EP1242458A2 - Elements de liaison specifiques du cerveau - Google Patents

Elements de liaison specifiques du cerveau

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Publication number
EP1242458A2
EP1242458A2 EP00979747A EP00979747A EP1242458A2 EP 1242458 A2 EP1242458 A2 EP 1242458A2 EP 00979747 A EP00979747 A EP 00979747A EP 00979747 A EP00979747 A EP 00979747A EP 1242458 A2 EP1242458 A2 EP 1242458A2
Authority
EP
European Patent Office
Prior art keywords
seq
specific binding
antibody
binding member
domain
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP00979747A
Other languages
German (de)
English (en)
Inventor
Carl Webster
Jane Osbourn
George Ward
Karen Miller
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MedImmune Ltd
Original Assignee
Cambridge Antibody Technology Ltd
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.)
Filing date
Publication date
Application filed by Cambridge Antibody Technology Ltd filed Critical Cambridge Antibody Technology Ltd
Publication of EP1242458A2 publication Critical patent/EP1242458A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present invention relates to specific binding members directed to the brain, including specific binding members able to pass through the blood brain barrier or which are directed to areas of brain inflammation or blood brain barrier breakdown.
  • Panels and mixtures of antibody are provided, also individual antibody molecules and VH and VL domains, along with methods of obtaining specific binding members by selection using libraries displayed on the surface of filamentous bacteriophage.
  • BBB blood brain barrier
  • the BBB presents a substantial hurdle for the delivery of many drugs, particularly proteins such as antibodies and cytokines or growth factors into the central nervous system.
  • drugs particularly proteins such as antibodies and cytokines or growth factors into the central nervous system.
  • lipid-soluble molecules which have a molecule weight from 400-600 Da
  • drugs that originate from either biotechnology or classical small molecule pharmacology undergo negligible transport through the intact BBB.
  • novel transporter molecules or receptors associated with the BBB represents a potential target for enhancing the uptake of drugs into the CNS, by for example conjugation to the natural ligands of those receptors or by selection of antibodies or drugs which bind directly to the receptors.
  • Central Nervous System (CNS) diseases that may be treated in this manner include sleep disorders (e.g. narcolepsy), affective disorders, schizophrenia, regenerative disorders such as Alzheimer's disease, Parkinson's disease, fronto- temporal dementia, Huntington's chorea, demyelination diseases (e.g. multiple sclerosis), spinal cord injury, brain tumours (primary and secondary), viral encephalopathies (e.g. rabies and cerebral HIV) , motor neurone diseases, prion diseases, hydrocephalus, stroke (primary haemorrhagic and ischaemic) , epilepsies, obsessive-compulsive disorders, anxieties and phobias, drug abuse, alcohol and other substance abuses, symptoms such as pain.
  • sleep disorders e.g. narcolepsy
  • affective disorders e.g. narcolepsy
  • schizophrenia e.g. narcolepsy
  • regenerative disorders such as Alzheimer's disease, Parkinson's disease
  • the present invention relates to the selection and characterisation of human antibodies from a phage display antibody library which have been demonstrated to cross the BBB or which localise to areas of inflammation in brain tissue.
  • Such antibodies have value and therapeutic potential as agents to enhance drug delivery across the blood brain barrier, and as tools to identify novel transporter or other blood brain barrier or inflamed tissues marker molecules which may themselves be targets for therapeutic intervention.
  • Some molecules on brain capillary endothelium are already known to be specific receptors for circulating proteins or plasma proteins.
  • Human microvessels have been used to characterise the human BBB receptors for insulin, insulin-like growth factor I, insulin-like growth factor-II, transferrin and leptin. (Reviewed by W Pardridge (1997) Journal of Cerebral Blood Flow and Metabolism, 17: 713-731).
  • the BBB insulin, insulin-like growth factor, and transferrin receptor systems have been shown to mediate the transcytosis of peptides through the BBB in vivo (e.g. Duffy and Pardridge, 1987, Brain Res. 420: 32-38).
  • the anti-human insulin receptor mAb 83-14 has been conjugated to streptavidin and shown to retain its ability to be transported (Wu et al, 1997, J Clin Invest, 100: 1804-12) allowing biotinylated compounds to be transported along with it.
  • the present invention provides a mixture or panel of at least 5, and preferably at least 10 different specific binding members each comprising an antibody VH variable domain and/or an antibody VL variable domain, wherein an antibody VH variable domain has an amino acid sequence selected from the group consisting of the G65 (SEQ ID NO. 2), G67 (SEQ ID NO. 6), G73 (SEQ ID NO. 10), G76 (SEQ ID NO. 14), G77 (SEQ ID NO. 18), G78 (SEQ ID NO. 22), G79 (SEQ ID NO. 24), G81 (SEQ ID NO. 30), G83 (SEQ ID NO. 34), G85 (SEQ ID NO. 38), G88 (SEQ ID NO. 42), G92 (SEQ ID NO.
  • G65 SEQ ID NO. 2
  • G67 SEQ ID NO. 6
  • G73 SEQ ID NO. 10
  • G76 SEQ ID NO. 14
  • G77 SEQ ID NO. 18
  • G78 SEQ ID NO. 22
  • G79 SEQ ID NO. 24
  • VH domain sequences disclosed herein, and/or an antibody VL variable domain has an amino acid sequence selected from the group consisting of the G65 (SEQ ID NO. 4), G67 (SEQ ID NO. 8), G73 (SEQ ID NO. 12), G76 (SEQ ID NO. 16), G77 (SEQ ID NO.20), G78 (SEQ ID NO. 24), G79 (SEQ ID NO. 28), G81 (SEQ ID NO. 32), G83 (SEQ ID NO.
  • each specific binding member being able, when displayed on the surface of filamentous bacteriophage particles, to pass through a mammalian blood brain barrier, preferably a human or rodent (e.g. rat) blood brain barrier.
  • the blood brain barrier may be an in vi tro barrier such as generated in accordance with Example 1 herein.
  • a mixture or panel of specific binding members may include at least 11, 12, 13, 14, 15, 16, 17 or 18 of the VH or VL domains.
  • VH and VL domains are paired according to corresponding nomenclature as used herein (e.g. G65 VH (SEQ ID NO. 2) with G65 VL (SEQ ID NO. 4), G81 VH (SEQ ID NO. 30) with G81 VL (SEQ ID NO. 32), and so on).
  • a panel or mixture of specific binding members provided by the present invention is useful for selection of specific binding members that cross the blood brain barrier. Additionally, a panel or mixture may be employed together to deliver different molecules or effector functions.
