WO2009114641A1 - Antibodies with enhanced adcc function - Google Patents
Antibodies with enhanced adcc function Download PDFInfo
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- WO2009114641A1 WO2009114641A1 PCT/US2009/036855 US2009036855W WO2009114641A1 WO 2009114641 A1 WO2009114641 A1 WO 2009114641A1 US 2009036855 W US2009036855 W US 2009036855W WO 2009114641 A1 WO2009114641 A1 WO 2009114641A1
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- 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
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- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/72—Increased effector function due to an Fc-modification
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- C12N2320/00—Applications; Uses
- C12N2320/10—Applications; Uses in screening processes
- C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids
Definitions
- the present invention concerns antibodies enhanced antibody-dependent cell mediated cytotoxicity (ADCC) and method for preparation thereof.
- ADCC antibody-dependent cell mediated cytotoxicity
- Antibody-dependent cell-mediated cytotoxicity is a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
- FcRs Fc receptors
- NK Natural Killer
- ADCC antigen- dependent cell-mediated cyotoxicity
- CDC complement dependent cytotoxicity
- Enhancement of effector functions may be achieved by various means, including introducing one or more amino acid substitutions in an Fc region of the antibody. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
- the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992).
- Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobi functional cross-linkers as described in Wolff et al, Cancer Research 53:2560-2565 (1993).
- an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al, Anti-Cancer Drug Design 3:219-230 (1989).
- Another approach to enhance the effector function of antbodies, including antibodies of the IgG class, is to engineer the glycosylation pattern of the antibody Fc region.
- An IgG molecule contains an N-linked oligosaccharide covalently attached at the conserved Asn297 of each of the CH2 domains in the Fc region.
- the oligosaccharides found in the Fc region of serum IgGs are mostly biantennary glycans of the complex type.
- a number of antibody glycoforms have been reported as having a positive impact on antibody effector function, including antibody-dependent cell mediated cytotoxicity (ADCC).
- ADCC antibody-dependent cell mediated cytotoxicity
- Antibodies with select glycoforms have been made by a number of means, including the use of glycosylation pathway inhibitors, mutant cell lines that have absent or reduced activity of particular enzymes in the glycosylation pathway, engineered cells with gene expression in the glycosylation pathway either enhanced or knocked out, and in vitro remodeling with glycosidases and glycosyltransferases.
- Rothman et al, 1989; Molecular Immunology 26: 1113-1123 expressed monoclonal IgG in the presence of the glucosidase inhibitors castano spermine and N-methyldeoxynojirimycin, and the mannosidase I inhibitor deoxymannojirimycin.
- the invention concerns a mammalian cell lacking GIcNAc Transferase I activity, engineered to express an antibody or a fragment thereof, or an immunoadhesin or a fragment thereof.
- the mammalian cell additionally has enhanced ⁇ -l,2-mannosidase (also referred to herein as ⁇ -mannosidase I) activity.
- the invention concerns a mammalian cell, in which GIcNAc Transferase I activity is diminished by RNAi knockdown engineered to express an antibody or a fragment thereof, or an immunoadhesin or a fragment thereof.
- the mammalian cell additionally has enhanced ⁇ - 1 ,2-mannosidase activity.
- the invention concerns a mammalian cell, in which GIcNAc Transferase I activity is diminished by RNAi knockdown, sufficient to result in a carbohydrate structure comprising 5% or greater, or 10% or greater, or 20% or greater, or 25% or greater, or 30% or greater, or 35% or greater Man5, Man6 glycans, and which may in addition have enhanced ⁇ -1,2 mannosidase activity, engineered to express an antibody or a fragment thereof, or an immunoadhesin or a fragment thereof, wherein said fragment comprises at least one glycosylation site.
- the invention concerns a mammalian cell, in which GIcNAc Transferase I activity is diminished by RNAi knockdown of the Golgi UDP-GIcNAc transporter, and which additionally may have enhanced ⁇ -1,2 mannosidase activity, engineered to express an antibody or a fragment thereof, or an immunoadhesin or a fragment thereof, wherein the fragment comprises at least one glycosylation site.
- the invention concerns a mammalian cell, in which GIcNAc Transferase I activity is diminished by RNAi knockdown of the Golgi UDP-GIcNAc transporter, and which also has GIcNAc transferase I knocked down by RNAi, engineered to express an antibody or a fragment thereof, or an immunoadhesin or a fragment thereof, wherein the fragment comprises at least one glycosylation site.
- the invention concerns a method for making an antibody or a fragment thereof, or an immunoadhesin or a fragment thereof, bearing predominantly Man5 glycans, comprising culturing a mammalian cell line according to claim 2 or claim 22 under conditions such that said antibody or a fragment thereof, or an immunoadhesin or a fragment thereof is produced.
- the invention concerns a method for recombinant production of an antibody, an immunoadhesin, or a fragment thereof with a controlled amount of Man5 glycans in the carbohydrate structure thereof, comprising expressing nucleic acid encoding the antibody or antibody fragment in a mammalian cell line which has a diminished GIcNAc Transferase I activity as a result of RNAi knockdown.
- the invention concerns a method for recombinant production of an antibody, an immunoadhesin, or a fragment thereof, bearing predominantly Man5 glycans in the carbohydrate structure thereof, comprising culturing a mammalian cell line lacking GIcNAc Transferase I activity engineered to express said antibody, immunoadhesin, or fragment thereof in the presence of an ⁇ -l,2-mannosidase, or contacting the expressed product with such ⁇ -l,2-mannosidase, wherein Man7,8,9 glycans are converted to Man5, 6 glycans.
- the invention concerns a method for making an antibody or a fragment thereof, or an immunoadhesin or a fragment thereof, bearing 5% or greater, or 10% or greater, or 20% or greater, or 25% or greater, or 30% or greater, or 35% or greater, Man5 glycans, comprising culturing a mammalian cell line according to claim 2 or claim 14 under conditions such that said antibody or a fragment thereof, or an immunoadhesin or a fragment thereof is produced, wherein said fragment comprises at least one glycosylation site.
- the invention further concerns a method for recombinant production of an antibody, an immunoadhesin, or a fragment thereof, bearing predominantly Man5 glycans in the carbohydrate structure thereof, comprising culturing a mammalian cell line with diminished GIcNAc Transferase I activity due to RNAi knockdown, engineered to express said antibody, immunoadhesin, or a fragment thereof, in the presence of an ⁇ -l,2-mannosidase, or contacting the expressed product with such ⁇ -l,2-mannosidase, wherein Man7,8,9 glycans are converted to Man5, 6 glycans.
