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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • 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
  • A61P25/28—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
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • 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
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • 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
• • 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
  • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
  • C07K16/241—Tumor Necrosis Factors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
• • 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
  • C07K2317/52—Constant or Fc region; Isotype
• • 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
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates to a simple method for generating antibody molecules suitable for in vivo use. Ln particular, the invention relates to a method for the generation of antibody molecules suitable for in vivo use which are based on antibody single variable domains.
• the antigen binding domain of an antibody comprises two separate regions: a heavy chain variable domain (VH) and a light chain variable domain (V : which can be either N K Nk or N ⁇ )-
• VH heavy chain variable domain
• V light chain variable domain
• the antigen binding site itself is formed by six polypeptide loops: three from V H domain (HI, H2 and H3) and three from V domain (LI, L2 and L3).
• V H gene
• the V H gene is produced by the recombination of three gene segments, V H , D and JH- hi humans, there are approximately 51 functional V H segments (Cook and Tomlinson (1995) Immunol Today, 16: 237), 25 functional D segments (Corbett et al.
• VH segment encodes the region of the polypeptide chain which forms the first and second antigen binding loops of the V H domain (HI and H2), whilst the V H , D and JH segments combine to form the third antigen binding loop of the V H domain (H3).
• the VL gene is produced by the recombination of only two gene segments, V L and J . Ln humans, there are approximately 40 functional VK segments (Schable and Zachau (1993) Biol. Chem.
• V L segment encodes the region of the polypeptide chain which forms the first and second antigen binding loops of the VL domain (LI and L2), whilst the V L and J segments combine to form the third antigen binding loop of the VL domain (L3).
• Antibodies selected from this primary repertoire are believed to be sufficiently diverse to bind almost all antigens with at least moderate affinity.
• High affinity antibodies are produced by "affinity maturation" of the rearranged genes, in which point mutations are generated and selected by the immune system on the basis of improved binding.
• the main-chain conformations are determined by (i) the length of the antigen binding loop, and (ii) particular residues, or types of residue, at certain key position in the antigen binding loop and the antibody framework.
• antibodies have been obtained from natural sources such as by the immunisation of rabbits and other such animals.
• molecular biology techniques may be employed and antibodies may be generated using techniques such as those involving the use of hybrid hydribomas.
• antibodies of a selected or desired antigen binding specificity can be generated.
• Such antibodies are of great therapeutic value as they can be designed against disease antigens, for instance.
• the method of production of these antibodies is laborious and prone to error, as well as being limited to diversity resulting from the immunisation history of the donor. It would be an advantage to generate increased diversity, e.g. using synthetic librarires. Therefore, there remains in the art a need for a simple method of generating functionally active antibody molecules of a desired or predetermined antigen binding specificity.
• Single heavy chain variable domains have been described, derived from natural antibodies which normally comprise light chains, from monoclonal antibodies or from repertoires of domains (EP-A-0368684). These heavy chain variable domains have been shown to interact specifically with one or more antigens (Ward et al,). However, these single domains have been shown to have a very short in vivo half-life. Therefore such domains are of limited therapeutic value.
• EP 0 656 946A1 describes dual-chain immunoglobulin molecules which bind antigen specifically, and in which the heavy polypeptide chains are devoid of CHI heavy chain domains, the immunoglobulin also being devioid of light polypeptide chains.
• Such antibodies are naturally occurring in Camelids, and therefore, as such the antigen specificity of the antibody is limited to those generated by the Camelid.
• Heavy Chain Disease immunoglobulin molecules are generated which comprise a heavy chain variable domain, CH2 and CH3 domains, but lack a CHI domain and light chains. Such molecules are found to accumulate in Heavy Chain Disease (Block et al, Am J. Med, 55, 61-70 (1973), Ellman et al, New Engl. J. Med, 278:95-1201 (1968)).
• Heavy Chain Disease prior art teaches that antibodies comprising a single antigen interaction domain type only (in this case heavy chain variable domains) are associated with disease. That is, the prior art teaches away from the use of antibodies based solely on human heavy chain variable domains for prophylactic and or therapeutic use.
• the present inventors have devised a simple and non-laborious method for the synthesis of antibody based molecules of a selected epitope binding specificity, which are suitable for in vivo prophylactic and/or therapeutic use.
• the method of the invention permits the synthesis of single chain antibody based molecules of a desired or predetermined epitope binding specificity.
• the use of this simple method is surprising in light of the Heavy Chain disease prior art which teaches away from the therapeutic use of heavy chain-only antibodies.
• the molecules of the present invention comprise an antibody single variable domain having a defined or predetermined epitope binding specificity and one or more antibody constant regions and/or hinge region (collectively termed "an effector group”).
• an effector group Such a molecule is referred to as a single domain-effector group immunoglobulin (dAb- effector group) and the present inventors consider that such a molecule will be of considerable therapeutic value.
• the present invention provides a method for synthesising a single- domain-effector group immunoglobulin (dAb-effector group) suitable for in vivo use comprising the steps of:
• step (b) attaching the single domain of step (a) to an immunoglobulin effector group
• the antibody single domain is a non- camelid antibody single domain.
• it is a single variable domain of human origin.
• the invention also described herein also contemplates CDR grafting non-camelid, for example human, CDRs onto Camelid framework regions. Techniques for CDR grafting of human CDRs to Camelid framework regions are known in the art. Such methods are described in European Patent Application 0 239 400 (Winter) and, may include framework modification [EP_ 0 239 400; Riechmann, L. et al, Nature, 332, 323- 327, 1988; Verhoeyen M. et al, Science, 239, 1534-1536, 1988; Kettleborough, C. A. et al., Protein Engng., 4, 773-783, 1991; Maeda, H. et al, Human Antibodies and
• the single variable domain comprises non-Camelid (eg, human) framework regions (eg, 1, 2, 3 or 4 human framework regions).
• human framework regions eg, 1, 2, 3 or 4 human framework regions.
• one or more of the human framework regions are identical on the amino acid level to those encoded by human germline antibody genes.
• Variable region sequences in, for example, the Kabat database of sequences of immunological interest, or other antibody sequences known or identifiable by those of skill in the art can be used to generate a dAb-effector group as described herein.
• the Kabat database or other such databases include antibody sequences from numerous species.
• CDRs and framework regions are those regions of an immunoglobulin variable domain as defined in the Kabat database of Sequences of Proteins of Immunological Interest.
• Preferred human framework regions are those encoded by germline gene segments DP47 and DPK9.
• FW1, FW2 and FW3 of a V H or VL domain have the sequence of FW1, FW2 or FW3 from DP47 or DPK9.
• the human frameworks may optionally contain mutations, for example up to about 5 amino acid changes or up to about 10 amino acid changes collectively in the human frameworks used in the ligands of the invention.
• the antibody single variable domains used according to the methods of the present invention are isolated, at least in part by human immunisation.
• they are not isolated by animal immunisation.
• the single variable domain comprises a binding site for a generic ligand as defined in WO 99/20749.
• the generic ligand is Protein A or Protein L.
• the term 'single-domain-effector group immunoglobulin molecule' describes an engineered immunoglobulin molecule comprising, a single variable domain capable of specifically binding one or more epitopes, attached to one or more constant region domains and/or hinge (collectively termed "an effector group").
• Each variable domain may be a heavy chain domain (V H ) or a light chain domain (V L ).
• Each light chain domain may be either of the kappa or lambda subgroup.
• an effector group as herein described comprises an Fc region of an antibody.
• dAb-effector groups may be combined to form multivalent structures, including any of those selected from the group consisting of the following: hornodimers, heterodimers and multimers.
• multimeric structures have improved avidity of antigen interaction by virtue of the multimeric structures having more than one epitope binding site where the epitopes are on the same antigen. Where the epitopes are on different antigens, eg those close together on the same cell surface, these epitopes may be bridged by dAb-effector groups.
• dAb-effector groups according to the invention do not include the dual-chain antibodies as described in EP-A-0656946 as well as single chain fragments disclosed therein, such as V HH -hinge fragments, based on camelid immunoglobulins.
• the term 'dAb-effector group' does not include within its scope the naturally occurring dual chain antibodies generated within Camelids.
• the term 'dAb- effector group' include within its scope the four-chain structure of IgG antibody molecules comprising two light and two heavy chains or single heavy or light chains derived therefrom.
• the term 'suitable for in vivo use' means that the 'dAb-effector group' according to the present invention has sufficient half-life such that the molecule is present within the body for sufficient time to produce one or more desired biological effects. Ln this regard the present inventors have found that the size and nature of the effector group influences the in vivo half-Jife of the dAb-effector groups according to the- invention.
• a preferred effector group according to the present invention is or comprises the Fc region of an antibody molecule. Such an effector group permits Fc receptor binding (e.g. to one or both of Fc receptors CD64 and CD32) and complement activation via the interaction with Clq, whilst at the same time providing the molecule with a longer half- life then a single variable heavy chain domain in isolation.
• the term 'epitope' is a unit of structure conventionally bound by anantigen binding site as provided by one or more variable domains, e.g. an immunoglobulin V H V L pair.
• Epitopes define the minimum binding site for an antibody, and thus represent the target of specificity of an antibody. Ln the case of a single domain antibody, an epitope represents the unit of structure bound by a variable domain in isolation of any other variable domain.
• the term 'select' includes within its scope the selection of (an antibody variable domain) from a number of different alternatives. Techniques for the 'selection' of antibody variable domains will be familiar to those skilled in the art.
• the term 'selection' (of an antibody variable domain) includes within its scope the 'selection' of one or more variable domains by library screening.
• the selection involves the screening of a repertoire of antibody variable domains displayed on the surfaces of bacteriophage within a phage display library (McCafferty et al, (1990) Nature 340, 662-654) or emulsion-based in vitro systems (Tawfik & Griffiths (1998) Nature Biotechnol 16(7), 652-6).
