Field of the invention

The present invention relates to the field of modulation of pharmacokinetic and pharmacodynamic properties of a target in vivo. It also relates to new methods, uses and compositions for pharmacokinetic and pharmacodynamic modulation in vivo.

Background

Serum albumin

Serum albumin is the most abundant protein in mammalian sera (40 g/l; approximately 0.7 mM in humans), and one of its functions is to bind molecules such as lipids and bilirubin (Peters T, Advances in Protein Chemistry 37:161 , 1985). Serum albumin is devoid of any enzymatic or immunological function. Furthermore, human serum albumin (HSA) is a natural carrier involved in the endogenous transport and delivery of numerous natural as well as therapeutic molecules (Sellers EM and Koch-Weser MD, Albumin Structure, Function and Uses, eds Rosenoer VM et al, Pergamon, Oxford, p 159, 1977). The half life of serum albumin is directly proportional to the size of the animal, where for example human serum albumin has a half life of 19 days and rabbit serum albumin has a half life of about 5 days (McCurdy TR et al, J Lab Clin Med 143:115, 2004). HSA is widely distributed throughout the body, in particular in the intestinal and blood compartments, where it is mainly involved in the maintenance of osmolarity. Structurally, albumins are single-chain proteins comprising three homologous domains and totaling 584 or 585 amino acids (Dugaiczyk L et al, Proc Natl Acad Sci USA 79:71 , 1982). Albumins contain 17 disulfide bridges and a single reactive thiol, C34, but lack N-linked and O-linked carbohydrate moieties (Peters, 1985, supra; Nicholson JP et al, Br J Anaesth 85:599, 2000).

Fusion or association with HSA results in increased in vivo half life of proteins Several strategies have been reported to either covalently couple proteins directly to serum albumins or to a peptide or protein that will allow in vivo association to serum albumins. Examples of the latter approach have been described e.g. in WO91/01743, in WO01/45746 and in Dennis et al, J Biol Chem 277:35035-43 (2002). The first document describes inter alia the use of albumin binding peptides or proteins derived from streptococcal protein G (SpG) for increasing the half life of other proteins. The idea is to fuse the bacterially derived, albumin binding peptide/protein to a therapeutically interesting peptide/protein, which has been shown to have a rapid elimination in blood. The thus generated fusion protein binds to serum albumin in vivo, and benefits from its longer half life, which increases the net half life of the fused therapeutically interesting peptide/protein. WO01 /45746 and Dennis et a/ relate to the same concept, but here, the authors utilize relatively short peptides to bind serum albumin. The peptides were selected from a phage displayed peptide library. In Dennis et al, earlier work is mentioned in which the enhancement of an immunological response to a recombinant fusion of the albumin binding domain of streptococcal protein G to human complement receptor Type 1 was found. US patent application published as

US2004/0001827 (Dennis) also discloses the use of constructs comprising peptide ligands, again identified by phage display technology, which bind to serum albumin and which are conjugated to bioactive compounds for tumor targeting.

Albumin binding domains of bacterial receptor proteins Streptococcal protein G (SpG) is a bi-functional receptor present on the surface of certain strains of streptococci and is capable of binding to both IgG and serum albumin (Bjόrck et al, MoI Immunol 24:1113, 1987). The structure is highly repetitive with several structurally and functionally different domains (Guss et al, EMBO J 5:1567, 1986), more precisely three Ig-binding motifs and three serum albumin binding domains (Olsson et al, Eur J Biochem 168:319, 1987). The structure of one of the three serum albumin binding domains has been determined, showing a three-helix bundle domain (Kraulis et al, FEBS Lett 378:190, 1996). This motif was named ABD (albumin binding domain) and is 46 amino acid residues in size. In the literature, it has subsequently also been designated G148-GA3.

Other bacterial albumin binding proteins than protein G from Streptococcus have also been identified, which contain domains similar to the albumin binding three-helix domains of protein G. Examples of such proteins are the PAB, PPL, MAG and ZAG proteins. Studies of structure and function of such albumin binding proteins have been carried out and reported e.g. by Johansson and co-workers (Johansson et al, J MoI Biol 266:859-865, 1997; Johansson et al, J Biol Chem 277:8114-8120, 2002), who introduced the designation "GA module" (protein G-related albumin binding module) for the three-helix protein domain responsible for albumin binding. Furthermore, Rozak et al have reported on the creation of artificial variants of the GA module, which were selected and studied with regard to different species specificity and stability (Rozak et al, Biochemistry 45:3263-3271 , 2006).

In addition to the three-helix containing proteins described above, other bacterial proteins exist that bind albumin. For example, the family of streptococcal proteins designated the "M proteins" comprises members that bind albumin (see e.g. Table 2 in Navarre & Schneewind, MMBR 63:174-229, 1999). Non-limiting examples are proteins M1/Emm1 , M3/Emm3, M12/Emm12, Emml_55/Emm55, Emm49/Emml_49, and H.

Neonatal Fc receptor (FcRn) mediated recycling of HSA

The MHC class l-related neonatal Fc receptor (FcRn) mediates cellular trafficking and recycling of albumin and IgG (Brambell et al, Nature 203:1352- 1354, 1964; Chaudhury et al, J Exp Med 197:315-322, 2003). The FcRn, also known as the Brambell receptor, specifically binds albumin and IgG at acidic endosomal pH (approximately a pH of 5.5) and thus protects pinocytosed albumin and IgG from lysosomal degradation by instead transporting these back to the cell surface and releasing them at neutral pH. The FcRn has an affinity for IgG and albumin in the low nanomolar and low micromolar range respectively at pH 5, but the affinity is about two orders of magnitude weaker at neutral pH (Chaudhury et al, Biochemistry 45:4983-4990, 2006; Andersen et al, Eur. J. Immunol. 36:3044-3051 , 2006). In this manner, the concentrations and the half lives of these two most abundant proteins in the plasma are regulated. Furthermore, the FcRn is responsible for actively transporting albumin and IgG over cellular barriers, e.g. the epithelium of the airways and the endothelium covering the intestines and the placenta.

The provision of new ways of altering the pharmacokinetics (PK) and/or pharmacodynamics (PD) for molecules in vivo is a key issue in the development of new and efficient therapeutics and treatment methods. There is therefore a need in the art of new ways of altering PK and/or PD of therapeutics. Disclosure of the invention

A first aspect of the invention meets the need for novel ways of modulating the pharmacokinetics (PK) and/or pharmacodynamics (PD) of a target having a biological function in a mammal, through the provision of a capture molecule comprising i) at least one target binding moiety capable of interacting with a target, said interaction being characterized by a first K ₀ value; ii) at least one albumin binding moiety capable of binding to albumin, said binding being characterized by a second K ₀ value; wherein said interaction with a target at a pH value of 5.5 modulates PK and/or PD of said target in said mammal.

The aim of the PK/PD modulation is to either shorten or extend the period during which the target may exhibit a biological effect in a mammal.

A capture molecule according to the invention, comprising at least one albumin binding moiety and at least one target binding moiety, provides a link between albumin and a target molecule. The capture molecule binds albumin via its albumin binding moiety. The binding between albumin and the albumin binding moiety of the capture molecule is characterized by a (second) K ₀ value, i.e. a dissociation constant, which describes the affinity between the albumin and the albumin binding moiety of the capture molecule. When albumin is present, a complex may form between albumin and the capture molecule. Thus, as albumin is one of the most abundant proteins in plasma, the capture molecule most frequently occurs in a complex with albumin in vivo.

The target binding moiety and albumin binding moiety in a capture molecule according to the invention may for example be connected by covalent coupling using known organic chemistry methods, or, if one or both moieties are polypeptides, expressed as one or more fusion polypeptides in a system for recombinant expression of polypeptides, or joined in any other fashion, directly or mediated by a linker comprising a number of amino acids.

The capture molecule interacts with said target via the at least one target binding moiety. This interaction is characterized by a (first) K ₀ value, which describes the affinity between the target and the target binding moiety of the capture molecule. At a pH value of 5.5, the interaction between the target and the capture molecule bound to albumin modulates the PK and/or PD of the target. PK and/or PD properties are hereinafter referred to as PK/PD.

The PK/PD modulating capture molecule is envisioned in different therapeutically relevant applications. At a pH value of 5.5, said capture molecule provides PK/PD modulation of a target by either forming (or remaining in) a complex with said target and albumin, or by releasing said target from said complex. When the target is released from said complex at said pH, PK/PD modulation is provided by elimination of the released target from the mammal. Thus, the target is directed to the subcellular lysosomes where it is degraded. Increased elimination of a target, such as a protein from a mammal, implies increased elimination rate of the biologically active target from the body of a mammal, as compared to a "normal" elimination rate of the target molecule per se, i.e. without previous interaction with the capture molecule. In this context, elimination should be understood as removal by lysosymal degradation.

On the other hand, when a complex between target, capture molecule and albumin is formed (or maintained) at said pH, the target is rescued from elimination by lysosomal degradation. Thus, target half life is extended. Half life extension implies that the elimination of a target having a biological function in a mammal is slower. The target molecule thus exhibits a half life in vivo when interacting with a capture molecule according to the invention, which is longer than the half life in vivo of the target molecule in the absence of capture molecule.

