EP2222700A2 - Anticorps anti-amyloïde bêta humanisés - Google Patents

Anticorps anti-amyloïde bêta humanisés

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Publication number
EP2222700A2
EP2222700A2 EP08854674A EP08854674A EP2222700A2 EP 2222700 A2 EP2222700 A2 EP 2222700A2 EP 08854674 A EP08854674 A EP 08854674A EP 08854674 A EP08854674 A EP 08854674A EP 2222700 A2 EP2222700 A2 EP 2222700A2
Authority
EP
European Patent Office
Prior art keywords
antibody
seq
amino acid
acid sequence
subject
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP08854674A
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German (de)
English (en)
Inventor
Lisa Lynn Shafer
Francis Josesph Carr
James Peter Gregson
Dawn Ann Bembridge
Tim Jones
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Medtronic Inc
Original Assignee
Medtronic Inc
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Filing date
Publication date
Application filed by Medtronic Inc filed Critical Medtronic Inc
Priority to EP13152910.9A priority Critical patent/EP2586797A3/fr
Publication of EP2222700A2 publication Critical patent/EP2222700A2/fr
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • This disclosure relates to antibodies to amyloid beta, more particularly to humanized antibodies to amyloid beta and compounds, compositions, and methods related thereto.
  • Amyloid beta or "A ⁇ ” is peptide of between about 39-43 amino acids that corresponds to a peptide formed in vivo upon cleavage of an amyloid beta A4 precursor protein (APP or ABPP) by beta-secretase (at the N-terminal portion of A ⁇ ) and gamma secretase (at the C- terminal portion of A ⁇ ).
  • the most common isoforms of A ⁇ are A ⁇ 40 and A ⁇ 42, 40 and 42 amino acids, respectively.
  • a ⁇ 42 is less common, but is thought to be more fibrillogenic than A ⁇ 40.
  • a ⁇ is the main constituent of amyloid plaques in brains of Alzheimer's disease patients. Similar plaques can also be found in some Lewy body dementia patients. Such plaques or A ⁇ aggregates are also found in the cerebral vasculature of cerebral amyloid angiopathy patients.
  • humanized anti-A ⁇ antibodies derived from a murine antibody directed to a N-terminal epitope of A ⁇ .
  • the humanized antibodies have reduced or no human T cell epitopes and bind A ⁇ with an affinity similar to that of the murine antibody.
  • an anti-amyloid beta antibody presented herein includes a variable heavy chain region having an amino acid sequence of SEQ ID NOs: 42, 44, 46, or 47-51, or an amino acid sequence having 90% or greater, 95% or greater, 98% or greater, or 99% or greater sequence identity to SEQ ID NOs: 42, 44, 46, or 47-51.
  • the antibody includes a heavy chain variable region having an amino acid sequence having 90% or greater, 95% or greater, 98% or greater, or 99% or greater sequence identity to SEQ ID NOs: 42, 44, 46, or 47-51, one or more or all of the amino acid differences are conservative substitutions.
  • an anti-amyloid beta antibody presented herein includes a light chain variable region amino acid sequence of SEQ ID NOs: 43, 45, or 52-54, or an amino acid sequence having 90% or greater, 95% or greater, 98% or greater, or 99% or greater sequence identity to SEQ ID NOs: 43, 45, or 52-54.
  • the antibody includes a light chain variable region having an amino acid sequence having 90% or greater, 95% or greater, 98% or greater, or 99% or greater sequence identity to SEQ ID NOs: 43, 45, or 52-54, one or more or all of the amino acid differences are conservative substitutions.
  • a humanized antibody includes (i) a heavy chain variable region having an amino acid sequence of any of SEQ ID NOs: Attorney Docket No. P0030103.02
  • a method for treating a disease associated with increased or aberrant soluble A ⁇ , A ⁇ fibrils or A ⁇ plaques in a subject in need thereof includes administering an effective amount of a humanized antibody as described herein to the subject.
  • the subject may be suffering from or at risk of, for example, Alzheimer's disease, Lewy body dementia, Down's syndrome, or cerebral amyloid angiopathy.
  • the antibody may be administered directly to the subject's central nervous system, e.g., intrathecally, intraventricularly, or intraparenchymally.
  • the antibody is delivered to the subject via an infusion device implanted in the patient.
  • an isolated polynucleotide encodes an amino acid of any of SEQ ID NOs: 42-62, or an amino acid sequence having 90% or greater, 95% or greater, 98% or greater, or 99% or greater sequence identity to any of SEQ ID NOs: 42-62.
  • the antibody includes a heavy chain variable region having an amino acid sequence having 90% or greater, 95% or greater, 98% or greater, or 99% or greater sequence identity to SEQ ID NOs: 42-62, one or more or all of the amino acid differences are conservative substitutions.
  • An expression vector may include the isolated polynucleotide.
  • a cell may include the expression vector.
  • FIG. 1 is a schematic diagram of light and heavy chain expression vectors.
  • FIG. 2 provides heavy chain and light chain amino acid sequences of a murine anti-A ⁇ antibody, a deimmunized variant of the murine antibody, humanized grafted variant of the murine antibody, and composite human antibodies derived from the murine antibody.
  • FIG. 3-5 provide nucleotide sequences corresponding to the amino acid sequences of the composite human antibodies shown in FIG. 2. Attorney Docket No. P0030103.02
  • FIG. 6 is a graph showing the frequency of HLA class II allotypes in the world population and a population studied.
  • FIGs. 7a-b are graphs of results of MDT-2007 and chimeric antibodies tested in EPISCREENTM (Antitope Ltd.) time course T cell assays using PBMC from 20 donors. Bulk cultures of PBMC incubated with test antibodies were sampled on days 5, 6, 7 and 8, and pulsed with 3H-Thymidine. Cells were harvested and incorporation of radioactivity measured by scintillation counting. Results for each triplicate sample were averaged and normalized by conversion to Stimulation Index (SI). The SI for each time point with each donor is shown above for (a) the chimeric antibody and (b) MDT-2007. The cut-off for determining positive responses with an SI > 2 is highlighted by the horizontal line at 2.0 and significant responses (p ⁇ 0.05 in a student's t-test) are indicated (*).
  • SI Stimulation Index
  • FIG. 8 is a graph of a comparison of immunogencity predicted using EPISCREENTM (Antitope Ltd.) technology and immunogenicity observed in a clinical setting. Thirteen therapeutic proteins were tested for their relative risk of immunogenicity using EPISCREENTM (Antitope Ltd.) technology. Results were plotted against the frequency of immunogenicity (antitherapeutic antibody responses) observed for each protein when used in the clinic (data sourced from PubMed). The line of regression and the correlation coefficient is shown.
  • FIGs. 9A-B are images of MDT-2007 amyloid positive plaques in a section of the frontal cortex of an aged Rhesus monkey (A) and a section of a temporal cortex of an 84 year-old human female diagnosed with Alzheimer's Disease (B).
  • FIG. 10 is a schematic drawing of a side view of a representative infusion device showing selected internal components in block form.
  • FIG. 11 is a schematic drawing of a section of a brain and portions of a spinal cord showing cerebrospinal fluid flow.
  • FIG. 12 is a schematic drawing of a view of an infusion device and associated catheter implanted in a patient. Attorney Docket No. P0030103.02
  • FIG. 13 is a schematic drawing of a view of a section of a patient showing an implanted infusion device and associated catheter implanted.
  • FIG. 14 is a schematic drawing of a view showing an implanted infusion device and associated catheter in the environment of a patient.
  • FIG. 15 is a schematic drawing of a view showing an injection port in the environment of a patient.
  • FIG. 16 is a schematic drawing of a side view of a representative system including an infusion device and a sensor, showing some internal components in block form.
