WO2008092209A1

WO2008092209A1 - Protein construct with improved properties

Info

Publication number: WO2008092209A1
Application number: PCT/AU2008/000116
Authority: WO
Inventors: Philip Anthony Jennings; Anthony Gerard Doyle; Robert Daniel Gay; Adam William Clarke
Original assignee: Arana Therapeutics Limited
Priority date: 2007-02-01
Filing date: 2008-02-01
Publication date: 2008-08-07

Abstract

The present invention provides a protein construct which binds to an antigen or ligand, the construct comprising: (a) an antigen- or ligand-binding region which is not a domain antibody (dAb) that binds human TNF-α; (b) a modified hinge region sequence; and (c) a human or primate heavy chain constant region sequence having a truncated CH1 domain of not more than 20 residues, preferably not more than 10 residues, preferably not more than 5 residues, and even more preferably not more than a single residue.

Description

PROTEIN CONSTRUCT WITH IMPROVED PROPERTIES

FIELD OF THE INVENTION

The present invention relates to a novel protein construct which binds to an antigen or a ligand.

BACKGROUND OF THE INVENTION

Antibodies are highly specific for their binding targets and conventionally comprise at least four polypeptide chains. For example, human IgG has two heavy chains and two light chains that are disulfide bonded to form the functional antibody. Because of their structure and relatively large size, complete antibodies are limited in their therapeutic and in vivo diagnostic usefulness due to problems in, for example, tissue penetration and their biological half life. Considerable efforts have focussed on identifying and producing smaller antibody fragments that retain antigen binding function and solubility.

The heavy and light polypeptide chains of antibodies comprise variable (V) regions that directly participate in antigen interactions, and constant (C) regions that provide structural support and function in non-antigen-specific interactions with immune effectors. The antigen binding domain of a conventional antibody is comprised of two separate domains: a heavy chain variable domain (V_H) and a light chain variable domain (V_L) which can be either kappa (VK) or lambda (V_\). The antigen binding site itself is formed by six polypeptide loops: three from the V_H domain and three from the V_L domain. A diverse primary repertoire of V genes that encode the V_H and V_L domains is produced by the combinatorial rearrangement of gene segments. C regions include the light chain C regions (C_L) and heavy chain C regions (C_H)- The constant region of an antibody heavy chain is comprised of three domains, CHI, C_H2 and C_H3. The constant region is responsible for prolonged serum half-life and for the provision of antibody-mediated effector functions such as complement binding, stimulation of phagocytosis, and triggering of mast cell granule release. Furthermore, there is evidence that effector function is a component of the anti-inflammatory mechanism of anti-TNF antibodies. Within each variable domain are regions of hypervariability, otherwise known as complementarity determining regions (CDRs) which are flanked by more conserved regions referred to as framework regions. Within each variable region there are three CDRs and four framework regions.

From work done with antibodies from old world primates (rhesus monkeys and chimpanzees) it has been postulated that these non-human primate antibodies will be tolerated in humans because they are structurally similar to human antibodies (Ehrlich et ai, Human and primate monoclonal antibodies for in vivo therapy. Clin Chem. 34:9 pg 1681-1688 (1988)). Furthermore, because human antibodies are non-immunogenic in Rhesus monkeys (Ehrlich et ah, Rhesus monkey responses to multiple injections of human monoclonal antibodies. Hybridoma; 6:151-60 (1987)), it is likely that the converse is also applicable and primate antibodies will be non-immunogenic in humans.

Evolutionarily distant primates, such as New World primates are not only sufficiently different from humans to allow antibodies against human antigens to be generated, but are sufficiently similar to human to have antibodies similar to human antibodies so that the host does not generate an anti-antibody immune response when such primate-derived antibodies or their components are introduced into a human. New World primates (infraorder-Platyrrhini) comprise at least 53 species commonly divided into two families, the Callithricidae and Cebidae. The Callithricidae consist of marmosets and tamarins. The Cebidae includes the squirrel monkey, titi monkey, spider monkey, woolly monkey, capuchin, night or owl monkey and the howler monkey.

Previous studies have characterised the expressed immunoglobulin heavy chain repertoire of the Callithrix jacchus marmoset (von Budingen et ah, Characterization of the expressed immunoglobulin IGFFV repertoire in the New World marmoset Callithrix jacchus. Immunogenetics; 53:557-563 (2001)). Six IGHV subgroups were identified which showed a high degree of sequence similarity to their human IGHV counterparts. The framework regions were more conserved when compared to the complementarity determining regions (CDRs), with the greatest degree of variability located in CDR3. The degree of similarity between C. jacchus and human IGHV sequences was less than between Old World monkeys and humans.

Domain antibodies

Domain antibodies (dAb) are the smallest functioning binding units which can be created using antibody frameworks, and correspond to a single variable region of either the heavy (V_H) or light (V_L) chains of antibodies. Domain antibodies have a molecular weight of approximately 13 kDa, or less than one tenth the size of a full antibody.

hi contrast to conventional antibodies, domain antibodies are well expressed in bacterial, yeast and mammalian systems. Their small size allows for higher molar quantities per gram of product, thus providing a significant increase in potency. In addition, domain antibodies can be used as a building block to create therapeutic products such as multiple targeting molecules in which a construct containing two or more dAbs bind to two or more distinct molecular targets, or dAbs may be designed for pulmonary or oral administration.

The present inventors have now devised a novel protein construct comprising a single immunoglobulin variable region sequence (otherwise known as a dAb) linked via a modified hinge region to a truncated and otherwise modified heavy chain constant region sequence. The inclusion of the constant region will assist in prolonging the in vivo half- life of the dAb which is typically of a short duration.

Other Ligand Binding Regions

It will be apparent to one skilled in the art that numerous receptor-ligand pairs are known, e.g. TNF-TNF receptor (US 5,605,690), IL-l-IL-1 receptor or RANK-RANKL. For the purposes of this description a ligand-binding domain is taken as the region of the protein primarily involved in binding its cognate partner: either of the partners is considered a ligand-binding domain for the purpose of this description. Any such ligand-binding domain might be beneficially fused to the modified CHrhinge-CEb-CEb chain described herein. SUMMARY OF THE INVENTION

in a first aspect, the present invention provides a protein construct which binds to an antigen or ligand, the construct comprising:

(a) an antigen- or ligand-binding region which is not a domain antibody (dAb) that binds human TNF-ας

(b) a modified hinge region sequence;

(c) a human or primate heavy chain constant region sequence having a truncated C_HI domain of not more than 20 residues, more preferably not more than 10 residues, still more preferably not more than 5 residues and even more preferably a single residue.

In a second aspect, the invention provides a pharmaceutical composition comprising an effective amount of the protein construct according to the first aspect, together with a pharmaceutically acceptable carrier or diluent.

In a third aspect, the present invention provides a nucleic acid sequence encoding a protein construct according to the first aspect of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the preferred embodiment of the protein construct according to the present invention as (A) a monomer and (B) a dimer.

Figure 2 shows the amount of purified serine substituted and cysteine containing anti-BSA domain antibody Fc constructs derived from mammalian cells.

Figure 3 shows the binding of serine substituted and cysteine containing HEL-4 domain antibody Fc constructs, present in mammalian transfection supernatant, to HEL in ELISA format.

Figure 4 shows the binding of serine substituted and cysteine containing HEL-4 domain antibody Fc constructs, present in partially purified bacterial lysate, to HEL in ELISA format. Figure 5 shows the binding of TNF-αto serine substituted and cysteine containing TNFRI Fc constructs, present in mammalian transfection tissue culture supernatant in a receptor binding assay.

Figure 6 shows an assay of the soluble fraction obtained from partially purified bacterial cell lysate determined that the amount of expressed RANK Fc constructs captured by a conformationally dependent anti-RANK antibody was similar for cysteine and serine variants.

Figure 7 shows blood levels of Compound 170 in male and female cynomolgus monkeys with time after escalating subcutaneous dosing.

Figure 8 shows blood levels of Compound 170 in male and female cynomolgus monkeys with time after escalating intravenous dosing.

Figure 9 shows Compound 170 concentration following subcutaneous administration of a single dose of Compound 170 administered at 0.5 mg/kg, 5 mg/kg and 50 mg/kg in (A) male cynomolgus monkeys and (B) female cynomolgus monkeys. Blood plasma samples were analysed using an anti- Compound 170 ELISA qualified for primate plasma samples.

Figure 10 shows a comparison of Compound 170 concentration following intravenous administration of a single dose of Compound 170 administered at 50 mg/kg in (A) male cynomolgus monkeys and (B) female cynomolgus monkeys. Blood plasma samples were analysed using an anti- Compound 170 ELISA qualified for primate plasma samples.

Figure 11 shows Compound 170 concentration following subcutaneous administration of a repeat dose of Compound 170 administered weekly at 0.5 mg/kg, 5 mg/kg and 50 mg/kg in (A) male cynomolgus monkeys and (B) female cynomolgus monkeys. Blood plasma samples were taken over a 7 day period after 4, 8 and 13 weeks of dosing, and samples were analysed using an anti- Compound 170 ELISA qualified for primate plasma samples.

Figures 12a-c show Compound 170 concentration following intravenous administration of a single dose of Compound 170 administered at 0.0625 mg/kg, 0.125 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 1.0 mg/kg and 2.0 mg/kg to human subjects. Blood plasma samples were taken pre-dose and then at 1, 2, 4, 6, 12, 18 and 24 hours post infusion, and then again at 2, 4, 6, 8, 10, 14, and 18 or 20 days post infusion, and analysed using a qualified anti- Compound 170 ELISA.

Figures 13a-b show Compound 170 concentration following subcutaneous administration of a single dose of Compound 170 administered at 0.25 mg/kg, 0.5 mg/kg, 1.0 mg/kg and 2.0 mg/kg to human subjects. Blood plasma samples were taken pre-dose and then at 1, 2, 4, 6, 12, 18 and 24 hours post infusion, and then again at 2, 4, 6, 8, 10, 12, 18, and 22 or 24 days post infusion, and analysed using a qualified anti- Compound 170 ELISA.

Figure 14 shows anti-TNFa antibodies binding to membrane bound human TNFα- expressing NSO cells. (A) 10 μg/ml IgGl Human Kappa isotype control (linear mean = 6), (B) 10 μg/ml Compound 170 (linear mean = 33), (C) 10 μg/ml Infliximab (linear mean = 40), (D) 10 μg/ml Etanercept (linear mean = 23).

