteases, hypersensitivity mediators, proinflammatory s, toxins such as bacterial toxins and snake venoms; pollutants, metals and anti—oxidants.

Under certain conditions, such as in certain cancer diseases, it is desired to remove endogenous molecules, for example VEGF, PDGF, HGF and other growth stimulatory hormones. Such molecules could also be targeted by a binding function in said fusion protein or conjugate.

Under other conditions, such as in certain immunological diseases, it may be desirable to remove nous molecules transiently, such as selected interieukines or TNF. Such molecules could also be targeted by a binding function in said fusion protein or conjugate.

In one embodiment, the second moiety having a desired biological activity is a therapeutically active polypeptide. miting examples of therapeutically active polypeptides are biomolecules, such as molecules selected from the group consisting of enzymes, for example algasidase d and B, glucocerebrosidase, laronidase, arylsulphatase, aglucosidase—o, asparaginase, Factor VII, Factor Vlll, Factor IX and Factor Xa; hormones and growth factors, for e growth hormone, transforming growth factor—82, erythropoietin, insulin, insulin-like growth —1, myostatin, bone—derived growth factor and glucagon—like peptide—1; chemokines, for example CCL17, CCL19, CCLZO, CCL21, CCL22, CCL27, XCL1 and CXCECL1; and cytokines, for example interleukin (lL)—2, lL—4, lL—7, lL—10, lL-12, lL-15, lL—18, lL—22, lL-27, interferon (lFN)—d, lFN—B, lFN—y, tumor necrosis factor, granulocyte-colony stimulating factor (G—CSF), macrophage—CSF, and granulocyte/macrophage—CSF.

As the skilled person understands, the FcRn binding polypeptide according to the first aspect may be useful in a fusion protein or as a conjugate partner to any other moiety. ore, the above lists of therapeutically active polypeptides should not be ued as limiting in any way.

Other possibilities for the creation of fusion polypeptides or conjugates are also contemplated. Thus, an FcRn binding polypeptide according to the first aspect of the invention may be ntly coupled to a second or further moiety or moieties, which in addition to or d of target g exhibit other functions. One example is a fusion between one or more FcRn binding polypeptide(s) and an enzymatically active polypeptide serving as a reporter or effector moiety.

With regard to the description above of fusion proteins or conjugates 3O incorporating an FcRn binding polypeptide according to the sure, it is to be noted that the designation of first, second and further moieties is made for y reasons to distinguish between FcRn binding ptide or polypeptides according to the disclosure on the one hand, and moieties exhibiting other functions on the other hand. These designations are not intended to refer to the actual order of the different domains in the polypeptide chain of the fusion protein or conjugate. Thus, for example, said first moiety may without restriction appear at the N—terminal end, in the middle, or at the inal end of the fusion protein or ate. in one embodiment, there is provided an FcRn binding polypeptide, fusion protein or conjugate, which binds to FcRn such that binding of lgG to FcRn is at least partially inhibited. This inhibition may be due to binding of the FcRn binding polypeptide, fusion protein or conjugate to the same, or an at least partially pping, region of FcRn as lgG. Alternatively, the FcRn binding polypeptide, fusion protein or conjugate may bind to a different region of FcRn than lgG but sterically hinder the binding of lgG to FcRn. Thus, the rate of elimination or clearance of lgG from the circulatory system would increase due to increased lysosomal degradation of lgG, because the FcRn mediated recycling of lgG would be wholly or partially unavailable due to the occupation of FcRn binding sites by the FcRn binding polypeptide according to the t disclosure. in other words, administration of FcRn binding polypeptide, fusion protein or conjugate or composition according to the present disclosure will act to increase the catabolism of circulating lgG antibodies. in one ment, the KB value of the interaction between the FcRn binding polypeptide, fusion protein or conjugate and FcRn is lower than the KB of the interaction between lgG and FcRn. This relationship may be true at both pH 6.0 and pH 7.4, or at pH 6.0 only.

The above aspects furthermore encompass polypeptides in which the FcRn g polypeptide according to the first aspect, or the FcRn g polypeptide as comprised in a fusion protein or conjugate according to the second aspect, further comprises a label, such as a label selected from the group ting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides and les. Such labels may for example be used for detection of the polypeptide. in other embodiments, the labeled FcRn g polypeptide is present as a moiety in a fusion n or conjugate also comprising a second moiety having a desired biological activity and/or comprising a binding on as described above. The label may in some instances be coupled only to the FcRn binding polypeptide, and in some instances both to the FcRn binding polypeptide and to the second moiety of the conjugate or fusion protein.

Furthermore, it is also possible that the label may be coupled to a second moiety only and not to the FcRn binding moiety. Hence, in yet another embodiment there is provided an FcRn binding polypeptide comprising a second moiety, wherein said label is coupled to the second moiety only.

When reference is made to a d polypeptide, this should be understood as a reference to all s of polypeptides as described herein, ing fusion proteins and conjugates comprising an FcRn binding polypeptide and a second and optionally further moieties. Thus, a labeled polypeptide may contain only the FcRn binding polypeptide and eg. a 1O therapeutic radionuclide, which may be chelated or covalently coupled to the FcRn binding polypeptide, or contain the FcRn binding ptide, a therapeutic radionuclide and a second moiety such as a small molecule having a desired biological activity, for example resulting in a therapeutic in embodiments where the FcRn binding polypeptide, fusion protein or conjugate is radiolabeled, such a radiolabeled polypeptide may comprise a radionuclide. A ty of radionuclides have a metallic nature, are used in the ionic form, and are typically incapable of g stable covalent bonds with elements presented in proteins and peptides. For this reason, labeling of ns and peptides with radioactive metals is performed with the use of chelators, i.e. multidentate s, which form non—covalent compounds, called chelates, with the metal ions. in an embodiment of the FcRn binding polypeptide, fusion protein or conjugate, the incorporation of a radionuclide is enabled through the ion of a chelating environment, through which the radionuclide may be coordinated, chelated or complexed to the polypeptide.

One example of a or is the polyaminopolycarboxylate type of chelator. Two classes of such inopolycarboxylate chelators can be distinguished: macrocyclic and acyclic chelators. in one embodiment, the FcRn g polypeptide, fusion protein or 3O conjugate comprises a chelating environment provided by a polyaminopolycarboxylate or conjugated to the FcRn binding polypeptide via a thiol group of a cysteine residue or an epsilon amine group of a lysine residue.

The most commonly used macrocyclic chelators for radioisotopes of indium, gallium, yttrium, bismuth, radioactinides and radiolanthanides are different derivatives of DOTA (1 ,4,7,10—tetraazacyclododecane—l ,4,7,10— tetraacetic acid). in one embodiment, a chelating environment of the FcRn binding polypeptide, fusion protein or conjugate is provided by DOTA or a derivative thereof. More specifically, in one embodiment, the chelating polypeptides encompassed by the present disclosure are obtained by reacting the DOTA derivative 1,4,7,10-tetraazacyclododecane-1,4,7—tris— acetic 0—maleimidoethylacetamide (maleimidomonoamide—DOTA) with said polypeptide.

Additionally, l,4,7—triazacyclononane~1,4,7—triacetic acid (NOTA) and derivatives thereof may be used as ors. Hence, in one embodiment, there is provided an FcRn g polypeptide, fusion protein or conjugate, wherein the poiyaminopolycarboxylate chelator is 1,4,7—triazacyclononane— 1,4,7—triacetic acid or a derivative thereof.

The most commonly used acyclic inopolycarboxylate chelators are different derivatives of DTPA ylenetriamine—pentaacetic acid).

Hence, polypeptides having a chelating environment provided by diethylenetriaminepentaacetic acid or derivatives thereof are also encompassed by the present disclosure. in a r embodiment, the FcRn binding polypeptide, ed recombinantly through expression of a polynucleotide or synthetically, is conjugated to one or more tic polymers, in order for example to increase its hydrodynamic radius. hylene glycol (PEG) is commonly used for this purpose, but other polymers have also been used in the art.

Such ation” may be used to increase the size of the FcRn binding polypeptide of any of the types described herein to a size above the threshold for effective renal excretion. in one embodiment, a tic polymer is conjugated to one or more chemically synthesized, monomeric FcRn binding ptides. Other functionalities may also be conjugated to the same synthetic polymer. if the FcRn binding polypeptide and other components are chemically synthesized, none of the components will have to be made in a ical system if this is not desired.

In a preferred embodiment, one or more synthetically or biologically manufactured FcRn binding polypeptides are conjugated to a synthetic polymer, to achieve a size exceeding the size associated with efficient renal clearance and used for blocking binding of lgG to FcRn. A unique cysteine in each FcRn binding polypeptide may be used for site specific conjugation, for example a C—terminaily located cysteine introduced for this purpose. With a branched synthetic polymer, more than two FcRn binding moieties may be conjugated to the same polymer, to enhance the avidity and therefore the ng potency.

In a third aspect of the present disclosure, there is provided a polynucleotide encoding an FcRn g ptide or a fusion protein as described herein. Also assed by this sure is a method of producing a polypeptide or fusion protein as described above comprising expressing a polynucleotide; an expression vector comprising the polynucleotide; and a host cell comprising the expression vector.

Also encompassed is a method of producing a polypeptide, comprising ing said host cell under conditions permissive of expression of said polypeptide from its expression vector, and isolating the polypeptide.

The FcRn binding polypeptide of the present disclosure may alternatively be produced by non—biological peptide synthesis using amino acids and/or amino acid derivatives having protected reactive side—chains, the non-biological peptide synthesis comprising — step—wise coupling of the amino acids and/or the amino acid derivatives to form a polypeptide according to the first aspect having protected reactive side-chains, — removal of the ting groups from the ve side-chains of the polypeptide, and — folding of the polypeptide in aqueous solution.

In a fourth aspect of the disclosure, there is provided a ition comprising an FcRn binding polypeptide, fusion protein or ate as described herein and at least one pharmaceutically acceptable excipient or carrier. In one embodiment thereof, said composition further comprises at least one onal active agent, such as at least two additional active 3O agents, such as at least three additional active agents. Non-limiting examples of additional active agents that may prove useful in such a combination are suppressing agents, anti—inflammatory agents, anti-microbial agents and enzymes.

In one embodiment of this aspect, said composition is adapted for administration by a route selected from the group consisting of oral stration, intranasal administration, pulmonar administration, vaginal administration, rectal administration, intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous ion and intradermal injection.

As used , the term “systemic administration” refers to a route of administration such the substance of interest enters into the circulatory system so that the entire body is affected. The skilled person is aware that systemic administration can take place via enteral administration (absorption of the drug through the gastrointestinal tract) or parenteral administration (generally injection, infusion or implantation). in one embodiment, said composition is adapted for administration systemically or locally. In certain embodiments, systemic administration of said compound may be used. in another embodiment, said composition is adapted for administration by a local route. For example, local administration may be topical in an ointment, paste, foam or cream. In r embodiment, said composition is adapted for administration across an endothelial or epithelial layer. Here, the composition may be transcytosed across said layer. in one embodiment, the rate of uptake of a composition sing a fusion protein or conjugate as described herein is higher than the rate of uptake of polypeptides corresponding to second or further moieties per se. in one embodiment, the rate of uptake is at least 2 times higher, such as at least 5 times higher, such as at least 10 times higher, such as at least 25 times higher than the rate of uptake of the at second or further moieties per se.

It should be understood from the above disclosure that the FcRn binding polypeptide fusion protein or conjugate or the composition as described herein may for example be useful as a eutic agent, and/or as a means for extending the in vivo half-life of a fusion partner, and/or as a means for sing the rate of elimination of undesirable targets.

Hence, in a fifth aspect of the present disclosure, there is provided an FcRn binding polypeptide, fusion protein, conjugate or composition as disclosed herein for use as a ment.

In a related, sixth, aspect of the present sure, there is provided a method of treatment of a t in need thereof, comprising the step of administrating a therapeutically active amount of an FcRn g polypeptide, fusion protein, conjugate or composition as disclosed herein. in one embodiment of any one of these two latter aspects, the medicament or method is ed for treatment in which the capacity of the FcRn binding polypeptide to at least partially block binding of lgG to FcRn is exploited, i.e. treatment in which increased catabolism of lgG antibodies is desired. In one embodiment, a condition in which such treatement may be indicated is an auto—immune condition. As miting examples of indicated conditions, mention is made of myasthenia gravis, in—Barré syndrome, autoimmune limbic encephalitis, pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection (PANDAS), neuromyotonia ’s syndrome), morvan syndrome, multiple sclerosis, pemphigus vulgaris, eus, bullous goid, epidermolysis bullosa acquisita, pemphigoid gestationis, mucous membrane pemphigoid, lichen sclerosus, antiphospholipid syndrome, erlapsing polychondritis, autoimmune anemia, idiopathic trombocytic purpura, autoimmune s disease, d cardiomyopathy, vasculitis, Goodpasture’s syndrome, idiopathic membranous nephropathy, rheumatoid arthritis and systemic lupus erythematosus. in another embodiment, there is provided an FcRn binding polypeptide, fusion protein, conjugate or composition as bed herein for use in blocking or removal of an undesirable target from the circulation. In one embodiment, said undesirable target is selected from the group comprising allergens, amyloids, antibodies, auto—antigens, blood clotting factors, hormones, tumor cells, drug molecules, cytokines, chemokines, hypersensitivity mediators, pro-inflammatory factors, toxins such as ial toxins and snake venoms, pollutants, metals and anti—oxidants.

While the invention has been described with reference to various exemplary s and embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for ts f without departing from the scope of the invention. ln addition, many modifications may be made to adapt a particular ion or molecule to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to any particular embodiment contemplated, but that the invention will include all embodiments falling within the scope of the appended claims.

Brief description of the figures Figure 1 is a g of the amino acid sequences of es of FcRn binding motifs comprised in FcRn binding polypeptides of the invention (SEQ ID NO:1-353), examples of 49—mer FcRn binding ptides according to the disclosure (SEQ ID NO:354—706), es of 58-mer FcRn binding polypeptides according to the disclosure (SEQ ID NO:707—1062) as well as the amino acid sequences of the albumin binding polypeptide variant PP013 (SEQ ID NO:1063), Taq polymerase binding Z variant 203638 (SEQ ID NO:1064), human chRn (SEQ ID NO:1065), murine chRn (SEQ ID NO:1070), human BZ—microglobulin (SEQ ID N011066), murine 62— microglobulin (SEQ ID NO:1067), human chRn (SEQ ID NO:1068) when in human FcRn—eGFP and murine chRn (SEQ ID NO:1069) when in murine FcRn—eGFP. s 2A-2E show the binding to human FcRn at pH 6.0 and dissociations at pH 6.0 and 7.4 for HISG-tagged Z variants and for lgG as described in Example 3. Overlays of sensorgrams obtained from a Biacore instrument representing injection at pH 6.0 followed by dissociation at pH 6.0 (solid line) and injection at pH 6.0 ed by dissociation at pH 7.4 (dashed line) are displayed for (A) ZO7918 (SEQ ID NO:707), (B) ZO7960 (SEQ ID NO:710), (C) Z10109 (SEQ ID ), (D) 210193 (SEQ ID NO:708) and (E) IgG.

Figure 3 shows dot plots from a flow cytometry analysis of binding of FcRn binding Z variant to human (upper panel) and mouse (lower panel) FcRn—eGFP HeLa cells, as described in Example 4. Due to heterogeneous expression of GFP by HeLa cells, cells were gated according to FcRn- eGFP expression level. Cells in gate H are considered to be FcRn-eGFP negative and cells in gate l are considered to be positive. tion with Alexa647 labeled Z variants resulted in a population positive both for Alexa647 and eGFP, s incubation with buffer (buffer control) did not.

The figure shows that the three variants 207960 (SEQ ID NO:710), 207930 (SEQ ID NO:712) and 207918 (SEQ ID ) bind to human FcRn and mouse FcRn. The y-axis shows Alexa647 intensity and the x—axis shows eGFP activity.

Figure 4 shows mean fluorescence intensity (MFI) values of Alexa647 labeled 207960 (SEQ ID NO:710), Z07930 (SEQ ID NO:712) and 207918 (SEQ ID NO:707), measured in the cell binding assay bed in Example 4. Diagram (A) shows MFI from HeLa cells transduced with human FcRn~ eGFP and diagram (B) shows MFI from HeLa cells transduced with mouse FcRn—eGFP.

Figure 5 shows dot plots from flow cytometry is of human or mouse IgG 47 binding to human (upper panel) and mouse (lower panel) GFP HeLa cells, as bed in Example 5. Due to heterogeneous expression of FcRn-eGFP by HeLa cells, cells were gated according to the abundance of FcRn—eGFP on the cell surface. Cells in gate M are considered to be FcRn-eGFP negative and cells in gate N are considered to be positive. Binding of 100 nM human or mouse lgG~Alexa647 to FcRn transduced HeLa cells are shown in the left panel (0 nM). The figure shows that lgG binding was blocked by tagged Z07918 (SEQ lD NO:707) in a dose dependent manner (1, 10, 100 and 1000 nM). The y—axis shows Alexa647 intensity and the x—axis shows eGFP activity. 1O Figure 6 shows mean fluorescence intensity (MFl) values resulting from FcRn binding of lgG Alexa647 in the presence of different concentrations of agged ZO7918 (SEQ lD NO:707) on (A) human FcRn— eGFP transduced HeLa cells and (B) mouse FcRn—eGFP transduced HeLa cells, as described in Example 5. The figure shows dose dependent ng of the lgG—FcRn binding by the Z variant.

Figures 7A—7C show kinetics of binding of three Z variants to human FcRn at pH 6.0, as described in Example 6, using a Biacore instrument.

Sensorgrams for a concentration series of (A) Z11948 (SEQ lD NO:1060), (B) Z11946 (SEQ lD NO:1061) and (C) Z11947 (SEQ ID NO:1062), respectively, in fusion with the albumin binding polypeptide PP013 (SEQ ID NO:1063) and the control Z variant molecule 203638 (SEQ lD NO:1064; not specific for FcRn), are displayed. Curves from 640 nM (dashed line), 160 nM (dotted line) and 40 nM (solid grey line) were subjected to kinetic analysis using the Langmuir 1:1 binding model. Kinetic parameters and affinities were calculated from fitted curves (solid black lines) and are shown in Table 5.

