Modified Host Cells and Uses Thereof
The present disclosure provides compositions and methods comprising cells producing glycoproteins with variant glycosylation patterns. The methods and compositions may be used in producing antibodies and proteins of therapeutic value.
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This application is a continuation of U.S. patent application Ser. No. 12/507,046, filed Jul. 21, 2009, which application claims priority to U.S. Ser. Nos. 61/194,292 filed on Sep. 26, 2008, 61/120,722 filed on Dec. 8, 2008, and 61/168,186 filed on Apr. 9, 2009, all of which are incorporated herein in ir entirety for all purposes.
BACKGROUNDVariations in composition of carbohydrate, saccharide, or sugar molecule, have been shown to affect affinity of IgG for three classes of FcγRs (FcγRI, FcγRII, and FcγRIII) that link IgG-mediated immune response with cellular effector functions (Wright and Morrison, Trends Biotechnol 15(1): 26-32; Gessner et al., Ann Hematol 76(6): 231-48 (1998); Jefferis et al., Immunol Rev 163: 59-76 (1998). Ravetch and Bolland, Annu Rev Immunol 19: 275-90 (2001)).
Sugar chains of glycoproteins are generally divided into following two broad types based on binding form to a proteinaceous moiety: namely a sugar chain which binds to asparagine (N-glycoside-linked sugar chain), and a sugar chain which binds to or amino acids, such as serine or threonine (O-glycoside-linked sugar chain). Typically, y have a basic common core structure shown by following structural formula (I):
The N-glycoside-linked sugar chains have various structures, with various sugar molecules. The sugar chain terminus which binds to asparagine is typically called a reducing end, and opposite side is called a non-reducing end. N-glycoside-linked sugar chains can include a high mannose type in which mannose alone binds to non-reducing end of core structure; a complex type in which non-reducing end side of core structure has at least one parallel branch of galactose-N-acetylglucosamine (Gal-GlcNAc) and non-reducing end side of Gal-GlcNAc has a structure of sialic acid, bisecting N-acetylglucosamine or like; a hybrid type in which non-reducing end side of core structure has branches of both of high mannose type and complex type; and like. The structure of a sugar chain can be determined by sugar chain genes, such as a gene for a glycosyltransferase which synsizes a sugar chain, and/or a gene for a glycolytic enzyme which hydrolyzes sugar chain.
Glycoproteins are typically modified with a sugar chain in endoplasmic reticulum (ER) lumen. For example, during biosynsis step of N-glycoside-linked sugar chain, a relatively large sugar chain is transferred to a polypeptide chain that is elongating in ER lumen. Sugar molecules can be added in succession to phosphate groups of a long chain lipid carrier comprising about 20 α-isoprene units, such as dolichol phosphate (P-Dol). For example, N-acetylglucosamine (GlcNAc) is transferred to P-Dol to form GlcNAc-P-P-Dol and n one more GlcNAc is transferred to form GlcNAc-GlcNAc-P-P-Dol. Next, five mannoses (Man) are transferred to reby form (Man)5-(GlcNAc)2-P-P-Dol and n four Man's and three glucoses (Glc) are transferred. As a result, a sugar chain precursor, (Glc)3-(Man)9-(GlcNAc)2-P-P-Dol, a core oligosaccharide, is formed. The sugar chain precursor comprising 14 sugars can n be transferred to a polypeptide having an asparagine-X-serine or asparagine-X-threonine sequence in ER lumen. The dolichol pyrophosphate (P-P-Dol) bound to core oligosaccharide is typically released and becomes dolichol phosphate (P-Dol) by hydrolysis with pyrophosphatase, and is recycled. Trimming of sugar chain typically starts after sugar chain binds to polypeptide. For example, 3 Glc's and 1 or 2 Man's are eliminated on ER, such as by action of α-1,2-glucosidase I, α-1,3-glucosidase II and α-1,2-mannosidase.
The glycoprotein which was subjected to trimming on ER can be transferred to Golgi body and furr modified. For example, present in cis part of Golgi body are N-acetylglucosamine phosphotransferase (which aids in addition of mannose phosphate), N-acetylglucosamine 1-phosphodiester α-N-acetylglucosamimidase and α-mannosidase I (which reduce Man residues to 5). Present in medium part of Golgi body are N-acetylglucosamine transferase I (GnTI) (which aids in addition of first outside GlcNAc of complex type N-glycoside-linked sugar chain), α-mannosidase II (which aids in removal of 2 Man's), N-acetylglucosamine transferase II (GnTII) (which aids in addition of second GlcNAc from outside) and α-1,6-fucosyltransferase (which aids in addition of fucose to reducing end N-acetylglucosamine). Present in trans part of Golgi body are galactose transferase, which aids in addition of galactose, and sialyltransferase, which relates to addition of sialic acid such as N-acetylneuraminic acid or like. Thus, various N-glycoside-linked sugar chains can be formed by activities of se various enzymes.
Sugar chain structure variations, or variant glycosylation patterns due to various sugar molecule content in such chains, plays an important role in effector function of glycoproteins, such as antibodies. For example, in Fc region of an antibody of an IgG type, two N-glycoside-linked sugar chain binding sites are typically present. In serum IgG, sugar chain binding site generally binds a complex type sugar chain having multiple branches, and in which addition of sialic acid or bisecting N-acetylglucosamine is low. There are a variety of manners and forms in which addition of galactose is made to non-reducing end of complex type sugar chain and addition of fucose to N-acetylglucosamine in reducing end (see for example, Leppanen et al., Biochemistry, 36, 7026-7036 (1997)).
Fucosylation is an example of a process in which newly synsized antibodies can be modified by addition of fucose saccharides in Golgi apparatus of a cell. This protein modification can be visualized by staining cells with fluorophore-conjugated LCA (Lens culimaris agglutinin-A), a chemical that preferentially binds to proteins modified with fucose. Recently, it has been observed that fucosylation of antibodies affects antibody binding to human FcγR and antibody-dependent cellular cytotoxicity (ADCC). Antibody-binding affinity and antibody-mediated ADCC is strongly enhanced when antibodies have low levels of fucose (Shields et al., J Biol Chem 277(30): 26733-4 (2002); Shinkawa et al., J Biol Chem 278(5): 3466-73 (2003)).
Protein fucosylation is a process that begins with uptake of free fucose, followed by phosphorylation by fucose kinase and conversion to GDP-fucose by GDP-fucose pyrophosphorylase. Fucosyltransferases transfer fucose residue to glycans or protein within secretary pathways, subsequently modified glycoproteins are delivered to cell surface for secretion. The fucosylation status of antibody-producing cells correlates with fucose content in antibody produced, and that absence of fucosyltransferase abrogated fucosylation at both cellular and antibody levels (Yamane-Ohnuki et al., Biotechnol Bioeng 87(5): 614-22 (2004)).
ADCC is an important mechanism of action by which rapeutic antibodies induce immune responses and mediate killing of cancer cells. Enhancement of ADCC by rapeutic antibodies can improve clinical responses and reduce rapeutic dosages, thus diminishing possible side effects (Adams and Weiner, Nat Biotechnol 23(9). 1147-57 (2005)). In vivo models and clinical trials have demonstrated that rapeutic antibodies, such as Herceptin, possess cytotoxic properties, including ADCC. These properties are main factors in Herceptin induced breast tumor regression and protection from lung metastasis (Carter et al., Proc Natl Acad Sci USA 89(10): 4285-9 (1992); Lewis et al., Cancer Immunol Immunor 37(4): 255-63 (1993); Cooley et al., Exp Hematol 27(10): 1533-41 (1999); Clynes et al., Nat Med 6(4): 443-6 (2000); Repka et al., Clin Cancer Res 9(7): 2440-6 (2003); Gennari et al., Clin Cancer Res 10(17): 5650-5 (2004); Nahta and Esteva, Cancer Lett 232(2): 123-38 (2006)). Furr, it was demonstrated that antibodies produced by fucosylation-low cells enhance ADCC activity (Shields et al., J Biol Chem 277(30): 26733-4 (2002); Shinkawa et al., J Biol Chem 278(5): 3466-73 (2003)).
Expression of ADCC activity of human IgG1 subclass antibodies typically requires binding of Fc region of an antibody to an antibody receptor existing on surface of an effector cell, such as a killer cell, a natural killer cell, an activated macrophage or like (FcγR) and various complement components. It has been suggested that several amino acid residues in second domain of antibody hinge region and C region (hereinafter referred to as “Cγ2 domain”) and a sugar chain linked to Cγ2 domain are important for this binding reaction.
Currently, several strategies have been proposed for enhancing monoclonal antibody-mediated ADCC against tumor cells, such as: 1) developing specific anti-cancer antibodies in which one arm of antibody binds to an IgG receptor in order to more efficiently recruit immune effector cells (Segal et al., J Immunol Methods 248(1-2). 1-6 (2001)); 2) using recombinant human cytokines to increase effector function of immune effector cells (Carson et al., Eur J Immunol 31(10): 3016-25 (2001); Repka et al., Clin Cancer Res 9(7): 2440-6 (2003)); 3) using IgG-cytokine fusion protein (Penichet and Morrison, J Immunol Methods 248(1-2): 91-101 (2001)); 4) altering Fc sequence of an antibody for improved binding to an IgG receptor (Shields et al., J Biol Chem 276(9): 6591-604 (2001)); and 5) optimization of levels of Asn297-linked carbohydrates (Umana et al., Nat Biotechnol 17(2): 176-80 (1999); Davies et al., Biotechnol Bioeng 74(4): 288-94 (2001); Shinkawa et al., J Biol Chem 278(5): 3466-73 (2003)).
One approach is to modify fucose content of anti-cancer antibodies to increase binding affinity for FcγRs and ADCC. IgG1 has two N-linked oligossacharide chains bound to Asn297, composed of a trimannosyl core structure with presence or absence of a core fucose, bisecting N-acetylglucosamine and terminal galactose (Rademacher et al., Biochem Soc Symp 51: 131-48 (1986)). The nature and importance of Asn297-linked carbohydrates in immunoglobulin G effector functions has long been recognized. It has been demonstrated that defucosylated Rituxan, an anti-CD20 antibody for lymphoma treatment, strongly binds to FcγRIIIa with high affinity and 100-fold enhanced ADCC activity (Shinkawa et al., J Biol Chem 278(5): 3466-73 (2003); Yamane-Ohnuki et al., Biotechnol Bioeng 87(5): 614-22 (2004); Kanda et al., Biotechnol Bioeng 94(4): 680-8 (2006)). Ors have shown that binding low-fucose Herceptin to FcγRIIIa was improved by about 50-fold over normal-fucose Herceptin, and as a result, Herceptin-mediated-ADCC was substantially improved (Shields et al., J Biol Chem 277(30): 26733-40 (2002)). This indicates that defucosylation of anti-cancer antibodies increases ir binding affinity to FcγR and enhances ADCC. Furr, it suggests that modification of fucose content represents a way to improve anti-cancer immune response of antibodies so as to augment ir rapeutic efficacy and expand treatment to cancer patients that are unresponsive to fucosylated antibodies.
However, re remains a need for alternative methods for modifying antibodies of high rapeutic potential.
SUMMARY OF THE INVENTIONThere exists a need to produce rapeutic proteins with increased effector functions. The present disclosure provides methods and compositions to meet this need, as well as related advantages, by producing cells lines and glycoproteins with variant glycosylation patterns, or glycoslyation patterns that are modified in comparison to a corresponding wildtype protein, or protein produced in a cell line with an unmodified glycosylation pattern. The differences in sugar chain content of glycoproteins expressed by host cells and development of a host cell which can be used for production of se glycoproteins with variant glycosylation patterns, such as an antibody, can have higher effector function.
In one embodiment, provided herein are methods and compositions for producing and selecting a host cell that is modified to yield a variant glycosylation pattern as compared to an unmodified parental host cell. The method of selecting a host cell with a variant glycosylation pattern may comprise providing a plurality of host cells; introducing random genetic mutation(s) to plurality of host cells; and selecting from plurality of cells at least one cell that exhibits a variant glycosylation pattern characterized by a change in level of at least one type of sugar molecules as compared to a corresponding parental cell that has not been subject to said random genetic mutation. Furrmore, genetic mutation(s) may be induced by a chemical mutagen.
The host cell may be a eukaryotic cell, such as a mammalian cell. The modified host cell can be a Chinese Hamster Ovarian (CHO) cell. The host cell may be a modified CHO cell, such as a CHO-1E5, CHO-3F, or CHO-2.6 cell. The host cell may also be a myeloma cell. The modified host cell can be an NS0, SP2/0, HEK293, PER.C6, or YB2/0 cell. The host cell that is modified exhibits a variant glycosylation pattern that can be characterized by a change in levels of at least two types of sugar molecules present on a surface of host cell as compared to an unmodified parental host cell. The change in level can be at least approximately one-, two-, three-, four-, five-, six-, seven-, eight-, nine-, ten-fold or higher. The variant glycosylation pattern can be evidenced by a change in level of fucosylation, mannosylation, N-acetylglucosaminylation, or combinations reof. In some embodiments, change can be in level of galactose, glucose, or both. The change can be an increase or a decrease in levels of galactose, glucose or both. For example, change can be an increase in glucose and a decrease in galactose.
In some embodiments, change in level may be a reduction in level of fucosylation of modified host cell as compared to unmodified parental host cell. The variant glycosylation pattern can also be evidenced by a change in level of mannosylation, such as level of α-linked mannose. The level of mannosylation may be increased in modified host cell. The variant glycosylation pattern can also be evidenced by a change in level of N-acetylglucosaminylation, such as a decrease or increase of N-acetylglucosaminylation in modified host cell. The N-acetylglucosaminylation may involve α- or β-linked N-acetylglucosamine. The host cell that is modified to yield a variant glycosylation pattern as compared to an unmodified parental host may have 1,6-fucosyltransferase activity comparable to that of unmodified parental host cell. In anor aspect, modified host cell can maintain its variant glycosylation pattern after at least approximately 30, 40, 50, 60, 80, 100, 120, 150, 200, 1000 or more passages. The modified host cell may also be grown in a serum-free medium, in suspension, and/or in a fermentor.
In anor aspect, present invention provides a population of antibodies produced by a modified non-lymphocytic host cell that produces antibodies exhibiting a substantially homogeneous pattern of N-linked glycan, wherein members of said population bind at least two distinct antigens.
In anor aspect, present invention provides a modified host cell that produces N-linked glycans having a variant glycosylation pattern characterized by a change in level of one or more sugar moieties selected from group consisting of glucose, galactose, mannose, and glucosamine. In some embodiments, variant glycosylation pattern is characterized by a reduction in level of galactose. In some embodiments, variant glycosylation pattern is characterized by a reduction in level of D-glucosamine. In some embodiments, variant glycosylation pattern is characterized by an increase in level of mannose. In some embodiments, variant glycosylation pattern is characterized by an increase in level of glucose. In some embodiments, modified host cell produces antibodies that exhibit ADCC activity higher than that of a corresponding unmodified host cell. In some embodiments, modified host cell produces antibodies having an increased binding affinity to FcγRIIIA receptor as compared to a corresponding antibody produced by a corresponding unmodified host cell.
