WO2014111550A1

WO2014111550A1 - Modified anti-serum albumin binding proteins

Info

Publication number: WO2014111550A1
Application number: PCT/EP2014/050961
Authority: WO
Inventors: Claire Ashman; Mary Birchler; Rudolf M T De Wildt; Sarah FRIEL; Marie Claire H HOLLAND; Alan Peter Lewis; Peter Joseph MORLEY; Thomas Sandal; Oliver Schon; Michael Steward
Original assignee: Glaxosmithkline Intellectual Property Development Limited
Priority date: 2013-01-17
Filing date: 2014-01-17
Publication date: 2014-07-24

Abstract

The invention relates to immunoglobulin single variable domain molecules which bind serum albumin comprising modifications and/or mutations which allow reduced binding to pre-existing ADAs and, in particular, wherein the immunoglobulin single variable domain molecules are human V_H or Camelid V_HH. Fusion proteins, ligands, compositions, nucleic acid, vectors and hosts are also described.

Description

Modified anti-serum albumin binding proteins

The disclosure relates to modified variants of anti-serum albumin immunoglobulin single variable domain proteins, as well as ligands and drug conjugates comprising such variants, compositions, nucleic acids, vectors and hosts.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.

Background to invention

Naturally occurring autoantibodies exist in humans that can bind to proteins such as host immunoglobulins or immunoglobulin fragments. These autoantibodies may be part of a polyclonal repertoire of anti-immunoglobulin (Ig) autoantibodies with specificity to epitopes throughout the Ig molecule that are present in both humans and non-human primates. Anti-IgG autoantibodies that bind epitopes within the intact Fc domain (e.g. the Rheumatoid factors (RF)) have been observed as well as anti-idiotypic autoantibodies that bind to antibody variable/CDR regions of IgG and anti- hinge antibodies that react with cryptic epitopes in the C terminal hinge regions of the Ig constant domain in Fab or F(ab')₂ fragments has also been observed.

The functional role of these different anti-IgG autoantibodies remains uncertain. Rheumatoid factor and anti-hinge autoantibodies have been linked with pathological conditions such as autoimmunity and certain infections, while anti-idiotypic antibodies may confer protection from autoantibodies in autoimmune diseases. Furthermore, an immunoregulatory role for anti-IgG autoantibodies, has been proposed wherein these autoantibodies control the stimulation of autoreactive B cells and regulate immune responses to foreign antigens. Anti-hinge antibodies are anti-IgG autoantibodies that react with cleaved but not intact IgG. Their high prevalence in the normal human population implicates previous exposure to IgG fragments, possibly as a result of cleavage of IgG by bacterial or endogenous proteases.

As well as binding to endogenous proteins present in naive subjects, autoantibodies can also bind to proteins or peptides which are administered to a subject for treatment. Pre-existing antibodies which bind to molecules such as therapeutic proteins and peptides, administered to a subject (i.e. therapeutic protein-binding autoantibodies) can affect the efficacy of the administered proteins or peptides and could result in administration reactions, hypersensitivity, altered clinical response in treated patients as well as altered bioavailability by sustaining, eliminating or neutralizing the molecule. The significance of these autoantibodies in a drug treatment varies.

Therapeutic protein-binding pre-existing autoantibodies and antibodies that are newly formed in response to drug treatment (such as administration of a therapeutic protein or peptide) are generally collectively termed "anti-drug antibodies (ADAs)". However, in the context of the present invention and application, reference to "ADAs" is to pre-existing ADAs (i.e. naturally occurring autoantibodies) unless specifically stated otherwise.

V_H and V_L domain antibodies (dAbs™) are derived from fully human framework sequences and, although in silico predictions describe a markedly low incidence of potentially immunogenic peptides, it is possible that these domain antibodies may bind to pre-existing ADAs depending on both sequence dependent and sequence independent factors.

Similarly, a number of single dAbs™ derived from the Camelid heavy chain (V_HH) are under investigation in the clinic and binding to pre-existing ADAs remains a possibility.

WO2004/003019 and WO2008/096158 disclose anti-serum albumin (SA) binding moieties, such as anti-SA immunoglobulin single variable domains (AlbudAbs™), 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™.

WO2005/118642 discloses 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 new chemical entity (NCE) drugs are disclosed and exemplified. 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, pp283-288, 2008.

It could thus be advantageous to provide anti-serum albumin binding molecules for therapy which have a reduced ability to bind to pre-existing ADAs when administered to a subject.

Summary of the invention

The present invention provides immunoglobulin single variable domain molecules which bind serum albumin comprising modifications and/or mutations which allow reduced binding to preexisting ADAs. Suitably, the immunoglobulin single variable domain is a human V_H or a Camelid V_HH- Accordingly, in a first aspect, there is provided an immunoglobulin single variable domain antibody which binds to serum albumin comprising a C-terminal amino acid sequence consisting of the sequence VTVS(S)_nX (SEQ ID NO: 7) wherein:

n represents an integer independently selected from 0 or 1;

X represents an amino acid extension of 1 to 5 amino acids residues.

Suitably, the immunoglobulin single variable domain antibody is a heavy chain single variable domain antibody such as a V_H single variable domain antibody or dAb™ or a V_HH nanobody.

In one embodiment, said amino acid extension, X, has the effect of reducing the amount of

ADA binding in the immunoglobulin single variable domain comprising the amino acid extension compared to an immunoglobulin single variable domain having the same amino acid sequence but lacking the C-terminal extension.

"Reducing the amount of ADA binding" or "reduced ADA binding" means that a molecule has a lower binding affinity and/or avidity for an ADA than an equivalent unmodified dAb™. This may be determined using surface plasmon resonance e.g. on a BIAcore™ apparatus using standard techniques. The skilled person will understand that the lower the K_D value the stronger the binding. Suitably, terms such as "reduce binding to pre-existing ADAs", "reduced ADA binding" or "reducing the amount of ADA binding" mean that a KD of binding to ADA which is 150% or more (e.g. 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650% or more) of the K_D of an equivalent but unmodified single immunoglobulin variable domain (dAb™) sequence is obtained, "reduced ADA binding" may also be determined using a confirmation assay as described in the Examples section herein and where said modified dAb™ has a mean % inhibition of signal which is less than 90%, e.g. less than 80%, e.g. less than 70%, e.g. less than 60% , e.g. less than 50%, e.g. less than 40%, e.g. less than 30%, e.g. less than 20% , e.g. less than 10% , in comparison with a control single immunoglobulin variable domain which has around 98%-100% inhibition of signal, and wherein said control (unmodified) single immunoglobulin variable domain has the same or similar sequence but is not modified to reduce ADA binding.