  • One or more specific binding members may be selected from a panel or mixture e.g. by binding with antigen, which may be on endothelial cells. Where the specific binding members are displayed on filamentous bacteriophage, this allows nucleic acid encoding a selected specific binding member, or at least a component of the member, to be readily isolated. Encoding nucleic acid may then be manipulated and may be used to produce more of the specific binding member or component thereof by means of recombinant expression.
  • the present invention provides a method of obtaining one or more specific binding members, the method including bringing into contact a library of specific binding members displayed on the surface of filamentous bacteriophage particles, and selecting one or more specific binding members of the library able when displayed on filamentous bacteriophage particles to pass through a mammalian blood brain barrier. Selection on ability to pass through a blood brain barrier may be followed by one or more further rounds of selection for ability to bind a type of cell of the brain or CNS, for instance endothelial cells, or cells of the meninges, parenchyma, choroid plexus, cerebrum, cerebellum, spinal cord or microglia.
  • a type of cell of the brain or CNS for instance endothelial cells, or cells of the meninges, parenchyma, choroid plexus, cerebrum, cerebellum, spinal cord or microglia.
  • a population or mixture of specific binding members which when displayed on filamentous bacteriophage are able to pass through a mammalian blood brain barrier is obtainable on selection from a phage display library as disclosed.
  • Such a population or mixture may comprise at least 10 6 different specific binding members.
  • One or more further rounds of selection may be used to provide a sub-population of about 10,000 different specific binding members, 1,000, 100, a mixture of 10-20 different specific binding members or fewer, and individual specific binding members.
  • the invention further provides a plurality of different antibody VH variable domains obtainable from a mixture, panel, population or library as disclosed, and further provides a plurality of different antibody VL variable domains obtainable from such a mixture, panel, population or library.
  • VH and VL domains of which sequences are shown herein each represent individual aspects of the present invention.
  • a VH domain of which the sequence is disclosed herein may be combined with a VL domain of which the sequence is disclosed herein, or other VL domain, to provide a VH/VL pairing representing an antigen-binding site of an antibody.
  • a VL domain of which the sequence is disclosed herein may be combined with a VH domain of which the sequence is disclosed herein, or other VH domain.
  • Pairings of VH and VL domains represent further aspects of the present invention.
  • Preferred pairings are as identified by corresponding nomenclature herein, e.g. G65 VH (SEQ ID NO. 2) with G65 VL (SEQ ID NO.
  • individual aspects of the present invention provide a specific binding member comprising a VH/VL pairing selected from the group consisting of G65, G67, G73, G76, G77, G78, G79, G81, G83, G85, G88, G92, G93, G95, G101, G102, G110 and G112 (SEQ ID NO' s are given above).
  • VH domain and each VL domain of each of these pairings represents a further aspect of the present invention.
  • G67, G73, G76, G78, G79, G83, G85, G88, G92, G93, G95 and G110 represent preferred embodiments, being cross-reactive with human tissue.
  • G93 VH SEQ ID NO. 50; VL SEQ ID NO. 52
  • G73 VH SEQ ID NO. 10; VL SEQ ID NO. 12
  • VH and VL domains may be obtained from a filamentous bacteriophage library, employing selection for ability to pass through a blood brain barrier.
  • D5 VH/VL is directed against human serum amyloid protein (SAP) which passes through the blood brain barrier.
  • SAP human serum amyloid protein
  • One or more CDRs may be taken from any VH or VL domain according to the present invention and incorporated into a suitable framework. This is discussed further below.
  • Variants of the VH and VL domains and CDRs of which the sequences are set out herein and which can be employed in specific binding members for brain and/or endothelial cell antigens can be obtained by means of methods of sequence alteration or mutation and screening. Such methods are also provided by the present invention.
  • the specific binding member may comprise other amino acids, e.g. forming a peptide or polypeptide, such as a folded domain, or to impart to the molecule another functional characteristic in addition to ability to bind antigen.
  • Specific binding members of the invention may carry a detectable label, or may be conjugated to a toxin or enzyme (e.g. via a peptidyl bond or linker) .
  • a VH domain or a VL domain according to the present invention may be provided in a fusion with additional amino acids .
  • a further aspect of the present invention provides a method of obtaining one or more specific binding members with a desired property, the method including bringing into contact a library or panel of specific binding members and selecting one or more with the desired property.
  • Such a method may employ phage display technology, wherein the specific binding members in the library or panel are displayed on the surface of bacteriophage particles, each particle containing nucleic acid encoding the specific binding member or a component thereof (e.g. VH domain) .
  • Nucleic acid may be taken from a bacteriophage particle containing nucleic acid encoding a selected specific binding member or component thereof, and nucleic acid with the sequence of the nucleic acid from the particle can be used to provide (by means of recombinant technology) the encoded product, or further nucleic acid with the sequence, or a variant or derivative.
  • the invention provides an isolated nucleic acid which comprises a sequence encoding a specific binding member as defined above, 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 (which may include prophylactic treatment) of a disease or disorder in a human patient which comprises administering to said patient an effective amount of a specific binding member of the invention.
  • the specific binding member may be unconjugated, or may be conjugated to an active agent.
  • Conditions treatable in accordance with the present invention include neurological diseases, including Alzheimer's disease, prion diseases, AIDS- related dementia, and any disease involving inflammation occurring within the brain or CNS .
  • 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 .
  • immunoglobulin whether natural or partly or wholly synthetically produced. 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.
  • Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains (Y. Reiter et al, Nature Biotech, 14, 1239-1245, 1996) .
  • Minibodies comprising a scFv joined to a CH3 domain may also be made (S. Hu et al, Cancer Res., 56, 3055-3061, 1996) .
  • 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)), 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.
  • 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.
  • Bispecific whole antibodies may be made by knobs-into-holes engineering (J. B. B. Ridgeway et al, Protein Eng., 9, 616-621, 1996).
  • 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 (e.g. a so- called Fd antibody fragment consisting of a VH domain) .
  • 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 (e.g. CHO or NSO (ECACC 85110503) cells, or they may be (for example if produced by expression in a prokaryotic cell) unglycosylated.
  • the structure for carrying a CDR of the invention will generally be of an antibody heavy or light chain sequence or substantial portion thereof in which the CDR is located at a location corresponding to the CDR 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. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof, now available on the Internet (http: //immuno .bme . nwu. edu) ) .
  • a CDR amino acid sequence substantially as set out herein is carried as a CDR in a human variable domain or a substantial portion thereof.
  • the VL CDR 3 and especially the VH CDR3 sequences substantially as set out herein represent preferred embodiments of the present invention and it is preferred that each of these is carried as a respective VL CDR3 or VH CDR3 in a respective human light or heavy chain variable domain or a substantial portion thereof.