Without wishing to be bound by any theory, the interaction between the FcRn receptor and albumin is considered to be involved in the PK/PD modulating effect of the capture molecule on the target. Thus, an albumin- bound capture molecule is thought to take advantage of the FcRn-albumin interaction and follow the transportation of the FcRn within the cell and out of the cell. Thus, to accomplish half life extension of a target, the complex of capture molecule, albumin and target is protected from intracellular degradation at a pH value of around 5-6 and is subsequently transported to the cell surface through interaction with the FcRn. This interaction between the complex and FcRn mimics the naturally occurring interaction between FcRn and albumin. Increased elimination, on the other hand, is achieved by release, within a cell, of the target from the complex of FcRn, albumin, and capture molecule and subsequent elimination of the target by intracellular degradation. The albumin bound capture molecule is transported to the cell surface through interaction with the FcRn.

Beneficially, the inventive capture molecule exhibits a half life in vivo similar to that of albumin. Interaction between the target and the capture molecule in vivo modulates PK/PD properties of the target while the properties of the capture molecule itself remain essentially unaffected.

The inventive capture molecule preferably binds endogenous albumin in vivo, such as HSA. Several advantages of exploiting target association with endogenous albumin via a capture molecule, rather than target-albumin fusion or direct coupling, are foreseen. For example, the manufacturing cost will be lower for the capture molecule as compared to a target-albumin fusion. Moreover, reducing the administered amount of synthetic substances, such as synthetic albumin fusions, decreases the risk of inducing an immune response in a mammal. Furthermore, the risk of breaking tolerance towards the endogenous protein is avoided by not incorporating endogenous protein in a fusion protein which creates new epitopes. A reduced size of a drug also allows alternative formulations and ways of administration.

The terms "binding" and "binding affinity" as used in this specification for target binding and albumin binding refer to a property of a moiety, such as a polypeptide, which may be tested for example by the use of surface plasmon resonance technology, such as in a Biacore instrument. The skilled person may then interpret the results obtained by such experiments to establish at least a qualitative measure, or to obtain a quantitative measure, for example to determine a K ₀ value for the interaction between an albumin binding moiety and albumin, or between a target binding moiety and a target. The term "interaction" as used in this specification refers to a pH sensitive binding between the capture molecule and the target, wherein binding is as defined above.

Aspects and embodiments of the invention which concern PK/PD modulation by increased elimination of a target are referred to as elimination related applications of the invention. Similarly, aspects and embodiments of the invention which concern PK/PD modulation by extended half life of a target are referred to as half life related applications of the invention. When reference is made only to PK/PD modulation or PK/PD modulating, both elimination and half life related applications are concerned. Thus, in one embodiment of the first aspect of the invention, said PK/PD modulation comprises increased elimination of an active target from a mammal. This embodiment concerns a capture molecule which enables release of said target from the capture molecule at a pH value of 5.5, and subsequent degradation of said target. An increased elimination of a target is typically necessary when the target molecule is undesired and the amount thereof has to be reduced in a mammal. Undesired targets may for example be foreign proteins, or naturally expressed proteins that display elevated levels in plasma following a medical disorder, so that a therapeutic effect may be achieved by reducing the biological effect of said protein. The undesired target is not necessarily evenly distributed in the plasma but may be concentrated in certain regions, for example around a tumor or at sites of inflammation.

More specifically, said interaction between a target and a capture molecule allows target binding at physiological pH but results in target dissociation at a pH value of 5.5. Increased elimination of a target in a mammal, such as a human being, is effected through capture of the target in the blood, or in the tissue, at physiological pH and subsequent transportation along with the albumin-bound capture molecule to an intracellular degradation system. As described above, FcRn receptors expressed in certain cells are thought to be involved in the underlying mechanism. The complex of albumin, capture molecule and target typically enters the cell by pinocytosis or endocytosis. The complex associates with FcRn in such cells and the target dissociates from the complex of FcRn, albumin, and capture molecule in the acidic environment of endosomal vesicles, i.e. an environment having a pH value of approximately 5-6, whereafter the target becomes destined for lysosomal degradation. However, albumin, with a firmly attached capture molecule, will remain bound to the FcRn, which will transport the albumin- bound capture molecule back to the cell surface for release into the extracellular space. Here, it will be available to bind another target molecule and to be reused in another elimination cycle, thereby catalyzing the elimination of the undesired target.

The most efficient elimination is obtained if the first K ₀ value, i.e. the affinity of the capture molecule for the target, measured at physiological pH, i.e. pH 7.4, and at pH 5.5, approximately representing the pH of the endosomes, display a favourable balance. Affinity at pH 7.4 should be sufficiently high to allow capture in circulation and affinity at pH 5.5 should be sufficiently low to allow release during transport inside the cell.

Thus, in one embodiment of an elimination related application of the present invention, the first K ₀ value, i.e. the measure of the affinity of the capture molecule for said target, is lower at physiological pH than at a pH of

5.5. For example, said first K ₀ value at a pH value of 5.5 is at least 25 times said first K ₀ value at a pH value of 7.4. Put differently, this means that the binding between the capture molecule and the undesired agent is at least 25 times stronger at physiological pH (e.g. in the extracellular space) than at a pH of 5.5 (e.g. within endosomal vesicles). Said first K ₀ value at a pH value of

5.5 is for example at least 100 times said first K ₀ value at a pH value of 7.4. In one embodiment of the capture molecule according to an elimination related application, said first K ₀ value is no more than 1 x 10 ^~7 M at a pH value of 7.4, such as no more than 1 x 10 ^"8 M at a pH value of 7.4, for example no more than 1 x 10 ^"9 M at a pH value of 7.4.

In one embodiment of an elimination related application of the inventive capture molecule, said first K ₀ value is at least 1 x 10 ^~5 M at a pH value of 5.5, for example at least 1 x 10 ^"4 M at a pH value of 5.5.

Optimally, both target affinity and target dissociation rate at a certain pH should be considered. The early endosomes have been identified as the major sorting site for FcRn-complexes in endothelial cells and the occupation time in this compartment may be as short as a few seconds (Ober et al J.

Immunol. 172: 2021 -2029, 2004; Prabhat et a/ PNAS 104:5889-5894, 2007).

Therefore, in the case where elimination of a target is desired, the pH dependent release of the target from the capture molecule should ideally occur within this timeframe. That is, the capture molecule should display fast off-rate kinetics for its target.

In one embodiment of an elimination related application of the inventive capture molecule, the interaction between said target binding moiety and said target is characterized by a k _O ff value at a pH value of 5.5, said k _O ff being at least 1 x 10 ^~3 s ^"1 , such as at least 1 x 10 ^~2 s ^"1 , for example at least 1 x 10 ^~1 s ^"1 . In one embodiment of an elimination related application of the invention, there is provided a capture molecule wherein said interaction between said target binding moiety and said target neutralizes a biological function of the target. Thus, PD properties are explicitly modulated by neutralization of a biological function of the target through interaction with the capture molecule. The neutralization may for instance be accomplished by blocking the target molecule's receptor binding site or catalytically active site.

In another embodiment of the first aspect of the invention, there is provided a capture molecule wherein said PK/PD modulation comprises half life extension of said target in said mammal. This half life related application concerns targets, for example proteins, for which the plasma levels may have been reduced following a medical disorder, or targets for which one wishes to increase the plasma level to achieve or enhance a therapeutic effect. In these scenarios, an extended half life of the target molecule is desired.

The half life related applications of the inventive capture molecule concern an interaction between a target and the inventive capture molecule that mimics the albumin-FcRn interaction. The capture molecule binds to albumin when albumin is present. The target, if not already pre-mixed with the capture molecule in vitro, is either captured by the albumin bound capture molecule in the blood, i.e. at physiological pH, or later in the endosomal compartment, where it has high enough affinity to remain bound at the acidic pH. Thereafter, the target, in complex with the capture molecule and albumin, is transported back to the cell surface by FcRn mediated recycling. Thus, in this way the target is rescued from being directed to the lysosome for degradation and an extended half life is obtained.

For extended half life of a target molecule, the interaction between the target molecule and the capture molecule should have a low dissociation constant, K ₀ , at pH 5.5 so that the half life is significantly extended. Beneficially, the target molecule remains fully functional while in circulation.

Thus, in one embodiment of a half life related application of the inventive capture molecule, said first K ₀ value is no more than 1 x 10 ^~6 M at a pH value of 5.5, such as no more than 1 x 10 ^"7 M at a pH value of 5.5, for example no more than 1 x 10 ^"8 M at a pH value of 5.5, for example no more than 1 x 10 ^"9 M at a pH value of 5.5.

While in the circulation of a mammal, the target can occur in a complex with the capture molecule, in a complex with the capture molecule and albumin, but it can also occur freely, i.e. not in complex with the capture molecule (and optionally albumin). In one embodiment of a half life related application of the capture molecule, said first K ₀ value is no more than 1 x 10 ^~6 M at a pH value of 7.4, such as no more than 1 x 10 ^"7 M at a pH value of 7.4, for example no more than 1 x 10 ^"8 M at a pH value of 7.4, for example no more than 1 x 10 ^"9 M at a pH value of 7.4.

The FcRn mediated recycling back to the cell surface may last for several minutes, during which the complex is situated in an acidic environment. The target molecule needs to remain bound to the capture molecule throughout the whole cycle, which implies slow off-rate kinetics. In one embodiment of a half life related application of the capture molecule, said interaction between target binding moiety and target is characterized by a k _O ff value at a pH value of 5.5, said k _O ff being no more than 1 x 10 ^"3 s ^"1 , such as no more than 1 x 10 ^"4 s ^"1 , for example no more than 1 x 10 ^"5 s ^"1 .

