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WO2007023298A2 - Anticorps - Google Patents

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Publication number
WO2007023298A2
WO2007023298A2 PCT/GB2006/003187 GB2006003187W WO2007023298A2 WO 2007023298 A2 WO2007023298 A2 WO 2007023298A2 GB 2006003187 W GB2006003187 W GB 2006003187W WO 2007023298 A2 WO2007023298 A2 WO 2007023298A2
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Prior art keywords
antibody
fragment
antibodies
ion channel
sodium ion
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PCT/GB2006/003187
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English (en)
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WO2007023298A3 (fr
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Ming Lei
Bin Gao
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Isis Innovation Limited
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Publication of WO2007023298A3 publication Critical patent/WO2007023298A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to antibodies and in particular antibodies that are directed against sodium ion channels and in particular are directed to specific sodium ion channel isoforms, and their uses.
  • Ion channels are integral membrane proteins that play important roles in various cell types.
  • Large numbers of ion channel genes have been discovered in the mammalian genome, each of these genes encoding a particular ion channel subunit with cell-specific expression patterns. To assign functional and pathophysiological roles to these genes are enormous tasks in the post genomic era.
  • Ion channels are important therapeutic drug targets.
  • Existing tools including both synthetic chemicals and natural compounds, are rarely ion channel isoform/gene specific. In particular, they generally do not discriminate between closely related members of the ion channel protein sub family.
  • numerous antibodies against specific ion channel isoforms have been developed, such antibodies are designed to target intracellular C-terminal or N-terminal regions of the channel protein. Such antibodies cannot be used as inhibitors in living cells.
  • Voltage-dependent Na + channels are composed of pore forming ⁇ subunit (nav) and associated auxiliary modulator ⁇ subunits. Each ⁇ subunit is approximately 260 kDa. The ⁇ subunits are approximately 36 kDa in size. All ⁇ subunits are predicted to have a similar structure comprising four homologous domains (termed DI-IV), each of which contains six membrane spanning segments, S1-S6. Ten ⁇ subunits and four ⁇ subunits have been identified so far (Golding 2001 Annu. Rev. Physio 63 871-894, Yu and Catterall 2003. Genome Biol. 4 2003, 207). Different ⁇ subunit isoforms are expressed in different tissues and have different pharmacological properties. Summary of the Invention
  • the present inventors have developed isoform-specif ⁇ c sodium ion channel antibodies. These antibodies specifically target an extracellular region of the sodium ion channel. Accordingly the invention provides an antibody or fragment thereof which binds to an extra cellular pore forming region of a sodium ion channel.
  • Figure 1 is a schematic drawing to show sodium ion channel ⁇ subunit domain structure.
  • Figure 2 sets out the sequence alignment for a number of sodium ion channel ⁇ subunits. The following Table indicates the SEQ ID NO. for each sequence shown in Figure 2.
  • Figure 3 shows Kyte Doolittle hydrophilicity analysis of "P" region of Dl of sodium channel isoforms.
  • ES5 the hydrophilicity region S5
  • ES6 the hydrophilicity region S6, P: pore region.
  • Figure 4 shows expression of NaV 1.5 in HEK-293 cells, Na+-current traces elicited by voltages steps ranging from -120 to +40 mV in 10-mV increments. The holding potential was -100 mV. The voltage step began at the start of the traces and the initial capacity current has been deleted. Lidocaine and antibody were bath- applied at 1 mM and 15 ⁇ g/ml respectively. 'Control Ab' was a non-Na v 1.5 affmity- purified rabbit antibody.
  • Figure 5 shows the effect of Anti-Navl.5 on Na+- current in isolated rat ventricular myocytes.
  • the current was elicited by voltage steps ranging from -100 to +40 mV in 10-mV increments.
  • the holding potential was -120 mV.
  • the isoform specific antibody only inhibits Navl.5 current. Multiple Na channel currents have identified in this cell type.
  • Figure 6 shows the effects of Anti-Navl.l on Na+-current in isolated rat ventricular myocytes.
  • the current was elicited by voltage steps from -100 to +40 mV in 10-mV increments.
  • the holding potential was -12OmV.
  • the isoform specific antibody only inhibits Navl.l current.
  • SEQ ID NOS. 1-16 are the full length sequences for the ⁇ subunit isoforms shown in Figure 2.
