[go: up one dir, main page]

WO2006014679A1 - Immunoglobulines contenant principalement un glycoforme de type glcnac2man3glcnac2 - Google Patents

Immunoglobulines contenant principalement un glycoforme de type glcnac2man3glcnac2 Download PDF

Info

Publication number
WO2006014679A1
WO2006014679A1 PCT/US2005/025652 US2005025652W WO2006014679A1 WO 2006014679 A1 WO2006014679 A1 WO 2006014679A1 US 2005025652 W US2005025652 W US 2005025652W WO 2006014679 A1 WO2006014679 A1 WO 2006014679A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
glcnac
immunoglobulins
glycan
antibody
Prior art date
Application number
PCT/US2005/025652
Other languages
English (en)
Inventor
Tillman U. Gerngross
Huijuan Li
Stefan Wildt
Original Assignee
Glycofi, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Glycofi, Inc. filed Critical Glycofi, Inc.
Priority to CA002573745A priority Critical patent/CA2573745A1/fr
Priority to AU2005269759A priority patent/AU2005269759A1/en
Priority to JP2007522673A priority patent/JP2008512353A/ja
Priority to EP05790298A priority patent/EP1776385A1/fr
Publication of WO2006014679A1 publication Critical patent/WO2006014679A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

  • the present invention relates to compositions and methods for producing glycoproteins having specific N-linked glycosylation patterns.
  • the present invention relates to compositions of immunoglobulin glycoproteins comprising a plurality of N-glycans having specific N-glycan structures, and more particularly, to compositions comprising immunoglobulin glycoproteins wherein within the plurality there are one or more predominant glycoform structures on the immunoglobulins that regulate, e.g., promote a specific effector function.
  • Glycoproteins mediate many essential functions in humans and other mammals, including catalysis, signaling, cell-cell communication, and molecular recognition and association. Glycoproteins make up the majority of non-cytosolic proteins in eukaryotic organisms (Lis and Sharon, 1993, Eur. J. Biochem. 218: 1-27). Many glycoproteins have been exploited for therapeutic purposes, and during the last two decades, recombinant versions of naturally-occurring glycoproteins have been a major part of the biotechnology industry.
  • glycosylated proteins used as therapeutics include erythropoietin (EPO), therapeutic monoclonal antibodies (mAbs), tissue plasminogen activator (tPA), interferon- ⁇ (IFN- ⁇ ), granulocyte-macrophage colony stimulating factor (GM-CSF), and human chorionic gonadotrophin (hCH) (Cumming et al, 1991, Glycobiology 1 :115-130). Variations in glycosylation patterns of recombinantly produced glycoproteins have recently been the topic of much attention in the scientific community as recombinant proteins produced as potential prophylactics and therapeutics approach the clinic.
  • EPO erythropoietin
  • mAbs therapeutic monoclonal antibodies
  • tPA tissue plasminogen activator
  • IFN- ⁇ interferon- ⁇
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • hCH human chorionic gonadotrophin
  • Antibodies or immunoglobulins are glycoproteins that play a central role in the humoral immune response. Antibodies may be viewed as adaptor molecules that provide a link between humoral and cellular defense mechanisms. Antigen- specific recognition by antibodies results in the formation of immune complexes that may activate multiple effector mechanisms, resulting in the removal and destruction of the complex.
  • immunoglobulins five classes of antibodies — IgM, IgD, IgG, IgA, and IgE — can be distinguished biochemically as well as functionally, while more subtle differences confined to the variable region account for the specificity of antigen binding. Amongst these five classes of Igs, there are only two types of light chain, which are termed lambda ( ⁇ ) and kappa (K).
  • immunoglobulin M immunoglobulin M
  • IgD immunoglobulin D
  • IgG immunoglobulin G
  • IgA immunoglobulin A
  • IgE immunoglobulin E
  • the immunoglobulin G (IgG) molecule comprises a Fab (/ragment antigen binding) domain with constant and variable regions and an Fc (/ragment crystallized) domain.
  • the CH2 domain of each heavy chain contains a single site for N-linked glycosylation at an asparagine residue linking an N-glycan to the Ig molecule, usually at residue Asn-297 (Kabat et al, Sequences of proteins of immunological interest, Fifth Ed., U.S. Department of Health and Human Services, ⁇ IH Publication No. 91- 3242).
  • compositions of fucosylated G2 (Gal 2 Glc ⁇ Ac 2 Man 3 Glc ⁇ Ac 2 ) IgG made in CHO cells reportedly increase complement-dependent cytotoxicity (CDC) activity to a greater extent than compositions of heterogenous antibodies (Raju, 2004, US Pat. Appl. No. 2004/0136986). It has also been suggested that an optimal antibody against tumors would be one that bound preferentially to activate Fc receptors (Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIII) and minimally to the inhibitory Fc ⁇ RIIb receptor (Clynes et al., 2000, Nature, 6:443-446). Therefore, the ability to enrich for specific glycoforms on Ig glycoproteins is highly desirable.
  • glycosylation structures on glycoprotein will vary depending upon the expression host and culturing conditions.
  • Therapeutic proteins produced in non-human host cells are likely to contain non-human glycosylation which may elicit an immunogenic response in humans — e.g. hypermannosylation in yeast (Ballou, 1990, Methods Enzymol. 185:440-470); ⁇ (l ,3)- fucose and ⁇ (l,2)-xylose in plants, (Cabanes-Macheteau et al, 1999, Glycobiology, 9: 365-372); N-glycolylneuraminic acid in Chinese hamster ovary cells (Noguchi et al., 1995. J. Biochem.
  • glycoforms can profoundly affect the properties of a therapeutic, including pharmacokinetics, pharmacodynamics, receptor-interaction and tissue-specific targeting (Graddis et al, 2002, Curr Pharm Biotechnol. 3: 285- 297).
  • the oligosaccharide structure can affect properties relevant to protease resistance, the serum half-life of the antibody mediated by the FcRn receptor, binding to the complement complex Cl, which induces complement-dependent cytoxicity (CDC), and binding to Fc ⁇ R receptors, which are responsible for modulating the antibody-dependent cell-mediated cytoxicity (ADCC) pathway, phagocytosis and antibody feedback.
  • ADCC antibody-dependent cell-mediated cytoxicity
  • glycoprotein compositions that are enriched for particular glycoforms are highly desirable.
  • the present invention provides a composition comprising a plurality of immunoglobulins each immunoglobulin comprising at least one N-glycan attached thereto wherein the composition thereby comprises a plurality of N-glycans in which the predominant N-glycan consists essentially of at least 75 mole percent GlcNAc 2 Man 3 GlcNAc 2 . In preferred embodiments, greater than 75 mole percent of said plurality of N-glycans consists essentially Of GIcNAc 2 MaH 3 GIcNAc 2 .
  • GlcNAc2Man 3 GlcNAc 2 -glycan structure is present at a level that is from about 50 mole percent more than the next most predominant N-glycan structure of said plurality of N-glycans.
  • the present invention also provides methods for increasing binding to Fc ⁇ RIIIa and Fc ⁇ RIIIb receptor and decreasing binding to Fc ⁇ RIIb receptor by enriching for a specific glycoform (e.g. GlcNAc 2 Man 3 GlcNAc 2 ) on an immunoglobulin.
  • a preferred embodiment provides a method for producing a composition comprising a plurality of immunoglobulins, each immunoglobulin comprising at least one N-glycan attached thereto wherein the composition thereby comprises a plurality of N-glycans consists essentially of at least GlcNAc 2 Man3GlcNAc 2 , said method comprising the step of culturing a host cell that has been engineered or selected to express said immunoglobulin or fragment thereof.
  • Another preferred embodiment provides a method for producing a composition comprising a plurality of immunoglobulins, each immunoglobulin comprising at least one N-glycan attached thereto wherein the composition thereby comprises a plurality of N-glycans consists essentially of at least 75 mole percent GlcNAc 2 Man 3 GlcNAc 2i said method comprising the step of culturing a lower eukaryotic host cell that has been engineered or selected to express said immunoglobulin or fragment thereof.
  • a host cell comprises an exogenous gene encoding an immunoglobulin or fragment thereof, said host cell is engineered or selected to express said immunoglobulin or fragment thereof, thereby producing a composition comprising a plurality of immunoglobulins, each immunoglobulin comprising at least one N-glycan attached thereto wherein the composition thereby comprises a plurality of N-glycans consists essentially of at least 75 mole percent GlcNAc 2 Man 3 GlcNAc 2 .
  • a lower eukaryotic host cell comprises an exogenous gene encoding an immunoglobulin or fragment thereof, said host cell is engineered or selected to express said immunoglobulin or fragment thereof, thereby producing a composition comprising a plurality of immunoglobulins, each immunoglobulin comprising at least one N-glycan attached thereto wherein the composition thereby comprises a plurality of N-glycans consists essentially of at least 75 mole percent GIcNAc 2 MaH 3 GIcNAc 2 .
  • a composition comprising a plurality of immunoglobulins each immunoglobulin comprising at least one N- glycan attached thereto wherein the composition thereby comprises a plurality of N- glycans in which the predominant N-glycan consists essentially of 75 mole percent GlcNAc 2 Man 3 GlcNAc 2 wherein said immunoglobulins exhibit decreased binding affinity to Fc ⁇ RIIb receptor.
  • a composition comprising a plurality of immunoglobulins each immunoglobulin comprising at least one N-glycan attached thereto wherein the composition thereby comprises a plurality of N-glycans in which the predominant N-glycan consists essentially of at least 75 mole percent GIcNAc 2 MaH 3 GIcNAc 2 wherein said immunoglobulins exhibit increased binding affinity to Fc ⁇ RIIIa and Fc ⁇ RIIIb receptor.
  • a composition comprising a plurality of immunoglobulins each immunoglobulin comprising at least one N-glycan attached thereto wherein the composition thereby comprises a plurality of N-glycans in which the predominant N-glycan consists essentially of 75 mole percent GlcNAc 2 Man 3 GlcNAc 2 wherein said immunoglobulins exhibit increased antibody-dependent cellular cytoxicity (ADCC).
  • ADCC antibody-dependent cellular cytoxicity
  • composition of the present invention comprises immunoglobulins which are essentially free of fucose. In another embodiment, the composition of the present invention comprises immunoglobulins which lack fucose.
  • the composition of the present invention also comprises a pharmaceutical composition and a pharmaceutically acceptable carrier.
  • the composition of the present invention also comprises a pharmaceutical composition of immunoglobulins which have been purified and incorporated into a diagnostic kit.
  • the present invention provides materials and methods for production of compositions of glycoproteins having predetermined glycosylation structures, in particular, immunoglobulin or antibody molecules having N-glycans consisting essentially of 75 mole percent GlcNAc 2 Man 3 GlcNAc 2 .
  • Figure 1 Schematic representation of IgG having GlcNAc 2 Man 3 GlcNAc 2 glycan structure.
  • FIG. 1 Coomassie blue stained SDS-PAGE gel of JC-IgG expressed in YAS309 (as described Example 2) and purified from the culture medium (Example 3) over a Protein A column (lane 1) and phenyl sepharose column (lane 2). 3.0 ⁇ g protein/lane.
  • SEQ ID NO: 1 nucleotide sequence corresponding to the murine IgGl variable region of the JC-IgGl light chain (GenBank #AF013576).
  • SEQ ID NO: 2 nucleotide sequence corresponding to the murine IgGl variable region of the JC-IgGl heavy chain (GenBank #AF013577).
  • SEQ ID NO: 3 to 14 encode 12 overlapping oligonucleotide sequences used to PCR- synthesize the murine light chain variable region of JC-IgGl.
  • SEQ ID NO: 15 to 26 encode 12 overlapping oligonucleotides used to PCR- synthesize the murine heavy chain Fab fragment of JC-IgGl.
  • SEQ ID NO: 27 to 38 encode 12 overlapping oligonucleotides used to PCR- synthesize the murine heavy chain Fc fragment of JC-IgG 1.
  • SEQ ID NO: 39 encodes a 3' Kpnl primer corresponding to the 3' end of the Fc fragment.
  • SEQ ID NO: 40 encodes the nucleotide sequence for human serum albumin (HSA).
  • SEQ ID NO: 41 encodes the nucleotide sequence for thrombin cleavage used in the present invention.
  • N-glycan oligosaccharide
  • glycan oligosaccharide
  • glycoproteins The predominant sugars found on glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine (Gal ⁇ Ac), N- acetylglucosamine (Glc ⁇ Ac) and sialic acid (e.g., N-acetyl-neuraminic acid
  • N-glycans have a common pentasaccharide core of Man 3 GlcNAc 2 ("Man” refers to mannose; “GIc” refers to glucose; and “NAc” refers to N-acetyl; Glc ⁇ Ac refers to N-acetylglucosamine).
  • ⁇ -glycans differ with respect to the number of branches (antennae) comprising peripheral sugars (e.g., Glc ⁇ Ac, galactose, fucose and sialic acid) that are added to the Ma ⁇ GIcNAc 2 (“Man3”) core structure which is also referred to as the "trimannose core", the "pentasaccharide core” or the "paucimannose core”.
  • N-glycans are classified according to their branched constituents (e.g., high mannose, complex or hybrid).
  • a "high mannose” type N- glycan has five or more mannose residues.
  • a "complex” type N-glycan typically has at least one Glc ⁇ Ac attached to the 1,3 mannose arm and at least one Glc ⁇ Ac attached to the 1,6 mannose arm of a "trimannose" core.
  • Complex N-glycans may also have galactose (“Gal”) or N-acetylgalactosamine (“Gal ⁇ Ac”) residues that are optionally modified with sialic acid or derivatives (e.g., "NANA” or “NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refers to acetyl).
  • Gal galactose
  • Gal ⁇ Ac N-acetylgalactosamine residues
  • sialic acid or derivatives e.g., "NANA” or “NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refers to acetyl
  • Complex N-glycans may also have intrachain substitutions comprising "bisecting" Glc ⁇ Ac and core fucose ("Fuc").
  • Complex N-glycans may also have multiple antennae on the "trimannose core,” often referred to as “multiple antennary glycans.”
  • a “hybrid” N-glycan has at least one Glc ⁇ Ac on the terminal of the 1,3 mannose arm of the trimannose core and zero or more mannoses on the 1,6 mannose arm of the trimannose core.
  • the various N-glycans are also referred to as "glycoforms.”
  • nucleic acid or polynucleotide e.g., an R ⁇ A, D ⁇ A or a mixed polymer
  • isolated or substantially pure nucleic acid or polynucleotide is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases and genomic sequences with which it is naturally associated.
  • the term embraces a nucleic acid or polynucleotide that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the "isolated polynucleotide” is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature.
  • isolated or substantially pure also can be used in reference to recombinant or cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems.
  • isolated does not necessarily require mat the nucleic acid or polynucleotide so described has itself been physically removed from its native environment.
  • an endogenous nucleic acid sequence in the genome of an organism is deemed “isolated” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered.
  • a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous (originating from a different host cell or progeny thereof).
  • a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern.
  • This gene would now become “isolated” because it is separated from at least some of the sequences that naturally flank it.
  • a nucleic acid is also considered “isolated” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome.
  • an endogenous coding sequence is considered “isolated” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention.
  • An "isolated nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.
  • an "isolated nucleic acid” can be substantially free of other cellular material, or substantially free of culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the phrase "degenerate variant" of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence.