In one embodiment of the invention as described above, there is provided a capture molecule wherein said interaction with a target does not affect the biological function of the target. Thus, PD properties are modulated by extending the period during which the target is active. Said interaction between the capture molecule and the target thus potentiates said target by potentiation of a biological function of the target. The target molecule is more likely to be fully functional when linked to endogenous or exogenous albumin via said capture molecule, as compared to situations wherein the target is covalently attached to albumin or an albumin binding molecule.

For optimal performance of the PK/PD modulating capture molecule the affinity between the albumin binding moiety (of the capture molecule) and albumin should be such that the capture molecule essentially occurs in a complex with albumin in vivo. If the complex should dissociate, re-association is most likely to take place due to the high abundance of albumin in vivo . Beneficially, the affinity of the albumin binding moiety (of the capture molecule) for albumin is of the same order as the affinity between the target binding moiety (of the capture molecule) and the target. For example, said second K ₀ value may be no more than said first K ₀ value. In one embodiment of the PK/PD modulating capture molecule, said second K ₀ value is no more than 1 x 10 ^"7 M, such as no more than 1 x 10 ^"8 M, for example no more than 1 x 10 ^"9 M. In a more specific embodiment of the invention, said second K ₀ value is no more than the values given above, when measured at a pH value of 5.5. This reflects the particular need for a strong affinity for albumin within the subcellular compartments. Where increased elimination of a target is desired, it is beneficial that the undesired target dissociates from the complex, while the capture molecule (via the albumin binding moiety) remains in association with albumin and hence with the FcRn receptor. Where extended half life of a target is desired, a strong affinity between the capture molecule and albumin rescues the target from degradation in the lysosymes. In one embodiment of the PK/PD modulating applications, the capture molecule comprises at least two target binding moieties. It may be of interest to obtain even stronger binding of the target than is possible with one target binding moiety. In this case, the provision of a multimer, such as a dimer, trimer or tetramer, of the target binding moiety may provide the necessary avidity effects. The multimer may consist of a suitable number of target binding moieties according to the inventive capture molecule. Preferentially, the target binding moieties in such a multimer may all be identical, for example have the same amino acid sequence. The linked target binding moieties in such a multimer may for example be connected by covalent coupling using known organic chemistry methods, or expressed as one or more fusion polypeptides in a system for recombinant expression of polypeptides, or joined in any other fashion, directly or mediated by a linker comprising a number of amino acids.

A capture molecule consisting of several identical domains of the target binding moiety linearly fused to one albumin binding moiety may, for example in an elimination related application of the inventive capture molecule, be beneficial, in particular if the target molecule is small. This may further increase the rate of elimination.

In one embodiment of the PK/PD modulating capture molecule, said albumin binding moiety is a polypeptide. In particular, said albumin binding moiety may be a naturally occurring polypeptide or an albumin binding fragment thereof. The albumin binding moiety may, as non-limiting examples, be selected from the group consisting of albumin binding proteins M1/Emm1 , M3/Emm3, M12/Emm12, Emml_55/Emm55, Emm49/Emml_49, H, G, MAG, ZAG, PPL and PAB. In a more specific embodiment, the albumin binding moiety is streptococcal protein G or an albumin binding fragment or derivative thereof. In an even more specific embodiment, the albumin binding moiety is selected from the group consisting of domain GA1 , domain GA2 and domain GA3 of protein G from Streptococcus strain G148, and may thus, for example, be the GA3 domain (SEQ ID NO:491 ).

In one embodiment of the PK/PD modulating capture molecule, said albumin binding moiety is an engineered polypeptide. An engineered polypeptide may be derived from a naturally occurring starting polypeptide through subjecting it to protein engineering techniques, such as mutations and alterations in a site-directed or randomized approach, with a view to create novel or enhanced properties, such as binding affinity for a molecule such as albumin.

In particular, said albumin binding moiety may comprise an albumin binding motif, which motif consists of the amino acid sequence:

GVSDX ₅ YKX ₈ X ₉ I Xi iXi ₂ AXi ₄ TVEGVX ₂ o ALX ₂₃ X ₂₄ X ₂₅ I

wherein, independently of each other,

X ₅ is selected from Y and F;

X ₈ is selected from N, R and S; Xg is selected from V, I, L, M, F and Y;

Xii is selected from N, S, E and D;

Xi ₂ is selected from R, K and N;

Xi ₄ is selected from K and R;

X ₂₀ is selected from D, N, Q, E, H, S, R and K; X ₂₃ is selected from K, I and T;

X ₂₄ is selected from A, S, T, G, H, L and D; and

X ₂₅ is selected from H, E and D.

Such a polypeptide capable of binding albumin can in particular be a variant of a protein scaffold, which variant has been selected for its specific binding affinity for albumin.

The above definition of a class of sequence related, albumin binding polypeptides is based on a statistical analysis of a large number of albumin binding polypeptides identified and characterized as described in Jonsson et a/, supra. The variants are selected from a large pool of random variants of a parent polypeptide sequence or basic structure or "scaffold", said selection being based on an interaction with albumin in phage display. The identified albumin binding motif, or "ABM", corresponds to the albumin binding region of the parent scaffold, which region constitutes two alpha helices within a three- helical bundle protein domain. While the original amino acid residues of the two ABM helices in the parent scaffold already constitute a binding surface for interaction with albumin, that binding surface is modified by the substitutions according to the invention to provide an alternative albumin binding ability.

As the skilled person will realize, the function of any polypeptide, such as the albumin binding capacity of an albumin binding moiety of a capture molecule according to this embodiment of the invention, is dependent on the tertiary structure of the polypeptide. It is therefore possible to make minor changes to the sequence of amino acids in a polypeptide without affecting the function thereof. Thus, the polypeptide moiety according to this embodiment encompasses modified variants of the ABM, which are such that the albumin binding characteristics are retained. 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 residue from the same functional group.

In one embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, X ₅ is Y.

In one embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, Xs is selected from N and R, and may in particular be R.

In one embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, Xg is L.

In one embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, Xn is selected from N and S, and may in particular be N.

In one embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, X12 is selected from R and K, such as X12 being R or Xi2 being K.

In one embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, Xi ₄ is K.

In one embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, X ₂ o is selected from D, N, Q, E, H, S and R, and may in particular be E.

In one embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, X23 is selected from K and I, and may in particular be K. In one embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, X ₂₄ is selected from A, S, T, G, H and L. In a more specific embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, X2 ₄ is L.

In an even more specific embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, X23X24 is KL. In another even more specific embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, X23X24 is TL.

In one embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, X2 ₄ is selected from A, S, T, G and H.

In a more specific embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, X2 ₄ is selected from A, S, T, G and H

In one embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, X25 is H.

The selection of polypeptide variants of albumin binding moieties led to the identification of a substantial amount of individual albumin binding motif (ABM) sequences. These sequences constitute individual embodiments of the ABM sequence in the above definition of possible albumin binding moieties according to the PK/PD modulating capture molecule. The sequences of individual albumin binding motifs are presented in Figure 1 and as SEQ ID NO:1 -245 and 496. In certain embodiments of the albumin binding moiety in the PK/PD modulating capture molecule according to the invention, the ABM consists of an amino acid sequence selected from SEQ ID NO:1 -245 and 496. In a more specific embodiment of the inventive capture molecule, the ABM sequence defining the albumin binding moiety is selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:155, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241 , SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244 and SEQ ID NO:245. In yet more specific embodiments of the albumin binding moiety in the PK/PD modulating capture molecule according to the invention, the ABM sequence is selected from SEQ ID NO:3, SEQ ID NO:35, SEQ ID NO:53 and SEQ ID NO:239.

In embodiments of the albumin binding moiety of the PK/PD modulating capture molecule, the ABM may form part of a three-helix bundle protein domain. For example, the ABM may essentially constitute or form part of two alpha helices with an interconnecting loop, within said three-helix bundle protein domain. In particular embodiments of the albumin binding moiety of the PK/PD modulating capture molecule, such a three-helix bundle protein domain is selected from the group consisting of three-helix domains of bacterial receptor proteins. Non-limiting examples of such bacterial receptor proteins may be selected from the group consisting of albumin binding receptor proteins from species of Streptococcus, Peptostreptococcus and Finegoldia, such as for example selected from the group consisting of proteins G, MAG, ZAG, PPL and PAB. In a specific embodiment of the albumin binding moiety of the PK/PD modulating capture molecule, the ABM forms part of protein G, such as for example protein G from Streptococcus strain G148. In different variants of this embodiment, the three-helix bundle protein domain of which the ABM forms a part is selected from the group consisting of domain GA1 , domain GA2 and domain GA3 of protein G from Streptococcus strain G148, in particular domain GA3. In alternative embodiments, the ABM forms part of one of the five three-helix domains of the bacterial receptor protein A from Staphylococcus aureus; i.e. the three-helix bundle protein domain is selected from the group consisting of protein A domains A, B, C, D and E. In other similar embodiments, the ABM forms part of protein Z, derived from domain B of protein A from Staphylococcus aureus.