  • SEQ ID NOS. 17-20 are fragments of the S5-S6 region of isoforms Nav 5, Navla, 6 and 4 respectively (corresponding to Nav 1.5, Nav 1.1, Nav 1.6 and Nav 1.4).
  • Polypeptides The present invention provides antibodies that bind to an extracellular pore- forming region of the sodium ion channel ⁇ subunit, referred to herein as nav.
  • nav an extracellular pore- forming region of the sodium ion channel ⁇ subunit
  • the term antibody unless explicitly specified to the contrary, includes antibody fragments.
  • an antibody of the invention is specific for a subset of sodium ion channel ⁇ subunit isoforms, for example, is specific for 1 to 3 ion channel isoforms and typically will bind to a single sodium ion channel ⁇ subunit isoform, and so is isoform-specific.
  • an antibody of the invention binds to an extracellular region of the sodium ion channel ⁇ subunit, in particular, to the pore-forming region thereof.
  • an antibody of the invention binds to S5-P-S6 region of one of the domains DI to DIV of the sodium ion channel.
  • the antibody binds to the S5-P-S6 region of the domain DI of nav, but may also be an antibody that binds to the S5-P-S6 region of any one of the domains DI to DIV, such as the Dili domain.
  • An antibody of the invention thus preferentially binds or specifically binds to the extracellular region of a sodium ion channel ⁇ subunit.
  • An antibody of the invention preferably binds or specifically binds to a single isoform.
  • the binding of the antibody to other isoforms of the sodium ion channel ⁇ subunit other than that to which the antibody is specifically directed is generally non-specific binding at a substantially lower affinity than the specific binding of the antibody to the selected isoform.
  • a substantially lower affinity is generally at least two fold, three fold, five fold, ten fold, fifty fold or hundred fold lower affinity.
  • An antibody "preferentially binds" or “specifically binds” to a selected sodium ion channel isoform when it binds with preferential or high affinity to the selected sodium ion channel isoform but does not substantially bind, does not bind or binds with only lower affinity to other polypeptides.
  • a variety of protocols for binding, competitive binding or immunoradiometic assays to determine the specific binding capability of an antibody are well known in the art (see for example Maddox et al, J. Exp. Med. 158, 1211-1226, 1993). Such immunoassays typically involve the formation of complexes between a specific protein and its antibody and the measurement of complex formation.
  • epitope refers to that portion of the molecule that makes contact with a particular binding polypeptide.
  • An epitope may be linear, comprising an essentially linear amino acid sequence from the antigen or conformational, comprising sequences that are separated by other sequences that come together structurally to form a binding site for the polypeptide.
  • the amino acid sequences of human sodium ion channel isoforms and some selected animal isoforms are shown in Figure 2. As is explained above and shown in Figure 1, each ⁇ subunit has the same basic structure comprising an intracellular N-terminal region, four repeat domains DI to DIV and an intracellular C-terminal region. Each of the domains DI to DIV are linked together via linkers.
  • Each domain is composed of six transmembrane segments Sl to S6, the section between S5 and S6 comprising part of a pore forming region including an extracellular region (P).
  • the antibodies of the present invention are directed against the S5-P-S6 extracellular region.
  • the antibody binds to at least a 3 amino acid fragment, preferably at least a 5, 6, 7 or 8 amino acid fragment such as a 10, 12, 14, 16 or 20 amino acid segment of the S5-P-S6 region of one of the domains of the sodium ion channel ⁇ subunit.
  • the antibodies bind to the S5-S6 region of domain Dl, such as this region of isoform nav 5 or nav 6, such as SEQ ID NO. 17 or 19.
  • the antibody is directed to a region in domain III such as domain III of isoform nav Ia such as SEQ ID NO. 18.
  • the antibody is directed against a polypeptide comprising part or all of the sequences of SEQ ID NOS. 17 to 20.
  • the antibody is directed against a polypeptide that comprises at least six, preferably at least eight, preferably at least ten or twelve contiguous amino acids comprising a fragment of SEQ ID NOS. 17, 18, 19 or 20.
  • Such a polypeptide may be flanked by sequences that are contiguous to SEQ ID NOS. 17, 18, 19 and 20 in the S5- P-S6 region of the relevant sodium ion channel isoform.
  • a sequence comprising part or all of the sequences of SEQ ID NOS.
