Glycoprotein compositions

专利摘要:
The present invention relates to a composition comprising a glycoprotein having an Fc region, wherein about 80-100% of the glycoprotein comprises a mature core carbohydrate structure lacking a fucose attached to the Fc region of the glycoprotein. . Preferred glycoproteins are antibodies or immunoadhesin.
公开号:KR20040066105A
申请号:KR10-2004-7006034
申请日:2002-10-22
公开日:2004-07-23
发明作者:레오나르드 지. 프레스타
申请人:제넨테크, 인크.；
IPC主号:

专利说明:

Glycoprotein composition {GLYCOPROTEIN COMPOSITIONS}
[2] Antibodies
[3] Antibodies are proteins that exhibit binding specificity for specific antigens. Natural antibodies are usually about 150,000 Daltons heterotetrameric glycoproteins consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by one covalent disulfide bond, with the number of disulfide linkage groups varying between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V _H ) followed by a number of constant domains. Each light chain has a variable domain (V _L ) at one end and a constant domain at the other end; The constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that certain amino acid residues form the interface between the variable domains of the light and heavy chains.
[4] The term "variable" refers to the fact that certain portions of the variable domains have widely differing sequences between antibodies and are responsible for the binding specificity of each particular antibody for a particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. Variability is concentrated in three segments called complementarity determining regions (CDRs) in both the light and heavy chain variable domains. The more highly conserved portions of variable domains are called framework regions (FRs). The variable domains of natural heavy and light chains are predominantly in β-sheet arrangements, each containing four FRs linked by three CDRs that link the β-sheet structure and in some cases form a loop that forms part of the structure. do. CDRs in each chain are closely held together by the FRs and serve to form the antigen binding site of the antibody with the CDRs of the other chain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Health Service, National Institutes of Health, Bethesda, MD. (1991)].
[5] The constant domains do not directly participate in binding the antibody to the antigen, but exhibit the function of various effectors. Depending on the amino acid sequence of the constant domain of the heavy chains, antibodies or immunoglobulins can fall into different classes. There are five major types of immunoglobulins (IgA, IgD, IgE, IgG and IgM), some of which are further classified as subspecies (isotypes) (eg, IgG1, IgG2, IgG3 and IgG4; IgA1 and IgA2). The heavy chain constant domains that correspond to the different kinds of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Of the various human immunoglobulin classes, only human IgG1, IgG2, IgG3 and IgM are known to activate complement, and human IgG1 and IgG3 mediate ADCC more effectively than IgG2 and IgG4.
[6] A schematic representation of the native IgG1 structure is shown in FIG. 1A, where the variable portion of the native antibody molecule is indicated. Papain digestion of antibodies results in two identical antigen binding fragments, called Fab fragments, each having a single antigen binding site, and the remaining “Fc” fragments, whose names indicate the ability to readily crystallize. The crystal structure of the human IgG Fc region was determined (Deisenhofer, Biochemistry 20: 2361-2370 (1981)). In human IgG molecules, the Fc region is produced by N-terminal papain cleavage for Cys226. The Fc region plays a central role in the functional function of the antibody.
[7] Other antibody-like molecules have also been described. For example, "immunoadhesin" has been reported in the literature that binds the binding domains of heterologous proteins such as receptors, ligands or enzymes with the functional function of the Fc region. An example of such a molecule is tumor necrosis factor receptor-IgG (TNFR-IgG) immunoadhesin described in US Pat. No. 5,610,297. Bispecific immunoadhesin and antibody-immuneadhesin chimeras have also been described. Stabila, P., Nature Biotech, 16: 1357 (1998) describe another Fc region-containing plasma membrane-fixed fusion protein. The fusion protein of this reference binds a type II transmembrane domain located into the plasma membrane, fused to the N-terminus of the Fc region.