In embodiments of the albumin binding moiety of the PK/PD modulating capture molecule wherein the ABM "forms part of a three-helix bundle protein domain, this is understood to mean that the sequence of the ABM is "inserted" into or "grafted" onto the sequence of the naturally occurring (or otherwise original) three-helix bundle domain, such that the ABM replaces a similar structural motif in the original domain. For example, without wishing to be bound by theory, the ABM is thought to constitute two of the three helices of a three-helix bundle, and can therefore 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 ABM helices has to be performed so as not to affect the basic structure of the polypeptide. That is, the overall folding of the Ca backbone of the polypeptide moiety capable of binding albumin will be 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 secondary structure in the same order etc. Thus, an ABM according to these embodiments of the albumin binding moiety of the PK/PD modulating capture molecule "forms part" of a three-helix bundle domain if the polypeptide according to this embodiment has the same fold as the original domain, implying that the basic structural properties are shared, those properties e.g. resulting in similar CD spectra. The skilled person is aware of other parameters that are relevant. In one embodiment of the PK/PD modulating capture molecule, the albumin binding polypeptide is a three-helix bundle protein domain, which comprises the albumin binding motif as defined above and additional sequences making up the remainder of the three-helix configuration. Thus, the PK/PD modulating capture molecule may comprise an albumin binding polypeptide, which comprises the amino acid sequence:

LAEAKX ₃ XbAXcXd E LX ₆ KY- [ABM]- LAAL P wherein

[ABM] is an albumin binding motif as defined above,

and, independently of each other,

Xa is selected from V and E; Xb is selected from L, E and D; X ₀ is selected from N, L and I; Xd is selected from R and K; and Xe is selected from D and K.

In one embodiment, X ₃ is V. In one embodiment, X _b is L.

In one embodiment, X ₀ is N. In one embodiment, X _d is R. In one embodiment, X _e is D.

As detailed in for example Jonsson et al, supra, the selection and sequencing of a number of albumin binding variants led to the identification of individual albumin binding polypeptide sequences. These sequences constitute individual examples of the albumin binding moiety according to the above embodiment of the capture molecule. The sequences of these individual albumin binding polypeptides are presented in Figure 1 and as SEQ ID NO:246-490. Also encompassed by these examples of an albumin binding moiety in the inventive capture molecule is an albumin binding polypeptide having an amino acid sequence with 85 % or greater identity to a sequence selected from SEQ ID NO:246-490. In particular embodiments, the sequence of the albumin binding polypeptide is selected from SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:254, SEQ ID NO:260, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:280, SEQ ID NO:291 , SEQ ID NO:294, SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:400, SEQ ID NO:484, SEQ ID NO:485, SEQ ID NO:486, SEQ ID NO:487, SEQ ID NO:488, SEQ ID NO:489 and SEQ ID NO:490 and sequences having 85 % or greater identity thereto. In more specific embodiments of the inventive capture molecule, the sequence of the albumin binding polypeptide is selected from SEQ ID NO:248, SEQ ID NO:280, SEQ ID NO:298 and SEQ ID NO:484 and sequences having 85 % or greater identity thereto.

As is evident from the above, in addition to a polypeptide whose amino acid sequence is selected from SEQ ID NO:246-490 or a subset thereof, the present invention also encompasses variants thereof. The amino acid sequences of such encompassed variants exhibit small differences only in comparison with SEQ ID NO:246-490. One definition of such variants is given above, i.e. an albumin binding polypeptide with an amino acid sequence having at least 85 % identity to a sequence selected from SEQ ID NO:246- 490. In some embodiments, the polypeptide may have a sequence which is at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % identical to the sequence selected from SEQ ID NO:246-490. The comparison may be performed over a window corresponding to the shortest of the sequences being compared, or over a window corresponding to an albumin binding motif in at least one of the sequences being compared.

In another embodiment of the PK/PD modulating capture molecule, the albumin binding moiety binds to human serum albumin.

In another embodiment of the PK/PD modulating capture molecule, the albumin binding moiety is capable of interacting with at least one of, and preferably all of, residues F228, A229, A322, V325, F326 and M329 in human serum albumin so as to enhance binding of the molecule to albumin. For example, the albumin binding moiety may include an amino acid residue which forms an interaction with the M329 residue in human serum albumin so as to enhance binding of the molecule to albumin. In addition, or alternatively, the albumin binding moiety may include an amino acid residue which forms an interaction with helix 7 in the human serum albumin domain MB so as to enhance binding of the molecule to albumin. In addition, or alternatively, the albumin binding moiety includes an amino acid residue which forms an interaction with residues in human serum albumin domain MA so as to enhance binding of the molecule to albumin. In addition, or alternatively, the albumin binding moiety includes an amino acid residue which forms an interaction with residues between helices 2 and 3 of human serum albumin so as to enhance binding of the molecule to albumin.

In one embodiment of the inventive capture molecule, the target binding moiety is a polypeptide. In particular, said target binding moiety may be a naturally occurring polypeptide or a target binding fragment thereof, such as the ectodomain of a receptor, such as VEGFr, or an enzyme, such as carboxypeptidase N. In some embodiments of the PK/PD modulating capture molecule, the target binding polypeptide may include a three-helix bundle protein domain. For example, the target binding motif of a target binding moiety may essentially constitute two alpha helices with an interconnecting loop, within said three-helix bundle protein domain.

In particular embodiments of the target binding moiety according to the inventive capture molecule, such a three-helix bundle protein domain is selected from the group consisting of three-helix domains of bacterial receptor proteins. Non-limiting examples of such bacterial receptor proteins are the five different three-helical domains of protein A from Staphylococcus aureus, and derivates thereof. In other similar embodiments, the polypeptide includes a variant of protein Z, derived from domain B of protein A from Staphylococcus aureus.

In one embodiment of the inventive capture molecule, said target binding moiety is an engineered polypeptide. The target binding moiety may for example be an engineered ectodomain of a receptor, such as VEGFr, or an engineered enzyme, such as carboxypeptidase N. In particular, said target binding moiety may be a variant of a protein scaffold, which variant has been selected for its specific binding affinity for said target. Non-limiting examples of polypeptide moieties capable of binding to a target are selected from the group consisting of antibodies and fragments and domains thereof substantially retaining antibody binding activity; microbodies, maxybodies, avimers and other small disulfide-bonded proteins; and variants of binding proteins derived from a scaffold selected from the group consisting of staphylococcal protein A and domains thereof, and lipocalins, ankyrin repeat domains, cellulose binding domains, Y crystallines, green fluorescent protein, human cytotoxic T lymphocyte-associated antigen 4, protease inhibitors, PDZ domains, peptide aptamers, staphylococcal nuclease, tendamistats, fibronectin type III domain, zinc fingers, conotoxins, and Kunitz domains. In other similar embodiments, said variants of a target binding protein derived from a scaffold include variants of protein Z, such as the variants presented in Figure 1 and also as SEQ ID NO:492 and 493, derived from a common scaffold and originating from domain B of protein A from Staphylococcus aureus. The generalized amino acid sequence of such a Z derived scaffold is presented in Figure 1 (denoted "Scaffold 1 ") and as SEQ ID NO:494, wherein each X individually corresponds to an amino acid residue which is varied. Thus, each X may be any amino acid residue independent of the identity of any other residue denoted X in the sequence. Alternatively, said Z variants can be derived from a scaffold variant which has been engineered in such a way as to provide novel or improved properties, such as improved structural and chemical stability. The generalized amino acid sequence of such a Z derived engineered scaffold is presented in Figure 1 (denoted "Scaffold 2") and as SEQ ID NO:495 wherein each X individually corresponds to an amino acid residue which is varied. Each X may be any amino acid residue as described above.

In a particular variant of the above described embodiments of the inventive capture molecule, said albumin binding moiety is a polypeptide and said target binding moiety is a polypeptide.

In another embodiment of the inventive capture molecule, said at least one target binding moiety and at least one albumin binding moiety are covalently coupled. Said target binding moiety and said albumin binding moiety may form, for example, a fusion complex or a conjugated complex.

Related aspects of the present invention provide a polynucleotide encoding a capture molecule as described above, i.e. when the albumin binding moiety and the target binding moiety respectively are polypeptides, as well as an expression vector comprising the polynucleotide and a host cell comprising the expression vector. The latter three aspects of the invention are tools for the production of a capture molecule comprising polypeptide moieties as described above, and the skilled person will be able to obtain them and put them into practical use without undue burden, given the information herein concerning the capture molecule that is to be expressed and given the current level of skill in the art of recombinant expression of proteins. Thus, another related aspect of the invention is a method of producing a capture molecule according to the invention, comprising expressing a polynucleotide as herein described, for example via the culturing of a host cell as herein defined under conditions permitting expression of the capture molecule from the expression vector, and isolating the capture molecule.

Yet another aspect of the present invention concerns a capture molecule as defined above for use as a medicament.

A further aspect of the present invention concerns a method of producing a capture molecule wherein said target binding moiety is a polypeptide, comprising i) providing a library of potential target binding moieties; ii) selecting a target binding moiety capable of a PK/PD modulating interaction with a target, wherein said selection comprises at least the steps of binding, wash and elution, and wherein said binding is performed at a first pH, said wash is performed at a second pH and said elution is performed at a third pH; iii) providing an albumin binding moiety; and iv) coupling said binding moieties.

The inventive method involves providing a library of polypeptide moieties potentially capable of binding a target. From the library of potential target binding moieties, actual binding moieties capable of PK/PD modulating interaction with a target are extracted. This is accomplished by selection of at least one such binding moiety. The selection comprises the steps of binding, wash and elution, wherein the first two steps, i.e. binding and wash, are performed at a first pH value and the last step, i.e. elution, is performed at a second pH value. After selection of a suitable binding moiety, an albumin binding moiety is provided and the two moieties are coupled to provide a capture molecule.

