HK1130811A

HK1130811A - Domain antibody construct

Info

Publication number: HK1130811A
Application number: HK09108801.3A
Authority: HK
Inventors: A．G．多伊尔; B．P．伍尔文; I.M.汤姆林森; J.A.李; P.A.詹宁斯
Original assignee: 赛法隆澳大利亚控股有限公司
Priority date: 2006-02-01
Filing date: 2007-02-01
Publication date: 2010-01-08

Description

Domain antibody constructs

Technical Field

The present invention relates to domain antibody constructs useful in human therapy. More specifically, the invention relates to domain antibody constructs that bind to human TNF- α and their use in treating disorders characterized by TNF- α activity.

Background

Tumor necrosis factor α (TNF- α) is a cytokine involved in mediating shock and the pathophysiology of a variety of human diseases and disorders, including sepsis, infections, autoimmune diseases such as rheumatoid arthritis, crohn's disease, ulcerative colitis and other intestinal conditions, psoriasis, toxic shock, transplant rejection and graft-versus-host disease. TNF- α is produced primarily by activated macrophages and T lymphocytes, but also by neutrophils, endothelial cells, keratinocytes and fibroblasts during acute inflammatory reactions.

Due to its role in inflammation, TNF- α has emerged as an important target for inhibition in efforts to alleviate the symptoms of inflammatory disorders. Various approaches to the inhibition of TNF- α for the clinical treatment of disease have been pursued, including in particular the use of soluble TNF- α receptors and antibodies specific for TNF- α.

Domain antibodies

Domain antibodies (dAbs) are the smallest functional binding units of antibodies and correspond to the heavy chain variable region (V) of antibodies_H) Or the variable region of the light chain (V)_L). The domain antibody has a molecular weight of about 13kDa, or less than one tenth of the size of the intact antibody.

Unlike conventional antibodies, domain antibodies are well expressed in bacterial, yeast and mammalian systems. Their small size allows for higher molar amounts per gram of product, providing a significant increase in potency per milligram dose. In addition, the dAb can be used as a building block to prepare therapeutic products such as multi-targeted dAb-containing molecules in which two or more dabs bind to two or more different molecular targets, or the dAb can be incorporated into a structure designed for pulmonary or oral administration.

The present inventors have now devised a novel domain antibody construct comprising an immunoglobulin variable domain linked to a polypeptide comprising a truncated C_H1 domain. It is postulated that the inclusion of a constant region helps to extend the in vivo half-life of a dAb which typically has a short duration.

New world primate immunoglobulins

Evolutionarily distant primates, such as new world primates, are sufficiently similar to humans to have antibodies similar to human antibodies such that when antibodies derived from such primates are introduced into humans, the host does not produce an anti-antibody immune response. New world primates (Platyrrhini) contain at least 53 species, which are generally divided into 2 families: marmoset (Callithridae) and Palmae (Cebidae). Marmoset family consists of marmoset andand (4) forming. The family Macacaceae includes squirrel monkey, spider monkey, fluffy monkey, monkey of the genus Macaca, night or owl monkey and monkey of the family Macaca.

Previous studies have characterized the immunoglobulin heavy chain repertoire of expressed common marmosets (Callithrix jacchus) (von Budinggen H-C et al, Characterisation of the expressed immunoglobulin IGHV reteirire in the New Worldmamoset Callithrix jacchus. Immunogenetics; 53: 557-. 6 subsets of IGHV were identified, which showed a high degree of sequence similarity to their human IGHV counterparts. The framework regions are more conserved when compared to the Complementarity Determining Regions (CDRs), with the highest degree of variability located in CDR 3. The degree of similarity between common marmoset and human IGHV sequences is less than that between old world primates and humans.

Summary of The Invention

In a first aspect, the present invention provides a domain antibody construct that binds to human TNF- α, said construct comprising:

(a) a domain antibody (dAb) that binds to human TNF- α;

(b) a modified hinge region sequence;

(c) a human or primate heavy chain constant region sequence having a truncated C of no more than 20 residues, more preferably no more than 10 residues, even more preferably no more than 5 residues and even more preferably a single residue_H1, a domain of a polypeptide having a sequence of,

wherein the modified hinge region sequence comprises a deletion or a single amino acid substitution of cysteine residues that normally promote the formation of heavy and light antibody interchain disulfide bonds.

In a second aspect, the invention provides a nucleic acid sequence encoding the domain antibody construct of the first aspect of the invention.

In a third aspect, the present invention provides an isolated nucleic acid molecule comprising a sequence encoding a domain antibody construct that binds to human TNF- α, wherein the nucleic acid molecule comprises a sequence identical to SEQ id no: 50 or SEQ ID No: 51, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99%, and most preferably a nucleic acid sequence comprising a sequence as set forth in SEQ ID No: 50 or SEQ ID No: 51, or a sequence shown in seq id no.

In a fourth aspect, the present invention provides an isolated nucleic acid molecule comprising a sequence encoding a domain antibody construct that binds to human TNF- α, wherein said nucleic acid molecule comprises a sequence identical to SEQ id no: 50 or SEQ ID No: 51 under high stringency conditions.

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

In a sixth aspect, the present invention provides the use of a domain antibody construct according to the first aspect of the invention in diagnostic applications for the detection of human TNF- α.

In a seventh aspect, the present invention provides a method for treating a disorder characterized by human TNF- α activity in a human subject, comprising administering to the subject a pharmaceutical composition according to the second aspect of the invention.

Brief Description of Drawings

FIG. 1 shows the amino acid sequence (SEQ ID No: 5) and nucleotide sequence (SEQ ID No: 6) of an acceptor (acceptor) dAb.

FIG. 2 shows the structure of a preferred embodiment of the domain antibody construct of the present invention as (A) monomer and (B) dimer.

FIG. 3 shows the nucleotide and amino acid sequences of eleven (11) marmosets and six (6) owl monkey V κ gene segments.

Figure 4 shows the amino acid sequence and nucleotide sequence (both strands) of the acceptor dAb. Restriction digestion sites for KpnI and SanDI are indicated in the figure, which excise the region containing CDR 2. The removed CDR2 residues are underlined.

FIG. 5 shows the ability of compound 170(SEQ ID No: 11) to neutralize TNF-. alpha.mediated cytotoxicity in murine L929 cell viability assay.

FIG. 6 shows that compound 170(SEQ ID No: 11) prevents the interaction of TNF- α with the human p55 or p75 TNF receptor.

FIG. 7 shows that staining of NSO 27D4 cells expressing transmembrane TNF-. alpha. (black solid line) with compound 170(SEQ ID No: 11) shows higher fluorescence intensity than an unrelated specific isotype-matched control (grey filled).

FIG. 8 shows that Compound 170(SEQ ID No: 11) produced in a bacterial expression system retained binding to TNF-. alpha.in an ELISA.

FIG. 9 shows the comparison of specific control human IgG₁In other words, the efficacy of Compound 170(SEQ ID NO: 11) in a TNF-mediated murine arthritis model. Mice were scored at weekly intervals (arthritis score) (a), and weighed (B).

FIG. 10 shows the effect on protein expression of compound 112(SEQ ID NO: 59) and compound 170(SEQ ID NO: 11).

FIG. 11 shows a non-reducing SDS PAGE analysis of compound 170(SEQ ID No: 11), which compound 170 was from a 4X 10L lead cell line (lead cell line) fermentation and purified by protein A. Lane 1 as an inter-assay control; lane 2 ═ molecular weight standards; lane 3 is blank; lane 4 ═ compound 170 purified from protein a in 10L fermentation ID (run 1) (SEQ ID No: 11); lane 5 ═ compound 170 purified from protein a in 10L fermentation ID (run 2); lane 6 ═ compound 170 purified from protein a in 10L fermentation ID (run 3); lane 7 is protein a purified compound 170 in 10L fermentation ID (run 4).

FIG. 12 shows a reducing SDS PAGE analysis of compound 170(SEQ ID No: 11) from a 4X 10L lead cell line (lead cell line) fermentation and protein A purification. Lane 1 as an inter-assay control; lane 2 ═ molecular weight standards; lane 3 is blank; lane 4 ═ compound 170 purified from protein a in 10L fermentation ID (run 1) (SEQ ID No: 11); lane 5 ═ compound 170 purified from protein a in 10L fermentation ID (run 2); lane 6 ═ compound 170 purified from protein a in 10L fermentation ID (run 3); lane 7 is protein a purified compound 170 in 10L fermentation ID (run 4).

Detailed Description

The present inventors have generated domain antibody constructs that bind to human TNF- α and are presumed to exhibit low immunogenicity when administered to humans. The domain antibody construct comprises a portion corresponding to an immunoglobulin heavy or light chain variable domain (i.e., a domain antibody (dAb)), a hinge region, and a portion corresponding to an antibody heavy chain constant region, but wherein the constant region has a truncated C_H1 domain.

The inclusion of a constant region moiety is presumed to increase the in vivo half-life of the dAb, as well as providing effector function that is believed to be part of the anti-inflammatory mechanism of the anti-TNF antibody.

(a) a domain antibody (dAb) that binds to human TNF- α;

(b) a modified hinge region sequence;

In a preferred embodiment, C_HThe 1 domain and hinge region are xepkzdkthtcppcpa, where X is valine, leucine or isoleucine, and Z is absent or is an amino acid other than cysteine. Preferably, X is valine and Z is serine.

In a preferred embodiment of the invention, the dAb comprises an immunoglobulin heavy or light chain variable domain, wherein the variable domain comprises at least one Complementarity Determining Region (CDR) having a sequence derived from a New world primate, wherein the CDR is selected from the group consisting of YAATKLQS (SEQ ID No: 1), YEASSLQS (SEQ ID No: 2), YEASKLQS (SEQ ID No: 3) and ASNLET (SEQ ID No: 4).

In another preferred embodiment, the CDR is CDR 2.

In a preferred embodiment, the dAb has a sequence selected from the group consisting of:

DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYSASNLETG

VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(Compound 145; SEQ ID No: 7)

DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYSASNLET

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(Compound 123; SEQ ID No: 8)

DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYSASNLETG

VPSRFSGSGSGTDFTLTISSLLPHDFATYYCQQVVWRPFTFGQGTKVEIKR

(Compound 100; SEQ ID No: 9)

DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYSASNLET

GVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(Compound 196; SEQ ID No: 10)

DIQMTQSPSSLSASVGDRYTITCRASQSIDSYLHWYQQKPGKPPKLLIYSASNLETG

VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(Compound 134; SEQ ID No: 52)

DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYSASNLETG

VPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(Compound 137; SEQ ID No: 53)

DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYSASNLETG

VPSRFSGSGSGTDFTLTISSLVPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(Compound 121; SEQ ID No: 54); and

a sequence which is at least 95%, more preferably at least 96%, 97%, 98% or 99% identical to one of these sequences.

