# Patent

## Title
Annexin 1 antibody

## Abstract

The present invention provides a specific binding molecule raised against the human Anx-A1 protein having the amino acid sequence shown in FIG. 2A. The present invention also relates to the sue of such a specific binding molecule in the treatment of T cell-mediated disease.

## Background
FIELD OF THE INVENTION
The present invention relates to specific binding molecules, particularly antibodies and fragments thereof, which bind to annexin-A1, and hybridoma cell lines which produce such specific binding molecules. Such specific binding molecules are useful in the treatment of T cell-mediated diseases.
BACKGROUND TO THE INVENTION
Glucocorticoids (GCs) are often used for the therapy of a variety of chronic autoimmune diseases because of their ability to simultaneously block both the innate and adaptive immune response. Studies over the last 10 years or so by the present inventors and other research groups have shown that some of the inflammatory effects of GCs on the innate immune response are mediated by a protein called Annexin-1 (Anx-A1). This protein has been proven to exert a homeostatic control over a number of cell types including neutrophils, macrophages and endothelial cells. However, one aspect that has always been neglected is the role of Anx-A1 in the adaptive immune response. This is surprising considering that Anx-A1 has been proposed as one of the second messengers of the pharmacological effects of GCs.
The present inventors have previously shown that Anx-A1 plays a homeostatic role in T cells by modulating the strength of T cell receptor (TCR) signaling (D'Acquisto et al., Blood 109: 1095-1102, 2007).
Furthermore, the inventors have shown that high levels of Anx-A1 lower the threshold of T cell activation and favour the differentiation into Th1 cells, whereas Anx-A1 deficient mice show impaired T cell activation and increased differentiation into Th2 cells (D'Acquisto et al., Eur. J. Immunol. 37: 3131-3142, 2007).
WO 2005/027965 describes the discovery of a mechanism by which apoptotic neutrophils deliver anti-inflammatory signals to dendritic cells and identifies an antibody that interferes with this process.
SUMMARY OF THE INVENTION
The present inventors have identified a monoclonal antibody that has excellent properties in terms of specific inhibition of T cell activation without any adverse cytotoxic effects. The antibody is useful in the treatment of T cell mediated disease, for example rheumatoid arthritis or multiple sclerosis.
Accordingly, the present invention provides a specific binding molecule raised against the human Anx-A1 protein having the amino acid sequence shown in FIG. 2A.
DEFINITIONS
As used herein, a “specific binding molecule” is a member of a pair of molecules which have binding specificity for one another. The members of a specific binding pair may be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, which may be a protrusion or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organisation of the other member of the pair of molecules. Thus, the members of the pair have the property of binding specifically to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The present invention is generally concerned with antigen-antibody type reactions. The specific binding molecule of the present invention binds with greater affinity to Anx-A1 than to other molecules, i.e. it binds specifically to Anx-A1. Specific binding molecules which bind to Anx-A1 include anti-Anx-A1 antibodies and aptamers. The specific binding molecule of the present invention is typically an antibody. The anti-Anx-A1 antibodies of the present invention function by blocking the activation of T cells and thus, when administered, can be used in the treatment of T cell-mediated diseases, which are typically caused by aberrant T cell activation.
The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced. Antibodies are polypeptides that typically contain two identical heavy chains and two identical light chains, which are smaller than the heavy chains. In mammals there are two types of light chain, which are called lambda (λ) and kappa (κ). Each of the heavy chains and each of the light chains are composed of a variable region and a constant region. The heavy chain variable region is referred to as the VH region and the light chain variable region is referred to as the VL region. For kappa light chains, the VL region can also be referred to as the VK region. Each of the variable regions of the heavy and light chains comprise three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3. These are named VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3 respectively. Examples of antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, F(ab′)2, Fv, scFv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to herein as “mAb”.
DETAILED DESCRIPTION OF THE INVENTION
Annexins are a group of calcium- and phospholipid-binding cellular proteins and are also known as lipocortins. The annexin family has 13 members, including Annexin A1, Annexin A2 and Annexin A5. Annexin-A1 is also known as Annexin-1 and is referred to herein as “Anx-A1”. Annexin-1 (Anx-A1) is a 37-kDa protein and was originally described as a mediator of the actions of glucocorticoids. Over the last few years evidence has shown than Anx-A1 plays a homeostatic role in the adaptive immune system, in particular T cells, by modulating the strength of T cell receptor (TCR) signalling. Anx-A1 acts as an endogenous down-regulator of inflammation in cells of the innate immune system in vivo. FIG. 1A is a ribbon diagram showing the three-dimensional structure of Anx-A1.
There are eight human nucleotide sequences which encode Anx-A1. Of these, only four are translated and thus there are four isoforms of Anx-A1, designated ANXA1-002, ANXA1-003, ANXA1-004 and ANXA1-006. These sequences are available from the Ensembl website (www.ensembl.org) and are designated OTTHUMT00000052664 (ANXA1-002), OTTHUMT00000052665 (ANXA1-003), OTTHUMT00000052666 (ANXA1-004) and OTTHUMT00000052668 (ANXA1-006). The amino acid and nucleotide sequences of one isoform of human Annexin-1 (Anx-A1), ANXA1-003, are shown in FIG. 2A. The amino acid sequences of isoforms ANXA1-002, ANXA1-004 and ANXA1-006 are shown in FIGS. 2B, 2C and 2D respectively. As can be seen from FIG. 2, isoforms ANXA1-002, ANXA1-004 and ANXA 1-006 are either short splice variants of ANXA 1-003 or variants of ANXA1-003 with a small number of amino acid changes.
A number of studies have shown that an N-terminal peptide of Anx-A1 named Ac.2-26 acts as a bioactive surrogate of the whole protein (see e.g. Lim et al., Proc Natl Acad Sci USA 95, 14535-9, 1998).
FIG. 1B is a schematic representation of the annexin repeats and the location of this bioactive sequence. Peptide Ac.2-26 is an acetylated peptide having the sequence of amino acid residues 2-26 of the full-length amino acid sequence of Anx-A1 shown in FIG. 2. The sequence of peptide Ac.2-26 is shown in FIG. 1C and is as follows:









(SEQ ID NO: 1)