  • 17, 18, 19 or 20 may be a variant of a fragment or all of SEQ ID NOS. 17, 18, 19 or 20.
  • One or more amino acids, such as 1, 2, 3, 4 or 5 conservative substitutions may be present in the sequence of SEQ ID NOS. 17, 18, 19 or 20 or a fragment thereof. Conservative substitutions are shown in the following table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.
  • the polypeptide to which the antibody binds may be a recombinant polypeptide.
  • the polypeptide may be in solution or may be attached a solid surface.
  • the polypeptide may be attached to beads such as magnetic beads.
  • the polypeptide may be biotinylated.
  • the biotin molecule conjugated to the peptide may be used to immobilize the polypeptide on a solid surface by coupling biotinstreptavidin on the solid surface.
  • antibody refers to a protein comprising at least one, and preferably two, heavy chain variable regions (VH) and/or at least one, preferably two, light chain variable regions (VL).
  • VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions (CDR)", interspersed with regions that are more conserved, termed “framework regions (FR)”.
  • CDR complementarity determining regions
  • FR framework regions
  • the extent of the FR and CDRs has been precisely defined (see, Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia et al. (1987) J. MoI. Biol.
  • Each VH and VL is composed of three CDRs and four FRs arranged from N-terminus to C-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • the VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain respectively.
  • the antibody is a tetramer of two heavy and two light chains, wherein the heavy and light chains are interconnected by, for example, disulphide bonds.
  • the heavy chain constant region is comprised of three domains, CHl, CH2 and CH3.
  • the light chain constant region is comprised of one domain, CL.
  • the variable region of the heavy and light chains contains a binding domain that interacts with antigen.
  • the constant regions of the antibodies typically mediate the binding of the antibody to host tissues and factors, including various cells of the immune system and the first component of the complement system.
  • the term "antibody” includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM and subtypes thereof.
  • a preferred immunoglobulin is IgG.
  • immunoglobulin refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes.
  • the recognised human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu conatant region genes as well as a myriad of immunoglobulin variable region genes.
  • Full-length immunoglobulin light chains (about 25 kD or 214 amino acids) are encoded by a variable region gene at the N-terminus (about 110 amino acids) and a kappa or lambda constant region at the C-terminus.
  • Full-length immunoglobulin heavy chains (about 50 kD or 446 amino acids) are encoded by a variable region gene at the N- terminus (about 116 amino acids) and one of the other aforementioned constant region genes at the C-terminus, e.g. gamma (encoding about 330 amino acids).
  • An antibody fragment of the invention is typically an antigen-binding fragment.
  • the term "antigen-binding fragment” refers to one or more fragments of a full-length antibody that are capable of specifically binding to an isoform of sodium ion channel ⁇ subunit.
  • binding fragments include (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab') 2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulphide bridge at the hinge region; (iii) a Fd fragment (consisting of the VH and CHl domains); (iv) a Fv fragment (consisting of the VH and VL domains of a single arm of an antibody); (v) a dAb fragment (consisting of the VH domain); (vi) an isolated CDR; (vii) a single chain Fv (scFv) (consisting of the VH and VL domains of a single arm of an antibody joined by a synthetic linker using recombinant means such that the VH and VL domains pair to form a monovalent molecule); (viii) diabodies (consisting of two scFvs in which the VH and VL domains
  • Variant antibodies may be obtained by any suitable method. Typically variants with improved binding characteristics are selected by affinity maturation.
  • the antibody is a recombinant or modified anti-nav antibody, e.g. a chimeric, humanised, deimmunised or an in vitro generated antibody.
  • recombinant or modified antibody as used herein is intended to include all antibodies that are prepared, expressed, created or isolated by recombinant means, such as (i) antibodies expressed using a recombinant expression vector transfected into a host cell; (ii) antibodies isolated from a recombinant, combinatorial antibody library; (iii) antibodies isolated from an animal (e.g.
  • Such recombinant antibodies include humanised, CDR grafted, chimeric, deimmunised, in vitro generated antibodies and may optionally include constant regions derived from human germline immunoglobulin sequences.
  • An antibody according to the invention may be a human antibody.
  • the antibody may be a chimeric antibody, a recombinant antibody, a humanised antibody, a monoclonal antibody or a polyclonal antibody.
  • Preferably the antibody is monoclonal.
