Binding molecule

The present relates to binding molecules, and in particular to molecules that bind to oxidised forms of low density lipoprotein (oxLDL).

Atherosclerosis is now widely seen as a chronic inflammatory disease, driven in large part by the deposition and oxidative modification of low density lipoprotein (LDL) in the walls of arteries (1 -3). Following oxidative modification, LDL becomes recognised by macrophage scavenger receptors, resulting in phagocytosis, foam cell formation, cell death and eventually to the generation of the lipid rich core that characterises atherosclerotic lesions (4;5). However, the humoral immune system provides an additional pathway for the disposal of modified LDL, mediated largely through the binding of IgM antibodies and complement (6;7). Besides contributing to oxidised LDL clearance, IgM "natural antibodies" can inhibit its uptake into macrophages and prevent foam cell formation in the arterial wall (8). The homeostatic role of IgM is well demonstrated by the marked acceleration of atherosclerosis caused by serum IgM deficiency in LDL receptor deficient (Ld/r ^_ ) mice (9).

Although IgG antibodies that react with oxidized LDL (oxLDL) can readily be detected in serum, relatively little is known about their precise epitope specificities and functions (10; 1 1). IgG anti-oxLDL in mice tend to be lgG1 , lgG3 and lgG2a/c isotypes, whilst in humans they are predominantly lgG1 and lgG3 (12). Theoretically, distinct IgG anti-oxLDL isotypes may have quite different consequences for atherogenesis, depending upon their relative capacities to activate complement and ligate proinflammatory Fey receptors (FcyR) (13-18). The proinflammatory potential of IgG antibodies in atherosclerosis was supported by the marked protection from lesion development in LDL receptor deficient mice lacking FcyRIII (CD16) (19). On the other hand, passive immunization with human lgG1 or mouse lgG2b mAb has been shown to be protective (20;21).

Antibodies that react with oxLDL antibodies probably recognize a range of epitopes, expressed at different levels of oxidative modification in plasma and in atherosclerotic lesions. Much of what we know about these antibodies has been discovered through the generation of monoclonal antibodies (mAb) in mice, either following immunization (22-27) or occurring spontaneously in hyperlipidaemic mice (28;29). So far the spontaneously-occurring autoantibodies that have been studied have been mostly, if not entirely, of the IgM isotype (28;29).

According to a first aspect of the present invention, there is provided an isolated IgG antibody capable of binding oxidised low density lipoprotein (oxLDL).

A second aspect of the invention provides an isolated specific binding member capable of binding an antigen on oxidised low density lipoprotein (oxLDL), which antigen is capable of being bound by a member comprising one or more binding domains selected from domains comprising an amino acid sequence substantially as set out as GYAFTNYL, INPGSGGT and ARSFKWKFDY. The binding domain may comprise an amino acid sequence substantially as set out as

ARSFKWKFDY. In one embodiment, the isolated specific binding member of the second aspect of the present invention comprises one or more binding domains selected from domains which comprise an amino acid sequence substantially as set out as GYAFTNYL, INPGSGGT and ARSFKWKFDY.

In one embodiment, the member comprises a binding domain which comprises an amino acid sequence substantially as set out as ARSFKWKFDY. In this embodiment, the isolated specific binding member may additionally comprise one or both, preferably both, of the binding domains substantially as set out as GYAFTNYL and INPGSGGT.

One isolated specific binding member of the second aspect of the invention comprises the amino acid sequence substantially as set out in Figure 2a.

In a third aspect, the present invention provides an isolated specific binding member capable of binding an antigen on oxidised low density lipoprotein (oxLDL), which antigen is capable of being bound by a member comprising one or more binding domains selected from domains comprising an amino acid sequence substantially as set out as ENVGTY, GAT and GQSYTYPYT.

The binding domain may comprise an amino acid sequence substantially as set out as GQSYTYPYT.

In one embodiment, the isolated specific binding member of the third aspect of the present invention comprises one or more binding domains selected from domains which comprise an amino acid sequence substantially as set out as residues ENVGTY, GAT and GQSYTYPYT.

In one embodiment, the member comprises a binding domain which comprises an amino acid sequence substantially as set out as GQSYTYPYT. In this embodiment, the isolated specific binding member may additionally comprise one or both, preferably both, of the binding domains substantially as set out as ENVGTY and GAT.

One isolated specific binding member of the third aspect of the invention comprises the sequence substantially as set out in Figure 2b.

Specific binding members which comprise a plurality of binding domains of the same or different sequence, or combinations thereof, are included within the present invention. The or each binding domain may be carried by a human antibody framework. For example, one or more binding regions may be substituted for the CDRs of a whole human antibody or of the variable region thereof. In a fourth aspect, the invention provides a specific binding member which comprises a binding member of the second aspect in combination or association with a binding member of the third aspect. Such a binding member may be in the form of a Fv, (Fab')2, scFv or other derivative antibody construct. Binding members of the first to fourth aspects of the invention are capable of binding an antigen on copper-oxidised low density lipoprotein (oxLDL) or malondialdehyde-conjugated LDL, both as generated by Palinski et al. (1990) Arteriosclerosis 10: 325-335. Low density lipoprotein is a 18-25 nm diameter particle (Mr 550 kD) consisting of a hydrophobic triglyceride and cholesterol ester core, surrounded by a phospholipid shell wrapped by a single apolipoprotein apoB molecule which in human has 4,536 amino acids (51). Preferably, these binding members do not bind or bind minimally to native LDL.

A fifth aspect of the invention provides an isolated specific binding member capable of binding an antigen on a specific binding member of the first, second, third or fourth aspects of the invention. In a sixth aspect, such an antigen may be capable of being bound by a member comprising one or more binding domains selected from domains comprising an amino acid sequence substantially as set out as GFTFSSYA, DISGSGNTTTYADSVKG and DDDAFDY.

The binding domain may comprise an amino acid sequence substantially as set out as DDDAFDY. In one embodiment, the isolated specific binding member of the sixth aspect of the present invention comprises one or more binding domains selected from domains which comprise an amino acid sequence substantially as set out as GFTFSSYA, DISGSGNTTTYADSVKG and DDDAFDY.

In one embodiment, the member comprises a binding domain which comprises an amino acid sequence substantially as set out as DDDAFDY. In this embodiment, the isolated specific binding member may additionally comprise one or both, preferably both, of the binding domains substantially as set out as GFTFSSYA and DISGSGNTTTYADSVKG.

One isolated specific binding member of the sixth aspect of the invention comprises the amino acid sequence substantially as set out in Figure 4a.

In a seventh aspect, such an antigen is capable of being bound by a member comprising one or more binding domains selected from domains comprising an amino acid sequence substantially as set out as QSISSY, YASALQS and QQGDAAPTT.

The binding domain may comprise an amino acid sequence substantially as set out as QQGDAAPTT.

In one embodiment, the isolated specific binding member of the seventh aspect of the present invention comprises one or more binding domains selected from domains which comprise an amino acid sequence substantially as set out as QSISSY, YASALQS and QQGDAAPTT. In one embodiment, the member comprises a binding domain which comprises an amino acid sequence substantially as set out as QQGDAAPTT. In this embodiment, the isolated specific binding member may additionally comprise one or both, preferably both, of the binding domains substantially as set out as QSISSY and YASALQS.

One isolated specific binding member of the sixth aspect of the invention comprises the sequence substantially as set out in Figure 4b. Specific binding members which comprise a plurality of binding domains of the same or different sequence, or combinations thereof, are included within the present invention. The or each binding domain may be carried by a human antibody framework. For example, one or more binding regions may be substituted for the CDRs of a whole human antibody or of the variable region thereof. In an eighth aspect, the invention provides a specific binding member which comprises a binding member of the sixth aspect in combination or association with a binding member of the seventh aspect. Such a binding member may be in the form of a F _v , (Fab') ₂ , or scFV antibody fragment.

Specific binding members of the invention may carry a detectable or functional label.

