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WO1999023222A1 - Bibliotheques d'anticorps a boucle de cysteine et procedes de fabrication et d'utilisation - Google Patents

Bibliotheques d'anticorps a boucle de cysteine et procedes de fabrication et d'utilisation Download PDF

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Publication number
WO1999023222A1
WO1999023222A1 PCT/GB1998/003255 GB9803255W WO9923222A1 WO 1999023222 A1 WO1999023222 A1 WO 1999023222A1 GB 9803255 W GB9803255 W GB 9803255W WO 9923222 A1 WO9923222 A1 WO 9923222A1
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WIPO (PCT)
Prior art keywords
cysteine
repertoire
noose
specific binding
nucleic acid
Prior art date
Application number
PCT/GB1998/003255
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English (en)
Inventor
Jane Katharine Osbourn
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Cambridge Antibody Technology Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Cambridge Antibody Technology Limited filed Critical Cambridge Antibody Technology Limited
Priority to AU97531/98A priority Critical patent/AU9753198A/en
Publication of WO1999023222A1 publication Critical patent/WO1999023222A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons

Definitions

  • the present invention relates to libraries which encode and express antibody variable domains containing modified complementarity determining regions (CDRs) and the use of such libraries for providing novel binding molecules.
  • CDRs complementarity determining regions
  • a wide range of ligands for G-protein linked receptors contain two cysteine residues separated by between four and ten amino acids.
  • the structure has been solved and the cysteines have been found to define the boundary of a surface oriented hydrophilic loop defining what is designated a cysteine-noose .
  • These nooses have been implicated by mutagenesis in defining the specificity of the binding of the polypeptides to their respective receptors (Nature Structural Biology 1995, 2 266-268) .
  • Other potential cysteine-nooses have been identified in gp41 of HIV, bovine serum RNase and endochitinase .
  • Many other receptor ligands have similar loops, even if not necessarily bound by cysteines, for example a neutralising epitope of the cytokine transforming growth factor beta (TGF ⁇ l) is thought to consist of an eight amino acid loop.
  • TGF ⁇ l cytokine transforming growth factor beta
  • ligands containing cysteine-nooses include G-protein linked receptor ligands such as endothelins ETl, ET2 , ET3 and SRTX-a, human chorionic gonadotropin (hCG) , C5a anaphylatoxin, calcitonin, calcitonin-gene related peptides a and b, amylin, vasopressin, oxytocin and somatostatin.
  • the cysteine nooses of these ligands comprise loops of from 4 to 10 amino acids between (and not including) two cysteine residues (i.e. providing nooses of from 6 to 12 amino acids in size) .
  • Tyrosine kinase receptor ligands also have disulphide bridge linked motifs, for example the epidermal growth factor family, all have the structural motif: CX T CX 3 _ ⁇ CX ⁇ _ 1 XCX 5 GX C
  • Cysteine bridged or cyclic peptides have been isolated against a variety of target molecules from constrained peptide libraries displayed on the gene III or gene VIII protein of filamentous bacteriophage (Wright et al , Biotechnology 1995, 13, 165-169; O'Neil et al , Proteins 1992, 14, 509-515; McLafferty et al , Gene, 1993, 128, 29-36; Koivunene et al, J. Biol. Chem., 1993, 268(27) 20205-20210 Livnah et al , Science, 1996, 273, 464-471; Wrighton et al, Science, 1996, 273, 458-464).
  • a cysteine bridged peptide with a loop of 8 amino acids was identified as a mimetic of erythropoietin (Wrighton et al, Livnah et al, ibid) .
  • cysteine-nooses in the CDR3 of antibody heavy chains is permitted, as demonstrated by a number of antibodies which have been isolated from phage display antibody libraries.
  • Such antibodies include specificities against carcinoembryonic antigen (CEA) and a human mab (Fog-1) , as reported in Griffiths et al . (1993), Human antibodies with high specificity from phage display libraries, EMBO 12, 725-734.) .
  • CCA carcinoembryonic antigen
  • Fog-1 human mab
  • a total of 52 examples of two cysteine residues within VH CDR3s have also been identified in naturally occurring antibodies
  • the present invention provides a repertoire of specific binding members, which binding members comprise a antibody variable domain, wherein said repertoire comprises at least 10 J members which each carry a cysteine noose in at least one of their complementarity determining regions (CDR) present in said variable domains.
  • the repertoire may comprise antibody Fab or scFv fragments.
  • the antibody variable domain is a heavy chain variable domain.
  • the cysteine noose comprises from 2 to 8 amino acids between its cysteine residues.
  • the cysteine noose is present in the third CDR of the variable domain and where this is the case it is preferred that at least about 30% of said members have a tyrosine residue immediately following the C-terminal cysteine of the noose.
  • a particularly preferred VH third CDR sequence comprises the consensus sequence GGXXC (X) n CX ⁇ XGG wherein G is glycine, X is any amino acid, C is cysteine, X y is tyrosine in at least about 30% of the members, and n is from 3 to 7.
  • the invention provides a nucleic acid library encoding the repertoire of any one of the preceding claims.
  • the invention also provides a method of selecting a specific binding member capable of binding a target antigen, which method comprises : screening said antigen against a repertoire of specific binding members which binding members comprise an antibody variable domain, and wherein said repertoire comprises at least 10 3 members which each carry a cysteine noose in at least one of their complementarity determining regions (CDR) present in said variable domains; and selecting a specific binding member which is able to bind to the target antigen.
  • CDR complementarity determining regions
  • Such nucleic acid may be expressed, optionally after being manipulated to provide, for example, co-expression of a complementary variable domain.
  • Methods of then invention may be used to select specific binding members for a range of antigens, including a cancer antigen, a cytokine or cytokine receptor, a growth factor or growth factor receptor, a hormone or hormone receptor, or a viral antigen or viral receptor.
