Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • 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
  • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • 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

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 ⁇ -glycans having specific ⁇ -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 ah, 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.
• IgM immunoglobulin M
• IgD immunoglobulin D
• IgG immunoglobulin G
• IgA immunoglobulin A
• IgE immunoglobulin E
• IgG is the most abundant immunoglobulin isotype in blood plasma, ⁇ See or example, Immunobiology, Janeway et al, 6 th Edition, 2004, Garland Publishing, New York).
• 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 (GaI 2 GIcNAc 2- Man 3 GlcNAc 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 ( ⁇ oguchi et al., 1995. J.
• 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 Gal 2 GlcNAc 2 Man3.GlcNAc 2 lacking fucose.
• the predominant N-glycan consists essentially of Gal 2 GlcNAc 2 Man3.GlcNAc 2 lacking fucose.
• greater than 50 mole percent of said plurality of N- glycans consists essentially of GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 lacking fucose.
• greater than 75 mole percent of said plurality of N-glycans consists essentially of GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 lacking fucose.
• greater than 90 percent of said plurality of N-glycans consists essentially of Gal 2 GlcNAc 2 Man 3 .GlcNAc 2 lacking fucose.
• said Gal 2 GlcNAc 2 Man 3 .GlcNAc 2 N-glycan structure lacking fucose is present at a level that is from about 5 mole percent to 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. Gal 2 GlcNAc 2 Man 3 GlcNAc 2 ) on an immunoglobulin.
• a specific glycoform e.g. Gal 2 GlcNAc 2 Man 3 GlcNAc 2
• 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 in which the predominant N-glycan consists essentially of Gal 2 GlcNAc 2 Man 3 GlcNAc 2 lacking fucose , 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 in which the predominant N- glycan consists essentially of GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 lacking fucose , 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 in which the predominant N-glycan consists essentially of Gal 2 GlcNAc 2 Man 3 GlcNAc 2 lacking fucose.
• 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 in which the predominant N-glycan consists essentially of GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 lacking fucose.
• 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 Gal 2 GlcNAc 2 Man 3 GlcNAc 2 lacking fucose 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 GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 lacking fucose wherein said immunoglobulins exhibit increased binding affinity to Fc ⁇ RIIIa and Fc ⁇ RJIIb 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 GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 lacking fucose wherein said immunoglobulins exhibit increased antibody-dependent cellular cytoxicity (ADCC).
• ADCC antibody-dependent cellular cytoxicity
• the 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.
• 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 GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 lacking fucose.
• Figure 1 Schematic representation of an IgG molecule having a GaI 2 GIcNAc 2- Man 3 GlcNAc 2 N-glycan structure.
• FIG. 1 Coomassie blue stained SDS-PAGE gel of DX-IgG expressed in YAS309 (as described in Example 2) and purified from the culture medium (as described in Example 3) over a Protein A column and a phenyl sepharose column (lane 1). (2.0 ⁇ g protein/lane).
• Figure 3. MALDI-TOF spectrum of DX-IgG expressed in YAS309, treated with galactosyltransferase showing predominantly Gal 2 GlcNAc 2 Man 3 GlcNAc 2 N-glycans.
• FIG. 4 ELISA binding assay of Fc ⁇ RIIIb with DX-IgG and Rituximab®.
• G2 Gal 2 GlcNAc 2 Man 3 GlcNAc 2 N-glycan).
• SEQ ID NO: 1 encodes the nucleotide sequence of the murine variable and human constant regions of DX-IgGl light chain.
• SEQ ID NO: 2 encodes the nucleotide sequence of the murine variable and human constant regions of DX-IgGl heavy chain.
• SEQ ID NO: 3 encodes the nucleotide sequence of the human constant region of an IgGl light chain.
• SEQ ID NO: 4 encodes the nucleotide sequence of the human constant region of an IgGl heavy chain.
• SEQ ID NO: 5 to 19 encode 15 overlapping oligonucleotides used to synthesize by polymerase chain reaction (PCR) the murine light chain variable region of DX-IgGl .
• SEQ ID NO: 20 to 23 encode four oligonucleotide primers used to ligate the DX- IgGl murine light chain variable region to a human light chain constant region.