In further aspects, the invention provides an isolated nucleic acid which comprises a sequence encoding a specific binding member of any one the first to eighth aspects of the invention, and methods of preparing specific binding members of the invention which comprise expressing said nucleic acids under conditions to bring about expression of said binding member, and recovering the binding member.

Specific binding members according to the invention may be used in a method of treatment or diagnosis of the human or animal body, such as a method of treatment of atherosclerosis or its clinical complications such as myocardial ischaemia or infarction, cerebral ischaemia or infraction, lower limb ischaemia or infarction or arterial aneurysm or for the treatment of other inflammatory diseases involving oxidative stress such as inflammatory arthritis in a patient (preferably human) which comprises administering to said patient an effective amount of a specific binding member of the invention. The invention also provides a specific binding member of the present invention for use in medicine, as well as the use of a specific binding member of the present invention in the manufacture of a medicament for the diagnosis or treatment of atherosclerosis or its clinical complications or for the treatment of other inflammatory diseases involving oxidative stress.

These and other aspects of the invention are described in further detail below. As used herein, a "specific binding member" 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, although it also concerns small molecules which bind to the antigen defined herein. Thus, preferred specific binding members of the invention are antibodies.

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).

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. 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, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. A

monoclonal antibody may be referred to herein as "mab".

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, US Patent No. 5225539

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 ai, 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 ai, Science 242:423-426 (1988); Huston et ai, 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 ai, Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)).

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 Biotechnol. 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 ai., 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.

An "antigen binding domain" is the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding domain may be provided by one or more antibody variable domains. An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

"Specific" is generally used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s), and, e.g., has less than about 30%, preferably 20%, 10%, or 1 % cross-reactivity with any other molecule. The term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case, the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.

"Isolated" refers to the state in which specific binding members of the invention or nucleic acid encoding such binding members will preferably be, in accordance with the present invention.

Members and nucleic acid will generally be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo. Specific binding members and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated - for example, the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy. Specific binding members may be glycosylated, either naturally or by systems of heterologous eukaryotic cells, or they may be (for example if produced by expression in a prokaryotic cell) unglycosylated.

By "substantially as set out" it is meant that the CDR regions of the invention will be either identical or highly homologous to the specified regions. By "highly homologous" it is contemplated that from 1 to 5, from 1 to 4, from 1 to 3, 2 or 1 substitutions may be made in the CDRs, although such substitutions will be conservative in the sense that binding to the specified antigen will be retained.

The invention also includes within its scope polypeptides having the amino acid sequence as set out in Figure 2a (LO-1 V _H ), Figure 2b (LO-1 V _L ), Figure 4 (VH and/or VL), and sequences having substantial identity thereto, for example, 70%, 80%, 85%, 90%, 95% or 99% identity thereto. Also included are polynucleotides comprising a sequence encoding such polypeptides, and sequences having substantial identity thereto, for example, 70%, 80%, 85%, 90%, 95% or 99% identity thereto. The percent identity of two amino acid sequences or of two nucleic acid sequences is generally determined by aligning the sequences for optimal comparison purposes (e.g. , gaps can be introduced in the first sequence for best alignment with the second sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is an alignment of two sequences that results in the highest percent identity. The percent identity is determined by comparing the number of identical amino acid residues or nucleotides within the sequences (i.e. , % identity = number of identical positions/total number of positions x 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 N BLAST 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 a 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 a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (Id.). When utilizing 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 utilized 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 GCG 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.

Isolated specific binding members of certain aspects of the present invention are capable of binding to oxLDL. In one embodiment, the CDR3 regions, comprising the amino acid sequences substantially as set out as ARSFKWKFDY and GQSYTYPYT are carried in a structure which allows the binding of these regions to oxLDL.

The structure for carrying the CDR3s of the invention will generally be of an antibody heavy or light chain sequence or substantial portion thereof in which the CDR3 regions are located at locations corresponding to the CDR3 region of naturally-occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. The structures and locations of immunoglobulin variable domains may be determined by reference to Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 4 ^th Edition, US Department of Health and Human Services, (1987), and updates thereof, now available on the Internet The amino acid sequence substantially as set out as ARSFKWKFDY may be carried as the CDR3 in a human heavy chain variable domain or a substantial portion thereof, and the amino acid sequence substantially as set out as GQSYTYPYT may be carried as the CDR3 in a human light chain variable domain or a substantial portion thereof. The variable domains may be derived from any germline or rearranged human variable domain, or may be a synthetic variable domain based on consensus sequences of known human variable domains. The CDR3-derived sequences of the invention may be introduced into a repertoire of variable domains lacking CDR3 regions, using recombinant DNA technology. For example, Marks et al {Bio/Technology 10:779-783 (1992)) describe methods of producing repertoires of antibody variable domains in which consensus primers directed at or adjacent to the 5' end of the variable domain area are used in conjunction with consensus primers to the third framework region of human VH genes to provide a repertoire of VH variable domains lacking a CDR3. Marks et al further describe how this repertoire may be combined with a CDR3 of a particular antibody. Using analogous techniques, the CDR3-derived sequences of the present invention may be shuffled with repertoires of VH or VL domains lacking a CDR3, and the shuffled complete VH or VL domains combined with a cognate VL or VH domain to provide specific binding members of the invention. The repertoire may then be displayed in a suitable host system such as the phage display system of WO92/01047 so that suitable specific binding members may be selected. A repertoire may consist of from anything from 10 ⁴ individual members upwards, for example from 10 ⁶ to 10 ⁸ or 10 ¹⁰ members.

Analogous shuffling or combinatorial techniques are also disclosed by Stemmer (Nature 370:389-391 (1994)) who describes the technique in relation to a β-lactamase gene but observes that the approach may be used for the generation of antibodies.

A further alternative is to generate novel VH or VL regions carrying the CDR3-derived sequences of the invention using random mutagenesis of, for example, the VH or VL genes of the antibodies of the invention to generate mutations within the entire variable domain. Such a technique is described by Gram et al (Proc. Natl. Acad. Sci. USA 89:3576-3580 (1992)), who used error-prone PCR.

Another method which may be used is to direct mutagenesis to CDR regions of VH or VL genes. Such techniques are disclosed by Barbas et al (Proc. Natl. Acad. Sci. USA 91 :3809-3813 (1994)) and Schier ef al (J. Mol. Biol. 263:551 -567 (1996)). A substantial portion of an immunoglobulin variable domain will generally comprise at least the three CDR regions, together with their intervening framework regions. The portion may also include at least about 50% of either or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Additional residues at the N-terminal or C-terminal end of the substantial part of the variable domain may be those not normally associated with naturally occurring variable domain regions. For example, construction of specific binding members of the present invention made by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps, including the introduction of linkers to join variable domains of the invention to further protein sequences including immunoglobulin heavy chains, other variable domains (for example in the production of diabodies) or protein labels as discussed in more detail below.

One embodiment of the invention provides specific binding members comprising a pair of binding domains based on the amino acid sequences for the VL and VH regions substantially as set out in Figures 2a and 2b. Single binding domains based on either of these sequences form further aspects of the invention. In the case of the binding domains based on the amino acid sequence for the VH region substantially set out in Figure 2a, such binding domains may be used as targeting agents since it is known that immunoglobulin VH domains are capable of binding target antigens in a specific manner.

In the case of either of the single chain specific binding domains, these domains may be used to screen for complementary domains capable of forming a two-domain specific binding member which has properties as good as or equal to the L01 antibody disclosed herein. This may be achieved by phage display screening methods using the so-called hierarchical dual combinatorial approach as disclosed in WO92/01047 in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the resulting two-chain specific binding member is selected in accordance with phage display techniques such as those described in that reference. This technique is also disclosed in Marks et al. ibid.