• the term 'attaching' (the single domain as herein described to an effector group) includes within its scope the direct attachment of a single domain as described herein to one or more constant regions as herein described. It also includes the indirect attachment of a single domain to an effector group via for example a further group and/or a linker region. Furthermore, the term 'attaching' includes within its scope an association of the respective groups such that the association is maintained in vivo such that the dAb- effector group is capable of producing biological effects, such as increasing half life (i.e., serum residence time of the variable domain) and allowing the functional attributes of, for example, constant regions, such as Fc regions, to be exploited in vivo.
• variable domain and the effector group are directly attached, without the use of a linker.
• the linker is advantageously a polypeptide linker.
• the length and composition of the linker may affect the physical characteristics of the dAb-effector group.
• a short linker may minimise the degree of freedom of movement exhibited by each group relative to one another, whereas a longer linker may allow more freedom of movement.
• bulky or charged amino acids may also restrict the movement of one domain relative to the other. Discussion of suitable linkers is provided in Bird et al. Science 242, 423-426.
• the attachment of a single variable domain to an effector group, as herein defined may be achieved at the polypeptide level, that is after expression of the nucleic acid encoding the respective domains and groups. Alternatively, the attachment step may be performed at the nucleic acid level.
• Methods of attachment an include the use of protein chemistry and/or molecular biology techniques which will be familiar to those skilled in the art and are described herein.
• non-camelid antibody single variable domain refers to an antibody single variable domain of non-camelid origin.
• the non-camelid antibody single variable domain may be selected from a repertoire of single domains, for example from those represented in a phage display library. Alternatively, they may be derived from native antibody molecules. Those skilled in the art will be aware of further sources of antibody single variable domains of non-camelid origin.
• Antibody single variable domains- may- be-light chain variable domains- (V L ) or heavy chain variable domains (V H ).
• V L chain variable domain is of the Vkappa (VK) or Nlambda (N ⁇ ) sub-group.
• those domains selected are light chain variable domains.
• single domain effector groups may comprise VH or V L domains as described above.
• an antibody variable domain (VH or V L ) is attached to one or more antibody constant region heavy domains.
• Such one or more constant heavy chain domains constitute an 'effector group' according to the present invention.
• each V H or V L domain is attached to an Fc region (an effector group) of an antibody.
• a dAb-effector group according to the invention is V L - Fc. Ln the case that the effector group is an Fc region of an antibody, then the CH3 domain facilitates the interaction of a dAb-effector group with Fc receptors whilst the CH2 domain permits the interaction of a dAb-effector group with Clq, thus facilitating the activation of the complement system.
• the Fc portion of the antibody stabilises the dAb-effector group and provides the molecule with a suitable half-life for in vivo therapeutic and/or prophylactic use.
• an effector group comprising at least an antibody light chain constant region (CL), an antibody CHI heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof.
• an effector group may also comprise a hinge region of an antibody (such a region normally being found between the CHI and CH2 domains of an IgG molecule).
• the effector group is a hinge region alone such that the dAb-effector group comprises a single variable domain attached to the hinge region of an antibody molecule.
• an effector group as herein described is or comprises the constant region domains CH2 and/or CH3.
• the effector group- comprises CH2 and/or CH3, preferably an effector group .as Jierein described consists of CH2 and CH3 domains, optionally attached to a hinge region of an antibody molecule as described herein.
• the present invention provides a 'dAb-effector group' obtainable using the methods of the present invention.
• 'dAb-effector groups' according to the present invention do not include within their scope the four- chain structure of IgG molecules nor the dual-chain structure of naturally occurring Camelid antibodies or those described in EP 0 656 946 Al.
• Antibody single variable domains may be light chain variable domains (V L ) or heavy chain variable domains (V H ).
• Each VL chain variable domain is of the Nkappa (Vk) or Vlambda (V ⁇ ) sub-group.
• those domains selected are light chain variable domains.
• the use of V L domains has the advantage that these domains unlike variable heavy chain domains (V H ) do not possess a hydrophobic interfaces which are 'sticky' and can cause solubility problems in the case of isolated N H domains.
• single domain effector group immunoglobulin molecules according to the present invention comprise either N H or N L domains as described above.
• the dAb-effector group obtained by the methods of the invention is an V H -FC or a V L -FC. More advantageously, the dAb-effector group is VL-FC. In an alternative embodiment of this aspect of the invention the dAb-effector group is V H -hinge. In an alternative embodiment still, the dAb-effector group is a Vk-Fc. The present inventors have found that the Fc portion of the antibody stabilises the dAb-effector group providing the molecule with a suitable half- life.
• the effector group is based on a Fab antibody fragment. That is, it comprises an antibody fragment comprising a H domain or a V L domain attached to one or more constant region domains making up a Fab fragment.
• a fragment comprises only one variable. domain.
• Such Fab effector-groups are illustrated in Fig Hi.
• Various preferred 'dAb-effector groups' prepared according to the methods of the present invention are illustrated in Fig 1.
• the dAb-effector groups of the present invention may be combined onto non- immunoglobulin multi-ligand structures so that they form multivalent structures comprising more than one antigen binding site. Such structures have an increased avidity of antigen binding. Ln an example of such multimers, the V regions bind different epitopes on the same antigen providing superior avidity.
• multivalent complexes may be constructed on scaffold proteins, as described in WO0069907 (Medical Research Council), which are based for example on the ring structure of bacterial GroEL or on other chaperone polypeptides.
• dAb-effector groups according to the present invention may be combined in the absence of a non-immunoglobulin protein scaffold to form multivalent structures which are solely based on immunoglobulin domains.
• Such multivalent structures may have increased avidity towards target molecules, by virtue of them comprising multiple epitope binding sites.
• Such multivalent structures may be homodimers, heterodimers or multimers.
• dAb-effector groups of the invention as well as such multivalent structures, will be of particular use for use in prophylactic and/or therapeutic uses.
• Antigens may be, or be part of, polypeptides, proteins or nucleic acids, which may be naturally occurring or synthetic.
• polypeptides proteins or nucleic acids
• They may be for instance human or animal proteins, cytokines, cytokine receptors, enzymes co-factors for enzymes or DNA binding proteins.
• Suitable cytokines and growth factors include but are not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF- ⁇ l, insulin, LL1R1, EFN- ⁇ , IGF-I, IGF-H, IL-l ⁇ , IL-l ⁇ , IL- 2, EL-3, IL-4, IL-5, IL-6, EL-7, EL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, EL-10, IL-.11, IL-12, LL- 13, EL-15, IL-16, IL-17, LL-18 (IGLF), Lnhibin ⁇ , Inhibi
• MDC (69 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.),
• MDC (69 a.a.), MIG, MLP-l ⁇ , MEP-l ⁇ , MLP-3 ⁇ , MLP-3 ⁇ , MLP-4, myeloid progenitor inhibitor factor-1 (MPLF-1), NAP-2, Neurturin, Nerve growth factor, ⁇ -NGF, NT-3, NT-
• Oncostatin M Oncostatin M, p55, TNFcarecognition site, pro-TNF- ⁇ -stalk, PDGF-AA, PDGF-AB,
• PDGF-BB PDGF-BB
• PF-4 RANTES
• SDFl ⁇ SDFl ⁇
• SCF SCGF
• SCF stem cell factor
• TACE enzyme recognition site TGF- ⁇ , TGF- ⁇ , TGF/31, TGF- ⁇ 2, TGF- ⁇ 3, tumour necrosis factor (TNF), TNF- ⁇ , TNF- ⁇ , TNF receptor I, TNF receptor ⁇ , TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA,
• Cytokine receptors include receptors for the foregoing cytokines. It will be appreciated that this list is not intended to be exhaustive.
• variable domains are derived from an antibody directed against one or more antigen/s or epitope/s.
• the dAb-effector group of the invention may bind the epiotpe/s or antigen/s and act as an antagonist or agonist (eg, EPO receptor agonist).
• variable domains are derived from a repertoire of single variable antibody domains.
• the repertoire is a repertoire that is not created in an animal or a synthetic repertoire.
• the single variable domains are not isolated (at least in part) by animal immunisation.
• the single domains can be isolated from a naive library.
• a library (eg, phage or phagemid library or using emulsion technology as described in WO 99/02671) is made wherein a population of library members each comprises a common construct encoding an effector group (eg, an Fc region). A diversity of sequences encoding single variable domains is then spliced in to form a library of members displaying a diversity of single variable domains in the context of the same effector group. dAb-effector group selection against antigen or epitope is then effected in the context of the common effector group, which may have been selected in the basis of its desirable effects on half life, for example. Ln a further aspect, the present invention provides one or more nucleic acid molecules encoding at least a dAb-effector group as herein defined.
• the dAb-effector group may be encoded on a single nucleic acid molecule; alternatively, different parts of the molecule may be encoded by separate nucleic acid molecules. Where the 'dAb-effector group' is encoded by a single nucleic acid molecule, the domains may be expressed as a fusion polypeptide, or may be separately expressed and subsequently linked together, for example using chemical linking agents. dAb-effector groups expressed from separate nucleic acids will be linked together by appropriate means.
• the nucleic acid may further encode a signal sequence for export of the polypeptides from a host cell upon expression and may be fused with a surface component(eg, at least part of the pill coat protein) of a filamentous bacteriophage particle (or other component of a selection display system) upon expression.
• a surface component(eg, at least part of the pill coat protein) of a filamentous bacteriophage particle (or other component of a selection display system) upon expression.
• the present invention provides a vector comprising nucleic acid according to the present invention.
• the present invention provides a host cell transfected with a vector according to the present invention.
• Expression from such a vector may be configured to produce, for example on the surface of a bacteriophage particle, dAb-effector groups for selection.