Figure 15 shows the antibody dependent cell mediated cytotoxicity activity of anti-TNF antibodies (Infliximab, Adalimumab), Receptor:Fc fusion protein (Etanercept) and the Domain Antibody :Fc construct (Compound 170) as measured by LDH release using a membrane bound TNFα expressing NSO cell line (mbTNF-NSO) as target cells and human PBMCs as effector cells at an effector-to-taget (E/T) ratio of 25.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have generated a protein construct which comprises a modified hinge region portion, and a portion corresponding to a truncated constant region of an antibody heavy chain.

The inclusion of the constant region portion is postulated to increase the in vivo half life of an antigen- or ligand-binding region.

(a) an antigen- or ligand-binding region which is not a domain antibody that binds human TNF-a; (b) a modified hinge region sequence;

In a preferred embodiment of the present invention, the modified hinge region sequence contains either a deletion or a single amino acid substitution of the cysteine residue which normally facilitates disulfide bond formation between heavy and light antibody chains. It is further preferred that the cysteine residue is substituted with a serine residue.

In another preferred embodiment of the present invention, the antigen- or ligand-binding region is an antibody variable region, m yet another preferred embodiment of the present invention, the antigen- or ligand-binding region is a receptor or a ligand-binding region thereof and wherein the receptor is not the TNFce receptor.

Where the antigen- or ligand-binding region is an antibody variable region, it is preferred that the antigen binding region is an antibody single chain variable domain (V_H or V_L) polypeptide that specifically binds antigen. Single variable domains are also referred to as domain antibodies (dAb). In yet a further preferred embodiment of the present invention, the antigen binding region is a human domain antibody.

It is also preferred that the variable region sequence is a light chain variable region, and preferably a kappa light chain.

The whole of the variable region sequence may be selected from the group consisting of human, mouse, New World primate and Old World primate. Alternatively, the variable region may be a chimeric variable region comprising sequences from at least two different species specified above.

In one embodiment, at least one portion of the variable region sequence is a New World primate sequence. In a further embodiment of the present invention, the at least one portion may be at least one New World primate complementarity determining region (CDR), and even three CDRs. In another preferred embodiment of the present invention, the New World primate sequence is a marmoset sequence.

When the antigen- or ligand-binding region is a receptor, it is preferred that the receptor is RANEC.

It will be appreciated by persons skilled in the art of the present invention that where the variable region comprises a portion of new World primate sequence, the remainder of the variable region sequence can be derived from another species, in particular a human, Old World primate or other New World primate sequence thus giving rise to a chimeric variable region sequence.

For example, a marmoset CDR may be grafted into a human or primate variable region acceptor sequence.

The term "binds to" as used herein, is intended to refer to the binding of an antigen by an immunoglobulin variable region with a dissociation constant (K_d) of lμM or lower as measured by surface plasmon resonance analysis using, for example a BIAcore™ surface plasmon resonance system and BIAcore™ kinetic evaluation software (e.g. version 2.1). The affinity or dissociation constant (EQ) for a specific binding interaction is preferably about 500 nM or lower, more preferably about 300 nM or lower and preferably at least 300 nM to 50 pM, 200 nM to 50 pM, and more preferably at least 100 nM to 50 pM, 75 nM to 5O pM, 1O nM to 5O pM.

In a further embodiment, the protein construct according to the first aspect of the invention may be multimerised, as for example, hetero- or homodimers, hetero- or honiotrimers, hetero- or homotetramers, or higher order hetero- or homomultimers. Multimerisation can increase the strength of antigen binding, wherein the strength of binding is related to the sum of the binding affinities of the multiple binding sites.

In a particularly preferred embodiment of the present invention, the protein construct forms a homodimer with an identical protein construct. In one embodiment, the invention is further based on a method for amplification of New World primate immunoglobulin variable region genes, for example by polymerase chain reaction (PCR) from nucleic acid extracted from New World primate lymphocytes using primers specific for heavy and light chain variable region gene families. For example, information regarding the boundaries of the variable domains of heavy and light chain genes (V_H and V_L respectively) can be used to design PCR primers that amplify the variable domain from a cloned heavy or light chain coding sequence encoding an antibody known to bind to human TNF-a. The amplified variable region is then inserted as a fusion with the polypeptide sequence for the human or primate constant region sequence of the invention into a suitable expression vector for production of the protein construct of the invention. Suitable expression vectors will be familiar to those skilled in the art.

The repertoire of V_H, V_L and constant region domains can be a naturally occurring repertoire of immunoglobulin sequences or a synthetic repertoire. A naturally occurring repertoire is one prepared, for example, from immunoglobulin expressing cells harvested from one or more primates. Such repertoires can be naϊve i.e. prepared from newborn immunoglobulin expressing cells, or rearranged i.e. prepared from, for example, adult primate B cells. If desired, clones identified from a natural repertoire, or any repertoire that bind the target antigen are then subject to mutagenesis and further screening in order to produce and select variants with improved binding characteristics.

Synthetic repertoires of single immunoglobulin variable domains are prepared by artificially introducing diversity into a cloned variable domain.

A repertoire of VH and V_L domains can be screened for desired binding specificity and functional behaviour by, for example, phage display. Methods for the construction of bacteriophage display libraries and lambda phage expression libraries are well known in the art. The phage display technique has been described extensively in the art and examples of methods and compounds for generating and screening such libraries and affinity maturing the products of them can be found in, for example, Barbas et al. (1991) PNAS 88:7978-7982; Clarkson et al. (1991) Nature 352:624-628; Dower et al. PCT WO 91/17271, U.S. Patent No. 5,427,908, U.S. Patent No. 5,580,717 and EP 527,839; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Garrad et al (1991) Bio/Technology 9:1373- 1377; Garrard et al. PCT WO 92/09690; Gram et al (1992) PNAS 89:3576-3580; Griffiths et al. (1993) EMBO J 12:725-734; Griffiths et al. U.S. Patent No. 5,885,793 and EP 589,877; Hawkins et al. (1992) J MoI Biol 226:889-896; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; Huse et al. (1989) Science 246:1275-1281; Knappik et al. (2000) J MoI Biol 296:57-86; Knappik et al. PCT WO 97/08320; Ladner et al. U.S. Patent No. 5,223,409, No. 5,403,484, No. 5,571,698, No. 5,837,500 and EP 436,597; McCafferty et al. (1990) Nature 348:552-554; McCafferty et al. PCT WO 92/01047, U.S. Patent No. 5,969,108 and EP 589,877; Salfeld et al. PCT WO 97/29131, U.S. Provisional Application No. 60/126,603; and Winter et al PCT WO 92/20791 and EP 368,684.

Recombinant libraries expressing the repertoire of V_H and VL domains can be expressed on the surface of microorganisms e.g. yeast or bacteria (see PCT publications WO 99/36569 and 98/49286).

The Selected Lymphocyte Antibody Method or SLAM as it is referred to in the state of the art, is another means of generating high affinity antibodies rapidly. Unlike phage display approaches all antibodies are fully divalent. In order to generate New World primate antibodies, New World primates are immunised with a human antigen e.g. a TNF-α polypeptide. Following immunisation cells are removed and selectively proliferated in individual micro wells. Supernatants are removed from wells and tested for both binding and function. Gene sequences can be recovered for subsequent manipulations e.g. humanisation, Fab fragment, scFv or dAb generation. Thus another example is the derivation of the antibody or antibody species of the invention by SLAM and its derivatives (Babcook et al 1996, Proc.Natl. Acad.Sci, USA 93; 7843-7848, US Patent 5,627,052 and PCT publication WO 92/02551). Adaptations of SLAM, such as the use of alternatives to testing supernatants such as panning, also lie within the scope of this invention.

In one expression system the recombinant peptide/protein library is displayed on ribosomes (for examples see Roberts and Szostak (1997) Proc.Natl. Acad.Sci.US A 94:12297-123202 and PCT Publication No. WO 98/31700). Thus another example involves the generation and in vitro transcription of a DNA library (eg of antibodies or derivatives preferably prepared from immunised cells, but not so limited), translation of the library such that the protein and "immunised" mRNAs stay on the ribosome, affinity selection (eg by binding to RSP), mRNA isolation, reverse translation and subsequent amplification (eg by polymerase chain reaction or related technology). Additional rounds of selection and amplification can be coupled as necessary to affinity maturation through introduction of somatic mutation in this system or by other methods of affinity maturation as known in the state of the art.

Another example sees the application of emulsion compartmentalisation technology to the generation of the antibodies of the invention, m emulsion compartmentalisation, in vitro and optical sorting methods are combined with co-compartmentalisation of translated protein and its nucleotide coding sequence in aqueous phase within an oil droplet in an emulsion (see PCT publications No's WO 99/026711 and WO 00/40712). The main elements for the generation and selection of antibodies are essentially similar to the in vitro method of ribosome display.

The variable region or ligand binding sequences according to the invention may be obtained from several sources, for example, databases such as The National Centre for Biotechnology Information protein and nucleotide databases www.ncbi.nlm.nih.gov, The Kabat Database of Sequences of Proteins of Immunological Interest www.kabatdatabase.com, or the IMGT database www.imgt.cines.fr. CDRs in the variable region which may be used for grafting into a variable region acceptor sequence, can be predicted from the V_H and V_L domain repertoire (see for example Kabat and Wu Attempts to locate complementarity determining residues in the variable positions of light and heavy chains. Ann. NY Acad. Sci. 190:382-93 (1971)) or derived from a database. The CDR sequence may be a genomic DNA or a cDNA.

For example, the New World primate variable region sequence may be used as an acceptor sequence for grafting non-New World primate sequences, in particular, CDR sequences using standard recombinant techniques. For example, US Patent No. 5,585,089 describes methods for creating low immunogenicity chimeric antibodies that retain the high affinity of the non-human parent antibody and contain one or more CDRs from a donor immunoglobulin and a framework region from a human immunoglobulin. United States publication No. 20030039649 describes a humanisation method for creating low immunogenicity chimeric antibodies containing CDR sequences from a non-human antibody and framework sequences of human antibodies based on using canonical CDR structure types of the non-human antibody in comparison to germline canonical CDR structure types of human antibodies as the basis for selecting the appropriate human framework sequences for a humanised antibody. Accordingly, these principles can be applied to the grafting of one or more non-New World primate CDRs into a New World primate acceptor variable region.

Variable region acceptor sequences derived from a New World primate may be grafted with one or more CDRs derived from a different New World primate, for example, a marmoset, from an Old World primate eg orang-utan, from a human or mouse CDR sequence. The CDR sequences may be obtained from the genomic DNA, or from sequences present in a database e.g. The National Centre for Biotechnology Information protein and nucleotide databases, The Kabat Database of Sequences of Proteins of Immunological Interest. The CDR sequence may be a genomic DNA or a cDNA.