Specific binding members of the present invention may further comprise antibody constant regions or parts thereof. For example, specific binding members based on the VL region shown in Figure 2b may be attached at their C-terminal end to antibody light chain constant domains including human CK or CX chains. Similarly, specific binding members based on VH region shown in Figure 2a or 2b may be attached at their C-terminal end to all or part of an immunoglobulin heavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE and IgM and any of the isotype sub-classes, particularly lgG1 and lgG4 As detailed in the examples below, the inventors have produced a monoclonal antibody - referred to as mAb L01 - which is an lgG3K monoclonal autoantibody that reacts with copper-oxidised LDL but not native LDL. The mAb L01 epitope on LDL appears after exposure to Cu ²⁺ ions for 2-3 hours, indicating that it is a feature of relatively heavily modified LDL particles. MAb L01 also reacts in vitro with MDA-conjugated LDL, which is known to a prominent epitope in copper-oxidised LDL

preparations. Sequencing of the mAb L01 variable heavy chain (VH) domain showed that it belongs to the VH1 family (V1 -54) and is germline. Binding of mAb L01 was not influenced by treatment of MDA-LDL with isopropanol, methanol or ethanol, suggesting that it does not recognize lipid. Staining of both mouse and human atherosclerotic tissue shows specific binding of mAb L01 to antigen within the lesions.

In addition, the inventors have provided an anti-idiotype scFv (designated H3) that neutralises mAb L01 binding to MDA-LDL. Amino acid substitutions between H3 and an irrelevant control scFv C12 showed that residues in the H3 CDRH2, CDRH3 and CDRL2 are all critical for mAb L01 binding, consistent with a conformational epitope on H3 involving both heavy and light chains. Comparison of amino acids in H3 CDRH2 and CDRL2 with apoB, the major LDL protein, showed homologous sequences, suggesting H3 has structural similarities to the mAb L01 binding site on MDA-LDL. The inventors looked to see whether H3 might recognise serum antibodies bearing the L01 idiotype, using an ELISA with H3 on the solid phase. It was found that serum IgG and IgM antibodies in both mouse and human react with H3, and that the level varied with age in mice and between human individuals. In both mouse and human, levels of anti-H3 antibodies correlated closely with levels of anti-MDA-LDL antibodies. Furthermore, human IgG isolated on an H3-affinity column showed increased binding to MDA-LDL compared with flow-through IgG, indicating that anti-H3 antibodies react with MDA-LDL. However, it is unlikely that H3 is reacting with serum antibodies in the same way as mAb L01 as (i) preincubation of H3 with mAb L01 failed to inhibit the binding of serum IgG; (ii) serum antibodies also react with C12, another scFv with which mAb L01 does not react. Thus the epitope on H3 with which serum antibodies interact is common to the scFv library. Interestingly, assays performed on sera taken from the PARSEC study showed that IgG anti-H3 negatively correlate with the severity of coronary artery disease. Specific binding members of the present invention can be used in methods of diagnosis and treatment of atherosclerosis or its clinical complications or other inflammatory diseases involving oxidative stress in human or animal subjects. Such subjects may be passively immunised with a specific binding member of the first, second, third or fourth aspects. Alternatively, subjects may be actively immunised with a specific binding member of the fifth, sixth, seventh or eight aspects, resulting in an immune response that protects from the conditions set out above. The active immunizing agent may be conjugated or complexed with an adjuvant, and/or an immune complex combined with antibody. The specific binding member of the fifth, sixth, seventh or eight aspects may be used as a monomer or a polymer. The specific binding member of the first, second, third or fourth aspects may be used for targeting of therapeutic constructs to sites of antigen deposition. Such constructs include enzymes that convert prodrugs to an active form or anti-inflammatory cytokines (such as interleukin-10 (IL-10) or transforming growth factor beta (TGFp)) or anti-inflammatory soluble receptors (such as a tumor necrosis factor receptor) or anti-thrombotic factors (such as tissue factor pathway inhibitor (TFPI) or thrombomodulin or enzyme inhibitors such as a tissue inhibitor of metalloproteinases (TIMP). When used in diagnosis, specific binding members of the invention may be labelled with a detectable label, for example a radiolabel such as ³ 1 or ⁹⁹ Tc, which may be attached to specific binding members of the invention using conventional chemistry known in the art of antibody imaging. Labels also include enzyme labels such as horseradish peroxidase. Labels further include chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin.

In particular embodiments, binding members of the invention may be used for in vivo diagnosis. For example, the rate of clearance of injected binding member/antibody may be a reflection of antigen load, and hence act as a marker of atherosclerosis or oxidative stress.

Binding members of the invention may be used for in vitro diagnostics. In particular, binding members of the fifth, sixth, seventh or eight aspects of the invention may be used for measuring serum antibodies as biomarkers of cardiovascular risk, and can be used in assays as a surrogate of oxidised LDL.

Furthermore, the specific binding members of the present invention may be administered alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated. Thus, the present invention further provides products containing a specific binding member of the present invention and an active agent as a combined preparation for simultaneous, separate or sequential use in the treatment of atherosclerosis or its clinical

complications or other inflammatory diseases involving oxidative stress. Active agents may include anti-inflammatory agents such as corticosteroids or non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) and anti-thrombotic agents such as heparin.

Specific binding members of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the specific binding member. The pharmaceutical composition may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, diluent, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. intravenous. It is envisaged that injections will be the primary route for therapeutic administration of the

compositions although delivery through a catheter or other surgical tubing is also used. Some suitable routes of administration include intravenous, subcutaneous and intramuscular administration. Liquid formulations may be utilised after reconstitution from powder formulations. For intravenous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Where the formulation is a liquid it may be, for example, a physiologic salt solution containing non-phosphate buffer at pH 6.8-7.6, or a lyophilised powder.

The composition may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood. Suitable examples of sustained release carriers include semi-permeable polymer matrices in the form of shared articles, e.g. suppositories or microcapsules. Implantable or microcapsular sustained release matrices include polylactides (US Patent No. 3, 773, 919; EP-A-0058481) copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers 22(1): 547-556, 1985), poly (2-hydroxyethyl- methacrylate) or ethylene vinyl acetate (Langer et al, J. Biomed. Mater. Res. 15: 167-277, 1981 , and Langer, Chem. Tech. 12:98-105, 1982). Liposomes containing the polypeptides are prepared by well- known methods: DE 3,218, 121 A; Epstein et al, PNAS USA, 82: 3688-3692, 1985; Hwang et al, PNAS USA, 77: 4030-4034, 1980; EP-A-0052522; E-A-0036676; EP-A-0088046; EP-A-0143949; EP-A-

0142541 ; JP-A-83-1 1808; US Patent Nos 4,485,045 and 4,544,545. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being adjusted for the optimal rate of the polypeptide leakage.

The compositions are preferably administered to an individual in a "therapeutically effective amount", this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated.

Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. The compositions of the invention are particularly relevant to the treatment of atherosclerosis or its clinical complications or other inflammatory diseases involving oxidative stress. Examples of the techniques and protocols mentioned above can be found in Remington's

Pharmaceutical Sciences, 16 ^th edition, Oslo, A. (ed), 1980. The optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration. In general, a serum concentration of polypeptides and antibodies that permits saturation of receptors is desirable. A concentration in excess of

approximately 0.1 nM is normally sufficient. For example, a dose of 100mg/m ² of antibody provides a serum concentration of approximately 20nM for approximately eight days.

As a rough guideline, doses of antibodies may be given weekly in amounts of 10-300mg/m ² .

Equivalent doses of antibody fragments should be used at more frequent intervals in order to maintain a serum level in excess of the concentration that permits saturation of receptors.

The dose of the composition will be dependent upon the properties of the binding member, e.g. its binding activity and in vivo plasma half-life, the concentration of the polypeptide in the formulation, the administration route, the site and rate of dosage, the clinical tolerance of the patient involved, the pathological condition afflicting the patient and the like, as is well within the skill of the physician. For example, doses of 30C^g of antibody per patient per administration are preferred, although dosages may range from about 1 C^g to 6 mg per dose. Different dosages are utilised during a series of sequential inoculations; the practitioner may administer an initial inoculation and then boost with relatively smaller doses of antibody.

