GB2427194A - Single domain Helicobacter pylori adhesin antibodies - Google Patents

Abstract

Single domain (dAb) Helicobacter pylori adhesin antibodies are claimed. Preferably these bind to BabA (blood group Ag-binding adhesion which interacts with the human fucosylated Lewis b (Le<b>) histo-blood group antigen. Specific antibody sequences are claimed, as are specific Koff and Kds. These antibodies are claims to inhibit the functional activity of H pylori adhesion of BabA. Pharmaceutical compositions are also claimed. Alternatively claimed is an antibody fragment which binds to an adhesion from H pylori.

Description

1 2427194 ANTIBODIES.

This invention relates to domain antibodies. In particular, it relates to domain antibodies that recognize adhesion molecules of Ilelicobacter pylon (H. pylon) and confer protection against or use to treat H. pylon infection.

The bacterium Helicobacter pylon (H. pylon) is a human-specific gastric pathogen.

This gram-negative, S-shaped microaerophilic bacterium was discovered and cultured from a human gastric biopsy specimen in 1983. It has been identified as the causative agent of chronic active gastritis and peptic ulcer disease. In addition, H. pylon is defined as a class 1 carcinogen, since it can cause the chronic infection that is associated with the development of gastric adenocarcinoma.

Progress has been achieved in determining the pathogenesis of this infection. However, although effective antimicrobial therapy is available, there is still no ideal treatment, and indications for therapy continue to evolve.

H. pylon colonizes the human gastric mucosa by adhering to the mucous epithelial cells and the mucus layer lining the gastric epithelium. This is mediated by the interaction between human fucosylated Lewis b (Le") histo-blood group antigen to the adhesion molecule (BabA) located on the bacterial cell outer membrane of H. pylon.

The Ka of the Le"-BabA complex is about lx 10 M'. The L&'/BabA interaction results in the adherence of H. pylon to human gastric epithelial cells in situ, which can cause the damage of host tissue and eventually leading to ulcer disease, either directly or through inflammatory or autoimmune reactions. Antibodies against BabA adhesin could compete for Leb and potentially reduce or prevent infection caused by H. pylon.

SUMMARY OF THE INVENTION.

The present inventors have generated a number of single domain antibodies (dAbs) which they have shown to be effective in the treatment of H. pylon infection. These S *S.

* . S * S * * * * * :. * * * * * S * * * S S S ** S S * S S S * * * S * I I.. S II single domain antibodies have been shown to bind andlor inhibit the functional activity of certain H. pylon adherence factors.

Thus in a first aspect the present invention provides a single domain antibody (dAb) which is capable of binding to andlor inhibiting the functional activity of an adherence factor from H. pylon.

In a preferred aspect of the invention the adherence factor is blood group Ag-binding adhesin (BabA).

According to the invention described herein those amino acid sequences designated ADR1-X in the sequence listing provided herein represent BabA binding dAb amino acid sequences.

Preferably it is a BabA binding dAb comprising, preferably consisting of, one or more sequences shown in the sequence listing provided herein and designated ADRI-i designated SEQ ID No ito ADR1-43 designated SEQ ID No 43.

In a most preferred embodiment of the above aspect of the invention, the dAb is a BabA binding dAb comprising, preferably consisting of, one or more sequences shown in the sequence listing provided herein and designated ADR1-5 represented by SEQ ID No 5, ADR1-9 represented by SEQ ID No 9, ADR1-19 represented by SEQ ID No 19, ADRI-25 represented by SEQ ID No 25, ADRI-26 represented by SEQ ID No 26, and ADR1-28 represented by SEQ ID No 28 herein.

Advantageously, the dAb according to the present invention binds to one or more BabA as herein defined with a rate constant of between 5 xl 0 and ix! 0 s1 More advantageously, the dAb according to the present invention binds to BabA with with a dissociation constant (Kd) of at least 100 j.tM to 1 pM.

In a further aspect the present invention provides a single domain antibody (dAb) I a..

* . . S * S * S * . S * * S * S * * * . *S S S * . S S S S S * . *.* S IS which is capable of binding to and/or inhibiting the functional activity of an adherence factor from H. pylon and which exhibits 80% identity to one or more BabA dAbs provided herein.

In a preferred embodiment of the above aspect of the invention, the dAb exhibits 82, 84, 86, 88, 90, 92, 94, 96, 98 or 99% identity to one or more BabA dAbs provided herein.

In a preferred embodiment of the above aspect of the invention, the dAb according to the present invention is capable of binding to one or more BabA proteins as herein defined with a K0ffrate constant of between 5 x10 and 1x107 s' More advantageously, the dAb according to the above aspect of the invention is capable of binding to BabA with a dissociation constant (Kd) of at least 1 OOiiM to I pM and/or a K0f1 rate constant of between 5 xIO' and 1x107 In a further aspect still the present invention provides a pharmaceutical composition comprising any one or more dAbs according to the invention and a pharmaceutically acceptable carrier, diluent and/or exipient.

In yet a further aspect the invention provides a method for the prophylaxis and/or treatment of H. pylon infection in a patient by administering to a patient in need of such treatment one or more dAbs or a composition according to the invention.

Methods for the administration of dAbs according to the invention are described in the detailed description of the invention. Preferably, the dAb is administered either by oral and/or systemic administration.

in a further embodiment of the above aspect of the invention, the administration of a pharmaceutical composition comprising dAbs for the treatment of a patient suffering from H. pylon infection includes a continuous infusion over a period of time, or a single dose or bolus administration. Further, it is envisaged that following a single 0 *I. * ::

dose or bolus administration a second bolus dose may be administered or a continuous infusion may be administered.

In a preferred embodiment of the invention, dAbs are human variable domains or comprise human framework regions (FWs) and one or more heterologous CDRs which bind specifically to one or more adhesion factors described herein. CDRs and framework regions are those regions of an immunoglobulin variable domain as defined in the Kabat database of Sequences of Proteins of Immunological Interest.

Preferred human framework regions are those encoded by germline gene segments DP47 and DPK9. Advantageously, FWI, FW2 and FW3 of a V or V1. domain have the sequence of FW1, FW2 or FW3 from DP47 or DPK9. The human frameworks may optionally contain mutations, for example up to about 5 amino acid changes or up to about 10 amino acid changes collectively in the human frameworks used in the dAbs of the invention.

In a further aspect the present invention provides a method for the prophylaxis and/or treatment of H. pylon infection in a patient wherein the method comprises the step of administering to the patient in need of such treatment one or more of: (i) An antibody which binds to a BabA adherence factor.

(ii) A fragment of an antibody which binds to a BabA adherence factor.

(iii) A dAb which binds to a BabA adhesion factor.

In a final aspect the present invention provides a method for the prophylaxis and/or treatment of H. pylon infection in a patient wherein the method comprises the step of administering to the patient in need of such treatment one or more of: (i) An antibody which inhibits the functional activity of a BabA adherence factor.

(ii) A fragment of an antibody which inhibits the functional activity of a BabA adherence factor.

(iii) A dAb which inhibits the functional activity of a BabA adherence factor.

In a preferred embodiment of the above aspect of the invention, preferably the * antibody or fragment thereof is mono-specific.

in a further aspect of the invention, the invention provides an antibody fragment which binds to an adhesin molecule from H. pylon. Preferably, the antibody fragment binds to the adhesin molecule BabA.

In a preferred embodiment of the above aspect of the invention the antibody fragment binds to a BabA molecule with a Koff rate constant of between 5 xlO-1 and lxlO-7 s- 1.

More advantageously, the antibody fragment according to the above aspect of the invention is capable of binding to BabA with a dissociation constant (Kd) of at least lOOp.Mto I pM.

In a preferred embodiment of the invention the antibody fragment is monovalent.

In a further aspect of the invention, the invention provides a multimeric composition which comprises two or more single domain antibodies or two or more antibody fragments antibody fragments. The multimeric composition may be a dimer, trimer or tetramer of the single domain antibody or antibody fragment, two or more being identical, and linked directly or with an optional spacer or linker. In addition, they can be fused to the Fe portion of an immunoglobulin molecule forming an Fe Fusion.

Furthermore, they can be optionally linked to a molecule to further enhance the property of the composition such as a PEG, albumin binding moiety (eg an affibody) or a muco-adhesive to increase the in vivo half life.

BRIEF DESCRIPTION OF THE DRAWINGS.

Figure 1 shows the effect of 1 pM dAbs + 1 ig!ml protein-L on the binding of 5 nM BabA to LebHSA (HSA on ref cell). Binding measured as binding of [BabA + dAb] / BabA binding xl 00. S **

* S S * * * * * * .:. * * * * S S S S * * * *, * , aS * * * , * a S * S S.. 0 Figure 2 shows the ranking inhibitors of 5 nM BabA binding to immobilized Leb Figure 3 shows analysis of three dAbs competing with BabA for binding to immobiljsed Leb on BlAcore.

DEFINITIONS.

BabA: Blood group Ag-binding adhesin.

Immunoglobulin: This refers to a family of polypeptides which retain the immunoglobulin fold characteristic of antibody molecules, which contains two sheets and, usually, a conserved disuiphide bond. Members of the immunoglobulin superfamily are involved in many aspects of cellular and non-cellular interactions in vivo, including widespread roles in the immune system (for example, antibodies, T-cell receptor molecules and the like), involvement in cell adhesion (for example the ICAM molecules) and intracellular signalling (for example, receptor molecules, such as the PDGF receptor). The present invention is applicable to all immunoglobulin superfamily molecules which possess binding domains. Preferably, the present invention relates to antibodies.

Domain: A domain is a folded protein structure which retains its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.

The term single antibody variable domain' (dAb) as used herein refers to a folded polypeptide domain comprising sequences characteristic of antibody variable domains.

It therefore includes complete antibody variable domains and modified variable domains, for example in which one or more loops have been replaced by further sequences, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least in part the binding activity and specificity of the full-length domain. Moreover, S *S.

* S * * S S * S * * * * S S S S * a * * * ** S * * S. S S ** S * * * * . . . S S ** the term dAb includes within its scope those single antibody variable domains in which one or more hypervariable loops andlor CDRs have been replaced with those from a second variable domain, which may be from the same or different origin.

Library: The term library refers to a mixture of heterogeneous polypeptides or nucleic acids. The library is composed of members, each of which have a single polypeptide or nucleic acid sequence. To this extent, library is synonymous with repertoire. Sequence differences between library members are responsible for the diversity present in the library. The library may take the form of a simple mixture of polypeptides or nucleic acids, or may be in the form of organisms or cells, for example bacteria, viruses, animal or plant cells and the like, transformed with a library of nucleic acids.