Methods for grafting one or more CDRs into an acceptor variable sequence will be familiar to persons skilled in the art of the present invention. The preferred method of the present invention involves replacement of the CDR2 in a variable region sequence via primer directed mutagenesis. The method consists of annealing a synthetic oligonucleotide encoding a desired mutation to a target region where it serves as a primer for initiation of DNA synthesis in vitro, extending the oligonucleotide by a DNA polymerase to generate a double-stranded DNA that carries the desired mutation, and ligating and cloning the sequence into an appropriate expression vector.

The chimeric variable region may be further subjected to affinity maturation in order to improve its antigen binding characteristics. This may necessitate the substitution of certain amino acid residues within CDRs and framework. Techniques for affinity maturation will be familiar to persons skilled in the art of the present invention.

The constant region sequence of the protein construct may be derived from a human or primate heavy chain constant region sequence. The primate sequence may be New World primate or an Old World primate sequence. Suitable Old World primates include chimpanzee, or other hominid ape e.g. gorilla or orang-utan, which because of their close phylo genetic proximity to humans, share a high degree of homology with the human constant region sequence. Preferably, the constant region is derived from a human heavy chain constant region sequence. Examples of such sequences can be found in The National Centre for Biotechnology Information protein and nucleotide databases, and The Kabat Database of Sequences of Proteins of Immunological Interest.

hi designing the protein construct of the present invention, the inventors have truncated the C_HI domain of the heavy chain constant (Fc) region. A minimal number of C_RI domain residues have been retained in order to provide flexibility in the protein construct around the hinge region. Preferably, the truncated C_HI domain is not more than 20 amino acid residues, more preferably not more than 10 amino acid residues, still more preferably not more than 5 amino acid residues, even more preferably a single amino acid residue.

Thus, in a preferred embodiment, the protein construct has a format comprising variable region-single CHI domain residue-hinge region-Cκ2 domain-C_H3 domain wherein the Cys residue which normally facilitates disulfide bond formation between heavy and light antibody chains is substituted with a residue which does not form a disulfide bond as illustrated schematically in Figure 1.

The hinge region of the naturally occurring immunoglobulin contains a cysteine (C) side chain which facilitates the formation of a disulfide bond between the C_RI domain of the antibody heavy chain and the constant domain of the antibody light chain. Because the construct comprises only a single variable domain and thus leaves a potentially reactive unpaired cysteine residue, the cysteine residue is preferably substituted with an amino acid residue which prevents disulfide bond formation. The potential consequences of having an unp aired cysteine may include reduced protein expression due to aggregation and misfolding of the construct.

It is to be understood that any hinge region sequence derived from any of the antibody classes would be appropriate for use in the present invention. It is preferred however, that the hinge region is derived from the antibody subclass IgGl . Preferably, the hinge region is based on the naturally occurring sequence of the hinge region of IgGl and comprises the sequence EPKSSDKTHTCPPCPA (SEQ ID No:l). In this sequence, the Cys which normally occurs at position 5 is replaced by the underlined bolded Ser residue.

Preferably, the C-terminal amino acid residue of the C_HI domain is derived from IgGl. More preferably, the C_HI residue is a valine (V) residue or a conservative amino acid substitution such as leucine (L) or isoleucine (I). This residue is located immediately proximal to the hinge region and assists in increasing the flexibility of the construct around the hinge region.

Sequences of the C_H2 and C_H3 domains are preferably derived from Swissprot database accession number POl 857:

PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID No: 12).

The protein construct may be derivatised or linked to another functional molecule. For example, the protein construct can be functionally linked by chemical coupling, genetic fusion, noncovalent association or otherwise, to one or more other molecular entities, such as another antibody, a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody with another molecule (such as a streptavidin core region or a polyhistidine tag).

Useful detectable agents with which the protein construct may be derivatised include fluorescent compounds. Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin and the like. The protein construct may also be derivatised with detectable enzymes such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When the protein construct is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. The protein construct may also be derivatised with biotin, and detected through indirect measurement of avidin or streptavidin binding.

The protein construct according to the invention may be linked to one or more molecules which can increase its half-life in vivo. These molecules may be linked to the protein construct via a linker so that they do not interfere/sterically hinder the antigen binding site. Typically, such molecules are polypeptides which occur naturally in vivo and which resist degradation or removal by endogenous mechanisms. Molecules which increase half life may be selected from the following:

(a) proteins from the extracellular matrix, e.g. collagen, laminin, integrin and fibronectin;

(b) proteins found in blood, e.g. fibrin α-2 microglobulin, serum albumin, fibrinogen A, fibrinogen B, serum amyloid protein A, heptaglobin, protein, ubiquitin, uteroglobulin, β-2 microglobulin, plasminogen, lysozyme, cystatin C, alpha- 1 -antitrypsin and pancreatic kypsin inhibitor; (c) immune serum proteins, e.g. IgE, IgG, IgM;

(d) transport proteins, e.g. retinol binding protein, α-1 microglobulin;

(e) defensins, e.g. beta-defensin 1, neutrophil defensins 1, 2 and 3;

(f) proteins found at the blood brain barrier or in neural tissues, e.g. melanocortin receptor, myelin, ascorbate transporter; (g) transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins (see US 5,977,307); brain capillary endothelial cell receptor, transferrin, transferrin receptor, insulin, insulin- like growth factor 1 (IGF 1) receptor, insulin-like growth factor 2 (IGF 2) receptor, insulin receptor; (h) proteins localised to the kidney, e.g. polycystin, type TV collagen, organic anion transporter Kl, Heymann's antigen; (i) proteins localised to the liver, e.g. alcohol dehydrogenase, G250;

(j) blood coagulation factor X;

(k) α- 1 antitrypsin;

(λ) HNF lα; (m) proteins localised to the lung, e.g. secretory component (binds IgA);

(n) proteins localised to the heart, e.g. HSP 27;

(o) proteins localised to the skin, eg, keratin;

(p) bone specific proteins, such as bone morphogenic proteins (BMPs) e.g.

BMP-2, -4, -5, -6, -7 (also referred to as osteogenic protein (OP-I) and -8 (OP-2);

(q) tumour specific proteins, e.g. human trophoblast antigen, herceptin receptor, oestrogen receptor, cathepsins eg cathepsin B (found in liver and spleen);

(r) disease-specific proteins, e.g. antigens expressed only on activated T- cells: including LAG-3 (lymphocyte activation gene); osteoprotegerin Iigand (OPGL) see Kong et al. Nature (1999) 402, 304-309; OX40 (a member of the TNF receptor family, expressed on activated T cells and the only costimulatory T cell molecule known to be specifically up-regulated in human T cell leukaemia virus type-I (HTLV-I)-producing cells - see Pankow et al. (2000) J. Immunol. 165(l):263-70; metalloproteases (associated with arthritis/cancers), including CG6512 Drosophila, human paraplegin, human FtsH, human AFG3L2, murine ftsH; angiogenic growth factors, including acidic fibroblast growth factor (FGF-I), basic fibroblast growth factor (FGF-2), Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), transforming growth factor-α (TGF-α), angiogenin, interleukin-3 (IL-3), interleukin-8 (IL-8), platelet derived endothelial growth factor (PD- ECGF), placental growth factor (PlGF), midkine platelet-derived growth factor-BB (PDGF), fractalkine;

(s) stress proteins (heat shock proteins); and

(t) proteins involved in Fc transport. The present invention also extends to a PEGylated protein construct which provides increased half-life and resistance to degradation without a substantial loss in activity (e.g. binding affinity) relative to non-PEGylated antibody polypeptides.

The protein construct can be coupled, using methods known in the art, to polymer molecules (preferably PEG) useful for achieving the increased half-life and degradation resistance properties. Polymer moieties which can be utilised in the invention can be synthetic or naturally occurring and include, but or not limited to straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymers, or a branched or unbranched polysaccharide such as a homo-or heteropolysaccharide. Preferred examples of synthetic polymers which can be used in the invention include straight or branched chain poly(ethylene glycol) (PEG), poly(propylene glycol), or polyvinyl alcohol) and derivatives or substituted forms thereof. Particularly preferred substituted polymers for linkage to the protein construct include substituted PEG, including methoxy(polyethylene glycol). Naturally occurring polymer moieties which can be used in addition to or in place of PEG include lactose, amylose, dextran, or glycogen, as well as derivatives thereof which would be recognised by persons skilled in the art.

The polymer (PEG) molecules useful in the invention can be attached to the construct using methods which are well known in the art. The first step in the attachment of PEG or other polymer moieties to the constructs of the invention is the substitution of the hydroxyl end-groups of the PEG polymer by electrophile-containing functional groups. Particularly, PEG polymers are attached to either cysteine or lysine residues present in the construct monomers or multimers. The cysteine and lysine residues can be naturally occurring, or can be engineered into the construct molecule.

Pegylation of the constructs of the invention may be accomplished by any number of means (see for example Kozlowski & Harris 2001 Journal of Controlled Release 72:217). PEG may be attached to the construct either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins is described in Delgado et ah, Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al, Intern. J. of Hematol. 68:1-18 (1998); US 4,002,531; US 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monomethoxy polyethylene glycol (MPEG) using tresylchloride.

Following reaction of amino acid residues with tresylated MPEG, polyethylene glycol is directly attached to the amine groups. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, US 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1 '-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p- nitrophenolcarbonate, and various MPEG-succinate derivatives. A number additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference.

In a particularly preferred embodiment of the present invention the construct is coupled directly to polyethylene glycol via a lysine residue, hi yet another preferred embodiment of the present invention, the construct is coupled directly to PEG by incorporating a cysteine residue in, for example, the C-terminus of the construct, with attachment of the PEG to the construct facilitated by a disulphide bond interaction such as that described in US 20060210526.

Derivatized forms of polymer molecules include, for example, derivatives which have additional moieties or reactive groups present therein to permit interaction with amino acid residues of the antibody polypeptides described herein. Such derivatives include N- hydroxylsuccinimide (NHS) active esters, succinimidyl propionate polymers, and sulfhydryl-selective reactive agents such as maleimide, vinyl sulfone, and thiol. PEG polymers can be linear molecules, or can be branched wherein multiple PEG moieties are present in a single polymer.

The reactive group (e.g., MAL, NHS, SPA, VS, or Thiol) maybe attached directly to the PEG polymer or may be attached to PEG via a linker molecule.

The size of polymers useful in the invention can be in the range of 500 Da to 60 kDa, for example, between 1000 Da and 60 kDa, 10 kDa and 60 kDa, 20 kDa and 60 kDa, 30 kDa and 60 kDa, 40 kDa and 60 kDa, and up to between 50 kDa and 60 kDa. The polymers used in the invention, particularly PEG, can be straight chain polymers or may possess a branched conformation.