[8] Antibodies and immunoadhesin have been used as therapeutics in human diseases (Glennie et al. Immunol. Today 21: 403-410 (2000); King et al. Curr. Opin. Drug Discovery Develop 2: 110-117 (1999); Vaswani et al. Ann.Allergy Asthma Immunol. 81: 105-119 (1998); and Abraham et al. Sec.Intern. Autumnal Them.Meeting on Sepsis, Deauville, France (1995). Portions of this antibody and immunoadhesin, such as those that bind to receptors or ligands to block ligand receptor interactions, can function without the use of antibody mechanism mechanisms. Others may need to recruit an immune system to kill target cells (Clynes et al. Nature Med. 6: 443-446 (2000); Clynes et al. PNAS (USA) 95: 652-656 (1998); And Anderson et al. Biochem. Soc. Trans. 25: 705-708 (1997)).
[9] Antibody Agonist Functions
[10] Agonist function mediated by antibody Fc regions can be divided into two categories: (1) Agonist function that acts after the antibody binds to the antigen (this function is the participation of complement cascades or Fc receptor (FcR) -bearing cells. Related to); And (2) an agent function that works independently of antigen binding, which imparts persistence to the ability and circulation to be delivered across the cell barrier by transcytosis. Ward and Ghetie, Therapeutic Immunology 2: 77-94 (1995).
[11] Binding of antibodies to essential antigens has a neutralizing effect that can prevent foreign antigens from binding to endogenous targets (eg, receptors or ligands), whereas binding alone cannot eliminate foreign antigens. In order to efficiently remove and / or destroy foreign antigens, an antibody must be endowed with both high affinity binding to its antigen and efficient agent function.
[12] The interaction of cells of the immune system with antibodies and antibody-antigen complexes results in a variety of responses including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) (Daeron, Annu. Rev. Immunol. 15: 203-234 (1997); Ward and Ghetie, Therapeutic Immunol. 2: 77-94 (1995); and Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991).
[13] Some antibody agonist functions are mediated by Fc receptors (FcRs) that bind to the Fc region of an antibody. FcRs are defined by specificity for immunoglobulin isotypes; Fc receptors for IgG antibodies are called FcγR, IgE for FcεR, IgA for FcαR and the like. Three subspecies of FcγR have been identified: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Because each FcγR subspecies is encoded by two or three genes, and alternative RNA splicing leads to multiple transcripts, there is a wide variety of FcγR isomers. Three genes encoding FcγRI subspecies (FcγRIA, FcγRIB and FcγRIC) are concentrated in the 1q21.1 region of the long arm of chromosome 1; The genes encoding the FcγRII isomers (FcγRIIA, FcγRIIB and FcγRIIC) and the two genes encoding FcγRIII (FcγRIIIA and FcγRIIIB) are all concentrated in the 1q22 region. These different FcR subtypes are expressed in different cell types (reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991)). For example, in humans, FcγRIIIB is found only in neutrophils, while FcγRIIIA is found in subpopulations of macrophages, monocytes, natural killer (NK) cells, and T-cells.
[14] Structurally, FcγR is a member of an immunoglobulin superfamily with IgG-binding α-chains containing an extracellular portion consisting of both (FcγRI and FcγRIII) or three (FcγRI) Ig-like domains. In addition, FcγRI and FcγRIII have auxiliary protein chains (γ, ζ) associated with α-chains that function in signal transduction. Receptors are also distinguished by their affinity for IgG. FcγRI exhibits high affinity for IgG (K _a = 10 ⁸ -10 ⁹ M ^-1 ) (Ravetch et al. Ann. Rev. Immunol. 19: 275-290 (2001)) and can bind to monomeric IgG. . In contrast, FcγRII and FcγRIII show relatively weak affinity for monomeric IgG (K _a ≦ 10 ⁷ M ⁻¹ ) (Ravetch et al. Ann. Rev. Immunol. 19: 275-290 (2001)). It only interacts effectively with the body's immune complex. FcγRII receptors mainly include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibiting receptor”) having similar amino acid sequences that differ only in the cytoplasmic domain. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic domain. Inhibitor receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic domain (see review by Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). NK cells contain only FcγRIIIA and binding of the antibody to FcγRIIIA induces ADCC activity by NK cells.