  • Variable domains employed in the invention 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.
  • a CDR-derived sequences of the invention e.g. CDR3
  • CDR3 may be introduced into a repertoire of variable domains lacking a CDR (e.g. CDR3), using recombinant DNA technology.
  • Marks et al (Bio/Technology, 1992, 10:779-783) describe methods of producting 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.
  • 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.
  • a further alternative is to generate novel VH or VL regions carrying a CDR-derived sequences of the invention using random mutagenesis of one or more selected VH and/or VL genes to generate mutations within the entire variable domain.
  • 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 , (1994, Proc . Natl . Acad. Sci . , USA, 91:3809- 3813) and Schier et al (1996, J. Mol . Biol . 263:551-567) .
  • a further aspect of the invention provides a method for obtaining an antibody antigen binding domain with a desired property, e.g.
  • the method comprising providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a VH domain set out herein a VH domain which is an amino acid sequence variant of the VH domain, combining the VH domain thus provided with one or more VL domains, and testing the VH/VL combination or combinations for to identify an antibody antigen binding domain with the desired property, e.g. ability to cross the blood brain barrier and/or specificity for the antigen, and optionally with one or more of preferred properties, e.g. ability to bind areas of inflammation in the brain, breakdown of the blood brain barrier.
  • Said VL domain may have an amino acid sequence which is substantially as set out herein.
  • An analogous method may be employed in which one or more sequence variants of a VL domain disclosed herein are combined with one or more VH domains .
  • a further aspect of the invention provides a method of preparing a specific binding member with a desired property, which method comprises:
  • VL CDR3 of the invention is combined with a repertoire of nucleic acids encoding a VL domain which either include a CDR3 to be replaced or lack a CDR3 encoding region.
  • one or more, or all three CDRs may be grafted into a repertoire of VH or VL domains which are then screened for a specific binding member or specific binding members with the desired property.
  • the desired property may be any one or more of ability to cross the blood brain barrier, bind an endothelial cell or other brain cell antigen, bind areas of inlammation in the brain or blood brain barrier breakdown, bind ICAM or bind transferrin receptor.
  • a substantial portion of an immunoglobulin variable domain will comprise at least the three CDR regions, together with their intervening framework regions.
  • the portion will 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.
  • construction of specific binding members of the present invention made by recombinant DNA techniques may result in the introduction of - or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps.
  • Other manipulation steps include 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 details below.
  • single binding domains based on either VH or VL domain sequences form further aspects of the invention. It is known that single immunoglobulin domains, especially 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 with the desired property.
  • phage display screening methods using the so-called hierarchical dual combinatorial approach as disclosed in WO 92/01047 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.
  • a VL domain may be attached at its C-terminal end to antibody light chain constant domains including human CK or C ⁇
  • Antibodies of the invention may be labelled with a detectable or functional label.
  • 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.
  • Antibodies of the present invention are designed to be used in methods of diagnosis or treatment in human or animal subjects, preferably human.
  • aspects of the invention provide methods of treatment comprising administration of a specific binding member as provided, pharmaceutical compositions comprising such a specific binding member, and use of such a specific binding member in the manufacture of a medicament for administration, for example in a method of making a medicament or other composition comprising formulating the specific binding member with at least one additional component, such as a pharmaceutically acceptable excipient.
  • compositions provided may be administered to individuals. Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom.
  • 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, eg decisions on dosage etc, is within the responsibility of general practioners and other medical doctors. Appropriate doses of antibody are well known in the art; see Ledermann J.A. et al. (1991) Int J. Cancer 47: 659-664; Bagshawe K.D. et al . (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915- 922.
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated.
  • Antibodies of the present invention may be administered to a patient in need of treatment via any suitable route, usually by intravenous injection into the bloodstream.
  • the precise dose will depend upon a number of factors, including whether the antibody is for diagnosis or for treatment, the size and location of the area to be treated (e.g. wound), the precise nature of the antibody (e.g. whole antibody, fragment or diabody) , and the nature of any detectable label or other molecule attached to the antibody.
  • a typical antibody dose will be in the range 0.5mg to lOOg for systemic applications, and lO ⁇ g to lmg for local applications.
  • the antibody will be a whole antibody, preferably the IgG4 subclass.
  • Treatments may be by continuous infusion or may be repeated at daily, twice-weekly, weekly or monthly intervals, as appropriate. Treatment may be repeated within the same day, e.g. for treatment of short-term problems such as epilepsy and traumatic brain injury.
  • a whole antibody of the IgG4 subclass is used for systemic and local applications but for local applications a scFv antibody may be particularly valuable .
  • 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.
  • 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.
  • a pharmaceutically acceptable excipient 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.
  • 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.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection solution, Ringer's Injection solution, or Lactated Ringer's Injection solution.
  • 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 or anti-emetics.
  • the present invention provides a method comprising causing or allowing binding of a specific binding member as provided herein to an antigen.
  • binding may take place in vivo, e.g. following administration of a specific binding member, or nucleic acid encoding a specific binding member, or it may take place in vi tro .
  • the amount of binding of specific binding member to an antigen may be determined. Quantitation may be related to the amount of the antigen in a test sample, which may be of diagnostic interest .
  • Radioimmunoassay is one possibility. Radioactive labelled antigen is mixed with unlabelled antigen (the test sample) and allowed to bind to the antibody. Bound antigen is physically separated from unbound antigen and the amount of radioactive antigen bound to the antibody determined. The more antigen there is in the test sample the less radioactive antigen will bind to the antibody.
  • a competitive binding assay may also be used with non-radioactive antigen, using antigen or an analogue linked to a reporter molecule.
  • the reporter molecule may be a fluorochrome, phosphor or laser dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine .
  • Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded.
  • These molecules may be enzymes which catalyse reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed.
  • the signals generated by individual antibody-reporter conjugates may be used to derive quantifiable absolute or relative data of the relevant antibody binding in samples (normal and test) .
  • the present invention also provides the use of a specific binding member as above for measuring antigen levels in a competition assay, that is to say a method of measuring the level of antigen in a sample by employing a specific binding member as provided by the present invention in a competition assay. This may be where the physical separation of bound from unbound antigen is not required.
  • Linking a reporter molecule to the specific binding member so that a physical or optical change occurs on binding is one possibility.
  • the reporter molecule may directly or indirectly generate detectable, and preferably measurable, signals.
  • the linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non-covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule .
  • the present invention also provides for measuring levels of antigen directly, by employing a specific binding member according to the invention for example in a biosensor system.