An "effective amount" may include a therapeutically effective amount or prophylactically effective amount of the protein construct of the present invention. A therapeutically effective amount refer to an amount effective at dosages and for periods of time necessary, to achieve the desired therapeutic result. A prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.

A "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like which are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like as well as combinations thereof. In many cases it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable substances such as wetting or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers. The composition may be in a variety of forms, including liquid, semi-solid and solid dosage forms, such as liquid solutions (eg injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. Preferably, the composition is in the form of an injectable solution for immunization. The administration may be intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the protein construct into the required amount in an appropriate solvent with one or a combination of ingredients listed above, followed by filtered sterilisation.

The composition may also be formulated as a sterile powder for the preparation of sterile injectable solutions. The proper fluidity of a solution can be maintained by for example, use of a coating such as lecithin and/or surfactants.

In certain embodiments, the protein construct may be prepared with a carrier that will protect it against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Compatible polymers may be used such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid.

The composition may also be formulated for oral administration. In this embodiment, the protein construct may be enclosed in a hard or soft shell gelatine capsule, compressed into tablets, or incorporated directly into the subject's diet.

Formulations that allow for rectal, transdermal, intrathecal and intraocular administration will be familiar to persons skilled in the art.

Supplementary active compounds can also be incorporated into the composition. The protein construct may be co-formulated with and/or co-administered with one or more additional therapeutic agents e.g. soluble TNF-α receptor or a chemical agent that inhibits human TNF-α production, or antibodies that bind other targets such as cytokines or cell surface molecules. Alternatively, it may be co-administered with a soluble immunochemical reagent such as protein A, C, G or L.

In a third aspect the present invention provides a nucleic acid sequence encoding a protein construct according to the first aspect of the invention.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following non-limiting examples. EXAMPLE 1

Methods

Constructs

A range of four different binding domains were formatted into Fc constructs with either a serine or cysteine present at the fifth amino acid position in the hinge region:

EPKSSDKTHTCPPCP (Ser-hinge; SEQ ID No:l) or EPKSCDKTHTCPPCP (Cys-hinge; SEQ ID No:2).

The Fc constructs were designed to carry the following binding domains: an anti-bovine serum albumin (BSA) domain antibody (anti-BSA dAb), the human TNFRI receptor, the human RANK receptor, and HEL-4 (anti-hen lysozyme (HEL)) domain antibody.

Sequences for HEL-4 and human RANK (Accession No: NP_003830 amino acids 29-213) were optimized by GeneOptimizer™ for E.coli expression and synthesized de novo at GeneArt GmbH. Synthesized genes were subcloned into the Invitrogen pBAD gill / His tagged expression vector creating Cys- and Ser-Fc constructs formatted for bacterial expression.

The different binding domain sequences used are as follows:

Anti-BSA dAb (SEQ ID No:3)

METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQSIRTGVVW YQQKPGKAPKLLIYSASHLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQIFT RPVTFGQGTKVEIKR

HEL-4 (SEQ ID No:4)

METDTLLLWVLLLWVPGSTGEVQLLESGGGLVQPGGSLRLSCAASGFRISDEDMG WVRQAPGKGLEWVSSΓYGPSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED

TAVYYCASALEPLSEPLGFWGQGTLVTVSS TNFRI (SEQ ID No:5)

MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSI CCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQ VEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVC TCHAGFFLRENECVSCSNCKKSLECT

RANK (SEQ ID No:6)

MASTQIAPPCTSEKHYEHLGRCCNKCEPGKYMSSKCTTTSDSVCLPCGPDEYLDS WNEEDKCLLHKVCDTGKALVAVVAGNSTTPRRCACTAGYHWSQDCECCRRNTE CAPGLGAQHPLQLNKDTVCKPCLAGYFSDAFSSTDKCRPWTNCTFLGKRVEHHG TEKSDAVCSSSLPARKPPNEPHVYLPG

All binding domains terminate with the serine/cysteine under investigation and are linked directly to the acceptor domain which is defined as follows:

Acceptor Domain: TruncatedC_Hl-hinge-C_H2-C_H3:Ser (SEQ ID No:7)

VEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHDWLNGKEYKCKVSN KALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

Acceptor Domain: TruncatedC_Hl-hinge-C_H2-CH3:Cys (SEQ ID No:8)

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHDWLNGKEYKCKVSN KALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

The protein sequence of anti-BSA domain antibody was obtained from the Domantis patent application 2004/0202995 Al. The protein sequence of the HEL-4 domain antibody was obtained from Jespers et a (Crystal structure of HEL4, a soluble, refoldable human V_H single domain with a germ-line scaffold. J. MoI Biol. 337, 893-903 (2004)). Each sequence was back translated into nucleotide sequence, optimized by GeneOptimizer™ for Chinese hamster ovary cell expression and synthesized de novo at GeneArt GmbH, Regensberg, Germany. Synthesized genes were subcloned into the Peptech vector pEE12.4 + 053893, creating Cys- and Ser-Fc constructs formatted for mammalian cell expression.

Mammalian Expression

The DNA was transfected into CHOKl SV cells using a standard lipofectamine based system (anti-BSA domain antibody Fc constructs; HEL-4 domain antibody constructs; TNFRI Fc constructs), culture medium harvested and used for analysis.

For the anti-BSA domain antibody construct culture media was purified using Protein A purification. SDS-PAGE was used to confirm the molecular weight and the purity of the resulting protein product. A BCA assay (Pierce) was used to determine the concentration of the total purified protein.

Bacterial Expression

TOPlO cells (Invitrogen) were transformed with the desired constructs by the heat shock method and glycerol stocks of single colonies generated. The induction conditions were 0.002% arabinose and 4hr induction period.

Samples were generated for binding analysis using the osmotic shock method as detailed in the pBAD bacterial expression system manual (Invitrogen). The BCA assay (Pierce) was used to determine the total protein concentration of the samples.

HEL ELISA

HEL (Sigma Aldrich) was diluted to 1 μg/mL in carbonate coating buffer (15 mM disodium carbonate, 20 mM sodium hydrogen carbonate pH 9.6). 100 μL of this solution was added to each well of a 96 well plate and incubated at 4⁰C overnight in a humidified container. The plate was then washed three times with wash buffer (1 x PBS pH 7.2, 0.05% Tween-20) and then three times with 1 x PBS pH 7.2. The wells were then blocked by adding 200 μL blocking buffer (1% w/v BSA in 1 x PBS pH 7.2) to each well and incubating the plate at 25⁰C, in a humidified container, for 1 hour. The HEL-4 transfection supernatant was diluted 1 in 2 in antibody diluent (1% w/v BSA, 0.05% Tween-20 in 1 x PBS pH 7.2) and then serial dilutions performed across the plate. The wells were incubated with the antibody for 1 hour at 25⁰C. The plate was then washed as previously described. 100 μL of Anti-IgG H + L antibody HRP conjugate (Zymed, Cat No: 81- 71200) at 1 :2000 in antibody diluent was used to detect bound antibody. Wells with antibody diluent only were used to measure the background absorbance. After incubation at 25⁰C, in a humidified container, for 1 hour the plate was washed again as previously described. 100 μL TMB substrate solution (Zymed, Cat No: 00-2023) was added to each well and the colour allowed to develop for 4 min. 100 μL of IM HCl was added to terminate the colour development reaction and absorbance was determined at 450 nm (ref. 620 nm).

For analysis of the bacterially expressed HEL-4 Fc contracts, detection was via anti-6HIS antibody HRP conjugate (Sigma A7058) at 1:2000 and the colour was allowed to develop for 20 min prior to the addition of stop solution.

Receptor neutralization assay

Transfection supernatant containing expressed TNFRI Fc-ser and TNFRI Fc-cys constructs were diluted 1 in 2 in carbonate coating buffer pH 9.2 (15 mM disodium carbonate, 20 niM sodium hydrogen carbonate pH 9.6) and coated onto Maxisorb plates (Nunc) with an overnight incubation at 4⁰C.

The following day the coated plates were washed 3 times with 1 x PBS pH 7.2 with 0.05% Tween-20 and then 3 times with 1 x PBS pH 7.2. The plates were then blocked with 200 ul/well of 1% BSA in 1 x PBS pH 7.2 for 45 minutes at room temperature. Following washing of the plate, 100 μ\ of TNF-α (Peprotech Cat No: 300-01 A) was added to triplicate wells starting at 50 ng/mL and serial dilutions performed across the plate. The plate was then incubated at room temperature for 1 hour. Following washing of the plate, a biotinylated anti-human TNF-a antibody (RnD Systems, Cat No: BAF210) was added at 0.6 μg/mL in antibody diluent to each well and allowed to incubate for 1 hour at room temperature. Following washing a Strepavidin-HRP conjugate (Zymed, Cat No: 43-4323) was added at 1 :2000 in antibody diluent and allowed to incubate at room temperature for 45 mins. Detection was performed using a single solution TMB substrate (Invitrogen, Cat No: 00-2023) for 4 mins then stopped with 1 M HCl. Absorbance readings were then measured at 450 nm using a reference of 620 nm.

Analysis was performed by calculating the average absorbance of the triplicates.

TNF-a binding assay

Recombinant human TNF-α (CytoLab) was diluted to 1 μg/mL in carbonate coating buffer (15 mM disodium carbonate, 20 mM sodium hydrogen carbonate pH 9.6). 100 μL of this solution was added to each well of a 96 well plate and incubated at 4⁰C overnight in a humidified container. The plate was then washed three times with wash buffer (1 x PBS pH 7.2, 0.05% Tween-20) and then three times with 1 x PBS pH 7.2. The wells were then blocked by adding 200 μL blocking buffer (1% w/v BSA in 1 x PBS pH 7.2) to each well and incubating the plate at 25⁰C, in a humidified container, for 1 hour. The wells were incubated for 1 hour at 25⁰C. The plate was then washed as previously described. 100 μL of Anti-6HIS antibody HRP conjugate (Sigma A7058) at 1 :2000 in antibody diluent was used to detect bound antibody. Wells with antibody diluent only were used to measure the background absorbance. After incubation at 25⁰C, in a humidified container, for 1 hour the plate was washed again as previously described. 100 μL TMB substrate solution (Zymed, Cat No: 00-2023) was added to each well and the colour allowed to develop for 15 min. 100 μL of 1 M HCl was added to terminate the colour development reaction and absorbance was then measured at 450 nm using a reference of 620 nm.

