US20160052998A1

US20160052998A1 - Anti-serum albumin binding variants

Info

Publication number: US20160052998A1
Application number: US14/633,463
Authority: US
Inventors: Haren Arulanantham; Thil Batuwangala; Elena De Angelis; Carolyn Enever; Haiqun Liu; Oliver Schon
Original assignee: GlaxoSmithKline Intellectual Property Development Ltd
Current assignee: GlaxoSmithKline Intellectual Property Development Ltd
Priority date: 2010-08-13
Filing date: 2015-02-27
Publication date: 2016-02-25
Also published as: EP2603522A1; US9012609B2; US20130202597A1; JP2013538566A; WO2012020143A1

Abstract

The invention relates to improved variants of the anti-serum albumin immunoglobulin single variable domains, as well as ligands and drug conjugates comprising such variants, compositions, nucleic acids, vectors and hosts.

Description

The invention relates to improved variants of anti-serum albumin immunoglobulin single variable domains, as well as ligands and drug conjugates comprising such variants, compositions, nucleic acids, vectors and hosts. The invention also relates to the identification of an epitope in serum albumin that is bound by these anti-serum albumin immunoglobulin single variable domains and the specific amino acid residues within those anti-serum albumin immunoglobulin single variable domains that make contact with serum albumin.

BACKGROUND OF THE INVENTION

WO04003019 and WO2008/096158 disclose anti-serum albumin (SA) binding moieties, such as anti-SA immunoglobulin single variable domains (dAbs), which have therapeutically-useful half-lives. These documents disclose monomer anti-SA dAbs as well as multi-specific ligands comprising such dAbs, e.g., ligands comprising an anti-SA dAb and a dAb that specifically binds a target antigen, such as TNFR1. Binding moieties are disclosed that specifically bind serum albumins from more than one species, e.g. human/mouse cross-reactive anti-SA dAbs.
WO05118642 and WO2006/059106 disclose the concept of conjugating or associating an anti-SA binding moiety, such as an anti-SA immunoglobulin single variable domain, to a drug, in order to increase the half-life of the drug. Protein, peptide and NCE (new chemical entity) drugs are disclosed and exemplified. WO2006/059106 discloses the use of this concept to increase the half-life of insulinotropic agents, e.g., incretin hormones such as glucagon-like peptide (GLP)-1.
Reference is also made to Holt et al, “Anti-Serum albumin domain antibodies for extending the half-lives of short lived drugs”, Protein Engineering, Design & Selection, vol 21, no 5, pp 283-288, 2008.
WO2008/096158 discloses the molecules given the name DOM7h-11 and DOM7h-14, which are good anti-SA dAbs. PCT/EP2010/060112 describes V_HAlbudAbs and affinity matured derivatives thereof. It would be desirable to provide improved dAbs that are variants of DOM7h-11 or DOM7h-14, or improved V_HAlbudAbs™, and that specifically bind serum albumin, preferably albumins from human and non-human species, which would provide utility in animal models of disease as well as for human therapy and/or diagnosis. It would also be desirable to provide for the choice between relatively modest- and high-affinity anti-SA binding moieties (dAbs). Such moieties could be linked to drugs, the anti-SA binding moiety being chosen according to the contemplated end-application. This would allow the drug to be better tailored to treating and/or preventing chronic or acute indications, depending upon the choice of anti-SA binding moiety. It would also be desirable to provide anti-SA dAbs that are monomeric or substantially so in solution. This would especially be advantageous when the anti-SA dAb is linked to a binding moiety, e.g., a dAb, that specifically binds a cell-surface receptor, such as TNFR1, with the aim of antagonizing the receptor. The monomeric state of the anti-SA dAb is useful in reducing the chance of receptor cross-linking, since multimers are less likely to form which could bind and cross-link receptors (e.g., TNFR1) on the cell surface, thus increasing the likelihood of receptor agonism and detrimental receptor signaling.
SA is an abundant plasma protein and human serum albumin (HSA) is known to bind to a number of commonly-used drugs (e.g. warfarin, diazepam, ibuprofen) (as described, for example, by Ghuman et al. J. Mol. Biol. 2005, 353, 38052). It would be advantageous to provide an anti-SA binding moiety which does not interfere with the known HSA-drug interactions.

SUMMARY OF THE INVENTION

Improved anti-SA dAbs are described in PCT/EP2010/052008 and PCT/EP2010/052007. PCT/EP2010/060112 describes V_HAlbudAbs and affinity matured derivatives thereof.
As described herein, binding interactions between anti-SA dAbs and SA have been identified using three different techniques. The present inventors have therefore identified specific interactions between improved anti-SA dAbs and domain II of HSA thus identifying the residues within HSA that are involved in binding by an anti-SA dAb and those residues of an anti-SA dAb that are involved in the binding interaction. The residues from the anti-SA dAbs which interact with SA are set out in Tables 22A and B. Significant interactions are identified in Table 22A while additional residues at the interface are identified in Table 22B. Any one of the residues identified in these tables may provide an interaction with SA. These residues may be modified in order to modify SA binding of the variants.
Accordingly, in a first aspect of the invention, there is provided an anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM 7h-11 (SEQ ID NO: 125) or DOM 7h-14 (SEQ ID NO: 123), or a derivative having an amino acid sequence that is at least 96, 97, 98 or 99% identical to the amino acid sequence of DOM 7h-11 (SEQ ID NO: 125) or DOM7h-14 (SEQ ID NO:123), wherein the variant comprises an amino acid substitution in at least one of positions 28, 29, 30, 31, 32, 36, 46, 49, 50, 51, 53, 67, 68, 90, 91, 92, 93 or 94 of the amino acid sequence. Positions 28, 29, 30, 31, 32, 36, 46, 49, 50, 51, 53, 67, 68, 90, 91, 92, 93 or 94 of the amino acid sequence are those positions in the sequences set out in the cited SEQ ID NOs: and relative to those residues set out in the sequences given in these SEQ ID NOs. Suitably, the variant comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acid mutations. Significant interactions are set out in Table 22A. Accordingly, in one embodiment, the variant comprises an amino acid substitution in at least one of positions 30, 31, 32, 49, 50, 51, 53, 67, 91 or 94 of the amino acid sequence. Suitably, the variant comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations. Such a substitution may be to provide the amino acid that is found at these positions in the anti-SA immunoglobulin single variable domain DOM7h-11-3 (SEQ ID NO:2), DOM7h-11-15 (SEQ ID NO:1) or DOM7h14-10 (SEQ ID NO:83) or an equivalent conservative substitution. Suitably, such a substitution may serve to improve binding affinity to SA. In one embodiment, the variant is not a single variable domain selected from DOM7h-11-3, DOM7h-11-15 or DOM7h-14-10. In another embodiment, the variant is not a single variable domain as described in PCT/EP2010/052008 and PCT/EP2010/052007.
In another aspect, there is provided an anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM 7r-31 (SEQ ID NO: 71) or DOM 7r-92 (SEQ ID NO: 75), or a derivative having an amino acid sequence that is at least 96, 97, 98 or 99% identical to the amino acid sequence of DOM 7r-31 (SEQ ID NO: 71) or DOM 7r-92 (SEQ ID NO: 75), wherein the variant comprises an amino acid substitution in at least one of positions 28, 29, 30, 31, 32, 36, 46, 49, 50, 51, 53, 67, 68, 90, 91, 92, 93 or 94 of the amino acid sequence. In one embodiment, the variant is not a single variable domain DOM7r-92-4. In another embodiment, the variant is not a single variant domain as described in PCT/EP2010/060112.
In another aspect, the invention provides an anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11-3 (SEQ ID NO: 2) or DOM7h-11-15 (SEQ ID NO: 1), or a derivative having an amino acid sequence that is at least 96, 97, 98 or 99% identical to the amino acid sequence of DOM7h-11-3 (SEQ ID NO: 2) or DOM7h-11-15 (SEQ ID NO: 1), wherein the variant comprises an amino acid substitution in at least one of positions 28, 29, 30, 31, 32, 36, 46, 49, 50, 51, 53, 67, 68, 90, 91, 92, 93 or 94 of the amino acid sequence. Suitably, the variant comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acid mutations. In one embodiment of this aspect, the variant comprises an amino acid substitution in at least one of positions 30, 31, 32, 49, 50, 51, 53, 67, 91 or 94 of the amino acid sequence. Suitably, the variant comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations.
Amino acid substitution at any of the residues identified in Tables 22A or 22B may enable modification of the binding properties of an anti-SA dAb. Importantly, substitutions can be made to modify the affinity of binding to SA to achieve the desired affinity for a particular application.
Thus embodiments of any aspect of the invention provide anti-SA dAb variants having good anti-serum albumin affinities. The choice of variant can allow for tailoring of half-life according to the desired therapeutic and/or prophylactic setting. For example, in one embodiment, the affinity of the variant for serum albumin is relatively high, such that the variant would be useful for inclusion in products that find utility in treating and/or preventing chronic or persistent diseases, conditions, toxicity or other chronic indications, for example. In one embodiment, the affinity of the variant for serum albumin is relatively modest, such that the variant would be useful for inclusion in products that find utility in treating and/or preventing acute diseases, conditions, toxicity or other acute indications, for example. In one embodiment, the affinity of the variant for serum albumin is intermediate, such that the variant would be useful for inclusion in products that find utility in treating and/or preventing acute or chronic diseases, conditions, toxicity or other acute or chronic indications, for example.
It is conceivable that a molecule with an appropriately high affinity and specificity for serum albumin would stay in circulation long enough to have the desired therapeutic effect (Tomlinson, Nature Biotechnology 22, 521-522 (2004)). Here, a high affinity anti-SA variant would stay in serum circulation matching that of the species' serum albumin (WO2008096158). Once in circulation, any fused therapeutic agent to the AlbudAb™ variant (an AlbudAb is an anti-serum albumin dAb or immunoglobulin single variable domain), be it NCE, peptide or protein, consequently would be able to act longer on its target and exhibit a longer lasting therapeutic effect. This would allow for targeting chronic or persistent diseases without the need of frequent dosing.
A variant with moderate affinity (but specificity to SA) would only stay in serum circulation for a short time (e.g., for a few hours or a few days) allowing for the specific targeting of therapeutic targets involved in acute diseases by the fused therapeutic agent.
This way it is possible to tailor the anti-SA-containing product to the therapeutic disease area by choosing an anti-SA variant with the appropriate albumin binding affinity and/or serum half-life.
In one embodiment, the invention provides an anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11-15 (SEQ ID NO: 1), or a derivative having an amino acid sequence that is at least 96, 97, 98 or 99% identical to the amino acid sequence of DOM7h-11-15 (SEQ ID NO: 1), comprising an amino acid substitution in at least one of positions Gly30, Thr31, Met32, Leu49, Ala50, Phe51, Arg53, Ser67, Ala91 or His94 of the amino acid sequence.
In one embodiment, there is provided a variant in accordance with any aspect or embodiment of the invention wherein the substitution is a conservative amino acid substitution. Suitable conservative amino acid substitutions are known by those skilled in the art and are exemplified herein in the following text. Suitably, a conservative amino acid substitution will maintain similar contact interactions with SA. Such conservative amino acid substitution may allow similar binding affinities to the parental molecule to be maintained.
Accordingly, in one embodiment, the variant comprises at least one mutation compared to DOM 7h-11-15 selected from the following: Position Gly 30=Pro, Ala
Position Thr 31=Ser
Position Thr 32=Ser
Position Leu 49=Norleucine, Ile, Val, Met, Ala, Phe
Position Trp 50=Tyr, Phe
Position Asn 51=Gln
Position Arg 53=Lys, Gln, Asn
Position Ser 67=Thr, Ala, Cys
Position Ala 91=Val, Leu, Ile
Position His 94=Asn, Gln, Lys, Arg
In another embodiment, there is provided an anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM 7h-11-3 (SEQ ID NO: 2), or a derivative having an amino acid sequence that is at least 96, 97, 98 or 99% identical to the amino acid sequence of DOM 7h-11-3 (SEQ ID NO: 2), wherein the variant comprises an amino acid substitution in at least one of positions Gly30, Thr31, Thr32, Leu49, Trp50, Asn51, Arg53, Ser67, Ala91 or His94 of the amino acid sequence.
Suitably, the variant comprises a conservative substitution such that the variant comprises at least one mutation compared to DOM7h-11-3 selected from the following:
Position Gly30=Pro, Ala
Position Thr31=Ser
Position Thr32=Ser
Position Leu49=Norleucine, Ile, Val, Met, Ala, Phe
Position Trp50=Tyr, Phe
Position Asn51=Gln
Position Arg53=Lys, Gln, Asn
Position Ser67=Thr, Ala, Cys
Position Ala91=Val, Leu, Ile
Position His94=Asn, Gln, Lys, Arg
In another embodiment of any aspect or embodiment of the invention, the substitution is not a conservative substitution. Introducing a non-conservative substitution/mutation at one of the residues known to be involved in binding to SA is one way in which affinity to SA may be decreased or otherwise altered.
In one embodiment of any aspect or embodiment of the invention SA is SA from an animal, e.g., a mammal, e.g., a non-human primate (such as a baboon, rhesus monkey or Cynomolgus monkey), mouse, human, rabbit, rat, dog, cat or pig. In one embodiment SA is human SA (HSA).
In another aspect, the invention provides an HSA binding moiety which binds to an epitope comprising at least part of the interface defined by amino acids 227, 228, 229, 230, 232, 233, 263, 307, 308, 309, 314, 317, 318, 321, 322, 325, 326, 329 and 333 of HSA (wherein sequence is given in SEQ ID NO:81). In one embodiment, the binding moiety binds to an epitope comprising at least part of the interface defined by amino acids 228, 230, 308, 309, 317, 318, 321, 322, 325, 326 and 329 of HSA. The residues of HSA which interact with the anti-HSA binding moieties exemplified by anti-SA dAbs herein are set out in Tables 22A and B. Significant interactions are identified in Table 22A while additional residues at the interface are identified in Table 22B. Any one of the residues identified in these tables may provide an interaction between HSA and the HSA binding moiety.
Suitably, the binding moiety may comprise amino acids identified in SEQ ID NOs: 1 or 2 at positions 28, 29, 30, 31, 32, 36, 46, 49, 50, 51, 53, 67, 68, 90, 91, 92, 93 or 94 of the amino acid sequence wherein these amino acids enable binding to SA. However, these amino acids may be part of a domain which is a derivative of a non-immunoglobulin protein scaffold. In one embodiment, the binding moiety is an antibody. Suitably, the binding moiety is an anti-SA immunoglobulin single variable domain antibody.
In one embodiment the variant or binding moiety in accordance with any aspect or embodiment of the invention comprises a binding site that specifically binds human SA with a dissociation constant (KD) of from about 0.1 to about 10000 nM, optionally from about 1 to about 6000 nM, as determined by surface plasmon resonance. In another embodiment, the variant or binding moiety in accordance with any aspect or embodiment of the invention comprises a binding site that specifically binds human SA with an off-rate constant (K_d) of from about 1.5×10⁻⁴to about 0.1 sec⁻¹, optionally from about 3×10⁻⁴to about 0.1 sec⁻¹as determined by surface plasmon resonance. In another embodiment the variant or binding moiety in accordance with any aspect or embodiment of the invention comprises a binding site that specifically binds human SA with an on-rate constant (K_a) of from about 2×10⁶to about 1×10⁴M⁻¹sec⁻¹, optionally from about 1×10⁶to about 2×10⁴M⁻¹sec⁻¹as determined by surface plasmon resonance. In a further embodiment, the variant or binding moiety in accordance with any aspect or embodiment of the invention comprises a binding site that specifically binds Cynomolgus monkey SA with a dissociation constant (KD) of from about 0.1 to about 10000 nM, optionally from about 1 to about 6000 nM, as determined by surface plasmon resonance. In yet a further embodiment, the variant or binding moiety in accordance with any aspect or embodiment of the invention comprises a binding site that specifically binds Cynomolgus monkey SA with an off-rate constant (K_d) of from about 1.5×10⁻⁴to about 0.1 sec⁻¹, optionally from about 3×10⁻⁴to about 0.1 sec⁻¹as determined by surface plasmon resonance. Another embodiment provides a variant or binding moiety in accordance with any aspect or embodiment of the invention, wherein the variant comprises a binding site that specifically binds Cynomolgus monkey SA with an on-rate constant (K_a) of from about 2×10⁶to about 1×10⁴M⁻¹sec⁻¹, optionally from about 1×10⁶to about 5×10³M⁻¹sec⁻¹as determined by surface plasmon resonance.
An aspect of the invention provides a multispecific ligand comprising any anti-SA variant or SA-binding moiety as described above and a binding moiety that specifically binds a target antigen other than SA.
An aspect of the invention provides fusion proteins, conjugates or compositions comprising any variant or binding moiety in accordance with the invention. For example, the invention provides e.g., a fusion protein or fusion with a peptide or NCE (new chemical entity) drug, comprising a polypeptide, protein, peptide or NCE drug fused or conjugated (for an NCE) to any variant or binding moiety as described above. In one embodiment, the variant or binding moiety gives only a modest drop in affinity when fused or conjugated to a partner making them useful in fusion products. An aspect of the invention provides a composition comprising a variant, fusion protein or ligand of any preceding aspect and a pharmaceutically acceptable diluent, carrier, excipient or vehicle.
Another aspect of the invention provides a nucleic acid comprising a nucleotide sequence encoding a variant, binding moiety, multispecific ligand or fusion protein in accordance with any aspect or embodiment of the invention.
Another aspect provides a nucleic acid comprising the nucleotide sequence of a DOM7h-11, DOM7h-14, DOM7h-11-3 or DOM7h-11-15 variant in accordance with the invention or a nucleotide sequence that is at least 80% identical to said selected sequence. Further aspects provide a vector comprising a nucleic acid of the invention and an isolated host cell comprising such a vector.
An aspect of the invention provides a method of treating or preventing a disease or disorder in a patient, comprising administering at least one dose of a variant or binding moiety according to any aspect or embodiment of the invention to said patient. The invention further provides a variant or binding moiety in accordance with the present invention for use as a medicament.
In another aspect of the invention there is provided a method for affinity maturation of an anti-SA immunoglobulin single variable domain comprising taking an anti-SA immunoglobulin single variable domain and introducing a mutation at an amino acid at any one of positions 28, 29, 30, 31, 32, 36, 46, 49, 50, 51, 53, 67, 68, 90, 91, 92, 93 or 94 of the amino acid sequence of an anti-SA immunoglobulin single variable domain. In one embodiment the anti-SA immunoglobulin single variable domain is, or is derived from, the amino acid sequence of DOM7h-11 or DOM7h-14. Methods for obtaining anti-SA immunoglobulin single variable domain molecules are described, for example, in PCT/EP2010/052008 and PCT/EP2010/052007. In one embodiment, the maturation may be in silico. Suitable in silico methods are described, for example, in Barderas et al. (2008), PNAS, 105, 26, p. 9029-9034. Preferably, the method comprises introducing a mutation at any one of the amino acids at positions 30, 31, 32, 49, 50, 51, 53, 67, 91 or 94 or the amino acid sequence.
Another aspect provides a method of modifying the binding affinity of an anti-SA immunoglobulin single variable domain comprising mutating an amino acid at any one of positions 30, 31, 32, 49, 50, 51, 53, 67, 91 or 94 of the amino acid sequence of an anti-SA immunoglobulin single variable domain. Suitably the anti-SA immunoglobulin single variable domain is a DOM7h-11 or DOM7h-14 derivative. Mutations may be to introduce conservative or non-conservative amino acid substitutions as described above.
In these aspects, suitably the mutation at any one of these positions is chosen to modify the affinity of binding to SA of the matured sequences when compared to the parental anti-SA immunoglobulin single variable domain.
The present inventors have identified a specific region of Serum Albumin that can be bound by an anti-SA binding moiety. Advantageously, this region is one which can be bound by a binding moiety, serving to enhance the half life of the bound moiety whilst not blocking any of the known drug binding sites such that the binding interaction does not have an effect on the other drug-binding properties of Serum Albumin. The binding portion identified can be used to identify binding moieties that preferentially bind to this region.
Accordingly, in another aspect of the invention, there is provided a method of identifying SA binding moieties comprising taking a portion of HSA defined by amino acids 213-229, 231-238, 321-331, 334-342 or 348-357; or 213-219, 222-228, 231-238, 311-218, 321-324, 329-333 or 347-357; or 321-326 or 329-331 wherein reference to amino acid residues are references to those amino acids set out in SEQ ID NO: 81, and using said portions in a binding assay or screen. In another aspect there is provided a method for generating an anti-HSA binding agent comprising taking a portion of HSA defined by amino acids 213-229, 231-238, 321-331, 334-342 or 348-357; or 213-219, 222-228, 231-238, 311-218, 321-324, 329-333 or 347-357; or 321-326 or 329-331 wherein reference to amino acid residues are references to those amino acids set out in SEQ ID NO: 81, and using these portions in a screen or assay.
Suitably an anti-HSA binding moiety may be derived using a part of HSA comprising the binding epitope described herein. In one embodiment, the method comprises providing an HSA polypeptide comprising at least part of the binding interface defined by amino acids 228, 230, 308, 309, 317, 318, 321, 322, 325, 326 and 329 of HSA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Amino-acid sequence alignment for DOM7h-11 variant dAbs. A “.” at a particular position indicates the same amino as found in DOM7h-11 at that position. The CDRs are indicated by underlining and bold text (the first underlined sequence is CDR1, the second underlined sequence is CDR2 and the third underlined sequence is CDR3). FIG. 2: Kinetic parameters of DOM7h-11 variants. KD units=nM; Kd units=sec⁻¹; Ka units=M⁻¹sec⁻¹. The notation A e−B means A×10^−Band C e D means C×10^D. The overall kinetic ranges in various species, as supported by the examples below, are indicated. Optional ranges are also provided for use in particular therapeutic settings (acute or chronic indications, conditions or diseases and “intermediate” for use in both chronic and acute settings). High affinity dAbs and products comprising these are useful for chronic settings. Medium affinity dAbs and products comprising these are useful for intermediate settings. Low affinity dAbs and products comprising these are useful for acute settings. The affinity in this respect is the affinity for serum albumin. Various example anti-serum dAbs and fusion proteins are listed, and these support the ranges disclosed. Many of the examples have favourable kinetics in human and one or more non-human animals (e.g., in human and Cynomolgus monkey and/or mouse). Choice of dAb or product comprising this can be tailored, according to the invention, depending on the setting (e.g., chronic or acute) to be treated therapeutically.
FIG. 3: Sequence segments of HSA identified as possible DOM7h-11-3 epitopes from H/D exchange data
FIG. 4: Overall structure of HSA in complex with DOM7h11-15. HSA and DOM7h11-15 are depicted schematically in ribbon representation. Chains A and C are HSA; Chain B and D are DOM7h-11-15. FIG. 4A shows the asymmetric unit; FIG. 4B shows biologically relevant complex in 3 different orientations; FIG. 4C shows electron density map for DOM7h-11-15 calculated from phases from the final model contoured at 2.06.
FIG. 5: Grid showing contacting residues between HSA and DOM7h-11-15.
FIG. 6: Alignment of DOM7h-11 lineage AlbudAbs.
FIG. 7: Detail of interactions between HSA and DOM7h-11-15.
FIG. 8: Comparison of epitope data from orthogonal techniques. HSA is rendered in surface representation and dark patches depict epitope regions identified using each technique.
FIG. 9: Positions of lipid carrier pockets relative to DOM7h-11-15 binding site. The HSA backbone is drawn in tube representation; lipid molecules are dark spheres; electron density for DOM7h-11-15 is drawn in dark mesh. 1E7G.pdb was structurally aligned to the peptide backbone of the HSA/DOM7h-11-15 crystal structure to superimpose lipid moieties to carrier pockets (FA1-FA7: fatty acid binding sites).
FIG. 10: Nucleic and amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION

Within this specification the invention has been described, with reference to embodiments, in a way which enables a clear and concise specification to be written. It is intended and should be appreciated that embodiments may be variously combined or separated without parting from the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4^thEd, John Wiley & Sons, Inc. which are incorporated herein by reference) and chemical methods.
A “patient” is any animal, e.g., a mammal, e.g., a non-human primate (such as a baboon, rhesus monkey or Cynomolgus monkey), mouse, human, rabbit, rat, dog, cat or pig. In one embodiment, the patient is a human.
As used herein an antibody refers to IgG, IgM, IgA, IgD or IgE or a fragment (such as a Fab, Fab′, F(ab′)₂, Fv, disulphide linked Fv, scFv, closed conformation multispecific antibody, disulphide-linked scFv, diabody) whether derived from any species naturally producing an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria.
As used herein, “antibody format” refers to any suitable polypeptide structure in which one or more antibody variable domains can be incorporated so as to confer binding specificity for antigen on the structure. A variety of suitable antibody formats are known in the art, such as, chimeric antibodies, humanized antibodies, human antibodies, single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy chains and/or light chains, antigen-binding fragments of any of the foregoing (e.g., a Fv fragment (e.g., single chain Fv (scFv), a disulfide bonded Fv), a Fab fragment, a Fab′ fragment, a F(ab′)₂fragment), a single antibody variable domain (e.g., a dAb, V_H, V_HH, V_L), and modified versions of any of the foregoing (e.g., modified by the covalent attachment of polyethylene glycol or other suitable polymer or a humanized V_HH).
The phrase “immunoglobulin single variable domain” refers to an antibody variable domain (V_H, V_HH, V_L) that specifically binds an antigen or epitope independently of different V regions or domains. An immunoglobulin single variable domain can be present in a format (e.g., homo- or hetero-multimer) with other variable regions or variable domains where the other regions or domains are not required for antigen binding by the single immunoglobulin variable domain (i.e., where the immunoglobulin single variable domain binds antigen independently of the additional variable domains). A “domain antibody” or “dAb” is the same as an “immunoglobulin single variable domain” as the term is used herein. A “single immunoglobulin variable domain” is the same as an “immunoglobulin single variable domain” as the term is used herein. A “single antibody variable domain” or an “antibody single variable domain” is the same as an “immunoglobulin single variable domain” as the term is used herein. An immunoglobulin single variable domain is in one embodiment a human antibody variable domain, but also includes single antibody variable domains from other species such as rodent (for example, as disclosed in WO 00/29004, the contents of which are incorporated herein by reference in their entirety), nurse shark and Camelid V_HHdAbs. Camelid V_HHare immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. The V_HHmay be humanized.
A “domain” is a folded protein structure which has tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins and, in many cases, may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain. A “single antibody variable domain” is a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains and modified variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain.
In the instant application, the term “prevention” and “preventing” involves administration of the protective composition prior to the induction of the disease or condition. “Treatment” and “treating” involves administration of the protective composition after disease or condition symptoms become manifest. “Suppression” or “suppressing” refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease or condition.
As used herein, the term “dose” refers to the quantity of ligand administered to a subject all at one time (unit dose), or in two or more administrations over a defined time interval. For example, dose can refer to the quantity of ligand (e.g., ligand comprising an immunoglobulin single variable domain that binds target antigen) administered to a subject over the course of one day (24 hours) (daily dose), two days, one week, two weeks, three weeks or one or more months (e.g., by a single administration, or by two or more administrations). The interval between doses can be any desired amount of time. The term “pharmaceutically effective” when referring to a dose means sufficient amount of the ligand, domain or pharmaceutically active agent to provide the desired effect. The amount that is “effective” will vary from subject to subject, depending on the age and general condition of the individual, the particular drug or pharmaceutically active agent and the like. Thus, it is not always possible to specify an exact “effective” amount applicable for all patients. However, an appropriate “effective” dose in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
Methods for pharmacokinetic analysis and determination of ligand (e.g., single variable domain, fusion protein or multi-specific ligand) half-life will be familiar to those skilled in the art. Details may be found in Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al, Pharmacokinetic analysis: A Practical Approach (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi & D Perron, published by Marcel Dekker, 2^ndRev. ex edition (1982), which describes pharmacokinetic parameters such as t alpha and t beta half lives and area under the curve (AUC). Optionally, all pharmacokinetic parameters and values quoted herein are to be read as being values in a human. Optionally, all pharmacokinetic parameters and values quoted herein are to be read as being values in a mouse or rat or Cynomolgus monkey.
Half lives (t½ alpha and t½ beta) and AUC can be determined from a curve of serum concentration of ligand against time. The WinNonlin analysis package, e.g. version 5.1 (available from Pharsight Corp., Mountain View, Calif. 94040, USA) can be used, for example, to model the curve. When two-compartment modeling is used, in a first phase (the alpha phase) the ligand is undergoing mainly distribution in the patient, with some elimination. A second phase (beta phase) is the phase when the ligand has been distributed and the serum concentration is decreasing as the ligand is cleared from the patient. The t alpha half life is the half life of the first phase and the t beta half life is the half life of the second phase. Thus, in one embodiment, in the context of the present invention, the variable domain, fusion protein or ligand has a t alpha half life in the range of (or of about) 15 minutes or more. In one embodiment, the lower end of the range is (or is about) 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition, or alternatively, the variable domain, fusion protein or ligand according to the invention will have a t alpha half life in the range of up to and including 12 hours (or about 12 hours). In one embodiment, the upper end of the range is (or is about) 11, 10, 9, 8, 7, 6 or 5 hours. An example of a suitable range is (or is about) 1 to 6 hours, 2 to 5 hours or 3 to 4 hours.
In one embodiment, the present invention provides the variable domain, fusion protein or ligand according to the invention has a t beta half life in the range of (or of about) 2.5 hours or more. In one embodiment, the lower end of the range is (or is about) 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours, or 12 hours. In addition, or alternatively, the t beta half life is (or is about) up to and including 21 or 25 days. In one embodiment, the upper end of the range is (or is about) 12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days, 19 days, 20 days, 21 days or 22 days. For example, the variable domain, fusion protein or ligand according to the invention will have a t beta half life in the range 12 to 60 hours (or about 12 to 60 hours). In a further embodiment, it will be in the range 12 to 48 hours (or about 12 to 48 hours). In a further embodiment still, it will be in the range 12 to 26 hours (or about 12 to 26 hours).
As an alternative to using two-compartment modeling, the skilled person will be familiar with the use of non-compartmental modeling, which can be used to determine terminal half-lives (in this respect, the term “terminal half-life” as used herein means a terminal half-life determined using non-compartmental modeling). The WinNonlin analysis package, e.g. version 5.1 (available from Pharsight Corp., Mountain View, Calif. 94040, USA) can be used, for example, to model the curve in this way. In this instance, in one embodiment the single variable domain, fusion protein or ligand has a terminal half life of at least (or at least about) 8 hours, 10 hours, 12 hours, 15 hours, 28 hours, 20 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days or 25 days. In one embodiment, the upper end of this range is (or is about) 24 hours, 48 hours, 60 hours or 72 hours or 120 hours. For example, the terminal half-life is (or is about) from 8 hours to 60 hours, or 8 hours to 48 hours or 12 to 120 hours, e.g., in man.
In addition, or alternatively to the above criteria, the variable domain, fusion protein or ligand according to the invention has an AUC value (area under the curve) in the range of (or of about) 1 mg·min/ml or more. In one embodiment, the lower end of the range is (or is about) 5, 10, 15, 20, 30, 100, 200 or 300 mg·min/ml. In addition, or alternatively, the variable domain, fusion protein or ligand according to the invention has an AUC in the range of (or of about) up to 600 mg·min/ml. In one embodiment, the upper end of the range is (or is about) 500, 400, 300, 200, 150, 100, 75 or 50 mg·min/ml. Advantageously the variable domain, fusion protein or ligand will have an AUC in (or about in) the range selected from the group consisting of the following: 15 to 150 mg·min/ml, 15 to 100 mg·min/ml, 15 to 75 mg·min/ml, and 15 to 50 mg·min/ml.
“Surface Plasmon Resonance”: Competition assays can be used to determine if a specific antigen or epitope, such as human serum albumin, competes with another antigen or epitope, such as cynomolgus serum albumin, for binding to a serum albumin binding ligand described herein, such as a specific dAb. Similarly competition assays can be used to determine if a first ligand, such as dAb, competes with a second ligand such as a dAb for binding to a target antigen or epitope. The term “competes” as used herein refers to substance, such as a molecule, compound, preferably a protein, which is able to interfere to any extent with the specific binding interaction between two or more molecules. The phrase “does not competitively inhibit” means that substance, such as a molecule, compound, preferably a protein, does not interfere to any measurable or significant extent with the specific binding interaction between two or more molecules. The specific binding interaction between two or more molecules preferably includes the specific binding interaction between a single variable domain and its cognate partner or target. The interfering or competing molecule can be another single variable domain or it can be a molecule that is structurally and/or functionally similar to a cognate partner or target.
The term “binding moiety” refers to a domain that specifically binds an antigen or epitope independently of a different epitope or antigen binding domain. A binding moiety may be a domain antibody (dAb) or may be a domain which is a derivative of a non-immunoglobulin protein scaffold, e.g., a scaffold selected from the group consisting of CTLA-4, lipocalin, SpA, an adnectin, affibody, an avimer, GroEI, transferrin, GroES and fibronectin, which binds to a ligand other than the natural ligand (in the case of the present invention, the moiety binds serum albumin). See WO2008/096158, which discloses examples of protein scaffolds and methods for selecting antigen or epitope-specific binding domains from repertoires (see Examples 17 to 25). These specific disclosures of WO2008/096158 are expressly incorporated herein by reference as though explicitly written herein and for use with the present invention, and it is contemplated that any part of such disclosure can be incorporated into one or more claims herein). In one aspect, the invention provides a binding moiety comprising the amino acids which interact with and/or enable binding to SA as described herein wherein the interacting amino acids are presented in the context of an alternative or non-immunoglobulin scaffold.
In one embodiment, the mutations at any of the positions identified in accordance with any aspect or embodiment of the invention are mutations to residues as exemplified in the Examples section herein. In another embodiment, mutations are to conservative amino acids substitutions of the exemplified residues.
Conservative amino acid substitutions are well know to the person skilled in the art and are exemplified by the following table:


Amino Acid
Substitution
Original Residues	Exemplary Substitutions	Preferred Substitutions

Ala	Val, Leu, Ile	Val
Arg	Lys, Gln, Asn	Lys
Asn	Gln	Gln
Asp	Glu	Glu
Cys	Ser, Ala	Ser
Gln	Asn	Asn
Glu	Asp	Asp
Gly	Pro, Ala	Ala
His	Asn, Gln, Lys, Arg	Arg
Ile	Leu, Val, Met, Ala, Phe,	Leu
	Norleucine
Leu	Norleucine, Ile, Val, Met,	Ile
	Ala, Phe
Lys	Arg, 1,4 Diamino-	Arg
	butyricAcid, Gln, Asn
Met	Leu, Phe, Ile	Leu
Phe	Leu, Val, Ile, Ala, Tyr	Leu
Pro	Ala	Gly
Ser	Thr, Ala, Cys	Thr
Thr	Ser	Ser
Trp	Tyr, Phe	Tyr
Tyr	Trp, Phe, Thr, Ser	Phe
Val	Ile, Met, Leu, Phe, Ala,	Leu
	Norleucine

Conservative amino acid substitutions may also relate to non-naturally occurring amino acid residues, such as peptidomimetics and other reversed or inverted forms of amino acid moieties which may be incorporated by chemical peptide synthesis.
In one embodiment, the variant comprises one or more of the following kinetic characteristics:—

- (a) The variant comprises a binding site that specifically binds human SA with a dissociation constant (KD) from (or from about) 0.1 to (or to about) 10000 nM, optionally from (or from about) 1 to (or to about) 6000 nM, as determined by surface plasmon resonance;
- (b) The variant comprises a binding site that specifically binds human SA with an off-rate constant (K_d) from (or from about) 1.5×10⁻⁴to (or to about) 0.1 sec⁻¹, optionally from (or from about) 3×10⁻⁴to (or to about) 0.1 sec⁻¹as determined by surface plasmon resonance;
- (c) The variant comprises a binding site that specifically binds human SA with an on-rate constant (K_a) from (or from about) 2×10⁶to (or to about) 1×10⁴M⁻¹sec⁻¹, optionally from (or from about) 1×10⁶to (or to about) 2×10⁴M⁻¹sec⁻¹as determined by surface plasmon resonance;
- (d) The variant comprises a binding site that specifically binds Cynomolgus monkey SA with a dissociation constant (KD) from (or from about) 0.1 to (or to about) 10000 nM, optionally from (or from about) 1 to (or to about) 6000 nM, as determined by surface plasmon resonance;
- (e) The variant of any preceding claim, wherein the variant comprises a binding site that specifically binds Cynomolgus monkey SA with an off-rate constant (K_d) from (or from about) 1.5×10⁻⁴to (or to about) 0.1 sec⁻¹, optionally from (or from about) 3×10⁻⁴to (or to about) 0.1 sec⁻¹as determined by surface plasmon resonance;
- (f) The variant of any preceding claim, wherein the variant comprises a binding site that specifically binds Cynomolgus monkey SA with an on-rate constant (K_a) from (or from about) 2×10⁶to (or to about) 1×10⁴M⁻¹sec⁻¹, optionally from (or from about) 1×10⁶to (or to about) 5×10³M⁻¹sec⁻¹as determined by surface plasmon resonance;
- (g) The variant comprises a binding site that specifically binds rat SA with a dissociation constant (KD) from (or from about) 1 to (or to about) 10000 nM, optionally from (or from about) 20 to (or to about) 6000 nM, as determined by surface plasmon resonance;
- (h) The variant comprises a binding site that specifically binds rat SA with an off-rate constant (K_d) from (or from about) 2×10⁻³to (or to about) 0.15 sec⁻¹, optionally from (or from about) 9×10⁻³to (or to about) 0.14 sec⁻¹as determined by surface plasmon resonance;
- (i) The variant comprises a binding site that specifically binds rat SA with an on-rate constant (K_a) from (or from about) 2×10⁶to (or to about) 1×10⁴M⁻¹sec⁻¹, optionally from (or from about) 1×10⁶to (or to about) 3×10⁴M⁻¹sec⁻¹as determined by surface plasmon resonance;
- (j) The variant comprises a binding site that specifically binds mouse SA with a dissociation constant (KD) from (or from about) 1 to (or to about) 10000 nM as determined by surface plasmon resonance;
- (k) The variant comprises a binding site that specifically binds mouse SA with an off-rate constant (K_d) from (or from about) 2×10⁻³to (or to about) 0.15 sec⁻¹as determined by surface plasmon resonance; and/or
- (l) The variant comprises a binding site that specifically binds mouse SA with an on-rate constant (K_a) from (or from about) 2×10⁶to (or to about) 1×10⁴M⁻¹sec⁻¹, optionally from (or from about) 2×10⁶to (or to about) 1.5×10⁴M⁻¹sec⁻¹as determined by surface plasmon resonance.

Optionally, the variant has

- I: a KD according to (a) and (d), a K_daccording to (b) and (e), and a K_aaccording to (c) and (f); or
- II: a KD according to (a) and (g), a K_daccording to (b) and (h), and a K_aaccording to (c) and (i); or
- III: a KD according to (a) and (j), a K_daccording to (b) and (k), and a K_aaccording to (c) and (I); or
- IV: kinetics according to I and II; or
- V: kinetics according to I and III; or
- VI: kinetics according to I, II and III.