[15] Several allelic variants of human FcγR have been found in the human population. This allelic variant form has been shown to show differences in the binding of human and murine IgG, and numerous binding studies have correlated the presence of certain allelic forms with clinical outcomes (Lehrnbecher et al. Blood 94 (12): 4220-4232 (1999). Several studies have investigated the association between two forms of FcγRIIA, R131 and H131, and clinical outcomes (Hatta et al. Genes and Immunity 1: 53-60 (1999); Yap et al. Lupus 8: 305-310 (1999). And Lorenz et al. European J. Imnunogenetics 22: 397-401 (1995). Two allelic forms of FcγRIIIA, F158 and V158 have not been investigated at this time (Lehrnbecher et al., Supra; and Wu et al. J. Clin. Invest. 100 (5): 1059-1070 (1997) ). FcγRIIIA (Val158) allotypes interact better with human IgG than FcγRIIIA (Phe158) allotypes (Shields et al. J. Biol. Chem. 276: 6591-6604 (2001); Koene et al. Blood 90: 1109 -1114 (1997); and Wu et al. J. Clin. Invest. 100: 1059-1070 (1997).
[16] Another type of Fc receptor is the neonatal Fc receptor (FcRn). FcRn is structurally similar to major histocompatibility complex (MHC) and consists of an α-chain that is covalently bound to β2-microglobulin. FcRn has been suggested to be capable of regulating transcytosis across tissues as well as regulating IgG homeostasis in the blood (Ghetie et al. Annu. Rev. Immunol. 18: 739-766 (2000)).
[17] The binding site on human and murine antibodies to FcγR is residue 233-239 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Previously mapped to a so-called "lower hinge region" consisting of EU index numbering as in [Woof et al. Molec. Immunol. 23: 319-330 (1986); Duncan et al. Nature 332: 563 (1988); Canfield and Morrison, J. Exp. Med. 173: 1483-1491 (1991); Chappel et al., Proc. Natl. Acad. Sci USA 88: 9036-9040 (1991). Of residues 233-239, it was mentioned that P238 and S239 are likely to be involved in binding.
[18] Regions previously mentioned as likely to be involved in binding to FcγR include G316-K338 (human IgG) for human FcγRI (by sequence comparison only; substitution mutations were not evaluated) (Woof et al. Molec. Immunol. 23: 319-330 (1986); K274-R301 (human IgG1) (based on peptide) against human FcγRIII (Sarmay et al. Molec. Immunol. 21: 43-51 (1984)); Y407-R416 (human IgG) (based on peptide) against human FcγRIII (Gergely et al. Biochem. Soc. Trans. 12: 739-743 (1984)); And N297 and E318 (murine IgG2b) for murine FcγRII (Lund et al., Molec. Immunol. 29: 53-59 (1992)). Armor et al. Eur. J. Immunol. 29: 2613-2624 (1999).
[19] WO00 / 42072 (Presta) describes polypeptide variants having enhanced or reduced binding capacity for FcRs. The contents of this patent document are specifically incorporated by reference herein. See also Shields et al. J. Biol. Chem. 9 (2): 6591-6604 (2001).
[20] C1q and the two serine proteases, C1r and C1s, form the complex C1, which is the first component of the complement dependent cytotoxicity (CDC) pathway. C1q is a hexavalent molecule having a molecular weight of approximately 460,000 and has a structure like a tulip bouquet in which six collagen "stems" are connected to six spherical head regions. Burton and Woof, Advances in Immunol. 51: 1-84 (1992). To activate the complement cascade, C1q needs to bind to at least two molecules of IgG1, IgG2, or IgG3 (IgG4 agrees that it does not activate complement), but only one of the IgMs attached to the antigen target It is necessary to bind to the molecule. Ward and Ghetie, Therapeutic Immunology 2: 77-94 (1995), page 80.