- the invention concerns a method for recombinant production of an antibody, an immunoadhesin, or a fragment thereof, bearing predominantly Man5 glycans in the carbohydrate structure thereof, comprising culturing a mammalian cell line in the presence of a toxic lectin to select for clones with diminished GIcNAc Transferase I activity, engineering one or more of said clones with diminished GIcNAc Transferase I activity to express said antibody, immunoadhesin, or a fragment thereof, in the presence of an ⁇ -1,2- mannosidase, or contacting the expressed product with such ⁇ -l,2-mannosidase, wherein Man7,8,9 glycans are converted to Man5 glycans, wherein said fragment comprises at least one glycosylation site.
- the mannosidase is endogenous in the cell used for recombinant production.
- the invention concerns a method for recombinant production of an antibody, an immunoadhesin, or a fragment thereof, bearing predominantly Man5 glycans in the carbohydrate structure thereof, comprising culturing a mammalian cell line lacking UDP-GIcNAc transporter activity engineered to express said antibody, immunoadhesin, or fragment thereof in the presence of an ⁇ -l,2-mannosidase, or contacting the expressed product with such ⁇ -l,2-mannosidase, wherein Man7,8,9 glycans are converted to Man5 glycans, wherein said fragment comprises at least one glycosylation site.
- the mannosidase is endogenous in the cell used for recombinant production.
- the mammalian cell line may, for example, be a Chinese Hamster Ovary (CHO) cell line.
- the cell lines and methods of the present invention can be used for the production of any antibody, including, without limitation, antibodies of diagnostic or therapeutic interest, such as, antibodies binding to one or more of the following antigens: CD3, CD4, CD8, CD19, CD20, CD22, CD34, CD40, EGF receptor (EGFR, HERl , ErbBl), HER2 (ErbB2), HER3 (ErbB3), HER4 (ErbB4), LFA-I, Macl, pl50,95, VLA-4, ICAM-I, VCAM , ⁇ v/ ⁇ 3 integrin, CDl Ia, CDl 8, CDl Ib, VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, DR5, EGFL7, neuropilins and receptors, netrins and receptors, slit and receptors, sema and receptors, semaphorins and receptor
- the antibodies and antibody fragments may be chimeric or humanized, and specifically include chimeric and humanized anti-CD20 antibodies, where, in a specific embodiment, the antibody is rituximab or ocrelizumab.
- the humanized antibody is an anti-HER2, anti-HERl , anti-
- VEGF or anti-IgE antibody including, without limitation, trastuzumab, pertuzumab, bevacizumab, ranibizumab, and omalizumab, as well as fragments, variants and derivatives of such antibodies.
- Antibody fragments include, for example, complementarity determining region (CDR) fragments, linear antibodies, single-chain antibody molecules, minibodies, diabodies, multispecific antibodies formed from antibody fragments, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide, provided that they are glycosylated.
- CDR complementarity determining region
- Figure 1 depicts a portion of the N-glycan biosynthetic pathway.
- FIG. 1 Plasmid vector used to add N-terminus FLAG ® tag to GIcNAc Transferase I (GnT-I) protein (Stratagene).
- Figure 3. Plasmid vector used to express small inhibitory RNA (Ambion, Austin. TX).
- siRNA probe sequences SEQ ID NOs: 2-6) and their relative positions (in parentheses) in full length GnT-I gene. Each siRNA probe sequence is underlined (a). The underlined sequence close to BamHI site is complementary to the GnT-I mRNA sequence. The two underlined sequences are complementary to each other resulting in formation of the hairpin loop siRNA.
- FIG. 1 Western blot analysis of lysates from the co-transfection of the individual siRNA probes and the FLAG -tagged GnT-I construct.
- Five individual siRNA expression constructs in addition to empty vector were transiently co-transfected with FLAG ® -tagged GnT-I construct.
- Cell lysates containing equal amounts of cellular protein were analyzed by Western blot with anti-FLAG ® antibody (Sigma MO).
- FIG. 6A Cell line generating ocrelizumab was transiently transfected with siRNA expression plasmids. Cell pellets from each sample condition were collected on dayl, 2 and 5 post transfection, and then mRNA was isolated for TaqMan analysis. GnT-I mRNA expression level of control was set to 100%.
- Figure 6B Man5 level of day 5 post transfection from each sample transfected with the indicated RNAi vector.
- FIG. 7 Transient transfection of scramble and RNAi 13 vectors into ocrelizumab- generating cell line for a 14-day experiment. Man5 level of HCCF collected at the indicated culture duration was determined using CE-glycan. Error bar represents standard deviation from duplicate runs.
- Figure 8A cDNA sequence of CHO ⁇ -mannosidase I.
- Figure 8B Amino acid sequence alignment between CHO and mouse ⁇ - mannosidase I.
- Figure 8C Configuration of the SV40GS.CMV.Manl .RNAil3 expression plasmid.
- Figure 9A Relative GnT-I mRNA level in stable clones determined by TAQMAN ® assay. Control represents the GnT-I level in untransfected baseline.
- Figure 9B Man5 level of stable clones at the end of 14 days production run. The Man5% is determined by CE-glycan analysis.
- Figure 1OA Man5 level at various days of culture duration. The Man5 level was determined by CE-glycan assay, and the errors bars represent standard deviations.
- Figure 1OB Comparison of Man5 level after 22 days culture. Four different osmolality in basal media was tested (300, 330, 360, 400 mOsm). The Man5 level was determined by CE-glycan assay.
- Man5 level with the addition Of MnCl 2 on various days of a total 14 day culture.
- the Man5 level was determined by CE-glycan assay.
- FIG. 10D Man5 level (CE-glycan assay) of GnT-I knockdown clone 6D at different cell culture conditions.
- Control represents standard production culture media.
- High osmo represents increased osmolality to 400 mOsm in basal media.
- Without Mn represents standard production media which lacks manganese.
- FIG. 1 Antibody binding to Fc gamma receptor IHa-V 158.
- Open circles represent HERCEPTIN (trastuzumab), open squares represent RITUXAN ® (rituximab), open triangles represent anti-receptor antibody with 5% Man5 (7-9% afucosyl glycans), open diamonds represent anti-receptor antibody with 16% Man5 (14.6% afucosyl glycans), and closed circles represent anti-receptor antibody with 62% Man5 (1 1% afucosyl glycans).