• the present invention further provides a kit suitable for the prophylaxis and/or treatment of disease comprising at least an dAb-effector group according to the present invention.
• the present invention provides a composition comprising a dAb- effector group, obtainable by a method of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
• a pharmaceutically acceptable carrier diluent or excipient.
• Half lives tYz alpha and t l ⁇ beta) and AUC can be determined from a curve of serum concentration of dAb-Effector Group against time (see, eg figure 6).
• the WinNonlin analysis package (available from Pharsight Corp., Mountain View, CA94040, USA) can be used, for example, to model the curve.
• a first phase the alpha phase
• a second phase (beta phase) is the terminal phase when the dAb-Effector Group has been distributed and the serum concentration is decreasing as the dAb-Effector Group is cleared from the patient.
• the t alpha half life is the half life of the first phase and the t beta half life is the half life of the second phase.
• the present invention provides a dAb-effector group or a composition comprising a dAb-effector group according to the invention having a t ⁇ half-life in the range of 15 minutes or more.
• the lower end of the range is 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours.
• a dAb-effector group or composition according to the invention will have a t ⁇ half life in the range of up to and including 12 hours.
• the upper end of the range is 11, 10, 9, 8, 7, 6 or 5 hours.
• An example of a suitable range is 1 to 6 hours, 2 to 5 hours or 3 to 4 hours.
• the present invention provides a dAb-effector group or a composition comprising a dAb-effector group according to the invention having a t ⁇ half-life in the range of 2.5 hours or more.
• the lower end of the range is 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours, or 12 hours.
• a dAb-effector group or composition according to the invention has a t ⁇ half-life in the range of up to and including 21 days.
• a dAb- effector group according to the invention has a t ⁇ half-life of any of those t ⁇ half-lifes selected from the group consisting of the following: 12 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more or 20 days or more .
• a dAb-effector group or composition according to the invention will have a t ⁇ half life in the range 12 to 60 hours. In a further embodiment, it will have a t ⁇ half-life of a day or more. . In a further embodiment still, it will be in the range 12 to 26 hours.
• a dAb-effector group according to the present invention comprises or consists of an V L -FC having a t ⁇ half-life of a day or more, two days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more or 7 days or more.
• a dAb-effector group according to the present invention comprises or consists of an V L -FC having a t ⁇ half-life of a day or more.
• a dAb-effector group comprises an effector group consisting of the constant region domains CH2 and or CH3, preferably CH2 and CH3, either with or without a hinge region as described herein, wherein the dAb-effector group has a t ⁇ half-life of a day or more, two days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more or 7 days or more.
• a dAb-effector group according to the present invention comprises an effector group consisting of the constant region domains CH2 and/or CH3 wherein the dAb-effector group has a t ⁇ half-life of a day or more.
• the present invention provides a dAb- effector group or a composition comprising a dAb-effector group according to the invention having an AUC value (area under the curve) in the range of 1 mg.min/ml or more.
• the lower end of the range is 5, 10, 15, 20, 30, 100, 200 or 300mg.min/ml.
• a dAb-effector group or composition according to the invention has an AUC in the range of up to 600 mg.min/ml.
• the upper end of the range is 500, 400, 300, 200, 150, 100, 75 or 50 mg.minml.
• a dAb-effector group according to the invention will have a AUC in the range selected from the group consisting of the following: 15 to 150mg.min/ml, 15 to 100 mg.min/ml, 15 to 75 mg.min/ml, and 15 to 50mg.min/ml.
• the present invention provides a method for the prophylaxis and/or treatment of disease using a dAb-effector group or a composition according to the present invention.
• the present invention provides a dAb-effector group according to the present invention or a composition thereof in the treatment of disease.
• a dAb-Fc specific for target human TNF alpha and designated TARl-5-19-Fc was shown to be a highly effective therapy in a model of arthritis.
• a TARl-5-19-effector group may be of particular use in the prophylaxis and/or treatment of one or more inflammatory diseases.
• the present invention provides a method for the treatment of one or more inflammatory diseases in a patient in need of such treatment which comprises the step of administering to that patient a therapeutically effective amount of a dAb-effector group according to the invention.
• the present invention provides the use of a dAb-effector group according to the invention in the preparation of a medicament for the prophylaxis and/or treatment of one or more inflammatory diseases.
• the dAb-effector group specifically binds to TNF alpha. More advantageously, the dAb-effector group specifically binds to human TNF alpha. More advantageously still, the dAb-effector group is a dAb-Fc and specifically binds to TNF alpha, preferably human TNFalpha. More advantageously still, the dAb-effector group comprises TARl-5-19 as effector group. Most advantageously, the dAb-effector group for use according to the above aspects of the invention is TARl-5-19-Fc. According to the above aspects of the invention, advantageously, the one or more inflammatory diseases are mediated by TNF-alpha.
• the one or more inflammatory diseases are mediated by TNFalpha and are selected from the group consisting of the following: rheumatoid arthritis, psoriasis, Crohns disease, inflammatory bowel disease (EBD), multiple sclerosis, septic shock, alzheimers, coronary thrombosis, chronic obstructive pulmonary disease (COPD) and glomerular nephritis.
• TNFalpha mediated by TNFalpha and are selected from the group consisting of the following: rheumatoid arthritis, psoriasis, Crohns disease, inflammatory bowel disease (EBD), multiple sclerosis, septic shock, alzheimers, coronary thrombosis, chronic obstructive pulmonary disease (COPD) and glomerular nephritis.
• the present invention provides a method for reducing and/or preventing and/or suppressing cachexia in a patient which is mediated by TNFalpha which method comprises the step of administering to a patient in need of such treatment a therapeutically effective amount of a dAb-effector group according to the present invention.
• the invention provides the use of a dAb-effector group according to the invention in the preparation of a medicament for reducing and/or preventing and/or suppressing cachexia in patient.
• the cachexia is associated with an inflammatory disease.
• the inflammatory disease is selected from the group consisting of the following: rheumatoid arthritis, psoriasis, Crohns disease, inflammatory bowel disease (EBD), multiple sclerosis, septic shock, alzheimers, coronary thrombosis, chronic obstructive pulmonary disease (COPD) and glomerular nephritis.
• the method or use may be used for the treatment of human or non-human patients.
• the patient is a human and the TNFalpha is human TNFalpha.
• Suitable dosages for the administration to a subject of dAb-effector group according to the invention will be familiar to those skilled in the art.
• the dose is in the range of between 0.5 to 20mg/Kg of dAb-effecor group. More advantageously, the dosage of dAb-effecotr group is in the range of between 1 to lOmg/Kg. Preferred, dosage ranges are between 1 and 5 mg/Kg. Most advantageously, dosages of lmg/Kg or 5mg/Kg dAb-effector group are administered.
• Suitable dosage regimes may be dependent upon certain subject characterisitics including age, severity of disease and so on.
• dAb-effector group may for example be administered, particularly when the subject is a human, daily, once a week, twice a week or monthly. Those skilled in the art will appreciate that this list is not intended to be exhaustive.
• Figure 1 shows various preferred dAb-effector groups according to the present invention.
• (a) shows V H or V L attached to the hinge region of an antibody molecule.
• step (f) Shows a dimer having the same components as step (e) but in which the point of attachment between the two components making up the dimer is the effector groups.
• step (g) Shows V H or V L attached to the Fc region of an antibody molecule.
• Figure 2 shows the signal plgplus vector used to create E5-Fc and VH2-Fc fusions. Details are given in Example 1.
• Figure 3a shows SDS page gels representing the purification of immunoglobulin effector groups according to the invention
• Lane 1 - MW marker (kDa) Lane 2 - Culture medium before Protein A purification
• Lane 3 - Culture medium after Protein A purification Lane 4 - Purified E5-Fc protein
• Lane 5 - Purified E5-Fc protein Lane 3b shows the glycosylation of immunoglobulin-effector groups according to the invention. Lanes are labelled on the figure.
• Figure 3c shows ELISA results demonstrating that Cos-1 cells, Cos-7 cells and CHO cells are capable of expressing dAb-Fc fusion proteins of the correct specificity and with no cross reactivity with irrelevant antigens.
• FIG. 4 shows that E5-Fc fusion protein is able to bind to the cell line expressing human Fc receptors.
• Purified E5-Fc protein was labelled with fluorescein at 3.3 / 1 ratio of Fluo/Protein. The labelled protein (491 ⁇ g/ml concentration) was then used for FACS analysis.
• Human monocyte-like U937 cells which express two types of human FcRs (CD 64 and CD32) were used to assess the ability of E5-Fc fusion protein to bind these receptors.
• FACS results indicate that E5-Fc fusion protein binds to the U937 cell line (5x10 ⁇ U-937 cells were incubated with 80ml of the 1 :50 dilution of the labelled protein and examined live). a.
• Fig 4a U-937 cells (control) b.
• Fig 4b U-937 cells incubated with anti CD64 antibody (positive control)
• Fig 4c U-937 cells incubated with anti CD32 antibody (positive control)
• Fig 4d U-937 cells incubated with anti CD 16 antibody (negative control)
• Fig 4e U-937 cells incubated with E5-Fc fusion protein
• FIG. 5 Raj 1 cells (expressing only CD32 receptor) were used for FACS analysis. FACS results demonstrate that E5-Fc chain binds to Raj 1 cells. 1. Raj 1 cells (control)
• Figure 6 shows the results of Pharmacokinetic Analysis.
• the figure shows the serum levels in mice following 50 ⁇ g bolus LV doses of HEL-4 or E5-Fc according to the invention.
• Figure 7 shows the effect of twice weekly injections of TARl-5-19 on the arthritic scores of the Tgl97 mice.
• Figure 8 shows histopathological scoring of the ankle joints from the different treatment groups.