In some embodiments, modified host cell is a Chinese Hamster Ovarian (CHO) cell. In some embodiments, modified host cell is selected from group consisting of NS0, SP2/0, HEK293, PER.C6 and YB2/0 cell. In some embodiments, modified host cell is a myeloma cell. In some embodiments, modified host cell maintains variant glycosylation pattern after at least approximately 60 passages. In some embodiments, modified host cell grows in a serum-free medium. In some embodiments, modified host cell grows in suspension. In some embodiments, modified host cell comprises a heterologuous sequence encoding a heterologous glycoprotein.
In yet anor aspect, present invention provides an isolated host cell that produces glycoproteins exhibiting a substantially homogeneous pattern of N-linked glycan. In some embodiments, substantially homogeneous pattern of N-linked glycan is evidenced by a single peak resolved by mass spectrometry. In some embodiments, N-linked pattern has a structure of Formula I or II, in which a fucose moiety can optionally be linked to first 4GlcNAc from right:
In yet anor aspect, modified host cell may comprise a heterologous sequence, wherein heterologous sequence may encode a heterologous glycoprotein, such as an antibody or an enzyme. As disclosed herein, a method of producing a modified glycoprotein comprising: providing a heterologous polynucleotide sequence that encodes modified glycoprotein; and causing modified glycoprotein to be expressed in a modified host cell is also provided. Thus, also provided herein is a glycoprotein, such as a heterologous glycoprotein encoded by a heterologous sequence, that exhibits a variant glycosylation pattern characterized by a change in levels of at least two types of sugar molecules as compared to a corresponding wildtype glycoprotein, such as produced by an unmodified parental host cell. The variant glycosylation pattern can be evidenced by a change in level of N-linked oligosaccharides. The heterologous glycoprotein may exhibit a reduced or increased level of glucose, galactose, fucose, mannose, and/or of N-acetylglucosamine content as compared to a corresponding wildtype glycoprotein produced by an unmodified parental host cell. The N-acetylglucosamine may be α- or β-linked N-acetylglucosamine.
In one aspect, glycoprotein is an antibody or antibody fragment. The antibody may bind a cancer antigen. For example, antigen may be selected from group consisting of HER2, CD20, EGF receptor, VEGF receptor, PDGF receptor, EpCam, CD3, CD4, CD19, CD30, CD33, CD40, CD51, CD55, CD80, CD95, CCR2, CCR3, CCR4, CCR5, folate receptor, CXCR4, insulin-like growth factor receptor, and integrin family members. The antibody may also exhibit increased antibody-dependent cellular cytotoxicity (ADCC) as compared to a corresponding antibody produced by an unmodified host cell. Furrmore, antibody may include an IgG antibody. As disclosed herein, antibody may also exhibit a variant glycosylation pattern characterized by a change in levels of at least two types of sugar molecules as compared to a corresponding wildtype antibody, such as produced by an unmodified parental host cell. The sugar molecules may be attached through an Fc region of antibody.
In anor aspect, present invention provides an N-linked glycan comprising one glucose molecule, four mannose molecules, and two N-acetylglycosamine molecules. In some embodiments, N-linked glycan comprises one or more fucose molecules. In some embodiments, N-linked glycan has a structure of formula (I), in which a fucose moiety can optionally be linked to first 4GlcNAc from right.
In some embodiments, N-linked glycan has a structure of formula (II), in which a fucose moiety can optionally be linked to first 4GlcNAc from right.
In anor aspect, present invention provides an isolated glycoprotein that contains an N-glycan disclosed herein. In some embodiments, glycoprotein is an antibody. In some embodiments, glycoprotein is an enzyme. In some embodiments, N-linked glycan is attached to an Fc region of antibody. In some embodiments, antibody binds to a cancer antigen. In some embodiments, cancer antigen is selected from group consisting of HER2, Immunoglobulin epsilon Fc receptor II, Alk-1, CD20, EGF receptor, VEGF receptor, FGF receptor, NGF receptor, PDGF receptor, EpCam, CD3, CD4, CD11a, CD19, CD22, CD30, CD33, CD38, CD40, CD51, CD55, CD80, CD95, CCR2, CCR3, CCR4, CCR5, CTLA-4, Mucin 1, Mucin 16, Endoglin, Mesolin receptor, Nogo receptor, folate receptor, CXCR4, insulin-like growth factor receptor, Ganglioside GD3, and alpha and beta Integrins. In some embodiments, antibody is produced by a modified host cell which produces a substantially homogeneous population of N-linked glycans. In some embodiments, antibody has an increased ADCC (antibody-dependent cellular cytotoxicity) activity as compared to a corresponding antibody produced by an unmodified CHO cell clone, CHO-K1 (ATCC #CCL-61 and CRL-9618) or CHO-DG44 (Invitrogen #12609-012). In some embodiments, antibody has an increased binding affinity to FcγRIIIA receptor as compared to a corresponding antibody produced by an unmodified CHO cell clone, CHO-K 1 (ATCC #CCL-61 and CRL-9618) or CHO-DG44 (Invitrogen #12609-012). In some embodiments, antibody is an inhibitory antibody. In some embodiments, antibody is a stimulatory antibody. In some embodiments, antibody is an IgG antibody.
In some embodiments, host cell that produces glycoproteins exhibiting a substantially homogeneous pattern of N-linked glycan is a non-lymphocytic cell. In some embodiments, host cell is a CHO cell. In some embodiments, host cell produces antibodies exhibiting a substantially homogeneous pattern of N-linked glycan.
In still anor aspect, present invention provides a method of producing a modified glycoprotein comprising: (a) providing a heterologous polynucleotide sequence that encodes modified glycoprotein; and (b) causing modified glycoprotein to be expressed in a host cell disclosed herein. In some embodiments, modified glycoprotein is secreted by host cell. In some embodiments, host cell is maintained in a serum free medium. In some embodiments, host cell is maintained in a suspension culture. In some embodiments, modified glycoprotein is an antibody.
Also provided in present invention is a culture medium comprising a host cell as disclosed herein. In some embodiments, culture medium is serum free. The present invention also discloses a culture fermentor comprising a plurality of host cells that produce N-glycan having a variant glycosylation pattern or glycoprotein exhibiting a substantially homogeneous pattern of N-glycan in a culture medium.
In anor aspect, present invention provides an antibody produced by a modified host cell, antibody comprising an N-linked glycan, wherein antibody has increased binding affinity to an Fc gamma receptor Ma (FcγRIIIa), and/or decreased binding affinity to an Fc gamma receptor IIb (FcγRIIb), as compared to a corresponding antibody produced by an unmodified host cell, reby enhancing ADCC (antibody-dependent cell-mediated cytotoxicity) activity against effector cells expressing FcγRIIIa and/or FcγRIIb. In anor aspect, present invention provides an antibody produced by a modified host cell, antibody comprising an N-linked glycan, wherein antibody exhibits an increased ADCC activity, as compared to a corresponding antibody produced by a corresponding unmodified host cell, and wherein increased ADCC activity is against effector cells expressing a high affinity Fc gamma receptor, FcγRIIIa 158V/V, and effector cells expressing a low affinity Fc gamma receptor, FcγRIIIa 158F/F or FcγRIIIa 158F/V.
In some embodiments of antibody of present invention, N-linked glycan comprises one glucose molecule, four mannose molecules, and two N-acetylglycosamine molecules. In some embodiments, N-linked glycan comprises one or more fucose molecules. In some embodiments, N-linked glycan having a structure of formula (I) or (II), in which a fucose moiety can optionally be linked to first 4GlcNAc from right.
In some embodiments, N-linked glycan is attached to an Fc region of antibody. In some embodiments, antibody binds to a cancer antigen. The cancer antigen can include but are not limited to HER2, Immunoglobulin epsilon Fc receptor II, Alk-1, CD20, EGF receptor, VEGF receptor, FGF receptor, NGF receptor, PDGF receptor, EpCam, CD3, CD4, CD11a, CD19, CD22, CD30, CD33, CD38, CD40, CD51, CD55, CD80, CD95, CCR2, CCR3, CCR4, CCR5, CTLA-4, Mucin 1, Mucin 16, Endoglin, Mesolin receptor, Nogo receptor, folate receptor, CXCR4, insulin-like growth factor receptor, Ganglioside GD3, and alpha and beta Integrins. The antibody of present invention can be an inhibitory antibody or a stimulatory antibody. In some embodiments, antibody is an IgG antibody. In some embodiments, antibody has an increased ADCC (antibody-dependent cell-mediated cytotoxicity) activity against effector cells expressing a low affinity FcR, FcγRIIIa 158F/F or FcγRIIIa 158 F/V, as compared to a corresponding antibody produced by an unmodified CHO cell clone, CHO-K1 (ATCC #CCL-61 and CRL-9618) or CHO-DG44 (Invitrogen #12609-012). In some embodiments, antibody has an increased ADCC (antibody-dependent cell-mediated cytotoxicity) activity against effector cells expressing a high affinity FcR, FcγRIIIa 158V/V, as compared to a corresponding antibody produced by an unmodified CHO cell clone, CHO-K1 (ATCC #CCL-61 and CRL-9618) or CHO-DG44 (Invitrogen #12609-012). In some embodiments, host cell is a Chinese Hamster Ovarian (CHO) cell. The host cell can include but are not limited to NS0, SP2/0, HEK293, PER.C6 and YB2/0 cell. The host cell can also be a myeloma cell. In some embodiments, effector cell is a human peripheral blood mononuclear cell (PBMC). In some embodiments, effector cell is an NK cell, a monocyte, a macrophage, or a polymorphonuclear neutraphils (PMN).
In anor aspect, present invention provides a modified host cell characterized in its ability to produce a modified antibody having an N-linked glycan, wherein antibody exhibiting an increased binding affinity to an Fc gamma receptor IIIa (FcγRIIIa), and/or decreased binding affinity to an Fc gamma receptor IIb (FcγRIIb), as compared to a corresponding antibody produced by an unmodified host cell, reby enhancing ADCC (antibody-dependent cell-mediated cytotoxicity) activity against effector cells expressing FcγRIIIa and/or FcγRIIb. In yet anor aspect, present invention provides a modified host cell characterized in its ability to produce a modified antibody having an N-linked glycan, wherein antibody exhibits an increased ADCC activity, as compared to a corresponding antibody produced by a corresponding unmodified host cell, and wherein increased ADCC activity is against effector cells expressing a high affinity Fc gamma receptor, FcγRIIIa 158V/V, and effector cells expressing a low affinity Fc gamma receptor, FcγRIIIa 158F/F or FcγRIIIa 158F/V.
In some embodiments of modified host cells of present invention, N-linked glycan exhibits a variant glycosylation pattern characterized by a change in level of one or more sugar moieties selected from group consisting of glucose, galactose, mannose, and glucosamine. In some embodiments, variant glycosylation pattern is characterized by a reduction in level of galactose. In some embodiments, variant glycosylation pattern is characterized by a reduction in level of D-glucosamine. In some embodiments, variant glycosylation pattern is characterized by an increase in level of mannose. In some embodiments, variant glycosylation pattern is characterized by an increase in level of glucose. In some embodiments, N-linked glycan has a structure of Formula I or II, in which a fucose moiety can optionally be linked to first 4GlcNAc from right:
In some embodiments, host cell produces antibodies that exhibit an increased ADCC activity against effector cells expressing a high affinity Fc receptor, FcγRIIIa 158V/V, and an increased ADCC activity against cells expressing a low affinity Fc receptor, FcγRIIIa 158F/F or FcγRIIIa 158F/V, as compared to a corresponding antibody produced by a corresponding unmodified host cell. In some embodiments, host cell produces antibodies that exhibit an increased binding affinity to an Fc gamma receptor IIIa (FcγRIIIa), and/or decreased binding affinity to an Fc gamma receptor IIb (FcγRIIb), as compared to a corresponding antibody produced by an unmodified host cell, reby enhancing ADCC activity against effector cells expressing FcγRIIIa and/or FcγRIIb. In some embodiments, antibody binds to a cancer antigen. The cancer antigen can include but are not limited to HER2, Immunoglobulin epsilon Fc receptor II, Alk-1, CD20, EGF receptor, VEGF receptor, FGF receptor, NGF receptor, PDGF receptor, EpCam, CD3, CD4, CD11a, CD19, CD22, CD30, CD33, CD38, CD40, CD51, CD55, CD80, CD95, CCR2, CCR3, CCR4, CCR5, CTLA-4, Mucin 1, Mucin 16, Endoglin, Mesolin receptor, Nogo receptor, folate receptor, CXCR4, insulin-like growth factor receptor, Ganglioside GD3, and alpha and beta Integrins. In some embodiments, host cell is a Chinese Hamster Ovarian (CHO) cell. The host cell can include but are not limited to NS0, SP2/0, HEK293, PER.C6 and YB2/0 cell. The host cell can also be a myeloma cell. In some embodiments, cell maintains variant glycosylation pattern after at least approximately 60 passages. In some embodiments, modified host cell grows in a serum-free medium. In some embodiments, modified host cell grows in suspension. In some embodiments, modified host cell comprises a heterologuous sequence encoding a heterologous glycoprotein.
Also provided by present invention is a method of preventing or treating a disorder, method comprising administering to a subject in need reof an effective amount of a subject antibody disclosed herein. In some embodiments, disorder is selected from group consisting of cancer, allergies, cardiovascular diseases, inflammatory diseases, metabolic diseases, neurological diseases, viral infections and/or bacterial infections. For example, disorder can be a cancer or an allergy. In some embodiments, subject is a mammal, for example, a human. In some embodiments, administration of subject antibody is via parenteral injection, infusion, oral administration or inhalation. The present invention also provides a method of producing a modified antibody comprising: (a) providing a heterologous polynucleotide sequence that encodes modified antibody; and (b) causing modified antibody to be expressed in a subject host cell disclosed herein. In some embodiments, modified antibody is secreted by host cell. In some embodiments, host cell is maintained in a serum free medium. In some embodiments, host cell is maintained in a suspension culture. The present invention also encompasses a culture medium comprising a subject host cell and a culture fermentor comprising a plurality of subject host cells in a culture medium. In some embodiments, culture medium is serum free.
INCORPORATION BY REFERENCEAll publications and patent applications mentioned in this specification are herein incorporated by reference to same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The novel features of disclosure are set forth with particularity in appended claims. A better understanding of features and advantages of present disclosure will be obtained by reference to following detailed description that sets forth illustrative embodiments, in which principles of disclosure are utilized, and accompanying drawings of which:
The compositions and methods of present disclosure provide cell lines that are modified to have variant glycosylation patterns. Such cell lines can be used to produce proteins, such as antibodies with enhanced effector functions, such as increased antibody-dependent cellular cytotoxicity (ADCC) activity. The proteins typically also have variant glycosylation patterns.
Host CellsThe host cell of present disclosure is modified to yield a variant glycosylation pattern as compared to an unmodified parental host cell. A host cell includes an individual cell, cell culture, and/or cell line. Host cells include progeny of a single host cell. A host cell can be transfected with a heterologous sequence of present disclosure. Host cells may be prokaryotic or eukaryotic, such as bacterial cells, fungal cells, animal cells, insect cells, plant cells and like that are capable of glycosylation.