In a further preferred embodiment, the constant region comprises C_H2 and C_H3 domains which together have the following sequence:

PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN

AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；

(SEQ ID NO：63)

or an amino acid sequence which is at least 60% identical thereto, preferably at least 80% identical thereto, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

In another preferred embodiment, the domain antibody construct comprises a heavy chain variable region identical to the heavy chain variable region of seq id No: 11, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99%, and most preferably comprises the amino acid sequence of SEQ ID No: 11, and (c) the sequence shown in fig. 11.

The term "and.. binding" as used herein means that the antigen is bound by the immunoglobulin variable region with a dissociation constant (K) of 1 μ M or less_d) Binding is carried out, e.g. using, for example, BIAcore^TMSurface plasmon resonance system and BIAcore^TMKinetic evaluation software (e.g., version 2.1) was measured by surface plasmon resonance analysis. Affinity or dissociation constant (K) of specific binding interactions_d) Preferably about 500nM or less, more preferably about 300nM or less, and preferably at least 300nM to 50pM, 200nM to 50pM, and more preferably at least 100nM to 50pM, 75nM to 50pM, 10nM to 50 pM. The term "dAb" as used herein refers to an antibody single variable domain (V) that specifically binds to an antigen_HOr V_L) A polypeptide.

In a further preferred embodiment of the invention, the domain antibody construct forms a homo-or heterodimer with another domain antibody construct according to the invention. Dimerization 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. To facilitate dimer formation, the hinge region of the domain antibody construct comprises at least one, and preferably two cysteine residues.

In a particularly preferred embodiment of the invention, the domain antibody construct and one and the same domain antibody construct form a homodimer.

Thus, in another aspect, the invention provides a dimeric domain antibody construct that binds to human TNF- α, wherein the dimer consists of two domain antibody constructs according to the invention.

Preferably, the dimeric domain antibody construct is a homodimer, and it is particularly preferred that the domain antibody constructs that make up the homodimer comprise an amino acid sequence identical to the amino acid sequence of SEQ ID No: 11, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99%, and most preferably comprises the amino acid sequence of SEQ id no: 11, and (c) the sequence shown in fig. 11.

In determining whether two polypeptide sequences fall within the limits of percent identity, one skilled in the art will recognize that a side-by-side comparison or multiple alignments of sequences must be performed. In such a comparison or alignment, differences will occur in the positioning of residues that are not identical, depending on the algorithm used to perform the alignment. In this context, reference to "percent identity" or "similarity" between two or more amino acid sequences shall be taken to refer to the number of residues that are the same or similar, respectively, between the sequences, as determined using any standard algorithm known to those of skill in the art. For example, amino acid sequence identity or similarity can be calculated using the GAP program and/or aligned using the PILEUP program (Computer Genetics Group, Inc., university research Park, Madison, Wisconsin, United States of America) (Devereaux et al, 1984). The GAP program uses the algorithm of Needleman and Wunsch (1970) to maximize the number of identical/similar residues and minimize the number and length of sequence GAPs in the alignment. Alternatively or additionally, when comparing more than two amino acid sequences, the Clustal W program of Thompson et al (1994) may be used.

In determining whether two nucleotide sequences fall within these percentage limits, one skilled in the art will recognize that a side-by-side comparison or multiple alignments of sequences must be performed. In such a comparison or alignment, differences may arise in the positioning of residues that are not identical, depending on the algorithm used to perform the alignment. In this context, reference to "percent identity" between two or more nucleotide sequences should be taken to refer to the number of residues that are identical between the sequences, as determined using any standard algorithm known to those skilled in the art. For example, the BESTFIT program or other suitable program (Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America) (Devereaux et al, Nucl. acids Res., 12: 387-.

High stringency preferably involves hybridization at 65 ℃ and 0.1 × SSC (1 × SSC ═ 0.15M NaCl, 0.015M sodium citrate, ph 7.0).

In one embodiment, the invention is also based on a method for amplifying new world primate immunoglobulin variable region genes, for example by Polymerase Chain Reaction (PCR) amplification from nucleic acids extracted from new world primate lymphocytes using primers specific for the heavy and light chain variable region gene families. For example, with respect to heavy and light chain variable domain genes (V, respectively)_HAnd V_L) Can be used to design PCR primers that amplify a variable domain from cloned heavy or light chain coding sequences that encode antibodies known to bind to a given antigen. The amplified variable regions are then inserted into a suitable expression vector, either alone or as a fusion with another human or primate constant region polypeptide sequence of the invention, to produceThe domain antibody constructs of the invention are generated. Suitable expression vectors will be well known to those skilled in the art.

V_H、V_LAnd the constant region domain repertoire can be a naturally occurring immunoglobulin sequence repertoire or a synthetic repertoire. Naturally occurring reservoirs are, for example, reservoirs prepared from immunoglobulin-expressing cells harvested from one or more primates. Such reservoirs may be virgin () I.e. from newborn immunoglobulin-expressing cells, or rearranged, i.e. from e.g. adult primate B cells. Clones identified from the natural depot or any depot that bind the target antigen are then subjected to mutagenesis and further screening, if necessary, to generate and select variants with improved binding characteristics.

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

V can be screened for desired binding specificity and functional behavior by, for example, phage display_HAnd V_LA domain repository. Methods for constructing phage display libraries and lambda phage expression libraries are well known in the art. Phage display techniques have been widely described in the art, and examples of methods and compounds for generating and screening such libraries and affinity maturation of their products can be found in, for example, the following references: barbas et al, (1991) PNAS 88: 7978-7982; clarkson et al (1991) Nature 352: 624-; dower et al, pct.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; garrrard et al, PCT WO 92/09690; gram et al, (1992) PNAS 89: 3576-3580; griffiths et al, (1993) EMBO J12: 725-; griffiths et al, U.S. Pat. No. 55,885,793 and EP 589,877; hawkins et al, (1992) J Mol Biol 226: 889-896; hay et al, (1992) Hum AutibodHybridomas 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 Mol Biol 296: 57-86; knappik et al, PCT WO 97/08320; ladner et al, U.S. Pat. nos. 5,223,409, 5,403,484, 5,571,698, 5,837,500 and EP 436,597; McCafferty et al, (1990) Nature 348: 552 and 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 EP368,684.

Expression of V_HAnd V_LRecombinant libraries of domain depots can be expressed on the surface of microorganisms such as yeast or bacteria (see PCT publications WO 99/36569 and 98/49286).

The domain antibody constructs of the invention can be produced by recombinant methods, including production from eukaryotic expression systems, including, for example, yeast, higher plant, insect and mammalian cells, as well as fungal expression systems and virally encoded expression systems, as described herein or as known in the art.

The domain antibody constructs of the invention may be prepared with S antibody encoding nucleic acids to provide transgenic plants and cultured plant cells (e.g., without limitation, tobacco and corn) that produce such constructs in certain parts of plants or in cells cultured therefrom. As a non-limiting example, transgenic tobacco leaves expressing recombinant proteins have been successfully used to provide large quantities of recombinant proteins, for example, using inducible promoters (see, e.g., Cramer et al, curr. Top. Microbol. Immunol.240: 95-1181999) and references cited therein. In addition, transgenic maize has also been used to express mammalian proteins at commercial production levels with biological activity equivalent to that produced in other recombinant systems or purified from natural sources (see, e.g., Hood et al, adv. exp. Med. biol. 464: 127-. Antibodies, including antibody fragments such as single chain antibodies (scFv's), have also been produced in large quantities from transgenic plant seeds, including tobacco seeds and potato tubers (see, e.g., Conrad et al, plantatMol. biol. 38: 101-109, 1998 and references cited therein). Thus, the domain antibody constructs of the invention may also be produced in transgenic plants according to known methods (see also, e.g., Fischer et al, Biotechnol. appl. biochem.30: 99-108 October, 1999; Ma & Hein., Trends Biotechnol. 13: 522-71995; Ma et al, Plant physiol.109: 341-61995; Whitelam et al, biochem. Soc. Trans.22: 940-9441994; and references cited therein; each of which is hereby incorporated herein by reference in its entirety).

The domain antibody constructs of the invention include naturally purified products, products of chemical synthetic methods, and products produced by recombinant techniques from eukaryotic hosts, including, for example, yeast, higher plant, insect, and mammalian cells. Depending on the host employed in the recombinant production method, the antibody construct of the invention may be glycosylated or non-glycosylated, with glycosylated being preferred. Such methods are described in many standard laboratory manuals, Sambrook et al, Molecular Cloning: a Laboratory Manual, 2 nd edition, Cold Spring Harbor, N.Y.1989, sections 17.37-17.42; ausubel et al, eds., Current Protocols in Molecular Biology 1987. 1993, chapters 10, 12, 13, 16, 18 and 20; colligan et al, Current Protocols in protein Science, John Wiley & Sons, NY, N.Y.1997-2001, protein Science, Chapter 12-14, all of which are hereby incorporated by reference in their entirety.

In one expression system, the recombinant peptide/protein library is displayed on ribosomes (see, e.g., Roberts, RW and Szostak, J.W.1997. Proc.Natl.Acad.Sci.USA.94: 12297-123202 and PCT publication No. WO 98/31700). Thus, another example involves the generation and in vitro transcription of a DNA library (e.g., preferably, but not limited to, a DNA library of antibodies or derivatives prepared from immunized cells), translation of the library such that proteins and "immunized" mrnas reside on ribosomes, affinity selection (e.g., by binding to RSP), mRNA isolation, reverse translation, and subsequent amplification (e.g., by polymerase chain reaction or related techniques). Additional selection and amplification cycles can be coupled if necessary to perform affinity maturation by introducing somatic mutations in such systems or by other affinity maturation methods known in the art.

Another example contemplates the use of emulsion compartmentalization technology (emulsion compartmentalization technology) to generate domain antibodies of the invention. In emulsion compartmentalization, in vitro and optical sorting methods are combined with co-compartmentalization of proteins translated in the aqueous phase within the oil droplets of the emulsion and their nucleotide coding sequences (see PCT publication nos. WO 99/026711 and WO 00/40712).

CDR sequences can be obtained from several sources, for example, databases such as The National center for Biotechnology Information protein and nucleotide Database (www.ncbi.nlm.nih.gov), The Kabat Database of sequences of Proteins of Immunological Interest (www.kabatdatabase.com) Or IMGT database (www.imgt.cines.fr). Alternatively, may be selected from V_HAnd V_LDomain libraries predict CDR regions (see, e.g., Kabat EA and Wu TT, Attempts to locate complementary locations in the variable positions of light and height chains, Ann. NY Acad. Sci.190: 382-393 (1971)). The CDR sequences may be genomic DNA or cDNA.