[21] Based on the results of chemical modifications and crystallographic studies, Burton et al. Nature, 288: 338-344 (1980), suggested that the position at which the complement subcomponent C1q binds to IgG comprises the last two (C-terminal) β-strands of the CH2 domain. Burton is then described in Molec. Immunol, 22 (3): 161-206 (1985), suggested that a region comprising amino acid residues 318 to 337 may be involved in complement fixation.
[22] Duncan and Winter Nature 332: 738-40 (1988) reported that Glu318, Lys320 and Lys322 form binding sites for C1q using positional mutations. Duncan and Winter data were generated by testing the binding of the mouse IgG2b isotype to guinea pig C1q. The role of the Glu318, Lys320 and Lys322 residues in the binding of Clq was confirmed by the ability of short synthetic peptides containing these residues to inhibit complement mediated degradation. Similar results are disclosed in US Pat. No. 5,648,260, issued July 15, 1997, and US Pat. No. 5,624,821, issued April 29,1997.
[23] Pro331 residues were linked to C1q binding by analysis of the ability of human IgG subspecies to perform complement mediated cell lysis. Mutation of Ser331 to Pro331 in IgG4 conferred the ability to activate complement. Tao et al., J. Exp. Med., 178: 661- 667 (1993); Brekke et al., Eur. J. Immunol., 24: 2542-47 (1994)].
[24] By comparing the data of the Winter Group with the papers of Tao et al. And Breke et al., Ward and Gettie found that there are at least two different areas involved in the binding of C1q in their review paper. Conclusions: One is on the β-strand of the CH2 domain with Glu318, Lys320 and Lys322 residues, the other is on a turn located proximate the same β-strand, and the major amino acid residue at position 331 It contains.
[25] Other reports suggest that human IgGl residues Lys235 and Gly237, located in the lower hinge region, play an important role in complement fixation and activation. Xu et al., J. Immunol. 150: 152 A (Abstract) (1993)]. W094 / 29351 published Dec. 22, 1994, reported that the amino acid residues required for C1q and FcR binding of human IgG1 are located in the N-terminal region of the CH2 domain, ie residues 231 to 238.
[26] It has also been shown that the ability of IgG to bind C1q and activate the complement cascade depends on the presence, absence or modification of carbohydrate moieties (usually anchored in Asn297) located between the two CH2 domains (Ward and Ghetie, Therapeutic Immunology 2: 77-94 (1995) p. 81).
[27] Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding capacity have been described in US Pat. Nos. 6,194,551B1 and WO99 / 51642. The contents of this patent publication are specifically incorporated by reference herein. See also Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
[28] Other methods of enhancing immune system recruitment include bispecific antibodies and IgG receptors to which one arm of the antibody binds (Segal et al. J. Immunol. Meth. 248: 1-6 (2001)); And cytokine-IgG fusion proteins (Penichet et al. J. Immunol. Meth. 248: 91-101 (2001)).
[29] Antibody Glycosylation
[30] Many polypeptides, including antibodies, undergo various post-translational modifications involving carbohydrate moieties such as glycosylation to oligosaccharides. Such glycosylated polypeptides are called "glycoproteins".
[31] There are several factors that can affect glycosylation. Species, tissues and cell types have all been shown to be important in the way glycosylation occurs. In addition, the extracellular environment through altered culture conditions, such as serum concentrations, may have a direct effect on glycosylation (Lifely et al. Glycobiology 5 (8): 813-822 (1995)). Various methods have been proposed for altering the glycosylation pattern achieved in certain host organisms, including introducing or overexpressing specific enzymes involved in oligosaccharide production (US Pat. No. 5,047,335; US Pat. No. 5,510,261). This method is not limited to intracellular methods (US Pat. No. 5,278,299).