  • the antibody may be conjugated to a functional moiety such as a drug, detectable moiety or a solid support.
  • the antibody is preferably monospecific, e.g. a monoclonal antibody, or antigen-binding fragment thereof.
  • an antibody of the invention may be joined to a binding moiety such as biotin.
  • an antibody preferably an IgG
  • an antibody may be biotinylated by incubation with sulfosuccinimidyl-2-(biotinamido) ethyl-l,3-dithiopropionate.
  • a biotinylated IgG preferably comprises from 1 to 5 such as 2, 3 or 4 biotin groups.
  • An antibody of the invention may be in substantially isolated form. They may be mixed with carriers or diluents which will not interfere with their intended use and still be regarded as substantially isolated. They may also be in a substantially purified form, in which case they will generally comprise at least 90%, e.g. at least 95%, 98% or 99%, of the polypeptides or dry mass of the preparation.
  • Antibodies of the invention can be produced by any suitable method.
  • Means for preparing and characterising antibodies are well known in the art, see for example Harlow and Lane (1988) "Antibodies: A Laboratory Manual", Coldspring Harbour Laboratory Press, Coldspring Harbour, NY.
  • an antibody may be produced by raising antibody in a host animal against the polypeptide or a fragment thereof, for example an antigenic epitope thereof, hereinafter the immunogen.
  • the fragment may be any of the fragments mentioned herein, typically at least 10 or at least 15 amino acids long.
  • a method for producing a polyclonal antibody comprises immunizing a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the animals serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG fraction purified.
  • a method for producing a monoclonal antibody comprises immortalizing cells which produce the desired antibody.
  • Hybridoma cells may be produced by using spleen cells from an inoculated experimental animal with tumor cells (Kohler and Milstein 1975) Nature 256, 495-497.
  • An immortalized cell producing the desired antibody may be selected by a conventional procedure.
  • the hybridomas may be grown in culture or injected intraperitranely for formation of ascites or into the blood stream of an allogenic host or immuno compromised host.
  • Human antibody may be prepared by in vitro immunization of human lymphocytes, followed by transformation of the lymphocytes epstein-barr virus.
  • the experimental animal is suitably a goat, rabbit, rat, mouse, guinea pig, chicken, sheep or horse.
  • the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier.
  • the carrier molecule is typically a physiologically acceptable carrier.
  • the antibody obtained may be isolated and if desired purified.
  • nucleic acid sequence encoding the antibody is cloned into nucleic acid expression vector. If the antibody includes multiple peptide chains, each chain must be cloned into an expression vector for example, the same or different vectors, that are expressed in the same or different cells. If the antibody fragment is sufficiently small, for example which has less than 50 amino acids, it can be synthesised using automated synthetic methods. Methods well known to those skilled in the art can be used to construct vectors encoding an antibody of the invention and an appropriate transcriptional/translational control signals.
  • Antibodies that bind to nav and described herein have in vitro and in vivo diagnostic, therapeutic and prophylactic utilities.
  • the present invention provides a diagnostic method for detecting the presence of a sodium ion channel isoform in vitro for example on a cellular sample, or in vivo for example in in vivo imaging in a subject.
  • the method includes: (i) contacting a sample with an antibody of the invention; and (ii) detecting formation of a complex between the antibody and the sample.
  • the method can also include contacting a reference sample, such as a control sample with the antibody, and determining the extent of formation of the complex between the antibody and the sample relative to the same for the reference sample.
  • a change for example a statistically significant change, in the formation of the complex in the sample or subject relative to the control sample or subject can be indicative of the presence of the selected nav isoform in the sample.
  • Another method includes: (i) administering an antibody of the invention to a subject; and (ii) detecting formation of a complex between the antibody and the subject.
  • the detection step can include determining location or time of formation of the complex.
  • the antibody ligand can be directly or indirectly labelled with a detectable substance to facilitate detection of the bound or unbound antibody.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
  • Complex formation between an antibody of the invention and a nav isoform can be detected by measuring or visualising either the antibody bound to the nav or unbound antibody.
  • Conventional detection assays can be used, e.g., an enzyme- linked immunosorbent assay (ELISA), a radioimmunoassay (RIA) or tissue immunohistochemistry.
  • ELISA enzyme- linked immunosorbent assay
  • RIA radioimmunoassay
  • tissue immunohistochemistry e.g., tissue immunohistochemistry.