Figure 8 shows the pharmacokinetic profiles for three FcRn binding Z variants fused to the albumin binding polypeptide PP013 obtained as described in e 6. The Z variants Z11947 (SEQ lD NO: 1062, open squares), 211946 (SEQ lD NO:1061, open triangles) and 211948 (SEQ ID 0, open diamonds) all yed prolonged half—life compared to the negative l PPO13—ZOB638 (open circles).

Figure 9 shows the blocking of human lgG to human FcRn by HiSs— 207918 (SEQ lD NO:707; black circles), lVlg (grey squares) and SClg (grey triangles), tively, assayed as described in Example 10.

Figure 10 shows that blocking of the lgG—FcRn interactions with FcRn specific Z variants in mice results in reduced levels of lgG. As further described in Example 11, mice were d with five daily injections of Vehicle (+), the ABD fused Z variant ZO7918—PP013 (open square) and 211948 (SEQ ID NO:1060; closed circle). The concentration of endogenous lgG was ed by ELISA. The concentration of lgG in individual mice at 24, 72, 120 and 168 h were related to the level at 0 h and the results are therefore presented as tage of lgG at O h.

Examples Summary 1O The following Examples disclose the development of novel Z variant molecules targeting the neonatal Fc receptor (FcRn). The Z variants were obtained using phage display technology. The genes encoding FcRn binding polypeptides bed herein were sequenced, and the corresponding amino acid sequences are listed in Figure 1, and d by the identifiers SEQ ID -1059. Also, the deduced binding motifs of these selected binding variants are listed in Figure 1 with sequence identifiers SEQ lD NO:1—353.

Example 1 Production of human chRn and human fi2-microglobulin (82M) in this Example, the extracellular domain (ECD) of human chRn (SEQ ID NO:1065) in complex with human BZ—microglobulin (SEQ lD NO:1066) (complex denoted FcRn) and human BZ-microglobulin in non—complexed form (denoted 82M) were produced as soluble ns. Human FcRn and 82M produced in this Example were used for phage selection, ELISA and Biacore assays in Examples 2 and 3. als and methods Construction of plasmids containing the genes for human chRn and human fiZ—microglobulin to be used for co-expression: The genes encoding human chRn (Genbank 800087342) and human BZ-microglobulin (82M) (Genbank BCO32589.1) were obtained from OpenBiosystems. Using PCR overlap ion, a gene fragment ng amino acids 24-290 of human chRn (chRnEco) (SEQ ID N021065) was amplified to a construct consisting of attBi—site/Kozak sequence followed by a gene encoding: an lg kappa chain leader sequence, hFCRnECD, a ker and a flag tag, followed by an attBZ site. A similar construct was made containing a gene fragment encoding amino acids 21—119 of human 82M (SEQ ID NO:1066), except that a Hi35 tag replaced the flag tag. The constructs were inserted into the d pDONOR221 (lnvitrogen, cat. no. 12536—017) by recombination using the Gateway system (lnvitrogen, cat. no. 11789020, Gateway® BP Clonase® l| Enzyme mix), according to the manufacturer’s recommendations. After verification of correct sequences, the human ochRnEco construct was inserted into 2K7bsd (Suter etal. (2006) Stem Cells 24:615—623) using multi- site gateway cloning together with the er—containing plasmid pENTR— CMV (Tai et al. (2012) PLoS One 7(9):e46269), ing in the vector 2K7bsd~ CMV-hFCRnECD. The human 82M gene uct was similarly inserted into 2K7neo (Suter et al., supra), giving the vector 2K7neo—CMV—hBZM.

Cell culture, preparation of recombinant lentiviral vectors and gene insertions into SKOV—3 cell line: The HEK293T and SKOV-3 cell lines were obtained from ATCC. Cells were grown at 37 °C in a humidified incubator in the presence of 5 % 002. Complete medium for the HEK293T cell line was Dulbeccos modified eagle medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 1 % Antibiotic Antimycotic Solution (AA) and 1 % MEM Non-essential Amino Acid on (NEAA). Complete medium for the SKOV— 3 cell line was McCoy’s 5A medium supplemented with 10 % FBS and 1 % The ds —CMV—hFcRnEco and 2K7neo-CMV-hB2M were separately co—transfected together with VSV—G envelope and gag/pol packaging plasmid into HEK293T cells using calcium chloride transfection (Zufferey et al. (1997) Nat Biotechnol 15(9):871-5; son etal. (2006) J Neurosci Res 84:58-67). HEK293 culture supernatants containing formed lentiviral particles with human chRnEco and human 82M transgenes, tively, were cleared from cell debris by centrifugation and filtration. The two types of iral particles were used to sequentially uce SKOV—3 cells. Successful double integrants containing both the human chRnECD and the 82M genes were selected for by the addition of blasticidin (Invitrogen) and G418 sulfate (lnvitrogen) to culture medium while passaging the cells for two weeks. The resulting, stably transduced SKOV-3 cell line was denoted SKOV- 3 hFCRnECD/hBZM.

Expression of recombinant human FcRn: SKOV—3 cells, co-expressing human chRnEcp and 82M resulting in human FcRn, were expanded and 1.5 x 107 cells were seeded in a HYPERFIask (Corning) in 560 ml complete growth medium. After five days, when the cells had settled and lied, the medium was changed to complete growth medium without FBS. After five days, the culture was terminated and the supernatant was collected, passed through a 45 um filter and frozen at ~80 °C. cation of recombinant human FcRn using human lgG chromatography: Protein purification was carried out in an AKTA Explorer system (GE Healthcare). Human lgG (Pharmacia), 1 ml in 0.2 M NaHCOa, 0.5 M NaCl pH 8.3 at a concentration of 10 mg/ml, was coupled to a 1 ml HiTrap NHS-activated HP column (GE Healthcare) ing to the manufacturer’s 1O instruction. The supernatant containing recombinant human FcRn from SKOV—3 cells was thawed and the pH was ed to 5.8 with HCl. The atant was subsequently loaded in batches of 100 ml onto the column previously brated with 20 mM Bis—Tris pH 5.8. The column was washed with 20 ml of 20 mM Bis-Tris pH 5.8 and eluted in fractions of 1 ml using 50 mM Tris, pH 8.1. Buffer exchange to PBS hate buffered saline, 10 mM phosphate, 137 mM NaCl, 2.68 mM KCI, pH 7.4) was performed using dialysis.

GE and Western blot: The purity of the eluted fractions from the protein purification was analyzed by SDS-PAGE and staining with GeICode Blue Stain Reagent (Pierce) and Xpress® Silver Staining Kit (lnvitrogen). Western blotting was carried out using an Amersham HybondTM— C Extra nitrocellulose membrane (GE Healthcare). The membrane was blocked with 5 % non—fat dry milk r) in TBS+T (50 mM Trizma base, 150 mM NaCl, 0.05 % Tween—20, pH 8) for 1 hour, then probed with a mixture of rabbit anti-FCGRT polyclonal antibody (Atlas Antibodies) at a concentration of 0.15 ug/ml and rabbit anti—82M polyclonal antibody (Atlas Antibodies) at a concentration of 0.23 ug/ml in TBS+T. The membrane was subsequently incubated with stabilized goat anti—rabbit antibody conjugated with horse radish dase (Pierce) diluted 1:10,000 in TBS+T. After addition of TMB Substrate (Pierce), an image of the membrane was acquired on Amersham Hyperfilm ECL (GE Healthcare). The Hyperfilm was processed using GBX developer and GBX fixer (Sigma-Aldrich).

Production ofa non—complexed form of human B2M: Human 82M was produced in E. coli. The sion and purification was performed essentially as described in Sandalova etal. (2005) Acta Chryst F6121090— 1093 and Michaelsson et al. (2001) J Immunol 166:7327—7334. The purified protein, consisting of amino acids 21—1 19 of human 82M, in urea was subjected to arginine refolding as follows; 0.5 mg of BZM was rapidly added to 2 ml refolding buffer (20 ml 1 M Tris—HCl pH 8.0, 16.87 g L—Arginine (buffered with HCl), 0.8 ml 0.5 M EDTA, 61 mg GSSG, 307 mg GSH and milli— Q water to a final volume of 200 ml, pH 8.0, and supplemented with protease tor (Roche, cat. no. 11 873 580 001)). The refolding procedure was performed at 4 °C during 4 hours. Refolded 82M protein was buffer ged to PBS using a PD—10 column (GE care).

Results Construction of plasmids containing the genes for human chRn and human @2—microglobulin to be used for ression: Genes encoding the ellular domain of the or—chain of human FcRn (chRnEco) and human B2M were inserted into the lentiviral transfer plasmids 2K7bsd and 2K7neo, respectively. In both cases, the inserted gene is under the control of a CMV promoter. The genes were extended so that the resulting proteins would have an lg kappa chain leader sequence in the N—terminus to target the protein for export through the endoplasmic reticulum to the culture medium (the signal sequence was cleaved upon secretion). In addition, chRneco had a C- terminal spacer ce followed by a FLAG—tag for potential detection.

Human 82M had a C—terminal spacer sequence followed by a Hi85 tag for potential detection. The spacer sequence was added to e accessibility of the tag. The lentiviral transfer plasmids also contained two different antibiotic resistance genes to allow selection of cells where both constructs had been inserted.

Expression and purification of recombinant human FcRn: The genes encoding ClFCRnECD and 82M were inserted into the genome of SKOV—3 by lentiviruses, and the resulting FcRn protein was secreted into the culture medium. To capture only FcRn having retained pH—dependent lgG g, affinity chromatography using immobilized lgG was used where the receptor was captured at pH 5.8 and eluted at pH 8.1. Captured protein was eluted in three fractions.

SDS—PAGE and Western blot: To investigate the ce of two peptide chains co and 82M) of the produced FcRn protein, and to analyze the purity of the eluted material, an SDS-PAGE analysis was performed on the eluted fractions. For the gel stained with GelCode Blue Stain, two bands were detected with molecular weights of 12 and 36 kDa, tively. This corresponds approximately to the theoretical molecular weights of the non—glycosylated peptide chains of 12 kDa for 82M and 31 kDa for GFCRnECD. The ochRnEco part of the protein contains one glycosylation site and it was therefore expected that its molecular mass would be higher than 31 kDa. The gel was also silver stained to se sensitivity and possibly detect impurities. A band of imately 66 kDa was detected in the first eluted fraction, which could correspond to BSA e serum albumin) originating from cell ment. The total amount of protein recovered in fraction 2 and 3 corresponded to 1.4 mg/l culture medium. A western blot analysis on the pooled material was carried out, which showed 1O essentially only the two major bands and in addition a very weak band below 12 kDa which might correspond to a degradation product.

Example 2 Selection and ELISA binding of FcRn g Z variants In this Example, human FcRn was used as target in phage display selections using a phage library of Z variants. Selected clones were DNA sequenced, produced in E. coli periplasmic fractions and assayed against FcRn in ELISA (enzyme-linked immunosorbent assay).

Materials and methods ylation of target protein FcRn and of 82M: Human FcRn and human 82M, produced as described in Example 1, were biotinylated using No-Weigh EZ—Link Sulfo-NHS-LC—Biotin (Pierce, cat. no. 21327) at a 31 x (FcRn) and 10 x (82M) molar excess, respectively, according to the manufacturer’s recommendations. The reactions were performed at room temperature (RT) for 30 min. uent buffer exchange to PBS was performed using Slide-a-lyzer dialysis cassettes (FcRn; Pierce, cat. no. 66380, 10,000 MWCO and 82M; Pierce, cat. no. 66333, 3,500 MWCO), according to the manufacturer’s ctions.

Phage display selection of FcRn binding Z variants: A library of random variants of protein Z displayed on bacteriophage, constructed in id pAY02592 ially as described in Gronwall et al. (2007) J Biotechnol, 128:162—183, was used to select FcRn binding Z variants. In this library, an albumin binding domain (ABD, GA3 of protein G from ococcus strain G148) is used as fusion partner to the Z variants. The library is denoted Zlib006Naive.ll and has a size of 1.5 x 1010 library members (Z variants).

E. coli RRIAM15 cells (R'L'Ither et a/., (1982) Nucleic Acids Res 10:5765—5772) from a glycerol stock containing the phagemid library Zlib006Naive.ll, were inoculated in 20 l of a defined proline free medium [dipotassium hydrogenphosphate 7 g/l, trisodium citrate dihydrate 1 g/l, uracil 0.02 g/l, YNB (DifcoTM Yeast en Base w/o amino acids, Becton Dickinson) 6.7 g/l, glucose drate 5.5 g/l, L—alanine 0.3 g/l, L-arginine monohydrochloride 0.24 g/l, L-asparagine monohydrate 0.11 g/l, L-cysteine 0.1 g/l, L—glutamic acid 0.3 g/l, L-glutamine 0.1 g/l, glycine 0.2 g/l, L-histidine 0.05 g/l, L— isoleucine 0.1 g/l, L—leucine 0.1 g/l, L—lysine monohydrochloride 0.25 g/l, L— methionine 0.1 g/l, L-phenylalanine 0.2 g/l, L-serine 0.3 g/l, onine 0.2 g/l, L-tryptophane 0.1 g/l, L—tyrosine 0.05 g/l, L—valine 0.1 g/l], supplemented with 100 ug/ml llin. The cultivations were grown at 37 °C in a fermenter (Belach Bioteknik, BR20). When the cells reached an optical density at 600 nm (ODsoo) of 0.75 2.6 l of the cultivation was infected using a , approximately x molar excess of M13K07 helper phage (New England Biolabs, cat. no.

N0315S). The cells were incubated for 30 minutes, whereupon the fermenter was filled up to 20 l with TSB—YE (Tryptic Soy Broth—Yeast t; 30 g/l TSB, 5 g/l yeast extract) supplemented with 100 pM isopropyl—B—D thiogalactopyranoside (IPTG) for induction of expression and with 25 pg/ml kanamycin and 12.5 ug/ml carbenicillin and grown at 30 °C for 22 h. The cells in the cultivation were pelleted by fugation at 15,900 g. The phage les were precipitated from the atant twice in PEG/NaCl (polyethylene glycol/sodium chloride), filtered and dissolved in PBS and glycerol as described in Gronwall et al., supra. Phage stocks were stored at — 80 °C before use.

Selections against biotinylated human FcRn were performed in four cycles divided in two different tracks. Phage stock preparation and selection ure were performed essentially as described for selection t another biotinylated target in W02009/077175. The amplification of phage between the selection cycles was performed by infecting E. coli RRIAM15 with phage, then performing cultivation in solution as follows. Eluted phage and 10 x excess of M13K07 helper phage compared to bacteria were allowed to simultaneously infect log phase bacteria at 37 °C for 30 min without rotation, followed by 30 min with slow rotation. Prior to infection, ia were grown to log phase in the defined proline free medium bed above.

Infected bacteria were pelleted by centrifugation at 4,300 g for 10 min and resuspended in 200 ml TSB+YE medium supplemented with 0.1 mM IPTG, pg/ml kanamycin and 100 pg/ml ampicillin and cultivated at 30 °C overnight for phage production.

The ion buffer consisted of 100 mM sodium phosphate and 150 mM sodium chloride adjusted to pH 5.5 with hydrogen chloride and supplemented with 0.1 % gelatin and 0.1 % Tween—20. At selection, human serum albumin (HSA, Albucult, mes) was added to the selection buffer to a final concentration of 1.5 pM. In order to reduce the amount of ound binders, pre-selection was performed by incubation of phage 1O stock with Dynabeads® M—280 Streptavidin (SA—beads, Dynal, cat. no. 112.06) for 1 hour at RT. A second pre—selection was performed during 30 min at RT t human BZM immobilized in immunotubes (Nunc, cat. no. 444474). 5 pg/ml of human 82M in carbonate buffer (Sigma, cat. no. 068K8214) was lized in the tube at 7 °C for >1 h. After washing twice with tap water, the tubes were blocked with PBS + 0.5 % casein (Sigma, cat. no. C8654) for 30 min at RT before use. All tubes and beads used in the selection were pre—blocked with PBS + 0.1 % gelatin. Selection was performed in solution at RT, followed by capture of target—phage complexes on SA—beads where 1 mg beads per 2.9 pg biotinylated FcRn were used. in cycle 1 of the selections, 100 nM biotinylated FcRn was used and two washes of two min each were performed using selection buffer. An increased stringency, using a lowered target concentration and an increased number of , was applied in the uent cycles: 50 nM/5 washes, 25 nM/8 washes and 10 nM/12 washes were applied in cycle 2, 3 and 4, respectively.

After the washes, bound phage was eluted from the two selection tracks using two different procedures; 1) 500 pl 0.1 M glycine—HCI, pH 2.2, followed by immediate lization with 50 pl 1 M Tris—HCI, pH 8.0, and 450 pl PBS, or; 2) 500 pl of 100 mM sodium phosphate and 150 mM sodium chloride, pH 8.0 and neutralization with 500 pl PBS.

Seguencing: PCR fragments were amplified from single colonies using a standard PCR program and the primers AFFl—21 (5’-tgcttccggctcgtatgttgtgtg (SEQ ID NO:1071)) and AFFl—22 (5’—cggaaccagagccaccaccgg (SEQ ID 2)). Sequencing of amplified fragments was performed using the biotinylated oligonucleotide AFFI—72 (5’—biotin—cggaaccagagccaccaccgg (SEQ lD NO:1073)) and a BigDye® ator v3.1 Cycle Sequencing Kit (Applied Biosystems), used in ance with the manufacturer’s protocol. The sequencing reactions were purified by binding to magnetic streptavidin coated beads (Detach Streptavidin Beads, Nordiag, cat. no. 1) using a Magnatrix 8000 (Magnetic Biosolution), and analyzed on ABI PRISM® 3130xl Genetic Analyzer (PE Applied Biosystems).

Production of Z variants for ELISA: Sequenced Z variants were produced by ating single colonies from the selections into 10 ml TSB— YE medium supplemented with 100 pg/ml ampicillin and 0.1 mM IPTG and incubating for 24 h at 37 °C. Cells were pelleted by centrifugation, re— ded in 2 ml PBST (PBS supplemented with 0.05 % Tween—20), frozen at ~80 °C and thawed in a water bath, to release the periplasmic fraction of 1O the cells. The freeze-thawing procedure was repeated seven times and cells were then pelleted by centrifugation. The supernatant of the periplasmic extract contained the Z variants as s to ABD, expressed as AQHDEALE—[Z#####]—VDYV—[ABD]-YVPG all et al., . Z##### refers to individual, 58 amino acid residue Z variants.