- FIG. 12 Antibody binding to Fc gamma receptor IIIa-F158.
- Open circles represent HERCEPTIN R (trastuzumab), open squares represent RITUXAN ® (rituximab) open triangles represent anti-receptor antibody with 5% Man5 (7-9% afucosyl glycans), open diamonds represent anti-receptor antibody with 16% Man5 (14.6% afucosyl glycans), and closed circles represent anti-receptor antibody with 62% Man5 (1 1% afucosyl glycans).
- Antibody-dependent cell-mediated cytotoxicity and “ADCC” refer to a cell- mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
- FcRs Fc receptors
- FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).
- ADCC activity of a molecule of interest may be assessed in vitro, such as that described in U.S. Patent Nos. 5,500,362 or 5,821,337.
- useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
- PBMC peripheral blood mononuclear cells
- NK Natural Killer
- ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al, PNAS (USA) 95:652-656 (1998).
- Human effector cells are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least Fc ⁇ RIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred.
- PBMC peripheral blood mononuclear cells
- NK natural killer cells
- monocytes cytotoxic T cells and neutrophils
- the effector cells may be isolated from a native source thereof, e.g., from blood or PBMCs as described herein.
- Fc receptor or “FcR” are used to describe a receptor that binds to the Fc region of an antibody.
- the preferred FcR is a native sequence human FcR.
- a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
- Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor") and Fc ⁇ RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
- Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
- Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)).
- FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al, Immunomethods 4:25-34 (1994); and de Haas et al, J. Lab. Clin. Med. 126:330-41 (1995).
- FcR FcR
- the term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al, J. Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)) and mediates slower catabolism, thus longer half-life.
- “Complement dependent cytotoxicity” or “CDC” refers to the ability of a molecule to lyse a target in the presence of complement.
- the complement activation pathway is initiated by the binding of the first component of the complement system (CIq) to a molecule ⁇ e.g., an antibody) complexed with a cognate antigen.
- CIq first component of the complement system
- a CDC assay e.g., as described in Gazzano-Santoro et al, J. Immunol. Methods 202: 163 (1996), may be performed.
- “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains. Each light chain has a variable domain at one end (V L ) and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
- variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs).
- the variable domains of native heavy and light chains each comprise four FRs, largely adopting a ⁇ -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
- the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen- binding site of antibodies (see Kabat et ah, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).
- the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
- hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
- the hypervariable region generally comprises amino acid residues from a "complementarity determining region” or "CDR" (e.g., residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et ah, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.
- CDR complementarity determining region
- residues from a "hypervariable loop” e.g., residues 26-32 (Ll), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. MoI. Biol. 196:901-917 (1987)).
- "Framework Region” or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.
- framework region refers to the art recognized portions of an antibody variable region that exist between the more divergent CDR regions. Such framework regions are typically referred to as frameworks 1 through 4 (FRl, FR2, FR3, and FR4) and provide a scaffold for holding, in three-dimensional space, the three CDRs found in a heavy or light chain antibody variable region, such that the CDRs can form an antigen-binding surface.
- frameworks 1 through 4 FRl, FR2, FR3, and FR4
- antibodies can be assigned to different classes. There are five major classes of antibodies IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.
- the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
- the "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
- monoclonal antibody is used to refer to an antibody molecule synthesized by a single clone.
- the modifier “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.
- monoclonal antibodies may be made by the hybridoma method first described by Kohler and Milstein, Nature 256:495 (1975); Eur. J. Immunol. 6:51 1 (1976), by recombinant DNA techniques, or may also be isolated from phage or other antibody libraries.
- polyclonal antibody is used to refer to a population of antibody molecules synthesized by a population of B cells.
- Antibody fragments comprise a portion of a full length antibody, generally the antigen binding domain(s) or variable domain(s) thereof.
- antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , scFv, (ScFv) 2 , dAb, and complementarity determining region (CDR) fragments, linear antibodies, single-chain antibody molecules, minibodies, diabodies, multispecific antibodies formed from antibody fragments, and, in general, polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
- bispecific antibody fragments are bispecific antibody fragments.
- Antibodies are glycoproteins, with glycosylation in the Fc region.
- the Fc region of an IgG immunoglobulin is a homodimer comprising interchain disulfide- bonded hinge regions, glycosylated CH2 domains bearing N-linked oligosaccharides at asparagine 297 (Asn-297), and non-covalently paired CH3 domains.
- Glycosylation plays an important role in effector mechanisms mediated Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII, and CIq.
- antibody fragments of the present invention must include a glycosylated Fc region and an antigen-binding region.
- bispecific antibody and "bispecific antibody fragment” are used herein to refer to antibodies or antibody fragments with binding specificity for at least two targets. If desired, multi-specificity can be combined by multi-valency in order to produce multivalent bispecific antibodies that possess more than one binding site for each of their targets. For example, by dimerizing two scFv fusions via the helix-turn-helix motif, (scFv)i-hinge-helix-turn-helix-(scFv) 2 , a tetravalent bispecific miniantibody was produced (M ⁇ lleret al., FEBS Lett. 432(l-2):45-9 (1998)). The so-called Mi-bi-miniantibody 1 possesses two binding sites to each of it target antigens.
- Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab') 2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
- Fv is the minimum antibody fragment which contains a complete antigen- recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen- binding site on the surface of the V H -V L dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
- the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain.
- Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region.
- Fab'-SH is the designation herein for Fab 1 in which the cysteine residue(s) of the constant domains bear at least one free thiol group.
- F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
- the "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
- Single-chain Fv or “scFv” antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
- the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the scFv to form the desired structure for antigen binding.
- HER2 antibody scFv fragments are described in WO93/16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458.
- diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a variable heavy domain (V H ) connected to a variable light domain (V L ) in the same polypeptide chain (V H - V L ).
- V H variable heavy domain
- V L variable light domain
- the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
- Diabodies are described more fully in, for example, EP 404,097; WO 93/1 1161 ; and Hollinger ef ⁇ /., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
- Humanized forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
- humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
- donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
- framework region (FR) residues of the human immunoglobulin are replaced by corresponding non- human residues.
- humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine 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 hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
- the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
- Fc immunoglobulin constant region
- a “naked antibody” is an antibody (as herein defined) that is not conjugated to a heterologous molecule, such as a cytotoxic moiety or radiolabel.