The binding members of the present invention may be generated wholly or partly by chemical synthesis. The binding members can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2 ^nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984); and Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California), or they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.

Another convenient way of producing a binding member according to the present invention is to express the nucleic acid encoding it, by use of nucleic acid in an expression system.

The present invention further provides an isolated nucleic acid encoding a specific binding member of the present invention. Nucleic acid includes DNA and RNA. In a preferred aspect, the present invention provides a nucleic acid which codes for a specific binding member of the invention as defined above. The skilled person will be able to determine substitutions, deletions and/or additions to such nucleic acids which will still provide a specific binding member of the present invention.

The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one nucleic acid as described above. The present invention also provides a recombinant host cell which comprises one or more constructs as above. As mentioned, a nucleic acid encoding a specific binding member of the invention forms an aspect of the present invention, as does a method of production of the specific binding member which method comprises expression from encoding nucleic acid therefor. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression, a specific binding member may be isolated and/or purified using any suitable technique, then used as appropriate.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common, preferred bacterial host is E. coli. The expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Pliickthun, Bio/Technology 9:545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of a specific binding member, see for recent review, for example Reff, Curr. Opinion Biotech. 4:573-576 (1993); Trill et al., Curr. Opinion Biotech. 6:553-560 (1995).

Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate. For further details see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual: 2 ^nd Edition, Cold Spring Harbor Laboratory Press (1989). Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Ausubel et al. eds., Short Protocols in Molecular Biology, 2 ^nd Edition, John Wiley & Sons (1992).

Thus, a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.

In one embodiment, the nucleic acid of the invention is integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.

The present invention also provides a method which comprises using a construct as stated above in an expression system in order to express a specific binding member or polypeptide as above.

Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis. The prior art documents mentioned herein are incorporated to the fullest extent permitted by law. EXAMPLES

The invention will now be described with reference to the following non-limiting examples.

With a view to better understanding the fine specificity and function of naturally occurring IgG anti- oxLDL autoantibodies, the inventors generated hybridomas from the spleen of an elderly LDL receptor deficient mouse that had not been immunized. The following examples describe the characterisation of a new antibody, together with an anti-idiotype single chain variable region antibody (scFv) with which it specifically reacts. Increasing evidence implicates IgG autoantibodies against oxidised forms of low density lipoprotein (oxLDL) in the pathophysiology of atherosclerotic arterial disease. Using an elderly LDL receptor deficient atherosclerotic mouse, the inventors isolated a novel lgG3K against oxLDL (designated mAb L01). L01 reacts with copper-oxidised LDL, but minimally with native LDL. Further analysis showed that mAb L01 also reacts in vitro with malondialdehyde-conjugated LDL (MDA-LDL), a known key epitope in copper-oxidised LDL preparations. By screening a phage library expressing single chain variable region antibodies (scFv), the inventors selected an anti-idiotype scFv (designated H3) that neutralises mAb L01 binding to MDA-LDL. Amino acid substitutions between H3 and an irrelevant control scFv C12 showed that residues in the H3 CDRH2, CDRH3 and CDRL2 are all critical for binding to mAb L01 , consistent with a conformational epitope on H3 involving both heavy and light chains. Comparison of amino acids in H3 CDRH2 and CDRL2 with apoB, the major LDL protein, showed homologous sequences, suggesting H3 has structural similarities to the mAb L01 binding site on MDA-LDL. Immunocytochemical staining showed that mAb L01 binds epitopes in mouse and human atherosclerotic lesions. The mAb L01 - H3 combination therefore provides a model for analysing in detail the structure and function of an individual IgG autoantibody in relation to atherosclerosis. Reference is made to the accompanying drawings in which:

Figure 1 relates to initial characterization of mAb L01 : (A) Reactivity by ELISA of mAb L01 culture supernatant and pooled sera from Ld\f' ^~ mice (diluted 1 :40) against Cu-oxLDL (black bars) or native LDL (white bars); (B) Competition ELISA showing binding of different concentrations of mAb L01 in the presence and absence of fluid phase Cu-oxLDL. Values are mean + SD of triplicates; (C) binding by ELISA of mAb L01 to LDL modified by MDA for durations up to 5 hours. Also shown is the lack of binding of control lgG3 HK-PEG-1 at 1 and 10 mg/ml (curves superimposed). Values are mean + SD of triplicates; (D) the extent of carbonyl adduction to LDL following incubation with MDA was estimated using a carbonyl assay, with results expressed as the number of nmoles of DNP/mg of protein (nmol/mg).

Figure 2 shows the amino acid sequence of the mAb L01 V _H and its complete homology with the germline V1 -54/JH2 sequence, and the amino acid sequence of the mAb L01 V _L and its complete homology with the germline V6-20/12 sequence. The CDRs are indicated by boxes.

Figure 3 relates to reactivity and sequence of anti-idiotype scFv H3: (A) shows specific binding of mAb L01 to scFv H3. Microtiter plates were coated with H3 or C12 and then tested for binding of mAb L01 or control lgG3k HK-PEG-1 (each at 2 μg/ml). Values are expressed as mean and upper value of duplicates; (B) shows neutralisation of mAb L01 binding to MDA-LDL by scFv H3. mAb L01 (2 μg/ml) were incubated with plates coated with MDA-LDL (10 μg/ml) in the presence of varying concentrations of scFv H3 or control scFv C12; (C,D) effects of changing (C) salt concentration and (D) pH on binding of mAb L01 to H3 and MDA-LDL. In (C) and (D) the data are expressed as percentage of the binding seen with PBS and percentage of maximal binding respectively.

Figure 4 shows similarities of sequences of LO-1 binding scFv and apoB-100: The figure shows the sequence of the V _H and V _L regions of scFv H3. Amino acids at each of the eighteen points at which the Tomlinson I scFv library is diverse are red. ApoB peptide sequences with similarities to H3 sequences are shown, with amino acids identical to H3 in green. Amino acid numbering is taken from NCBI protein sequences AAA51752.1 and NP_ Q33823.2 for human and mouse apoB respectively.

Figure 5 shows effects of V _H and V _L domain swops between scFv H3 and C12: H3, C12, H3V _H /C12V _L , and H3V _L /C12V _H were coated at 1 (^g/ml. Binding of mAb L01 was tested by ELISA across a concentration range of 0.1 - 2000 ng/ml.

Figure 6 shows immunocytochemical staining of Ldl ^' mouse aortic root with mAb L01 : (A) shows an aortic valve cusp from a Ld\f' ^~ mouse stained with Giemsa, a general purpose tissue stain; in (B) the region indicated by the arrow is stained with the macrophage-specific marker CD68- AlexaFluor488 (green) and the DNA-binding dye TOPRO (blue), revealing the localized area of macrophage infiltration and the unstained media (Med). Diffuse staining of the whole valve cusp is seen with (C) mAb L01 but not (D) control lgG3. In (E) no equivalent staining was seen with mAb L01 applied to the aortic root of a wild-type mouse. Preincubation of mAb L01 with (F) MDA-LDL or (G) scFv H3, but not (H) scFv C12 inhibited staining, demonstrating specificity of mAb L01 binding. Blue = TOPRO counterstain. Red = AlexaFluor 568 fluorescence showing specific binding of mAb L01 . Ld\f' ^~ mice fed a low fat diet to the age of 22 weeks were chosen because their lesions are composed almost all exclusively of macrophages, simplifying interpretation. Med = tunica media (vascular smooth muscle cell layer); Le = lesion (composed of macrophages); Open arrow points to lesion.

Figure 7 shows immunocytochemical staining of human carotid atherosclerotic plaque with mAb L01 : for orientation (A) shows a low power view of a human carotid atherosclerotic plaque stained with Giemsa, with the area within the box magnified in (B). (C-E) show confocal imaging of consecutive serial sections in the vicinity of the boxed area in (B): (C) single stained with mAb L01 (red AlexaFluor 568), (D) dual-stained by mAb L01 (red AlexaFluor 568) and CD68-AlexaFluor488

(green) with brilliant yellow-white indicating colocalisation, and (E) single stained with control lgG3 with protocol and settings identical to (C). Lu = lumen; LC = lipid core; Med = media. Blue = TOPRO = nuclei (DNA). Fine arrowheads, inflammatory cells at edge of lipid core. Scalebars = distances indicated.