ELISA KD analysis of Z variants: The binding of Z ts to FcRn was analyzed in ELISA assays. Half—area 96—well ELISA plates were coated with 2 pg/ml of an anti—ABD goat antibody ced in—house) diluted in coating buffer (50 mM sodium ate, pH 9.6) at 4 °C overnight. The antibody solution was poured off and the wells were blocked with 100 pl of PBSC (PBS supplemented with 0.5 % casein) for 1.5 h at RT. The blocking solution was discarded and 50 pl periplasmic solution, diluted 1:4, was added to the wells and ted for 1.5 h at RT under slow shaking. The solutions were poured off and the wells were washed four times with either 0.05% PCT buffer, pH 6.0 (Mcllvaines phosphate-citrate buffer, pH 6.0, supplemented with 0.05 % Tween—20) or 0.05% PCT buffer, pH 7.4 (Mcllvaines phosphate—citrate buffer, pH 7.4, supplemented with 0.05 % Tween—20). The target protein, biotinylated human FcRn, was added to the wells in a 1:3 diluted concentration series from 2 ug/ml (45 nM) to 0.3 ng/ml (6.9 pM) diluted in PCC buffer, pH 6.0 or pH 7.4, (Mcllvaines phosphate—citrate buffer, pH 6.0 or pH 7.4, supplemented with 0.5 % ), respectively. The plates were incubated for 1.5 h at RT followed by washes as described above. Streptavidin conjugated HRP (Thermo Scientific, cat. no. N100) was diluted 1:30 000 in PCC buffer, pH 6.0 or pH 7.4, respectively, and added to the wells followed by 45 min incubation.

After washing as described above, 50 pl ImmunoPure TMB substrate (Thermo Scientific, cat. no. 34021) was added to the wells and the plates were treated according to the manufacturer’s endations. Absorbance was measured at 450 nm using a multi—well plate reader, Victor3 (Perkin Elmer). A Z variant binding an vant protein was used as negative control and a blank was created by ng the periplasmic step. A Z t which bound to FcRn in a pre—experiment (207918, SEQ lD NO:707) was used as positive control. Measured values were analyzed using GraphPad Prism 5 (GraphPad Software, LaJoIla, CA, USA) and non—linear regression in order to determine the affinities (Kn) of the interactions.

ELISA specificity analysis of Z variants: in another ELlSA experiment, the specificities of the Z variants were tested by assaying them against 2 ug/ml ylated human proteins 82M, PSMA (in house produced) and lgG lonal, Pharmacia, Sweden) and against PCC buffer pH 6.0 or pH 7.4, respectively. The assay was performed at pH 6.0 and at pH 7.4, respectively, as described above. The biotinylated proteins or buffer were added to the wells instead of FcRn in the target protein step.

Results Phage display ion of FcRn binding Z variants: Individual clones were obtained after four cycles of phage display selections against biotinylated human FcRn.

Seguencing: Sequencing was performed on clones picked at random from selection round four. Each Z t was given a unique identification number ##### and individual variants are referred to as 2#####. The amino acid sequences of the 58 amino acid residues long Z variants are listed in Figure 1 as SEQ ID NO:707—722 and SEQ lD NO:1059.

The deduced FcRn g motifs of these Z ts are listed in Figure 1 as SEQ lD 6 and SEQ ID NO:353. The amino acid sequences of the 49 amino acid residues long polypeptides predicted to constitute the complete three-helix bundle within each of these Z variants are listed in Figure 1 as SEQ lD NO:354-369 and SEQ lD NO:706. 3O ELlSA assays with Z variants: Sixteen clones were produced as ABD fusion proteins in E. coli. The periplasmic fractions were used in an ELISA against a dilution series of human FcRn. The clones were: 207909 (SEQ ID NO:719), 207918 (SEQ ID NO:707), 207930 (SEQ lD NO:712), 207960 (SEQ lD NO:710), 210109 (SEQ ID NO:709), 210111 (SEQ lD NO:714), 210127 (SEQ lD NO:718), 210129 (SEQ ID NO:715), 210140 (SEQ ID ), 210141 (SEQ ID NO:716), 210145 (SEQ ID NO:721), 210152 (SEQ lD NO:720), 210156 (SEQ lD NO:717), 210161 (SEQ ID NO:722), 210183 (SEQ ID NO:713) and 210193 (SEQ ID ). Kn values were determined for all variants at pH 6.0 and for three variants at pH 7.4 (Table 1). For thirteen ts, data was not obtained for a KD analysis at pH 7.4. None of the sixteen variants displayed non—specific binding when d against human B2M, lgG or PSMA.

Table 1. ELISA KD analysis of 2~ABD variants in E. coliperiplasmic fractions.

Z variant SEQ ID NO: Ko pH 6.0 (M) Kn pH 7.4 (M) 207909 719 24.5 x10“9 n.d. 207918 707 2.0 x10“9 10.9 x10‘9 207930 712 10.4 x10“9 n.d. 207960 710 6.0 x10"9 n.d. 210109 709 3.9 x10'9 23.9 x10“9 210111 714 11.4 x10'9 n.d. 210127 718 21.3 x10‘9 n.d. 210129 715 17.6 x10'9 n.d. 210140 711 8.8 x109 n.d. 210141 716 21.2 x10'9 n.d. 210145 721 42.0 x10“9 n.d. 210152 720 24.6 x109 n.d. 210156 717 21.3 x10”9 n.d. 210161 722 163.0 x10”9 n.d. 210183 713 10.9 x10“9 n.d. 210193 708 2.3 x10"9 25.9 x109 n.d.= not determinable Example 3 Production and characterization of FcRn binding 2 variants In this Example, seventeen 2 variants were produced in E. coli, purified and assayed against human FcRn in Biacore. A subset of said variants was also assayed t mouse FcRn. Circular dichroism (CD) spectroscopy was performed for a subset of 2 variants for investigation of their secondary structure.

Materials and methods Subcloning of Z variants: The DNA of seventeen FcRn binding Z variants (SEQ lD NO:707—722 and SEQ lD NO:1059) was amplified from the library vector pAY02592. A ning strategy for uction of monomeric Z variant molecules with N—terminal His6 tag was applied using standard molecular biology techniques (essentially as described in detail in W02009/077175 for Z variants binding another ). The Z gene fragments were subcloned into the expression vector pAY01448 resulting in the encoded sequence MGSSHHHHHHLQ-[Z#####]—VD. 1O In addition, the FcRn binding variant 207918 (SEQ ID N02707), but ng with the amino acids AE instead of VD and denoted 211948 (SEQ ID NO:1060), was cloned as homodimeric constructs with two different linkers between the Z ts and followed by a C~terminal Hl86 tag. This was performed using conventional molecular biology s including DNA amplification, restriction with suitable restriction enzymes and ligation of the DNA. The two linkers were obtained from Thermo Fisher Scientific. The Z gene fragments were subcloned into the sion vector (pET—26 origin, Novagen) resulting in the encoded sequence [Z#####]~GT—(G4S)-PR— [Z#####]—LEHHHHHH and [Z#####]-GT-(G4S)3—[Z#####]-LEHHHHHH, respectively.

Cultivation and purification: E. coli BL21(DE3) cells (Novagen) were transformed with plasmids containing the gene fragment of each respective FcRn binding Z variant and cultivated at 37 °C in 800 or 1000 ml of TSB—YE medium mented with 50 ug/ml kanamycin. At ODsoo = 2, lPTG was added to induce expression at a final tration of 0.17 or 0.2 mM and the culture was incubated at 37 °C for another 5 h. The cells were harvested by centrifugation.

Approximately 2—5 g of each cell pellet was resuspended in 10—25 ml binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) supplemented with Benzonase® (Merck, cat. no. 1016540001) to a concentration of 15 U/ml and Lysozyme (Sigma, cat. no. ) to a concentration of 0.5 mg/ml. After cell disruption by three —thawing cycles or sonication, cell debris was removed by centrifugation and each supernatant was applied on a 1 ml His GraviTrap lMAC column (GE care, cat. no. 11-0033—99). Contaminants were d by washing with wash buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 or 60 mM imidazole, pH 7.4), and the FcRn binding Z variants were subsequently eluted with elution buffer 1 (20 mM sodium phosphate, 0.5 M sodium chloride, 250 mM imidazole, pH 7.4) or n buffer 2 (0.1 M acetic acid, 0.5 M sodium chloride, pH 4.5). Purified Z variants were buffer exchanged to PBS using PD- columns (GE Healthcare), according to the manufacturer’s protocol.

Protein concentrations were determined by measuring the absorbance at 280 nm, using a NanoDrop® ND—1000 spectrophotometer, and using the tion coefficient of the respective protein. The purity of the FcRn binding Z variants was analyzed by SDS—PAGE stained with Coomassie Blue. The identity of each purified FcRn binding Z variant was med using LC/MS analysis. 1O CD analysis: Purified HiSG—tagged Z variants were diluted to 0.5 mg/ml in PBS. For each diluted Z variant, a CD spectrum at 250—195 nm or 250—190 nm was obtained at 20 °C. In addition, a variable temperature ement (VTM) was performed to determine the melting temperature (Tm). In the VTM, the absorbance was ed at 221 nm while the ature was raised from 20 to 90 °C, with a temperature slope of 5 °C/min. A new CD spectrum was obtained at 20 °C after the heating procedure in order to study the refolding ability of the Z variants. The CD measurements were performed on a Jasco J—810 spectropolarimeter (Jasco Scandinavia AB) using a cell with an optical path—length of 1 mm.

Biacore binding and kinetic analysis: The interaction of FcRn binding HiSe-tagged Z variants with human FcRn was analyzed in a Biacore 2000 instrument (GE care). Human FcRn was immobilized in a flow cell on the carboxylated n layer of a CM5 chip surface (GE care). The immobilization was performed using amine coupling chemistry according to the manufacturer’s protocol and using HBS—EP (GE Healthcare) as g buffer. One flow cell surface on the chip was activated and deactivated for use as blank during analyte injections. In the two binding experiments presented below, Mcllvaines phosphate-citrate buffer pH 6.0 supplemented with 0.005 % Tween-20 (0.005% PCT) was used as running buffer. In all experiments, a flow rate of 50 ul/min was used.

In one experiment, the dissociation at pH 6.0 was compared to the dissociation at pH 7.4. HISG-tagged Z ts and a human monoclonal IgG1 were diluted in running buffer to a final concentration of 250 nM or 2.5 nM, respectively, and ed over the FcRn chip for 1 minute using the co—inject procedure. The second ion of the co~inject procedure, representing the dissociation phase of the interactions, ned either running buffer (pH 6.0) or 0.005% PCT pH 7.4. The Z variants were allowed to dissociate for 1 minute, except for 207918 and 210193, which were allowed to iate for 4 minutes, before a surface equilibration during 5 s in running buffer. lgG was allowed to dissociate for 4 minutes before bration. Buffer injections were performed in a similar way; co—injection of buffer pH 6.0 followed by pH 6.0 or ection of buffer pH 6.0 followed by pH 7.4. The results were analyzed in BiaEvaluation software 4.1 (GE Healthcare). Curves of the blank surface were subtracted from the curves of the ligand surface. In addition, curves of buffer injections were subtracted from the Z variant curves and from the lgG curves to adjust for the buffer s.

In another experiment, approximate kinetic constants (kon and kof‘f) and affinities (KD) were determined for a subset of HiSs~tagged Z variants. Three concentrations of the Z variants were injected for 1 minute followed by dissociation in running buffer for 1 minute. The surfaces were equilibrated with running buffer during 7.5 minutes before the start of next cycle. lnjected concentrations were either 675 nM, 225 nM and 75 nM (210140, 210156 and 210183) or 225 nM, 75 nM and 25 nM 8 and 210193). c constants were calculated from the sensorgrams using the Langmuir 1:1 model of BiaEvaluation software 4.1 (GE Healthcare).

In a separate ment, the affinity of the interactions of 2 variants to hFcRn (SEQ ID 5) and chRn (SEQ ID NO:1070), respectively, was measured at both pH 6.0 and pH 7.4 on a Biacore 3000 instrument (GE Healthcare). hFcRn and chRn were produced essentially as described in Example 1 but using mouse 3T3 cells instead of human SKOV—3 cells for production of chRn, and lized on separate flow cells on a CM5 chip in acetate buffer at pH 4.65. The immobilization level was approximately 1000 RU for both receptors. A reference flow cell was created by tion and deactivation. 0.005% PCT pH 6.0 or 7.4 was used as running buffer and for dilution of the analytes. All analyses were performed at 25 °C. The affinity constants for the HiSs—tagged Z variants 207918 (SEQ lD NO:707), 207960 (SEQ ID NO:710) and 210193 (SEQ ID NO:708) were determined by injecting a dilution series from 1024 nM to 0.5 nM (pH 6.0) or from 10240 nM to 5 nM (pH 7.4). The affinities were derived using ad Prism 5 software, using a one site binding saturation model.

AlphaLlSA blocking assay: The potential of 2 variants to inhibit binding of lgG to FcRn was analyzed in an AlphaLlSA assay with an EnSpire multiplate reader 2300 (Perkin Elmer). Human lgG (Roactemra) was lized on AlphaLlSA acceptor beads (Perkin Elmer, cat. no. 6772002) according to the manufacturer’s recommendations. Stepwise serial dilutions 1:3 of His-tagged Z variants to final trations of 250 nM to 38 pM were made in a 384—well plate (Perkin Elmer, cat. no. G6005350) and incubated for 45 min with 10 nM biotinylated human FcRn (Biorbyt, cat. no. orb84388; biotinylated essentially as described in Example 2) in ISA buffer (Perkin Elmer, cat. no. AL000F) adjusted to pH 6.0 using HCl. lgG-coated Acceptor beads were added to a final concentration of 10 uM and incubated for 45 min. y, streptavidin coated Donor beads n Elmer, cat. no. 6772002) were added to a final concentration of 40 pg/ml and incubated for 1O 30 min. All incubations were performed at RT in the dark. The plate was analyzed in the EnSpire instrument and the 1050 values were calculated using GraphPad Prism 5.

Results Cultivation and purification: The seventeen FcRn binding Z ts (SEQ lD NO: 707—722 and SEQ lD N011059), constructed with an N—terminal His6 tag, were produced in E. coli. The amount of lMAC-purified protein from approximately 2—5 g bacterial pellets, determined spectrophotometrically by measuring the absorbance at 280 nm, ranged from approximately 10 mg to 20 mg for the different FcRn binding Z variants. SDS—PAGE analysis of each final protein preparation showed that these predominantly contained the FcRn binding Z variant. The correct identity and lar weight of each FcRn binding Z variant was confirmed by S analysis.

CD analysis: The CD spectra determined for six Z variants showed that each had an a—helical structure at 20 °C. This result was also verified in the variable ature measurements, wherein melting temperatures (Tm) were ined (Table 2). A reversible folding was seen for the six Z variants when ying spectra measured before and after heating to 90 °C. 3O Table 2. Meltingtemperatures for a selection of Z variants.

Z variant SEQ lD NO: Tm (°C) 207909 719 56 207918 707 49 207930 712 56 207960 710 58 210109 709 61 Z10193 708 59 e binding and kinetic analyses: The binding of seventeen Z variants to human FcRn and the dissociation at different pH were tested in a Biacore instrument by tially injecting each of the Z variants at pH 6.0 and either buffer pH 6.0 or pH 7.4 over a chip surface containing FcRn. The ligand immobilization level of the e was 1668 RU human FcRn. The een Z variants showed binding to FcRn at pH 6.0, and for all variants, faster off—rates were seen at pH 7.4 compared to pH 6.0. The result for lgG was similar, displaying a faster off-rate at pH 7.4. The variants 207918 and 210193 showed the slowest dissociation curves. Sensorgrams for a subset of variants and lgG are yed in Figure 2 A—E.

Table 3. Biacore kinetic constants and affinities for hFcRn binding at pH 6.0. z variant SEQ ID NO: 1S'1) kofr(S'1) KD (M) 207918 707 1.4 x 106 0.022 1.6 x10-8 210140 711 1.4 x106 0.12 8.8 x10“8 210158 717 7.6 x 106 0.28 3.7 x10”7 210183 713 1.0x106 0.13 1.3x10-7 210193 708 1.5 x106 0.033 2.2 x10-8 The kinetic constants of five Z variants interacting with FcRn at pH 6.0 were determined (see Table 3). The immobilization level of the surface was 2015 RU human FcRn. For each Z variant, kinetic constants were calculated using a curve set of three injected trations.

Affinity (K0) constants were also determined for HiSs-tagged Z variants ZO7918 (SEQ ID NO:707), 207960 (SEQ ID NO:710) and Z10193 (SEQ ID NO:708) interacting with human and mouse FcRn at pH 6.0 and pH pH 7.4 (Table 4). For all three variants, KD values were lower at pH 6.0 compared to pH 7.4.

Table 4. Biacore affinities for hFcRn and chRn at pH 6.0 and pH 7.4.

SEQ ID KD (M) hFcRn KD 1M) chRn Z variant NO: pH 6.0 pH 7.4 pH 6.0 pH 7.4 207918 707 1.2 X 10'8 >5 X 10'7 9.0 X 10'8 >5 X 10'7 207960 710 5.0 X 10'8 >1 X 10'6 3.5 X 10'7 >5 X 10'5 Z10193 708 1.4 X 10‘8 >5 X 10~7 9.5 X 10'8 >5 X 10‘7 Table 5: Calculated lCSO values from Al haLlSA blockin assa . 210193 — 12x108 213993 1059 1.3 x107 Z11948—G4S—Z1 1948 1060 3.8 X 10'10 Z11948— G48 3—Z11948 1060 4.1 X 1O~10 lSA blocking assay: The ability of seventeen HiS5—tagged monomeric Z variants (SEQ ID NO:707—722 and SEQ ID NO:1059) and two dimeric variant, Z11948—G4S—Z11948 and Z11948—(G4S)3—Z11948to inhibit lgG binding to FcRn was tested in an AlphaLlSA blocking assay. Serial dilutions of the Z variants were ted with biotinylated human FcRn and the blocking ability of each respective variant was measured after addition of lgG 1O coated Acceptor beads and subsequently streptavidin coated Donor beads.