For the purpose of the Sequence Listing filed herewith, the sequence VTVS(S)_nX is indicated as VTVS (SEQ ID NO: 7) (S)_nX. Suitably, X represents an amino acid extension selected from the group consisting of A, AAA, T, or conservative amino acid substitutions thereof. Co-pending application PCT/EP2012/065782 describes additional modifications which reduce ADA binding in immunoglobulin single variable domain molecules compared to un-modified versions otherwise having the same amino acid sequence. In particular, co-pending application PCT/EP2012/065782 describes V_H or V_HH immunoglobulin single variable domains wherein X represents an amino acid extension selected from the group consisting of (a) AS, (b) AST, (c) ASTK (SEQ ID NO: 8), (d) ASTKG (SEQ ID NO: 9). Accordingly, these C-terminal extensions when applied to immunoglobulin single variable domains which bind to serum albumin are also envisaged as falling within the scope of the present invention.

Co-pending application PCT/EP2012/065782 also describes molecules in which a C-terminal extension is present or absent and where the immunoglobulin single variable domain comprises one or more amino acid substitutions in the human germline framework sequence. In particular, one or more amino acid substitutions at positions 14, 41, 108, 110 or 112 result in molecules with reduced ADA binding. Accordingly, the present invention also provides an immunoglobulin single variable domain which binds to serum albumin and which comprises one or more amino acid substitution in the human germline framework at these residues, wherein amino acid numbering is in accordance with Kabat. Preferred human framework regions are those encoded by germ line gene segments DP47 and DPK9. Advantageously, FW1, FW2 and FW3 of a V_H or V_L domain have the sequence of FW1, FW2 or FW3 from DP47 or DPK9. Suitably, an immunoglobulin single variable domain in accordance with the invention has similar biophysical properties to a single variable domains having the same amino acid sequence but lacking the C-terminal amino acid extension. Such biophysical properties may be, for example, monomeric/dimeric state which may be assessed by SEC-MALLS and thermostability which may be assessed by DSC. In one embodiment, the binding properties to serum albumin are substantially the same as the unmodified molecule.

In one embodiment, the immunoglobulin single variable domain in accordance with the invention has a therapeutically useful half-life. In another embodiment, the immunoglobulin single variable domain in accordance with the invention can confer an increased half-life to an additional moiety when linked to that moiety.

In one embodiment, the immunoglobulin single variable domain binds to serum albumin (SA) which is human serum albumin. Suitably, an immunoglobulin single variable domain in accordance with the invention comprises a binding site that specifically binds human SA with a dissociation constant (K_D) 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 immunoglobulin single variable domain in accordance with the invention comprises a binding site that specifically binds human SA with an off-rate constant (K_d) of from about 1.5 x 10^"4 to about 0.1 sec^"1, optionally from about 3 x 10^"4 to about 0.1 sec^"1 as determined by surface plasmon resonance. In another embodiment, the immunoglobulin single variable domain in accordance with the invention comprises a binding site that specifically binds human SA with an on-rate constant (K_a) of from about 2 x 10⁶ to about 1 x 10⁴ M^sec^"1, optionally from about 1 x 10⁶ to about 2 x 10⁴ M^sec^"1 as determined by surface plasmon resonance.

Advantageously, the immunoglobulin single variable domain in accordance with the invention is cross-reactive with serum albumin from a number of different species such as, for example, monkey e.g. Cynomolgus monkey, Suncus (shrew), marmoset, ferret, rat, mouse, pig and dog SA. Such cross reactivity provides utility in animal models of disease.

Accordingly, in one embodiment, the immunoglobulin single variable domain in accordance with the invention comprises a binding site that specifically binds Cynomolgus monkey SA with a dissociation constant (K_D) 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 immunoglobulin single variable domain 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 x 10^"4 to about 0.1 sec^"1 , optionally from about 3 x 10^"4 to about 0.1 sec^"1 as determined by surface plasmon resonance. In another embodiment, the immunoglobulin single variable domain of the invention comprises a binding site that specifically binds Cynomolgus monkey SA with an on-rate constant (K_a) of from about 2 x 10⁶ to about 1 x 10⁴ M^sec^"1 , optionally from about 1 x 10⁶ to about 5 x 10³ M^" ^ec^"1 as determined by surface plasmon resonance. In one embodiment, said immunoglobulin single variable domain has the sequence as set out in SEQ ID NO: 3 or an amino acid sequence that is at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to such sequence, and comprises a C-terminal amino acid sequence consisting of the sequence VTVS(S)_nX as defined above.

In another embodiment, said immunoglobulin single variable domain has the amino acid sequence as set out in SEQ ID NO: 5 or an amino acid sequence that is at least 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to such sequence.

The invention also provides a V_HH immunoglobulin single variable domain which binds serum albumin with any one of the modifications described herein to reduce binding to ADAs. Suitable V_HH sequences are described in WO2003/035694, WO2004/041865, WO2004/041867, WO2006/122787, WO2008028977 and WO2008/043821, for example.

In another embodiment, there is provided an immunoglobulin single variable domain according to any aspect or embodiment of the invention wherein the immunoglobulin single variable domain is present as a fusion or conjugate with one or more additional molecules. Suitably, said one or more additional molecule may be selected from an additional immunoglobulin single variable domain or protein or polypeptide or fragment thereof, a further therapeutic or active molecule, a PEG molecule, an antibody or a fragment thereof or an Fc region. In one embodiment, where said immunoglobulin single variable domain is present with one or more additional immunoglobulin single variable domains, said additional immunoglobulin single variable domain may bind to a target other than SA.

A single immunoglobulin variable domain in accordance with the invention may be formatted as part of a larger molecule. For example, the single immunoglobulin variable domain (dAb™) may be present as a mAbdAb molecule, an inline fusion with another protein or polypeptide or as part of a dumbbell format.

In one embodiment, the single immunoglobulin variable domain in accordance with the invention may be conjugated or fused to a drug moiety such as an NCE (new chemical entity), a biopharmaceutical or a polypeptide or peptide.

Suitably, the immunoglobulin single variable domain in accordance with the invention confers an increased or improved half-life of the additional molecule to which it is fused or conjugated compared to the half-life of the additional molecule alone.

In one aspect there is provided a conjugate comprising a single immunoglobulin variable domain in accordance with the invention.

In one aspect there is provided a fusion protein comprising a single immunoglobulin variable domain in accordance with the invention fused to a drug moiety such as an NCE, or protein or polypeptide drug or biopharmaceutical. Suitably, there is provided a fusion product, e.g., a fusion protein or fusion with a peptide or NCE drug, comprising a polypeptide, protein, peptide or NCE drug fused or conjugated to any anti-SA variable domain of the invention. In one embodiment, the immunoglobulin single variable domain in accordance with the invention shows no effect on, or only a modest drop in, affinity for its binding partner when fused or conjugated to a partner making it useful in fusion products.