METHODS

LDL modification

Human LDL (Calbiochem, Beeston.UK) was dialyzed against PBS overnight at 4°C to remove EDTA. Following dialysis, copper oxidised LDL (Cu-oxLDL) was prepared by incubating LDL with freshly prepared copper sulphate (10 μΜ) at 37°C for 24 hours. The oxidization reaction was stopped by adding an excess amount of chelating resin Chelex 100 (Sigma-Ald ich, Pooie, UK). The extent of LDL modification was estimated by the relative electrophoretic mobility of the LDL particles using pre-cast 0.6% agarose, 1 .0% Barbital Buffer gels (Beckman Coulter, Fullerton, CA). Malondialdehyde-conjugated LDL (MDA-LDL) was prepared as described by Palinski et al (30).

Briefly, MDA was synthesized by the acid hydrolysis of malondialdehyde bisdimethylacetal. Dialysed human LDL was incubated for 3 hrs at 37°C with 0.5M MDA at a ratio of 100μΙ MDA/mg LDL. The extent of modification was estimated with a carbonyl assay (31). Briefly, 0.1 mg of modified LDL was added to 500μΙ of 4mg/ml bovine serum albumin (BSA). 700μΙ of 0.1 % (wt/vol) 2,4- dinitrophenylhydrazine (DNP) in 2M HCI was then added and incubated for 1 hour at RT. Following addition of 500μΙ 30% trichloroacetic acid, the mixture was centrifuged for 5 min at 18,000 g at 4°C, and then left on ice for 30 minutes. The precipitate was then washed three times with ethanol/ethyl acetate (1 :1 , vol/vol). The pellet was finally dissolved in 1 ml of 8M guanidine hydrochloride, 13mM EDTA and 133mM Tris pH 7.4 and the OD read at 365nm. The results were expressed as moles dinitrophenol (DNP)/mg of protein (mol/mg) using an extinction coefficient of 21 mM-1/cm ^"1 . The synthesis and analysis of MDA conjugated to human serum albumin (HSA) was prepared using similar protocols.

Trypsinisation of LDL was performed by adding 10 μΙ washed and diluted (60 μΙ beads in 100 μΙ of PBS) bovine pancreatic trypsin-conjugated agarose beads (Sigma-Aldrich), to 0.5 ml of 342μg/ml LDL. 45μΙ aliquots were removed at intervals between one minute and overnight, added to 3 μΙ of 1 mg/ml bovine pancreas-derived trypsin inhibitor (Sigma Aldrich) and centrifuged at 9100g for 5 min. Control LDL was processed in the same way but without the addition of trypsin. The extent of LDL modification was confirmed by electrophoresis as above.

Hypochlorite modification of LDL (Calbiochem, Beeston, UK) was achieved by incubating LDL (1 mg protein/ml in PBS) with reagent-grade sodium hypochlorite (1 mM, Sigma-Aldrich) to a final concentration of 1 mg LD /ml of 1 μΜ hypochlorite solution for 24h. Hybridomas and selection of mAb

Hybridomas were generated by fusing Sp2/0 myeloma cells with splenocytes from a one-year-old female LDL receptor deficient mouse that had been fed a high fat diet from the age of 6 weeks old to give a serum cholesterol 25-30 mmol/l. Hybridoma culture supernatants were screened by ELISA for the presence of antibodies that bound with native LDL or oxLDL (each coated onto plates at ^g/ml). Hybridomas with differential reactivity to native LDL and oxLDL were then subcloned twice prior to further characterization. The isotypes of mAb were determined using a mouse mAb isotyping kit (IsoStrip, Roche Applied Science, Burgess Hill, UK). The hybridoma secreting an lgG3K isotype control mAb HK-PEG-1 (anti-influenza virus) was purchased from the European Collection of Cell Cultures, Porton Down, Salisbury, UK). MAb were purified from culture supernatant using a protein-G affinity chromatography column.

Enzyme-linked immunosorbent assay (ELISA)

Maxisorb 96-well plates (Nunc, ThermoFisher Scientific, Waltham, MA) were coated with 50 μΙ antigen/well at 4°C overnight. Non-adherent material was washed out, and then the plates were blocked with 2% BSA/PBS for 1 hour at room temperature (RT). Appropriately diluted culture supernatant or purified mAb was added and incubated for 1 hour at RT. Plates were then washed, and wells incubated with goat anti-mouse Ig (SouthernBiotech, Birmingham, AL) at 1 :5000 dilution. After further washing, antibody binding was detected with 3,3',5,5'-tetramethylbenzidine (Sigma), and the reaction was stopped with 0.5M H ₂ S0 ₄ . The optical density was then measured with a Synergy HT microplate reader (Biotek, USA) at wavelength 450nm.

Variation in salt concentration was achieved by diluting mAb L01 in different concentrations of NaCI ranging from 2.15 M to 18mM. For testing the effect of altering pH, mAb L01 was dialysed into 0.1 M sodium acetate buffer using Slide-A-Lyzer Dialysis Cassettes (Thermo Scientific, Rockford, IL) and then mixed with 0.1 M sodium acetate solutions prepared using combinations of 0.1 M sodium carbonate and 0.05M sodium hydroxide solutions to produce working buffers of pH ranging from 3 to 11 .

Equal loading of ELISA plates with native or modified LDL was confirmed in parallel wells using goat polycolonal anti-apoB (Abeam, Cambridge, UK). In the case of assessing mAb L01 binding to immobilized scFv constructs, similar coating of wells with different constructs was checked by measuring binding of anti-myc mAb 9E10 (Sigma-Aldrich) in parallel wells.

Sequencing of mAb L01 V _H variable region

The mAb L01 V _H variable region was amplified from hybridoma mRNA by RT-PCR using a series of degenerate oligonucleotide primers, which had been prepared by Eurofin MWG Operon (Ebersberg, Germany). The sense primer was based upon the leader sequence (32), and used in combination with a universal antisense constant region oligonucleotide (33;34) to amplify the leader, VDJ and start of the constant region. PCR products were cloned into pCRII (Invitrogen) and transformed into TOP10 E.coli. Individual colonies were selected and DNA isolated by miniprep (Qiagen) prior to sequence analysis using an ABI 3730 automated DNA analyzer. The VH-(D)-JH was analyzed by comparison with the International MunoGeneTics information system® (IMGT) (http://www.imgt.org) (35). The veracity of the heavy chain sequence was confirmed by mass-spectrometry of tryptic peptides of purified mAb L01 IgG. The mAb L01 V _L variable region was sequenced in a similar manner.

Isolation of an anti-idiotype scFv antibody

The Tomlinson I library (Medical Research Council, Cambridge, UK) is a non-immunized human scFv phagemid library that was constructed in plT2 (HIS6 myc tag) from V _H (V3-23/DP-47 and JH4b) and VK (012/02/DPK9 and JK1). The diversified residues were based mainly on positions that are diverse in the primary repertoire and which are known from crystallographic studies to make contact with antigen. These are: V _H amino acids H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97; and V _L amino acids L50, L53, L91 , L92, L93, L94, L95, and L96. The total diversity of the library is about 1 .4 x 10 ⁸ (36;37). The functional scFvs bind both protein A and protein L, and either one of these or the myc or 6 histidine tags can be used for detection, purification or immobilization.