Inhibition could be measured as a decrease in AlphaLlSA counts for positive Z variants. The calculated lC5O values for the ten monomeric variants and the two dimeric variants that were shown to block lgG binding to FcRn in this assay are shown in Table 5.

Example 4 Binding of FcRn binding Z variants to human or mouse FcRn/eGFP transfected HeLa cells In this example, the binding ability of FcRn binding Z ts was investigated. The tion of HeLa cells expressing human and murine FcRn—eGFP gene transgene and the use of these cells for flow cytometry analysis with 47 d Z variants is described.

Materials and methods Cloning of FcRn-eGFP and 82M viral vectors: The genes encoding murlne FcRn (chRn, Genbank BC003786.1, OpenBiosystems) and murine BZM (mBZM, Genbank 641 OpenBiosystems) were ied in a similar way as the genes for human FcRn and human 82M as described in Example 1. Human and murlne FcRn and 82M genes were amplified as follows: for hFcRn, the sequence encoding amino acids 1-365 (SEQ ID NO:1068) was amplified; for hBZM, the sequence encoding amino acids 21 — 1O 119 (SEQ lD NO:1066) was ied; for chRn, the sequence ng amino acids 1-369 (SEQ ID NO:1069) was amplified; and for mBZM, the sequence ng amino acids 21-119 (SEQ ID NO:1067) was amplified.

The vector PT—CMV—EGFP sson et al. (2003) J Neurosci Res 73:876—85) and FcRn PCR amplicons (human and murine) were cut using the restriction enzymes BamHl (human) or Bc/l (murlne) and Mlul (New England Biolabs, cat. nos. R0136M, R0160L and RO198L, respectively), and ligated using T4 DNA Ligase (New England Biolabs, cat. no. M0202M). The ligation mix was chemically transformed into E. coli RRIAM15 and spread on ampicillin plates. Colonies were picked and screened with suitable primer 2O pairs. The construct encoding the original signal peptide, human or murlne FcRn and eGFP at the cytoplasmic tail were verified by sequencing and denoted pHR—cPPT—CMV-hFcRn—eGFP and pHR—cPPT—CMV—chRn-eGFP, respectively.

The human and murlne 82M PCR amplicons were inserted into the plasmid pDONOR221 (lnvitrogen, cat. no. 12536—017) by ination using the Gateway system (lnvitrogen, cat. no. 11789020, Gateway® BP Clonase® ll Enzyme mix) according to the manufacturer’s recommendations. After verification of correct sequences, human or murlne B2M was inserted into p2k7_gtc (Suter et al., supra) using a multi-site gateway cloning system (lnvitrogen, cat. no. 11791020, Gateway® LR Clonase® ll Enzyme mix) together with the promoter containing plasmid pENTR-CMV (Tai et al. supra), resulting in the s —CMV—hBZM and 2k7neo-CMV—mBZM, respectively.

Lentiviral transduction of HeLa cells: The vector pairs 2k7neo—CMV— hBZM and pHR—cPPT—CMV—hFcRn-eGFP or 2k7neo—CMV—mBZM and pHR- cPPT—CMV—chRn-eGFP were co-transfected together with VSV-G envelope and gag/pol packaging plasmid into HEK293T cells using calcium chloride transfection (Zufferey et al., supra; Jakobsson er al. (2006) supra). HEK293T culture supernatants containing formed lentiviral particles with FcRn and 82M transgenes tively were used to sequentially transduce HeLa Cervix adenocarcinoma cells (Cell Line Service) at low passage number. The resulting two stably uced HeLa cell lines are in the following denoted hFcRn—eGFP (transduced with genes for human FcRn—eGFP and hBZM) and chRn—eGFP (transduced with genes for mouse FcRn—eGFP and mBZM).

Alexa647 labeling of FcRn binding Z variants: The three HiSs—tagged Z ts 207918, 207930 and 207960 were d with Alexa Fluor® 647 Carboxylic Acid Succinimidyl Ester (lnvitrogen cat. no. A20106). Before labeling, buffer was exchanged to 0.2 M ate buffer, pH 8.3, using Vivaspin500 centrifugal filter units (10 kDa MWCO, Vivaproducts cat. no. 512— 2838) spun at 10,000 g. The labeling was performed in the Vivaspin500 and 1 pl of Alexa647 Succinimidyl Ester dye (40 pg/pl in DMSO corresponding to 1.3 x molar excess) was added to 200 pg/25 pl Z variant. The mixes were incubated at RT in the dark for 40 minutes in a wiggling rota mixer. The reaction mixes were subsequently put on ice for 3.5 hours and free dye was d by washing with 15 x 100 pl PBS in the Vivaspin500. fluorescence staining of human and mouse FcRn—eGFP transfected HeLa-cells with FcRn binding Z variants: hFcRn—eGFP and chRn—eGFP HeLa cells were harvested by nation and washed twice in PBS at pH 6.0 before counting. 100,000 cells were pipetted per well of a v— bottomed 96 well plate (Nunc, cat no 277143) and the cells in the plate were subsequently pelleted at 1,700 rpm for 4 min at 4 °C. The supernatants were removed and the cells were fixed with 50 pl of 2 % formaldehyde (Sigma Aldrich, cat. no. F8775) in PBS at pH 6.0 for 10 min at RT. Cells were thereafter washed with 2 x 100 pl PBS pH 6.0, saturated with casein (PBSC), and resuspended in PBSC plus 0.1 % saponin Chem, cat no A4518.0100) containing 620 nM of Alexa647 labeled HiSG—tagged Z variants; 207960, 207930 and 207918. Transduced HeLa cells, incubated with buffer alone, were used as control. The cells were ted for 1 h at 8 °C on a shaker in the dark, washed with 2 x 100 pl PBSC and ended in 180 pl of PBS pH 6.0 plus 1 % BSA (fraction V, Merck, cat. no. 1.120180100). ,000 cells/well were analyzed in a Gallios Flow Cytometer (Beckman Coulter) and the data was analyzed using Kaluza software (Beckman Coulter).

Results Flow cytometry analysis was utilized to determine r the FcRn binding Z variants could bind to human and/or mouse FcRn on human or mouse FcRn/eGFP transduced HeLa cells. The experiment was performed at pH 6.0 with Alexa647 labeled 207960, 207930 and 207918 (SEQ ID N02710, 712 and 707, respectively). Dot plot analysis (y—axis: Alexa647, : eGFP) showed that the transduced cell population could be divided into FcRn-eGFP negative and positive population (Figure 3, gate H and l, respectively) indicating heterogeneous expression of the FcRn-eGFP fusion protein by HeLa cells (Figure 3). Accordingly, the mean fluorescence intensity (MFI) values for Alexa647 in gate l were cted by background MFI values of Alexa647 in gate H. The calculated MFI values are presented in Figure 4. The results show that 207960, 207930 and 207918 are capable of binding HeLa cells displaying human (Figure 4A) or murine (Figure 4B) FcRn—eGFP.

Example 5 ng of lgG binding to FcRn with the FcRn binding Z variant 207918 In this example, the potential competition of FcRn binding Z variants with lgG for binding to FcRn was investigated in a cell based assay. Such binding will result in ng of the lgG-FcRn interaction. als and methods Blocking of Rn immunofluorescence staining: Human or murine FcRn-eGFP transduced HeLa cells were prepared as described in Example 4. Fixed cells were resuspended in 50 pl of a mix of either 100 nM 47— conjugated human or mouse lgG on laboratories, cat. no. 0—003 and 015—600—003, respectively) and 1000, 100, 10, 1 or 0 (buffer control) nM His6-tagged 207918 diluted in PBS-casein, pH 6.0, plus 0.1 % n (AppliChem). The cells were incubated for 1 h at 37 °C on a shaker in the dark, washed with 2 x 100 pl PBS—casein pH 6.0 and re-suspended in 180 pl of PBS, pH 6.0, plus 1 % BSA. Data from 10,000 cells/well (except somewhat fewer cells for mouse 100 nM mlgG-Alexa647) were obtained using a Gallios Flow Cytometer (Beckman Coulter) and the data was analyzed using Kaluza software (Beckman Coulter).

Results The experiment was med to determine if the FcRn binding Z variant 207918 (SEQ ID ) blocks the IgG—FcRn ction. Human or murine FcRn-eGFP transduced HeLa cells were incubated with human or mouse 47-conjugated IgG. The binding was blocked with unlabeled 207918 at ent concentrations. Due to the geneous expression of FcRn by the transduced HeLa cells ibed in Example 4), the MFI values for Alexa647 in gate N of each sample was subtracted by the corresponding MFI values in gate M (Figure 5). The percent IgG Alexa647 binding was ated by dividing the different MFI values with the MFI for the blank control. The results showed that 207918 effectively blocked hlgG binding to hFcRn (Figure 6A) in a dose dependent manner. Furthermore, 207918 also blocked mlgG binding to chRn (Figure 68) although less efficiently compared to hlgG-binding.

Example 6 Pharmacokinetic study of three FcRn binding Z variants In this example, the ability of FcRn binding Z variants to prolong serum half—life of a non-specific Z variant was investigated by a pharmacokinetic study performed in mice.

Materials and methods Subcloning of Z variants: A subset of Z variants (207918, 207960 and 210193) was submitted to a second subcloning. DNA from the subcloned HiSs-tagged variants in Example 3 was used as template. First, PCR amplification using suitable primer pairs was performed to create genes encoding 2 ts starting with the amino acids AE instead of VD. The mutated Z variants are listed in Figure 1 and were denoted 211948 (SEQ ID NO:1060), 211946 (SEQ ID NO:1061) and 211947 (SEQ ID NO:1062), corresponding to mutated 207918, 207960 and 210193, tively. Genes encoding the new 2 variants were restriction cleaved and ligated into a vector harboring the genes encoding albumin binding variant PPO13 (SEQ ID NO:1063) and 203638 (SEQ ID NO:1064) with spacer sequences resulting in a gene fusion encoding [2#####]—GAP(G4S)4TS—[PP013]—GT(G4S)4PR- 8] (also denoted “2#####-PP013-203638” or “Z variant in fusion with PP013-ZO3638"). The ve l molecule [ZO3638]—GAP(G4S)4TS— [PP013] was subcloned in a similar way by ligating 203638 into a vector containing a (G4S)4 linker and the sequence for PP013. The subsequent steps for vector transformation into E. coli were performed as in e 3.

Cultivation and purification: Z variants in fusion with PP013-ZO3638 were produced in E. coli as described in e 3. Approximately 3 g of each cell pellet was re-suspended in 30 ml TST—buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05 % Tween20, pH 8.0) supplemented with Benzonase® (Merck). After cell disruption by sonication and clarification by centrifugation, each supernatant was applied on a gravity flow column with 5 ml e immobilized with an anti—ABD ligand (produced in—house). After washing with TST-buffer and 5 mM NH4Ac buffer, pH 5.5, the Z variants were eluted with 0.1 M HAc. Acetonitrile (ACN) was added to a final concentration of 10 % to the eluted fractions from the anti—ABD agarose affinity chromatography cation step and the samples were loaded on a 3 ml Resource 15RPC column (GE Healthcare), previously equilibrated with RPC t A (0.1 % trifluoroacetic acid (TFA), 10 % ACN, 90 % water). After column wash with RPC solvent A, bound protein was eluted with a linear gradient 0—50 % RPC solvent B (0.1 % TFA, 80 % ACN, 20 % water) during 60 ml. Fractions containing pure Z variant were fied by SDS~PAGE analysis and pooled. After the RPC purification, the buffer of the pools was exchanged to PBS using a HiPrep 26/10 Desalting column (GE Healthcare).

Finally, the Z variants were purified on 1 ml EndoTrap red columns (Hyglos, cat. no. 321063) to ensure low endotoxin content.

Protein concentrations, purities and the ty of each purified Z variant were analyzed as described in Example 3.

Biacore is: Expressed and purified Z variants fused to PP013— 203638 were assayed against human FcRn at pH 6.0 essentially as described for the kinetic is in Example 3. The Z variants and the negative control ZO3638—PP013 were injected at 40 nM, 160 nM and 640 nM during 1 minute ed by dissociation for 2.5 minutes and equilibration for 1 minute. Kinetic constants and ties were determined for the Z variants using the BiaEvaluation software.

Pharmacokinetic study: Z11947, Z11946 and 211948 fused to PP013— 203638 were administered intravenously (i.v.) to male NMRl mice (Charles River, Germany) at a dose of 92 nmol/kg body weight. Sera from groups of three mice were obtained at 0.08, 6, 18, 78, 120, 168 and 240 hours. The concentration of respective Z variant was determined by ELISA.

ELISA: Half—area 96—well ELISA plates were coated at 4 °C overnight with 50 pl/well of an Z specific goat antibody (produced in—house) d to 4 ug/ml in coating buffer (50 mM sodium carbonate, pH 9.6). The antibody solution was poured off and the wells were blocked with 100 pl of PBSC for 1.5 h at RT. The sera were diluted in PBSC plus 1 % mouse serum x) from 1:100 to 1:51,200 in a two—fold dilution series in a dilutions plate. A standard titration for respective Z variant and four quality ls (very low, 1O low, medium and high control) diluted in matrix were included on each plate. 50 pl of the dilutions were transferred per well and the ELISA plates were incubated for 1.5 h at RT. The plates were washed four times with PBST.

Bound Z variants were detected with 50 pl/well of rabbit anti—PP013 lg (produced in—house) diluted to 4 ug/ml in PBSC. The plates were uently incubated for 1.5 h at RT followed by washes as described above. HRP conjugated donkey anti—rabbit HRP obtained from Jackson laboratories (cat. no. 711—035—152), diluted 120,000 in PBSC, was added and the plates were incubated for 1 hour. After washing as bed above, 50 pl of ImmunoPure TMB substrate was added to the wells and the plates were ped according to the manufacturer’s recommendations. After 15 minutes of development, the absorbance was measured at 450 nm using a multi—well plate reader r3). The absorbance values were analyzed using GraphPad Prism 5 to determine the concentrations (cubic—spline curve fit) and area under curve (AUC). The concentrations were then d as their natural logarithms against time. The resulting curves followed a two compartment model and the terminal half—life was calculated as ln2 divided by the slope based on the last three time points.

Results Cultivation and Qurification: The three FcRn binding Z variants 211947, 211946 and 211948 (SEQ ID NO:1062, 1061 and 1060), constructed as Z#####—PP013—ZOB638, and the negative control 203638-PP013, were produced in E. coli. The amount of purified protein from approximately 3 g bacterial pellets, determined spectrophotometrically by ing the absorbance at 280 nm, ranged from approximately 10 to 25 mg for the different FcRn binding Z variants. GE analysis of each final n ation showed that they predominantly contained respective FcRn binding Z variant. The correct molecular weight of each FcRn binding Z t was confirmed by LC/MS analysis.

Table 6. c constants and affinities for FcRn at pH 6.0 of Z variants produced as fusions to PP013—ZOS638. z t SEQ lD NO: kon (M‘s‘) kofi (3") K6 (M) Z11948 1060 773 X105 0.047 62 x168 211946 1061 3.35 x105 0.275 82 x107 211947 1062 554 x105 0.064 98 x108 Biacore analysis: The binding to FcRn was analyzed for the three PP013—203638 fused Z variants. The immobilization level of the surface was 548 RU of human FcRn. The resulting rough kinetic constants and affinities 1O for the target binding at pH 6.0 are displayed in Table 6. Fitted curves are displayed in Figure 7A—C. The negative control ZOS638—PPO13 was negative against FcRn.

Pharmacokinetic study: The pharmacokinetic profiles of the above— mentioned constructs of Z variants fused to PPO13-ZOS638 were compared to the negative control ZO3638—PP013 in a mouse pharmacokinetic study. In previous work, eg. as described in PCT application WO2009/O16043, it is shown that ABD fusion proteins have a long half—life in serum, caused by ABD binding to serum albumin. in ance with the previous results, terminal half—life of ABD—fused Z variant molecule (ZOB638—PPO13) was approximately 43 hours, which is comparable to half-life of mouse albumin (35 hours). The al half—lives of the constructs containing FcRn binding Z t le in addition to ABD were two— to three—fold longer (Figure 8). The calculated al half—lives were 99 hours (211947), 69 hours (211946) and 58 hours (211948), suggesting that FcRn binding of the Z variants contributed to the ged half—life.

Examgle 7 Design and construction of a maturation library of FcRn binding Z variants in this Example, a maturated library was constructed. The library was used for selections of FcRn binding Z variants. Selections from maturated libraries are usually expected to result in binders with increased affinity (Orlova et al., (2006) Cancer Res 66(8):4339—48). In this study, randomized single stranded linkers were generated using split—pool synthesis enabling incorporation of defined codons in d positions in the synthesis.

Materials and methods Library design: The library was based on the sixteen sequences of the human FcRn binding Z variants in Table 1 and further described in Examples 2—6. In the new library, 13 le positions in the Z le scaffold were biased s certain amino acid es, according to a strategy mainly based on the binding motifs of the Z variants defined in SEQ lD NO:707-722.

A DNA linker was generated using split—pool synthesis containing the 147 bp partially randomized helix 1 and 2 of the amino acid sequence: 5’— AA ATA AAT CTC GAG GTA GAT GCC AAA TAC GCC AAA GAA NNN NNN NNN GCG NNN NNN GAG ATC NNN NNN TTA CCT AAC TTA ACC NNN NNN CAA NNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTA TTT A ~3’ (SEQ ID NO:1074; ized codons are illustrated as NNN) flanked by restriction sites Xhol and Sacl, was ordered from DNA 2.0 (Menlo Park, CA, USA). The theoretical distributions of amino acid residues in the new library, ing eight variable amino acid positions (9, 10, 11, 13, 14, 24, 32 and 35) and five constant amino acid positions (17, 18, 25, 27 and 28) in the Z molecule scaffold are given in Table 8. The ing theoretical library size is 5.3 x 108 variants.

Table 7: Desin of libra for maturation.