  • the invention also provides methods of preparing a cysteine noose repertoire of the invention which methods include: a) providing a source of nucleic acid encoding an antibody variable domain; b) mutating said variable domain so as to introduce two codons which encode cysteine within a CDR of said domain, said codons being separated by one or more codons selected at random; and c) recovering the mutated nucleic acid encoding said repertoire .
  • the invention also provides certain specific binding members per se, which are novel by virtue of their noose size, location or target antigen, including binding members which are specific for
  • the invention also provides a novel method of selecting peptide ligand mimetics capable of binding a target antigen, including the various antigens mentioned herein.
  • this aspect of the invention provides a method for obtaining a peptide ligand mimetic capable of binding a target antigen which comprises:
  • step (d) making at least one peptide mimetic comprising a sequence determined in step (c) .
  • the process further includes the step of:
  • the process may optionally further include one or more of the following steps, in any order:
  • Figure 1 is a bar chart showing the length of amino acid sequences between two cysteine residues of VH CDR3s identified in naturally occurring antibodies.
  • Figure 2 shows the locations within the antibodies analysed in Figure 1 in relation to the start and end of CDR3.
  • Figure 3 shows the frequency of amino acid usage within the VH CDR3s analysed N-terminal and C-terminal to the cysteine noose.
  • Figure 4 shows the strategy for the construction of a cysteine noose library of the invention.
  • Figure 5 shows inhibition of MlP-l binding to CD4 cells by specific binding members of the invention.
  • Figure 6 shows inhibition of binding of MlP-l ⁇ by a cysteine noose peptide mimetic (ML2CA5) obtained in accordance with the invention (panel a) , together with a control (panel b) .
  • ML2CA5 cysteine noose peptide mimetic
  • Figure 7 shows inhibition of binding of MlP-l ⁇ by a cysteine noose peptide mimetic (ML4CA11) obtained in accordance with the invention (panel a) , together with a control (panel b) .
  • ML4CA11 cysteine noose peptide mimetic
  • 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, 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.
  • the members of the pair have the property of binding specifically to each other.
  • types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate . This application is concerned with antigen-antibody type reactions.
  • An tibody is concerned with antigen-antibody type reactions.
  • immunoglobulin 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 and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies .
  • antibody should be construed as covering any specific binding member or substance having a binding domain with the required specificity.
  • this term covers antibody fragments, derivatives, functional equivalents and homologues of 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.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S.
  • 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 associate 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) .
  • bispecific antibodies may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol . 4, 446-449 (1993)), eg 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, A) . , 3655-3659, (1991) .
  • Bispecific diabodies as opposed to bispecific whole antibodies, may also be particularly 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.
  • variable domains of antibodies will comprise three CDRs and sufficient N- and C- terminal residues to form an an antigen binding domain, if need be in association with a complementary variable domain.
  • an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen.
  • 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 comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) .
  • 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.
  • binding members of the invention or nucleic acid encoding such binding members will be, in accordance with the present invention.
  • Members and nucleic acid will 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 vi tro or in vivo .
  • 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.
  • a repertoire is a collection of either nucleic acid sequences encoding specific binding members or specific binding members themselves which sequences or specific binding members share common structural features, which features serve to carry a collection of varying sequences.
  • the common structural features will be antibody framework regions which will be broadly similar in length and sequence to each other within each member of the repertoire .
  • the varying structural features will generally be one or more of the CDRs carried by the antibody framework regions.
  • a repertoire will comprise at least 10 3 , preferably at least 10 5 , for example at least 10 6 or 10 7 members.
  • the maximum size of a repertoire is generally governed only by the technology available to make them but it is certainly possible to obtain repertoires of up to 10 14 , for example up to 10 12 individual members .
  • a repertoire of specific binding members in which at least 10 3 members comprise a cysteine noose in at least one of their CDRs. It is not essential for all members of the repertoire to comprise a cysteine noose.
  • the repertoire could comprise 10 4 members of which 10% each carried the cysteine noose. However it is preferred that a substantial portion of the members, preferably at least 90% and more preferably at least 95%, 98% or 99% of the members carry a cysteine noose.
  • the repertoire may comprise a single antibody chain or two antibody chains, for example a light chain and a heavy chain. In the case of two antibody chains, these chains may be linked in-frame so as to provide for expression of single chain Fv molecules .
  • the antibody heavy chain variable domain is particularly important in providing antigen binding properties of an antibody and therefore it is preferred that the members of the repertoire carry a cysteine noose in a heavy chain CDR. However this is not to exclude the presence of cysteine nooses in light chain CDRs. " Cys teine Noose " and " Cysteine Loop "
  • cysteine noose refers to a sequence of amino acids which include an N-terminal and C-terminal cysteine residue
  • a cysteine loop, or "loop” refers to a sequence of amino acids bounded by, but not including, the terminal cysteines.
  • the cysteine noose may be present in any CDR of an antibody heavy or light chain.
  • the third CDR is preferred and particularly preferred is the third CDR of the heavy chain variable domain.
  • Cysteine nooses may be of any size subject only to the maximum size of the CDR.
  • CDRs may be defined by reference to Kabat et al , Sequences of Proteins of Immunological Interest, Fourth Edition, US Department of Health and Human Services, 1987, and updates thereof, now available on the Internet (http://immuno.bme.nwu.edu).
  • the maximum and minimum sizes (number of amino acids) of CDRs may therefore be as follows:
  • CDR2 of the VL domain is generally 7 amino acids in size. Cysteine nooses may therefore be present in any of the above CDRs ranging from a loop size (ie length of sequence between and not including the two cysteine residues) of from 1 to n-2 where n is the maximum size of the CDR as set out above.
  • the location of the cysteine noose within a CDR may be varied.
  • the N-terminal cysteine of the noose will be the second, third, fourth or fifth residue of the CDR.
  • the C-terminal cysteine will desirably be at least one to five, for example preferably 2, 3 or 4 residues away from the end of the CDR.
  • the skilled person may either select N- and C- terminal locations for cysteines by reference to the start and finish points of a particular CDR, or by reference to only one point and a selected loop size, or a combination of both.