CH3CO-AMVSEFLKQAWFIENEEQEYVQTVK






Anx-A1 and its N-terminal derived bioactive peptides mediate their biological effects through members of the formyl peptide receptor (FPR) family. Anx-A1 exerts its counterregulatory actions on neutrophil extravasation and innate immunity by direct binding and activation of one member of this family, formyl peptide receptor like-1 (FPRL-1). The present inventors have previously found that stimulation of T cells in the presence of hrAnx-A1 increases T cell activation via stimulation of FPRL-1 (D'Acquisto et al., Blood 109: 1095-1102, 2007).
The specific binding molecule of the present invention binds to Annexin-1 (Anx-A1). The Anx-A1 to which the specific binding molecule binds is human Anx-A1 having the polypeptide sequence shown in FIG. 2A.
In a first aspect, the present invention provides a specific binding molecule raised against the human Anx-A1 protein having the amino acid sequence shown in FIG. 2A.
This aspect of the invention also extends to a specific binding molecule comprising the Complementarity Determining Regions (CDRs) VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3 of the specific binding molecule of the first aspect of the invention or an amino acid sequence at least 70% identical to each of the respective CDRs. The specific binding molecule is typically an antibody.
In one embodiment, the specific binding molecule comprises Complementarity Determining Regions (CDRs) VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3, each having a respective amino acid sequence as follows in which









(SEQ ID NO: 2)



VLCDR1 is KASENVVTYVS



 



(SEQ ID NO: 3)



VLCDR2 is GASNRYT



 



(SEQ ID NO: 4)



VLCDR3 is GQGYSYPYT



 



(SEQ ID NO: 5)



VHCDR1 is GYTFTNYWIG



 



(SEQ ID NO: 6)



VHCDR2 is DIYPGGDYTNYNEKFKG



 



(SEQ ID NO: 7)



VHCDR3 is WGLGYYFDY






or an amino acid sequence at least 70% identical thereto.

The CDRs are designated according to a combination of conserved sequence definition (Kabat et al in “Sequences of Proteins of Immunological Interest”, Nat'l. Inst. Health, Bethesda, Md. (1987)), and structural definition (Chothia and Lesk J. Mol Biol. 196:901-17(1987)). These definitions were also subsequently described in Carter et al, Proc Nat'l Acad Sci USA. 89:4285-9 (1992).
The present invention also extends to variants of peptide sequences referred to above. As used herein the term “variant” relates to proteins which have a similar amino acid sequence and/or which retain the same function. For instance, the term “variant” encompasses proteins or polypeptides which include one or more amino acid additions, deletions, substitutions or the like. An example of a variant of the present invention is a protein, such as a fusion protein, comprising a peptide as defined above, apart from the substitution of one or more amino acids with one or more other amino acids. The skilled person is aware that various amino acids have similar properties. One or more such amino acids of a substance can often be substituted by one or more other such amino acids without eliminating a desired activity of that substance.
Thus the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic). Other amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains).
Substitutions of this nature are often referred to as “conservative” or “semi-conservative” amino acid substitutions. The present invention therefore extends to a specific binding molecule comprising CDRs having the amino acid sequences described above but with one or more conservative substitutions in the CDRs, such that the amino acid sequences of the CFRs have at least 70% identity to those described above. For example, each CDR may have 1, 2, 3, 4 or 5 conservative substitutions (depending on the CDR) compared to the amino acid sequences of the CDRs set out above. For example, there can be 1, 2 or 3 conservative substitutions in the amino acid sequence of VLCDR1 set out above, 1 or 2 conservative substitutions in the amino acid sequence of VLCDR2 set out above, 1 or 2 conservative substitutions in the amino acid sequence of VLCDR3 set out above, 1, 2 or 3 conservative substitutions in the amino acid sequence of VHCDR1 set out above, 1, 2, 3, 4 or 5 conservative substitutions in the amino acid sequence of VHCDR2 set out above and 1, 2 or 3 conservative substitutions in the amino acid sequence of VHCDR3 set out above, and the sequence will still retain at least 70% identity to the CDR sequences set out above.
Using the three letter and one letter codes the amino acids may be referred to as follows: glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), proline (P or Pro), phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp), lysine (K or Lys), arginine (R or Arg), histidine (H or His), aspartic acid (D or Asp), glutamic acid (E or Glu), asparagine (N or Asn), glutamine (Q or Gln), cysteine (C or Cys), methionine (M or Met), serine (S or Ser) and Threonine (T or Thr). Where a residue may be aspartic acid or asparagine, the symbols Asx or B may be used. Where a residue may be glutamic acid or glutamine, the symbols Glx or Z may be used. References to aspartic acid include aspartate, and glutamic acid include glutamate, unless the context specifies otherwise.
Amino acid deletions or insertions can also be made relative to the amino acid sequence for the protein, such as a fusion protein, referred to above. Thus, for example, amino acids which do not have a substantial effect on the activity of the polypeptide, or at least which do not eliminate such activity, can be deleted. Such deletions can be advantageous since the overall length and the molecular weight of a polypeptide can be reduced whilst still retaining activity. This can enable the amount of polypeptide required for a particular purpose to be reduced—for example, dosage levels can be reduced.
Amino acid insertions relative to the sequence of the fusion protein above can also be made. This can be done to alter the properties of a substance of the present invention (e.g. to assist in identification, purification or expression, as explained above in relation to fusion proteins).
Amino acid changes relative to the sequence given above can be made using any suitable technique e.g. by using site-directed mutagenesis or solid state synthesis.
It should be appreciated that amino acid substitutions or insertions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids. Whether or not natural or synthetic amino acids are used, it is preferred that only L-amino acids are present.