In another aspect, there is provided a multispecific ligand comprising an anti-SA immunoglobulin single variable domain in accordance with any aspect or embodiment of the invention and a binding moiety that specifically binds a target antigen other than SA. In one embodiment, the binding moiety that specifically binds a target antigen may be another single domain immunoglobulin molecule. In another embodiment, the binding moiety that specifically binds a target antigen may be a monoclonal antibody. Suitable formats and methods for making dual specific molecules, such as mAbdAb molecules are described, for example in WO2009/068649.

Target antigens other than SA 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. One skilled in the art will appreciate that the choice is large and varied. Target antigens may be, for instance, human or animal proteins, cytokines, cytokine receptors, where cytokine receptors include receptors for cytokines, enzymes, co-factors for enzymes or DNA binding proteins although this list is by no means exhaustive.

Suitably, a fusion protein or multi-specific ligand in accordance with the invention comprises a linker (e.g., AST, a GlySer linker or a linker comprising the amino acid sequence TVA, optionally TVAAPS (SEQ ID NO: 10)) between the immunoglobulin single variable domain and the drug.

In another aspect, there is provided a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin single variable domain, a fusion protein or a multispecific ligand in accordance with any aspect or embodiment or the invention. Suitably said nucleic acid is isolated and/or recombinant.

In one embodiment, there is provided a nucleic acid comprising the nucleotide sequence set out in SEQ ID NO: 6 or a nucleotide sequence that is at least 80% identical to said selected sequence.

In another aspect there is provided a vector comprising a nucleic acid in accordance with the invention. In a further aspect there is provided an isolated host cell comprising the vector of the invention. Also provided is a method of producing a polypeptide such as an immunoglobulin single variable domain, fusion protein or multispecific ligand in accordance with the invention wherein said method comprises maintaining a host cell under conditions suitable for expression of said nucleic acid or vector, whereby a polypeptide is produced.

In another aspect, the invention provides a composition, suitably a pharmaceutical composition, comprising an immunoglobulin single variable domain, fusion protein or ligand of any aspect or embodiment of the invention and a pharmaceutically acceptable diluent, carrier, excipient or vehicle. The invention also provides a method of treating or preventing a disease or disorder in a patient, comprising administering at least one dose of an immunoglobulin single variable domain, fusion protein or ligand of any aspect or embodiment of the invention to said patient. In another aspect, there is provided an immunoglobulin single variable domain, fusion protein or ligand in accordance with any aspect or embodiment of the invention for use in medicine.

Further provided is an immunoglobulin single variable domain, fusion protein or ligand of any aspect or embodiment of the invention, or a pharmaceutical composition, for use in a method of therapy or in medicine. In addition, there is provided an immunoglobulin single variable domain, fusion protein or ligand of any aspect or embodiment of the invention for use in the preparation of a medicament for use in therapy or treatment. Suitably, the immunoglobulin single variable domain, fusion protein or ligand of any aspect or embodiment of the invention, or pharmaceutical composition is for use in a method of preventing or reducing side effects. In particular, the immunoglobulin single variable domain, fusion protein or ligand of any aspect or embodiment of the invention, or pharmaceutical composition are for use in a method in which fewer or a reduced severity of side effects are observed compared to a method which uses an immunoglobulin single variable domain which has not been modified to reduce ADA binding. 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 etal., 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^th Ed, John Wiley & Sons, Inc. which are incorporated herein by reference) and chemical methods.

The term "single variable domain" or "immunoglobulin single variable domain" (or "single immunoglobulin variable domain") refers to a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains such as V_H, V_HH and V_L and modified antibody 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 fragments of variable domains which retain at least the binding activity and specificity of the full-length domain. A single variable domain is capable of binding an antigen or epitope independently of a different variable region or domain. A "domain antibody™" or "dAb™" may be considered the same as a "single variable domain". A single variable domain may be a human single variable domain, but also includes single variable domains from other species such as rodent (for example, as disclosed in WO 00/29004), nurse shark and Camelid V_HH including nanobodies™. Camelid V_HH are immunoglobulin single variable domains that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such V_HH domains may be humanised according to standard techniques available in the art, and such domains are considered to be "single variable domains". As used herein, V_H includes Camelid V_HH domains. Also within the scope of the present invention are human dAbs™ which have been modified so as to be not fully human, for example modifications which are made to reduce aggregation, including mutation of the same residues which are Camelid motifs.

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).

An unmodified immunoglobulin single variable domain (i.e. unmodified dAb™), for example a dAb™ that binds a target, comprises three complementarity determining regions (CDRs) within a framework structure. Whereas in the genetics of naturally occurring immunoglobulin chains the V region terminates at the beginning of CDR3, with the remainder of CDR3 being provided by the D and J regions (resulting in a V-D-J fusion), for the purposes of the present invention a dAb™ includes all of CDR3 and terminates in framework 4 residue at its C-terminus. A V_H dAb™ terminates in residues LVTVSS (SEQ ID NO: 11) at its C-terminus. A V_HH dAb™ terminates in residues VTVSS (SEQ ID NO: 12) at its C-terminus.

A "modified dAb™" is a dAb™ as described herein which additionally has a modification which alters the three dimensional conformation of the dAb C-terminus. A modified dAb™ includes a dAb™ which comprises C-terminal additions, extensions or tags and/or certain amino acid substitutions as disclosed herein.

The invention additionally provides a dAb™ which has reduced binding to ADA in human sera (e.g. does not bind to pre-existing ADA in human sera) and wherein the epitope on the dAb™ to which the ADA binds is masked (i.e. the epitope is no longer available to bind to ADA as it has been covered or masked by another molecule so preventing binding or its steric conformation has been changed so preventing binding for example). The epitope on the dAb™ can be masked by any of the modifications described herein to reduce ADA binding, for example adding a chemical entity to the C terminus of the dAb™ or by framework substitutions, or deletions as described herein. The chemical entity added to the C terminus of the dAb™ can be an extension (e.g. an amino acid extension) or a tag or it can be a chemical modification such as PEGylation or amidation. The modification to the C terminus can be one which either directly or indirectly changes the conformation of the epitope on the dAb™ which binds to ADAs thereby reducing the ability of the dAb™ to bind to ADAs. In one embodiment the invention the immunoglobulin single variable domain can be modified to prevent binding to ADAs such that the modification comprises a tag present at the C terminus. This tag can be present as a fusion or conjugate with the molecule. The tag can be any tag known in the art for example affinity tags such as myc-tags, FLAG tags, his-tags, chemical modification such as PEG, or protein domains such as the antibody Fc domain.

A pre-existing ADA is an ADA already present in the subject to which the drug is to be administered. A pre-existing ADA may be present in a naive subject (i.e. a subject to which the drug has never been administered before).