Examples of bacterial host cells include microorganisms belonging to genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas and like. For example, bacterial host cells may include, but not be limited to, Escherichia coli XL1-Blue, XL2-Blue, DH1, MC1000, KY3276, W1485, JM109, HB101, No. 49, i W3110, NY49, G 1698, or TB 1. Or bacterial host cells may include, but not be limited to, Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium ammoniagenes, Brevibacterium immariophilum ATCC 14068, Brevibacterium saccharolyticum ATCC 14066, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium acetoacidophilum ATCC 13870, Microbacterium ammoniaphilum ATCC 15354, Pseudomonas putida, Pseudomonas sp. D-0110 and like.
Yeast host cells may include microorganisms belonging to genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, Candida and like, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Candida utilis and like.
Or examples of eukaryotic cells include animal cells such as mammalian cells. For example, host cells preferably include, but are not limited to, Chinese hamster ovary cells (CHO) or monkey cells, such as COS cells. The CHO cells may include, but not be limited to, CHO/dhfr− or CHO/DG44 cells. The Chinese hamster ovary tissue-derived CHO cell includes any cell which is a cell line established from an ovary tissue of Chinese hamster (Cricetulus griseus). Examples include CHO cells described in documents such as Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics, 55, 513 (1968); Chromosoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260 (1997); Proc. Natl. Acad. Sci. USA, 77, 4216 (1980); Proc. Natl. Acad. Sci., 60, 1275 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics, Appendix I, II (pp. 883-900); and like. In addition, CHO-K1 (ATCC CCL-61), DUXB11 (ATCC CCL-9096) and Pro-5 (ATCC CCL-1781) registered in ATCC (The American Type Culture Collection) and a commercially available CHO-S (Life Technologies, Cat #11619) or sub-cell lines obtained by adapting cell lines using various media can also be exemplified.
In an alternative embodiment parent cell line is derived from a lymphocytic lineage cell line, such as a B cell line. The host cell may be from cell lines used in hybridoma production. They can be myeloma cells, such as from murine myeloma lines, such as, but not limited to, MOPC-21, MPC-11, NS0, SP-2, Sp2/0, S194, and X63-Ag8-653 cells; human myeloma cell lines, such as, but not limited to, Namalwa, Karpas 707H, RPMI 8226, 8226 AR/NIP4-1, KM-2R, and U-266; or rat myeloma cell lines, such as, but not limited to, YB2/0, YB2/3.0.Ag.20, Y3-Ag1.2.3, IR983F. Cell lines, such as HeLa, HEK-293, NIH3T3, COS, CHO, NS0, PER.C6, K562, L1.2, JY, BHK, K562, 293F, 3T3, and Jurkat may also be used in present disclosure. For example, in some embodiments, host cell is a CHO-1E5, CHO-3F, or CHO-2.6 clone.
Examples of insect host cells include Spodoptera frugiperda ovary cells, such as Sf9 and Sf21 (Baculovirus Expression Vectors, A Laboratory Manual, W. H Freeman and Company, New York (1992)); a Trichoplusia ni ovary cell such as High 5 (manufactured by Invitrogen); and like. Examples of plant host cells include plant cells of tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat, barley and like.
The modified host cells of present disclosure may be grown in cultures, and in any apparatus that may be used to grow cultures, including fermetors. They may be grown as monolayers or attached to a surface. Alternatively, host cells may be grown in suspension. The cells can be grown in a culture medium that is serum-free. The media can be a commercially available media, such as, but not limited to, Opti-CHO (Invitrogen, Catalogue #12681) supplemented with glutamine, such as 8 mM L-glutamine. The modified host cells can maintain its variant glycosylation pattern for a number of passages in culture. For example, modified host cell can maintain its variant glycosylation pattern after at least approximately 20, 30, 40, or 50 passages. In some embodiments, modified host cell can maintain its variant glycosylation pattern after at least approximately 60 passages. In yet or embodiments, modified host cell can maintain its variant glycosylation pattern after at least approximately 100, 150, 200, 500, or 1000 or more passages.
In some embodiments, host cell is a non-lymphocytic cell. A lymphocyte is a type of white blood cell in vertebrate immune system. Lymphocytes typically include T cells, B cells and natural killer (NK) cells. A non-lymphocytic cell encompasses any type of cell that is not a lymphocyte. The host cell of invention may have a species origin selected from group consisting of human, mouse, rat, fruit fly, worm, yeast and bacterium. The host cell may be derived from a suitable tissue including but not limited to blood, muscle, nerve, brain, heart, lung, liver, pancreas, spleen, thymus, esophagus, stomach, intestine, kidney, testis, ovary, hair, skin, bone, breast, uterus, bladder, spinal cord, or various kinds of body fluids. The host cells producing N-glycan may be derived from any developmental stage including embryo and adult stages, as well as developmental origin such as ecotodermal, mesodermal, and ectodermal origin. In some embodiments, host cells are CHO, NS0, SP2/0, HEK293, PER.C6 or YB2/0 cells.
Generating Modified Cell LinesThe present disclosure provides methods for generating and selecting host cells that are modified to yield a variant glycosylation pattern. The method of selecting a host cell with a modified glycosylation pattern can comprise providing a plurality of host cells; introducing random genetic mutation(s) to plurality of host cells; and selecting from plurality of cells at least one cell that exhibits a variant glycosylation pattern characterized by a change in level of at least one type of sugar molecules as compared to a corresponding parental cell that has not been subject to random genetic mutation. The host cell may be any of those described herein, including eukaryotic cells such as CHO cells or YB2.0 cells.
The genetic mutation(s) can be induced by mutagens. The mutagens may be, but are not limited to, genetic, chemical or radiation agents. For example, mutagen may be ionizing radiation, such as, but not limited to, ultraviolet light, gamma rays or alpha particles. Or mutagens may include, but not be limited to, base analogs, which can cause copying errors; deaminating agents, such as nitrous acid; intercalating agents, such as ethidium bromide; alkylating agents, such as bromouracil; transposons; natural and syntic alkaloids; bromine and derivatives reof; sodium azide; psoralen (for example, combined with ultraviolet radiation). The mutagen may be a chemical mutagen such as, but not limited to, ICR191, 1,2,7,8-diepoxy-octane (DEO) or ethyl methane sulfonate (EMS). Different mutagens may be combined, eir sequentially or concurrently, when introduced into a host cell.
Methods may include those as shown in
The glycosylation patterns of parental host cell can be compared to variant glycosylation pattern of a modified host cell, and can be evidenced by a change in level of one or more types of sugar molecules. For example, variant glycosylation pattern can be characterized by a change in at least two types of glycosylation selected from group consisting of: fucosylation, mannosylation, and N-acetylglucosaminylation, as furr described below. Furrmore, variant glycosylation pattern can be evidenced by a change in level of glucose and/or galactose, such as an increase in glucose and a decrease in galactose. Additional variations are described as follows.
Glycosylation PatternsThe host cell of present disclosure is modified to yield a variant glycosylation pattern characterized by, e.g., a change in levels of at least two types of sugar molecules present on a surface of host cell as compared to unmodified parental host cell. The host cells of present disclosure may also be used to produce proteins or glycoproteins. The proteins may be antibodies or enzymes, and may be used as rapeutics. Furrmore, glycoproteins may be secreted. The proteins may be heterologous proteins, such as proteins produced from introduction of heterologous sequences into modified host cell exhibiting a variant glycosylation pattern. Heterologous means derived from a genotypically distinct entity from rest of entity to which it is being compared and can be applied to a polynucleotide, such as a nucleic acid sequence, or a polypeptide, which means that polynucleotide or polypeptide or protein.
The proteins or glycoproteins of present disclosure can be endogenous or heterologous, and are produced in a host cell that has been modified to exhibit a variant glycosylation pattern. The glycoproteins may also exhibit a variant glycosylation pattern, which may be characterized by a change in levels of at least two types of sugar molecules as compared to a corresponding wildtype glycoprotein (ie. a glycoprotein produced in an unmodified host cell). The sugar molecules may be directly attached to glycoprotein (for example, N- or O-linked to glycoprotein), or indirectly (for example, linked through or sugars that are N- or O-linked to glycoprotein). Sugar chain structure variations, or variant glycosylation patterns, due to various sugar molecule content in such chains play an important role in effector function of glycoproteins. For example, variant glycosylation patterns of a glycoprotein can increase effector function of glycoprotein, such as increase antibody-dependent cellular cytotoxicity (ADCC) activity of an antibody.
The glycosylation pattern of a host cell may be N- or O-glycosylation of any proteineous moiety, wherein addition of one or more sugar molecules may be at amide nitrogen of asparagine or hydroxyloxygen of hydroxylysine, hydroxyproline, serine, or threonine, respectively. The glycosylation pattern may be characterized by a change levels of at least two or more sugar molecules or saccharides, such as monosaccharides, disaccharides, polysaccharides or oligosaccarhides. For example, sugar molecules may be trioses, tetrososes, pentoses, hexoses, heptoses, octoses, nonoses, or derivatives reof, such as deoxy sugars, such as deoxyhexoses; N- or O-substituted derivatives, such as sialic acid; or sugars with amino groups. The sugar molecules may include, but not be limited to, galactose (Gal), glucose (Glc), mannose (Man), N-acetylneuraminic acid (NeuAc), fucose (Fuc), N-Acetylgalactoseamine (GalNAc), N-Acetylglucosamine (GlcNAc); and Xylose (Xyl). The sugar molecules may be linked to or sugar molecules via α or β linkage.
The variant glycosylation pattern of present disclosure may be evidenced by a change in level of at least two types of sugar molecules and can be evidenced by change of different types of glycosylation of host cell or glycoprotein. For example, level of fucosylation, mannosylation, N-acetylglucosaminylation and/or combinations reof may be used as evidence of a variant glycosylation pattern. For example, level of fucosylation, mannosylation, and/or N-acetylglucosaminylation can be reduced or increased in a modified host cell as compared to an unmodified parental cell. The level of fucosylation, mannosylation, and/or N-acetylglucosaminylation of a glycoprotein can be reduced or increased as compared to a corresponding wildtype glycoprotein (ie. produced in an unmodified parental host cell). For example, heterologous glycoprotein may exhibit a reduced level of fucose, mannose, and/or of N-acetylglucosamine content as compared to a corresponding wildtype glycoprotein produced by said unmodified host cell. The glycosylation can involve α- or β-linked sugars, such as α- or β-linked mannose or α- or β-linked N-acetylglucosamine. Furrmore, host cell with a variant glycosylation pattern may still contain 1,6-fucosyltransferase gene activity and/or maintain it; as compared to that of unmodified parental host cell.
The variant glycosylation pattern of present disclosure can also be evidenced by a change in level of glucose, galactose, or both, as compared to an unmodified parental cell. For example, change can be an increase or decrease in galactose levels. The change can also be an increase or decrease in glucose levels. In some embodiments, change can be an increase in glucose levels, a decrease in galactose levels, or an increase in glucose levels and a decrease in galactose levels, such as shown in
The variant glycosylation pattern may be evidenced by a change in level of at least two types of sugar molecules, wherein change in level of at least one type of sugar molecule is at least approximately two fold. Alternatively change in level is at least approximately two fold for at least two types of sugars. The change may be an increase or decrease in level of sugar molecules. The change in level may be at least approximately 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 75, 100, 500, 1000 fold, or even higher.
In some embodiments, present invention provides compositions comprising a subject N-linked glycan, when exhibited on an antibody, results in an enhanced antibody-dependent-cell-cytotoxicity (ADCC) activity of antibody. N-linked glycans are attached to amide nitrogen of asparagine side chains of a protein. There are three major types of N-linked saccharides: high-mannose oligosaccharides, complex oligosaccharides and hybrid oligosaccharides. High-mannose N-linked glycan typically comprises two N-acetylglucosamines with many mannose residues. Complex oligosaccharides typically contain almost any number of or types of saccharides. Proteins can be glycosylated by both types of oligos on different portions of protein. N-linked glycans can be modified with a variety of different monosaccharides including glucose, mannose, galactose, N-acetylglucosamine, N-acetylgalactosamine, fucose and sialic acid.
The subject N-linked glycan composition and structure of N-linked oligosaccharide can be produced by an exemplary modified CHO cell clone designated as CHO-1E5. A single N-glycan structure on antibody is produced by CHO-1E5. The CHO-1E5 has altered N-glycan biosynsis and antibodies produced by this host cell clone exhibit a glycosylation profile consisting of glucose, mannose, and N-acetylglucosamine with or without fucose. The structural formula of N-glycan has been demonstrated as Glu-GlcNAC2-Man4 (+/−Fuc). This glycan has basic structure of core N-linked oligosaccharide: 3Man-2 GluNAc with or without fucose but lacks two outside acetylglucosamine. There is a glucose molecule and an extra mannose molecule attached to one of side chains of core oligosaccharide (
In some embodiments, glycosylation pattern represents a composition of structural formula (II), in which a fucose moiety can optionally be linked to first 4GlcNAc from right.
The structural formulae (I) and (II) depicted hereinabove and in
In some embodiments, glycosylation pattern represents a mixed composition of structural formulas (I) and (II). In some embodiments, N-glycan synsized by a modified host cell of present invention designated as CHO-1E5 lacks galactose, has reduced levels of fucose and N-acetylglucosamine, and contains a terminal glucose as compared to N-glycan produced by wild type parental host cells (
The glycosylation pattern of a host cell or glycoprotein can be detected using agents that specifically, or preferentially recognizes or binds specific sugar molecules or proteinaceous moieties that are associated or modified with specific sugar molecules. Agents may include, but not be limited to, Lens culimaris agglutinin-A (LCA) is a chemical that specifically binds to proteins modified with fucose; wheat germ agglutinin (WGA), which has preferential binding to N-acetylglucosamine; concanavalin A (Con A), which recognizes α-linked mannose; and Griffonia (Bandeiraea) Simplicifolia Lectin II (GS-II), which binds to α- or β-linked N-acetylglucosamine residues.
The agents may be detected directly or indirectly. For example, agents may be conjugated to a label. Any detectable label can be used, such as a fluorescent label. Detections methods known in arts, such as flow cytometry may be used to detect and/or separate labeled cells. For example, detection and/or separation may be by fluorescence activate cell sorting (FACS). Labels may include fluorescein or its derivatives, such as fluorescein isothiocyanate (FITC), Oregon Green, Tokyo Green, SNAFL, carboxynaphthofluorescein (CFSE), Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), DyLight 488, Alexa Fluor 488, green fluorescent protein (GFP), phycoerythrin (PE), Peridinin Chlorophyll protein (PerCP), PE-Alexa Fluor 700, PE-Cy5 (TRI-COLOR), PE-Cy5.5, PE-Alexa Fluor 750, PE-Cy7, allophycocyanin (APC), APC-Cy7, and derivatives reof. The aforementioned labels may also be used to analyze glycosylation patterns of glycoproteins. Alternatively, agents may be detected directly, for example, by using antibodies to detect agents, such as by Western blotting and or methods well known in arts.