• Figure 9 shows the effect of twice weekly injections of TARl-5-19 on the group average weights of Tgl97 mice.
• Figure 10 Nucleotide sequence of the alpha factor dAb Fc fusion protein from the start of the alpha factor leader sequence to the EcoRI cloning site.
• Figure 11 Amino acid sequence of the alpha factor dAb Fc fusion protein, as encoded by the sequence shown in figure 10.
• Antigen binding activity was determined using a TNF receptor binding assay.
• a 96 well Nunc Maxisorp plate is coated with a mouse anti- human Fc antibody, blocked with 1% BSA, then TNF receptor 1-Fc fusion is added.
• the dAb-Fc fusion protein at various concentrations is mixed with lOng/ml TNF protein and incubated at room temperature for >lhour. This mixture is added to the TNF receptor 1- Fc fusion protein coated plates, and incubated for lhour at room temperature. The plates are then washed to remove unbound free dAb-Fc fusion, TNF and dAb-Fc/TNF complexes.
• the plate was then incubated sequentially with a biotinylated anti-TNF antibody and streptavidin-horse radish peroxidase.
• the plate was then incubated with the chromogenic horse radish peroxidase substrate TMB.
• the colour development was stopped with the addition of 1M hydrochloric acid, and absorbance read at 450nm.
• the absorbance read is proportional to the amount of TNF bound, hence, the TARl-5-19Fc fusion protein will compete with the TNF receptor for binding of the TNF, and reduce the signal in the assay.
• the P. pastoris produced protein had an equivalent activity to the mammalian protein in the vitro TNF receptor assay described above.
• Figure 13 shows a 15% non-reducing SDS-PAGE gel showing comparison between TARl-5-19 Fc fusion protein produced in mammalian cells (lanes 1 and 2) and P. pastoris
• the minor band below the 80kDa marker represents free Fc protein, without dAb attached, produced through proteolytic attack of the polypeptide linking the dAb and Fc domains.
• Immunoglobulin This refers to a family of polypeptides which retain the immunoglobulin fold characteristic of antibody molecules, which contains two ⁇ 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).
• Domain A domain is a folded protein structure which retains its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function.
• single antibody variable domain we mean a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes antibody variable domains, for example in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions.
• a library used in the present invention will encompass a repertoire of polypeptides comprising at least 1000 members.
• Library The term library refers to a mixture of heterogeneous polypeptides or nucleic acids.
• the library is composed of members, which have a single polypeptide or nucleic acid sequence. To this extent, library is synonymous with repertoire. 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 or a limited number of library members.
• 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 population of host organisms has the potential to encode a large repertoire of genetically diverse polypeptide variants.
• a single-domain-effector group as herein defined describes an engineered synthetic structure comprising a single variable domain capable of specifically binding one or more epitopes, attached to one or more constant region domains and/or hinge (collectively termed an "effector group").
• Each variable domain may be a heavy chain domain (V H ) or a light chain domain (V L ).
• an effector group as herein described comprises an Fc region of an antibody. dAb-effector groups may be combined to form multivalent structures, thus improving the avidity of antigen interaction.
• dAb-effector group immunoglobulin molecules according to the invention are single chain molecules, they are not dual-chain antibodies (for example those described in EP 0 656 946A1). Ln addition, the term 'dAb-effector group does not include within its scope the naturally occurring dual chain antibodies generated within Camelids nor the four chain structure of IgG molecules. 'dAb-effector groups' according to the present have a half-life which is of sufficient length such that it can produce an in vivo biological effect. The present inventors have found that it is the size and nature of the effector group which determines the effector functions of the dAb- effector group as herein defined as well as the in vivo half-life of the molecule.
• Antibody An antibody (for example IgGl, 2, 3 and 4; IgM; IgA; IgD; or IgE) or fragment (such as a Fab, Dab, F(ab') 2 , Fv, disulphide linked Fv, scFv, diabody) whether derived from any species naturally producing an antibody, or created by recombinant
• DNA technology whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria).
• TARl-5-19 is a Dab which specifically binds to the target human TNF alpha (TAR1).
• Antigen A ligand that is bound by a dAb-effector group according to the present invention.
• single domains may be selected according to their antigen- binding specificity for use in the present invention.
• the antigen may be a polypeptide, protein, nucleic acid or other molecule.
• the antibody binding site defined by the variable loops (LI, L2, L3 and HI, H2, H3) is capable of binding to the antigen.
• An epitope as referred to herein is a unit of structure conventionally bound by one or more immunoglobulin variable domains, for example an immunoglobulin V H /V pair.
• Epitopes define the minimum binding site for an antibody, and thus represent the target of specificity of an antibody. Ln the case of a single domain antibody, an epitope represents the unit of structure bound by a variable domain in isolation of another variable domain.
• selecting means choosing from a number of different alternatives. Those skilled in that art will be aware of methods of selecting one or more antibody single variable domains.
• the method involves selecting from a library.
• the library is a phage display library.
• Universal framework A single antibody framework sequence corresponding to the regions of an antibody conserved in sequence as defined by Kabat ("Sequences of Proteins of Immunological Interest", US Department of Health and Human Services) or structure as defined by Chothia and Lesk, (1987) J. Mol. Biol. 196:910-917.
• the invention provides for the use of a single framework, or a set of such frameworks, which has been found to permit the derivation of virtually any binding specificity though variation in the hypervariable regions alone.
• Specific generic ligand A ligand that binds to all members of a repertoire. Generally, not bound through the antigen binding site. Examples include protein A and protein L.
• human origin means that at some point in the derivation of a sequence in question, a human sequence was used as a source of nucleic acid sequence. An analogous meaning applies to the term “Camelid origin.”
• the phrase "increased half-life" means that a given dAb-effector group has at least a 25% longer serum half-life relative to the same dAb without the effector. Increased half-lives are preferably at least 30% longer, 40% longer, 50% longer, 75% longer, 100% longer, 3X longer, 5X longer, 10X longer, 20X longer, 50X longer or more.
• selecting is to be understood to require the application of a technique or selective pressure, thus permitting the isolation of one or more items from among a population based on one or more characteristics possessed by the selected item(s) that is/are not possessed by the other members of the population.
• dAb-effector groups may be prepared according to previously established techniques, used in the field of antibody engineering, for the preparation of scFv, "phage” antibodies and other engineered antibody molecules. Techniques for the preparation of antibodies, and in particular bispecific antibodies, are for example described in the following reviews and the references cited therein: Winter & Milstein, (1991) Nature 349:293-299; Plueckthun (1992) Immunological Reviews 130:151-188; Wright et al, (1992) Crti. Rev. Immunol.l2:125-168; Holliger, P. & Winter, G. (1993) Curr. Op. Biotechn. 4, 446-449; Carter, et al. (1995) J. Hematother.
• VH and/or V L libraries may be selected against target antigens or epitopes separately, in which case single domain binding is directly selected for, or together.
• a preferred method for synthesising a 'dAb-effector group' comprises using a selection system in which a repertoire of variable domains is selected for binding to an antigen or epitope. The single domains selected are then attached to an effector group.
• Suitable effector groups include any of those selected from the group consisting of the following: an effector group comprising at least an antibody light chain constant region (CL), an antibody CHI heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof.
• an effector group may also comprise a hinge region of an antibody (such a region normally being found between the CHI and CH2 domains of an IgG molecule).
• the 'dAb- effector group' is a single variable domain attached to the hinge region derived from and antibody molecule.
• the effector group is based on a Fab antibody fragment. That is, it comprises an antibody fragment comprising a N ⁇ domain or a V L domain attached to one or more constant region domains making up a Fab fragment.
• a fragment comprises only one variable domain.
• Such Fab effector groups are illustrated in Fig lg.
• the single variable domains each forms a respective epiope or antigen binding site.
• the single variable domains do not together form a single binding site.
• the eiptiope or antigen specificity of the variable domains may be the same or different.
• an effector group according to the present invention is an Fc region of an IgG molecule.
• Bacteriophage lambda expression systems 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. Whilst such expression systems can be used to screening up to 10 6 different members of a library, they are not really suited to screening of larger numbers (greater than 10 6 members).
• selection display systems which enable a nucleic acid to be linked to the polypeptide it expresses.
• a selection display system is a system that permits the selection, by suitable display means, of the individual members of the library by binding the generic and/or target ligands.
• 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.
• 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).
• a similar technique may be used to identify DNA sequences which bind a predetermined human transcription factor (Thiesen and Bach (1990) Nucleic Acids Res., 18: 3203; Beaudry and Joyce (1992) Science, 257: 635; WO92/05258 and WO92/14843).
• Ln Ln a similar way, in vitro translation can be used to synthesise polypeptides as a method for generating large libraries.
• These methods which generally comprise stabilised polysome complexes, are described further in WO88/08453, WO90/05785, WO90/07003, WO91/02076, WO91/05058, and WO92/02536.
• Alternative display systems which are not phage-based, such as those disclosed in WO95/22625 and WO95/11922 (Affymax) use thepolysomes to display polypeptides for selection.
• a still further category of techniques involves the selection of repertoires in artificial compartments, which allow the linkage of a gene with its gene product.
• a selection system in which nucleic acids encoding desirable gene products may be selected in microcapsules formed by water-in-oil - emulsions is described in WO99/02671, .
• WO00/40712 and Tawfik & Griffiths (1998) Nature Biotechnol 16(7), 652-6.
• Genetic elements encoding a gene product having a desired activity are compartmentalised into microcapsules and then transcribed and/or translated to produce their respective gene products (RNA or protein) within the microcapsules.
• Genetic elements which produce gene product having desired activity are subsequently sorted. This approach selects gene products of interest by detecting the desired activity by a variety of means.