  • Cysteine noose repertoires of the invention may comprise members with uniform cysteine loop sizes or ranges of cysteine loop sizes either within a single or a mixture of CDRs. Loops sizes may be selected subject to the maximum CDR sizes and may therefore range from 1 to 28, preferably from 3 to 15, for example from 2 to 8 such as 3 , 4, 5, 6 or 7 residues in size, subject to the total noose size being no greater than that of the CDR in which it is located.
  • the cysteine noose is present in the third CDR of a heavy chain variable domain.
  • a repertoire of such domains may be prepared which is biased to favour the presence of a tyrosine residue immediately adjacent and following the C-terminal residue of the cysteine noose.
  • VH CDR3 libraries Another feature which may be incorporated into VH CDR3 libraries is the presence of a pair of glycine residues at the N- and/or C- termini of the CDR3 , and preferably both termini. These glycines may be present in the repertoire in conjunction with or as an alternative to the tyrosine bias mentioned above.
  • Repertoires according to the invention may comprise a diverse range of binding members which are capable of binding a diverse range of target antigens. Such libraries may be used for screening against any desired antigen.
  • a repertoire of the invention may comprise a preselected subgroup of binding members with a non- random bias towards a particular target antigen. For example, this may be brought about by providing a repertoire in which one or more of the CDRs are of a defined sequence and only one CDR is varied between members of the repertoire.
  • the repertoire could be derived from specific binding members with a particular ligand noose and the members of the repertoire would be varied in the noose sequence .
  • Repertoires according to the invention may be used to screen and select specific binding members capable of binding in a specific manner to a target antigen. Once a specific binding member has been isolated from the repertoire the nucleic acid encoding the specific binding member may be obtained and used for expression of the nucleic acid to obtain further copies of the selected specific binding member. Nucleic acid from the specific binding member may be manipulated in any manner known per se in the art.
  • variable domain of the specific binding member may be isolated and linked to additional antibody sequences such as sequences encoding all or part of a light or heavy chain constant region.
  • sequences may be linked to a complementary variable domain so that on expression an antibody two chain variable region is obtained.
  • Manipulation also includes methods in which the CDRs of the selected antibody are combined with other framework regions so as to produce a reshaped antibody. This may be accomplished by CDR grafting as shown in for example EP-A-239400 or framework grafting as shown in EP-B-549581.
  • Specific binding members may be selected by any suitable system.
  • the preparation of repertoires of specific binding members and the use of the repertoires for selecting antigens is as such well known in the art and this underlying technology is not part of the present invention as such.
  • the above references also disclose in particular repertoires prepared in phage or phagemid libraries, and such type of libraries may conveniently be used in the present invention.
  • the above references may also be used by way of guidance to those of skill in the art in methods of preparing repertoires according to the present invention.
  • the preparation of repertoires according to the present invention will differ primarily by virtue of the use of techniques specifically designed to introduce cysteine nooses into a CDR of a variable domain.
  • a suitable and preferred technique is the use of oligonucleotide mutagenesis in which oligonucleotides are used to amplify nucleic acid encoding all or part of an antibody variable domain wherein the oligonucleotide comprises a CDR replacement sequence comprising two codons for cysteine separated by one or more codons selected at random.
  • the oligonucleotide comprises a CDR replacement sequence comprising two codons for cysteine separated by one or more codons selected at random.
  • one or more codons selected at random it is meant that of the codons separating the two cysteine residues at least one but not necessarily all are selected at random such that a repertoire of specific binding members according to the invention is obtained.
  • One or more of the codons flanking the N-terminal and/or C-terminal may also be selected at random.
  • Random selection also includes partially random selection, for example to bias a codon in favour of a particular residue or group of residues.
  • a cysteine noose replacement primer will additionally comprise sufficient sequence flanking either or both of the codons encoding the cysteine noose such that the oligonucleotide may be used as a primer on a source of nucleic acid sequences in which members of the source comprise target sequences substantially homologous to said flanking regions of the oligonucleotide. This allows the preparation of a repertoire according to the invention by schemes such as that illustrated in Figure 4 or methods analogous thereto.
  • the source nucleic acid may comprise any suitable source from which binding members of the invention can be made through methods such as that described herein. Suitable sources include existing repertoires of specific binding members or primary sources of nucleic acid such as spleen cell mRNA.
  • the source of nucleic acid may be a selected group of specific binding members which are specific for a target antigen.
  • a group may for example be a group in which no cysteine nooses are present and the method of the invention is carried out in order to introduce a cysteine noose in a defined CDR.
  • the source nucleic acid may also be one which encodes a single specific binding member including a specific binding member comprising a cysteine noose.
  • Such a source may be used for introducing additional cysteine nooses into the nucleic acid or to introduce variations in an existing cysteine noose.
  • the methods of preparing repertoires of specific binding members of the invention and methods of screening such repertoires may be used to select specific binding members for a variety of antigens.
  • Specific binding members may be selected to bind to a target to antagonise or agonise the function of that target.
  • target antigens include cytokines, growth factors, hormones and viral antigens, or any of their cognate receptors, or a cancer antigen.
  • a cancer antigen is an antigen expressed by tumour cells which is otherwise normally absent or expressed only at a low level in or on the surface of cells normally present in the human body.
  • Cytokines include MlP-l ⁇ , MlP-l ⁇ , MIP-2, RANTES , tumour necrosis factor (eg TNF ⁇ ) , interferons ⁇ , ⁇ or ⁇ , interleukins (including any of IL-1 to IL-18, such as IL-2, IL-6 and IL-12) , MCP-1, 2 and
  • TGF ⁇ macrophage inhibitory factor
  • Epo Erythropoietin
  • Tpo growth factor
  • Growth factors include TGF ⁇ , TGF ⁇ , CSF, GCSF, PDGF, FGF, EGR, VEGF, BDNF and SCF .
  • Viral antigens include HIV antigens such as gpl60/l20, HBV surface or core antigens, HAV antigens, HCV antigens, HPV (eg HPV16) antigens, HSV-1 or -2 antigens, Epstein Barr virus (EBV) antigens, Kaposi ' s sarcoma virus antigens, neurotropic virus antigens, adenovirus antigens, cytomegalovirus antigens, polio myelitis virus antigens.