Preferably, each individual organism or cell contains only one or a limited number of library members. Advantageously, the nucleic acids are incorporated into expression vectors, in order to allow expression of the polypeptides encoded by the nucleic acids.

In a preferred aspect, therefore, a library may take the form of a population of host organisms, each organism containing one or more copies of an expression vector containing a single member of the library in nucleic acid form which can be expressed to produce its corresponding polypeptide member. Thus, the population of host organisms has the potential to encode a large repertoire of genetically diverse polypeptide variants.

Antibody An antibody (for example IgG, 1gM, IgA, IgD or IgE) or fragment (such as a Fab, F(ab')2, Fv, disuiphide linked Fv, scFv, closed conformation multispecific antibody, disulphide-linked scFv, diabody, dAb) whether derived from any species naturally producing an antibody, or created by recombinant DNA technology; whether isolated from serum, Bcells, hybridomas, transfectomas, yeast or bacteria).

Antigen A molecule that is bound by a ligand according to the present invention.

Typically, antigens are bound by antibody ligands and are capable of raising an antibody response in vivo. It may be a polypeptide, protein, nucleic acid or other molecule. Generally, the dAbs according to the invention are selected for target * * * * a.: * S * * * * **. S * * * S S S * * * * * S. * * ** S S * S * , * a *.* * *S specificity against a particular antigen. In the case of conventional antibodies and fragments thereof, the antibody binding site defined by the variable loops (LI, L2, L3 and HI, H2, H3) is capable of binding to the antigen.

Epitope A unit of structure conventionally bound by an immunoglobulin VH/VI, pair. Epitopes define the minimum binding site for an antibody, and thus represent the target of specificity of an antibody. In the case of a single domain antibody, an epitope represents the unit of structure bound by a variable domain in isolation.

Universal framework A single antibody framework sequence corresponding to the regions of an antibody conserved in sequence as defined by Kabat ("Sequences of Proteins of Immunological Interest", US Department of Health and Human Services) or corresponding to the human germline immunoglobulin repertoire or structure as defined by Chothia and Lesk, (1987) J. Mol. Biol. 196:910-917. The invention provides for the use of a single framework, or a set of such frameworks, which has been found to permit the derivation of virtually any binding specificity though variation in the hypervariable regions alone.

Substantially identical: A first amino acid or nucleotide sequence that contains a sufficient number of identical amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have similar activities, in the case of antibodies, the second antibody has the same binding specificity and has at least 50% of the affinity of the same.

DETAILED DESCRIPTION OF THE INVENTION.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridisation techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d

S

* S * S * . I * I *** * * S * S * * * * I S * SI S S S I S *

S S S S S *

ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc. which are incorporated herein by reference) and chemical methods.

(A) H. Pylon Infection.

In 1982, Warren and Marshall (NIH Consensus Development Panel on Helicobacter pylon in Peptic Ulcer Disease. Helicobacter pylon in peptic ulcer disease. JAMA 1994; 272: 65-9) provided the first insight into a new pathogenic factor in peptic ulcer disease. They isolated an urease-producing organism, later identified as Helicobacter pylon, nestling in the narrow interface between the gastric epithelial cell surface and the overlying mucus gel. In their early studies, the presence of this organism was shown to be highly correlated with antral gastritis as well as with gastric and duodenal ulcers, and eradication of this organism effectively eliminated ulcer recurrences.

Furthermore, an epidemiological relationship between H. pylon infection and gastric malignancies was reported.

Current medical treatments.

It has been clearly shown that eradication of H. pylon dramatically changes the natural history of duodenal ulcer disease. Several studies have demonstrated that ulcers recur in only a small percentage of cases (0-10%) following successful H. pylon eradication, compared with a recurrence rate 50% within the course of 1 year when the organism persists.

H. pylon has proved difficult to eradicate. Numerous treatment trials of H. pylon eradication using many different antibiotics, either singly or in combination.

Single therapy eradication rates with amoxicillin and bismuth compounds were equivalent, and because H. pylon did not seem to develop resistance to bismuth S *S * * * S t * : * : : : * *4 S I SI * * * I a *mI I compounds, it appeared very important to use these bismuth preparations in combination with another antibiotic.

Double therapy with bismuth and metronidazole was better than bismuth and amoxicillin. However, triple-antibiotic therapy emerged in an effort to prevent antibiotic resistance. Therapy with bismuth, metronidazole and tetracycline or bismuth, metronidazole and amoxicillin are common. Further, other triple therapy combinations have been used including tetracycline triple therapy. In addition, new regimes include the use of omeprazole, although probably, the most effective combination is bismuth subcitrate (120 mg q.i.d.), metronidazole (500 mg t.i.d.) and either tetracycline (500 mg q.i.d.) or amoxicillin (500 mg t.i.d.).

However, triple therapy is a complicated regimen to follow and is associated with low compliance in some series, frequent adverse reactions, and a reduced efficacy in populations with a high primary resistance to nitroimidazoles.

(B) H. pylon Adensin binding dAbs accordin2 to the invention.

In a first aspect the present invention provides a single domain antibody (dAb) which is capable of binding to andlor inhibiting the functional activity of a BabA from H. pylon.

(Bi) H. pylon Adensin factors.

The gastric mucosa is well protected against bacterial infections. However, H. pylon is highly adapted to this ecologic niche, with a unique array of features that permit entry into the mucus, swimming and spatial orientation in the mucus, attachment to epithelial cells, evasion of the immune response, and, as a result, persistent colonization and transmission. o *.

* .,*a. * $ * * $10 * * * * * * s a * * I. 0 * * p $ *4* I II The H. pylon genome (1.65 million bp) codes for about 1500 proteins, which includes a large family of 32 related outer-membrane proteins (Hop proteins) that includes most known H. pylon adhesins.

After being ingested, the bacteria have to evade the bactericidal activity of the gastric luminal contents and enter the mucous layer. Urease production and motility are essential for this first step of infection. Urease hydrolyzes urea into carbon dioxide and ammonia, thereby permitting H. pylon to survive in an acidic milieu. Motility is essential for colonization, and H. pylon flagella have adapted to the gastric niche.

H. pylon can bind tightly to epithelial cells by multiple bacterialsurface components.

The Blood group Ag-binding adhesin, BabA, is a 78-kD outer-membrane protein that binds to the fucosylated Lewis B blood-group antigen. It is believed to play a role in the initial steps of the colonization of the gastric mucosa allowing the bacteria to bind to the epithelial surface.

(Bii) BabA binding dAbs according to the invention.

According to the present invention, those sequences designated ADR1-X in the sequence listing provided herein represent the sequences of BabA binding dAbs (dAbs).

According to the present invention, advantageously the dAb is a BabA binding dAb comprising, preferably consisting of, one or more sequences shown in the sequence listing provided herein and designated ADR1-X, and represented as SEQ ID No I to SEQ ID No 43 respectively.

According to the above aspect of the invention, more advantageously the dAb is a BabA binding dAb comprising, preferably consisting of, one or more sequences shown in the sequence listing provided herein and designated ADRI-5 sequence designated SEQ ID No.5, ADR1-9 sequence designated SEQ ID No.9, ADRI-19 sequence

S 4. *4

4 S * d * S S ö * S S S S * S S S

S S

4.44 5 55 designated SEQ ID No.19, ADRI-25 sequence designated SEQ ID No. 25, ADRI-26 designated SEQ ID No 26 or ADRI-28 designated SEQ ID No 28.

Advantageously, the dAb according to the present invention is capable of binding to one or more BabA as herein defined with a rate constant of between 5 xl 0 and lxl17 s More advantageously, the dAb according to the present invention is capable of binding to BabA with a dissociation constant (Kd) of at least IOOIIM to 1 pM.

In a preferred embodiment of the above aspect of the invention, the dAb exhibits 82, 84, 86, 88, 90, 92, 94, 96, 98 or 99% identity to one or more of those dAbs identified as ADR1-5, ADR1-9, ADR1-19, ADRI-25, ADR1-26 or ADRI-28 designated SEQ ID No 5, 9, 19, 25, 26, and 28 respectively.

Calculation of amino acid sequence identity.

Sequences similar or homologous (e.g., at least about 80% sequence identity) to the sequences disclosed herein are also part of the invention. In some embodiments, the sequence identity at the amino acid level can be about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the sequence identity can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, substantial identity exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., very high stringency hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

Calculations of "homology" or "sequence identity" or "similarity" between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e. g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison a a S * : : * * *** : : LI I * . * * i a a a ILL * S purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

Advantageously, the BLAST algorithm (version 2.0) is employed for sequence alignment, with parameters set to default values. The BLAST algorithm is described in detail at the world wide web site ("www") of the National Center for Biotechnology Information (".ncbi") of the National Institutes of Health ("nih") of the U.S. government (".gov"), in the "/Blast/" directory, in the "blast_help.html" file. The search parameters are defined as follows, and are advantageously set to the defined default parameters.

BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87(6):2264-8 (see the "blast_help.html" file, as described above) with a few enhancements. The BLAST programs were tailored for sequence similarity searching, for example to identify homologues to a query sequence. The programs are not generally useful for motif-style searching. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al. (1994).

* * S * *SS * S S S S * S S S S S S * * *S* * S * SI * S 55 S S S S * S S S S S S S SSS S 55 0 5 The five BLAST programs available at the National Center for Biotechnology Information web site perform the following tasks: "blastp" compares an amino acid query sequence against a protein sequence database; "blastn" compares a nucleotide query sequence against a nucleotide sequence database; "blastx" compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database; "tblastn" compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames (both strands).

"tblastx" compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

BLAST uses the following search parameters: HISTOGRAM Display a histogram of scores for each search; default is yes.

(See parameter H in the BLAST Manual).

DESCRIPTIONS Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter V in the manual page). See also EXPECT and CUTOFF.

ALIGNMENTS Restricts database sequences to the number specified for which high-scoring segment pairs (HSPs) are reported; the default limit is 50. If more database sequences than this happen to satisfy the statistical significance threshold for reporting (see EXPECT and CUTOFF below), only the matches ascribed the greatest statistical significance are reported. (See parameter B in the BLAST Manual).

EXPECT The statistical significance threshold for reporting matches against database sequences; the default value is 10, such that 10 matches are expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance ascribed to a match is greater than the EXPECT threshold, the match will not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Fractional values are acceptable. (See parameter E in the BLAST Manual).

CUTOFF Cutoff score for reporting high-scoring segment pairs. The default value is calculated from the EXPECT value (see above). HSPs are reported for a * * a * *.s * * a * a I * * S * I S I * *I* S S S *I I S IS S S S S * S S * S S S S I.. I S. S S database sequence only if the statistical significance ascribed to them is at least as high as would be ascribed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, leading to fewer chance matches being reported.