In one embodiment of the inventive method, said first pH is physiological pH, i.e. a pH value of around 7.4. The wash and elution conditions are adapted so as to obtain binding moieties that have either a strong affinity or a low to no affinity for the target at pH 5.5. For example, said second pH (i.e. wash pH) may be 7.4. The third pH

(i.e. elution pH) may be 5.5. Using a combination of wash at pH 7.4 and elution at pH 5.5 will yield a fraction eluted at pH 5.5 comprising target binding moieties that may be suitable for elimination applications.

In an alternative embodiment, said second pH (i.e. wash pH) is 5.5. The third pH (i.e. elution pH) may be lower than 5, such as lower than 3, such as 2.2. Using a combination of wash at pH 5.5 and elution at an even more acidic pH will yield a fraction eluted at very low pH comprising target binding moieties that may be suitable for extended half life applications.

Alternatively, a target binding moiety may be selected in parallel selections depending on the application of the capture molecule. Thus, the selections are performed in parallel, with buffers having pH values mimicking the extracellular and the endosomal environment respectively. That is, target binding moieties enriched in selections performed at pH 5.5 may be suitable for extended half life applications, whereas target binding moieties enriched in selections performed at pH 7.4 may be suitable for elimination applications. Selection of pH sensitive binders for the target of interest is typically performed using a phage display library of potential target binding moieties, such as for example a library of Z variants as described in Example 2 and 3.

A PK/PD modulating interaction between a target binding moiety and a target may for example originate from a pH sensitive binding interface having one or more histidine residues either in the target molecule or in the target binding moiety of the capture molecule. If a target binding moiety for the target of interest is available, for instance a Z variant e.g. derived from a phage display selection, a pH-sensitive interaction may be designed on the amino acid sequence level by rational introduction of one or more histidine residues situated at the binding interface. Selection of candidate residues is simplified if a three-dimensional model structure of the complex of target and target binding moiety is available. For instance, charge-charge interactions at the binding surface will guide the choice of where to introduce histidine residues causing either electrostatic repulsion or attraction as the pH is lowered. Replacements of amino acid residues are readily achieved by standard mutagenesis techniques. For each target binding variant, the binding kinetics and affinity for the target molecule would be measured at pH 5.5 and 7.4, for instance by biosensor analysis. If no capture molecule is available, then such a molecule can be developed de novo using phage display selection at different pH conditions, for example as described in Examples 2 and 3. This would be followed by screening for pH-sensitive binding moieties as above, followed by additional biosensor analysis to assess functional interference, for instance blocking of the target molecule's receptor binding site. This may be preferred in an elimination application but is preferably avoided if an extended half life of the target molecule is desired. As for the overall structure, the capture molecule is suitably designed in a way such that no interference occurs with the albumin-FcRn interaction. That is, sterical hindrance or allosteric effects are avoided. In Example 1 , a set of Z variants (as target binding moieties) fused to ABD variants (as albumin binding moieties) were tested in binding experiments with albumin and FcRn. The binding experiments showed that the ABD fused Z variants do not interfere with the albumin-FcRn interaction.

In another aspect of the present invention, there is provided a pharmaceutical preparation for the elimination of an undesired target from a mammal through transport of said target to an intracellular degradation system, said preparation comprising i) a capture molecule as described above in connection with elimination related applications of the invention; and ii) pharmaceutically acceptable excipients.

The capture molecule comprised in the inventive pharmaceutical preparation will, upon administration to a mammalian host, associate with albumin already present in the host via the binding affinity of the albumin binding moiety to form a non-covalent complex. Alternatively, said complex is formed in vitro through the addition of exogenous albumin to the inventive pharmaceutical preparation itself, which then comprises albumin in a non- covalent complex with the capture molecule as described above.

In one embodiment of this aspect of the invention, the pharmaceutical preparation may be in a form suitable for injection. Alternatively, the pharmaceutical preparation may be in a form suitable for uptake over an epithelial barrier. As non-limiting examples, it may take the form of an aerosol formulation, for oral or nasal inhalation, or an oral formulation enabling intestinal absorption.

In related aspects of the present invention, there is provided a method of treatment of a mammal through the elimination of an undesired target from said mammal, comprising administering a pharmaceutical preparation as described immediately above to said mammal, whereby i) said capture molecule captures said target, and optionally associates with albumin via said albumin binding moiety, in an extracellular space at a first pH value, thus forming a complex of target, capture molecule and albumin, ii) said complex is transported into a cell expressing FcRn receptors, thus becoming located in an intracellular space at a second pH value; iii) said complex is bound to said FcRn receptor at said second pH value, said target dissociates from said complex and is transported to an intracellular degradation system.

Beneficially, the capture molecule will escape degradation and will be safely recycled back to the cell surface along with the albumin-FcRn complex.

Furthermore, the capture molecule preferably neutralizes a biological function of the target, for example by blocking the receptor binding site or catalytically active site on the target.

In one embodiment of this aspect, said first pH value is physiological pH, i.e. a pH of around 7.4, such as is typical for the extracellular space of a mammalian host.

In one embodiment of this aspect, said second pH value is in the range of 5-6, such as is typical for intracellular vesicles (endosomes) involved in the processes of endocytosis and pinocytosis. Another elimination related aspect of the invention provides a capture molecule, as described in connection with elimination related applications of the invention, for treatment of a mammal through the elimination of an undesired target from said mammal.

Another elimination related aspect of the invention provides the use of a capture molecule, as described in connection with elimination related applications of the invention, for the preparation of a medicament for treatment of a mammal through the elimination of an undesired target from said mammal.

Some elimination related aspects of the present invention, such as a capture molecule, method or use as described immediately above, concern removal of undesired targets from a mammal, such as from the circulation or tissue, by increased elimination of such undesired targets. This may for example be foreign proteins, or naturally expressed proteins that display elevated levels in plasma following a medical disorder and where a therapeutic effect may be achieved by, optionally, neutralizing the biological effect of said protein followed by elimination of said protein. The undesired target is not necessarily evenly distributed in the plasma but may be concentrated in certain regions, for example around a tumor or at sites of inflammation. Thus, the target with which the capture molecule interacts (via the target binding moiety) may be any molecule having an undesired biological function or effect in a mammal. Non-limiting examples of such target molecules are selected from the group consisting of TGFβ

(transforming growth factor beta); Aβ peptide; other disease-associated amyloid peptides; toxins, such as bacterial toxins and snake venoms; blood clotting factors, such as von Willebrand factor; interleukins, such as IL-13 and IL-2; myostatin; pro-inflammatory factors, such as TNF-α (tumor necrosis factor alpha), TNF-α receptor and IL-8 (interleukin 8); complement factors, such as C3a and C5a; hypersensitivity mediators, such as histamine and IgE; hGH (human growth hormone).

Such inventive method or use can be applied when said mammal for example suffers from a condition selected from cardiovascular disease, Alzheimer's disease, growth deficiencies, hypersensitivity, anaphylactic shock, muscle wasting disease, renal dysfunction, hemophilia, diabetes, sepsis and cancer diseases. As the skilled person will understand, the inventive method or use are suitable for any medical or other condition where elimination of a target is beneficial to the host.

In a further, half life related aspect of the invention there is provided a pharmaceutical preparation for extending the half life of a target in a mammal through transport of said target from an intracellular space, said preparation comprising i) a capture molecule as described above in connection with half life related applications of the invention; and ii) pharmaceutically acceptable excipients. The capture molecule comprised in the inventive pharmaceutical preparation according to this aspect will, upon administration to a mammalian host, associate with any target already present in the host via the binding affinity of the target binding moiety to form a non-covalent complex. Alternatively, said complex is formed in vitro through the addition of said target to the inventive pharmaceutical preparation itself, which then comprises said target in a non-covalent complex with the capture molecule described above. Beneficially, the target and the capture molecule remain in a complex upon administration to avoid a situation where the target dissociates from the capture molecule and is not recaptured in vivo due to dilution effects. In this case, the capture molecule may exhibit a sufficiently strong affinity for the target molecule for the capture molecule to remain associated with the target at physiological pH, such as in the circulation.

Furthermore, the capture molecule will, upon administration of the pharmaceutical preparation, associate with albumin already present in the mammalian host via the albumin binding moiety of the capture molecule.

Alternatively, said association of albumin and capture molecule can take place in vitro through the addition of albumin to the inventive pharmaceutical preparation. Thus, in combination with what is described above, it is understood that the capture molecule optionally may associate with the target and/or albumin in vitro, and/or optionally may associate with the target and/or albumin in vivo.

In one embodiment of this aspect of the invention, the pharmaceutical preparation may be in a form suitable for injection. Alternatively, the pharmaceutical preparation may be in a form suitable for uptake over an epithelial barrier. As non-limiting examples, it may take the form of an aerosol formulation, for oral or nasal inhalation, or an oral formulation enabling intestinal absorption.

In a related aspect of the invention, there is provided a method of treatment of a mammal through extending the half life of a target in vivo, comprising administering a pharmaceutical preparation, as described immediately above, to said mammal, whereby i) said preparation optionally captures said target via said capture molecule, and optionally associates with albumin via said albumin binding moiety, thus forming a complex of target, capture molecule and albumin, ii) if said complex is located in a cell at a first pH value, it is transported out of the cell, via interaction between said albumin and an FcRn receptor, thus becoming located in an extracellular space at a second pH value, and optionally iii) said target dissociates from said complex in said extracellular space.