The invention also provides a ligand comprising a variant of any preceding aspect or embodiment of the invention. For example, the ligand can be a dual-specific ligand (see WO04003019 for examples of dual-specific ligands). In one aspect, the invention provides a multispecific ligand comprising an anti-SA variant of any preceding aspect or embodiment of the invention and a binding moiety that specifically binds a target antigen other than SA. The binding moiety can be any binding moiety that specifically binds a target, e.g., the moiety is an antibody, antibody fragment, scFv, Fab, dAb or a binding moiety comprising a non-immunoglobulin protein scaffold. Such moieties are disclosed in detail in WO2008/096158 (see examples 17 to 25, which disclosure is incorporated herein by reference). Examples of non-immunoglobulin scaffolds are CTLA-4, lipocallin, staphylococcal protein A (spA), Affibody™, Avimers™, adnectins, GroEL and fibronectin.
In one embodiment, a linker is provided between the anti-target binding moiety and the anti-SA single variant, the linker comprising the amino acid sequence AST, optionally ASTSGPS. Alternative linkers are described in WO2007085814 (incorporated herein by reference), WO2008/096158 (see the passage at page 135, line 12 to page 140, line 14, which disclosure and all sequences of linkers are expressly incorporated herein by reference as though explicitly written herein and for use with the present invention, and it is contemplated that any part of such disclosure can be incorporated into one or more claims herein) and WO2009/068649.
In one embodiment of the multispecific ligand, the target antigen may be, or be part of, polypeptides, proteins or nucleic acids, which may be naturally occurring or synthetic. In this respect, the ligand of the invention may bind the target antigen and act as an antagonist or agonist (e.g., EPO receptor agonist). One skilled in the art will appreciate that the choice is large and varied. They may be for instance, human or animal proteins, cytokines and growth factors, cytokine receptors, where cytokine receptors include receptors for cytokines, enzymes, co-factors for enzymes or DNA binding proteins.
As used herein, the term “antagonist of Tumor Necrosis Factor Receptor 1 (TNFR1)” or “anti-TNFR1 antagonist” or the like refers to an agent (e.g., a molecule, a compound) which binds TNFR1 and can inhibit a (i.e., one or more) function of TNFR1. For example, an antagonist of TNFR1 can inhibit the binding of TNF alpha to TNFR1 and/or inhibit signal transduction mediated through TNFR1. Accordingly, TNFR1-mediated processes and cellular responses (e.g., TNF alpha-induced cell death in a standard L929 cytotoxicity assay) can be inhibited with an antagonist of TNFR1.
In one embodiment, the multispecific ligand comprises an anti-SA dAb variant of the invention and an anti-TNFR1 binding moiety, e.g., an anti-TNFR1 dAb. Optionally, the ligand has only one anti-TNFR1 binding moiety (e.g., dAb) to reduce the chance of receptor cross-linking. Anti-TNFR1 dAbs are described, for example, in WO2006/038027, WO2007/049017, WO2008149148 and WO2010/081787 (the amino acid sequences of which and the nucleotide sequence of which, as disclosed in those PCT applications, are expressly incorporated herein by reference as though explicitly written herein and for use with the present invention, and it is contemplated that any part of such disclosures can be incorporated into one or more claims herein).
In one embodiment, the ligand of the invention is a fusion protein comprising a variant of the invention fused directly or indirectly to one or more polypeptides. For example, the fusion protein can be a “drug fusion” as disclosed in WO2005/118642 (the disclosure of which is incorporated herein by reference), comprising a variant of the invention and a polypeptide drug as defined in that PCT application.
As used herein, “drug” refers to any compound (e.g., small organic molecule, nucleic acid, polypeptide) that can be administered to an individual to produce a beneficial, therapeutic or diagnostic effect through binding to and/or altering the function of a biological target molecule in the individual. The target molecule can be an endogenous target molecule encoded by the individual's genome (e.g. an enzyme, receptor, growth factor, cytokine encoded by the individual's genome) or an exogenous target molecule encoded by the genome of a pathogen (e. g. an enzyme encoded by the genome of a virus, bacterium, fungus, nematode or other pathogen). Suitable drugs for use in fusion proteins and conjugates comprising an anti-SA dAb variant of the invention are disclosed in WO2005/118642 and WO2006/059106 (the entire disclosures of which are incorporated herein by reference, and including the entire list of specific drugs as though this list were expressly written herein, and it is contemplated that such incorporation provides disclosure of specific drugs for inclusion in claims herein). For example, the drug can be glucagon-like peptide 1 (GLP-1) or a variant, interferon alpha 2b or a variant or exendin-4 or a variant.
In one embodiment, the invention provides a drug conjugate as defined and disclosed in WO2005/118642 and WO2006/059106, wherein the conjugate comprises a variant of the invention. In one example, the drug is covalently linked to the variant (e.g., the variant and the drug are expressed as part of a single polypeptide). Alternatively, in an example, the drug is non-covalently bonded or associated with the variant. The drug can be covalently or noncovalently bonded to the variant directly or indirectly (e.g., through a suitable linker and/or noncovalent binding of complementary binding partners (e.g., biotin and avidin)). When complementary binding partners are employed, one of the binding partners can be covalently bonded to the drug directly or through a suitable linker moiety, and the complementary binding partner can be covalently bonded to the variant directly or through a suitable linker moiety. When the drug is a polypeptide or peptide, the drug composition can be a fusion protein, wherein the polypeptide or peptide, drug and the polypeptide binding moiety are discrete parts (moieties) of a continuous polypeptide chain. As described herein, the polypeptide binding moieties and polypeptide drug moieties can be directly bonded to each other through a peptide bond, or linked through a suitable amino acid, or peptide or polypeptide linker.
A ligand which contains one single variable domain (monomer) variant of the invention or more than one single variable domain (multimer, fusion protein, conjugate, and dual specific ligand as defined herein) which specifically binds to serum albumin, can further comprise one or more entities selected from, but preferably not limited to a label, a tag, an additional single variable domain, a dAb, an antibody, an antibody fragment, a marker and a drug. One or more of these entities can be located at either the COOH terminus or at the N terminus or at both the N terminus and the COOH terminus of the ligand comprising the single variable domain, (either immunoglobulin or non-immunoglobulin single variable domain). One or more of these entities can be located at either the COOH terminus, or the N terminus, or both the N terminus and the COOH terminus of the single variable domain which specifically binds serum albumin of the ligand which contains one single variable domain (monomer) or more than one single variable domains (multimer, fusion protein, conjugate, and dual specific ligand as defined herein). Non-limiting examples of tags which can be positioned at one or both of these termini include a HA, his or a myc tag. The entities, including one or more tags, labels and drugs, can be bound to the ligand which contains one single variable domain (monomer) or more than one single variable domain (multimer, fusion protein, conjugate, and dual specific ligand as defined herein), which binds serum albumin, either directly or through linkers as described above.
Also encompassed herein is an isolated nucleic acid encoding any of the variants, fusion proteins, conjugates or ligands described herein, e.g., a ligand which contains one single variable domain (monomer) variant of the invention or more than one single variable domain (e.g., multimer, fusion protein, conjugate, and dual specific ligand as defined herein) variant which specifically binds to serum albumin, or which specifically binds both human serum albumin and at least one non-human serum albumin, or functionally active fragments thereof. Also encompassed herein is a vector and/or an expression vector, a host cell comprising the vector, e.g., a plant or animal cell and/or cell line transformed with a vector, a method of expressing and/or producing one or more variants, fusion proteins or ligands which contains one single variable domain (monomer) variant or more than one single variable domain variants (e.g., multimer, fusion protein, conjugate, and dual specific ligand as defined herein) which specifically binds to serum albumin, or fragment(s) thereof encoded by said vectors, including in some instances culturing the host cell so that the one or more variants, fusion proteins or ligands or fragments thereof are expressed and optionally recovering the ligand which contains one single variable domain (monomer) or more than one single variable domain (e.g., multimer, fusion protein, conjugate, and dual specific ligand as defined herein) which specifically binds to serum albumin, from the host cell culture medium. Also encompassed are methods of contacting a ligand described herein with serum albumin, including serum albumin and/or non-human serum albumin(s), and/or one or more targets other than serum albumin, where the targets include biologically active molecules, and include animal proteins, cytokines as listed above, and include methods where the contacting is in vitro as well as administering any of the variants, fusion proteins or ligands described herein to an individual host animal or cell in vivo and/or ex vivo. Preferably, administering ligands described herein which comprises a single variable domain (immunoglobulin or non-immunoglobulin) directed to serum albumin and/or non-human serum albumin(s), and one or more domains directed to one or more targets other than serum albumin, will increase the half life, including the T beta and/or terminal half life, of the anti-target ligand. Nucleic acid molecules encoding the variants, fusion proteins or single domain containing ligands or fragments thereof, including functional fragments thereof, are contemplated herein. Vectors encoding the nucleic acid molecules, including but preferably not limited to expression vectors, are contemplated herein, as are host cells from a cell line or organism containing one or more of these expression vectors. Also contemplated are methods of producing any variant, fusion protein or ligand, including, but preferably not limited to any of the aforementioned nucleic acids, vectors and host cells.
An aspect of the invention provides a nucleic acid comprising a nucleotide sequence encoding a variant according to the invention or a multispecific ligand of the invention or fusion protein of the invention or a nucleotide sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to said selected sequence.
An aspect of the invention provides a vector comprising the nucleic acid of the invention. An aspect of the invention provides an isolated host cell comprising the vector.
Reference is made to WO2008/096158 for details of library vector systems, combining single variable domains, characterization of dual specific ligands, structure of dual specific ligands, scaffolds for use in constructing dual specific ligands, uses of anti-serum albumin dAbs and multispecific ligands and half-life-enhanced ligands, and compositions and formulations of comprising anti-serum albumin dAbs. These disclosures are incorporated herein by reference to provide guidance for use with the present invention, including for variants, ligands, fusion proteins, conjugates, nucleic acids, vectors, hosts and compositions of the present invention.

Sequences

TABLE 1

Amino Acid Sequences of DOM7h-11 Variant dAbs

DOM7h-11-15 (SEQ ID NO: 1)

DIQMTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLLIL

AFSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTF

GQGTKVEIKR

DOM7h-11-3 (SEQ ID NO: 2)

DIQMTQSPSSLSASVGDRVTITCRASRPIGTTLSWYQQKPGKAPKLLIL

WNSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTF

GQGTKVEIKR

DOM7h-11-12 (SEQ ID NO: 157)

DIQMTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLLIL

FGSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTF

GQGTKVEIKR

TABLE 2

Nucleotide Sequences of DOM7h-11 Variant dAbs

DOM7h-11-15 (SEQ ID NO: 3)

GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT

CTGTAGGAGA CCGTGTCACC ATCACTTGCC GGGCAAGTCG

TCCGATTGGG ACGATGTTAA GTTGGTACCA GCAGAAACCA

GGGAAAGCCC CTAAGCTCCT GATCCTTGCT TTTTCCCGTT

TGCAAAGTGG GGTCCCATCA CGTTTCAGTG GCAGTGGATC

TGGGACAGAT TTCACTCTCA CCATCAGCAG TCTGCAACCT

GAAGATTTTG CTACGTACTA CTGCGCGCAG GCTGGGACGC

ATCCTACGAC GTTCGGCCAA GGGACCAAGG TGGAAATCAA

ACGG

DOM7h-11-3 (SEQ ID NO: 4)

GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT

CTGTAGGAGA CCGTGTCACC ATCACTTGCC GGGCAAGTCG

TCCGATTGGG ACGACGTTAA GTTGGTACCA GCAGAAACCA

GGGAAAGCCC CTAAGCTCCT GATCCTTTGG AATTCCCGTT

TGCAAAGTGG GGTCCCATCA CGTTTCAGTG GCAGTGGATC

TGGGACAGAT TTCACTCTCA CCATCAGCAG TCTGCAACCT

GAAGATTTTG CTACGTACTA CTGTGCGCAG GCTGGGACGC

ATCCTACGAC GTTCGGCCAA GGGACCAAGG TGGAAATCAA

ACGG

DOM7h-11-12 (SEQ ID NO: 158)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG

ACCGTGTCACCATCACTTGCCGGGCAAGTCGTCCGATTGGGACGATGTT

AAGTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTTG

TTTGGTTCCCGGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTG

GATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGA

TTTTGCTACGTACTACTGTGCGCAGGCTGGGACGCATCCTACGACGTTC

GGCCAA GGGACCAAGGTGGAAATCAAACGG

TABLE 5

Anti-serum albumin dAb (DOM7h) fusions

(used in Rat studies): -
DOM7h-14/Exendin-4 fusion DMS number 7138
Amino acid sequence (SEQ ID NO: 5)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGSGGGGSGGGGSDIQ
MTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMWRSSLQSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCAQGAALPRTFGQGTKVEIKR

Nucleotide sequence (SEQ ID NO: 6)
CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAGGCAGTG
CGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGGGCACCTCCGCCA
TCGGGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGGACA
TCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCAT
CACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTATCTTGGTACCAGCAGAAACCA
GGGAAAGCCCCTAAGCTCCTGATCATGTGGCGTTCCTCGTTGCAAAGTGGGGTCCCA
TCACGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGC
AACCTGAAGATTTTGCTACGTACTACTGTGCTCAGGGTGCGGCGTTGCCTAGGACGTT
CGGCCAAGGGACCAAGGTGGAAATCAAACGG

DOM7h-14-10/Exendin-4 fusion DMS number 7139
Amino acid sequence (SEQ ID NO: 7)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGSGGGGSGGGGSDIQ
MTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMWRSSLQSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQGTKVEIKR

Nucleotide sequence (SEQ ID NO: 8)
CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAGGCAGTG
CGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGGGCACCTCCGCCA
TCGGGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGGACA
TCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCAT
CACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTATCTTGGTACCAGCAGAAACCA
GGGAAAGCCCCTAAGCTCCTGATCATGTGGCGTTCCTCGTTGCAAAGTGGGGTCCCA
TCACGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGC
AACCTGAAGATTTTGCTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTT
CGGCCAAGGGACCAAGGTGGAAATCAAACGG

DOM7h-11/Exendin-4 fusion DMS number 7142
Amino acid sequence (SEQ ID NO: 9)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGSGGGGSGGGGSDIQ
MTQSPSSLSASVGDRVTITCRASRPIGTTLSWYQQKPGKAPKLLIWFGSRLQSGVPSRFS
GSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQGTKVEIKR

Nucleotide sequence (SEQ ID NO: 10)
CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAGGCAGTG
CGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGGGCACCTCCGCCA
TCGGGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGGACA
TCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCAT
CACTTGCCGGGCAAGTCGTCCGATTGGGACGACGTTAAGTTGGTACCAGCAGAAACC
AGGGAAAGCCCCTAAGCTCCTGATCTGGTTTGGTTCCCGGTTGCAAAGTGGGGTCCC
ATCACGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTG
CAACCTGAAGATTTTGCTACGTACTACTGTGCGCAGGCTGGGACGCATCCTACGACGT
TCGGCCAAGGGACCAAGGTGGAAATCAAACGG

DOM7h-11-15/Exendin-4 fusion DMS number 7143
Amino acid sequence (SEQ ID NO: 11)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGSGGGGSGGGGSDIQ
MTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLLILAFSRLQSGVPSRFS
GSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQGTKVEIKR

Nucleotide sequence (SEQ ID NO: 12)
CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAGGCAGTG
CGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGGGCACCTCCGCCA
TCGGGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGGACA
TCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCAT
CACTTGCCGGGCAAGTCGTCCGATTGGGACGATGTTAAGTTGGTACCAGCAGAAACC
AGGGAAAGCCCCTAAGCTCCTGATCCTTGCTTTTTCCCGTTTGCAAAGTGGGGTCCCA
TCACGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGC
AACCTGAAGATTTTGCTACGTACTACTGCGCGCAGGCTGGGACGCATCCTACGACGTT
CGGCCAAGGGACCAAGGTGGAAATCAAACGG

DOM7h14-10/ G4SC-NCE fusion
Amino acid sequence (SEQ ID NO: 13) encoding DOM7h14-10/G4SC
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMWRSSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQGTKVEIKRGGGGSC

The C-terminal cysteine can be linked to a new chemical entity
(pharmaceutical chemical compound, NCE), eg using maleimide linkage.
Nucleotide sequence (SEQ ID NO: 14) encoding DOM7h14-10/G4SC
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCA
CCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTATCTTGGTACCAGCAGAA
ACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGGCGTTCCTCGTTGCAAAGTGGGGT
CCCATCACGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGT
CTGCAACCTGAAGATTTTGCTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGA
CGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGGTGGCGGAGGGGGTTCCTGT

DOM7h14-10/TVAAPSC fusion
Amino acid sequence (SEQ ID NO: 15)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMWRSSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQGTKVEIKRTVAAPSC

The C-terminal cysteine can be linked to a new chemical entity
(pharmaceutical chemical compound, NCE), eg using maleimide linkage.
Nucleotide sequence (SEQ ID NO: 16)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCA
CCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTATCTTGGTACCAGCAGAA
ACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGGCGTTCCTCGTTGCAAAGTGGGGT
CCCATCACGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGT
CTGCAACCTGAAGATTTTGCTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGA
CGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGACCGTCGCTGCTCCATCTTGT

(used in mouse studies): -
DOM7h-11/DOM1m-21-23 fusion DMS number 5515
Amino acid sequence (SEQ ID NO:17)
EVQLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRIDSYGRGT
YYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQFGSNAFDYWGQGTQVT
VSSASTSGPSDIQMTQSPSSLSASVGDRVTITCRASRPIGTTLSWYQQKPGKAPKLLIWFG
SRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQGTKVEIKR

Amino acid plus myc tag sequence (SEQ ID NO: 18)
EVQLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRIDSYGRGT
YYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQFGSNAFDYWGQGTQVT
VSSASTSGPSDIQMTQSPSSLSASVGDRVTITCRASRPIGTTLSWYQQKPGKAPKLLIWFG
SRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQGTKVEIKRAAA
EQKLISEEDLN

Nucleotide sequence (SEQ ID NO: 19)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCG
TCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATGGGGTGGCTCCGC
CAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATTGATTCTTATGGTCGTGGT
ACATACTACGAAGACCCCGTGAAGGGCCGGTTCAGCATCTCCCGCGACAATTCCAAG
AACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCCGTATATTACT
GTGCGAAAATTTCTCAGTTTGGGTCAAATGCGTTTGACTACTGGGGTCAGGGAACCCA
GGTCACCGTCTCGAGCGCTAGCACCAGTGGTCCATCGGACATCCAGATGACCCAGTC
TCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGT
CGTCCGATTGGGACGACGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAG
CTCCTGATCTGGTTTGGTTCCCGGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCA
GTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGC
TACGTACTACTGTGCGCAGGCTGGGACGCATCCTACGACGTTCGGCCAAGGGACCAA
GGTGGAAATCAAACGG

Nucleotide plus myc tag sequence (SEQ ID NO: 20)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCG
TCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATGGGGTGGCTCCGC
CAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATTGATTCTTATGGTCGTGGT
ACATACTACGAAGACCCCGTGAAGGGCCGGTTCAGCATCTCCCGCGACAATTCCAAG
AACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCCGTATATTACT
GTGCGAAAATTTCTCAGTTTGGGTCAAATGCGTTTGACTACTGGGGTCAGGGAACCCA
GGTCACCGTCTCGAGCGCTAGCACCAGTGGTCCATCGGACATCCAGATGACCCAGTC
TCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGT
CGTCCGATTGGGACGACGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAG
CTCCTGATCTGGTTTGGTTCCCGGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCA
GTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGC
TACGTACTACTGTGCGCAGGCTGGGACGCATCCTACGACGTTCGGCCAAGGGACCAA
GGTGGAAATCAAACGGGCGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAA
TTAA

DOM7h-11-15/DOM1m-21-23 fusion DMS number 5517
Amino acid sequence (SEQ ID NO: 21)
EVQLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRIDSYGRGT
YYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQFGSNAFDYWGQGTQVT
VSSASTSGPSDIQMTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLLILAF
SRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQGTKVEIKR

Amino acid plus nucleotide plus myc tag sequence(SEQ ID NO: 22)
EVQLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRIDSYGRGT
YYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQFGSNAFDYWGQGTQVT
VSSASTSGPSDIQMTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLLILAF
SRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQGTKVEIKRAAA
EQKLISEEDLN

Nucleotide sequence (SEQ ID NO: 23)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCG
TCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATGGGGTGGCTCCGC
CAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATTGATTCTTATGGTCGTGGT
ACATACTACGAAGACCCCGTGAAGGGCCGGTTCAGCATCTCCCGCGACAATTCCAAG
AACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCCGTATATTACT
GTGCGAAAATTTCTCAGTTTGGGTCAAATGCGTTTGACTACTGGGGTCAGGGAACCCA
GGTCACCGTCTCGAGCGCTAGCACCAGTGGTCCATCGGACATCCAGATGACCCAGTC
TCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGT
CGTCCGATTGGGACGATGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAG
CTCCTGATCCTTGCTTTTTCCCGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCA
GTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGC
TACGTACTACTGCGCGCAGGCTGGGACGCATCCTACGACGTTCGGCCAAGGGACCAA
GGTGGAAATCAAACGG

Nucleotide plus myc tag sequence (SEQ ID NO: 24)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCG
TCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATGGGGTGGCTCCGC
CAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATTGATTCTTATGGTCGTGGT
ACATACTACGAAGACCCCGTGAAGGGCCGGTTCAGCATCTCCCGCGACAATTCCAAG
AACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCCGTATATTACT
GTGCGAAAATTTCTCAGTTTGGGTCAAATGCGTTTGACTACTGGGGTCAGGGAACCCA
GGTCACCGTCTCGAGCGCTAGCACCAGTGGTCCATCGGACATCCAGATGACCCAGTC
TCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGT
CGTCCGATTGGGACGATGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAG
CTCCTGATCCTTGCTTTTTCCCGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCA
GTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGC
TACGTACTACTGCGCGCAGGCTGGGACGCATCCTACGACGTTCGGCCAAGGGACCAA
GGTGGAAATCAAACGGGCGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAA
TTAA

Where a myc-tagged molecule is indicated in this table, this was the version used in PK studies in the examples. Where no myc-tagged sequences are given, the PK studies in the examples were not done with myc-tagged material, ie, the studies were done with the non-tagged constructs shown.

EXEMPLIFICATION

All numbering in the experimental section is according to Kabat (Kabat, E.A. National Institutes of Health (US) & Columbia University. Sequences of proteins of immunological interest, edn 5 (US Dept. Of Health and Human Services Public Health Service, National Institutes of Health, Bethesda, Md., 1991)).
Derivation of DOM7h-11, DOM7h-14 and DOM7r variants is described.