There are many ways in which alternative CDRs can be grafted into variable region sequences, and such methods will be familiar to those skilled in the art. Preferred methods of the invention involve replacement of CDR2 in the variable region (or dAb) via primer directed mutagenesis. The method comprises the following steps: annealing a synthetic oligonucleotide encoding the desired mutation to the target region, wherein it serves as a primer for initiating in vitro DNA synthesis; extending the oligonucleotide by a DNA polymerase to produce a double stranded DNA carrying the desired mutation; and ligating and cloning the sequence into a suitable expression vector.

In a preferred embodiment of the invention, the CDR sequences of new world primates are grafted into variable region sequences with low immunogenicity in humans.

The term "low immunogenicity" refers to the efficacy of the domain antibody construct or antigen-binding portion thereof in humans without producing an antibody response of sufficient magnitude to reduce the duration of continuous administration of the domain antibody construct for a therapeutic effect.

Preferably, the variable region sequence in which the new world primate CDR is grafted is the "dAb acceptor sequence" (designated compound 128) in fig. 1. The dAb acceptor sequence consists of seq id No: 5, and the amino acid sequence shown in the specification comprises:

DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYSASELQSG

VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(SEQ ID No：5)。

the sequence is represented by SEQ ID No: 6 encodes:

GAC ATC CAG ATG ACC CAG TCT CCA TCC TCT CTG TCT GCA TCT GTA GGA

GAC CGT GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC ATT GAT AGT TAT

TTA CAT TGG TAC CAG CAG AAA CCA GGC AAA GCC CCT AAG CTC CTG ATC

TAT AGT GCA TCC GAG TTG CAA AGT GGG GTC CCA TCA CGT TTC AGT GGC

ACT GGA TCT GGG ACA GAT TTC ACT CTC ACC ATC AGC AGT CTG CAA CCT

GAA GAT TTT GCT ACG TAC TAC TGT CAA CAG GTT GTG TGG CGT CCT TTT

ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA CGG

(SEQ ID No：6)。

in a preferred embodiment of the invention, the New world Primates marmoset CDR sequence YSASNLET (SEQ ID No: 4) is grafted into the variable region dAb acceptor sequence so as to replace the CDR2 sequence (YSASELQS; SEQ ID No: 55) of the dAb acceptor sequence, thereby producing the following dAb (designated Compound 145):

compound 145

DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYSASNLETG

VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(SEQ ID No：7)。

Thus, in a preferred embodiment, the dAb of the domain antibody construct that binds human TNF- α comprises the amino acid sequence of SEQ ID No: 7.

It is within the scope of the present invention that the variable region sequences (dabs) of the domain antibody constructs may further undergo affinity maturation in order to improve their antigen binding characteristics. This may require modification of certain amino acid residues in the CDRs 1, CDR3, or framework regions of the domain antibody constructs.

For example, as described in "materials and methods," for SEQ ID nos: 7 affinity maturation and testing for TNF-alpha binding. In a further preferred embodiment, the variable region (dAb) of the domain antibody construct that binds human TNF- α comprises the amino acid sequence of SEQ ID No: 8 or SEQ ID No: 9. These were designated compound 123 and compound 100, respectively, and their sequences are shown below:

compound 123

DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLTYSASNLET

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(SEQ ID No：8)

Compound 100

DIQMTQSPSSLSASVGDRVTTTCRASQSIDSYLHWYQQKPGKAPKLLTYSASNLETG

VPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(SEQ ID No：9)。

In a particularly preferred embodiment, the variable region (dAb) of the domain antibody construct that binds human TNF- α comprises the amino acid sequence of SEQ ID No: 10. This was designated compound 196 and the sequence is provided below:

compound 196

DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYSASNLET

GVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(SEQ ID No：10)。

Those skilled in the art will appreciate that the constant region sequences of the domain antibody constructs may be derived from human or primate sequences. The primate sequence can be a new world primate or an old world primate sequence. Suitable old world primates include chimpanzees, or other apes such as gorillas or orangutans, which share a high degree of homology with human constant region sequences due to their close, phylogenetic proximity to humans. Preferably, the constant region is derived from a human antibody sequence. Examples of such sequences can be found in: national center 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 IMGT database (www.imgt.cines.fr)。

In designing the domain antibody constructs of the present invention, the present inventors truncated the C of the constant (Fc) region_H1 domain. A minimum number of C is reserved_H1 domain residues to provide flexibility to the domain antibody construct near the hinge region. Preferably, retention C_H1 domain of at least 20C-terminal amino acid residues, more preferably at least 10 amino acids, even more preferably at least 5 amino acids, even more preferably a single amino acid residue.

Thus, in a preferred embodiment, the domain antibody construct has a form comprising: dAb-C terminal C_H1 Domain residue-hinge region-C_H2 Domain-C_H3 domains as schematically illustrated in figure 2.

In a particularly preferred embodiment, the domain antibody construct has the amino acid sequence of SEQ ID No: 11, or a pharmaceutically acceptable salt thereof. This was designated compound 170.

Compound 170

DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYSASNLET

GVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQGTKVEIKRVEPKS

SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP

APLEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG

QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK (SEQ ID No：11)。

The hinge region of naturally occurring immunoglobulins contains cysteine (C) side chains, which contribute to heavy chain C_H1 domain and light chain constant region domainFormation of disulfide bonds between them. Since the construct contains only a single variable domain, leaving one potentially active unpaired cysteine residue, this cysteine residue is replaced with an amino acid residue that prevents disulfide bond formation. Possible consequences of having unpaired cysteines may include reduced protein expression due to aggregation and misfolding of the construct.

It is understood that any hinge region sequence derived from any antibody class may be suitable for use in the present invention. However, the hinge region is preferably from the antibody subclass IgG₁. Preferably, the hinge region is based on IgG₁Naturally occurring sequence of the hinge region and comprising the sequence EPKSSDKTHTCPPCPA (SEQ ID No: 12). In this sequence, the Cys, which is usually present at position 5, is underlined for a Ser residue substitution.

Preferably, C_HThe C-terminal amino acid residue of domain 1 is derived from IgG 1. More preferably, C_HResidue 1 is a valine (V) residue or a conservative amino acid substitution such as leucine (L) or isoleucine (I). This residue is immediately adjacent to the hinge region and helps to increase the flexibility of the construct near the hinge region.

C_H2 and C_HThe sequence of domain 3 is preferably derived from Swissprot database accession No. PO 1857:

PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN

AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID No：63)。

the domain antibody construct may be derivatized or linked to another functional molecule. For example, a domain antibody construct may be functionally linked by chemical coupling, genetic fusion, non-covalent binding, or other means 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 the binding of the antibody or antibody binding moiety to another molecule (e.g., a streptavidin core region or a polyhistidine tag).

Useful detectable agents from which the domain antibody constructs may be derived include fluorescent compounds. Exemplary fluorescently detectable reagents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. The domain antibody constructs may also be derivatized with detectable enzymes such as alkaline phosphatase, horseradish peroxidase, glucose oxidase, and the like. When the domain antibody construct is derivatized with a detectable enzyme, it is detected by the addition of an additional reagent for the enzyme to produce a detectable reaction product. The domain antibody construct may also be derivatized with biotin and detected by indirect measurement of avidin or streptavidin binding.

The domain antibody constructs according to the invention may be linked to one or more molecules capable of providing increased half-life and resistance to degradation in vivo without loss of activity (e.g., binding affinity). These molecules may be linked to the domain antibody construct by a linker so that they do not interfere with/sterically hinder the antigen binding site. These adducted molecules include dAbs directed against endogenous molecules as described in U.S. patent application 20050271663. Typically, such adducting molecules are polypeptides or polypeptide fragments that occur naturally in vivo and are resistant to degradation or removal via endogenous mechanisms. The half-life increasing molecule may be selected from the following:

(a) proteins from the extracellular matrix, for example, collagen, laminin, integrin, and fibronectin;

(b) proteins found in blood, such as fibrin, alpha-2 macroglobulin, serum albumin, fibrinogen a, fibrinogen B, serum amyloid a, heptaglobin (heptaglobin), proteins, ubiquitin, uteroglobin (uteroglobulin), beta-2 microglobulin, plasminogen, lysozyme, cystatin C, alpha-1-antitrypsin and pancreatic trypsin inhibitor;

(c) immune serum proteins, such as IgE, IgG, IgM;

(d) transport proteins, such as retinol binding protein, alpha-1 microglobulin;

(e) defensins, such as beta-defensin 1, neutrophil defensins 1, 2 and 3;

(f) proteins found at the blood brain barrier or in neural tissue, such as melanocortin receptors, myelin, ascorbic acid transporters;

(g) transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins (see US 5977307); brain capillary endothelial cell receptor, transferrin receptor, insulin-like growth factor 1(IGF1) receptor, insulin-like growth factor 2(IGF2) receptor, insulin receptor;

(h) kidney-localized proteins such as polycystic protein, type IV collagen, organic anionic transporter K1, Heymann's antigen;

(i) liver-localized proteins, such as alcohol dehydrogenase, G250;

(j) a blood coagulation factor X;

(k) alpha-1 antitrypsin;

(l)HNF 1α；

(m) proteins localized to the lung, such as secretory components (binding IgA);

(n) cardiac-localized proteins, such as HSP 27;

(o) proteins that localize to the skin, such as keratin;

(p) bone-specific proteins, such as Bone Morphogenic Proteins (BMPs), e.g., BMP-2, -4, -5, -6, -7 (also known as osteogenic protein (OP-1)) and-8 (OP-2);

(q) tumor-specific proteins, such as human trophoblast antigens, herceptin (herceptin) receptors, estrogen receptors, cathepsins such as cathepsin B (found in liver and spleen);

(r) disease-specific proteins, such as antigens expressed only on activated T-cells: including LAG-3 (lymphocyte activation gene); osteoprotegerin ligand (OPGL), see KongYY et al, Nature (1999)402, 304-; OX40 (a member of the TNF receptor family, expressed on activated T cells, and being the only co-stimulatory T cell molecule known to be specifically upregulated in cells producing human T cell leukemia virus type I (HTLV-I) -see Pankow R et al, J.Immunol. (2000) Jul 1; 165 (1): 263-70); metalloproteases (associated with arthritis/cancer), including CG6512 drosophila, human paraplegia protein, human FtsH, human AFG3L2, murine FtsH; angiogenic growth factors, including acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), transforming growth factor-alpha (TGF- α), tumor necrosis factor- α (TNF- α), angiogenin, interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derived endothelial growth factor (PD-ECGF), placental growth factor (PlGF), midkine (midkine) platelet-derived growth factor-BB (PDGF), CXXXC chemotactic molecule (fractalkine);

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

(t) proteins involved in Fc transport.