(See parameter S in the BLAST Manual). Typically, significance thresholds can be more intuitively managed using EXPECT. MATRIX Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and

TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992, Proc. Nati. Aacad. Sci. USA 89(22):10915-9). The valid alternative choices include: PAM4O, PAM 120, PAM25O and IDENTITY. No alternate scoring matrices are available for BLASTN; specifying the MATRIX directive in BLASTN requests returns an error response.

STRAND Restrict a TBLASTN search to just the top or bottom strand of the database sequences; or restrict a BLASTN, BLASTX or TBLASTX search to just reading frames on the top or bottom strand of the query sequence.

FILTER Mask off segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton & Federhen (1993) Computers and Chemistry 17:149-163, or segments consisting of short-periodicity internal repeats, as determined by the XNU program of Claverie & States, 1993, Computers and Chemistry 17:191- 201, or, for BLASTN, by the DUST program of Tatusov and Lipman (see the world wide web site of the NCBI). Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.

Low complexity sequence found by a filter program is substituted using the letter "N" in nucleotide sequence (e.g., "N" repeated 13 times) and the letter "X" in protein sequences (e.g., "X" repeated 9 times).

Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default filtering is DUST for BLASTN, SEG for other programs.

* S S S Sal * S * S * I * S * S S S * I *5* S I * Sl a I el * I * I * a * * . S * * S.. * IS S S It is not unusual for nothing at all to be masked by SEG, XNU, or both, when applied to sequences in SWISS-PROT, so filtering should not be expected to always yield an effect. Furthermore, in some cases, sequences are masked in their entirety, indicating that the statistical significance of any matches reported against the unfiltered query sequence should be suspect.

NCBI-gi Causes NCBI gi identifiers to be shown in the output, in addition to the accession andlor locus name.

Most preferably, sequence comparisons are conducted using the simple BLAST search algorithm provided at the NCBI world wide web site described above, in the "IBLAST" directory.

(C) Preparation of dAbs acccordin to the invention.

DAbs according to the invention, may be prepared according to previously established techniques, used in the field of antibody engineering, for the preparation of scFv, "phage" antibodies and other engineered antibody molecules. Techniques for the preparation of antibodies, are for example described in the following reviews and the references cited therein: Winter & Milstein, (1991) Nature 349:293-299; Plueckthun (1992) Immunological Reviews 130:151-188; Wright et al., (1992) Crti. Rev.

Immunol.12:125-168; Holliger, P. & Winter, G. (1993) Cun. Op. Biotechn. 4, 446- 449; Carter, et al. (1995) J. Hematother. 4, 463-470; Chester, K.A. & Hawkins, R.E.

(1995) Trends Biotechn. 13, 294-300; Hoogenboom, H.R. (1997) Nature Biotechnol. 15, 125-126; Fearon, D. (1997) Nature Biotechnol. 15, 618-619; Plückthun, A. & Pack, P. (1997) Immunoteclmology 3, 83-105; Carter, P. & Merchant, A. M. (1997) Curr. Opin. Biotechnol. 8, 449-454; Holliger, P. & Winter, G. (1997) Cancer Immunol. Tmmunother. 45,128-130.

The techniques employed for selection of the variable domains employ libraries and selection procedures which are known in the art. Natural libraries (Marks et a!. (1991) I Mo!. Biol., 222: 581; Vaughan et a!. (1996) Nature Biotech., 14: 309) which use * S * S Sal * * S I a S S * * * S * I S **S * I * IS S I a* S * S S

S I S I I S S I

rearranged V genes harvested from human B cells are well known to those skilled in the art. Synthetic libraries (Hoogenboom & Winter (1992) J. Mo!. Biol., 227: 381; Barbas et a!. (1992) Proc. Nail. Acad. Sci. USA, 89: 4457; Nissim et a!. (1994) EMBO 1, 13: 692; Griffiths et a!. (1994) EMBO.1, 13: 3245; De Kruif et a!. (1995) 1 Mol. Biol., 248: 97) are prepared by cloning immunoglobulin V genes, usually using PCR.

Errors in the PCR process can lead to a high degree of randomisation. VH andlor V1 libraries may be selected against target antigens or epitopes separately.

Library vector systems A variety of selection systems are known in the art which are suitable for use in the present invention. Examples of such systems are described below.

Bacteriophage lambda expression systems may be screened directly as bacteriophage plaques or as colonies of lysogens, both as previously described (Huse et a!. (1989) Science, 246: 1275; Caton and Koprowski (1990) Proc. Nat!. Acad. Sci. US.A., 87; Mullinax et a!. (1990) Proc. Nat!. Acad. Sci. US.A., 87: 8095; Persson et a!. (1991) Proc. Nat!. Acad. Sci. US.A., 88: 2432) and are of use in the invention. Whilst such expression systems can be used to screen up to 106 different members of a library, they are not really suited to screening of larger numbers (greater than 106 members).

Of particular use in the construction of libraries are selection display systems, which enable a nucleic acid to be linked to the polypeptide it expresses. As used herein, a selection display system is a system that permits the selection, by suitable display means, of the individual members of the library by binding the generic andlor target ligands.

Selection protocols for isolating desired members of large libraries are known in the art, as typified by phage display techniques. Such systems, in which diverse peptide sequences are displayed on the surface of filamentous bacteriophage (Scott and Smith (1990) Science, 249: 386), have proven useful for creating libraries of antibody fragments (and the nucleotide sequences that encoding them) for the in vitro selection * * S S *SI * * * * * e S S S * I I I S *** S I S

II S S IS S S S S

* S S I S S S * S.. S IS S * and amplification of specific antibody fragments that bind a target antigen (McCafferty ci' at., WO 92/01047). The nucleotide sequences encoding the V and VL regions are linled to gene fragments which encode leader signals that direct them to the periplasmic space of E. coli and as a result the resultant antibody fragments are displayed on the surface of the bacteriophage, typically as fusions to bacteriophage coat proteins (e.g., pill or pVIII). Alternatively, antibody fragments are displayed externally on lambda phage capsids (phagebodies). An advantage of phage-based display systems is that, because they are biological systems, selected library members can be amplified simply by growing the phage containing the selected library member in bacterial cells. Furthermore, since the nucleotide sequence that encode the polypeptide library member is contained on a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation is relatively straightforward.

Methods for the construction of bacteriophage antibody display libraries and lambda phage expression libraries are well known in the art (McCafferty et at. (1990) Nature, 348: 552; Kang et at. (1991) Proc. Nail. Acad. Sc US.A., 88: 4363; Clackson et at.

(1991) Nature, 352: 624; Lowman et al. (1991) Biochemistry, 30: 10832; Burton et at.

(1991) Proc. Nail. Acad. Sci USA., 88: 10134; Hoogenboom et at. (1991) Nucleic Acids Res., 19: 4133; Chang et at. (1991) J. Immunol., 147: 3610; Breitling et at.

(1991) Gene, 104: 147; Marks ci' at. (1991) supra; Barbas et at. (1992) supra; Hawkins and Winter (1992) .1 Immunol., 22: 867; Marks et at., 1992, .1. Biol. Chem., 267: 16007; Lerner et at. (1992) Science, 258: 1313, incorporated herein by reference).

One particularly advantageous approach has been the use of phagelibraries (Huston et a!., 1988, Proc. Nat!. Acad. Sci U.S.A., 85: 58795883; Chaudhary ci at. (1990) Proc. Natl. Acad. Sci U.S.A., 87: 1066-1070; McCafferty ci at. (1990) supra; Clackson et at.

(1991) Nature, 352: 624; Marks et al. (1991) J. Mot. Biol., 222: 581; Chiswell et at.

(1992) Trends Biotech., 10: 80; Marks ci at. (1992) 1 Biol. Chem., 267). Various embodiments of scFv!ibraries displayed on bacteriophage coat proteins have been described. Refinements of phage display approaches are also known, for example as described in W096/06213 and WO92/01047 (Medical Research Council et al.) and W097/08320 (Morphosys), which are incorporated herein by reference.

a * * *Ia * a a a a a a a S * S I I I *S* a * a SI I I SI 1 1 1 S S S S * a a a * aaa S *I S * Other systems for generating libraries of polypeptides involve the use of cell- free enzymatic machinery for the in vitro synthesis of the library members. In one method, RNA molecules are selected by alternate rounds of selection against a target ligand and PCR amplification (Tuerk and Gold (1990) Science, 249: 505; Ellington and Szostak (1990) Nature, 346: 818). A similar technique may be used to identify DNA sequences which bind a predetermined human transcription factor (Thiesen and Bach (1990) Nucleic Acids Res., 18: 3203; Beaudry and Joyce (1992) Science, 257: 635; W092/05258 and W092/14843). In a similar way, in vitro translation can be used to synthesise polypeptides as a method for generating large libraries. These methods which generally comprise stabilised polysome complexes, are described further in W088/08453, WO9O/05785, WO9O/07003, WO9 1/02076, W09 1/05058, and W092/02536. Alternative display systems which are not phage-based, such as those disclosed in W095/22625 and WO95/1 1922 (Affymax) use the polysomes to display polypeptides for selection.

A still further category of techniques involves the selection of repertoires in artificial compartments, which allow the linkage of a gene with its gene product. For example, a selection system in which nucleic acids encoding desirable gene products may be selected in microcapsules formed by water-in-oil emulsions is described in W099/0267 1, W000/407 12 and Tawfik & Griffiths (1998) Nature Biotechnol 16(7), 652-6. Genetic elements encoding a gene product having a desired activity are compartmentalised into microcapsules and then transcribed andlor translated to produce their respective gene products (RNA or protein) within the microcapsules.

Genetic elements which produce gene product having desired activity are subsequently sorted. This approach selects gene products of interest by detecting the desired activity by a variety of means.

Lihrar Construction.

Libraries intended for selection, may be constructed using techniques known in the art, for example as set forth above, or may be purchased from commercial sources.

* * * * *** * * 4 S S S S * I * I S * S eSS S S S I* * a.* * S I I S * S I S I I I *SS I.5 5 a Libraries which are useful in the present invention are described, for example, in W099/20749. Once a vector system is chosen and one or more nucleic acid sequences encoding polypeptides of interest are cloned into the library vector, one may generate diversity within the cloned molecules by undertaking mutagenesis prior to expression; alternatively, the encoded proteins may be expressed and selected, as described above, before mutagenesis and additional rounds of selection are performed. Mutagenesis of nucleic acid sequences encoding structurally optimised polypeptides is carried out by standard molecular methods. Of particular use is the polymerase chain reaction, or PCR, (Mullis and Faloona (1987) Methods Enzymol., 155: 335, herein incorporated by reference). PCR, which uses multiple cycles of DNA replication catalysed by a thermostable, DNA-dependent DNA polymerase to amplify the target sequence of interest, is well known in the art. The construction of various antibody libraries has been discussed in Winter et a!. (1994) Ann. Rev. Immunology 12, 433-55, and

references cited therein.