- An “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
- the antibody will be purified to greater than 95% by weight of antibody as determined by non-reducing SDS-PAGE, CE-SDS, or Bioanalyzer.
- Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
- immunoadhesin designates antibody-like molecules which combine the "binding domain" of a heterologous protein (an “adhesin”, e.g. a receptor, ligand or enzyme) with the effector functions of immunoglobulin constant domains.
- adhesin e.g. a receptor, ligand or enzyme
- the immunoadhesins comprise a fusion of the adhesin amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site (antigen combining site) of an antibody (i.e. is "heterologous") and an immunoglobulin constant domain sequence.
- the immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgGl, IgG.2, IgG3, or IgG4 subtypes, IgA, IgE, IgD or IgM.
- immunoadhesins ligand binding domains and receptor binding domains see, e.g. U.S. Patent Nos. 5,116,964; 5,714,147; and 6,406,604, the disclosures of which are hereby expressly incorporated by reference.
- the present invention provides a method for preparing antibodies and antibody-like molecules, such as Fc fusion proteins (immunoadhesins), bearing predominantly Man5 glycans, but with decreased amounts of Man7, Man8, and Man9, in a mammalian host cell, by manipulating the glycosylation machinery of the recombinant mammalian host cell producing the antibody or antibody-like molecule.
- Fc fusion proteins immunoadhesins
- the antibodies and other recombinant proteins herein can be produced by well known techniques of recombinant DNA technology.
- the skilled practitioner could generate antibodies directed against an antigen of interest, e.g., using the techniques described below.
- the antibodies produced in accordance with the present invention are directed against an antigen of interest.
- the antigen is a biologically important polypeptide and administration of the antibody to a mammal suffering from a disease or disorder can result in a therapeutic benefit in that mammal.
- antibodies directed against nonpolypeptide antigens are also contemplated.
- the antigen is a polypeptide, it may be a transmembrane molecule (e.g. receptor) or ligand such as a growth factor.
- Exemplary molecular targets for antibodies encompassed by the present invention include CD proteins such as CD3, CD4, CD8, CD19, CD20, CD22, CD34, CD40; members of the ErbB receptor family such as the EGF receptor (EGFR, HERl, ErbBl), HER2 (ErbB2), HER3 (ErbB3) or HER4 (ErbB4) receptor; cell adhesion molecules such as LFA-I, Macl, pl50,95, VLA-4, ICAM-I, VCAM and ⁇ v/ ⁇ 3 integrin including either ⁇ or ⁇ subunits thereof (e.g.
- anti-CDl la, anti-CD18 or anti-CDl lb antibodies growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA -4; protein C, neutropilins and receptors, EGF-C, ephrins and receptors, netrins and receptors, slit and receptors, anti-Mi, or any of the other antigens mentioned herein.
- Antigens to which the antibodies listed above bind are specifically included within the scope herein.
- the nucleic acid encoding it may be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
- the antibody may be produced by homologous recombination, e.g. as described in U.S. Pat. No. 5,204,244, specifically incorporated herein by reference.
- DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available.
- the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, e.g., as described in U.S. Pat. No. 5,534,615 issued JuI. 9, 1996 and specifically incorporated herein by reference.
- the antibodies of the present invention must be glycosylated, and thus suitable host cells for cloning or expressing the DNA encoding antibody chains or other antibody-like molecules include mammalian host cells. Interest has been great in mammalian host cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.
- Examples of useful mammalian host cell lines are monkey kidney CV 1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells ⁇ DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod.
- COS-7 monkey kidney CV 1 line transformed by SV40
- human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)
- baby hamster kidney cells BHK, ATCC CCL 10
- Chinese hamster ovary cells ⁇ DHFR CHO, Urlaub et al., Proc. Natl.
- monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (Wl 38, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
- Host cells are transformed with expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
- the mammalian host cells may be cultured in a variety of media.
- Commercially available media such as Ham's FlO (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI- 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
- any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
- the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
- the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the primary purification step.
- affinity chromatography is the primary purification step.
- the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
- Protein A can be used to purify antibodies that are based on human ⁇ l, human ⁇ 2, or human ⁇ 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human ⁇ 3 (Guss et al., EMBO J.
- the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
- the antibody comprises a CHBdomain
- the BAKERBOND ABXTM resin J. T. Baker, Phillipsburg, NJ. is useful for purification.
- the mixture comprising the antibody of interest and contaminants may be subjected to additional purification steps to achieve the desired level of purity.
- a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al, Nature, 321 :522-525 (1986); Riechmarm et al, Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized” antibodies are chimeric antibodies (U.S. Patent No.
- humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
- variable domains both light and heavy
- sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
- the human sequence which is closest to that of the rodent is then accepted as the human FR for the humanized antibody (Sims et ah, J. Immunol., 151 :2296 (1993)).
- Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et ah, Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta e/ ⁇ /., J. Immnoh, 151 :2623 (1993)).
- humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
- Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
- Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
- FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
- the CDR residues are directly and most substantially involved in influencing antigen binding.
- transgenic animals ⁇ e.g., mice
- transgenic animals that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production.
- J H antibody heavy-chain joining region
- Human antibodies can also be derived from phage-display libraries (Hoogenboom et al, J. MoI. Biol, 227:381 (1991); Marks et al, J. MoI. Biol, 222:581-597 (1991); Vaughan et al Nature Biotech 14:309 (1996)).
- Multispecific antibodies have binding specificities for at least two different antigens. While such molecules normally will only bind two antigens (i.e. bispecific antibodies, BsAbs), antibodies with additional specificities such as trispecific antibodies are encompassed by this expression when used herein.
- bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al, EMBO J., 10:3655-3659 (1991).
- the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
- the preferred interface comprises at least a part of the C H 3 domain of an antibody constant domain.
- one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
- Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
- Bispecific antibodies include cross-linked or "heteroconjugate” antibodies.
- one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
- Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (US Patent No. 4,676,980), and for treatment of HIV infection (WO
- Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in US Patent No. 4,676,980, along with a number of cross-linking techniques.
- Antibodies with more than two valencies are contemplated.
- trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).
- the simplest and most straightforward immunoadhesin design combines the binding domain(s) of the adhesin (e.g. the extracellular domain (ECD) of a receptor) with the hinge and Fc regions of an immunoglobulin heavy chain.