The invention also extends to pegylated domain antibody constructs that provide increased half-life and resistance to degradation without loss of activity (e.g., binding affinity) relative to non-pegylated antibody polypeptides.

The domain antibody constructs may be coupled to polymer molecules (preferably PEG) useful for achieving increased half-life and degradation resistance properties using methods known in the art. The polymer moieties that may be used in the present invention may be synthetic or naturally occurring and include, but are not limited to, linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymers, or branched or unbranched polysaccharides such as, for example, homopolysaccharides or heteropolysaccharides. Preferred examples of synthetic polymers that may be used in the present invention include linear or branched poly (ethylene glycol) (PEG), poly (propylene glycol), or poly (vinyl alcohol), and derivatives or substituted forms thereof. Particularly preferred substituted polymers for attachment to the domain antibody construct include substituted PEGs, including methoxy (polyethylene glycol). Naturally occurring polymer moieties that may be used in addition to, or in place of, PEG include lactose, amylose, dextran, or glycogen, as well as derivatives thereof, as will be recognized by those skilled in the art.

Polymer (PEG) molecules useful in the present invention can be linked to the domain antibody construct using methods well known in the art. The first step in the attachment of a PEG or other polymer moiety to a domain antibody construct of the invention is to replace the hydroxyl end group of the PEG polymer with a functional group comprising an electrophile. In particular, the PEG polymer is linked to cysteine or lysine residues present in the monomer or multimer of the domain antibody construct. The cysteine and lysine residues may be naturally occurring or may be engineered into the domain antibody construct molecule.

PEGylation of the domain antibody constructs of the invention can be accomplished by a variety of methods (see, e.g., Kozlowski-A & Harris-JM (2001) Journal of controlledRelease 72: 217). The PEG may be linked to the domain antibody construct directly or through an intervening linker. Linker-free systems for attaching polyethylene glycol to proteins have been described in the following documents: delgado et al (1992), crit.rev.thera.drug carrier sys.9: 249-304; francis et al, (1998), lntern.j.hematol.68: 1 to 18; US 4,002,531; US5,349,052; WO 95/06058; and WO98/32466, the disclosure of each of which is incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acid residues of proteins without the use of an intervening linker employs tresylated MPEG, which is produced by modifying monomethoxypolyethylene glycol (MPEG) with tresylchloride. After the amino acid residue was reacted with triflated MPEG, polyethylene glycol was attached directly to the amine group. Thus, the present invention includes protein-polyethylene glycol conjugates produced by reacting a protein of the present invention with a polyethylene glycol molecule having a 2, 2, 2-trichloroethylsulfonyl group.

A number of different intervening linkers can also be used to attach the polyethylene glycol to the protein. For example, US5,612,460 discloses a urethane linker for attaching polyethylene glycol to a protein. Protein-polyethylene glycol conjugates, wherein polyethylene glycol is attached to the protein via a linker, can also be produced by reacting the protein with a compound such as MPEG-succinimidyl succinate, MPEG activated with 1, 1 '-carbonyldiimidazole (1, 1' -carbonyldiimidazole), MPEG-2, 4, 5-trichloropentylcarbonate, MPEG-p-nitrophenol carbonate, and various MPEG-succinate derivatives. Many other polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO98/32466, the entire disclosure of which is incorporated herein by reference.

In a particularly preferred embodiment of the invention, the domain antibody construct is directly coupled to polyethylene glycol via a lysine residue. In another preferred embodiment of the invention, the domain antibody construct is directly coupled to PEG via a cysteine residue. Unpaired cysteine residues may be present in the sequence beforehand or may be added by incorporating cysteine residues, for example, in the C-terminus of the domain antibody construct. Alternatively, the attachment of PEG to the domain antibody construct may be facilitated by the formation of disulfide bond forming cysteine residues, as described in US 20060210526.

Other derivatized forms of the polymer molecule include, for example, derivatives in which additional moieties or reactive groups are present to allow interaction with the amino acid residues of the domain antibody constructs described herein. Such derivatives include N-hydroxysuccinimide (NHS) active esters, succinimidyl propionate polymers, and thiol-selective reactive reagents such as maleimides, vinylsulfones and thiols. The PEG polymer may be a linear molecule, or may be branched, with multiple PEG moieties present in a single polymer.

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

The size of the polymers useful in the present invention may be from 500Da to 60kDa, e.g., 1000Da to 60kDa, 10kDa to 60kDa, 20kDa to 60kDa, 30kDa to 60kDa, 40kDa to 60kDa, and up to 50kDa to 60 kDa. The polymers used in the present invention, particularly PEG, may be linear polymers or may have a branched conformation.

In a further embodiment, the domain antibody construct according to the first aspect may be multimerized, such as e.g. an xor homodimer, an xor homotrimer, an xor homotetramer, or higher order xor homomultimers. Multimerization can increase the strength of antigen binding, where the strength of binding is related to the sum of the binding affinities of the multiple binding sites.

"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, and 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 minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffering substances.

The compositions may be in various forms, including liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., inhalable, injectable, and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, or suppositories. Preferably, the composition is in the form of an injectable solution for immunization. Administration may be intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular.

Generally, the therapeutic compositions must be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high drug concentrations. Proper fluidity of the solution can be maintained, for example, by the use of coating agents such as lecithin and/or surfactants. Sterile injectable solutions can be prepared by: the active compound (i.e., the domain antibody construct) is incorporated in the desired amount in a suitable solvent with one or a combination of the ingredients listed above, followed by filter sterilization.

The compositions may also be formulated as sterile powders for the preparation of sterile injectable solutions.

In certain embodiments, the active compound may be prepared with carriers that will protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches and microencapsulated delivery systems. Compatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, or polylactic acid may be used.

The compositions may also be formulated for oral administration. In this embodiment, the domain antibody construct may be encapsulated in a hard or soft shell gelatin capsule, compressed into a tablet, or incorporated directly into the diet of the subject.

Formulations allowing pulmonary, rectal, transdermal, intrathecal, intraocular administration will be well known to those skilled in the art.

Supplementary active compounds may also be incorporated into the compositions. The domain antibody constructs may be co-formulated and/or co-administered with one or more additional therapeutic agents, such as soluble TNF-alpha receptors or chemical agents that inhibit human TNF-alpha production, or antibodies that bind other targets, such as cytokines or cell surface molecules. Alternatively, it may be co-administered with a soluble immunochemical agent such as protein A, C, G or L.

An effective amount may include a therapeutically effective amount or a prophylactically effective amount of a domain antibody construct of the invention. A therapeutically effective amount is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A prophylactically effective amount is an amount that is effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Because prophylactic doses are administered prior to onset or at an early stage of the disease, a prophylactically effective amount can be less than a therapeutically effective amount.

For example, the anti-human TNF-alpha domain antibody constructs according to the present invention may be used for the detection of human TNF-alpha in, for example, a biological sample such as serum or plasma using conventional immunoassays, such as enzyme linked immunosorbent assay (ELISA), Radioimmunoassay (RIA) or tissue immunohistochemistry. Anti-human TNF-alpha domain antibody constructs according to the invention can be assayed in biological fluids by competitive immunoassays using recombinant human TNF-alpha standards labeled with a detectable substance and unlabeled anti-human TNF-alpha antibodies.

The anti-human TNF-alpha domain antibody constructs according to the present invention may also be used for the detection of TNF-alpha from other species than humans, such as non-human primates (including macaques, chimpanzees, marmosets, rhesus monkeys) and other species such as dogs, rats, mice, rabbits, cats, pigs, cows.

Anti-human TNF-alpha domain antibody constructs according to the invention may also be used in cell culture applications where inhibition of TNF-alpha activity is desired.

Disorders characterized by human TNF- α activity are intended to include diseases and other disorders in which the presence of TNF- α in a subject suffering from the disorder has been shown or suspected to be responsible for the pathophysiology of the disorder, or has been shown or suspected to be a factor contributing to the exacerbation of the disorder. Preferably, the condition characterised by human TNF- α activity is selected from the group consisting of inflammation, inflammatory disease, sepsis, including septic shock, endotoxic shock, gram-negative sepsis and toxic shock syndrome; autoimmune diseases, including rheumatoid arthritis, juvenile arthritis, rheumatoid spondylitis, ankylosing spondylitis, sjogren's syndrome, osteoarthritis and gouty arthritis, allergies, multiple sclerosis, autoimmune diabetes, autoimmune uveitis, psoriasis, pemphigoid and nephrotic syndrome; inflammatory conditions of the eye including macular degeneration, uveitis, behcet's disease; infectious diseases, including fever and myalgia due to infection and cachexia secondary to infection; graft versus host disease; tumor growth or metastasis, hematological malignancies (hematogenous malignancies); pulmonary disorders including asthma, adult respiratory distress syndrome, shocked lung, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis, and silicosis; inflammatory bowel disease, including crohn's disease and ulcerative colitis; cardiac disorders, congestive heart failure; vascular disorders including Wegener's disease, giant cell arteritis; inflammatory bone disease; central nervous system disorders, such as alzheimer's disease; peripheral nervous system disorders, such as sciatica; hepatitis, blood coagulation disorders, burns, reperfusion injury, endometriosis, keloid formation, and scar tissue formation.

In a particularly preferred embodiment, the disorder characterized by human TNF- α activity is age-related macular degeneration. TNF- α is involved in stimulating VEGF production and promoting intraocular neovascularization (Oh-H et al, 1999 research optics & Visualscience 40: 1891-98), and inhibitors of TNF- α activity, such as the domain antibody constructs described herein, are therefore useful in the treatment of ocular disorders associated with angiogenesis, including age-related macular degeneration.

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.

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

Example 1

Materials and methods

New world primate V_LIsolation of genes

Marmoset (caliuthrix species unknown) and owl monkey (nocturnal monkey (aotsustrivirgatus)) genomic DNA were obtained from the european collection of cell cultures (ECACC) under catalogue numbers 85011419 and 90110510, respectively. Marmoset DNA was derived from cell line B95-8, while owl monkey DNA was derived from cell line OMK 637-69.