  • antibody is used in the broadest sense and specifically includes, for example, single anti-A ⁇ monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), anti-A ⁇ antibody compositions with polyepitopic specificity, single chain anti- anti-A ⁇ antibodies, and fragments of anti-A ⁇ antibodies (see below).
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies forming the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
  • An antibody may include an immunoglobulin constant domain from any immunoglobulin, such as IgG-I, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-I and IgA-2), IgE, IgD or IgM.
  • immunoglobulin such as IgG-I, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-I and IgA-2), IgE, IgD or IgM.
  • an "antibody fragment” means a portion of an intact antibody, most typically the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diaries; linear antibodies (Zapata et al., Protein Eng. 8 (10): 1057-1062 [1995]); and single-chain antibody molecules.
  • Fv is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy-and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain.
  • Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region.
  • F(ab') 2 antibody fragments originally were Attorney Docket No. P0030103.02
  • the "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.
  • Single-chain Fv or “sFv” antibody fragments include the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains, which enables the sFv to form the desired structure for antigen binding.
  • antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred.
  • peptide molecules can be designed that retain the ability to bind the target protein sequence.
  • Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).
  • hybridoma describes the combination of a B cell that can recognize a particular antigen and a myeloma cell that lives indefinitely to make the hybridoma cell a kind of perpetual antibody-producing factory.
  • the antibodies are therefore generated from animal cells, typically the mouse.
  • treat means alleviating, slowing the progression, preventing, attenuating, or curing the treated disease.
  • subject means a mammal to which an agent, such as an antibody, is administered for the purposes of treatment or investigation.
  • Mammals include mice, rats, cats, guinea pigs, hampsters, dogs, monkeys, chimpanzees, and humans.
  • isolated in the context of an antibody or nucleic acid molecule encoding an antibody, means the antibody or nucleic acid has been removed from its natural milieu or has been altered from its natural state. As such "isolated” does not necessarily reflect the extent to which the antibody or nucleic acid molecule has been purified. However, it will be understood that an antibody or nucleic acid molecule that has been purified to some degree is “isolated”. If the antibody or nucleic acid molecule does not exist in a natural milieu, i.e. it does not exist in nature, the molecule is "isolated” regardless of where it is present. By way of example, a humanized antibody that does not naturally exist in humans is “isolated” even when it is present in humans.
  • a “conservative amino acid substitution” refers to substituting an amino acid of a polypeptide for another amino acid that is functionally similar. Such conservative amino acids may be substituted for each other in a polypeptide with a minimal disturbance to the structure or function of the polypeptide according to well-known techniques.
  • a humanized antibody includes a heavy chain variable region having an amino acid sequence of SEQ ID NOs: 42, 44, 46, or 47-51 (see FIG. T), or an amino acid sequence having 90% or greater, 95% or greater, 98% or greater, or 99% or greater sequence identity to SEQ ID NOs: 42, 44, 46, or 47-51.
  • the antibody includes a heavy chain variable region having an amino acid sequence having 90% or greater, 95% or greater, 98% or greater, or 99% or greater sequence identity to SEQ ID NOs: 42, 44, 46, or 47-51, one or more or all of the amino acid differences are conservative substitutions.
  • the humanized antibody may further includes a light chain variable region amino acid sequence of SEQ ID NOs: 43, 45, or 52-54 (see FIG. 2), or an amino acid sequence having 90% or greater, 95% or greater, 98% or greater, or 99% or greater sequence identity to SEQ ID NOs: 43, 45, or 52-54.
  • a light chain variable region amino acid sequence having 90% or greater, 95% or greater, 98% or greater, or 99% or greater sequence identity to SEQ ID NOs: 43, 45, or 52-54 one or more or all of the amino acid differences are conservative substitutions. Any possible combination of such heavy chain and light chain amino acid sequences are contemplated.
  • a humanized antibody includes (i) a heavy chain variable region having an amino acid sequence of any of SEQ ID NOs: 47-51 and (ii) a light chain variable region having an amino acid sequence of any of SEQ ID NOs: 52-54.
  • a humanized antibody includes (i) a variable heavy chain region comprising an amino acid sequence of SEQ ID NO:47 and (ii) a variable light chain region comprising an amino acid sequence of SEQ ID NO:52.
  • a humanized antibody includes (i) a variable heavy chain region comprising an amino acid sequence of SEQ ID NO:48 and (ii) a variable light chain region comprising an amino acid sequence of SEQ ID NO:54.
  • a humanized antibody includes (i) a variable heavy chain region comprising an amino acid sequence of SEQ ID NO:50 and (ii) a variable light chain region comprising an amino acid sequence of SEQ ID NO:52.
  • a variable heavy chain region comprising an amino acid sequence of SEQ ID NO:50
  • a variable light chain region comprising an amino acid sequence of SEQ ID NO:52.
  • a humanized antibody includes (i) a variable heavy chain region comprising an amino acid sequence of SEQ ID NO:51 and (ii) a variable light chain region comprising an amino acid sequence of SEQ ID NO:52.
  • a humanized antibody includes (i) a variable heavy chain region comprising an amino acid sequence of SEQ ID NO:50 and (ii) a variable light chain region comprising an amino acid sequence of SEQ ID NO:53.
  • a humanized antibody includes (i) a variable heavy chain region comprising an amino acid sequence of SEQ ID NO:51 and (ii) a variable light chain region comprising an amino acid sequence of SEQ ID NO:53.
  • a polynucleotide includes a sequence that encodes a humanized antibody as described herein.
  • the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 55, 56, 57, 58, 59, 60, 61, and 62.
  • an expression vector includes a polynucleotide as described above.
  • a cell contains such an expression vector.
  • the expression of the expression vector results in production of an antibody as described above.
  • Humanized antibodies as described herein, may be prepared according to any known or developed method. Typically, the humanized antibodies will be expressed from expression vectors transfected or transformed into cells; e.g., as described the Examples provided below.
  • Host cells may be transfected or transformed with cloning vectors or expression vectors for humanized antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting trans formants, or amplifying the genes encoding the desired sequences.
  • the culture conditions such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of Attorney Docket No. P0030103.02
  • Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to those of ordinarily skill in the art and include, for example, CaCl 2 , CaPO 4 , liposome- mediated and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. For example, calcium treatment employing calcium chloride, as described Molecular Cloning, A Laboratory Manual, 3 rd edition, vols 1-3, eds. Sambrook and Russel (2001) Cold Spring Harbor Laboratory Press, or electroporation may be used for prokaryotes.
  • DNA into cells such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used.
  • polycations e.g., polybrene, polyornithine.
  • Suitable host cells for cloning the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells.
  • Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli.
  • Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635).
  • Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E.
  • Suitable host cells for the expression of glycosylated humanized antibodies include those derived from multicellular organisms.
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells.
  • useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol, 36:59 (1977)); Chinese hamster ovary cells/-DHFP (CHO, Urlaub and Chasin, Proc. Natl. Acad.
  • mice Sertoli cells TM4, Mather, Biol. Reprod., 23:243-251 (1980)
  • human lung cells W138, ATCC CCL 75
  • human liver cells Hep G2, HB 8065
  • mouse mammary tumor MMT 060562, ATCC CCL51. The selection of the appropriate host cell is within the routine ability skill in the art.
  • the nucleic acid (e.g., cDNA or genomic DNA) encoding a humanized antibody (or portion thereof) may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression.
  • a replicable vector for cloning (amplification of the DNA) or for expression.
  • the vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage.
  • the appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art.
  • Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to those of skill in the art.
  • the humanized antibody (or portion thereof) may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • a heterologous polypeptide which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the signal sequence may be a component of the vector, or it may be a part of the humanized antibody (or portion thereof)-encoding DNA that is inserted into the vector.
