WO2004046187A2 - Anticorps destines a une utilisation in vitro - Google Patents

Anticorps destines a une utilisation in vitro Download PDF

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Publication number
WO2004046187A2
WO2004046187A2 PCT/GB2003/004944 GB0304944W WO2004046187A2 WO 2004046187 A2 WO2004046187 A2 WO 2004046187A2 GB 0304944 W GB0304944 W GB 0304944W WO 2004046187 A2 WO2004046187 A2 WO 2004046187A2
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amino acid
identity
seq
acid sequence
framework region
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PCT/GB2003/004944
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WO2004046187A3 (fr
Inventor
Terence Howard Rabbitts
Tomoyuki Tanaka
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Medical Research Council
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Publication of WO2004046187A2 publication Critical patent/WO2004046187A2/fr
Publication of WO2004046187A3 publication Critical patent/WO2004046187A3/fr

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    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C07K2317/60—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 the characteristics of antibodies that function within an intracellular environment (intrabodies).
  • the invention relates to the use of methods used in the generation of intracellularly functioning antibodies in the simple and efficient production of antibodies for in vitro use.
  • Intracellular antibodies or intrabodies have been demonstrated to function in antigen recognition in the cells of higher organisms (reviewed in Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Austin and Springer- Nerlag). This interaction can influence the function of cellular proteins which have been successfully inhibited in the cytoplasm, the nucleus or in the secretory pathway. This efficacy has been demonstrated for viral resistance in plant biotechnology (Tavladoraki, P., et al. (1993) Nature 366: 469-472) and several applications have been reported of intracellular antibodies binding to HIN viral proteins (Mhashilkar, A.M., et al.
  • the present inventors have devised a technique for the selection of immunoglobulins which are capable of f nctioning within an intracellular environment. That is they are correctly folded and are functional with respect to the selective binding of their ligand within that environment.
  • the antibody-antigen interaction method uses antigen linked to a DNA-binding domain as a bait and the scFv linked to a transcriptional activation domain as a prey. Specific interaction of the two facilitates transcriptional activation of a selectable reporter gene.
  • An initial in-vitro binding step is performed in which an antigen is assayed for binding to a repertoire of immunoglobulin molecules.
  • Those immunoglobulins which are found to bind to their ligand in vitro assays are then assayed for their ability to bind to a selected antigen in an intracellular environment, generally in a cytoplasmic environment.
  • the inventors found using this method that antibodies which function intracellularly comprise variable heavy chain domains which fall within the NHIII subgroup of heavy chain domains. In addition they found that such antibodies comprise light chain variable domains which fall within the N ⁇ l subgroup of variable domains. The inventors have used such antibodies as a model to evaluate a consensus scaffold derived from IAC methods. This method is described in PCT/GB02/003512.
  • antibodies are used extensively in bioscience as in vitro tools for recognising target antigens and for medical applications such as diagnosis or therapeutics.
  • Methods currently employed for the production of antibodies for in vitro use include the isolation of antibodies from body fluids of animals immunised with one or more particular antigens.
  • an antibody of a desired antigen binding specificity may be generated by expressing it within hybrid hybridomas.
  • Antibodies or fragments thereof are frequently generated using molecular biology techniques known to those skilled in the art and/or selected by phage display. All of the above techniques involve multiple steps and are therefore laborious.
  • intracellularly functioning antibodies isolated using IAC as discussed above that comprise variable heavy chain domains and/or light chain variable domains described by the consensus shown therein and depicted in fig 3 are also functional in vitro. This is surprising in light of prior art studies, discussed above, which show that antibodies that function in vitro often do not function in vivo. That is, prior art studies teach away from the use of intrabodies (intracellular functioning antibodies) for in vitro use.
  • the inventors have surprisingly found that intracellularly functioning antibodies comprising one variable domain type only are capable of specifically binding ligand within an in vitro environment. Furthermore, the inventors have found that there is no requirement for antibodies according to the invention to possess a heavy chain variable domain intra domain disulphide bond in order to bind specifically to ligand within such an environment.
  • the present invention provides the use of an immunoglobulin molecule comprising at least one N ⁇ framework region amino acid sequence showing at least 85% identity with the framework region consensus sequence depicted in SEQ ID no 1 and shown in fig 3, and/or a molecule comprising at least one N H framework region amino acid sequence showing at least 85% identity with the framework region consensus sequence depicted in SEQ ID no 1 and shown in fig 3 wherein one or more of amino acids 22 and 92 are not cysteine residues, in the specific binding of a ligand within an in vitro environment.
  • the use is of an immunoglobulin molecule comprising at least one VH framework region amino acid sequence showing at least 86% identity with the framework region consensus sequence depicted in SEQ ID no 1 and shown in fig 3 and/or a molecule comprising one VH framework region amino acid sequence showing at least 85% identity with the framework region consensus sequence depicted in SEQ ID no 1 and shown in fig 3 wherein one or more of amino acids 22 and 92 are not cysteine residues, and a J region which is a member of the JH5 and/or Jkl subgroup of J regions.
  • the immunoglobulin has a V H framework region amino acid sequence which shows at least 88% identity with the V H framework consensus sequence identified by SEQ ID no 1 and shown in fig 3 or the molecule comprises at least one VH framework region amino acid sequence showing at least 88% identity with the framework region consensus sequence depicted in SEQ ID no 1 and shown in fig 3 wherein one or more of amino acids 22 and 92 are not cysteine residues.
  • the immunoglobulin molecule has a V H framework region amino acid sequence shows at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identity with the V H framework consensus sequence identified by SEQ ID no 1 and shown in fig 3 or the molecule comprises at least one V H framework region amino acid sequence showing at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identity with the framework region consensus sequence depicted in SEQ ID no 1 and shown in fig 3 wherein one or more of amino acids 22 and 92 are not cysteine residues.
  • the immunoglobulin has a VH framework region amino acid sequence which shows at least 99% identity with the VH framework consensus sequence identified by SEQ ID no 1 and shown in fig 3 or a V H framework region amino acid sequence which shows at least 99% identity with the framework region consensus sequence depicted in SEQ ID no 1 and shown in fig 3 wherein one or more of amino acids 22 and 92 are not cysteine residues.
  • the immunoglobulin has a VH framework region amino acid sequence which shows 100% identity with the VH framework consensus sequence identified by SEQ ID no 1 and shown in fig 3 or the VH framework region amino acid sequence exhibits 100% identity with the framework region consensus sequence depicted in SEQ ID no 1 and shown in fig 3 wherein one or more of amino acids 22 and 92 are not cysteine residues.
  • the use is of an immunoglobulin molecule having a VH framework region amino acid sequence depicted in SEQ ID nos 2 to 10.
  • Immunoglobulins for use according to the above aspect of the invention may comprise a single domain type only or more than one domain type.
  • the use according to the above aspect of the invention is of one or more single variable domain type antibodies (single variable domain antibodies).
  • a single variable domain type antibody means an antibody as herein defined which comprises either one or more heavy chain variable domains or one or more light chain domains but not both heavy and light chain variable domains.
  • a single variable domain type antibody according to the invention is a Dab.
  • a 'Dab' is a single variable heavy chain domain or a single variable light chain domain optionally attached to a 'bulking group'.
  • the 'bulking group' as herein defined may comprise one or more antibody constant region domains.
  • the 'bulking group' may comprise components of non-immunoglobulin origin. These may include cytotoxins, fluorescent or other forms of labels.
  • a Dab as herein defined may comprise a light chain variable domain or a heavy chain variable domain in isolation.
  • the term 'in vitro ' means outside a cell.
  • the term 'an in vitro environment' means an extracellular environment.
  • the term 'in vitro environment' as herein defined does not include within its scope environments which mimic an in vivo environment, but which are in fact in vitro.
  • the term ' vitro ' as herein defined does not include within its scope in vitro transcription and translation systems.
  • the term c in vitro use ' therefore refers to any use which is not inside the cell, and which does not include the use in in vitro transcription and translation systems.
  • the term 'immunoglobulin' refers to a member of the immunoglobulin superfamily as herein described.
  • the immunoglobulin molecule is an antibody.
  • the antibody is an scFv or a Dab as herein defined.
  • the term 'specific binding' in the context of the present invention means that the interaction between the immunoglobulin and the ligand is selective, that is, in the event that a number of molecules are presented to the immunoglobulin, the latter will only bind to one or a few of those molecules presented.
  • the immunoglobulin ligand interaction will be of high affinity.
  • the interaction between immunoglobulin and ligand is mediated by non-covalent interactions such as hydrogen bonding and Van der Waals forces. Generally, the interaction will occur in the cleft between the heavy and the light chains of the immunoglobulin.
  • variable domain of an immunoglobulin molecule has a particular 3 dimensional conformation characterised by the presence of an immunoglobulin fold. Certain amino acid residues present in the variable domain are responsible for maintaining this characteristic immunoglobulin domain core structure. These residues are known as framework residues and tend to be highly conserved.
  • the present invention provides the use of an immunoglobulin molecule comprising at least one VL framework region amino acid sequence showing at least 85% identity with the framework region consensus sequence depicted in SEQ ID no 11 and shown in fig 3, in the specific binding of a ligand within an in vitro environment.
  • the use is of an immunoglobulin molecule comprising at least one VL framework region amino acid sequence showing at least 86% identity with the framework region consensus sequence depicted in SEQ ID no 11 and shown in fig 3, and a J region which is a member of the JH5 and/or Jkl subgroup of J regions.