• SEQ ID NO: 24 to 40 encode 17 overlapping oligonucleotides used to synthesize by PCR the murine heavy chain variable region of DX-IgGl.
• SEQ ID NO: 41 to 44 encode four oligonucleotide primers used to ligate the DX- IgGl murine heavy chain variable region to a human heavy chain constant region.
• SEQ ID NO: 45 encodes the nucleotide sequence encoding the Kar2 (Bip) signal sequence with an N-terminal EcoRI site.
• SEQ ID NO: 46 to 49 encode four oligonucleotide primers used to ligate the Kar2 signal sequence to the light and heavy chains of DX-IgGl .
• N-glycan As used herein, the terms "N-glycan” , “glycan” and “glycoform” are used interchangeably and refer to an N-linked oligosaccharide, e.g., one that is or was attached by an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in a protein.
• 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 (NANA)).
• the processing of the sugar groups occurs cotranslationally in the lumen of the ER and continues in the Golgi apparatus for N-linked glycoproteins.
• N-glycans have a common pentasaccharide core of Ma ⁇ GIcNAc 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 Man 3 GlcNAc 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 that 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.
• the term "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.
• sequence identity 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.
• polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin.
• 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) (hereby incorporated by reference in its entirety).
• 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 provided in GCG Version 6.1, herein incorporated by reference.
• sequences can be compared using 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)).
• 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
• 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 m 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
• 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 URA3.
• 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 RNA 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.
• 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 pastoris, Pichia ⁇ nlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thertnotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis andPichia methanolica; .
• Pichia sp. such as Pichia pastoris, Pichia ⁇ nlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia the
• 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 monomelic 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 ah, 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.
• antibody As used herein, the terms “antibody”, “immunoglobulin”,”Ig” and “Ig molecule” are used interchangeably. 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
• 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.
• the carboxy-terminal portion of 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, 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.
• 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.
• IgGs are 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 C H 3 domains ( Figure 1).
• Fab fragment refers to the 'fragment antigen binding' region of the antibody containing the VH, CHl, VL and CL domains ( Figure 1).
• monoclonal antibody as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.
• 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 ef ⁇ /.).
• 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).
• Target molecules can be human, mammalian or bacterial.
• Other targets are 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 arenatal receptors.
• Fc ⁇ RI can refer to any one of
• Fc ⁇ RII 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.
• Stereoisomers ⁇ e.g., D-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.
• Examples of unconventional amino acids include: 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ -N,N,N-trimethyllysine, ⁇ -N-acetyllysine, O-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” or is “homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein.
• 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%, 98%, 99% or 99.9% sequence identity.
• homology between two regions of amino acid sequence is interpreted as implying similarity in function.
• 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).
• R group side chain
• 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.
• 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.
• GCG Genetics Computer Group
• Protein analysis software matches similar sequences using a measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
• GCG contains programs such as "Gap” and "Bestfit” 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. See, e.g., GCG Version 6.1.
• 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 En ⁇ ymol. 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.
• affinity or avidity of a specific binding reaction 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 fiicose, 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 GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 N-linked glycoform lacking fucose.
• the present invention also provides Igs and Ig compositions having a predominant Gal 2 GlcNAc 2 Man 3 GlcNAc 2 N-linked glycoform lacking fucose 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 GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 glycoforms lacking fucose 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 Gal 2 GlcNAc 2 Man 3 GlcNAc 2 glycan structure lacking fucose at Asn-297 of a C H 2 domain of a heavy chain on the Fc region mediating antibody effector function in an Ig molecule.
• the GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 glycan structure lacking fucose 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 Gal 2 GlcNAc 2 Man 3 GlcNAc 2 glycan structure lacking fucose at Asn-297 ( Figure 1).
• an N-glycan consisting essentially of Gal 2 GlcNAc 2 Man 3 GlcNAc 2 glycan structure lacking fucose 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 an Ig molecule can be varied by introducing asparagines (Asn) or N-glycosylation sites at varying locations within the molecule.
• 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.