  • Expression vectors or cloning vectors may incude a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such 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 ⁇ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
  • Expression and cloning vectors may contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • Expression and cloning vectors usually contain a promoter operably linked to the humanized antibody (or portion thereofj-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems [Chang et al, Nature, 275:615 (1978); Goeddel et al, Nature, 281 :544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgamo (S. D.) sequence operably linked to
  • Humanized antibody (or portion thereof) transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published JuI. 5, 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published JuI. 5, 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytome
  • Enhancers are cis-acting Attorney Docket No. P0030103.02
  • enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5' or 3' to the humanized antibody (or portion thereof) coding sequence, but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding a humanized antibody (or portion thereof).
  • a viral vector such as an adeno-associated viral (AAV) vector may be operatively linked components of control elements.
  • AAV adeno-associated viral
  • a typical vector includes a transcriptional initiation region, a nucleotide sequence of the protein to be expressed, and a transcriptional termination region.
  • a transcriptional initiation region Typically, such an operatively linked construct will be flanked at its 5 and 3 regions with AAV ITR sequences, which are viral cis elements.
  • the control sequences can often be provided from promoters derived from viruses such as, polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40.
  • Viral regulatory sequences can be chosen to achieve a high level of expression in a variety of cells.
  • the vector contains the proximal human brain natriuretic brain (hBNP) promoter that functions as a cardiac-specific promoter.
  • hBNP proximal human brain natriuretic brain
  • Expression vectors containing DNA encoding a humanized antibody (or portion thereof) may be administered in vivo in any known or future developed manner.
  • the expression vectors are packaged into viruses, such as adenoviruses, and are delivered in proximity to targeted cells, tissue or organs.
  • the expression vectors are packaged into adenoviruses, such as helper-dependent adeno virus (HDAd) or adeno-assocated virus pseudo-type 9 (AAV2/9).
  • HDAd virus packaging typically illicits less of an immunogenic response in vivo compared to some other adenoviruses and thus allows for longer term expression.
  • AAV2/9 packaging can result in cardiac tropism as well as a prolonged expression time frame.
  • non-viral delivery systems are employed.
  • liposomes, DNA complexes, plasmis, liposome complexes, naked DNA, DNA-coated particles, or polymer based systems may be used to deliver the desired sequence to the cells.
  • the above- mentioned delivery systems and protocols therefore can be found in Gene Targeting Protocols, Kmeic 2ed., pages 1-35 (2002) and Gene Transfer and Expression Protocols, Vol. 7, Murray ed., Pages 81-89 (1991).
  • An expression vector including a polynucleotide encoding a humanized antibody (or portion thereof) can be delivered into a cell by, for example, transfection or transduction procedures.
  • Transfection and transduction refer to the acquisition by a cell of new genetic material by incorporation of added nucleic acid molecules. Transfection can occur by physical or chemical methods. Many transfection techniques are known to those of ordinary skill in the art including, without limitation, calcium phosphate DNA co- precipitation, DEAE-dextrin DNA transfection, electroporation, naked plasmid adsorption, and cationic liposome-mediated transfection.
  • Transduction refers to the process of transferring nucleic acid into a cell using a DNA or RNA virus.
  • Suitable viral vectors for use as transducing agents include, but are not limited to, retroviral vectors, adeno associated viral vectors, vaccinia viruses, an Semliki Foret virus vectors. Attorney Docket No. P0030103.02
  • Humanized anti-A ⁇ antibodies can be administered for the treatment of various disorders in the form of pharmaceutical compositions.
  • Pharmaceutical compositions may be used for the purposes of treatment or for investigation.
  • Antibodies as described herein can be administered as pharmaceutical compositions the antibody and a variety of other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa. (1980)). The preferred form depends on the intended mode of administration and therapeutic application.
  • the compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • the diluent is selected so as not to adversely affect the biological activity of the antibody. Examples of such diluents are distilled water, physiological phosphate- buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SEPHAROSETM (GE Healthcare Bio-Sciences Ltd.), agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SEPHAROSETM (GE Healthcare Bio-Sciences Ltd.), agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • compositions may be injectable compositions.
  • injectable compositions include solutions, suspensions, dispersions, and the like.
  • injectable solutions, suspensions, dispersions, and the like may be formulated according to techniques well-known in the art (see, for example, Remington's Pharmaceutical Sciences, Chapter 43, 14th Ed., Mack Publishing Co., Easton, Pa.), using suitable dispersing or wetting and suspending agents, Attorney Docket No. P0030103.02
  • sterile oils including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • compositions that include an anti-A ⁇ antibody may be prepared in water, saline, isotonic saline, phosphate-buffered saline, citrate-buffered saline, and the like and may optionally mixed with a nontoxic surfactant.
  • Dispersions may also be prepared in glycerol, liquid polyethylene, glycols, DNA, vegetable oils, triacetin, and the like and mixtures thereof. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • compositions suitable for injection or infusion include sterile, aqueous solutions or dispersions or sterile powders comprising an active ingredient which powders are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions.
  • the ultimate dosage form is a sterile fluid and stable under the conditions of manufacture and storage.
  • a liquid carrier or vehicle of the solution, suspension or dispersion may be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol such as glycerol, propylene glycol, or liquid polyethylene glycols and the like, vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • Proper fluidity of solutions, suspensions or dispersions may be maintained, for example, by the formation of liposomes, by the maintenance of the desired particle size, in the case of dispersion, or by the use of nontoxic surfactants.
  • the prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • Isotonic agents such as sugars, buffers, or sodium chloride may be included.
  • Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of agents delaying absorption— for example, aluminum monosterate hydrogels and gelatin. Solubility enhancers may be added.
  • Sterile injectable compositions may be prepared by incorporating an anti-A ⁇ antibody in the desired amount in the appropriate solvent with various other ingredients, e.g. as enumerated above, and followed by sterilization, as desired, by, for example filter sterilization.
  • methods of preparation include vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in a previously sterile- Attorney Docket No. P0030103.02
  • Any suitable sterilization process may be employes, such as filter sterilization, e.g. 0.22 micron filter or nanofiltration, gamma or electron beam sterilization, or pused white light.
  • Other suitable sterilization processes include UtiSter (Pegasus Biologies, Irvinie CA) and those described in, e.g., U.S. Patent No. 6,946,098 and U.S. Patent No. 5,730,933.
  • the final solution is adjusted to have a pH between about 4 and about 9, between about 5 and about 7, between about 5.5 and about 6.5, or about 6.
  • the pH of the composition may be adjusted with a pharmacologically acceptable acid, base or buffer.
  • Hydrochloric acid is an example of a suitable acid
  • sodium hydroxide is an example of a suitable base.
  • the hydrochloric acid or sodium hydroxide may be in any suitable form, such as a IN solution
  • a resultant injectable solution preferably contains an amount of one or more anti- A ⁇ antibodies effective to treat a disease associated with increase or aberrant soluble A ⁇ or to allow meaningful study of a subject to which the solution is injected.
  • an anti-A ⁇ antibody is present in an injectable composition at a concentration between about 0.0001 mg/ml and about 50 mg/ml.
  • an anti-A ⁇ antibody is present in an injectable composition at a concentration between about .01 mg/mL and about 10 mg/mL.
  • an anti-A ⁇ antibody is present in an injectable composition at a concentration of about 1 mg/mL.
  • anti-A ⁇ antibodies may be administered to the CNS in the form of a depot injection or implant preparation, which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • formulations discussed above or other formulations containing an anti-A ⁇ antibody may be administered to a subject via any acceptable route.
  • a formulation comprising an antibody agent capable of binding A ⁇ is chronically administered directly to the CNS of a subject.