Or methods to analyze glycosylation pattern may include compositional analysis of different types of sugars, such as neutral sugars and sugars with amino groups. For example, neutral sugars, such as galactose, mannose, fucose or like, and sugars with amino groups, such as N-acetylglucosamine or like, and an acidic sugar, such as sialic acid or like can be analyzed. The compositional ratio can be analyzed by releasing neutral sugars or amino sugars by acid hydrolysis of sugar chain. Methods man include, but not be limited to, a method using a sugar composition analyzer (BioLC) manufactured by Dionex. The BioLC is an apparatus for analyzing sugar composition by HPAEC-PAD (high performance anion-exchange chromatography-pulsed amperometric detection) method (Rocklin et al., J. Lig. Chromatogr. 6(9), 1577-1590 (1983)). The compositional ratio can also be analyzed by a fluorescence labeling method using 2-aminopyridine (PA). Specifically, compositional ratio can be calculated by fluorescence-labeling an acid-hydrolyzed sample with 2-aminopyridine in accordance with a known method (Kondo et al., Agric. Biol. Chem., 55(1), 283-284 (1991)) and carrying out HPLC analysis.
The glycosylation pattern can also be analyzed by a two-dimensional sugar chain mapping method (Tomiya et al., Anal. Biochem., 171, 73-80 (1988); Biochemical Experimentation Method 23—Method for Studying Glycoprotein Sugar Chains (Gakkai Shuppan Center), edited by Reiko Takahashi (1989)). The two-dimensional sugar chain mapping method is a method in which sugar chain structure is estimated, for example, by plotting retention time or eluting position of sugar chain by reverse phase chromatography as X axis and retention time or eluting position of sugar chain by a normal phase chromatography as Y axis, and comparing results with those of known sugar chains.
The sugar chain can be released by hydrazinolysis and n fluorescence labeling of sugar chain with 2-aminopyridine (Hase et al., J. Biochem., 95, 1973203 (1984)) is carried out. The sugar chain is separated from an excess PA reagent and like by gel filtration and subjected to reverse phase chromatography. Subsequently, each peak of fractionated sugar chain is analyzed by normal phase chromatography. Based on se results, sugar chain structure can be estimated by plotting spots on a two-dimensional sugar chain map and comparing m with those of sugar chain standards (manufactured by Takara Shuzo) or a reference (Tomiya et al., Anal. Biochem., 171, 73-80 (1988)).
In addition, glycosylation pattern can be analyzed by mass spectrometry, such as MALDI-TOF-MS or like, of each sugar chain.
Heterologous SequencesThe host cells of present disclosure modified to yield variant glycosylation patterns may comprise a heterologous sequence. The heterologous sequence may comprise a nucleic acid sequence. As used herein, nucleic acid sequence is used interchangeably with polynucleotide, nucleotide, nucleotide sequence, nucleic acid and oligonucleotide. They refer to a polymeric form of nucleotides of any length, eir deoxyribonucleotides or ribonucleotides, or analogs reof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to nucleotide structure may be imparted before or after assembly of polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be furr modified after polymerization, such as by conjugation with a labeling component.
The heterologous sequence of present disclosure can encode a proteinaceous moiety, which refers to proteins, polypeptides, peptides, amino acid sequences, which encompasses polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The proteinaceous moiety encompasses an amino acid polymer that has been modified, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any or manipulation, such as conjugation with a labeling component. Furrmore, amino acid refers to eir natural and/or unnatural or syntic amino acids, including but not limited to glycine and both D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to designate amino acids.
The proteins encoded by heterologous sequences of present disclosure are typically glycoproteins, and may include, but not be limited to, enzymes or immunologically functional molecules, such as antibodies. A modified cell line yielding a variant glycosylation pattern may express a heterologous sequence that produces an antibody (humanized or chimeric), antibody fragment, cytokine, hormone or any or protein of interest. The protein produced by cell line may be secreted by cells and harvested. Alternatively, cells may be harvested and protein extracted from cells. The proteins, or glycoproteins, may be used rapeutically, to effect beneficial or desired results.
The heterologous sequence may comprise a vector, which is a nucleic acid molecule, preferably self-replicating, which transfers an inserted nucleic acid molecule into and/or between host cells. Vectors may include those that function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for replication of DNA or RNA, and expression vectors that function for transcription and/or translation of DNA or RNA. Also included are vectors that provide more than one of above functions. An expression vector is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s).
The heterologous sequence encoding a protein can be expressed by a single or multiple vectors. The nucleic acid sequences can be arranged in any order in a single operon, or in separate operons that are placed in one or multiple vectors. Where desired, two or more expression vectors can be employed, each of which contains one or more heterologous sequences operably linked in a single operon. Linked refers to joining toger of two more chemical elements or components, by whatever means including chemical conjugation or recombinant means. Operably-linked refers to a juxtaposition wherein components so described are in a relationship permitting m to function in ir intended manner. For instance, a promoter sequence is linked, or operably linked, to a coding sequence if promoter sequence promotes transcription of coding sequence.
While choice of single or multiple vectors and use of single or multiple promoters may depend on size of heterologous sequences and capacity of vectors, it will largely dependent on overall yield of a given glycoprotein that vector is able to provide when expressed in a selected host cell. In some instances, two-operon expression system provides a higher yield of glycoproteins. The subject vectors can stay replicable episomally, or as an integral part of host cell genome.
The heterologous sequences of present disclosure can be under control of a single regulatory element. In some cases, heterologous nucleic acid sequences are regulated by a single promoter. In or cases, heterologous nucleic acid sequences are placed within a single operon. In still or cases, heterologous nucleic acid sequences are placed within a single reading frame.
Preparation of subject nucleic acids can be carried out by a variety of routine recombinant techniques and syntic procedures. Standard recombinant DNA and molecular cloning techniques are well known in art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987). Briefly, subject nucleic acids can be prepared genomic DNA fragments, cDNAs, and RNAs, all of which can be extracted directly from a cell or recombinantly produced by various amplification processes including but not limited to PCR and rt-PCR.
Direct chemical synsis of nucleic acids typically involves sequential addition of 3′-blocked and 5′-blocked nucleotide monomers to terminal 5′-hydroxyl group of a growing nucleotide polymer chain, wherein each addition is effected by nucleophilic attack of terminal 5′-hydroxyl group of growing chain on 3′-position of added monomer, which is typically, a phosphorus derivative, such as a phosphotriester, phosphoramidite, or like. Such methodology is known to those of ordinary skill in art and is described in pertinent texts and literature (for example, Matteuci et al., Tet. Lett. 521:719 (1980); U.S. Pat. No. 4,500,707 to Carurs et al.; and U.S. Pat. Nos. 5,436,327 and 5,700,637 to Sourn et al.).
Regulatory elements include, for example, promoters and operators, which can also be engineered to increase expression of one or more heterologous sequences encoding a glycoprotein. A promoter is a sequence of nucleotides that initiates and controls transcription of a nucleic acid sequence by an RNA polymerase enzyme. An operator is a sequence of nucleotides adjacent to promoter that functions to control transcription of desired nucleic acid sequence. The operator contains a protein-binding domain where a specific repressor protein can bind. In absence of a suitable repressor protein, transcription initiates through promoter. In presence of a suitable repressor protein, repressor protein binds to operator and reby inhibits transcription from promoter.
In some embodiments of present disclosure, promoters used in expression vectors are inducible. In or embodiments, promoters used in expression vectors are constitutive. In some embodiments, one or more nucleic acid sequences are operably linked to an inducible promoter, and one or more or nucleic acid sequences are operably linked to a constitutive promoter. Non-limiting examples of suitable promoters for use in eukaryotic host cells include, but are not limited to, a CMV immediate early promoter, an HSV thymidine kinase promoter, an early or late SV40 promoter, LTRs from retroviruses, and a mouse metallothionein-I promoter.
The genes in expression vector typically will also encode a ribosome binding site to direct translation (that is, synsis) of any encoded mRNA gene product. Or regulatory elements that may be used in an expression vector include transcription enhancer elements and transcription terminators. See, for example, Bitter et al., Methods in Enzymology, 153:516-544 (1987).
An expression vector may be suitable for use in particular types of host cells and not ors. One of ordinary skill in art, however, can readily determine through routine experimentation wher a particular expression vector is suited for a given host cell. For example, expression vector can be introduced into host organism, which is n monitored for viability and expression of any genes contained in vector.
The expression vector may also contain one or more selectable marker genes that, upon expression, confer one or more phenotypic traits useful for selecting or orwise identifying host cells that carry expression vector. Non-limiting examples of suitable selectable markers for eukaryotic cells include dihydrofolate reductase and neomycin resistance.
The subject vectors can be introduced into a host cell stably or transiently by variety of established techniques. For example, one method involves a calcium chloride treatment wherein expression vector is introduced via a calcium precipitate. Or salts, for example calcium phosphate, may also be used following a similar procedure. In addition, electroporation (that is, application of current to increase permeability of cells to nucleic acids) may be used. Or transformation methods include microinjection, DEAE dextran mediated transformation, and heat shock in presence of lithium acetate. Lipid complexes, liposomes, and dendrimers may also be employed to transfect host cells.
Upon introduction of heterologous sequence into a host cell, a variety of methods can be practiced to identify host cells into which subject vectors have been introduced. One exemplary selection method involves subculturing individual cells to form individual colonies, followed by testing for expression of desired protein prodcut. Anor method entails selecting host cells containing heterologous sequence based upon phenotypic traits conferred through expression of selectable marker genes contained within expression vector. Those of ordinary skill can identify genetically modified host cells using se or or methods available in art.
For example, introduction of various heterologous sequences of disclosure into a host cell can be confirmed by methods such as PCR, Sourn blot or Norrn blot hybridization. For example, nucleic acids can be prepared from resultant host cells, and specific sequences, of interest can be amplified by PCR using primers specific for sequences of interest. The amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, followed by staining with ethidium bromide, SYBR Green solution or like, or detection of DNA with a UV detection. Alternatively, nucleic acid probes specific for sequences of interest can be employed in a hybridization reaction. The expression of a specific gene sequence can be ascertained by detecting corresponding mRNA via reveres-transcription coupled PCR, Norrn blot hybridization, or by immunoassays using antibodies reactive with encoded gene product. Exemplary immunoassays include but are not limited to ELISA, radioimmunoassays, and sandwich immunoassays.
Furrmore, introduction of various heterologous sequences of disclosure into a host cell can be confirmed by enzymatic activity of an enzyme that heterologous sequence encodes. The enzyme can be assayed by a variety of methods known in art. In general, enzymatic activity can be ascertained by formation of product or conversion of a substrate of an enzymatic reaction that is under investigation. The reaction can take place in vitro or in vivo.
Antibody ProductionIn one aspect, present disclosure provides host cells, modified to yield a variant glycosylation pattern, that produce antibodies. Antibody, as used herein, includes all forms of antibodies such as recombinant antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, fusion antibodies, monoclonal antibodies, polyclonal antibodies and like. The antibodies may be fragments. The antibodies may also be conjugated with drugs, toxins or rapeutic radioisotopes. Bispecific antibody fusion proteins can also be produced by host cells of present disclosure, including hybrid antibodies which bind to more than one antigen. Therefore, antibody encompasses naked antibodies and conjugated antibodies and antibody fragments, which may be monospecific or multispecific.
The antibodies can also have a variant glycosylation pattern, which may be characterized by a change in levels of at least two types of sugar molecules as compared to a corresponding wildtype antibody (ie. an antibody produced in an unmodified parental host cell). The sugar molecules may be directly attached to glycoprotein (for example, N- or O-linked to glycoprotein), or indirectly (for example, linked through or sugars that are N- or O-linked to glycoprotein). Sugar chain structure variations, or variant glycosylation patterns, due to various sugar molecule content in such chains, play an important role in effector function of glycoproteins. For example, variant glycosylation patterns, such as described above, of an antibody can increase effector function of antibody, such as increase antibody-dependent cellular cytotoxicity (ADCC) activity of an antibody. The variant glycosylation pattern can be evidenced by a change in levels of at least two types of sugar molecules that are attached through an Fc region of antibody, as compared to a corresponding antibody produced in an unmodified parental host cell.
In some embodiments, antibodies exhibit an N-glycan that has structural formula Glu-GlcNAC2-Man4(+/−Fuc). In some embodiments, antibody exhibits a glycosylation pattern that has structural formula (I), in which a fucose moiety can optionally be linked to first 4GlcNAc from right:
In some embodiments, antibody exhibits a glycosylation pattern that has structural formula (II), in which a fucose moiety can optionally be linked to first 4GlcNAc from right:
The structural formulae (I) and (II) encompass both scenarios, in which one or more fucose molecules are present or absent in subject N-glycan. In some embodiments, fucose is present in N-glycan. In or embodiments, fucose is absent in N-glycan.
The antibodies produced can be from one of five major classes of intact antibodies: e.g., IgA, IgD, IgE, IgG, and IgM. The antibody produced is a member of one of subclasses selected from IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
The antibodies of present disclosure may be monoclonal, which refers to an antibody from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising population are identical and/or bind same epitope(s), except for possible variants that may arise during production of monoclonal antibody, such variants generally being present in minor amounts. Such monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein target-binding polypeptide sequence was obtained by a process that includes selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, selection process can be selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones.
It should be understood that selected target binding sequence can be furr altered, for example, to improve affinity for target, to humanize target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising altered target binding sequence is also a monoclonal antibody of this disclosure. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to ir specificity, monoclonal antibody preparations are advantageous in that y are typically uncontaminated by or immunoglobulins. The modifier “monoclonal” indicates character of antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of antibody by any particular method. For example, monoclonal antibodies to be used in accordance with present disclosure may be made by a variety of techniques, including, for example, hybridoma method (e.g., Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681, (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al. J. Immunol. Methods 284(1-2):119-132 (2004)), and technologies for producing human or human-like antibodies in animals that have parts or all of human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806; 5,569,825; 5,591,669 (all of GenPharm); U.S. Pat. No. 5,545,807; WO 1997/17852; U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio/Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995)).
Monoclonal antibodies herein include chimeric antibodies in which a portion of heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while remainder of chain(s) is identical with or homologous to corresponding sequences in antibodies derived from anor species or belonging to anor antibody class or subclass, as well as fragments of such antibodies, so long as y exhibit desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey, Ape etc) and human constant region sequences.
The host cells of present disclosure can also be used to make hybridomas for production of antibodies. A mouse or or appropriate host animal, (such as a hamster, goat, sheep, dog, horse, pig, rat, rabbit, dog, cat, or gerbil, amongst ors) can be immunized as to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes n are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).
The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit growth or survival of unfused, parental myeloma cells. For example, if parental myeloma cells lack enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), culture medium for hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent growth of HGPRT-deficient cells.
In one embodiment, myeloma cells are used that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. In some embodiments myeloma cell lines are murine myeloma lines, (including, but not limited to, MOPC-21, MPC-11, SP-2 or X63-Ag8-653 cells), human myeloma cell lines (including, but not limited to, Karpas 707H, RPMI 8226, 8226 AR/NIP4-1, KM-2R, or U-266), or rat myeloma cell lines (including, but not limited to, YB2/3.0.Ag.20, YB2/0, Y3-Ag1.2.3, IR983F).
Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). The binding affinity of monoclonal antibody can, for example, be determined by Scatchard analysis. (see for example, Munson et al., Anal. Biochem. 107:220 (1980)).
After hybridoma cells are identified that produce antibodies of desired specificity, affinity, and/or activity, clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells may be grown in vivo as ascites tumors in an animal.