Degenerate primers based on the human vk leader and Recombination Signal Sequence (RSS) were obtained from Walter and Tomlinson, Antibody Engineering: a Practical Approach (1996). Primers used to amplify germline vk DNA were as follows:

primer VK1BL

AATCKCAGGTKCCAGATG(SEQ ID No：13)

Primer VK1BL35a

GTTYRGGTKKGTAACACT(SEQ ID No：14)

Primer VK1BL35b

ATGMCTTGTWACACTGTG(SEQ ID No：15)。

PCR was performed using Taq polymerase with primer pairs VK1BL x VK1BL35a or VK1BL x VK1BL35b (30 cycles). There was an overlap between the cloned sequence and the 2 sets of primers used.

The genomic PCR product was cloned into the TOPO TA cloning kit from Invitrogen (catalog number K4500-01) and sequenced with M13 forward and pUC reverse primers. The sequence is acknowledged from the forward and reverse directions. To further confirm that no PCR errors occurred in the critical sequence, the PCR and cloning procedure was repeated twice for the marmoset sequence. The nucleotides (SEQ ID Nos: 16-26 and 38-43) and amino acids (SEQ ID Nos: 27-37 and 44-49) are given in FIG. 3. Marmoset sequences 1, 2 and 3 were confirmed. Sequences 4, 5,6, 7 and 8 were only reviewed in the initial PCR. Sequences 9,10 and 11 were only reviewed in duplicate (i.e., second) PCR and cloning.

Oligonucleotides were synthesized and cloned into acceptor sequences

The four CDR sequences, YAATKLQS (SEQ ID No: 1) from owl monkey sequence 1(SEQ ID No: 44), YEASSLQS (SEQ ID No: 2) from owl monkey sequence 2(SEQ ID No: 45), YEASKLQS (SEQ ID No: 3) from marmoset sequence 1(SEQ ID No: 27) and YSASNLET (SEQ ID No: 4) from marmoset sequence 2(SEQ ID No: 28) are selected from the amino acid sequences shown in FIG. 3. Owl monkey sequence 5, YYASSLQS (SEQ ID No: 56), was found to be identical to the cDNA sequence GI6176295 of Peru nyu (Aotus nanctycaae) (Ma's nocturnal monkey), while all other sequences were unique.

The acceptor variable region (anti-TNF domain antibody) sequence in this expression vector (domentis proprietary vector) was sequentially digested with KpnI and SanDI (25 μ g), which excise most of FR2 and CDR2, as shown on the restriction digestion map (fig. 4). The vector was then gel purified to remove the excised wild type FR2 and CDR2 sequences.

Annealing of the oligonucleotides is performed by: oligonucleotide pairs (500pmol, based on the sequence shown in FIG. 3) were incubated at 95 ℃ for 5 minutes, followed by 65 ℃ for 5 minutes, and then allowed to slowly reach room temperature on a hot block (hot block). Then, the overlapping part was filled up in the Klenow reaction in the presence of dNTPs. Cloning of the synthesized double-stranded DNA (derived from oligonucleotide annealing and end-filling) molecules into the acceptor variable region sequences is accomplished using standard methods.

Affinity maturation

Affinity maturation of the marmoset CDR grafted dAb compound 145(SEQ ID No: 7) was performed by constructing 14 separate libraries each having the amino acid sequence of SEQ ID No: 7 is diverse. Selected residues are shaded below.

DIQMTQSPSSLSASVGDRVTITCRASQWYQQKPGKPKLLIYSASNLETG

VPSRFSGGSGTFTLTISSLPEDFATYYCQQPTFGQGTKVEIKR。

The selection was based on residues in CDR1 and CDR3 that are known to be diversified in the mature human Ig repertoire, as well as the framework residues that were observed to produce functional proteins following mutagenesis in the relevant dAb. Complementary forward and reverse PCR primer pairs were designed with NKK degeneracy for each selected residue, and two initial PCR reactions were performed, with each PCR reaction employing a single mutagenic primer and flanking primers. After clean-up, the two PCR products were annealed and subsequently amplified using separate flanking primers (splicing by overlap extension of PCR; Lowman H.L. & Clackson T. (eds.), phase Display: A pructicallappacach, Oxford University Press, Oxford, UK). Clones were initially screened by ELISA using solid phase TNF and positive clones were sequenced. dAb proteins were purified from the best clones and evaluated for potency in receptor binding assays and L929 cytotoxicity assays. Compounds 100(SEQ ID No: 9) and 123(SEQ ID No: 8) were found to have improved TNF neutralization relative to the parent dAb, compound 145(SEQ ID No: 7).

The combination of the enhanced affinity substitutions of compounds 100 and 123 resulted in an anti-TNF dAb (compound 196; SEQ ID No: 10) with further improved potency in the L929 cytotoxicity assay.

Cell culture

CHOK1SV cells (Lonza Biologics, UK), a suspension variety of CHOK1, were maintained in logarithmic growth phase in CD CHO medium supplemented with 6mM L-glutamine (Invitrogen Cat. Nos. 10743-029 and 25030-081). Culture at 36.5 deg.C with 10% CO₂And incubated with shaking at 140 rpm. Number and viability were assessed 24 hours prior to transfection by trypan blue exclusion on a hemocytometer (Sigma catalog number T8154). 8X 10 by 5 minutes at 200 Xg⁶Viable cells were pelleted and resuspended in 8ml of CM25 medium (Lonza Biologics, UK) supplemented with 10% heat-inactivated dialyzed fetal bovine serum (Invitrogen Cat. No. 26400-044) and 6mM L-glutamine. Cells were plated in 24-well plates at 500. mu.l per well and 10% CO at 36.5 ℃₂The following incubation was performed.

3 hours prior to transfection, the medium was supplemented with 500 μ L fresh aliquots of CM25 medium supplemented with 10% heat-inactivated dialyzed fetal bovine serum and 6mM L-glutamine.

Expression vector

The gene sequences for compound 112(SEQ ID No: 50) and compound 170(SEQ ID No: 51) were optimized for mammalian cell expression and synthesized.

Compound 112

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACC

ATCACCTGCAGAGCCAGCCAGGCCATCGACAGCTACCTGCACTGGTATCAGCAGAAGCCT

GGCAAGGCCCCTAAGCTGCTGATCTACAGCGCCAGCAATCTGGAGACCGGCGTGCCTAGC

AGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCTGCCT

GAGGATTTCGCCACCTACTACTGCCAGCAGGTGGTGTGGAGACCTTTCACCTTCGGCCAG

GGCACCAAGGTGGAGATCAAGCGGGTGGAGCCCAAGAGCTGCGATAAGACCCACACCTGC

CCCCCCTGCCCTGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAG

CCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTGGATGTG

AGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAAT

GCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTG

ACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAG

GCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCC

CAGCTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACC

TGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAG

CCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTG

TACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGC

GTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGC

AAG(SEQ ID No：50)

Compound 170

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACC

ATCACCTGCAGAGCCAGCCAGGCCATCGACAGCTACCTGCACTGGTATCAGCAGAAGCCT

GGCAAGGCCCCTAAGCTGCTGATCTACAGCGCCAGCAATCTGGAGACCGGCGTGCCTAGC

AGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCTGCCT

GAGGATTTCGCCACCTACTACTGCCAGCAGGTGGTGTGGAGACCTTTCACCTTCGGCCAG

GGCACCAAGGTGGAGATCAAGCGGGTGGAGCCCAAGAGCAGCGATAAGACCCACACCTGC

CCCCCCTGCCCTGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAG

CCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTGGATGTG

AGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAAT

GCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTG

ACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAG

GCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCC

CAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACC

TGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAG

CCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTG

TACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGC

GTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGC

AAG(SEQ ID No：51)

Each sequence was flanked at the 5' end by a Hind III site, a Kozak sequence (GCCACC; SEQ ID NO: 57) and a human IgG kappa leader sequence (amino acid sequence MSVPTQVLGLLLLWLTDARC; SEQ ID NO: 58). At the 3' end, two stop codons and one EcoRI site were added to each sequence. After synthesis, the genes were supplied as cloned into the pCRScript vector (Stratagene) and released by HindIII/EcoRI digestion in an appropriate restriction enzyme buffer (Roche Diagnostics Cat. Nos. 10656313001, 10703737001 and 11417967001, respectively). Similarly, the GS expression vector pee12.4(Lonza Biologics, UK) was digested and dephosphorylated with bovine small intestine alkaline phosphatase (Roche Diagnostics cat # 10713023001). Each gene was ligated into a prepared pEE12.4 backbone using the Promega's Ligafast Rapid DNA ligation System (catalog No. M8221). The ligation products were then transformed into chemically obtained competent cells (Invitrogen Cat. No. C4040-03) of One Shot Top10 and positive clones were identified by standard techniques. A large number of the resulting vectors (pEE12.4-PNO621 and pEE12.4-PNO521-S114C) were prepared by medium preparation of overnight cultures using QIAfilter medium preparation columns (QIAgen catalog No. 12243). Vectors were prepared for transfection by precipitating 20 μ g in 100% ethanol (Sigma Cat. Nos. S2889 and E7023, respectively) containing 1/10 volumes of 3M sodium acetate (pH 5.2). After washing with 70% ethanol, the vector was resuspended at a working concentration of 0.5. mu.g/. mu.l in 40. mu.l T.E.pH8.0(Sigma Cat. No. T9285).

Transfection

For each transfection, 2. mu.l Lipofectamine 2000 was added to 50. mu.l Optimem I medium (Invitrogen catalog numbers 11668-027 and 31985-062) in one well of a 96-well plate. In well 2, 1.6. mu.l of expression vector (0.8. mu.g) was added to 50. mu.l of Optimem I medium. After 5 minutes of incubation at room temperature, the contents of the 2 wells were mixed together and further incubated for 20 minutes. After this second incubation, the entire transfection mixture was added to the wells of a 24-well plate containing CHOK1SV cells. Cells were incubated for at least 72 hours and supernatants were harvested. The supernatant was centrifuged at 4,000Xg for 5 minutes to pellet the cell debris and stored at-20 ℃ until the expression of compound 112(SEQ ID No: 59) and compound 170(SEQ ID No: 11) was assayed by TNF ELISA.