Thus, if not already added and associated with the capture molecule in the pharmaceutical preparation in vitro, said target is captured by the capture molecule in vivo. Similarly, if said capture molecule is not already associated with exogenous albumin in vitro, such association with endogenous albumin occurs upon administration of the preparation to said mammal. The target, associated in said complex with capture molecule and albumin, is rescued from intracellular degradation through transport out of the cell by interaction between albumin and FcRn, in a manner mimicking the interaction between FcRn and albumin (or IgG).

In one embodiment of this aspect, said first pH value is in the range of 5-6, such as is typical for intracellular vesicles (endosomes) involved in the processes of endocytosis and pinocytosis.

In one embodiment of this aspect, said second pH value is physiological pH, i.e. a pH of around 7.4, such as is typical for the extracellular space of a mammalian host. In a related aspect of the present invention, there is provided a capture molecule, as described in the half life related applications of the invention, for treatment of a mammal through extending the half life of a target in said mammal.

In a related aspect of the present invention, there is provided use of a capture molecule as described in the half life related applications of the invention, for the preparation of a medicament for treatment of a mammal through extending the half life of a target in said mammal.

In situations relevant to half life extension, targets, such as proteins, typically exhibit reduced plasma levels as a result of a medical disorder in a mammalian host. Alternatively, target plasma levels need to be increased in order to achieve or potentiate a therapeutic effect, or short-lived therapeutics need extended circulation time. Clinically relevant targets and conditions where a method or use as described immediately above are beneficial are, for example, hGH (see Example 7 below) to treat growth deficiencies, Turner syndrome and AIDS wasting; TGFβ (see Example 8 below); G-CSF for treatment of neutropaenia in AIDS or after chemotherapy or bone-marrow transplantation; insulin and GLP-1 for treatment of type I and type Il diabetes respectively; IFN-α for treatment of chronic hepatitis C; and interleukins such as interleukin-2 for treatment of cancers such as malignant melanoma and renal cell cancer.

Brief description of the figures

Figure 1 shows the sequences of individual albumin binding motifs SEQ ID NO:1 -245 and 496, individual albumin binding polypeptides SEQ ID NO:246-491 , Z variants SEQ ID NO:492-493 and Z scaffold variants SEQ ID NO:494-495. Figures 2A-F show the results of the additive ELISA analysis performed at pH 6.0 (squares) and pH 7.4 (triangles) as described in Example 1 , using an albumin binding moiety alone or comprised in a capture molecule. The results are shown as absorbance at a wavelength of 620 nm. Wells were coated with increasing concentrations of albumin binding moieties ABDwt (A) and ABD035 (B) alone; and with increasing concentrations of capture molecules Z00342-ABDwt (C), ABDwt-Z00342 (D), (Z00342) ₂ -ABDwt (E) and Z02891-ABD035-cys-Mal-DOTA (F).

Figures 3A-E show the results of SPR analysis performed at pH 6.0 as described in Example 1 , using an albumin binding moiety alone or comprised in a capture molecule. The analysis was performed using ABDwt (A), Z00342- ABDwt (B), ABDwt-Z00342 (C), (Z00342) ₂ -ABDwt (D) and Z02891 -ABD035- cys-Mal-DOTA (E). HSA (broken line), HSA and capture molecule (solid line) and albumin binding moiety alone (dotted line), were injected over a surface with immobilized FcRn.

Examples

Example 1 : Effect of different ABD-fused Z variants on the albumin-FcRn interaction

A set of capture molecules consisting of target binding Z variants fused to albumin binding domains (ABD) were tested in interaction studies between albumin and FcRn performed with ELISA and Biacore at pH 6 and at pH 7.4.

Materials and methods

The following molecules were selected for analysis:

1. 6 x His-ABDwt

2. ABD035 3. Z00342-ABDwt

4. ABDwt-Z00342

5. (Z00342) ₂ -ABDwt

6. Z02891-ABD035-cys-Mal-DOTA

Z00342 (SEQ ID NO:492) and Z02891 (SEQ ID NO:493) are two Z variants with an affinity for the Her2 receptor. A detailed description of the Z00342 molecule is given in Orlova et al, Cancer Res 66:4339-4348, 2006, where it is denoted Z _Hβ r2342- Z02891 is identical to Z00342 at the randomized positions but differs in the scaffold residues. ABD035 (SEQ ID NO:280) is an affinity matured variant of wild-type ABD (ABD _w t, SEQ ID NO:491 ) as described in Jonsson et al, supra. Fusion proteins were produced with recombinant DNA technology and purification was performed using affinity capture on HSA-sepharose (GE Healthcare) and reverse phase chromatography. Maleimido-mono-amide- DOTA (Macrocyclics, cat. no. B-272) conjugation of ABD035 was performed in 0.2 M NaAc, pH 5.5, for 60 min at 37 ⁰ C and with 3x molar excess of MaI- DOTA. Conjugated molecules were separated from unconjugated molecules and free MaI-DOTA by reverse phase chromatography.

Construction and production of soluble human FcRn (shFcRn), fused to glutathione S-transferase, GST, in HEK293 cells were carried out as described in Berntzen et al, J Immunol Methods, 298:93-104, 2005.

Additive ELISA analysis: Wells of MaxiSorp™ ELISA plates (Nunc) were coated with serial dilutions (2 μg/ml-0.027 μg/ml) of molecules 1-6 listed above, and incubated overnight at 4 ⁰ C. The wells were blocked with 4 % skimmed milk (Acumedia) diluted in PBS for 1 h at room temperature and washed four times with PBS-T, pH 6.0 (PBS with 0.005 % Tween 20 (Sigma- Aldrich)). Monomeric HSA (50 μg/ml, Sigma-Aldrich), diluted in PBS-T, pH 6.0, was added to each well. The wells were incubated for 1 h at room temperature and washed four times with PBS-T, pH 6.0. Purified shFcRn- GST (0.25 μg/ml) was pre-incubated with an HRP-conjugated anti-GST IgG (GE Healthcare; diluted 1 :5000) in PBS-T, pH 6.0, and added to each well. After incubation for 1 h at room temperature the wells were washed four times with PBS-T, pH 6.0. Bound receptors were detected by adding 100 μl of the substrate TMB (Calbiochem) to each well. The absorbance was measured at 620 nm using a Sunrise TECAN spectrophotometer (TECAN). The same ELISA was repeated using PBS buffer at pH 7.4 in all steps.

Interaction studies using surface plasmon resonance: A Biacore 3000 instrument (GE Healthcare) was used. CM5 sensor chips were coupled with ShFcRn-GST (-600-1000 RU) using amine coupling chemistry according to the manufacturer's instructions. The coupling was performed by injecting 10- 12 μg/ml of each protein into 10 mM sodium acetate, pH 4.5 (GE Healthcare). All experiments were performed in phosphate buffer (67 mM phosphate buffer, 0.15 M NaCI, 0.005% Tween20) at pH 6.0. Portions of 1 μM of size exclusion chromatography isolated monomeric HSA (Sigma-Aldhch) or mouse serum albumin (MSA, Calbiochem) were injected, alone or together with one of the molecules 1 , 3, 4, 5 and 6 (1 -2 μM), over immobilized receptors with a flow rate of 40 μl/min at 25 ⁰ C. The surfaces were gently regenerated by dissociation of bound molecules using buffer at pH 6.0. Albumin binding moieties alone were injected over immobilized receptors as controls. In all experiments, data were zero adjusted and the reference cell value subtracted. Binding analyses were performed using the BIAevaluation wizard (GE Healthcare).

Results

The result of the additive ELISA is presented in Figure 1. At pH 6.0, shFcRn is still capable of binding to HSA that has formed a complex with ABD alone or with ABD fused to a Z variant. Fusions with ABDwt and fusions with the affinity matured ABD035 gave the same result. Fusions with the Z variant at either the N or the C terminus of ABD was indiscriminate, and use of a dimeric Z variant (designated no. 5) did not affect the result. No interaction between HSA and FcRn was seen at pH 7.4, since the affinity of FcRn for albumin is lower at neutral pH as compared to at a pH of 6.0.

The result of the Biacore analysis performed at pH 6.0 is presented in Figure 2, and confirmed the above results. The analysis showed an increased signal for the complex of HSA and the various capture molecules compared to for HSA alone, the increase corresponding to the additional mass of the complex. It was also shown that no cross reactivity is present between the capture molecules and the FcRn.

In summary, Z variants fused to ABD do not interfere with the albumin- FcRn interaction and allow a flexible design of the capture molecule in terms of the arrangement of the ABD relative to the Z variant. The same results were obtained when using mouse FcRn and rat FcRn (data not shown). This validates the molecules for in vivo studies in these animals in order to evaluate the half life of capture molecules according to the present invention. Example 2: Phage display selection of Z variants having a low affinity for target at pH 5.5

Materials and methods

Biotinylation of target: A target protein, or a domain or a peptide thereof, is biotinylated for 30 min at room temperature using EZ-LinkTM-Sulfo-NHS-LC- Biotin (Pierce, cat. no. 21335) in a 10-fold molar excess of biotin in PBS (2.68 mM KCI, 1.47 mM KH ₂ PO ₄ , 137 mM NaCI, 8.1 mM Na ₂ HPO ₄ , pH 7.4). Buffer exchange and removal of excess biotin is performed on a protein desalting column (Pierce, cat. no. 89849) using the PBS buffer above.