• Libraries intended for use in selection may be constructed using techniques known in the art, for example as set forth above, or may be purchased from commercial sources. Libraries which are useful in the present invention are described, for example, in WO99/20749.
• PCR polymerase chain reaction
• PCR is performed using template DNA (at least lfg; more usefully, 1-1000 ng) and at least 25 pmol of oligonucleotide primers; it may be advantageous to use a larger amount of primer when the primer pool is heavily heterogeneous, as each sequence is represented by only a small fraction of the molecules of the pool, and amounts become limiting in the later amplification cycles.
• a typical reaction mixture includes: 2 ⁇ l of DNA, 25 pmol of oligonucleotide primer, 2.5 ⁇ l of 10X PCR buffer 1 (Perkin-Elmer, Foster City, CA), 0.4 ⁇ l of 1.25 ⁇ M dNTP, 0.15 - ⁇ l (or 2.5 units) of Taq DNA polymerase (Perkin Elmer, Foster City, CA) and deionized water to a total volume of 25 ⁇ l.
• Mineral oil is overlaid and the PCR is performed using a programmable thermal cycler. The length and temperature of each step of a PCR cycle, as well as the number of cycles, is adjusted in accordance to the stringency requirements in effect.
• Annealing temperature and timing are determined both by the efficiency with which a primer is expected to anneal to a template and the degree of mismatch that is to be tolerated; obviously, when nucleic acid molecules are simultaneously amplified and mutagenized, mismatch is required, at least in the first round of synthesis.
• the ability to optimise the stringency of primer annealing conditions is well within the knowledge of one of moderate skill in the art.
• An annealing temperature of between 30 °C and 72 °C is used.
• Initial denaturation of the template molecules normally occurs at between 92°C and 99°C for 4 minutes, followed by 20-40 cycles consisting of denaturation (94-99°C for 15 seconds to 1 minute), annealing (temperature determined as discussed above; 1-2 minutes), and extension (72°C for 1-5 minutes, depending on the length of the amplified product).
• Final extension is generally for 4 minutes at 72°C, and may be followed by an indefimte (0-24 hour) step at 4°C.
• Domains according to the invention may be attached to effector groups as herein described by a variety of methods known in the art, including covalent and non- covalent methods.
• Preferred methods include the use of polypeptide linkers, as described, for example, in connection with scFv molecules (Bird et al, (1988) Science 242:423-426).
• Linkers may be flexible, allowing the two single domains to interact.
• the linkers used in diabodies, which are less flexible, may also be employed (Holliger et al, (1993) PNAS (USA) 90:6444-6448).
• Variable domains may be attached to effector groups using methods other than linkers. For example, the use of disulphide bridges, provided through naturally-occurring or engineered cysteine residues, may be exploited. Other techniques for attaching variable domains of immunoglobulins to effector groups of the present invention may be employed as appropriate.
• linker may affect the physical characteristics of the dAb- effector molecule.
• the linkers may facilitate the association of the domains, such as by incorporation of small amino acid residues in opportune locations.
• a suitable rigid structure may be designed which will keep the effector group and the variable domain in close physical proximity to one another.
• single V H and single V L variable domains are attached to an effector group via means herein described.
• a dAb-effector group according to the present invention may be derived from any species naturally producing an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria.
• the single variable domain and the effector group according to the present invention may be on the same polypeptide chain. Alternatively, they may be on separate polypeptide chains. In the case that they are on the same polypeptide chain they may be linked by a linker.
• the linker is a peptide sequence, as described above.
• the single variable domain and the effector group may be covalently or non-covalently associated.
• the covalent bonds may be disulphide bonds.
• variable domains are selected from V-gene repertoires selected for instance using phage display technology as herein described, then these variable domains comprise a universal framework region, such that is they may be recognised by a specific generic ligand as herein defined.
• the use of universal frameworks, generic ligands and the like is described in WO99/20749.
• prefe ⁇ ed germ-line gene segments for preparation of dAB-effector groups according to the invention include any of those selected from the group consisting of the following: DP38, DP45, DP47 and
• variable domain sequence is preferably located within the structural loops of the variable domains.
• the polypeptide sequences of either variable domain may be altered by DNA shuffling or by mutation in order to enhance the interaction of each variable domain with its complementary epitope.
• the present invention provides nucleic acid encoding at least a single domain-effector group antibody as herein defined.
• variable regions may be derived from antibodies directed against target antigens or epitopes. Alternatively they may be derived from a repertoire of single antibody domains such as those expressed on the surface of filamentous bacteriophage. Selection may be performed as described below.
• nucleic acid molecules and vector constructs required for the performance of the present invention may be constructed and manipulated as set forth in standard laboratory manuals, such as Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, USA.
• nucleic acids in the present invention is typically carried out in recombinant vectors.
• the present invention provides a vector comprising nucleic acid encoding at least a single domain-effector group as herein defined.
• vector refers to a discrete element that is used to introduce heterologous DNA into cells for the expression and/or replication thereof.
• Methods by which to select or construct and, subsequently, use such vectors are well known to one of ordinary skill in the art.
• Numerous vectors are publicly available, including bacterial plasmids, bacteriophage, artificial chromosomes and episomal vectors. Such vectors may be used for simple cloning and mutagenesis; alternatively gene expression vector is employed.
• a vector of use according to the invention may be selected to accommodate a polypeptide coding sequence of a desired size, typically from 0.25 kilobase (kb) to 40 kb or more in length
• a suitable host cell is transformed with the vector after in vitro cloning manipulations.
• Each vector contains various functional components, which generally include a cloning (or "polylinker") site, an origin of replication and at least one selectable marker gene. If given vector is an expression vector, it additionally possesses one or more of the following: enhancer element, promoter, transcription termination and signal sequences, each positioned in the vicinity of the cloning site, such that they are operatively linked to the gene encoding a polypeptide repertoire member according to the invention.
• Both cloning and expression vectors generally contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells.
• this sequence is one that enables the vector to replicate independently of the host chromosomal DNA and includes origins of replication or autonomously replicating sequences.
• origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast and viruses.
• the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 micron plasmid origin is suitable for yeast, and various viral origins (e.g. SV 40, adenovirus) are useful for cloning vectors in mammalian cells.
• the origin of replication is not needed for mammalian expression vectors unless these are used in mammalian cells able to replicate high levels of DNA, such as COS cells.
• a cloning or expression vector may contain a selection gene also referred to as selectable marker.
• This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will therefore not survive in the culture medium.
• Typical selection genes encode proteins that confer resistance to antibiotics and-other toxins, e.g. ampicillin, neomycin, methotrexate . or tetracycline, . complement auxofrophic deficiencies, or supply critical nutrients not available in the growth media.
• an E. co/z ' -selectable marker for example, the -lactamase gene that confers resistance to the antibiotic ampicillin.
• E. coli plasmids such as pBR322 or a pUC plasmid such as pUC18 or pUC19 or pUCl 19.
• Expression vectors usually contain a promoter that is recognised by the host organism and is operably linked to the coding sequence of interest. Such a promoter may be inducible or constitutive.
• operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
• a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
• Promoters suitable for use with prokaryotic hosts include, for example, the /3-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence operably linked to the coding sequence.
• the prefe ⁇ ed vectors are expression vectors that enable the expression of a nucleotide sequence co ⁇ esponding to a polypeptide library member.
• selection with the first and or second antigen or epitope can be performed by separate propagation and expression of a single clone expressing the polypeptide library member or by use of any selection display system.
• the prefe ⁇ ed selection display system is bacteriophage display.
• phage or phagemid vectors may be used.
• the prefe ⁇ ed vectors are phagemid vectors which have an E. coli. origin of replication (for double stranded replication) and also a phage origin of replication (for production of single- stranded DNA).
• the vector - contains a /3-lactamase gene to confer selectivity on the phagemid and a lac promoter upstream of a expression cassette that consists (N to C terminal) of a pelB leader sequence (which directs the expressed polypeptide to the periplasmic space), a multiple cloning site (for cloning the nucleotide version of the library member), optionally, one or more peptide tag (for detection), optionally, one or more TAG stop codon and the phage protein pIIL
• the vector is able to replicate as a plasmid with no expression, produce large quantities of the polypeptide library member only or produce phage, some of which contain at least one copy of the polypeptide-pIII fusion on their surface.
• vectors employs conventional ligation techniques. Isolated vectors or DNA fragments are cleaved, tailored, and religated in the form desired to generate the required vector. If desired, analysis to confirm that the co ⁇ ect sequences are present in the constructed vector can be performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing expression and function are known to those skilled in the art.
• telomere sequence The presence of a gene sequence in a sample is detected, or its amplification and/or expression quantified by conventional methods, such as Southern or Northern analysis, Western blotting, dot blotting of DNA, RNA or protein, in situ hybridisation, immunocytochemistry or sequence analysis of nucleic acid or protein molecules. Those skilled in the art will readily envisage how these methods maybe modified, if desired.
• a single-domain antibody-effector group as herein defined describes an engineered antibody molecule comprising a single variable domain capable of specifically binding one or more epitopes, attached to one or more constant region domains (effector groups).
• Each variable domain may be a heavy chain domain (V H ) or a light chain domain (V L ).
• Suitable effector groups include any of those selected from the group, consisting of the following: an effector group comprising at least an antibody light chain constant region (CL), an antibody CHI heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof.
• an effector group may also comprise a hinge region of an antibody (such a region normally being found between the CHI and CH2 domains of an IgG molecule).
• an effector group as herein described comprises an Fc region of an antibody. More advantageously a dAb-effector group according to the present invention is a VL-FC.
• the effector group is based on a Fab antibody fragment. That is, it comprises an antibody fragment comprising a VH domain or a V L domain attached to the constant region domains making up a Fab fragment.
• a Fab antibody fragment comprises only one variable domain.