  • HIV antigens such as gpl60/l20, HBV surface or core antigens, HAV antigens, HCV antigens, HPV (eg HPV16) antigens, HSV-1 or -2 antigens, Epstein Barr virus (EBV) antigens, Kaposi ' s sarcoma virus antigens, neurotropic virus antigens, adenovirus antigens, cytomegalovirus antigens, polio myelitis virus antigen
  • Hormones include steriod hormones such as estradiol, hCG, endothelins such as ETl ET2 ET3 and SRTX-a, thyroid stimulating hormone, growth hormone, adrenocorticotropin, follicle stimulating hormone, prolactin, luteinizing hormone, insulin, glucagon, amylin, calcitonin CGRP a and b, oxytocin and somatostatin.
  • cysteine noose specific binding members of the invention which bind specifically to cell surface receptors including those in the categories mentioned above form another important and preferred aspect of the invention in view of the presence of cysteine nooses in the ligands which bind some of these receptors.
  • Cancer antigens include CEA, alpha fetal protein (AFP) , neu/HER2 , polymorphic endothelia mucin (PEM) , N-CAM and Lewis Y.
  • AFP alpha fetal protein
  • PEM polymorphic endothelia mucin
  • N-CAM Lewis Y.
  • cysteine-nooses Some enzymes, for example bovine serum Rnase and endochitinase, and viral coat proteins, for example gp41 of HIV, also possess cysteine-nooses.
  • cysteine-nooses in molecules such as these suggests a potential for generating antibodies with surface loops which mimic these which could block viral infection events, or inhibit enzymic reactions.
  • the invention provides a cysteine noose specific binding member, in particular a VH domain comprising a cysteine noose in its third CDR, and optionally in association with a cognate VL domain, capable of binding in a specific manner to MlP-l ⁇ receptor.
  • the binding member may be in the form of an antibody fragment, for example an scFv or Fab fragment.
  • compositions of such specific binding members particularly pharmaceutical compositions as defined below. Such compositions may be of use in therapies of the human or animal body, for treating or alleviating conditions which benefit from anti-MIP-l ⁇ receptor therapy. Such conditions include HIV infection.
  • compositions may also be of use in vi tro for investigating mechanisms of infection of HIV on CD4+ cells to which such specific binding members bind, or for investigating potential HIV therapeutics by determining the degree to which such therapeutics modulate (e.g. agonise or antagonise) the binding of such specific binding members to CD4+ cells .
  • the present invention provides for the first time a repertoire of such antibodies and the ability to select antibodies against a whole range of antigens. Accordingly, in a further aspect of the invention individual specific binding members are provided apart from those already in the art. Thus specific binding members of the invention include those against the target antigens mentioned above. Specific binding members may be provided in isolated form.
  • Detectable labels include radiolabels such as 131 I or 99 Tc, which may be attached to antibodies 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.
  • Functional labels include substances which are designed to be targeted to the site of a tumour to cause destruction of tumour tissue.
  • Such functional labels include toxins such as ricin and enzymes such as bacterial carboxypeptidase or nitroreductase, which are capable of converting prodrugs into active drugs at the site of a tumour.
  • Binding members of the invention comprising such labels may be used in methods of diagnosis and treatment of tumours in human or animal subjects, particularly solid tumours which have a necrotic centre. These tumours may be primary or secondary solid tumours of any type including, but not limited to, cervical, ovarian, prostate, lung, liver, pancreatic, colon and stomach tumours .
  • Specific binding members obtained or obtainable from the repertoires or by the methods of the present invention when designed for therapeutic use may be administered to a patient in need of treatment via any suitable route, for example usually by injection into the bloodstream or directly into the a particular location such as the site of the tumour.
  • F(ab') 2 antibody fragments may be used for both tumour imaging and tumour treatment .
  • Specific binding members obtained or obtainable by the present invention will usually be prepared for administration in the form of a pharmaceutical composition, which may comprise at least one component in addition to the specific binding member.
  • compositions according to the present invention may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, 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 .
  • 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.
  • 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.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • 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.
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • Other treatments may include the administration of suitable doses of pain relief drugs such as non-steroidal anti-inflamatory drugs (e.g. asprin, paracetamol, ibuprofen or ketoprofen) or opitates such as morphine, or anti-emetics.
  • pain relief drugs such as non-steroidal anti-inflamatory drugs (e.g. asprin, paracetamol, ibuprofen or ketoprofen) or opitates such as morphine, or anti-emetics.
  • the present invention further provides an isolated nucleic acid encoding a specific binding member of the present invention.
  • Nucleic acid includes DNA and RNA.
  • the present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise isolated nucleic acid of the invention encoding a single specific binding member.
  • the present invention also provides a recombinant host cell which comprises one or more constructs as above.
  • a nucleic acid encoding any specific binding member as provided itself 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.
  • the isolation and purification of the cysteine noose antibodies will be carried out under conditions in which the cysteine bonds of the CDR will form disulphide bridges. These conditions are normally found when the protein is exported from cells. For example in E. coli , the periplasm is an oxidising environment which promotes disulphide bridge formation. Antibodies and their fragments, such as ScFvs contain a number of internal disulphide bridges encoded within the framework regions, and these are formed when scFv are expressed in cells such as by bacteria. Disulphide bridges formed within the noose will be more exposed to the oxidising environment than internal disulphide bridges, since they are present on the surface of the specific binding partner.
  • nucleic acid molecules and vectors according to the present invention may be provided isolated and/or purified, e.g. from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes origin other than the sequence encoding a polypeptide with the required function.
  • Nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic .
  • 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 and many others.
  • a common, preferred bacterial host is E . coli .
  • 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.
  • Mol ecular Cloning a Labora tory Manual : 2nd edition, Sambrook et al . ,
  • 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.
  • 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 .
  • 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.
  • 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 .