• the skilled artisan can alter 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 Gal 2 GlcNAc 2 Man 3 GlcNAc 2 N-glycan structure lacking fucose, wherein said Gal 2 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 Gal 2 GlcNAc 2 Man3GlcNAc2 glycan structure lacking fucose, wherein said
• Gal 2 GlcNAc 2 Man3GlcNAc 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 Gal 2 GlcNAc 2 Man 3 GlcNAc 2 glycan structure lacking fucose, wherein said
• Gal 2 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 Gal 2 GlcNAc 2 Man 3 GlcNAc 2 glycan structure lacking fucose, wherein said
• GaI 2 GIcNAc 2 Ma ⁇ GIcNAc 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 Gal 2 GlcNAc 2 Man3GlcNAc 2 glycan structure lacking fucose, wherein said Gal 2 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 Gal2GlcNAc 2 Man3GlcNAc 2 glycan structure lacking fucose wherein said
• Gal 2 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 DX-IgG having a predominant (approximately 62 mole %) Gal 2 Glc ⁇ Ac 2 Man 3 Glc ⁇ Ac 2 lacking fucose 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 GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 N-glycan lacking fucose capable of carrying out an effector function.
• the Fc region having a predominant GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 N-glycan lacking fucose confers an increase in binding to Fc ⁇ RIIIa ( Figure 5) and Fc ⁇ RIIIb ( Figure 4) receptors.
• an Fc has a predominant Gal 2 GlcNAc 2 Man 3 GlcNAc2 N-glycan lacking fucose.
• 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 ⁇ Ri ⁇ 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.
• DX-IgG (an Ig made according to the present invention) having predominantly Gal 2 GlcNAc 2 Man3GlcNAc 2 N-glycans lacking fucose has 50-100 fold increased binding activity to Fc ⁇ RIIIb compared with Rituximab® as shown in Figure 4, and at
• 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, Clin. Cane Res. 10: 6248-6255).
• the antibodies of 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 and 2).
• 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®. Decreased binding of Ig- GabGlcNAc ⁇ MamGlcNAc? to Fc ⁇ RIIb receptor
• the effector functions of Ig binding to Fc ⁇ RIIb 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 Gal 2 GlcNAc 2 Man 3 GlcNAc 2 N-glycan lacking fucose capable of carrying out an effector function. In one embodiment, an Fc region of an Ig having a predominant GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 N-glycan lacking fucose confers a decrease in binding to an Fc ⁇ RIIb receptor.
• 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.
• DX-IgG (anlg of the present invention) having predominantly Gal 2 GlcNAc 2 Man 3- GlcNAc 2 N-glycans lacking fucose , has a 2-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 an Ig molecule or composition having afucosylated Gal 2 Glc ⁇ Ac 2 Man 3 Glc ⁇ Ac 2 as the predominant N-glycan may confer an increase in Fc ⁇ RIII-mediated ADCC. It is well
• an Ig molecule or composition of the present invention exhibits increased ADCC activity conferred by the presence of a predominant GaI 2 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 use in conjunction with assays for any Ig 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.
• induced 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
• 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 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 exerts regulatory functions by competitive inhibition of Fc ⁇ R-dependent effector functions and via binding to the complement receptor CR3, leading to production of inflammatory mediators (Sautes-Fridman et al, 2003, ASHI Quarterly, 148-151).
• the present invention thus provides an immunoglobulin molecule comprising an N-glycan consisting essentially of afucosylated Gal 2 GlcNAc 2 Man3GlcNAc 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 GaUGIcNAc 2 MaH 3 GIcNAc 2 lacking fucose.
• the predominance of said afucosylated Gal 2 GlcNAc 2 Man 3 GlcNAc 2 N- glycan on an immunoglobulin preferably confers desired therapeutic effector activity in addition to the improved binding to Fc ⁇ RIIIa and Fc ⁇ RIIIb and decreased binding
• the present invention provides an IgGl composition that comprises afiicosylated Gal 2 GlcNAc 2 Man 3 GlcNAc 2 as the predominant N-glycan attached to IgGl molecules.
• the present invention comprises an IgG2 composition that comprises afiicosylated Gal 2 Glc ⁇ Ac 2 Man 3 Glc ⁇ Ac 2 as the predominant N-glycan attached to IgG2 molecules.