The phrase, "half-life," refers to the time taken for the serum or plasma concentration of the fusion or conjugate to reduce by 50%, in vivo, for example, due to degradation and/or clearance or sequestration by natural mechanisms. The compositions of the invention are stabilized in vivo and their half-life increased by binding to serum albumin molecules e.g. human serum albumin (HSA) which resist degradation and/or clearance or sequestration. These serum albumin molecules are naturally occurring proteins which themselves have a long half-life in vivo. The half-life of a molecule is increased if its functional activity persists, in vivo, for a longer period than a similar molecule which is not specific for the half-life increasing molecule.

Affinity is the strength of binding of one molecule, e.g. an antigen binding protein of the invention, to another, e.g. its target antigen, at a single binding site. The binding affinity of an antigen binding protein to its target may be determined by standard equilibrium methods (e.g. enzyme-linked immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (such as surface plasmon resonance e.g. BIACORE™ analysis).

K_D or dissociation constant refers to the strength of a two molecule interaction. A skilled person will appreciate that the smaller the K_D numerical value, the stronger the binding. The reciprocal of K_D (i.e. 1/ K_D) is the equilibrium association constant (K_A) having units M^"1. A skilled person will appreciate that the larger the K_A numerical value, the stronger the binding.

The dissociation rate constant (k_d) or "off-rate" describes the stability of an antigen binding protein-target complex, i.e. the fraction of complexes that decay per second. For example, a k_d of 0.01 s^"1 equates to 1% of the complexes decaying per second. The association rate constant (k_a) or "on-rate" describes the rate of antigen binding protein-target complex formation.

"Percent identity" between a query nucleic acid sequence and a subject nucleic acid sequence is the "Identities" value, expressed as a percentage, that is calculated by the BLASTN algorithm when a subject nucleic acid sequence has 100% query coverage with a query nucleic acid sequence after a pair-wise BLASTN alignment is performed. Such pair-wise BLASTN alignments between a query nucleic acid sequence and a subject nucleic acid sequence can be performed by using the default settings of the BLASTN algorithm available on the National Center for Biotechnology Information's website with the filter for low complexity regions turned off. Importantly, a query nucleic acid sequence may be described by a nucleic acid sequence identified in one or more claims herein or elsewhere in this application. "Percent identity" between a query amino acid sequence and a subject amino acid sequence is the "Identities" value, expressed as a percentage, that is calculated by the BLASTP algorithm when a subject amino acid sequence has 100% query coverage with a query amino acid sequence after a pair-wise BLASTP alignment is performed. Such pair-wise BLASTP alignments between a query amino acid sequence and a subject amino acid sequence can be performed by using the default settings of the BLASTP algorithm available on the National Center for Biotechnology Information's website with the filter for low complexity regions turned off. Importantly, a query amino acid sequence may be described by an amino acid sequence identified in one or more claims herein or elsewhere in this application.

As described herein, a conjugate refers to a composition comprising an immunoglobulin single variable domain in accordance with the invention to which a further molecule is chemically conjugated by means of a covalent or noncovalent linkage, preferably a covalent linkage. Such covalent linkage could be through a peptide bond or other means such as via a modified side chain. The noncovalent bonding may be direct (e.g., electrostatic interaction, hydrophobic interaction) or indirect (e.g., through noncovalent binding of complementary binding partners (e.g., biotin and avidin), wherein one partner is covalently bonded to drug and the complementary binding partner is covalently bonded to the immunoglobulin single variable domain). 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 immunoglobulin single variable domain directly or through a suitable linker moiety.

As used herein, a fusion protein comprising an anti-SA immunoglobulin single variable domain refers to a fusion protein that comprises an anti-SA immunoglobulin single variable domain and one or more additional molecules such as an additional immunoglobulin single variable domain, a protein, polypeptide or fragment thereof, a polypeptide drug, active molecule, antibody or antibody fragment, wherein the immunoglobulin single variable domain and the additional molecule are present as discrete parts (moieties) of a single continuous polypeptide chain.

For example a dAb™ can be present as a formatted dAb™ (e.g. the dAb can be present as a dAb-Fc fusion or conjugate as described in for example WO 2008/149148) or it can be present as a mAbdAb (as described in WO 2009/068649) or in combination with further therapeutic or active molecules. A fusion or conjugate with an immunoglobulin single variable domain in accordance with the invention may be linked to via either the C-terminal extension or the N-terminus of the immunoglobulin single variable domain. In one embodiment one or more therapeutic molecules are present as a fusion (or conjugate) at the N terminus of the immunoglobulin single variable domain.

As used herein, the term mAbdAb refers to a monoclonal antibody linked to a further binding domain, in particular a single variable domain such as a domain antibody in accordance with the invention. A mAbdAb has at least two antigen binding sites, at least one of which is from a domain antibody, and at least one is from a paired V_H/V_L domain. Such mAbdAbs are described for example in WO 2009/068649. As used herein, the term "Fc region" refers to a single heavy chain Fc region of an IgG, such as an IgGl, IgG2, IgG3, iGG4 or IgG4PE, or an IgA antibody. A single heavy chain Fc region may comprise one or more of the CHI, CH2 and CH3 constant region antibody domains, for example all three constant region antibody domains or just the CH2 and CH3 domains. In addition to comprising one or more of the CHI, CH2 and CH3 constant region antibody domains, the single heavy chain Fc region of an antibody may further comprise a hinge region of an antibody (such a region normally found between the CHI and CH 2 domains).

As used herein, "target ligand" or "target antigen" refers to a ligand or antigen which is specifically or selectively bound by a polypeptide or peptide. For example, when a polypeptide is an antibody, antigen-binding fragment thereof, or immunoglobulin single variable domain, the target ligand can be any desired antigen or epitope. Suitably, binding to the target antigen is dependent upon the polypeptide or peptide being functional.

As used herein an antibody refers to IgG, IgM, IgA, IgD or IgE or a fragment (such as a 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.

The invention also relates to a recombinant host cell e.g. mammalian or microbial, which comprises a (one or more) recombinant nucleic acid or expression construct comprising nucleic acid(s) encoding an immunoglobulin single variable domain, fusion protein, multi-specific ligand or composition of the invention as described herein. There is also provided a method of preparing a molecule such as an immunoglobulin single variable domain, fusion protein, multi-specific ligand or composition in accordance with the invention as described herein, comprising maintaining a recombinant host cell e.g. mammalian or microbial, of the invention under conditions appropriate for expression of the molecule. The method can further comprise the step of isolating or recovering the expressed molecule, if desired.

For example, a nucleic acid molecule {i.e., one or more nucleic acid molecules) encoding a molecule of the invention can be introduced into a suitable host cell to create a recombinant host cell using any method appropriate to the host cell selected {e.g., transformation, transfection, electroporation, infection), such that the nucleic acid molecule(s) are operably linked to one or more expression control elements {e.g., in a vector, in a construct created by processes in the cell, integrated into the host cell genome). The resulting recombinant host cell can be maintained under conditions suitable for expression {e.g., in the presence of an inducer, in a suitable animal, in suitable culture media supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), whereby the encoded peptide or polypeptide is produced. If desired, the encoded peptide or polypeptide can be isolated or recovered {e.g., from the animal, the host cell, medium, milk). This process encompasses expression in a host cell of a transgenic animal (see, e.g., WO 92/03918, GenPharm International).