The monoclonal antibodies secreted by subclones can n be suitably separated from culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
In some embodiments, monoclonal antibody can be purified (1) to greater than 95% by weight of antibody as determined by Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
In an alternative embodiment, cell population produced by method of present disclosure is used for production of antibodies by recombinant DNA methods (U.S. Pat. No. 4,816,567, which is herein incorporated by reference in its entirety). The modified host cell exhibiting a variant glycosylation pattern can be produced from a parent cell line such as HeLa, HEK-293, NIH3T3, COS, CHO, NS0, PER.C6, K562, L1.2, JY, BHK, K562, 293F, 3T3, or Jurkat. In an alternative embodiment, modified host cell is derived from a parent cell line derived from a lymphocytic lineage cell line, such as a B cell line.
In one embodiment, messenger RNA (mRNA) coding for heavy or light chain is isolated from a suitable source, such as mature B cells or a hybridoma culture, is obtained by employing standard techniques of RNA isolation purification and optionally size based isolation. cDNAs corresponding to mRNAs coding for heavy or light chain are n produced and isolated using techniques known in art, such as cDNA library construction, phage library construction and screening or RT-PCR using specific relevant primers. In some embodiments, cDNA sequence may be one that is wholly or partially manufactured using known in vitro DNA manipulation techniques to produce a specific desired cDNA. The cDNA sequence can n be positioned in a vector which contains a promoter in reading frame with gene and compatible with low-modified host cell. Numerous plasmids that contain appropriate promoters, control sequences, ribosome binding sites, and transcription termination sites, and optionally convenient markers are known in art, se include but are not limited to, vectors described in U.S. Pat. Nos. 4,663,283 and 4,456,748. In one embodiment, cDNA coding for light chain and that coding for heavy chain may be inserted into separate expression plasmids. In an alternative embodiment, cDNA coding for light chain and that coding for heavy chain may be inserted toger in same plasmid, so long as each is under suitable promoter and translation control.
The expression vectors constructed above can n be used to transform modified host cells of present disclosure. In one embodiment, light and heavy chains may be transformed into separate modified host cell cultures, eir of same or of differing species. In an alternative embodiment, separate plasmids for light and heavy chain may be used to co-transform a single modified host cell culture. In anor embodiment, a single expression plasmid containing both genes and capable of expressing genes for both light and heavy chain may be transformed into a single modified host cell culture.
When heavy and light chains are coexpressed in same host, isolation procedure is designed so as to recover reconstituted antibody. This can be accomplished by conventional antibody purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
In some embodiments, monoclonal antibody purified by recombinant methods can be purified (1) to greater than 95% by weight of antibody as determined by Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
The antibody produced by recombinant method set forth above can be a humanized antibody, such as a human chimeric antibody, or a human complementary determining region (CDR) grafted antibody. Humanized forms of non-human (e.g., mouse, rat, hamster, goat, sheep, horse, cattle or rabbit) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies are typically human immunoglobulins (recipient antibody) in which residues from a hypervariable region of recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, goat, sheep, horse, rabbit or nonhuman primate having desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. Furrmore, humanized antibodies may comprise residues that are not found in recipient antibody or in donor antibody. These modifications are made to furr refine antibody performance. In general, humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of 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. For furr details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
The term variable refers to certain portions of variable domains differing extensively in sequence among antibodies and are used in binding and specificity of each particular antibody for its particular antigen. However, variability is typically not evenly distributed throughout variable domains of antibodies. It is usually concentrated in three segments called hypervariable regions both in light chain and heavy chain variable domains. The more highly conserved portions of variable domains are called framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, β-sheet structure. The hypervariable regions in each chain are held toger in close proximity by FRs and, with hypervariable regions from or chain, contribute to formation of antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Besda, 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 antibody in antibody dependent cellular cytotoxicity (ADCC).
The term hypervariable region when used herein refers to amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a CDR (e.g residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in light chain variable domain and 31-35 (HI), 50-65 (H2) and 95-102 (H3) in heavy chain variable domain; Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Besda, Md. (1991)) and/or those residues from a “hypervariable loop” (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in light chain variable domain and 26-32 (HI), 53-55 (H2) and 96-101 (H3) in heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework Region” or “FR” residues are those variable domain residues or than hypervariable region residues as herein defined.
A human chimeric antibody is an antibody which comprises an antibody heavy chain variable region (hereinafter referred to as “HV” or “VH”, heavy chain being “H chain”) and an antibody light chain variable region (hereinafter referred to as “LV” or “VL”, light chain being “L chain”), both of an animal or than human, a human antibody heavy chain constant region (hereinafter also referred to as “CH”) and a human antibody light chain constant region (hereinafter also referred to as “CL”). As animal or than human, any animal such as mouse, rat, hamster, rabbit or like can be used, so long as a hybridoma can be prepared re from.
A human chimeric antibody can be produced by obtaining cDNA's encoding heavy chain variable region (VH) and light chain variable region (VL) from a monoclonal antibody-producing hybridoma, inserting m into an expression vector for a host cell having genes encoding human antibody CH and human antibody CL to reby construct a human chimeric antibody expression vector, and n introducing vector into a low-fucosylation cell to express antibody.
In regards to heavy chain constant region (CH) of a human chimeric antibody, any CH can be used, so long as it is in human immunoglobulin (hereinafter referred to as “hIg”) class. In some embodiments, CH belongs to hIgG class or one of subclasses belonging to hIgG class, such as hIgG1, hIgG2, hIgG3 and hIgG4. Likewise, in regards to light chain constant region (CL) of human chimeric antibody, any CL can be used, so long as it belongs to hIg class. In some embodiments, light chain constant region (CL) of human chimeric antibody belongs to keppa class or lambda class.
A human CDR-grafted antibody can be produced by constructing cDNA's encoding variable regions in which CDR's of VH and VL of an antibody derived from an animal or than human are grafted into CDR's of VH and VL of a human antibody, inserting m into an expression vector for host cell having genes encoding human antibody CH and human antibody CL to reby construct a human CDR-grafted antibody expression vector, and n introducing expression vector into a modified host cell of present disclosure to express human CDR-grafted antibody.
Preferably antibody of present invention essentially retains ability to bind antigen compared to parental antibody. In some embodiments, antibody of present invention exhibits higher binding affinity to an antigen, for example, at least 1.1, 1.2, 1.3. 1.4, 1.5, 2, 3, 4, or 5 fold higher than a parental antibody. In or embodiments, antibody of present invention exhibits lower binding affinity to an antigen, for example, no less than 5%, 10%, 20%, 30%, 40%, or 50% of binding affinity of a parental antibody to antigen. The binding capability of antibody of present invention may be determined using techniques such as fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA), for example.
The antibody with variant glycosylation patterns and/or produced by modified host cells of present disclosure may bind an antigen such as a cancer antigen. The antigen may be selected from group consisting of The glycoprotein of claim 9 wherein cancer antigen is selected from group consisting of HER2, Immunoglobulin epsilon Fc receptor II, Alk-1, CD20, EGF receptor, VEGF receptor, FGF receptor, NGF receptor, PDGF receptor, EpCam, CD3, CD4, CD11a, CD19, CD22, CD30, CD33, CD38, CD40, CD51, CD55, CD80, CD95, CCR2, CCR3, CCR4, CCR5, CTLA-4, Mucin 1, Mucin 16, Endoglin, Mesolin receptor, Nogo receptor, folate receptor, CXCR4, insulin-like growth factor receptor, Ganglioside GD3, and alpha and beta integrins.
In some embodiments, humanized HER2 antibodies are produced in a modified host cell that exhibits a variant glycosylation pattern. Humanized HER2 antibodies include, but are not limited to, huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 or trastuzumab (HERCEPTIN®) as described in Table 3 of U.S. Pat. No. 5,821,337 expressly incorporated herein by reference; humanized 520C9 (WO93/21319); and humanized 2C4 antibodies such as pertuzumab. Such humanized antibodies produced by modified host cells of present disclosure can mselves have variant glycosylation patterns, as compared to antibodies if produced in an unmodified parental cell.
In anor embodiment, a vector encoding a HER inhibitor such as an EGFR, HER2, HER3 or HER4 antibody is introduced into modified host cell line of present disclosure. HER inhibitor antibodies include trastuzumab, pertuzumab, cetuximab, ABX-EGF, ABX0303, EMD7200, C11033, IMC-11F8, and IMC-11F5. The HER inhibitor produced in a modified host cell can exhibit a variant glycosylation pattern, such as a change in levels of at least 2 sugar molecules as described above, in comparison to a HER inhibitor produced in an unmodified parental host cell.
The HER antibodies or inhibitors of present disclosure can be used to treat cancer cells which display HER expression, amplification, or activation. HER activation refers to activation, or phosphorylation, of any one or more HER receptors. Generally, HER activation results in signal transduction (e.g. that caused by an intracellular kinase domain of a HER receptor phosphorylating tyrosine residues in HER receptor or a substrate polypeptide). HER activation may be mediated by HER ligand binding to a HER dimer comprising HER receptor of interest. HER ligand binding to a HER dimer may activate a kinase domain of one or more of HER receptors in dimer and reby results in phosphorylation of tyrosine residues in one or more of HER receptors and/or phosphorylation of tyrosine residues in additional substrate polypeptides(s), such as Akt or MAPK intracellular kinases.
HER inhibitors include dimerization inhibitors which are agents that inhibit formation of a HER dimer. The HER dimerization inhibitor can be an antibody, for example an antibody which binds to HER2 at heterodimeric binding site reof. The antibody may be those described herein, such as those exhibiting a variant glycosylation pattern. A dimerization inhibitor contemplated herein is pertuzumab or monoclonal antibody 2C4 (MAb 2C4). Or examples of HER dimerization inhibitors include antibodies which bind to EGFR and inhibit dimerization reof with one or more or HER receptors (for example EGFR monoclonal antibody 806, MAb 806, which binds to activated or untered EGFR; see Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)); antibodies which bind to HER3 and inhibit dimerization reof with one or more or HER receptors; antibodies which bind to HER4 and inhibit dimerization reof with one or more or HER receptors; peptide dimerization inhibitors. See, U.S. Pat. No. 6,417,168 hereby incorporated by reference in its entirety. Antibodies that inhibit HER dimerization, such as pertuzumab, may be used to treat cancer cells, which does not over express or amplify HER2 receptor.
The modified host cell can also be used to produce a recombinant protein or than a full-length antibody, with a variant glycosylation pattern. A vector encoding a recombinant protein (such as a plasmid, or virus) using techniques standard in art can be introduced into a modified host cell and recombinant protein may be any protein of interest, including hormones, cytokines and antibody fragments, such as Fv, Fab, scFV or diabody fragments.
The Fv fragment is 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 three hypervariable regions of each variable domain interact to define an antigen-binding site on surface of VH-VL dimer. Collectively, six hypervariable regions confer antigen-binding specificity to antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has ability to recognize and bind antigen, although at a lower affinity than entire binding site.
The Fab fragment also contains constant domain of light chain and first constant domain (CH1) of heavy chain. Fab′ fragments differ from Fab fragments by addition of a few residues at carboxy terminus of heavy chain CH1 domain including one or more cysteines from antibody hinge region. Fab′-SH is designation herein for Fab′ in which cysteine residue(s) of 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 m. Or chemical couplings of antibody fragments are also known.
Single-chain Fv (scFv) antibody fragments comprise VH and VL domains of antibody, wherein se domains are present in a single polypeptide chain. In some embodiments Fv polypeptide furr comprises a polypeptide linker between VH and VL domains which enables scFv to form desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). HER2 antibody scFv fragments are described in WO93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458, which are hereby incorporated by reference in ir entirety, and may be produced by modified host cells of present disclosure.
The term diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a VH domain connected to a VL domain in same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between two domains on same chain, domains are forced to pair with complementary domains of anor chain and create two antigen-binding sites. Diabodies are described more fully in, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), which are hereby incorporated by reference in ir entirety.
In some embodiments, antibody is an inhibitory antibody. Inhibitory antibody may inhibit one or more biological activities of antigen to which antibody binds. For example, an inhibitory antibody can downregulate signal transduction of corresponding antigen by inhibiting activity of antigen or inhibit expression of antigen. In some embodiments, antibody is a neutralizing antibody. A neutralizing antibody reduces or abolishes some biological activity of a soluble antigen or of a living microorganism, such as an infectious agent. Neutralizing antibodies may compete with natural ligand or receptor for its antigen. In some embodiments, antibody is a stimulatory or activating antibody. A stimulatory or activating antibody may be an agonist antibody which may activate signal transduction of corresponding antigen upon binding of antigen reby activating or upregulating activity of antigen, or upregulate expression of antigen to which antibody binds.
Antibodies that may comprise N-glycan of invention include, but are not limited to, abciximab (ReoPro®), adalimumab (Humira®), alemtuzumab (Campath®), basiliximab (Simulect®), bevacizumab (Avastin®), cetuximab (Erbitux®), daclizumab (Zenapax®), dacetuzumab, eculizumab (Soliris®), efalizumab (Raptiva®), Edrecolomab (Panorex®), epratuzumab, ibrituthomab (Zevalin®), tiuxetan, infliximab (Remicade®), muromonab-CD3 (OKT3), natalizumab (Tysabri®), omalizumab (Xolair®), palivizumab (Synagis®), panitumumab (Vectibix®), ranibizumab (Lucentis®), gemtuzumab ozogamicin (Mylotarg®), oregovomab (OvaRex®), rituximab (Rituxan®), tositumomab (Bexxar®), trastuzumab (Herceptin®), MetMAb, ocrelizumab, pertuzumab, Raptiva® (efalizumab), hu M195Mab, MDX-210, BEC2, anti-Abeta, anti-CD4, anti-IL-13, anti-oxLDL, trastuzumab-DM1, apomab, rhuMAb beta7, rhuMAb1F/Valpha, GA101, anti-OX40L, ipilimumab, Valortim, ustekinumab, golimumab, ofatumumab, zalutumumab, tremelimumab, motavizumab, mitumomab, ecromeximab, ABX-EGF, MDX010, XTL 002, H11 SCFV, 4B5, XTL001, MDX-070, TNX-901, IDEC-114, and any antibody fragments specific for antigens including but not limited to complement C5, CBL, CD147, gp120, VLA4, CD11a, CD18, VEGF, CD40L, anti-Id, ICAM1, CD2, EGFR, TGF-β2, TNF-α, TNF receptor, E-selectin, FactIl, Her2/neu, F gp, CD11/18, CD14, CD80, ICAM3, CD4, CD23, β2-integrin, α4β7, CD52, CD22, OX40L, IL-5 receptor, GM-CSF receptor, GM-CSF, HLA-DR, oxLDL, CD64 (FcR), TCRαβ, CD3, Hep B, CD125, DR5, EpCAM, gpIIbIIIa, IgE, beta 7 integrin, CD20, IL1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-12/IL-23, IL-15, IFN-α, VEGFR-1, platelet-derived growth factor receptor α (PDGFRα), vascular adhesion protein 1 (VAP1), connective tissue growth factor (CTGF), Apo2/TRAIL, CD25, CD33, FILA, F gp, IgE, CTLA-4, IP-10, anti-C. difficile Toxin A and Toxin B, B. anthracia PA, respiratory syncytial virus (RSV), mannose receptor/hCGβ, integrin receptors, PD1, PDL-1, CD19, CD70, and VNR integrin.