RANKELISA

Goat anti-human RANK antibody (R & D systems Cat No: AF683) was diluted to 1 μg/mL in carbonate coating buffer (15 mM disodium carbonate, 20 mM sodium hydrogen carbonate pH 9.6). 100 μL of this solution was added to each well of a 96 well plate and incubated at 4⁰C overnight in a humidified container, The plate was then washed three times with wash buffer (1 x PBS pH 7.2, 0.05% Tween-20) and then three times with 1 x PBS pH 7.2. The wells were then blocked by adding 200 μL blocking buffer (1% w/v BSA in 1 x PBS pH 7.2) to each well and incubating the plate at 25⁰C, in a humidified container, for 1 hour. The soluble bacterial extract containing expressed RANK-Fc was diluted 1 in 5 in antibody diluent (1% w/v BSA, 0.05% Tween-20 in 1 x PBS pH 7.2) and then serial dilutions performed across the plate. The wells were incubated for 1 hour at 25⁰C. The plate was then washed as previously described. 100 μL of Anti-6HIS antibody HRP conjugate (Sigma A7058) at 1 :2000 in antibody diluent was used to detect bound antibody. Wells with antibody diluent only were used to measure the background absorbance. After incubation at 25⁰C, in a humidified container, for 1 hour the plate was washed again as previously described. 100 μL TMB substrate solution (Zymed, Cat No: 00-2023) was added to each well and the colour allowed to develop for 15 min. 100 μL of IM HCl was added to terminate the colour development reaction and absorbance was then measured at 450 nm using a reference of 620 nm.

Results

The effect of a cysteine to serine substitution was evaluated in a range of Fc constructs which were formatted to carry the following binding domains: an anti-bovine serum albumin (BSA) domain antibody, the human TNFRI receptor, the human RANK receptor and HEL-4 (anti-hen lysozyme (HEL)) domain antibodies.

Anti-BSA Fc constructs

Following transfection and protein A purification the amount of purified anti-BSA domain antibody Fc construct was similar for the cysteine and the serine containing variants (Figure 2).

HEL-4 domain antibody Fc constructs

Mammalian Expression

After transfection, an assay of the transfection supernatant determined that the amount of domain antibody present that bound to HEL was similar for the serine and the cysteine Fc variants (Figure 3). Bacterial Expression

After induction and osmotic shock treatment to release soluble expressed HEL-4 Fc constructs, an assay of the soluble fraction determined that the amount of domain antibody Fc construct available to bind HEL was similar for the serine and the cysteine variants (Figure 4). In addition, a BCA total protein assay was performed to show that the amount of protein in both samples was not significantly different (data not shown).

TNFRIFc constructs

After transfection an assay of the transfection supernatant determined that the amount of receptor Fc construct present that bound to TNF-α was similar for the serine and the cysteine variants (Figure 5).

RANK Constructs

Bacterial Expression

After induction and osmotic shock treatment to release soluble RANK Fc constructs, an assay of the soluble fraction determined that the amount of expressed protein captured by a conformationally dependent anti-RANK antibody was similar for the serine and the cysteine variants (Figure 6). hi addition, a BCA total protein assay was performed and showed that the amount of protein in both samples was not significantly different (data not shown).

EXAMPLE 2

Human and animal studies were conducted using a protein construct "Compound 170" comprising a domain antibody which binds to human TNF-O, a modified hinge region sequence and a human heavy chain constant region sequence (SEQ ID No: 9; described in WO 2007/087673 and incorporated herein by reference).

Compound 170 (SEQ ID No:9) DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYSASNLET GVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQGTKVEIKRVEPKS SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

Compound 170 demonstrated improved biological properties, including improved kinetics, efficacy and bio-distribution, compared to constructs lacking the heavy chain constant region sequence. The results of these studies are summarised as follows:

Animal (Primate) Studies

Study 1 — Escalating Dose Study

Non-GLP escalating dose studies were performed on groups of cynomolgus monkeys by both subcutaneous and intravenous routes of administration.

One group of two male and two female monkeys were subcutaneously administered escalating doses of 0.5, 2.25, 10 and 50mg/kg of Compound 170 (formulated in 25mM histidine, 125mM sodium chloride, pH 6.0) with each escalation taking place after a 7 day observation period. Blood plasma samples were taken across the 7 day inter-dose period after the first three doses and over a 14 day period following the final highest dose.

For the intravenous route of administration one group of two male and two female monkeys was dosed with escalating doses of Compound 170 and blood plasma samples were taken as detailed above.

Each set of blood plasma samples derived from the escalating dose studies were analysed using an anti- Compound 170 ELISA qualified for primate plasma samples and the data is presented in Figures 7a and 7b.

Analysis of these data allowed for the calculation of toxicokinetic parameters which are presented and discussed for both subcutaneous and intravenous routes of administration as follows: (i) Subcutaneous administration

Table 1: Toxicokinetic parameters of Compound 170 in male monkeys following subcutaneous administration of Compound 170 at 0.5, 2.25, 10 and 50 mg/kg

Compound 170

Animal AUCo.168h AUCo-168h Cmax Cmax tmax ti/2 Dose Level Number (ng.h/mL) (norm) (ng/mL) (norm) (mg/kg) (h) (h)

1 218000 436000 1710 3410 48.0 NC

0.5 2 159000 319000 2160 4330 48.0 NC

N 2 2 2 2 2 0 Mean 189000 377000 1940 3870 48.0 NC

1 1490000 663000 11900 5280 48.0 NC

2.25 2 1800000 799000 17600 7820 24.0 NC

N 2 2 2 2 2 0 Mean 1650000 731000 14700 6550 36.0 NC

1 5510000 551000 44800 4480 48.0 NC

10 2 5270000 527000 50100 5010 24.0 NC

N 2 2 2 2 2 0 Mean 5390000 539000 47500 4750 36.0 NC

1 31400000 629000 258000 5160 24.0 74.5

50 2 26300000 525000 219000 4380 48.0 55.9

N 2 2 2 2 2 2 Mean 28900000 577000 239000 4770 36.0 65.2

Table 2: Toxicokinetic parameters of Compound 170 in female monkeys following subcutaneous administration of Compound 170 at 0.5, 2.25, 10 and 50 mg/kg

Compound 170 Animal Dose Level AUC₀.iβ8h AUCo-168 h Cmax Cmax ϊnjiax tie Number (ng.h/mL) (norm) (ng/mL) (norm) (mg/kg) (h) <h)

3 268000 536000 2120 4230 48.0 NC

0.5 4 206000 412000 1740 3490 48.0 NC

N 2 2 2 2 2 0 Mean 237000 474000 1930 3860 48.0 NC

5 1680000 746000 13500 6000 48.0 NC

2.25 6 1470000 654000 12500 5540 24.0 NC

N 2 2 2 2 2 0 Mean 1580000 700000 13000 5770 36.0 NC

5 4410000 441000 53700 5370 6.00 77.5

10 6 3670000 367000 35800 3580 24.0 NC

N 2 2 2 2 2 1 Mean 4040000 404000 44700 4470 15.0 77.5

5 19800000 396000 229000 4580 24.0 98.3

50 6 9920000 198000 155000 3100 48.0 NC

N 2 2 2 2 2 1 Mean 14900000 297000 192000 3840 36.0 98.3

Following single subcutaneous administration of Compound 170 at 0.5 to 50 mg/kg dose levels, plasma drug concentrations increased slowly to reach maximum levels (C_max) at blood sampling times 24 and 48 hours post-dose, with the exception of one female animal at the 10 mg/kg dose level who had a t_max value of 6 hours post-dose. No change in t_max was noted with increasing dose.

The elimination half-life was only calculable in four animals at the 10 and 50 mg/kg dose levels, and appeared to be independent of dose level, with values ranging from 55.9 to 98.3 hours in individual animals. This compares favourable to that seen following intravenous dosing (see below).

To assess dose proportionality the increases in AUC_0-168 and C_maχ with increase in dose based upon the preceding dose were calculated and are presented in Table 3. Table 3: Dose proportionality as measured by AUCo-iβs and C_max levels

Following single subcutaneous dosing, both AUC_0-168 and C_max increased in a greater than dose proportional manner in males and a less than dose proportional manner in females, across the 0.5 to 50 mg/kg dose range, hi males, a 100-fold increase in dose (0.5 to 50 mg/kg) resulted in 152.9 and 123.2-fold increases in AUC_{0-168 h} and C_max, whilst in females respective 62.9-fold and 99.5-fold increases were observed. It is of interest to note that marked supra-proportional increases were seen in males and females upon increasing the dose from 0.5 to 2.25 mg/kg, with sub-proportional and proportional increases being observed from 2.25 to 50 mg/kg.

The systemic exposure of monkeys to Compound 170 following subcutaneous administration was generally independent of sex with AUCo_-168 and C_max values being similar in both sexes across the dose range 0.5 to 50 mg/kg, with the exception of mean AUC_0-168 at the 50 mg/kg dose level, which was 1.9-fold higher in males when compared to females. (U) Intravenous administration

Table 4: Toxicokinetic parameters of Compound 170 in male monkeys following intravenous administration of Compound 170 at 0.5, 2.25, 10 and 50 mg/kg

Compound 170 Animal AUCo.1β8h AUC₀.iβ8h Cmax Cmax C₀ trπax UlZ CL v_z Dose Level Number (ng.h/mL) (norm) (ng/mL) (norm) (ng/mL) (h) (h) (mL/hr/kg) (mL/kg) (mL/kg) (mg/kg)

3 1580000 3150000 41300 82700 42200 0.500 62.9 0.276 25.0 19.7

0.5

4 2030000 4070000 39300 78600 NC 1.00 70.0 0.205 20.7 17.3

N 2 2 2 2 1 2 2 2 2 2 Mean 1810000 3610000 40300 80600 42200 0.750 66.4 0.240 22.8 18.5

3 3250000 1440000 82400 36600 90000 0.500 NC NC NC NC

2.25

4 3520000 1560000 67800 30100 67900 0.500 78.4 0.511 57.8 49.2

N 2 2 2 2 2 2 1 1 1 1 Mean 3390000 1500000 75100 33400 79000 0.500 78.4 0.511 57.8 49.2

3 6890000 689000 220000 22000 260000 0.500 76.1 1.21 133 100

10

4 5360000 536000 163000 16300 145000 2.00 76.5 1.52 168 135

N 2 2 2 2 2 2 2 2 2 2 Mean 6130000 613000 192000 19200 203000 1.25 76.3 1.37 150 118

3 30300000 606000 718000 14400 NC 1.00 85.9 1.27 158 136

50

4 28500000 569000 919000 18400 NC 1.00 78.8 1.42 162 131

N 2 2 2 2 0 2 2 2 2 2 Mean 29400000 588000 819000 16400 NC 1.00 82.4 1.35 160 133