- ECD extracellular domain
- nucleic acid encoding the binding domain of the adhesin will be fused C-terminally to nucleic acid encoding the N-terminus of an immunoglobulin constant domain sequence, however N-terminal fusions are also possible.
- the encoded chimeric polypeptide will retain at least functionally active hinge, C H 2 and C H 3 domains of the constant region of an immunoglobulin heavy chain. Fusions are also made to the C-terminus of the Fc portion of a constant domain, or immediately N-terminal to the C H I of the heavy chain or the corresponding region of the light chain.
- the precise site at which the fusion is made is not critical; particular sites are well known and may be selected in order to optimize the biological activity, secretion, or binding characteristics of the immunoadhesin.
- the adhesin sequence is fused to the N-terminus of the Fc domain of immunoglobulin Gi (IgGi). It is possible to fuse the entire heavy chain constant region to the adhesin sequence. However, more preferably, a sequence beginning in the hinge region just upstream of the papain cleavage site which defines IgG Fc chemically (i.e. residue 216, taking the first residue of heavy chain constant region to be 1 14), or analogous sites of other immunoglobulins is used in the fusion.
- the adhesin amino acid sequence is fused to (a) the hinge region and C H 2 and C H 3 or (b) the C H 1 , hinge, C H 2 and C H 3 domains, of an IgG heavy chain.
- the immunoadhesins are assembled as multimers, and particularly as heterodimers or heterotetramers.
- these assembled immunoglobulins will have known unit structures.
- a basic four chain structural unit is the form in which IgG, IgD, and IgE exist.
- a four chain unit is repeated in the higher molecular weight immunoglobulins; IgM generally exists as a pentamer of four basic units held together by disulfide bonds.
- IgA globulin, and occasionally IgG globulin may also exist in multimeric form in serum. In the case of multimer, each of the four units may be the same or different.
- the immunoadhesin structures of the present invention must have an Fc region.
- Various exemplary assembled immunoadhesins within the scope herein are schematically diagrammed below:
- each A represents identical or different adhesin amino acid sequences
- V L is an immunoglobulin light chain variable domain
- V H is an immunoglobulin heavy chain variable domain
- C L is an immunoglobulin light chain constant domain
- Cu is an immunoglobulin heavy chain constant domain
- n is an integer greater than 1 ;
- Y designates the residue of a covalent cross-linking agent.
- the adhesin sequences can be inserted between immunoglobulin heavy chain and light chain sequences, such that an immunoglobulin comprising a chimeric heavy chain is obtained.
- the adhesin sequences are fused to the 3' end of an immunoglobulin heavy chain in each arm of an immunoglobulin, either between the hinge and the C H 2 domain, or between the C H 2 and C H 3 domains. Similar constructs have been reported by Hoogenboom, et ah, MoI. Immunol. 28:1027-1037 (1991).
- an immunoglobulin light chain might be present either covalently associated to an adhesin-immunoglobulin heavy chain fusion polypeptide, or directly fused to the adhesin.
- DNA encoding an immunoglobulin light chain is typically coexpressed with the DNA encoding the adhesin-immunoglobulin heavy chain fusion protein.
- the hybrid heavy chain and the light chain will be covalently associated to provide an immunoglobulin-like structure comprising two disulfide- linked immunoglobulin heavy chain-light chain pairs.
- Immunoadhesins are most conveniently constructed by fusing the cDNA sequence encoding the adhesin portion in-frame to an immunoglobulin cDNA sequence.
- fusion to genomic immunoglobulin fragments can also be used (see, e.g. Aruffo et ah, Cell 61 :1303-1313 (1990); and Stamenkovic et ah, Cell 66:1133-1 144 (1991)).
- the latter type of fusion requires the presence of Ig regulatory sequences for expression.
- cDNAs encoding IgG heavy-chain constant regions can be isolated based on published sequences from cDNA libraries derived from spleen or peripheral blood lymphocytes, by hybridization or by polymerase chain reaction (PCR) techniques.
- the cDNAs encoding the "adhesin" and the immunoglobulin parts of the immunoadhesin are inserted in tandem into a plasmid vector that directs efficient expression in the chosen host cells.
- glycosylation Following the expression of proteins in eukaryotic, e.g. mammalian host cells, the proteins undergo post-translational modifications, often including the enzymatic addition of sugar residues, generally referred to as "glycosylation".
- N-linked refers to the attachment of the carbohydrate moiety to the side-chain of an asparagine residue.
- the tripeptide sequences, asparagine (Asn)-X-serine (Ser) and asparagine (Asn)-X- threonine (Thr), wherein X is any amino acid except proline, are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
- O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, fucose, N-acetylglucosamine, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be involved in O-linked glycosylation.
- N-linked glycans there is an amide bond connecting the anomeric carbon (C-I) of a reducing-terminal N-acetylglucosamine (GIcNAc) residue of the oligosaccharide and a nitrogen of an asparagine (Asn) residue of the polypeptide.
- C-I anomeric carbon
- GIcNAc reducing-terminal N-acetylglucosamine
- O-linked glycans are attached via a glycosidic bond between N-acetylgalactosamine (GaINAc), galactose (Gal), fucose, N-acetylglucosamine, or xylose and one of several hydroxyamino acids, most commonly serine (Ser) or threonine (Thr), but also hydroxyproline or hydroxyzine in some cases.
- the biosynthetic pathway of O-linked oligosaccharides consists of a step-by-step transfer of single sugar residues from nucleotide sugars by a series of specific glycosyltransferases.
- the nucleotide sugars which function as the monosaccharide donors are uridine-diphospho-GalNAc (UDP-GaINAc), UDP-GIcNAc, UDP-GaI, guanidine- diphospho-fucose (GDP-Fuc), and cytidine-monophospho-sialic acid (CMP-SA).
- N-linked oligosaccharide assembly In N-linked oligosaccharide synthesis, initiation of N-linked oligosaccharide assembly does not occur directly on the Asn residues of the protein, but involves preassembly of a lipid-linked precursor oligosaccharide which is then transferred to the protein during or very soon after its translation from mRNA.
- This precursor oligosaccharide (Glc 3 Man 9 GlcNAc 2 ) is synthesized while attached via a pyrophosphate bridge to a polyisoprenoid carrier lipid, a dolichol, with the aid of a number of membrane -bound glycosyltransferases.
- another membrane-bound enzyme transfers it to sterically accessible Asn residues which occur as part of the sequence -Asn-X-Ser/Thr-.