“Identity” as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polypeptide or two polynucleotide sequences, methods commonly employed to determine identity are codified in computer programs. Preferred computer programs to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)).
One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present invention.
The percent identity of two amino acid sequences or of two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The “best alignment” is an alignment of two sequences which results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., % identity=number of identical positions/total number of positions×100).
The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporated such an algorithm. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilised as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilising BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm utilised for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the CGC sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.
Typically, the amino acid sequence of the CDRs of the specific binding molecule of the invention have at least 70% identity, using the default parameters of the BLAST computer program (Atschul et al., J. Mol. Biol. 215, 403-410 (1990)) provided by HGMP (Human Genome Mapping Project), at the amino acid level, to the amino acid sequences of the CDRs described above. More typically, the CDR sequence has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity, at the amino acid level, to the sequences shown above. Typically, each of the CDR sequences of the specific binding molecule of the invention has this level of identity to the amino acid sequences of the CDRs set out above. Alternatively, any 1, 2, 3 4 or 5 of the CDRs of the specific binding molecule of the invention has this level of identity to the amino acid sequences of the CDRs set out above.
The specific binding molecule of the invention is typically an antibody, more typically a monoclonal antibody. In one embodiment, the monoclonal antibody of the present invention is humanised.
The monoclonal antibody of the present invention can be humanised by modifying the amino acid sequence of the antibody. Methods to reduce the immunogenicity of the specific binding molecules of the invention include CDR grafting on to a suitable antibody framework scaffold or variable surface residues remodelling, e.g. by site-directed mutagenesis or other commonly used molecular biological techniques (Roguska et al Protein Eng. 9 895-904 (1996)).
Other methods applicable can include the identification of potential T-cell epitopes within the molecule, and the subsequent removal of these e.g. by site-directed mutagenesis (de-immunisation). Humanisation of the specific binding molecule may be desired where the molecule is to be used as a therapeutic agent. Humanisation of the CDR regions or of the surrounding framework sequence can be carried out as desired.
It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementary determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP-A-184187, GB 2188638A or EP-A-239400. A hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
As antibodies can be modified in a number of ways, the term “antibody” should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023. A humanised antibody may be a modified antibody having the variable regions of a non-human, e.g. murine, antibody and the constant region of a human antibody. Methods for making humanised antibodies are described in, for example, U.S. Pat. No. 5,225,539
The specific binding molecule of the invention can be a fragment of an antibody. It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E. S. et al., Nature 341:544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., Science 242:423-426 (1988); Huston et al., PNAS USA 85:5879-5883 (1988)); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) “diabodies”, multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Hollinger et al., Proc. Natl Acad. Sci. USA 90: 6444-6448 (1993)). Typically, the fragment is a Fab, F(ab′)2 or Fv fragment or an scFv molecule.
Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associated with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Current Opinion Biotechnot 4:446-449 (1993)), e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain “Janusins” described in Traunecker et al., EMBO Journal 10:3655-3659 (1991).
Bispecific diabodies, as opposed to bispecific whole antibodies, may also be useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
Monoclonal antibody VJ-4B6 is secreted by the hybridoma cell line VJ-4B6-E5-B10-D4 deposited on 3 Jun. 2010 with the European Collection of Cell Cultures (ECACC), Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury, SP4 0JG, United Kingdom, under the Budapest Treaty, and designated by the accession no. 10060301.
The deposit was made by Fulvio D'Acquisto, Queen Mary and Westfield College, Centre for Biochemical Pharmacology, Charterhouse Square, London EC1M 6BQ.
The depositor has authorised the applicant to refer to the deposited material in the application and has given his unreserved and irrevocable consent to the deposited material being made available to the public in accordance with Rule 31(1)(d) of the European Patent Convention.
The hybridoma cell line VJ-4B6-E5-B10-D4 produces the monoclonal antibody VJ-4B6 that specifically binds to Annexin-A1. The monoclonal antibody VJ-4B6 of the present invention is of the IgG2b isotype.
The antibody VJ-4B6 was raised against the full-length human Anx-A1 protein having the amino acid sequence shown in FIG. 2A.
The DNA and amino acid sequence of the light chain variable region of the antibody VJ-4B6 are shown in FIG. 11. FIG. 12 shows the amino acid sequence of the light chain variable region of VJ-4B6 with the CDRs annotated. FIG. 12 also shows the first few amino acids of the light chain constant region of VJ-4B6.
The DNA and amino acid sequence of the heavy chain variable region of the antibody VJ-4B6 are shown in FIG. 13. FIG. 14 shows the amino acid sequence of the heavy chain variable region of VJ-4B6 with the CDRs annotated. FIG. 14 also shows the first few amino acids of the heavy chain constant region of VJ-4B6.
The CDRs of the antibody VJ-4B6 are as follows:









(SEQ ID NO: 2)



VLCDR1 is KASENVVTYVS



 



(SEQ ID NO: 3)



VLCDR2 is GASNRYT



 



(SEQ ID NO: 4)



VLCDR3 is GQGYSYPYT



 



(SEQ ID NO: 5)



VHCDR1 is GYTFTNYWIG



 



(SEQ ID NO: 6)



VHCDR2 is DIYPGGDYTNYNEKFKG



 



(SEQ ID NO: 7)



VHCDR3 is WGLGYYFDY






The present invention extends to specific binding molecules having the CDRs of the antibody VJ-4B6, as described herein, and also to specific binding molecules having CDRs with at least 70% identity to one or more of the CDRs of the antibody VJ-4B6, as described herein.
The present invention also extends to specific binding molecules having either the light chain variable region, the heavy chain variable region or both the light chain variable region and the heavy chain variable region of the antibody VJ-4B6.
In a specific embodiment, the present invention therefore provides a specific binding molecule in accordance with the first aspect of the invention comprising a polypeptide having an amino acid sequence as shown in FIG. 11 and/or FIG. 13.
This embodiment of the invention also extends to certain antibody fragments which contain the light chain variable region having the amino acid sequence shown in FIG. 11 and/or the heavy chain variable region having the amino acid sequence shown in FIG. 13. For example, this embodiment extends to Fab, F(ab′)2 or Fv fragments and scFv molecules.
The present invention also encompasses specific binding molecules in accordance with the first aspect of the invention comprising a polypeptide having an amino acid sequence as shown in FIG. 12 and/or FIG. 14.
In a specific embodiment, the present invention provides a specific binding molecule in accordance with the first aspect of the invention produced by the hybridoma cell line deposited with the European Collection of Cell Cultures (ECACC) on 3 Jun. 2010 as Accession No. 10060301.
In a second aspect, the present invention provides a hybridoma cell line which produces a specific binding molecule raised against the human Anx-A1 protein having the amino acid sequence shown in FIG. 2A.
In a specific embodiment, the present invention provides a hybridoma cell line in accordance with the second aspect of the invention deposited with the European Collection of Cell Cultures (ECACC) on 3 Jun. 2010 as Accession No. 10060301.
In a third aspect, the present invention provides a pharmaceutical composition comprising a specific binding molecule of the invention.
The composition in accordance with this aspect of the invention can be formulated for use by any convenient route. The pharmaceutical composition of the invention will normally include a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle, buffer or stabiliser in addition to a specific binding molecule of the invention. Such carriers include, but are not limited to, saline, buffered saline, dextrose, liposomes, water, glycerol, polyethylene glycol, ethanol and combinations thereof. This pharmaceutical composition may be in any suitable form depending upon the desired method of administering it to a patient.
It can be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It can include a plurality of said unit dosage forms.
The pharmaceutical composition can be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such compositions can be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
Pharmaceutical compositions adapted for oral administration can be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions)
Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof.
Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.
For the preparation of solutions and syrups, excipients which can be used include for example water, polyols and sugars. For the preparation of suspensions, oils (e.g. vegetable oils) can be used to provide oil-in-water or water in oil suspensions.
Pharmaceutical compositions adapted for transdermal administration can be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient can be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6):318 (1986).
Pharmaceutical compositions adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For infections of the eye or other external tissues, for example mouth and skin, the compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient can be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient can be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.
Pharmaceutical compositions adapted for rectal administration can be presented as suppositories or enemas.
Pharmaceutical compositions adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.
Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists that can be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators.
Pharmaceutical compositions adapted for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.
Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which can contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. Excipients which can be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The compositions can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets.
The pharmaceutical compositions can contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts (substances of the present invention can themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants.
The pharmaceutical compositions of the invention can also contain one or more other therapeutically active agents in addition to the molecule of the present invention.
In some embodiments, the formulation of the active drug concentrate can comprise a pharmaceutically acceptable tonicity agent, a buffering agent, and a pharmaceutically acceptable surfactant.
Alternatively, the formulation can comprise the active ingredient plus sodium phosphate, monobasic, sodium phosphate dibasic, sodium chloride, polysorbate 80 or polysorbate 20 (surfactant to minimise risk of agitation-induced aggregation) and water (USP/Ph.Eur), optionally with a pH adjusted to about 6.0 to 7.0, e.g. around 6.5.
The active drug concentrate may or may not be lyophilised.
Other formulations can comprise sodium acetate trihydrate as a buffering agent, sodium chloride as a tonicity modifier, acetic acid for pH adjustment, and water for injection.
Dosages of the composition of the present invention can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.
This dosage can be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be reduced, in accordance with normal clinical practice.
For administration to mammals, and particularly humans, it is expected that the daily dosage of the active agent will be from 1 μg/kg to 10 mg/kg body weight, typically around 10 μg/kg to 1 mg/kg body weight. The physician in any event will determine the actual dosage which will be most suitable for an individual which will be dependent on factors including the age, weight, sex and response of the individual.
The above dosages are exemplary of the average case. There can, of course, be instances where higher or lower dosages are merited, and such are within the scope of this invention.
The specific binding molecules of the invention can be used in medicine, for example in the treatment of T cell-mediated diseases.
In a fourth aspect, the present invention therefore provides a specific binding molecule of the invention for use in medicine.
In a fifth aspect, the present invention provides a specific binding molecule of the invention for use in the treatment of a T cell-mediated disease. This aspect of the invention therefore also includes a method for the treatment of a T cell-mediated disease in a subject, typically a subject in need thereof, comprising administering to the subject a specific binding molecule of the invention. The invention therefore also extends to the use of a specific binding molecule of the invention in the manufacture of a medicament for use in the treatment of a T cell-mediated disease or alternatively to the use of a specific binding molecule of the invention in the manufacture of a medicament for the treatment of a T cell-mediated disease. The method of treatment can be of a human or an animal subject and the invention extends equally to uses in both human and/or veterinary medicine. The specific binding molecule of the invention is preferably administered to an individual in a “therapeutically effective amount”, this being sufficient to show benefit to the individual. As used herein, “treatment” includes any regime that can benefit a human or non-human animal, preferably mammal. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment).
In this aspect of the invention, the specific binding molecule of the invention can be used to treat a wide range of diseases which are mediated by T cells. In the present context, “T cell-mediated disease” means any disease or condition in which T cells play a role in pathogenesis or development of the disease or condition. T cell-mediated diseases are typically caused by aberrant T cell activation. Accordingly, such diseases can be treated by preventing the activation of T cells by blocking the activity of Anx-A1, which as demonstrated herein can be effected by the monoclonal antibody VJ-4B6. Typically, the T cell-mediated diseases treated in the present invention are diseases in which Th1 cells play a role.
T cell-mediated diseases include but are not limited to graft-versus-host disease, graft rejection, atherosclerosis, HIV and/or AIDS, psoriasis, miscarriage, and some autoimmune diseases. Autoimmune diseases which can be treated according to the present invention include rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), Addison's disease, Grave's disease, scleroderma, polymyositis, diabetes and in particular some forms of diabetes mellitus (for example juvenile onset diabetes), autoimmune uveoretinitis, ulcerative colitis, pemphigus vulgaris, inflammatory bowel disease, autoimmune thyroiditis, uveitis, Behcet's disease and Sjögren's syndrome. The T cell-mediated disease is typically rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, atherosclerosis, HIV, AIDS or psoriasis. More typically, the T cell-mediated disease is rheumatoid arthritis or multiple sclerosis,
T cell-mediated diseases as defined herein also include miscarriage. As defined herein, treatment may be prophylactic (preventative treatment). The specific binding molecule of the invention can thus be used to prevent miscarriage. An uncontrolled Th1 response is known to be implicated in miscarriage, and increasing a Th2 response favours pregnancy.
The T cell-mediated disease is typically rheumatoid arthritis. In rheumatoid arthritis (RA), it is thought that T cells recognise and interact with antigen presenting cells in the synovium. Once activated, these cells produce cytokines and effector molecules; this sequential, expanded production of cytokines constitutes the “cytokine cascade” that results in the activation of macrophages and induction of the inflammatory process, culminating in degradation and resorption of cartilage and bone. Over time, bone erosion, destruction of cartilage, and complete loss of joint integrity can occur. Eventually, multiple organ systems may be affected.
In another embodiment, the T cell-mediated disease is multiple sclerosis (MS).
In another embodiment, the T cell-mediated disease is atherosclerosis. Inflammation plays a key role in coronary artery disease and other manifestations of atherosclerosis. Immune cells dominate early atherosclerotic lesions, their effector molecules accelerate progression of the lesions, and activation of inflammation can elicit acute coronary syndromes. Adaptive immunity is highly involved in atherogenesis since it has been shown to interact with metabolic risk factors to initiate, propagate, and activate lesions in the arterial tree.
In another embodiment, the T cell-mediated disease is systemic lupus erythematosus (SLE).
In relation to the ability of Anx-A1 to favour differentiation of Th1 cells, the present invention can also be used, for example, to limit uncontrolled protective cellular (Th1) responses against intracellular pathogens and to treat extracellular infection (Th2 response) by suppressing Th1 differentiation and favouring Th2 differentiation.
Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.
In one embodiment, the present invention provides a specific binding molecule raised against the full-length human Anx-A1 protein having the amino acid sequence shown in FIG. 2A.
One example of such a specific binding molecule is the monoclonal antibody VJ-4B6 produced by the hybridoma cell line deposited with the European Collection of Cell Cultures (ECACC) on 3 Jun. 2010 as Accession No. 10060301.
In one embodiment, the present invention provides a specific binding molecule having the CDRs of the monoclonal antibody VJ-4B6, which have the following amino acid sequences:









(SEQ ID NO: 2)



VLCDR1 is KASENVVTYVS



 



(SEQ ID NO: 3)



VLCDR2 is GASNRYT



 



(SEQ ID NO: 4)



VLCDR3 is GQGYSYPYT



 



(SEQ ID NO: 5)



VHCDR1 is GYTFTNYWIG



 



(SEQ ID NO: 6)



VHCDR2 is DIYPGGDYTNYNEKFKG



 



(SEQ ID NO: 7)