Example 1

Vk Affinity Maturation

Selections:

HSA (Human Serum Albumin) and RSA (Rat Serum Albumin) antigens were obtained from Sigma (essentially fatty acid free, ˜99% (agarose gel electrophoresis), lyophilized powder Cat. No. A3782 and A6414 respectively)
Biotinylated products of above two antigens were made by using EZ Link Sulfo-NHS-SS-Biotin (Pierce, Cat. No. 21331). Free biotin reagent was removed by passing the samples twice through PD10 desalting column followed by overnight dialysis against 1000× excess volume of PBS at 4° C. Resulting product was tested by mass spec and 1-2 biotins per molecule were observed.

Affinity Maturation Libraries:

Both error-prone and CDR libraries were created using DOM7h-11 and DOM7h-14 parental dAbs (see WO2008/096158 for the sequences of DOM7h-11 and DOM7h-14). The CDR libraries were generated in the pDOM4 vector and the error prone libraries were generated in the pDOM33 vector (to allow for selection with or without protease treatment). Vector pDOM4, is a derivative of the Fd phage vector in which the gene III signal peptide sequence is replaced with the yeast glycolipid anchored surface protein (GAS) signal peptide. It also contains a c-myc tag between the leader sequence and gene III, which puts the gene III back in frame. This leader sequence functions well both in phage display vectors but also in other prokaryotic expression vectors and can be universally used. pDOM33 is a modified version of the pDOM4 vector where the c-myc tag has been removed which renders the dAb-phage fusion resistant to the protease trypsin. This allows the use of trypsin within the phage selection to select for dAbs that are more protease stable (see WO2008149143).
For error-prone maturation libraries, plasmid DNA encoding the dAb to be matured was amplified by PCR, using the GENEMORPH® II RANDOM MUTAGENESIS KIT (random, unique mutagenesis kit, Stratagene). The product was digested with Sal I and Not I and used in a ligation reaction with cut phage vector pDOM33. For the CDR libraries, PCR reactions were performed using degenerate oligonucleotides containing NNK or NNS codons to diversify the required positions in the dAb to be affinity matured. Assembly PCR was then used to generate a full length diversified insert. The insert was digested with Sal I and Not I and used in a ligation reaction with pDOM4 for mutagenesis of multiple residues and pDOM5 for mutagenesis of single residues. The pDOM5 vector is a pUC119-based expression vector where protein expression is driven by the LacZ promoter. A GAS1 leader sequence (see WO 2005/093074) ensures secretion of isolated, soluble dAbs into the periplasm and culture supernatant of E. coli. dAbs are cloned SalI/NotI in this vector, which appends a myc tag at the C-terminus of the dAb. This protocol using SalI and Not I results in inclusion of an ST amino acid sequence at the N-terminus.
The ligation produced by either method was then used to transform E. coli strain TB1 by electroporation and the transformed cells plated on 2×TY agar containing 15 μg/ml tetracycline, yielding library sizes of >5×10⁷clones.
The error-prone libraries had the following average mutation rate and size: DOM7h-11 (2.5 mutations per dAb), size: 6.1×10⁸, DOM7h-14 (2.9 mutations per dAb), size: 5.4×10⁸.
Each CDR library has four amino acid diversity. Two libraries were generated for each of CDRs 1 and 3, and one library for CDR2. The positions diversified within each library are as follows (amino acids based on VK dummy DPK9 sequence):


		Library size

		DOM7h-11	DOM7h-14

1—Q27, S28, S30, S31 (CDR1)	8.8 × 10⁷	5.8 × 10⁷
2—S30, S31, Y32, N34 (CDR1)	4.6 × 10⁸	4.2 × 10⁸
3—Y49, A50, A51, S53 (CDR2)	3.9 × 10⁸	2.4 × 10⁸
4—Q89, S91, Y92, S93 (CDR3)	1.8 × 10⁸	2.5 × 10⁸
5—Y92, Y93, T94, N96 (CDR3)	4.0 × 10⁸	3.3 × 10⁸

Example 2

Selection Strategies

Three phage selection strategies were adopted for Vκ AlbudAb™ (anti-serum albumin dAb) affinity maturation:
1) Selections Against HSA Only:

- Three rounds of selection against HSA were carried out. The error prone libraries and each CDR library were selected as an individual pool in all rounds. The first round of selection was performed against HSA passively coated onto an immunotube at 1 mg/ml. Round 2 was performed against 100 nM HSA and round 3 against 10 nM (CDR selections) or 20 or 100 nM (Error prone selections) HSA, both as soluble selections followed by a fourth round of selection with the error prone libraries against 1.5 nM HSA as a soluble selection. The error prone libraries were eluted with 0.1M glycine pH 2.0 before neutralisation with 1M Tris pH 8.0 and the CDR libraries were eluted with 1 mg/ml trypsin before infection into log phase TG1 cells. The third round of each selection was subcloned into pDOM5 for screening. Soluble selections used biotinylated HSA.

2) Trypsin Selections Against HSA:

- In order to select dAbs with increased protease resistance compared to the parental clone and with potentially improved biophysical properties, trypsin was used in phage selections (see WO2008149143). Four rounds of selection were preformed against HSA. The first round of selection of error prone libraries was performed against passively coated HSA at 1 mg/ml without trypsin; the second round against passively coated HSA at 1 mg/ml with 20 μg/ml trypsin for 1 hour at 37° C.; the third round selection was performed by soluble selection using biotinylated HSA against 100 nM HSA with 20 μg/ml or 100 μg/ml trypsin for 1 hour at 37° C. The final round of selection was performed by soluble selection using biotinylated HSA against 100 nM HSA with 100 μg/ml trypsin overnight at 37° C.

3) Cross-Over Selections Against HSA (Round 1) and RSA (Rounds 2-4):

- The first round selection was carried out against 1 mg/ml passively coated HSA or 1 μM HSA (soluble selection), followed by a further three rounds of soluble selections against biotinylated RSA at concentrations of 1 μM for round 1, 100 nm for round 2 and 20 nM, 10 nM or 1 nM for round 3.

Screening Strategy and Affinity Determination:

In each case after selection a pool of phage DNA from the appropriate round of selection is prepared using a QIAfilter midiprep kit (Qiagen), the DNA is digested using the restriction enzymes Sal1 and Not1 and the enriched V genes are ligated into the corresponding sites in pDOM5 the soluble expression vector which expresses the dAb with a myc tag (see PCT/EP2008/067789). The ligated DNA is used to electro-transform E. coli HB 2151 cells which are then grown overnight on agar plates containing the antibiotic carbenicillin. The resulting colonies are individually assessed for antigen binding. In each case at least 96 clones were tested for binding to HSA, CSA (Cynomolgus monkey Serum Albumin), MSA (mouse serum albumin) and RSA by BIAcore™ (surface plasmon resonance). MSA antigen was obtained from Sigma (essentially fatty acid free, ˜99% (agarose gel electrophoresis), lyophilized powder Cat. No. A3559) and CSA was purified from Cynomolgus serum albumin using prometic blue resin (Amersham). Soluble dAb fragments were produced in bacterial culture in ONEX culture media (Novagen) overnight at 37° C. in 96 well plates. The culture supernatant containing soluble dAb was centrifuged and analysed by BIAcore for binding to high density HSA, CSA, MSA and RSA CM5 chips. Clones were found to bind to all these species of serum albumin by off-rate screening. The clones were sequenced revealing unique dAb sequences.
DOM7h11-15 had 96.3% identity to parent (at the amino acid level). DOM7h-11-3 had 97.2% identity to parent (at the amino acid level).
DOM7h-14-10 had 96.3% identity to parent (at the amino acid level).
dAbs were expressed as bacterial supernatants in 2.5 L shake flasks in Onex media at 30° C. for 48 hrs at 250 rpm. dAbs were purified from the culture media by absorption to protein L agarose followed by elution with 10 mM glycine pH2.0. Binding to HSA, CSA, MSA and RSA by BIAcore was confirmed using purified protein at 3 concentrations 1 μM, 500 nM and 50 nM. To determine the binding affinity (K_D) of the AlbudAbs to each serum albumin; purified dAbs were analysed by BIAcore over albumin concentration range from 5000 nM to 39 nM (5000 nM, 2500 nM, 1250 nM, 625 nM, 312 nM, 156 nM, 78 nM, 39 nM).

TABLE 6

	Affinity (K_D)
AlbudAb	to SA (nM)	Kd	Ka

	Rat
DOM7h-14	60	2.095E−01	4.00E+06
DOM7h-14-10	4	9.640E−03	4.57E+06
DOM 7h-11	2100	1.00E−01	4.80E+04
DOM 7h-11-3	10000	(7.18e−1)	(8.11e3)
	(88000)
DOM 7h-11-15	20	2.10E−02	1.10E+06
	Cyno
DOM 7h-14	66	9.65E−02	1.50E+06
DOM 7h-14-10	9	1.15E−02	1.60E+06
DOM 7h-11	1000	6.82E−01	8.00E+05
DOM 7h-11-3	670	9.6E−02	2.90E+05
	(200)	(1.5e−1)	(7.26e5)
DOM 7h-11-15	3	5.57E−03	5.80E+06
	Mouse
DOM 7h-14	12	4.82E−02	4.10E+06
DOM 7h-14-10	30	3.41E−02	1.29E+06
DOM 7h-11	5000	9.00E−01
DOM 7h-11-3	≧10000	(6.12e−1)	(1.67e4)
	(36000)
DOM 7h-11-15	10	9.40E−03	1.10E+06
	Human
DOM 7h-14	33	4.17E−02	1.43E+06
DOM 7h-14-10	12	1.39E−02	1.50E+06
DOM 7h-11	2800	6.41E−01	7.00E+05
DOM 7h-11-3	32	1.6E−02	6.50E+05
	(130)	(2.35e−2)	(1.86e5)
DOM 7h-11-15	1	1.84E−03	2.00E+06

*: values in brackets were derived from a second, independent SPR experiment.

All DOM7h-14 derived variants are cross-reactive to mouse, rat, human and cyno serum albumin. DOM7h-14-10 has improved affinity to rat, cyno and human serum albumin compared to parent.
DOM7h-11-3 has improved affinity to CSA and HSA. DOM7h-11-15 has improved affinity to RSA, MSA, CSA and HSA.

Example 3

Origins of Key DOM7h-11 Lineage Clones

DOM7h-11-3: From affinity maturation performed against HSA using the CDR2 library (Y49, A50, A51, S53), round 3 output 10 nM HSA
DOM7h-11-15: From cross-over selections performed against HSA as round 1 followed by additional 3 rounds of selections against RSA using the CDR2 library (Y49, A50, A51, S53) at round 3 selection with 1 nM of RSA.

TABLE 7

CDR sequences (according to Kabat; ref. as above)

CDR

AIbudAb	CDR1	CDR2	CDR3

DPK9 Vk dummy	SQSISSYLN	YAASSLQS	QQSYSTPNT
	(SEQ ID NO: 25)	(SEQ ID NO: 26)	(SEQ ID NO: 27)

DOM7h-11	SRPIGTTLS	WFGSRLQS	AQAGTHPTT
	(SEQ ID NO: 28)	(SEQ ID NO: 29)	(SEQ ID NO: 30)

DOM 7h-11-15	SRPIGTMLS	LAFSRLQS	AQAGTHPTT
	(SEQ ID NO: 31)	(SEQ ID NO: 32)	(SEQ ID NO: 33)

DOM 7h-11-3	SRPIGTTLS	LWFSRLQS	AQAGTHPTT
	(SEQ ID NO: 34)	(SEQ ID NO: 35)	(SEQ ID NO: 36)

Example 4

Origins of Key DOM7h-14 Lineage Clones

DOM7h-14-10: From affinity maturation performed against HSA using CDR3 library (Y92, Y93, T94, N96), round 3 output.

TABLE 8

CDR sequences (according to Kabat; ref. as above)

CDR

AIbudAb	CDR1	CDR2	CDR3

DOM 7h-14	SQWIGSQLS	MWRSSLQS	AQGAALPRT
	(SEQ ID NO: 37)	(SEQ ID NO: 38)	(SEQ ID NO: 39)

DOM 7h-14-10	SQWIGSQLS	MWRSSLQS	AQGLRHPKT
	(SEQ ID NO: 40)	(SEQ ID NO: 41)	(SEQ ID NO: 42)

Example 5

Expression and Biophysical Characterisation

The routine bacterial expression level in 2.5 L shake flasks was determined following culture in Onex media at 30° C. for 48 hrs at 250 rpm. The biophysical characteristics were determined by SEC MALLS and DSC.
SEC MALLS (size exclusion chromatography with multi-angle-LASER-light-scattering) is a non-invasive technique for the characterizing of macromolecules in solution. Briefly, proteins (at concentration of 1 mg/mL in buffer Dulbecco's PBS at 0.5 ml/min are separated according to their hydrodynamic properties by size exclusion chromatography (column: TSK3000 from TOSOH Biosciences; S200 from Pharmacia). Following separation, the propensity of the protein to scatter light is measured using a multi-angle-LASER-light-scattering (MALLS) detector. The intensity of the scattered light while protein passes through the detector is measured as a function of angle. This measurement taken together with the protein concentration determined using the refractive index (RI) detector allows calculation of the molar mass using appropriate equations (integral part of the analysis software Astra v.5.3.4.12).
DSC (Differential Scanning calorimetry): briefly, the protein is heated at a constant rate of 180° C./hrs (at 1 mg/mL in PBS) and a detectable heat change associated with thermal denaturation measured. The transition midpoint (_appT_m) is determined, which is described as the temperature where 50% of the protein is in its native conformation and the other 50% is denatured. Here, DSC determined the apparent transition midpoint (appTm) as most of the proteins examined do not fully refold. The higher the Tm, the more stable the molecule. Unfolding curves were analysed by non-2-state equations. The software package used was Origin® v7.0383.

	TABLE 9

		Biophysical parameters

AlbudAb	SEC MALLS	DSC Tm(° C.)

DOM7h-14	M	60
DOM 7h-14-10	M	59
DOM 7h-11	M	66.9-72.2
DOM 7h-11-3	M (95%)*	66.6/70.5
DOM 7h-11-15	M (<5% D)	58.5-60.5

*in one other trial, monomer was primarily seen by SEC MALLS, although lower than 95%

We observed expression levels for all clones in Table 9 in the range from 15 to 119 mg/L in E. coli.
For DOM7h-14 and DOM7h-11 variants, favorable biophysical parameters (monomeric in solution as determined by SEC MALLs and appTm of >55° C. as determined by DSC) and expression levels were maintained during affinity maturation. Monomeric state is advantageous because it avoids dimerisation and the risk of products that may cross-link targets such as cell-surface receptors.

Example 6

Determination of Serum Half Life in Rat, Mouse and Cynomolgus Monkey

AlbudAbs DOM7h-14-10, DOM7h-11 and DOM7h-11-15 were cloned into the pDOM5 vector. For each AlbudAb™, 20-50 mg quantities were expressed in E. coli and purified from bacterial culture supernatant using protein L affinity resin and eluted with 100 mM glycine pH2. The proteins were concentrated to greater than 1 mg/ml, buffer exchanged into PBS and endotoxin depleted using Q spin columns (Vivascience). For Rat pharmacokinetic (PK) analysis, AlbudAbs were dosed as single i.v injections at 2.5 mg/kg using 3 rats per compound. Serum samples were taken at 0.16, 1, 4, 12, 24, 48, 72, 120, 168 hrs. Analysis of serum levels was by anti-myc ELISA as per the method described below.
For Mouse PK, DOM7h-11 and DOM7h-11-15 were dosed as single i.v injections at 2.5 mg/kg per dose group of 3 subjects and serum samples taken at 10 mins; 1 h; 8 h; 24 h; 48 h; 72 h; 96 h. Analysis of serum levels was by anti-myc ELISA as per the method described below.
For Cynomolgus monkey PK DOM7h-14-10 and DOM7h-11-15 were dosed as single i.v injections at 2.5 mg/kg into 3 female Cynomolgus monkeys per dose group and serum samples taken at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, 144, 192, 288, 336, 504 hrs. Analysis of serum levels was by anti-myc ELISA as per the method described below.

Anti-Myc ELISA Method

The AlbudAb concentration in serum was measured by anti-myc ELISA. Briefly, goat anti-myc polyclonal antibody (1:500; Abcam, catalogue number ab9132) was coated overnight onto Nunc 96-well Maxisorp plates and blocked with 5% BSA/PBS+1% Tween. Serum samples were added at a range of dilutions alongside a standard at known concentrations. Bound myc-tagged AlbudAb was then detected using a rabbit polyclonal anti-Vk (1:1000; in-house reagent, bleeds were pooled and protein A purified before use) followed by an anti-rabbit IgG HRP antibody (1:10,000; Sigma, catalogue number A2074). Plates were washed between each stage of the assay with 3×PBS+0.1% Tween20 followed by 3×PBS. TMB (SureBlue TMB 1-Component Microwell Peroxidase Substrate, KPL, catalogue number 52-00-00) was added after the last wash and was allowed to develop. This was stopped with 1M HCl and the signal was then measured using absorbance at 450 nm.
From the raw ELISA data, the concentration of unknown samples was established by interpolation against the standard curve taking into account dilution factors. The mean concentration result from each time point was determined from replicate values and entered into WinNonLin analysis package (e.g. version 5.1 (available from Pharsight Corp., Mountain View, Calif. 94040, USA). The data was fitted using a non-compartmental model, where PK parameters were estimated by the software to give terminal half-lives. Dosing information and time points were selected to reflect the terminal phase of each PK profile.

TABLE 10

Single AlbudAb ™ PK

PK parameters

		Albumin	AUC	CL	t½	Vz
Species	AlbudAb	K_D(nM)	h × μg/ml	ml/h/kg	h	ml/kg

Rat	DOM7h-14*	60
	DOM7h-14-10	4	2134.6	1.2	42.1	71.2
	DOM 7h-11	2100	320.1	7.8	23.3	263.9
	DOM 7h-11-15	20	843.4	3.0	30.3	130.7
mouse	DOM 7h-11	5000	304.7	8.2	18.3	216.8
	DOM 7h-11-15	10	499.2	5.0	33.7	243.4
Cyno	DOM 7h-14*	66			217.5
	DOM 7h-14-10	9	6174.6	0.4	200.8	117.8
	DOM 7h-11*	3300			135.1
	DOM 7h-11-15	3	4195	0.6	198.1	170.3

*Historical data

Pharmacokinetic parameters derived from rat, mouse and cynomolgus monkey studies were fitted using a non-compartmental model. Key: AUC: Area under the curve from dosing time extrapolated to infinity; CL: clearance; t½: is the time during which the blood concentration is halved; Vz: volume of distribution based on the terminal phase.
DOM7h-11-15 has an improved AUC and t½ in rat and mouse compared to parent. DOM7h-11-15 also has an improved AUC and t½ in cyno compared to parent. This improvement in AUC/t½ correlates with an improved in vitro KD to serum albumin.

Example 7

AlbudAb™ IFN Fusions

Cloning and Expression

As well as single AlbudAbs, the affinity matured Vk Albudabs were linked to Interferon alpha 2b (IFNα2b) to determine whether a useful PK of the AlbudAb was maintained as a fusion protein.

Interferon alpha 2b amino acid sequence:

(SEQ ID NO: 43)

CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKA

ETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVI

QGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRS

FSLSTNLQESLRSKE

Interferon alpha 2b nucleotide sequence:

(SEQ ID NO: 44)

TGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCT

CCTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGAC

ATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCT

GAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTCTT

CAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAAT

TCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATA

CAGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCT

GGCTGTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGA

AATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCT

TTTTCTTTGTCAACAAACTTGCAAGAAAGTTTAAGAAGTAAGGAA

IFNa2b was linked to the AlbudAb via a TVAAPS linker region (see WO2007085814). The constructs were cloned by SOE-PCR (single overlap extension according to the method of Horton et al. Gene, 77, p 61 (1989)). PCR amplification of the AlbudAb and IFN sequences were carried out separately using primers with a ˜15 base pair overlap at the TVAAPS linker region. The primers used are as follows:—

	IFNα2b SOE fragment 5′
	(SEQ ID NO: 45)
	GCCCGGATCCACCGGCTGTGATCTG

	IFNα2b SOE fragment 3′
	(SEQ ID NO: 46)
	GGAGGATGGAGACTGGGTCATCTGGATGTC

	Vk SOE fragment 5′
	(SEQ ID NO: 47)
	GACATCCAGATGACCCAGTCTCCATCCTCC

	Vk SOE fragment 3′ to also introduce a myc tag
	(SEQ ID NO: 48)
	GCGCAAGCTTTTATTAATTCAGATCCTCTTC

	TGAGATGAGTTTTTGTTCTGCGGCCGCCCGT

	TTGATTTCCACCTTGGTCCC

The fragments were purified separately and subsequently assembled in a SOE (single overlap extension PCR extension) reaction using only the flanking primers.