The phagemid-containing TG1 (T-phage resistant E. coli) cells were amplified in a 2xYT microbial medium (Sigma) containing ampicillin (100 μg/ml) and glucose (1 % w/v) and grown with shaking at 37°C until the OD ₆₀₀ was about 0.4. The exponentially grown library-containing TG1 was then infected with KM13 helper phage and selected by ampicillin (100 μg/ml) and kanamycin (50 μg/ml, Sigma). The expanded phagemid library was then precipitated with PEG/NaCI (20% w/v polyethylene glycol 6000, 2.5 M NaCI) and titered on TYE plates (15 g Bacto-Agar, 8 g NaCI, 10 g Tryptone, 5 g Yeast Extract in 1 L dH ₂ 0) containing ampicillin (100 μg/ml) and glucose (1 %).

A magnetic bead based method was employed to select L01 binding phage-scFv. This was carried out in 3 steps. Initially an aliquot of the library phage (5x10 ¹² PFU) was pre-cleared of irrelevant binding phage-scFv by rotating with blank human anti-mouse magnetic beads (Invitrogen) for 1 hour at room temperature. The unbound fraction was then further pre-cleared against human anti-mouse Ig beads preincubated with control lgG3K (1 ^g/20C^I beads). For positive selection of L01 binding phage-scFv, the unbound fraction was then transferred to magnetic beads coated with L01

(1 .^g/20C^I beads) and incubated rotating for 1 hour at room temperature. Selected phages were then eluted with trypsin (1 mg/ml in PBS) for 15 minutes at RT, and used to infect logarithmic grown TG1 (OD ₆₀₀ about 0.4) by incubating at 37°C in a water bath without shaking. One tenth of the infected TG1 were then titrated in serial 10-fold dilution for phage titrating. The remaining infected TG1 were spread on a 15cm x 15cm TYE plates containing ampicillin (100μg/ml) and grown at 37°C overnight. Phagemid-containing bacteria were scraped from the agar surface into 2xYT media containing ampicillin (100 μg/ml) and glucose (1 %) and grown at 37°C with shaking until OD ₆₀₀ reached 0.4. The propagated phagemid was then rescued by MK13 helper phage and precipitated with PEG/NaCI (20% (w/v) polyethylene glycol 6000, 2.5M NaCI). The phage produced was then titrated for the next round of selection. In total three rounds of selection were carried out with a decreased concentration of LO-1 (^g/200ml beads) used in rounds 2 and 3. Following incubation of human anti-mouse beads with LO-1 , potential non specific binding sites on the beads were blocked with 3%BSA/PBS. Beads were washed between stages with 0.05 % Tween 20-PBS

After each round of selection, 95 colonies on titrating plates were randomly chosen and grown in 100 μΙ 2xYT containing 100 Dg/ml ampicillin and 1 % glucose in microtiter plates. The ELISA plates were coated with L01 or control lgG3 KH-PEG-1 (10 μg/ml in PBS) overnight at 4°C and were then blocked with 2% BSA/PBS for 2 hours at RT. 10 μΙ PEG precipitated phage in 100DI of 2% BSA/PBS was added into each well for 1 hour of incubation. After 3 washes with PBS-Tween 20, the bound phage was probed with a horseradish peroxidase conjugated monoclonal antibody that reacts specifically with the M13 phage major coat protein product of gene VIII, followed by TMB substrate. Phages giving an OD450 in mAb L01 coated wells three times higher than those in the control lgG3 coated wells were considered to be specifically reactive with mAb L01 .

All clones positive in the ELISA screen were sequenced. Phagemid double stranded DNA was purified by miniprep kits (Qiagen cat# 27104) and subjected to sequencing on an automated ABI3730 sequencing system operated by Imperial Central Sequencing Service. The primers used for sequencing were: VH: CGA CCC GCC ACC GCC GCT G and VK: CTA TGC GGC CCC ATT CA (Eurofins MWG Operon, Ebersberg Germany). The data were analyzed with open source software eBioX ( UD.//www ¾biok:fo;:naHc¾.oiryebiox/). Sequence-comparisons of the CDR regions of the H3 anti-idiotype with human and mouse apolipoprotein B-100 (Swiss-Prot: P041 14.1) was performed with Clustal W algorithm to create multiple alignments.

As the TAG stop codon is suppressed in TG1 E. coli, both scFv and pill are expressed. To produce scFv alone, selected phages were transferred to HB2151 (a non-suppressor strain) which was induced with 1 mM isopropyl β-D-thiogalactoside (Sigma). Antibodies were then isolated on Ni-NTA agarose (Qiagen, Hamburg, Germany) and eluted with imidazole (250 mM). Purity was confirmed by SDS- PAGE stained with Coomassie blue and a His-tag ln-gel Staining kit (Invitrogen, Paisley, UK).

Generation of domain swaps between scFv

To construct the H3 V _H /C12 V _L and C12 V _H / H3 V _L , the light chains of each construct were swapped. To release the light chain from the vector, purified DNA from both H3 and C12 scFv was digested with Xho1 (Promega) and Not1 (Promega) in the following reaction: 5Dg DNA, 2n\ 10x buffer, 2n\ BSA (1 mg/ml), 2.5U Xho1 , 2.5U Not1 adjusted to 20μΙ with dH ₂ 0. Samples were incubated at 37°C for 1 hour. Vector samples were subsequently treated with CIAP in the following reaction: 10x buffer 4μΙ, restriction digested DNA 20μΙ, 0.01 U CIAP, volume adjusted to 40μΙ with dH ₂ 0. Samples were incubated for 1 hour at 37°C. Fragments were separated on 1 % agarose gels and the appropriate bands gel purified before ligation using the following reaction: 4μΙ 5x ligation reaction buffer, 45fmol ends of insert, 15fmol ends of insert DNA and 0.1 U T4 ligase (Invitrogen), with the final volume adjusted to 20μΙ with dH ₂ 0. After overnight incubation at 16°C, DNA was purified by

phenol/chloroform/IAA extraction and ethanol precipitation. Purified, ligated DNA was transformed into TOP10 E.coli and selected on ampicillin (100μg/ml) plates.

Generation of mutant H3

Mutant constructs were created using "Site Directed Mutagenesis™" kit (Agilent Technologies, Santa Clara, USA) on the C12V _L /H3V _H domain swap background. Mutagenic primers were designed by hand as specified in the kit's manual for single and multiple mutations. Forward and reverse mutagenic primers contained the desired mutation and annealed to the same sequence on opposite strands of the plasmid carrying the C12V _L /H3V _H domain swap insert. The primers were designed to be 25-45 bases in length with melting temperature (T _m ) below 78°C to prevent secondary structure formation. The T _m for each primer was estimated using the formula: T _m = 81 .5+0.41 (%GC) - 675/Λ/ - %mismatch, where N = the primer length; %GC and %mismatch are whole numbers. Optimally primers contained a minimum 40 % GC content and ended in two G or C bases. The desired mutation was located in the middle with 10-15 bases of correct sequence on both sides. All primers were manufactured by Eurofins MWG Operon, (Ebersberg, Germany). To ensure that the mutagenic primers functioned adequately through annealing and amplification at the correct temperature, 25 cycles of PCR were performed for each primer pair on vector dsDNA containing the C12V _L /H3V _H domain swap scFv sequence. The results were analysed through gel electrophoresis (0.7% agarose) for the presence of a band approximately 5600bp in size that represented the amplified construct with the desired mutation. Finally, the fidelity of the mutation was validated by sequencing. Mutant scFv were amplified and isolated as above.