- Glycosylated Asn residues of newly-synthesized glycoproteins transiently carry only one type of oligosaccharide, GIc 3 MaUgGIcNAc 2 . Processing of this oligosaccharide structure generates the great diversity of structures found on mature glycoproteins.
- N-linked oligosaccharides is accomplished by the sequential action of a number of membrane-bound enzymes and includes removal of the three glucose residues, removal of a variable number of mannose residues, and addition of various sugar residues to the resulting trimmed core.
- MansGlcNAc 2 can serve as a substrate for GIcNAc transferase I (GIcNAcT-I), which transfers a ⁇ l ⁇ 2-linked GIcNAc residue from UDP-GIcNAc to the ⁇ l-»3-linked mannose residue to form GlcNAcMansGlcNAc 2 , which is further trimmed by ⁇ -mannosidase II, which removes two mannose residues to generate a protein-linked oligosaccharide with the composition GlcNAcMan 3 GlcNAc 2 .
- This structure is a substrate for GIcNAc transferase II (not shown).
- This stage is followed by a complex series of processing steps, including sequential addition of monosaccharides to the oligosaccharide chain by a series of membrane-bound glycosyltransferases, which differ between various cell types.
- a diverse family of "complex" oligosaccharides is produced, including various branched, such as biantennary (two branches), triantennary (three branches) or tetraantennary (four branches) structures.
- ADCC antibody-dependent cell mediated cytotoxicity
- the present invention discloses methods for producing antibodies having an increased amount of the Man5 glycoform while diminishing the amount of Man7,8,9 relative to what has been previously described. It also describes a method for modulating the amount of the Man5 glycoform produced.
- GIcNAc Transferase I adds a GlcNac moiety to the terminal ⁇ -1 ,3 arm of Man5, which can then be acted on by ⁇ -mannosidase II.
- GIcNAc Transferase I 5 By abrogating or modulating the activity of GIcNAc Transferase I 5 the proportion of antibodies bearing Man5 glycans can be increased.
- Man7,8,9 glycoforms can be diminished by enhancing ⁇ -1,2 mannosidase activity.
- ⁇ -1,2 mannosidase either in vivo or in vitro, the more rapidly cleared Man7,8,9 glycans can be converted to Man5.
- the present invention also provides a method for producing antibodies with a variable amount of Man5 using RNA interference (RNAi) knockdown.
- RNAi RNA interference
- RNA interference is a method for regulating gene expression.
- RNA molecules can bind to single-stranded mRNA molecules with a complementary sequence and repress translation of particular genes.
- the RNA can be introduced exogenously (small interfering RNA, or siRNA), or endogenously by RNA producing genes (micro RNA, or miRNA).
- siRNA small interfering RNA
- miRNA miRNA producing genes
- double-stranded RNA complementary to GIcNAc Transferase I can decrease the amount of this glycosyltransferase expressed in an antibody expressing cell line, resulting in an increased level of the Man5 glycoform in the antibody produced.
- RNAi knockdown approach rather than a complete knockout, allows the fine tuning of th amount of Man5 glycan to an optimal level, which may be of great benefit, if the production of antibodies bearing less than 100% Man5 glycans is desirable.
- ⁇ -1,2 mannosidase activity can be enhanced in a variety of ways.
- ⁇ -1,2 mannosidase activity can be enhanced by providing additional copies of the ⁇ - mannosidase I present in the recombinant host cell used for antibody production.
- an ⁇ -1,2 mannosidase from a microbial cell line may be transfected into the expressing cell line.
- Alpha- 1 ,2-mannosidase from different species have different specificity toward the various high mannose glycans.
- a commercially available ⁇ - mannosidase I, ⁇ -l,2-mannosidase from Aspergillus saitoi has demonstrated robust in vitro trimming of highly-enriched Man9 glycoform to Man5. Contreras et.al. have showed that the ⁇ -l,2-mannosidase from Trichoderma reesei alone can trim all four mannoses from Man9 to yield homogenous Man5 glycan (Maras el ah, J.
- the A Saitoi or T. reesei ⁇ -1,2- mannosidases can be used with the protein A-purified ocrelizumab with high level of Man 9 as a substrate.
- an ⁇ -1,2 mannosidase from other mammalian species may be transfected into the expressing cell line. It is also apparent in higher organisms that different endogenous mannosidases are involved in the trimming of each mannose to convert Man9 to Man5. In fact, most species utilize two mannosidases, one in the endoplasmic reticulum(ER) and another one in the golgi apparatus, to trim Man9 to Man5 in a two-step reaction (Gonzalez et ah, J. Biol. Chem., 274(30): 21375-21386 (1999); Mast and Moremen, Methods Enzymoh, 415: 31-46 (2006)).
- Man5 glycoform Another approach toward generating homogenous Man5 glycoform involves combining the RNA interference technology and the in vitro trimming reaction discussed above. Since CHO cells use two mannosidases to convert Man9 to Man5, the CHO golgi mannosidase can be knocked-down using RNAi which would lead to the accumulation of Man8B. The Man8B-enriched antibodies can subsequently be purified, and then converted to Man5 by the same in vitro trimming reaction using ⁇ -l,2-mannosidase from Aspergillus saitoi or Trichoderma reesei.
- the in vitro trimming reaction may be incorporated in vivo by expressing the ⁇ -l,2-mannosidase in the same cell line where the CHO golgi mannosidase is knockdown specifically. This will eliminate a purification step prior to the conversion from Man8B to Man5.
- any of the previously described mannosidases may be used post expression in vitro to trim Man6,7,8,9 to Man5.
- RNAi approach was employed to knock down the expression of the endogenous GnT-I gene.
- a 1.3 kb fragment of GnT-I coding sequence (NCBI Accession No: U65791) was cloned by reverse transcription polymerase chain reaction (RT-PCR) using total RNA purified from CHO DP 12 cells. The PCR fragment was then cloned into pCMV-3Tag-6 vector (Cat # 240195) from Strategene ( Figure 2). The DNA sequence encoding the full-length GnT-I protein was cloned in the BamHI and HindIII sites.
- siRNA Small inhibitory RNA
- siRNA probes were constructed using annealed synthetic oligonucleotides independently cloned into the pSilencer 3.1 -Hl hygro plasmid ( Figure 3) from Ambion, Inc. (Austin, TX) to produce short hairpin siRNAs.
- the DNA sequences encoding siRNA probes were cloned into BamHI and HindIII sites under the control of PoIIII type Hl promoter.