	IFNα2b SOE fragment 5′
	(SEQ ID NO: 49)
	GCCCGGATCCACCGGCTGTGATCTG

	Vk SOE fragment 3′ to also introduce a myc tag
	(SEQ ID NO: 50)
	GCGCAAGCTTTTATTAATTCAGATCCTCTTC

	TGAGATGAGTTTTTGTTCTGCGGCCGCCCGT

	TTGATTTCCACCTTGGTCCC

The assembled PCR product was digested using the restriction enzymes BamHI and HindIII and the gene ligated into the corresponding sites in the pDOM50, a mammalian expression vector which is a pTT5 derivative with an N-terminal V-J2-C mouse IgG secretory leader sequence to facilitate expression into the cell media.

Leader sequence (amino acid):

(SEQ ID NO: 51)

METDTLLLWVLLLWVPGSTG

Leader sequence (nucleotide):

(SEQ ID NO: 52)

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCGG

ATCCACCGGGC

Plasmid DNA was prepared using QIAfilter megaprep (Qiagen). 1 μg DNA/ml was transfected with 293-Fectin into HEK293E cells and grown in serum free media. The protein is expressed in culture for 5 days and purified from culture supernatant using protein L affinity resin and eluted with 100 mM glycine pH2. The proteins were concentrated to greater than 1 mg/ml, buffer exchanged into PBS and endotoxin depleted using Q spin columns (Vivascience).

TABLE 11

Interferon alpha 2b-AIbudAb sequences with and without myc-taq (as
amino acid- and nucleotide sequence)
The Interferon alpha 2b is N-terminal to the AIbudAb in the following fusions.

	aa + myc	nt + myc	aa no tag	nt no tag

DMS7321	CDLPQTHSLGSRRTL	TGCGACTTGCCA	CDLPQTHSLGS	TGCGACTTGCCA
(IFNα2b-	MLLAQMRRISLFSCL	CAGACACATAGT	RRTLMLLAQM	CAGACACATAGT
DOM7h-	KDRHDFGFPQEEFG	TTGGGATCAAGA	RRISLFSCLKD	TTGGGATCAAGA
14)	NQFQKAETIPVLHEMI	AGAACATTGATG	RHDFGFPQEE	AGAACATTGATG
	QQIFNLFSTKDSSAA	TTATTAGCACAAA	FGNQFQKAETI	TTATTAGCACAA
	WDETLLDKFYTELYQ	TGCGTAGAATTT	PVLHEMIQQIF	ATGCGTAGAATT
	QLNDLEACVIQGVGV	CTTTGTTCTCTTG	NLFSTKDSSAA	TCTTTGTTCTCTT
	TETPLMKEDSILAVRK	TCTAAAGGACCG	WDETLLDKFYT	GTCTAAAGGACC
	YFQRITLYLKEKKYSP	TCACGACTTCGG	ELYQQLNDLEA	GTCACGACTTCG
	CAWEVVRAEIMRSFS	ATTCCCTCAGGA	CVIQGVGVTET	GATTCCCTCAGG
	LSTNLQESLRSKETV	AGAGTTTGGAAA	PLMKEDSILAV	AAGAGTTTGGAA
	AAPSDIQMTQSPSSL	CCAATTCCAAAA	RKYFQRITLYLK	ACCAATTCCAAA
	SASVGDRVTITCRAS	AGCAGAAACTAT	EKKYSPCAWE	AAGCAGAAACTA
	QWIGSQLSWYQQKP	TCCTGTCTTGCA	VVRAEIMRSFS	TTCCTGTCTTGC
	GKAPKLLIMWRSSLQ	CGAAATGATCCA	LSTNLQESLRS	ACGAAATGATCC
	SGVPSRFSGSGSGT	GCAAATATTCAAT	KETVAAPSDIQ	AGCAAATATTCA
	DFTLTISSLQPEDFAT	TTGTTTTCTACAA	MTQSPSSLSAS	ATTTGTTTTCTAC
	YYCAQGAALPRTFGQ	AGGACTCATCAG	VGDRVTITCRA	AAAGGACTCATC
	GTKVEIKR	CCGCTTGGGATG	SQWIGSQLSW	AGCCGCTTGGGA
	AAAEQKLISEEDLN*	AAACTCTGTTAG	YQQKPGKAPK	TGAAACTCTGTT
	(SEQ ID NO: 53)	ATAAATTCTACAC	LLIMWRSSLQS	AGATAAATTCTA
		TGAACTATATCAA	GVPSRFSGSG	CACTGAACTATA
		CAACTGAACGAT	SGTDFTLTISSL	TCAACAACTGAA
		CTAGAGGCTTGC	QPEDFATYYCA	CGATCTAGAGGC
		GTTATTCAGGGT	QGAALPRTFG	TTGCGTTATTCA
		GTAGGAGTTACT	QGTKVEIKR	GGGTGTAGGAGT
		GAAACTCCCCTA	(SEQ ID NO: 55)	TACTGAAACTCC
		ATGAAAGAAGAT		CCTAATGAAAGA
		TCAATTCTAGCC		AGATTCAATTCTA
		GTTAGAAAATACT		GCCGTTAGAAAA
		TTCAGCGTATCA		TACTTTCAGCGT
		CATTGTATTTAAA		ATCACATTGTATT
		GGAAAAGAAATA		TAAAGGAAAAGA
		CTCCCCATGTGC		AATACTCCCCAT
		ATGGGAGGTGGT		GTGCATGGGAG
		TAGAGCAGAAAT		GTGGTTAGAGCA
		TATGAGGTCCTT		GAAATTATGAGG
		CTCTCTTTCTACG		TCCTTCTCTCTTT
		AATTTGCAAGAAT		CTACGAATTTGC
		CTTTGAGATCTAA		AAGAATCTTTGA
		GGAAACCGTCGC		GATCTAAGGAAA
		TGCTCCATCTGA		CCGTCGCTGCTC
		CATCCAGATGAC		CATCTGACATCC
		CCAGTCTCCATC		AGATGACCCAGT
		CTCCCTGTCTGC		CTCCATCCTCCC
		ATCTGTAGGAGA		TGTCTGCATCTG
		CCGTGTCACCAT		TAGGAGACCGTG
		CACTTGCCGGGC		TCACCATCACTT
		AAGTCAGTGGAT		GCCGGGCAAGT
		TGGGTCTCAGTT		CAGTGGATTGGG
		ATCTTGGTACCA		TCTCAGTTATCTT
		GCAGAAACCAGG		GGTACCAGCAGA
		GAAAGCCCCTAA		AACCAGGGAAAG
		GCTCCTGATCAT		CCCCTAAGCTCC
		GTGGCGTTCCTC		TGATCATGTGGC
		GTTGCAAAGTGG		GTTCCTCGTTGC
		GGTCCCATCACG		AAAGTGGGGTCC
		TTTCAGTGGCAG		CATCACGTTTCA
		TGGATCTGGGAC		GTGGCAGTGGAT
		AGATTTCACTCTC		CTGGGACAGATT
		ACCATCAGCAGT		TCACTCTCACCA
		CTGCAACCTGAA		TCAGCAGTCTGC
		GATTTTGCTACG		AACCTGAAGATT
		TACTACTGTGCT		TTGCTACGTACT
		CAGGGTGCGGC		ACTGTGCTCAGG
		GTTGCCTAGGAC		GTGCGGCGTTG
		GTTCGGCCAAGG		CCTAGGACGTTC
		GACCAAGGTGGA		GGCCAAGGGAC
		AATCAAACGGGC		CAAGGTGGAAAT
		GGCCGCAGAAC		CAAACGG (SEQ
		AAAAACTCATCT		ID NO: 56)
		CAGAAGAGGAT
		CTGAATTAA
		(SEQ ID NO: 54)

DMS732	CDLPQTHSLGSRRTL	TGCGACTTGCCA	CDLPQTHSLGS	TGCGACTTGCCA
(IFNα2b-	MLLAQMRRISLFSCL	CAGACACATAGT	RRTLMLLAQM	CAGACACATAGT
DOM7h-	KDRHDFGFPQEEFG	TTGGGATCAAGA	RRISLFSCLKD	TTGGGATCAAGA
14-10)	NQFQKAETIPVLHEMI	AGAACATTGATG	RHDFGFPQEE	AGAACATTGATG
	QQIFNLFSTKDSSAA	TTATTAGCACAAA	FGNQFQKAETI	TTATTAGCACAA
	WDETLLDKFYTELYQ	TGCGTAGAATTT	PVLHEMIQQIF	ATGCGTAGAATT
	QLNDLEACVIQGVGV	CTTTGTTCTCTTG	NLFSTKDSSAA	TCTTTGTTCTCTT
	TETPLMKEDSILAVRK	TCTAAAGGACCG	WDETLLDKFYT	GTCTAAAGGACC
	YFQRITLYLKEKKYSP	TCACGACTTCGG	ELYQQLNDLEA	GTCACGACTTCG
	CAWEVVRAEIMRSFS	ATTCCCTCAGGA	CVIQGVGVTET	GATTCCCTCAGG
	LSTNLQESLRSKETV	AGAGTTTGGAAA	PLMKEDSILAV	AAGAGTTTGGAA
	AAPSDIQMTQSPSSL	CCAATTCCAAAA	RKYFQRITLYLK	ACCAATTCCAAA
	SASVGDRVTITCRAS	AGCAGAAACTAT	EKKYSPCAWE	AAGCAGAAACTA
	QWIGSQLSWYQQKP	TCCTGTCTTGCA	VVRAEIMRSFS	TTCCTGTCTTGC
	GKAPKLLIMWRSSLQ	CGAAATGATCCA	LSTNLQESLRS	ACGAAATGATCC
	SGVPSRFSGSGSGT	GCAAATATTCAAT	KETVAAPSDIQ	AGCAAATATTCA
	DFTLTISSLQPEDFAT	TTGTTTTCTACAA	MTQSPSSLSAS	ATTTGTTTTCTAC
	YYCAQGLRHPKTFG	AGGACTCATCAG	VGDRVTITCRA	AAAGGACTCATC
	QGTKVEIKR	CCGCTTGGGATG	SQWIGSQLSW	AGCCGCTTGGGA
	AAAEQKLISEEDLN*	AAACTCTGTTAG	YQQKPGKAPK	TGAAACTCTGTT
	(SEQ ID NO: 57)	ATAAATTCTACAC	LLIMWRSSLQS	AGATAAATTCTA
		TGAACTATATCAA	GVPSRFSGSG	CACTGAACTATA
		CAACTGAACGAT	SGTDFTLTISSL	TCAACAACTGAA
		CTAGAGGCTTGC	QPEDFATYYCA	CGATCTAGAGGC
		GTTATTCAGGGT	QGLRHPKTFG	TTGCGTTATTCA
		GTAGGAGTTACT	QGTKVEIKR	GGGTGTAGGAGT
		GAAACTCCCCTA	(SEQ ID NO: 59)	TACTGAAACTCC
		ATGAAAGAAGAT		CCTAATGAAAGA
		TCAATTCTAGCC		AGATTCAATTCTA
		GTTAGAAAATACT		GCCGTTAGAAAA
		TTCAGCGTATCA		TACTTTCAGCGT
		CATTGTATTTAAA		ATCACATTGTATT
		GGAAAAGAAATA		TAAAGGAAAAGA
		CTCCCCATGTGC		AATACTCCCCAT
		ATGGGAGGTGGT		GTGCATGGGAG
		TAGAGCAGAAAT		GTGGTTAGAGCA
		TATGAGGTCCTT		GAAATTATGAGG
		CTCTCTTTCTACG		TCCTTCTCTCTTT
		AATTTGCAAGAAT		CTACGAATTTGC
		CTTTGAGATCTAA		AAGAATCTTTGA
		GGAAACCGTCGC		GATCTAAGGAAA
		TGCTCCATCTGA		CCGTCGCTGCTC
		CATCCAGATGAC		CATCTGACATCC
		CCAGTCTCCATC		AGATGACCCAGT
		CTCCCTGTCTGC		CTCCATCCTCCC
		ATCTGTAGGAGA		TGTCTGCATCTG
		CCGTGTCACCAT		TAGGAGACCGTG
		CACTTGCCGGGC		TCACCATCACTT
		AAGTCAGTGGAT		GCCGGGCAAGT
		TGGGTCTCAGTT		CAGTGGATTGGG
		ATCTTGGTACCA		TCTCAGTTATCTT
		GCAGAAACCAGG		GGTACCAGCAGA
		GAAAGCCCCTAA		AACCAGGGAAAG
		GCTCCTGATCAT		CCCCTAAGCTCC
		GTGGCGTTCCTC		TGATCATGTGGC
		GTTGCAAAGTGG		GTTCCTCGTTGC
		GGTCCCATCACG		AAAGTGGGGTCC
		TTTCAGTGGCAG		CATCACGTTTCA
		TGGATCTGGGAC		GTGGCAGTGGAT
		AGATTTCACTCTC		CTGGGACAGATT
		ACCATCAGCAGT		TCACTCTCACCA
		CTGCAACCTGAA		TCAGCAGTCTGC
		GATTTTGCTACG		AACCTGAAGATT
		TACTACTGTGCT		TTGCTACGTACT
		CAGGGTTTGAGG		ACTGTGCTCAGG
		CATCCTAAGACG		GTTTGAGGCATC
		TTCGGCCAAGGG		CTAAGACGTTCG
		ACCAAGGTGGAA		GCCAAGGGACC
		ATCAAACGGGCG		AAGGTGGAAATC
		GCCGCAGAACA		AAACGG (SEQ ID
		AAAACTCATCTC		NO: 60)
		AGAAGAGGATCT
		GAATTAA (SEQ
		ID NO: 58)

DMS7325	CDLPQTHSLGSRRTL	TGCGACTTGCCA	CDLPQTHSLGS	TGCGACTTGCCA
(IFNα2b-	MLLAQMRRISLFSCL	CAGACACATAGT	RRTLMLLAQM	CAGACACATAGT
DOM7h-	KDRHDFGFPQEEFG	TTGGGATCAAGA	RRISLFSCLKD	TTGGGATCAAGA
11)	NQFQKAETIPVLHEMI	AGAACATTGATG	RHDFGFPQEE	AGAACATTGATG
	QQIFNLFSTKDSSAA	TTATTAGCACAAA	FGNQFQKAETI	TTATTAGCACAA
	WDETLLDKFYTELYQ	TGCGTAGAATTT	PVLHEMIQQIF	ATGCGTAGAATT
	QLNDLEACVIQGVGV	CTTTGTTCTCTTG	NLFSTKDSSAA	TCTTTGTTCTCTT
	TETPLMKEDSILAVRK	TCTAAAGGACCG	WDETLLDKFYT	GTCTAAAGGACC
	YFQRITLYLKEKKYSP	TCACGACTTCGG	ELYQQLNDLEA	GTCACGACTTCG
	CAWEVVRAEIMRSFS	ATTCCCTCAGGA	CVIQGVGVTET	GATTCCCTCAGG
	LSTNLQESLRSKETV	AGAGTTTGGAAA	PLMKEDSILAV	AAGAGTTTGGAA
	AAPSDIQMTQSPSSL	CCAATTCCAAAA	RKYFQRITLYLK	ACCAATTCCAAA
	SASVGDRVTITCRAS	AGCAGAAACTAT	EKKYSPCAWE	AAGCAGAAACTA
	RPIGTTLSWYQQKPG	TCCTGTCTTGCA	VVRAEIMRSFS	TTCCTGTCTTGC
	KAPKLLIWFGSRLQS	CGAAATGATCCA	LSTNLQESLRS	ACGAAATGATCC
	GVPSRFSGSGSGTD	GCAAATATTCAAT	KETVAAPSDIQ	AGCAAATATTCA
	FTLTISSLQPEDFATY	TTGTTTTCTACAA	MTQSPSSLSAS	ATTTGTTTTCTAC
	YCAQAGTHPTTFGQ	AGGACTCATCAG	VGDRVTITCRA	AAAGGACTCATC
	GTKVEIKR	CCGCTTGGGATG	SRPIGTTLSWY	AGCCGCTTGGGA
	AAAEQKLISEEDLN*	AAACTCTGTTAG	QQKPGKAPKLL	TGAAACTCTGTT
	(SEQ ID NO: 61)	ATAAATTCTACAC	IWFGSRLQSGV	AGATAAATTCTA
		TGAACTATATCAA	PSRFSGSGSG	CACTGAACTATA
		CAACTGAACGAT	TDFTLTISSLQP	TCAACAACTGAA
		CTAGAGGCTTGC	EDFATYYCAQA	CGATCTAGAGGC
		GTTATTCAGGGT	GTHPTTFGQG	TTGCGTTATTCA
		GTAGGAGTTACT	TKVEIKR (SEQ	GGGTGTAGGAGT
		GAAACTCCCCTA	ID NO: 63)	TACTGAAACTCC
		ATGAAAGAAGAT		CCTAATGAAAGA
		TCAATTCTAGCC		AGATTCAATTCTA
		GTTAGAAAATACT		GCCGTTAGAAAA
		TTCAGCGTATCA		TACTTTCAGCGT
		CATTGTATTTAAA		ATCACATTGTATT
		GGAAAAGAAATA		TAAAGGAAAAGA
		CTCCCCATGTGC		AATACTCCCCAT
		ATGGGAGGTGGT		GTGCATGGGAG
		TAGAGCAGAAAT		GTGGTTAGAGCA
		TATGAGGTCCTT		GAAATTATGAGG
		CTCTCTTTCTACG		TCCTTCTCTCTTT
		AATTTGCAAGAAT		CTACGAATTTGC
		CTTTGAGATCTAA		AAGAATCTTTGA
		GGAAACCGTCGC		GATCTAAGGAAA
		TGCTCCATCTGA		CCGTCGCTGCTC
		CATCCAGATGAC		CATCTGACATCC
		CCAGTCTCCATC		AGATGACCCAGT
		CTCCCTGTCTGC		CTCCATCCTCCC
		ATCTGTAGGAGA		TGTCTGCATCTG
		CCGTGTCACCAT		TAGGAGACCGTG
		CACTTGCCGGGC		TCACCATCACTT
		AAGTCGTCCGAT		GCCGGGCAAGT
		TGGGACGACGTT		CGTCCGATTGGG
		AAGTTGGTACCA		ACGACGTTAAGT
		GCAGAAACCAGG		TGGTACCAGCAG
		GAAAGCCCCTAA		AAACCAGGGAAA
		GCTCCTGATCTG		GCCCCTAAGCTC
		GTTTGGTTCCCG		CTGATCTGGTTT
		GTTGCAAAGTGG		GGTTCCCGGTTG
		GGTCCCATCACG		CAAAGTGGGGTC
		TTTCAGTGGCAG		CCATCACGTTTC
		TGGATCTGGGAC		AGTGGCAGTGGA
		AGATTTCACTCTC		TCTGGGACAGAT
		ACCATCAGCAGT		TTCACTCTCACC
		CTGCAACCTGAA		ATCAGCAGTCTG
		GATTTTGCTACG		CAACCTGAAGAT
		TACTACTGTGCG		TTTGCTACGTAC
		CAGGCTGGGAC		TACTGTGCGCAG
		GCATCCTACGAC		GCTGGGACGCAT
		GTTCGGCCAAGG		CCTACGACGTTC
		GACCAAGGTGGA		GGCCAAGGGAC
		AATCAAACGGGC		CAAGGTGGAAAT
		GGCCGCAGAAC		CAAACGG (SEQ
		AAAAACTCATCT		ID NO: 64)
		CAGAAGAGGAT
		CTGAATTAA
		(SEQ ID NO: 62)