VHCDR3 is WGLGYYFDY






In one embodiment, the present invention provides a specific binding molecule having the VH and/or VL regions of the monoclonal antibody VJ-4B6, which are shown in FIGS. 11 and 13 respectively.
In one embodiment, the present invention provides the hybridoma cell line VJ-4B6-E5-B10-D4 deposited with the European Collection of Cell Cultures (ECACC) on 3 Jun. 2010 as Accession No. 10060301.
The monoclonal antibody VJ-4B6 was produced by a method described in FIG. 3A. Briefly, cDNA encoding full-length human Anx-A1 having the sequence shown in FIG. 2A was cloned into an expression vector and cell-surface expression confirmed. Several intradermal applications of vector DNA adsorbed to gold particles were then administered to mice. The mouse cells took up the immunization vector and expressed the cDNA-encoded protein, stimulating an immune response. Mouse lymphocytes were then fused with murine myeloma cells to produce hybridomas. Specificity testing was then carried out, the hybridomas cloned and the monoclonal antibody produced.
The approach used to produce the monoclonal antibody VJ-4B6 thus uses Anx-A1 cDNA-transfected cells which express the whole molecule on the cell surface and hence the protein is in the same form as would be found in vivo, i.e. in its quaternary native structure. This approach for the production of antibodies differs from the production of other commercially-available antibodies to Anx-A1, which have been generated by immunizing mice with Anx-A1 peptide or denatured full-length protein.
The antibody of the invention is particularly useful in the treatment of T cell-mediated diseases such as rheumatoid arthritis and multiple sclerosis.



The present invention will now be further described by way of reference to the following Examples which are present for the purposes of illustration only. In the Examples, reference is made to a number of Figures in which:
FIG. 1A is a ribbon diagram of annexin-1 structure showing the four annexin repeats and the N-terminal domain. FIG. 1B is a schematic representation of the annexin repeats and the location of the bioactive sequence, Annexin-1 peptide Ac,2-26. FIG. 1C shows the amino acid sequence of peptide Ac.2-26, which is an acetylated N-terminal peptide fragment of Anx-A1.
FIG. 2A shows (i) the amino acid sequence (SEQ ID NO:8) and (ii) the nucleotide sequence (SEQ ID NO:9) of human Annexin-1 (Anx-A1), isoform ANXA1-003. FIG. 2B shows the amino acid sequence of human Annexin-1 (Anx-A1), isoform ANXA1-002 (SEQ ID NO:10). FIG. 2C shows the amino acid sequence of human Annexin-1 (Anx-A1), isoform ANXA1-004 (SEQ ID NO:11). FIG. 2D shows the amino acid sequence of human Annexin-1 (Anx-A1), isoform ANXA1-006 (SEQ ID NO:12).
FIG. 3 shows the generation of VJ-4B6. (A) Schematic representation of the strategy used to isolate and produce VJ-4B6. (B) The histogram shows the staining of cell lines stably transfected with Annexin-1 cDNA (green line; right-hand peak) or an irrelevant control cDNA (red line; left-hand peak) with VJ-4B6.
FIG. 4 shows validation of VJ-4B6. (A) Staining of permeabilised human Peripheral Blood Mononuclear Cells (PBMC) or Polymorph Mononuclear Cells (PMN) with VJ-4B6 (50 ng/ml). (B) Staining of permeabilized murine splenocytes or resident peritoneal macrophages with VJ-4B6 (50 ng/ml). In all tests, cells were fixed with 4% paraformaldehyde and then permeabilised with 0.02% saponin in FACS buffer. Cells were incubated with biotinylated VJ-4B6 overnight at 4° C. and then stained for 1 hour using a streptavidin-FITC conjugated antibody (dil. 1:200). In all graphs, left-hand peak represents control and right-hand peak represents VJ-4B6. The data shown are from a single experiment and are representative of other two experiments with similar results.
FIG. 5 shows that VJ-4B6 inhibits anti-CD3-induced T cell proliferation. T cells from spleen and lymph nodes of AnxA1+/+ (A) and AnxA1−/− (B) mice were stimulated with plate-bound anti-CD3(1 μg/ml) in presence of the indicated concentrations of VJ-4B6. After 18-20 hours, cells were pulsed with 3H-thymidine (1 μCi) for 12 hours and then processed by cell-harvester to measure the levels of 3H-thymidine incorporation. The data shown are from a single experiment and are representative of other two experiments with similar results.
FIG. 6 shows that VJ-4B6 inhibits anti-CD3/CD28-induced T cell proliferation. T cells from spleen and lymph nodes of AnxA1+/+ (A) and AnxA1−/− (B) mice were stimulated with plate-bound anti-CD3(1 μg/ml) and anti-CD28 (1 μg/ml) in presence of the indicated concentrations of VJ-4B6. After 18-20 hours, cells were pulsed with 3H-thymidine (1 μCi) for 12 hours and then processed by cell-harvester to measure the levels of 3H-thymidine incorporation. The data shown are from a single experiment and are representative of other two experiments with similar results.
FIG. 7 shows that VJ-4B6 inhibits anti-CD3-induced IL-2 production. T cells from spleen and lymph nodes of AnxA1+/+ (A) and AnxA1−/− (B) mice were stimulated with plate-bound anti-CD3 (1 μg/ml) in presence of the indicated concentrations of VJ-4B6. After 20-24 hours, cell supernatants were collected and analyzed for the levels of IL-2 by ELISA. The data shown are from a single experiment and are representative of other two experiments with similar results.
FIG. 8 shows that VJ-4B6 inhibits anti-CD3/CD28-induced IL-2 production. T cells from spleen and lymph nodes of AnxA1+/+ (A) and AnxA1−/− (B) mice were stimulated with plate-bound anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) in presence of the indicated concentrations of VJ-4B6. After 20-24 hours, cell supernatants were collected and analyzed for the levels of IL-2 by ELISA. The data shown are from a single experiment and are representative of other two experiments with similar results.
FIG. 9 shows that VJ-4B6 inhibits anti-CD3-induced CD25/CD69 upregulation. T cells from spleen and lymph nodes of AnxA1+/+ (A) and AnxA1−/− (B) mice were stimulated with plate-bound anti-CD3 (1 μg/ml) in presence of the indicated concentrations of VJ-4B6. After 18-20 hours, cells were collected and stained with anti-CD25 FITC plus anti-CD69 PE. Samples were acquired by FACScalibur and analyzed by FlowJo software. The data shown are from a single experiment and are representative of other two experiments with similar results.
FIG. 10 shows that VJ-4136 inhibits anti-CD3/CD28-induced CD25/CD69 upregulation. T cells from spleen and lymph nodes of AnxA1+/+ (A) and AnxA1−/− (B) mice were stimulated with plate-bound anti-CD3(1 μg/ml) and anti-CD28 (1 μg/ml) in presence of the indicated concentrations of VJ-4B6. After 18-20 hours, cells were collected and stained with anti-CD25 FITC plus anti-CD69 PE. Samples were acquired by FACScalibur and analyzed by FlowJo software. The data shown are from a single experiment and are representative of other two experiments with similar results.
FIG. 11 shows the DNA (SEQ ID NO:13 and SEQ ID NO:14) and amino acid sequence (SEQ ID NO:15) of the light chain variable region of VJ-4B6.
FIG. 12 shows the amino acid sequence (SEQ ID NO:16) of the light chain variable region of VJ-4B6 with the CDRs annotated. CDR1, CDR2, CDR3 and the beginning of the constant region are highlighted. Numbering and CDRs according to Kabat.
FIG. 13 shows the DNA (SEQ ID NO:17 and SEQ ID NO:18) and amino acid sequence (SEQ ID NO:19) of the heavy chain variable region of VJ-4B6.
FIG. 14 shows the amino acid sequence (SEQ ID NO:20) of the heavy chain variable region of VJ-4B6 with the CDRs annotated. CDR1, CDR2, CDR3 and the beginning of the constant region are highlighted. Numbering and CDRs according to Kabat. In the heavy chain variable region residues 26 to 29, although not part of the hypervariable region as defined by Kabat, are part of the CDR loop defined by Chothia (Chothia and Lesk, 1987). Positions at insertions 52, 52a, 82, 82a, 82b, 82c, 100 and 100a, are indicated as 52a, 82abc, 100a.
FIG. 15 shows that VJ-4B6 inhibits the development of EAE. C57BL/6 mice were immunized with MOG35-55 in CFA and monitored daily for signs of EAE for 22 days. At day 6 after the immunization, mice received an i.p. injection of 100 ng of IgG (A) or 5 (B), 50 (C) 100 ng (D) of VJ-4B6 (in 100 μl of PBS) every six days. Results are means±SEM (n=6/group). *p<0.05, ***p<0.001.
FIG. 16 shows that VJ-4B6 reduces the weight loss associated with the development of EAE. C57BL/6 mice were immunized with MOG35-55 in CFA and monitored daily for weight gain/loss for 22 days. At day 6 after the immunization, mice received an i.p. injection of 100 ng of IgG (A) or 5 (B), 50 (C) 100 ng (D) of VJ-4B6 (in 100 μl of PBS) every six days. Results are means±SEM (n=6/group). *p<0.05, ***p<0.001.
FIG. 17 shows that VJ-4B6 reduces the development of CIA. DBA/1 mice were immunized with bovine type II collagen in CFA. At 21 days after the primary immunization, mice were boosted (s.c.) with 200 μg type II in complete CFA and monitored daily for signs of disease for 20 days. The day of the boosting mice received an i.p. injection of 100 ng of VJ-4B6 (in 100 μl of PBS) every six days. Results are means±SEM (n=6/group). ***p<0.001.