Antibodies comprising N-glycan of present invention have a good half-life comparable to that of native antibodies or antibodies produced by unmodified host cells. Antibody half-life typically refers to a measure of mean survival time of antibody molecules following ir formation, usually expressed as time required to eliminate 50% of a known quantity of immunoglobulin from animal body. Half-life varies from one immunoglobulin class to anor, and also can vary from species to species. In some embodiments, antibody expressing subject N-glycan produced by CHO-1E5 cells has a comparable terminal half-life in vivo as that of an antibody produced by parental CHO cells. In some embodiment, subject antibodies have a terminal half-life in a test animal for at least 4, 8, 12, 24, 48, 72 hours, or even longer. Terminal half-life can be assessed by quantitating amount of antibodies remained in subject's biological fluid (e.g., blood or serum) after initial administration and over a course of defined period of time. Initial administration can be carried out intravenously, subcutaneously or any or suitable route known in art or disclosed herein.
In some embodiments, subject antibodies comprising N-glycan of present invention having enhanced ADCC activity but do not elicit substantially higher immunogenicity as compared to a parental antibody produced by an unmodified host cell or antibodies lacking unique N-glycan of present invention. Immunogenicity typically refers to ability of a particular substance, such as an antigen or epitope, to provoke an immune response, humoral and/or cell-mediated immune responses. Proteins and polysaccharides can be immunogenic. In one embodiment, antibody comprising subject N-glycan exhibits comparable immunogenicity or lack reof in vivo as that of an antibody produced by parental CHO cells. Methods for assessing immunogenicity or lack reof are known in art. For example, a conventional approach is to measure its capacity to induce antibody responses, such as serum antibody titer. As described in Example 14 and
Assays suitable for measuring IgM or or antibody isotype titers for assessing immunogenicity of subject antibodies include but are not limited to direct binding assays, bridging assays, capture (sandwich) assays and competitive immunoassays using radioligand, enzymatic, fluorescent, chemiluminescent or electrochemical luminescence detection systems. One alternative method for assessing in vivo immunogenicity of a target antibody is a magnetic bead based immunoprecipitation method followed by quantitative liquid chromatography-mass spectrometry (LC/MS) to determine anti-drug antibodies (ADA) in human and cynomolgus monkey serum in presence of high circulating concentrations of target antibody, i.e. ET101-CHO-1E5 antibody. Available ADA binding sites are saturated by addition of excess target antibody followed by magnetic bead based protein G isolation of IgG antibodies and ir bound antigens before elution and digestion. Peptides of target antibodies are n quantified by LC/MS using stable isotope labeled standards inferring presence of total ADA. This approach complements established methodologies for assessment of immunogenicity response (Hendrik N. et al. Analytical chemistry, 2008 vol. 80, no 18, pp. 6907-6914).
Unwanted immunogenicity induced by target antibodies can comprise humoral and cellular immune responses. Where desired, one can measure both humoral and cellular immune responses. In most cases, development of a mature IgG response implies underlying antigen specific helper T-cell involvement. Examples of assays for detecting/assessing cell-mediated responses upon administration of subject antibodies of present invention include but are not limited to T-cell stimulation/proliferation assays, cytokine (e.g. IL2, IL4, IFN-gamma) production/release methods, measurement of receptor phosphorylation status, or modulation of one or more T cell or B cell intracellular markers. These involve use of T-cell preparations sometimes co-cultured with preparations of or cell types, e.g. dendritic cells. Elispot and flow cytometry procedures are commonly used for se assays. Memory B-cell (and sometimes memory T-cell) assays can provide useful information regarding nature of immune response and may contribute to prediction of development of immunogenicity. Studies using peptides or full-length target protein, e.g. ET101-CHO-1E5 antibody (depending on assays and purpose of assays) and Elispot methodologies can be used (See “GUIDELINE ON IMMUNOGENICITY ASSESSMENT OF BIOTECHNOLOGY-DERIVED THERAPEUTIC PROTEINS”, 2007).
Fc Receptor (FcR) BindingIn one aspect, present invention provides an antibody produced by a modified host cell, wherein antibody has increased binding affinity to an Fc receptor, FcγIIIa, and/or decreased binding affinity to anor Fc receptor, FcγIIb, as compared to a corresponding antibody produced by an unmodified host cell, reby enhancing antibody-dependent cell-mediated cytotoxicity against effector cells expressing such Fc receptors. In anor aspect, present invention provides a modified host cell characterized in its ability to produce a modified antibody, wherein antibody exhibits increased binding affinity to FcγIIIa, and/or decreased binding affinity to FcγIIb, as compared to a corresponding antibody produced by an unmodified host cell.
The interaction of antibodies and antibody-antigen complexes with cells of immune system effects a variety of responses, including antibody-dependent cell-mediated cytotoxicity (ADCC) (reviewed in Daeron, Annu. Rev. Immunol. 15:203-234 (1997); Ward and Ghetie, Therapeutic Immunol. 2:77-94 (1995); as well as Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991)).
The term “Fc receptor” or “FcR” is used to describe a receptor that binds to Fc region of an antibody. An exemplary FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of Fc γRI, Fc γRII, and Fc γRIII subclasses, including allelic variants and alternatively spliced forms of se receptors. Fc γRII receptors include Fc γRIIA (an “activating receptor”) and Fc γRIIB (an “inhibiting receptor”), which have similar amino add sequences that differ primarily in cytoplasmic domains reof. Activating receptor Fc γRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor Fc γRIIB 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). Or FcRs, including those to be identified in future are encompassed by term “FcR” herein. The term also includes neonatal receptor, FcRn, which is responsible for transfer of materal IgGs to fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).
Several antibody effector functions are mediated by Fc receptors (FcRs), which bind Fc region of an antibody. FcRs are defined by ir specificity for immunoglobulin isotypes; Fc receptors for IgG antibodies are referred to as Fc γR, for IgE as Fc εR, for IgA as Fc αR and so on. Three subclasses of Fc γR have been identified: Fc γRI (CD64), Fc γRII (CD32) and Fc γRIII (CD16). Because each Fc γR subclass is encoded by two or three genes, and alternative RNA spicing leads to multiple transcripts, a broad diversity in Fc γR isoforms exists. The three genes encoding Fc γRI subclass (Fc γRIA, Fc γRIB and Fc γRIC) are clustered in region 1q21.1 of long arm of chromosome 1; genes encoding Fc γRII isoforms (Fc γRIIA, Fc γRIIB and Fc γRIIC) and two genes encoding Fc γRIII (Fc γRIIIA and Fc γRIIIB) are all clustered in region 1q22. These different FcR subtypes are expressed on different cell types (reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991)). For example, in humans, Fc γRIIIB is found only on neutrophils, whereas Fc γRIIIA is found on macrophages, monocytes, natural killer (NK) cells, and a subpopulation of T-cells. Notably, Fc γRIIIA is only FcR present on NK cells, one of cell types implicated in ADCC. Fc γRIIIA is an activating transmembrane receptor expressed on macrophages and NK cells. It is also a neutrophil opsonin receptor (Ravetch, J. and L. Lanier, 2000, Science 290:84). Fcγ RIA is transmembrane protein with three extracellular Ig-like domains. Fcγ RI is expressed constitutively on monocytes and macrophages and can be induced on neutrophils and eosinophils. FcR expression on hematopoietic cells is reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), which is herein incorporated by reference in its entirety.
In one embodiment, present invention provides an antibody with altered FcR binding affinity. An antibody with “altered” FcR binding affinity is one which has eir enhanced or diminished FcR binding activity and/or ADCC activity compared to a parent antibody or to an antibody comprising a native sequence Fc region. The antibody of present invention which “displays increased binding” to an FcR binds at least one FcR with better affinity than parent antibody. The antibody of present invention which “displays decreased binding” to an FcR, binds at least one FcR with lower affinity than a parent antibody. Such variants which display decreased binding to an FcR may possess little or no appreciable binding to an FcR, e.g., 0-20% binding to FcR compared to a native sequence IgG Fc region.
The antibody of present invention which binds an FcR with higher affinity than a parent antibody is one which binds any one or more of above identified FcRs with substantially higher binding affinity than parent antibody, when antibody having unique N-linked glycan and parent antibody are applied in comparable amounts. For example, antibody with improved FcR binding affinity may display from about 1.1 fold to about 10,000 fold, for example, 1.15 fold, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1,000, 10,000 fold or more in FcR binding affinity as compared to parent antibody, where FcR binding affinity is determined, for example, as disclosed in Examples herein.
The phrase “low affinity receptor” denotes a receptor that has a weak binding affinity for a ligand of interest, e.g. having a binding constant of about 50 nM or worse affinity. Exemplary low affinity receptors include but are not limited to Fc γRIIIa 158 F/F and Fc γRIIIa 158 F/V.
In some embodiments, antibody produced by CHO 1E5 cells displays increased binding to Fc γRIIIa, an activating Fc γR, and may furr display decreased binding to Fc γRIIb, an inhibitory Fc γR.
Antibody-Dependent Cellular Cytotoxicity (ADCC) ActivityThe modified host cells of present disclosure can be used to produce antibodies or functional antibody fragments as described above. The antibodies or functional antibody fragments produced by se cells can also exhibit a variant glycosylation as described herein, such as a change in levels of at least 2 sugar molecules as compared to corresponding antibodies or functional antibody fragments produced in an unmodified parental host cells. Furrmore, se antibodies or fragments with a variant glycosylation pattern can have improved antibody-dependent cellular cytotoxicity (ADCC) or or antibody effector functions in comparison to corresponding antibodies or antibody fragments produced in unmodified parental host cells. Alternatively, antibody or antibody fragments with variant glycosylation patterns exhibits increased ADCC as compared to a corresponding antibody lacking said variant glycosylation pattern. The antibody or antibody fragments with improved or increased ADCC activity may also have a variant glycosylation pattern.
For example, a parental CHO cell line can be modified to yield a modified CHO cell line with a variant glycosylation pattern. The modified CHO cell line can n produce an antibody having higher ADCC activity than that of an antibody produced by parental CHO cell. The antibody produced by modified CHO cell line can also have a variant glycosylation pattern.
“Antibody-dependent cell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction in which effector cells that express FcRs (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of target cell. The primary cells for mediating ADCC comprise NK cells, monocytes, and macrophages. NK cells typically express FcγRIII predominantly, whereas monocytes express Fc γRI, Fc γRII and Fc γRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).
“Effector cells” are leukocytes which express one or more FcRs and perform effector function(s). Preferably, cells express at least Fc γRIII 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. The effector cells may be isolated from a native source reof, e.g. from blood or PBMCs as described herein, or may propagate in vitro using known methods in art.
In one embodiment, antibody of present invention mediates antibody-dependent cell-mediated cytotoxicity (ADCC) in presence of human effector cells more effectively than a corresponding antibody produced by parental host cells, when subject antibodies are applied in comparable amounts. Generally, ADCC activity can be ascertained using assays disclosed herein, but or assays or methods for determining ADCC activity, e.g. in an animal model etc, are contemplated. The antibody of present invention is from about 1.1 fold to about 10,000 fold, e.g., 1.15, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1,000, 10,000 fold or more, more effective at mediating ADCC than parent antibody, e.g. in in vitro assay disclosed herein.
In some embodiments, antibody of present invention having unique N-linked glycan exhibits increased ADCC against effector cells expressing Fc γRIIIa of different genotypes, as compared with parental antibody. In one example, antibody having unique N-linked glycan exhibits increased ADCC, as compared to parental antibody, against effector cells expressing Fc γRIIIa 158V/V (when amino acid 158 is valine at two alleles), which has high affinity to Fc fragment of IgG. In anor example, antibody having unique N-linked glycan exhibits increased ADCC, as compared to parental antibody, against effector cells expressing Fc γRIII 158F/F or Fc γRIII 158F/V (when amino acid 158 is phenylalanine at both or one of alleles), which has low affinity to Fc fragment of IgG. It is known that genomic polymorphism corresponding to phenotype of valine (V) or phenylalanine (F) at amino acid 158 on Fc γRIIIa greatly influences affinity of IgG1 to Fc γR (Koene H R, et al. Blood 90:1109-1114, 1997). In addition, immune effector cells bearing Fc γRIIIa V allele mediate ADCC of anti-HER-2/neu IgG1 variants better than cells bearing F allele (Shields R L, et al. J Biol Chem 9:6591-6604, 2001). The anti-HER-2/neu monoclonal antibody trastuzumab has been shown to engage both activatory (fragment C receptor (Fc γRIIIa; Fc γRIIa) and inhibitory (Fc γRIIb) antibody receptors and Fc γR polymorphisms have been identified that may affect antibody-dependent cell-mediated cytotoxicity (ADCC) of natural-killer cells/monocytes. Studies have investigated wher Fc γR polymorphisms are associated with clinical outcome of patients with breast cancer who received trastuzumab. It was found that Fc γRIIIa-158 V/V genotype was significantly correlated with objective response rate (ORR) and progression-free survival (PFS). Also, re was trend significance in ORR and PFS for Fc γRIIa-131 H/H genotype. The combination of two favorable genotypes (VV and/or H/H) was independently associated with better ORR and PFS compared with or combinations. The ADCC analysis showed that V/V and/or H/H PBMCs had a significantly higher trastuzumab-mediated cytotoxicity than PBMCs harboring different genotypes. The PFS estimate of patients with Fc γRIIIa 158 V/V was significantly longer than for patients with 158 V/F, 158 F/F, or F carriers (V/F+F/F combined) (Antonino Musolino, et al. Journal of Clinical Oncology, Volume 26, No. 11, Apr. 10, 2008). Thus, antibody of present invention, having increased ADCC activity against cells expressing Fc γRIII 158V/V and cells expressing Fc γRIII 158F/F, as compared to an unmodified antibody, can be very useful in enhancing clinical outcome of patients with various disorders including cancer treated with antibody-based rapy.
ADCC activity as used herein also encompasses an activity to injure a cell, for example, a tumor cell, by activating an effector cell via binding of Fc region of an antibody to an Fc receptor existing on surface of an effector cell such as a killer cell, a natural killer cell, an activated macrophage or like (Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Chapter 2.1 (1995)). For example, ADCC activity can be a cytotoxic activity in which an antibody bound to a cell surface antigen on a tumor cell in living body activates an effector cell that express Fc receptors (FcRs) (e.g. peripheral blood mononuclear cells (PBMC), monocytes, cytotoxic T cells Natural Killer (NK) cells, neutrophils, and macrophages), leading to recognition of bound antibody on a target cell and subsequently cause lysis of target cell. (Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Chapter 2.1 (1995)). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. USA 95:652-656 (1998).
Antibody effector functions also include, but are not limited to, C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc. Complement dependent cytotoxicity (CDC) refers to ability of a molecule to lyse a target in presence of complement. The complement activation pathway is initiated by binding of first component of complement system (C1q) to a molecule (e.g. an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.
The antibody of present invention may be subjected to one or more assays to evaluate any change in biological activity compared to parental antibody.
The ability of antibody of present invention to bind an FcR may be evaluated. Where FcR is a high affinity Fc receptor, such as Fc γRIIIA-158 V/V, binding can be measured by titrating subject antibody and measuring bound antibody using an antibody which specifically binds to antibody of present invention in a standard ELISA format.
To assess ADCC activity of antibody of present invention, an in vitro ADCC assay, such as that described in Example 12 may be performed using varying effector:target ratios. Useful “effector cells” for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of antibody of present invention may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
Formulation, Administration and TreatmentAn antibody of present invention which has increased ADCC activity can be useful in prevention and treatment of various disorders.