DMS7327	CDLPQTHSLGSRRTL	TGCGACTTGCCA	CDLPQTHSLGS	TGCGACTTGCCA
(IFNα2b-	MLLAQMRRISLFSCL	CAGACACATAGT	RRTLMLLAQM	CAGACACATAGT
DOM7h-	KDRHDFGFPQEEFG	TTGGGATCAAGA	RRISLFSCLKD	TTGGGATCAAGA
11-15)	NQFQKAETIPVLHEMI	AGAACATTGATG	RHDFGFPQEE	AGAACATTGATG
	QQIFNLFSTKDSSAA	TTATTAGCACAAA	FGNQFQKAETI	TTATTAGCACAA
	WDETLLDKFYTELYQ	TGCGTAGAATTT	PVLHEMIQQIF	ATGCGTAGAATT
	QLNDLEACVIQGVGV	CTTTGTTCTCTTG	NLFSTKDSSAA	TCTTTGTTCTCTT
	TETPLMKEDSILAVRK	TCTAAAGGACCG	WDETLLDKFYT	GTCTAAAGGACC
	YFQRITLYLKEKKYSP	TCACGACTTCGG	ELYQQLNDLEA	GTCACGACTTCG
	CAWEVVRAEIMRSFS	ATTCCCTCAGGA	CVIQGVGVTET	GATTCCCTCAGG
	LSTNLQESLRSKETV	AGAGTTTGGAAA	PLMKEDSILAV	AAGAGTTTGGAA
	AAPSDIQMTQSPSSL	CCAATTCCAAAA	RKYFQRITLYLK	ACCAATTCCAAA
	SASVGDRVTITCRAS	AGCAGAAACTAT	EKKYSPCAWE	AAGCAGAAACTA
	RPIGTMLSWYQQKP	TCCTGTCTTGCA	VVRAEIMRSFS	TTCCTGTCTTGC
	GKAPKLLILAFSRLQS	CGAAATGATCCA	LSTNLQESLRS	ACGAAATGATCC
	GVPSRFSGSGSGTD	GCAAATATTCAAT	KETVAAPSDIQ	AGCAAATATTCA
	FTLTISSLQPEDFATY	TTGTTTTCTACAA	MTQSPSSLSAS	ATTTGTTTTCTAC
	YCAQAGTHPTTFGQ	AGGACTCATCAG	VGDRVTITCRA	AAAGGACTCATC
	GTKVEIKR	CCGCTTGGGATG	SRPIGTMLSWY	AGCCGCTTGGGA
	AAAEQKLISEEDLN*	AAACTCTGTTAG	QQKPGKAPKLL	TGAAACTCTGTT
	(SEQ ID NO: 65)	ATAAATTCTACAC	ILAFSRLQSGV	AGATAAATTCTA
		TGAACTATATCAA	PSRFSGSGSG	CACTGAACTATA
		CAACTGAACGAT	TDFTLTISSLQP	TCAACAACTGAA
		CTAGAGGCTTGC	EDFATYYCAQA	CGATCTAGAGGC
		GTTATTCAGGGT	GTHPTTFGQG	TTGCGTTATTCA
		GTAGGAGTTACT	TKVEIKR (SEQ	GGGTGTAGGAGT
		GAAACTCCCCTA	ID NO: 67)	TACTGAAACTCC
		ATGAAAGAAGAT		CCTAATGAAAGA
		TCAATTCTAGCC		AGATTCAATTCTA
		GTTAGAAAATACT		GCCGTTAGAAAA
		TTCAGCGTATCA		TACTTTCAGCGT
		CATTGTATTTAAA		ATCACATTGTATT
		GGAAAAGAAATA		TAAAGGAAAAGA
		CTCCCCATGTGC		AATACTCCCCAT
		ATGGGAGGTGGT		GTGCATGGGAG
		TAGAGCAGAAAT		GTGGTTAGAGCA
		TATGAGGTCCTT		GAAATTATGAGG
		CTCTCTTTCTACG		TCCTTCTCTCTTT
		AATTTGCAAGAAT		CTACGAATTTGC
		CTTTGAGATCTAA		AAGAATCTTTGA
		GGAAACCGTCGC		GATCTAAGGAAA
		TGCTCCATCTGA		CCGTCGCTGCTC
		CATCCAGATGAC		CATCTGACATCC
		CCAGTCTCCATC		AGATGACCCAGT
		CTCCCTGTCTGC		CTCCATCCTCCC
		ATCTGTAGGAGA		TGTCTGCATCTG
		CCGTGTCACCAT		TAGGAGACCGTG
		CACTTGCCGGGC		TCACCATCACTT
		AAGTCGTCCGAT		GCCGGGCAAGT
		TGGGACGATGTT		CGTCCGATTGGG
		AAGTTGGTACCA		ACGATGTTAAGT
		GCAGAAACCAGG		TGGTACCAGCAG
		GAAAGCCCCTAA		AAACCAGGGAAA
		GCTCCTGATCCT		GCCCCTAAGCTC
		TGCTTTTTCCCGT		CTGATCCTTGCT
		TTGCAAAGTGGG		TTTTCCCGTTTG
		GTCCCATCACGT		CAAAGTGGGGTC
		TTCAGTGGCAGT		CCATCACGTTTC
		GGATCTGGGACA		AGTGGCAGTGGA
		GATTTCACTCTCA		TCTGGGACAGAT
		CCATCAGCAGTC		TTCACTCTCACC
		TGCAACCTGAAG		ATCAGCAGTCTG
		ATTTTGCTACGTA		CAACCTGAAGAT
		CTACTGCGCGCA		TTTGCTACGTAC
		GGCTGGGACGC		TACTGCGCGCAG
		ATCCTACGACGT		GCTGGGACGCAT
		TCGGCCAAGGGA		CCTACGACGTTC
		CCAAGGTGGAAA		GGCCAAGGGAC
		TCAAACGGGCGG		CAAGGTGGAAAT
		CCGCAGAACAA		CAAACGG (SEQ
		AAACTCATCTCA		ID NO: 68)
		GAAGAGGATCTG
		AATTAA (SEQ ID
		NO: 66)

The amino acid and nucleotide sequences highlighted in bold represents the cloning site and MYC tag. * represents the stop codon at the end of the gene.

Affinity Determination and Biophysical Characterisation:

To determine the binding affinity (K_D) of the AlbudAb-IFNα2b fusion proteins to each serum albumin; purified fusion proteins were analysed by BIAcore over albumin (immobilised by primary-amine coupling onto CM5 chips; BIAcore) using fusion protein concentrations from 5000 nM to 39 nM (5000 nM, 2500 nM, 1250 nM, 625 nM, 312 nM, 156 nM, 78 nM, 39 nM) in HBS-EP BIAcore buffer.

TABLE 12

Affinity to SA

		Affinity to
AlbudAb	Fusion	SA (nM)	Kd	Ka

		Rat
DOM7h-14	IFNα2b	350	4.500E−02	1.28E+05
DOM7h-14-10	IFNα2b	16	4.970E−03	5.90E+05
DOM 7h-11	IFNα2b	6000	7.500E−01	nd
DOM 7h-11-15	IFNα2b	200	1.660E−02	1.50E+05
		Cyno
DOM 7h-14	IFNα2b	60	1.32E−02	5.0E+05
DOM 7h-14-10	IFNα2b	19	7.05E−03	4.50E+05
DOM 7h-11	IFNα2b	3300	3.59E−01	1.20E+05
DOM 7h-11-15	IFNα2b	15	4.86E−03	3.60E+05
		Mouse
DOM 7h-14	IFNα2b	240	3.21E−02	1.50E+06
DOM 7h-14-10	IFNα2b	60	3.45E−02	6.86E+05
DOM 7h-11	IFNα2b	6000	1.55E−01	nd
DOM 7h-11-15	IFNα2b	28	6.69E−03	2.80E+05
		Human
DOM 7h-14	IFNα2b	244	2.21E−02	9.89E+04
DOM 7h-14-10	IFNα2b	32	6.58E−03	3.48E+05
DOM 7h-11	IFNα2b	670	2.02E−01	7.00E+05
DOM 7h-11-15	IFNα2b	10	1.87E−03	3.50E+05

When IFNa2b is linked to the AlbudAb variants, in all cases the affinity of AlbudAb binding to serum albumin is reduced. DOM7h-14-10 and DOM7-11-15 retain improved binding affinity to serum albumin across species compared to parent.

TABLE 13

Biophysical Characterisation
Biophysical Characterisation was carried out by SEC MALLS
and DSC as described above for the single AlbudAbs.

Biophysical parameters

		DMS	SEC	DSC
AlbudAb	Fusion	number	MALLS	Tm(° C.)

DOM 7h-14	IFNα2b	DMS7321	M/D	58-65
DOM 7h-14-10	IFNα2b	DMS7322	M/D	55-65
DOM 7h-11	IFNα2b	DMS7325	M/D	65.8-66.2
DOM 7h-11-15	IFNα2b	DMS7327	M/D	56.3-66.2

M/D Indicates a Monomer/Dimer Equilibrium as Detected by SEC MALLS

We observed expression for all clones in Table 13 in the range of 17.5 to 54 mg/L in HEK293.
For IFNα2b-DOM7h-14 and IFNα2b-DOM7h-11 variants, favorable biophysical parameters and expression levels were maintained during affinity maturation.
PK Determination for AlbudAb-IFNα2bfusions
AlbudAbs IFNα2b fusions DMS7321 (IFNα2b-DOM7h-14) DMS7322 (IFNα2b-DOM7h-14-10), DMS7325 (IFNα2b-DOM7h-11), DMS7327 (IFNα2b-DOM7h-11-15) were expressed with the myc tag at 20-50 mg quantities in HEK293 cells and purified from culture supernatant using protein L affinity resin and eluted with 100 mM glycine pH2. The proteins were concentrated to greater than 1 mg/ml, buffer exchanged into Dulbecco's PBS and endotoxin depleted using Q spin columns (Vivascience).
For Rat PK, IFN-AlbudAbs were dosed as single i.v injections at 2.0 mg/kg using 3 rats per compound. Serum samples were taken at 0.16, 1, 4, 8, 24, 48, 72, 120, 168 hrs. Analysis of serum levels was by EASY ELISA according to manufacturer's instructions (GE Healthcare, catalogue number RPN5960).
For Mouse PK, DMS7322 (IFN2b-DOM7h-14-10) DMS7325 (IFN2b-DOM7h-11), DMS7327 (IFN2b-DOM7h-11-15) all with myc tags were dosed as single i.v injections at 2.0 mg/kg per dose group of 3 subjects and serum samples taken at 10 mins; 1 h; 8 h; 24 h; 48 h; 72 h; 96 h. Analysis of serum levels was by EASY ELISA according to manufacturer's instructions (GE Healthcare, catalogue number RPN5960).

TABLE 14

				PK parameters (mean results)

				AUC	CL
			Albumin	h ×	ml/	t½	Vz
Species	AlbudAb	Fusion	K_D(nM)	ug/ml	h/kg	h	ml/kg

Rat	7h-14	IFNα2b	350	832.1	2.4	27	94.5
	7h-14-10	IFNα2b	16	1380.7	1.5	35.8	75.2
	7h-11	IFNα2b	6000	327.9	6.5	11	101.9
	7h-11-15	IFNα2b	200	1118.7	1.8	39.5	103.6
	7h-11-12	IFNα2b	1700	747.1	2.8	25.8	104.7
mouse	7h-14	IFNα2b	240	761.2	2.6	30.4	115.3
	7h-14-10	IFNα2b	60	750.5	2.7	30.9	118.6
	7h-11	IFNα2b	6000	493.9	4.0	8.8	51.2
	7h-11-15	IFNα2b	28	971.8	2.1	33.6	99.6

Pharmacokinetic parameters derived from rat and mouse studies were fitted using a non-compartmental model. Key: AUC: Area under the curve from dosing time extrapolated to infinity; CL: clearance; t½: is the time during which the blood concentration is halved; Vz: volume of distribution based on the terminal phase.
IFNα2b-AlbudAbs were tested in rat and mouse. For all IFNα2b-DOM7h-11 variant fusion proteins in both rat and mouse, t½ is improved compared to parent. The improvement in t½ correlates with the improved in vitro K_Dto serum albumin. For IFNα2b-DOM7h-14-10 variants, the improvement in in vitro K_Dto serum albumin also correlated to an improvement in t½ in rat.
All IFNα2b-AlbudAb fusion proteins exhibit a 5 to 10-fold decrease in the binding to RSA compared to the single AlbudAb. This effect is more pronounced (i.e. 10-fold) for the DOM7h-14 series than the DOM7h-11 series (only 5-fold decrease).

Example 8

Further AlbudAb Fusions with Proteins, Peptides and NCEs

Various AlbudAbs fused to other chemical entities namely domain antibodies (dAbs), peptides and NCEs were tested. The results are shown in table 15.

TABLE 15

				PK
				parameters
			Albumin	AUC	CL	t½	Vz
Species	AlbudAb	Fusion	K_D(nM)	h × ug/ml	ml/h/kg	h	ml/kg

Rat	DOM7h-14	Exendin-4	2400	18	57.1	11	901.9
	DOM7h-14-	Exendin-4	19	43.6	23.1	22.1	740.3
	10
	DOM7h-11	Exendin-4	2400	6.1	168	7.1	1684.1
	DOM7h-11-	Exendin-4	273	36.3	27.6	19.3	765.7
	15
In a	DOM7h-11-	Exendin-4	130	not tested	not	not	not
different	15				tested	tested	tested
experiement
	DOM7h14-	NCE-	62
	10	GGGGSC
	DOM7h14-	NCE-	35
	10	TVAAPSC
Human	DOM7h-14	NCE	204
mouse	DOM7h-11	DOM1m-21-		234	10.7	4.7	72.5
		23
	DOM7h-11-	DOM1m-21-		1008	2.5	17.4	62.4
	15	23

Key: DOM1m-21-23 is an anti-TNFR1 dAb, Exendin-4 is a peptide (a GLP-1 agonist) of 39 amino acids length. NCE, NCE-GGGGSC and NCE-TVAAPSC are described below.

Previously we have described the use of genetic fusions with an albumin-binding dAb (AlbudAb) to extend the PK half-life of anti-TNFR1 dAbs in vivo (see, e.g., WO04003019, WO2006038027, WO2008149148). Reference is made to the protocols in these PCT applications. In the table above, DOM1m-21-23 is an anti-mouse TNFR1 dAb.
To produce genetic fusions of exendin-4 or with DOM7h-14 (or other AlbudAb) which binds serum albumin, the exendin-4-linker-AlbudAb sequence was cloned into the pTT-5 vector (obtainable from CNRC, Canada). In each case the exendin-4 was at the 5′ end of the construct and the dAb at the 3′ end. The linker was a (G₄S)₃linker. Endotoxin-free DNA was prepared in E. coli using alkaline lysis (using the endotoxin-free plasmid Giga kit, obtainable from Qiagen CA) and used to transfect HEK293E cells (obtainable from CNRC, Canada). Transfection was into 250 ml/flask of HEK293E cells at 1.75×10⁶cells/ml using 333 ul of 293fectin (Invitrogen) and 250 ug of DNA per flask and expression was at 30° C. for 5 days. The supernatant was harvested by centrifugation and purification was by affinity purification on protein L. Protein was batch bound to the resin, packed on a column and washed with 10 column volumes of PBS. Protein was eluted with 50 ml of 0.1M glycine pH2 and neutralized with Tris pH8. Protein of the expected size was identified on an SDS-PAGE gel.

NCE Albudab™ Fusions:

A new chemical entity (NCE) AlbudAb fusion was tested. The NCE, a small molecule ADAMTS-4 inhibitor was synthesised with a PEG linker (PEG 4 linker (i.e. 4 PEG molecules before the maleimide) and a maleimide group for conjugation to the AlbudAb. Conjugation of the NCE to the AlbudAb is via an engineered cysteine residue at amino acid position R108C, or following a 5 amino acid (GGGGSC) or 6 amino acid (TVAAPSC) spacer engineered at the end of the AlbudAb. Briefly, the AlbudAb was reduced with TCEP (Pierce, Catalogue Number 77720), desalted using a PD10 column (GE healthcare) into 25 mM Bis-Tris, 5 mM EDTA, 10% (v/v) glycerol pH6.5. A 5 fold molar excess of maleimide activated NCE was added in DMSO not to exceed 10% (V/V) final concentration. The reaction was incubated over night at room temperature and dialysed extensively into 20 mM Tris pH7.4

PEG Linker:

Sequences:

DOM7h-14 R108C:

(SEQ ID NO: 69)

DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW

RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQ

GTKVEIKC

Nucleotide:

(SEQ ID NO: 70)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG

CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAA

GGGACCAAGGTGGAAATCAAATGC

NCE-AlbudAbs DOM7h-14-10 GGGGSC and DOM7h14-10 TVAAPSC, exhibit a 5 to 10 fold decrease in in vitro affinity (K_D) to RSA as determined by BIAcore when fused to the chemical entity.

dAb-Albudab fusion: the 2 DOM7h-11 AlbudAbs with the highest affinity to RSA experience a 2-fold decrease in affinity to RSA as on BIAcore when fused to a therapeutic domain antibody (DOM1 m-21-23) compared to the unfused AlbudAb. The DOM7h-11 clone shows a micromolar K_Dwhen fused (2.8 uM) as well as when unfused (˜5 uM).
Exendin 4-AlbudAb fusion: the effect of fusing the AlbudAbs to a peptide on the binding ability to RSA is about 10-fold, apart from DOM7h-14-10, which only shows a 4-fold decrease in binding. The effect, however, is more pronounced for the DOM7h-14 series (except DOM7h-14-10) than it appears to be for the DOM7h-11 series.
For all the above data, the T½ of the fusion increased with improved affinity to the species' SA.
We generally classify Albudab-therapeutics as being therapeutically amenable (for treatment and/or prophylaxis of diseases, conditions or indications) when the AlbudAb-drug fusions show an affinity range (K_D) of from 0.1 nM to 10 mM for serum albumin binding.
We define the therapeutic ranges of AlbudAbs and AlbudAb fusions (Protein-AlbudAbs for example IFNa2b-DOM7h-14-10; Peptide-AlbudAbs for example Exendin-4-DOM7h-14-10; dAb-AlbudAbs for example DOM1m21-23-DOM7h11-15; NCE-AlbudAb for example ADAMTS-4-DOM7h-14-10) as follows: Affinity (K_D) ranges that are useful for therapy of chronic or acute conditions, diseases or indications are shown. Also shown are affinity ranges marked as “intermediate”. AlbudAbs and fusions in this range have utility for chronic or acute diseases, conditions or indications. In this way, the affinity of the AlbudAb or fusion for serum albumin can be tailored or chosen according to the disease, condition or indication to be addressed. As described above, the invention provides AlbudAbs with affinities that allow for each AlbudAb to be categorised as “high affinity”, “medium affinity” or “low affinity”, thus enabling the skilled person to select the appropriate AlbudAb of the invention according to the therapy at hand. See FIG. 2.

Example 9

PCT/EP2010/060112 describes V_HAlbudAbs and affinity matured derivatives thereof. V_HAlbudAb sequences are as follows:

DOM7r31 amino acid

SEQ ID NO: 71

EVQLLESGGGLVQPGGSLRLSCTASGFTFRHYRMGWVRQAPGKGLEWVSW

IRPDGTFTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSY

MGDRFDYWGQGTLVTVSS

DOM7r31 nucleic acid

SEQ ID NO: 72

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC

CCTGCGTCTCTCCTGTACAGCCTCCGGATTCACCTTTAGGCATTATCGTA

TGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCATGG

ATTCGTCCGGATGGTACGTTTACATACTACGCAGACTCCGTGAAGGGCCG

GTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGA

ACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAATCTTAT

ATGGGTGATAGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTC

GAGCG

DOM7r-31-14 amino acid

SEQ ID NO: 73

EVQLLESGGGLVQPGGSLRLSCTASGFTFRHYRMGWVRQAPGKGLEWVSW

IRPDGTFTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSY

MADRFDYWGQGTLVTVSS

DOM7r-31-14 nucleic acid

SEQ ID NO: 74

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC

CCTGCGTCTCTCCTGTACAGCCTCCGGATTCACCTTTAGGCATTATCGTA

TGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCATGG

ATTCGTCCGGATGGTACGTTTACATACTACGCAGACTCCGTGAAGGGCCG

GTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGA

ACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAATCTTAT

ATGGCTGATAGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTC

GAGC

DOM7h-92 amino acid

SEQ ID NO: 75

EVQLLESGGGLVQPGGSLRLSCAASGFTFANATMSWVRQAPGKGLEWVSD

IDQVGHATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYS

WHPDLFDYWGQGTLVTVSS

DOM7r-92 nucleic acid

SEQ ID NO: 76

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC

CCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGGGAATTATAGGA

TGACTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAACT

ATTTCTCCTTTGGGTACGTATACATACTACGCAGACTCCGTGAAGGGCCG

GTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGA

ACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAGGGCGT

TGGTCGATTTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAG

C

DOM7r-92-4 amino acid

SEQ ID NO: 77

EVQLLESGGGLVQPGGSLRLSCAASGFTFDTSSMLWVRQAPGKGLEWVSV

IHQSGTPTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFP

STHGKFDYWGQGTLVTVSS

DOM7r-92-4 nucleic acid

SEQ ID NO: 78

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC

CCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGATACGAGTAGTA

TGTTGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGTT

ATTCATCAGAGTGGTACGCCTACATACTACGCAGACTCCGTGAAGGGCCG

GTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGA

ACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAATTTCCG

TCTACTCATGGTAAGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGT

CTCGAGC

Affinity Determination:

To determine the binding affinity (K_D) of the VH AlbudAbs to each serum albumin; purified fusion proteins were analysed by BIAcore over albumin (immobilised by primary-amine coupling onto CM5 chips; BIAcore) using fusion protein concentrations from 5000 nM to 39 nM (5000 nM, 2500 nM, 1250 nM, 625 nM, 312 nM, 156 nM, 78 nM, 39 nM) in HBS-EP BIAcore buffer, as described above. MALLS data were obtained as described above.
Results are shown in the following tables:

TABLE 16A

	RSA	HSA	CSA	MSA*
	KD (M)	KD (M)	KD (M)	KD (M)

DOM7r-92-4	2.6E−07	1.3E−07	9.8E−08	1.1E−07

TABLE 16B

	HSA	CSA	RSA	MSA	MALLS

DOM7r-92	200 nM	170 nM	500 nM	2000 nM	13 + 22 kDa dimer
FLAG OD

TABLE 16 C

			CSA	MSA
	HSA	RSA KD	KD	KD
	KD (nM)	(nM)	(nM)	(nM)	MALLS

	KD	KD	KD	KD
7r-31	fast on/off	fast	fast	fast
		on/off	on/	on/off	M
			off
7r31-	208, 360, 1330,	103, 90.2, 370	no binding	6, 12, 12	M
14	1950			14.2

Values above represent multiple, independent measurements.