Table 5: Toxicokinetic parameters of Compound 170 in female monkeys following intravenous administration of Compound 170 at 0.5, 2.25, 10 and 50 mg/kg

Compound 170 Animal AUCθ.168 h AUC<j.168 h Cmax Cmax C₀ tmax ti/2 CL V₂ Vss Dose Level Number (ng.h/mL) (norm) (ng/mL) (norm) (ng/mL) (h) (h) (mL/hr/kg) (mUkg) (mL/kg) (mg/kg)

7 1250000 2500000 44400 88800 NC 1.00 NC NC NC NC

0.5 8 1080000 2170000 46400 92700 43700 6.00 NC NC NC NC

N 2 2 2 2 1 2 0 0 0 0 Mean 1170000 2330000 45400 90800 43700 3.50 NC NC NC NC

7 3310000 1470000 100000 44400 151000 0.500 68.6 0.570 56.4 46.7

2.25 8 3120000 1390000 85400 38000 115000 0.500 73.6 0.583 61.8 53.2

N 2 2 2 2 2 2 2 2 2 2 Mean 3220000 1430000 92700 41200 133000 0.500 71.1 0.576 59.1 49.9

7 6710000 671000 193000 19300 247000 0.500 75.8 1.22 134 106

10 8 5270000 527000 142000 14200 177000 0.500 NC NC NC NC

N 2 2 2 2 2 2 1 1 1 1

Mean 5990000 599000 168000 16800 212000 0.500 75.8 1.22 134 106

7 33100000 662000 889000 17800 934000 0.500 87.7 1.17 149 121

50 8 34100000 683000 1070000 21400 1200000 0.500 96.2 1.09 152 128

N 2 2 2 2 2 2 2 2 2 2 Mean 33600000 673000 980000 19600 1070000 0.500 91.9 1.13 150 125

Following a single intravenous administration of 0.5, 2.25, 10 and 50 mg/kg of Compound 170, maximum plasma concentrations generally occurred at the first or second sampling occasions (30 minutes or 1 hour post-dose), with the exception of two animals; one male animal at the 10 mg/kg dose level and one female animal at the 0.5 mg/kg dose level, which had C_max values at 2 and 6 hours post-dose, respectively. Slight dose dependency in apparent terminal elimination half-life was observed, with individual values increasing from 63 to 70 hours at the lowest dose level (0.5 mg/kg) to 79 to 96 hours at the highest dose level (50 mg/kg). However, this may be artefactual as sampling time was extended to 336 hours at the highest dose level, thereby allowing a more accurate assessment of the elimination phase.

The total plasma clearance (CL) for Compound 170, where calculable, appeared to be dose dependent, increasing from 0.21 to 0.28 mL/hr/kg at the 0.5 mg/kg dose level to 1.09 to 1.42 mL/hr/kg at the 50 mg/kg dose level.

The apparent volumes of distribution (Vz and Vss) also showed dose dependency, rising from 17 to 25 mL/kg at the low dose to 121 to 162 mL/kg at the top dose. These values suggest that the distribution of Compound 170 is limited and may mainly be confined to the central compartment.

To assess dose proportionality the increases in AUC_0-168 and C_max with increase in dose based upon the preceding dose were calculated and are presented in the Table 7 below.

Following single intravenous dosing, both AUCo_-16S and C_max increased in a less than dose proportional manner between the 0.5 and 10 mg/kg dose levels, with a 20-fold increase in dose resulting in 3.4 to 5.1 -fold increases in mean AUCo_{-168 h} and C_max respectively. AUCo_-168 and C_max increased in a generally dose proportional manner between the 10 and 50 mg/kg dose levels, with a 5-fold increase in dose resulting in 4.8 and 4.3-fold increases in males and 5.6 and 5.8-fold increases in females, respectively.

The systemic exposure of monkeys to Compound 170 following intravenous administration was generally independent of sex with AUC_0-168 and C_max values being similar in both sexes across the dose range 0.5 to 50 mg/kg, with the exception of mean AUCo_-168 values at the 0.5 mg/kg dose level, which were 1.5 -fold higher in males when compared to females.

(Hi) Absolute bioavailability

Comparison of mean AUCo_-168 following intravenous and subcutaneous doses can provide an indication of absolute bioavailability. Estimates of absolute bioavailability are presented below:

Table 8: Estimates of bioavailability

At the lowest dose level, approximately 10 to 20% of the subcutaneous dose appears to reach the systemic circulation. Thereafter the absolute bioavailability of Compound 170 appears to increase with increasing dose, with a maximum bioavailability of 98% in males at the 50 mg/kg dose level.

Study 2 - Single Dose: Subcutaneous

Good Laboratory Practice single dose studies were performed on groups of cynomolgus monkeys by both subcutaneous and intravenous routes of administration. All studies detailed below were performed using Compound 170 derived from GMP batch LB45855 which was used in the phase I clinical trial and will be used in the proposed phase II clinical trial.

Groups of three male and three female monkeys were dosed subcutaneously with 0.5, 5 or 50mg/kg of Compound 170 and blood plasma samples were taken over a 14 day period. Blood plasma samples were analysed using an anti- Compound 170 ELISA qualified for primate plasma samples and the data is presented in Figure 8. These data show the mean concentrations of Compound 170 detected in plasma samples from male and female monkeys after a single dose administered subcutaneously. Doses were administered to six different groups of monkeys at three different dose levels, namely 0.5 mg/kg, 5 mg/kg and 50mg/kg.

The toxicokinetic parameters of Compound 170 following subcutaneous administration at the various dose levels are summarised in Tables 9-14:

Table 9: Toxicokinetic parameters of Compound 170 in male monkeys following subcutaneous administration of Compound 170 at 0.5 mg/kg

Compound 170 Animal AUCθ-336 h AUCθ-336 Cmax Cmax Dose Level Number (ng.h/mL) h (norm) (ng/ (norm) ti/2 tmax (h) (mg/kg) mL) (h)

4 507000 1010000 3370 6740 6.00 120

0.5 5 957000 1910000 3740 7480 48.0 NC

6 566000 1130000 2440 4890 96.0 120

N 3 3 3 3 3 2

Mean 676000 1350000 3190 6370 50.0 120

SD (n-1) 245000 490000 669 1340 45.0 NC

CV% 36.2 36.2 21.0 21.0 90.1 NC

Cmax (norm) = Cmax [ng/mL] / dose [mg/kg] AUCo-336h (norm) = AUC [ng.h/mL] / dose [mg/kg] NC not calculable

Table 10: Toxicokinetic parameters of Compound 170 in female monkeys following subcutaneous administration of Compound 170 at 0.5 mg/kg

Compound 170 Cmax

Animal AUCθ-336 h AUCθ-336 h Cmax ti/2 Dose Level Number (ng.h/mL) ( ) ( mg/kg) (norm ng/ mL) (norm) Tmax (h)

(h)

16 362000 725000 2500 4990 48.0 125

0.5 17 470000 940000 3080 6150 48.0 108 18 502000 1000000 3060 6110 48.0 91.6

N 3 3 3 3 3 3

Mean 445000 890000 2880 5750 48.0 108 SD (n-1) 73200 146000 329 658 0.00 16.8 CV% 16.5 16.5 11.4 11.4 0.0 15.5

Table 11: Toxicokinetic parameters of Compound 170 in male monkeys following subcutaneous administration of Compound 170 at 5 mg/kg

Compound 170 Animal AUCθ-336 h AUCθ-336 Cmax Cmax Dose Level Number (ng.h/mL) h (norm) (ng/m (norm) ti/2 tmax (h) (mg/kg) L) (h)

7 4660000 933000 25500 5100 48.0 123

5 8 3440000 688000 21100 4230 48.0 113

9 3720000 744000 21500 4300 24.0 116

N 3 3 3 3 3 3

Mean 3940000 788000 22700 4540 40.0 117

SD(n-1) 642000 128000 2410 482 13.9 5.05

CV% 16.3 16.3 10.6 10.6 34.6 4.3

Cmax (norm) = Cmax [iig/mL] / dose [mg/kg] AUCo-336h (norm) = AUC [ng.h/mL] / dose [mg/kg] NC not calculable

Table 12: Toxicokinetic parameters of Compound 170 in female monkeys following subcutaneous administration of Compound 170 at 5 mg/kg

Compound 170 Cmax

Animal AUCθ-336 h AUCθ-336 Cmax ti/2 Dose Level tmax (h) Number (ng.h/mL) (mg/kg) h (norm) (ng/m

(norm) L) (h)

19 3650000 730000 23300 4660 6.00 132

20 4020000 803000 22900 4580 6.00 108

21 3850000 769000 25100 5030 24.0 87.6

3 3 3 3 3 3

Mean 3840000 767000 23800 4760 12.0 109

SD (n-1) 184000 36800 1190 238 10.4 22.1

CV% 4.8 4.8 5.0 5.0 86.6 20.3

Table 13: Toxicokinetic parameters of Compound 170 in male monkeys following subcutaneous administration of Compound 170 at 50 mg/kg

Compound 170 Animal AUCθ-336 h AUCθ-336 Cmax Cmax Dose Level Number (ng.h/mL) h (norm) (ng/mL) (norm) ti/2 tmax (h) (mg/kg) (H)

10 34700000 693000 203000 4060 48.0 119

50 11 47400000 948000 294000 5880 48.0 125

12 42800000 856000 319000 6380 24.0 121

N 3 3 3 3 3 3

Mean 41600000 832000 272000 5440 40.0 122

SD(n-1) 6450000 129000 61000 1220 13.9 2.89

CV% 15.5 15.5 22.4 22.4 34.6 2.4

Table 14: Toxicokinetic parameters of Compound 170 in female monkeys following subcutaneous administration of Compound 170 at 50 mg/kg

Compound 170

Animal AUCθ-336 h AUCo-336 Cmax Cmax ti/2 Dose Level tmax (h)

Number (ng.h/mL) h (norm) (ng/mL) (norm) (mg/kg) (h)

22 43900000 879000 331000 6620 24.0 110

50 23 26100000 521000 133000 2660 48.0 124 24 26900000 538000 172000 3440 24.0 86.8

N 3 3 3 3 3 3

Mean 32300000 646000 212000 4240 32.0 107 SD (n-1) 10100000 202000 105000 2100 13.9 18.9 CV% 31.2 31.2 49.5 49.5 43.3 17.6

Cmax (norm) = Cmax [ng/mL] / dose [mg/kg] AUCo-336h (norm) = AUC [ng.h/mL] / dose [mg/kg] NC not calculable Table 15: Summary details of the animals used in Study 2

Toxicokinetic analysis was performed on the data and the following conclusions drawn:

1. Following single subcutaneous dosing to the monkey across the dose range 0.5 to 50 mg/kg, Compound 170 was slowly absorbed with maximum plasma concentrations generally occurring within 24 to 48 hours.