  • degenerate oligonucleotide or “degenerate primer” is used to signify an oligonucleotide capable of hybridizing with target nucleic acid sequences that are not necessarily identical in sequence but that are homologous to one another within one or more particular segments.
  • percent sequence identity or “identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence.
  • the length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.
  • FASTA FASTA
  • Gap or Bestfit programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin.
  • GCG Genetics Computer Group
  • percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as etal
  • sequences can be compared using the computer program, BLAST (Altschul et ah, J. MoI. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden , Meth. Enzymol. 266: 131 - 141 ( 1996); Altschul et ah, Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et ah, Nucleic Acids Res. 25:3389-3402 (1997)).
  • nucleic acid or fragment thereof indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 50%, more preferably 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well- known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.
  • nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions.
  • Stringent hybridization conditions and “stringent wash conditions” in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization.
  • “stringent hybridization” is performed at about 25 0 C below the thermal melting point (T m ) for the specific DNA hybrid under a particular set of conditions.
  • “Stringent washing” is performed at temperatures about 5°C lower than the T n , for the specific DNA hybrid under a particular set of conditions.
  • the T n is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • stringent conditions are defined for solution phase hybridization as aqueous hybridization ⁇ i.e., free of formamide) in 6X SSC (where 2OX SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65 0 C for 8-12 hours, followed by two washes in 0.2X SSC, 0.1% SDS at 65 0 C for 20 minutes. It will be appreciated by the skilled worker that hybridization at 65 °C will occur at different rates depending on a number of factors including the length and percent identity of the sequences which are hybridizing.
  • mutated when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence.
  • a nucleic acid sequence may be mutated by any method known in the art including but not limited to mutagenesis techniques such as "error- prone PCR" (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989) and Caldwell and Joyce, PCR Methods Applic.
  • mutagenesis techniques such as "error- prone PCR” (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989) and Caldwell and Joyce, PCR Methods Applic.
  • oligonucleotide-directed mutagenesis a process which enables the generation of site-specific mutations in any cloned DNA segment of interest; see, e.g., Reidhaar-Olson and Sauer, Science 241 :53-57 (1988)).
  • vector as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC).
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • viral vector Another type of vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below).
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced ⁇ e.g., vectors having an origin of replication which functions in the host cell).
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome.
  • certain preferred vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors" (or simply, “expression vectors”).
  • sequence of interest or “gene of interest” refers to a nucleic acid sequence, typically encoding a protein, that is not normally produced in the host cell.
  • the methods disclosed herein allow one or more sequences of interest or genes of interest to be stably integrated into a host cell genome.
  • sequences of interest include sequences encoding one or more polypeptides having an enzymatic activity, e.g., an enzyme which affects N-glycan synthesis in a host such as mannosyltransferases, N-acetylglucosaminyltransferases, UDP-N-acetylglucosamine transporters, galactosyltransferases, UDP-N- acetylgalactosyltransferase, sialyltransferases and fucosyltransferases.
  • an enzyme which affects N-glycan synthesis in a host such as mannosyltransferases, N-acetylglucosaminyltransferases, UDP-N-acetylglucosamine transporters, galactosyltransferases, UDP-N- acetylgalactosyltransferase, sialyltransferases and fucosyltransferases
  • marker sequence refers to a nucleic acid sequence capable of expressing an activity that allows either positive or negative selection for the presence or absence of the sequence within a host cell.
  • the P. pastoris URA5 gene is a marker gene because its presence can be selected for by the ability of cells containing the gene to grow in the absence of uracil. Its presence can also be selected against by the inability of cells containing the gene to grow in the presence of 5-FOA. Marker sequences or genes do not necessarily need to display both positive and negative selectability.
  • ⁇ on-limiting examples of marker sequences or genes from P. pastoris include ADEl, ARG4, HIS4 and URAS.
  • For antibiotic resistance marker genes kanamycin, neomycin, geneticin (or G418), paromomycin and hygromycin resistance genes are commonly used to allow for growth in the presence of these antibiotics.
  • “Operatively linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.
  • expression control sequence refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient R ⁇ A processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.
  • the nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence.
  • control sequences is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • recombinant host cell ("expression host cell”, “expression host system”, “expression system” or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • a recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.
  • eukaryotic refers to a nucleated cell or organism, and includes insect cells, plant cells, mammalian cells, animal cells and lower eukaryotic cells.
  • lower eukaryotic cells includes yeast, fungi, collar-flagellates, microsporidia, alveolates (e.g., dinoflagellates), stramenopiles (e.g, brown algae, protozoa), rhodophyta (e.g., red algae), plants (e.g., green algae, plant cells, moss) and other protists.
  • Yeast and fungi include, but are not limited to: Pichia sp., such as Pichia pasto ⁇ s, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis andPichia methanolica; .
  • Pichia sp. such as Pichia pasto ⁇ s, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia
  • Saccharomyces sp. such as Saccharomyces cerevisiae; Hansenula polymorpha, Kluyveromyces sp., such as Kluyveromyces lactis; Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., such as Fusarium gramineum, Fusarium venenatum; Physcomitrella patens and Neurospora crassa.
  • peptide refers to a short polypeptide, e.g., one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long.
  • the term as used herein encompasses analogs and mimetics that mimic structural and thus biological function.
  • polypeptide encompasses both naturally-occurring and non- naturally-occurring proteins, and fragments, mutants, derivatives and analogs thereof.
  • a polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities.
  • isolated protein or "isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds).
  • polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components.
  • a polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
  • isolated does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from its native environment.
  • polypeptide fragment refers to a polypeptide that has a deletion, e.g., an amino-terminal and/or carboxy-terminal deletion compared to a full-length polypeptide.
  • the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long.
  • a “modified derivative” refers to polypeptides or fragments thereof that are substantially homologous in primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate amino acids that are not found in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those skilled in the art.
  • a variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as 125 1, 32 P, 35 S, and 3 H, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand.
  • the choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation.
  • Methods for labeling polypeptides are well known in the art. See, e.g., Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002) (hereby incorporated by reference).
  • fusion protein refers to a polypeptide comprising a polypeptide or fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins.
  • a fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, yet more preferably at least 75, 100 or 125 amino acids. Fusions that include the entirety of the proteins of the present invention have particular utility.
  • the heterologous polypeptide included within the fusion protein of the present invention is at least 6 amino acids in length, often at least 8 amino acids in length, and usefully at least 15, 20, and 25 amino acids in length.
  • Fusions that include larger polypeptides, such as an immunoglobulin Fc fragment, or an immunoglobulin Fab fragment or even entire proteins, such as the green fluorescent protein ("GFP") chromophore-containing proteins or a full length immunoglobulin having particular utility.
  • Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein.
  • a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein.
  • each antibody molecule has a unique structure that allows it to bind its specific antigen, but all antibodies/immunoglobulins have the same overall structure as described herein.
  • the basic antibody structural unit is known to comprise a tetramer of subunits. Each tetramer has two identical pairs of polypeptide chains, each pair having one "light” chain (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • each chain defines a constant region primarily responsible for effector function.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM 5 IgA, IgD and IgE, respectively.
  • the light and heavy chains are subdivided into variable regions and constant regions (See generally, Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7 (incorporated by reference in its entirety for all purposes).
  • the variable regions of each light/heavy chain pair form the antibody binding site.
  • an intact antibody has two binding sites.
  • the chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs.
  • the CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope.
  • the terms include naturally occurring forms, as well as fragments and derivatives. Included within the scope of the term are classes of Igs, namely, IgG, IgA, IgE, IgM, and IgD. Also included within the scope of the terms are the subtypes of IgGs, namely, IgGl, IgG2, IgG3 and IgG4.
  • the term is used in the broadest sense and includes single monoclonal antibodies (including agonist and antagonist antibodies) as well as antibody compositions which will bind to multiple epitopes or antigens.
  • the terms specifically cover monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they contain or are modified to contain at least the portion of the CH2 domain of the heavy chain immunoglobulin constant region which comprises an N-linked glycosylation site of the CH2 domain, or a variant thereof.
  • molecules comprising the Fc region such as immunoadhesins (US Pat. Appl. No. 2004/0136986), Fc fusions and antibody- like molecules.
  • these terms can refer to an antibody fragment of at least the Fab region that at least contains an N-linked glycosylation site.
  • Fc refers to the 'fragment crystallized' C-terminal region of the antibody containing the C H 2 and CH3 domains ( Figure 1).
  • Fab refers to the 'fragment antigen binding' region of the antibody containing the VH, CHl, VL and CL domains ( Figure 1).
  • mAb monoclonal antibody
  • monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes)
  • each mAb is directed against a single determinant on the antigen.
  • monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins.
  • the term "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al, (1975) Nature, 256:495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al).
  • the monoclonal antibodies herein include hybrid and recombinant antibodies produced by splicing a variable (including hypervariable) domain of an antibody with a constant domain (e.g.
  • humanized antibodies or a light chain with a heavy chain, or a chain from one species with a chain from another species, or fusions with heterologous proteins, regardless of species of origin or immunoglobulin class or subclass designation, (See, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.; Mage and Lamoyi, in Monoclonal Antibody Production Techniques and Applications, pp.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a first species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from a different species or belonging to a different antibody class or subclass, as well as fragments of such antibodies, so long as they contain or are modified to contain at least one C H 2 .
  • chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a first species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from a different species or belonging to a different antibody class or subclass, as well as fragments of such antibodies, so long as they contain or
  • “Humanized” forms of non-human (e.g., murine) antibodies are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 , or other antigen-binding subsequences of antibodies) which contain sequences derived from human immunoglobulins.
  • An Fv fragment of an antibody is the smallest unit of the antibody that retains the binding characteristics and specificity of the whole molecule.
  • the Fv fragment is a noncovalently associated heterodimer of the variable domains of the antibody heavy chain and light chain.
  • the F(ab)'2 fragment is a fragment containing both arms of Fab fragments linked by the disulfide bridges.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary- determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementary- determining region
  • donor antibody non-human species
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the CDR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • “Fragments” within the scope of the terms antibody or immunoglobulin include those produced by digestion with various proteases, those produced by chemical cleavage and/or chemical dissociation and those produced recombinantly, so long as the fragment remains capable of specific binding to a target molecule.
  • fragments include Fc, Fab, Fab', Fv, F(ab') 2 , and single chain Fv (scFv) fragments.
  • Targets of interest for antibodies of the invention include growth factor receptors (e.g., FGFR, PDGFR, EGFR, NGFR, and VEGF) and their ligands.
  • Other targets are G protein receptors and include substance K receptor, the angiotensin receptor, the ⁇ - and ⁇ -adrenergic receptors, the serotonin receptors, and PAF receptor. See, e.g., Gilman, Ann. Rev.Biochem. 56:625-649 (1987).
  • Other targets include ion channels (e.g., calcium, sodium, potassium channels), muscarinic receptors, acetylcholine receptors, GABA receptors,glutamate receptors, and dopamine receptors (see Harpold, U.S.
  • cytokines such as interleukins IL-I through IL-13, tumor necrosis factors ⁇ & ⁇ , interferons ⁇ , ⁇ and ⁇ , tumor growth factor Beta (TGF- ⁇ ), colony stimulating factor (CSF) and granulocyte monocyte colony stimulating factor (GMCSF). See Human Cytokines: Handbook for Basic & Clinical Research (Aggrawal et ⁇ /.eds., Blackwell Scientific, Boston, MA 1991).
  • targets are hormones, enzymes, and intracellular and intercellular messengers, such as, adenyl cyclase, guanyl cyclase, and phospholipase C.
  • targets of interest are leukocyte antigens, such as CD20, and CD33.
  • Drugs may also be targets of interest.
  • Target molecules can be human, mammalian or bacterial.
  • antigens such as proteins, glycoproteins and carbohydrates from microbial pathogens, both viral and bacterial, and tumors. Still other targets are described in U.S. 4,366,241.
  • Immune Fc receptors discussed herein may include: Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa, Fc ⁇ RIIIb and FcRn (neonatal receptor).
  • Fc ⁇ RI can refer to any Fc ⁇ RI subtype unless specified otherwise.
  • Fc ⁇ RII can refer to any Fc ⁇ RII