  • the mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge.
  • the present invention further extends to a specific binding member which competes for binding to an antigen with any specific binding member which both binds the antigen and comprises a V domain including a CDR with amino acid substantially as set out herein or a V domain with amino acid sequence substantially as set out herein.
  • Competition between binding members may be assayed easily in vi tro, for example by tagging a specific reporter molecule to one binding member which can be detected in the presence of other untagged binding member (s), to enable identification of specific binding members which bind the same epitope or an overlapping epitope.
  • Competition may be determined for example using the ELISA, e.g. a whole cell ELISA using immobilised brain endothelial cells, looking for inhibition of signal when cells are pre-incubated with a potential competitor antibody.
  • a peptide fragment of the antigen may be employed, especially a peptide including an epitope of interest.
  • a peptide may have the epitope sequence plus one or more amino acids at either end, may be used.
  • Such a peptide may be said to "consist essentially" of the specified sequence.
  • Specific binding members according to the present invention may be such that their binding for antigen is inhibited by a peptide with or including the sequence given. In testing for this, a peptide with either sequence plus one or more amino acids may be used.
  • Specific binding members which bind a specific peptide may be isolated for example from a phage display library by panning with the peptide (s).
  • 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 provides a nucleic acid which codes for a CDR or VH or VL domain of the invention as defined above .
  • the present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise least one polynucleotide as above.
  • the present invention also provides a recombinant host cell which comprises one or more constructs as above.
  • a nucleic acid encoding any CDR, VH or VL domain, or specific binding member as provided itself forms an aspect of the present invention, as does a method of production of the encoded product, 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 VH or VL domain, or specific binding member may be isolated and/or purified using any suitable technique, then used as appropriate.
  • Specific binding members, VH and/or VL domains, and encoding 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.
  • nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise .
  • 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 .
  • a suitable host where a coding sequence includes a TAG suppressible stop codon is a suppressor strain such as the sup44 strain of TGI. In such a strain, TAG codons are read as encoding glutamine.
  • Embodiments of the present invention where a suppressor strain may be used in production of the encoded polypeptide by expression from encoding nucleic acid comprising a TAG codon include where the coding sequence of any of SEQ ID NO' s 37, 43 and 69 are used (respectively encoding G85 VH SEQ ID NO. 38, G88 VL SEQ ID NO. 44, and G112 VH SEQ ID NO. 70) .
  • 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.
  • plasmids viral e.g. 'phage, or phagemid, as appropriate.
  • 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 .
  • ICC Interleukin 1- ⁇
  • IL-l ⁇ Enzyme linked immunosorbent assay
  • SAP Serum amyloid protein
  • BSA Bovine serum albumin
  • BBB Blood brain barrier
  • EXAMPLE 1 Generation of in vi tro blood brain barrier cell culture chambers.
  • EXAMPLE 2 Selection of antibody-expressing phage which cross in vi tro blood brain barriers.
  • EXAMPLE 3 Characterisation of selected antibodies by endothelial cell ELISA and sequencing.
  • EXAMPLE 4 Demonstration of transport of clonal phage.
  • EXAMPLE 5 Rat brain immunocytochemistry of endothelial-cell binding antibody clones.
  • EXAMPLE 6 Examination of ICC cross-reactivity of rat endothelial cell-binding antibodies with a panel of human tissues .
  • EXAMPLE 7 Selection of SAP-binding antibodies.
  • EXAMPLE 8 Characterisation of SAP-binding antibodies on the basis of sequence and ability to cross a blood brain barrier in the presence or absence of SAP.
  • the protocol used for the preparation of separate cultures of astrocytes was an adaptation of the method described by K.D. McCarthy and J. De Vellis (J. Cell. Biol., 1980, vol. 85, 890- 902). Using similar methods, astroglial cultures obtained are reported to appear to be >98% pure by immunocytochemistry using an antibody to glial fibrillary acidic protein (GFAP) . A summary of this protocol for astrocyte cell culture has been published (G.M.A. Wells., 1996, in Glia, vol. 18, p. 332 340).
  • the use of 12 to 24 hour old rat pups ensured the absence of viable neurons in the cell suspension obtained from dissociating the cerebral cortex and the ready dissociation of cells by mechanical sieving techniques rather than using trypsinisation.
  • the length of the initial culture period was kept to between 7 and 9 days to ensure stratification of the astrocytes, microglia and oligodendrocytes .
  • the procedure for the separation of individual glial cell cultures relies upon the selective detachment of other cells from the layer of firmly attached type 1 astrocytes when the primary cell culture is exposed to sheer forces on shaking the cultures in an orbital incubator overnight (approx. 16 hours) at 37°C.
  • the separated astroglial cell cultures can be maintained for several weeks enabling investigation of this cell type.
  • the optic nerves were detached with a spatula, and the cerebral hemispheres removed (without the cerebellum) from the cavity with the same spatula and placed in a sterile 50 ml tube containing 20 ml DMEM (containing 4.5 g/1 glucose and 110 mg/1 sodium pyruvate - from Life Technologies) + L glutamine (Life Technologies) + 20% foetal bovine serum (FBS) (Life Technologies) + penicillin / streptomycin (Life Technologies) .
  • 20 ml DMEM containing 4.5 g/1 glucose and 110 mg/1 sodium pyruvate - from Life Technologies
  • L glutamine Life Technologies
  • FBS foetal bovine serum
  • penicillin / streptomycin Life Technologies
  • the cerebral tissue in DMEM was poured from the 50 ml tube into a sterile petri dish and each individual brain tissue was removed with forceps onto a pad of sterile filter paper.
  • the cerebral hemispheres of each brain were parted and individually rolled across the length of a fresh filter paper to remove the meninges and blood vessels. These were then placed in another petri dish containing 20 ml of fresh DMEM containing 20% FBS.
  • the brain tissue was chopped into very small pieces using a single edged razor blade whilst immersed in the DMEM in the petri dish.
  • a 230 ⁇ m re-usable metal sieve in its cup shaped holder was placed on the top of a Sterilin pot and 20 ml of fresh DMEM containing 20% FBS was pipetted through it.
  • the 20 ml of DMEM containing the chopped brain tissue was then taken up from the petri dish in a 25 ml pipette and pipetted onto the sieve.
  • the chopped tissue was forced through the sieve using the glass tissue.
  • the filtered cell suspension was divided equally between two 50 ml centrifuge tubes and centrifuged for 10 minutes at 1000 rpm. Supernatants were removed and the cell pellets resuspended by gentle pipetting in a final volume of 20 ml DMEM containing 20% FBS.