Phage display selection: A phage library, prepared essentially as described in Nord et a/ (Prot Eng 8:601-608, 1995), is used for selection. Alternatively, a library enriched for histidine residues in the variable positions is likewise created and used. The latter may be accomplished by designing the oligonucleotides for the initial library construction such that the allowed codon combination results in a bias, such as 15 %, for histidine residues in each of the variable positions. Preparation of phage stocks from the library and between selections is performed according to previously described procedures (Nord et al, Nat. Biotechnol. 15:772-777, 1997) using the helper phage M13K07 (New England Biolabs, Beverly, MA, USA) and PBS, pH 7.4.

Selection and wash are performed at physiological pH, but the elution conditions are adapted so as to obtain binders that have low to no affinities for the target at pH 5.5. Several rounds of selection, using increasingly stringent conditions, such as lowering the target concentration and harsher washing conditions, are performed in solution and the bound phages are captured on streptavid in-coated paramagnetic beads (Dynabeads M-280 Streptavidin; Dynal cat. no. 112.06). To avoid nonspecific binders, all tubes used in this procedure are pretreated with PBS-T (0.5 % Tween 20 in PBS) supplemented with 0.1 % gelatin, and the phage stocks are pre-incubated with streptavidin beads for the first two rounds of selection. In each cycle of selection, phages are eluted in a buffer at pH 5.5 (e.g. 50 mM acetate, 150 mM NaCI) for 10 minutes, after which the pH is adjusted to 7.4. In each round of selection, the eluted fraction is kept and used for the next round. Eluted phages are subsequently used to infect cultures of log phase E. coli RR1ΔM15 cells. After 30 minutes of incubation, the cells are pelleted by centrifugation, dissolved in a small volume of TSB-YE and spread on TYE agar plates (15 g/l agar, 3 g/l NaCI, 10 g/l tryptone and 5 g/l yeast extract), supplemented with 2 % glucose and 100 mg/l ampicillin, and incubated over night at 37 ⁰ C.

Identification of target binders: Randomly picked colonies from the final round of selections are expressed and screened for target binding using ELISA, for instance as described in Grόnwall et al (J Biotechnol 128:162-183, 2007), using a PBS buffer at pH 7.4. ELISA positive clones are sequenced using ABI PRISM dGTP, BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's recommendations. Purified sequencing reactions are analyzed on an ABI Prism® 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and cluster analysis is performed to identify relevant variants, targeting one or more epitopes.

Z variants enriched in fractions eluted at pH 5.5 may be suitable for elimination applications.

Z variants of interest are cloned as fusions to an appropriate albumin binding domain and recombinantly expressed, purified and characterized further as described in Example 4. These candidates do not necessarily contain histidine residues, since a pH sensitive interaction may also originate from histidine residues in the target molecule.

Example 3: Phage display selection of Z variants having a high affinity for target at pH 5.5

Materials and methods

Biotinylation of target: Target protein, or domain or peptide thereof, is biotinylated as described in Example 2.

Phage display selection: Phage libraries and phage stocks are prepared essentially as described in Example 2. The selection is performed at physiological pH, but the washing and elution conditions are adapted so as to obtain binders that have high affinities for the target at pH 5.5. Several rounds of selection, using increasingly stringent conditions such as lowering the target concentration and harsher washing conditions, are performed in solution and the bound phages are captured on streptavid in-coated paramagnetic beads (Dynabeads M-280 Streptavidin; Dynal cat. no. 112.06). To avoid nonspecific binders, all tubes used in this procedure are pretreated with PBS-T (0.5 % Tween 20 in PBS) supplemented with 0.1 % gelatin and the phage stocks are pre-incubated with streptavidin beads for the first two rounds of selection. In each cycle of selection, phages are washed in a buffer at pH 5.5 (e.g. 50 mM acetate, 150 mM NaCI) followed by elution in Glycine-HCI, pH 2.2, for 10 min, after which the pH is adjusted to pH 7.4.

Eluted phages are subsequently used to infect cultures of log phase E. coli RR1ΔM15 cells. After 30 min of incubation, the cells are pelleted by centhfugation, dissolved in a small volume of TSB-YE and spread on TYE agar plates (15 g/l agar, 3 g/l NaCI, 10 g/l tryptone and 5 g/l yeast extract), supplemented with 2 % glucose and 100 mg/l ampicillin, and incubated over night at 37 ⁰ C.

Identification of target binders: Randomly picked colonies from the final round of selections are expressed and screened for target binding using ELISA as described in Example 2, but using an acetate buffer at pH 5.5. Positive clones are sequenced, analyzed and identified as described in Example 2.

Z variants enriched in the fraction resisting elution at pH 5.5 may be suitable for extended half life applications.

Z variants of interest are cloned as fusions to an appropriate albumin binding domain and recombinantly expressed, purified and characterized further as described in Example 4. These candidates do not necessarily contain histidine residues, since a pH sensitive interaction may origin from histidine residues in the target molecule.

Example 4: Biosensor characterization of pH sensitive interactions

Materials and methods

Z variants selected as described in Examples 2 or 3, or previously selected variants that have been subjected to rational mutagenesis, are fused to an appropriate albumin binding moiety, for example as described in Example 1.

The resulting capture molecules are characterized by biosensor analysis on a Biacore 2000 instrument (GE Healthcare). The target protein is immobilized on CM-5 chips (GE Healthcare) by amine coupling chemistry according to the manufacturer's instructions. Buffers (50 mM sodium phosphate, 150 mM NaCI, 0.005 % surfactant P20) at pH 7.4 or pH 5.5 are used as running buffers for the analysis. The candidate capture molecules diluted in either buffer are injected over the flow cells in concentration ranges as appropriate. To study the off-rate kinetics when the pH is shifted from 7.4 to 5.5, the candidate capture molecule is first injected in the buffer at pH 7.4, and about 10 min post injection the buffer is changed to pH 5.5 for up to 15 min. For a desired elimination application, the capture molecule should ideally dissociate immediately when the pH is changed from 7.4 to 5.5, and the K ₀ should be no more than 1 x 10 ^~7 M at pH 7.4, with no detectable binding in biosensor analysis at pH 5.5. In this application, fast off-rate kinetics are also desired. For a desired extended half life application, capture molecules that have a K ₀ of no more than 1 x 10 ^~6 at pH 5.5 will be selected for further analysis. In order to fulfill the criteria outlined above, promising candidates may be further modified by site-directed mutagenesis and reanalyzed for their pH-sensitive binding.

Example 5: Investigating the in vitro half life of a target molecule having a pH sensitive interaction with an albumin binding capture molecule

To test whether the half life of a target molecule, for instance a target protein, is affected in vitro, the target molecule is labeled with radioactivity and added to cell cultures, alone or together with the capture molecule pre-incubated with albumin.

Materials and methods

Radiolabeling of target molecule: Radiometal labeling may be the method of choice since radiolabeled catabolites will be trapped in the cell upon lysosomal degradation. The target molecule is dissolved in, or buffer exchanged into, 0.07 M borate buffer, pH 9.2. Freshly prepared CHX-A"- DTPA (Macrocyclics, Dallas, US, Cat No B-355) 1 mg/ml, in 0.7 M borate buffer pH 9.2, is added to target at a 1 :1 molar ratio and the mixture is incubated at 37 ⁰ C for 3 h. Buffer exchange to 0.2 M ammonium acetate, pH 5.5, is performed on a NAP-5 column (GE Healthcare, cat. no. 17-0853-02) according to the manufacturer's instructions. The CHX-A"-DTPA conjugated target molecule is mixed with a radionuclide such as ¹¹¹ In (for example ¹¹¹ InCI ₃ , Malinckrodt/Covidien, Hazelwood, US, Cat No N132F0), diluted in hydrochloric acid. Approximately 4 MBq per nmol of the target molecule is used and the mixture is incubated at room temperature for 30 min. A 1 μl aliquot is analysed by ITLC SG (instant thin layer chromatography, Gelman Sciences Inc.), eluted with 0.2 M citric acid and analyzed in a phosphor imager (for example Cyclone™ Storage Phosphor System, PerkinElmer, Waltham, US). If the concentration of labeled target molecule exceeds 95 % of the population, the sample is used directly. Otherwise, free radiometals are removed using a NAP-5 column as described above.

In vitro cell assay: Antigen presenting cells (APC), such as the monocytic cell lines U293 or THP- 1 , are selected for the study, since these cells readily engulf extracellular components by non-specific pinocytosis and have been shown to express FcRn. Cell cultures are established in RPMI 1640 medium (GIBCO, cat. no. 3187-25) with 10 % FCS (Fetal calf serum; GIBCO, cat. no. 10108-165), but this is changed to serum free medium just before the start of the experiment. For each candidate capture molecule, two cell populations will be compared: cultures with 1 ) labeled target molecule and 2) labeled target molecule and a 10-fold molar excess of the capture molecule pre- incubated in a 10-fold molar excess of albumin. 10 pmol/ml of the target molecule is added in the initial experiment. After, for instance, 1 , 12, 24 and 48 h (separate cell populations are prepared for each time point) the medium is withdrawn and the cells are harvested and washed 3 times with fresh medium. Radioactivity in the medium and the cells is measured using an automated gamma counter equipped with a 3-inch NaI (Tl) detector (1480 WIZARD OY, Wallac, Turku, Finland). In addition, the medium is applied to a NAP-5 column where low and high molecular weight fractions are separated and analyzed individually. Elution is performed stepwise with PBS containing 2.5 % BSA (bovine serum albumin) with 1 ) 0.5 ml = void volume, 2) 1 ml = high molecular weight fraction considered as intact target molecule, and 3) 1.5 ml = low molecular weight fraction considered as catabolites. The fractions are analyzed anew using the automated gamma counter. Samples from the population to which the capture molecule was added are compared to samples from control cultures and assessed in terms of the capture molecule's effect on the half life of the target molecule. For instance, higher levels of the target molecule in the population to which the capture molecule was added compared to levels in control cultures indicate an extended half life of the target molecule, achieved by a pH resistant binding to the capture molecule within the acidic endosome and by albumin-FcRn mediated recycling.