PCR is performed using template DNA (at least lfg; more usefully, 1-1000 ng) and at least 25 pmol of oligonucleotide primers; it may be advantageous to use a larger amount of primer when the primer pool is heavily heterogeneous, as each sequence is represented by only a small fraction of the molecules of the pooi, and amounts become limiting in the later amplification cycles. A typical reaction mixture includes: 2jil of DNA, 25 pmol of oligonucleotide primer, 2.5.tl of lox PCR buffer 1 (Perkin-Elmer, Foster City, CA), 0.4 p1 of 1.25 tiM dNTP, 0.15 j.tl (or 2. 5 units) of Taq DNA polymerase (Perkin Elmer, Foster City, CA) and deionized water to a total volume of jil. Mineral oil is overlaid and the PCR is performed using a programmable thermal cycler. The length and temperature of each step of a PCR cycle, as well as the number of cycles, is adjusted in accordance to the stringency requirements in effect. Annealing temperature and timing are determined both by the efficiency with which a primer is expected to anneal to a template and the degree of mismatch that is to be tolerated; obviously, when nucleic acid molecules are simultaneously amplified and niutagenised, mismatch is required, at least in the first round of synthesis. The ability to optimise the stringency of primer annealing conditions is well within the knowledge of one of moderate skill in the art. An annealing temperature of between 30 C and 72 * * * * I** * * a * a a a a a * * * a a a.. a a.

a. . a as * a a.

* a S * * S * S **a a *a a a C is used. Initial denaturation of the template molecules normally occurs at between 92 C and 99 C for 4 minutes, followed by 20-40 cycles consisting of denaturation (94- 99 C for 15 seconds to 1 minute), aimealing (temperature determined as discussed above; 1-2 minutes), and extension (72 C for 1-5 minutes, depending on the length of the amplified product). Final extension is generally for 4 minutes at 72 C, and may be followed by an indefinite (0- 24 hour) step at 4 C.

i. Selection of the main-chain conformation The members of the immunoglobulin superfamily all share a similar fold for their polypeptide chain. For example, although antibodies are highly diverse in terms of their primary sequence, comparison of sequences and crystallographic structures has revealed that, contrary to expectation, five of the six antigen binding loops of antibodies (Hi, H2, Li, L2, L3) adopt a limited number of main-chain conformations, or canonical structures (Chothia and Lesk (1987) J. Mo!. Biol., 196: 901; Chothia et a!.

(1989) Nature, 342: 877). Analysis of loop lengths and key residues has therefore enabled prediction of the main-chain conformations of Hi, H2, Li, L2 and L3 found in the majority of human antibodies (Chothia et a!. (1992) J. Mo!. Biol., 227: 799; Tomlinson et a!. (1995) EMBO J., 14: 4628; Williams et a!. (1996) J. Mo!. Biol., 264: 220). Although the H3 region is much more diverse in terms of sequence, length and structure (due to the use of D segments), it also forms a limited number of main-chain conformations for short loop lengths which depend on the length and the presence of particular residues, or types of residue, at key positions in the loop and the antibody framework (Martin et a!. (1996) J. Mol. Biol., 263: 800; Shirai et a!. (1996) FEBS Letters, 399: 1).

The dAbs of the invention may themselves be provided in the form of libraries. In one aspect of the present invention, libraries of dAbs andlor domains are designed in which certain loop lengths and key residues have been chosen to ensure that the main-chain conformation of the members is known. Advantageously, these are real conformations of immunoglobulin superfamily molecules found in nature, to minimise the chances that they are non-functional, as discussed above. Germline V gene segments serve as one suitable basic framework for constructing antibody or T-cell receptor libraries; * * S S uS * S * S S S S S * * S S S a *.. S S S *5 S 0 *S S I S S * S S S S S S I., S 55 5 5 other sequences are also of use. Variations may occur at a low frequency, such that a small number of functional members may possess an altered main-chain conformation, which does not affect its function.

Canonical structure theory is also of use to assess the number of different main-chain conformations encoded by ligands, to predict the main-chain conformation based on ligand sequences and to choose residues for diversification which do not affect the canonical structure. It is known that, in the human VK domain, the Li loop can adopt one of four canonical structures, the L2 loop has a single canonical structure and that 90% of human VK domains adopt one of four or five canonical structures for the L3 loop (Tom linson et at. (1995) supra); thus, in the VK domain alone, different canonical structures can combine to create a range of different main-chain conformations. Given that the V domain encodes a different range of canonical structures for the Li, L2 and L3 loops and that VK and V, domains can pair with any V domain which can encode several canonical structures for the HI and H2 loops, the number of canonical structure combinations observed for these five loops is very large. This implies that the generation of diversity in the main-chain conformation may be essential for the production of a wide range of binding specificities. However, by constructing an antibody library based on a single known main-chain conformation it has been found, contrary to expectation, that diversity in the main-chain conformation is not required to generate sufficient diversity to target substantially all antigens. Even more surprisingly, the single main-chain conformation need not be a consensus structure - a single naturally occurring conformation can be used as the basis for an entire library.

Thus, in a preferred aspect, the dAbs of the invention possess a single known main- chain conformation.

The single main-chain conformation that is chosen is preferably commonplace among molecules of the immunoglobulin superfamily type in question. A conformation is commonplace when a significant number of naturally occurring molecules are observed to adopt it. Accordingly, in a preferred aspect of the invention, the natural occurrence of the different main-chain conformations for each binding loop of an * * * * .4q * I I S * S C * * S S * SIC S S II a S SC C S I * * S S S S * ô S *. I 55 5 5 immunoglobulin domain are considered separately and then a naturally occurring variable domain is chosen which possesses the desired combination of main-chain conformations for the different loops. If none is available, the nearest equivalent may be chosen. It is preferable that the desired combination of main-chain conformations for the different loops is created by selecting germline gene segments which encode the desired main-chain conformations. It is more preferable, that the selected germline gene segments are frequently expressed in nature, and most preferable that they are the most frequently expressed of all natural germline gene segments.

In designing dAbs or libraries thereof the incidence of the different main-chain conformations for each of the six antigen binding loops may be considered separately.

For Hi, H2, Li, L2 and L3, a given conformation that is adopted by between 20% and 100% of the antigen binding loops of naturally occurring molecules is chosen.

Typically, its observed incidence is above 35% (i.e. between 35% and 100%) and, ideally, above 50% or even above 65%. Since the vast majority of H3 loops do not have canonical structures, it is preferable to select a mainchain conformation which is commonplace among those loops which do display canonical structures. For each of the loops, the conformation which is observed most often in the natural repertoire is therefore selected. In human antibodies, the most popular canonical structures (CS) for each loop are as follows: HI - CS 1 (79% of the expressed repertoire), H2 - CS 3 (46%), Li - CS 2 of VK (39%), L2 - CS 1 (100%), L3 - CS 1 of VK (36%) (calculation assumes a i<:?. ratio of 70:30, Hood et al. (1967) Cold Spring Harbor Symp. Quant.

Biol., 48: 133). For H3 loops that have canonical structures, a CDR3 length (Kabat et a!. (1991) Sequences ofproteins of immunological interest, U.S. Department of Health and Human Services) of seven residues with a salt-bridge from residue 94 to residue 101 appears to be the most common. There are at least 16 human antibody sequences in the EMBL data library with the required H3 length and key residues to form this conformation and at least two crystallographic structures in the protein data bank which can be used as a basis for antibody modelling (2cgr and I tet). The most frequently expressed germline gene segments that this combination of canonical structures are the VH segment 3-23 (DP-47), the JH segment JH4b, the VK segment 02/012 (DPK9) and thejK segment 1K1. VHsegments DP45 and DP38 are also suitable. These segments can therefore be used in combination as a basis to construct a library with the desired single main-chain conformation.

Alternatively, instead of choosing the single main-chain conformation based on the natural occurrence of the different main-chain conformations for each of the binding loops in isolation, the natural occurrence of combinations of main-chain conformations is used as the basis for choosing the single main-chain conformation. In the case of antibodies, for example, the natural occurrence of canonical structure combinations for any two, three, four, five or for all six of the antigen binding ioops can be determined.

Here, it is preferable that the chosen conformation is commonplace in naturally occurring antibodies and most preferable that it observed most frequently in the natural repertoire. Thus, in human antibodies, for example, when natural combinations of the five antigen binding loops, ill, H2, LI, L2 and L3, are considered, the most frequent combination of canonical structures is determined and then combined with the most popular conformation for the H3 loop, as a basis for choosing the single main-chain conformation.

ii. Diversification of the canonical sequence Having selected several known main-chain conformations or, preferably a single known main-chain conformation, dAbs according to the invention or libraries for use in the invention can be constructed by varying the binding site of the molecule in order to generate a repertoire with structural andlor functional diversity. This means that variants are generated such that they possess sufficient diversity in their structure andlor in their function so that they are capable of providing a range of activities.

The desired diversity is typically generated by varying the selected molecule at one or more positions. The positions to be changed can be chosen at random or are preferably selected. The variation can then be achieved either by randomisation, during which the resident amino acid is replaced by any amino acid or analogue thereof, natural or synthetic, producing a very large number of variants or by replacing the resident amino acid with one or more of a defined subset of amino acids, producing a more limited number of variants.

I I I I

* S S S I S S

S S SI I S S I I

SI I S S S S I I

I SI I I

ISI S II S S

Various methods have been reported for introducing such diversity. Errorprone PCR (Hawkins et a!. (1992) J. Mo!. Biol., 226: 889), chemical mutagenesis (Deng et al. (1994) .1 Bio!. Chem., 269: 9533) or bacterial mutator strains (Low et a!. (1996) 1 Mo!. Biol., 260: 359) can be used to introduce random mutations into the genes that encode the molecule. Methods for mutating selected positions are also well known in the art and include the use of mismatched oligonucleotides or degenerate oligonucleotides, with or without the use of PCR. For example, several synthetic antibody libraries have been created by targeting mutations to the antigen binding loops. The H3 region of a human tetanus toxoid-binding Fab has been randomised to create a range of new binding specificities (Barbas et a!. (1992) Proc. Nat!. Acad. Sci. USA, 89: 4457). Random or semi-random H3 and L3 regions have been appended to germline V gene segments to produce large libraries with unmutated framework regions (Hoogenboom & Winter (1992) 1 Mo!. Biol., 227: 381; Barbas et a!. (1992) Proc. Nat!. Acad. Sci. USA, 89: 4457; Nissim et al. (1994) EMBO 1, 13: 692; Griffiths et a!. (1994) EMBO 1, 13: 3245; De Kruifet a!. (1995) J. Mo!. Biol., 248: 97). Such diversification has been extended to include some or all of the other antigen binding loops (Crameri et a!. (1996) Nature Med., 2: 100; Riechmann et al. (1995) Bio/Techno!ogy, 13: 475; Morphosys, W097/08320, supra).