  • the presence of a nav isoform can be assayed in a sample by a competition immunoassay utilising standards labelled with a detectable substance and an unlabelled antibody.
  • the biological sample, the labelled standards and the antibody are combined and the amount of labelled standard bound to the unlabeled ligand is determined.
  • the amount of nav isoform in the sample is inversely proportional to the amount of labelled standard bound to antibody.
  • Fluorophore and chromophore labelled antibodies can be prepared. Since antibodies absorb light having wavelengths up to about 310 nm, the fluorescent moieties should be selected to have substantial absorption at wavelengths above 310 nm and preferably above 400 nm. A variety of suitable fluorescers and chromophores are described by Stryer (1968) Science 162:526 and Brand, L. at al. (1972) Annual Review of Biochemistry 41 :843-868. The antibodies can be labelled with fluorescent chromophore groups by conventional procedures such as those disclosed in U.S. Patent Nos. 3,940,475, 4,289,747 and 4,376,110.
  • fluorescers having a number of the desirable properties described above is the xanthene dyes, which include the fluoresceins and rhodamines.
  • Another group of fluorescent compounds are the naphylamines.
  • the antibody can be used to detect the presence or localisation of the nav in a sample, e.g., using fluorescent microscopy (such as confocal or deconvolution microscopy).
  • Immunohistochemistry can be performed using the antibodies described herein.
  • the antibody can synthesised with a label (such as a purification or epitope tag), or can be detectably labelled, e.g., by conjugating a label or label- binding group.
  • a chelator can be attached to the antibody.
  • the antibody is then contacted to a histological preparation, e.g., a fixed section of tissue that is on a microscope slide. After an incubation for binding, the preparation is washed to remove unbound antibody. The preparation is then analysed, e.g., using microscopy, to identify if the antibody bound to the preparation.
  • the antibody can be unlabelled at the time of binding. After binding and washing, the antibody is labelled in order to render it detectable.
  • the antibodies of the present invention are particularly useful to study pore function and to localise ion channel distribution patterns and pore location in living cells.
  • the antibodies can also be used as ion channel blockers, in particularly, to block specific sodium ion channel isoforms in assays, for example to monitor and determine the effects on activity by blocking a specific isoform, or to inhibit one type of isoform activity to allow analysis of the activity associated with a second sodium channel isoform.
  • the antibodies of the invention may also be used in methods of therapy.
  • the antibodies of the invention may be used to ameliorate or reduce the effects and symptoms of a disorder.
  • the antibodies of the invention may be used in the treatment of cancer and in particularly, to reduce mestastasis or spread of the tumor.
  • Sodium ion currents have been shown to play a role in the invasion capacity, for example in a breast cancer cell line.
  • Recent studies have demonstrated that Na channels are involved in several types of cancers and in particular their metastatic activity.
  • Neonatal Navl .7 (fNavl .7) has been shown to be strongly expressed in matastatic prostate cancer cells (Diss et ah, Prostate, 2001; 48: 165-78).
  • fNavl.5 has also been shown to be significantly overexpressed in metastatic breast cancer cells (Fraser et ah, Clin Cancer Res, 2005; 15:5381-5389).
  • a polyclonal antibody specific for fNavl.5 has been shown to inhibit migration of MDA-MB-231 human breast cancer cells in vitro (Brackenbury et ah, Breast Cancer Res Treat, 2006; electronic publication ahead of printed publication).
  • the antibodies of the invention may be used to reduce the potential of invasion of the tumor.
  • the amount of the antibody effective to treat a disorder or a therapeutically effective amount refers to the amount of the antibody which is effective, upon single and multiple dose administration to a subject to alleviate, relieve or improve the symptoms of the disorder, and in particular, to reduce symptoms such as invasion in the treatment of cancer.
  • compositions e.g. pharmaceutically acceptable compositions, which include an antibody of the invention formulated together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any physiologically compatible solvents, dispersion media, coatings, and the like.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).
  • the active compound i.e. polypeptide may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
  • compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-sold and solid dosage forms, such as liquid solutions (e.g. injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • liquid solutions e.g. injectable and infusible solutions
  • dispersions or suspensions tablets, pills, powders, liposomes and suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for administration of humans with antibodies.
  • the preferred mode of administration is parental (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular).
  • Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.