  • Fc ⁇ RIII refers to any Fc ⁇ RIII subtype unless specified otherwise.
  • “Derivatives” within the scope of the term include antibodies (or fragments thereof) that have been modified in sequence, but remain capable of specific binding to a target molecule, including: interspecies chimeric and humanized antibodies; antibody fusions; heteromeric antibody complexes and antibody fusions, such as diabodies (bispecific antibodies), single-chain diabodies, and intrabodies (see, e.g., Intracellular Antibodies: Research and Disease Applications, (Marasco, ed., Springer-Verlag New York, Inc., 1998).
  • non-peptide analog refers to a compound with properties that are analogous to those of a reference polypeptide.
  • a non-peptide compound may also be termed a "peptide mimetic” or a "peptidomimetic”. See, e.g., Jones, Amino Acid and Peptide Synthesis, Oxford University Press (1992); Jung, Combinatorial Peptide and Nonpeptide Libraries: A Handbook, John Wiley (1997); Bodanszky et al., Peptide Chemistry--A Practical Textbook, Springer Verlag (1993); Synthetic Peptides: A Users Guide, (Grant, ed., W. H. Freeman and Co., 1992); Evans et al., J. Med. Chem.
  • Amino acid substitutions can include those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity or enzymatic activity, and (5) confer or modify other physicochemical or functional properties of such analogs.
  • the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2 nd ed. 1991), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as ⁇ -, ⁇ -disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present invention.
  • Stereoisomers e.g., D-amino acids
  • unnatural amino acids such as ⁇ -, ⁇ -disubstituted amino acids, N-alkyl amino acids
  • other unconventional amino acids may also be suitable components for polypeptides of the present invention.
  • Examples of unconventional amino acids include: 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ -N,N,N-trimethyllysine, ⁇ -N-acetyllysine, (9-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
  • the left-hand end corresponds to the amino terminal end and the right-hand end corresponds to the carboxy-terminal end, in accordance with standard usage and convention.
  • a protein has homology to a second protein if the two proteins have "similar” amino acid sequences.
  • the term "homologous proteins” is defined to mean that the two proteins have similar amino acid sequences.
  • a homologous protein is one that exhibits at least 65% sequence homology to the wild type protein, more preferred is at least 70% sequence homology. Even more preferred are homologous proteins that exhibit at least 75%, 80%, 85% or 90% sequence homology to the wild type protein.
  • a homologous protein exhibits at least 95%, i 98%, 99% or 99.9% sequence identity.
  • homology between two regions of amino acid sequence is interpreted as implying similarity in function.
  • residue positions that are not identical often differ by conservative amino acid substitutions.
  • a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, 1994, Methods MoI Biol 24:307-31 and 25:365-89 (herein incorporated by reference).
  • the following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
  • Sequence homology for polypeptides is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wisconsin 53705. Protein analysis software matches similar sequences using a measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap” and "Bestf ⁇ t" which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof.
  • GCG Genetics Computer Group
  • Bestf ⁇ t programs
  • a preferred algorithm when comparing a particular polypepitde sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al., J. MoI. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131- 141 (1996); Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).
  • Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.
  • the length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences.
  • polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1.
  • FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (herein incorporated by reference).
  • percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, herein incorporated by reference.
  • Specific binding refers to the ability of two molecules to bind to each other in preference to binding to other molecules in the environment.
  • “specific binding” discriminates over adventitious binding in a reaction by at least two-fold, more typically by at least 10-fold, often at least 100-fold.
  • the affinity or avidity of a specific binding reaction, as quantified by a dissociation constant is about 10 "7 M or stronger ⁇ e.g., about 10 "8 M, 10 "9 M or even stronger).
  • region refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein.
  • domain refers to a structure of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof; domains may also include distinct, non ⁇ contiguous regions of a biomolecule.
  • molecule means any compound, including, but not limited to, a small molecule, peptide, protein, glycoprotein, sugar, nucleotide, nucleic acid, lipid, etc., and such a compound can be natural or synthetic.
  • the term “consisting essentially of” will be understood to imply the inclusion of a stated integer or group of integers; while excluding modifications or other integers which would materially affect or alter the stated integer.
  • the term “consisting essentially of a stated N-glycan” will be understood to include the N-glycan whether or not that N- glycan is fucosylated at the N-acetylglucosamine (GIcNAc) which is directly linked to the asparagine residue of the glycoprotein.
  • the term “predominantly” or variations such as “the predominant” or “which is predominant” will be understood to mean the glycan species that has the highest mole percent (%) of total N-glycans after the glycoprotein has been treated with P ⁇ Gase and released glycans analyzed by mass spectroscopy, for example, MALDI-TOF MS.
  • the phrase “predominantly” is defined as an individual entity, such as a specific glycoform, is present in greater mole percent than any other individual entity. For example, if a composition consists of species A in 40 mole percent, species B in 35 mole percent and species C in 25 mole percent, the composition comprises predominantly species A, and species B would be the next most predominant species.
  • the term "essentially free of a particular sugar residue, such as fucose, or galactose and the like, is used to indicate that the glycoprotein composition is substantially devoid of N-glycans which contain such residues.
  • essentially free means that the amount of N-glycan structures containing such sugar residues does not exceed 10%, and preferably is below 5%, more preferably below 1%, most preferably below 0.5%, wherein the percentages are by weight or by mole percent.
  • substantially all of the N-glycan structures in a glycoprotein composition according to the present invention are free of fucose, or galactose, or both.
  • a glycoprotein composition "lacks” or “is lacking” a particular sugar residue, such as fucose or galactose, when no detectable amount of such sugar residue is present on the N-glycan structures at any time.
  • the glycoprotein compositions are produced by lower eukaryotic organisms, as defined above, including yeast [e.g., Pichia sp.; Saccharomyces sp.; Kluyveromyces sp.; Aspergillus sp.], and will "lack fucose," because the cells of these organisms do not have the enzymes needed to produce fucosylated N-glycan structures.
  • a composition may be "essentially free of fucose” even if the composition at one time contained fucosylated N-glycan structures or contains limited, but detectable amounts of fucosylated N-glycan structures as described above.
  • the phrase “increased binding activity” is used interchangeably with “increased binding affinity” referring to an increase in the binding of the IgG molecule with a receptor—or otherwise noted molecule.
  • the phrase “decreased binding activity” is used interchangeably with “decreased binding affinity” referring to a decrease in the binding of the IgG molecule with a receptor—or otherwise noted molecule.
  • phagocytosis is defined to be clearance of immunocomplexes. Phagocytosis is an immunological activity of immune cells — including but not limited to, macrophages and neutrophils.
  • ADCC antibody-dependent cell- mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • phagocytosis clearance of immunocomplexes
  • B cells IgG serum half-life
  • the present invention provides compositions comprising a population of glycosylated Igs having a predominant GlcNAc 2 Man 3 GlcNAc 2 N-linked glycoform.
  • the present invention also provides Igs and Ig compositions having a predominant GlcNAc 2 Man 3 GlcNAc 2 N-linked glycoform that mediates antibody effector functions, such as receptor binding.
  • the interaction between an Ig of the present invention and an Fc ⁇ RIII receptor provides an increase in direct binding activity.
  • the interaction between an Ig of the present invention and the Fc ⁇ RJIb receptor provides a decrease (or lack of) direct binding activity.
  • an Ig or Ig composition of the present invention exhibits increased binding activity conferred by the enrichment/predominance of a glycoform structure.
  • a salient feature of the present invention is that it provides Igs and Ig compositions having a predominant, specific glycoform that mediates antibody effector functions, such as an increase in ADCC activity or an increase in antibody production by B cells.
  • an Ig or Ig composition of the present invention exhibits increased ADCC activity or antibody production by B cells conferred by the enrichment/predominance of one glycoform.
  • Ig compositions having a predominant glycoform avoids production of Igs having undesired glycoforms and/or production of heterogeneous mixtures of Igs which may induce undesired effects and/or dilute the concentration of the more effective Ig glycoform(s). It is, therefore, contemplated that a pharmaceutical composition comprising Igs having predominantly GlcNAc 2 Man 3 GlcNAc 2 glycoforms will have beneficial features, including but not limited to, decreased binding to Fc ⁇ RIIb and increased binding to Fc ⁇ RIIIa and Fc ⁇ RIIIb, and therefore may well be effective at lower doses, thus having higher efficacy/potency.
  • an Ig molecule of the present invention comprises at least one GlcNAc 2 Man3GlcNAc 2 glycan structure at Asn-297 of a CH2 domain of a heavy chain on the Fc region mediating antibody effector function in an Ig molecule.
  • the GlcNAc 2 Man3GlcNAc 2 glycan structure is on each Asn-297 of each CH2 region in a dimerized Ig ( Figure 1).
  • the present invention provides compositions comprising Igs which are predominantly glycosylated with an N-glycan consisting essentially of GlcNAc 2 Man 3 GlcNAc 2 glycan structure at Asn-297 ( Figure 1).
  • one or more carbohydrate moieties found on an Ig molecule may be deleted and/or added to the molecule, thus adding or deleting the number of glycosylation sites on an Ig.
  • the position of the N-linked glycosylation site within the C H 2 region of a Ig molecule can be varied by introducing asparagines (Asn) or N-glycosylation sites at varying locations within the molecule. While Asn-297 is the N-glycosylation site typically found in murine and human IgG molecules (Kabat et al., Sequences of Proteins of Immunological Interest, 1991), this site is not the only site that can be envisioned, nor does this site necessarily have to be maintained for function.
  • a D ⁇ A molecule encoding an Ig of the present invention so that the N-glycosylation site at Asn-297 is deleted, and can further alter the D ⁇ A molecule so that one or more ⁇ -glycosylation sites are created at other positions within the Ig molecule. It is preferred that ⁇ -glycosylation sites are created within the C H 2 region of the Ig molecule.
  • glycosylation of the Fab region of an Ig has been described in 30% of serum antibodies — commonly found at Asn-75 (Rademacher et al, 1986, Biochem. Soc. Symp., 51: 131-148).
  • Glycosylation in the Fab region of an Ig molecule is an additional site that can be combined in conjunction with N-glycosylation in the Fc region, or alone.
  • the present invention provides a recombinant Ig composition having a predominant GIcNAc 2 MaH 3 GIcNAc 2 N-glycan structure, wherein said GlcNAc 2 Man 3 GlcNAc 2 glycan structure is present at a level that is at least about 5 mole percent more than the next predominant glycan structure of the recombinant Ig composition.
  • the present invention provides a recombinant Ig composition having a predominant GlcNAc 2 Man 3 GlcNAc 2 glycan structure, wherein said GlcNAc 2 Man 3 GlcNAc 2 glycan structure is present at a level of at least about 10 mole percent to about 25 mole percent more than the next predominant glycan structure of the recombinant Ig composition.
  • the present invention provides a recombinant Ig composition having a predominant GlcNAc 2 Man 3 GlcNAc 2 glycan structure, wherein said GlcNAc 2 Man 3 GlcNAc 2 glycan structure is present at a level that is at least about 25 mole percent to about 50 mole percent more than the next predominant glycan structure of the recombinant Ig composition.
  • the present invention provides a recombinant Ig composition having a predominant GlcNAc 2 Man 3 GlcNAc 2 glycan structure, wherein said GIcN Ac 2 Man 3- GlcN Ac 2 glycan structure is present at a level that is greater than about 50 mole percent more than the next predominant glycan structure of the recombinant Ig composition.
  • the present invention provides a recombinant Ig composition having a predominant GIcNAc 2 MaH 3 GIcNAc 2 glycan structure, wherein said GlcNAc 2 Man 3 GlcNAc 2 glycan structure is present at a level that is greater than about 75 mole percent more than the next predominant glycan structure of the recombinant Ig composition.
  • the present invention provides a recombinant Ig composition having a predominant GIcNAc 2 MaH 3 GIcNAc 2 glycan structure, wherein said GlcNAc 2 Man3GlcNAc 2 glycan structure is present at a level that is greater than about 90 mole percent more than the next predominant glycan structure of the recombinant Ig composition.
  • MALDI-TOF analysis of N-glycans of JC-IgG having a predominant Glc ⁇ Ac 2 Man 3 Glc ⁇ Ac 2 ⁇ -glycan (89.7%) is shown in Figure 3.
  • the effector functions of Ig binding to Fc ⁇ RIIIa and Fc ⁇ RIIIb are mediated by the Fc region of the Ig molecule. Different functions are mediated by the different domains in this region. Accordingly, the present invention provides Ig molecules and compositions in which an Fc region on an Ig molecule has a predominant GIcNAc 2 MaH 3 GIcNAc 2 N-glycan capable of carrying out an effector function. In one embodiment, the Fc region having a predominant GlcNAc 2 Man 3 .GlcNAc 2 N-glycan confers an increase in binding to Fc ⁇ RIIIa ( Figure 6) and Fc ⁇ RIIIb ( Figure 5) receptors.
  • an Fc has a predominant GIcNAc 2 MaH 3- GIcNAc 2 N-glycan. It will be readily apparent to the skilled artisan that molecules comprising the Fc region, such as immunoadhesions (Chamow and Ashkenazi, 1996, Trends Biotechnol. 14: 52-60; Ashkenazi and Chamow, 1997, Curr Opin. Immunol. 9: 195-200), Fc fusions and antibody-like molecules are also encompassed in the present invention.
  • Binding activity (affinity) of an Ig molecule to an Fc receptor may be determined by an assay.
  • An example of an Fc ⁇ RIII binding assay with IgG is described in Example 6.
  • this assay can be easily adapted for use in conjunction with assays for any immunoglobulin molecule.
  • JC-IgG an Ig made according to the present invention having predominantly GlcNAc 2 Man 3 GlcNAc 2 N-glycans has 50-100 fold increased binding activity to Fc ⁇ RIIIb and Fc ⁇ RIIIa compared with Rituximab® as shown in Figure 4 and Figure 5.
  • Fc ⁇ RIIIa gene dimorphism generates two allotypes:
  • a Rituximab®-like anti-CD20 antibody when expressed in a host cell which lacks fucosyltransferase activity, this antibody is equally effective for enhancing ADCC through both Fc ⁇ RIIIa -158F and Fc ⁇ RIIIa-158V ( ⁇ iwa et al, 2004, CHn. Cane Res. 10: 6248-6255).
  • the antibodies of certain preferred embodiments of the present invention are expressed in host cells that do not add fucose to ⁇ -glycans (e.g., P. pastoris, a yeast host lacking fucose; see Examples 1, 2). Therefore, it is contemplated that the antibodies of the present invention that lack fucose and have enhanced binding to Fc ⁇ RIIIa- 158F may be especially useful for treating many patients exhibiting a reduced clinical response to Rituximab®.