The molecules of the invention as described herein can also be produced in a suitable in vitro expression system, e.g. by chemical synthesis or by any other suitable method.

The molecules of the invention can be expressed in £ coii or in Pichia species (e.g., P. pastoris). In one embodiment, the dAb™ is secreted in £ coii or in Pichia species (e.g., P. pastoris); or in mammalian cell culture (e.g. CHO, or HEK 293 cells). Although, the molecules described herein can be secretable when expressed in £ coii or in Pichia species or mammalian cells they can be produced using any suitable method, such as synthetic chemical methods or biological production methods that do not employ £ coii or Pichia species.

The skilled person will appreciate that, upon production of a molecule as described herein, in particular depending on the cell line used and particular amino acid sequence of the molecule, post- translational modifications may occur. For example, this may include the cleavage of certain leader sequences, the addition of various sugar moieties in various glycosylation and phosphorylation patterns, deamidation, oxidation, disulfide bond scrambling, isomerisation, C-terminal lysine clipping, and N-terminal glutamine cyclisation. The present invention encompasses the use of such molecules, which have been subjected to, or have undergone, one or more post-translational modifications. Thus an dAb™ of the invention includes an a dAb™ which has undergone a post- translational modification such as described as follows: Glycosylation of antibodies at conserved positions in their constant regions is known to have a profound effect on antibody function, particularly effector functioning, see for example, Boyd et al. (1996) Mol. Immunol. 32: 1311-1318. Glycosylation variants of the antigen binding proteins of the invention wherein one or more carbohydrate moiety is added, substituted, deleted or modified are contemplated. Introduction of an asparagine-X-serine or asparagine-X-threonine motif creates a potential site for enzymatic attachment of carbohydrate moieties and may therefore be used to manipulate the glycosylation of an antibody. In Raju et al. (2001) Biochemistry 40: 8868-8876 the terminal sialyation of a TNFR-IgG immunoadhesin was increased through a process of regalactosylation and/or resialylation using beta-1, 4-galactosyltransferace and/or alpha, 2,3 sialyltransferase. Increasing the terminal sialylation is believed to increase the half-life of the immunoglobulin. Antibodies, in common with most glycoproteins, are typically produced as a mixture of glycoforms. This mixture is particularly apparent when antibodies are produced in eukaryotic, particularly mammalian cells. A variety of methods have been developed to manufacture defined glycoforms, see Zhang et al. (2004) Science 303: 371: Sears et al. (2001) Science 291: 2344; Wacker et al. (2002) Science 298: 1790; Davis et al. (2002) Chem. Rev. 102: 579; Hang et al. (2001) Acc. Chem. Res 34: 727. The antibodies (for example of the IgG isotype, e.g. IgGl) as herein described may comprise a defined number (e.g. 7 or less, for example 5 or less, such as two or a single) of glycoform(s); Deamidation is an enzymatic reaction primarily converting asparagine (N) to iso-aspartic acid and aspartic acid (D) at approximately 3: 1 ratio. To a much lesser degree, deamidation can occur with glutamine residues in a similar manner. Deamidation in a CDR results in a change in charge of the molecule, but typically does not result in a change in antigen binding, nor does it impact on PK/PD; Oxidation can occur during production and storage (i.e. in the presence of oxidizing conditions) and results in a covalent modification of a protein, induced either directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidation happens primarily with methionine residues, but occasionally can occur at tryptophan and free cysteine residues; disulfide bond scrambling can occur during production and basic storage conditions. Under certain circumstances, disulfide bonds can break or form incorrectly, resulting in unpaired cysteine residues (-SH). These free (unpaired) sulfhydryls (-SH) can promote shuffling; Isomerization typically occurs during production, purification, and storage (at acidic pH) and usually occurs when aspartic acid is converted to isoaspartic acid through a chemical process; N-terminal glutamine in the heavy chain and/or light chain is likely to form pyroglutamate (pGlu). Most pGlu formation happens in the production bioreactor, but it can be formed non-enzymatically, depending on pH and temperature of processing and storage conditions. pGlu formation is considered as one of the principal degradation pathways for recombinant mAbs; C-terminal lysine clipping is an enzymatic reaction catalyzed by carboxypeptidases, and is commonly observed in recombinant mAbs. Variants of this process include removal of lysine from one or both heavy chains. Lysine clipping does not appear to impact bioactivity and has no effect on mAb effector function.

In an embodiment the molecules of the invention including the immunoglobulin single variable domains of the invention which have reduced ability to bind ADAs can have expression levels which are at least 3%, e.g. 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of those shown by a dAb™ of the same or similar amino acid sequence which is not modified as described herein to reduce binding to ADAs. In a further embodiment the molecules of the invention which have reduced ability to bind ADAs can have expression levels of at least O. lg/Litre.

The invention also provides pharmaceutical compositions comprising the molecules of the invention. The invention further provides a pharmaceutical composition of the invention for use in medicine, e.g. for use in the treatment or prevention of e.g. disease or condition or disorder and which comprises administering to said individual a therapeutically effective amount of a pharmaceutical composition of the invention. Generally, the molecules of the invention will be utilised in purified form together with pharmacologically or physiologically appropriate carriers. Typically, these carriers can include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition). A variety of suitable formulations can be used, including extended release formulations.

As used herein, the term "dose" refers to the quantity of fusion or conjugate 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 fusion or conjugate 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 invention also provides a method for treating (therapeutically or prophylactically) a patient or subject having a disease or disorder, such as those described herein, and which comprises administering to said individual a therapeutically effective amount of a pharmaceutical composition of the invention.

Additionally, the immunoglobulin single variable domain molecules, conjugates, fusion proteins or ligands described herein and pharmaceutical compositions comprising these molecules may be useful in the prevention or reduction of side effects. By prevention is meant that the use of the molecules of the invention abrogates to a complete or partial level binding of pre-existing anti drug antibodies as compared to the equivalent molecule which has not been modified. The reduction in binding of ADAs leads to a reduction in the level of unwanted pharmacological effects. Thus the molecules of the invention can have an enhanced safety profile and fewer side effects than the unmodified molecules e.g. unmodified immunoglobulin single variable domains, which do not comprise a C terminal extension, addition, deletion or tag and/or other framework modification, to reduce pre-existing ADA binding. Similarly, administration of the modified molecules described herein or of pharmaceutical compositions comprising these modified molecules (which have reduced ability to bind to pre-existing ADA) can lead to modified immunogenicity. This is because when the unmodified molecules bind to ADAs they form immune complexes and such immune complexes could then generate an immune response. In addition administration of the modified molecules described herein or of pharmaceutical compositions comprising these modified molecules can also result in improved efficacy and an improved safety profile and e.g. can be advantageously used for repeat dosing to patients who could develop autoantibodies to the unmodified molecules. In addition, the dAb™ molecules of the invention are able to be administered to a patient population without the need for pre-screening for ADA titres to remove subjects at risk of an adverse reaction.