  • the first step to obtain such mimetics is the same as the first step of obtaining specific binding members of the invention, namely screening a target antigen against a library of the invention.
  • One or more of the selected binding members which bind the target will then be selected, and the sequence of the cysteine noose determined. Most conveniently, the sequence will be determined by sequencing the nucleic acid encoding the specific binding member, although other methods may be used if required. Once the sequence of the cysteine noose is determined, a peptide mimetic comprising said sequence may be made. Conveniently, this may be done by solid phase peptide synthesis although recombinant production may also be used, utilising methods known as such in the art .
  • the peptide mimetic will consist of the cysteine noose, and optionally include at its N- and/or C-terminii, additional amino acid residues.
  • the total size of the mimetic will be comparable to a the size of a CDR, i.e. from 5 to 30 amino acids in size. More preferably, the total size of the mimetic will be from 8 to 20 amino acids in length.
  • the location of the N- terminal cysteine is preferably at position 2 to 5 of the mimetic, where 1 is the N-terminal residue.
  • the location of the C-terminal cysteine is usally at X-l to X-4, where X is the C- terminal residue.
  • the peptide mimetics of Example 8 below have glycine a their N- terminal and C-terminal positions. This is because the mimetics are derived from a library where the noose CDR was designed to contain this feature. Those of skill in the art will appreciate that this is not essential and that other peptide mimetics may be made, based on the production of other cysteine noose specific binding members .
  • Peptide mimetics selected in accordance with the invention may be screened against the target antigen for ligand binding activity.
  • the present method of selecting peptide ligand mimetics particularly when selected from CDR3 cysteine noose libraries (more particularly VH cysteine noose libraries) , provide a means to select a different and potentially more effective population of peptide ligands than direct display of similar cysteine noose ligands on the surface of bacteriophage.
  • the CDR3 of an antibody chain projects above the surface of the bulk of the antibody molecule and thus may project the cysteine noose more effectively than when such a noose is directly linked to a surface component of a bacteriophage .
  • a peptide mimetic may be made for any desired application.
  • Applications incude formulation of the mimetic into a pharmaceutial composition comprising the mimetic and one or more suitable carriers or diluents, such as those discussed above.
  • the mimetic may be used in monomeric form or covalently linked to other compounds, for example compounds such as biologically compatible polymers, for example polyethylene glycol based molecules designed to enhance stability of the molecules.
  • the peptides may also be polymerised to provide multiple copies of the mimetic in a single molecule, for example in the form of tandem repeats.
  • the mimetic may also be used as a basis for preparing further mimetics, for example by preparing variants which include an altered loop length, or one or more amino acid substitutions. Deletions and insertions in the cysteine loop will alter the loop length. Such alterations may be made in any part of the loop.
  • these are desirably made at either the N- terminus or C-terminus of the loops, adjacent to the cysteine residues.
  • These may be made in an iteratitve process, e.g. by inserting a range of amino acids at a first position, selecting a mimetic variant with improved properties (e.g. binding affinity or biological stability) compared to the starting variant, and then repeating this process with said variant one or more times.
  • one or more deletions may be made in the loop. Again, it may be desirable to make single deletions, select a deleted variant, and then repeat the process to obtain variants with one or more deletions.
  • substitutions, deletions or insertions will be constrained by the size of the loop, although generally from 1 to 4 , such as 1 or 2 may be made. Such altered mimetics may then be tested for binding against the target antigen, and any with a desired binding activity may be selected, manufactured and used as described above.
  • Mimetic variants which utilize an alternative loop constraint may also be made.
  • Various means of constraint are known as such in the art, for example the use of a beta-turn loop (Saragovi et al , Science, 1991, 253, 792-795) or other means (e.g. see Kieber- Emmons et al , ibid) such as a cyclic lactam.
  • EXAMPLE 1 Design of a cysteine-noose scFv repertoire.
  • EXAMPLE 2 Construction of a cysteine-noose scFv repertoire.
  • EXAMPLE 3 Selection of the cysteine-noose repertoire on cell membranes .
  • EXAMPLE 4 Selection of the cysteine-noose repertoire on whole cells .
  • EXAMPLE 5 Selection of ligand inhibitors from the cysteine-noose repertoire .
  • EXAMPLE 6 Demonstration of the importance of the cysteine noose in antibody binding to antigen.
  • EXAMPLE 7 Construction of further cysteine noose repertoires with loop sizes of 4 to 10 amino acids.
  • EXAMPLE 8 Generation of peptide mimetics from a cysteine noose library.
  • the human antibody sequence database (V-BASE sequence directory, Tomlinson, Williams, Corbett, Cox and Winter (1995) MRC Centre for Protein Engineering, Cambridge UK) was surveyed for the presence of naturally occurring antibodies which contain two cysteines within the CDRs. No such motifs were found in any light chain CDRs, or in CDR1 and CDR2 of heavy chains. 52 examples of the occurrence of two cysteines were found in heavy chain CDR3 ' s from 1200 surveyed. The number of amino acid residues present between the two cysteines in each case was found and the majority of the antibodies contained four residues between the two cysteines. The distribution of the number of residues is shown in Figure 1.
  • the positioning of the C(X) n C motif within the CDR3 was also assessed.
  • the number of residues between the end of the framework 3 region of the heavy chain gene segment and the first cysteine was most commonly 3 or 4 , ( Figure 2a) .
  • the number of residues between the second cysteine residue and the start of the JH region of the heavy chain gene segment ranged from 0 to 9 , and was most commonly between 2 or 4 residues ( Figure 2b) .
  • X is any amino acid and X ⁇ is any amino acid, but with a bias designed to favour the presence of a tyrosine residue.
  • FR3 represents the end of the framework three region of the heavy chain gene segment, and JH represents the start of the JH region of the heavy chain gene segment .
  • EXAMPLE 2 CONSTRUCTION OF A CYSTEINE-NOOSE SCFV LIBRARY
  • the cysteine-noose library construction was designed using an exisiting scFv antibody repertoire as a starting template. This template was then PCR amplified to give a VL gene segment repertoire and a VH gene segment repertoire .