EXAMPLE 1
VJ-4B6 Specifically Inhibits T Cell Activation in Annexin-1 Containing T Cells
Materials and Methods
Mice (mice used in FIGS. 4B, 5, 6, 7, 8, 9 and 10)
Balb/C mice were obtained from B&K Universal (Grimston, England). AnxA1−/− mice were generated in the inventors' lab and bred in pathogen free conditions at B&K Universal. All mice used in these studies were aged between 6 and 8 weeks. Animal work was performed according to United Kingdom Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act 1986) and regulations of the European Union directives.
Murine T Cell Extraction (Cells Used in FIGS. 4B, 5, 6, 7, 8, 9 and 10)
Spleen and lymph nodes (axillary, inguinal and intestinal) were removed from 6 to 8 week old mice and prepared by gentle disaggregation of tissue through a 50 μm cell strainer (BD), with a syringe plunger as previously described. Cell suspensions were layered over Ficoll to obtain the mononuclear cells and then collected and washed with RPMI medium (Gibco).
T Cell Proliferation Assay (Data in FIGS. 5 and 6)
Purified lymph node T cells (105 cells/ml) were stimulated by plate-bound anti-CD3 (clone 145-2C11; eBioscience) or anti-CD3 plus anti-CD28 (clone 37.51; eBioscience) in 96 well plates. After 18-20 h, cultures were pulsed for 12 h with 1 μCi of [3H]-thymidine (Amersham Pharmacia Biotech) and incorporated radioactivity was measured by automated scintillation counter (Packard).
IL-2 Production (Data in FIGS. 7 and 8)
Purified lymph node T cells (105 cells/ml) were stimulated by plate-bound anti-CD3 (clone 145-2C11; eBioscience) or anti-CD3 plus anti-CD28 (clone 37.51; eBioscience) in 96 well plates. After 20-24 h, culture supernatants were collected and analyzed for IL-2 content using mouse IL-2 ELISA kit (eBioscience) according to the manufacturer's instructions.
Flow Cytometric Analysis (FIGS. 9 and 10)
Cells were resuspended in FACS buffer (PBS containing 1% FCS and 0.02% NaN2). Lymphocytes were stained at 1×106/ml in 100 μl of FACS buffer and acquired on a FACScalibur with the CellQuestTM software (Becton Dickinson). The antibodies used were anti-CD25 FITC (clone PC61, eBioscience) and PE-conjugated anti-CD69 (clone H1.2F3). Cells were preincubated in FACS buffer containing anti-CD16/32 for 30 min at 4° C. to avoid non-specific binding and then labeled with the appropriate concentration of conjugated antibodies for 30 minutes at 4° C. After labeling, cells were washed and analyzed. Forward and side scatters were set to exclude erythrocytes and dead cells, and at least 2×104 lymphocytes were analyzed per sample. In all the experiments stained cells were acquired with a FACScalibur flow cytometer and analyzed using FlowJo software.
Validation of VJ-4B6 by Flow Cytometric Analysis (FIG. 4)
Human PBMC and PMN or murine splenocytes and peritoneal macrophages were resuspended first in FACS buffer containing 4% paraformaldehyde for 10 minutes and thereafter in FACS buffer containing 4% paraformaldehyde and 0.02% saponin for 15 minutes. Cells were incubated with 50 ng/ml of biotinylated VJ-4B6 for 12 hours at 4° C. Thereafter, cells were incubated with streptavidin-FITC (dil.1:200; eBioscience) for 1 hour at room temperature. After labeling, cells were washed and analyzed. Forward and side scatters were set to exclude erythrocytes and dead cells, and at least 2×104 lymphocytes were analyzed per sample. In all the experiments stained cells were acquired with a FACScalibur flow cytometer and analyzed using FlowJo software.
Human Peripheral Blood Leukocytes (Cells Used in FIG. 4A)
Blood donors were 20- to 35-year-old healthy men and women who were tested to be negative for HIV, hepatitis B virus, and hepatitis C virus. Further exclusion criteria were manifest infections during the last 4 weeks, fever, symptomatic allergies, abnormal blood cell counts, increased liver enzymes, or medication of any kind. Fresh venous blood was collected from healthy volunteers and immediately transferred to a tube containing 3.2% sodium citrate (1:10 dilution). Cells were then separated using the density gradient method: 3 ml of Ficoll Histopaque-10771 was layered on top of 3 ml of Ficoll Histopaque-11911 (both from Sigma) to create discrete layers and 6 ml of blood (diluted 1:1 with RPMI medium) layered on top of the Histopaques. After centrifugation at 1500 RPM at room temperature for 30 minutes, PBMCs and PMNs were aspirated from their appropriate layer using a sterile Pasteur pipette and washed with RPMI three times.
Statistical Analysis
All statistical analysis was performed with Prism software (GraphPad software). All values are expressed as mean±SE. Statistical analysis was assessed either by Student's t test or one-way ANOVA where appropriate. A probability of P<0.05 was considered significant.
Results
A novel anti-AnxA1 antibody was generated by genetic immunisation as indicated in the scheme in FIG. 3A (Genovac GmbH, Germany). Serum from several immunized mice were tested and three resulted positive for IgG recognizing cells transfected with AnxA1 cDNA. Splenocytes from these mice were fused to myeloma cells to generate hybridoma cells. Only one of the three hybridoma cell clones were successfully subcloned and expanded. These hybridoma cells are called VJ-4B6-E5-B10-D4. Purified IgG2b fraction from the hybridoma cells recognizes cells transfected with AnxA1 cDNA (FIG. 3B, green line; right-hand peak) but not cell transfected with an irrelevant cDNA (FIG. 3B, red line; left-hand peak).