A “disorder” as used herein refers to any condition that would benefit from treatment with antibody produced by modified host cells of present invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose mammal to disorder in question. Examples of disorders include but are not limited to cancer, allergies, cardiovascular diseases, inflammatory diseases, metabolic diseases, neurological diseases, viral infections and/or bacterial infections. In one embodiment, disorder is cancer. Examples of cancer include, but are not limited to, carcinorma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include but are not limited to adrenal cortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, central nervous system (CNS) cancers, peripheral nervous system (PNS) cancers, breast cancer, Castleman's Disease, cervical cancer, childhood Non-Hodgkin's lymphoma, colon and rectum cancer, endometrial cancer, esophagus cancer, Ewing's family of tumors (e.g. Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, hairy cell leukemia, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, children's leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, liver cancer, lung cancer, lung carcinoid tumors, Non-Hodgkin's lymphoma, male breast cancer, malignant mesolioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (adult soft tissue cancer), melanoma skin cancer, non-melanoma skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine cancer (e.g. uterine sarcoma), vaginal cancer, vulvar cancer, and Waldenstrom's macroglobulinemia. One example of a cancer can be a “HER2-expressing cancer”, which comprises cells having HER2 receptor protein (Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al. Nature 319:230-234 (1986) (Genebank accession number X03363)) present on ir cell surface, such that an anti-HER2 antibody is able to bind to cancer.
For example, in cancer, such as a malignant tumor, an antibody having high ADCC activity can treat cancers by injuring proliferation of cancer cells through its cytotoxic effect. Antitumor effect of antibody on various tumor cells can be analyzed by methods described above. For example, in vitro tests such as, but not limited to, CDC activity measuring method, ADCC activity measuring methods and like, and in vivo tests, such as an antitumor experiment using a tumor system in an experimental animal such as a mouse or like may be performed. CDC activity and ADCC activity measurements and antitumor experiments can be carried out in accordance with methods as described in shitara et al., Cancer Immunology Immunorapy, 36, 373-380 (1993); Nakamura et al., Cancer Research, 54, 1511-1516 (1994) and like.
For treatment of allergies, where an allergic reaction is typically induced by release of a mediator molecule from immune cells, allergic reaction can be inhibited by removing immune cells using an antibody having high ADCC activity. Cardiovascular diseases can be prevented and treated by inhibiting proliferation of arterial cells in re-stricture after treatment, by using an antibody having high ADCC activity. Various diseases including viral or bacterial infections can be prevented and treated by inhibiting proliferation of virus- or bacterium-infected cells using an antibody having high ADCC activity.
In some embodiments, antibody exhibiting glycosylation pattern Glu-GlcNAC2-Man4 (+/−Fuc) has increased ADCC activity against various cancer cells including but not limited to breast cancer cells, ovarian cancer cells, and lung cancer cells, as compared to a corresponding antibody produced by unmodified parental host cell. In some embodiments, antibody exhibiting glycosylation pattern Glu-GlcNAC2-Man4(+/−Fuc) has increased binding affinity to Fc γRIIIA receptor as compared to a corresponding antibody produced by unmodified parental host cell. The antibody exhibiting Glu-GlcNAC2-Man4 (+/−Fuc) has similar in vivo pharmacokinetic characteristics as a corresponding antibody produced by unmodified parental host cell.
The subject antibody having an N-linked glycosylation pattern disclosed herein exhibits a pharmacokinetic profile that is comparable to that of antibody produced by wild-type parental host cells. The pharmacokinetic profile describes parameters of how a drug is absorbed, distributed, metabolized, and eliminated by body. Pharmacokinetics is a study of action of drugs in body over a period of time, including processes of absorption, distribution, localization in tissues, biotransformation, and excretion. Pharmacokinetic parameters include but are not limited to route of drug administration, first order kinetics including volume of distribution (Vd), clearance (Cl) of a drug, elimination constant (kel), elimination half life (t½), serum concentration, bioavailability, in vivo solubility, zero order elimination, dosing regimen, hepatic drug clearance, drug distribution, protein binding, and degree of ionization. Drug pharmacokinetics determines onset, duration, and intensity of a drug's effect.
Therapeutic formulations of antibody of present invention are prepared for storage by mixing antibody having desired degree of purity with optional physiologically acceptable-carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at dosages and concentrations employed, and include buffers such as phosphate, citrate, and or organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and or carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as Tween, Pluronics™ or polyethylene glycol (PEG).
The formulation herein may also contain more than one active compound as necessary for particular indication being treated, preferably those with complementary activities that do not adversely affect each or. Such molecules are suitably present in combination in amounts that are effective for purpose intended.
The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibody of present invention, which matrices are in form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron Depot™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in body for a long time, y may denature or aggregate as a result of exposure to moisture at 37° C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on mechanism involved. For example, if aggregation mechanism is discovered to be intermolecular S—S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
It is contemplated that antibody produced by 1E5 cells of present invention may be used to treat a mammal e.g. a patient suffering from, or predisposed to, a disease or disorder, who could benefit from administration of antibody. The conditions which can be treated with antibody of present invention are many and include cancer (e.g. where antibody of present invention binds HER2 receptor, CD20 or vascular endolial growth factor (VEGF)); allergic conditions such as asthma (with an anti-IgE antibody); and LFA-1-mediated disorders (e.g. where antibody of present invention is an, anti-LFA-1 or anti-ICAM-1 antibody) etc.
Where antibody binds HER2 receptor, disorder preferably is HER2-expressing cancer, e.g. a benign or malignant tumor characterized by overexpression of HER2 receptor. Such cancers include, but are not limited to, breast cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, hepatoma, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer. According to teachings herein, one may prepare a polypeptide with a variant Fc region which has improved, or diminished, ADCC activity. Such molecules will find applications in treatment of different disorders.
For example, antibody with improved ADCC activity may be employed in treatment of diseases or disorders where destruction or elimination of tissue or foreign micro-organisms is desired. For example, antibody of present invention may be used to treat cancer; inflammatory disorders; infections (e.g. bacterial, viral, fungal or yeast infections); and or conditions (such as goiter) where removal of tissue is desired, etc.
Where antibody has diminished ADCC activity, such antibodies may be used to treat diseases or disorders where an Fc region-containing polypeptide with long halt-life is desired, but antibody of present invention preferably does not have undesirable effector function(s). For example, Fc region-containing polypeptide may be an anti-tissue factor (TF) antibody; anti-IgE antibody; and anti-integrin antibody (e.g. an anti-β4-β7 antibody). The desired mechanism of action of such Fc region-containing polypeptides may be to block ligand-receptor binding pairs. Moreover, Fc-region containing polypeptide with diminished ADCC activity may be an agonist antibody.
The antibody of present invention is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local immunosuppressive treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, antibody of present invention is suitably administered by pulse infusion, particularly with declining doses of antibody of present invention. Preferably dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on wher administration is brief or chronic.
For prevention or treatment of disease, appropriate dosage of antibody of present invention will depend on type of disease to be treated, severity and course of disease, wher antibody of present invention is administered for preventive or rapeutic purposes, previous rapy, patient clinical history and response to antibody of present invention, and discretion of attending physician. The antibody of present invention is suitably administered to patient at one time or over a series of treatments.
Depending on type and severity of disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of antibody of present invention is an initial candidate dosage for administration to patient, wher, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on factors mentioned above. For repeated administrations over several days or longer, depending on condition, treatment is sustained until a desired suppression of disease symptoms occurs. However, or dosage regimens may be useful. The progress of this rapy is easily monitored by conventional techniques and assays.
The antibody composition will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include particular disorder being treated, particular mammal being treated, clinical condition of individual patient, cause of disorder, site of delivery of agent, method of administration, scheduling of administration, and or factors known to medical practitioners. The “rapeutically effective amount” of antibody to be administered will be governed by such considerations, and is minimum amount necessary to prevent, ameliorate, or treat a disease or disorder. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat disorder in question. The effective amount of such or agents depends on amount of antibody present in formulation, type of disorder or treatment, and or factors discussed above. These are generally used in same dosages and with administration routes as used hereinbefore or about from 1 to 99% of heretofore employed dosages.
In some embodiments, antibody and/or host cell of present invention may be used to prevent or treat a disorder in a mammal. The mammal selected from group consisting of a human, non-human primate, rodent, dog, cat, horse, cow, pig, sheep, rabbit, guinea pig, or goat.
Fermentation and Method of ProductionIn one aspect, present invention provides a method of producing a modified glycoprotein comprising providing a heterologous polynucleotide sequence that encodes modified glycoprotein and causing modified glycoprotein to be expressed in a host cell disclosed herein. In some embodiments, present invention encompasses a culture medium comprising a host cell disclosed herein. In or embodiments, present invention furr includes a culture fermentor comprising a plurality of host cells of invention in a culture medium.
Fermentation is a standard process well known in relevant art for breakdown and re-assembly of biochemicals and biological molecules including proteins for industry, often in aerobic growth conditions. A fermentor or a bioreactor as used herein refers to an apparatus that maintains optimal conditions for growth of microorganisms, e.g. host cells of present invention, used in large-scale or small-scale fermentation and in commercial production of biological macromolecules including but not limited to glycoproteins, for example, antibodies.
The production of recombinant monoclonal antibodies involves technologies, referred to as “repertoire cloning” or “phage display/yeast display”. Recombinant antibody engineering involves use of viruses or yeast to create antibodies, rar than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected. These techniques can be used to enhance specificity with which antibodies recognize antigens, ir stability in various environmental conditions, ir rapeutic efficacy, and ir detectability in diagnostic applications. Fermentation chambers have been used to produce se antibodies on a large scale. In some embodiments, antibody expressing unique N-glycan of present invention can be produced in a culture fermentor.
EXAMPLES Example 1 Selection of Optimal Concentration of Killing AgentTo acquire a cell population with a variant glycosylation pattern, chemicals were applied to cells to cause random genomic mutations that would also select cells with a variant glycosylation pattern. Chemical-induced random mutagenesis can lead to mutations of genes that control or regulate sugar biogenesis and protein glycosylation processes, such as fucosylation. Stable clones with low fucosylation activity after mutagenesis can be enriched and isolated by targeting cells with LCA, which binds to fucosylation proteins on cell surface, with a toxin (see
LCA-biotinwas added to bind to cell surface. Streptavidin-Saporin was n added to medium. Biotin-streptavidin interaction brings Saporin to proximity of cell membrane so that complex could be internalized into cells. Once inside cells, Saporin can break away from targeting agent and inactivate ribosomes, leading to death.
To determine optimal concentrations of LCA-biotin and streptavidin-saporin, to kill cells with high fucosylation activity efficiently, reduce nonspecific killing, thus resulting in cells with a variant glycosylation patter, effects of different combinations of LCA-biotin and streptavidin-saporin concentrations on killing of parental CHO-K1 cells was performed. LCA-biotin at a concentration of approximately 10 μg/ml and streptavidin-saporin at approximately 2 μg/ml was optimal combination to kill a majority of parental CHO-K1 cells (
Two chemical mutagens, ICR191 and ethyl methane sulfonate (EMS), were used to introduce random genomic mutations. CHO-K1 cells were treated with ICR191 or EMS, which typically cause frame shift mutations and guanine alkylation respectively. The chemicals were washed after 16 hours. The cells were allowed to recover for 5 days. The survived cells were selected with LCA-biotin at a concentration of 10 μg/ml and streptavidin-Saporin at 2 μg/ml for 4 weeks. The cells were routinely stained with FITC-LCA followed by FACS analysis. The sorted cells were cultured at a high density to expand ir population and to obtain cells for subsequent LCA labeling and FACS sorting.
The selection gradually enriched a low-fucosylation population after ICR191 or EMS-induced mutagenesis (
Low fucosylation cell clones have a unique and consistent profile of a glycosylation pattern that is varied in comparison to an unmodified or parental cell line. This profile was established by staining parental cells and low fucosylation clones with FITC-conjugated wheat germ agglutinin (WGA), which has preferential binding to N-acetylglucosamine, concanavalin A (Con A), which recognizes α-linked mannose, and Griffonia (Bandeiraea) Simplicifolia Lectin II (GS-II), which binds to α- or β-linked N-acetylglucosamine residues). The labeled cells were analyzed by FACS. The low fucosylation clones also have low binding affinity to WGA, suggesting that se clones have low levels of N-acetylglucosamine (
Antibodies expressed in cell lines with a variant glycosylation pattern have low fucose content. They also have a variant glycosylation pattern as shown in
The antibody was obtained from conditioned media of mutant clone 1E5. The expression of antibody was determined by SDS-PAGE and Coomassie blue staining (
The results show that antibodies produced by clone 1E5 were modified to have a variant glycosylation pattern with significantly reduced binding affinity to LCA, WGA, GSII, and increased binding affinity to ConA, as compared to control (FIG. 9B)/Results indicate that fucose content of antibodies was drastically decreased.
The monosaccharide profile of antibodies ET101 and ET201 produced by different CHO lines was also determined, as shown in
The monosaccharide composition analysis of human IgG1 produced by Fut8−/− knockout CHO cells as disclosed in Yamane-Ohnuki et al., Biotechnol. Biogeng. 87:614-622 (2004) is shown in
Humanized anti-ErbB2 antibody was expressed in a mutant clone with a variant glycosylation pattern and compared to antibody produced in a parental cell to demonstrate ErbB2-blocking antibody synsized by cell lines possessed enhanced ADCC activity. To demonstrate enhanced ADCC activity, antibody-mediated lysis of A549, SKBR3, SKOV3, MDA-MB-361, and MDA-MB-231 cells were assessed to compare efficiency of cytotoxcity. The target cells were pre-incubated with ErbB2-blocking antibodies (ET101 or ET201) produced from clone 1E5, 2.6, or 3F to allow antibodies to bind with its target. Human PBMC were n incubated with target cells. As shown in
Expressed antibody ET101 from 1 milliliter of conditioned media containing 10% FBS was precipitated by protein L beads, separated by a reducing SDS-PAGE gel and stained with Coomassie blue as shown in
For. ADCC assays as shown in
ErbB2-overexpressing breast cancer cell line SKBR3 is plated in 96-well plates for real time proliferation assay or in 24-well plates at a low density for colony formation assay. After allowing cells to attach overnight, various concentrations of antibodies, control or various antibodies produced from cell clones with variant glycosylation patterns are added to medium to test ir inhibition on cell proliferation. The ErbB2 blocking antibodies produced from cell clones with variant glycosylation patterns inhibit cell proliferation comparable to, or to a greater extent than, control antibody (antibodies produced by parental cells). Cell proliferation inhibition is ascertained by methods such as colony formation assays, real time proliferation assays, or or methods known in arts.