  • the VH gene segment repertoire was amplified using a primer which was designed to incoporate a cysteine-noose into the VHCDR3.
  • the two repertoires were then PCR assembled to give full length scFv gene segments which were cloned into a phagemid vector.
  • the PCR strategy is outlined in Figure 4.
  • the starting material for construction of the cysteine-noose scFv library was DNA prepared from the large scFv antibody library as described in Vaughan et al . , Nature Biotechnology 1996, 14, 309-314 (Human Antibodies with sub-nanomolar affinities isolated from a large non-immunised phage display library) .
  • Cells from 200 ⁇ l of a bacterial glycerol stock of this library were pelleted at 10 000 x g for 2 minutes in a microcenrifuge .
  • Miniprep DNA was then prepared from these cells using a Promega Wizard Miniprep kit.
  • a vacuum was applied to pull the resin/lysate mix into the minicolumn and 2 ml of Column Wash Solution (80mM potassium acetate, 8.3mM Tris-HCl, pH 7.5, 40mM EDTA in 55% ethanol) was then added and drawn through the vacuum.
  • the resin was dried under vacuum for 30 seconds and the minicolumn was then removed from the vacuum apparatus.
  • the minicolumn was centrifuged at 10 000 x g in a microcentrifuge for 2 minutes to remove any residual Column Wash Solution. 50 ⁇ l of water was then added to the minicolumn and the minicolumn was centrifuged at 10 000 x g to elute the DNA. DNA was stored at -20°C.
  • This primer was used in conjuction with the primer LMB3 to amplify a repertoire of heavy chains which were mutated in the VH CDR3.
  • LMB3 CAGGAAACAGCTATGAC 3' (SEQ ID NO : 5 )
  • the JH Cys Back primer has been designed to prime on the VH JH region with enough homology to VH Cys loop For to allow PCR assembly:
  • Oligonucleotide JH Cys Back (as coding squence) : 5' TGGGGCCA(AG)GG(AG) ACCCTGGTC 3' (SEQ ID NO : 8 )
  • Primer 1 (10 mM) 2.5 ⁇ l
  • Primer 2 (10 mM) 2.5 ⁇ l dH 2 0 34.5 ⁇ l
  • VH and VL repertoires generated by PCR were electrophoresed on a 1.5% low melting point agarose gel. Bands corresponding to the VH and VL gene segments were excised from the gel and the DNA extracted from the bands using Promega Wizard DNA purification columns, exactly as described in the manufacturer's instructions. DNA was eluted from the column in 50 ⁇ l of dH 2 0.
  • amplified CDR3 mutated heavy chain repertoire was combined. This was used in an assembly amplification after the addition of reaction buffer to IX, dNTP's to 200nM and 5 units Taq polymerase. Amplifications consisted of 7 cycles of 94 °C for 1 min, 65°C for 4 min. 5 ⁇ l of each assembly was used as the template in a 'pull-through' amplification using the primers FTAG and LMB3. Amplification conditions consisted of 25 cycles of 94°C for 1 min, 55°C for 2 min and 72°C for 1 min, followed by 10 min at 72°C.
  • the pull-through amplification product was separated through 1.5% agarose-TAE.
  • a PCR product of the expected size (1.1 Kb) was visualised and excised from the gel.
  • the gel fragment was then melted by incubation at 70 °C for 15 min and the DNA purified from the gel using a Promega Magic PCR DNA purification column. 1ml of resin was added to the melted gel, mixed by inversion and then passed down a DNA minicolumn.
  • the column was washed with 2ml of 80% isopropanol and spun at top speed in a minifuge for 20 seconds to dry the column. DNA was then eluted from the column in 50 ⁇ l dH 2 0 and the DNA recovered by centrifugation in a minifuge at top speed for 20 seconds.
  • the purified DNA fragment was digested with the restriction endonucleases Sfi I and Not 1 (NEB) then ligated (Amersha ligation system) into Not I/Sfi I digested phagemid vector pCANTAB6 (McCafferty, J. et al . , 1994. Selection and rapid purification of murine antibody fragments that bind a transition- state analog by phage display. Applied Biochemistry and Biotechnology, 47: 157-173) .
  • the ligation product was used to transform electrocompetent TGI cells, plated out on 2YTAG (2YT media supplemented with 100 ⁇ g/ml ampicillin and 2% glucose) plates and incubated overnight at 30°C. Approximately 7.2 x 10 8 individual clones were generated. Plates were scraped into a total of 9ml of 2TY containing 15% glycerol and 1.5 ml aliquots were made and stored at -70°C.
  • the cysteine-noose repertoire was selected to isolate antibodies which bind cell membranes of a specific cell type. Phagemid particles were recovered from the repertoire as follows. 500 ml prewarmed (37°C) 2TYAG in a 21 concical flask was inoculated with approximately 3 x 10 10 cells from a glycerol stock culture of the library. The culture was grown at 37°C with good aeration until the OD 600nm reached 0.7. M13K07 helper phage (Stratagene) was added to the culture to a multiplicity of infection (moi) of approximately 10 (assuming that an OD 600nm of 1 is equivalent to 5 x 10 8 cells per ml of culture) .
  • moi multiplicity of infection
  • the culture was incubated stationary at 37°C for 15 minutes followed by 45 minutes with light aeration (200rpm) at the same temperature.
  • the culture was centrifuged and the supernatant drained from the cell pellet.
  • the cells were resuspended in 500 ml 2YTAK (2YT media supplemented with 100 ⁇ g/ml amplicillin and 50 ⁇ g/ml kanamycin, and the culture incubated overnight at 30°C with good aeration (300 rpm) .
  • Phage particles were purified and concentrated by three polyethylene glycol (PEG) precipitations (Sambrook, J. , Fritsch, E.F., & ⁇ Maniatis, T. (1990). Molecular Cloning - A Laboratory Manual. Cold Spring Harbour, New York) and resuspended in PBS to 10 13 transducing units (tu)/ml.