Example 6: Investigating the in vivo half life of a target molecule with a pH sensitive interaction with an albumin binding capture molecule

For target molecules exceeding the size for excretion by glomerular filtration in the kidney (>60 kDa), a comparative study of the half life, modulated by interaction with capture molecules as described herein, may also be performed in vivo. If the size of the target molecule is <60 kDa the half life may be investigated per se.

Materials and methods

Radiolabeling of target molecule: Radiolabeling of target molecule is carried out as described in Example 5.

Administration of molecules: In a comparative study, the target molecule is radiolabeled and administrated to mice with or without (control) the albumin- binding capture molecule. For each capture molecule, two populations of mice (n>6) are injected via the tail vein according to the following suggested scheme:

Population A: 1 ) radiolabeled target molecule, 1 -3 μg

Population B: Premix of: 1 ) non-labeled albumin binding capture molecule, 10-fold molar excess compared to target molecule 2) radiolabeled target molecule, 1 -3 μg

If the target molecule is <60 kDa, the animals are treated as in population B only. Blood sample analysis: Blood samples are withdrawn, e.g. at 0.25, 0.5, 1 , 2 , 4, 8, 24, 40 and 48 h post injection, and collected in pre-weighed heparin treated vials. The samples are weighed and radioactivity in whole blood is measured using an automated gamma counter equipped with a 3-inch NaI (Tl) detector (1480 WIZARD OY, Wallac, Turku, Finland). The percent of injected activity per gram (% lA/g) is calculated for each sample. In addition, the blood samples are centrifuged at 6000 rpm for 5 min and the serum is collected and applied to a NAP-5 column where low and high molecular weight fractions are separated and analyzed individually. Elution is performed stepwise with PBS containing 2.5 % BSA with: 1 ) 0.5 ml = void volume, 2) 1 ml = high molecular weight fraction considered as intact target molecule, and 3) 1.5 ml = low molecular weight fraction considered as catabolites. The fractions are analyzed anew using the automated gamma counter. Samples from the two populations are compared and assessed in terms of the capture molecule's effect on the half life of the target molecule. For instance, higher serum levels of the target molecule in the population co-administered with the capture molecule, compared to in the control population, indicates an extended circulation time of the target molecule achieved by a pH resistant binding to the capture molecule within the acidic endosome and by albumin- FcRn mediated recycling. Alternatively, if the target molecule is <60 kDa, the half life calculated in a pharmacokinetic analysis is compared to the half life limited by renal clearance.

Example 7: Exploiting an albumin binding capture molecule to regulate the plasma levels of active human growth hormone

The human growth hormone (hGH) is produced in the pituitary gland and functions to regulate tissue growth and metabolism, mainly by stimulating the production of IGF-I and IGF-II (insulin-like growth factors). hGH exists in several isoforms, the predominant form being a 22 kDa protein that can cause medical disorders both when it is hypersecreted and hyposecreted. For instance, increased secretion may cause insulin resistance, hyperglycemia, pituitary tumors and growth deficiencies such as acromegaly and giantism, whereas reduced secretion may result in growth deficiencies and dwarfism (Rodriguez et al Human Genet, 122: 1 -21 , 2007). Therefore, this is a target molecule that is interesting both in an elimination and an extended half life application as described in the present invention. In an elimination application, the therapeutic effect would be enhanced by a simultaneous functional impairment, for instance by interfering with GH receptor interaction. Each hGH contains two distinct sites for cooperative binding of two receptors (de Vos et al, Science 255:306-312, 1992). Blocking either of these sites may prevent GH receptor dimerisation and subsequent inhibition of IGF production. The FDA approved Pegvisomant (Somavert®, Pfizer; PEGylated recombinant hGH with a single Gly120Arg mutation) was shown to prevent such a dimerisation and is successfully used to treat acromegaly. There are also several recombinant forms of hGH (under the generic name Somatropin) on the market, which are used as GH replacement drugs to treat patients with growth failure, Turners syndrome, Prader-Willi syndrome, AIDS wasting etc (Leader et al, Nat Rev Drug Discov 7:21 -39, 2008). A capture molecule developed as described herein may be used in combination with such drugs to extend their half lives which would allow less frequent administration and thus reduced risk of administration-related infections as well as discomfort for patients.

Materials and methods

Recombinantly produced full-length hGH (Sigma-Aldhch, cat no S4776) is biotinylated and used in a phage display selection as described in Examples 2 and/or 3. Candidate capture molecules are evaluated in terms of their pH sensitive interaction with the target molecule by biosensor analysis as described in Example 4. Successful candidates are further assessed in terms of their ability to interfere with GH receptor interactions. This is performed by a competition assay, for instance by biosensor analysis using a Biacore2000 instrument (Biacore). The hGH is immobilized on a CM-5 chip (Biacore) by amine coupling chemistry according to the manufacturer's instructions. HBS- EP, pH 7.4, is used as running buffer for the analysis. Injections with ligands are performed as follows: a) hGH receptor, b) the candidate capture molecule c) hGH receptor followed by the candidate capture molecule. Alternatively, the hGH receptor is immobilized on the chip and ligands are injected as follows: 1 ) hGH, 2) the candidate capture molecule (control experiment), and 3) a premix of hGH and the candidate capture molecule. The sensograms are evaluated as being additive or displaying competition / displacement events. Promising candidates are defined as follows: for an extended half life application the capture molecules have a K ₀ of no more than 1 x 10 ^~6 for hGH at pH 5.5, and do not interfere with the interaction between hGH and the hGH receptor; for an elimination application the capture molecules have a K ₀ value of no more than 1 x 10 ^~7 M for hGH at pH 7.4 and with no detectable binding at pH 5.5, and when pH is changed from 7.4 to 5.5 the capture molecule rapidly dissociates from hGH. Ideally, capture molecules in the latter case also prevent binding of hGH to its receptor, by blocking one of the two interaction sites. Such candidates are further assessed in terms of their half life in vitro and in vivo as described in Example 5 and 6. In addition, the pharmacodynamic effect in vivo may be investigated for instance by analyzing the serum IGF-1 levels in normal mice injected with the capture molecule or vehicle only; or study of the growth effects in hypophysectomized rats treated with the capture molecule or vehicle only.

Example 8: Exploiting an albumin binding capture molecule to regulate the plasma levels of active TGF31

Transforming growth factor beta (TGFβ) is a family of ubiquitously expressed cytokines that regulate the proliferation and differentiation of cells and are important for embryonic development, wound healing and angiogenesis. Activation of the TβRII/TβRI receptor complex predominantly induces the SMAD signaling pathway, a major regulator of transcription, but activation of other pathways such PI3K (phosphatidylinositol 3-kinase) and MAPK (mitogen-activated protein kinase) signaling has also been reported (reviewed in Gordon et al, Biochim Biophys Acta 1782:197-228, 2008). TGFβ has been implicated in both tumor suppressor and tumor promoter functions. Reduced serum levels of TGFβ are found in patients with arthrosclerosis, autoimmune and inflammatory diseases, whereas elevated levels may promote metastasis formation, stimulate angiogenesis and cause fibrosis (Blobe et al, N Engl J Med 342:1350-1358, 2000). Thus, TGFβ is a molecule that may be targeted both in extended half life and elimination applications. The role of TGFβ in tumor development is of particular interest. Overexpression of TGFβ is found in most cancers, sometimes elevated by autocrine signaling, and high serum levels of the protein is associated with recurrent metastasis and poor prognosis. Furthermore, anticancer treatments such as radiation and chemotherapy may increase the serum levels of TGFβ and thus accelerate tumor progression (Biswas et al, J Clin Invest 117:1305-1313, 2007).

In an increased elimination application, the therapeutic effect may be enhanced if the capture molecule also blocks the TGFβ interaction with TβRII (which after binding to TGFβ recruits TβRI). To achieve this, a separate phage selection using only the receptor-interacting region of TGFβ will be pursued in parallel. Three isoforms of TGFβ have been identified. These are produced as a precursor from which the C-terminal 112 amino acids are cleaved and the polypeptides dimerise to form a 25 kDa active protein. The TβRII-interacting region of TGFβ has been mapped to the C-terminal 83-112 amino acids and in particular, residues 91-96 were identified as key residues (Qian et al, J. Biol. Chem, 29:30656).

Materials and methods

Here, focus is on the TGFβi isoform, although a capture molecule simultaneously targeting isoform 2 and 3 may be equally, or even more, interesting. Recombinantly produced human TGFβ1 i-n2 (purchased from R&D systems cat. no. 100-B) and a synthetic peptide, TGFβ1 _84- n2, are biotinylated and used for phage display selection as described in Example 2 and/or 3. Candidate capture molecules are evaluated in terms of their pH sensitive interaction with the TGFβi I-H ₂ by biosensor analysis as described in Example 4. Successful candidates are further assessed in terms of their ability to interfere with the TβRII receptor interaction in a similar manner as described in Example 7 and promising candidates are selected and tested in vitro and in vivo as outlined in Example 5 and 6. In addition, the pharmacodynamic effect in vivo may be investigated for instance by comparing the tumor progression in radiation treated mice injected with the capture molecule or vehicle only.