  • the immunoglobulin has a VL framework region amino acid sequence which shows at least 87% identity with the VL framework consensus sequence identified by SEQ ID no 11 and shown in fig 3 or the molecule comprises at least one V framework region amino acid sequence showing at least 88% identity with the framework region consensus sequence depicted in SEQ ID no 11 and shown in fig 3.
  • the immunoglobulin molecule has a V framework region amino acid sequence shows at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identity with the V L framework consensus sequence identified by SEQ ID no 11 and shown in fig 3.
  • the immunoglobulin has a VL framework region amino acid sequence which shows at least 99% identity with the VL framework consensus sequence identified by SEQ ID no 11 and shown in fig 3.
  • the immunoglobulin has a V L framework region amino acid sequence which shows 100% identity with the V L framework consensus sequence identified by SEQ ID no 11 and shown in fig 3.
  • the use is of an immunoglobulin molecule having a VL framework region amino acid sequence depicted in SEQ ID nos 12 to 20.
  • Immunoglobulins for use according to the above aspect of the invention may comprise a single domain type only or more than one domain type.
  • the use according to the above aspect of the invention is of one or more single variable domain type antibodies (single variable domain antibodies), or one or more scFv's.
  • the present invention provides the use of an immunoglobulin molecule comprising at least one VH amino acid sequence showing at least 85% identity with the consensus sequence depicted in SEQ ID no 1 and shown in fig 3, and/or an immunoglobulin molecule comprising at least one VH amino acid sequence showing at least 85% identity with the consensus sequence depicted in SEQ ID no 1 and shown in fig 3 wherein one or more of amino acids 22 and 92 are not cysteine residues, in the specific binding of a ligand within an in vitro environment.
  • the use is of an immunoglobulin molecule comprising at least one V H amino acid sequence showing at least 86% identity with the consensus sequence depicted in SEQ ID no 1 and shown in fig 3, and a J region which is a member of the JH5 and/or Jkl subgroup of J regions.
  • the immunoglobulin has a VH amino acid sequence which shows at least 88% identity with the V H consensus sequence identified by SEQ ID no 1 and shown in fig 3 or the molecule comprises at least one V H amino acid sequence showing at least 88% identity with the consensus sequence depicted in SEQ ID no 1 and shown in fig 3 wherein one or more of amino acids 22 and 92 are not cysteine residues.
  • the immunoglobulin molecule has a V H amino acid sequence shows at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identity with the V H consensus sequence identified by SEQ ID no 1 and shown in fig 3 or the molecule comprises at least one V H amino acid sequence showing at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%», 98% identity with the consensus sequence depicted in SEQ ID no 1 and shown in fig 3 wherein one or more of amino acids 22 and 92 are not cysteine residues.
  • the immunoglobulin has a VH amino acid sequence which shows at least 99% identity with the VH consensus sequence identified by SEQ ID no 1 and shown in fig 3 or a VH region amino acid sequence which shows at least 99% identity with the consensus sequence depicted in SEQ ID no 1 and shown in fig 3 wherein one or more of amino acids 22 and 92 are not cysteine residues.
  • the immunoglobulin has a VH amino acid sequence which shows 100% identity with the VH consensus sequence identified by SEQ ID no 1 and shown in fig 3 or the VH amino acid sequence exhibits 100% identity with the consensus sequence depicted in SEQ ID no 1 and shown in fig 3, except wherein one or more of amino acids 22 and 92 are not cysteine residues.
  • the use is of an immunoglobulin molecule having a VH amino acid sequence depicted in SEQ ID nos 2 to 10 as shown in fig 3 and/or an immunoglobulin molecule comprising at least one of the latter sequences wherein one or more of amino acids 22 and 92 are not cysteine residues.
  • the present invention provides the use of an immunoglobulin molecule comprising at least one V L amino acid sequence showing at least 85% identity with the consensus amino acid sequence depicted in SEQ ID nos 11 and shown in fig 3, in the specific binding of a ligand within an in vitro environment.
  • an immunoglobulin molecule comprising at least one VL amino acid sequence showing at least 86% identity with the consensus sequence depicted in SEQ ID nos 11 and shown in fig 3, and a J region which is a member of the JH5 and/or Jkl subgroup of J regions.
  • the immunoglobulin has a VL amino acid sequence which shows at least 87% identity with the VL consensus sequence identified by SEQ ID no 11 and shown in fig 3 or the molecule comprises at least one VL amino acid sequence showing at least 88% identity with the consensus sequence depicted in SEQ ID no 11 and shown in fig 3.
  • the immunoglobulin molecule has a V L amino acid sequence shows at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identity with the V consensus sequence identified by SEQ ID no 11 and shown in fig 3.
  • the immunoglobulin has a V amino acid sequence which shows at least 99% identity with the VL consensus sequence identified by SEQ ID no 11 and shown in fig 3.
  • the immunoglobulin has a VL amino acid sequence which shows 100% identity with the V consensus sequence identified by SEQ ID no 11 and shown in fig 3.
  • the use is of an immunoglobulin molecule having a VL amino acid sequence described in SEQ ID nos 12 to 20.
  • the consensus sequences have been generated using multiple sequence alignments, which can be performed using methods familiar to those skilled in the art, as herein described.
  • the residue which is most common in any one given position, when the sequences of those immunoglobulins according to the invention are compared is chosen as the consensus residue for that position.
  • the consensus sequence is generated by comparing the residues for all the intracellularly binding immunoglobulins, at each position in turn, and then collating the data.
  • the ligand may be any selected from a polypeptide, protein, or nucleic acid.
  • polypeptide refers to a number of amino- acids joined together via peptide bonds to form single chain.
  • 'protein' refers to one or more polypeptide chains which are associated with one another to form a structure with a defined secondary and tertiary conformation.
  • a significant advantage in the generation of libraries from VH and V L amino acid sequences described herein is that their production obviates the use of phage scFv libraries and will reduce the necessary library size required for use in intracellular antibody capture technology as described in WOOO/54057.
  • intracellularly binding antibodies selected using this method also function in vitro, as herein defined, then the use of such libraries provides a simple and efficient method for the production of antibodies of in vitro use.
  • the present invention provides a library for producing immunoglobulins suitable for in vitro use, wherein the library is generated using any one or more of the following group consisting of: a VH framework region amino acid sequence showing at least 85% identity with the framework region consensus sequence depicted in SEQ ID no 1 and shown in fig 3, a VH amino acid sequence showing at least 85% identity with the consensus sequence depicted in SEQ ID no 1 and shown in fig 3, a VH framework region amino acid sequence showing at least 85% identity with the framework region consensus sequence depicted in SEQ ID no 1 and shown in fig 3 wherein one or more of amino acids 22 and 92 are not cysteine residues, a VH amino acid sequence showing at least 85% identity with the consensus sequence depicted in SEQ ID no 1 and shown in fig 3 wherein one or more of amino acids 22 and 92 are not cysteine residues, a VH amino acid sequence showing at least 85% identity with the consensus sequence depicted in SEQ ID no 1 and shown in fig 3 wherein one or more of
  • variable chains described in the above aspect of the invention show at least 86% identity with the respective amino acid sequences identified by SEQ ID no 1 and 11 and shown in fig 3.
  • the immunoglobulin molecule shows at least 87% identity.
  • the immunoglobulin shows at least 88%, 89%, 90%, 91, 92, 93, 94, 95, 96, 97% identity, 98% identity, 99% identity with the respective amino acid sequences identified by SEQ ID no 1 and 11 and shown in fig 3. In a most preferred embodiment it shows 100% identity with the respective sequences as herein described.
  • the library is generated from both one or more VH and one or more V L chain sequence/s as herein described. In an especially preferred embodiment it is generated from one or more VH and/or one or more V L chain sequences described by SEQ ID nos 2 to 10 and 12 to 20.
  • the library is generated from one or more variable heavy chain and variable light chain sequences/s as herein described and one or more J regions which is a member of the JH5 and/or Jkl subgroup of J regions.
  • the term 'library' refers to a mixture of polypeptides or nucleic acids coding for these polypeptides.
  • the library is composed of members. Sequence differences between library members are responsible for the diversity present in the library.
  • the library may take the form of a simple mixture of polypeptides or nucleic acids, or may be in the form organisms or cells, for example bacteria, viruses, animal or plant cells and the like, transformed with a library of nucleic acids.
  • each individual organism or cell contains only one member of the library.
  • each individual organism or cell may contain two or more members of the library.
  • the nucleic acids are incorporated into expression vectors, in order to allow expression of the polypeptides encoded by the nucleic acids.
  • a library may take the form of a population of host organisms, each organism containing one or more copies of an expression vector containing a single member of the library in nucleic acid form which can be expressed to produce its corresponding polypeptide member.
  • the library encodes a repertoire of immunoglobulin molecules.
  • a "repertoire” refers to a set of molecules generated by random, semi-random or directed variation of one or more template molecules, at the nucleic acid level, in order to provide a multiplicity of binding specificities sufficient to include individual specificities.
  • the template molecule is one or more of the V H and/or V L amino acid sequences herein described. Methods for generating repertoires are well characterised in the art.
  • the library expresses immunoglobulin molecules in which a substantial proportion of those molecules expressed are capable of selectively binding to a ligand within an intracellular environment
  • the immunoglobulin molecules are antibody molecules.