Example 10

Summary of HSA Epitope Mapping with AlbudAbs

The epitopes of AlbudAb Vk molecules on HSA were determined using three orthogonal techniques: hydrogen deuterium exchange mass spectrometry, site directed mutagenesis and structure determination by x-ray crystallography.

1.0 EPITOPE MAPPING BY HYDROGEN DEUTERIUM (H/D) EXCHANGE

1.1 Protein Preparation

Domain 2 of HSA (defined as amino acid residues 188-384 of HSA; SEQ ID NO: 79 and 80 for amino acid and DNA sequences for HSA Domain 2 respectively; SEQ ID NO: 81 and 82 for amino acid and DNA sequence of full length HSA respectively) were expressed in Pichia using the pPICZα expression system (Invitrogen) and purified using Prometic Technologies Mimetic Blue™ according to the manufacturer's instructions. DOM7h-11-3, DOM7h-14-10 and DOM7r-92-4 (SEQ ID NOs: 2, 45, 46 for amino acid and 4, 47, 48 for DNA sequences respectively). was expressed in E. coli strain HB2151 using an auto-induction expression system. In some instances, the cloning strategy resulted in additional N and C terminal residues (see SEQ ID NOs: 121 and 122, for example). The expressed AlbudAbs were purified from clarified supernatants by Protein-L (DOM7h-11-3 and DOM7h-14-10) or Protein-A (DOM7h-92-4) affinity chromatography using established protocols.
Expression and purification of final protein preparations of HSA Domain 2 and AlbudAbs were confirmed by SDS-PAGE analysis.

1.2 Experimental Method and General Principles

Methods and principles on using H/D exchange perturbation for epitope mapping are discussed in a review by Hamuro et al (2003), J. Biomol. Tech. 2003, 14, 171-182; and Coales et al (2009), Rapid Communications in Mass Spectrometry 2009 March; 23(5):639-47. For the epitope mapping of HSA, H/D exchange analysis of the antigen in the presence and absence of AlbudAb was carried out. The regions of HSA which exchange slower in the presence of each AlbudAb compared to speed of exchange when the AlbudAb is absent is considered to define the epitope on HSA. To identify the epitope one requires firstly the identification of proteolytic fragments of the antigen and secondly the determination of the perturbation of the H/D exchange reaction. Suitable methods are described, for example, in U.S. Pat. No. 6,291,189, U.S. Pat. No. 6,331,400 and U.S. Pat. No. 7,280,923.
After each H/D exchange reaction HSA was digested with pepsin. The digested mixture was separated by HPLC. Each HSA peptic fragment was then analyzed by mass spectrometry to determine the degree of deuterium incorporation upon the H/D exchange reaction. To obtain optimal coverage of the HSA sequence a maximum possible number of peptic fragments were followed. For the H/D exchange experiments in the presence of AlbudAb, the mixture of antigen and antibody was digested together. The peptic digest fragment mixture of the complex contained both AlbudAb and HSA fragments.
As an excess amount of an AlbudAb over HSA was used, a large amount of AlbudAb originated peptides may interfere with the mass detection of antigen originated peptides by ion competition. For this reason the least possible amount of excess antibody was used Stock solutions of 199 μM DOM7h-11-3, 199 μM DOM7h-14-10, 547 μM DOM7r-92-4 and 45 μM HSA were used in the H/D exchange experiments. 24 μl of HSA stock+36 μl of DOM7h-11-3; 49.5 μl of HSA stock+10.5 μl of DOM7r-92-4; 74 μl of HSA stock+20 μl of DOM7h-14-10 were used to make a complexation mixtures (final concentrations of HSA:DOM7h-11-3=17.9 μM:119.4 μM; HSA:DOM7h-14-10=35.0 μM:42.0 μM; HSA:DOM7r-92-4=37.1 μM:95.7 μM equivalent to 1:6.7, 1:1.2, 1:2.6 ratio respectively). For the control reactions either HSA or AlbudAb were replaced with PBS.
10 μl of HSA+DOM7h-11-3 complexation mixture was added to 10 μl of PBS made with D₂O; 5 or 8 μl of HSA:DOM7h-14-10 complexation mixture was added to 12 or 15 μl of PBS made with D₂O; 5 μl of HSA+DOM7r-92-4 complexation mixture was added to 15 μl of PBS made with D₂O. All deuteration reactions were incubated for 500 seconds at 0° C. After incubation all 20 μl of the complexation mixture was mixed with 30 μl of quenching solution (2M Urea, 1M TCEP pH3.0). 45 μl of the quenched reaction mixture was injected onto a proprietary proteolysis/HPLC system and fragments analysed by mass spectrometry

1.3 Perturbation Results

Fragments were identified, data deconvoluted and visualized using proprietary software (ExSAR). A summary of epitope hits based on H/D exchange perturbation data are highlighted in Table 17 below.

TABLE 17

Difference in deuteration levels in each segment of HSA compared with and
without (A) DOM7h-11-3, (B) DOM7h-14-10 and (C) DOM7r-92-4 complexation

(A)	Incubation Time (Seconds)

Peptide	30	100	300	1000	3000	10000	Average

189-198	9%	−7%	7%	−1%	−2%	6%	2%

189-200	2%	5%	3%	3%	−2%	2%	2%

203-210	−2%	2%	0%	0%	5%	0%	1%

203-213	8%	5%	1%	1%	−1%	6%	3%

213-219	−9%	−25%	−32%	−35%	−39%	−32%	−29%

216-219	−12%	−20%	−28%	−36%	−43%	−42%	−30%

220-228	−35%	−31%	−31%	−18%	−19%	−8%	−24%

222-228	−38%	−36%	−40%	−26%	−15%	−3%	−26%

231-238	−17%	−41%	−51%	−54%	−58%	−33%	−42%

231-250	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected

237-250

−4%

−8%

−5%

−3%

−1%

−4%

237-251

0%

−5%

−8%

−4%

−1%

3%

−3%

237-252

−1%

−5%

−4%

−1%

−2%

−3%

253-260

10%

−6%

5%

−3%

−1%

1%

266-284	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected
269-289	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected
269-292	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected

292-302

−2%

−5%

−2%

−3%

2%

−9%

−3%

292-305

5%

1%

3%

−1%

−5%

1%

295-305

−2%

0%

4%

−2%

2%

0%

301-305

2%

−1%

−11%

−1%

6%

−1%

311-318

−24%

−18%

−10%

−1%

0%

−9%

311-326

−24%

−27%

−19%

−13%

−7%

−19%

312-326

−28%

−24%

−29%

−18%

−15%

−9%

−21%

321-326

−20%

−29%

−25%

−23%

−17%

−22%

329-330

−1%

−7%

−18%

−29%

−46%

−56%

−26%

329-331

−4%

−8%

−9%

−26%

−39%

−48%

−22%

332-333

2%

5%

−4%

−2%

−14%

−27%

−7%

334-342

−8%

−20%

−15%

−22%

−31%

−21%

336-345

0%

−2%

−8%

−12%

−9%

−11%

−7%

344-346

−2%

4%

−1%

−9%

−5%

−8%

−4%

345-346

2%

0%

1%

−7%

−6%

−15%

−4%

347-349	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected

348-349	−10%	−18%	−30%	−31%	−30%	−20%	−23%

348-357	−24%	−33%	−41%	−39%	−37%	−28%	−34%

349-357	−27%	−33%	−43%	−41%	−40%	−30%	−36%

373-384	2%	1%	0%	3%	−2%	6%	2%

376-384	4%	3%	1%	−1%	1%	3%	2%

380-384	−5%	−2%	−4%	8%	−3%	5%	0%


(B)	Incubation Time (Seconds)

Peptide	30	100	300	1000	3000	10000	Average

189-198

−6%

2%

6%

9%

−4%

8%

3%

189-200

−4%

3%

4%

6%

−6%

4%

1%

203-210

−4%

1%

4%

6%

−6%

3%

1%

203-213

1%

3%

5%

−6%

4%

2%

213-219

−14%

−26%

−32%

−50%

−51%

−49%

−37%

216-219

−17%

−28%

−36%

−55%

−60%

−65%

−44%

220-228

−63%

−47%

−51%

−42%

−25%

−46%

222-228

−62%

−55%

−52%

−55%

−40%

−26%

−48%

231-238

−23%

−28%

−43%

−56%

−55%

−48%

−42%

231-250

−4%

−15%

−23%

−26%

−22%

−18%

237-250

−9%

−3%

−1%

−3%

−4%

3%

−3%

237-251

−3%

−1%

−2%

−1%

−6%

2%

−2%

237-252

−5%

−4%

5%

−1%

5%

−1%

253-260

−5%

1%

−1%

−7%

−5%

7%

−2%

266-284

−13%

−7%

2%

5%

−2%

10%

−1%

269-289

−9%

−4%

−8%

0%

−7%

4%

−4%

269-292

0%

2%

−3%

6%

−3%

3%

1%

292-302

−9%

−4%

−3%

12%

−5%

6%

−1%

292-305

−5%

1%

−4%

5%

−5%

3%

−1%

295-305

−7%

−1%

13%

−8%

7%

1%

301-305

5%

−3%

1%

10%

−2%

−5%

1%

311-318

−27%

−23%

−17%

−5%

−7%

7%

−12%

311-326

−30%

−26%

−25%

−19%

−7%

−21%

312-326

−27%

−22%

−18%

−20%

−9%

−20%

321-326

−9%

−13%

−18%

−13%

−18%

−11%

−14%

329-330

−5%

−15%

−13%

−35%

−43%

−67%

−30%

329-331

−3%

−7%

−10%

−23%

−31%

−56%

−22%

332-333

−2%

0%

−2%

−14%

−18%

−55%

−15%

334-342

−7%

−11%

−18%

−23%

−35%

−17%

336-345

−5%

−7%

−5%

−9%

−15%

−13%

−9%

344-346

4%

5%

−1%

−7%

−9%

−14%

−4%

345-346

3%

−2%

2%

−2%

−8%

−29%

−6%

347-349

−11%

−15%

−41%

−47%

−50%

−30%

348-349

−7%

−18%

−20%

−40%

−43%

−35%

−27%

348-357

−28%

−36%

−43%

−60%

−50%

−44%

349-357

−31%

−33%

−41%

−62%

−57%

−54%

−46%

373-384

−5%

−1%

5%

−5%

7%

0%

376-384

−1%

1%

4%

9%

−4%

4%

2%

380-384

−3%

−2%

8%

−7%

0%

−1%


(C)	Incubation Time (Seconds)

Peptide	30	100	300	1000	3000	10000	Average

189-198	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected

189-200

1%

−6%

−4%

1%

−2%

−1%

−2%

203-210

−20%

−21%

−12%

−16%

−14%

−17%

203-213	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected

213-219

−5%

−9%

−15%

−21%

−28%

−32%

−18%

216-219

−8%

−11%

−16%

−24%

−30%

−19%

220-228	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected

222-228

−29%

−28%

−22%

−16%

−3%

−5%

−17%

231-238

−1%

2%

−6%

−8%

−11%

−13%

−6%

237-250		2%		4%		3%		0%		−1%		1%		2%

237-251	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected

237-252	0%	−4%	−2%	−4%	1%	−2%	−2%

253-260	1%	−11%	2%	−6%	−3%	4%	−2%

292-302

1%

−5%

−10%

−6%

6%

5%

−2%

292-305

−4%

0%

2%

15%

−4%

1%

295-305

6%

−1%

−4%

3%

−1%

301-305

−8%

−4%

8%

4%

−12%

−1%

311-318

−25%

−19%

−9%

−6%

−8%

−1%

−11%

311-326

−26%

−28%

−23%

−21%

−9%

−6%

−19%

312-326

−24%

−25%

−20%

−15%

−7%

−6%

−16%

321-326

−14%

−22%

−27%

−30%

−16%

−15%

−21%

329-330

−26%

−23%

−19%

−36%

−33%

−47%

−31%

329-331

−7%

−8%

−10%

−18%

−44%

−16%

332-333

−1%

5%

−4%

4%

−1%

−15%

−2%

334-342

14%

9%

2%

−5%

4%

−6%

3%

336-345

2%

4%

−3%

3%

−1%

0%

1%

344-346	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected

345-346		4%		4%		3%		3%		4%		−11%		1%

348-349

2%

−8%

−14%

2%

−2%

−13%

−6%

348-357	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected

349-357	−1%	−7%	−2%	−3%	−2%	−6%	−4%

373-384	6%	0%	4%	6%	3%	5%	4%

376-384	2%	1%	−1%	3%	0%	−3%	0%

380-384	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected	Not detected

Based on data summarized in Table 17(A), (B) and (C) it was concluded that segments which showed significant perturbation (on average >20%) of deuteration could define the epitope. The sequence segments identified are highlighted in FIG. 3.

2.0 ALANINE SCANNING SITE DIRECTED MUTAGENESIS (SDM)

Target residues for SDM for Alanine scanning were selected based on 3 criteria: (1) H/D exchange perturbation data above, (2) surface accessibility of side chains based on a previously published crystal structure of HSA (1BKE.pdb (RCSB Protein DataBank)) and (3) charge or size of side chains.

2.1 Cloning and Expression of Alanine Mutants

The wild type template of HSA was PCR-cloned into a mammalian expression vector using standard molecular biology protocols. A 6-Histidine tag was fused to the C-terminus of the sequence (SEQ ID NOS: 25 and 34 for amino acid and DNA sequences respectively for the WT HSA-His₆construct) for nickel affinity purification. Primer pairs used for PCR amplification to make the WT expression construct were TB147 and TB148 (SEQ ID NOS: 85 and 86 respectively).
Mutants were made following standard molecular biology protocols using the WT HSA-His₆construct as a template for mutagenesis. The list of Alanine mutants and mutagenesis oligo pairs used to construct them are listed in Table 18 below.

TABLE 18

Primers pairs for making HSA mutants

K225A	TB153	GAGCCAGAGATTTCCCGCCGCTGAGTTT	sense	SEQ ID NO:
		GCAGAAG		87
	TB154	CTTCTGCAAACTCAGCGGCGGGAAATCT	anti-	SEQ ID NO:
		CTGGCTC	sense	88

E227A	TB155	GAGATTTCCCAAAGCTGCCTTTGCAGAA	sense	SEQ ID NO:
		GTTTCCAAG		89
	TB156	CTTGGAAACTTCTGCAAAGGCAGCTTTG	anti-	SEQ ID NO:
		GGAAATCTC	sense	90

E230A	TB157	CCAAAGCTGAGTTTGCAGCCGTTTCCAA	sense	SEQ ID NO:
		GTTAGTGAC		91
	TB158	GTCACTAACTTGGAAACGGCTGCAAACT	anti-	SEQ ID NO:
		CAGCTTTGG	sense	92

D314A	TB159	GATTTTGTTGAAAGTAAGGCCGTTTGCA	sense	SEQ ID NO:
		AAAACTATG		93
	TB160	CATAGTTTTTGCAAACGGCCTTACTTTCA	anti-	SEQ ID NO:
		ACAAAATC	sense	94

K317A	TB161	GAAAGTAAGGATGTTTGCGCCAACTATG	sense	SEQ ID NO:
		CTGAGGCAAAGG		95
	TB162	CCTTTGCCTCAGCATAGTTGGCGCAAAC	anti-	SEQ ID NO:
		ATCCTT AC TTTC	sense	96

V325A	TB163	GCTGAGGCAAAGGATGCCTTCCTGGGC	sense	SEQ ID NO:
		ATGTTTTTG		97
	TB164	CAAAAACATGCCCAGGAAGGCATCCTTT	anti-	SEQ ID NO:
		GCCTCAGC	sense	98

M329A	TB165	GGATGTCTTCCTGGGCGCCTTTTTGTAT	sense	SEQ ID NO:
		GAATATG		99
	TB166	CATATTCATACAAAAAGGCGCCCAGGAA	anti-	SEQ ID NO:
		GACATCC	sense	100

K351A	TB167	GCTGCTGCTGAGACTTGCCGCCACATAT	sense	SEQ ID NO:
		GAAACCACTCTAG		101
	TB168	CTAGAGTGGTTTCATATGTGGCGGCAAG	anti-	SEQ ID NO:
		TCTCAGCAGCAGC	sense	102

Sequence verified clones were selected from plasmid DNA minipreps made using Millipore Montage kits following the manufacturers protocols. Amino acid and DNA sequences of constructs are summarized in Table 19 below.

TABLE 19

List of HSA mutants made

HSA-His6	Amino Acid	DNA

WT	SEQ ID 103	SEQ ID 112
K225A	SEQ ID 104	SEQ ID 113
E227A	SEQ ID 105	SEQ ID 114
E230A	SEQ ID 106	SEQ ID 115
D314A	SEQ ID 107	SEQ ID 116
K317A	SEQ ID 108	SEQ ID 117
V325A	SEQ ID 109	SEQ ID 118
M329A	SEQ ID 110	SEQ ID 119
K351A	SEQ ID 111	SEQ ID 120

His₆-tagged WT HSA and mutants were expressed in mammalian HEK293-6E cells using transient transfection techniques. Mutants and WT HSA were purified from clarified expression supernatants using nickel affinity chromatography according to established protocols. SDS-PAGE analysis of the purified mutants showed >95% purity.

2.2 BIAcore Analysis of Alanine Mutants

2.2.1 Experimental Method for BIAcore

Briefly, WT HSA and mutants were immobilised onto CM5 Biacore chips on a Biacore 3000 (GE Healthcare). This was performed by first activating all four flow cells with EDC/NHS and then injecting WT HSA or mutants in acetate buffer pH 4.5. Any free sites on the chip were then blocked with an injection of ethanolamine across all four flow cells. Levels of immobilization are for each sample are summarized in Table 20 below.

TABLE 20

BIAcore CM5 chip preparation.

Chip1	Fc2	E227A HSA	312	RUs
	Fc4	WT HSA	293	RUs
Chip2	Fc2	D314A HSA	369	RU
	Fc3	K225A HSA	143	RU
	Fc4	E230A HSA	339	RU
Chip3	Fc2	M324A HSA	167	RU
	Fc3	V325A HSA	147	RU
	Fc4	WT HSA	304	RU
Chip4	Fc2	K317A HSA	305	RU
	Fc3	K351A HSA	165	RU
	Fc4	WT HSA	223	RU

2.2.2 BIAcore Data

Flow rate using HBS-EP buffer was 40 uL/min and the purified dAb proteins were injected for 1 minute at concentrations 5000 nM followed by 7 further injections at a sequential 1:2 dilution in mobile phase.
Analysis and determination of equilibrium binding constants (KD) was performed using standard procedures.