[32] All antibodies contain carbohydrates in conserved positions within the constant region of the heavy chain. Each antibody isotype has a unique variety of N-linked carbohydrate structures. In addition to carbohydrates attached to the heavy chain, up to 30% of human IgG has a glycosylated Fab region. IgG has a single N-linked biantenary carbohydrate at Asn297 of the CH2 domain. The fully processed carbohydrate structure attached to Asn297 is shown in FIG. 2 herein. For IgG produced ex vivo or from serum in hybridomas or engineered cells, IgG is heterogeneous for Asn297 linked carbohydrates. Jefferis et al. Immunol. Rev. 163: 59-76 (1998); And Wright et al. Trends Biotech 15: 26-32 (1997). Human IgG has core oligosaccharides usually composed of GlcNAc ₂ Man ₃ GlcNAc and have different numbers of external residues. FIG. 2 of the present application shows the pathway by which oligosaccharides are processed into mature carbohydrates. The initially synthesized species Glu ₃ Man ₉ GlcNac ₂ is delivered to Asn297 in the CH2 domain of the antibody when it exits the ribosome. After the three terminal glucoses are cut off as the glycoprotein passes through the endoplasmic reticulum, the glycoprotein moves to the cis Golgi apparatus, where the mannose residues are enzymatically removed by α-mannosidase. As the over-mannosylated glycoproteins are obtained, processing can stop at this point. Processing may stop at this point, resulting in a hyper-mannosylated glycoprotein. Otherwise, processing is continued to obtain Man ₅ GlcNac ₂ . The action of N-acetylglycosaminyltransferase I in medial Golgi apparatus is an obvious step in complex oligosaccharide synthesis. In media and trans Golgi apparatus, oligosaccharides undergo additional processing steps to remove mannose residues and add sugar residues sequentially. The newly synthesized glycoprotein then exits the Golgi apparatus and is either delivered or secreted to the cell membrane.
[33] Variation between each IgG occurs through the addition of galactose and / or galactose-sialic acid at two terminal GlcNac or the addition of a third GlcNac arm (bisecting GlcNAc). Carbohydrates linked to Asn297 in IgG were studied. The absence of carbohydrates affects the binding to C1q and FcγR (resultingly, complement activation and ADCC). See Leatherbarrow et al. Molec. Immunol. 22: 407-415 (1985); Duncan et al. Nature 332: 738-740 (1988); Walker et al. Biochem. J. 259: 347-353 (1989); Dorai et al. Hybridoma 10: 211-217 (1990); And Horan Hand et al. Cancer Immunol. Immunother. 35: 165-174 (1992). While binding to FcRn does not appear to be affected by the lack of carbohydrates (Hobbs et al. Molec. Immunol. 29: 949-956 (1992); and Kim et al. Eur. J. Immunol. 24: 542-548 (1994)), the effect on clearance is uncertain (Dorai et al. Hybridoma 10: 211-217 (1990); Horan Hand et al. Cancer Immunol. Immunother. 35: 165-174 (1992); Hobbs et al. Molec. Immunol. 29: 949-956 (1992); Kim et al. Eur. J. Immunol. 24: 542-548 (1994); Wawrzynczak et al. Biochem. Soc. Trans. 17: 1061-1062 (1989); and Tao et al. J. Immuno. 143: 2595-2601 (1989). When carbohydrates are present, the nature of the sugar residues can also affect IgG agonist function. The presence or absence of terminal galactose residues has been reported to affect function (Wright et al. J. Immunol. 160: 3393-3402 (1998)) and seems to be associated with rheumatoid arthritis (Parekh et al. Nature 316: 452-457 (1985)). Human IgG isolated from the sera of patients with multiple myeloma showed the opposite behavior by the presence / absence of fucose, galactose, and bipartite N-acetylglycosamine (Parekh et al. Nature 316: 452-457 (1985)). . Raju et al. Describe variations in glycosylation of IgG from different species (Raju et al. Glycobiology 10 (5): 477-486 (2000)).
[34] Boyd et al. Found that the removal of terminal sialic acid from CHO-derived CAMPATH-1H via glycopeptidase F degradation did not affect any of the IgG activity tested, whereas in desialylated CAMPATH-1H. Removing most of the galactose residues has been shown to reduce (but not completely eliminate) complement lytic activity. Other activities were not affected by degalactosylation. Boyd et al. Molec. Immunol. 32 (17/18): 1311-1318 (1995). Kumpel et al., Hum. Antibod. Hybridomas, 5 (3-4): 143-151 (1994), reported that galactosylation of human IgG monoclonal antibodies affects Fc receptor-mediated functional activity.