  • the antibody molecules are scFv or Dab/s as herein defined.
  • the library is a phage display library.
  • the present invention provides a method for designing an immunoglobulin molecule capable of specifically binding to a ligand within an in vitro environment comprising the steps of: (a) providing an immunoglobulin variable chain library, comprising essentially of VHIII and VKJ amino acid sequences. (b) selecting an immunoglobulin molecule from the library, wherein the molecule comprises at least one immunoglobulin variable chain selected from the group consisting of:
  • an immunoglobulin molecule comprising a VH framework region amino acid sequence showing at least 85% identity with the framework region consensus sequence depicted in SEQ ID no 1 and shown in fig 3
  • an immunoglobulin molecule comprising a VH framework region amino acid sequence showing at least 85% identity with the framework region consensus sequence depicted in SEQ ID no 1 and shown in fig 3.
  • an immunoglobulin molecule comprising a V H amino acid sequence showing at least 85% identity with the consensus sequence depicted in SEQ ID no 1 and shown in fig 3, wherein at least one of residues 22 and 92 (according to kabat numbering) are not cysteine residues.
  • an immunoglobulin molecule comprising a VH amino acid sequence showing at least 85% identity with the consensus sequence depicted in SEQ
  • an immunoglobulin molecule comprising a V L framework region amino acid sequence showing at least 85% identity with the framework region consensus sequence depicted in SEQ ID nos 11 and shown in fig 3.
  • an immunoglobulin molecule comprising a V amino acid sequence showing at least 85% identity with the VL consensus amino acid sequence depicted in SEQ ID nos 11 and shown in fig 3.
  • the library provided in step (a) is one of the variable chain immunoglobulin libraries of the present invention.
  • the library provided in step (a) comprises either VHIII or Vkl amino acid sequences and either JH5 and/or JRI amino acid sequences.
  • the step of selecting an immunoglobulin molecule described in step (b) may be performed using methods known to those skilled in the art as herein described.
  • the immunoglobulin molecules selected according to step (b) of the above aspect of the invention exhibit at least 86% identity with the respective amino acid sequences identified by SEQ ID no 1 and 11 and shown in fig 3. In an especially preferred embodiment, the immunoglobulin molecule shows at least 87% identity.
  • the immunoglobulin shows at least 88% identity 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identity, 98% identity or 99% identity with the respective amino acid sequences identified by SEQ ID no 1 and 11 and shown in fig 3, or those respective heavy chain amino acid sequences wherein at least one of amino acids 22 or 92 (according to Kabat numbering) are not cysteine residues. In a most preferred embodiment it shows 100% identity with the respective sequences as herein described.
  • the present invention provides the use of a library according to the present invention for producing immunoglobulin molecules suitable for in vitro use.
  • the term (antibody) 'suitable for in vitro use' means that the antibody according to the present invention is capable of binding specifically to a ligand /antigen within an in vitro environment and on specific ligand binding may initiate one or more downstream effects such as receptor binding and/or complement activation.
  • ligand /antigen within an in vitro environment and on specific ligand binding may initiate one or more downstream effects such as receptor binding and/or complement activation.
  • the immunoglobulin molecules expressed are antibody molecule/s.
  • the antibody molecule/s are scFv or Dab/s as herein defined.
  • Antibodies according to the present invention may be used for in vitro uses such as protein purification, for example antibody affinity purification, molecule detection, for example in Western blotting and ELISA, as blocking molecules, for example antibodies lacking Fc portions and therefore lack the ability to interact with Fc receptors and activate complement are particularly useful for this purpose.
  • Antibodies according to the invention which comprise an Fc antibody portion are particularly useful for initiating one or more biological responses such as complement activation and receptor binding in vitro.
  • Antibodies according to the invention may also be useful as cross-linking reagents, of particular use for this purpose are antibodies comprising two or more ligand binding specificitys.
  • antibodies according to the invention may be used in in vitro diagnosis. Details are described herein. Those skilled in the art will be aware of other suitable in vitro uses, and will be aware that this list is not intended to be exhaustive.
  • the present invention provides, the use of an immunoglobulin molecule comprising any one or more of the variable chains selected from the group consisting of the following: VH framework region amino acid sequence showing at least 85% identity with the framework region consensus sequence depicted in SEQ ID no 1 and shown in fig 3, a V H amino acid sequence showing at least 85% identity with the consensus sequence depicted in SEQ ID no 1 and shown in fig 3, a V framework region amino acid sequence showing at least 85% identity with the framework region consensus sequence depicted in SEQ ID nos 4 and shown in fig 3, and a V amino acid sequence showing at least 85% identity with the V L consensus amino acid sequence depicted in SEQ ID nos 4 and shown in fig 3, in one or more in vitro uses
  • the immunoglobulin molecules in the final aspect of the invention referred to above exhibit at least 86% identity with the respective amino acid sequences identified by SEQ ID no 1 and 11 and shown in fig 3.
  • the immunoglobulin molecule shows at least 87% identity.
  • the immunoglobulin shows at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identity, 98% identity, 99% identity with the respective amino acid sequences identified by SEQ ID no 1 and 11 and shown in fig 3 or the respective heavy chain amino acid sequences wherein one or more of amino acid residues 22 and 92 are not cysteine residues. In a most preferred embodiment it shows 100% identity with the respective sequences as herein described.
  • the immunoglobulin is an scFv or a Dab as herein defined.
  • the immunoglobulin molecule comprises a variable chain amino acid sequence which is described by any one of the sequences depicted by SEQ 2 to 10 and 12 to 20.
  • Immunoglobulins molecules refer to any moieties which are capable of binding to a target.
  • they include members of the immunoglobulin superfamily, a family of polypeptides which comprise the immunoglobulin fold characteristic of antibody molecules, which contains two beta sheets and, usually, a conserved disulphide bond.
  • Members of the immunoglobulin superfamily are involved in many aspects of cellular and non-cellular interactions in vivo, including widespread roles in the immune system (for example, antibodies, T-cell receptor molecules and the like), involvement in cell adhesion (for example the ICAM molecules) and intracellular signalling (for example, receptor molecules, such as the PDGF receptor).
  • the present invention is applicable to all immunoglobulin superfamily molecules which are capable of binding to target molecules.
  • the present invention relates to antibodies.
  • the antibody molecule is an scFv molecule or a Dab as herein defined.
  • Antibodies as used herein refers to complete antibodies or antibody fragments capable of binding to a selected target, and including Fv, ScFv, Fab' and F(ab') 2 , Dab, monoclonal and polyclonal antibodies, engineered antibodies including chimeric, CDR-grafted and humanised antibodies, and artificially selected antibodies produced using phage display or alternative techniques. Small fragments, such as Fv and ScFv, possess advantageous properties for diagnostic and therapeutic applications on account of their small size and consequent superior tissue distribution.
  • the antibody is a Dab as herein defined or an scFv.
  • a 'Dab' is a single variable heavy chain domain or a single variable light chain domain optionally attached to a 'bulking group'.
  • the 'bulking group' as herein defined may comprise one or more antibody constant region domains.
  • the 'bulking group' may comprise components of non-immunoglobulin origin. These may include cytotoxins, fluorescent or other forms of labels. Those skilled in the art will appreciate that this list is not intended to be exhaustive.
  • a Dab as herein defined may comprise a light chain variable domain or a heavy chain variable domain in the absence of a bulking group.
  • Heavy chain variable domain refers to that part of the heavy chain of an immunoglobulin molecule which forms part of the antigen binding site of that molecule.
  • the VHIII subgroup describes a particular sub-group of heavy chain variable regions (the VHIII).
  • immunoglobulin molecules having a variable chain amino acid sequence falling within this group possess a VH amino acid sequence which can be described by the VHIII consensus sequence in the Kabat database .
  • Light-chain variable domain refers to that part of the light chain of an immunoglobulin molecule which forms part of the antigen binding site of that molecule.
  • the Vkl subgroup of immunoglobulin molecules describes a particular subgroup of variable light chains.
  • immunoglobulin molecules having a variable chain amino acid sequence falling within this group possess a VL amino acid sequence which can be described by the V ⁇ l consensus sequence in the Kabat database.
  • variable domain of an immunoglobulin heavy and light chain variable domain has a particular 3 dimensional conformation characterised by the presence of an immunolgobulin fold. Certain amino acid residues present in the variable domain are responsible for maintaining this characteristic immunoglobulin domain core structure. These residues are known as framework residues and tend to be highly conserved.
  • CDR (complementarity determining region) of an immunoglobulin molecule heavy and light chain variable domain describes those amino acid residues which are not framework region residues and which are contained within the hypervariable loops of the variable regions. These hypervariable loops are directly involved with the interaction of the immunoglobulin with the ligand. Residues within these loops tend to show less degree of conservation than those in the framework region.
  • Intracellular means inside a cell and the present invention is directed to those immunoglobulins which will bind to ligands/targets selectively within a cell.
  • the cell may be any cell, prokaryotic or eukaryotic, and is preferably selected from the group consisting of a bacterial cell, a yeast cell and a higher eukaryote cell. Most preferred are yeast cells and mammalian cells.
  • "intracellular" immunoglobulins and targets or ligands are immunoglobulins and targets/ligands which are present within a cell.
  • the term 'Intracellular' refers to environments which resemble or mimic an intracellular environment.