Compound 112

DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYSASNLET

GVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQGTKVEIKRVEPKS

CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKPN

WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG

QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK(SEQ ID No：59)

TNF ELISA

Microtiter plates (e.g., Sarstedt 82.9923-148) were coated with 100. mu.l/well of a solution of 1. mu.l/mL human recombinant TNF- α (Peprotech Cat # 300-01A) in carbonate/bicarbonate coating buffer (pH 9.6). After overnight incubation at 4 ℃, the plates were washed three times with PBS containing 0.05% Tween-20 (0.01M, pH7.2), and three times with PBS. Add 200. mu.l blocking buffer (containing 1% BSA [ bovine serum Albumin, Sigma Cat # A-9647) to each well]PBS) and incubated at 25 ℃ for 1 hour. The plates were washed as described above and a 100 μ l volume of sample or compound 170 standard diluted in antibody diluent (PBS with 1% BSA and 0.05% Tween 20) was added to each well. After 1 hour incubation at 25 ℃, the plates were washed as described above and a 100 μ Ι volume of a 1: 1000 secondary antibodies (peroxidase-conjugated goat anti-human immunoglobulin, Zymed, Cat # 81-7120) diluted in antibody diluent. Plates were washed and 100. mu.l volume of ABTS substrate (2, 2' -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, Sigma Cat # A-1888, 0.5mg/mL in a solution containing 0.03% H₂O₂In a citrate buffer (pH 4.4). The substrate was developed for 30 minutes at room temperature and the absorbance read at 405nm (reference 620 nm). The sample concentration was determined relative to the standard curve and expressed as the average concentration relative to the expressed compound 112.

Results

Comprising a truncated C_H1

Comprising truncated C in a Domain antibody construct_H1 results in a linkage between the variable domain and the hinge that is as compared to the absence of the truncated C_H1 (83.3%; calculated with a gap opening penalty of 1 using Align X on Vector NTI (Invitrogen)), with a higher affinity for regular IgG between the variable domain and the hinge₁C_HHomology of 1-hinge connection (91.7%). Increased homology may translate into increased similarity to conventional immunoglobulin peptide sequences for which human patients should be immunologically tolerated, thereby reducing immunogenic potential.

The sequence is as follows:

compound 170 variable region-truncated C_H1-hinge connection:

TKVEIKREPKS

IgG₁ C_H1-hinge connection (NCBI accession number AAG 00909):

EPKS

compound 170 variable region-hinge connection (without truncated C)_H1)：

TKVEIKREPKS

C_HThe 1 sequence is indicated by double underlining.

Obtained from NCBI protein database ((II))http://www.ncbi.nlm.nih.gov) C of (A)_H1 domain (SEQ ID No: 60) AAG 00909:

1 STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEFVTVSW NSGALTSGVH TFPAVLQSSG

6.1 LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPELLGGP

121 SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPKEEQYNS

181 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM

241 TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ

301 QCNVFSCSVM HEALHNHYTQ KSLSLSPGK

C_Hthe 1-hinge connection is marked with underlining.

Neutralizing TNF-alpha induced cytotoxicity

The ability of domain antibody construct compound 170(SEQ ID No: 11) to neutralize TNF-. alpha.mediated cytotoxicity was evaluated using the murine L929 cell viability assay. Serial dilutions of compound 170 in RPMI medium containing 10% fetal bovine serum (RPMI-FBS) were prepared in flat bottom 96 well plates in 50 μ L volumes. To each of these wells 50. mu.l of recombinant human TNF-. alpha.was added at a concentration of 360pg/mL (Strathmann Biotec, Hamburg, Germany) followed by 2.5X 10 in 50. mu.L⁴L929 cells and 25 u L40 u g/mL actinomycin D, all in RPMI-FBS preparation. Controls included wells without TNF (for determination of 100% viability), wells without cells (0% viability) and wells with a TNF-. alpha.standard curve ranging from 2pg/mL to 4200 pg/mL. Plates were incubated at 5% CO₂Incubation at 37 ℃ for 20 hours under an atmosphere followed by addition of 25. mu.l of 3- (4, 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS)/Phenazine Ethosulfate (PES) (Promega, CellTiter96 AQ_ueousOne, Madison USA) and then incubated for 3 hours. The absorbance was measured at 492nm for a reference wavelength of 630nm and the viability curve was plotted using the average values calculated from triplicate test wells. The ability of compound 170 to neutralize TNF- α is demonstrated by the increase in cell viability with increasing concentration of compound 170 (fig. 6).

Neutralizing TNF-alpha binding to human p55 and p75 TNF receptors

Domain antibody construct the ability of compound 170(SEQ ID No: 11) to inhibit TNF- α binding to human p55 and p75 TNF receptors was evaluated by a receptor binding assay. Human p55(RnD systems, Cat. No. 372-RI) or p75(RnD systems, Cat. No. 762-R2) was coated onto Maxisorb plates (Nunc) by incubation at 4 ℃ overnight at 0.1. mu.g/mL in carbonate coating buffer (pH 9.2). Serial half-log dilutions of compound 170 (and a "blank" control without compound 170) ranging from 100 μ g/mL down to 3.15ng/mL were prepared in antibody diluent (PBS ph7.2, 0.05% Tween-20, 1% BSA) and mixed with an equal volume of 60ng/mL human TNF- α in antibody diluent. Blanks without PN0621 and without TNF-alpha were also prepared to measure background binding. All mixtures were incubated at room temperature for exactly 1 hour with gentle agitation. During this incubation, the coated plate was washed 3 times with PBS, 0.05% Tween-20, and then three times with PBS. The plate was then blocked with 200. mu.l/well PBS, 1% BSA for 45 min at room temperature. After washing the plates, 100. mu.l of compound 170/TNF-. alpha.mixture was added to triplicate wells for each concentration of test compound 170, and all controls were added. The plates were then incubated at room temperature for 1 hour. After washing the plates, biotinylated anti-human TNF-. alpha.antibody (RnD Systems, Cat. No. BAF210) was added to each well at 0.6. mu.g/mL in antibody diluent and incubated for 1 hour at room temperature. After washing, streptavidin-HRP conjugate (Zymed, catalog No. 43-4323) was added at 1:2000 in antibody diluent and incubated for 45 minutes at room temperature. Color development was performed using TMB substrate (Invitrogen, Cat. No. 00-2023) and after 4 minutes the reaction was stopped with 1M HCl. Then, absorbance readings were measured at 450nm using a 620nm reference. Analysis was performed by calculating the average absorbance of the triplicates. The average value of non-specific binding (no TNF-. alpha.) was subtracted from each absorbance value.

The results are shown in figure 7 and show that compound 170 prevents TNF-alpha interaction with the human p55 or p75 TNF receptor.

Binding of cell-bound TNF-alpha

Binding to cell-bound (transmembrane) TNF-. alpha.was analyzed using NS0 cell line 27D4, which was stably transfected with a gene encoding a human TNF-. alpha.protein lacking the TACE cleavage site, so that TNF-. alpha.remained cell membrane-bound because it could not be cleaved. Similar cell lines based on another murine myeloma (SP2/0) have also been described (Scallon et al, (1995) Cytokine 7251-259).

At 5X 10 per sample⁵Flow cytometry analysis was performed on individual 27D4 cells, with all steps performed at 4 ℃ or on ice. The cell pellet was resuspended in PBS containing 2% FBS at 100. mu.g/mL with test sample (Compound 170; SEQ ID No: 11) or an unrelated specific isotype-matched control (Sigma, cat # 15154) and incubated on ice for 1 hour. Two cell wash cycles were performed, each involving centrifugation at 1000 Xg for 10 minutes and resuspension of the cell pellet in PBS/2% FBS. After another centrifugation step, the cell pellet was resuspended in 100 μ l of secondary antibody conjugate (anti-human Fc FITC conjugate, Sigma, catalog No. F9512) and incubated for 30 minutes. The sample was then washed twice as described above and the cell pellet resuspended in 500. mu.l PBS/2% FBS containing 5. mu.g/mL propidium iodide (Sigma, Cat. No. P4864). Cells were analyzed for fluorescent staining on a BeckmanCoulter Cell Lab Quanta flow cytometer and data were processed using WinMDI.

The results appear in fig. 8 and show that compound 170 staining of NSO 27D4 cells expressing transmembrane TNF- α (black solid line) shows higher fluorescence intensity than the unrelated specific isotype-matched control (grey filled).

Establishment of cell line highly expressing Compound 170

The expression vector described in the "materials and methods" section was used to establish a stable CHOK1SV cell line expressing Compound 170(SEQ ID No: 11). Brief description of the drawingsIn the presence of 40. mu.g of linearized plasmid DNA, in a protein-free CDCHO medium without glutamine, 1X 10⁷Several cells in logarithmic growth phase were electroporated. 24 hours after transfection, a selection pressure of 50. mu.M methionine sulfoximine (Sigma) was applied and resistant cells were allowed to form colonies in 96-well plates. When near confluence, single colonies were transferred to 24-well plates, T25 bottles and then T75 bottles. Once confluent in T75 flasks, the cell lines were progressed to culture in E125 erlenmeyer flasks and adapted to suspension growth through 6 subcultures. Once adapted for suspension growth, the cell lines were grown in 92.5% CDCHO medium: refrigerated in a frozen mixture of 7.5% DMSO.

While the cell line was expanded through different well and vial sizes, many productivity (productivity) evaluations were performed in parallel with the progress of the cell line into the next stage. Therefore, productivity evaluations were performed at the 24-well plate and E125 erlenmeyer flask stages. In each case, cells were allowed to grow for 14 days and supernatants were evaluated for levels of compound 170 by the TNF ELISA method described in example 1. Cell lines were ranked by productivity and the top10 were selected for evaluation in proprietary fed-batch productivity assessment by Lonza Biologics. The productivity obtained was between 700mg/L and 3371 mg/L. A2724 mg/L productivity lead cell line was selected for evaluation in a 10L laboratory scale fermentor.

Four separate 10L laboratory scale fermentors were run for 15 days with a lead cell line and proprietary universal fed-batch process based on protein-free CDCHO medium. The resulting average productivity of 4 fermentations was 4851mg/L with a maximum productivity of 4925mg/L (the highest productivity level previously reported by Lonza Biologics in 15 days fermentations was 3560mg/L for the non-clonal cell line). The 10L laboratory scale fermentor used was designed to simulate the fermentation conditions found in larger scale fermenters up to 2000L, and therefore this lead cell line is expected to be suitable for commercial scale production. Indeed, similar expression levels were observed in the 200L fermentor.

The product harvested from a 4X 10L fermentation of a lead cell line expressing Compound 170(SEQ ID No: 70) was purified by protein A affinity chromatography and analyzed by SDS PAGE under reducing and non-reducing conditions. As shown in fig. 11, compound 170 is expressed as a monomer of about 90 kDa. This monomer consists of two subunits of approximately 40kDa, which are visible in figure 12 when running SDS PAGE under reducing conditions. Further analysis of compound 170 monomer was performed since SDS PAGE was not suitable for accurate protein sizing. ESI-MS (electrospray ionization mass spectrometry) determined the size of compound 170 monomer to be 78.739 kDa. This is consistent with the predicted molecular weight of 2 subunits (2 x 38.058 ═ 76.116kDa), each of which also carries a biantennary (biantennary) core fucosylated glycan structure.