[35] Rothman et al. Tested the ADCC function of monoclonal IgG purified in hybridomas treated with glycosidase inhibitors that act at different stages of the carbohydrate processing pathway. Rothman et al. Molecular Immunol. 26 (12): 1113-1123 (1989). Treatment with castanospermin that inhibits the removal of glucose residues from natural oligosaccharides (Kaushal et al. Meth. Enzymol. 230: 316-329 (1994)) shows elevated ADCC by NK cells expressing only FcγRIII but There was no elevated ADCC by other types of effector cells, such as monocytes. It has been suggested that castanospermin-treated IgG lacks fucose by lectin-binding assays, but IgG produced by castanospermin treatment may have other carbohydrate structures such as terminal glucose residues and hyper-mannosylation ( Kaushal et al. Meth.Enzymol. 230: 316-329 (1994); Hashim et al. Immunology 63: 383-388 (1988); Hashim et al. Molec. Immunol. 24: 1087-1096 (1987)) The structure is one that is not routinely found in IgG secreted from untreated cells or human serum.
[36] WO97 / 30087 describes the preparation of glycosylated antibodies in which the N-glycosylation site of the Fc domain of the antibody is substituted with biantennary oligosaccharides.
[37] Umana et al. Described the β (1,4) -N-acetylglucosaminyltransferase III (GcTIII) gene that catalyzes the addition of bisected GlcNAc to the carbohydrate core attached to Asn297 of the antibody. Introduced into ovarian (CHO) cells. Glycoforms produced by engineered CHO cells were believed to have optimized ADCC. See WO 99/54342 and Umana et al., Nature Biotechnology, 17: 176-180 (1999).
[38] W098 / 58964 (Raju et al.) Describe antibody compositions in which substantially all N-linked oligosaccharides are G2 oligosaccharides. G2 refers to a biantennary structure having two terminal Gals and no NeuAc. WO 99/22764 (Raju et al.) Refers to antibody compositions substantially free of glycoproteins having G1, G0 or G-1 oligosaccharides N-linked to the CH2 domain. G1 refers to a biantennary structure having one Gal and no NeuAc, G0 refers to a structure without terminal NeuAc or Gal present, and G-1 refers to a core unit minus one GlcNac.
[39] In WO00 / 61739, 47% of anti-hIL-5R antibodies expressed by YB2 / 0 (rat myeloma) cells were compared with 73% of anti-hIL-5R antibodies expressed by NSO (mouse myeloma) cells. reported to have α 1-6 fucose-linked sugar chains. The fucose relative ratio of α-hIL-5R antibodies expressed by various host cells was YB2 / 0 <CHO / d <NSO.
[40] Rutier et al studied the glycosylation pattern of humanized IgG1 antibody (D1.3) expressed in CHO-DUKX cells. The structure of the N-glycans of the CHO-expressed was biantennary N-glycans with core fucose but without dichotomous GlcNAc and sialic acid. The structure was called G ₂ , G ₁ and G _{0 since it} was heterogeneous for terminal glycosylation. Routier et al. Glycoconjugate J. 14: 201-207 (1997).
[41] O-linked fucose has been found in many polypeptides and previously attached fucose was previously reported to be important for the proper activity of the polypeptide. See WO98 / 33924, which describes a glycosylation method with an O-fucose moiety. Stankova et al. J. Immunol. Lett. 11: 39-44 (1985) found that fucose significantly enhanced the cytolytic ability of mixed leukocyte culture (MCL) -induced or preincubated effector cells. Cameron et al. Immunol. Lett. 11: 39-44 (1985) found that α-L-fucose appears to play an important role in macrophage-tumor cell interactions.
[42] There is a continuing need in the art to produce glycoproteins such as antibodies with improved biological activity.