  • the cell suspension was then passed through a 100 ⁇ m disposable sieve placed on top of a 50 ml tube and washed through with 10 ml DMEM containing 20% FBS.
  • the filtered cell suspension was then passed through a 70 ⁇ m disposable sieve placed on top of a fresh 50 ml tube and washed through with 10 ml DMEM containing 20% FBS.
  • the resulting filtered cell suspension was pipetted into a 75 cm tissue culture flask and enough DMEM containing 20% FBS added so that there was a final volume of 20 ml per brain used.
  • the cell suspension was mixed and divided into the relevant number of 75 cm flasks so that each flask contained 20 ml of cell suspension.
  • the top of each flask was loosened and the flasks placed in a 37°C incubator with a 5% CO atmosphere.
  • the flasks were left undisturbed for 4 days and then inspected. The old medium and tissue debris was removed, the cells fed with 20 ml of fresh DMEM containing 20% FBS and left for a further 3 days in the incubator. On day 7 of the primary culture flasks were checked for good coverage of total cells (by this stage the astrocytes should be approaching confluency) . The medium was removed and replaced with 15 ml of DMEM containing 10% FBS. The flasks were returned to the incubator with loosened tops and left for a further 24-48 hours. Astrocytes were passaged, with a 1:4 dilution of cells, approximately every week (or upon reaching confluency) over a period of several weeks using DMEM containing 10% FBS as the standard growth medium.
  • Tissue dissected from two Wistar rats results, digested and purified using this protocol results in a primary cultures of microvessels set up in two 6 well dishes from which individual colonies of endothelial cells can be trypsinised out after 7 days in culture.
  • the cerebral tissue in DMEM was poured into a sterile petri dish and each piece of tissue was removed onto a pad of sterile filter paper. The cerebral hemispheres were parted and the meninges and large blood vessels were removed by peeling off the outer layer of the tissue and rolling the tissue along the length of the filter paper pad.
  • the grey matter was dissected away from the white matter, pooled together and placed into a fresh petri dish containing sterile serum free DMEM containing penicillin and streptomycin. This was then chopped up into fine pieces using a single sided razor blade. The chopped up tissue was then placed into a sterile 50 ml centrifuge tube and pelleted at 200 g for 10 minutes at room temperature.
  • the supernatant was removed and the tissue was resuspended in 10 ml of serum free DMEM containing penicillin and streptomycin plus 1 mg/ml collagen (Boehringer Mannheim, cat. no. 269 638) and 10 ⁇ g/ml DNase I (Sigma Type II, cat. no. D4263) . This was then placed on a shaking incubator at 37°C for 90 minutes. The tissue digest was then pelleted at 200 g for 10 minutes at room temperature and resuspended in 40 ml of DMEM containing 25% (w/v) bovine serum albumin (BSA, Fraction V from Sigma, cat. no. A9647) with trituration until it had a creamy texture.
  • BSA bovine serum albumin
  • ECGM endothelial cell growth medium
  • rat astrocytes and endothelial cells were set up set up several days/weeks in advance from frozen stocks stored in liquid nitrogen. These were kept at as low passage number as possible to preserve the normal characteristics of the primary cells (astrocytes at less than passage 6, and endothelial cells at less than passage 10) .
  • astrocytes were cultured in astrocyte growth medium (AGM) consisting of DMEM containing 4.5 g/1 glucose and 110 mg/1 sodium pyruvate (Life Technologies) supplemented with 2mM L-glutamine (Sigma) , 10% foetal bovine serum (FBS) (Life Technologies) , 100 units/ml penicillin plus 100 ⁇ g/ml streptomycin (Sigma) .
  • AGM astrocyte growth medium
  • ECGM endothelial cell growth medium
  • astrocytes were placed in the lower chamber and allowed to adhere before placing the tissue culture insert into the plate and adding the endothelial cells to the upper chamber.
  • a T 175 flask of astrocytes was trypsinised and the number of cells counted in a haemocytometer . These were then diluted to 1.0 x 10 5 cells/ml in AGM and 1 ml of cell suspension was added to the wells of the 24 well lower plate. The astrocytes were allowed to adhere to the surface of the lower chamber for at least 2 hours in a C0 2 incubator.
  • the appropriate number of 8 ⁇ m inserts were coated with 100 ⁇ g/ml rat tail type 1 collagen before adding the endothelial cells.
  • the inserts were placed in a separate lower plate and 1 ml of diluted collagen was added to the lower chamber and 0.5 ml of collagen was added to the upper chamber.
  • the inserts were then left for at least 1 hour at room temperature in a class II tissue culture cabinet. The collagen was then removed and the inserts were rinsed in PBS.
  • a T 175 flask of EC was trypsinised and the number of cells counted in a haemocytometer. These were then diluted to 2.0 x 10 5 cells/ml in AGM. The lower plate containing the cultures of astrocytes was removed from the incubator and the medium on the astrocytes was replaced with 1 ml of fresh AGM.
  • the coated inserts were then placed in the lower plate containing the astrocyte cultures using forceps sterilised in 70% ethanol and 0.5ml of the 2.0 x 10 5 EC/ml suspension was added to the upper chamber.
  • the plates were placed back in the C0 2 incubator and not disturbed for at least 24 hours.
  • the barrier cultures were inspected under the microscope.
  • the EC should have formed a near confluent coating of the upper chamber, and, likewise, the astrocytes in the lower chamber should have spread out over most of the surface area of the lower chamber.
  • the electrical resistance of EC barrier can be measured, however, it takes several days before the resistance rises as the cells need to reach confluency and then form tight junctions.
  • the medium in both the upper and lower chambers was replaced with fresh AGM every 7 days and electrical resistance monitored with time. High levels of electrical resistance are obtained after at least 2 weeks in culture. These cultures have been maintained for up to 8 weeks before any fall in electrical resistance is observed.
  • Measurement of electrical resistance across the tissue culture insert membrane can be performed under sterile conditions to monitor the progress of the endothelial cell cultures in forming a good model of the blood brain barrier in the inserts.
  • the use of the Endohm chamber electrodes for the 24 well inserts with its concentric electrodes situated above and beneath the membrane reduces the background resistance to between 20 and 30 ⁇ for an empty 8 ⁇ m tissue culture insert. This background figure was measured on each occasion and subtracted from actual readings obtained with inserts containing EC.
  • the Endohm electrode was sterilised using 70% ethanol. The cup shaped electrode was filled with 70% ethanol and the upper electrode placed back in place. The ethanol was then tipped out and the electrode drained.