The pharmaceutical compositions, of the invention may be administered alone or in combination with other molecules or moieties e.g. polypeptides, therapeutic proteins and/or molecules (e.g., other proteins (including antibodies), peptides, or small molecule drugs.

Treatment or therapy performed using the compositions described herein is considered

"effective" if one or more symptoms or signs are reduced or alleviated (e.g., by at least 10% or at least one point on a clinical assessment scale), relative to such symptoms present before treatment, or relative to such symptoms in an individual (human or model animal) not treated with such composition or other suitable control. Symptoms will obviously vary depending upon the precise nature of the disease or disorder targeted, but can be measured by an ordinarily skilled clinician or technician.

Similarly, prophylaxis performed using a composition as described herein is "effective" if the onset or severity of one or more symptoms or signs is delayed, reduced or abolished relative to such symptoms in a similar individual (human or animal model) not treated with the composition.

In one aspect, the invention provides the molecules and compositions of the invention for delivery by parenteral administration e.g. by subcutaneous, intramuscular or intravenous injection, inhalation, nasal delivery, transmucosal delivery, oral delivery, delivery to the GI tract of a patient, rectal delivery or ocular delivery. In one aspect, the invention provides the use of the molecules and compositions of the invention in the manufacture of a medicament for delivery by subcutaneous injection, inhalation, intravenous delivery, nasal delivery, transmucosal delivery, oral delivery, delivery to the GI tract of a patient, rectal delivery, transdermal or ocular delivery.

In one aspect, the invention provides a method for delivery to a patient by subcutaneous injection, pulmonary delivery, intravenous delivery, nasal delivery, transmucosal delivery, oral delivery, delivery to the GI tract of a patient, rectal or ocular delivery, wherein the method comprises administering to the patient a pharmaceutically effective amount of a molecule of the invention.

In one aspect, the invention provides an oral, injectable, inhalable, nebulisable formulation comprising a molecule of the invention. The formulation can be in the form of a tablet, pill, capsule, liquid or syrup.

The term "subject" or "individual" is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent or murine species.

The invention also provides a kit for use in administering molecules and compositions according to the invention to a subject (e.g., human patient), comprising a molecule or composition of the invention, a drug delivery device and, optionally, instructions for use. The composition can be provided as a formulation, such as a freeze dried formulation or a slow release formulation. In certain embodiments, the drug delivery device is selected from the group consisting of a syringe, an inhaler, an intranasal or ocular administration device (e.g., a mister, eye or nose dropper), and a needleless injection device.

The molecules and compositions of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilization method (e.g., spray drying, cake drying) and/or reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of antibody activity loss and that use levels may have to be adjusted to compensate. In a particular embodiment, the invention provides a composition comprising a lyophilized (freeze dried) composition as described herein. Preferably, the lyophilized (freeze dried) composition loses no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50% of its activity (e.g., binding activity for serum albumin) when rehydrated. Activity is the amount of composition required to produce the effect of the composition before it was lyophilized. The activity of the composition can be determined using any suitable method before lyophilization, and the activity can be determined using the same method after rehydration to determine amount of lost activity.

The invention also provides sustained or slow release formulations comprising the molecules of the invention. Such sustained release formulations can comprise the molecules of the invention in combination with, e.g. hyaluronic acid, microspheres or liposomes and other pharmaceutically or pharmacologically acceptable carriers, excipients and/or diluents.

In one aspect, the invention provides a pharmaceutical composition comprising a molecule of the invention, and a pharmaceutically or physiologically acceptable carrier, excipient or diluent. dAb™ and AlbudAb™ are trademarks registered in the name of Domantis Limited.

Examples Example 1: Frequency of healthy subjects with pre-existing ADA to VH dAb

Anti Drug Antibody assay procedure

Materials

DOM 1H-131-206 (amino acid) (SEQ ID NO: 1)

EVQLLESGGGLVQPGGSLRLSCAASGFTFAHETMVWVRQAPGKGLEWVSHIPPDGQDPFYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAVYHCALLPKRGPWFDYWGQGTLVTVSS

DOM 1H-131-206 (nucleic acid) (SEQ ID NO: 2)

G GTACAACTGCTGGAGAGCGGTGGCGGCCTGGTTCAACCGGGTGGTTCCCTGCGCCTGTCCTGTGCGGC ATCTGGTTTCACCTTCGCACACGAAACGATGGTGTGGGTTCGCCAAGCTCCGGGCAAAGGCCTGGAATGGGT AAGCCACATTCCTCCAGATGGCCAGGACCCATTCTATGCGGATTCCGTT GGGTCGCTTTACCATTTCTCGT GATAACTCCA AACACCCTGTACCTGCAGATGAACTCCCTGCGCGCCGAGGATACTGCGGTGTACCATTGT GCGCTGCTGCCTAAACGTGGCCCGTGGTTCGATTACTGGGGTCAGGGTACTCTGGTCACCGTAAGCAGC

The V_H dAb, DOM1H-131-206 (SEQ ID NO 1), is biotinylated at a biotin molar challenge ratio of 8: 1. After labelling, biotinylated DOM1H-131-206 (SEQ ID NO 1) is buffer-exchanged and stored in a formulation buffer containing 14mM sodium phosphate, 8.4% sucrose, 0.35% glycine, 0.014% polysorbate 80 at pH 7.4. DOM1H-131-206 is ruthenium labelled at a Sulfo-Tag™ molar challenge ratio of 5: 1. After labelling, Sulfo-Tag™ labelled DOM1H-131-206 is buffer-exchanged and stored in a formulation buffer containing 14mM sodium phosphate, 8.4% sucrose, 0.35% glycine, 0.014% polysorbate 80 at pH 7.4.

The anti-drug antibody (ADA) assay is a bridging assay performed on the MSD™ ECL

(electrochemiluminescence) technology platform (available from Meso Scale Discovery, Maryland, USA). The MSD™ technology utilizes a ruthenium metal chelate as the ECL label in conjunction with carbon electrodes placed within the wells of a microtiter plate that are coated with streptavidin. The bound Sulfo-Tag™ used in the assay produces a chemiluminescence signal that is triggered when voltage is applied by the instrument (Meso Scale Discovery Sector™ Imager 6000). The resulting luminescence signal is measured in ECL™ units. The intensity of the signal is directly proportional to the quantity of detected antibodies in the sample. Negative (normal human serum; NHS) and positive (PC; mouse anti- DOM1H-131-206 idiotypic antibody spiked in normal human serum) control samples (QCs) are run on each assay plate.