Since ioop randomisation has the potential to create approximately more than 1015 structures for H3 alone and a similarly large number of variants for the other five loops, it is not feasible using current transformation technology or even by using cell free systems to produce a library representing all possible combinations. For example, in one of the largest libraries constructed to date, 6 x 1010 different antibodies, which is only a fraction of the potential diversity for a library of this design, were generated (Griffiths et a!. (1994) supra).

In a preferred embodiment, only those residues which are directly involved in creating or modifying the desired function of the molecule are diversified. For many molecules, the function will be to bind a target and therefore diversity should be concentrated in the target binding site, while avoiding changing residues which are crucial to the * * S * *S* * S S * * P S * * * * * * * * r S S. S S SI S * I S

I I S S S S S S

S.. S *S * . overall packing of the molecule or to maintaining the chosen main-chain con formation.

Diversification of the canonical sequence as it apilies to antibody domains In the case of antibody dAbs, the binding site for the target is most often the antigen binding site. These residues are extremely diverse in the human antibody repertoire and are known to make contacts in highresolution antibody/antigen complexes. For example, in L2 it is known that positions 50 and 53 are diverse in naturally occurring antibodies and are observed to make contact with the antigen. In contrast, the conventional approach would have been to diversify all the residues in the corresponding Complementarity Determining Region (CDR1) as defined by Kabat et a!. (1991, supra), some seven residues compared to the two diversified in the library for use according to the invention. This represents a significant improvement in terms of the functional diversity required to create a range of antigen binding specificities.

In nature, antibody diversity is the result of two processes: somatic recombination of germline V, D and J gene segments to create a naive primary repertoire (so called germline and junctional diversity) and somatic hypermutation of the resulting rearranged V genes. Analysis of human antibody sequences has shown that diversity in the primary repertoire is focused at the centre of the antigen binding site whereas somatic hypermutation spreads diversity to regions at the periphery of the antigen binding site that are highly conserved in the primary repertoire (see Tomlinson et a!.

(1996) J. Mol. Biol., 256: 813). This complementarity has probably evolved as an efficient strategy for searching sequence space and, although apparently unique to antibodies, it can easily be applied to other polypeptide repertoires. The residues which are varied are a subset of those that form the binding site for the target. Different (including overlapping) subsets of residues in the target binding site are diversified at different stages during selection, if desired.

In the case of an antibody repertoire, an initial naive' repertoire is created where some, but not all, of the residues in the antigen binding site are diversified. As used herein in this context, the term "naive" refers to antibody molecules that have no pre- * * S * **.

* S * I S * S * S * S * S * 555 5 I I SI * S SI S S S I * S S S S S S SS.. S 55 5 1 determined target. These molecules resemble those which are encoded by the immunoglobulin genes of an individual who has not undergone immune diversification, as is the case with fetal and newborn individuals, whose immune systems have not yet been challenged by a wide variety of antigenic stimuli. This repertoire is then selected against a range of antigens or epitopes. If required, further diversity can then be introduced outside the region diversified in the initial repertoire.

This matured repertoire can be selected for modified function, specificity or affinity.

In the construction of libraries for use in the invention, diversification of chosen positions is typically achieved at the nucleic acid level, by altering the coding sequence which specifies the sequence of the polypeptide such that a number of possible amino acids (all 20 or a subset thereof) can be incorporated at that position.

Using the IUPAC nomenclature, the most versatile codon is NNK, which encodes all amino acids as well as the TAG stop codon. The NNK codon is preferably used in order to introduce the required diversity. Other codons which achieve the same ends are also of use, including the NNN codon, which leads to the production of the additional stop codons TGA and TAA.

A feature of side-chain diversity in the antigen binding site of human antibodies is a pronounced bias which favours certain amino acid residues. If the amino acid composition of the ten most diverse positions in each of the V11, VK and V regions are summed, more than 76% of the side-chain diversity comes from only seven different residues, these being, serine (24%), tyrosine (14%), asparagine (11%), glycine (9%), alanine (7%), aspartate (6%) and threonine (6%). This bias towards hydrophilic residues and small residues which can provide main-chain flexibility probably reflects the evolution of surfaces which are predisposed to binding a wide range of antigens or epitopes and may help to explain the required promiscuity of antibodies in the primary repertoire.

Since it is preferable to mimic this distribution of amino acids, the distribution of amino acids at the positions to be varied preferably mimics that seen in the antigen binding site of antibodies. Such bias in the substitution of amino acids that permits S * *I * S S S I S I S S S S I S S ISS * S S *5 5 S IS S I S S S a * * * * a * S.. S *S S * selection of certain polypeptides (not just antibody polypeptides) against a range of target antigens is easily applied to any polypeptide repertoire. There are various methods for biasing the amino acid distribution at the position to be varied (including the use of tri-nucleotide mutagenesis, see W097/08320), of which the preferred method, due to ease of synthesis, is the use of conventional degenerate codons. By comparing the amino acid profile encoded by all combinations of degenerate codons (with single, double, triple and quadruple degeneracy in equal ratios at each position) with the natural amino acid use it is possible to calculate the most representative codon. The codons (AGT)(AGC)T, (AGT) (AGC)C and (AGT)(AGC)(CT) - that is, DVT, DVC and DVY, respectively using IUPAC nomenclature - are those closest to the desired amino acid profile: they encode 22% serine and 11% tyrosine, asparagine, glycine, alanine, aspartate, threonine and cysteine. Preferably, therefore, libraries are constructed using either the DVT, DVC or DVY codon at each of the diversified positions.

D. Characterisatjon of dAbs accordin2 to the present invention.

The binding of dAbs according to the invention to its specific antigens (or epitopes can be tested by methods which will be familiar to those skilled in the art and include ELISA. In a preferred embodiment binding is tested using monoclonal phage ELISA.

Phage ELISA may be performed according to any suitable procedure: an exemplary protocol is set forth below.

Populations of phage produced at each round of selection can be screened for binding by ELISA to the selected antigen or epitope, to identify "polyclonal" phage antibodies.

Phage from single infected bacterial colonies from these populations can then be screened by ELISA to identify "monoclonal" phage antibodies. It is also desirable to screen soluble antibody fragments for binding to antigen or epitope, and this can also be undertaken by ELISA using reagents, for example, against a C- or N-terminal tag * I I $ *** * * S I I I I * * * I I $ S **l I * $ II I I It * I * I * * I * I * * * I.. I SI * I (see for example Winter et al. (1994) Ann. Rev. Immunology 12, 433-55 and

references cited therein).

The diversity of the selected phage monoclonal antibodies may also be assessed by gel electrophoresis of PCR products (Marks et a!. 1991, supra; Nissim et a!. 1994 supra), probing (Tomlinson et al., 1992) J. Mol. Biol. 227, 776) or by sequencing of the vector DNA.

E. Treatment of H. pylon infection using dAbs according to the invention DAbs selected according to the method of the present invention may be employed in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, and the like. For example DAbs may be used in antibody based assay techniques, such as ELISA techniques, according to methods known to those skilled in the art.

As alluded to above, dAbs according to the invention are of use in diagnostic, prophylactic and therapeutic procedures. Single domaineffector group antibodies (dAbs) selected according to the invention are of use diagnostically in Western analysis and in situ protein detection by standard immunohistochemical procedures; for use in these applications, the antibodies of a selected repertoire may be labelled in accordance with techniques known to the art. In addition, such antibody polypeptides may be used preparatively in affinity chromatography procedures, when complexed to a chromatographic support, such as a resin. All such techniques are well known to one of skill in the art.

Substantially pure dAbs according to the present invention of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more homogeneity is most preferred for pharmaceutical uses, especially when the mammal is a human. Once purified, partially or to homogeneity as desired, the selected dAbs may be used diagnostically and/or therapeutically (including extracorporeally) or in developing and performing assay procedures, immunofluorescent stainings and the * . S * *** * * I S S I I * * * S I S * *I* I * S SI S I *S S S I I * S I * . S a S I.. S ** S * like (Lefkovite and Pemis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).

The dAbs of the present invention will typically find use in preventing, suppressing or treating H. pylon infection.

In the instant application, the term "prevention" involves administration of the protective composition prior to the induction of the disease. "Suppression" refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease. "Treatment" involves administration of the protective composition after disease symptoms become manifest.

Animal model systems which can be used to screen the dAbs in protecting against or treating H. pylon infection are available and will be familiar to those skilled in the art.

Generally, the dAbs will be utilised in purified form together with pharmacologically appropriate carriers. Typically, these carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or buffered media.

Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington s Pharmaceutical Sciences, 16th Edition).

The dAbs of the present invention may be used as separately administered compositions or in conjunction with other agents. These can include various immunotherapeutic drugs. Pharmaceutical compositions can include "cocktails" of * * * * *** * I * * I S I S * S I a I *** S I * *. a S ** S * S S * a a S * * * S a.. I 5 various cytotoxic or other agents in conjunction with the dAbs of the present invention, or even combinations of dAbs according to the present invention having different speci ficities.

Further this may be in combination with existing regimes such as the double or triple therapy combinations are envisaged. This includes the use of combinations of antibiotics and also proton pump therapies.

The proton pump inhibitors eg omeprazole. may be used in neutral form or in the form of an alkaline salt, such as for instance the Mg2+, Ca2+, Na+, K+ or Li+ salts, preferably the Mg2+ salts. Further where applicable, they may be used in racemic form or in the form of a substantially pure enantiomer thereof, or alkaline salts of the single enantiomers.

Suitable proton pump inhibitors are for example disclosed in EP-Al0005l29, EP-Al- 174 726, EP-Al-166 287, GB 2 163 747 and W090/06925, W091/19711, W09l/l 9712, and further especially suitable compounds are described in W095/01977 and W094/27988 and these are incorporated by reference in their entirety.