Amino acid Randomization (amino acid on in the abbreviations) Z variant A,D,E,F,H,l,K,L,N,Q,R,S,T,V,W,Y 16 1/17 A D E F H I K,L,N,Q,R,S,T,V,W,Y - 1/16 A D E F G H l,K,L,N,Q,R,S,T,V,W,Y - 1/17 A,F H 25 % l,K,L,N,Q,R,S,T,V,W,Y - 3/52, 13/52 H 1/16 16 1/16 Libram uction: The library was amplified using AmpliTaq Gold polymerase (Applied Biosystems, cat. no. 4311816) during 12 cycles of PCR and pooled products were purified with QlAquick PCR Purification Kit (QlAGEN, cat. no. 28106) according to the supplier’s recommendations. The purified pool of randomized library fragments was digested with restriction enzymes Xhol and Sacl—HF (New England Biolabs, cat. no. R0146L, and cat. no. R3156M) and concentrated using a PCR Purification Kit. uently, the product was subjected to preparative 2.5 % agarose (Nuisieve GTC agarose, Cambrex, lnvitrogen) gel electrophoresis and ed using QIAGEN gel extraction Kit (QlAGEN, cat. no. 28706) ing to the supplier's recommendations.

The phagemid vector pAY02592 (essentially as pAffi1 bed in Gronwall et al., supra) was restricted with the same enzymes, purified using phenol/chloroform extraction and l precipitation. The restricted fragments and the restricted vector were ligated in a molar ratio of 5:1 with T4 DNA ligase (Fermentas, cat. no. ELOO11) for 2 hours at RT, followed by ght incubation at 4 °C. The ligated DNA was recovered by phenol/chloroform extraction and ethanol precipitation, followed by dissolution in 10 mM Tris—HCl, pH 8.5. Thus, the resulting y in vector pAYO2592 encoded Z variants, each fused to an albumin binding domain (ABD) derived from streptococcal protein G.

The ligation reactions ximately 160 ng DNA/transformation) were oporated into electrocompetent E. coli ER2738 cells (50 pl, Lucigen, Middleton, Wl, USA). Immediately after oporation, approximately 1 ml of recovery medium ied with the ER2738 cells) was added. The transformed cells were incubated at 37 °C for 60 min. Samples were taken for titration and for determination of the number of transformants. The cells were 1O thereafter pooled and cultivated overnight at 37 °C in 1 l of TSB—YE medium, supplemented with 2 % glucose, 10 ug/ml tetracycline and 100 pg/ml ampicillin. The cells were pelleted for 7 min at 4,000 g and resuspended in a PBS/glycerol solution (approximately 40 % glycerol). The cells were aliquoted and stored at ~80 °C. Clones from the y of Z variants were sequenced in order to verify the content and to te the outcome of the constructed library vis-a-vis the library design. Sequencing was performed as described in Example 1 and the amino acid distribution was verified.

Preparation of phage stock: Phage stock containing the phagemid library was prepared in a 20 l fermenter (Belach Bioteknik). Cells from a 2O ol stock containing the phagemid library were inoculated in 10 l of TSB— YE (Tryptic Soy Broth-Yeast Extract; 30 g/l TSB, 5 g/l yeast extract) mented with 1 g/l glucose, 100 mg/l ampicillin and 10 mg/l tetracycline.

When the cells reached an optical density at 600 nm (0D600) of 0.6, approximately 1.5 l of the cultivation was infected using a 5 x molar excess of M13K07 helper phage. The cells were incubated for 30 min, whereupon the fermenter was filled up to 10 lwith complex fermentation medium [2.5 g/l (NH4)2SO4; 5.0 g/l yeast extract; 30 g/l ne, 2 g/l K2HP04; 3 g/l KH2P04, 1.25 g/l; NasCeH507 ' 2 H20; Breox FMT30 antifoaming agent 0.1 ml/l]. The following components were added: 10 ml carbenicillin 25 mg/ml; 5 ml kanamycin 50 mg/ml; 1 ml 1 M isopropyl—B—D—1—thiogalactopyranoside (lPTG); 17.5 ml/l of 300 g/l M9804, and 5 ml of a trace element solution [35 g/l FeCls ' 6 H20; 10.56 g/l ZnSO4 ' 7 H20; 2.64 g/l Cu804 ° 5 H20; 13.2 g/l MnSO4 - H20; 13.84 g/l CaCl2 - 2 H20, ved in 1.2 M HCl]. A glucose limited fed— batch cultivation was started where a 600 g/l glucose solution was fed to the reactor (3.5 g/h in the start, 37.5 g/h after 20 h and until the end of the ation). pH was controlled at pH 7 through the automatic addition of 25 % NH40H, air was supplemented (5 l/min), and the stirrer was set at 500 rpm.

After 24 h of fed—batch cultivation the OD600 was 33.2. The cells in the cultivation were pelleted by centrifugation at 15,900 g. The phage particles were precipitated from the supernatant twice in PEG/NaCl, filtered and dissolved in PBS and glycerol astin Example 2. Phage stocks were stored at —80 °C until use in selection.

Results Library construction: The new library was designed based on a set of 16 FcRn binding Z variants with verified binding ties (Example 2—6).

The theoretical size of the designed library was 5.3 x 108 Z variants. The actual size of the library, determined by titration after transformation to E. coli ER2738 cells, was 4.5 x 109 transformants.

The library quality was tested by sequencing of 96 transformants and by comparing their actual sequences with the theoretical design. The ts of the actual library ed to the designed library were shown to be ying. A maturated library of potential s to FcRn was thus successfully constructed.

Example 8 ion and ing of Z variants from a maturated libram Materials and methods Phage display selection of matured FcRn binding Z variants: The target proteins human FcRn (Biorbyt, cat. no. orb84388) and murine FcRn (Biorbyt, cat. no. orb99076) were biotinylated essentially as described in e 2 using biotin at 10x molar excess. Phage display ions, using the new library of Z variant molecules described in Example 7, were performed in four cycles against human FcRn or murine FcRn essentially as in Example 2 but with the following exceptions. Selection buffers were 0.1% PCTG , pH .5 (Mcllvaines phosphate—citrate buffer, pH 5.5, supplemented with 0.1 % Tween—20 and 0.1% gelatin) or 0.1% PCTG buffer, pH 7.4, (Mcllvaines phosphate—citrate buffer, pH 7.4, supplemented with 0.1 % Tween-20 and 0.1% gelatin) respectively. Prior to selection, HSA was added to the selection buffers to a final concentration of 1.5 pM. All tubes and beads used in the selection were pre-blocked with either of the two ent selections buffers.

A pre—selection step, by incubation of phage stock with SA-beads for 45 min, was med in cycle 1. For capture of phage—target complexes, 1 mg beads per 1.1 ug ylated human FcRn or 1.6 pg biotinylated murine FcRn was used. Washes were performed with 0.1% PCT buffer pH 5.5 or pH 7.4 except for tracks 2—1—2—1 and 22—2 where 0.1 % PCT supplemented with 25 nM lgG (Herceptin®) or 10 nM lgG, respectively, was used as outlined in Table 7.

The five tracks (1—5) in cycle 1 were divided in the second to fourth cycles, resulting in totally seven tracks (1-1 to 5—1) in cycle 2, eleven tracks (1-1—1 to 5—1—1) in cycle 3 and fourteen tracks (1—1—1—1 to 5—1-1—1) in cycle 4. 1 O The bound phage particles were eluted as described in Example 2.

An overview of the ion strategy, describing an increased stringency in subsequent cycles, using a lowered target concentration and an increased number of washes, is shown in Table 8. 1 5 Table 8. Overview of the maturation selection data.

Cycle ion Phage stock Target Target Selection Wash Number from library or selection track human murine human human 2 murine Cycle Selection Phage stock from library or selection track Amplification of phage les: Amplification of phage particles between selection cycle 1 and 2 was performed essentially as described in Example 2, with the following exceptions. E. coli ER2738 was used for phage amplification and M13KO7 helper phage was used in 5 x excess. The amplification of phage particles between the selection cycles 2 and 4 was done by performing infection of bacteria in solution as follows. After infection of log phase E. coli ER2738 with phage particles, TSB supplemented with 2 % glucose, 10 pg/ml tetracycline and 100 ug/ml ampicillin was added, followed by incubation with rotation for 30 min at 37 °C. Thereafter, the ia were infected with M13K07 helper phage in 5 x excess. The ed bacteria were pelleted by centrifugation, re—suspended in TSB-YE medium supplemented with 100 uM lPTG, 25 ug/ml cin and 100 ug/ml ampicillin, and grown overnight at 30 °C. The overnight es were pelleted in a centrifuge, and phage les in the supernatant were precipitated twice with PEG/NaCl buffer. Finally, the phage particles were re—suspended in selection buffer before entering the next selection cycle.

In the final selection cycle, log phase bacteria were infected with eluate and diluted before spreading onto TBAB plates (30 g/l tryptose blood agar base, Oxoid cat. no. CMOZBSB) supplemented with 0.2 g/I ampicillin in order to form single colonies to be used in ELISA screening.

Sequencing of potential binders: Individual clones from the different selection tracks were picked for sequencing. All clones run in the ELISA screening were sequenced. ication of gene fragments and sequence is of gene nts were performed essentially as described in Example 2.

ELISA screening of Z variants: Single colonies containing Z variants (expressed as Z variant ABD fusion proteins as described in Example 2) were randomly picked from the selected clones of the FcRn maturated library and grown in 1 ml cultivations essentially as described in Example 2. Preparation of the periplasmic supernatants was performed as in Example 2 with eight freeze g cycles and the periplasmic fractions were used undiluted in the ELISA screening. ELISA screenings were performed at both pH 6.0 and pH 7.4 ially as described in Example 2 using ylated human FcRn at a concentration of 2 nM in each well. The periplasmic fraction of the primary FcRn binder 210193 (SEQ ID N01708; assayed in above experiments) was used as a positive control. Periplasm containing the ABD moiety only was used as a negative control.

ELISA KD analysis of FcRn binding Z variants: A ion of FcRn binders was subjected to an analysis of the response against a dilution series of biotinylated human FcRn using ELISA at both pH 6.0 and pH 7.4 as described above. Biotinylated human FcRn was added at a concentration of nM and diluted stepwise 1:3 down to 14 pM. As a ound control, all Z ts were also assayed with no target protein added. Periplasm samples containing the primary FcRn binder 207918 (SEQ I‘D.NO:707) was included and analyzed as a ve control. Periplasm containing the ABD moiety only was used as a negative control. Data were analyzed using GraphPad Prism 5 and non—linear regression and KD values (the half maximal effective concentration) were calculated.

Results Phage display selection of maturated FcRn binding Z variants: Selection was performed in totally 14 parallel tracks containing four cycles each. The different selection tracks differed in target tration, target type (human FcRn or murine FcRn), selection time, and wash conditions.

Seguencing of potential binders: Randomly picked clones were sequenced. Each individual Z variant was given an identification number, Z#####, as described in Example 2. In total, 445 new unique Z variant molecules were identified.

The amino acid sequences of the 58 amino acid residues long Z variants are listed in Figure 1 and in the sequence listing as SEQ ID NO:723— 1058. The deduced FcRn binding motifs of these Z variants are listed in Figure 1 and in the ce g as SEQ ID NO:17-352. The amino acid sequences of the 49 amino acid residues long polypeptides predicted to 1O constitute the te three-helix bundle within each of these Z variants are listed in Figure 1 and in the ce listing as SEQ ID NO:370-705.

ELISA screening of Z variants: Clones obtained after four ion cycles were produced in 96-well plates and screened for FcRn binding activity using ELISA. All randomly picked clones were analyzed. At pH 6.0, 333 of the 445 unique Z variants were found to give a response of 0.3 AU or higher sponding to at least 3x the negative control) against human FcRn at a concentration of 2 nM. At pH 7.4, 278 of the 445 unique Z variants were found to give a response of 0.3 AU or higher (corresponding to at least 3x the negative control) against human FcRn at a concentration of 2 nM. Clones with a positive signal against human FcRn were found in all tracks (including those with murine target) except 1—1—1—1. The negative controls had absorbances of 0.070—0.096 AU (pH 6.0) and 1 12 AU (pH 7.4), respectively. The average response of the blank controls was 0.070 AU (pH 6.0) and 0.062 (pH 7.4).

ELISA KD analysis of FcRn binding Z variants: A subset of Z variants was selected based on the result in the ELISA experiment described above (highest ELISA value at pH 6.0 and/or pH 7.4) and subjected to a target titration in ELISA format. Periplasm s were incubated with a serial dilution of biotinylated human FcRn. A periplasm sample with the primary binder 207918 (SEQ ID NO:707) was also d as a positive l.

Obtained values were analyzed and their respective KD values were calculated (Table 9).

Table 9: Calculated KD values from ELISA titration anaiysis of Z—ABD variants from the maturation.

K0 K9 K9 K0 2 2 pH 6.0 pH 7.4 pH 6.0 pH 7.4 t variant M M M M 820 2.2x109 K0 Ko K9 K0 Z 2 pH 6.0 pH 7.4 pH 6.0 pH 7.4 t variant M M M M 784 846 Example 9 Production and characterization of Z variants from a maturated library in this Example, twelve Z variants were produced in E. coli, purified and assayed for binding to FcRn as well as for inhibition of lgG binding to FcRn.

Materials and methods Subcloning of Z variants into expression vectors: The DNA of twelve 1O FcRn binding Z variants (213577 (SEQ ID ), 213578 (SEQ lD NO:726), 213583 (SEQ ID NO:729), 213592 (SEQ ID NO:734), 213616 (SEQ ID NO:747), 213621 (SEQ ID NO:750), 213654 (SEQ ID NO:771), 213663 (SEQ lD NO:776), 213669 (SEQ ID NO:779), 213674 (SEQ ID NO:781), 213675 (SEQ ID NO:782) and 213676 (SEQ ID NO:783)) were amplified from the library vector pAY02592. The subcloning was performed as bed in Example 3. The 2 gene fragments were subcloned into the expression vector pAYO1448 resulting in the encoded ce MGSSHHHHHHLQ—[Z#####]— Production of Z variants: Cultivation and purification of the Hi35—tagged Z ts was performed essentially as described in Example 3. e g and kinetic analyses: The interaction of FcRn binding HiSG—tagged Z variants with human FcRn was analyzed in a Biacore 2000 instrument ially as described in Example 3. Human FcRn purchased from Biorbyt (cat. no. orb84388) was used as target protein. The analytes were ed during 2 minutes at 30 pl/min. The dissociation phase was 4 s and the equilibration time between the analyte injections was 30 minutes.

In one ment, the 2 variants were injected at pH 6.0 followed by dissociation in buffers of pH 6.0 or pH 7.4, respectively, using the co~inject procedure. The concentration of the 2 variants was 100 nM. in another experiment, approximate kinetic constants (km and koff) and affinities (Ko) were determined for a subset of Z variants. Injected concentrations were 540 nM, 180 nM, 60 nM, 20 nM and 6.7 nM.

AlphaLlSA blocking assay: The potential of 2 variants to inhibit binding of lgG to FcRn was analyzed in the AlphaLlSA assay bed in Example 3.

Results Production of Z ts: The twelve FcRn binding Z ts constructed with an N-terminai HiS6 tag were produced in E. coli. SDS—PAGE analysis of each final protein preparation showed that these predominantiy ned the FcRn binding Z variant. The t identity and molecular weight of each FcRn binding Z variant was confirmed by HPLC—MS analysis.

Biacore binding and kinetic analyses: The binding of the twelve Z variants to human FcRn and the dissociation at different pH were tested in a Biacore instrument by sequentially injecting each of the Z variants at pH 6.0 and either buffer pH 6.0 or pH 7.4 over a chip surface containing FcRn. The ligand immobilization level of the surface was 890 RU human FcRn. The twelve Z ts showed binding to FcRn at pH 6.0, and for all variants, faster off—rates were seen at pH 7.4 compared to pH 6.0.

The kinetic nts of the Z variants Z1357? (SEQ iD N02725) and Z13621 (SEQ ID NO:750) interacting with FcRn at pH 6.0 were determined (see Table 10). Kinetic constants were calculated using curve sets of two or four injected concentrations of Z1357? and , respectively.

Table 10. Biacore kinetic constants and affinities for FcRn binding at pH 6.0.

Z variant SEQ ID NO: kon (M434) koff(S'1) Kn (M) 213577 725 3.0 X 105 4.0 X 10’3 13 X 10'9 213621 750 6.4 X 105 3.7 X 10'3 6 X 10'9 AlphaLiSA ng analysis: The ability of twelve maturated Hiss— tagged ric Z variants to inhibit lgG binding to FcRn was tested in an iSA blocking assay. Serial dilutions of the Z variants were incubated with biotinylated human FcRn and the blocking ability of each respective variant was measured after addition of igG coated Acceptor beads and subsequently streptavidin coated Donor beads. inhibition could be measured as a decrease in AlphaLiSA counts for positive Z variants. All twelve tested Z variants were shown to block 196 binding to FcRn and the calculated IC50 values are shown in Table 11.

Table 11: Calculated lC50 values from Al haLlSA blockino assa . lC50 725 _1.2 x 10'8 726 -1.2x 10'8 729 -2.7 X 10'9 213654 771 -3.5 X 10'9 776 —1.1 x10'8 779 _5.2 x109 781 _2.5 x109 782 _8.2 x109 783 _3.9 x109 Example 10 Comparison of ng capacity of lgG binding to FcRn In this example, the lgG blocking capacity of the FcRn binding Z variant Hi36-ZO7918 (SEQ ID ) was ed to Intravenous immunoglobulin (Mg) and Subcutaneous immunoglobulin (SClg) currently 1O used in the treatment of some autoimmune disorders.