• a dAb-effector group according to the invention has a t ⁇ half-life of any of those t ⁇ half-lifes selected from the group consisting of the following: 12 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more or 20 days or more .
• a dAb-effector group or composition according to the invention will have a t ⁇ half life in the range 12 to 60 hours. In a further embodiment, it will have a t ⁇ half-life of a day or more. . In a further embodiment still, it will be in the range 12 to 26 hours.
• a dAb-effector group according to the present invention comprises or consists of an VL-FC having a t ⁇ half-life of a day or more, two days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more or 7 days or more.
• a dAb-effector group according to the present invention comprises or consists of an V L -FC having a t ⁇ half-life of a day or more.
• a dAb-effector group comprises an effector group consisting of the constant region domains CH2 and/or CH3, preferably CH2 and CH3, either with or without a hinge region as described herein, wherein the dAb-effector group has a t ⁇ half-life of a day or more, two days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more or 7 days or more. More advantageously, a dAb-effector group according to the present invention comprises an effector group consisting of the constant region domains CH2 and/or CH3 wherein the dAb-effector group has a t ⁇ half-life of a day or more.
• Each single variable domain comprises an immunoglobulin scaffold and one or more CDRs which are involved in the specific interaction of the domain with one or more epitopes.
• the members of the immunoglobulin superfamily all share a similar fold for their polypeptide chain.
• antibodies are highly diverse in terms of their primary sequence
• comparison of sequences and crystallographic structures has revealed that, contrary to expectation, five of the six antigen binding loops of antibodies (HI, H2, LI, L2, L3) adopt a limited number of main-chain conformations, or canonical structures (Chothia and Lesk (1987) J. Mol. Biol, 196: 901; Chothia et al (1989) Nature, 342: 877).
• Analysis of loop lengths and key residues has therefore enabled prediction of the main- chain conformations of HI, H2, LI, L2 and L3 found in the majority of human antibodies (Chothia et al. (1992) J.
• H3 region is much more diverse in terms of sequence, length and structure (due to the use of D segments), it also forms a limited number of main-chain conformations for short loop lengths which depend on the length and the presence of particular residues, or types of residue, at key positions in the loop and the antibody framework (Martin et al. (1996) J. Mol. Biol, 263: 800; Shirai et al. (1996) FEBS Letters, 399: 1).
• the dAb-effector groups of the present invention are advantageously assembled from libraries of domains, such as libraries of N H domains and libraries of N L domains.
• libraries of antibody polypeptides are designed in which certain loop lengths and key residues have been chosen to ensure that the main-chain conformation of the members is known.
• these are real conformations of immunoglobulin superfamily molecules found in nature, to minimise the chances that they are non-functional.
• Germline N gene segments serve as one suitable basic framework for constructing antibody or T-cell receptor libraries; other sequences are also of use. Variations may occur at a low frequency, such that a small number of functional members may possess an altered main-chain conformation, which does not affect its function.
• Canonical structure theory is also of use in the invention to assess the number of different main-chain conformations encoded by antibodies, to predict the main-chain conformation based on antibody sequences and to chose residues for diversification which do not affect the canonical structure. It is known that, in the human V domain, the LI loop can adopt one of four canonical structures, the L2 loop has a single canonical structure and that 90% of human N K domains adopt one of four or five canonical structures for the L3 loop (Tomlinson et al. (1995) supra); thus, in the N K domain alone, different canonical structures can combine to create a range of different main-chain conformations.
• the single main-chain conformation need not be a consensus structure - a single naturally occurring conformation can be used as the basis for an entire library.
• the dAb-effector groups of the invention possess a single known main-chain conformation.
• the single main-chain conformation that is chosen is preferably commonplace among molecules of the immunoglobulin superfamily type in question.
• a conformation is commonplace when a significant number of naturally occurring, molecules are observed to adopt it.
• the natural occu ⁇ ence of the different main-chain conformations for each binding loop of an immunoglobulin superfamily molecule are considered separately and then a naturally occurring immunoglobulin superfamily molecule is chosen which possesses the desired combination of main-chain conformations for the different loops. If none is available, the nearest equivalent may be chosen.
• the desired combination of main- chain conformations for the different loops is created by selecting germline gene segments which encode the desired main-chain conformations. It is more preferable, that the selected germline gene segments are frequently expressed in nature, and most preferable that they are the most frequently expressed of all natural germline gene segments.
• the incidence of the different main-chain conformations for each of the six antigen binding loops may be considered separately.
• HI, H2, LI, L2 and L3 a given conformation that is adopted by between 20% and 100% of the antigen binding loops of naturally occurring molecules is chosen.
• its observed incidence is above 35% (i.e. between 35% and 100%) and, ideally, above 50% or even above 65%.
• the conformation which is observed most often in the natural repertoire is therefore selected.
• the natural occu ⁇ ence of combinations of main-chain conformations is used as the basis for choosing the single main-chain conformation.
• the natural occu ⁇ ence of canonical structure combinations for any two, three, four, five or for all six of the antigen binding loops can be determined.
• the chosen conformation is commonplace in naturally occurring antibodies and most preferable that it observed most frequently in the natural repertoire.
• the desired diversity is typically generated by varying the selected molecule at one or more positions.
• the positions to be changed can be chosen at random or are preferably selected.
• the variation can then be achieved either by randomisation, during which the resident amino acid is replaced by any amino acid or analogue thereof, natural or synthetic, producing a very large number of variants or by replacing the resident amino acid with one or more of a defined subset of amino acids, producing a more limited number of variants.
• E ⁇ or-prone PCR Hawkins et al. (1992) J. Mol. Biol, 226: 889
• chemical mutagenesis (Deng et al. (1994) J Biol. Chem., 269: 9533) or bacterial mutator strains (Low et al. (1996) J. Mol. Biol, 260: 359)
• Methods for mutatmg selected positions are also well known in the art and include the use of mismatched oligonucleotides or degenerate oligonucleotides, with or without the use of PCR.
• H3 region of a human tetanus toxoid-binding Fab has been randomised to create a range of new binding specificities (Barbas et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457). Random or semi-random H3 and L3 regions have been appended to germline V gene segments to produce large libraries with unmutated framework regions (Hoogenboom & Winter (1992) J. Mol. Biol, 227: 381; Barbas et al. (1992) Proc. Natl Acad. Sci. USA, 89: 4457;
• loop randomisation has the potential to create approximately more than 10 15 structures for H3 alone and a similarly large number of variants for the other five loops, it is not feasible using cu ⁇ ent transformation technology or even by using cell free systems to produce a library representing all possible combinations.
• 6 x 10 10 different antibodies which is only a fraction of the potential diversity for a library of this design, were generated (Griffiths et al. (1994) supra).
• binding of single domain antibody-effector groups (dAb-effector group) according to the invention to its specific antigens or epitopes can be tested by methods which will be familiar to those skilled in the art and include ELISA.
• binding is tested using monoclonal phage ELISA.
• Phage ELISA may be performed according to any suitable procedure: an exemplary protocol is set forth below.
• phage produced at each round of selection can be screened for binding by ELISA to the selected antigen or epitope, to identify "polyclonal" phage antibodies. Phage from single infected bacterial colonies from these populations can then be screened by ELISA to identify "monoclonal” phage antibodies. It is also desirable to screen soluble antibody fragments for binding to antigen or epitope, and this can also be undertaken by ELISA using reagents, for example, against a C- or N-terminal tag (see for example Winter et al. (1994) Ann. Rev. Immunology 12, 433-55 and references cited therein).
• the diversity of the selected phage monoclonal antibodies may also be assessed by gel electrophoresis of PCR products (Marks et al. 1991, supra; Nissim et al. 1994 supra), probing (Tomlinson et al, 1992) J. Mol. Biol. 227, 776) or by sequencing of the vector DNA.
• nucleic acid molecules and vector constructs required for the performance of the present invention may be constructed and manipulated as set forth in standard laboratory manuals, such as Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, USA.
• nucleic acids in the present invention is typically carried out in recombinant vectors.
• vector refers to a discrete element that is used to introduce heterologous DNA into cells for the expression and/or replication thereof.
• Methods by which to select or construct and, subsequently, use such vectors are well known to one of moderate skill in the art.
• Numerous vectors are publicly available, including bacterial plasmids, bacteriophage, artificial chromosomes and episomal vectors. Such vectors may be used for simple cloning and mutagenesis; alternatively gene expression vector is employed.
• a vector of use according to the invention may be selected to accommodate a polypeptide coding sequence of a desired size, typically from 0.25 kilobase (kb) to 40 kb or more in length.
• a suitable host cell is transformed with the vector after in vitro cloning manipulations.
• Each vector contains various functional components, which generally include a cloning (or "polylinker") site, an origin of replication and at least one selectable marker gene. If a given vector is an expression vector, it additionally possesses one or more of the following: enhancer element, promoter, transcription termination and signal sequences, each positioned in the vicinity of the cloning site, such that they are operatively linked to the gene encoding a polypeptide .
• Both cloning and expression vectors generally contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells.
• this sequence is one that enables the vector to replicate independently of the host chromosomal DNA and includes origins of replication or autonomously replicating sequences.
• origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast and viruses.
• the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 micron plasmid origin is suitable for yeast, and various viral origins (e.g. SV 40, adeno virus) are useful for cloning vectors in mammalian cells.
• the origin of replication is not needed for mammalian expression vectors unless these are used in mammalian cells able to replicate high levels of DNA, such as COS cells.
• a cloning or expression vector may contain a selection gene also refe ⁇ ed to as selectable marker.
• This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will therefore not survive in the culture medium.
• Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available in the growth media.
• an E. coli- selectable marker for example, the /3-lactamase gene that confers resistance . to the antibiotic ampicillin, is of use.