  • chronically it is meant over the course or days, weeks or months. In some circumstances, chronic administration may occur at various times or continuously over the life of the patient after therapy is initiated, or until Attorney Docket No. P0030103.02
  • direct administration to the CNS means delivery of an agent via a delivery region infusion portion of a catheter, where the delivery portion of the catheter is located within the CNS.
  • Example routes of direct CNS administration include intraparenchymal, intrathecal, intracerebroventricular, epidural, or the like.
  • catheters include cannulas, needles, tubes, and the like.
  • a delivery portion of a catheter is typically located at or near a distal end portion of a catheter and may include an opening in fluid communication with a lumen of a catheter.
  • formulations containing an anti-A ⁇ antibody are administered to the CNS via an implantable infusion device, for example, as described below with regard to FIGs. 10-16 and associated text under the heading "delivery device".
  • delivery device Suitable systems and conditions for delivering anti-A ⁇ antibodies via an implantable infusion device are described in, e.g., U.S. Patent Application No. 12/120,269, filed May 14, 2008, which application is hereby incorporated herein by reference in its entirety to the extent that it does not conflict with the present disclosure.
  • An injectable composition including an anti-A ⁇ antibody may be delivered at any suitable rate.
  • the composition may be delivered at a rate of 0.01-15 ml/day, 0.01-5 ml/day, or 0.01-1 ml/day.
  • concentration of the anti-A ⁇ antibody in the composition may be adjusted, based on the delivery rate, to achieve a desired daily dose.
  • the rate of delivery may be constant or may be variable. In various embodiments, delivery includes periods of increased delivery rate (e.g., pulsed boluses) on top of a constant lower rate.
  • Such pulsed boluses may readily be achieved with a programmable infusion device such as the SYNCHROMED II® (Medtronic, Inc.) infusion device.
  • a programmable infusion device such as the SYNCHROMED II® (Medtronic, Inc.) infusion device.
  • Such pulsed bolus administration may result in improved distribution of the delivered antibody relative to constant rate delivery due to increased convection.
  • the antibody may be delivered for a period of time, the delivery may then be halted, and then resumed. For example, the antibody may be delivered for one hour to one week, delivery may then be halted for one hour to one week, and so on.
  • Such a dosing scheme may serve to prolong the delivery life of the antibody, as the half life of the delivered antibody may be considerably shorter than the half life of an antibody housed in a reservoir of an infusion device.
  • the mode of delivery may be altered during the course of treatment.
  • the antibody may be delivered i.c.v. at one stage of treatment and delivered intraparenchymally (Lp. a) at another stage of treatment, as conditions warrant.
  • Lp. a intraparenchymally
  • an antibody may be delivered i.c.v. and i.p.a at the same time.
  • a delivery region of a catheter intraparenchymally may be desirable to implant a delivery region of the same (e.g., split catheter) or different catheter in the CSF at the time of initial implant.
  • an antibody may be conjugated with poly-sialic acid, dextran, polyethylene glycol, or the like.
  • Polysialylation may result in reduced proteolysis, retention of binding and biological activity, prolongation of half-life in the circulation, or reduction in immunogenicity of the antibody.
  • Studies have been performed that show that mouse 6E10 anti-amyloid beta IgG and F(ab') 2 antbodies are capable of being sialylated, that sialylation does not adversely affect the ability of the antibody to bind amyloid beta, and that bioavailability (plasma half-life) is markedly enhanced by between about three- and forty- fold (data not shown).
  • humanized antibodies described herein are to be conjugated with polysialic acid, significantly less antibody may need to be delivered to achieve a desired effect, which could result in reduced toxicity. Such effects may be most desirable for systemic administration, where dosages tend to be greater than with direct central administration. However, conjugation with polysialic acid or other polymers may be desirable and beneficial with intracerebroventricular or intraparenchymal administration, whether delivered via an implantable infusion system or via bolus infusion.
  • Administering an antibody capable of binding A ⁇ in the CNS of a subject as described herein or according to any other known or developed technique may be used to treat or prevent a disease associated with increased or aberrant soluble A ⁇ , amyloid fibrils or amyloid plaques. Examples of disease associated with increased or aberrant soluble A ⁇ , Attorney Docket No. P0030103.02
  • amyloid fibrils or amyloid plaques include Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Lewy body dementia, and Down's Syndrome (DS).
  • AD Alzheimer's disease
  • CAA cerebral amyloid angiopathy
  • DS Down's Syndrome
  • any amount of antibody effective to bind A ⁇ , whether soluble, in fibrils, plaques, or the like may be employed.
  • a daily dose of between about 0.0001 and about 1 mg of the antibody per kg of the subject's body weight will be effective.
  • daily doses of between about 0.001 and about 1, between about 0.01 and about 0.1, or between about 0.1 and about 1 mg of the antibody per kg of the subject's body weight are administered to the subject's CNS.
  • the above daily doses should be generally effective for i.c.v. delivery to a lateral ventrical of the subject.
  • increased dosages may be warranted.
  • daily dosages may be increased by about 20% relative to i.c.v.
  • any other suitable modification in dosing based on route of administration in comparison to i.c.v. may be employed.
  • the antibody need not be administered daily, but may be administered only once, once a week, once a month, every other day, or according to any other suitable regimen.
  • a method includes identifying a subject suffering from or at risk of AD and chronically delivering to the CNS of the subject an antibody directed to A ⁇ .
  • Those at risk of AD include those of advancing age, family history of the disease, mutations in APP or related genes, having heart disease risk factors, having stress or high levels of anxiety. Identification of those suffering from or at risk of AD can be readily accomplished by a physician. Diagnosis may be based on mental, psychiatric and neuropyschological assessments, blood tests, brain imaging (PET, MRI, CT scan), urine tests, tests on the cerebrospinal fluid obtained through lumbar puncture, or the like.
  • a method includes identifying a patient suffering from or at risk of CAA and chronically delivering to the CNS of the patient a humanized antibody directed to A ⁇ .
  • Symptoms of CAA include weakness or paralysis of the limbs, difficulty speaking, loss of sensation or balance, or even coma. If blood leaks out to the sensitive tissue around the brain, it can cause a sudden and severe headache. Other symptoms sometimes caused by irritation of the surrounding brain are seizures (convulsions) or short spells of temporary neurologic symptoms such as tingling or weakness in the limbs or face.
  • CAA patients can be identified by, e.g., examination of an evacuated hematoma or brain biopsy specimen, the Attorney Docket No. P0030103.02
  • APOE ⁇ 2 or ⁇ 4 alleles with clinical or radiographic (MRI and CT scans) grounds according the Boston Criteria (Knudsen et al, 2001, Neurology;56:537-539), or the like.
  • Those at risk of CAA include those of advancing age, those having the APOE genotype, and those having other risk factors associated with AD.
  • a method includes identifying a patient suffering from or at risk of Down Syndrome and chronically delivering to the CNS of the patient a humanized antibody directed to A ⁇ .
  • a newborn with Down syndrome can be identified at birth by a physician's physical exam. The diagnosis may be confirmed through kariotyping. Multiple screening tests may be used to test or diagnosis a patient prior to birth (biomarkers, nuchal translucency, amniocentesis, etc.).
  • a Down's syndrome patient may be diagnosis with AD using diagnostic criteria relevant for AD.
  • a method includes identifying a patient suffering from or at risk of Lewy body dementia and chronically delivering to the CNS of the patient a humanized antibody directed to A ⁇ .
  • Those suffering from or at risk of Lewy body dementia can be identified by mental, psychiatric or neuropyschological assessments, blood tests, brain imaging (PET, MRI, CT scan), urine tests, tests on the cerebrospinal fluid obtained through lumbar puncture, or the like.