Materials and methods Blocking of lgG-FcRn immunofluorescence staining: Human or murine FcRn-eGFP transduced HeLa cells were prepared as described in Example 4. Fixed cells were resuspended in 50 pl of a mix of 50 nM Alexa647— conjugated human lgG (Jackson laboratories, cat. no. 009-600—003) and HlSB- tagged 207918, lVlg (Octagam®, Octapharma) or SClg (Gammanorm®, Octapharma), respectively, d at concentrations of 1000, 100, 10, 1, 0.1 or 0 (buffer control) nM in nes, pH 6.0, plus 2.5 % FCS (Ultra low lgG, Life technologies) and 0.1 % saponin (AppliChem). The cells were incubated for 1 h at 37 °C in the dark, washed with 2 x 100 pl Mcllvanes, pH 6.0, plus 2.5 % FCS (Ultra low lgG) pH 6.0 and re—suspended in 180 pl of Mcllvanes, pH 6.0, plus 1 % BSA. Data from 10,000 GFP/FcRn positive cells were ed using a FACS Calibur (Beckman Coulter) and the data was analyzed using Flowing software 2.5.0 (Turku University).

Results The experiment was performed to determine if the FcRn binding Z variant HiSe-ZO7918 (SEQ ID NO:707) blocks the lgG-FcRn interaction and compare the blocking effect to Mg and SClg . Human or murine FcRn—eGFP transduced HeLa cells were incubated with human Alexa647—conjugated lgG. 1O The binding was blocked with led Hise—ZO7918, IVIg or SClg at different trations. The results showed that HISe-ZO7918 ively blocked hlgG binding to hFcRn to a similar extent as IVlg or SClg (Figure 9).

Example 11 Increased lgG catabolism by FcRn binding Z variants in mice The ability of the FcRn binding Z variant 207918 to block lgG g to FcRn in vitro was shown in Example 10. In this example, the blocking ability of the same Z variant was evaluated in vivo. Blocking of lgG—FcRn interactions in vivo will lead to increased [96 catabolism and concomitant d levels of lgG (Mezo 2008, supra).

Materials and methods Animal study: The inding Z variants 211948 (SEQ ID NO:1060) and ZO7918-PP013 (Z07918 (SEQ ID NO:707) identical to 211948 but with the N—terminus starting with the amino acids VD instead of AE, in fusion with the ABD variant PP013 (SEQ ID NO:1063)) or vehicle (PBS buffer), were administered to male NMRI (Charles River), at a dose of 16.3 pmol/kg. The 3O mice were treated with five intravenous injections given at 0, 24, 48, 72 and 96 h. Serum samples were taken at 0, 72, 120 and 168 h (termination of study) and stored at —20 °C. The concentration of mouse IgG in serum was quantified by ELISA.

Mouse lgG ELISA: The concentration of mouse lgG in mouse serum samples was analyzed by a mouse lgG ELISA kit (Mabtech 3825—1AD—6) and performed as described by the manufacturer. The tration of mlgG was ated from a rd curve provided and GraphPad prism5 using a non— linear regression formula. The concentration of lgG in individual mice at 24, 72, 120 and 168 h were related to the level at 0 h and the results are therefore presented as percentage of lgG (O h).

Results The results showed a reduction of mouse lgG concentration in mice treated with FcRn-specific Z variants. Both 211948 and the ABD—fused variant ZO7918—PP013 lowered the concentration of endogenous lgG in mice in vivo.

Most pronounced effects were obtained with the ABD—fused variant and after 1O 120 hours. Thus, the results indicates that the FcRn—specific Z variants blocked recycling of lgG resulting in increased lgG catabolism and subsequent lower levels of lgG in mice.

Exam le 12 In vitro transcytosis of FcRn binding Z variants in this example, the FcRn binding Z variants are tested for their ability to be transported through lial or elial cells or recycled by FcRn in vitro. A drug containing a Z variant with the power of ytosis will facilitate drug uptake after for example oral or pulmonary administration.

Materials and methods Cells, for example T84, MDCK, HeLa, CaCo2, CaLu-1 and/or CaLu—3 cells, with or without endogenous or inant expression of FcRn, are grown in tive growth medium on a membrane in a ell to form a monolayer. The integrity of monolayers can be evaluated by measuring the ical resistance or adding a probe that is not able to penetrate or being actively transported over the cell monolayer. A defined yer of cells is pulsed from the apical or basolateral side with ligand such as FcRn binding Z variants, HSA or lgG in a buffer such as HBSS (Hanks’ Balanced Salt Solution, SigmaAldrich, cat. no. H9269) or growth medium at a suitable pH and ature, and chased with buffers such as HBSS or growth medium at a suitable pH and ature on the opposite side.

In a variant of this assay, ligands can be chased with buffers such as HBSS or growth medium at suitable pH and temperature on the same side as administration to measure recycled ligand as well. This can be done in a transwell or in a cell culture dish. Cells are seeded into transwell or cell culture dishes and pulsed with ligands such as FcRn binding Z variants, HSA or lgG. Endocytosed ligands will bind to FcRn and return to the cell surface at the same or opposite side as they were loaded. After g, free s are removed by washing the cells with cold buffer. To chase s, warm buffer or medium is added to the cells and, after a period in the range from 10 minutes to several hours, the buffer or medium is removed and assayed for the presence of ligands. ln a variant of this assay, ligands such as FcRn binding Z variants, HSA or lgG can be used to block the binding to FcRn by s such as other FcRn binding Z variants, HSA or lgG by administering them at the same time or sequentially to the cells.

The amount of ligand can be quantified by methods such as ELISA, HPLC—MS, fluorescent dye or radio labeling.

The s of the experiment described above are expected to show that the FcRn—specific Z variants can be transcytosed and/or recycled in vitro.

ITEMlZED LISTING OF EMBODIMENTS 1. FcRn binding polypeptide, comprising an FcRn binding motif BM, which motif consists of the amino acid sequence EX2 X3 X4 AXs X7 EIR WLPNLX16X17 X18 QR X21 AFIX25 X26LX28 X29 wherein, independently from each other, X2 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y; X3 is selected from A, D, E, F, G, H, l, K, L, M, N, Q, R, S, T, V, Wand X4 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W and Y; X6 is selected from A, E, F, G, H, l, K, Q, R, S and V; X7 is selected from A, F, H, K, N, Q, R, S and V; X15 is selected from N and T; X17 is ed from F, W and Y; X18 is selected from A, D, E and N; X21 is selected from A, S, V and W; X25 is ed from D, E, G, H, l, K, L, N, Q, R, S, T, V, W and Y; X26 is selected from K and S; X28 is selected from A, D, E, F, H, i, K, L, N, Q, R, S, T, V, W and Y; X29 is selected from D and R. 2. FcRn binding polypeptide according to item 1, wherein, ndently from each other, X2 is selected from A, D, E, F, H, l, K, L, N, Q, R, S, T, V, W and Y; X3 is selected from A, D, E, F, H, l, K, L, M, N, Q, R, S, T, V, W and Y; X4 is selected from A, D, E, F, H, l, K, L, N, Q, R, S, T, V, W and Y; X8 is selected from A, E, F, G, H, l, K, Q, R and S; X7 is selected from A, F, H, K, N, Q, R, S and V; X16 is selected from N and T; X17 is selected from F and Y; X18 is D; X21 is V; X25 is selected from D, E, H, l, K, L, N, Q, R, S, T, V, W and Y; X26 is selected from K and S; X28 is selected from A, D, E, F, H, l, K, L, N, Q, R, S, T, V and W; and X29 is selected from D and R. 3. FcRn binding polypeptide according to item 1, wherein the BM ts of an amino acid sequence selected from i) EX2 X3 X4 AX5 HElR WLPNLTX17 X18 QR X21 AF|X25 KLX28 D wherein, independently from each other, X2 is selected from A, D, E, F, H, l, K, L, N, Q, R, S, T, V, W and Y; X3 is selected from A, D, E, G, H, K, L, M, N, Q, R, S, T, V and Y; X4 is selected from A, D, E, F, G, l, K, L, N, Q, R, S, T, V and Y; X6 is selected from A, G, K, R, S and V; X17 is selected from F, W and Y; X18 is selected from A, D, E and N; X21 is selected from A, S, V and W; X25 is selected from D, G, H, K, L, N, R, V and W; X28 is selected from A, D, E, H, K, L, N, Q, R, S, T, W and Y; ii) an amino acid ce which has at least 96 % identity to a sequence defined by i). 4. FcRn binding polypeptide according to any preceding item, wherein X2 is selected from A, D, E, F, l, L, N, Q, R, S, T, V, W and Y.

. FcRn binding ptide according to item 4, wherein X2 is selected from A, D, F, l, L, N, Q, R, S, T, V, W and Y. 6. FcRn binding ptide according to item 5, wherein X2 is ed from A, D, F, l, L, N, Q, R, S, V and W. 7. FcRn binding polypeptide according to item 5, wherein X2 is selected from A, l, L, N, Q, R, S, T, V, W and Y. 8. FcRn binding ptide according to item 7, wherein X2 is selected from A, l, L, N, Q, S, T, Vand W. 9. FcRn binding polypeptide according to item 6 or 8, wherein X2 is selected from A, l, L, N, Q, V and W.

. FcRn binding polypeptide according to item 9, wherein X2 is selected from A, l, L, Q, V and W. 11. FcRn binding polypeptide according to item 10, wherein X2 is selected from A, l, L and Q. 12. FcRn binding polypeptide according to item 11, wherein X2 is selected from i, L and Q. 13. FcRn binding polypeptide according to item 12, wherein X2 is selected from i and Q. 14. FcRn binding polypeptide according to item 13, wherein X2 is I.

. FcRn binding polypeptide according to item 13, wherein X2 is Q. 16. FcRn binding ptide according to any one of items 1 and 3—15, wherein X3 is selected from A, D, E, G, H, K, L, M, N, Q, R, S, T, V and Y. 17. FcRn binding polypeptide according to item 2 or 16, wherein X3 is 1O selected from A, D, E, H, K, L, M, N, Q, R, S, T, V and Y. 18. FcRn binding polypeptide according to item 16, n X3 is selected from A, D, E, G, H, K, L, M, N, Q, R, S and T. 19. FcRn binding polypeptide according to item 18, wherein X3 is selected from A, D, E, G, H, K, M, N, Q, S and T.

. FcRn binding polypeptide according to item 19, wherein X3 is selected from A, D, E, G, H, M, N, Q, S and T. 21. FcRn binding polypeptide according to item 19, wherein X3 is selected from A, D, E, K, N, Q, S and T. 22. FcRn binding ptide according to item 21, wherein X3 is selected from A, D, E, K, Q and T. 23. FcRn binding ptide according to item 22, n X3 is selected from A, D, E, Q and T. 24. FcRn binding polypeptide according to item 23, wherein X3 is selected from D, E and T.

. FcRn binding polypeptide according to item 24, wherein X3 is selected from D and E. 26. FcRn binding ptide according to item 25, wherein X3 is D. 27. FcRn binding polypeptide ing to item 25, wherein X3 is E. 28. FcRn binding polypeptide according to any one of items 1 and 3-27, wherein X4 is selected from A, D, E, F, G, l, K, L, N, Q, R, S, T, V and Y. 29. FcRn binding polypeptide according to item 28, wherein X4 is selected from A, D, E, G, N, Q, R, S, T and V.

. FcRn binding polypeptide according to item 2 or 28, wherein X4 is 1O selected from A, D, E, F, l, K, L, N, Q, R, S, T and V. 31. FcRn binding polypeptide according to item 30, wherein X4 is selected from A, D, E, l, K, N, Q, R, S and T. 32. FcRn binding polypeptide according to item 31, wherein X4 is selected from A, D, E, l, K, Q, S and T. 33. FcRn binding polypeptide according to item 32, wherein X4 is selected from A, D, l, K, Q and S. 34. FcRn binding polypeptide according to item 32, wherein X4 is selected from A, D, E, K and S.