• E. coli plasmids such as pBR322 or a pUC plasmid such as pUC18 or pUC19 or pUCl 19.
• Expression vectors usually contain a promoter that is recognised by the host organism and is operably linked to the coding sequence of interest. Such a promoter may be inducible or constitutive.
• operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
• a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
• Promoters suitable for use with prokaryotic hosts include, for example, the /3-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence operably linked to the coding sequence.
• the prefe ⁇ ed vectors are expression vectors that enables the expression of a nucleotide sequence co ⁇ esponding to a polypeptide. Thus, selection with antigen can be performed by separate propagation and expression of a single clone expressing the polypeptide or by use of any selection display system. As described above, the prefe ⁇ ed selection display system is bacteriophage display. Thus, phage or phagemid vectors may be used.
• the prefe ⁇ ed vectors are phagemid vectors which have an E. coli. origin of replication (for double stranded replication) and also a phage origin of replication (for production of single-stranded DNA).
• the vector contains a /3-lactamase gene to confer selectivity on the phagemid and a lac promoter upstream of a expression cassette that consists (N to C terminal) of a pelB leader sequence (which directs the expressed polypeptide to the periplasmic space), a multiple cloning site (for cloning the nucleotide version of the polypeptide), optionally, one or more peptide tag (for detection), optionally, one or more TAG stop codon and the phage protein pEII.-
• a pelB leader sequence which directs the expressed polypeptide to the periplasmic space
• a multiple cloning site for cloning the nucleotide version of the polypeptide
• optionally, one or more peptide tag for detection
• TAG stop codon optionally, one or more TAG stop codon and the phage protein pEII.
• the vector is able to replicate as a plasmid with no expression, produce large quantities of the polypeptide only or produce phage, some of which contain at least one copy of the polypeptide-pIII fusion on their surface.
• vectors employs conventional ligation techniques. Isolated vectors or DNA fragments are cleaved, tailored, and religated in the form desired to generate the required vector. If desired, analysis to confirm that the co ⁇ ect sequences are present in the constructed vector can be performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing expression and function are known to those skilled in the art.
• telomere sequence The presence of a gene sequence in a sample is detected, or its amplification and/or expression quantified by conventional methods, such as Southern or Northern analysis, Western blotting, dot blotting of DNA, RNA or protein, in situ hybridisation, immunocytochemistry or sequence analysis of nucleic acid or protein molecules. Those skilled in the art will readily envisage how these methods may be modified, if desired.
• dAb-effector groups selected according to the method of the present invention may be employed in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, and the like.
• the dAb-effector groups may be used in antibody based assay techniques, such as ELISA techniques, according to methods known to those skilled in the art.
• the dAb-effector groups of the invention can be prepared according to a desired or predetermined antigen binding specificity.
• the method of the invention permits the synthesis of dAb-effector groups with a desired effector group. In this way the effector functions can be designed into the dAb- effector group.
• the present inventors have found that the presence of the effector group increases the in vivo half life of the molecule.
• the dAb-effector groups according to the invention are of use in diagnostic, prophylactic and therapeutic procedures.
• Single domain-effector group antibodies selected according to the invention are of use diagnostically in Western analysis and in situ protein detection by standard immunohistochemical procedures; for use in these applications, the antibodies of a selected repertoire may be labelled in accordance with techniques known to the art.
• antibody polypeptides may be used preparatively in affinity chromatography procedures, when complexed to a chromatographic support, such as a resin. All such techniques are well known to one of skill in the art.
• Substantially dAb-effector groups according to the present invention of at least 90 to 95% homogeneity are prefe ⁇ ed for administration to a mammal, and 98 to 99% or more homogeneity is most prefe ⁇ ed for pharmaceutical uses, especially when the mammal is a human.
• the selected dAb-effector groups may be used diagnostically and/or therapeutically (including exfracorporeally) or in developing and performing assay procedures, immunofluorescent stainings and the like (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).
• the dAb-effector groups of the present invention will typically find use in preventing, suppressing or treating inflammatory states, allergic hypersensitivity, cancer, bacterial or viral infection, and autoimmune disorders (which include, but are not limited to, Type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and myasthenia gravis).
• inflammatory states allergic hypersensitivity, cancer, bacterial or viral infection
• autoimmune disorders which include, but are not limited to, Type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and myasthenia gravis.
• prevention involves administration of the protective composition prior to the induction of the disease.
• suppression refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease.
• Treatment involves administration of the protective composition after disease symptoms become manifest.
• EAE in mouse and rat serves as a model for MS in human. Ln this model, the demyelinating disease is induced by administration of myelin basic protein (see Paterson (1986) Textbook of Immunopathology, Mischer et al, eds., Grune and Sfratton, New York, pp. 179-213; McFarlin et al. (1973) Science, 179: 478: and Satoh et al (1987) J. Immunol, 138: 179).
• the dAb-effector groups will be utilised in purified form together with pharmacologically appropriate carriers.
• these carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or buffered media.
• Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's.
• Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension may be chosen from thickeners such as carboxymethylcellulose, polyvinylpy ⁇ olidone, gelatin and alginates.
• Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).
• the dAb-effector groups of the present invention may be used as separately administered compositions or in conjunction , with other agents. These can include various immunotherapeutic drugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in conjunction with the dAb-effector groups of the present invention, or even combinations of dAb-effector groups according to the present invention having different specificities, such asdAb-effector groups having variable domains selected using different target ligands, whether or not they are pooled prior to administration.
• various immunotherapeutic drugs such as cylcosporine, methotrexate, adriamycin or cisplatinum
• Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in conjunction with the dAb-effector groups of the present invention, or even combinations of dAb-effector groups according to the present invention having different specificities
• the route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art.
• the dAb-effector groups and compositions of the invention can be administered to any patient in accordance with standard techniques.
• the admimsfration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermalry, via the pulmonary route, or also, appropriately, by direct infusion with a catheter.
• the dosage and frequency of administration will depend on the age, sex and condition of the patient, concu ⁇ ent administration of other drugs, counterindications and other parameters to be taken into account by the clinician.
• the dAb-effector groups of this invention can be lyopbilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-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 antibody activity loss (e.g. with conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted upward to compensate.
• compositions containing the dAb-effector groups or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments.
• an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a "therapeutically-effective dose”. Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 0.005 to 5.0 mg of selected antibody, receptor (e.g. a T-cell receptor) or binding protein thereof per kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used.
• compositions containing the dAb-effector groups or cocktails thereof may also be administered in similar or slightly lower dosages.
• a composition containing a dAb-effector group or cocktail thereof according to the present invention may be utilised in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal.
• the dAb-effector groups described herein may be used extraco ⁇ oreally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells.
• Blood from a mammal may be combined extraco ⁇ oreally with the selected antibodies, cell-surface receptors or binding proteins thereof whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques.
• a /3-galactosidase binding N ⁇ dAb E5 was used to create V ⁇ -Fc fusion and an alkaline phosphatase (APS) binding V H dAb VH2 was used to create N H -Fc fusion (sequences of N ⁇ dAb E5 and N ⁇ dAb VH2 are shown in Table la).
• Hind III and Not /restriction sites were introduced onto the 5 'and 3 'ends, respectively, of the E5 and VH2 dAbs using oligonucleotides VK5Hind, VH5Hind and VH3Not (Table la, note that there was no need to introduce Not /site onto the 3' end of the E5 dAb, as it already exists).
• Hind ////Not /digested fragments containing E5 V ⁇ dAb and VH2 V H dAb were then ligated into Hind ////Not / digested Signal plgplus vector (R&D Systems Europe Ltd, Figure 2).
• Ligation mixtures were transformed into competent E.coli TGI cells and recombinant clones were verified by colony PCR screening and sequencing using PIG5SEQ and PIG3SEQ oligonucleotides (Table lb).
• VK5HIND CCC AAG CTT GAC ATC CAG ATG ACC CAG TCT CC
• VH5HIND CCC AAG CTT GAG GTG CAG CTG TTG GAG TCT GG
• E5-Fc and VH2-Fc fusions (Example 1) could be expressed in mammalian cells and that the produced proteins retain antigen specificity of the parental dAbs.
• Three mammalian cell lines (COSl, COS7 and CHO) were transfected with E5 dAb in plgplus and NH2 dAb in plgplus plasmid D ⁇ A (Example 1) using FuGENE 6 Transfection Reagent (Roche).
• Stably transformed cell lines were generated using selection medium containing G418 (lmg/ml for COSl and COS7 cells and 0.5mg/ml for CHO cells).
• dAb-Fc fusion proteins 25ml of the spent tissue culture medium from transfected cells were collected, filtered using 0.45 ⁇ filter and then passed through Protein A Sepharose column. dAb-Fc fusions were eluted using 1.6ml 0.1M Glycine pH 2.0 into 0.4ml 1M Tris, pH 9.0. 50 ⁇ l of the resulting 2ml sample was tested in ELISA (standard ELISA protocol was followed) 96- well plates were coated with 100 ⁇ l of APS and /3-galactosidase at 10 ⁇ g/ml concentration in PBS overnight at 4C. Detection was performed using anti human IgG (Fc specific) -HRP conjugate (Sigma).
• E5-Fc fusion protein Following optimisation of expression and purification procedures, the yield of E5-Fc fusion protein from COSl cells is 20mg/l. The expression level of VH2-Fc fusion is lower.
• Example 3 Binding of the E5-Fc fusion protein to the cell line expressing human Fc receptors
• E5-Fc fusion protein is able to bind to the cell line expressing human Fc receptors.
• Purified E5-Fc protein was labelled with fluorescein at 3.3 / 1 ratio of Fluo/Protein. The labelled protein (491 ⁇ g/ml concentration) was then used for FACS analysis.