The summary of assay procedures is described below:

1. A MSD™ streptavidin plate is blocked with 150μί εΙΙ blocking casein in PBS (1%) at room temperature (RT) for 1-2 hours. The blocker is removed without washing.

2. After a 1 hour pre-incubation, a homogeneous mixture containing O.^g/mL biotinylated DOM1H-131-206 (drug), O.^g/mL ruthenylated (Sulfo-Tag™) DOM1H-131-206 (drug), and

2% serum sample in assay diluent (1% Casein in PBS) is transferred to the MSD™ plate and incubated for 1 hour ± 5 minutes at RT.

3. The MSD™ plate is then washed 3 times with PBST.

4. 150μΙ_ ννβΙΙ read buffer is added and the plate is read.

A panel of 60 healthy human donor serum samples was screened. It was determined that approximately 45% of serum samples from these subjects had detectable V_H-reactive autoantibodies.

Example 2: Generation of modified VH serum albumin binding molecules

The serum albumin binding immunoglobulin single variable domain, DOM7r-92-100 (SEQ ID NO: 3) is described in WO2012/072731 (as SEQ ID NO: 18 on page 20).

DOM7r-92-100 (amino acid) (SEQ ID NO: 3)

EVQLLESGGGLVQPGGSLRLSCAASGFTFDTSSMLWVRQAPGKGLEWVSVIHQSGTPTYYADSVKGRFTISRDN SKNTLYLQMNSLRAEDTAVYYCAKFPSSRMKFDYWGQGTLVTVSS DOM7r-92-100 (nucleic acid) (SEQ ID NO: 4)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC CTCCGGATTCACCTTTGATACGAGTAGTATGTTGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG TCTCAGTTATTCATCAGAGTGGTACGCCTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT GTGCGAAATTTCCG TCTTCTAGGATG GTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC

The modified version, DOM7r-92-104 (SEQ ID NO: 5), contains a single alanine extension at the C- terminal end of the molecule and was generated by site directed mutagenesis using standard procedures.

DOM7r-92-104 (amino acid) (SEQ ID NO: 5)

EVQLLESGGGLVQPGGSLRLSCAASGFTFDTSSMLWVRQAPGKGLEWVSVIHQSGTPTYYADSVKGRFTISRDN SKNTLYLQMNSLRAEDTAVYYCAKFPSSRMKFDYWGQGTLVTVSSA

DOM7r-92-104 (nucleic acid) (SEQ ID NO: 6)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGC CTCCGGATTCACCTTTGATACGAGTAGTATGTTGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGG TCTCAGTTATTCATCAGAGTGGTACGCCTACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC GCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT GTGCGA TTTCCGTCTTCTAGGATG GTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCG CT

Example 3: Confirmation of properties of modified VH serum albumin binding molecules

Expression: 5 uL DNA miniprep-ed stocks are used to transform into 50uL BL21(DE3) capside k/o chemically competent host cells via heat shock at 42°C for 45 sec followed by feed with 500uL 2TY and incubation at 37°C for 4 hours before 200uL is plated out onto triple selective LB + tetracycline + chloramphenicol + kanamycin (100 ug.ml) agar selective plates, and incubated overnight at 37°C.

Glycerol stocks are prepared and 10 ml. 2TY starter cultures which are used to inoculate

500ml_ cultures in overnight express media with lOug/ml kanamycin and a drop of antifoam. These are incubated over two nights (48 hours) at 30°C.

Cultures are harvested and supernatants batch bound to protein-A resin overnight before being washed with lOmM TrisHCI pH8.0, eluted with lOmM glycine pH2.0 and neutralised to pH5.0 with 1M TrisHCI pH8.0.

Proteins are checked on SDS PAGE. BIAcore™ to confirm SA binding: To determine the binding affinity (K_D) of the modified variants of DOM7h-92-100 to serum albumin; purified serum albumin binding immunoglobulin single variable domain antibodies (AlbudAbs™) are analysed by BIAcore™ over albumin (immobilised by primary-amine coupling onto CM5 chips; BIAcore™) using AlbudAb™ concentrations from 5000nM to 39nM (5000nM, 2500nM, 1250nM, 625nM, 312nM, 156nM, 78nM, 39nM) in HBS-EP BIAcore™ buffer.

aSEC: Analytical size exclusion chromatography is performed on an Agilent 1100 HPLC system. The chromatography column is a TSKGel S2000SWXL column from Tosoh Bioscience (cat #08540). The mobile phase is lOOmM NaP04 pH6.8; 200mM NaCI and 15% n-Propanol at a flow rate of 0.5mL/min. IOUL test compound at lmg/mL is injected and its migration through the column monitored at 215nm.

SEC-MALLS: 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 lmg/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; or a S200 column 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 at 3 different angles 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 lmg/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 (_appT_m) as most of the proteins examined do not fully refold. The higher the Tm, the more stable the molecule. Unfolding curves are analysed by non-2-state equations. The software package used is Origin^™ v7.0383. Example 4: Screening of VH molecules for binding to pre-existing anti-V^ ADA'S using an anti-V^ ADA screening assay

Pre-existing ADA binding: Screening Assay

The anti-drug antibody (ADA) assay is performed as described above.

V_H-dummy2 (described, for example in WO2008/096158 and pAC36 in Jespers et al. Nature Biotechnology, Vol. 22 (9), Sept 2004, pages 1161-1165) (in PBS buffer) is biotinylated at a biotin molar challenge ratio of 4: 1 using standard procedures. V_H-dumnny2 (in PBS buffer) is ruthenium labelled at a Sulfo-Tag™ (Meso Scale Discovery) molar challenge ratio of 4: 1 according to the manufacturer's instructions.

The summary of assay procedures is briefly described below:

1. A standard MSD™ 96-well plate is coated with 2μg/mL Neutravidin™ (Pierce) and incubated overnight at 4°C. The MSD™ plate is then washed 3 times with wash buffer PBST (PBS/0.1% Tween-20™).

2. The MSD™ plate is blocked with 150μί-/ννβΙΙ assay buffer (PBS/1% BSA/0.1% Tween-20™) at room temperature (RT) for 1 hour. The MSD™ plate is then washed 3 times with PBST. 3. After a 1 hour pre-incubation, a homogeneous mixture containing 0.05μg/mL biotinylated V_H dummy-2 and 0.05μg/mL ruthenylated (Sulfo-Tag™) V_H dummy-2 and 20% serum sample in assay buffer is transferred to the MSD™ plate and incubated for 1 hour at room temperature.

4. The MSD™ plate is then washed 3 times with PBST.

5. 150μΙ_/ννβΙΙ read buffer is added and the plate is read.