  • the present invention provides Ig molecules and compositions in which an Fc region on an Ig molecule has a predominant GlcNAc 2 Man 3 GlcNAc 2 N- glycan capable of carrying out an effector function.
  • an Fc region of an Ig having a predominant GIcNAc 2 MaH 3 GIcNAc 2 N-glycan confers a decrease in binding to an Fc ⁇ RIIb receptor. It will be readily apparent to the skilled artisan that molecules comprising an Fc region, such as immunoadhesions (Chamow and
  • Binding activity (affinity) of an Ig molecule to an Fc receptor may be determined by an assay.
  • An example of an Fc ⁇ RIIb binding assay with IgGl is disclosed in Example 6.
  • This disclosed assay can be easily adapted for use in connection to any immunoglobulin molecule.
  • JC-IgG (an Ig of the present invention) having predominant GlcNAc 2 Man 3 .
  • GIcNAc 2 N-glycans has 2-3 fold decreased binding activity to Fc ⁇ RIIb compared with Rituximab® as shown in Figure 6.
  • the increase in Fc ⁇ RIIIa or Fc ⁇ RIIIb binding of the Ig having Glc ⁇ Ac 2 Man 3 Glc ⁇ Ac 2 as the predominant N-glycan confers an increase in Fc ⁇ RIII-mediated ADCC. It is well established that the Fc ⁇ RIII (CD 16) receptor is responsible for ADCC activity (Daeron et al, 1997, Annu. Rev. Immunol. 15: 203- 234).
  • the decrease in Fc ⁇ RIIb binding of the Ig having Glc ⁇ Ac 2 Man 3- Glc ⁇ Ac 2 as the predominant N-glycan confers an increase in ADCC (Clynes et al., 2000).
  • An Ig molecule of the present invention exhibits increased ADCC activity conferred by the presence of a predominant GIcNAc 2 MaH 3 GIcNAc 2 glycan.
  • Example 7 An example of in vitro assays measuring B-cell depletion and fluorescence release ADCC assays are disclosed in Example 7.
  • these disclosed assays can be easily adapted for requirements pertaining to any immunoglobulin molecule.
  • an in vivo ADCC assay in an animal model can be adapted for any specific IgG from Borchmann et al., 2003, Blood, 102: 3737- 3742, Niwa et al., 2004, Cancer Research, 64: 2127-2133 and Example 7.
  • ITAM motifs
  • an Ig molecule of the present invention can mediate a decrease in Fc ⁇ RIIb receptor binding resulting in the activation of B cells which in turn, catalyzes antibody production by plasma cells (Parker, D.C. 1993, Annu. Rev. Immunol. 11 : 331 -360).
  • An example of an assay measuring antibody production by B cells with IgGl is described in Example 6.
  • TNF- ⁇ also increases the ability of neutrophils to bind and phagocytize IgG- coated erythrocytes (Capsoni et al, 1991, J. CHn. Lab Immunol. 34: 115-124). It is therefore contemplated that the Ig molecules and compositions of the present invention that show an increase in binding to Fc ⁇ RIII, may confer an increase in expression of TNF- ⁇ .
  • Fc ⁇ RIII receptor activity has been shown to increase the secretion of lysosomal beta-glucuronidase as well as other lysosomal enzymes (Kavai et al, 1982, Adv. Exp Med. Biol. 141: 575-582; Ward and Ghetie, 1995, Therapeutic Immunol, 2: 77-94). Furthermore, an important step after the engagement of immunoreceptors by their ligands is their internalization and delivery to lysosomes (Bonnerot et al, 1998, EMBOJ., 17: 4906-4916). It is therefore contemplated that an Ig molecule or composition of the present invention that shows an increase in binding to Fc ⁇ RIIIa and Fc ⁇ RIIIb may confer an increase in the secretion of lysosomal enzymes.
  • Fc ⁇ RIIIb Present exclusively on neutrophils, Fc ⁇ RIIIb plays a predominant role in the assembly of immune complexes, and its aggregation activates phagocytosis, degranulation, and the respiratory burst leading to destruction of opsonized pathogens. Activation of neutrophils leads to secretion of a proteolytically cleaved soluble form of the receptor corresponding to its two extracellular domains. Soluble Fc ⁇ RIIIb
  • the present invention thus provides an immunoglobulin molecule comprising an N-glycan consisting essentially of GlcNAc 2 Man 3 GlcNAc 2 ; and provides a composition comprising immunoglobulins and a plurality of N-glycans attached thereto, wherein the predominant N-glycan within said plurality of N-glycans consists essentially of GIcNAc 2 Ma ⁇ GIcNAc 2 .
  • the predominance of said GlcNAc 2 Man3.G IcNAc 2 N-glycan on an immunoglobulin preferably confers desired therapeutic effector activity in addition to the improved binding to Fc ⁇ RIIIa
  • the present invention provides an IgGl composition that comprises GIcNAc 2 MaH 3 GIcNAc 2 as the predominant N-glycan attached to IgGl molecules.
  • the present invention comprises an IgG2 composition that comprises GIcNAc 2 Ma ⁇ GIcNAc 2 as the predominant N-glycan attached to IgG2 molecules.
  • the present invention comprises an IgG3 composition that comprises Glc ⁇ Ac 2 Man 3 Glc ⁇ Ac 2 as the predominant N-glycan attached to IgG3 molecules.
  • the present invention comprises an IgG4 composition that comprises GlcNAc 2 Man 3 GlcNAc 2 as the predominant N- glycan attached to IgG4 molecules.
  • the present invention can be applied to all of the five major classes of immunoglobulins: IgA, IgD, IgE, IgM and IgG.
  • a preferred immunoglobulin of the present invention is a human IgG and preferably from one of the subtypes IgGl, IgG2, IgG3 or IgG4. More preferably, an immunoglobulin of the present invention is an IgGl molecule.
  • the invention provides a method for producing a recombinant Ig molecule having an N-glycan consisting essentially of a GIcNAc 2 MaH 3 GIcNAc 2 glycan structure at Asn-297 of the CH2 domain, wherein the Ig molecule mediates antibody effector function and activity, and similarly, an immunoglobulin composition wherein the predominant N-glycan attached to the immunoglobulins is GIcNAC 2 Ma ⁇ GIcN Ac 2 .
  • the heavy and light chains of the Ig are synthesized using overlapping oligonucleotides and are separately cloned into an expression vector (Example 1) for expression in a host cell.
  • recombinant Ig heavy and light chains are expressed in a host strain which catalyzes predominantly the addition of GIcNAc 2 Ma ⁇ GIcNAc 2 .
  • this glycoform structure is more specifically denoted as [(GlcNAc ⁇ l,2-
  • this predominant glycan can be added to an asparagine at a different site within the Ig molecule (other than Asn-297), or in combination with the N-glycosylation site in the Fab region.
  • One aspect of the present invention provides recombinant lower eukaryotic host cells which may be used to produce immunoglobulin or antibody molecules with predominantly the GIcNAc 2 MaH 3 GIcNAc 2 glycoform, which is an advantage compared with compositions of glycoproteins expressed in mammalian cells which naturally produce said glycoform in low yield. It is another advantage of the present invention that compositions of glycoproteins are provided with predetermined glycosylation patterns that are readily reproducible. The properties of such compositions are assessed and optimized for desirable properties, while adverse effects may be minimized or avoided altogether.
  • the present invention also provides methods for producing recombinant host cells that are engineered or selected to express one or more nucleic acids for the production of Ig molecules comprising an N-glycan consisting essentially of GlcNAc 2 Man 3 GlcNAc 2 and Ig compositions having predominantly a GlcNAc 2 Man 3 GlcNAc 2 glycan structure.
  • recombinant host cells preferably recombinant lower eukaryotic host cells, are used to produce said Ig molecules and compositions having predominantly GIcNAc 2 MaH 3 GIcNAc 2 glycan.
  • the invention comprises the glycoproteins obtainable from recombinant host cells or by the methods of the present invention.
  • the host cells of the invention may be transformed with vectors encoding the desired Ig regions, and with vectors encoding one or more of the glycosylation-related enzymes described herein, and then selected for expression of a recombinant Ig molecule or composition having a predominant GIcNAc 2 Ma ⁇ GIcNAc 2 N-glycan.
  • the recombinant host cell of the present invention may be a eukaryotic or prokaryotic host cell, such as an animal, plant, insect, bacterial cell, or the like which has been engineered or selected to produce an Ig composition having predominantly GlcNAc 2 Man 3 GlcNAc 2 N-glycan structures.
  • the recombinant host cell of the present invention is a lower eukaryotic host cell which has been genetically engineered as described in the art (WO 02/00879, WO 03/056914, WO 04/074498, WO 04/074499, Choi etal., 2003, PNAS, 100: 5022-5027; Hamilton et al, 2003, Nature, 301 : 1244-1246 and
  • WO 02/00879 discloses the teachings for expressing a glycoprotein having specifically Glc ⁇ Ac 2 Man 3 Glc ⁇ Ac 2 N-glycan structures in lower eukaryotic hosts. More specifically, WO 03/056914 discloses methods to obtain at least 75 mole percent Glc ⁇ Ac 2 Man 3 Glc ⁇ Ac 2 N-glycan structures on a glycoprotein (Fig. 22), as well as disclosure of immunoglobulins in Fig. 30, 31 and paragraphs 207-211.
  • a vector encoding an IgGl for example an AOXl/pPICZA vector containing JC-IgG (Example 1) is introduced into the yeast P. pastoris YAS309 strain.
  • This YAS309 strain is similar to the YSH44 strain with the K3 reporter protein removed (Hamilton et al, 2003, Science, 301 :
  • Mannosidase II activity was then reintroduced at the AMR2 locus, resulting in the reintroduction of the mannosidase II activity and the loss of the AMR2 gene, thus eliminating ⁇ - mannosylation as described (US Pat. Appl. No. 1 1/118008).
  • Glycoproteins from this YAS309 strain have predominantly GlcNAc 2 Man 3 GlcNAc 2 N-glycans, and with additional ⁇ -galactosidase treatment (Example 3), N-glycans on JC-IgG have 89.7% GlcNAc 2 Man 3 GlcNAc 2 ( Figure 3).
  • an antibody of the present invention can be expressed using several methods known in the art ⁇ Monoclonal Antibody Production Techniques and Applications, pp. 79-97 (Marcel Dekker, Inc., New York, 1987).
  • the immunoglobulin of the present invention can be expressed in a lower eukaryotic host which synthesizes the GlcNAc 2 Man 3 GlcNAc 2 N-glycans in vivo. Such host would be engineered in an ⁇ alg3 mutant as described (WO
  • 6,051,419 describe a selection system based on disrupting the URA3 gene in P. pastoris.
  • the PpURA3- or Pp[/&45-blaster cassettes are used to disrupt the URA3, URA5 or any gene in the uracil biosynthesis pathway, allowing for both positive and negative selection, based on auxotrophy for uracil and resistance to 5-fluoroorotic acid (5FOA) (Boeke, et al, 1984, MoI Gen. Genet, 197: 345-346).
  • 5FOA 5-fluoroorotic acid
  • Appl. No. 11/1 18008 discloses a method for the elimination of ⁇ -mannosylation.
  • organ is selected for heterologous protein expression that may or may not need to be engineered to express Igs having a predominant glycan structure.
  • the Examples provided herein are examples of one method for carrying out the expression of Ig with a particular glycan at Asn-297 or another N-glycosylation site, or both.
  • One skilled in the art can easily adapt these details of the invention and examples for any protein expression host system (organism).
  • protein expression host systems including animal, plant, insect, bacterial cells and the like may be used to produce Ig molecules and compositions according to the present invention.
  • Such protein expression host systems may be engineered or selected to express a predominant glycoform or alternatively may naturally produce glycoproteins having predominant glycan structures.
  • engineered protein expression host systems producing a glycoprotein having a predominant glycoform include gene knockouts/mutations (Shields et al, 2002, JBC, 277: 26733-26740); genetic engineering in (Umana et al, 1999, Nature Biotech., 17: 176-180) or a combination of both.
  • glycoproteins naturally express a predominant glycoform — for example, chickens, humans and cows (Raju et al, 2000, Glycobiology, 10: 477-486).
  • the expression of an Ig glycoprotein or composition having predominantly one specific glycan structure according to the present invention can be obtained by one skilled in the art by selecting at least one of many expression host systems.
  • Further expression host systems found in the art for production of glycoproteins include: CHO cells: Raju WO9922764A1 and Presta WO03/035835A1; hybridroma cells: Trebak et al, 1999, J. Immunol Methods, 230: 59-70; insect cells: Hsu et al, 1997, JBC, 272:9062-970, and plant cells: Gerngross et al., WO04/074499A2.
  • antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al. (1990) Nature, 348:552-554 (1990), using the antigen of interest to select for a suitable antibody or antibody fragment.
  • Recombinant Ig molecules produced according to the methods of the present invention can be purified according to methods outlined in Example 3.
  • Figure 2 shows an SDS-PAGE Coomassie stained gel of JC-IgG purified from YAS309.
  • a purified Ig antibody has GlcNAc 2 Man 3 GlcNAc 2 as the predominant N-glycan.
  • the glycan analysis and distribution on any Ig molecule can be determined by several mass spectroscopy methods known to one skilled in the art, including but not limited to: HPLC, NMR, LCMS and MALDI-TOF MS.
  • the glycan distribution is determined by MALDI-TOF MS analysis as disclosed in Example 5.
  • Figure 3 shows a MALDI-TOF spectra of JC- IgG purified from YAS309 and treated with ⁇ -galactosidase (Example 3).
  • This MALDI-TOF shows approximately 89.7 mole % of the total N-glycans are GlcNAc 2 Man 3 GlcNAc 2 .
  • Antibodies of the invention can be incorporated into pharmaceutical compositions comprising the antibody as an active therapeutic agent and a variety of other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pennsylvania, 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 affect the biological activity of the combination. 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 can also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • compositions for parenteral administration are sterile, substantially isotonic, pyrogen-free and prepared in accordance with GMP of the FDA or similar body.
  • Antibodies can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water, oils, saline, glycerol, or ethanol.
  • a pharmaceutical carrier can be a sterile liquid such as water, oils, saline, glycerol, or ethanol.
  • auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions.
  • Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil.
  • glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • Antibodies can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient.
  • compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above (see Langer, Science 249, 1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97-119 (1997).
  • Antibodies of the invention can also be incorporated into a variety of diagnostic kits and other diagnostic products such as an array.
  • Antibodies are often provided prebound to a solid phase, such as to the wells of a microtiter dish. Kits also often contain reagents for detecting antibody binding, and labeling providing directions for use of the kit. Immunometric or sandwich assays are a preferred format for diagnostic kits (see US 4,376,110, 4,486,530, 5,914,241, and 5,965,375).
  • Antibody arrays are described by e.g., US 5,922,615, US 5,458,852, US 6,019,944, and US 6,143,576.
  • the present invention provides glycoprotein compositions which comprise predominantly a particular glycoform on the glycoprotein. It is a feature of the present invention that when administered to mammals including humans, pharmaceutical compositions comprising the novel glycoprotein compositions, in preferred embodiments, advantageously exhibit superior in vivo properties when compared to other glycoprotein compositions having similar primary structure.
  • the novel compositions of the invention may be used wherever the glycoprotein pharmaceutical agent is presently used and may advantageously provide improved properties as well as increased uniformity between and throughout production lots.
  • the preparations of the invention can be incorporated into solutions, unit dosage forms such as tablets and capsules for oral delivery, as well as into suspensions, ointments and the like, depending on the particular drug or medicament and its target area.