• the present invention comprises an IgG3 composition that comprises afiicosylated
• the present invention comprises an IgG4 composition that comprises afiicosylated GaI 2 GIcNAc 2 -MaH 3 GIcNAc 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 ⁇ -glycan consisting essentially of a Gal 2 Glc ⁇ Ac 2 Man 3 Glc ⁇ Ac 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 Gal 2 GlcNAc 2- Man 3 GlcNAc 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 Gal 2 GlcNAc 2 Man 3 GlcNAc 2 .
• this glycoform structure is more specifically denoted as [(GaI ⁇ 1,4-
• 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 afucosylated GaI 2 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.
• 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 afucosylated Gal 2 GlcNAc 2 Man 3 GlcNAc 2 and Ig compositions having predominantly afucosylated GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 glycan structure.
• recombinant host cells preferably recombinant lower eukaryotic host cells, are used to produce said Ig molecules and compositions having predominantly afucosylated Gal 2 GlcNAc 2 Man 3 GlcNAc 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 afucosylated
• 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 afucosylated Gal 2 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 et al, 2003, PNAS, 100: 5022-5027; Hamilton et al, 2003, Nature, 301 : 1244-1246 and Bobrowicz et al, 2004, Glycobiology, 14: 757-766).
• WO 03/056914 discloses methods to obtain at least 50% GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 in Fig. 23, 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 DX-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 : 1244-1246), and has had the PNOl and MNN4b genes disrupted as described (US Pat. Appl. No. 11/020808), as well as a ⁇ -1,4 galactosyltransferase I gene introduced as
• 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. 11/118008).
• Glycoproteins from this YAS309 strain upon further in vitro treatment with ⁇ -l,4-galactosyltransferase have predominantly GaI 2 GIcNAc 2 MaH 3 GIcNAc 2 N-glycans.
• DX-IgG expressed in YAS309 and treated with ⁇ - 1 ,4-galactosyltransferase has predominantly Gal 2 GlcNAc 2 Man 3 GlcNAc 2 N-glycans ( 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).
• an Ig of the present invention can be expressed in a lower eukaryotic host which synthesizes the Gal 2 GlcNAc 2 Man3GlcNAc 2 N-glycans in vivo.
• a lower eukaryotic host which synthesizes the Gal 2 GlcNAc 2 Man3GlcNAc 2 N-glycans in vivo.
• Such host would be engineered in an ⁇ alg3 mutant as described in WO 03/056914 with an ⁇ -1,2 mannosidase, N-acetylglucosaminyltransferase and a ⁇ -1,4 galactosyltransferase gene introduced as also described.
• An immunoglobulin introduced into such a host would express predominantly Gal 2 GlcNAc 2 Man 3 GlcNAc 2 N-glycans by in vivo methods.
• the PpURA3- or PpURA5-b ⁇ aste ⁇ cassettes are used to disrupt the JJRAS, 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 ⁇ l, 1984, Mo/. Gen. Genet., 197: 345-346).
• 5FOA 5-fluoroorotic acid
• an expression host system 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 DX-IgG purified from YAS309.
• a purified Ig antibody has afucosylated GaI 2 GIcNAc 2 Ma ⁇ GIcNAc 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. In a preferred embodiment, the glycan distribution is determined by MALDI-TOF MS analysis as disclosed in Example 5.
• FIG. 3 shows a MALDI- TOF spectra of DX-IgG purified from YAS309 and treated with ⁇ -1,4 galatosyltransferase (Example 3).
• This MALDI-TOF shows approximately 62 mole % of the total N-glycans are afucosylated Gal 2 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 afucosylated Gal 2 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.
• compositions comprising the glycoform preparations of the invention.
• the compositions are preferably sterile. Where the composition is an aqueous solution, preferably the glycoprotein is soluble. Where 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 afucosylated GaI 2 GIcN Ac 2 Ma ⁇ GIcN Ac 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 DX-IgGl (an anti-CD20 IgGl) consists of mouse variable regions and human constant regions.
• the light chain is disclosed as SEQ ID NO: 1 and heavy chain as SEQ ID NO: 2.
• the heavy and light chain sequences were synthesized using overlapping oligonucleotides purchased from Integrated DNA Technologies (IDT).