  • Those at risk of Lewy body dementia include those of advancing age.
  • cerebral plaques may be cleared or prevented from forming by administering anti-A ⁇ antibodies to a subject's CNS. It will be understood that achieving any level of clearing of a plaque or plaques will constitute clearing of the plaque or plaques. It will be further understood that achieving any level of prevention of formation of a plaque or plaques will constitute preventing formation of the plaque or plaques. Accordingly, in various embodiments, methods for clearing plaques include delivering, to a subject in need thereof, an amount of a humanized anti-A ⁇ antibody effective in clearing the plaques. In various embodiments, methods for preventing the formation of plaques include delivering, to a subject in need thereof, an amount of a humanized anti-A ⁇ antibody effective in preventing the formation the plaques. The methods may further include clearing or preventing parenchymal amyloid plaques or soluble forms of A ⁇ . The methods may further include improving cognitive aspects of the subject. Attorney Docket No. P0030103.02
  • cognitive abilities of a subject are improved by administering anti- A ⁇ antibodies to a subject's CNS.
  • parenchymal amyloid plaques or soluble forms of A ⁇ are cleared in a subject by administering anti-A ⁇ antibodies to a subject's CNS.
  • a therapy described herein to treat a disease may be evaluated through medical examination, e.g. as discussed above, or by diagnostic or other tests.
  • a method as described in WO 2006/107814 is performed.
  • a subject may be administered radiolabeled leucine.
  • Samples, such as plasma or CSF, may then be obtained to quantify the labeled-to-unlabeled leucine in, for example, amyloid beta or other key disease related biomarkers, to determine the production and clearance rate of such proteins or polypeptides.
  • Clearing of, or formation of, amyloid beta can be evaluated in vivo by structural or functional neuro-imaging techniques. For example, diffusion tensor MRI (reviewed in Parente et al., 2008; Chua et al., 2008), PET imaging with the A ⁇ binding compound, Pittsburgh Compound B (PiB, Klunk et al., 2004; Fagan et al., 2006; Fagan et al 2007) or other SPECT based imaging of fibrillar A ⁇ structures and measurement of CSF levels of A ⁇ 42 or tau may be employed. Distribution of vascular A ⁇ may be evaluated using differential interpretation of PET imaging of PiB (Johnson et al., 2007). Additionally, a cerebral microhemorrahage may be recognized by on gradient-echo or T-2 weighted MRI sequences (Viswanathan and Chabriat, 2006).
  • ICH intracerebral hemorrhage
  • CT computed tomography
  • T-2 weighted MRI computed tomography
  • Cerebral microhemorrhage results from underlying small vessel pathologies such as hypertensive vasculpathy or CAA. Cerebral microhemorrhages, best visualized by MRI, result from rupture of small blood vessels.
  • the MRI diagnosis can be variable as described by Orgagozo et al., 2003 (Subacute meningoencephalitis in a subset of patients with AD after A ⁇ 42 immunization-Elan Trial). For instance, patients showing signs and symptoms of aseptic meningoencephalitis MRIs showed only meningeal enhancement, whereas others Attorney Docket No. P0030103.02
  • a humanized antibody is used to detect A ⁇ ; e.g., in a diagnostic assay or as part of a diagnostic kit.
  • the humanized antibody may be used to determine the ability of a therapeutic agent or putative therapeutic agent to affect processing of APP to produce soluble A ⁇ .
  • it may be desirable for the antibody to be labeled; e.g., with a fluorescent tag, a paramagnetic tag, or the like.
  • formulations containing an anti-A ⁇ antibody are administered to the CNS via an infusion device.
  • a system including an infusion device may be used to deliver a composition containing an anti-A ⁇ antibody to the CNS of a subject.
  • the system may further includes a catheter operably couplable to the infusion device.
  • the infusion device may include a drive mechanism.
  • drive mechanisms include peristaltic pumps, osmotic pumps, piston pumps, pressurized gas mechanisms, and the like. Devices including such drive mechanisms may be fixed-rate pumps, variable rate pumps, selectable rate pumps, programmable pumps and the like.
  • Each of the aforementioned infusion systems contain a reservoir for housing a fluid composition containing the anti-A ⁇ antibody.
  • the catheter includes one or more delivery regions, through which the fluid may be delivered to one or more target regions of the subject.
  • the infusion device may be implantable or may be placed external to the subject.
  • FIG. 10 An infusion device 30 according to various embodiments is shown in FIG. 10 and includes a reservoir 12 for housing a composition and a drive mechanism 40 operably coupled to the reservoir 12.
  • the catheter 38 shown in FIG. 11 has a proximal end 35 coupled to the therapy delivery device 30 and a distal end 39 configured to be implanted in a target location of a subject. Between the proximal end 35 and distal end 39 or at the distal end 39, the catheter 38 has one or more delivery regions (not shown), such as openings, through Attorney Docket No. P0030103.02
  • the infusion device 30 may have a port 34 into which a hypodermic needle can be inserted to inject a composition into reservoir 12.
  • the infusion device 30 may have a catheter port 37, to which the proximal end 35 of catheter 38 may be coupled.
  • the catheter port 37 may be operably coupled to reservoir 12.
  • a connector 14, such as a barbed connector or sutureless connector, may be used to couple the catheter 38 to the catheter port 37 of the infusion device 30.
  • the infusion device 30 may be operated to discharge a predetermined dosage of the pumped fluid into a target region of a subject.
  • the infusion device 30 may contain a microprocessor 42 or similar device that can be programmed to control the amount of fluid delivery. The programming may be accomplished with an external programmer/control unit via telemetry.
  • a controlled amount of fluid may be delivered over a specified time period.
  • dosage regimens may be programmed and tailored for a particular patient. Additionally, different therapeutic dosages can be programmed for different combinations of fluid comprising therapeutics.
  • a programmable infusion device 30 allows for starting conservatively with lower doses and adjusting to a more aggressive dosing scheme, if warranted, based on safety and efficacy factors.
  • device 30 may include a catheter access port to allow for direct delivery of a composition including an anti-A ⁇ antibody via catheter 38.
  • other components such as one-way valves, that may be included at one or more locations along the fluid flow path of the device 30. It will be understood that the components and the configuration of the components depicted in FIG. 10 may be readily modified to achieve a suitable infusion device 30 for delivering an injectable composition including a humanized anti-A ⁇ antibody.
  • a composition comprising a humanized anti-A ⁇ antibody may be delivered directly to cerebrospinal fluid of a subject.
  • cerebrospinal fluid (CSF) 6 exits the foramen of Magendie and Luschka to flow around the brainstem and cerebellum.
  • the arrows within the subarachnoid space 3 in FIG. 11 indicate cerebrospinal fluid 6 flow.
  • the subarachnoid space 3 is a compartment within the central nervous system that contains cerebrospinal fluid 6.
  • the cerebrospinal fluid 6 is produced in the ventricular system of the brain and communicates freely with the subarachnoid space 3 Attorney Docket No. P0030103.02
  • a composition containing an anti-A ⁇ antibody may be delivered to cerebrospinal fluid 6 of a subject anywhere that the cerebrospinal fluid 6 is accessible.
  • the composition may be administered intrathecally or intracerebroventricularly.
  • FIG. 12 illustrates a representative implantable system configured for intrathecal delivery of a composition containing an anti-A ⁇ antibody.
  • a system or device 30 may be implanted below the skin of a patient.
  • the device 30 is implanted in a location where the implantation interferes as little as practicable with activity of the subject in which it is implanted.
  • One suitable location for implanting the device 30 is subcutaneously in the lower abdomen.