  • the present invention provides novel compositions for glycoprotein pharmaceutical agents, drugs or medicaments wherein the glycoprotein comprises an immunoglobulin molecule and the composition comprises predominantly particular glycoforms of the glycoprotein agent.
  • compositions are provided comprising an immunoglobulin glycoprotein having predominantly an N-linked oligosaccharide of the Glc ⁇ Ac 2 Man 3 Glc ⁇ Ac 2 glycan structure as described herein.
  • the glycoprotein is an antibody and especially may be a monoclonal antibody.
  • the invention further provides methods and tools for producing the compositions of the invention.
  • the invention further encompasses pharmaceutical compositions comprising the glycoform preparations of the invention.
  • the compositions are preferably sterile.
  • the composition is an aqueous solution, preferably the glycoprotein is soluble.
  • the composition is a lyophilized powder, preferably the powder can be reconstituted in an appropriate solvent.
  • the invention involves a method for the treatment of a disease state comprising administering to a mammal in need thereof a therapeutically effective dose of a pharmaceutical composition of the invention. It is a further object of the invention to provide the glycoform preparations in an article of manufacture or kit that can be employed for purposes of treating a disease or disorder.
  • the Ig molecules of the present invention having predominantly GlcNAc2Man3.GlcNAc 2 N-glycans have many therapeutic applications for indications such as cancers, inflammatory diseases, infections, immune diseases, autoimmune diseases including idiopathic thrombocytopenic purpura, arthritis, systemic lupus erythrematosus, and autoimmune hemolytic anemia.
  • the light (L) and heavy (H) chains of the JC-IgGl consists of mouse variable regions and human constant regions.
  • the mouse variable light chain is disclosed as SEQ ID NO: 1 (GenBank #AF013576) and mouse variable heavy chain as SEQ ID NO: 2 (GenBank #AF013577).
  • the heavy and light chain sequences were synthesized using overlapping oligonucleotides purchased from Integrated DNA Technologies (IDT).
  • the Fc fragment was synthesized using 12 overlapping oligonucleotides (SEQ ID NOs: 27-38) which were annealed in a similar overlapping PCR reaction. Both Fab and Fc fragments of the heavy chain were then annealed using a 5' EcoRI primer (SEQ ID NO: 15) corresponding to the 5' end of the heavy Fab fragment and a 3' Kpnl primer (SEQ ID NO: 39) corresponding to the 3' end of the Fc fragment using pFU Turbo polymerase (Stratagene) producing the 1,330 base pair heavy chain. Using 5' EcoRI and 3' Kpnl sites encoded in the primers, the heavy chain was cloned into a pPICZa vector.
  • the A0X2 promoter sequence which functions as an integration locus, was subcloned into a final pPICZa vector.
  • a Bglll-BstBl fragment containing the AOXl promoter and a BstBl-BamHI fragment containing an HSA sequence from a human liver cDNA library (SEQ ID NO:40), thrombin site (SEQ ID NO:41) and JC- IgG light chain were both subcloned into the BamHI site of this AOX2/pPICZa vector.
  • RituximabWRituxan® is an anti-CD20 mouse/ human chimeric IgGl purchased from Biogen-IDEC/Genentech, San Francisco, CA.
  • PCR amplification An Eppendorf Mastercycler was used for all PCR reactions. PCR reactions contained template DNA, 125 ⁇ M dNTPs, 0.2 ⁇ M each of forward and reverse primer, Ex Taq polymerase buffer (Takara Bio Inc.), and Ex Taq polymerase or pFU Turbo polymerase buffer (Stratagene) and pFU Turbo polymerase. The DNA fragments were amplified with 30 cycles of 15 sec at 97°C, 15 sec at 55°C and 90 sec at 72°C with an initial denaturation step of 2 min at 97°C and a final extension step of 7 min at 72°C.
  • PCR samples were separated by agarose gel electrophoresis and the DNA bands were extracted and purified using a Gel Extraction Kit from Qiagen. All DNA purifications were eluted in 10 mM Tris, pH 8.0 except for the final PCR (overlap of all three fragments) which was eluted in deionized H 2 O.
  • the vector DNA of pJC140 was prepared by adding sodium acetate to a final concentration of 0.3 M. One hundred percent ice cold ethanol was then added to a final concentration of 70% to the DNA sample. The DNA was pelleted by centrifugation (1200Og x lOmin) and washed twice with 70% ice cold ethanol. The DNA was dried and resuspended in 50 ⁇ l of 1OmM Tris, pH 8.0.
  • the YAS309 yeast culture (supra) to be transformed was prepared by expanding a smaller culture in BMGY (buffered minimal glycerol: 100 mM potassium phosphate, pH 6.0; 1.34% yeast nitrogen base; 4xlO "5 % biotin; 1% glycerol) to an O.D. of -2-6.
  • BMGY bouffered minimal glycerol: 100 mM potassium phosphate, pH 6.0; 1.34% yeast nitrogen base; 4xlO "5 % biotin; 1% glycerol
  • the yeast cells were then made electrocompetent by washing 3 times in IM sorbitol and resuspending in ⁇ l-2 mis IM sorbitol.
  • Vector DNA (1-2 ⁇ g) was mixed with 100 ⁇ l of competent yeast and incubated on ice for 10 min.
  • Yeast cells were then electroporated with a BTX Electrocell Manipulator 600 using the following parameters; 1.5 kV, 129 ohms, and 25 ⁇ F.
  • YPDS 1% yeast extract, 2% peptone, 2% dextrose, IM sorbitol
  • Transformed yeast was subsequently plated on selective agar plates containing zeocin. Culture conditions for IsGl production in P. vastoris
  • a single colony of YAS309 transformed with pJC140 was inoculated into 10ml of BMGY media (consisting of 1% yeast extract, 2% peptone, 10OmM potassium phosphate buffer (pH 6.0), 1.34% yeast nitrogen base, 4 ⁇ 10 ⁇ 5 % biotin, and 1% glycerol) in a 50ml Falcon Centrifuge tube. The culture was incubated while shaking at 24°C/170-190 rpm for 48 hours until the culture was saturated. 100ml of BMGY was then added to a 500ml baffled flask. The seed culture was then transferred into a baffled flask containing the 100ml of BMGY media.
  • BMGY media consisting of 1% yeast extract, 2% peptone, 10OmM potassium phosphate buffer (pH 6.0), 1.34% yeast nitrogen base, 4 ⁇ 10 ⁇ 5 % biotin, and 1% glycerol
  • This culture was incubated with shaking at 24°C/170-190rpm for 24 hours.
  • the contents of the flask was decanted into two 50ml Falcon Centrifuge tubes and centrifuged at 3000rpm for 10 minutes.
  • the cell pellet was washed once with 20ml of BMGY without glycerol, followed by gentle resuspension with 20ml of BMMY (BMGY with 1% MeOH instead of 1% glycerol).
  • the suspended cells were transferred into a 250ml baffled flask.
  • the culture was incubated with shaking at 24°C/170-190rpm for 24 hours.
  • the contents of the flask was then decanted into two 50ml Falcon Centrifuge tubes and centrifuged at 3000rpm for 10 minutes.
  • the culture supernatant was analyzed by ELISA to determine approximate antibody titer prior to protein isolation. Quantification of antibody in culture supernatants was performed by enzyme linked immunosorbent assays (ELISAs): High binding microtiter plates (Costar) were coated with 24 ⁇ g of goat anti-human Fab (Biocarta, Inc, San Diego, CA) in 10 ml PBS, pH 7.4 and incubate over night at 4° C. Buffer was removed and blocking buffer (3% BSA in PBS), was added and then incubated for 1 hour at room temperature. Blocking buffer was removed and the plates were washed 3 times with PBS.
  • ELISAs enzyme linked immunosorbent assays
  • Monoclonal antibodies were captured from the culture supernatant using a Streamline Protein A column. Antibodies were eluted in Tris-Glycine pH 3.5 and neutralized using IM Tris pH 8.0. Further purification was carried out using hydrophobic interaction chromatography (HIC). The specific type of HIC column depends on the antibody. For the JC-IgG a phenyl sepharose column (can also use octyl sepharose) was used with 2OmM Tris (7.0), IM (NH 4 ) 2 S ⁇ 4 buffer and eluted with a linear gradient buffer of IM to OM (NH ⁇ 2 SO 4 .
  • HIC hydrophobic interaction chromatography
  • the antibody fractions from the phenyl sepharose column were pooled and exchanged into 5OmM NaOAc/Tris pH 5.2 buffer for final purification through a cation exchange (SP Sepharose Fast Flow) (GE Healthcare) column.
  • Antibodies were eluted with a linear gradient using 5OmM Tris, IM NaCl (pH 7.0)
  • JC-IgG Purified JC-IgG was mixed with an appropriate volume of sample loading buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with precast gels according to the manufacturer's instructions (NuPAGE bis-Tris electrophoresis system; Invitrogen Corporation, Carlsbad, Calif.). The gel proteins were stained with Coomassie brilliant blue stain (Bio-Rad, Hercules, CA). See Figure 2.
  • the concentration of protein chromatography fractions were determined using a Bradford assay (Bradford, M. 1976, Anal. Biochem. (1976) 72, 248-254) using albumin as a standard (Pierce, Rockford, IL)
  • MALDI-TOF MS MALDI-TOF MS.
  • MALDI-TOF analysis of aspargine-linked oligosaccharides N-linked glycans were released from JC-IgG using a modified procedure of Papac et al., Glycobiology 8, 445-454 (1998). A sample of the antibodies was reduced and carboxymethylated and the membranes were blocked, the wells were washed three times with water. The IgG proteins were deglycosylated by the addition of 30 ul of 10 mM Nh4HCO3 (pH 8.3) containing 1 mU of N-glycanase (EMD Biosciences, La Jolla, CA).
  • the solution containing the glycans was removed by centrifugation and evaporated to dryness.
  • the dried glycans from each well were dissolved in 15 ⁇ l of water, and 0.5 ⁇ l was spotted on stainless- steel sample plates and mixed with 0.5 ⁇ l of S-DHB matrix (9 mg/ml of dihydroxybenzoic acid/1 mg/ml of 5-methoxy-salicylic acid in 1:1 water/acetonitrile/0.1% trifluoroacetic acid) and allowed to dry.
  • Ions were generated by irradiation with a pulsed nitrogen laser (337 nm) with a 4-ns pulse time.
  • the instrument was operated in the delayed extraction mode with a 125-ns delay and an accelerating voltage of 20 kV.
  • the grid voltage was 93.00%
  • guide wire voltage was 0.1%
  • the low mass gate was 875 Da.
  • Spectra were generated from the sum of 100-200 laser pulses and acquired with a 500-MHz digitizer.
  • (Man)s(GlcNAc) 2 oligosaccharide was used as an external molecular weight standard. All spectra were generated with the instrument in the positive-ion mode .
  • High binding microtiter plates (Costar) were coated with lOug of antigen in PBS, pH 7.4 and incubate over night at 4° C. Buffer was removed and blocking buffer (3% BSA in PBS), was added and then incubated for 1 hour at room temperature. Blocking buffer was removed and the plates were washed 3 times with PBS. After the last wash, increasing amounts of purified antibody were added from 0.2ng to lOOng and incubated for 1 hour at room temperature. Plates were then washed with PBS + 0.05% Tween20. After the last wash, anti-human Fc-HRP was added in a 1 :2000 PBS solution, and then incubated for 1 hour at room temperature. Plates were then washed 4 times with PBS-Tween20. Plates were analyzed using TMB substrate kit following manufacturer's instructions (Pierce Biotechnology).
  • Fc receptor binding assays for Fc ⁇ RIIb, Fc ⁇ RIIIa and Fc ⁇ RIIIb were carried out according to the protocols previously described (Shields et al. , 2001 , J.Biol. Chem, 276: 6591-6604).
  • Fc ⁇ RIII binding Fc ⁇ RIIIb ( Figure 4) and FyRIIb ( Figure 6) fusion proteins at 1 ⁇ g/ml or Fc ⁇ RIIIa-LF ( Figure 5) fusion proteins at 0.8 ⁇ g/m in PBS, pH 7.4, were coated onto ELISA plates (Nalge-Nunc, Naperville, IL) for 48 h at 4 0 C.
  • JC-IgG or DX-IgG dimeric complexes were prepared in 1% BSA in PBS by mixing 2:1 molar amounts of JC-IgG or DX-IgG and HRP-conjugated F(Ab')2anti- F(Ab')2 at 25°C for 1 h. Dimeric complexes were then diluted serially at 1:2 in 1% BSA/PBS and coated onto the plate for 1 hour at 25°C.
  • the substrate used is 3,3',5,5'-tetramethylbenzidine (TMB) (Vector Laboratories). Absorbance at 450 nm was read following instructions of the manufacturer (Vector Laboratories).
  • BSA bovine serum albumin
  • B-cell depletion assay 10 ⁇ l of 100 ⁇ g/ml solution of antibody or stain buffer is added to 90 ⁇ l of SB matrix and incubated for 1 hour at 37°C. Samples are stained immediately with anti-CD 19-FITC and anti-CD45-PE for 30 minutes at 25 0 C. Samples are then fixed in 1% formaldehyde and run in triplicate. Quantification of B-cell depletion is obtained by flow cytometry. Flow cytometric analysis of B-cell depletion: A FACS Calibur (BD Biosciences) instrument equipped with an automated FACS Loader and Cell Quest Software is used for acquisition and analysis of all samples.
  • FACS Calibur BD Biosciences
  • Cytometer QC and setup include running CaliBrite beads and SpheroTech rainbow beads (BD Biosciences) to confirm instrument functionality and detector linearity. Isotype and compensation controls are run with each assay to confirm instrument settings. Percent of B cells of total lymphocytes is obtained by the following gating strategy. The lymphocyte population is marked on the forward scatter/side scatter scattegram to define Region 1 (Rl). Using events in Rl, fluorescence intensity dot plots are displayed for CD 19 and CD45 markers. Fluorescently labeled isotype controls are used to determine respective cutoff points for CD 19 and CD45 positivity. %B is determined using CellQuest as a fraction of cells in Rl region that have CD19-positive, CD45-positive phenotype.
  • PBMC isolation Peripheral venous blood from healthy individuals or blood donors (10-20) is collected into heparinised vacutainer tubes (Becton Dickinson Vacutainer Systems, Rutherford, NJ, USA). Approximately 5ml of blood is required for implanting 2 mice. Peripheral blood mononuclear cells (PBMCs) are separated by centrifugation using OptiPrep following manufacturer's instructions.
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs are washed once with complete culture media (CM) consisting of RPMI 1640, 2mM L-glutamine, 100 IU/ml penicillin, 100g/ml streptomycin (Gibco/BRL) and supplemented with 20% fetal calf serum, and then resuspended at a concentration of lxioVml CM and transfered to a 250 ml culture flask (Falcon, NJ, USA) for monocyte depletion.
  • CM complete culture media
  • non-adherent cells are recovered, washed once with culture media and the peripheral blood lymphocytes (PBLs) are adjusted to a concentration of 2.5x10 7 /ml CM.
  • Fluorescent dye-release ADCC The premise behind the ADCC assay is that antibody binding to CD20 or CD40 antigen presenting target cells (Raji cell line or BCLl -3B3 cells, respectively) stimulates target cell binding to Fc ⁇ receptors on the effector cells. This in turn promotes lysis of the target cells presenting the antigen, releasing an internal fluorescent dye that can be quantified. Alamar-blue fluorescence is used in place of 51 Cr labeling of the target cells.
  • effector to target cell ratio can be 100: 1, 50:1. 25:1 and 12.5:1) in 96 well tissue culture plates and incubated for 4h hours at 37 temperature and 5% CO2 to facilitate lysis of the Raji or BCLl -3B3 cells. 50 ⁇ l of Alamar blue is added and the incubation is continued for another 5 hours to allow for uptake and metabolism of the dye into its fluorescent state.
  • In vivo ADCC activity can be assayed using a mouse model engrafted with human peripheral blood mononuclear cells (PMBCs) from healthy donors which include heterozygous (Fc ⁇ RIIIa-LF/Fc ⁇ RIIIa-LV) and homozygous (Fc ⁇ RIIIa-LV/Fc ⁇ RIIIa-LV and Fc ⁇ RIIIa-LF/Fc ⁇ RIIIa-LF) genotypes.
  • PMBCs peripheral blood mononuclear cells
  • Igs having a predominant N-glycan are assayed for enhanced ADCC activity compared with Rituximab® or any other control antibody.
  • a detailed and sufficient protocol for this in vivo ADCC assay is found in Niwa et al., 2004, supra.
  • SEQ ID NO: 6 mh285L-4 caaagtgaaatcagtaccagaaccagaaccagaaaatctatctggaacaccagaaaatctgttagaaactctgta
  • SEQ ID NO: 7 mh285L-5 tctggtactgatttcactttgaagatttctagagttgaagctgaagatttgggtgtttacttctgtttgcaagttac
  • SEQ ID NO: 8 mh285L-6 caacagttctctttaatttccaaagtagtaccaccaccaaaaagtgtatggaacatgagtaacttgcaaacagaagtaa
  • SEQ ID NO: 20 mh285H-6 agtagaagcagaagaaacagtaacagaagtaccttgaccccagtaatccaatctagcacagtagtaagtagcagta
  • SEQ ID NO: 21 mh285H-7 ctgtttcttctgcttctactaagggtccatctgtttttccactttgctccatcttctaagtctacttctggtggta
  • SEQ ID NO: 38 Fc-12 5' ttatttacctggagacagggaaagagacttctgagtgtaatggttatgcaaag 3'