• IDCT Integrated DNA Technologies
• 15 overlapping oligonucleotides SEQ ID NOs: 5-19
• Extaq Teakada
• This light chain variable fragment was then joined with the light chain constant region (SEQ ID NO: 3) (Gene Art, Toronto, Canada) by overlapping PCR using the 5' MIyI primer CD20L/up (SEQ ID NO: 20), the 3' variable/5' constant primer LfusionRTVAAPS/up (SEQ ID NO: 21), the 3' constant region primer Lfusion RTVAAPS/lp (SEQ ID NO: 22) and 3 ' CD20L/lp (SEQ ID NO: 23).
• the final Mlyl-light chain fragment (which included 5 'AG base pairs) was then inserted into pCR2.1 topo vector (Invitrogen) resulting in pDX343.
• overlapping oligonucleotides SEQ ID NOs: 24-40 corresponding to the mouse heavy chain variable region were purchased from IDT and annealed using Extaq.
• This heavy chain variable fragment was then joined with the heavy chain constant region (SEQ ID NO: 4) (Gene Art) by overlapping PCR using the 5' MIyI primer CD20H/up (SEQ ID NO: 41), the 5' variable/constant primer HchainASTKGPS/up (SEQ ID NO: 42), the 3' variable/constant primer HchainASTKGPS/l ⁇ (SEQ ID NO: 43) and the 3' constant region primer HFckpnl/lp (SEQ ID NO: 44).
• the final Mlyl-heavy chain fragment (which included 5'AG base pairs) was inserted into pCR2.1 topo vector (Invitrogen) resulting in pDX360.
• the full length light chain and full length heavy chain were isolated from the respective topo vectors as Mlyl and Notl fragments.
• a Bglll-BamHI fragment from pDX344 was then subcloned into pBK85 containing the A0X2 promoter gene for chromosomal integration, resulting in pDX458.
• a Bglll-BamHI fragment from pDX468 carrying the heavy chain was then subcloned into pDX458, resulting in pDX478 containing both heavy and light chains of CD20 under the AOXl promoter.
• This plasmid was then linearized with Spel prior to transformation for integration into the A0X2 locus with transformants selected using Zeocin resistance. (See Example 2)
• Rituximab®/Rituxan® 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 0 C and 90 sec at 72 0 C with an initial denaturation step of 2 min at 97 0 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.
• Example 2
• the vector DNA of pDX478 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 -1-2 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.
• 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
• the culture was incubated while shaking at 24 0 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. 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 microliter 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 lM 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 DX-IgG a phenyl sepharose column (can also use octyl sepharose) was used with 2OmM Tris (7.0), IM (NH 4 ) 2 SO 4 buffer and eluted with a linear gradient buffer of IM to OM (NH 4 ) 2 S ⁇ 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)
• Purified DX-IgG was mixed with an appropriate volume of sample loading buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
• the gel proteins were stained with Coomassie brilliant blue stain (Bio-Rad, Hercules,
• 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 Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry
• 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.
• 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 F ⁇ RIIb ( 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.
• ELISPOT assay for antibody feedback in B cells This assay is conducted as described in Westman, et al., 1997, Scand. J.