A wide variety of antibacterial compounds may be used in combination with a suitable proton pump inhibitor. Such antibacterial compounds include for example nitroiridazole antibiotics, tetracyclines, penicillins, cephalosporins, carbopenems, aminoglycosides, macrolide antibiotics, lincosamide antibiotics, 4-quinolones, rifamycins and nitrofurantoin. Examples of such antibacterial compounds are: ampicillin, amoxicillin, benzylpeni cillin, phenoxymethylpeniclllin, bacampicillin, pivampicillln, carbenicillin, cloxacillin, cyclacillin, dicloxacillin, methicillin, oxacillin, piperaci Ilin, ticarcillin, fi ucloxacillin, cefuroxime, cefetamet, cefetrame, cefixime, cefoxitin, ceftazidime, ceftizoxime, latamoxef, cefoperazone, ceftriaxone, cefsulodin, cefotaxime, cephalexin, cefaclor, cefadroxil, cefalothin, cefazolin, cefpodoxime, ceftibuten, aztreonam, tigemonam, erythromycin, dirithromycin, roxithromycin, azithromycin, clarithromycin, clindamycin, paldimycin, lincomycin, vancomycin, * * I * *S* * * S I I I S I I * * a S S *I* S * I a. a I ** 5 * S S * S S I S 5*5 I II S I spectinomycin, tobramycin, paromomycin, metronidazole, tinidazole, ornidazole, amifloxacin, cinoxacin, ciprofloxacin, difloxacin, enoxacin, fleroxacin, norfioxacin, ofloxacin, temafloxacin, doxycycline, minocycline, tetracycline, chlortetracycline, oxytetracycline, methacycline, rolitetracyclin, nitrofurantoin, nalidixic acid, gentamicin, rifampicin, amikacin, netilmicin, imipenem, cilastatin, chioramphenicol, furazolidone, ni furoxazide, sul fadiazin, sul fametoxazol, bismuth subsalicylate, colloidal bismuth subcitrate, gramicidin, mecillinam, cloxiquine, chiorhexidine, dichlorobenzylalcohol, methyl-2-pentylphenol.

The active antibacterial agents could be in standard forms or used as salts, hydrates, esters etc. A combination of two or more of the above listed drugs may be used, for example to minimize the risk for developing resistance. Preferable antibacterial compounds are clarithromycin, erythromycin, roxithromycin, azithromycin, amoxicillin, metronidazole, tinidazole and tetracycline. Clarithromycin and metronidazole alone or in combination are especially suitable.

The route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, including without limitation immunotherapy, the dAbs and compositions of the invention can be administered to any patient in accordance with standard techniques.

The administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician.

In a preferred embodiment of the invention, therapeutic compositions described herein are administered by either topically or systemically.

In a preferred embodiment of the invention, the administration of a pharmaceutical composition comprising dAbs for the treatment of a patient suffering from Candidiasis * * * * *** * S S I S S I * S * * * a S III I S S S. I S *5 * I * S

S S S S S S I S

555 5 5* 5 5 includes a continuous infusion over a period of time, or a single dose or bolus administration. Further, it is envisaged that following a single dose or bolus administration a second bolus dose may be administered or a continuous infusion may be administered.

The dAbs of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of antibody activity loss (e.g. with conventional immunoglobulins, 1gM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted upward to compensate.

The compositions containing the dAbs or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a "therapeutically-effective dose". Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 0.005 to 5.0 mg of selected dAb per kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. For prophylactic applications, compositions containing the dAbs or cocktails thereof may also be administered in similar or slightly lower dosages.

A composition containing a dAb or cocktail thereof according to the present invention may be utilised in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal. In addition, the dAbs described herein may be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells. Blood from a mammal may be combined extracorporeally with the selected antibodies, cell-surface receptors or binding proteins * * * . a..

* . a 0 I * * S * * S * a S all a a I *1 a a II * a a * a * * a.. I ** . . thereof whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques.

The invention is further described, for the purposes of illustration only, in the

following examples.

EXAMPLES.

The antigen, BabA, obtained from Dr Thomas Borén (Umeà University, Sweden) was purified from H. pylon and displayed an apparent molecular mass of 75 kD by SDS- PAGE.

Purification of native BabA protein from strain CCUG17875 and strain J99.

BabA was purified from two reference strains.

Reference strain CCUG17875 is described in the paper of Ilver et. a!., 1998 (lIver D, Arnqvist A, Ogren J, Frick IM, Kersulyte D, Incecik ET, Berg DE, Covacci A, Engstrand L, Boren T. 1998. Science. Jan 16; 279 (5349): 373-7.) and purified as described in the reference.

Reference strain J99 is described in the paper of Mahdavi et. a!., 2002 (Mahdavi J, Sonden B, Hurtig M, Olfat FO, Forsberg L, Roche N, Angstrom J, Larsson T, Teneberg S, Karisson KA, Altraja 5, Wadstrom T, Kersulyte D, Berg DE, Dubois A, Petersson C, Magnusson KE, Norberg T, Lindh F, Lundskog BB, Arnqvist A, Hammarstrom L, Boren T (2002) Science 297 (5581) : 573-578 and purified as described.

Generation of phage display dAb libraries.

The dAb phage libraries were based on a single human antibody framework for VH * a a * *e.

* * S * * S * S S ft * I S I Ste S * S IS S S 55 I I I S * * S S S S I S aSS S SI S S (V3-23 [locus] DP47 [V Base entry] and JH4b) and for VL (012/02 [locus] DPK9 [V Base entry] and JK1) with diversity incorporated using DNA nucleotide diversification to generate amino acid side chain diversity at positions that are known to make protein contacts with antigen in known molecular structures and are naturally diversified in the mature human repertoire. The dAb variable domains were cloned into the phage vector pDOM4 which is based on the fd-phage genome and therefore contains all the necessary phage genes to produce infective phage particles used during the selection process.

dAb selection by phage display.

Selections were performed against passively coated BabA on Maxisorp ImmunoTM Tubes (Nunc) Four mi llilitres of antigens at 15 tg/ml in BupH carbonate-bicarbonate buffer (0.2M Carbonate-Bicarbonate Buffer, pH 9.4) (Perbio, UK) was used to coat immunotubes overnight at 4 C. The immunotubes were subsequently blocked with PBS containing 2% skimmed milk powder (PBSM) for lh at room temperature and washed 3 times with PBS. Approximately lx 10" phage from each dAb library, in total volume of lml 2% PBSM, was incubated in the antigen coated tube for lh at room temperature (RT) by rotating at 50rpm. After ten washes with PBS supplemented with 0.1% Tween-20 (Sigma-Aldrich) (PBST) and ten washes with PBS for round I selection (twenty washes for round 2 and 3 selections respectively), bound phage were eluted with 500p1 of PBS containing 1mg/mi trypsin (Sigma-Aldrich) supplemented with 0.1mM CaC12 (Sigma-Aldrich) with rotation at 50rpm at RT for 10mm. The trypsin solution containing the eluted phage was recovered and 250ji1 used to infected 1.75m1 of log phase E. coil TGI cells at 37 C for 30mm. Library plating and serial log dilutions (for phage titre) and were done on 2xTYE-agar plates (15g Bacto-Agar, 8g NaCI, I Og tryptone, 5g Yeast Extract in 1 liter water) supplemented with 15 j.xg/ml tetracycline (Sigma-Aldrich). For subsequent selection rounds, cells were recovered from the growth plates by scraping into 2ml 2xTY (16g tryptone, lOg yeast extract and 5g NaCl in I liter water) media supplemented with 20% glycerol (Sigma- Aldrich) of which 50 jil was used to inoculate 50m1 of 2xTYE-Tet at 37 C for phage - t S * * a * I S S S S S S S I *$* S - I SI S IS S P 5

I S I S S I S

S SI * I amplification.

Following the desired number of selection rounds (typically 2 to 3) the enriched dAb genes were recovered by digesting 20tg of purified (QIAgen Midiprep) dAb phage DNA from the desired selection output with Sail and NotT (NEB) restriction enzymes in NEB3 buffer (1.tg/ml BSA, 100mM NaC1, 50mM Tris-HC1, 10mM MgC12, 1mM dithiothreitol, pH 7.9@25 C). The excised dAb genes were electrophoresed on a I % agarose (Sigma-Aldrich) gel in TBE (Sigma-Aldrich) (44.5mM Tris-borate and 1mM EDTA, pH8.3) at 150V for lh. Following electorophoresis the dAb genes were excised and purified using the QIAgen gel purification kit (QiAgen). One hundred nano-grams of the purified digested dAb genes were then ligated with 400ng of SalI/NotI digested soluble expression vector using T4 DNA ligase (New England Biolabs) in ligation buffer (50mM Tris-HC1, 10mM MgCI2, 1mM ATP, 10 mM dithiothreitol, 25pg/mI BSA, pH7.5@ 25 c) at RT for 1 hour. Three microlitres of ligation product were used to transform 5Ojil of electrocompetent E. co/i HB2151 (200[, 25jiFd, 2.5kV, 0.2cm gap cuvettes) and the desired amount of transformed cells plated onto agar plates supplemented with 5% glucose and 501g/ml carbenicillin (Sigma-Aldrich); followed by incubation at 37 C for overnight.

Expression and purification of dAb protein Individual fresh colonies corresponding to each soluble dAb ELISA positive clone was inoculated into 5m1 of 2xTY supplemented with 50tg/m1 carbenicillin and 5% glucose and incubated overnight at 37 C with shaking at 250rpm. Five millilitres of this culture was used to inoculate 500m1 of fresh pre-warmed media (2xTY supplemented with 50tg!ml carbenicillin and 0.1% glucose) and incubated at 37 C with shaking at 250rpm until the 0D600 reached 0.8, when the culture was induced through the addition of IPTG to a final concentration of 1mM and incubated overnight at 30 C with shaking at 250rpm. The cells were then pelleted by centrifugation at 3450xg for 10 minutes and the resulting clarified supernatant filtered through a 0. 45 jtm sterile filter. Fifty millilitres of this supernatant was then mixed by rotation at 50rpm with * p * a * I I I & I * I I * I a *14 e * I *4 * I * I S I & S S *

I I I SI P

2O0il of Protein-L-sepharose (Sigma-Aldrich) for VL dAbs or Protein-A streamline (Amersham Pharmacia) for VH dAbs at RT for 1 hour. The resins were recovered by centrifugation at 220xg for 1 mm, followed by washing twice with imi of 0.5M NaC1 in PBS and twice with imi of PBS in 96 well paper- filter plate (Whatman) then the dAb eluted with 2lOpJ of 0.IM Glycine (pH2.0) and neutralized by adding 40p1 of IM Tris-HC1 (pH8.0). The purity of the dAbs was determined by SDS-PAGE performed under reducing conditions using 12% NuPAGE Bis-Tris gels run in MES buffer (Novex gel system, Invitrogen). Gels were stained with Coomassie blue (Simply Blue Safestain, Invitrogen). The concentration of the dAb samples were determined by absorbance at 280nm. For larger culture volumes the method was scaled appropriately.

Specific dAb binders screening by ELISA.

Fresh E. coli HB2 151 clones were analysed for BabA binding dAbs by ELISA.