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Cette invention se rapporte à des compositions de glycoprotéines d’immunoglobulines à prédominance de structures N-glycanes sur une glycoprotéine d’immunoglobuline, qui confèrent une fonction effectrice spécifique. Cette invention concerne également des compositions pharmaceutiques contenant un anticorps ayant une structure N-glycane enrichie donnée, ladite structure N-glycane étant GlcNAc2Man3GlcNAc2.
PCT/US2005/025652 2004-07-21 2005-07-19 Immunoglobulines contenant principalement un glycoforme de type glcnac2man3glcnac2 WO2006014679A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002573745A CA2573745A1 (fr) 2004-07-21 2005-07-19 Immunoglobulines contenant principalement un glycoforme de type glcnac2man3glcnac2
AU2005269759A AU2005269759A1 (en) 2004-07-21 2005-07-19 Immunoglobulins comprising predominantly a GlcNAc2Man3GlcNAc2 glycoform
JP2007522673A JP2008512353A (ja) 2004-07-21 2005-07-19 主にＧｌｃＮＡｃ２Ｍａｎ３ＧｌｃＮＡｃ２グリコフォームを含むイムノグロブリン
EP05790298A EP1776385A1 (fr) 2004-07-21 2005-07-19 Immunoglobulines contenant principalement un glycoforme de type glcnac2man3glcnac2