  • intracellular may refer to an environment which is not within the cell, but is in vitro.
  • the method of the invention may be performed in an in vitro transcription and/or translation system, which may be obtained commercially, or derived from natural systems.
  • the term 'in vitro' means outside a cell.
  • the term 'an in vitro environment' means an extracellular environment.
  • the term 'in vitro environment' as herein defined does not include within its scope environments which mimic an in vivo environment, but which are in fact in vitro.
  • the term 'in vitro ' as herein defined does not include within its scope in vitro transcription and translation systems.
  • Consensus sequence of VH and N chains in the context of the present invention refers to the consensus sequences of those VH and V L chains from immunoglobulin molecules which can bind selectively to a ligand in an intracellular environment.
  • the residue which is most common in any one given position, when the sequences of those immunoglobulins which can bind intracellularly are compared is chosen as the consensus residue for that position.
  • the consensus sequence is generated by comparing the residues for all the intracellularly binding immunoglobulins, at each position in turn, and then collating the data. In this case the sequences of 11 immunoglobulins was compared.
  • a consensus residue is only conferred if a residue occurred greater than 5 times at any one position.
  • VH and VL consensus sequences does not include the sequences of the J regions.
  • first two residues (methionine and alanine ) are not part of the consensus. They are derived from an NCI1 restriction site.
  • Specific binding in the context of the present invention means that the interaction between the immunoglobulin and the ligand are selective, that is, in the event that a number of molecules are presented to the immunoglobulin, the latter will only bind to one or a few of those molecules presented.
  • the immunoglobulin ligand interaction will be of high affinity.
  • the interaction between immunoglobulin and ligand will be mediated by non-covalent interactions such as hydrogen bonding and Van der Waals. Generally, the interaction will occur in the cleft between the heavy and the light chains of the immunoglobulin.
  • a repertoire in the context of the present invention refers to a set of molecules generated by random, semi-random or directed variation of one or more template molecules, at the nucleic acid level, in order to provide a multiplicity of binding specificities.
  • the template molecule is one or more of the VH and/or VL domain sequences herein described.
  • a library according to the present invention refers to a mixture of polypeptides or nucleic acids.
  • the library is composed of members. Sequence differences between library members are responsible for the diversity present in the library. As defined herein, substantially all library members have been generated from the VH and/or VL sequences shown in SEQ ID nos 1 to 40.
  • the library may take the form of a simple mixture of polypeptides or nucleic acids, or may be in the form organisms or cells, for example bacteria, viruses, animal or plant cells and the like, transformed with a library of nucleic acids. Typically, each individual organism or cell contains only one member of the library. In certain applications, each individual organism or cell may contain two or more members of the library.
  • a library may take the form of a population of host organisms, each organism containing one or more copies of an expression vector containing a single member of the library in nucleic acid form which can be expressed to produce its corresponding polypeptide member.
  • RASG12V bait 428 colonies grew on histidine selective plates and showed strong activation of the lacZ gene, determined by ⁇ -gal filter assay. All prey plasmids were isolated from histidine-independent and ⁇ -gal positive yeast colonies and were fingerprinted by digestion with restriction enzymes, BstNl, Mspl, Mbol, Rsal or
  • FIG. 1 Interaction of anti-RAS scFv with RAS protein in mammalian cells.
  • A. Luciferase Assay COS7 cells were transiently co-transfected with various scFv- VP16 activation domain fusions and the GAL4 -DBD bait plasmid ⁇ Ml-HRASG12V (closed boxes) or pMl-lacZ (open boxes), together with the firefly luciferase reporter plasmid pG5-Luc and an internal Renilla luciferase control plasmid pRL-CMV.
  • scFv- VP16 prey vectors were used expressing anti-RAS scFv33, J48 and 121 or anti- -gal scFvR4 (Martineau et al, 1998).
  • the luciferase activities were measured 48 hours after transfection using Dual Luciferase Assay System (Promega) and a luminometer.
  • the luciferase activities of each assay were normalised to the Renilla luciferase activity (used as internal control for the transfection efficiency).
  • the fold luciferase induction level is shown with the activity of each scFv-VP16 with non-relevant bait taken as baseline.
  • Figure 3 Sequence of anti-RAS intracellular scFv.
  • the nucleotide sequences were obtained and the derived protein translations (shown as single letter code) were aligned.
  • Dashes in framework (FR) represent identities with the consensus (CON) sequence (derived from anti-BCR and anti-ABL scFv isolated by the IAC method (Tse et al, 2002)).
  • the numbers indicate the reference positions of the residues, according to the system by Lefranc et al (Lefranc and Lefranc, 2001) (top column number, indicated as IMGT) and Kabat et al (Kabat et al., 1991) (second column, Kabat).
  • the 15 residues of the linker, (GGGGS) 3 between the heavy chain of variable domain (VH) and light chain (VL) are not shown.
  • the complementarily determining regions (CDR) are highlighted on grey background and demarcated from framework regions (FR).
  • Three anti-RAS intracellular scFv are designed as 33, J48 and 121. All anti-RAS scFv belong to the VH3 subgroup of heavy chain and V 1 subgroup of light chain shown in the middle (designed VH3 or V I) from the Kabat database (Kabat et al., 1991) or IGVH3 and IGVK1 from the Lefranc database(Lefranc and Lefranc, 2001).
  • I21K33 comprises the six CDRs of scFv33 in the 121 framework and I21R33 is identical except for a mutation Lys94Arg;
  • I21R33VH21 VL comprises the VH domain of I21R33 fused to the VL domain of 121;
  • con33 has all six CDRs of scFv33 in the canonical consensus framework (Tse et al., 2002);
  • I21R33VH (C22S;C92S) is a mutant of clone I21R33 with the mutations CYS22SER and CYS92SER of the VH domain.
  • FIG. 4 Periplasmic expression and purification of anti-RAS scFv.
  • the scFv with pelB leader sequence at N-terminal and His6-tag and myc-tag at C- terminal were expressed periplasmically from the pHEN2-scFv vector in E.coli HB2151 using ImM IPTG for 2 hour at 30°C in 1 litre of 2 X TY medium including lOO ⁇ g/ml ampicillin and 0.1% glucose. After induction, the cells were harvested and extracted in 4 ml of ice cold 1 X TES buffer (0.2 M Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.5 M sucrose) and a further 6ml of 1 : 5 TES buffer was added.
  • 1 X TES buffer 0.2 M Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.5 M sucrose
  • the supernatants of cell extracts were used as the soluble periplasmic fraction.
  • the his- tagged scFv were purified by immobilised Ni 2+ ion chromatography and fractionated by 15% SDS-PAGE and proteins revealed by Coomassie blue staining.
  • the approximate yields of purified anti-RAS scFv33 and J48 were less than lOO ⁇ g per 1 litre culture; scFvI21R33, 121R-33VHI21L and 121 more than 3mg per litre; con33, 1 mg per litre.
  • scFv were pre-incubated with HRASG12V-GppNp (8 ⁇ g/ml; approx. 400nM) for 30 min at room temperature before addition to ELISA well.
  • Biosensor measuremenst were made using the BIAcore 2000. Purified scFv from bacterial cultures were used. A. Sensograms showing the binding of anti-RAS scFv with HRASG12V-GppNp antigen (immobilised 1500 RU). An injection volumes of 40 ⁇ l and flow rates of 20 ⁇ l/min were used. The purified scFv (10-2000nM) were loaded on 2 channels of the chip, containing either immobilised HRASG12V-GppNp or no antigen. The sensograms of each measurement were normalised by the resonance of the channel without antigen.
  • COS7 cells were transiently transfected with pEF-myc-cyto-scFv expression clones as indicated. Soluble and insoluble proteins were extracted, as described in materials and methods, and fractionated on 15% SDS-PAGE. After electrophoresis, protein were transferred to membranes and incubated with the anti-myc tag monoclonal antibody 9E10. The migration molecular weight markers (in kDa) are shown on the left. Arrows on the right indicate to the scFv fragment band.
  • Figure 8 Improvement of intracellular interaction between anti-RAS ICAbs and RAS antigen by the mutation of framework sequences.
  • COS7 Mammalian two-hybrid antibody-antigen interaction assays were performed in COS7 cells.
  • the upper panel represents normalised fold induction of luciferase signals (zero being taken as signal from prey plasmid without scFv) for scFv-VP16 binding RAS antigen bait.
  • the lower panel shows a Western blot of COS7 cell extracts after the expression of scFv-VP16 fusion proteins.
  • ScFv-VP16 fusion proteins were detected by Western-blot using anti-VP16 (Santa Cruz Biotechnology, 14-5) monoclonal antibody and horseradish peroxidase (HRP)- conjugated anti-mouse IgG antibody.
  • ICAb scFv used as a control was anti- -gal R4 (Martineau et al, 1998).
  • scFv33 mutants were (using Kabat et al (Kabat et al., 1991) and number in parenthesis also indicate numbering by Lefranc et al (Lefranc and Lefranc, 2001)) (see Fig.
  • VH(A74S+S77T) substitutions Ala74(83)Ser and Ser77(86)Thr of VH
  • VH(D84A) substitution Asp84(96)Ala of VH
  • VH(R94K) substitution Arg94(106)Lys ofVH
  • VL(0T+V3Q) addition Thr between linker and NL domain plus substitution
  • NL(I84T) substitution Ile84(100)Thr of NL NH(Q1E+V5L+A7S+S28T) +VL(G100Q+V104L): substitutions Glnl(l)Glu, Val5(5)Leu, Ala7(7)Ser, Ser28(29)Thr of VH plus GlylOOGln and Vall04Leu of VL.