  • Sterile injectable solutions can be prepared by incorporating the active compound (i.e. the polypeptide) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • the antibodies of the present invention can be administered by a variety of methods known in the art, although for many applications, the preferred route/mode of administration is intravenous injection or infusion.
  • the antibody can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5 or 1 mg/min to reach a dose of about 1 to 100 mg/m 2 such as 7 to 25 mg/m 2 .
  • the route and/or mode of administration will vary depending upon the desired results.
  • An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody of the invention is 0.1-20 mg/kg, more preferably 1- 10 mg/kg. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
  • Voltage-dependent Na + channels are composed of pore forming ⁇ (Nav) subunits, which is approximately 260 kDa, and associated auxiliary modulate ⁇ subunits (approximately 36 kDa). All ⁇ subunits are predicted to have four homologous domains (DI-IV), which each contain six transmembrane spanning segments (S1-S6). Ten ⁇ subunits and four ⁇ subunits have been identified so far (Goldin, 2001 Annu Rev Physiol 63, 871-894; Yu & Catterall, 2003 Genome Biol 4 207). Different ⁇ subunit isoforms are expressed in different tissues and have different pharmacological properties (Goldin, 2001).
  • the "pore” consisting of two transmembrane segments (S5 and S6) and a hydrophobic region is responsibility for Na + selectivity (P region).
  • P region Such "membrane-pore-membrane” structure is highly conserved within Nav Na + channel subfamily. With Kyte-Doolittle hydrophilicity analysis, as showed in Figure 3, Nav Na + channel isoforms exhibit a recognised pattern in the P region structure, which lead us to identify the common localisation of P region.
  • hydrophilicity pattern allows ready identification of P region
  • a comparison of sequences of extracellular hydrophilicity peak between S 5 and S 6 reveals sufficient diversity to discriminate different Nav isoforms, the fact which allows us to design specific peptide for raising isoform specific Ab as inhibitors to target P region of a particular Nav channel.
  • Peptides of SEQ ID NOS. 17-20 were supplied by UCL peptide facility and were synthesised using standard Fmoc chemistry. The peptides were purified by HPLC and verified by both mass spectrometry and HPLC. Individual peptide was coupled onto carrier protein keyhole limpet hemocyanin (KLH) using standard m- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) method.
  • KLH carrier protein keyhole limpet hemocyanin
  • Rabbits were immunised subcutaneously using standard protocol.
  • the antibodies were purified using ammonia sulphate precipitation and further purified by protein-A affinity chromatography as necessary.
  • HEK-293 cells were transiently transfected with Scn5a (NM021544) encoding mouse NaVl.5. All cells were grown in DMEM-F 12 (Gibco, UK) medium containing 10% fetal calf serum, 100 units/ml penicillin and 100 ⁇ g/ml streptomycin, and maintained at 37°C under 95% air and 5% CO2.
  • HEK-293 cells were transiently transfected with mouse Navl .4 or rat Navl .1.
  • rat ventricular cells were isolated as described below.
  • Antibodies were affinity-purified on columns containing immobilized peptides.
  • NaVl.5-E3 were dialysed in phosphate-buffered saline (PBS: 137 niM NaCl, 10 mM Na2HPO4, 2 mM NaH2PO4, pH 7.4) to remove elution buffer.
  • PBS phosphate-buffered saline
  • the series resistance was electronically compensated (>80 %) and the current signal was filtered by a low pass Bessel filter with a cut-off frequency of 5 kHz (-3 dB). Data were digitised at 1-2 kHz using a Digidata 1200A AID converter (Axon Instruments Inc.) and stored on a computer for later analysis using pCLAMP version 8.2 software (Axon Instruments Inc.).
  • the figures show the molecular weight (KD) of protein labelled by specific Ab in different tissue.
  • Muscle type Navl.4 expresses in mucle, but also in the brain.
  • Brain type Navl.9 presents in all tissues examined, non-specific labelling of this Ab may exist.
  • Navbl is the bl subunit and it should present in all tissue.
  • Anti-Navl.5 only label the ventricular tissue, which is agreeable to the imrnunostaining in Navl.5 in HEK-293 cells.
  • Anti-Navl.5 inhibits Na+-current in HEK-293 cells transfected with NaV 1.5 ( Figure 4).
  • Anti-Navl.5 antibody positively labels EGFP-mNavl.5 expressed in HEK- 293 cells (results not shown).