Peptide synthesis

Peptides representing sequences of H3 were synthesized by Cambridge Peptides (Cambridge, UK). All peptides were purified by HPLC and quality controlled by mass spectroscopy. Immunocytochemistry

Sections of aortic roots from wild-type (WT) and Ld\f' ^~ mice fed a low fat diet to the age of 22 weeks were prepared as described (9). Human carotid endarterectomy specimens were collected with consent and Institutional and National Ethical approval. Specimens were transported and dissected on ice, with a transport time of under 30 mins. The tissue was inspected and areas of classical morphology with fibrous cap, lipid core and shoulder identified and snap-frozen in liquid nitrogen and stored at -80°C. Then the sections were cryosectioned at -20° C, embedded in OCT and fixed in isopropanol. Cryosections were equilibrated in double-distilled water, then PBS, blocked in 10% normal goat serum for 30 mins, and then incubated in 10 μg/ml biotinylated L01 or biotinylated isotype control at 4°C overnight in a humidified chamber. Antibody was then decanted, followed by a rinsing in PBS. Then bound antibody was detected with goat anti-mouse AlexaFluor 568 (Molecular Probes, Invitrogen Paisley, UK) at 1 :200 for 120 min at 21 °C. TOPRO-3 (Molecular Probes, Invitrogen Paisley, UK) was used as a nuclear counterstain for general inspection, and macrophages were detected by 1 :100 anti-mouse CD68-AlexaFluor488 (MCA1957, Serotec, Oxford, UK) or 1 :50 anti- human CD68-FITC (green) (clone KP-1 , F7135, Dako, Ely, UK). Sections were mounted in 80% glycerol /PBS and imaged by confocal microscopy (Zeiss LSM510 Meta) under standard settings as before (38).

EXAMPLE 1 - Generation and characterization of mAb L01

Hybridomas were generated using splenocytes from a one-year-old female LDL receptor deficient mouse that had not been immunized. Of 950 clones screened, only ten IgG antibodies with differential reactivity with native versus Cu-oxLDL were identified. From these, an lgG3K mAb, designated L01 , was selected for further characterization. As shown in Figure 1 A and B, mAb L01 reacted with Cu- oxLDL but only minimally with native LDL, and reactivity with Cu-OxLDL was inhibited by including 50 μg/ml Cu-oxLDL in the buffer. mAb L01 reactivity with malondialdehyde (MDA)-conjugated LDL, which is known to be a prominent epitope in copper-oxidised LDL preparations (30), was then tested. As shown in Figures 1 C, mAb L01 showed increasing binding over time of LDL exposure to MDA, in parallel with carbonyl adduction (Figure 1 D). The failure of mAb L01 to react with MDA conjugated to human serum albumin (not shown) signifies that it does not simply react with MDA as a hapten.

Whether LDL stored over two months in the absence of EDTA would become "automodified" in the presence of atmospheric oxygen to reveal the L01 epitope was checked and it was found that this was not the case (not shown). mAb L01 also failed to react with LDL incubated with hypochlorite for 24 hours (not shown). Furthermore, trypsin treatment for up to 2 hours did not reveal the L01 epitope, despite clear evidence for proteolysis upon electrophoresis (not shown). Trypsin treatment of MDA- LDL for up to two hours failed to influence mAb L01 binding (not shown).

The immunoglobulin variable region of the heavy chain of L01 was sequenced and then examined by searching the IMGT database. It belongs to the VH1 family (V1 -54) and is germline (Figure 2). EXAMPLE 2 - Generation ofscFv anti-idiotype H3

Failure to western blot MDA-LDL with mAb L01 suggested that it may react with a conformational epitope. In view of the challenge of defining a conformational epitope on a molecule as complex as

LDL, it was decided to explore L01 binding further by developing an anti-idiotype antibody against the antigen binding site (36;39;40). Affinity-purified mAb L01 was used to select human scFv antibodies from an unbiased phage library. Three rounds of selection resulted in the isolation of a single scFv antibody, designated H3, which reacted with mAb L01 but not with lgG3KControl mAb HK-PEG-1 (Figure 3A). Also shown in Figure 3A is the failure of mAb L01 to bind a control scFv, designated C12, isolated from the same scFv library. As shown in Figure 3B, H3 but not C12 was able to neutralize the binding of mAb L01 to MDA-LDL with an IC50 of 0.8-4.0 g/ml, indicating that H3 binds mAb L01 at or close to its recognition site for MDA-LDL. Changing the salt concentration had a very similar effect on binding of mAb L01 to H3 as to MDA-LDL (Figure 3C). There was also a similar profile of pH sensitivities with optimal binding at pH 6. Interestingly mAb L01 binding to H3 had a slightly broader pH range compared to mAb L01 binding to MDA-LDL (Figure 3D).

The CDR sequences of H3 were compared with the linear sequences of human and mouse apoB-100 using the Clustal W algorithm. As shown in detail in Figure 4, there are significant homologies between H3 CDRH2 and CDRL2 and peptide sequences of mouse and human ApoB. Surprisingly, the relevant amino acids in H3 are situated not just amongst those that are diverse within the scFv library but also amongst constant determinants, particularly in the CDRL2 region.

As a prelude to site-directed mutagenesis of H3, the V _H and V _L domains were swapped between H3 and C12. This established that the binding of mAb L01 to H3V _H chain paired with C12V _L was similar to native H3, although the signal was slightly reduced at all concentrations of mAb L01 (Figure 5A). In contrast, no binding of L01 occurred to a construct containing the H3V _L chain paired with the C12V _H (Figure 5A). Specificity of the L01 -H3 interaction was demonstrated by the virtual absence of binding of control lgG3 mAb HK-PEG-1 to any of the four constructs (Figure 5B).

The H3 and C12 scFv differ in 15 out of the 18 amino acids which are diverse in the Tomlinson I library (Table 1 A). Since the V _H of H3 was dominant for mAb L01 binding, a series of individual point mutations were carried out on the H3V _H /C12V _L background, changing amino acids in the H3V _H to those of C12, and amino acids in the C12V _L to those of H3. Thus, this strategy looked for mutations in the H3 V _H that that might reduce mAb L01 binding and mutations in the C12V _L as that might increase it. As summarized in Table 1 B, mAb L01 binding was severely reduced or completely abrogated by any change in the five H3 CDRH2 or four H3 CDRH3 amino acids to those found in C12. Although substituting the whole H3V _L with that of C12 had only a minor effect (Figure 5A), individual amino acid swops were surprisingly influential, with significant and paradoxical decreases in mAb L01 binding caused by changing either of two amino acids in C12 CDRL2 to those on H3, but significant increases in binding of mAb L01 to constructs with individual H3 substitutions at three of the four C12 CDRL3 sites (Table 1 C). Taken together, the data strongly suggest that mAb L01 recognises H3 as a

conformational structure rather than binding a particular linear peptide motif. This view is further supported by failed attempts by ELISA to bind mAb L01 to linear peptides, alone or in combination, covering CDRH2 (VSDISGSNTITYA), CDRH2-FR2 interface (YADVKGRFTIS), CDRH3

(CAKDDDAFDYWG) or CDRL2 (IYYASALQSGVP) (data not shown). Furthermore, these same peptides, either alone or in combination, failed to inhibit mAb L01 binding to H3 (data not shown).

Table 1. Differences between scFv H3 and scFv C12 and effects of amino acid substitutions

A. VH and VL CDR sequences of scFv H3 and C12 with divergent residues in bold

B. Effects of exchanging individual H3V _H amino acids in the H3V _H /C12V _L construct with the amino acids that differ in C12. Binding by mAb L01 was tested by ELISA.

C. Effects of exchanging individual C12V _L amino acids in the H3V _H /C12V _L construct with the amino acids th differ in H3. Binding by mAb L01 was tested by ELISA.

EXAMPLE 3 - Immunocytochemistry of atherosclerosis

Preliminary ELISAs established that mAb L01 binding was unaffected by treatment of MDA-LDL with isopropanol and this was therefore used as fixative for immunostaining arterial tissue. As shown in Figure 6, mAb L01 but not lgG3 control showed obvious staining of both the macrophage rich developing atherosclerotic lesion and the media of the aortic root of hyperlipidaemic Ldlr mice but not normocholesterolaemic WT mice, consistent with tissue accumulation of oxidised LDL in Ldlr mice. Importantly, staining of Ldlr ' ^' mouse aortic root was inhibited by MDA-LDL or scFv H3 but not by scFv C12 (Figures 6D-F). Taken together with the inhibition of mAb L01 binding to MDA/Cu-oxLDL by Cu- oxLDL (Figure 1 B) or H3 (Figure 3B), these data support the specific recognition of oxidized LDL by mAb L01 through cognate antibody-antigen recognition rather through non-specific binding.