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US59005104P 2004-07-21 2004-07-21
US59001104P 2004-07-21 2004-07-21
US60/590,011 2004-07-21
US60/590,051 2004-07-21

Publications (1)

Publication Number Publication Date
WO2006014679A1 true WO2006014679A1 (fr) 2006-02-09

Family

ID=35262113

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2005/025652 WO2006014679A1 (fr) 2004-07-21 2005-07-19 Immunoglobulines contenant principalement un glycoforme de type glcnac2man3glcnac2

Country Status (5)

Country Link
EP (1) EP1776385A1 (fr)
JP (1) JP2008512353A (fr)
AU (1) AU2005269759A1 (fr)
CA (1) CA2573745A1 (fr)
WO (1) WO2006014679A1 (fr)

Cited By (48)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007084926A2 (fr) * 2006-01-17 2007-07-26 Biolex Therapeutics, Inc. Compositions et méthodes destinées à l'humanisation et à l'optimisation de n-glycanes chez des plantes
US7326681B2 (en) 2000-06-28 2008-02-05 Glycofi, Inc. Methods for producing modified glycoproteins
WO2009016164A1 (fr) * 2007-08-01 2009-02-05 Glaxo Group Limited Nouveaux anticorps
WO2010049177A1 (fr) * 2008-10-31 2010-05-06 Lonza Ltd. Nouveau outils pour produire des protéines glycosylées dans des cellules hôtes
WO2010070094A1 (fr) 2008-12-19 2010-06-24 Glaxo Group Limited Protéines de liaison à la myostatine
WO2010075249A2 (fr) 2008-12-22 2010-07-01 Genentech, Inc. Méthode de traitement de la polyarthrite rhumatoïde avec des antagonistes de cellules b
US7923011B2 (en) 2006-10-12 2011-04-12 Genentech, Inc. Antibodies to lymphotoxin-alpha
WO2011094259A2 (fr) 2010-01-28 2011-08-04 Glaxo Group Limited Protéines de liaison à cd127
WO2011098424A2 (fr) 2010-02-09 2011-08-18 Glaxo Group Limited Traitement d'un trouble du métabolisme
WO2011107480A1 (fr) 2010-03-03 2011-09-09 Glaxo Group Limited Protéines de liaison à un antigène spécifiques pour un composant p de substance amyloïde sérique
US8025879B2 (en) 2008-09-26 2011-09-27 Eureka Therapeutics, Inc. Modified glycoproteins and uses thereof
WO2011151432A1 (fr) 2010-06-03 2011-12-08 Glaxo Group Limited Protéines humanisées de liaison à un antigène dirigées contre la myostatine
WO2012069557A1 (fr) 2010-11-24 2012-05-31 Glaxo Group Limited Protéines multispécifique se liant à un antigène et ciblant hgf
WO2012069433A2 (fr) 2010-11-23 2012-05-31 Glaxo Group Limited Protéines de liaison à l'antigène
WO2013014208A2 (fr) 2011-07-27 2013-01-31 Glaxo Group Limited Constructions de liaison à un antigène
WO2014029752A1 (fr) 2012-08-22 2014-02-27 Glaxo Group Limited Anticorps anti-lrp6
WO2014140180A1 (fr) 2013-03-15 2014-09-18 Glaxosmithkline Intellectual Property Development Limited Protéines de liaison anti-lag-3
US8877462B2 (en) 2000-06-28 2014-11-04 Glycofi, Inc. Combinatorial DNA library for producing modified N-glycans in lower eukaryotes
US8986949B2 (en) 2003-02-20 2015-03-24 Glycofi, Inc. Endomannosidases in the modification of glycoproteins in eukaryotes
WO2016028656A1 (fr) 2014-08-19 2016-02-25 Merck Sharp & Dohme Corp. Anticorps anti-tigit
WO2016059602A2 (fr) 2014-10-16 2016-04-21 Glaxo Group Limited Méthodes de traitement du cancer et compositions associées
WO2017075124A1 (fr) 2015-10-29 2017-05-04 Merck Sharp & Dohme Corp. Anticorps neutralisant le virus syncytial respiratoire humain
WO2017175145A1 (fr) 2016-04-05 2017-10-12 Glaxosmithkline Intellectual Property Development Limited Inhibition de tgfbêta en immunothérapie
WO2018025178A1 (fr) 2016-08-02 2018-02-08 Aduro Biotech Holdings, Europe B.V. Anticorps contre ctla-4 humain
WO2018058022A1 (fr) 2016-09-26 2018-03-29 Merck Sharp & Dohme Corp. Anticorps anti-cd27
WO2018102746A1 (fr) 2016-12-02 2018-06-07 Rigel Pharmaceuticals, Inc. Molécules de liaison à l'antigène destinées à la tigit
WO2020076969A2 (fr) 2018-10-10 2020-04-16 Tilos Therapeutics, Inc. Variants d'anticorps anti-lap et leurs utilisations
EP3693394A1 (fr) 2011-05-27 2020-08-12 Glaxo Group Limited Protéines se liant à des antigènes
WO2021207449A1 (fr) 2020-04-09 2021-10-14 Merck Sharp & Dohme Corp. Anticorps anti-lap à maturation d'affinité et leurs utilisations
US11230601B2 (en) 2017-10-10 2022-01-25 Tilos Therapeutics, Inc. Methods of using anti-lap antibodies
WO2022117526A1 (fr) 2020-12-02 2022-06-09 Glaxosmithkline Intellectual Property Development Limited Protéines de liaison à il-7 et leur utilisation en thérapie médicale
WO2022248870A1 (fr) 2021-05-28 2022-12-01 Glaxosmithkline Intellectual Property Development Limited Polythérapies pour traiter le cancer
WO2023017483A1 (fr) 2021-08-13 2023-02-16 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour des cellules exprimant ccr2
WO2023017484A1 (fr) 2021-08-13 2023-02-16 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité
WO2023057893A1 (fr) 2021-10-05 2023-04-13 Glaxosmithkline Intellectual Property Development Limited Polythérapies pour le traitement du cancer
US11680104B2 (en) 2015-09-02 2023-06-20 Immutep S.A.S. Anti-LAG-3 antibodies
WO2023161876A1 (fr) 2022-02-25 2023-08-31 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour des cellules exprimant cxcr3
WO2023161879A1 (fr) 2022-02-25 2023-08-31 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour des cellules exprimant des protéines d'activation de fibroblastes
WO2023161878A1 (fr) 2022-02-25 2023-08-31 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour cellules exprimant le récepteur du folate
WO2023161877A1 (fr) 2022-02-25 2023-08-31 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour des cellules exprimant l'intégrine avb6
WO2023161875A1 (fr) 2022-02-25 2023-08-31 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour des cellules exprimant l'antigène membranaire spécifique de la prostate
WO2023161874A1 (fr) 2022-02-25 2023-08-31 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour des cellules exprimant le récepteur 2 de la chimiokine c-c
WO2023161881A1 (fr) 2022-02-25 2023-08-31 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour des cellules exprimant ccr2
WO2023212304A1 (fr) 2022-04-29 2023-11-02 23Andme, Inc. Protéines de liaison à un antigène
WO2023227641A1 (fr) 2022-05-27 2023-11-30 Glaxosmithkline Intellectual Property Development Limited Utilisation de protéines de liaison au tnf-alpha et de protéines de liaison à l'il-7 dans un traitement médical
WO2024042112A1 (fr) 2022-08-25 2024-02-29 Glaxosmithkline Intellectual Property Development Limited Protéines de liaison à l'antigène et leurs utilisations
WO2024218345A1 (fr) 2023-04-21 2024-10-24 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour conjugués anticorps-médicament et anticorps bispécifiques
WO2024218361A1 (fr) 2023-04-21 2024-10-24 Glaxosmithkline Intellectual Property Development Limited Chimères de ciblage de cytotoxicité bispécifique

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1853566A1 (fr) * 2005-03-01 2007-11-14 Wyeth Composes de cinnoline et leur utilisation comme modulateurs du recepteur hepatique x
SG177764A1 (en) * 2009-07-30 2012-03-29 Hoffmann La Roche Enzymatic antibody processing

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999022764A1 (fr) * 1997-10-31 1999-05-14 Genentech, Inc. Compositions renfermant des glycoformes de glycoproteine et methodes afferentes
WO2004074499A2 (fr) * 2003-02-20 2004-09-02 Gerngross Tillman U Banques adn combinatoires destinees a produire des n-glycanes modifies dans des eucaryotes inferieurs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999022764A1 (fr) * 1997-10-31 1999-05-14 Genentech, Inc. Compositions renfermant des glycoformes de glycoproteine et methodes afferentes
WO2004074499A2 (fr) * 2003-02-20 2004-09-02 Gerngross Tillman U Banques adn combinatoires destinees a produire des n-glycanes modifies dans des eucaryotes inferieurs

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BOBROWICZ PIOTR ET AL: "Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose.", GLYCOBIOLOGY. SEP 2004, vol. 14, no. 9, 9 June 2004 (2004-06-09), pages 757 - 766, XP002354412, ISSN: 0959-6658 *
SHIELDS R L ET AL: "Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcgammaRIII and antibody-dependent cellular toxicity", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,, US, vol. 277, no. 30, 26 July 2002 (2002-07-26), pages 26733 - 26740, XP002964542, ISSN: 0021-9258 *
SHINKAWA T ET AL: "The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,, US, vol. 278, no. 5, 31 January 2003 (2003-01-31), pages 3466 - 3473, XP002965857, ISSN: 0021-9258 *
UMANA P ET AL: "ENGINEERED GLYCOFORMS OF AN ANTINEUROBLASTOMA IGG1 WITH OPTIMIZED ANTIBODY-DEPENDENT CELLULAR CYTOTOXIC ACTIVITY", NATURE BIOTECHNOLOGY, NATURE PUBLISHING, US, vol. 17, February 1999 (1999-02-01), pages 176 - 180, XP002921620, ISSN: 1087-0156 *