. FcRn binding ptide according to item 33 or 34, wherein X4 is selected from A, D, K and S. 36. FcRn binding ptide according to item 34, wherein X4 is selected from A, D, E and K. 37. FcRn binding polypeptide ing to item 35 or 36, wherein X4 is selected from A, D and K. 38. FcRn binding polypeptide according to item 37, wherein X4 is selected from A and D. 39. FcRn g polypeptide according to item 36, n X4 is selected from A and E. 40. FcRn binding polypeptide according to item 38 or 39, wherein X4 is A. 41. FcRn binding polypeptide according to item 38, n X4 is D. 42. FcRn binding polypeptide according to item 39, wherein X4 is E. 43. FcRn binding polypeptide ing to any one of items 1 and 442, wherein X5 is selected from A, G, K, O, R, S and V. 44. FcRn binding polypeptide according to item 3 or 43, wherein X6 is selected from A, G, K, R, S and V. 45. FcRn binding polypeptide according to item 2 or 44, n X5 is selected from A, G, K, R and S. 46. FcRn binding polypeptide according to item 44, wherein X6 is ed from A, G, K, S and V. 47. FcRn binding polypeptide according to item 46, n X5 is selected from A, G, K and V. 48. FcRn binding polypeptide according to item 45 or 46, wherein X6 is selected from A, G, K and S. 49. FcRn binding polypeptide according to item 47 or 48, wherein X6 is selected from A, G and K. 50. FcRn binding polypeptide according to item 47, wherein X5 is selected 3O from A, G and V. 51. FcRn binding polypeptide according to item 49 or 50, n X6 is selected from A and G. 52. FcRn binding polypeptide according to item 51, wherein X6 is A. 53. FcRn binding polypeptide according to item 51, wherein X6 is G. 54. FcRn binding polypeptide according to any one of items 1, 2 and 4—53, wherein X7 is selected from A and H. 55. FcRn binding polypeptide according to item 54, wherein X7 is H. 56. FcRn binding polypeptide according to any one of items 1, 2 and 4—55, wherein X15 is T. 1O 57. FcRn binding polypeptide according to any one of items 1, 2 and 4—55, wherein X16 is N. 58. FcRn binding polypeptide according to any one of items 1 and 3-57, wherein X17 is selected from F and Y. 59. FcRn binding polypeptide according to any preceding item, wherein X17 is 60. FcRn g polypeptide ing to any one of items 1 and 3—59, wherein X18 is selected from A, D and E. 61. FcRn binding polypeptide according to item 60, wherein X18 is selected from A and D. 62. FcRn binding polypeptide according to item 61, n X18 is D. 63. FcRn binding polypeptide ing to any one of items 1 and 3—62, wherein X21 is ed from V and W. 64. FcRn binding ptide according to item 63, wherein X21 is V. 65. FcRn binding polypeptide according to any one of items 1 and 4—64, wherein X25 is selected from D, E, G, H, K, L, N, Q, R, V and W. 66. FcRn binding polypeptide according to item 65, wherein X25 is selected from D, G, H, K, L, N, R, V and W. 67. FcRn binding polypeptide according to any one of items 2, 3 and 66, n X25 is selected from H, L, R, V and W. 68. FcRn binding ptide according to item 67, wherein X25 is selected from H, R, V and W. 69. FcRn binding polypeptide according to item 68, wherein X25 is selected from H, R and V. 70. FcRn binding polypeptide ing to item 67, wherein X25 is selected from H, L and R. 71. FcRn binding ptide according to item 69 or 70, wherein X25 is selected from H and R. 72. FcRn binding polypeptide according to item 69, wherein X25 is selected from H and V. 73. FcRn binding polypeptide according to item 71 or 72, wherein X25 is H. 74. FcRn g polypeptide according to any one of items 1, 2 and 4—73, wherein X25 is K. 75. FcRn binding polypeptide according to any one of items 1, 2 and 4—73, wherein X25 is S. 76. FcRn binding polypeptide according to any one of items 1 and 3-75, wherein X28 is selected from A, D, E, H, K, L, N, Q, R, S, T, W and Y. 77. FcRn binding polypeptide according to item 76, wherein X23 is selected from A, D, E, K, L, N, Q, R, S, T, W and Y. 78. FcRn binding polypeptide according to item 77, wherein X28 is selected from A, D, E, L, R, S, T, W and Y. 79. FcRn g polypeptide according to item 2 or 77, wherein X28 is selected from A, D, K, L, N, Q, R, S, T and W. 80. FcRn binding polypeptide according to item 78 or 79, wherein X28 is selected from A, D and R. 81. FcRn binding polypeptide according to item 80, wherein X28 is selected from A and R. 82. FcRn binding polypeptide according to item 80, wherein X28 is selected from D and R. 83. FcRn binding polypeptide ing to item 81, wherein X28 is A. 84. FcRn binding polypeptide according to item 81 or 82, wherein X28 is R. 85. FcRn binding polypeptide ing to item 82, wherein X28 is D. 86. FcRn binding polypeptide according to any one of items 1, 2 and 4-85, wherein X29 is D. 87. FcRn binding polypeptide according to any one of items 1, 2 and 4-85, wherein X29 is R. 88. FcRn binding polypeptide according to any one of items 1, 2 and 4-87, wherein X6X7 is selected from AH and GH. 89. FcRn binding polypeptide according to item 88, wherein X6X7 is AH. 90. FcRn binding polypeptide according to item 88, wherein X6X7 is GH. 3O 91. FcRn binding polypeptide according to any ing item, n X17X18 is selected from FD and YD. 92. FcRn binding ptide according to item 91, wherein X17X18 is FD. 93. FcRn binding ptide according to any preceding item, wherein the sequence fulfills at least three of the six conditions l—Vl: I. X6 is selected from A, G, K and S, such as in particular A; II. X7 is H; III. X17 is selected from F and Y, such as in particular F; IV. X18 is D; V. X21 is selected from V and W, such as in particular V; VI. X25 is selected from H and R, such as in particular H. 94. FcRn binding ptide according to item 93, wherein the sequence fulfills at least four of the six conditions l-VI. 95. FcRn binding polypeptide according to item 94, wherein the sequence fulfills at least five of the six ions I—Vl. 96. FcRn binding polypeptide according to item 95, wherein the sequence fulfills all of the six conditions |~Vl. 97. FcRn binding polypeptide according to any preceding item, wherein the sequence is selected from the group consisting of SEQ ID NO:1—353. 98. FcRn binding ptide according to item 97, wherein the sequence is selected from the group consisting of SEQ ID NO:1—15, SEQ ID NO:17-14O and SEQ ID N02353. 99. FcRn binding polypeptide according to item 98, wherein the sequence is selected from the group consisting of SEQ ID Nng1—2 and SEQ ID NO:17— 140. 100. FcRn binding polypeptide ing to item 99, wherein the sequence is selected from the group consisting of SEQ ID NO:1—2, SEQ ID NO:17—92, SEQ ID NO:94—103, SEQ ID NO:105-125 and SEQ ID NO:127-140. 101. FcRn binding polypeptide ing to item 98, wherein the sequence is ed from the group consisting of SEQ ID NO:1—8, SEQ ID NO:13, SEQ ID NO:19-20, SEQ ID N023, SEQ ID N028, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:75-77 and SEQ ID NO:353. 102. FcRn binding ptide according to item 100 or 101, n the sequence is selected from the group consisting of SEQ ID N021, SEQ ID NO:23, SEQ ID N0228, SEQ ID NO:41, SEQ ID N0:44, SEQ ID N0:65, SEQ ID N0273 and SEQ ID NO:75-77. 103. FcRn binding polypeptide according to item 102, wherein the ce is selected from the group consisting of SEQ ID N0:1, SEQ ID NO:23, SEQ ID N0z44, SEQ ID N0165, SEQ ID NO:75 and SEQ ID NO:77. 104. FcRn binding polypeptide according to item 103, wherein the sequence is selected from the group consisting of SEQ ID N021, SEQ ID N023 and SEQ ID NO:75. 105. FcRn binding polypeptide according to item 104, wherein the ce is SEQ ID N021. 106. FcRn binding polypeptide according to any preceding item, wherein said FcRn binding motif forms part of a three—helix bundle n domain. 107. FcRn binding ptide according to item 106, wherein said FcRn binding motif essentially forms part of two s with an interconnecting loop, within said three-helix bundle protein domain. 108. FcRn binding polypeptide according to item 107, wherein said three—helix bundle protein domain is selected from bacterial receptor domains. 109. FcRn binding polypeptide according to item 108, wherein said three—helix bundle protein domain is selected from domains of protein A from Staphylococcus aureus or derivatives thereof. 110. FcRn binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from: iii) K—[BM]—DPSQS xaxbLLxc EAKKL xdxexfo; wherein [BM] is an FcRn binding motif as defined herein, provided that X29 is D; Xa is selected from A and S; Xb is selected from N and E; Xc is selected from A, S and C; Xd is selected from E, N and S; Xe is selected from D, E and S; Xf is selected from A and S; iv) an amino acid ce which has at least 93 % identity to a sequence defined by iii). 111. FcRn binding polypeptide according to any one of items 1—109, which comprises an amino acid sequence selected from v) K—[BMJ—QPEQS XaXbLLXc EAKKL XdXeXfQ; [BM] is an FcRn binding motif as defined herein, provided that X29 is R; Xa is selected from A and S; Xb is selected from N and E; Xc is selected from A, S and C; Xd is selected from E, N and S; Xe is selected from D, E and S; Xr is selected from A and S; vi) an amino acid sequence which has at least 93 % identity to a sequence d by v). 112. FcRn binding ptide according to item 110 or 111, wherein Xa in sequence iii) or v) is A. 113. FcRn binding polypeptide according to item 110 or 111, wherein Xa in sequence iii) or v) is S. 114. FcRn binding polypeptide according to any one of items 110-113, wherein Xb in sequence iii) or v) is N. 115. FcRn binding polypeptide according to any one of items 110—113, wherein Xb in sequence iii) or v) is E. 116. FcRn binding polypeptide according to any one of items 110-115, wherein Xc in sequence iii) or v) is A. 1O 117. FcRn binding polypeptide according to any one of items 110—115, wherein X0 in sequence iii) or v) is S. 118. FcRn binding polypeptide according to any one of items 110-115, wherein Xc in ce iii) or v) is C. 119. FcRn binding ptide according to any one of items 8, wherein Xd in ce iii) or v) is E. 120. FcRn g polypeptide according to any one of items 110-118, wherein Xd in sequence iii) or v) is N. 121. FcRn binding polypeptide according to any one of items 110-118, wherein Xd in sequence iii) or v) is S. 122. FcRn binding ptide according to any one of items 110—121, wherein Xe in sequence ill) or v) is D. 123. FcRn binding polypeptide according to any one of items 110-121, wherein Xe in sequence iii) or v) is E. 124. FcRn binding polypeptide according to any one of items 110-121, wherein Xe in sequence iii) or v) is S. 125. FcRn binding polypeptide according to any one of items 110-119, 121, 123 and 124, wherein XdXe in sequence iii) or v) is selected from EE, ES, SE and SS. 126. FcRn binding polypeptide according to item 125, wherein XdXe in sequence ill) or v) is ES. 127. FcRn binding polypeptide according to item 125, wherein XdXe in 1O sequence iii) or v) is SE. 128. FcRn binding ptide according to any one of items 110—127, wherein Xr in sequence iii) or v) is A. 129. FcRn binding polypeptide according to any one of items 110—127, wherein Xf in sequence iii) or v) is S. 130. FcRn binding polypeptide according to item 110 or 111, wherein in sequence iii) or v), Xa is A; Xb is N; X0 is A and Xf is A. 131. FcRn binding polypeptide according to item 110 or 111, wherein in sequence iii) or v), Xa is A; Xb is N; Xc is C and Xr is A. 132. FcRn g polypeptide according to item 110 or 111, wherein in sequence iii) or v), X3 is S; Xb is E; Xc is S and Xf is S. 133. FcRn binding polypeptide according to item 110 or 111, wherein in sequence iii) or v), Xa is S; Xb is E; Xc is C and Xf is S. 134. FcRn g polypeptide according to item 110 or 111, wherein in sequence iii) or v), Xa is A; Xb is N; Xc is A; XdXe is ND and Xr is A. 135. FcRn binding polypeptide according to item 110 or 111, wherein in sequence ill) or v), Xa is A; Xb is N; Xc is C; XdXe is ND and Xf is A. 136. FcRn binding polypeptide according to item 110 or 111, wherein in sequence iii) or v), Xa is S; Xb is E; Xc is S; XdXe is ND and Xf is S. 137. FcRn binding polypeptide according to item 110 or 111, wherein in sequence iii) or v), Xa is S; Xb is E; Xc is C; XdXe is ND and Xf is S. 138. FcRn binding polypeptide according to item 110 or 111, wherein in sequence iii) or v), Xa is A; Xb is N; Xc is A; XdXe is SE and Xr is A. 139. FcRn g polypeptide according to item 110 or 111, wherein in sequence iii) or v), Xa is A; Xb is N; Xc is C; XdXe is SE and Xf is A. 140. FcRn g polypeptide according to item 110 or 111, wherein in sequence iii) or v), Xa is S; Xb is E; X0 is S; XdXe is SE and Xf is S. 141. FcRn binding polypeptide according to item 110 or 111, wherein in sequence iii) or v), X3 is S; Xb is E; Xc is C; XdXe is SE and Xf is S. 142. FcRn binding polypeptide ing to item 110 or 111, wherein in ce iii) or v), Xa is A; Xb is N; Xc is A; XdXe is ES and Xr is A. 143. FcRn binding polypeptide according to item 110 or 111, n in sequence iii) or v), Xa is A; Xb is N; X0 is C; XdXe is ES and Xf is A. 144. FcRn binding polypeptide according to item 110 or 111, wherein in sequence iii) or v), Xa is S; Xb is E; Xc is S; XdXe is ES and Xf is S. 145. FcRn binding polypeptide according to item 110 or 111, wherein in sequence iii) or v), Xa is S; Xb is E; Xc is C; XdXe is ES and Xf is S 146. FcRn binding polypeptide according to any one of items 110 and 112- 145, wherein sequence iii) is selected from the group consisting of SEQ ID NO:354-706. 147. FcRn binding polypeptide according to item 146, wherein sequence iii) is selected from the group consisting of SEQ ID NO:354~368, SEQ ID N02370- 493 and SEQ ID NO:706. 148. FcRn binding polypeptide according to item 147, wherein ce iii) is selected from the group ting of SEQ ID —355 and SEQ ID NO:370-493. 149. FcRn binding polypeptide according to item 148, wherein sequence iii) is 1O selected from the group consisting of SEQ ID NO:354—355, SEQ ID - 445, SEQ ID NO:447-456, SEQ ID NO:458—478 and SEQ ID NO:480-493. 150. FcRn binding ptide according to item 147, wherein sequence iii) is selected from the group consisting of SEQ ID NO:354-361, SEQ ID N01366, SEQ ID NO:372—373, SEQ ID NO:376, SEQ ID NO:381, SEQ ID NO:394, SEQ ID NO:397, SEQ ID NO:418, SEQ ID NO:423, SEQ ID NO:426, SEQ ID NO:428—430 and SEQ ID NO:706. 151. FcRn binding polypeptide according to item 149 or 150, wherein sequence iii) is selected from the group consisting of SEQ ID NO:354, SEQ ID NO:376, SEQ ID NO:381, SEQ ID N02394, SEQ ID NO:397, SEQ ID NO:418, SEQ ID NO:426 and SEQ ID NO:428—430. 152. FcRn binding polypeptide according to item 151, wherein sequence iii) is selected from the group consisting of SEQ ID NO:354, SEQ ID NO:376, SEQ ID N02397, SEQ ID NO:418, SEQ ID NO:428 and SEQ ID NO:430. 153. FcRn binding polypeptide according to item 152, wherein ce iii) is selected from the group consisting of SEQ ID , SEQ ID N02376 and SEQ ID NO:428. 154. FcRn binding polypeptide according to item 153, wherein sequence iii) is SEQ ID NO:354. 155. FcRn binding polypeptide according to any one of items 1—109, which ses an amino acid sequence selected from: vii) YAK—[BAfl-DPSQS SELLXc EAKKL NDSQA P; wherein [BM] is an FcRn g motif as d in any one of items 1-105 and Xc is selected from A, S and C; and viii) an amino acid sequence which has at least 94 % identity to a sequence d by vii). 156. FcRn binding polypeptide according to any one of items 1—109, which comprises an amino acid sequence selected from: ix) FNK-[BW—DPSQS ANLLXc EAKKL NDAQA P; wherein [BM] is an FcRn binding motif as defined in any one of items 1—105 and X0 is selected from A and C; and x) an amino acid sequence which has at least 94 % identity to a sequence defined by ix). 157. FcRn g polypeptide according to item 109, which comprises an amino acid sequence selected from: ADNNFNK—[BMl—DPSQSANLLSEAKKLNESQAPK; ADNKFNK-[BM]~DPSQSANLLAEAKKLNDAQAPK; ADNKFNK—[BM]~DPSVSKElLAEAKKLNDAQAPK; ADAQQNNFNK—[BAfl—DPSQSTNVLGEAKKLNESQAPK; AQHDE-[BW—DPSQSANVLGEAQKLNDSQAPK; VDNKFNK—[BW—DPSQSANLLAEAKKLNDAQAPK; AEAKYAK—[BAfl—DPSESSELLSEAKKLNKSQAPK; VDAKYAK—[BM-DPSQSSELLAEAKKLNDAQAPK; VDAKYAK-[BM-DPSQSSELLAEAKKLNDSQAPK; AEAKYAK—[BM-DPSQSSELLSEAKKLNDSQAPK; AEAKYAK-[BM-DPSQSSELLSEAKKLSESQAPK AEAKYAK—[BM]-DPSQSSELLSEAKKLESSQAPK VDAKYAK-[BM-DPSQSSELLSEAKKLNDSQAPK; VDAKYAK-[BM]—DPSQSSELLSEAKKLSESQAPK; VDAKYAK—[BW—DPSQSSELLSEAKKLESSQAPK; VDAKYAK—[BW—DPSQSSELLAEAKKLNKAQAPK; and AEAKYAK-[BM1—DPSQSSELLAEAKKLNKAQAPK; wherein [BM] is an FcRn binding motif as d in any one of items 1—105. 158. FcRn binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from: Xi) AEAKYAK—[BM]—DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is an FcRn g motif as defined in any one of items 1—105; 1O and xii) an amino acid sequence which has at least 94 % identity to the sequence defined in xi). 159. FcRn binding polypeptide according to item 158, in which sequence xi) is selected from the group consisting of SEQ ID NO:1060-1062. 160. FcRn binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from: xiii) VDAKYAK—[BAfl—DPSQSSELLSEAKKLNDSQAPK; n [BM] is an FcRn binding motif as d in any one of items 1-105; xiv) an amino acid sequence which has at least 94 % identity to the sequence defined in xiii). 161. FcRn binding polypeptide according to item 160, in which sequence xiii) is selected from the group consisting of SEQ lD NO: 707—1059. 162. FcRn binding ptide according to item 161, in which sequence xiii) is selected from the group consisting of SEQ lD NO:707—721, SEQ ID NO:723—846 and SEQ lD NO:1059. 163. FcRn binding polypeptide according to item 162, in which sequence xiii) is selected from the group consisting of SEQ ID NO:707-708 and SEQ ID NO:723—846. 164. FcRn g polypeptide according to item 163, in which sequence xiii) is ed from the group consisting of SEQ ID NO:707-708, SEQ ID NO:723-798, SEQ ID NO:800—809, SEQ ID NO:811—831 and SEQ ID N01833— 846. 1O 165. FcRn binding polypeptide according to item 162, in which sequence xiii) is selected from the group consisting of SEQ ID NO:707—714, SEQ ID NO:719, SEQ ID NO:725—726, SEQ ID NO:729, SEQ ID NO:734, SEQ ID NO:747, SEQ ID NO:750, SEQ ID NO:771, SEQ ID NO:776, SEQ ID NO:779, SEQ ID NO:781—783 and SEQ ID 9. 166. FcRn binding polypeptide ing to item 163 or 165, in which sequence xiii) is selected from the group consisting of SEQ ID NO:707, SEQ ID NO:729, SEQ ID NO:734, SEQ ID NO:747, SEQ ID NO:750, SEQ ID NO:771, SEQ ID NO:779 and SEQ ID -783. 167. FcRn g polypeptide according to item 166, in which ce xiii) is selected from the group consisting of SEQ ID NO:707, SEQ ID NO:729, SEQ ID NO:750, SEQ ID NO:771, SEQ ID NO:781 and SEQ ID NO:783. 168. FcRn binding polypeptide according to item 167, in which sequence xiii) is selected from the group consisting of SEQ ID NO:707, SEQ ID NO:729 and SEQ ID NO:781. 169. FcRn binding polypeptide according to item 168, in which sequence xiii) is SEQ ID NO:707. 170. FcRn binding polypeptide ing to any preceding item, which is capable of binding to FcRn at pH 6.0 such that the KB value of the interaction is at most 1 x 10'6 M, such as at most 1 x10‘7 M, such as at most 1 x10“8 M, such as at most 1 x10“9 M, such as at most 1 x10'10 M. 171. FcRn binding polypeptide according to any ing item, wherein the K0 value of the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is higher than the KB value of said interaction at pH 6.0, such as at least 2 times higher, such as at least 5 times higher, such as at least 10 times , such as at least 50 times higher, such as at least 100 times higher than the KD value of said interaction at pH 6.0. 172. FcRn binding polypeptide according to any preceding item, wherein the K0 value of said interaction at pH 7.4 is at least 1 x 10'8 M, such as at least 1O 1 x107 M, such as at least 1 x 10'6 M, such as at least 1 x 10'5 M. 173. FcRn binding polypeptide according to any one of items 1-170, wherein the KB value of said interaction at pH 7.4 is the same as or lower than the K13 value of said interaction at pH 6.0. 174. FcRn binding polypeptide ing to any one of items 1-170, wherein the K0 value of said interaction at pH 7.4 is at most 1 x 10'6 M, such as at most 1 x10‘7 M, such as at most 1 x10"8 M, such as at most 1 x10'9 M, such as at most 1 x10'10 M. 175. FcRn binding polypeptide according to any preceding item, which comprises at least one additional amino acid at the C—terminal and/or N— terminal end. 176. FcRn binding polypeptide ing to item 175, wherein said at least one additional amino acid extension improves production, purification, ization in vivo or in vitro, coupling or detection of the polypeptide. 177. FcRn binding polypeptide according to any preceding item in multimeric form, comprising at least two FcRn binding polypeptide r units, whose amino acid sequences may be the same or different. 178. FcRn binding polypeptide according to item 177, wherein said FcRn binding ptide monomer units are covalently coupled together. 179. FcRn binding polypeptide according to item 177, wherein the FcRn binding polypeptide monomer units are sed as a fusion protein. 180. FcRn binding polypeptide according to any one of items 9, in dimeric form. 181. Fusion protein or conjugate comprising - a first moiety consisting of an FcRn binding polypeptide according to any preceding item; and - a second moiety consisting of a polypeptide having a desired biological activity. 182. Fusion protein or ate according to item 181, wherein the in vivo half—life of said fusion protein or conjugate is longer than the in vivo half-life of the polypeptide having a desired biological activity per 39. 183. Fusion protein or conjugate according to any one of items 2, wherein said desired biological activity is a therapeutic ty. 184. Fusion protein or ate according to any one of items 181—182, wherein said desired biological activity is a binding activity to a selected target. 185. Fusion protein or conjugate according to item 184, wherein said ed target is albumin. 186. Fusion n or conjugate according to item 185, wherein said albumin binding activity is provided by the albumin binding domain of streptococcal protein G, or a derivative thereof. 187. Fusion protein or conjugate according to any one of items 185—189, wherein said albumin binding activity increases in vivo ife of the fusion protein or conjugate. 188. Fusion protein or conjugate according to any one of items 181—182, wherein said d biological activity is an enzymatic activity. 189. Fusion protein or conjugate according to any one of items 181-183, n the second moiety having a desired biological activity is a eutically active polypeptide. 190. Fusion protein or conjugate according to any one of items 181—183 and 188—189, wherein the second moiety having a desired biological activity is selected from the group consisting of enzymes, hormones, growth factors, ines and cytokines. 1O 191. FcRn binding polypeptide, fusion protein or conjugate according to any preceding item, which inhibits binding of lgG to FcRn. 192. FcRn binding polypeptide, fusion protein or ate according to item 191, wherein the KB value of the interaction between said FcRn binding polypeptide, fusion protein or conjugate and FcRn is lower than the KB value of the interaction between lgG and FcRn. 193. FcRn binding polypeptide, fusion protein or conjugate according to any preceding item, further comprising a label. 194. FcRn binding polypeptide, fusion n or ate according to item 193, wherein said label is selected from the group consisting of fluorescent dyes and , chromophoric dyes, chemiluminescent compounds and inescent proteins, enzymes, radionuclides and particles. 195. FcRn binding polypeptide, fusion protein or conjugate according to any preceding item, comprising a chelating environment provided by a polyaminopolycarboxylate chelator conjugated to the FcRn g polypeptide via a thiol group of a cysteine residue or an amine group of a 3O lysine e. 196. FcRn binding polypeptide, fusion n or conjugate according to item 195, wherein the polyaminopolycarboxylate chelator is 1,4,7,10— tetraazacyclododecane—1,4,7,10-tetraacetic acid or a derivative thereof. 197. FcRn binding polypeptide, fusion protein or conjugate according to item 196, wherein the 1 ,4,7,10-tetraazacyclododecane—1,4,7,10—tetraacetic acid derivative is 10—tetraazacyclododecane—1,4,7-tris—acetic acid—10— idoethylacetamide. 198. FcRn binding polypeptide, fusion protein or conjugate according to item 195, wherein the polyaminopolycarboxylate or is 1,4,7— triazacyclononane-1,4,7-triacetic acid or a derivative thereof. 199. FcRn binding polypeptide, fusion protein or conjugate according to item 195, n the polyaminopolycarboxylate or is 1O diethylenetriaminepentaacetic acid or derivatives thereof. 200. A poiynucleotide encoding a polypeptide according to any one of items 1-192. 201. Expression vector comprising a polynucleotide according to item 200. 202. Host cell sing an expression vector ing to item 201. 203. Method of producing a polypeptide according to any one of items 1—192, comprising — culturing a host cell according to item 202 under ions permissive of expression of said polypeptide from said expression vector, and — isolating said polypeptide. 204. Composition comprising an FcRn binding polypeptide, fusion protein or conjugate according to any one of items 1—199 and at least one pharmaceutically acceptable excipient or carrier. 205. Composition according to item 204, further comprising at least one additional active agent. 206. Composition according to any one of items 204—205, which is adapted for administration by a route selected from the group consisting of oral stration, intranasal administration, pulmonar administration, vaginal administration, rectal administration, intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection and intradermal injection. 207. FcRn binding polypeptide, fusion protein or conjugate according to any one of items 1—199 or composition according to any one of items 204—206 for use as a medicament. 208. FcRn binding polypeptide, fusion protein, conjugate or composition for use according to item 207, wherein said medicament is intended for treatment of an mmune condition. 1O 209. FcRn binding polypeptide, fusion protein, conjugate or composition for use according to item 207 or 208, wherein said ment is intended for treatment of a condition selected from the group consisting of myasthenia gravis, Guillain—Barré syndrome, autoimmune limbic encephalitis, pediatric mune neuropsychiatric disorders associated with streptococcal infection (PANDAS), neuromyotonia (lsaac’s syndrome), morvan syndrome, multiple sis, pemphigus vulgaris, foliaceus, bullous pemphigoid, epidermolysis bullosa acquisita, goid gestationis, mucous ne pemphigoid, lichen sclerosus, antiphospholipid syndrome, erlapsing polychondritis, mune anemia, idiopathic trombocytic purpura, autoimmune Grave’s disease, dilated cardiomyopathy, vasculitis, Goodpasture’s syndrome, idiopathic membranous pathy, rheumatoid arthritis and systemic lupus erythematosus. 210. Method of treatment of a subject in need f, comprising administering to the subject a therapeutically active amount of an FcRn binding polypeptide, fusion protein or conjugate according to any one of items 1—199 or composition according to any one of items 6. 211. Method according to item 210, for treatment of an auto—immune condition. 212. Method according to item 210 or 211, wherein said subject is suffering from a condition selected from the group consisting of myasthenia gravis, Guillain—Barré syndrome, autoimmune limbic encephalitis, pediatric autoimmune neuropsychiatric ers associated with ococcal infection (PANDAS), neuromyotonia (Isaac’s syndrome), morvan syndrome, multiple sclerosis, gus vulgaris, foliaceus, bullous pemphigoid, molysis bullosa acquisita, pemphigoid gestationis, mucous membrane pemphigoid, lichen sclerosus, ospholipid syndrome, erlapsing polychondritis, autoimmune anemia, idiopathic trombocytic purpura, autoimmune Grave’s disease, dilated cardiomyopathy, vasculitis, sture’s syndrome, idiopathic membranous nephropathy, rheumatoid arthritis and systemic lupus erythematosus.