• 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°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 ofB-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, lOOg/ml streptomycin (Gibco/BRL) and supplemented with 20% fetal calf serum, and then resuspended at a concentration of lxlO 6 /ml 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 2.5xlO 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 5 ' 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: 01 (mouse/human chimeric IgGl light chain) caaatcgtcttgtctcaatccccagctattttgtctgcttcccctggagagaaggtcaccatgacttgtagagcctcttcctctgt ctcttacattcactggttccagcaaaagccaggttcctctccaaagccatggatctacgctacttccaacttggcttccggtgtt ccagttagattctctggttctggttccggtacctcctactctcttaccatctccagagttgaagccgaggacgctgctacttact actgtcagcaatggacttctaacccaccaactttcggtggtggtaccaaattggagattaagagaact
• SEQ ID NO: 02 (mouse/human chimeric IgGl heavy chain) caagtccagttgcaacagcctggtgccgagttggtcaagccaggtgcttctgttaagatgtcctgtaaggcttctggttacact ttcacctcctacaacatgcactgggtcaagcaaactccaggtagaggtttggagtggttggtggtgccatctacccaggtaacgg tgacacttcttacaaccaaaaattcaagggaaaggctactctttaccgctgataagtcctctcaccgcctacatgcaattgtct t tgacttctgaagattctgctgtgtttactactgtgctagatccacctactacggtgga
• SEQ ID NO: 12 (CD20LF8) aaattggagattaagagaactgttgctgctccatcc
• SEQ ID NO: 13 (CD20LR1) caacagttctcttaatctccaatttggtaccaccaccaccgaagttg
• SEQ ID NO: 14 (CD20LR2) gtgggttagaagtccattgctgacagtagtaagtagcagcgtcct
• SEQ ID NO: 17 (CD20LR5) tagcgtagatccatggctttggagaggaacctggcttttgctgga
• SEQ ID NO: 18 (CD20LR6) ccagtgaatgtaagagacagaggaagaggctctacaagtcatgg
• SEQ ID NO: 19 (CD20LR7) tgaccttctctccaggggaagcagacaaaatagctggggattgag
• SEQ ID NO: 24 (CD20HF1) aggagtcgtattcaagtccagttgcaacagcctggtgccgagttg
• SEQ ID NO: 25 (CD20HF2) gtcaagccaggtgcttctgttaagatgtcctgtaaggcttctggt
• SEQ ID NO: 26 (CD20HF3) tacactttcacctcctacaacatgcactgggtcaagcaaactcca
• SEQ ID NO: 27 (CD20HF4) ggtagaggtttggagtggattggtgccatctacccaggtaacggt
• SEQ ID NO: 28 (CD20HF5) gacacttcttacaaccaaaattcaagggaaggctactcttacc
• SEQ ID NO: 29 (CD20HF6) gctgataagtcctcttccaccgcctacatgcaattgtcttccttg
• SEQ ID NO: 30 (CD20HF7) acttctgaagactctgctgttactactactgtgctagatccacctac SEQ ID NO: 31 (CD20HF8) tacggtggagactggtacttcaacgtttggggtgctggtaccact
• SEQ ID NO: 33 (CD20HR1) tagtagaagcagcggaaacggtgacagtggtaccagcaccccaaa
• SEQ ID NO: 34 (CD20HR2) cgttgaagtaccagtctccaccgtagtaggtggatctagcacag
• SEQ ID NO: 35 (CD20HR3) agtaaacagcagagtcttcagaagtcaaggaagacaattgcatgt SEQ ID NO: 36 (CD20HR4) aggcggtggaagaggacttatcagcggtaagagtagcctttcctt
• SEQ ID NO: 37 (CD20HR5) tgaatttttggttgtaagaagtgtcaccgttacctgggtagatgg SEQ ID NO: 38 (CD20HR6) caccaatccactccaaacctctacctggagtttgcttgacccagt
• SEQ ID NO: 39 (CD20HR7) gcatgttgtaggaggtgaaagtgtaaccagaagccttacaggaca
• SEQ ID NO: 40 (CD20HR8) tcttaacagaagcacctggcttgaccaactcggcaccaggctgtt SEQ ID NO: 41 (CD20H/up) Aggagtcgtattcaagtccag
• SEQ ID NO: 45 Kar2 signal sequence with EcoRD gaattcgaaacgatgctgtcgttaaaccatcttggctgacttttggcggcattaatgtatgccatgctattggtcgtagtgccat ttgctaaacctgttagagct
• SEQ ID NO: 46 (P.BiPss/UPl-EcoRI) aattcgaaacgatgctgtctttgaagccatcttggcttactttggctgctttgatgtacgctatgcttttt
• SEQ ID NO: 47 (P.BiPss/LPl) ccaaagtaagccaagatggcttcaaagacagcatcgtttcg
• SEQ ID NO: 48 (P.BiPss/UP2) ggttgttgttccatttgctaagccagttagagct SEQ ID NO: 49 (P.BiPss/LP2) agctctaactggcttagcaaatggaacaacaaccaaaagcatagcgtacatcaaagcag

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  • 2005-07-19 CA CA002573842A patent/CA2573842A1/en not_active Abandoned
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