Long serum half-life in non-human primates

Compound 170(SEQ ID No: 11) was administered subcutaneously to macaques at doses of 0.5, 5 and 50mg/kg and blood samples were drawn at 0.5, 1, 2, 6 and 24 hours, followed by 1, 2,4, 7, 10 and 14 days. These samples were analyzed for quantification of compound 170 levels using the anti-TNF ELISA method described in example 1. Elimination half-life was determined by analyzing the level of compound 170 in these samples. At 0.5mg/kg, the elimination half-life of 110.5. + -. 13.9 hours was calculated. At 5mg/kg and 50mg/kg, elimination half-lives of 110.9. + -. 10.4 and 103.5. + -. 11.5 hours were calculated.

When compound 170 was administered by the intravenous route at 50mg/kg, blood samples were drawn at 10, 30 and 60 minutes, 4 and 24 hours, 2,4, 7, 10 and 14 days. These samples were analyzed for quantification of compound 170 levels using the anti-TNF ELISA method described in example 1. Elimination half-life was determined by analyzing the level of compound 170 in these samples. After 50mg/kg intravenous administration, an elimination half-life of 109.6 ± 10.7 hours was calculated.

Advantageous safety features

Compound 170(SEQ ID No: 11) produced according to GMP standards was evaluated in animal safety and toxicity studies.

Safety of single dose

Single doses of compound 170 were administered to different monkeys at 0.5, 5, and 50mg/kg by subcutaneous or intravenous routes of administration, which did not show effects associated with their treatment with compound 170. In these studies, a series of organs were examined microscopically and no effect was observed.

Safety of escalated and repeated doses

Starting from a dose of 0.5mg/kg (subcutaneous or intravenous administration), macaques are administered escalating doses every 7 days up to 50 mg/kg. Animals were evaluated for various physiological and behavioral parameters, including hematology, clinical chemistry, body weight and organ weight, and gross examination of organs after necropsy. Throughout these studies, no adverse effects were reported for treatment with compound 170. After the escalating dose phase of the study, those animals that received the escalated dose subcutaneously were further administered 4 further doses of 50mg/kg over a further 4 week period. No effects associated with treatment with compound 170 were observed with various parameters.

Cardiovascular safety

In macaques fitted with radio telemetry monitors, 50mg/kg of compound 170 was evaluated for cardiovascular safety. These monitors are capable of reporting a range of respiratory and cardiovascular parameters directly from conscious monkeys. No adverse treatment-related clinical observations were reported after administration of compound 170.

Bacterial expression

Compound 170(SEQ ID NO: 11) in the previous examples was produced in a mammalian expression system. Bacterial expression systems are also used to produce functional compound 170.

The amino acid sequence of Compound 170 minus the Signal sequence was translated back and used with the GeneOptimizer^TMOptimized for e.coli (e.coli) expression and synthesized de novo in GeneArt GmbH. The synthesized genes were subcloned into pBAD gIII/His-tagged expression vector (Invitrogen) via NcoI and HindIII restriction sites (Roche) to generate vectors ready for bacterial expression. TOP10 cells (Invitrogen) were transformed with the vector by heat shock and single colony glycerol stocks were generated. Induction conditions were 0.002% arabinose (Sigma; final concentration) and an induction period of 4 hours. Compound 170 protein samples were generated using the osmotic shock method detailed in the pBAD bacterial expression systems manual (Invitrogen). The total protein concentration of the sample was determined using the BCA assay (Pierce). Compound 170 expressed by the bacteria was detected for its binding to TNF- α in an ELISA as described in example 1.

Figure 9 shows that compound 170 produced in the bacterial system retained binding to TNF-a in an ELISA assay.

The DNA sequence for bacterial expression of compound 170 is as follows:

ATGGCGAGCACCGATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGT

GATCGTGTGACCATTACCTGCCGTGCGAGCCAGGCGATTGATAGCTATCTGCATTGGTAT

CAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATAGCGCGAGCAACCTGGAAACC

GGCGTGCCGAGCCGTTTTAGCGGCAGCGGTAGCGGCACCGATTTTACCCTGACCATTAGC

AGCCTGCTGCCGGAAGATTTTGCGACCTATTATTGCCAGCAGGTGGTGTGGCGTCCGTTT

ACCTTTGGCCAGGGCACCAAAGTGGAAATTAAACGCGTGGAACCGAAAAGCAGCGATAAA

ACCCACACGTGCCCGCCGTGTCCGGCGCCGGAACTGCTGGGTGGCCCGAGCGTGTTTCTG

TTTCCGCCGAAACCGAAAGATACCCTGATGATTAGCCGTACCCCGGAAGTGACCTCCCTC

GTGGTGGATGTGAGCCATGAAGATCCGGAAGTGAAATTCAACTGGTATGTGGATGGCGTG

GAAGTGCATAACGCGAAAACCAAACCGCGTGAAGAACAGTATAACAGCACCTATCGTGTG

GTGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAACGGCAAAGAATACAAATGCAAA

GTGTCTAACAAAGCGCTGCCGGCGCCGATTGAAAAAACCATCAGCAAAGCGAAAGGCCAG

CCGCGTGAACCGCAGGTGTATACCCTGCCGCCGAGCCGTGATGAACTGACCAAAAACCAG

GTGAGCCTGACCTGCCTGGTGAAAGGCTTTTATCCGAGCGATATTGCGGTGGAATGGGAA

AGCAACGGCCAGCCGGAAAACAACTATAAAACCACCCCGCCGGTGCTGGATAGCGATGGC

AGCTTTTTCCTGTATAGCAAACTGACCGTGGATAAAAGCCGTTGGCAGCAGGGCAACGTG

TTTAGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTATACCCAGAAAAGCCTGAGC

CTGAGCCCGGGTAAAGCGGCGGCG

(SEQ ID No：61)。

consisting of SEQ ID No: 61 the amino acid sequence is as follows:

MASTDIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYSASNLET

GVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQGTKVEIKRVEPKSSDK

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ

PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG

SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAAAVDHHHHHH

(SEQ ID NO：62)。

in vivo utility of compound 170 in a murine arthritis model mediated by human TNF

The human TNF Transgenic mouse line Tg197 displays deregulated TNF expression and develops chronic inflammatory polyarthritis (Keffer, J. et al, (1991), Transgenic microxpressing human tumor necrosis factor: a predictive genetic model of arthritis. EMBO Journal 10: 4025-31). Administration of Compound 170(SEQ ID No: 11) in these mice prevented the development of arthritis and associated weight loss (FIGS. 10A and B). 8 heterozygous Tg197 groups (each group containing 4 males and 4 females) were injected intraperitoneally twice weekly with Compound 170 and an unrelated specific control human IgG₁(palivizumab [ alpha ], [ beta ]]MedImmune/Abbott) (in PBS) at 10 mg/kg. Treatment was initiated at 3 weeks of age of the mice. At weekly intervals, mice were weighed and scored based on visual ankle morphology (swelling, deformation and degree of mobility) (arthritis score).

Replacement of Cys at position 114 in Compound 112 results in increased protein expression

Compound 112(SEQ ID No: 59) is a modification of compound 170(SEQ ID No: 11) and comprises a cysteine residue instead of a serine residue at position 114, which is present at this position in compound 170. The effect of cysteine 114 substitution to serine on protein expression was evaluated by comparison with compound 170. Multiple cultures of host cells transfected with the gene constructs for compounds 112 and 170 were tested for protein expression by ELISA using solid phase TNF as described in the materials and methods section. The results are shown in FIG. 11.

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.

Sequence listing

<110>Peptech Limited

<120> Domain antibody constructs

<130>WJP GHE 03 1411 5717

<160>64

<170>PatentIn version 3.3

<210>1

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<213> night monkey

<400>1

<210>2

<211>8

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<213> night monkey

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<213> Calmoset genus, species unknown

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<213> Calmoset genus, species unknown

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<213> night monkey

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<211>89

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<213> night monkey

<400>44

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<211>89

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<213> night monkey

<100>45

<210>46

<211>89

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<400>46

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<400>47

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<211>89

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<213> night monkey

<400>48

<210>49

<211>89

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<213> night monkey

<400>49

<210>50

<211>1023

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<210>51

<211>1023

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<400>51

<210>52

<211>108

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<400>52

<210>53

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<400>53

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<400>54

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<211>8

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<213> synthetic

<400>55

<210>56

<211>8

<212>PRT

<213> night monkey

<400>56

<210>57

<211>6

<212>PRT

<213> synthetic

<400>57

<210>58

<211>20

<212>PRT

<213> synthetic

<400>58

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<211>341

<212>PRT

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<400>59

<210>60

<211>329

<212>PRT

<213> synthetic

<400>60

<210>61

<211>1044

<212>DNA

<213> synthetic

<400>61

<210>62

<211>356

<212>PRT

<213> synthetic

<400>62

<210>63

<211>216

<212>PRT

<213> CH2/CH3 Domain sequences from Swissprot database accession number P01857

<400>63

<210>64

<211>17

<212>PRT

<213> synthetic

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> X represents valine, leucine or isoleucine

<220>

<221>MISC_FEATURE

<222>(6)..(6)

<223> X is absent or is an amino acid other than cysteine

<400>64

Claims

1. A domain antibody construct that binds to human TNF- α, said construct comprising:

(a) a domain antibody (dAb) that binds to human TNF- α;

(b) a modified hinge region sequence;

(c) a human or primate heavy chain constant region sequence having a truncated C of no more than 20 residues_H1, a domain of a polypeptide having a sequence of,

wherein the modified hinge region sequence comprises a deletion or a single amino acid substitution of at least one cysteine residue that normally facilitates the formation of heavy and light antibody interchain disulfide bonds.

2. The domain antibody construct of claim 1, wherein said C with truncation_H1 domain comprises no more than 10 residues.

3. The domain antibody construct of claim 2, wherein said C with truncation_H1 domain comprises no more than 5 residues.

4. The domain antibody construct of claim 3, wherein said C with truncation_H1 domain comprises no more than a single residue.

5. The domain antibody construct of any one of claims 1 to 4, wherein C_H1 the sequence of the domain and the hinge region is XEPKSZDKTTCPPCPA (SEQ ID No: 64), wherein X is valine, leucine or isoleucine and Z is absent or is an amino acid other than cysteine.