- the transcript from Hl promoter forms a hairpin-loop siRNA, consisting of a 19 nucleotide sense sequence specific to the GnT-I gene, linked to its reverse complement antisense sequence by a 9 nucleotide hairpin-look sequence.
- Each siRNA probe consisted of a 19 nucleotide sense sequence specific to the GnT-I gene, linked to its reverse complement anti-sense sequence by a 9 nucleotide hairpin-loop sequence and followed by 5 6U's at the 3' end ( Figure 3).
- Figure 4 shows the 5 siRNA sequences targeting the GnT-I gene. The ability of these siRNA probes to cleave the GnT-I transcript was tested by transient cotransfection of each siRNA expression probe plasmid with the FLAG ® -tagged GnT-I plasmids into CHO cells.
- RNAiI and RNAi3 which demonstrated markedly stronger inhibitory effects than the rest of the RNAi 's, were chosen for further evaluation.
- RNAiI siRNA expression plasmids
- RNAi2, RNAi3, RNA ⁇ 4, and RNAi5 a combination siRNA plasmid containing the sequences of RNAiI and RNAi3 (RNAJ13) were transiently transfected into the cell line for ocrelizumab production.
- RNAJ13 combination siRNA plasmid containing the sequences of RNAiI and RNAi3
- RNAJ13 combination siRNA plasmid containing the sequences of RNAiI and RNAi3
- HCCF Harvested cell culture fluid
- HCCF was analyzed using a CE-glycan assay to determine levels of different glycoforms, and cell pellets were used for quantitative qPCR analysis to measure the endogenous mRNA level of GnT-I.
- mRNA was isolated by RNeasy ® 96 well kit (Qiagen) or MagMAXTM -96 total RNA isolation kit (Ambion).
- a TAQMAN ® analysis was performed to measure GnT-I mRNA expression level during the course of the experiment ( Figure 6A).
- the sequences of the primers and probe, which cover the 3' end of the cDNA (bp 1260- 1324) are as follows:
- RNAi plasmids targeting GnT-I were able to knock down GnT-I mRNA significantly, with a maximum of 80% knockdown compared to control (transfected with scramble plasmid) 5 days post transfection.
- GnT-I expression level of control was set to 100%.
- Knockdown activity in the TAQMAN ® assay correlated well with western blot analysis of FLAG ® -tagged GnT-I, in which RNAiI and RNAi3 seemed to be the strongest inhibitors in both assays.
- RNAi 13 provided additional inhibition compared to RNAi 1 and RNAi3 individually, and was chosen to be the primary RNAi vector for all subsequent studies.
- CE-glycan capillary electrophoresis
- the antibodies from HCCF were purified using a preparative protein-A purification method. Then the N-linked glycan attached to the Fc region is cleaved off by peptide-N-glycosidase F (PNGase F) with an overnight incubation at 37 0 C.
- PNGase F peptide-N-glycosidase F
- the protein was precipitated after the reaction to separate it from the cleaved glycans, which were then labeled with 8-aminopyrene-l,3,6-trisulfonate (APTS) by reductive amination.
- APTS 8-aminopyrene-l,3,6-trisulfonate
- the labeled glycans were then analyzed using capillary electrophoresis against APTS-labeled glycan standards with specific elution profile. The details of the assay can be found on the Beckman Coulter website.
- the Man5 content of the antibodies assayed at Day 5 correlated well with TAQMAN ® data ( Figure 6A), with RNAi 13 having the highest Man5 content at approximately 9% ( Figure 6B).
- RNAi 13 plasmid In order to increase the Man5 level with transient expression of the RNAi 13 plasmid, longer cell culture duration was tested in the same cell line (up to 14 days). Experience with other antibodies indicated that there was an increased Man5 level with increased production culture duration (Figure 10A). A similar transient transfection protocol was used in the 14- day experiment. The cell line was transfected with scrambled or RNAi 13 vectors using LIPOFECTAMINETM. HCCF was collected at various day post transfection, and samples were analyzed using a CE-glycan assay to determine the Man5 level. Figure 7 shows the Man5 level at the indicated culture duration, with the RNAi 13 plasmid resulted in roughly 10-fold higher Man5 level than the control condition, and the level appeared to be stable throughout the entire run. In addition, the GnT-I mRNA level for this particular experiment was similar to the 5 day culture (data not shown).
- ⁇ -mannosidase I is another important enzyme in the glycosylation pathway. It is responsible for converting the high mannose structures Man7,8,9 into Man5,6. By overexpressing this protein, it could potentially result in a more uniform conversion to Man5.
- coding sequences of homologue from homo sapien, Mus musculus, Rattus norvegecus were aligned to uncover conserved regions that could be used to clone out the CHO gene. A conserved area upstream of the 5' end of the coding sequence and a small region after the stop codon was cloned out the CHO ⁇ -mannosidase I.
- the cDNA has a size of 1.9kB ( Figure 8A).
- RNAi 13 vector into ocrelizumab resultsed in a roughly 10- fold increase in Man5 levels, from 0.5-1% to 9%.
- stable cell line development was undertaken to create stable clones with the shRNA incorporated into the genome and therefore is expected to provide a stable expression level to knockdown GnT-I in a more consistent fashion.
- the standard protocol for developing stable cell clones was done with the RNAi 13 plasmid (Shen et al. (2007), Metabolic engineering to control glycosylation In M. Butler (Ed.), Cell culture and Upstream Processing (pp.131- 148).
- transfection was done in the same fashion as transient transfection experiment using LIPOFECTAMINETM. Instead of being exchanged into production media 24 hours post transfection, the cells were exchanged into selection media containing 0.5 mg/mL hygromycin selective pressure, and then plated onto petri dishes at various seeding densities. The dishes (20-50 dishes total) were incubated in a CO 2 humidified incubator at 37 0 C for 2-3 weeks until clones were observed. The individual clones were transferred into 96-well plates (1 clone/well), and approximately 200- 300 clones were picked at the first stage.
- GnT-I mRNA level of all clones were determined using TAQMAN ® assay to select for clones with lowest GnT-I mRNA level. Subsequently, selected clones were scaled up to 48-well plate, 24-well plate, 6-well plate, T75 cultures flask, and then finally shake flasks. Roughly 12 clones were selected to perform an initial production culture, which is a 14 day culture in production media with the addition of 10% nutrient supplement on day 3. The top clones with the highest amount of Man5 were banked and stored for future use.