2. Following single subcutaneous dosing, systemic exposure to Compound 170 generally increased in a less than dose proportional manner across the dose range 0.5 to 50 mg/kg. The effect was more pronounced in male animals at the 0.5 and 5 mg/kg dose levels where a 10-fold increase in dose gave a 5.8 and 7.1-fold increase in AUC_0-336 and C_max, respectively.

3. Elimination half-life ranged between 87 and 132 hours in individual animals and was independent of dose.

4. The systemic exposure of monkeys to Compound 170 was sex independent, being similar for both genders following single subcutaneous dosing.

Study 3 - Single Dose: Intravenous

For the intravenous route of administration one group of three male and three female monkeys was dosed with 50mg/kg of Compound 170 and blood plasma samples were taken over a 14 day period. Each set of blood plasma samples derived from the single dose studies were analysed using an anti- Compound 170 ELISA qualified for primate plasma samples and the data is presented in Figure 9. These data show the mean concentrations of Compound 170 detected in plasma samples from male and female monkeys after a single dose administered intravenously. Doses were administered to two different groups of monkeys at one dose levels, namely 50mg/kg. The results of dosing at a similar level subcutaneously are overlayed in grey for comparison.

The toxicokinetic parameters of Compound 170 following intravenous administration is summarised in Tables 16-18: Table 16: Toxicokinetic parameters of Compound 170 in male monkeys following intravenous (Bolus) administration of Compound 170 at 50 mg/kg

Compound Animal AUCθ-336h Cmax (ng/mL) Co CL Vz Vss 170 Dose Number (ng.h/mL) (ng/mL) ti/2 (mL/hr/kg) (mL/kg) (mL/kg) tmax(h) Level (h) (mg/kg)

50 4 44500000 1290000 1840000 0.167 104 1.03 154 124

^' 5 45600000 1310000 2120000 0.167 110 0.974 154 132

6 47800000 1360000 1920000 0.167 99.3 0.955 137 118

N 3 3 3 3 3 3 3 3

Mean 46000000 1320000 1960000 0.167 104 0.985 148 125

SD (n-1 ) 1650000 36100 148000 0.00 5.33 0.0365 10.1 7.12

CV% 3.6 2.7 7.5 0.0 5.1 3.7 6.8 5.7

Table 17: Toxicokinetic parameters of Compound 170 in female monkeys following intravenous (Bolus) administration of Compound 170 at 50 mg/kg

Compound 170 Dose Animal AUCθ-336ll Cmax Co ti/2 CL Vz Vss tmax (h) Level Number (ng.h/mL) (ng/mL) (ng/mL) (h) (mL/hr/kg) (mL/kg) (mL/kg) (mg/kg)

50 10 42900000 999000 1040000 0.300 86.7 1.10 138 107

11 44900000 1150000 1340000 0.167 101 1.02 147 120

12 42200000 923000 1220000 0.167 105 1.08 163 135

N 3 3 3 3 3 3 3 3

Mean 43300000 1020000 1200000 0.211 97.4 1.06 149 120

SD (n-1) 1410000 116000 148000 0.0770 9.52 0.0433 12.7 13.9

CV% 3.3 11.3 12.3 36.5 9.8 4.1 8.5 11.6

Table 18: Summary details of the animals used in Study 2

Toxicokinetic analysis was performed on the data and the conclusions drawn from this analysis were:

1. Following single intravenous dosing to the monkey at 50 mg/kg Compound 170, plasma concentrations of Compound 170 declined in a generally bi-phasic manner with an elimination half-life of approximately 86.7 to 110 hours in individual males and females. Distribution of Compound 170was limited, and may mainly be confined to the central compartment.

2. Total systemic exposure of monkeys to Compound 170 was sex independent, with

AUC_0-336 being similar for both genders, however the mean C_maχ was 22.7% lower in females when compared to males.

Analysis of the blood level data allows for the elimination half life, clearance and volume of distribution to be calculated. These data are presented in Tables 9a and 9b for subcutaneous and intravenous routes of administration respectively.

Study 4 — Repeat Dose: Subcutaneous

A Good Laboratory Practice 13 week repeat dose study was undertaken using Compound 170 from batch LB45855. Groups of 4 male and 4 female cynomolgus monkeys were dosed subcutaneously with either 0.5 or 5mg/kg with Compound 170 every week, and a group of 6 male and 6 female monkeys were dosed subcutaneously with either 50mg/kg of Compound 170 or lml/kg of formulation buffer every week.

Plasma blood samples were taken over a 7 day period after 4, 8 and 13 weeks of dosing. Concentrations of Compound 170 were measured using the qualified ELISA and summary data is presented in the Figure 10. The key conclusions drawn from analysis of the toxicokinetic data were as follows:

1. Following multiple subcutaneous dosing across the dose range 0.5 to 50 mg/kg/week, Compound 170 was slowly absorbed with maximum plasma concentrations occurring within 6 h to 48 h.

2. Total systemic exposure to Compound 170 generally increased in a dose proportional manner across the 0.5 to 50 mg/kg/week dose range in males but in a supra-proportional manner in females, with up to a 231 -fold increase in AUCo_-16S for the overall 100-fold increase in dose. C_max increased in a greater than dose proportional manner in both sexes, although the effect was more pronounced in female animals where there was up to 213-fold increase.

3. Systemic exposure was generally sex independent, being similar for both genders following multiple subcutaneous dosing, with the exception of the 5 and 50 mg/kg/week dose levels on Week 13, where exposure was up to 2-fold higher in females when compared to males.

4. There was no consistent trend in exposure between the dosing occasions in male ' animals, however, in female animals at the 5 and 50 mg/kg/week dose levels exposure increased by up to 3.6-fold between Weeks 4 and 13.

The data in Figure 10 show the mean concentrations of Compound 170 detected in plasma samples from male and female monkeys dosed weekly for 13 weeks. Samples were taken after 4, 8 and 13 weeks of dosing at three different dose levels - 0.5, 5 and 50mg/kg. Hu man Phase I Clinical Trials

Intravenous Dosing

18 healthy subjects, 10 male and 8 female, entered the study and as in-patients were intravenously administered a single dose of Compound 170. Blood plasma samples were taken over a 20 day period as detailed below. The subjects were divided into cohorts of 3 with each cohort receiving double the dose that the previous cohort received. Starting at 0.0625mg/kg, which is approximately l/8th the putative therapeutic dose, dosing up to 2.0mg/kg, which is approximately 4 times the putative therapeutic dose, was undertaken across the 6 cohorts.

Blood plasma samples were taken pre-dose and then at 1, 2, 4, 6, 12, 18 and 24 hours post infusion before the subjects were discharged. Following discharge further samples were taken after 2, 4, 6, 8, 10 and 14 days with a final sample taken at the completion follow up visit after a further 4 to 6 days. Each subject's blood plasma samples were analysed using a validated anti- Compound 170 ELISA and the data is presented in the Figure 11. These data show the concentrations of Compound 170 detected in plasma samples from male and female human subjects after a single dose administered intravenously. Groups of three subjects were dosed at each level, commencing at 0.0625mg/kg and escalating to 2.0mg/kg.

Toxicokinetic parameters were calculated and are presented in Table 19 as follows:

NC Not calculated Unacceptably high

Subcutaneous Dosing

12 healthy subjects, 7 male and 5 female, entered the study and as in-patients were subcutaneously administered a single dose of Compound 170. Blood plasma samples were taken over a 24 day period as detailed below. The subjects were divided into cohorts of 3 with each cohort receiving double the dose that the previous cohort received. Starting at 0.25mg/kg, which is approximately one half the putative therapeutic dose, dosing up to 2.0mg/kg, which is approximately 4 times the putative therapeutic dose, was undertaken across the 4 cohorts.

Blood plasma samples were taken pre-dose and then at 2, 4, 6, 12, 18 and 24 hours post injection before the subjects were discharged. Following discharge further samples were taken after 2, 4, 6, 8, 10, 12 and 18 days with a final sample taken at the completion follow up visit after a further 4 to 6 days. Each subject's blood plasma samples were analysed using a validated anti- Compound 170 ELISA and the data is presented in Figure 12. These data show the concentrations of Compound 170 detected in plasma samples from male and female human subjects after a single dose administered subcutaneously. Groups of three subjects were dosed at each level, commencing at 0.25mg/kg and escalating to 2.0mg/kg.

Toxicokinetic parameters were calculated and are presented in Table 20 as follows:

EXAMPLE 3

Antibody Dependent Cell Mediated Cytotoxicity (ADCC) Study

Antibody dependent cell mediated cytotoxicity (ADCC) is a mechanism of cell-mediated immunity whereby an effector cell of the immune system actively lyses a target cell that has been bound by specific antibodies. It is one of the mechanisms through which antibodies, as part of the humoral immune response, can act to limit and contain infection. Classical ADCC is mediated by natural killer (NK) cells, however, monocytes and eosinophils can also mediate ADCC. ADCC is part of the adaptive immune response due to its dependence on a prior antibody response.

The aim of the following study is to determine if Compound 170 can mediate ADCC using a cell line expressing membrane bound TNFα as the target cell and Proliferal Blood Mononuclear Cells (PMBC) as the effector cell. The study also compares the ADCC activity of Compound 170 against other anti-TNF antibodies, including Infliximab, Adalimumab and Etanercept.

Methods

Binding of compounds to membrane bound human TNF-a expressing NSO cells

To assess the binding of various compounds to membrane bound human TNFα-expressing NSO cells, indirect immunofluorescence staining and flow cytometry were used. Cells were washed with PBS containing 2% foetal bovine serum (FBS) (Bovogen, Cat No. SFBS) then incubated with 10 μg/ml of each compound in 100 μl volume. After 1 hour at 4oC, cells were washed and resuspended in 100 μl FITC-conjugated anti-human IgG (Fc- specific) secondary antibody (Sigma Aldrich, Cat No. F9512) diluted 1 :100 in PBS. After incubating for 30 minutes at 4⁰C, cells were washed and analysed on the CellLab Quanta (Beckman Coulter). ADCC assays

ADCC assays were performed using the lactate dehydrogenase (LDH) release assay method. Human proliferal blood mononuclear cells (PBMCs) prepared from healthy donors by Lymphoprep® (Axis Shield, Oslo, Norway) were used as effector cells and a stably transfected NSO cell line expressing a membrane bound form of human TNFo; (mbTNF-NSO) were used as target cells. The target cells were plated at a density of 2 x 10 cells per well into 96 well round-bottom microplates and then freshly isolated PBMCs were added to achieve a 25:1 effector: target (E/T) ratio. Dilutions of the anti-TNFce constructs were added to the plates to start the reaction. After incubation at 37°C for 4 hours, the supernatants were recovered by centrifugation at 1500rpm for 2 mins. The LDH activities of each supernatant were measured using a nonradioactive cytotoxicity assay kit (Roche Applied Science). The absorbance at 492nm was determined using a plate reader.