Claims (62)

1. FcRn binding polypeptide, comprising an FcRn binding motif BM, which motif ts of the amino acid sequence EX2 X3 X4 AX6 X7 EIR WLPNLX16X17 X18 QR X21 AFIX25 X26LX28 X29 wherein, independently from each other, 10 X2 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y; X3 is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and X4 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, W and Y; X6 is selected from A, E, F, G, H, I, K, Q, R, S and V; 15 X7 is ed from A, F, H, K, N, Q, R, S and V; X16 is selected from N and T; X17 is selected from F, W and Y; X18 is selected from A, D, E and N; X21 is selected from A, S, V and W; 20 X25 is selected from D, E, G, H, I, K, L, N, Q, R, S, T, V, W and Y; X26 is selected from K and S; X28 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y; X29 is selected from D and R.

2. FcRn binding polypeptide according to claim 1, wherein the BM consists of an amino acid sequence selected from i) EX2 X3 X4 AX6 HEIR WLPNLTX17 X18 QR X21 AFIX25 KLX28 D wherein, independently from each other, X2 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y; X3 is selected from A, D, E, G, H, K, L, M, N, Q, R, S, T, V and Y; 35 X4 is selected from A, D, E, F, G, I, K, L, N, Q, R, S, T, V and Y; X6 is ed from A, G, K, R, S and V; X17 is selected from F, W and Y; X18 is selected from A, D, E and N; X21 is selected from A, S, V and W; X25 is selected from D, G, H, K, L, N, R, V and W; X28 is selected from A, D, E, H, K, L, N, Q, R, S, T, W and Y.

3. FcRn binding polypeptide according to claim 1, wherein the BM ts of the amino acid sequence EX2 X3 X4 AX6 HEIR WLPNLTX17 X18 QR X21 AFIX25 KLX28 D wherein, ndently from each other, X2 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W and Y; X3 is selected from A, D, E, G, H, K, L, M, N, Q, R, S, T, V and Y; 15 X4 is selected from A, D, E, F, G, I, K, L, N, Q, R, S, T, V and Y; X6 is selected from A, G, K, R, S and V; X17 is selected from F, W and Y; X18 is selected from A, D, E and N; X21 is selected from A, S, V and W; 20 X25 is selected from D, G, H, K, L, N, R, V and W; and X28 is ed from A, D, E, H, K, L, N, Q, R, S, T, W and Y.

4. FcRn binding polypeptide according to any one of claims 1-3, wherein sequence i) fulfills at least three of the six conditions I-VI: I. X6 is selected from A, G, K and S, such as in particular A; II. X7 is H; III. X17 is selected from F and Y, such as in particular F; IV. X18 is D; 30 V. X21 is selected from V and W, such as in ular V; VI. X25 is selected from H and R, such as in particular H.

5. FcRn binding polypeptide according to any one of claims 1-4, wherein the sequence is selected from the group consisting of SEQ ID NO:1-353.

6. FcRn binding polypeptide according to any one of claims 1-5, wherein the sequence is selected from the group consisting of SEQ ID NO: 1-15, SEQ ID NO: 17-140 and SEQ ID NO:353. 5

7. FcRn binding polypeptide according to any one of claims 1-6, wherein the sequence is ed from SEQ ID NO:1-2 and SEQ ID NO:17-140.

8. FcRn binding polypeptide according to any one of claims 1-6, wherein the sequence is selected from SEQ ID NO:1-2, SEQ ID NO:17-92, SEQ ID 10 NO:94-103, SEQ ID NO:105-125 and SEQ ID NO:127-140 or selected from the group consisting of SEQ ID NO:1-8, SEQ ID NO:13, SEQ ID NO:19-20, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:75-77 and SEQ ID

9. FcRn g polypeptide according to any one of claims 1-8, wherein the sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:73 and SEQ ID 77.

10. FcRn binding polypeptide according to any one of claims 1-9, n the sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:23, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:75 and SEQ ID NO:77. 25

11. FcRn binding polypeptide ing to any one of claims 1-10, wherein the sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:23 and SEQ ID NO:75.

12. FcRn binding polypeptide according to any one of claims 1-11, wherein 30 the sequence is SEQ ID NO:1.

13. FcRn binding polypeptide according to any one of claims 1-12, wherein said FcRn binding motif forms part of a three-helix bundle protein domain. 35

14. FcRn binding polypeptide ing to any one of claims 1-13, which comprises an amino acid sequence selected from: iii) K-[BM]-DPSQS XaXbLLXc EAKKL XdXeXfQ; wherein [BM] is an FcRn binding motif as defined in any one of claims 1-12, 5 provided that X29 is D; Xa is selected from A and S; Xb is selected from N and E; Xc is selected from A, S and C; Xd is selected from E, N and S; 10 Xe is selected from D, E and S; Xf is selected from A and S.

15. FcRn binding polypeptide according to claim 14, wherein sequence iii) is selected from the group consisting of SEQ ID NO:354-706.

16. FcRn binding ptide according to claim 15, n the sequence is selected from the group consisting of SEQ ID NO:354-368, SEQ ID NO:370- 493 and SEQ ID NO:706. 20

17. FcRn binding polypeptide according to claim 16, wherein the ce is selected from the group consisting of SEQ ID NO:354-355 and SEQ ID -493.

18. FcRn binding ptide according to claim 16, wherein the sequence is 25 selected from the group consisting of SEQ ID NO:354-355, SEQ ID NO:370- 445, SEQ ID NO:447-456, SEQ ID -478 and SEQ ID NO:480-493 or selected from the group consisting of SEQ ID NO:354-361, SEQ ID NO:366, SEQ ID NO:372-373, SEQ ID NO:376, SEQ ID NO:381, SEQ ID , SEQ ID NO:397, SEQ ID NO:418, SEQ ID , SEQ ID NO:426, SEQ ID 30 NO:428-430 and SEQ ID NO:706.

19. FcRn binding polypeptide according to claim 18, wherein the sequence is selected from the group consisting of SEQ ID NO:354, SEQ ID NO:376, SEQ ID NO:381, SEQ ID NO:394, SEQ ID NO:397, SEQ ID NO:418, SEQ ID 35 NO:426 and SEQ ID NO:428-430.

20. FcRn binding polypeptide according to claim 19, wherein the sequence is selected from the group consisting of SEQ ID NO:354, SEQ ID , SEQ ID NO:397, SEQ ID NO:418, SEQ ID NO:428 and SEQ ID NO:430. 5

21. FcRn binding polypeptide according to claim 20, n the sequence is selected from the group consisting of SEQ ID NO:354, SEQ ID NO:376 and SEQ ID NO:428.

22. FcRn binding polypeptide according to claim 21, wherein the sequence is 10 SEQ ID NO:354.

23. FcRn binding polypeptide according to any one of claims 1-22, which comprises an amino acid sequence selected from: 15 xi) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is an FcRn g motif as defined in any one of claims 1-12.

24. FcRn binding polypeptide according to claim 23, in which ce xi) is 20 selected from the group consisting of SEQ ID NO:1060-1062.

25. FcRn g polypeptide according to any one of claims 1-22, which ses an amino acid sequence selected from: 25 xiii) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is an FcRn binding motif as defined in any one of claims 1-12.

26. FcRn binding polypeptide according to claim 25, in which sequence xiii) is 30 selected from the group consisting of SEQ ID NO:707-1059.

27. FcRn binding polypeptide according to claim 26, wherein the sequence is selected from the group consisting of SEQ ID NO:707-721, SEQ ID NO:723- 846 and SEQ ID NO:1059.

28. FcRn binding polypeptide according to claim 27, wherein the sequence is selected from the group ting of SEQ ID NO:707-708 and SEQ ID NO:723-846. 5

29. FcRn binding polypeptide according to claim 27, wherein the sequence is selected from the group consisting of SEQ ID NO:707-708, SEQ ID NO:723- 798, SEQ ID NO:800-809, SEQ ID NO:811-831 and SEQ ID NO:833-846 or selected from the group consisting of SEQ ID NO:707-714, SEQ ID NO:719, SEQ ID NO:725-726, SEQ ID NO:729, SEQ ID , SEQ ID NO:747, 10 SEQ ID NO:750, SEQ ID , SEQ ID NO:776, SEQ ID NO:779, SEQ ID -783 and SEQ ID NO:1059.

30. FcRn binding polypeptide according to claim 29, wherein the sequence is selected from the group consisting of SEQ ID NO:707, SEQ ID NO:729, SEQ 15 ID , SEQ ID NO:747, SEQ ID NO:750, SEQ ID NO:771, SEQ ID NO:779 and SEQ ID NO:781-783.

31. FcRn g polypeptide according to claim 30, wherein the sequence is ed from the group consisting of SEQ ID NO:707, SEQ ID NO:729, SEQ 20 ID NO:750, SEQ ID , SEQ ID NO:781 and SEQ ID NO:783.

32. FcRn binding polypeptide according to claim 31, wherein the sequence is selected from the group consisting of SEQ ID NO:707, SEQ ID NO:729 and SEQ ID NO:781.

33. FcRn g polypeptide according to claim 32, wherein the sequence is SEQ ID NO:707.

34. FcRn binding polypeptide according to any one of claims 1-33, which has 30 the ability to bind to FcRn at pH 6.0 such that the KD value of the interaction is at most 1 x 10-6 M.

35. FcRn binding polypeptide according to claim 34, wherein the KD value of the interaction is at most 1 x 10-7 M.

36. FcRn binding polypeptide according to claim 35, wherein the KD value of the interaction is at most 1 x 10-8 M.

37. FcRn binding polypeptide according to claim 36, wherein the KD value of the interaction is at most 1 x 10-9 M. 5

38. FcRn binding polypeptide according to claim 37, wherein the KD value of the interaction is at most 1 x 10-10 M.

39. FcRn binding ptide according to any one of claims 1-38, wherein the KD value of the interaction between FcRn binding polypeptide and FcRn 10 at pH 7.4 is higher than the KD value of said interaction at pH 6.0.

40. FcRn binding polypeptide according to claim 39, wherein the KD value of the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is at least 2 times higher than the KD value of said interaction at pH 6.0.

41. FcRn binding polypeptide according to claim 40, n the KD value of the interaction between FcRn binding ptide and FcRn at pH 7.4 is at least 5 times higher than the KD value of said interaction at pH 6.0. 20

42. FcRn binding polypeptide according to claim 41, wherein the KD value of the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is at least 10 times higher than the KD value of said interaction at pH 6.0.

43. FcRn binding ptide according to claim 42, n the KD value of 25 the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is at least 50 times higher than the KD value of said interaction at pH 6.0.

44. FcRn binding polypeptide according to claim 43, wherein the KD value of the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is at 30 least 100 times higher than the KD value of said interaction at pH 6.0.

45. FcRn binding polypeptide according to any one of claims 1-44, n the KD value of the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is at least 1 x 10-8 M.

46. FcRn binding polypeptide according to claim 45, wherein the KD value of the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is at least 1 x 10-7 M. 5

47. FcRn binding polypeptide according to claim 46, wherein the KD value of the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is at least 1 x 10-6 M.

48. FcRn binding polypeptide according to claim 47, wherein the KD value of 10 the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is at least 1 x 10-5 M.

49. FcRn binding ptide according to any one of claims 1-33, n the KD value of the interaction between FcRn binding polypeptide and FcRn 15 at pH 7.4 is at most 1 x 10-6 M.

50. FcRn binding polypeptide according to claim 49, wherein the KD value of the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is at most 1 x 10-7 M.

51. FcRn binding polypeptide according to claim 50, wherein the KD value of the interaction n FcRn binding polypeptide and FcRn at pH 7.4 is at most 1 x 10-8 M. 25

52. FcRn binding polypeptide ing to claim 51, wherein the KD value of the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is at most 1 x 10-9 M.

53. FcRn binding polypeptide according to claim 52, wherein the KD value of 30 the interaction between FcRn binding polypeptide and FcRn at pH 7.4 is at most 1 x 10-10 M.

54. Fusion n or conjugate sing: - a first moiety consisting of an FcRn binding polypeptide according to 35 any one of claims 1-53; and - a second moiety consisting of a polypeptide having a desired biological activity.

55. FcRn binding ptide, fusion protein or conjugate according to any one of claims 1-54, which ts binding of IgG to FcRn. 5

56. A polynucleotide encoding a polypeptide according to any one of claims 1-53.

57. A composition comprising an FcRn binding polypeptide, fusion protein or conjugate according to any one of claims 1-55 and at least one 10 pharmaceutically acceptable excipient or carrier.

58. FcRn binding polypeptide, fusion protein or conjugate according to any one of claims 1-55 or a composition according to claim 57 formulated for use as a medicament.

59. FcRn binding polypeptide, fusion protein, conjugate or composition according to claim 58, wherein said medicament is formulated for the treatment of an auto-immune condition. 20

60. The use of an FcRn binding polypeptide, fusion protein or conjugate according to any one of claims 1-55 or a composition ing to claim 57 for the preparation of a medicament, wherein said medicament is formulated for treatment of an auto-immune ion. 25

61. The use of an FcRn binding polypeptide, fusion protein or conjugate according to any one of claims 1-55 or a composition according to claim 57 for the preparation of a medicament, wherein said medicament is formulated for inhibiting binding of IgG to FcRn. 30

62. The use ing to claim 60 or 61, wherein said medicament is formulated for treatment of a condition selected from the group consisting of myasthenia gravis, Guillain–Barré syndrome, autoimmune limbic encephalitis, pediatric mune neuropsychiatric disorders associated with streptococcal infection (PANDAS), neuromyotonia (Isaac’s syndrome), 35 morvan syndrome, le sclerosis, gus vulgaris, foliaceus, bullous pemphigoid, epidermolysis bullosa acquisita, pemphigoid ionis, mucous membrane pemphigoid, lichen sus, antiphospholipid syndrome, erlapsing ondritis, autoimmune anemia, idiopathic trombocytic purpura, autoimmune Grave’s disease, dilated cardiomyopathy, vasculitis, Goodpasture’s syndrome, idiopathic membranous nephropathy, rheumatoid arthritis and ic lupus erythematosus.