  • Mutant HRAS G12V cDNA were subcloned into the mammalian expression vector pZIPneoSV(X) and anti-RAS scFv into pEF-FLAG-Memb vector which has plasma membrane targeting signal at C-terminal of scFv and FLAG-tag at N-terminal to scFv.
  • 100 ng of pZIPneoSV(X)-RASG12V and 2 ⁇ g of pEF-FLAG-Memb-scFv were co- transfected into NIH 3T3 cells cloneD4. Two days later, the cells were transferred to 10 cm plates and grown for 14 days in DME medium containing 5 % donor calf serum and penicillin and streptomycin. Finally, the plates were stained with crystal violet and foci of transformed cells were counted.
  • the IAC technology described in WOOO/54057 includes one round of scFv phage display library screening in vitro with a recombinant bacterial protein, followed by selection in a yeast in vivo antibody-antigen interaction screening of the in vitro enriched scFv repertoire (Visintin, M.., Tse, E., Axelson, H., Rabbitts, T.H. and Cattaneo, A. (1999) Proc. Natl. Acad. Sci. USA 96 11723-11728)
  • Immunoglobulin molecules used according to the present invention include members of the immunoglobulin superfamily, which are a family of polypeptides which comprise the immunoglobulin fold characteristic of antibody molecules. The fold contains two beta sheets and, usually, a conserved disulphide bond. Members of the immunoglobulin superfamily are involved in many aspects of cellular and non-cellular interactions in vivo, including widespread roles in the immune system (for example, antibodies, T-cell receptor molecules and the like), involvement in cell adhesion (for example the ICAM molecules) and intracellular signalling (for example, receptor molecules, such as the PDGF receptor).
  • the present invention is applicable to all immunoglobulin superfamily molecules which are capable of binding to target molecules.
  • the present invention relates to antibodies.
  • the antibodies according to the present invention relates to scFv fragments and Dabs.
  • immunoglobulins molecules used according to the present invention all possess the requisite activity of being capable of specifically binding to a ligand within an intracellular environment and within an in vitro environment.
  • the immunoglobulins used according to the invention all share a VH amino acid sequence which is a member of the VHIII subgroup of heavy chains.
  • Heavy chains described by this group generally possess a VH amino acid sequence which show a very high degree of homology with the Kabat database VHIII consensus sequence shown in SEQ ID no 1 and in fig 3. This suggests that immunoglobulins having a heavy chain which falls within the VHIII sub-group have particularly high efficacy in an in vivo environment and surprisingly also within an in vitro enviroment.
  • the immunoglobulin molecules according to the present invention have a VHIII subgroup joined to JH5 or Jkl region.
  • the present inventors have shown that the framework regions of depicted as 121 in figure 3 and designated SEQ ID No: 4 and 14 and the framework regions of the consensus designated SEQ ID No: 1 and 11 in figure 3, and SEQ ID No: 7 and 17 in fig 3 confer upon the an antibody comprising them the solubility required to function within an intracellular environment and also within an in vitro environment.
  • sequences which do not comprise cysteine residues at one or more of positions 22 and 92 according to Kabat may also be used to confer upon the an antibody comprising them the solubility required to function within an in vitro environment.
  • amino acid sequences wherein the cysteine at one or both positions is replaced by serine may be used to confer upon the an antibody comprising them the conformational stability and solubility required to function within an in vitro environment.
  • an immunoglobulin molecule comprising any of the framework sequences above may be used to generate an scFv, a Dab or an antibody comprising both light and heavy chains which can specifically bind to one or more ligands within an in vitro environment.
  • Recombinant DNA technology may be used to produce the immunoglobulins for in vitro use according to the present invention using an established procedure, in bacterial or preferably mammalian cell culture.
  • the selected cell culture system preferably secretes the immunoglobulin product.
  • Multiplication of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which are the customary standard culture media, for example
  • Dulbecco's Modified Eagle Medium or RPMI 1640 medium
  • a mammalian serum e.g. foetal calf serum
  • trace elements and growth sustaining supplements e.g. feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoprotein, oleic acid, or the like.
  • Multiplication of host cells which are bacterial cells or yeast cells is likewise carried out in suitable culture media known in the art, for example for bacteria in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x YT, or M9 Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.
  • In vitro production provides relatively pure immunoglobulin preparations and allows scale-up to give large amounts of the desired immunoglobulins.
  • Techniques for bacterial cell, yeast or mammalian cell cultivation are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilised or entrapped cell culture, e.g. in hollow fibres, microcapsules, on agarose microbeads or ceramic cartridges.
  • the desired immunoglobulins can also be obtained by multiplying mammalian cells in vivo.
  • hybridoma cells producing the desired immunoglobulins are injected into histocompatible mammals to cause growth of antibody-producing tumours.
  • the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection.
  • pristane tetramethyl-pentadecane
  • hybridoma cells obtained by fusion of suitable myeloma cells with antibody-producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneally into Balb/c mice optionally pre-treated with pristane, and, after one to two weeks, ascitic fluid is taken from the animals.
  • the cell culture supernatants are screened for the desired immunoglobulins, preferentially by immunofluorescent staining of cells expressing the desired target by immunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.
  • an enzyme immunoassay e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.
  • those present in the culture supernatants or in the ascitic fluid may be concentrated, e.g. by precipitation with ammonium sulphate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes, or the like.
  • the antibodies are purified by the customary chromatography methods, for example gel filtration, ion- exchange chromatography, chromatography over DEAE-cellulose and/or (immuno- )affinity chromatography, e.g. affinity chromatography with the target molecule or with Protein-A.
  • the invention employs recombinant nucleic acids comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies.
  • nucleic acids comprise coding single stranded nucleic acids, double stranded nucleic acids consisting of said coding nucleic acids and of complementary nucleic acids thereto, or these complementary (single stranded) nucleic acids themselves.
  • nucleic acids encoding a heavy chain variable domain and/or for a light chain variable domain of antibodies can be enzymatically or chemically synthesised from nucleic acids having the authentic sequence coding for a naturally-occurring heavy chain variable domain and/or for the light chain variable domain, or a variant or derivitive thereof as herein described.
  • said modification(s) are outside the CDRs of the heavy chain variable domain and/or of the light chain variable domain of the antibody.
  • IAC libraries may be screened using methods described herein and those antibodies which bind within an intracellular and extracellular environment selected. Details of suitable libraries are described herein.
  • polypeptide sequences of the invention are not limited to the particular sequences set forth in SEQ. ID. No. 1 to 20 or fragments thereof, but also include homologous sequences obtained from any source, for example related cellular homologues, homologues from other species and variants or derivatives thereof.
  • the present invention encompasses variants, homologues or derivatives of the amino acid sequences set forth in SEQ. ID. No. 1 to SEQ 20 as long as when said variants, homologues or derivatives of the amino acid sequences set forth in SEQ. ID. No. 1 to SEQ 20 are one or more components of a immunoglobulin molecule, they possess the requisite activity of binding specifically to a ligand within an intracellular environment and also within an in vitro environment.
  • a homologous sequence is taken to include an amino acid sequence which is at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical at the amino acid level over at least 30, preferably 50, 70, 90 or 100 amino acids.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
  • % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • % homology preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • the present invention provides a method for designing an immunoglobulin molecule capable of specifically binding to a ligand within an in vitro environment comprising the steps of:
  • an immunoglobulin variable chain library comprising essentially of VHIII and V R I amino acid sequences.
  • an immunoglobulin molecule comprising a V H framework region amino acid sequence showing at least 85% identity with the framework region consensus sequence depicted in SEQ ID no 1 and shown in fig 3
  • an immunoglobulin molecule comprising a V H framework region amino acid sequence showing at least 85% identity with the framework region consensus sequence depicted in SEQ ID no 1 and shown in fig 3
  • an immunoglobulin molecule comprising a V H amino acid sequence showing at least 85% identity with the consensus sequence depicted in SEQ
  • an immunoglobulin molecule comprising a V H amino acid sequence showing at least 85% identity with the consensus sequence depicted in SEQ ID no 1 and shown in fig 3 wherein at least one of residues 22 and 92
  • an immunoglobulin molecule comprising a V L framework region amino acid sequence showing at least 85% identity with the framework region consensus sequence depicted in SEQ ID nos 11 and shown in fig 3.
  • an immunoglobulin molecule comprising a V L amino acid sequence showing at least 85% identity with the V L consensus amino acid sequence depicted in SEQ ID nos 11 and shown in fig 3.
  • the library screened according to the above aspect of the invention is one as herein described.
  • the library may be a Dab library.
  • the IAC method can equally be applied to the selection of single heavy chain variable domain only antibodies (Dabs) which are specific for one or more ligands.
  • Dabs single heavy chain variable domain only antibodies
  • Methods for the screening of Dab libraries are described in GBXXXX . An brief description of a suitable method is provided below:
  • the screening of synthetic Dab libraries may be performed according to the protocol of intracellular antibody capture (IAC) technology as described REF (see also a link within the Laboratory of Molecular Biology website http://mrc-lmb.cam.ac.uk) but excluding the phage panning step.