• Human monocyte-like U937 cells which express two types of human FcRs (CD 64 and CD32) were used to assess the ability of E5-Fc fusion protein to bind these receptors.
• HEL-4 an anti-hen egg lysozyme dAb named HEL-4 which has a C-terminal HA epitope tag, see below for the amino acid sequence.
• HEL-4 was expressed in the E.coli strain HB2151 and purified from the periplasmic fraction by standard chromatography using protein A and anion exchange. The protein was dialysed twice for >2h against 500 volumes of phosphate buffered saline. The amino acid sequence of HEL-4.
• Example 5 Efficacy study of TARl-5-19 in a human TNF transgenic model of arthritis.
• TARl-5-19 is a Dab which specifically binds to the target human TNF alpha (TAR1).
• Tgl97 mice are transgenic for the human TNF-globin hybrid gene and heterozygotes at 4-7 weeks of age develop a chronic, progressive polyarthritis with histological features in common with rheumatoid arthritis.
• Transgenic mice expressing human tumor necrosis factor a predictive genetic model of arthritis. EMBO J., Vol. 10, pp. 4025-4031.
• VK dAb Fc fusion dAb fused to IgGl CH2-CH3 regions, the dAb being TARl-5-19
• heterozygous transgenic mice were divided into 5 groups of 10 animals with equal numbers of male and females. Treatment commenced at 3 weeks of age with twice weekly infraperitoneal injections of test items. The treatment groups are listed in Table 1.
• the control dAb-Fc was a fusion between the Fc region of human IgGl and an anti-bgalactosidase dAb (termed E5) and was expressed in the supernatant of a stably transfected COS-7 cell line.
• the TARl-5-19-Fc fusion was expressed by transient transfection of COS-7. Both Fc fusion proteins were purified by protein A chromatography. TARl-5-19 monomer was expressed in E.coli and purified by protein L chromatography and LEX. All protein preparations were in phosphate buffered saline and were tested for acceptable levels of endotoxins.
• TARl-5-19-Fc was shown to be a highly effective therapy in the Tgl97 model of arthritis.
• Example 6 Expression of a dAb-Fc fusion protein in Pichia pastoris.
• the vector for the methanol inducible, secreted, expression of dAb Fc fusion proteins in Pichia was constructed based on the expression vector pPICZalpha (Invitrogen).
• the vector was modified to remove the Xhol site at nucleotide 1247 by digestion with Xbal and Kpnl, blunt ending wih Pfu polymerase and relegation.
• the Sail site at nucleotide 1315 was removed by digestion with Sail, blunt ending with Pfu polymerase and relegation.
• a VK dAb-Fc fusion was then PCR amplified from a mammalian expression construct described above using the primers below and PfuTurbo DNA polymerase (Stratagene):
• PVKF2 5'-TCTCTCGAGAAAAGAGACATCCAGATGACCCAGTCTCC-3'
• the PCR product was digested with Xhol and EcoRI, then cloned into EcoRI/XhoI digested expression vector. This gave the construct pPICZalpha-TARl-5-19Fc which would produce an anti-TNF Fc fusion protein.
• the Xhol- Notl dAb fragment was excised from pPICZalpha-TARl-5-19Fc, and replaced with a Xhol-Notl linker which contained an in frame Sail site (sequence of the fragment, including restriction sites: 5'- CTCGAGAAAAGAGCGTCGACATCTAGATCAGCGGCCGC-3 ').
• VH dAbs PVHF1 5'-TCTCTCGAGAAAAGAGAGGTGCAGCTGTTGGAGTCTG-3'
• PVKF2 5'-TCTCTCGAGAAAAGAGACATCCAGATGACCCAGTCTCC-3' PVKRl 5'-TAGAATTCTTATTACCGTTTGATTTCCACCTTGGTC-3'
• This vector when integrated into the P. pastoris genome will express the anti-TNF recombinant dAb-Fc fusion protein TARl-5-19Fc on induction with methanol.
• the protein will be produced with an amino terminal yeast alpha mating factor secretion signal, which will direct secretion to the culture medium, during which it will be cleaved off by the Kex2 protease, to leave a homogenous dAb-Fc fusion protein which can be purified from the culture supernatant.
• the protein produced here has a Factor Xa protease cleavage site between the dAb and the Fc region. This aids in functional analysis of the protein, but could be replaced by either: a flexible polypeptide linker, a rigid polypeptide linker, or other specific protease cleavable sequence.
• protease cleavage site would give advantages in reducing the amount of protein binding non-specifically to an antigen in a non-target tissue which also expressed the chosen protease, where the target tissue did not express. This could be useful in targeted immunotoxins, drug conjugates or prodrug activating enzymes.
• amino acid sequence of the alpha factor dAb Fc fusion protein as encoded by the nucleotide sequence above is shown below and also in fig 11 :
• sequence of the yeast alpha mating-factor leader In italics is the sequence of the dAb.
• the Fc portion is in bold.
• the dAb and the Fc region are separated by a polypeptide spacer, in this case containing a Factor Xa protease cleavage site.
• Pichia were made competent for electroporation by growing P. pastoris KM71H in 0.51
• YPD 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose
• the cells were then washed twice with ice cold water, once with 20ml ice cold 1M sorbitol, and resuspended in 1ml 1M sorbitol.
• Cells were recovered in 1ml 1M sorbitol, then plated onto YPDS plates (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, 1M sorbitol in 1.5% (w/v) agar) supplemented with 100, 500, 1000, or 2000microgram/ml zeocin. Plates were grown for 2-3 days at 30°C, and then colonies were re-streaked to isolate clonal populations. Clones were then characterised for expression levels as described below.
• Pichia pastoris strains such as X33, or the protease deficient strains smdll63, smdll65 or smdll68 which will be advantageous in reducing proteolytic cleavage of the dAb-Fc fusion protein dusing expression.
• Pichia species such as Pichia methanolica, or other yeast and fungal species such as Hansenula polymorpha, Saccharomyces cerevisiae, Candida boidinii, or Aspergillus awamorii, would be suitable for the expression of dAb Fc fusion proteins.
• Expression was carried out in baffled shake flasks in complex BMGY medium containing glycerol as a carbon source (1% (w/v) yeast extract, 2% (w/v) peptone, 1% (v/v) glycerol, 1.34%) (w/v) yeast nitrogen base, 4xl0 "5 % (w/v) biotin, lOOmM KPO buffer ⁇ H6.0).
• Growth and expression could also be performed in other media including minimal or chemically defined medium, as well as in complex media, with equivalent results. Growth to higher cell densities under conditions of controlled carbon source feeding, controlled methanol induction levels and controlled oxygen levels in a fermenter using fed batch or continuous processes, would lead to higher expression levels.
• glycosylation pattern is required that is closer to that seen in humans, mammalian like glycosylation could be obtained using modifications of the glycosylation enzymes in Pichia, such as that described in: Hamilton SR, Bobrowicz P, Bobrowicz B, Davidson RC, Li H, Mitchell T, Nett JH, Rausch S, Stadheim TA, Wischnewski H, Wildt S, Gerngross TU (2003). Production of complex human glycoproteins in yeast. Science. 29;301(5637):1171. This would yield a homogenously glycosylated product. Purification.
• Pure dAb-Fc fusion was further purified from this material by ion exchange chromatography on a 5ml Resource Q column (Amersham Biotech) 20mM Tris-HCl buffer at pH8.5 using a 0 to 0.5M NaCl gradient over 30 column volumes.
• Amino terminal sequencing showed that the protein had been processed as predicted by the P. pastoris Kex2 protease to give the amino-terminal sequence of NH 2 -EDQTM after 5 cycles of Edman degradation.
• Non-reduced and reduced SDS-PAGE analysis showed that the protein was the same size as that produced in mammalian cells using the same TARl-5-19-Fc fusion protein construct in a mammalian expression vector.
• Antigen binding activity was determined using a TNF receptor binding assay (fig 12).
• a 96 well Nunc Maxiso ⁇ plate is coated with a mouse anti-human Fc antibody, blocked with 1% BSA, then TNF receptor 1-Fc fusion is added.
• the dAb-Fc fusion protein at various concentrations is mixed with lOng/ml TNF protein and incubated at room temperature for >lhour. This mixture is added to the TNF receptor 1-Fc fusion protein coated plates, and incubated for lhour at room temperature. The plates are then washed to remove unbound free dAb-Fc fusion, TNF and dAb-Fc/TNF complexes.
• the plate was then incubated sequentially with a biotinylated anti-TNF antibody and sfreptavidin-horse radish peroxidase.
• the plate was then incubated with the chromogenic horse radish peroxidase substrate TMB.
• the colour development was stopped with the addition of 1M hydrochloric acid, and absorbance read at 450nm.
• the absorbance read is proportional to the amount of TNF bound, hence, the TARl-5-19Fc fusion protein will compete with the TNF receptor for binding of the TNF, and reduce the signal in the assay.
• the P. pastoris produced protein had an equivalent activity to the mammalian protein in the vitro TNF receptor assay described above.
• the protein produced was effective at activation of human complement after antigen binding, as measured by the following assay:
• 96-well Maxisop plates (Nunc) were coated with human TNF at lmicrogram/ml.
• the dAb-Fc fusion or control antibody was bound to the TNF coated plates, which were washed with phosphate buffered saline to remove unbound antibody, then pre-incubated with human complement Cl at lmicrogram/ml (Merck Biosciences, consisting of a complex of the stoichiomety: (Clr) 2 (Cls) 2 Clq) in complement fixation diluent.
• complement activation is important for dAb-Fc fusion functionality, such as complement lysis of target tumour cells, this activity is advantageous. If the Fc fusion is for other reasons, where complement activation is not required or is deleterious to function, removal of the glycosylated Asparagine residue would remove the glycosylation site, and a homogenous aglycosyl protein could be produced.

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