Negative (normal human serum; NHS) and positive (negative human serum spiked with Rabbit anti-V_H polyclonal antibody, generated in-house (#AC080408) using standard methods) were included as control samples (QCs). In addition to this, normal human serum containing pre-existing anti-V_H ADAs, as determined in Example 1, were also included as positive controls and are used to confirm pre-existing ADA binding in the confirmatory assay. It was determined that free, unlabelled V_H-dummy 2 competed for pre-existing ADA binding and resulted in reduced signal intensity in the pre-existing ADA assay. This assay was used to determine whether modified versions of V_H antibody based molecules could also be bound by ADA.

Amino acid extensions to the VH framework of DOM7r-92-100

Test materials e.g. DOM7r-92-100 and a modified version of this molecule, DOM7r-92-104 were tested in the screening assay described above. Pre-existing ADA binding: Confirmatory assay

2. The MSD™ plate is blocked with 150μί-/ννβΙΙ assay buffer (PBS/1% BSA/0.1% Tween-20™) at room temperature (RT) for 1 hour. The MSD™ plate is then washed 3 times with PBST.

3. In a microtitre assay plate, pre-existing ADA positive and negative serum (as determined in Example 1) is incubated with DOM7r-92-100 or DOM7r-92-104 (test molecules) or unlabelled VH-dummy 2 (positive control) at a final concentration of 0.25μς/ηηί. In addition to this, a homogeneous mixture containing 0.(^g/ml_ Biotinylated V_H-dummy2 and 0.(^g/ml_ ruthenylated (Sulfo-Tag™) V_H-dummy2 in assay buffer is added. The assay plate is incubated for 1 hour at RT. (Final serum concentration is at 20%).

4. After the 1 hour pre-incubation, the mixture is added to the MSD™ plate, and the plate is incubated for 1 hour at RT.

5. The MSD™ plate is then washed 3 times with PBST

6. 150μΙ_ ννβΙΙ read buffer is added and the plate is read.

Using this confirmatory assay, it was determined that alanine extension on the DOM7r-92-104 molecule reduced pre-existing ADA binding compared to the unmodified version DOM7r-92-100.

Assessment of ADA binding of VH domain antibodies with a C-terminal extension

% inhibition = ADA binding (% inhibition of signal when the given protein is competed in the ADA bridging assay (confirmation assay)).

TABLE OF SEQUENCES

Claims

1. An innnnunoglobulin single variable domain antibody which binds to serum albumin and comprises a C-terminal amino acid sequence consisting of the sequence VTVS(S)_nX wherein: n represents an integer independently selected from 0 or 1;

X represents an amino acid extension of 1 to 5 amino acids residues.

2. An immunoglobulin single variable domain antibody as claimed in any preceding claim wherein said amino acid extension, X, has the effect of reducing the amount of anti-drug antibody binding in the immunoglobulin single variable domain antibody comprising the amino acid extension compared to an immunoglobulin single variable domain antibody having the same amino acid sequence but lacking the C-terminal extension.

3. An immunoglobulin single variable domain antibody as claimed in any preceding claim wherein X represents an amino acid extension selected from the group consisting of A, AAA, T.

4. An immunoglobulin single variable domain antibody as claimed in claim 1 wherein said immunoglobulin single variable domain is a heavy chain single variable domain antibody such as a V_H immunoglobulin single variable domain antibody or a V_HH nanobody.

5. An immunoglobulin single variable domain antibody as claimed in claim 1 or claim 2 which binds to human serum albumin.

6. An immunoglobulin single variable domain antibody as claimed in any preceding claim which comprises a binding site that specifically binds human serum albumin with a K_D of from about 0.1 to about 10000 nM or from about 1 to about 6000 nM, as determined by surface plasmon resonance.

7. An immunoglobulin single variable domain as claimed in any preceding claim which comprises a binding site that specifically binds human serum albumin with a K_d of from about 1.5 x 10^"4 to about 0.1 sec^"1 or from about 3 x 10^"4 to about 0.1 sec^"1 as determined by surface plasmon resonance.

8. An immunoglobulin single variable domain as claimed in any preceding claim which comprises a binding site that specifically binds human serum albumin with a K_a of from about 2 x 10⁶ to about 1 x 10⁴ i sec^"1 or from about 1 x 10⁶ to about 2 x 10⁴ i sec^"1 as determined by surface plasmon resonance.

9. An immunoglobulin single variable domain as claimed in any preceding claim wherein said immunoglobulin single variable domain is cross-reactive with serum albumin from a monkey, shrew, marmoset, ferret, rat, mouse, pig and dog.

10. An immunoglobulin single variable domain antibody as claimed in any preceding claim having the sequence as set out in SEQ ID NO: 3 or an amino acid sequence that is 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to such sequence, and comprises a

C-terminal amino acid sequence consisting of the sequence VTVS(S)_nX as defined above.

11. An immunoglobulin single variable domain as claimed in any preceding claim having the amino acid sequence as set out in SEQ ID NO: 5 or an amino acid sequence that is 100%, 99.5%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to such sequence.

12. A fusion protein comprising an immunoglobulin single variable domain antibody as claimed in any preceding claim fused to a protein or polypeptide drug.

13. A multi-specific ligand comprising an immunoglobulin single variable domain antibody as claimed in any preceding claim and a binding moiety that specifically binds a target antigen other than serum albumin.

14. A multi-specific ligand as claimed in claim 13 wherein said binding moiety that specifically binds a target antigen is an immunoglobulin single variable domain antibody, wherein said target antigen is a target other than serum albumin.

15. A fusion protein as claimed in claim 12 or a multi-specific ligand as claimed in claim 13 or claim 14 further comprising a linker.

16. A nucleic acid comprising a nucleotide sequence encoding an immunoglobulin single variable domain antibody, a fusion protein or a multispecific ligand in accordance with any preceding claim.

17. A nucleic acid as claimed in claim 16 comprising the nucleotide sequence set out in SEQ ID NO:

6 or a nucleotide sequence that is at least 80% identical to said selected sequence.

18. A vector comprising a nucleic acid as claimed in claim 16 or 17.

19. An isolated host cell comprising a vector as claimed in claim 18.

20. A method of producing an immunoglobulin single variable domain antibody, fusion protein or multispecific ligand as claimed in any of claims 1 to 15 wherein said method comprises maintaining a host cell under conditions suitable for expression of said nucleic acid or vector, whereby a polypeptide is produced.

21. A pharmaceutical composition comprising an immunoglobulin single variable domain antibody, fusion protein or multi-specific ligand as claimed in any of claims 1 to 15 and a pharmaceutically acceptable diluent, carrier, excipient or vehicle.

22. A method of treating or preventing a disease or disorder in a patient, comprising administering at least one dose of an immunoglobulin single variable domain antibody, fusion protein or multi- specific ligand as claimed in any of claims 1 to 15 to said patient.

23. An immunoglobulin single variable domain antibody, fusion protein or multi-specific ligand as claimed in any of claims 1 to 15 for use in medicine.