  • Phage induced from the cysteine-noose antibody repertoire were initially selected on intact cells. 100 ⁇ l of PBS containing 10 6 cells prepared from the breast of a 42 year old female were added to 100 ml of phage (2 x 10 12 ) prepared from the cysteine-noose library which had been premixed with 800 ⁇ l of 3% marvel in PBS (MPBS) . Phage and cells were incubated at 37°C for 1 hour with occasional mixing. This cell type floats to the top of the liquid allowing unbound phage to be removed from under the cells with a pipette.
  • Glycerol stock cultures from the first round of selection were rescued using helper phage to derive phagemid particles for the second round of selection.
  • 250 ⁇ l of glycerol stock was used to inoculate 50 ml 2TYAG broth, and incubated in a 250 ml conical flask at 37°C with good aeration until the OD 600nm reached 0.7.
  • the cells were resuspended in 50 ml prewarmed 2TYAK, and the culture incubated overnight at 30°C with good areation. Phage particles were purified and concentrated by PEG precipitation (Sambrook et al . , 1990) and resuspended in PBS to 10 13 tu/ml .
  • Phage induced from the first round of selection were selected a second time as described above, except that for the second round of selection cell membrane preparations immobilised on a Nunc immunosorb tube were used as the selection surface. Immobilisation was carried out overnight at 4°C, using 1 ml of membrane preparation at a total protein concentration of 10 ⁇ g/ml in PBS. c) Growth of individual colonies for immunoassay. Individual colonies from the third round of selection were used to inoculate lOO ⁇ l of 2TYAG into individual wells of 96 well tissue culture plates (Corning) . Plates were incubated at 30°C overnight with moderate shaking (200 rpm) . Glycerol to 15% was added to each well and these master plates stored at -70°C until ready for analysis.
  • M13K07 was added to each well to an moi of 10 and incubated stationary for 15 min then 45 min with gentle shaking (100 rpm) , both at 37°C.
  • the plates were centrifuged at 2000 rpm for 10 min and the supernatant removed. Each cell pellet was resuspended in lOO ⁇ l 2TYAK and incubated at 30°C overnight.
  • Each plate was centrifuged at 2000 rpm and the 100 ⁇ l supernatant from each well recovered and blocked in 20 ⁇ l 18%M6PBS (18% skimmed milk powder, 6 X PBS) , stationary at room temperature for 1 hour. Meanwhile, flexible microtitre plates which had been blocked overnight stationary at 37°C with either 100 ⁇ l 10 ⁇ g/ml of plasma membrane preparation from either the cell type used for the selection, or an unrelated cell type, were washed in PBS and blocked for 2 hours stationary at room temperature in MPBS. These plates were then washed in PBS and 50 ⁇ l of preblocked phage added to each well . The plates were incubated stationary at room temperature, after which the phage was poured off.
  • the nucleotide sequences of the membrane-specific antibodies were determined by first using vector specific primers to amplify the inserted DNA from each clone. Cells from an individual colony on a 2TYAG agar plate were used as the template for a PCR amplification of the inserted DNA using the primers LMB3 and fdtetseq. Amplification conditions consisted of 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2 min, followed by 10 min at 72 °C. The PCR products were purified using a PCR Clean-up Kit (Promega) into a final volume of 50 ⁇ l H 2 0.
  • each insert preparation was used as the template for sequencing using the Taq Dye-terminator cycle sequencing system (Applied Biosystems) .
  • the primers mycseqlO and PCR-L-LINK were used to sequence the light chain of each clone and PCR-H-Link and pUC19reverse to sequence the heavy chain.
  • CD4+ cells Preparation of human CD4+ cells from blood. Mononuclear cells were prepared from a 50ml buffy coat using Ficoll-Paque (Pharmacia) density gradient centrifugation (600g for 20 min at 20°C) . CD4+ cells were then isolated from the 1.5 x 10 8 recovered cells using a Biotex CD4 column, following the manufacturer's instructions, although PBS /2% foetal calf serum (FCS) was used throughout. Eluted cells were pelleted at 600g for 5 min and resuspended in 300 ⁇ l PBS / 2% FCS. 8.3 x 10 c cells were recovered using this procedure. The recovered cells were analysed by flow cytometry and approximately 59% of the cells were found to be CD4+.
  • FCS foetal calf serum
  • the cysteine-noose phage antibody library was induced as described in Example 2 part a) . Approximately 1 x 10 12 phage induced from the cysteine-noose antibody library were then selected on 1 x 10 6 CD4+ cells in a total volume of 1ml MPBS.
  • Phage and cells were incubated at room temperature for 1 hour, and cells were then pelleted at low speed (2000 rpm) in a minifuge for 2 min. The cells were washed three times in 1 ml
  • Lysates were used to directly infect 5 ml of an exponentially growing culture of E coli in 2TY. Infection and plating out was carried out as previously described. Phage selected from the first round of cell selection were induced as described and second and third rounds of selection were performed exactly as the first round.
  • EXAMPLE 5 SELECTION OF LIGAND INHIBITORS FROM THE CYSTEINE-NOOSE REPERTOIRE .
  • This example describes use of the biotin tyramine signal transfer selection procedure (as described in patent application GB 9712818.5 and in Osbourn, J.K. et al . , Pathfinder selection; in vivo isolation of novel antibodies. Immunotechnology, submited) , in a two step manner to isolate antibodies from the cysteine-noose antibody repertoire which inhibit binding of the initial guide molecule to cells.
  • the cysteine-noose repertoire has been designed to potentially provide a population of scFv's which are biased towards binding cellular receptors. This selection procedure could be applied to the generation of inhibitors to any ligand, small molecule, or antibody.
  • the process involves an initial first stage of the selection to biotinylate and capture phage antibodies which bind around the site of ligand binding.
  • the biotinylated phage are then used directly, without amplification, to guide a second stage of selection using cells in the absence of ligand.
  • antibodies which bind in the ligand binding site can be biotinylated by signal transfer procedure, then captured and screened for inhibition of ligand binding.