  • Anti-Navl.4 and anti-Navl.l antibodies positively label HEK-293 cells expressing mouse Navl.4 and rat Navl.l respectively (results not shown).
  • Anti-Navl.l positively labels Navl.l channel in rat ventricular myocytes with permeabilization and without permeabilization (results not shown). Without permeabilization, the antibody is able to detect the extracellular distribution pattern of Navl .1 in this cell type.
  • Such antibodies can be used for detecting cell subtypes in living cells.
  • Anti-Navl.5 antibody positively labels Navl.5 channels in rat ventricular myocytes with permeabilization and without permeabilization (results not shown). Navl.5 shows a very different membrane distribution pattern to that of Navl.l.

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  • Pharmacology & Pharmacy (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne un anticorps ou un fragment de celui-ci qui se lie à une région extracellulaire formant un pore d'un canal ionique sodique. Les anticorps de l'invention peuvent être utilisés dans des méthodes de traitement.
PCT/GB2006/003187 2005-08-26 2006-08-25 Anticorps WO2007023298A2 (fr)

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GBGB0517487.5A GB0517487D0 (en) 2005-08-26 2005-08-26 Antibodies

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JP2013507980A (ja) * 2009-10-27 2013-03-07 ユセベ ファルマ ソシエテ アノニム 機能改変するＮａｖ１．７抗体
JP2013508443A (ja) * 2009-10-27 2013-03-07 ユセベ ファルマ ソシエテ アノニム イオンチャネルに対する抗体
WO2014159595A2 (fr) 2013-03-14 2014-10-02 Regeneron Pharmaceuticals, Inc. Anticorps humains dirigés contre nav1.7
WO2014207401A1 (fr) * 2013-06-28 2014-12-31 Galderma Research & Development Modulateurs du canal sodique nav1.9 pour le traitement d'une maladie cutanée inflammatoire et méthodes diagnostiques
WO2014207400A1 (fr) * 2013-06-28 2014-12-31 Galderma Research & Development Anticorps dirigés contre la canal sodique nav1.9 humain et leurs utilisations pour le diagnostic des maladies cutanées inflammatoires
WO2014207402A1 (fr) * 2013-06-28 2014-12-31 Galderma Research & Development Anticorps dirigés contre le canal sodique nav1.9 humain et leurs utilisations en diagnostic
JP2016020396A (ja) * 2009-10-27 2016-02-04 ユセベ ファルマ ソシエテ アノニム イオンチャネルに対する抗体
US9340590B2 (en) 2011-03-16 2016-05-17 Amgen Inc. Potent and selective inhibitors of NaV1.3 and NaV1.7
US9636418B2 (en) 2013-03-12 2017-05-02 Amgen Inc. Potent and selective inhibitors of NAV1.7
US9956274B2 (en) 2009-10-27 2018-05-01 Ucb Biopharma Sprl Method to generate antibodies to ion channels
US10344060B2 (en) 2013-03-12 2019-07-09 Amgen Inc. Potent and selective inhibitors of Nav1.7
US11753479B2 (en) 2014-03-04 2023-09-12 Kymab Limited Nucleic acids encoding anti-OX40L antibodies
US11779604B2 (en) 2016-11-03 2023-10-10 Kymab Limited Antibodies, combinations comprising antibodies, biomarkers, uses and methods
US11932673B2 (en) 2017-07-12 2024-03-19 Maxion Therapeutics Limited Sodium channel inhibitors
US12209128B2 (en) 2016-06-20 2025-01-28 Kymab Limited Anti-PD-L1 antibodies

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US8986694B1 (en) 2014-07-15 2015-03-24 Kymab Limited Targeting human nav1.7 variants for treatment of pain
US8992927B1 (en) 2014-07-15 2015-03-31 Kymab Limited Targeting human NAV1.7 variants for treatment of pain

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US6573067B1 (en) * 1998-01-29 2003-06-03 Yale University Nucleic acid encoding sodium channels in dorsal root ganglia