Cited By (74)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8877462B2 (en) 2000-06-28 2014-11-04 Glycofi, Inc. Combinatorial DNA library for producing modified N-glycans in lower eukaryotes
US8883483B2 (en) 2000-06-28 2014-11-11 Glycofi, Inc. Combinatorial DNA library for producing modified N-glycans in lower eukaryotes
US7326681B2 (en) 2000-06-28 2008-02-05 Glycofi, Inc. Methods for producing modified glycoproteins
US8986949B2 (en) 2003-02-20 2015-03-24 Glycofi, Inc. Endomannosidases in the modification of glycoproteins in eukaryotes
US8716557B2 (en) 2006-01-17 2014-05-06 Synthon Biopharmaceuticals B.V. Compositions and methods for inhibition of fucosyltransferase and xylosyltransferase expression in plants
WO2007084926A2 (fr) * 2006-01-17 2007-07-26 Biolex Therapeutics, Inc. Compositions et méthodes destinées à l'humanisation et à l'optimisation de n-glycanes chez des plantes
WO2007084926A3 (fr) * 2006-01-17 2008-04-24 Biolex Therapeutics Inc Compositions et méthodes destinées à l'humanisation et à l'optimisation de n-glycanes chez des plantes
US7884264B2 (en) 2006-01-17 2011-02-08 Biolex Therapeutics, Inc. Compositions and methods for inhibition of fucosyltransferase and xylosyltransferase expression in duckweed plants
AU2007205939B2 (en) * 2006-01-17 2012-12-13 Synthon Biopharmaceuticals B.V. Compositions and methods for humanization and optimization of N-glycans in plants
US7923011B2 (en) 2006-10-12 2011-04-12 Genentech, Inc. Antibodies to lymphotoxin-alpha
US8541552B2 (en) 2006-10-12 2013-09-24 Genetech, Inc. Antibodies to lymphotoxin-α
US8216807B2 (en) 2006-10-12 2012-07-10 Genentech, Inc. Antibodies to lymphotoxin-α
US8642740B2 (en) 2006-10-12 2014-02-04 Genentech, Inc. Antibodies to lymphotoxin-alpha
WO2009016164A1 (fr) * 2007-08-01 2009-02-05 Glaxo Group Limited Nouveaux anticorps
US8025879B2 (en) 2008-09-26 2011-09-27 Eureka Therapeutics, Inc. Modified glycoproteins and uses thereof
US8084222B2 (en) 2008-09-26 2011-12-27 Eureka Therapeutics, Inc. Methods for generating host cells
US8080415B2 (en) 2008-09-26 2011-12-20 Eureka Therapeutics, Inc. Modified host cells and uses thereof
WO2010049177A1 (fr) * 2008-10-31 2010-05-06 Lonza Ltd. Nouveau outils pour produire des protéines glycosylées dans des cellules hôtes
WO2010070094A1 (fr) 2008-12-19 2010-06-24 Glaxo Group Limited Protéines de liaison à la myostatine
WO2010075249A2 (fr) 2008-12-22 2010-07-01 Genentech, Inc. Méthode de traitement de la polyarthrite rhumatoïde avec des antagonistes de cellules b
WO2011094259A2 (fr) 2010-01-28 2011-08-04 Glaxo Group Limited Protéines de liaison à cd127
WO2011098424A2 (fr) 2010-02-09 2011-08-18 Glaxo Group Limited Traitement d'un trouble du métabolisme
EP3025729A1 (fr) 2010-03-03 2016-06-01 Glaxo Group Limited Protéines de liaison à un antigène spécifiques pour un composant p de substance amyloïde sérique
WO2011107480A1 (fr) 2010-03-03 2011-09-09 Glaxo Group Limited Protéines de liaison à un antigène spécifiques pour un composant p de substance amyloïde sérique
WO2011151432A1 (fr) 2010-06-03 2011-12-08 Glaxo Group Limited Protéines humanisées de liaison à un antigène dirigées contre la myostatine
WO2012069433A2 (fr) 2010-11-23 2012-05-31 Glaxo Group Limited Protéines de liaison à l'antigène
EP2853542A1 (fr) 2010-11-24 2015-04-01 Glaxo Group Limited Protéines multispécifique se liant à un antigène et ciblant HGF
WO2012069557A1 (fr) 2010-11-24 2012-05-31 Glaxo Group Limited Protéines multispécifique se liant à un antigène et ciblant hgf
EP4338754A2 (fr) 2011-05-27 2024-03-20 Glaxo Group Limited Protéines de liaison à l'antigène
EP3693394A1 (fr) 2011-05-27 2020-08-12 Glaxo Group Limited Protéines se liant à des antigènes
WO2013014208A2 (fr) 2011-07-27 2013-01-31 Glaxo Group Limited Constructions de liaison à un antigène
WO2014029752A1 (fr) 2012-08-22 2014-02-27 Glaxo Group Limited Anticorps anti-lrp6
WO2014140180A1 (fr) 2013-03-15 2014-09-18 Glaxosmithkline Intellectual Property Development Limited Protéines de liaison anti-lag-3
US10344088B2 (en) 2013-03-15 2019-07-09 Glaxosmithkline Intellectual Property Development Limited Antigen binding proteins
EP3712177A1 (fr) 2013-03-15 2020-09-23 GlaxoSmithKline Intellectual Property Development Limited Protéines de liaison anti-lag-3
US10280221B2 (en) 2013-03-15 2019-05-07 Glaxosmithkline Intellectual Property Development Limited Anti-LAG-3 binding proteins
WO2016028656A1 (fr) 2014-08-19 2016-02-25 Merck Sharp & Dohme Corp. Anticorps anti-tigit
WO2016059602A2 (fr) 2014-10-16 2016-04-21 Glaxo Group Limited Méthodes de traitement du cancer et compositions associées
US11680104B2 (en) 2015-09-02 2023-06-20 Immutep S.A.S. Anti-LAG-3 antibodies
US9963500B2 (en) 2015-10-29 2018-05-08 Merck Sharp & Dohme Corp. Antibody neutralizing human respiratory syncytial virus
US10072072B2 (en) 2015-10-29 2018-09-11 Merck Sharp & Dohme Corp. Antibody neutralizing human respiratory syncytial virus
US11981726B2 (en) 2015-10-29 2024-05-14 Merck Sharp & Dohme Llc Antibody neutralizing human respiratory syncytial virus
US10323079B2 (en) 2015-10-29 2019-06-18 Merck Sharp & Dohme Corp. Antibody neutralizing human respiratory syncytial virus
US10358480B2 (en) 2015-10-29 2019-07-23 Merck Sharp & Dohme Corp. Antibody neutralizing human respiratory syncytial virus
WO2017075124A1 (fr) 2015-10-29 2017-05-04 Merck Sharp & Dohme Corp. Anticorps neutralisant le virus syncytial respiratoire humain
US11566065B2 (en) 2015-10-29 2023-01-31 Merck Sharp & Dohme Llc Antibody neutralizing human respiratory syncytial virus
US11008380B2 (en) 2015-10-29 2021-05-18 Merck Sharp & Dohme Corp. Antibody neutralizing human respiratory syncytial virus
WO2017175145A1 (fr) 2016-04-05 2017-10-12 Glaxosmithkline Intellectual Property Development Limited Inhibition de tgfbêta en immunothérapie
WO2018025178A1 (fr) 2016-08-02 2018-02-08 Aduro Biotech Holdings, Europe B.V. Anticorps contre ctla-4 humain
US10808030B2 (en) 2016-08-02 2020-10-20 Aduro Biotech Holdings, Europe B.V. Anti-HCTLA-4 antibodies
WO2018058022A1 (fr) 2016-09-26 2018-03-29 Merck Sharp & Dohme Corp. Anticorps anti-cd27
US10759855B2 (en) 2016-12-02 2020-09-01 Rigel Pharmaceuticals, Inc. Antigen binding molecules to TIGIT
WO2018102746A1 (fr) 2016-12-02 2018-06-07 Rigel Pharmaceuticals, Inc. Molécules de liaison à l'antigène destinées à la tigit
US11230601B2 (en) 2017-10-10 2022-01-25 Tilos Therapeutics, Inc. Methods of using anti-lap antibodies
WO2020076969A2 (fr) 2018-10-10 2020-04-16 Tilos Therapeutics, Inc. Variants d'anticorps anti-lap et leurs utilisations
US11130802B2 (en) 2018-10-10 2021-09-28 Tilos Therapeutics, Inc. Anti-lap antibody variants
WO2021207449A1 (fr) 2020-04-09 2021-10-14 Merck Sharp & Dohme Corp. Anticorps anti-lap à maturation d'affinité et leurs utilisations
WO2022117526A1 (fr) 2020-12-02 2022-06-09 Glaxosmithkline Intellectual Property Development Limited Protéines de liaison à il-7 et leur utilisation en thérapie médicale
WO2022248870A1 (fr) 2021-05-28 2022-12-01 Glaxosmithkline Intellectual Property Development Limited Polythérapies pour traiter le cancer
WO2023017483A1 (fr) 2021-08-13 2023-02-16 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour des cellules exprimant ccr2
WO2023017484A1 (fr) 2021-08-13 2023-02-16 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité
WO2023057893A1 (fr) 2021-10-05 2023-04-13 Glaxosmithkline Intellectual Property Development Limited Polythérapies pour le traitement du cancer
WO2023161879A1 (fr) 2022-02-25 2023-08-31 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour des cellules exprimant des protéines d'activation de fibroblastes
WO2023161877A1 (fr) 2022-02-25 2023-08-31 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour des cellules exprimant l'intégrine avb6
WO2023161875A1 (fr) 2022-02-25 2023-08-31 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour des cellules exprimant l'antigène membranaire spécifique de la prostate
WO2023161874A1 (fr) 2022-02-25 2023-08-31 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour des cellules exprimant le récepteur 2 de la chimiokine c-c
WO2023161881A1 (fr) 2022-02-25 2023-08-31 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour des cellules exprimant ccr2
WO2023161878A1 (fr) 2022-02-25 2023-08-31 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour cellules exprimant le récepteur du folate
WO2023161876A1 (fr) 2022-02-25 2023-08-31 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour des cellules exprimant cxcr3
WO2023212304A1 (fr) 2022-04-29 2023-11-02 23Andme, Inc. Protéines de liaison à un antigène
WO2023227641A1 (fr) 2022-05-27 2023-11-30 Glaxosmithkline Intellectual Property Development Limited Utilisation de protéines de liaison au tnf-alpha et de protéines de liaison à l'il-7 dans un traitement médical
WO2024042112A1 (fr) 2022-08-25 2024-02-29 Glaxosmithkline Intellectual Property Development Limited Protéines de liaison à l'antigène et leurs utilisations
WO2024218345A1 (fr) 2023-04-21 2024-10-24 Glaxosmithkline Intellectual Property Development Limited Chimères ciblant la cytotoxicité pour conjugués anticorps-médicament et anticorps bispécifiques
WO2024218361A1 (fr) 2023-04-21 2024-10-24 Glaxosmithkline Intellectual Property Development Limited Chimères de ciblage de cytotoxicité bispécifique

Also Published As

Publication number Publication date
JP2008512353A (ja) 2008-04-24
EP1776385A1 (fr) 2007-04-25
CA2573745A1 (fr) 2007-01-12
AU2005269759A1 (en) 2006-02-09

Similar Documents

Publication Publication Date Title
US20060029604A1 (en) Immunoglobulins comprising predominantly a GlcNAc2Man3GlcNAc2 glycoform
US20100184143A1 (en) Immunoglobulins comprising predominantly a Man3GlcNAc2 glycoform
US20090162377A1 (en) Immunoglobulins comprising predominantly a Gal2GlcNAc2Man3GlcNAc2 glycoform
WO2006014679A1 (fr) Immunoglobulines contenant principalement un glycoforme de type glcnac2man3glcnac2
US20060034830A1 (en) Immunoglobulins comprising predominantly a GalGlcNAcMan5GLcNAc2 glycoform
US20060034828A1 (en) Immunoglobulins comprising predominantly a GlcNAcMAN5GLCNAC2 glycoform
US20060257399A1 (en) Immunoglobulins comprising predominantly a Man5GIcNAc2 glycoform
US20060024304A1 (en) Immunoglobulins comprising predominantly a Man5GlcNAc2 glycoform
EP1771477A2 (fr) Immunoglobulines contenant principalement une glycoforme gal2glcnac2man3glcnac2
EP1942935A2 (fr) Immunoglobulines comprenant principalement une glycoforme glcnacman3glcnac2
WO2006071856A2 (fr) Immunoglobulines comprenant principalement une glycoforme man5glcnac2
US20090226464A1 (en) Immunoglobulins comprising predominantly a glcnacman3glcnac2 glycoform
EP1771480A1 (fr) Immunoglobulines comprenant principalement une glycoforme de type man3glcnac2
WO2006071280A1 (fr) Immunoglobulines comprenant principalement un glycoforme galglcnacman5glcnac2
WO2006014725A1 (fr) Immunoglobulines comprenant principalement une glycoforme glcnacman5glcnac2
EP1771478A2 (fr) Immunoglobulines comprenant principalement une glycoforme man5glcnac2
CN101001875A (zh) 主要包含man5glcnac2糖形的免疫球蛋白
CN101001876A (zh) 包含占优势的GlcNacMAN5GLCNAC2糖形的免疫球蛋白

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2573745

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2005790298

Country of ref document: EP

Ref document number: 2007522673

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 200580024584.5

Country of ref document: CN

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2005269759

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 1208/DELNP/2007

Country of ref document: IN

ENP Entry into the national phase

Ref document number: 2005269759

Country of ref document: AU

Date of ref document: 20050719

Kind code of ref document: A

WWP Wipo information: published in national office

Ref document number: 2005269759

Country of ref document: AU

WWP Wipo information: published in national office

Ref document number: 2005790298

Country of ref document: EP