6. The domain antibody construct of claim 5, wherein X is valine and Z is serine.

7. The domain antibody construct of any one of claims 1 to 6, wherein said dAb comprises an immunoglobulin heavy or light chain variable domain, wherein said variable domain comprises at least one Complementarity Determining Region (CDR) having a sequence derived from a New world primate, wherein said CDR is selected from the group consisting of YAATKLQS (SEQ ID No: 1), YEASSLQS (SEQ ID No: 2), YEASKLQS (SEQ ID No: 3) and YSASNLET (SEQ ID No: 4).

8. The domain antibody construct of claim 7, wherein said CDR is CDR 2.

9. The domain antibody construct of any one of claims 1 to 8, wherein the domain antibody has a sequence selected from the group consisting of:

DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYSASNLETG

VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(SEQ ID No：7)；

DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYSASNLET

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(SEQ ID No：8)；

DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYSASNLETG

VPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(SEQ ID No：9)；

DIQMTQSPSSLSASVGDRVTITCRASQAIDSYLHWYQQKPGKAPKLLIYSASNLET

GVPSRFSGSGSGTDFTLTISSLLPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(SEQ ID No：10)；

DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKPPKLLIYSASNLETG

VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(SEQ ID No：52)；

DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYSASNLETG

VPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(SEQ ID No：53)；

DIQMTQSPSSLSASVGDRVTITCRASQSIDSYLHWYQQKPGKAPKLLIYSASNLETG

VPSRFSGSGSGTDFTLTISSLVPEDFATYYCQQVVWRPFTFGQGTKVEIKR

(SEQ ID No: 54); and

a sequence that is at least 95% identical to one of these sequences.

10. The domain antibody construct of any one of claims 1 to 9 wherein cysteine residues within the hinge region that normally promote the formation of heavy and light antibody interchain disulfide bonds are replaced with serine residues.

11. The domain antibody construct of any one of claims 1 to 10, wherein said hinge region comprises the sequence EPKSSDKTHTCPPCPA (SEQ ID No: 12).

12. The domain antibody construct of any one of claims 1 to 11, wherein the constant region comprises a sequence identical to SEQ ID No: 63 is at least 60% identical to the sequence shown in_H2 and C_H3 domain.

13. The domain antibody construct of claim 12, wherein said C_H2 and C_H3 domain and SEQ ID No: 63 is at least 80% identical.

14. The domain antibody construct of claim 13, wherein said C_H2 and C_H3 domain and SEQ ID No: 63 is at least 90% identical.

15. The domain antibody construct of claim 14, wherein said C_H2 and C_H3 domain and SEQ ID No: 63 is at least 95% identical.

16. The domain antibody construct of claim 15, wherein said C_H2 and C_H3 domain and SEQ ID No: 63 is at least 96% identical.

17. The domain antibody construct of claim 16, wherein said C_H2 and C_H3 domain and SEQ ID No: 63 is at least 97% identical.

18. The domain antibody construct of claim 17, wherein said C_H2 and C_H3 domain and SEQ ID No: 63 is at least 98% identical.

19. The domain antibody construct of claim 18, wherein said C_H2 and C_H3 domain and SEQ ID No: 63 is at least 99% identical.

20. The domain antibody construct of any one of claims 1 to 19, wherein the CDR1, CDR3, or at least one framework region is modified to improve antigen binding.

21. The domain antibody of any one of claims 1 to 20, wherein the amino acid sequence is identical to SEQ ID No: 11 is at least 60% identical.

22. The domain antibody of claim 21, wherein said amino acid sequence is identical to SEQ ID No: 11 is at least 80% identical.

23. The domain antibody of claim 22, wherein said amino acid sequence is identical to SEQ ID No: 11 is at least 90% identical.

24. The domain antibody of claim 23, wherein said amino acid sequence is identical to SEQ ID No: 11 is at least 95% identical.

25. The domain antibody of claim 24, wherein said amino acid sequence is identical to SEQ ID No: 11 is at least 96% identical.

26. The domain antibody of claim 25, wherein said amino acid sequence is identical to SEQ ID No: 11 is at least 97% identical.

27. The domain antibody of claim 26, wherein the amino acid sequence is identical to SEQ ID No: 11 is at least 98% identical.

28. The domain antibody of claim 27, wherein said amino acid sequence is identical to SEQ ID No: 11 is at least 99% identical.

29. The domain antibody of claim 28, wherein said amino acid sequence is identical to SEQ ID No: 11 are identical.

30. The domain antibody construct of any one of claims 1 to 29, wherein said domain antibody has low immunogenicity in humans.

31. A dimeric domain antibody construct that binds human TNF- α, wherein the dimer consists of two domain antibody constructs of any one of claims 1 to 30.

32. The dimerization domain antibody construct of claim 31, wherein the dimerization domain antibody construct is a homodimer.

33. The dimeric domain antibody construct of claim 32, wherein said domain antibody construct constituting a homodimer comprises a heavy chain variable region identical to the heavy chain variable region of SEQ ID No: 11 is at least 60% identical to the sequence shown in figure 11.

34. The dimeric domain antibody construct of claim 33, wherein said domain antibody construct constituting a homodimer comprises a heavy chain variable region identical to the heavy chain variable region of SEQ ID No: 11 is at least 80% identical to the sequence shown in figure 11.

35. The dimeric domain antibody construct of claim 34, wherein said domain antibody construct constituting a homodimer comprises a heavy chain variable region identical to the heavy chain variable region of SEQ ID No: 11 is at least 90% identical to the sequence shown in figure 11.

36. The dimeric domain antibody construct of claim 35, wherein said domain antibody construct constituting a homodimer comprises a heavy chain variable region identical to the heavy chain variable region of SEQ ID No: 11 is at least 95% identical to the sequence shown in fig. 11.

37. The dimeric domain antibody construct of claim 36, wherein said domain antibody construct constituting a homodimer comprises a heavy chain variable region identical to the heavy chain variable region of SEQ ID No: 11 is at least 96% identical.

38. The dimeric domain antibody construct of claim 37, wherein said domain antibody construct constituting a homodimer comprises a heavy chain variable region identical to the heavy chain variable region of SEQ ID No: 11 is at least 97% identical.

39. The dimeric domain antibody construct of claim 38, wherein said domain antibody construct constituting a homodimer comprises a heavy chain variable region identical to the heavy chain variable region of SEQ ID No: 11 is at least 98% identical to the sequence shown in figure 11.

40. The dimeric domain antibody construct of claim 39, wherein said domain antibody construct constituting a homodimer comprises a heavy chain variable region identical to the heavy chain variable region of SEQ ID No: 11 is at least 99% identical.

41. The dimeric domain antibody construct of claim 40, wherein said domain antibody construct that makes up a homodimer comprises the amino acid sequence of SEQ ID No: 11, or a pharmaceutically acceptable salt thereof.

42. An isolated nucleic acid molecule encoding the domain antibody construct of any one of claims 1 to 30.

43. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a domain antibody construct that binds human TNF- α, wherein said nucleic acid sequence is identical to SEQ ID No: 50 or SEQ ID No: 51 is at least 60% identical.

44. The isolated nucleic acid molecule of claim 43, wherein the nucleic acid sequence is identical to SEQ ID NO: 50 or SEQ ID No: 51 is at least 80% identical.

45. The isolated nucleic acid molecule of claim 44, wherein the nucleic acid sequence is identical to SEQ ID NO: 50 or SEQ ID No: 51 is at least 90% identical.

46. The isolated nucleic acid molecule of claim 45, wherein the nucleic acid sequence is identical to SEQ ID NO: 50 or SEQ ID No: 51 is at least 95% identical.

47. The isolated nucleic acid molecule of claim 46, wherein the nucleic acid sequence is identical to SEQ ID NO: 50 or SEQ ID No: 51 is at least 96% identical.

48. The isolated nucleic acid molecule of claim 47, wherein the nucleic acid sequence is identical to SEQ ID NO: 50 or SEQ ID No: 51 is at least 97% identical.

49. The isolated nucleic acid molecule of claim 48, wherein said nucleic acid sequence is identical to SEQ ID NO: 50 or SEQ ID No: 51 is at least 98% identical.

50. The isolated nucleic acid molecule of claim 49, wherein said nucleic acid sequence is identical to SEQ ID NO: 50 or SEQ ID No: 51 is at least 99% identical.

51. The isolated nucleic acid molecule of claim 50, wherein the nucleic acid sequence is identical to SEQ ID NO: 50 or SEQ ID No: 51 are identical.

52. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a domain antibody construct that binds to human TNF- α, wherein said nucleic acid sequence hybridizes under high stringency conditions with the complement of SEQ ID No: 50 or SEQ ID No: 51, under conditions which are not identical to those of the standard nucleotide sequence.

53. A pharmaceutical composition comprising an effective amount of a domain antibody construct according to any one of claims 1 to 30, and a pharmaceutically acceptable carrier or diluent.

54. A pharmaceutical composition comprising an effective amount of the dimeric domain antibody construct of any one of claims 31 to 41, and a pharmaceutically acceptable carrier or diluent.

55. A method for detecting human TNF- α in a sample, comprising contacting the sample with an effective amount of a domain antibody construct according to any of claims 1 to 41, and detecting the amount of bound domain antibody construct.

56. The method of claim 55, wherein the sample is a biological sample.

57. A method for treating a disorder characterized by human TNF- α activity in a human subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 55 or claim 56.

58. The method of claim 57, wherein said condition characterized by human TNF- α activity is selected from the group consisting of inflammation, inflammatory disease, sepsis, including septic shock, endotoxic shock, gram negative sepsis and toxic shock syndrome; autoimmune diseases, including rheumatoid arthritis, juvenile arthritis, rheumatoid spondylitis, ankylosing spondylitis, sjogren's syndrome, osteoarthritis and gouty arthritis, allergies, multiple sclerosis, autoimmune diabetes, autoimmune uveitis, psoriasis, pemphigoid and nephrotic syndrome; inflammatory conditions of the eye including macular degeneration, uveitis, behcet's disease; infectious diseases, including fever and myalgia due to infection and cachexia secondary to infection; graft versus host disease; tumor growth or metastasis, hematologic malignancy; pulmonary disorders including asthma, adult respiratory distress syndrome, shocked lung, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis, and silicosis; inflammatory bowel disease, including crohn's disease and ulcerative colitis; cardiac disorders, congestive heart failure; vascular disorders including wegener's disease, giant cell arteritis; inflammatory bone disease; central nervous system disorders, such as alzheimer's disease; peripheral nervous system disorders, such as sciatica; hepatitis, blood coagulation disorders, burns, reperfusion injury, endometriosis, keloid formation, and scar tissue formation.

59. The method of claim 57 or claim 58, wherein the disorder characterized by human TNF- α activity is an angiogenesis-related ocular disorder.

60. The method of claim 59, wherein the angiogenesis-related eye disorder is age-related macular degeneration.