Individual clones were grown in 200pd of 2xTY containing 50j.g!ml carbenicillin, 0.1% glucose in 96-well microtitre plates (Coming) at 37 C with shaking at 250rpm for 6 hours and induced with IPTG at final concentration of 1mM, incubation continued at 30 C/2SOrpm overnight to express soluble dAb. The plates were centrifuged at 870xg for 1 0mm at RT and the supernatant analysed by ELISA as follows (unless stated all volumes were 50.tl/wel1). Ninety-six well ELISA plates (Nunc Maxisorp) were coated overnight at 4 C with 1 tg/ml BabA in BupH carbonatebicarbonate buffer then washed three times by immersion in PBST. Plates were then blocked with 200tl/well PBS supplemented with 2% (v/v) Tween-20 for lh at room temperature, then washed 3 times with PBST. Twenty-five microlitres of supematant from the overnight culture was diluted 1:1 in 2% protein L in PBST and added into each well and incubated for I h at RT. Following three washes with PBST, bound BabA specific dAbs were detected by using 1:2000 dilution of mouse 9E10 antibody (anti-myc, Sigma-Aldrich, Cat No M5546) in PBST for lh at RT, washed 3 times with PBST then a 1:2000 dilution of anti-mouse Fc-specific Horse Radish Peroxidase conjugate (Sigma-Aldrich, Cat No A0168) in PBST add and incubated at RT for 1 hour. Following incubation with the appropriate detection ligand, BabA assays were a a * *i* * a i * * * a,ia I I

I I I I

a a a * lab *** * * I a washed 3 times with PBST followed by 3 washes with PBS. All plates were then developed by adding 50l/well of TMB substrate (SureBlue, KPL, MD. USA). The reaction was allowed to develop for several minutes until a sufficient signal had appeared relative to the control wells, then the reaction stopped by the addition of l00zl/well 1M HCI. Absorbance was read at 450nm on a Victor2 microtitre plate reader (Perkin Elmer).

Radioimmunoassay (RIA) A radioimmunoassay (RIA) was performed as described by liver et. a!., 1998 (lIver D, Arnqvist A, Ogren J, Frick IM, Kersulyte D, Incecik ET, Berg DE, Covacci A, Engstrand L, Boren T. (1998) Science 279(5349): 373-377.). Briefly, 251-labeled Leb conjugate was incubated with the bacteria, strain CCUG17875, and then this mixture was competed with the desired concentration of dAb. The amount of radioactivity remaining in the pellet was determined.

Surface Plasmon Resonance (SPR) Analysis of dAbs Binding to Immobilised BabA The ability of the dAbs to bind to BabA was assessed using Surface Plasmon Resonance (SPR) technology. SPR is a standard well known technology for measuring the interaction between molecules. The method is well known to those skilled in the art, who would be able to perform the technique without undue experimentation.

BabA was immobilised onto a BlAcore chip using standard conditions and the individual dAbs in solution were passed over the chip. The interactions were measured automatically by the BlAcore and the ability of the dAbs to bind BabA recorded.

BlAcore Analysis of dAbs Competing with BabA for binding to immobilised Le" The ability of dAbs to compete with BabA for the binding site on Leb was analysed using surface plasmon resonance (SPR) as above.

* * * * S..

* S S S * S S S * * S S S S *SS S S S S. S S 55 5 5 5 * * S S S S * S * S.. * *5 S S Briefly, Leb was immobilised on a BlAcore chip using standard conditions. The dAbs to be analysed were mixed with BabA just prior to being added to the BlAcore test.

Control solutions of dAbs only and BabA only were also passed over the Leb chip.

The ability of dAbs to compete with BabA was assessed by comparison of the results without and with BabA in the dAb solution.

Tissue Adhesion Analysis The effect of dAbs on the ability of H. pylon to bind to tissue was studied using the methods as described by O'Mahony, R. et al., 2005 (Journal of Microbiological Methods, 61, 1, 105-126).

RESULTS

43 unique dAbs were identified by antigen binding ELISA.

These dAbs were shown to be functionally active against BabA in both a cell surface BabA test and a BlAcore analysis. Further, ADR1-5, ADR1-9, ADR1-19 and ADR1- 28 were shown to inhibit Leb binding to H. pylon cells (CCUG17875 strain) by radioimmunoassay.

ADR1-25 ADR1-26 and ADR1-.28 were identified to inhibit BabA-Leb binding by BlAcore competition.

ADRI -25 was confirmed to inhibit H. pylon binding to human stomach tissue in tissue adhesion study.

All publications mentioned in the present specification and references cited in said publications are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.

* S S S *SS * S S S S I S 5 * * S S * * *S* S S S S. S S 5. S S S S S S S S S S * S S.. S 55 0 5 Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the field or related fields are intended to be within the scope of the following claims.

* * * S **I * * * S S S * S * * S S I I 155 I S * S. I S IS S S S S * I I S I S * S SI. I IS I S Sequence Listing ADRI-1 - SEQ ID No.!

DIQMTQSPSSLSASVGDRVTITCRASQSIGSSLRWYQQKPGKAYKLLIYRASQL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRKRRPKTFGQGTKVEIK

ADR1-2- SEQ ID No.2

DTQMTQSPSSLSASVGDRVTITCRASQSIKKHLRWYQQKPGKAPKLLIYGASRL

QSGVPSRFSGSGSGTDLTLTISSLQPEDFATYYCQQRFVEPKTFGQGTKVEIK

ADR1-3- SEQ ID No.3

DIQMTQSPSSLSASVGDRVTITCRASQSIKRRLKWYQQKPGKAPKLLIYYASKL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTRIQPYTFGQGTKVEIK

ADRI-4- SEQ ID No.4 DIQMTQSPSSLSASVGDRVTITCRASQSINKHLHWYQQKPG1C&PKLLJYRASKL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNFNRPHTFGQGTKVEIK

ADR1-5- SEQ ID No.5 DIQMTQSPSSL5A5VGDRVTJTCRASQ5IIKKHLRWYQQKPGKAPKLLIYGA5RL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRFVEPKTFGQGTKVEIK

ADR1-6- SEQ ID No.6

DIQMTQSPSSLSASVGDRVTITCRASQSIIKKLRWYQQKPGKAPKLLIYHASKL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRTYPPTFGQGTKVEIK

ADR1-7- SEQ ID No.7

DIQMTQSPSSLSASVGDRVTITCRASQSISRSLHWYQQKPGKAPKLLIYRASRL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNRLRPHTFGQGTKvEIK ADR1-8- SEQ ID No.8

DIQMTQSPSSLSASVGDRVTITCRASQSIKKHLRWYQQKPGKAPKWYGASRL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRTYPPTFGQGTKVEJK

ADR1-9- SEQ ID No.9

DIQMTQSPSSLSASVGDRVTITCRASQSISDFLAWYQQKPGKAPKLLIYLASRL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYFDYPTTFGQGTKVEIK

ADR1-lO- SEQ ID No.10 DIQMTQSPSSLSASVGDRVTITCRA5Q5JGKRLRWYQQKPGKAJKWyyASRL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ5RNRPQTFGQGTKVEJK ADR1-l 1-SEQ ID No. 11 DIQMTQSPSSLSASVGDRVTITCRA5Q5fNKKLRwyQQG}yKLLJyHA5KL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYISPPRTFGQGTKVEIK

* S 0 Sos * * 0 0 5 0 S 0 S * S S * S *** * * * *S S S ** S S * S * S S S S S S 0 SOS S S. S S ADR1-12- SEQ ID No.12 D1QMTQSPSSLSASVGDRVTITCRASQSITRJLLWYQQKPGJCkPKLLIYICkFRL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYVRPFTFGQGTKVEIK

ADRI-13-SEQIDNo.13 DIQMTQSPSSLSASVGDRVT1TCRASQSIGSKLSWYQQKPGKAPKLLIYISRL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKLFRPKTFGQGTKVEIK

ADR1-14- SEQ ID No.14

DIQMTQSPSSLSASVGDRVTITCRASQSISRSLHWYQQKPGKAPKLLIYRASRL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNRLRPHTFGQGTKVEIK

ADRI-15- SEQ ID No.15 DIQMTQSPSSLSASVGDRVTJTCRA5Q5ILLRwyQQG}}jjjy5KL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSVRPSTFGQGTKVEIK

ADR1-16- SEQ ID No.16

DIQMTQSPSSLSASVGDRVTITCRSQSIIJSLSWYQQKPGKAPKLLIYKASRL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRRMWPYTFGQGTKVEIK

ADR1-17- SEQ ID No.17 DIQMTQSPSSLSASVGDRVTJTCRASQ5INLHwyQQGKpKLLIyGA5KL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNRLRPHTFGQGTKVEIK

ADR1-18- SEQ ID No.18 D1QMTQSPSSLSASVGDRVTITCRASQSIRNRLRWYQQKPGKAPKLLIYISVL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNRVRPNTFGQGTKVEIK

ADRI-19- SEQ ID No.19

DIQMTQSPSSLSASVGDRVTITCRASQSIIQSVAWYQQKPGKAPKLLIYNASILQ

SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYVTYPPTFGQGTKVEIK

ADR1-20-- SEQ ID No.20 DIQMTQSPSSLSASVGDRVTITCRASQSWKJ. LNWYQQKPGKAPKLLIYSASKL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYIAYPPTFGQGTKVEIK

ADR1-21- SEQ ID No.2 1

DIQMTQSPSSLSASVGDRVTITCRASQSIGSSLRWYQQKPGKAYKLLIYRASQL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRKRPYKTFGQGTKVEIK

ADR1 -22- SEQ ID No.22 DIQMTQSPSSLSASVGDRVTITCRSQSILRKLRWYQQGJpKLLIYySKL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLDYPPTFGQGTKVEIK

ADR1-23--- SEQ ID No.23 DIQMTQSPSSLSASVGDRVTITCRASQSIIQSVAWYQQICJGJ&YKLLIYNASILQ

SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRTYPPTFGQGTKVEIK

* S S S *55 * S S S * I I S * * 5 * S S *S5 S I I S. I I S* S 5 5 I S I S 5 5 a S S *5* * S. S S ADR1-24- SEQ ID No.24 QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNAVPpHTFGQGTKVEIK ADR1-25-- SEQ ID No. 25

DIQMTQSPSSLSASVGDRVTITCRASQSIGVKLKWYQQGPKLLIYSRL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNTJJyFTFGQGTKVEIK ADRI -26- SEQ ID No. 26 DJQMTQSPSSLSASVGDRVTITCSQSITRKLCWYQQJGIC&YKLLIYJSRL

QSGVPSRLSGSGSGTDFTLTISSLQPEDFATYYCQQYSRSPSTFGQGTKVEIK

ADRI-27- SEQ ID No.27 DJQMTQSPSSLSASVGDRVTITCRSQSISSSLAWYQQJG1pKLLIYKA5YL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYPPTFGQGTKVEIK