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JP2016026211A (ja) * 2009-10-27 2016-02-12 ユセベ ファルマ ソシエテ アノニム 機能改変するＮａｖ１．７抗体
JP2013508443A (ja) * 2009-10-27 2013-03-07 ユセベ ファルマ ソシエテ アノニム イオンチャネルに対する抗体
EP2493928B1 (fr) * 2009-10-27 2020-04-15 UCB Biopharma SRL Anticorps dirigés contre les canaux ioniques
EP2493926B1 (fr) * 2009-10-27 2020-03-11 UCB Biopharma SRL Modification fonctionnelle des anticorps anti-nav 1.7
US10112996B2 (en) 2009-10-27 2018-10-30 Ucb Biopharma Sprl Function modifying NAv1.7 antibodies
US9956274B2 (en) 2009-10-27 2018-05-01 Ucb Biopharma Sprl Method to generate antibodies to ion channels
JP2018064588A (ja) * 2009-10-27 2018-04-26 ユセベ ファルマ ソシエテ アノニム 機能改変するＮａｖ１．７抗体
JP2013507980A (ja) * 2009-10-27 2013-03-07 ユセベ ファルマ ソシエテ アノニム 機能改変するＮａｖ１．７抗体
US9738710B2 (en) 2009-10-27 2017-08-22 Ucb Biopharma Sprl Method of treating a patient for pain by administering an anti-ion channel antibody
JP2016020396A (ja) * 2009-10-27 2016-02-04 ユセベ ファルマ ソシエテ アノニム イオンチャネルに対する抗体
US9796766B2 (en) 2011-03-16 2017-10-24 Amgen Inc. Potent and selective inhibitors of NAV1.3 and NAV1.7
US9340590B2 (en) 2011-03-16 2016-05-17 Amgen Inc. Potent and selective inhibitors of NaV1.3 and NaV1.7
US9636418B2 (en) 2013-03-12 2017-05-02 Amgen Inc. Potent and selective inhibitors of NAV1.7
US10344060B2 (en) 2013-03-12 2019-07-09 Amgen Inc. Potent and selective inhibitors of Nav1.7
WO2014159595A2 (fr) 2013-03-14 2014-10-02 Regeneron Pharmaceuticals, Inc. Anticorps humains dirigés contre nav1.7
WO2014207400A1 (fr) * 2013-06-28 2014-12-31 Galderma Research & Development Anticorps dirigés contre la canal sodique nav1.9 humain et leurs utilisations pour le diagnostic des maladies cutanées inflammatoires
FR3007656A1 (fr) * 2013-06-28 2015-01-02 Galderma Res & Dev Modulateurs du canal sodique nav1.9 pour le traitement d'une maladie cutanee inflammatoire et methodes diagnostiques
WO2014207402A1 (fr) * 2013-06-28 2014-12-31 Galderma Research & Development Anticorps dirigés contre le canal sodique nav1.9 humain et leurs utilisations en diagnostic
US10067144B2 (en) 2013-06-28 2018-09-04 Galderma Research & Development Antibodies against human sodium channel nav 1.9
US9944699B2 (en) 2013-06-28 2018-04-17 Galderma Research & Development In vitro method for identifying compounds of the Nav 1.9 sodium channel intended for the treatment of an inflammatory skin disease
FR3007762A1 (fr) * 2013-06-28 2015-01-02 Galderma Res & Dev Anticorps diriges contre le canal sodique nav1.9 humain et leurs utilisations pour le diagnostic des maladies cutanees inflammatoires
WO2014207401A1 (fr) * 2013-06-28 2014-12-31 Galderma Research & Development Modulateurs du canal sodique nav1.9 pour le traitement d'une maladie cutanée inflammatoire et méthodes diagnostiques
FR3007763A1 (fr) * 2013-06-28 2015-01-02 Galderma Res & Dev Anticorps diriges contre le canal sodique nav1.9 humain et leurs utilisations en diagnostic
US11753479B2 (en) 2014-03-04 2023-09-12 Kymab Limited Nucleic acids encoding anti-OX40L antibodies
US11773175B2 (en) 2014-03-04 2023-10-03 Kymab Limited Antibodies, uses and methods
US12209128B2 (en) 2016-06-20 2025-01-28 Kymab Limited Anti-PD-L1 antibodies
US11779604B2 (en) 2016-11-03 2023-10-10 Kymab Limited Antibodies, combinations comprising antibodies, biomarkers, uses and methods
US11932673B2 (en) 2017-07-12 2024-03-19 Maxion Therapeutics Limited Sodium channel inhibitors

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GB0517487D0 (en) 2005-10-05

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