When applied to human carotid endarterectomy tissue, mAb L01 showed more restricted staining compared with staining of the Ld\f' ^~ mouse tissue. Thus, mAb L01 but not lgG3 control stained intracellular deposits in macrophages, and occasional extracellular deposits adjacent to the edge of the lipid necrotic core, but medial tissue was not stained (Figure 7). This pattern is consistent with that expected of the distribution of heavily oxidized LDL in a human atherosclerotic plaque. DISCUSSION

These examples describe the characterization of mAb L01 , a novel IgG autoantibody that was selected as reacting with oxidized LDL. The approach of generating mAb from the spleens of atherosclerotic mice provides a means to dissect the naturally occurring autoantibody repertoire against modified forms of LDL. Earlier reports have highlighted the high frequency of hybridomas secreting IgM germ-line encoded antibodies reacting with phosphorylcholine and other oxidation- specific determinants (28;29). Out of an interest in the IgG response, the inventors deliberately fused splenocytes from a relatively old (ie one year) mouse and screened for clones secreting IgG rather than IgM antibodies. The low frequency of hybridomas secreting IgG autoantibodies against oxidized LDL is similar to the experience of Witztum et al who failed to isolate such clones (28). Quite why hybridomas making IgG anti-oxidised LDL antibodies are hard to generate is not clear, as these antibodies are readily detected in serum. Clearly, however, the antibodies found following splenocyte fusion are dependent upon the antigens used for screening, and ongoing work in our laboratory is also focused on other novel autoantibodies derived from Ldlr' ^' mice with specificities distinct from mAb L01 .

The copper-oxidized LDL preparation that was used to screen the hybridomas is a complex preparation likely to contain MDA-LDL as well as other oxidative modifications (30). Whilst mAb L01 was subsequently found to react well, if not better, with LDL conjugated selectively with MDA compared to crude Cu-oxLDL, there was no reactivity with other forms of modified LDL tested, such as by trypsin digestion or incubation with hypochlorite. The inventors found that mAb L01 did not bind MDA-conjugated human serum albumin, indicating that the LDL carrier is as important as the MDA hapten.

Staining of atherosclerotic plaques has been used by several groups to validate the binding of antibodies against modified LDL with naturally occurring epitopes in tissues (22-24;26;28;29). In a similar way, the inventors have found that mAb L01 specifically binds antigen accumulated in the aortic root of Ld\f' ^~ mice and in foam cells and extracellular aggregates adjacent to the necrotic core within clinical pathological carotid tissue (AHA Type IV or so-called vulnerable plaque with an advanced lipid necrotic core and thin fibrous cap). In this area, it is thought that macrophages further modify mildly modified LDL, and are killed by their load of heavily modified LDL. The selectivity of L01 staining for this zone of the plaque is consistent with current ideas about plaque LDL handling. Most of the extracellular L01 staining (i.e. not obviously around nuclei) was also cell-shaped, reflecting either that the nucleus was out of section (in the z-plane) or reflecting release from dying cells (41). Importantly, the isotype control did not share this pattern. Corroborating this interpretation was the colocalisation of L01 specific staining (not found in the isotype control) in the Alexafluor 568 (red emission) channel with classical oxidized LDL-associated autofluorescence in the green emission channel (nor shown) (42). It was notable that the diffuse staining including the media that was observed in the Ld\f' ^~ mouse tissue was not seen in the human carotid, perhaps reflecting a more widespread microscopic accumulation of oxidised LDL in the hypercholesterolaemic mouse.

It is interesting that mAb L01 is of the lgG3 isotype, as mouse lgG3 is a relatively minor isotype in mouse serum (0.1 -0.2 mg/ml, ~2% of total IgG; compared with lgG2a/c ~0.4 mg/ml). Mouse lgG3 activates complement well (lgG3,2a,2b> lgG1), probably as cooperative binding of its Fc domains gives lgG3 a functional polyvalency for C1 q similar to IgM (43-45). In contrast to other mouse IgG isotypes, lgG3 does not ligate proinflammatory Fey receptors (lgG1 ,2a/c,2b» lgG3 in mice)

(13;46;47). Thus, lgG3 antibodies may play a similar homeostatic role to IgM in scavenging modified LDL, and, furthermore, are able to perform feedback enhancement of antibody responses (48). By virtue of smaller monomeric size (~150 kD versus ~900 kD for IgM), lgG3 is likely to have greater tissue penetration and distribution than IgM (49). Secretion of lgG3 is thought to be by B1 cells and to occur early in the adaptive immune response by thymus-independent (Tl) class-switching with minimal hypermutations (45;50).

Low density lipoprotein is a 18-25 nm diameter particle (Mr 550 kD) consisting of a hydrophobic triglyceride and cholesterol ester core, surrounded by a phospholipid shell wrapped by a single apolipoprotein apoB molecule which in human has 4,536 amino acids (51). The failure of isopropanol to influence the antigenicity of MDA-LDL for mAb L01 suggests that the epitope is made up from protein rather than lipid determinants. To gain more insight into the binding interaction between mAb L01 and the large and complex LDL particle, the inventors screened an scFv library for an antiidiotype antibody that might mimic some or all of the epitope on MDA-LDL by carrying an "internal image" of the antigen. It should be noted that mutating amino acids in the binding site of a mAb against ApoB has previously been used to gain insight into the molecular basis of LDL interaction with the LDL receptor (52), but an anti-idiotype approach to dissecting an anti-oxidised LDL antoantibody does not appear to have been previously adopted. The inventors succeeded in isolating H3, a scFv antibody that specifically neutralizes the binding of mAb L01 to MDA-LDL and which has amino acid sequences in the H3 CDRH2 and CDRL2 with strong similarity to peptide stretches in mouse and human ApoB. It is notable that whilst mAb L01 showed similar physico-chemical constraints in binding H3 as MDA-LDL, the pH tolerance of H3 binding was greater, suggesting that H3 may actually be a better fit for the mAb L01 antigen binding site than MDA-LDL. This may be related to mAb L01 V _H being germline and lacking hypermutated sequences.

Further dissection of mAb L01 -H3 interactions in comparison with a non-binding control scFv C12 showed that the mAb L01 bound well to H3V _H paired with C12V _L , demonstrating the importance of V _H residues. Site-directed mutagenesis failed to reveal particular H3V _H amino acids critical for binding, and the major reduction in binding that occurred with any substitution suggests that CDRH2 and CDRH3 are both involved. It is notable that CDRH3 contains a stretch of six amino acids with four aspartates (DDDAFD) and this may well supply a critical negative charge. It should be noted that the failure of mAb L01 to bind to H3V _L paired with C12V _H does not exclude involvement of the V _L , since amino acid substitutions in the two diverse amino acids at CDRL2 abolished binding. Furthermore, SALQSG, which are the amino acids in CDRL2 with homology to apoB, are conserved between H3 and C12. Thus residues on H3V _L , particularly in CDRL2, may contribute to the putative

conformational epitope that mAb L01 reacts with.

Taken together, these observations raise the possibility that H3 has focal sequence and charge similarities to the site on oxidised LDL that binds mAb L01 . Linear peptides spanning H3 CDRH2 CDRH3 and CDRL2 were not able to inhibit mAb L01 binding to H3, consistent with the importance of conformation. Therefore definitive evidence for the residues on H3 that mAb L01 reacts with will require further studies. Whilst there are precedents for co-crystallization of Fab with anti-idiotype (53), this would be technically extremely difficult for mAb L01 bound to oxidized LDL. However an alternative approach for directly comparing mAb L01 binding to H3 and oxidized LDL is the use of hydrogen-deuterium exchange mass spectrometry (54).

Whilst phage display techniques have previously been used to isolate human IgG single-chain autoantibodies reacting with oxidized LDL (20;55), mAb L01 is the first spontaneously arising IgG anti- oxidised LDL autoantibody to be reported from mice.

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