  • the electrode was then rinsed in sterile astrocyte growth medium (AGM) a couple of times. 1 ml of fresh AGM was added to the lower chamber of the electrode and the electrode was ready for measuring the resistances of any inserts. The resistance of an empty insert was measured at the same time as the inserts containing EC and this was subtracted from all the barrier insert resistances. To obtain standardised resistance measurements, the resistance values recorded on the volt/ohm meter were divided by the surface area of the membrane (for the 24 well sized inserts used here this was 0.3cm) to give values measured in ohms/cm 2 . Resistances were generally in the 500-600 ohm/cm 2 range.
  • Polyadenylated RNA was prepared from the B-cells of various lymphoid tissues of 43 non-immunised donors using the "Quickprep mRNA Kit” (Pharmacia) .
  • First-strand cDNA was synthesized from mRNA using a "First-strand cDNA synthesis” kit (Pharmacia) using random hexamers to prime synthesis.
  • V-genes were amplified using family-specific primers for VH, V ⁇ and VK genes as previously described (Marks et al . , (1991) J. Mol. Biol. 222:581-597) and subsequently recombined together with the (Gly 4 , Ser) 3 scFv linker by PCR assembly.
  • VH-linker-VL antibody constructs were cloned into the Sfi I and Wot I sites of the phagemid vector, pCantab6. Ligation, electroporation and plating out of the cells was as described previously (Marks et al, supra) .
  • the library was made ca. lOOOx larger than that described previously by bulking up the amounts of vector and insert used and by performing multiple electroporations . This generated a scFv repertoire that was calculated to have ca. 1.3 x 10 10 individual recombinants which by BstNI fingerprinting were shown to be extremely diverse.
  • the phage antibody repertoire above was selected for antibodies which cross the blood brain barrier.
  • the scFv repertoire was treated as follows in order to rescue phagemid particles.
  • 500 ml prewarmed (37 2 C) 2YTAG (2YT media supplemented with 100 ⁇ g/ml ampicillin and 2% glucose) in a 21 conical flask was inoculated with approximately 3xl0 10 cells from a glycerol stock (-70 2 C) culture of the library.
  • the culture was grown at 37 e C with good aeration until the OD 600nm reached 0.7 (approximately 2 hours).
  • M13K07 helper phage (Stratagene) was added to the culture to a multiplicity of infection (moi) of approximately 10 (assuming that an 600nm of 1 is equivalent to 5 x 10 8 cells per ml of culture) .
  • the culture was incubated stationary at 37 2 C for 15 minutes followed by 45 minutes with light aeration (200 rpm) 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 ampicillin and 50 ⁇ g/ml kanamycin) , and the culture incubated overnight at 30 2 C with good aeration (300 rpm) .
  • Phage particles were purified and concentrated by one polyethylene glycol (PEG) precipitation (Sambrook, J., Fritsch, E.F., & Maniatis, T. (1990) Molecular Cloning - A Labora tory Manual Cold Spring Harbor, New York) and resuspended in 9ml lOmM Tris containing 1 mM EDTA (TE) . . Og of CsCl was added to the phage stock and mixed gently to dissolve. A 11.5ml ultracentrifuge tube was filled with phage at centrifuged at 40 000 rpm at 25 2 C for 24 hr. The ultracentrifuge was stopped with the brake off and the clear opalescent phage band collected using a pasteur pipette. Phage were dialysed at 4 2 C overnight against two changes of 11 of TE, titred and stored at 4 2 C.
  • PEG polyethylene glycol
  • the in vi tro BBB chambers provide a valuable system which can be utilised as a means to functionally select for phage antibodies which cross the in vi tro barriers.
  • the phage antibody library can be placed in the top chamber of the in vi tro barrier and phage which are capable of passing through the barrier and reaching the bottom reservoir can then be collected, and the process repeated. This is the basis on which the following selections were carried out.
  • the number of phage present in the media in the lower chamber (i.e. the number of transported phage) was titred. Titring was achieved by adding 0.5 ml of the media recovered from the bottom chamber (out of a total of 1ml) to a 5ml culture of exponentially growing E. coli TGI with light aeration in 2TY broth at 37 e C for 1 hour. Infected TGI ' s were plated on 2TYAG medium in 243mm x 243mm dishes. Dilutions of infected TGls were also plated out and incubated at 30 2 C overnight. Colony counts gave the phage output titre.
  • the phage population from well 7a was chosen as the input population for a second round of selection.
  • Colonies were scraped off the 7a 243mm x 243mm plate into 3 ml of 2TY broth and 15% (v/v) glycerol added for storage at -70 S C. Glycerol stock solutions from the first round of selection were rescued using helper phage to derive phagemid particles for the second round of selection. 250 ⁇ l of the 7a glycerol stock was used to inoculate 50 ml 2YTAG broth, and incubated in a 250 ml conical flask at 37 2 C with good aeration until the OD 600 nM reached 0.7 (approximately 2 hours).
  • Library phage, pCantab ⁇ phage (which is identical library except it has only myc and his tags fused with the gene III protein and no scFv) and the second round 7a phage were added to top reservoirs of in vi tro blood brain barrier culture chambers using lxlO 12 phage per chamber in 50 ⁇ l TE . Chambers were incubated at 37 2 C in a C0 2 incubator. The lower reservoir was removed at 30 min and 500 ⁇ l of this used to replace the top reservoir of a fresh barrier culture. After a further 30 min the lower reservoir of this barrier culture was removed and 500 ⁇ l of the media used to replace the top reservoir of a fresh barrier culture. This was repeated one final time after a further 30 min.
  • Colonies were plated out and titred as described above, and the results of the titration experiments from the second round of selection are shown in Table 2.
  • Population 24 was chosen as the starting population for a third round of selection.
  • Population 24 from the second round selection regime was rescued and caesium banded as described above for the 7a second round population.
  • Library phage, pCANTAB6 phage, population 7a and population 24 phage were added to the top reservoir of precooled barrier cultures using 1 x 10 12 phage per chamber in 50 ⁇ l TE . Chambers were incubated for 30 min on ice and washed 3 times with 0.5 ml of ice cold media. Insert and liquid within the upper chamber were then transferred to a fresh chamber well containing 1 ml of media at 37 2 C and the chambers incubated at 37 S C in a C0 2 incubator. The lower reservoir was removed at 30 min and 500 ⁇ l of this used to replace the top reservoir of a fresh barrier culture. After a further 30 min the lower reservoir of this barrier culture was removed and 500 ⁇ l of the media used to replace the top reservoir of a fresh barrier culture. This was repeated one final time after a further 30 min and the barrier culture incubated for 30 min at 37 2 C
  • Colonies were plated out and titred as described above (Table 3) .