- the 18 clones were further evaluated in a 14-day production run, and then the HCCF was analyzed at the end of the run using CE-glycan analysis.
- the Man5 levels are shown in Figure 9B. Again the results indicated that the percentage of Man5 glycoform (Man5%) of the stable clones is similar to those obtained with the transient transfection experiment. A roughly 5-fold increase in Man5 level was observed, with the highest level of Man5 at 6% for clone P2-1 OC.
- Manipulating cell culture conditions to increase Man5 level The use of optimized cell culture parameters in conjunction with RNAi knockdown of GnT-I can increase the amount of Man5 obtained. Longer culture duration and increased osmolality media have been found to be beneficial with another antibody evaluated, and results by others (US patent application US2007/0190057-A1 Figure 2, Figure 4) have also shown that increasing osmolality can increase the proportion of antibodies with high mannose glycoforms.
- Figure 1OA is an example of a production run of the antibody evaluated, which clearly shows that a large amount of Man5 antibodies were produced toward the end of the 14 days culture.
- increased NaCl (or osmolality) concentration in basal media was also tested with respect to level of Man5.
- increasing basal osmolality from 300 to 400 mOsm can further increase Man5 content.
- high osmolality nutrient supplement solution does not enhance the Man5 level beyond the benefit of the high osmolality basal media (data not shown).
- the high osmolality and longer culture duration effect can be used in combination in order to increase the Man5 level for other molecules. Due to these findings, an experiment was designed to test these conditions with the cell line generating ocrelizumab and the top 5 GnT-I knockdown stable clones of ocrelizumab described in the previous section.
- Figure 1OC summarizes the results of a 14 day production run with the same antibody, where 1 ⁇ M of manganese chloride was fed on either day 3, day 3 & 6, or day 3, 6, &9. In all cases, the Man5 level was decreased by 50% compared to the control. To increase the Man5 level, conditions which lower manganese concentration would be expected to be beneficial.
- Man5 level increases as culture duration increases for all conditions. High osmolality in basal media appears to have the strongest effect in enhancing the Man5 level, and the absence of manganese has a slight benefit as compared to the control.
- the Man5 level can be increased up to 2-fold. Therefore, by manipulating cell culture conditions, the Man5 level can be further enhanced in conjunction with the RNAi knockdown approach.
- GnT-I activity in the cell may be used separately or in combination with GnT-I knockdown.
- Cell lines with a high level of Man5 can also be selected by screening for cell clones with GnT-I mutation, which would lead to activity loss of GnT-I and accumulation of Man5 glycoform.
- Lectin-resistant methods have been studied by Stanley et al. (Stanley et al, Proc. Nat. Acad. ScL USA, 72(9): 3323-3327 (1975); Patnaik and Stanley, Methods Enzymol, 416: 159-182 (2006)).
- a lectin which binds to glycans which are generated downstream of GnT-I can select for cells having a high level of RNAi knockdown.
- Phytohemagglutinin (PHA) a toxic plant lectin
- PHA a toxic plant lectin
- PHA a toxic plant lectin
- Cells which lack GnT-I activity will result in defective lectin-binding glycoproteins present on the cell surface, which in turns allow the cells to survive in a PHA-containing environment.
- This approach can be used in conjunction with RNAi knockdown of GnT-I in order to increase the probability of cells survived under the lectin stress condition. This can also increase the efficiency of finding mutants with a high level of knockdown.
- GnT-I requires UDP-GIcNAc as a substrate.
- UDP-GIcNAc is synthesized in the cytosol, and transported to the lumen of the golgi. Guillen et al (PNAS 95: 7888-7892, 1998) cloned the mammalian Golgi membrane transporter. Knocking down or knocking out this transporter is expected to eliminate or greatly diminish the pool of UDP-GIcNAc in the Golgi apparatus. Accordingly, reducing the level of a substrate for GnT-I, UDP-GIcNAc, is expected to result in higher Man5 levels.
- Antibody enriched in the Man5 glycoform was purified by Con A Sepharose chromatography from harvested, clarified cell culture fluid (HCCF) from a CHO cell fermentation of a humanized IgGl which binds to a soluble receptor.
- the cell line expressing this antibody produced a higher than usual amount of Man5 bearing glycans (5-20%).
- 2L of HCCF (1.29 g/L mAb) was purified on a PROSEPTM A column (2.5 x 14cm, Millipore) equilibrated in 25mM Tris, 25mM NaCl, 5mM EDTA pH 7.1. After a series of post load wash steps using equilibration buffer and a 0.4M Potassium Phosphate buffer, bound antibody was eluted using 0.1 M Acetic Acid, pH 2.9, and adjusted to pH 7.4 with 1.5M Tris base.
- the eluted protein A pool was then processed over a Con A SEPHAROSETM column (2.5 x 5 cm, GE Healthcare), equilibrated in 1 mM MnCl 2 , 1 mM CaCl 2 , 0.5 M NaCl, 25 mM Tris, pH 7.4. Bound antibody was eluted with 0.5M alpha-D- mannopyranoside, 0.5 M NaCl, 25 mM Tris, pH 7.4.
- Antibody in the Con A SEPHAROSETM pool was recovered on the protein A column, and then subjected to chromatography on Con A SEPHAROSETM a second time. After recovery on protein A, the pool was rechromatographed on Con A SEPHAROSETM a third time, and this time elution was carried out with a 15 column volume gradient of equilibration buffer and elution buffer. The product was again isolated by protein A chromatography.
- Figure 1 1 shows antibody binding to Fc gamma receptor IHa-Vl 58.
- Open circles represent HERCEPTIN ® (trastuzumab, open squares represent RITUXAN ® (rituximab), open triangles represent anti-receptor antibody with 5% Man5 (7-9% afucosyl glycans), open diamonds represent anti-receptor antibody with 16% Man5 (14.6% afucosyl glycans), and closed circles represent anti-receptor antibody with 62% Man5 (11% afucosyl glycans).
- Figure 12 shows antibody binding to Fc gamma receptor IHa-F 158.
- Open circles represent HERCEPTIN ® (trastuzumab), open squares represent RITUXAN ® (rituximab), open triangles represent anti-receptor antibody with 5% Man5 (7-9% afucosyl glycans), open diamonds represent anti-receptor antibody with 16% Man5 (14.6% afucosyl glycans), and closed circles represent anti-receptor antibody with 62% Man5 (11% afucosyl glycans).
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