  • catheter 38 is positioned so that the distal end 39 of catheter 38 is located in the subarachnoid space 3 of the spinal cord such that a delivery region (not shown) of catheter is also located within the subarachnoid space 3.
  • the delivery region can be placed in a multitude of locations to direct delivery of an agent to a multitude of locations within the cerebrospinal fluid 6 of the patient.
  • the location of the distal end 39 and delivery region(s) of the catheter 38 may be adjusted to improve therapeutic efficacy.
  • a composition containing a humanized anti-A ⁇ antibody may be delivered intraparenchymally directly to brain tissue of a subject.
  • An infusion device may be used to deliver the agent to the brain tissue.
  • a catheter may be operably coupled to the infusion device and a delivery region of the catheter may be placed in or near a target region of the brain.
  • a system or infusion device 10 may be implanted below the skin of a subject.
  • the device 10 may have a port 14 into which a hypodermic needle can be inserted through the skin to inject a quantity of a composition comprising a therapeutic agent.
  • the composition is delivered from device 10 through a catheter port 20 into a catheter 22.
  • Catheter 22 is positioned to deliver the agent to specific infusion sites in a brain (B).
  • Device 10 may take the form of the like-numbered device shown in U.S. Pat. No. 4,692,147 (Duggan), assigned to Medtronic, Inc., Minneapolis, Minn, or take the form of a SYNCHROMED II® (Medtronic, Inc.) infusion device.
  • distal end of catheter 22 terminates in a cylindrical hollow tube 22A having a distal end 115 implanted into a target portion of the brain by conventional stereotactic surgical techniques. Additional details about end 115, according to various embodiments, may be obtained from U.S. application Ser. No. 08/430,960 entitled “Intraparenchymal Infusion Catheter System,” filed Apr. 28, 1995 in the name of Dennis Elsberry et al. and assigned to the same assignee as the present application. Tube 22A is surgically implanted through a hole in the skull 123 and catheter 22 is implanted between the skull and the scalp 125 as shown in FIG. 13. Catheter 22 may be coupled to implanted device 10 in the manner shown or in any other suitable manner.
  • a therapy delivery device 10 is implanted in a human body 120 in the location shown or may be implanted in any other suitable location.
  • Body 120 includes arms 122 and 123.
  • catheter 22 is divided into twin or similar tubes 22A and 22B that are implanted into the brain bilaterally.
  • tube 22B is supplied with a composition from a separate catheter and pump.
  • unilateral delivery may be performed in accordance with the teachings presented herein.
  • a humanized anti-A ⁇ antibody may be delivered to a subject's CNS via an injection port 10 implanted subcutaneous Iy in the scalp of a patient 1, e.g. as described in US Patent No. 5,954,687 or otherwise known in the art.
  • a guide catheter 10 may be used to guide an infusion catheter through port 10 to a target location.
  • an infusion catheter may be directly be inserted through port 10 to the target location.
  • a representative system including an infusion device 30 and a sensor 500 is shown.
  • the sensor 500 may detect an attribute of the nervous system, which attribute may reflect a pathology associated with a disease to be treated or studied or the amount of antibody already in the targeted region.
  • a microprocessor 42 may analyze output from the sensor 50 and regulate the amount of antibody delivered to the brain.
  • Sensor 500 may be operably coupled to processor 42 in any manner.
  • sensor 500 may be connected to processor via a direct electrical connection, such as through a wire or cable, or Attorney Docket No. P0030103.02
  • Sensed information may be recoded by device 30 and stored in memory (not shown).
  • the stored sensed memory may be relayed to an external programmer, where a physician may modify one or more parameter associated with the therapy based on the relayed information.
  • processor 42 may adjust one or more parameters associated with therapy delivery. For example, processor 42 may adjust the amount or timing of the infusion of an anti-A ⁇ antibody. It will be understood that two or more sensors 500 may be employed.
  • Sensor 500 may detect a polypeptide associated with a CNS disorder to be treated or investigated; a physiological effect, such as a change in membrane potential; a clinical response, such as blood pressure; or the like.
  • a component of an infused composition such as the anti-A ⁇ antibody or other component, which may be added specifically for the purpose of detection by the sensor 500, is detected.
  • Any suitable sensor 500 may be used.
  • a biosensor may be used to detect the presence of a polypeptide or other molecule in a patient. Any known or future developed biosensor may be used.
  • the biosensor may have, e.g., an enzyme, an antibody, a receptor, or the like operably coupled to, e.g., a suitable physical transducer capable of converting the biological signal into an electrical signal.
  • a suitable physical transducer capable of converting the biological signal into an electrical signal.
  • receptors or enzymes that reversibly bind the molecule being detected may be preferred.
  • sensor 500 is a sensor as described in, e.g., U.S. Pat. No. 5,978,702, entitled TECHNIQUES OF TREATING EPILEPSY BY BRAIN STIMULATION AND DRUG INFUSION, or U.S. patent application Ser. No. 10/826,925, entitled COLLECTING SLEEP QUALITY INFORMATION VIA A MEDICAL DEVICE, filed Apr. 15, 2004, or U.S. patent application Ser. No. 10/820,677, entitled DEVICE AND METHOD FOR ATTENUATING AN IMMUNE RESPONSE, filed Apr. 8, 2004.
  • Examples of sensor technology that may be adapted for use in some embodiments include those disclosed in: (i) U.S. Patent No. 5,640,764 for "Method of forming a tubular feed- through hermetic seal for an implantable medical device;" (ii) U.S. Patent No. 5,660,163 for "Glucose sensor assembly;” (iii) U.S. Patent No. 5,750,926 for "Hermetically sealed electrical feedthrough for use with implantable electronic devices;” (iv) U.S. Patent No. 5,791,344 for "Patient monitoring system;” (v) U.S. Patent No. 5,917,346 for "Low power Attorney Docket No. P0030103.02
  • mRNA was extracted from the hybridoma 6E10 cells (Covance, Inc., Emeryville, CA) using a Poly A Tract System 1000 mRNA extraction kit (Promega Corp. Madison WI) according to manufacturer's instructions. mRNA was reverse transcribed as follows. For the kappa light chain, 5.0 microliter of mRNA was mixed with 1.0 microliter of 20 pmol/ microliter MuIgG ⁇ V L -3' primer OL040 (Table 2) and 5.5 microliter nuclease free water (Promega Corp. Madison WI).
  • Amplification products were cloned into pGEM-T easy vector using the pGEM-T easy Vector System I (Promega Corp. Madison WI) kit and sequenced.
  • the resultant mouse VH (SEQ. ID. NO. 36) and VL (SEQ. ID. NO. 37) sequences are shown in FIG. 2 ("Mouse").
  • VH region genes were amplified by PCR using the primers OL330, GATCACGCGTGTCCACTCCGAAGTGCAGCTGGTGGAGTC (SEQ ID NO. 63), and OL331, GTACAAGCTTACCTGAGGAGACGGTGACTGAGG (SEQ ID NO. 64); these were designed to engineer in a 5' MIuI and a 3' HindIII restriction Attorney Docket No. P0030103.02
  • VL regions were amplified in a similar method using the oligonucleotides OL332, CATGGCGCGCGATGTGACATCCAGATGACTCAGTC (SEQ ID NO. 65), and OL333, TGCGGGATCCAACTGAGGAAGCAAAGTTTAAATTCTACTCACGTCTCAGCTCCA GCTTGGTCC (SEQ ID NO. 66), to engineer in BssHII and BamHI restriction enzyme sites. Reactions were placed in the block of the thermal cycler and heated to 95°C for 2 minutes. The polymerase chain reaction (PCR) reaction was performed for 30 cycles of 94°C for 30 seconds, 55°C for 1 minute and 72°C for 30 seconds.