Specific cytotoxicity was calculated using the following formula:

% Cytotoxicity = sample value - spontaneous lysis control high control - low control where,

(a) spontaneous lysis control = target cells + effector cells only

(b) high control = target cells incubated with 1 % Triton® X- 100

(c) low control = target cells only

The specific methods may be summarised as follows:

Preparation of Proliferal Blood Mononuclear Cells from Buffy Coat

(Adapted from http://www.dynalbiotech.com/samplepreparation for the preparation of proliferal blood mononuclear cells from Buffy Coat to obtain low platelet numbers):

1. dilute 10- 18 ml buffy coat with sterile calcium and magnesium free phosphate buffered saline (10 x PBS from Cellgro. Cat # 46-013-CM diluted 1 in 10 with sterile water) to a total volume of 35 ml at room temperature; 2. add the diluted buffy coat on top of 15 ml of Lymphoprep™;

3. centrifuge at 160 x g for 20 min at room temperature, and allow to decelerate without brakes;

4. remove 20 ml of supernatant to eliminate platelets; 5. centrifuge at 350 x g for 20 min at room temperature and allow to decelerate without brakes;

6. recover mononuclear cells (MNC) from the plasma/Lymphoprep interface and transfer the cells to a 50 ml tube;

7. wash MNC once with PBS containing 0.1 w/v bovine serum albumin and 2mM EDTA (pH 7.4) by centrifugation at 400 x g for 8 min at 2-8°C;

8. wash MNC twice with PBS/BSA/EDTA by centrifugation at 225 x g for 8 min at 2- 8°C; and

9. resuspend the MNC in phenol red free RPMI containing 0.5% FCS at 1 x 10⁷ viable cells/per ml and allow to rest at 37⁰C for 30 min prior to use.

ADCC Assay - Detection via LDH Release

(Adapted from Cytotoxicity Detection Kit (LDH) manual, Roche Applied Science, Cat No: 11 644793 001)

1. prepare PBMC (effector cells) as described above. Ensure a cell count of 1 x 10⁷ viable cells/mL in LDH assay media (Phenol Red free RPMI containing 0.5% FCS);

2. prepare mbTNF expressing NSO's (Target cells) by tapping flask firmly to detach cells and resuspending at 2x10⁵ viable cells/mL in LDH assay media;

3. prepare dilutions of the anti-TNFα and control proteins in LDH assay media;

4. using a round bottom, TC treated, 96 well plate (Corning, Cat No: CP 3799) dispense 100 μL target cells to required wells;

5. add 50 μL of effector cells to wells as required, followed by addition of 50 μL protein solution to the appropriate wells; make all wells to a final volume of 200 μL by addition of LDH assay media as required;

6. controls required include: i. background control - 200 μL LDH assay media only, ii. low control - 100 μL target + 100 μL LDH assay media iii. high control - 100 μL target cells + 100 μL 2% Triton X-100 in LDH assay media iv. spontaneous lysis control - 100 μL target cells + 50 μL effector cells + 50 μL LDH assay media

7. spin the plate for 2 min @ 1500 rpm;

8. incubate the plate in a humidified incubator @ 37°C for 4 hrs;

9. harvest the supernatant samples by spinning the plate for 2 min at 1500 rpm and transferring 100 μL into the corresponding wells of a flat bottomed 96 well plate; and

10. seal and store supernatant samples at 4°C for up to 3 days prior to analysis.

LDH Release Assay

(As per Cytotoxicity Detection Kit (LDH) manual, Roche Applied Science, Cat No: 11 644 793 001)

1. reconstitute kit solution 1 with 1 ml Milli Q Water;

2. create working solution by mixing 250 μL of solution 1 with 11.25 mLs of solution 2 and mix well;

3. gently add 100 μL of working solution to 100 μL supernatant samples (vigorous dispensing causes air bubbles which can interfere with measurement);

4. incubate the samples for 30 min at room temperature in the dark; and

5. read samples at 492 run.

ADCC Assay — Detection via Flow Cytometry

(Adapted from ACT1™ Assay for CytoToxicity protocol, Cell Technology, Cat No: ACTlOO-2)

1. harvest mbTNF expressing NSO's (target cells) by tapping the flask firmly to detach cells - usually T 175 flask passaged 24-48 hr before use; 2. adjust to a concentration of 1 x 10⁶ cells/ml in 1 x PBS (without calcium and magnesium);

3. add 10 μL membrane stain CFSE (neat) per 1 mL target cells required;

4. vortex and incubate for 15 minutes at room temperature (keep protected from light);

5. centrifuge cells and resuspend in 3 mL of media containing FBS then incubate at 37°C for 30 minutes;

6. centrifuge and wash the labelled target cells twice in media containing FBS;

7. re-count cells and adjust to a concentration of 1 x 10⁵ cells/mL; 8. take the required number of cells for the number of sample wells and dilute anti-

TNFo; and control proteins;

9. add 100 μL target cells in antibody solution (1 x 10⁴ cells) per well of round bottom, TC-treated, 96 well plate;

10. count PBMC (effector cells) and adjust to a concentration of 5 x 10⁶ cells/mL then add 100 μL to appropriate wells for an E/T ratio of 50:1;

11. incubate the plate in a humidified incubator at 37⁰C for 2-3 hrs;

12. prepare controls and setup flow cytometer: i. mix NO CFSE + CFSE stained live cells ii. mix NO CFSE + CFSE + NO CFSE [dead + 7-AAD (8O⁰C for 5 min) cells iii. mix NO CFSE + CFSE + NO CFSE (dead) + CFSE (dead cells)

13. add 0.5 μg/mL 7-AAD (BD Biosciences, Cat No. 559925) per well of 200 μL volume and incubate on ice for 15-30 min; and

14. analyse by flow cytometry using protocol ADCC - CFSE & 7AAD.

Results

To assess the binding of various compounds to membrane bound human TNFα-expressing NSO cells, indirect immunofluorescence staining and flow cytometry were used. The results are presented in Figure 14 and show that Compound 170 has a reduced binding affinity for membrane bound TNF-α compared to Infliximab and Etanercept. The results presented in Figure 15 show the antibody dependent cell mediated cytotoxicity activity of anti-TNF antibodies (Infliximab, Adalimumab), Receptor:Fc fusion protein (Etanercept) and the Domain Antibody:Fc construct (Compound 170) as measured by LDH release using a membrane bound TNFα expressing NSO cell line (mbTNF-NSO) as target cells and human PBMCs as effector cells at an effector-to-taget (FVT) ratio of 25. These data show that Compound 170 demonstrated the ability to induce ADCC in a recombinant cell line expressing a membrane bound form of TNFα at a similar level to Adalimumab. The level of ADCC for Compound 170 was also similar to that of Infliximab at higher concentrations.

Using the LDH release as a measure of cellular cytotoxicity, other anti-TNF antibodies were also compared, with Adalimumab demonstrating a similar response to that of Compound 170, while Etanercept showed no ADCC activity at the concentrations tested.

Claims

1. A protein construct which binds to an antigen or ligand, the construct comprising: (a) an antigen- or ligand-binding region which is not a domain antibody (dAb) that binds human TNF-α; (b) a modified hinge region sequence; and

(c) a human or primate heavy chain constant region sequence having a truncated C_HI domain of not more than 20 residues, preferably not more than 10 residues, preferably not more than 5 residues, and even more preferably not more than a single residue.

2. The protein construct according to claim 1 wherein the single C_HI domain residue is derived from IgGl .

3. The protein construct according to claim 1 or claim 2 wherein the single C_HI domain residue is selected from the group consisting of valine (V), leucine (L) and isoleucine (I).

4. The protein construct according to claim 3 wherein the single C_HI domain residue is valine (V).

5. The protein construct according to any one of claims 1 to 4 wherein the human or primate heavy chain constant region sequence comprises a C_H2 and C_H3 domain comprising the sequence:

PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID No: 12).

6. The protein construct according to any one of claims 1 to 5 wherein the modified hinge region is derived from IgGl.

7. The protein construct according to claim 6 wherein the modified hinge region comprises the sequence EPKSCDKTHTCPPCPA (SEQ ID No:2)

8. The protein construct according to any one of claims 1 to 6 wherein the modified hinge region sequence contains either a deletion or a single amino acid substitution of the cysteine residue which normally facilitates disulfide bond formation between heavy and light chain antibodies.

9. The protein construct according to claim 8 wherein the cysteine residue is substituted with a serine residue.

10. The protein construct according to claim 8 or claim 9 wherein the modified hinge region comprises the sequence EPKSSDKTHTCPPCPA (SEQ ID No: 1).

11. The protein construct according to any one of claims 1 to 10 wherein the antigen- or ligand-binding region is an antibody variable region sequence.

12. The protein construct according to any one of claims 1 to 11 wherein the antigen- or ligand-binding region is domain antibody (dAb).

13. The protein construct according to claim 12 wherein the domain antibody is a light chain variable region.

14. The protein construct according to any one of claims 11 to 13 wherein the variable region sequence is selected from the group consisting of human, mouse, New World primate and Old World primate variable region sequences.

15. The protein construct according to any one of claims 11 to 13 wherein the variable region sequence is a chimeric variable region sequence comprising sequences from at least two different species selected from the group consisting of human, mouse, New World primate and Old World primate.

16. The protein construct according to any one of claims 1 to 10 wherein the antigen- or ligand-binding region is a receptor or a ligand-binding region thereof and wherein the receptor is not the TNFo; receptor.

17. The protein construct according to any one of claims 1 to 16 wherein the protein construct is PEGylated.

18. The protein construct according to any one of claims 1 to 17 which forms part of a homo- or hetero-multimer in vivo.

19. A homo- or hetero-multimer comprising two or more protein constructs according to any one of claims 1 to 17.

20. The homo- or hetero-multimer according to claim 19 which is a homo-dimer.

21. A pharmaceutical composition comprising an effective amount of a protein construct according to any one or claims 1 to 17 together with a pharmaceutically acceptable carrier.

22. A nucleic acid molecule encoding a protein construct according to any one of claims 1 to 16.