Example 7 Determination of Composition and Structure of N-glycanTo determine components of monosaccharides of N-glycan, antibody produced CHO-1E5 cells and antibody produced by parental host cells in serum-free suspension were hydrolyzed with Trifluoroacetic Acid (TFA). The purified antibodies (100 μg) were mixed with Trifluoroacetic Acid (TFA) to final concentration of TFA at 4M. The mixtures were boiled for two hours and solutions were dried under vacuum. The pellets were dissolved in 200 μl of deionized water. 100 μl of resuspended samples were injected into PA1 column to analyze compositions of monosaccharides by Dionex ICS-3000 system (Dionex, Sunnyvale, Calif.). As shown in
To furr establish composition of N-glycan of invention, N-glycan was cleaved from antibodies produced by CHO-1E5 and its parental host cells by PNGase F (New England Biolabs, Ipswich, Mass.). N-glycan was released by digesting 200 μg of antibody with PNGase F for 72 hours at 37° C. The protein was precipitated by 70% ethanol at −20° C. overnight and removed by centrifugation. The supernatants were dried under vacuum and resuspended in 200 μl of deionized water. The samples were loaded onto microcolumns packed with C18 (Waters, Milford, Mass.), AG50WX8 and AG4×4 (BioRad Laboratories, Hercules, Calif.). The columns were n washed with 300 μl of deionozed water. The flowthrough was collected, dried under vacuum, and N-glycan was n analyzed for MALDI-TOF MS using a MALDI-TOF MS spectrometer. The m/z value corresponds to sodium-associated oligosaccharide ion. Each peak corresponds to [M+Na]+. Two peaks were observed in N-glycan from antibody produced by parental cells, which corresponds to mass of G1 (Gal1-Fuc1-GlcNAC4-Marb) and G0 (Fuc1-GlcNAC4-Man3) N-glycan (
The single population of N-glycan synsized by CHO-1E5 was furr determined by oligosaccharide analysis. The N-glycan was released from antibody and purified for MALDI-TOF MS analysis. Briefly, N-glycan was released by digesting 200 μg of antibody with PNGase F for 72 hours at 37° C. The protein was precipitated by 70% ethanol at −20° C. overnight and removed by centrifugation. The supernatants were dried under vacuum and resuspended in 200 μl of deionized water. The samples were loaded onto microcolumns packed with C18, AG50WX8 and AG4×4. The columns were n washed with 300 μl of deionozed water. The flowthrough was collected and oligosaccharide was injected into PA200 column and analyzed by Dionex ICS-3000 system. Consistent with findings by MALDI-TOF MS analysis, oligosaccharide from antibody produced by CHO-1E5 exists as a single peak and a substantially homogeneous population that is different from heterogeneous profile of oligosaccharides produced by parental host cells (
Based on composition of monosaccharides and molecular weight of N-glycan synsized by CHO-1E5, 6 possible structures of N-glycan were deduced (
As alterations of protein glycosylation may affect protein folding and functional assembly, this issue was addressed by separating antibody on SDS-PAGE electrophoresis. The purified antibodies produced by parental host cells and CHO-1E5 were subjected to SDS-PAGE electrophoresis under reducing and non-reducing conditions, and stained with Coomassie Blue. As shown in
To determine if modification of antibody with N-glycan of invention could improve its biologic functions such as ADCC activity, two purified antibodies targeting ErbB2 and EGFR were used to test ir ADCC activity in vitro (ET101 and ET201, respectively). ET101 antibody is an ErbB2-blocking IgG1. ET101 produced by CHO-1E5 cells was purified from conditioned medium by Protein A chromatography; and quantified by UV280. Parental unmodified ET101 was expressed in wild-type CHO cells and purified in same way. For ADCC assay, 100 μl of target cell suspension was pre-incubated with 50 μl of expressed ErbB2-blocking antibody ET101 in 96-well plate at 37° C. for half an hour. 50 μl of PBMCs were n added at effector/target cell ratio of 20:1. After incubation for 16 hours, plate was spun down and 50 μl of cell-free supernatants were transferred to a new plate. The released LDH was measured by CytoTox96 Non-radioactive Cytotoxicity Assay (Promega, Madison, Wis.). The cell lysis was calculated by formula (E-S)/(M-S) (E: experimental release, S: spontaneous release, M: maximal release). PBS or nonspecific antibody was used as a negative control. The ADCC activity of ET101 exhibiting N-glycan produced by CHO-1E5 cell clone in serum-free medium against ovarian cancer cell line (SKOV3) and breast cancer cell line (MDA-MB-231) was significantly enhanced as compared to that of unmodified ET101 produced by parental CHO cells (
ET201 is an anti-EGFR antibody. ET201 expressed in CHO-1E5 cells was purified from conditioned medium by Protein A chromatography and quantified by UV280. The ADCC assay was performed using same method as described in
To furr identify how N-glycan of invention improves ADCC activity, recombinant FcγRIa and FcγRIIIa (R&D Systems, Minneapolis, Minn.) and ForteBio system (ForteBio, Menlo Park, Calif.) were used to measure binding affinity of antibody to se two Fc receptors. The antibodies were first biotinylated and loaded onto streptavidin-coated biosensor (ForteBio, Menlo Park, Calif.). Recombinant FcγRI and FcγRIIIb proteins were suspended at concentration of 100-400 nM (R&D Systems, Minneapolis, Minn.). The binding affinity (KD, nM) was assessed according to ForteBio's standard kinetics protocol. The antibody produced by CHO-1E5 cells had increased binding affinity to FcγRIIIa as compared to antibody produced by parental CHO cells, although re was no significant difference in binding to FcγRIa (
To furr determine if glycosylation by N-glycan synsized by CHO-1E5 cells could affect pharmacokinetics of antibody in vivo, antibodies produced by eir parental host cells or by CHO-1E5 cells were injected into 13-week-old female Balb/c mice through tail vein at 10 mg/kg, 3 mice for each antibody. 50 of blood were collected at 5 minutes, 1 hour, 6 hours, 72 hours and 120 hours. Serum concentration of antibodies was monitored at 1, 6, 72 and 120 hours after injection. The antibody concentration in serum was measured by OCTET (ForteBio, Menlo Park, Calif.). The antibody concentration at time of 5 minutes was considered as 100%. The pharmacokinetics of CHO-1E5-produced antibody is comparable to that of antibody produced by parental host cells (
To furr validate structures of N-glycan synsized by 1E5 cells, high-quality N-glycan released by PNGase F was purified in large quantity, and purified N-glycan and a positive control (Oligimannose 9, Glyko, San Leandro, Calif.) were digested with α1,2,3 mannosidase or α1,2,3,6 mannosidase, and products of digestion were analyzed by PA1 column and Dionex ICS-3000 system (Dionex, Sunnyvale, Calif.).
The antibodies (ET101) synsized by 1E5 (A and C) and a positive control N-glycan (Band D) (oligomannose 9, Glyko, San Leandro, Calif.) were incubated with α1,2,3 mannosidase (A and B) or α1,2,3,6 mannosidase (C and D) at 37° C. for 24 hours. To remove antibody and enzyme, digested antibody solution was first passed through microcolumns packed with C18, AG50WX8 and AG4×4. The columns were n washed with 300 μl of deionozed water. The flowthrough was collected, dried under vacuum and analyzed by PA1 column/Dionex ICS-3000 system.
Two peaks were observed when product of digestion was eluted by 18 mM NaOH. One peak was at 17 minutes, which corresponds to elution time of a mannose standard. Anor was at 24 minutes, which is similar to a disachamide standard (
Human FCγR IIIc has a polymorphism at amino acid 158. When amino acid 158 is Valine at two alleles, FCγR IIIa has high affinity to Fc fragment of IgG (termed FCγR IIIa 158V/V). When it is a Phenylalanine-carrier at amino acid 158, binding affinity of FCγR IIIa to Fc fragment of IgG is low (FCγR IIIc 158F/F or 158F/V). The ADCC activity correlates positively with FCγR IIIa binding affinity with Fc fragment. The FCγR IIIa polymorphism of PBMCs used in ADCC assays was genotyped as shown previously.
For ADCC assay, 100 μl of target cell suspension was pre-incubated with 50 μl of expressed ErbB2-blocking antibody ET101 in 96-well plate at 37° C. for half an hour. 50 μl of PBMCs were n added at effector/target cell ratio of 20:1. After incubation for 16 hours, plate was spun down and 50 μl of cell-free supernatants were transferred to a new plate. The released LDH was measured by CytoTox96 Non-radioactive Cytotoxicity Assay (Promega, Madison, Wis.). The cell lysis was calculated by formula (E-S)/(M-S) (E: experimental release, S: spontaneous release, M: maximal release). PBS or nonspecific antibody was used as a negative control. The genomic DNA was isolated from PMBCs used for se ADCC assays. The PCR assay to genotype polymorphism of FcγR IIIa is according to standard protocols in art. The ADCC activity was measured against ovarian cancer cell line (SKOV3) and breast cancer cell line (MDA-MB-231). It was found that antibody expressing unique N-glycan produced by CHO-1E5 cells enhanced ADCC activity equally efficiently when PBMCs of different genetic backgrounds expressing eir high-affinity FCγR IIIa 158V/V or low-affinity FCγR IIIa 158F/F were used as effector cells (
ADCC is a process that a specific antibody targeting a cell surface antigen first binds to cell and Fc fragment of that antibody n recruits effector cells such as natural killer cells and monocytes to target cells through Fc binding to Fc receptors expressed on cell surface of effector cells. The recruitment brings target and effector cells to proximity in order to kill target cells by effector cells. The Fc receptors (FCγR) consist of three types, I, II and III. FCγR Ia, FCγR IIa and FCγR IIIa are activating receptors that mediate and enhance ADCC when activated. FCγR IIb is an inhibitory receptor that blocks ADCC when activated. So an antibody with high affinity to FCγR Ia, FCγR IIa or FCγR IIIa has enhanced ADCC activity. However, an antibody with high affinity to FCγR IIb may inhibit ADCC activity and vice versa (
In order to determine mechanism of this enhanced ADCC activity by antibodies produced by 1E5 cells, binding affinity of antibodies to FCγRs was assessed. Human full length FcγRIIIA and FcγRIIb were cloned into expression vector and stably expressed in CHO-K1 cells. The cells were released from tissue culture dishes by 20 mM EDTA in PBS and disbursed into single cells. The cells were incubated with biotinylated antibodies at 10 μg/ml in PBS with 5% FBS on ice for 30 minutes. After washing with PBS for three times, cells were incubated with FITC-conjugated streptadividin, analyzed by FACS and mean fluorescence intensity (MFI) was acquired. The antibody binding affinity with inhibitory FcγRIIb (A) and activating FcγRIIIA (B) (at 10 μg/ml) and mechanistic schemes are shown in
CHO-K1 cell lines that stably overexpressed human Fcγ receptors including FCγRIIb and FCγR IIIa 158 (V/V type-high affinity) on cell surface were established. By using FACS approach, binding affinities of antibodies produced by parental CHO cells and 1E5 cells with se Fcγ receptors were measured by quantifying mean fluorescence intensity (MFI). It was found that antibody produced by 1E5 cells has significantly reduced binding affinity with inhibitory receptor, FCγR IIb, when compared to antibody produced by parental CHO cells (
To assess immunogenicity of antibody produced by CHO-1E5 cells exhibiting enhanced ADCC activity as compared to that of an antibody produced by unmodified parental CHO cells, in vivo immunogenicity of subject antibodies is assessed by measuring titer of antibody specific against subject antibodies in primates. Specifically, female cynomolgus monkeys of 3-5 years old weighing 4-6 kilograms (kg) receive anti-Her2 antibody ET101 produced by wild type CHO cells, or ET101 produced by CHO-1E5 cells at a dose of 8 mg/ml/kg of body weight. Two monkeys are in each group. 0.5 ml of blood is collected 7 days before antibody injection and on days 3, 5, 7, 14, 21, 28, 35 after antibody injection. Serum samples are isolated and frozen at −80° C. (
The present disclosure is not limited to embodiments described above, but is capable of modification within scope of appended claims. Those skilled in art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of specific embodiments of present disclosure described herein.
Claims
1-17. (canceled)
18. An N-linked glycan comprising one glucose molecule, four mannose molecules, and two N-acetylglycosamine molecules.
19. The N-linked glycan of claim 18, wherein glucose is located at a terminal end of N-linked glycan, and wherein N-linked glycan optionally comprises one or more fucose molecules.
20. The N-linked glycan of claim 18 having a structure of formula (I)
21. The N-linked glycan of claim 18 having a structure of formula (II)
22. An isolated glycoprotein that contains an N-glycan of any one of claims 18-21.
23. The glycoprotein of claim 22 wherein glycoprotein is an enzyme.
24. The glycoprotein of claim 22 wherein glycoprotein is an antibody.
25. The glycoprotein of claim 24 wherein N-linked glycan is attached to an Fc region of antibody.
26. The glycoprotein of claim 24 wherein antibody binds to a cancer antigen.
27. The glycoprotein of claim 26 wherein cancer antigen is selected from group consisting of HER2, Immunoglobulin epsilon Fc receptor II, Alk-1, CD20, EGF receptor, VEGF receptor, FGF receptor, NGF receptor, PDGF receptor, EpCam, CD3, CD4, CD11a, CD19, CD22, CD30, CD33, CD38, CD40, CD51, CD55, CD80, CD95, CCR2, CCR3, CCR4, CCR5, CTLA-4, Mucin 1, Mucin 16, Endoglin, Mesolin receptor, Nogo receptor, folate receptor, CXCR4, insulin-like growth factor receptor, Ganglioside GD3, and alpha and beta Integrins.
28. The glycoprotein of claim 24 wherein antibody is produced by a modified host cell which produces a substantially homogeneous population of N-linked glycans.
29. The glycoprotein of claim 24 wherein antibody has an increased ADCC (antibody-dependent cellular cytotoxicity) activity as compared to a corresponding antibody produced by an unmodified CHO cell clone, CHO-K1 (ATCC #CCL-61 and CRL-9618) or CHO-DG44 (Invitrogen #12609-012).
30. The glycoprotein of claim 24 wherein antibody has an increased binding affinity to FcγRIIIA receptor as compared to a corresponding antibody produced by an unmodified CHO cell clone, CHO-K1 (ATCC #CCL-61 and CRL-9618) or CHO-DG44 (Invitrogen #12609-012).
31. The glycoprotein of claim 24 wherein antibody is an inhibitory antibody.
32. The glycoprotein of claim 24 wherein antibody is an IgG antibody.
33. A method of producing a modified glycoprotein comprising:
- (a) providing a heterologous polynucleotide sequence that encodes said modified glycoprotein; and
- (b) causing said modified glycoprotein to be expressed in a host cell, wherein host cell produces N-linked glycan having a variant glycosylation pattern characterized by a change in level of one or more sugar moieties selected from group consisting of glucose, galactose, mannose, and glucosamine.
34. The method of claim 33, wherein said host cell is maintained in a serum free medium.
35. The method of claim 33, wherein said host cell is maintained in a suspension culture.
36. The method of claim 33, wherein said modified glycoprotein is an antibody.
37. The method of claim 33, wherein host cell produces N-linked glycan having a structure of formula (I) or (II):
Type: Application
Filed: Nov 29, 2011
Publication Date: May 3, 2012
Applicant: Eureka Therapeutics, Inc. (Richmond, CA)
Inventors: Cheng Liu (Richmond, CA), Jingyi Xiang (Walnut Creek, CA), Su Yan (Albany, CA), Pei Wang (Fremont, CA), Wei Chan Tai (Emeryville, CA)
Application Number: 13/306,777
International Classification: C12P 21/00 (20060101); C07K 14/00 (20060101); C07K 16/30 (20060101); C07K 16/00 (20060101); C07K 16/18 (20060101); C07K 16/40 (20060101); C08B 37/00 (20060101); C12N 9/96 (20060101);