ADR1-28- SEQ ID No.28 DIQMTQSPSSLSASVGDRVTITCRASQSIIQSVAWYQQKPGICyKLLIYNASILQ

SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLDYPPTFGQGTKVEIK

ADR1-29- SEQ ID No.29 DIQMTQSPSSLSASVGDRVTITCSQS(LAWYQQKPGJpKLLIYISIL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYTQYPPTFGQGTKVEIK

ADR1-30- SEQ ID No.30 DIQMTQSPSSLSASVGDRVTITCRASQSILNKLKWYQQKpGICAYKLLIYI5KL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRNP.PHTFGQGTKVEIK ADR1-31- SEQ ID No. 3 1

DIQMTQSPSSLSASVGDRVTITCRASQSISRSLHWYQQKPGKMKLLIYRASRL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNRLRPHTFGQGTKVEIK

ADR1-32- SEQ ID No.32

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVRRWPFTFGQGTKVEIK

ADR1-33- SEQ ID No.33 DIQMTQSPSSLSASVGDRVTITCRASQS1FWYQQJGKJKLLIYJSHL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPJCJYYTFGQGTKVEIK

ADR1-34- SEQ ID No.34 DIQMTQSPSSLSASVGDRVTITCRSQSWLLNWYQQKJGIpKLLIYRA5KL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGKLJ<pYTFGQGTKVE ADR1-35- SEQ ID No.35 * S S S **S * S S * a S S S S * S a * S ate S S a SI I * *a S S S * * * a * S S I S a.. a a. . a

DIQMTQSPSSLSASVGDRVTITCRASQSIRRSLGWYQQKPGKAPKLLIYRASRL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRRYKPWTFGQGTKVEIK

ADRI-36- SEQ ID No.36 DIQMTQSPSSLSASVGDRVTITCRSQSISILNWYQQJ(pGIcjwKLLIYpSRL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTNLKPNTFGQGTKVEIK

ADRI -37- SEQ ID No.37

DIQMTQSPSSLSASVGDRVTITCRASQSIVRQLKWYQQKPGKJAJKLLIYKASKL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKFKLPFTFGQGTKVEIK

ADRI-38- SEQ ID No.38 DIQMTQSPSSLSASVGDRVTJTCRA5Q5IKRKL5wyQQgpGKyKLLIysAsRL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRIRJ-IPKTFGQGTKVEIK

ADR1-39- SEQ ID No.39 DIQMTQSPSSLSASVGDRVTITCRASQSISK] (LDWYQQKYGKAPKLLIYSASHL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRRRKPTTFGQGTKVEIK

ADR1-40- SEQ ID No.40 DIQMTQSPSSLSASVGDRVTJTC5QSI5R5LHwyQQj<juKJpKLLIyJsRL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNRLRPHTFGQGTKVEIK

ADR1-41- SEQ ID No.41

DIQMTQSPSSLSASVGDRVTITCRASQSIGKSLRWYQQKPGKAPKLLIYHASKL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRKSPPKTFGQGTKVEIK

ADR1-42-- SEQ ID No.42 DIQMTQSPSSLSASVGDRVTITCRASQSIGKRLRWYQQKPGIC&PKLLWYASRL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRTYPPTFGQGTKVEIK

ADR1-43- SEQ ID No.43

DIQMTQSPSSLSASVGDRVTITCRASQSISRSLHWYQQKPGKAPKLLIYRASRL

QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYAGRPTTFGQGTKVEIK

* * * 4 *II * * I * 4 S I * * S I 4 * * *I. S S * ** 4 S ** * S S I * * S S * * a S I.. S ** S S

Claims (17)

CLAIMS. 1. A single domain antibody (dAb) which binds to an adhesin molecule from H. pylon. 2. A single domain antibody (dAb) according to claim 1 which binds to BabA. 3. A single domain antibody (dAb) according to claim 2 which comprises preferably consists of any sequence in the group consisting of the following: ADRI-1 to ADR1-43 and designated SEQ ID No 1 to SEQ ID No 43 respectively. 4. A single domain antibody (dAb) according to claim 2 or claim 3, which comprises, preferably consists of the ADRI-5 sequence designated SEQ ID No.5, ADR1-9 sequence designated SEQ ID No.9, ADR1-19 sequence designated SEQ ID No.19, ADR1-25 sequence designated SEQ ID No. 25, ADRI-26 designated SEQ ID No 26 or ADRI-28 designated SEQ ID No 28. 5. A single domain antibody (dAb) according to any preceding claim wherein the dAb binds to an adhesin molecule as herein defined with a K4,, ff rate constant of between 5 x101 and 1x107 s1 6. A single domain antibody (dAb) according to any of claim 1 which binds to an adhesin molecule with a dissociation constant (Kd) of at least 100 j.tM to 1 pM. 7. A single domain antibody according to claim 4, 5 or claim 6 which binds to BabA. 8. A single domain antibody (dAb) which binds to an adhesin molecule from H. pylon and which exhibits at least 80% amino acid sequence identity to the sequence designated ADR1-5 sequence designated SEQ ID No.5, ADR1-9 sequence designated SEQ ID No.9, ADR1-19 sequence designated SEQ ID * S * . *** * * S S a * * * * S * * S *** * S * S. S * 55 S 5 5 I * S S S S * S S 0*S * 55 5 S No.19, ADR1-25 sequence designated SEQ ID No. 25, ADR1-26 designated SEQ ID No 26 or ADRI -28 designated SEQ ID No 28. 9. A single domain antibody (dAb) which inhibits the functional activity of an adhesin molecule from H. pylon. 10. A single domain antibody (dAb) according to claim 9 which inhibits the functional activity of BabA. 11. A single domain antibody (dAb) according to claim 10 which comprises, preferably consists of any sequence in the group consisting of the following: ADR1-1 to ADRI-43 and designated SEQ ID No 1 to SEQ ID No 43 respectively. 12. A single domain antibody (dAb) according to any preceding claim, which comprises, preferably consists of the sequence designated ADR1-5 sequence designated SEQ ID No.5, ADRI-9 sequence designated SEQ ID No.9, ADR1- 19 sequence designated SEQ ID No.19, ADR1-25 sequence designated SEQ ID No. 25, ADR1-26 designated SEQ ID No 26 or ADR1-28 designated SEQ ID No 28. 13. A pharmaceutical composition comprising any one or more dAbs according any preceding claim and a pharmaceutically acceptable carrier, diluent and/or exipient. 14. A pharmaceutical composition according to claim 13 comprising one or more BabA binding dAbs. 15. A pharmaceutical composition according to claim 14 comprising one or more dAbs which inhibits the functional activity of BabA. 16. An antibody fragment which binds to an adhesin molecule from H. pylon. * * * * *** * * I I * * S I * S I S S *SS I S * S. I S S. I I I I S S I S * S I S S.. * ** I * 17. An antibody fragment according to claim 16 wherein the antibody fragment binds to BabA. 18. An antibody fragment according to claim 17 wherein the antibody fragment binds to a BabA as herein defined with a K0ff rate constant of between 5 xlO' and 1x107 s1 19. An antibody fragment according to claim 17 which binds to an adhesin molecule with a dissociation constant (Kd) of at least 1O0tM to 1 pM. 20. An antibody fragment according to any of claims 16, 17, 18 or 19 wherein the antibody fragment is monovalent. 21. A multimeric composition wherein the multimeric composition comprises two or more single domain antibodies according to claims 1 to 12 or two or more antibody fragments according to claims 16 to 20. * * * . I.. * * S * * S S S S * S S S S *S. S S S 5 5 5* * * S S * S * S S S S S *** S *S S S Amendments to the claims have been filed as follows L4, CLAIMS.

1. A single domain antibody (dAb) which binds to an adhesin molecule from H. pylon.

2. A single domain antibody (dAb) according to claim I which binds to BabA.

3. A single domain antibody (dAb) according to claim 2 which comprises preferably consists of any sequence in the group consisting of the following: ADR I -1 to ADRI-43 and designated SEQ ID No ito SEQ ID No 43 respectively.

4. A single domain antibody (dAb) according to claim 2 or claim 3, which comprises, preferably consists of the ADRI -5 sequence designated SEQ ID No.5, ADRI -9 sequence designated SEQ ID No.9, ADRI -19 sequence designated SEQ ID No.19, ADR 1-25 sequence designated SEQ ID No. 25, ADR 1-26 designated SEQ ID No 26 or ADR1-28 designated SEQ ID No 28.

5. A single domain antibody (dAb) according to any preceding claim wherein the dAb binds to an acihesin molecule as herein defined with a K rate constant of between 5 x10' and 1xl07 s'

6. A single domain antibody (dAb) according to any of claim 1 which binds to an adhesin molecule with a dissociation constant (Kd) of at least I OOjiM to 1 pM.

7. A single domain antibody according to claim 4, 5 or claim 6 which binds to BabA.

8. A single domain antibody (dAb) which binds to an adhesin molecule from H. pylon and which exhibits at least 80% amino acid sequence identity to the sequence designated ADRI-5 sequence designated SEQ ID No.5, ADRI-9 sequence designated SEQ ID No.9, ADRI -19 sequence designated SEQ ID No. 19, ADR1-25 sequence designated SEQ ID No. 25, ADR1-26 designated SEQ ID No 26 or ADR1-28 designated SEQ ID No 28.

9. A single domain antibody (dAb) according to any preceding claim, which comprises, preferably consists of the sequence designated ADR1-5 sequence designated SEQ ID No.5, ADR1-9 sequence designated SEQ ID No.9, ADRI-19 sequence designated SEQ ID No.19, ADR1-25 sequence designated SEQ ID No. 25, ADRI-26 designated SEQ ID No 26 orADRI-28 designated SEQ ID No 28.

10. A pharmaceutical composition comprising any one or more dAbs according any preceding claim and a pharmaceutically acceptable carrier, diluent andlor exipient.

11. A pharmaceutical composition according to claim 10 comprising one or more BabA binding dAbs.

12. An antibody fragment which binds to an adhesin molecule from H. pylon.

13. An antibody fragment according to claim 12 wherein the antibody fragment binds to BabA.

14. An antibody fragment according to claim 13 wherein the antibody fragment binds to a BabA as herein defined with a K{)ff rate constant of between 5 x10' and 1x10 7 s-i.

15. An antibody fragment according to claim 13 which binds to an adhesin molecuI with a dissociation constant (Kd) of at least 1 00tM to 1 pM.

16. An antibody fragment according to any of claims 12, 13, 14 or 15 wherein the antibody fragment is monovalent.

17. A multimeric composition wherein the multimeric composition comprises two or more single domain antibodies according to claims I to 9 or two or more antibody fragments according to claims 12 to 16.