  • PCR polymerase chain reaction
  • PCR products were heated at 72°C for 5 minutes, and then held at 4°C.
  • VH and VL region PCR products were then cloned into the vectors pANT15 and pANT13 respectively (FIG. 1) at the MluI/HinDIII and BssHII/BamHI sites respectively.
  • Both pANT15 and pANT13 are pAT 153 -based plasmids containing a human Ig expression cassette.
  • the heavy chain cassette in pANT15 consists of a human genomic IgGl constant region gene driven by hCMVie promoter, with a downstream human IgG polyA region.
  • pANT15 also contains a hamster dhfr gene driven by the SV40 promoter with a downstream SV40 polyA region.
  • the light chain cassette of pANT13 includes the genomic human kappa constant region driven by hCMVie promoter with downstream light chain polyA region. Cloning sites between a human Ig leader sequence and the constant regions allow the insertion of the variable region genes.
  • NSO cells (ECACC 85110503, Porton, UK) were co-transfected with these two plasmids via electroporation and selected in DMEM (Invitrogen, Paisley UK) + 5% FBS (Ultra low IgG Cat No. 16250-078 Invitrogen, Paisley UK) + Penicillin/Streptomycin (Invitrogen, Paisley UK) + 10OnM Methotrexate (Sigma, Poole UK). Methotrexate resistant colonies were isolated and antibody was purified by Protein A affinity chromatography using a ImI HiTrap MabSelect Sure column (GE Healthcare, Amersham UK) following the manufacturers recommended conditions. Attorney Docket No. POOiOl 03.02
  • NSO supernatants were quantified for antibody expression in IgG Fc/Kappa ELISA using purified human IgGl/Kappa (Sigma, Poole UK) as standards.
  • Immunosorb 96 well plates (Nalge nunc Hereford, UK) were coated with mouse anti-human IgG Fc-specific antibody (16260 Sigma, Poole UK) diluted at 1 :1500 in IxPBS (pH 7.4) at 37 0 C for 1 hour. Plates were washed three times in PBS+0.05% Tween 20 before adding samples and standards, diluted in 2% BSA/PBS.
  • the chimeric antibody was tested in an ELISA-based competition assay using 6E10 mouse antibody, biotinylated using Biotintag micro biotinylation kit (Sigma, Poole UK).
  • Biotinylated mouse 6E10 antibody was diluted to 0.25 ⁇ g/ml and mixed with equal volumes of competing antibody at concentrations ranging from 3ng/ml-16 ⁇ g/ml. lOO ⁇ l of the antibody mixes were transferred into the wells of Immulon Maxisorb plates precoated with 0.06125 ⁇ g/ml Abeta 42 beta amyloid peptide coated plates and incubated at room temperature for 1 hour. The plate was washed, and bound biotinylated mouse 6E10 was detected by adding a strepavidin-HRP conjugate (Sigma, Poole UK) (diluted at 1 :500) and OPD substrate (Sigma, Poole UK). The assay was developed in the dark for 5 minutes before being stopped by the addition of 3M HCl. The assay plate was then read in a MRX TCII plate reader at absorbance 490nm.
  • VH79-93 TAYLHLNSLTSEDTA amino acids 79-93 of SEQ ID NO:36
  • VL10-24 SLTVTAGEKVALTCK amino acids 10-24 of SEQ ID NO:37
  • VL79-93 LTISSVQAEDLAVYY amino acids 79-93 of SEQ ID NO:37.
  • Deimmunized VH and VL sequences were designed to incorporate the variant peptide sequences as above. Sequences of the deimmunized variants (SEQ ID NO:42 and SEQ ID NO:43) are given in FIG. 2 ('Del').
  • Deimmunized V region genes were constructed using the mouse 6E10 VH and VL templates for PCR using long overlapping oligonucleotides to introduce amino acids corresponding to the variant peptide sequences as above.
  • Variant genes were cloned directly into the expression vectors pANT15 and pANT13 and transfected into NSO as detailed in Example 1. Binding of deimmunized antibody was tested in the competition binding ELISA described in Example 1.
  • Humanized VH and VL sequences were designed by comparison of mouse 6E10 sequences and homologous human VH and VL sequences. From this analysis, the human VH region VH 1-46 was chosen to provide frameworks for grafting of 6E10 VH CDRs as shown in FIG. 2 ('Graftl ', SEQ ID NO:44). The human VL region B3 was chosen to provide frameworks for grafting of 6E10 VL CDRs as shown in FIG. 2 ('Graft', SEQ ID NO:45). A variant of the humanized VH sequence was designed as shown in FIG. 2 ('Graft2', SEQ Attorney Docket No. P0030103.02
  • Humanized V region genes were constructed using the mouse 6E10 VH and VL templates for PCR using long overlapping oligonucleotides to introduce amino acids from homologous human VH and VL sequences. Variant genes were cloned directly into the expression vectors pANT15 and pANT13 and transfected into NSO as detailed in Example 1. Binding of humanized antibody variants were tested in the competition binding ELISA described in Example 1.
  • EXAMPLE 4 Composite human antibody variants of 6E10
  • Composite human VH and VL sequences were designed by comparison of mouse 6E10 sequences and fragments of different naturally occurring human VH and VL sequences and selection of such human fragments to build the composite human sequences.
  • the choice of human VH and VL sequence fragments was constrained for the presence of certain amino acids at corresponding positions in 6E10 which were considered to be potentially important for antibody binding in conjunction with CDRs from 6E10.
  • these amino acids were 1, 11, 23, 41, 48, 67, 71, 73, 76, 78 and 93 (Kabat numbering).
  • VL these amino acids were 12, 63 and 70 (Kabat numbering).
  • VH and VL sequence fragments were chosen for homology to certain human germline framework regions of closest homology to the 6E10 VH and VL.
  • VH these were VH1-46 for framework 1 (VHFRl), VH1-58 for VHFR2, VH1-69 for VHFR3 and J4 for the J region.
  • VL these were A14 for VLFRl, B3 for VLFR2, B3 for VHFR3 and J4 for the J region.
  • FIG. 2 'CHAB', SEQ ID NOs: 47-54, corresponding nucleotide sequences are shown in FIGs.
  • Variant genes were cloned directly into the expression vectors pANT15 and pANT13 and transfected into NSO as detailed in Example 1. All combinations of composite heavy and light chains (i.e. a total of 15 pairings) were stably transfected into NSO cells by electroporation and selected in media (high glucose DMEM with L-glutamine and Na pyruvate, 5% ultra-low IgG FCS, pen/strep - all from Invitrogen, Paisley, UK) containing 20OnM methotrexate. Several drug resistant colonies for each construct were tested for expression levels and the best expressing lines were selected and frozen under liquid nitrogen.
  • Binding of humanized variants were tested in the competition binding ELISA described in Example 1. Briefly, supernatants from the best expressing lines for each combination were quantified using an Fc capture, Kappa light chain detection ELISA in comparison to a IgGl /kappa standard. The quantified supernatants were then tested in a competition ELISA for binding to their target antigen, beta amyloid. 96 well maxisorb plates (Nunc - Fisher Scientific, Loughborough, UK) were coated overnight at 4°C with 50 ⁇ l/well of 0.06125 ⁇ g/ml beta amyloid (Covance, UK) in carbonate buffer pH 9.6.
  • Duplicate titrations of mouse MDT-2007 antibody and COMPOSITE HUMAN ANTIBODYTM (Antitope Ltd.) samples were generated (in the range 0.003 ⁇ g/ml to 16 ⁇ g/ml) and mixed with a constant concentration (0.25 ug/ml) of biotinylated mouse MDT-2007 antibody in PBS pH 7.4/0.5% BSA/ 0.05%Tween.