  • Phage ELISAs were carried out as follows: individual clones were picked into a 96 well tissue culture plate containing 100 ⁇ l 2YTAG. Plates were incubated at 37 2 C for 6 hours. M13K07 helper phage was added to each well to an moi of 10 and incubated with gentle shaking for 45 min at 37 2 C. The plates were centrifuged at 2000 rpm for 10 min and the supernatant removed. Cell pellets were resuspended in 100 ⁇ l 2TYA with kanamycin (50 ⁇ g/ml) and incubated at 30 S C overnight.
  • the nucleotide sequences of the rat endothelial cell binding antibodies were determined by first using vector-specific primers to amplify the inserted DNA from each clone.
  • Cells from an individual colony on a 2YTAG agar plate were used as the template for a polymerase chain reaction (PCR) amplification of the inserted DNA using the primers pUC19reverse and fdtetseq.
  • Amplification conditions consisted of 30 cycles of 94 2 C for 1 min, 55 2 C for 1 min and 72 2 C for 2min, followed by 10 min at 72 2 C.
  • the PCR products were purified using a PCR Clean-up Kit (Promega) in to a final volume of 50 ⁇ lH 2 0.
  • each insert preparation was used as the template for sequencing using the Taq Dye-terminator cycle sequencing system (Applied Biosystems) .
  • the primers pUC19reverse and fdtseq were used to sequence the heavy and light chain of each clone respectively.
  • the diversity of the CDR3s and the germline families is shown in Table 4. Detailed sequence data is included below.
  • phage antibody clones that gave positive endothelial cell ELISA results and gave endothelial specific staining by ICC on rat brain, were tested for their ability to cross the in vi tro blood brain barrier as clonal preparations. Phage were prepared by rescue and caesium banding as described Example 2. lxlO 8 phage in 50 ⁇ l TE were added to the upper reservoir of in vi tro blood brain barrier chamber cultures which had been pre-chilled on ice. The phage were allowed to bind to the endothelial cell layer for 30 min on ice and the cell layer then washed 3 x with 1ml ice cold media.
  • the chamber inserts were transferred to media prewarmed to 37 2 C and incubated at 37 2 C in a C0 2 incubator to allow transport to proceed. Phage crossing the barrier were titred, as described above, following the 30 min on ice then following 30 at 37 2 C (Table 5) .
  • the twenty clones identified by cell ELISA as binding to activated rat endothelial cells were also tested by immunocytochemistry (ICC) for their ability to bind to inflamed rat brain sections.
  • 3 week-old Lewis rats were anaesthetised with avertin (1ml / lOOg) and placed in a sterotaxic frame. Injections of l ⁇ l of interleukin-l ⁇ (10 units) were placed into, the striatum. An incision was made in the scalp to expose bone and a 2-mm diameter burr hole was drilled through the skull to allow the tip of a finely drawn calibrated glass capillary tube to be inserted.
  • Rats were deeply anaesthetised with sodium pentobarbitone and transcardialy perfused with 100ml of saline followed by 200ml of Karnovsky's fixative (1.25% gluteraldehyde and 1.25% paraformaldehyde in phosphate buffer) .
  • the animals were recovered from the anaesthetic before being killed.
  • the brain was removed and cryoprotected in 30% sucrose overnight at 4 2 C before being embedded in Tissue-Tek (Miles Inc, Elkhart, USA) and quickly frozen in liquid nitrogen. 5 micron cryosections were taken of treated brains .
  • Phage clones were inoculated into 1ml 2TYGA in a deep well microtitre plate and grown at 37 2 C with aeration for 5 hours. M13K07 helper phage was added to each well at an moi of 10 and incubated with gentle shaking for 45 min at 37 2 C. The plates were centrifuged at 2000 rpm for 10 min and the supernatant removed. Cell pellets were resuspended in 1ml 2TYKA and incubated at 25 2 C overnight. Plates were centrifuged at 2000 rpm for 10 min and the phage supernatant collected from each well ready for use in ICC.
  • Rat brain sections were fixed by immersion in acetone at ambient temperature for 15 min, and washed twice for 3 min in PBST. Sections were blocked in 5 ⁇ g/ml streptavidin in PBST for 15 min, washed 3 times 3 min in PBST and incubated in 10 ⁇ g/ml biotin in PBST for 15 min. Sections were washed 5 times 3 min in PBST, then twice for 10 min in wash containing 1% BSA. 1% BSA was added to the phage supernatants and phage incubated on the sections for 2 hr at ambient temperature.
  • Sections were then incubated for 30 min in SABC-HRP complex (DAKO K0377) diluted in PBST, and then washed 5 times for 3 min in PBST. Sections were stained by incubation with 3-amino-9-ethyl-carbazole (AEC, Sigma) at a concentration of 2.4 mg/ml in dimethylformamide . ACE incubation was for 3 min followed by washing in 0.1% tween, and this was repeated twice. Slides were further washed in 0.1% tween twice for 5 min and the slides then counterstaining with haemotoxylin (DAKO) for 10 sec. Seven changes of 3 min washing in water were then carried out and the sections coated in aqueous mount.
  • SABC-HRP complex DAKO K0377
  • rat endothelial cell clones positive by ICC on rat brain were tested on a range of normal and diseased human tissues for cross-reactivity.
  • Phage were prepared for ICC as described in Example 5.
  • the panel of human tissues prepared for ICC consisted of:
  • ICC was carried out exactly as described in Example 5. Six (G65, G77, G102, G81, GlOl, G112) out of the 18 clones tested gave no specific staining on the panel of human tissues.
  • G73 was cerebellum specific; GllO stained adenocarcinoma and striated muscle in addition to cerebellum; G95 stained smooth muscle in addition to cerebellum; G93 stained spinal cord in addition to cerebellum; G92 stained spinal cord and cerebrum in addition to cerebellum.
  • G76 stained peripheral nerve and cell bodies in spinal cord.
  • G88 gave specific staining of lung carcinoma.
  • G83 stained kidney glomeruli and leydig cells of the testis.
  • G92 gave staining of the leydig cells of the testis, plus other punctate staining of the testis, and some staining of striated muscle.
  • Serum Amyloid Protein is specifically transported across the blood brain barrier. Phage antibodies binding to human SAP (hSAP) were isolated, as outlined below, and a phage antibody clone (D5) which gave a strong signal by ELISA on hSAP was examined for its ability to be transported across an in vi tro BBB.