To validate the specificity of VJ-4B6, the expression of AnxA1 in permeabilised human and murine cells known to express different levels of protein was analysed by FACS. As shown in FIG. 4A, human PMN were highly positive for VJ-4B6 staining compared with human PBMC (FIG. 4A, right-hand peak, right and left panel, respectively). This is consistent with previous observations (D'Acquisto et al, unpublished results) that PMN express significantly higher levels of AnxA1 compared to PBMC. Similarly, staining of murine macrophages with VJ-4B6 showed higher levels of expression of AnxA1 compared to splenocytes (FIG. 4B, right-hand peak, right and left panel, respectively). Collectively, these results show that VJ-4B6 recognize both human and murine AnxA1.
Next, the effects and the specificity of VJ-4B6 on T cell activation were tested. To this aim, first the effects of VJ-4B6 on anti-CD3-induced cell proliferation (as mean of 3H-thymidine incorporation) were measured using T cells from AnxA1+/+ or AnxA1−/− mice. FIG. 5 shows an increased incorporation of 3H-thymidine in both AnxA1+/+ and AnxA1−/− T cells following stimulation with anti-CD3 (FIGS. 5A and 5B, respectively). Addition of VJ-4B6 to the stimulated cultures, caused a concentration-dependent inhibition of 3H-thymidine incorporation in AnxA1+/+ but not AnxA1−/− T cells. Similar results were obtained with T cells stimulated with anti-CD3 plus anti-CD28 (FIGS. 6A and 6B, respectively).
To further confirm these results, the effect of VJ-4B6 on other classical markers of T cell activation, i.e. interleukin-2 (IL-2) production and CD25/CD69 upregulation, were tested. T cells stimulated with either anti-CD3 alone or anti-CD3 plus anti-CD28 produced large amount of IL-2 (FIGS. 7 and 8, respectively). Addition of VJ-4B6 inhibited significantly and in a concentration-dependent manner IL-2 production in AnxA1+/+ but not AnxA1−/− T cells. Similar results were obtained on CD25/CD69 upregulation. Activation of T cells with either anti-CD3 alone or anti-CD3 plus anti-CD28 induced a marked increase in the percentage of CD25/CD69 double positive T cells (second panels from the left, FIGS. 9 and 10, respectively). Stimulation in presence of VJ-4B6 inhibited significantly and in a concentration-dependent manner CD25/CD69 induction (and expression) in AnxA1+/+ but not AnxA1−/− T cells.
Together these results show that VJ-4B6 significantly inhibits T cell proliferation, IL-2 production and CD25/CD69 upregulation induced by signalling elicited by either anti-CD3 or anti-CD3 plus anti-CD28. In addition this effect is specifically due to the neutralization of AnxA1 by VJ-4B6 since its effect is lost in AnxA1−/− T cells. This is consistent with the inventors' previous observations that activated T cells release endogenous AnxA1 that would be—in turn—required for proper T cell activation (D'Acquisto et al., Blood 109: 1095-1102, 2007; D'Acquisto et a, Eur. J. Immunol. 37: 3131-3142, 2007).
In summary, these data show that VJ-4B6 inhibits anti-CD3-induced upregulation of CD25 and CD69 (markers of T cell activation) in a concentration dependent manner. Similar inhibitory effect was observed on IL-2 production and on T cell proliferation. Most importantly, this effect was not observed in Annexin-1-deficient T cells, demonstrating that the inhibition is specific and that the antibody does not cause any adverse cytotoxic effects.
EXAMPLE 2
Sequencing of VJ-4B6
The aim of this Example was to clone the antibody heavy and light chain variable region genes from the hybridoma cells and to determine the DNA sequence and location of the complementarity determining regions (CDRs) and other features.
Cloning and Sequencing of Antibody Variable Regions
Total RNA was prepared from 1 vial of hybridoma cells using the Qiagen RNeasy mini kit (Cat No: 74104). RNA was eluted in 504, water and checked on a 1.2% agarose gel.
VH and VK (variable kappa light chain) cDNAs were prepared using reverse transcriptase with IgG and kappa constant region primers. The first strand cDNAs were amplified by PCR using a large set of signal sequence primers. The amplified DNAs were gel-purified and cloned into the vector pGem® T Easy (Promega). The VH and VK clones obtained were screened for inserts of the expected size. The DNA sequence of selected clones was determined in both directions by automated DNA sequencing. The locations of the complementarity determining regions (CDRs) in the sequences were determined with reference to other antibody sequences (Kabat E A et al., 1991).
Results
VJ-4B6 Light Chain
A single VK sequence was identified. The DNA sequence and deduced amino acid sequence for the VJ-4B6 VK is shown in FIG. 11. The deduced protein sequence with CDRs annotated is shown in FIG. 12, Nine clones (seven independent) from two separate amplification steps gave identical V region sequence. The non-productive aberrant VK sequence that arises from the hybridoma fusion partner was also present in a number of clones and there was one clone with a deletion within the sequence.
VJ-4B6 Heavy Chain
A single VH sequence was identified. The DNA sequence and deduced amino acid sequence for the VJ-4B6 VH is shown in FIG. 13. The same V region sequence was found in nine independent clones. Two clones had a single base pair change, one clone had a single base pair deletion and a single base pair change, and one clone had two single base pair changes. Each of the five single base pair changes occurred in only one clone. The remaining five clones had identical sequence. The deduced protein sequence with CDRs annotated is shown in FIG. 14.
REFERENCES