  • the titrations, 50 ⁇ l/well, were added to washed (3x with PBS pH 7.4/0.5%BSA/0.05% Tween 20) assay plates and incubated at room temperature for 1 hour.
  • Table 4 summarizes the results for the combinations of the composite VH and VK variant sequences obtained from a competition assay performed over a wide range of concentrations (0.003 ⁇ g/ml to 16 ⁇ g/ml) for the anti-beta amyloid antibodies.
  • Three of the humanized antibodies have an IC50 that is improved compared to the mouse reference, particularly VH4/VK2 that has a measured 1.72 fold increase in binding in this assay.
  • Tables 5-8 show the human sequences from which the humanized antibodies were derived.
  • Table 8 VK2 sequences from which humanized antibodies were derived Attorney Docket No. POOiOl 03.02
  • MDT-2007 Sequence variant VH4/VK2 (see Table 4) has been designated MDT-2007 and will be referred to hereinafter as MDT-2007.
  • MDT-2007 was subjected to a pre-clinical ex vivo T cell assay (EPISCREENTM, Antitope Ltd.).
  • the EPISCREENTM (Antitope Ltd.) assay provides an effective technology for predicting T cell immunogenicity by quantifying T cell responses to protein therapeutics. Using a cohort of community blood donors carefully selected based on MHC class II haplotypes, purified antibodies are tested for T cell immunogenicity in vitro using the EPISCREENTM (Antitope Ltd.) time course T cell assay format.
  • This assay provides a method by which the immunogenicity of whole proteins can be assessed both in terms of magnitude and frequency of T cell responses (Jones et al, J Interferon Cytokine Res. 2004 24(9):560-72; Jones et al., J Thromb Haemost. 2005 3(5):991-1000).
  • Standard T cell assays provide a single time point 'snapshot' of the immune response and do not allow for natural variation in individual donors' response to antigens.
  • the EpiScreenTM time course T cell assay samples donors' T cell responses over a four day period and the high degree of sensitivity along with the robust nature of the assay allows an accurate pre-clinical assessment of the potential for immunogenicity of the biologic being studied.
  • MDT-2007 (Lot No. MEDOl) was used as test antibody in the EPISCREENTM (Antitope Ltd.) assay.
  • Control chimeric antibody was prepared fresh as follows: a IL culture of chimeric antibody expressing cell-line was grown to saturation. Supernatant was separated from cells and debris, adjusted to pH 7.4, filter sterilized and run through 2x ImI Hi-Trap Mab Select Pure affinity columns (GE Healthcare, Amersham, UK) at a flow rate of 1 ml/min. The columns were washed with 20 ml PBS pH 7.4 and eluted with sodium citrate buffer pH 3.0. Fractions were immediately neutralized with 0.1 volumes IM Tris buffer pH 9.0. The protein content of each fraction was measured by UV absorption at 280 nm and protein containing fractions were pooled, buffer exchanged into PBS pH 7.4 and Attorney Docket No. P0030103.02
  • the antibody was further purified by size exclusion chromatography using a 16/60 Sephacryl S200 column (GE Healthcare, Amersham, UK). The major peak fractions were collected, pooled, filter sterilized and stored at +4°C.
  • PBMC Peripheral blood mononuclear cells
  • Donors were characterized by identifying HLA-DR haplotypes using a Biotest SSP- PCR based tissue-typing kit (Biotest, Landsteinerstrafie, Denmark) as well as determining T cell responses to a control antigen keyhole limpet haemocyanin (KLH) (Pierce, Rockford, USA). PBMC were then frozen and stored in liquid nitrogen until required.
  • KLH keyhole limpet haemocyanin
  • a cohort of 20 donors was selected to best represent the number and frequency of HLADR allotypes expressed in the world population. Analysis of the allotypes expressed in the cohort against those expressed in the world population revealed that coverage of >80% was achieved and that all major HLA-DR alleles (individual allotypes with a frequency >5% expressed in the world population) were well represented. Details of individual donor haplotypes and a comparison of the frequency of MHC class II allotypes expressed in the world population and the sample population can be found in Table 9 and FIG. 6, respectively.
  • PBMCs from each donor were thawed, counted and viability assessed. Cells were revived in room temperature AIMV culture medium (Invitrogen, Paisley, UK) and resuspended in Attorney Docket No. P0030103.02
  • AIMV to 4-6x106 PBMC/ml.
  • bulk cultures were established in which a total of ImI proliferation cell stock was added a 24 well plate.
  • a total of ImI of each diluted test sample was added to the PBMC to give a final concentration of 50 ⁇ g/ml per sample.
  • a positive control (cells incubated with lOO ⁇ g/ml KLH) and a negative control (cells incubated with culture media only) were also included. Cultures were incubated for a total of 8 days at 37°C with 5% CO 2 .
  • the two test samples were tested against a cohort of 20 healthy donors using EPISCREENTM (Antitope Ltd.) time course T cell assays in order to determine the relative risk of immunogenicity.
  • the cohort was selected to best represent the number and frequency of HLA-DR allotypes expressed in the world population (FIG. 6).
  • Donor haplotypes and the results of the control antigen tests are shown in Table 1.
  • the samples were tested at a final concentration of 50 ⁇ g/ml based on Antitope' s previous experience showing that this saturating concentration is sufficient to stimulate detectable protein- specific T cell responses.
  • the results from the current EPISCREENTM (Antitope Ltd.) time course proliferation assay with MDT-2007 and chimeric antibody are shown in FIG. 7 and summarised in Table 10.
  • FIG. 8 shows a clear correlation between the level of immunogenicity observed using the EPISCREENTM (Antitope Ltd.) assay and the level of immunogenicity (anti-protein therapeutic antibody responses) that has been actually observed in the clinic against a large panel of therapeutic proteins (Baker and Jones, Curr. Opin. Drug. Disc. Dev. 2007. 10:219- 217). High levels of immunogenicity were observed in both the clinical data and EPISCREENTM (Antitope Ltd.) assays for proteins such as Infliximab and Campath, whereas relatively low levels of immunogenicity were observed for proteins such as Xolair, Herceptin, and Avastin. It is clear that MDT-2007 would therefore be considered as having a low potential risk of immunogenicity.
  • Table 10 Summary of positive donor proliferation responses to chimeric anti-A ⁇ 42 and MDT-2007.
  • EXAMPLE 6 Binding of MDT-2007 to brains of AD human and aged non-human primate Attorney Docket No. P0030103.02
  • Sections were washed with TBS and then incubated again with ABC 135ul of A+ 135ul of B /5OmL TBS (1 :500) for 1 hr. Sections were then rinsed with Imidizole Acetate Buffer 3 times(0.68g Imidizole, 6.8g. NaAc Trihydrate/1L dH 2 ⁇ , pH w/glacial Acetic Acid to 7.4)
  • EXAMPLE 7 Stability of MDT-2007 in infusion system under implant conditions
  • MDT-2007 was found to be stable with 80% or greater recovery of active MDT-2007 (based on ELISA) infused from the infusion system. The 20% loss is believed to be due to adsorption of the antibody to components of the system, as steady state delivery was reached quickly.

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Abstract

La présente invention concerne des anticorps anti-Aβ humanisés dérivés d'un anticorps murin dirigé contre un épitope N-terminal de Aβ. Les anticorps humanisés présentent peu ou pas d'épitopes T et se lient à Aβ avec une affinité similaire à celle de l'anticorps murin.
EP08854674A 2007-11-27 2008-11-26 Anticorps anti-amyloïde bêta humanisés Ceased EP2222700A2 (fr)

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EP2755688A4 (fr) 2011-10-27 2014-08-06 Nkt Therapeutics Inc Anticorps humanisés dirigés contre inkt

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