  • IAC intracellular antibody capture
  • 500 ⁇ g of pBTM-antigen and lmg of pVP16-Dab library 1 or pVP16-Dab library 2 are co-transfected into S. cerevisiae L40.
  • Positive clones are selected by using auxotrophic markers, Tip, Leu and His.
  • Positive colonies are selected for His prototropy and confirmed by ⁇ -galactosidase ( ⁇ -gal) activity by filter assay.
  • false positive clones are eliminated and true positive clones are confirmed by re-testing of His independent growth and ⁇ -gal activation, using relevant and non-relevant bait vectors .
  • ligands include polypeptides and proteins, particularly nascent polypeptides and proteins or intracellular polypeptide or protein precursors, which are present in the cell.
  • the ligand is a mutant polypeptide or protein, such as a polypeptide or protein generated through genetic mutation, including point mutations, deletions and chromosomal translocations. Such polypeptides are frequently involved in tumourigenesis. Examples include the gene product produced by the spliced BCR- ABL genes.
  • the invention is moreover applicable to all mutated oncogene products, all chromosomal translocated oncogene products (especially fusion proteins), aberrant proteins in expressed in disease, and viral or bacerial specific proteins expressed as a result of infection.
  • the ligand may alternatively be an RNA molecule, for example a precursor RNA or a mutant RNA species generated by genetic mutation or otherwise.
  • the ligand may be inserted into the cell, for example as described below, or may be endogenous to the cell.
  • the present invention provides a library for the generation of antibodies suitable for in vitro use which is generated using any one or more of the variable domain amino acid sequences described herein.
  • These libraries may encode, express, and/or present immunoglobulin molecules or fragments thereof which may be tested for their ability to interact with a ligand within an intracellular environment.
  • the library of the present aspect of the invention may be tested for the binding of immunoglobulin molecules expressed using the intracellular antibody capture method described in WOOO/54057.
  • libraries of the present invention will encode or express VH and VL chains which when incorporated within an immunoglobulin molecule will bind selectively to a ligand within an intracellular environment.
  • the immunoglobulin is scFv or a Dab.
  • phagebodies lambda phage capsids
  • An advantage of phage-based display systems is that, because they are biological systems, selected library members can be amplified simply by growing the phage containing the selected library member in bacterial cells. Furthermore, since the nucleotide sequence that encode the polypeptide library member is contained on a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation is relatively straightforward.
  • Alternative library selection technologies include bacteriophage lambda expression systems, which may be screened directly as bacteriophage plaques or as colonies of lysogens, both as previously described (Huse et al. (1989) Science, 246: 1275; Caton and Koprowski (1990) Proc. Natl. Acad. Sci. U.S.A., 87; Mullinax et al. (1990) Proc. Natl. Acad. Sci. U.S.A., 87: 8095; Persson et al. (1991) Proc. Natl. Acad. Sci. U.S.A., 88: 2432) and are of use in the invention.
  • a significant improvement of the bead-based methods involves tagging each bead with a unique identifier tag, such as an oligonucleotide, so as to facilitate identification of the amino acid sequence of each library member.
  • a unique identifier tag such as an oligonucleotide
  • Another chemical synthesis method involves the synthesis of arrays of peptides (or peptidomimetics) on a surface in a manner that places each distinct library member (e.g., unique peptide sequence) at a discrete, predefined location in the array.
  • the identity of each library member is determined by its spatial location in the array.
  • the locations in the array where binding interactions between a predetermined molecule (e.g., a receptor) and reactive library members occur is determined, thereby identifying the sequences of the reactive library members on the basis of spatial location.
  • RNA molecules are selected by alternate rounds of selection against a target ligand and PCR amplification (Tuerk and Gold (1990) Science, 249: 505; Ellington and Szostak (1990) Nature, 346: 818).
  • Immunoglobulin molecules according to the present invention preferably scFv molecules may be employed in any in vitro use, for example in vitro assay and reagent applications, in functional genomics applications and the like.
  • antibodies according to the invention may be used in in vitro diagnosis.
  • such antibodies may be used in detecting levels of proteins, nucleic or other molecules within samples, such as the body fluids of individuals.
  • antibodies according to the invention may be generated against the hormone HCG and may be used in the diagnosis/detection of pregnancy.
  • Antibodies according to the invention for diagnostic and other uses may be utilised in solution or bound to a solid support.
  • suitable supports may be natural or synthetic. Synthetic supports include SephadexTM (useful for affinity purification), and SephagelTM (also useful for affinity purification) and plastic (useful for ELISA). Natural supports include cellulose. Those skilled in the art will be aware of other suitable supports. Supports may be any suitable shape. In particular, supports used for diagnosis may be in the form of a dipstick for easy sampling of fluids. Those skilled in the art will be aware of other suitable in vitro uses, and will be aware that this list is not intended to be exhaustive.
  • the selected immunoglobulins of the present invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use.
  • Known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of functional activity loss and that use levels may have to be adjusted upward to compensate.
  • Recombinant oncogenic HRAS (G12V; residues 1 - 166) was expressed in bacterial cells harbouring expression plasmids based on pETl la (Novagen) and purified by ion- exchange chromatography and gel-filtration described elsewhere (Pacold et al., 2000).
  • 3 mg of purified HRASG12V protein was loaded with 2 mM of 5'-guanylylimidodi-phosphate (GppNp, Sigma), non- hydrolysable analogue of GTP, using the alkaline phosphatase protocol (Herrmann et al., 1996). This GppNp-bound HRASG12V was used as antigen throughout.
  • In vitro scFv phage library screening and preparation of specific scFv-VP16 yeast library was used as antigen throughout.
  • the scFv DNA fragments were sub-cloned into the pVP16 yeast vector and 4.13 x 10 clones used for yeast screening.
  • the RAS bait was prepared by cloning truncated HRASG12V cDNA into the ⁇ coRl-BamHl site of pBTMl l ⁇ vector.
  • the pBTMl 16-RASG12V bait vector (tryp+) was transfected into S. cerevisiae L40 using the lithium acetate / polyethylene glycol method (Tse et al., 2000), and colonies growing on Tip- plates were selected.
  • the expression of LexA-RAS fusion protein was confirmed by Western blot using anti-pan RAS (Ab-3, Oncogene Research Product).
  • yeast scFv-VP16 library DNA 100 ⁇ g of yeast scFv-VP16 library DNA were transformed into L40 clone stably expressing antigen. Positive colonies were selected for His prototropy and confirmed by ⁇ -galactosidase ( ⁇ -gal) activity by filter assay. For the isolated individual clones, false positive clones were eliminated and true positive clones were confirmed by re-testing of His independent growth and ⁇ -gal activation.
  • Periplasmic bacterial expression of scFv was as described (Tse et al, 2000).
  • the scFv were cloned into pH ⁇ N2 vector (see www.mi-c-cpe.cam.ac.uk for map) and expressed for 2 hours at 30°C with 1 mM ITPG in 1 litre culture of E. coli HB2151 cells.
  • the cells were harvested and extracted periplasmic proteins with TES buffer (Tris-HCl (pH 7.5), EDTA, sucrose). The periplasmic proteins were dialyzed overnight against 2.5 litre of PBS including 10 mM imidazole.
  • Immobilised metal ion affinity chromatograpy of periplasmic scFv was carried out at 4°C for 1 hour with 4 ml of Ni- NTA agarose (QIAGEN). The agarose was washed 4 times with 20 ml of PBS with 20 mM imidazole. The polyhistidine-tagged scFv were eluted with 4 ml of 250 mM imidazole in PBS. The eluate was dialyzed overnight against 2.5 liter of 20 mM Tris- HCl (pH 7.5) including 10% glycerol at 4 °C.
  • Purified scFv was concentrated to 1 to 5 mg/ml using Centricon concentrator (YM-10, Amicon) and the aliquots were stored at -70°C. Protein concentration of purified scFv were measured using Bio-Rad Protein assay Kit (Bio-Rad).
  • the ELISA plate wells were coated with lOO ⁇ l of purified HRASG12V-GppNp antigen (4 ⁇ g/ml, approximately 200nM) in PBS overnight at 4°C. Wells were blocked with 3% bovine serum albumin (BSA)-PBS for 2 hours at room temperature. The respective purified scFv (approximately 450ng) were diluted in 90 ⁇ l in 1% BSA- PBS and allowed to bind for 1 hour at 37°C.
  • BSA bovine serum albumin
  • HRP horseradish peroxidase conjugated anti- polyhistidine
  • HIS-1 horseradish peroxidase conjugated anti- polyhistidine
  • HRP activity was visualised using 3,3',5,5-tetramethylbenzidine (TMB) liquid substrate system according to manufacturer's instruction. The reaction was stopped with 0.5M hydrosulphate and data collected with a microtiter plate reader (450 - 650nm filter).
  • TMB 3,3',5,5-tetramethylbenzidine
  • the BIAcore 2000 (Pharmacia Biosensor) was used to measure the binding kinetics of scFv with antigen.
  • the sensorchip was first activated by flowing 40 ⁇ l of the mixture of EDC/NHS (N-ethyl-N- (dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide) at lO ⁇ l/min flow rate.
  • EDC/NHS N-ethyl-N- (dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide
  • lOO ⁇ g/ml of purified HRASG12V-Gp ⁇ Np in lOmM sodium acetate, pH 3.5 was injected and immobilised until approximately 1500 RU.