    Chothia C and Lesk A M. Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol. 196: 901-17, 1987.
    Kabat E A, Wu T T, Perry H M, Gottesman K S, Foeller C. Sequences of proteins of Immunological Interest, US Department of Health and Human Services, 1991


EXAMPLE 3
Immunosuppressive Effects of VJ-4B6 In Vivo
To test the immunosuppressive effects of VJ-4B6 in vivo, we chose two classical models of autoimmune diseases: the MOG33-55-induced experimental autoimmune encephalomyelitis (EAE; mouse model of multiple sclerosis) and the collagen-induced arthritis (CIA; mouse model of rheumatoid arthritis).
Methods
Experimental autoimmune encephalomyelitis. Mice were immunized subcutaneously on day 0 with 300 μl of emulsion consisting of 300 μg of MOG35-55 in PBS combined with an equal volume of CFA containing 300 μg heat-killed M. tuberculosis H37Ra. The emulsion was injected in both flanks and followed by an intraperitoneal injection of B. pertussis toxin (500 ng/100 μl) in 100 μl of saline on days 0 and 2. Mice were observed daily for signs of EAE and weight loss. Disease severity was scored on a 6-point scale: 0=no disease; 1=partial flaccid tail; 2=complete flaccid tail; 3=impaired righting reflex; 4=partial hind limb paralysis; 5=complete hind limb paralysis; 6=moribund or dead animal.
Collagen Induced Arthritis. Six to eight male DBA/1 mice (8-12 weeks old) were injected intradermally at the base of the tail with 200 μg collagen type II emulsified in complete Freund's adjuvant (CFA; Hooke labs). At 21 days after the primary immunization, mice were boosted (s.c.) with 200 μg type II in IFA (Hooke labs). Mice were monitored for signs of arthritis onset using two clinical parameters: paw swelling and clinical score. Paw swelling was assessed by measuring thickness of the affected hind paws with plethysmometer. Clinical arthritis was assessed as recommended by the manufacturer (http://hookelabs.com/protocols/ciaInductionDBA1/ciaInduction_DBA1.html). Each limb was graded, giving a maximum possible score of 12 per animal
Results
Male C57/BL6 mice were immunized with MOG33-55/CFA as previously described (Paschalidis et al., J Neuroinflammation. 2009; 6:33). Mice received an intraperitoneal (i.p.) administration of VJ-4B6 (5, 50 and 100 ng/100 μl), IgG control (100 ng/μl) or PBS vehicle (control) every six days starting at day 6 after the immunization with MOG33-55/CFA. As shown in FIG. 15, mice treated with 5, 50 and 100 ng of VJ-4B6 showed a statistically significant dose-dependent reduction of signs of disease compared with control mice (FIGS. 15B, C and D, respectively) while administration of IgG control had no effects (FIG. 15A). The area under the curve (AUC) for each treatment and the percentage of inhibition versus the control (27.29 AUC) were the following: IgG100 ng, 24.17 AUC, 11.4%; VJ-4B6 5 ng, 15.04 AUC, 44.8%; VJ-4B6 50 ng, 5.87 AUC, 78.5%; VJ-4B6 100 ng, 7.04 AUC, 74.2%.
Studies on animal models of EAE have demonstrated that the acute phase of the disease coincides with weight loss, probably due to anorexia and deficient fluid uptake. Weight measurement of treated mice correlated with the severity of the clinical score and showed a dose-dependent reduced weight loss—from day 18 onwards—in the VJ-4B6-treated but not IgG-treated mice compared to controls (FIG. 16).
To confirm the therapeutic potential of VJ-4B6 as immunosuppressant in vivo, we tested its effects in the CIA model. Male DBA/1 mice were immunized with bovine type II collagen in CFA as previously described (D'Acquisto et al., Blood. 2007; 109(3):1095-102). Mice received an i.p. injection of VJ-4B6 (100 ng/100 μl) or PBS vehicle (control) every six days starting at day 0 after the boost with collagen. Consistent with the data obtained on the EAE, administration of VJ-4B6 significantly reduced (AUC 26.75; 67.9%) the development of sign of disease compared to control mice (AUC 83.50) (FIG. 17).

## Claims

1. An antibody or fragment thereof that binds human Anx-A1 having the amino acid sequence of SEQ ID NO:8, said antibody or fragment thereof comprising Complementarity Determining Regions (CDRs) VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3, each having a respective amino acid sequence as follows in which









(SEQ ID NO: 2)



VLCDR1 is KASENVVTYVS



 



(SEQ ID NO: 3)



VLCDR2 is GASNRYT



 



(SEQ ID NO: 4)



VLCDR3 is GQGYSYPYT



 



(SEQ ID NO: 5)



VHCDR1 is GYTFTNYWIG



 



(SEQ ID NO: 6)



VHCDR2 is DIYPGGDYTNYNEKFKG



 



(SEQ ID NO: 7)



VHCDR3 is WGLGYYFDY.

2. An antibody or fragment thereof as claimed in claim 1 wherein the antibody is a monoclonal antibody.

3. An antibody or fragment thereof as claimed in claim 2 wherein the monoclonal antibody is humanized.

4. An antibody or fragment thereof as claimed in claim 1 wherein the fragment is a Fab, F(ab′)2 or Fv fragment or an scFv molecule.

5. An antibody or fragment thereof as claimed in claim 1 comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:19 and/or a light chain variable region having the amino acid sequence of SEQ ID NO:15.

6. An antibody or fragment thereof as claimed in claim 1 produced by the hybridoma cell line deposited with the European Collection of Cell Cultures (ECACC) on 3 Jun. 2010 as Accession No. 10060301.

7. A hybridoma cell line which produces an antibody or fragment thereof that binds the human Anx-A1 protein having the amino acid sequence of SEQ ID NO:8, said antibody or fragment thereof comprising Complementarity Determining Regions (CDRs) VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3, each having a respective amino acid sequence as follows in which









(SEQ ID NO: 2)



VLCDR1 is KASENVVTYVS,


 



(SEQ ID NO: 3)



VLCDR2 is GASNRYT,


 



(SEQ ID NO: 4)



VLCDR3 is GQGYSYPYT,


 



(SEQ ID NO: 5)



VHCDR1 is GYTFTNYWIG,


 



(SEQ ID NO: 6)



VHCDR2 is DIYPGGDYTNYNEKFKG, and


 



(SEQ ID NO: 7)



VHCDR3 is WGLGYYFDY.

8. A hybridoma cell line as claimed in claim 7 deposited with the European Collection of Cell Cultures (ECACC) on 3 Jun. 2010 as Accession No. 10060301.

9. A pharmaceutical composition comprising an antibody or fragment thereof as claimed in claim 1.

10. A pharmaceutical composition as claimed in claim 9, further comprising another therapeutically active agent.