  • chemokine MlP-l ⁇ which is a ligand for CCRK1, CCRK4 and CCRK5 chemokine receptors which are found on CD4+ cells
  • the beads were washed three times in 1ml PBS / 0.1% Tween 20 (PBST) .
  • the beads were then resuspended in 100 ⁇ l lOOmM triethlyamine for 10 minutes at 37°C, which elutes phage from the beads.
  • TEA was then neutralised with 50 ⁇ l 1M Tris-HCl pH 7.4. After elution the beads were taken out of solution and the supernatant containing the biotinylated phage was taken for use in the second phase of the selection .
  • the population of biotinylated phage which had been recovered from the first stage of the selection were added directly to 1 x 10 fj CD4+ lymphocytes in a total volume of 200 ⁇ l in MPBS. Phage were allowed to bind to the cells for 1 hr at room temperature, and cells were then washed 3 times in 1 ml PBS. Cells were pelleted at 4000 rpm for 2 min in a minifuge between washes. A further aliquot of the scFv phage library (2 x 10 12 phage) was then added to the cells in 1ml MPBS and allowed to bind for 1 hr at room temperature .
  • Antibody sequences were determined as previously described.
  • MI4 GGLRCSTTRCYYGG SEQ ID NO: 39
  • MI5 GGNSCCPQBCYNGG SEQ ID NO: 40
  • MI2 has a truncated three residue loop, rather than four residues, demonstrating the potential for variation in loop length, whilst retaining cell binding activity.
  • EXAMPLE 6 DEMONSTRATION OF THE IMPORTANCE OF THE CYSTEINE NOOSE IN ANTIBODY BINDING TO ANTIGEN.
  • cysteine noose in antibody-antigen interactions is demonstrated by in vi tro mutagenesis of one or both of the cysteines which contribute to the noose.
  • oligonucleotides reverse complement sequences
  • mutagenesis mutated residues are marked in bold
  • a R C amino acid (SEQ ID NO: 43)
  • a R amino acid (SEQ ID NO: 45)
  • oligonucleotides are used individually in combination with LMB3 (SEQ ID NO : 5 ) in a PCR, using conditions exactly as described in Example 2b, to generate VH gene segments with either one, or both cysteines mutated to serine (s) .
  • Template for this PCR is minipreped DNA from phagemid clone CL1.
  • the CL1 VL gene segment is PCR amplified from minipreped DNA from phagemid clone CL1 using the primers JH (SEQ ID NO : 8 ) and Fdtseqtag (SEQ ID NO: 9) .
  • VH and VL gene segments are then PCR assembled using a pull through reaction exactly as described in Example 2b, using the primers FTAG (SEQ ID NO:10) and LMB3 (SEQ ID NO : 5 ) .
  • the resultant PCR products are digested with the restriction endonucleases Sfi I and Not I, then ligated into Not I/Sfi I difested phagemid vector pCANTAB 6.
  • the clones are designated:
  • Phage ELISA is carried out exactly as described in Example 2d.
  • EXAMPLE 7 CONSTRUCTION OF AN EXPANDED CYSTEINE-NOOSE REPERTOIRE.
  • Example 1 above describes the design of a cysteine-noose scFv repertoire which includes in the VH CDR3 a cysteine noose of 6 amino acids in size (i.e. a 4 amino acid loop) .
  • the cysteine-noose repertoire was expanded to cover a wider range of potential noose lengths.
  • the PCR template used was DNA prepared from the large scFv antibody library exactly as described in Example 2.
  • the repertoire of VL chains generated using primers JH Cys Back (SEQ ID NO: 8) and Fdtseq tag (SEQ ID NO: 9) as described in Example 2 were used as partners for the new set of mutated VH chains .
  • Cysteine noose-encoding sequences containing open reading frames included sequences coding for the following:
  • EXAMPLE 8 USE OF THE CYSTEINE NOOSE LIBRARY TO GENERATE PEPTIDE LIGAND MIMETICS.
  • Example 5 described the selection of ligand inhibitors of MlP-l ⁇ from the cysteine-noose repertoire.
  • Six different scFv antibody clones were isolated which gave varying degrees of inhibition of MlP-l ⁇ binding to CD4+ cells.
  • the VH CDR3 ' s of five of these clones were taken and used as the basis for the design of peptides covering the cysteine-noose region.
  • Peptides were synthesised which included the cysteine-noose region of the VH CDR3. Analagous peptides were also generated which contained serine residues in place of the noose cysteines. Serine was chosen because it possesses a similar R group to cysteine except that the SH group is replaced by OH, hence serine is incapable of forming a di-sulphide cross-linked noose.

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Abstract

L'invention concerne des bibliothèques qui comprennent un répertoire d'éléments liants spécifiques, lesdits éléments liants comprenant un domaine variable d'anticorps. Ce répertoire comprend au moins 103 éléments dont chacun comporte une boucle de cystéine dans au moins une des régions déterminant la complémentarité (CDR) présentes dans lesdits domaines variables. Ces éléments liants, que l'on peut utiliser pour fournir des agonistes ou des antagonistes de cibles telles que les cytokines ou d'autres protéines, peuvent aussi servir de base pour obtenir des peptides mimétiques de boucle de cystéine.
PCT/GB1998/003255 1997-10-31 1998-10-30 Bibliotheques d'anticorps a boucle de cysteine et procedes de fabrication et d'utilisation WO1999023222A1 (fr)

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EP2075256A2 (fr) 2002-01-14 2009-07-01 William Herman Ligands ciblés
US20140303084A1 (en) * 2011-06-10 2014-10-09 Biogen Idec Ma Inc. Pro-Coagulant Compounds and Methods of Use Thereof
US9486507B2 (en) * 2011-06-10 2016-11-08 Biogen Ma Inc. Pro-coagulant compounds and methods of use thereof
AU2012267484B2 (en) * 2011-06-10 2017-03-23 Bioverativ Therapeutics Inc. Pro-coagulant compounds and methods of use thereof

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