  • hSAP was coated onto the surface of a Maxisorp tube using 1 ml of lO ⁇ g/ml in PBS overnight at 4 2 C. After coating the Maxisorp tube was rinsed three times with PBS and filled to the brim with 3% (w/v) skimmed milk powder in PBS (3MPBS) and blocked for lh at 37 2 C. The library phage were blocked at room temperature for lh with 3MPBS . The coated Maxisorp tube was rinsed three times with PBS, and 1 ml of pre-blocked phage was added to the Maxisorp tube.
  • a single colony of E . coli . TGI taken from a minimal agar plate, was used to inoculate 50 ml of 2TY broth and incubated with shaking at 37 2 C until an A.
  • the colonies were scraped into 10 ml of 2TY broth in a 50 ml Falcon tube, 5 ml of sterile 50% v/v glycerol was added and mixed by placing tube on an end-over-end rotator for 10 min at room temperature.
  • 25 ml of 2TYG + lOO ⁇ g/ml ampicillin was inoculated with around 50 ⁇ l of the above plate scrape and grown at 37 2 C to an A 600 of 0.5 to 1.0.
  • M13K07 helper phage was added to a final concentration of 5 x 10 8 pfu/ml and the cells infected at 37 2 C for 30 min stationary and for 30 min shaking ( ⁇ 200 r.p.m.).
  • the cells were transferred to a 50 ml Falcon tube and centrifuged at 3500 r.p.m. for 10 min then the bacterial pellet resuspended in 25 ml prewarmed 2TY with kanamycin (50 ⁇ g/ml) and ampicillin (lOO ⁇ g/ml) . This was transferred to a fresh 250 ml flask and grown overnight with rapid shaking (300 r.p.m.) at 30 2 C to produce phage particles. Approximately 1 ml of culture was transferred to a 1.5 ml microcentrifuge tube and centrifuged at 13000 r.p.m. in a microfuge. The phage-containing supernatant was transferred to a fresh tube and used as the input population for a second round of selection which was performed exactly as described above .
  • the plate was incubated for 30 min without shaking at 37 2 C and then for 30 min with shaking (100 r.p.m.) at 37 2 C to allow superinfection of the helper phage.
  • the cultures were pelleted at 2,000 rpm for 10 min and the supernatant discarded.
  • the bacterial pellets were resuspended in lOO ⁇ l of 2TYAK and grown overnight at 30 2 C shaking at 100 rpm.
  • the cultures were centrifuged at 2,000 rpm for 10 min and the supernatant transferred to a fresh plate.
  • the phage was blocked by the addition of 20 ⁇ l of 6 x PBS/18% Marvel, mixed by pipetting and incubated for 1 h at room temp.
  • Antigen plates were prepared by adding lOO ⁇ l of lO ⁇ g/ml hSAP in PBS to microtitre plate wells and incubating overnight at 4 2 C. Plates were washed 3 x in PBS and blocked for 1 h in 3% MPBS at 37 2 C. Phage ELISAs were performed as described in Example 3.
  • EXAMPLE 8 CHARACTERISATION OF THE SAP-BINDING PHAGE ANTIBODY D5 ON THE BASIS OF ITS ABILITY TO CROSS AN IN VITRO BBB IN THE PRESENCE OR ABSENCE OF SAP
  • Phage either D5 or pCANTAB6, were added to the upper reservoir of ice cold BBB cultures in the presence or absence of lO ⁇ g/ml hSAP at 10 8 , 10 s , or 10 4 phage per chamber. The cultures were incubated on ice for 30 min and the liquid in the lower reservoir then removed for titring. The upper chamber was washed 3 x with 0.5 ml of ice-cold media then transferred to a fresh well and incubated at 37 S C in a C0 2 incubator for 30 min. Liquid from the lower reservoir was again removed and the phage titre present determined by titration (Table 7) .
  • Transport of the D5 phage is increased approximately two fold in the presence of hSAP whereas the transport of pCANTAB6 is reduced slightly in the presence of hSAP.
  • Overall the D5 phage were transported 10-fold more efficiently than the pCantba6 phage in the absence of SAP, and 100-fold more efficiently in the presence of SAP. This provides indication that the D5 phage are being actively transported, and the higher level of D5 transport in the absence of SAP may be a result of endogenous SAP which is pre-bound to the rat endothelial cells .
  • RB rat brain
  • RB Asy asymmetric staining of IL-l ⁇ -treated rat brain
  • C cerebellum
  • Tes testis
  • Ton tonsil
  • Kid kidney
  • Ad C adenocarcinoma
  • Lu C lung carcinoma
  • SM striated muscle

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Abstract

L'invention concerne des éléments de liaisons spécifiques dirigés vers le cerveau comprenant des éléments de liaisons spécifiques capables de passer la barrière hémato-encéphalique, ou qui sont dirigés vers des zones à affection cérébrale de caractère inflammatoire ou de rupture de la barrière hémato-encéphalique. L'invention concerne aussi des listes et des mélanges d'anticorps, des molécules d'anticorps individuel et des domaines VH et VL, ainsi que des procédés d'obtention d'éléments de liaisons spécifiques par sélection, c'est à dire par l'utilisation de bibliothèques présentées sur la surface du bactériophage filamenteux.
EP00979747A 1999-12-13 2000-11-27 Elements de liaison specifiques du cerveau Withdrawn EP1242458A2 (fr)

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PT1846451E (pt) * 2005-01-24 2013-08-28 Elan Pharma Int Ltd Membros de ligação específica para ngf
NZ706751A (en) 2010-11-30 2016-10-28 Genentech Inc Low affinity blood brain barrier receptor antibodies and uses therefor
ES2924282T3 (es) * 2014-12-19 2022-10-05 Medimmune Ltd Moléculas de transporte a través de la barrera hematoencefálica y usos de las mismas

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EP0773441A1 (fr) * 1995-09-11 1997-05-14 La Jolla Cancer Research Foundation Molécules qui s'adressent in vivo vers un organe ou tissu prélevé et méthodes pour leur identification

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US5527527A (en) * 1989-09-07 1996-06-18 Alkermes, Inc. Transferrin receptor specific antibody-neuropharmaceutical agent conjugates
AU705816B2 (en) * 1995-11-10 1999-06-03 Elan Corporation, Plc Peptides which enhance transport across tissues and methods of identifying and using the same
AU8269898A (en) * 1997-06-27 1999-01-19 Regents Of The University Of California, The Drug targeting of a peptide radiopharmaceutical through the primate blood-brain barrier in vivo with a monoclonal antibody to the human insulin receptor

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EP0773441A1 (fr) * 1995-09-11 1997-05-14 La Jolla Cancer Research Foundation Molécules qui s'adressent in vivo vers un organe ou tissu prélevé et méthodes pour leur identification

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