  • scFv 10-500nM were loaded at flow rate of 20 ⁇ l/minute at 25°C (running buffer HBS-EP (0.01 M HEPES, pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% v/v polysorbate 20) plus 2mM MgCl 2 ,) on 2 channels of the chip containing either immobilised HRASG12V-GppNp or no antigen, for the determination of the binding affinity of scFv. Each determination was performed in duplicate.
  • the antigen immobilised surface on the sensorchip after binding scFv was regenerated by rinsing with lOmM HC1 until the starting baseline was achieved.
  • the scFv were cloned into Sfil and Notl site of pEF-BOS-VP16 expression vector (manuscript in preparation).
  • the HRAS expression plasmid (pMl-RASG12V) expressing RASG12V in-frame with Gal4 DBD, was made by sub-cloning HRASG12V cDNA (codons 1-166) into EcoRl / BamHl site of pMl vector (Sadowski et al., 1992).
  • COS7 cells were transiently co-transfected with 500ng of pG5-Luc reporter plasmid (de Wet et al., 1987), 50ng of pRL-CMV (Promega), 500ng of pEF-BOS- NP16/scFv and 500ng of pMl/antigen bait with 8 ⁇ l of LipofectAMI ⁇ ETM transfection reagent (Invitrogen, according to manufacture's instruction). Forty-eight hours after transfection, the cells were washed once with PBS and lysed in 500 ⁇ l of IX passive lysis buffer (Promega) at room temperature for 15 min with gently shaking.
  • IX passive lysis buffer Promega
  • scFv DNA fragments were cloned into the Ncol-Notl site of pEF-nuc-myc (Invitrogen) with nuclear localisation signal (nls) at N-terminal and myc-tag at C-terminal of exprssed scFv.
  • nls nuclear localisation signal
  • full length RASG12V cDNA was cloned into the Kpnl-EcoRl site of pHM6 vector (Boehringer Mannheim) to encode RAS with HA-tag at N-terminal and His6-tag at C-terminal.
  • pHM6 vector Boehringer Mannheim
  • the plasmids were co-transfected using Lipofectamine and forty-eight hours after transfection, cells were washed twice with PBS, permeabilised with 0.5% Triton X in PBS and fixed with 4% paraformaldehyde in PBS. Cells were stained with anti c-myc mouse monoclonal antibody (Santa Cruz; 9E10) and anti-HA rabbit polyclonal serum (Santa Cruz; sc-805) both at dilutions of 1:100. Secondary antibodies, fluorescein-linked sheep anti-mouse antibody and Cy 3 -linked goat anti- rabbit antibody (Amersham Pharmacia Biotech (APB)), were used at dilutions of 1:200 for staining. After several washes with PBS, the slides were overlaid with cover-slips and staining patterns were studied using a Bio-Radiance confocal microscope (Bio-Rad).
  • the scFv or scFv-VP16 fusion proteins were expressed in COS7 cells.
  • scFv expression scFv DNA fragments were cloned into Ncol / Notl sites of pEF-myc-cyto expression vector (Invitrogen). The day before transfection, COS7 cells were seeded at about 2 x 10 5 per well in 6-well culture plate (Nunc). l ⁇ g of pEF-myc-cyto-scFv or pEF-BOS- scFv-VP16 were transiently transfected with 8 ⁇ l of LipofectAMINE.
  • the cells were washed once with PBS, lysed for 30 minutes in ice cold extraction buffer (lOmM HEPES, pH 7.6, 250mM NaCl, 5mM EDTA, 0.5% NP-40, 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml pepstatin A, O.lmg/ml aprotinin, lmM phenylmethanesulsonyl fluoride (PMSF)) and centrifuged for 10 minutes at 13,000 rpm at 4°C.
  • ice cold extraction buffer lOmM HEPES, pH 7.6, 250mM NaCl, 5mM EDTA, 0.5% NP-40, 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml pepstatin A, O.lmg/ml aprotinin, lmM phenylmethanesulsonyl fluoride (PMSF)
  • the pellets ("insoluble” fraction) and the supernatants ("soluble” fraction) were analysed by SDS-PAGE, followed by Western blot using anti-myc (9E10) monoclonal antibody (for detection of scFv) or anti-VP16 (14-5, Santa-Cruz) monoclonal antibody (for scFv-VP16AD fusion) as primary antibody and HRP- conjugated rabbit anti-mouse IgG antibody (APB) as secondary antibody.
  • the blots were visualised by enhanced chemiluminescence (ECL) detection kit (ABP)
  • I21R33 (sequence shown in Figure 3), which comprises FRs of anti-RAS scFvI21 and the CDRs of anti-RAS scFv33, was constructed using step-by-step site-specific mutagenesis of scFv33 as primary template using footprint mutagenesis.
  • I21R33 (VHC22S;C92S), con33 and I21R33VHI21VL ( Figure 3) were also constructed by mutations of I21R33 using footprint mutagenesis with appropriate oligonucleotides.
  • scFv constructs were digested with Sfil or ⁇ col, and ⁇ otl and subcloned into pEF-BOS-VP16 (for in vivo antigen antibody interaction assay) and pEF-myc-cyto vector (for expression of scFv in mammalian cells). All mutated scFv constructs were verified by D ⁇ A sequencing.
  • RAS protein is localsied to the plasma membrane of cells and therefore to localise scFv to cell membrane we used the pEF-Memb vector (Invitrogen).
  • the scFv expression plasmid was constructed by introducing carboxyl terminal 20 amino acid residues of HRAS into the ⁇ otl-Xbal site of pEF-myc-cyto vector.
  • This expression vector also was introduced FLAG-tag peptide (MDYKDDDDK) and alternative Sfil cloning site into blunt-ended Sfil site of pEF-Memb vector named pEF-FLAG-Memb.
  • the scFv were sub-cloned into Sfil -Not 1 of pEF-FLAG-Memb.
  • HRASG12V mutant cDNA were subcloned into expression vector pZIPneoSV(X) REF. .Low passage NIH3T3 cells clone D4 (a gift from Dr Chris Marshall) were seeded at 2 x 10 5 cells per well in 6-well plates the day before transfection, For transfection, 2 ⁇ g of each pEF-FLAG-Memb-scFv plus 100 ng of pZIPneoSV(X)-HRASG12V vector was used, using 12 ⁇ l of LipofectAMINETM.
  • the cells were transferred to 10 cm plates and grown for two weeks in DME medium containing 5 % donor calf serum (Invitrogen) and penicillin and streptomycin. The plates were finally stained with crystal violet and the number of foci counted.
  • DME medium containing 5 % donor calf serum (Invitrogen) and penicillin and streptomycin. The plates were finally stained with crystal violet and the number of foci counted.
  • the sub-library was prepared as phagemid DNA and cloned into a yeast VP16 transcriptional activation domain vector to make an anti-RAS scFv-VP16-AD library (about 4 x 10 6 clones).
  • This yeast sub-library was transfected into a yeast strain (L40 with his and - gal reporter genes) expressing the fusion protein bait comprising the LexA-DBD fused to RAS-G12V. A total of approximately 8.45 x 10 7 yeast colonies were screened (Fig. 1).
  • the scFv-VP16-AD plasmids were isolated from the histidine- independent, ⁇ -gal positive clones and assorted by their DNA restriction patterns. More than 90 % of these scFv-VP16-AD plasmids had an identical DNA finger printing pattern and twenty were sequenced and found to have identical DNA sequences. Those scFv with differing DNA finger print patterns were co-transformed with the pBTM/RASG12V bait in fresh yeast and assayed for histidine-independent growth and ⁇ -gal activation.
  • scFv Three anti-RAS scFv, designated 33, J48 and 121, were thus identified (Fig. 1 ). The specificity of these scFv for binding to RAS in yeast was further verified by their lack of interaction with the LexA DBD (made from the empty pBTMl 16 vector) and a non-relevant antigen ( ⁇ -galactosidase) (data not shown).
  • the efficacy of the anti-RAS ICAbs was confirmed using a mammalian cell reporter assay and in vivo antigen co-location assays (Fig. 2).
  • the mammalian cell assay used was luciferase production from a luciferase reporter gene.
  • the three scFv were shuttled into a mammalian expression vector, pEF-BOS-VP16, which has the elongation factor- la promoter (Mizushima and Nagata, 1990) and the VP16 transcriptional activation domain (AD).
  • the scFv were cloned in frame with the VP16 segment, on its N-terminal side (Triezenberg et al, 1988).
  • the RASG12V antigen was cloned into the pM vector (Sadowski et al, 1992) which has the GAL4-DBD as an N- terminal fusion with antigen ( ⁇ M-RASG12V).
  • ⁇ EFBOSVP16-scFv and pM- RASG12V were co-transfected into COS7 cells with the luciferase reporter plasmid. More than 10-fold activation was observed when scFv33 or J48 ICAb-VP16 fusion were expressed with the bait antigen RASG12V (Fig. 2 A) but none with a non- relevant antigen ⁇ -galactosidase.
  • ICAb 121 was co-expressed with RASG12V bait (Fig. 2A). Similar results were obtained in other mammalian cell lines viz. Hela and CHO cells.
  • RAS antigen was detected with anti-HA tag Ab and scFv with anti-myc tag Ab (Fig. 2B).