TABLE 21 A

	DOM7h-	DOM7h-
	14-10myc	11-15myc	DOM7h-11-3myc

		x-fold		x-fold		x-fold
		decrease		decrease		decrease
HSA		in		in		in
mutant	nM	binding	nM	binding	nM	binding

K225A	2.34E−	0.31	1.17E−	0.53	2.41E−08	0.90375
	09		09
E227A	3.33E−	4.44	7.91E−	3.58	1.46E−07	5.475
	08		09
E230A	1.50E−	2.00	2.45E−	11.09	8.00E−07	30
	08		08
D314A	5.35E−	0.71	4.45E−	2.01	1.78E−08	0.6675
	09		09
M329A	5.65E−	0.75	5.72 E−	2.59	6.52E−07	24.45
	09		09
WT	7.5E−		2.21E−		2.66667E−
	09		09		08

	TABLE 21 B

			DOM7r31-14myc

DOM7r92-4myc

x-fold

		x-fold		decrease
HSA		decrease		in
mutant	nM	in binding	nM	binding

K225A	3.49E−08	0.71	2.49E−08	0.44
E227A	1.28E−07	2.62	no	88.81
			binding
E230A	4.06E−06	83.03	3.97E−07	7.05
D314A	3.45E−08	0.71	5.20E−08	0.92
M329A	2.75E−08	0.56	4.93E−07	8.76
WT	4.89E−08		5.63E−08

2.2.3 BIAcore Analysis and Conclusions

DOM7h-14-10: No significant decrease in binding was observed upon mutagenesis of any of the above residues in isolation.
DOM7h-11-15: Some significant decrease in binding of DOM7h-11-15 to E230A is observed (11-fold decrease in binding over WT). This suggests that residue 230 on HSA plays a significant contribution in the specific binding to human serum albumin.
DOM7h-11-3: A significant decrease in binding was observed for two residues (E230 and M324) upon mutagenesis to alanine. This suggests that these two residues play an important contribution to the Antibody/Antigen interaction.
DOM7r92-4: A significant decrease in binding for E230A was observed.
DOM7r31-14: A significant decrease in binding for E227A was observed.

3 CRYSTAL STRUCTURE OF THE DOM7H-11-15/HSA COMPLEX

3.1 Protein Preparation

Fatty acid free HSA from a commercial source was purified by size exclusion chromatography to >95% purity as judged by SDS-PAGE.
DOM7h-11-15 (SEQ ID NO: 1 and SEQ ID NO: 2 for amino acid and DNA sequences respectively) was expressed in E. coli strain BL21 DE3 using an auto-induction expression system. DOM7h-11-15 was purified from clarified supernatants by Protein-L affinity chromatography using established protocols. It was further purified by ion exchange chromatography using a Hi-Trap SP column using established protocols.
HSA was mixed with DOM7h-11-15 and the complex purified by size exclusion chromatography. Protein was concentrated in 20 mM Tris-Cl pH 8.0 prior to crystallization screening.

3.2 Crystallization

The HSA/DOM7h-11-15 complex was put into a crystallization screen with approximately 1200 conditions using the sitting drop method.
3.3 X-ray Diffraction Data Collection and Processing
HSA/DOM7h-11-15 crystals were flash frozen in liquid nitrogen after cryoprotection. The crystal was maintained at 100K during data collection. X-ray diffraction data were collected at the SWISS LIGHT SOURCE (SLS, Villigen, Switzerland).
Data were processed using XDS and XSCALE (Kabsch) reviewed, for example, in Acta Crystallogr D Biol Crystallogr. 2010 Feb. 1; 66(Pt 2): 125-132). The crystals belonged to the space group P2₁2₁2₁with two complexes HSA/DOM7h-11-15 in the asymmetric unit.
Data collection statistics are summarised in Table 22 below.

TABLE 22

Data Collection and Processing Statistics for HSA/DOM7h-11-15
complex Crystal

	Complex	HSA-DOM7h11-15
	X-ray source	PXI/X06SA (SLS ¹)
	Wavlength [Å]	1.0000
	Detector	PILATUS 6M
	Temperature [K]	100
	Space group	P 2₁2₂2₃
	Cell: a; b; c [Å]	102,15; 110.00; 141.34
	α:β:γ [°]	90.0; 90,0; 90.0
	Resolution [Å] ²	2.50 (2.91-2.66)
	Unique reflections ²	55,052 (10,796)
	Multiplicity ²	5.3 (5.5)
	Completeness [%] ²	98.7 (100.0)
	R_sym[%] ^{2, 3}	10.0 (43.9)
	R_meas[%] ^2,4	11.1 (48.3)
	mean(I)/sigma ^2,5	12.17 (4.63)

	¹SWISS LIGHT SOURCE (SLS, Villigen, Switzerland)
	²Numbers in brackets correspond to the resolution bin with R_sym= 43.9%.
	$^{3} R_{sym} = \frac{\sum_{h}^{} \sum_{i}^{n_{h}} \langle {\hat{I}}_{h} - I_{h, i} \rangle}{\sum_{h}^{} \sum_{i}^{n_{h}} I_{h, i}} with {\hat{I}}_{h} = \frac{1}{n_{h}} \sum_{i}^{n_{h}} I_{h, i}$ where I_h,iis the intensity value of the ith measurement of h
	${}^{4}R_{meas} = \frac{\sum_{h}^{} \sqrt{\frac{n_{h}}{n_{h} - 1}} \sum_{i}^{n_{h}} \langle {\hat{I}}_{h} - I_{h, i} \rangle}{\sum_{h}^{} \sum_{i}^{n_{h}} I_{h, i}} with {\hat{I}}_{h} = \frac{1}{n_{h}} \sum_{i}^{n_{h}} I_{h, i}$ where I_h,iis the intensity value of the ith measurement of h
	⁵Calculated from independent reflections

3.4 Structure Determination and Model Refinement

Structure determination and model refinement was carried out to generate a representation of HSA in complex with DOM 7h-11-15.
The structure of the complex was determined by molecular replacement. DOM7h11-15 bound to HSA showed clear electron density in the initial maps from phases determined from the HSA molecules only and allowed unambiguous placement of the antibody domain using a difference maps. Subsequent model building and refinement was performed according to standard protocols with the software packages in CCP4 and COOT (see Collaborative Computational Project, Number 4. 1994.
“The CCP4 Suite: Programs for Protein Crystallography”. Acta Cryst. D50, 760-763; and “Coot: model-building tools for molecular graphics” Emsley P, Cowtan K Acta Crystallographica Section D-Biological Crystallography 60: 2126-2132 Part 12 Sp. Iss. 1 Dec. 2004, for example). Refinement statistics are summarized in Table 23
The Ramachandran Plot of the final model shows 91.5% of all residues in the most favoured region, 8.1% in the additionally allowed region, 0.4% of the residues in the generously allowed, and no residues in the disallowed regions (Modelling statistics are summarized in Table 23).

TABLE 23

Refinement and Modelling Statistics for the HSA/DOM7h-11-15 Structure

Complex	HSA-DOM7h11-15

Resolution [Å]	86.71-2.50
Number of reflections (working/test)	54,062/989
R_cryst[%]	23.8
R_free ²[%]	28.5
Total number of atoms:
Protein	10,881
Water	103
Ligand	—
Sulphate	60
1,2-Ethanediol	16
Deviation from ideal geometry: ³
Bond lengths [Å]	0.008
Bond angles [°]	1.06
Bonded B's ⁴[Å²]	1.7
Ramachandran Plot: ⁵
Most flavoured regions	92.0
Additional allowed regions	7.7
Generously allowed regions	0.3
Disallowed regions	0.0

¹Vaules as defined in REFMAC5, without sigma cut-off
²Test-set contains 1.8% of measured reflections
³Root mean square deviations from geometric target values
⁴Calculated with programme MOLEMAN
⁵Calculated with programme PROCHECK

3.5 Structure Analysis

3.5.1 Overall Structure

The structure of HSA in complex with DOM7h11-15 is represented in FIG. 4. FIG. 4A shows the asymmetric unit containing 2 copies each of HSA and DOM7h-11-15. The biologically relevant unit which consists of one molecule each of HSA and DOM7h-11-15 is shown in two orientations in FIG. 4B.

3.5.2 The Epitope and Paratope

Residues on the HSA-Albudab binding interface are displayed in a format by residue number in FIG. 5 (this figure lists all residue to residue contacts within 4.5 A between chains A (HSA) and chain B (DOM7h-11-15)). All significant interaction pairs are marked as solid diamonds. Types of interactions for those considered to significant are listed in Table 22.

TABLE 22A

List of interactions between HSA and DOM7h-11-15

HSA

DOM7h-11-15

	Residue	Residue		Residue	Residue
Chain	Number	Type	Chain	Number	Type	Interaction

A	228	(PHE)	B	49	(LEU)	hydrophobic
A	228	(PHE)	B	91	(ALA)	hydrophobic
A	230	(GLU)	B	94	(HIS)	1 H-bond SC-SC
A	308	(ASP)	B	53	(ARG)	3 H-bonds all SC-
						SC
A	309	(PHE)	B	51	(PHE)	hydrophobic
A	317	(LYS)	B	67	(SER)	1 H-bond SC-MC
A	318	(ASN)	B	31	(THR)	2 H-bond MC-
						SC, SC-SC
A	321	(GLU)	B	30	(GLY)	1 H-bond MC-SC
A	322	(ALA)	B	32	(MET)	hydrophobic
A	325	(VAL)	B	32	(MET)	hydrophobic
A	325	(VAL)	B	91	(ALA)	hydrophobic
A	326	(PHE)	B	32	(MET)	hydrophobic
A	326	(PHE)	B	51	(PHE)	hydrophobic
A	329	(MET)	B	32	(MET)	hydrophobic
A	329	(MET)	B	49	(LEU)	hydrophobic
A	329	(MET)	B	50	(ALA)	hydrophobic
A	329	(MET)	B	51	(PHE)	hydrophobic
A	329	(MET)	B	91	(ALA)	hydrophobic

All but two DOM7h-11-15 residues binding HSA are from CDR1, 2 and 3. Residues forming the paratope are showing in the alignment in FIG. 6 below where DOM7h-11-3, DOM7h11-15 are aligned against Vk dummy (VKDUM-1). Table 22B below lists additional residues on the AlbudAb-HSA interface within 4.5 A.

TABLE 22B

Table of additional residues on interface

HSA

DOM7h-11-15

	Residue	Residue		Residue	Residue
Chain	number	number	Chain	number	number

A	227	(GLU)	B	36	(TYR)
A	227	(GLU)	B	46	(LEU)
A	228	(PHE)	B	92	(GLY)
A	229	(ALA)	B	36	(TYR)
A	229	(ALA)	B	92	(GLY)
A	229	(ALA)	B	93	(THR)
A	229	(ALA)	B	94	(HIS)
A	232	(SER)	B	92	(GLY)
A	233	(LYS)	B	94	(HIS)
A	263	(TYR)	B	94	(HIS)
A	307	(ALA)	B	53	(ARG)
A	308	(ASP)	B	51	(PHE)
A	314	(ASP)	B	31	(THR)
A	317	(LYS)	B	30	(GLY)
A	317	(LYS)	B	68	(GLY)
A	318	(ASN)	B	29	(ILE)
A	318	(ASN)	B	30	(GLY)
A	318	(ASN)	B	32	(MET)
A	318	(ASN)	B	51	(PHE)
A	321	(GLU)	B	28	(PRO)
A	321	(GLU)	B	29	(ILE)
A	321	(GLU)	B	68	(GLY)
A	322	(ALA)	B	29	(ILE)
A	325	(VAL)	B	90	(GLN)
A	332	(TYR)	B	49	(LEU)
A	333	(GLU)	B	49	(LEU)

Significant interactions listed in Table 22 are detailed further in FIG. 7 and panels within. In these figures, interacting residues are drawn in stick representation with any hydrogen bonds drawn as dashed lines. Corresponding electron density for those interacting side chains are also show depicted in mesh (contoured at 1.56).

4.0 CONCLUSIONS

Three orthogonal techniques have been used to determine the epitope of the DOM7h-11 lineage on HSA. The results from all techniques provide information about the region of HSA which forms the epitope. Whilst H/D exchange perturbation data give a range of possible residues, Alanine scanning data and the crystal structure provide more detailed information on a single residue level. FIG. 8 below summarizes level of detail and specificity the data from each technique has provided.
Based on the crystal structure, it is also possible to state that the bind of DOM7h-11-15 to HSA does not block or obstruct any lipid carrier pockets on HSA. This is particularly relevant for therapeutic applications since these binding pockets are utilized by a number of therapeutic compounds for systemic transport. Therefore any potential biopharmaceutical formatted with DOM7h-11-15 would not be expected to interfere with HSA-drug interactions and transport. Drug/lipid carrier pocket positions relative to the DOM7h-11-15 epitope are detailed in FIG. 9. Based on the similar epitope observed with DOM7h-14-10 and DOM7r-92-4 H/D exchange perturbation data, these AlbudAbs™ would also not be expected to interfere with HSA-drug interactions and transport.

TABLE OF SEQUENCES

SEQ ID NO:

IDENTIFIER	Amino acid	Nucleic acid

DOM7h-11-15	1	3
DOM7h-11-3	2	4
DOM7h-14/Exendin-4 fusion	5	6
DOM7h-14-10/Exendin-4 fusion	7	8
DOM7h-11/Exendin-4 fusion	9	10
DOM7h-11-15/Exendin-4 fusion	11	12
DOM7h14-10/G4SC-NCE fusion	13	14
DOM7h14-10/TVAAPSC fusion	15	16
DOM7h-11/DOM1m-21-23 fusion	17	19
DOM7h-11/DOM1m-21-23 fusion + myc tag	18	20
DOM7h-11-15/DOM1m-21-23 fusion	21	23
DOM7h-11-15/DOM1m-21-23 fusion + myc tag	22	24
DPK9 Vk dummy CDRs 1-3	25-27	—
DOM7h-11 CDRs 1-3	28-30	—
DOM 7h-11-15 CDRs 1-3	31-33	—
DOM 7h-11-3 CDRs 1-3	34-36	—
DOM 7h-14 CDRs 1-3	37-39	—
DOM 7h-14-10 CDRs 1-3	40-42	—
Interferon alpha 2b	43	44
IFNα2b SOE fragment 5′	—	45
IFNα2b SOE fragment 3′	—	46
Vk SOE fragment 5′	—	47
Vk SOE fragment 3′ to also introduce a myc tag	—	48
IFNα2b SOE fragment 5′	—	49
Vk SOE fragment 3′ to also introduce a myc tag	—	50
Leader sequence	51	52
DMS7321	53	54
(IFNα2b-DOM7h-14) + myc
DMS7321 (IFNα2b-DOM7h-14) no tag	55	56
DMS732 (IFNα2b-DOM7h-14-10) + myc	57	58
DMS732 (IFNα2b-DOM7h-14-10) no tag	59	60
DMS 7325 (IFNα2b-DOM7h-11) + myc	61	62
DMS 7325 (IFNα2b-DOM7h-11) no tag	63	64
DMS 7327 (IFNα2b-DOM7h-11-15) + myc	65	66
DMS 7327 (IFNα2b-DOM7h-11-15) no tag	67	68
DOM7h-14 R108C	69	70
DOM7r31	71	72
DOM7r-31-14	73	74
DOM7h-94	75	76
DOM7r-92-4	77	78
DOMAIN 2 OF HSA	79	80
FULL LENGTH HSA	81	82
DOM 7H-14-10	83	84
PRIMER TB147	—	85
PRIMER TB148	—	86
TB153	—	87
TB154	—	88
TB155	—	89
TB156	—	90
TB157	—	91
TB158	—	92
TB159	—	93
TB160	—	94
TB161	—	95
TB162	—	96
TB163	—	97
TB164	—	98
TB165	—	99
TB166	—	100
TB167	—	101
TB168	—	102
HSA-His6 WT	103	112
HSA-His6 K225A	104	113
HSA-His6 E227A	105	114
HSA-His6 E230A	106	115
HSA-His6 D314A	107	116
HSA-His6 K317A	108	117
HSA-His6 V325A	109	118
HSA-His6 M329A	110	119
HSA-His6 K351A	111	120
DOM 7h-11-13	121	122
DOM7H-14	123	124
DOM7H-11	125	126

Example 11

Sequences of DOM7h-14-10 Variants

In another embodiment of the invention, listed below are the amino acid and nucleotide sequences for some variants of the anti-serum albumin immunoglobulin single variable domain DOM7h-14.

DOM7h-14-56.

(SEQ ID NO: 127)

DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPMLLIMW

SSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQ

GTKVEIKW

DOM7h-14-65.

(SEQ ID NO: 128)

DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW

RSALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQ

GTKVEIKW

DOM7h-14-74.

(SEQ ID NO: 129)

DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW

RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTYGK

GTKVENKW

DOM7h-14-76.

(SEQ ID NO: 130)

DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW

RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLKHPKTYGQ

GTKVEIKW

DOM7h-14-82.

(SEQ ID NO: 131)

DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW

RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGMRHPKTFGQ

GTKVEIKW

DOM7h-14-100.

(SEQ ID NO: 132)

DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW

RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTYGQ

GTKVENKW

DOM7h-14-101.

(SEQ ID NO: 133)

DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW

RSALQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQ

GTKVEIKW

DOM7h-14-109.

(SEQ ID NO: 134)

DIQMTQSPSSLFASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW

RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRKPKTFGQ

GTKVKIKW

DOM7h-14-115.

(SEQ ID NO: 135)

DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW

RSALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTYGQ

GTKVEIKW

DOM7h-14-116.

(SEQ ID NO: 136)

DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW

RSALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRYPKTFGQ

GTKVEIKW

DOM7h-14-119.

(SEQ ID NO: 137)

DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW

RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTYGQ

GTKVEIKR

DOM7h-14-120.

(SEQ ID NO: 138)

DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW

RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTYGQ

GTKVENKR

DOM7h-14-121.

(SEQ ID NO: 139)

DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW

RSALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQ

GTKVEIKR

DOM7h-14-122.

(SEQ ID NO: 140)

DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW

RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTYGK

GTKVEIKR

DOM7h-14-123.

(SEQ ID NO: 141)

DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW

RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTYGK

GTKVENKR

DOM7h-14-56.

(SEQ ID NO: 142)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTATGCTCCTGATCATGTGG

AGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAA

GGGACCAAGGTGGAAATCAAATGG

DOM7h-14-65.

(SEQ ID NO: 143)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG

CGTTCCGCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAA

GGGACCAAGGTGGAAATCAAATGG

DOM7h-14-74.

(SEQ ID NO: 144)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG

CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTACGGCAAA

GGGACCAAGGTGGAAAACAAATGG

DOM7h-14-76.

(SEQ ID NO: 145)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG

CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTTTGAAGCATCCTAAGACGTACGGCCAA

GGGACCAAGGTGGAAATCAAATGG

DOM7h-14-82.

(SEQ ID NO: 146)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG

CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTATGAGGCATCCTAAGACGTTCGGCCAA

GGGACCAAGGTGGAAATCAAATGG

DOM7h-14-100.

(SEQ ID NO: 147)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG

CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTTTGCGGCATCCTAAGACGTACGGCCAA

GGGACCAAGGTGGAAAACAAATGG

DOM7h-14-101.

(SEQ ID NO: 148)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG

CGTTCCGCGTTACAAAATGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAA

GGGACCAAGGTGGAAATCAAATGG

DOM7h-14-109.

(SEQ ID NO: 149)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTTTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG

CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTTTGAGGAAACCTAAGACTTTCGGCCAA

GGGACCAAGGTGAAAATCAAATGG

DOM7h-14-115.

(SEQ ID NO: 150)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG

CGTTCCGCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAAACGTACGGCCAA

GGGACCAAGGTGGAAATCAAATGG

DOM7h-14-116.

(SEQ ID NO: 151)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG

CGTTCCGCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTTTGAGGTATCCTAAGACGTTCGGCCAA

GGGACCAAGGTGGAAATCAAATGG

DOM7h-14-119.

(SEQ ID NO: 152)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG

CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTTTGCGGCATCCTAAGACGTACGGCCAA

GGGACCAAGGTGGAAATCAAACGG

DOM7h-14-120.

(SEQ ID NO: 153)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG

CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTTTGCGGCATCCTAAGACGTACGGCCAA

GGGACCAAGGTGGAAAACAAACGG

DOM7h-14-121.

(SEQ ID NO: 154)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG

CGTTCCGCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAA

GGGACCAAGGTGGAAATCAAACGG

DOM7h-14-122.

(SEQ ID NO: 155)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG

CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTACGGCAAA

GGGACCAAGGTGGAAATCAAACGG

DOM7h-14-123.

(SEQ ID NO: 156)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA

CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT

CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG

CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC

TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG

CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTACGGCAAA

GGGACCAAGGTGGAAAACAAACGG

Claims

1. A nucleic acid comprising a nucleotide sequence encoding an anti-serum albumin (SA) immunoglobulin single variable domain variant of DOM7h-11-15 (SEQ ID NO: 1), wherein the variant comprises an amino acid substitution in at least one position selected from the group consisting of: Leu49, Ala50, and Phe51,

wherein position Met32 is changed to Leu, Phe, or Ile,

position Leu49 is changed to Ile, Val, or Met,

position Ala50 is changed to Val, Leu, or Ile, and/or

position Phe51 is changed to Leu, Val, Ile, Ala, or Tyr.

2. The nucleic acid comprising a nucleotide sequence encoding an anti-serum albumin (SA) immunoglobulin single variable domain variant as claimed in claim 1, wherein

position Met32 is changed to Leu,

position Leu49 is changed to Ile,

position Ala50 is changed to Val, and/or

position Phe51 is changed to Leu.