  • the antigen was detected in the cytoplasm and antibody in the nucleus (Fig. 2B, lower panels), whereas if the antigen was co-expressed with the anti-RAS ICAb 33 with a nls, co-location of RAS antigen and scFv was observed in the nucleus.
  • the anti-RAS ICAbs 33 have sufficient expression and affinity to bind RAS antigen in vivo and cause re-location within the cell (similar results were found with anti-RAS scFv J48, data not shown).
  • the anti-RAS scFv (33, J48 and 121) were sequenced and derived protein sequence aligned (Fig. 3). All three scFv belong to VH3 subgroup joined to the JH5 and to the V 1 subgroup.
  • Our previous data on anti-BCR and anti-ABL scFv (Tse et al., 2002), which were isolated only from the library of Sheets et al (Sheets et al., 1998), also belong to VH3 and V 1 subgroup.
  • scFv The levels of expression of three anti-RAS scFv were initially examined by bacterial periplasmic expression. These scFv were sub-cloned into pHEN2, which has the PelB leader sequence 5' to the scFv, allowing the periplasmic expression of soluble scFv protein (see www.mrc-cpe.cam.ac.uk for map). Periplasmic scFv extracts were purified by immobilised metal ion affinity chromatography (IMAC) and protein preparations separated by SDS-PAGE (Fig. 4).
  • IMAC immobilised metal ion affinity chromatography
  • the scFvI21 accumulated mainly in the soluble fraction, when secreted to the periplasm at 30°C and the periplasmic expression yield was approximately 3 mg per litre culture.
  • the other anti-RAS scFv 33 and J48 were expressed at less than 0.1 mg per litre.
  • Comparison of the anti-RAS scFv sequences with the consensus ICAb sequence reveals only four differences in the VH framework residues of 33 and J48, one of which is position 7 in VH FR1. This residue is one of three which influence conformation of this region (Jung et al, 2001) and may thus influence ICAb 33 and J48 solubility.
  • 121 conforms to the consensus in positions VH FR1 6, 7 and 10.
  • the properties of the ICAbs isolated in our work were aslo characterised using two in vitro assays.
  • the interaction of the scFv with RAS antigen was investigated with ELISA and biosensor assays using purified scFv made in bacteria.
  • RASG12V-GppNp was coated as antigen onto ELISA plates, challenged with purified scFv and bound scFv was detected using HRP conjugated anti-His tag antibody (Fig. 5). All three anti- RAS scFv produced significant signals with RAS antigen compared with BSA and the signals were inhibited by pre-incubation with RASG12V antigen, as a measure of specificity of the interaction.
  • the affinities of binding anti-RAS scFv to antigen were measured by binding kinetics in the BIAcore (Fig. 6).
  • the Kd of scFv33 and J48 were determined to be 1.39 ⁇ 1.3 InM, 3.63 ⁇ 0.15 nM (Fig. 6B).
  • the affinity difference of their scFv may reflect the differences of CDR1 sequence in VH domain.
  • the scFvI21 had a Kd of 2.16 ⁇ 0.25 ⁇ M, about three order of magnitude weaker than scFv33 or J48. This weak affinity of scFvI21, in the micromolar range, is consistent with its weak ⁇ -gal reporter gene activation in the yeast in vivo antigen-antibody interaction assay and lack of detectable binding in mammalian cell assays.
  • Example 5 The functional improvement of anti-RAS scFv by modification of the scFv framework sequences
  • FIG. 8 shows expression and luciferase reporter data of various modifications of the scFv33 framework compared to levels with scFv33 itself or scFvR4 (anti -gal negative control, (Martineau et al., 1998)) and scFvI21 which does not give significant luciferase activity.
  • scFv33 One notable mutation of scFv33 is Arg94Lys (numbering according to Kabat et al (Kabat et al, 1991), position 106 according to IMGT, Lefranc et al (Lefranc and Lefranc, 2001)) which completely eliminated reporter response (Fig. 8 A) even though the expression of this scFv-VP16 is increased compared with original scFv 33 (Fig. 8A).
  • the arginine residue at position 94 is very close to the antigen binding site (CDR3 of heavy chain) and may be involved in interaction with RAS antigen directly.
  • the residue at this position may form a surface bridge across the CDR3 loop through its positively charged side chain with the carboxyl group of the aspartic acid at position HI 01 (Morea et al., 1998), and the substitution (Arg to Lys) may affect the critical conformation of CDR3.
  • the other mutant scFv33 variants generally maintained their binding ability with RAS antigen as judged by the luciferase reporter assay (Fig. 8A).
  • the mutated anti-RAS scFv I21R33 interacts specifically with RAS antigen in COS7 cells, even though, in this reducing environment, scFv mostly cannot form disulphide bonds (Biocca et al, 1995; Tavladoraki et al, 1993). Perhaps a small population of over-expressed scFv does form disulphide bonds in the cytoplasm and interact with antigen in vivo, such as the anti- ⁇ galactosidase scFvR4, some of which is disulphide bounded in cytoplasm of bacteria (Martineau et al, 1998).
  • This scFv based on the I21R33 sequence, had the two cys codons were mutated to serine (clone I21R33(VHC22S;C92S).
  • a vector encoding this protein was tested in our mammalian reporter assay (Fig. 8B).
  • the scFv protein was expressed at high levels and roughly comparable with those of I21R33 and 121 and the ability to activate the luciferase reporter was similar to the 12R33 scFv.
  • 121 framework with VH and VL CDRs of scFv33 gives a well expressed protein able to activate the luciferase reporter (Fig. 8B).
  • NIH3T3 cells were transfected with a plasmid expressing activated HRAS alone (RASG12V ) to yield transformed, foci (non-contact inhibited colonies) which can grow in multilayers and show a swirling appearance of spindle-shaped cells (Fig. 9A, RASG12V + empty scFv vector).

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Abstract

L'invention concerne des molécules qui peuvent fonctionner dans un environnement intracellulaire. L'invention concerne en particulier une séquence consensus pour la région variable de la chaîne lourde et la région variable de la chaîne légère de molécules d'immunoglobuline qui peuvent sélectivement se lier à un ligand dans un environnement intracellulaire. L'invention concerne également les utilisations de ces molécules dans la préparation de banques, dans des applications génomiques fonctionnelles et dans le traitement de maladies.
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WO2016077526A1 (fr) 2014-11-12 2016-05-19 Siamab Therapeutics, Inc. Composés interagissant avec le glycane et procédés d'utilisation
WO2017083582A1 (fr) 2015-11-12 2017-05-18 Siamab Therapeutics, Inc. Composés interagissant avec le glycane et méthodes d'utilisation
US9758576B2 (en) 2013-05-06 2017-09-12 Scholar Rock, Inc. Compositions and methods for growth factor modulation
US9879087B2 (en) 2014-11-12 2018-01-30 Siamab Therapeutics, Inc. Glycan-interacting compounds and methods of use
US11253609B2 (en) 2017-03-03 2022-02-22 Seagen Inc. Glycan-interacting compounds and methods of use
US11401330B2 (en) 2016-11-17 2022-08-02 Seagen Inc. Glycan-interacting compounds and methods of use

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WO2000054057A1 (fr) * 1999-03-10 2000-09-14 Medical Research Council Selection d'immunoglobulines intracellulaires
WO2003014960A2 (fr) * 2001-08-03 2003-02-20 Medical Research Council Anticorps intracellulaires
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WO2003014960A2 (fr) * 2001-08-03 2003-02-20 Medical Research Council Anticorps intracellulaires
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TSE ERIC ET AL: "Intracellular antibody capture technology: Application to selection of intracellular antibodies recognising the BCR-ABL oncogenic protein" JOURNAL OF MOLECULAR BIOLOGY, vol. 317, no. 1, 15 March 2002 (2002-03-15), pages 85-94, XP002247076 ISSN: 0022-2836 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9758576B2 (en) 2013-05-06 2017-09-12 Scholar Rock, Inc. Compositions and methods for growth factor modulation
US10597443B2 (en) 2013-05-06 2020-03-24 Scholar Rock, Inc. Compositions and methods for growth factor modulation
US10981981B2 (en) 2013-05-06 2021-04-20 Scholar Rock, Inc. Compositions and methods for growth factor modulation
US11827698B2 (en) 2013-05-06 2023-11-28 Scholar Rock, Inc. Compositions and methods for growth factor modulation
WO2016077526A1 (fr) 2014-11-12 2016-05-19 Siamab Therapeutics, Inc. Composés interagissant avec le glycane et procédés d'utilisation
US9879087B2 (en) 2014-11-12 2018-01-30 Siamab Therapeutics, Inc. Glycan-interacting compounds and methods of use
EP4183806A2 (fr) 2014-11-12 2023-05-24 Seagen Inc. Composés interagissant avec le glycane et procédés d'utilisation
WO2017083582A1 (fr) 2015-11-12 2017-05-18 Siamab Therapeutics, Inc. Composés interagissant avec le glycane et méthodes d'utilisation
US11028181B2 (en) 2015-11-12 2021-06-08 Seagen Inc. Glycan-interacting compounds and methods of use
US11401330B2 (en) 2016-11-17 2022-08-02 Seagen Inc. Glycan-interacting compounds and methods of use
US11253609B2 (en) 2017-03-03 2022-02-22 Seagen Inc. Glycan-interacting compounds and methods of use

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