[43] Summary of the Invention
[44] The present application provides a glycoprotein having an Fc region, wherein about 80-100% (preferably about 90-99%) of the glycoprotein comprises a mature core carbohydrate structure that lacks a fucose attached to the Fc region of the glycoprotein. It relates to a glycoprotein composition of. It has been demonstrated herein that the composition is surprisingly 100-fold improved in binding to FcγRIIIA (F158), which is not as effective as FcγRIIIA (V158) in interacting with human IgG. Accordingly, the compositions herein are expected to be superior to the compositions previously described for treating human patients expressing FcγRIIIA (F158) in particular. FcγRIIIA (F158) is more common than FcγRIIIA (V158) in normal healthy African American and Caucasian species. Lehrnbecher et al. Blood 94: 420 (1999).
[45] Further demonstrated herein is a synergistic increase in FcγRIII binding and / or ADCC function produced by combining amino acid sequence modifications and glycosylation variations in the Fc region of the glycoprotein. In order to generate Fc region amino acid sequence variants with improved ADCC activity, one will design Fc region variants with improved binding affinity for FcγRIII, which is generally considered to be an important FcR for mediating ADCC. For example, to generate such variants, amino acid modifications (eg substitutions) at one or more of the 256, 290, 298, 312, 326, 330, 333, 334, 360, 378 or 430 amino acid positions into the parent Fc region Can be introduced. Variants with improved binding affinity for FcγRIII may additionally have a reduced binding affinity for FcγRII, in particular affinity for inhibitory FcγRIIB receptors. In a preferred embodiment, the Fc region has amino acid substitutions at positions 298, 333 and 334, eg, at S298A / E333A / K334A. Fc regions with altered amino acid sequences further comprise glycosylation mutations that further elevate ADCC. For example, the variant Fc region may have a mature core carbohydrate structure that lacks fucose attached thereto.
[46] Accordingly, the present invention encompasses a mature core carbohydrate structure that lacks fucose, wherein about 51-100% of the glycoproteins in the composition are attached to the Fc region of the glycoprotein, wherein the Fc region comprises an amino acid sequence different from the native sequence Fc region. Provided is a composition comprising a glycoprotein having an Fc region. More preferably, about 80-100% of the glycoproteins in the composition comprise mature core carbohydrate structures lacking fucose, and most preferably, about 90-99% of the glycoproteins in the composition adhere to the mature core carbohydrate structures. Is deficient in fucose.
[47] Glycoproteins include, for example, antibodies or immunoadhesin. Glycoproteins generally comprise an Fc region, preferably a human Fc region, such as a human IgGl, IgG2, IgG3 or IgG4 Fc region. Glycoproteins have increased binding to FcγRIII (eg, FcγRIIIA (F158) and / or FcγRIIIA (V158)) and exhibit improved ADCC for glycoproteins with fucose attached to mature core carbohydrate structures.
[48] The invention also provides a pharmaceutical formulation comprising a glycoprotein and optionally a pharmaceutically acceptable carrier or diluent. This formulation for possible therapeutic use may be sterile and lyophilized.
[49] Diagnostic and therapeutic uses for the glycoproteins disclosed herein are contemplated. In one diagnostic application, the present invention provides a method of determining the presence of an antigen of interest comprising exposing a sample that is expected to contain an antigen to glycoproteins and measuring the binding of the glycoprotein to the sample.
[50] In one therapeutic application, the invention treats a mammal suffering from or susceptible to a disease or disorder in which such treatment would benefit, including administering to the mammal a therapeutically effective amount of a composition herein, in particular the composition is a pharmaceutical formulation. Provide a way to.
[51] The invention also provides a nucleic acid encoding a glycoprotein comprising an Fc region comprising a mature core carbohydrate structure lacking a fucose, wherein about 80-100% of the glycoprotein produced by the host cell is attached to the Fc region of the glycoprotein. It provides a host cell comprising a. The invention also provides a method for producing a glycoprotein comprising culturing the host cell so that the nucleic acid is expressed and optionally recovering the glycoprotein from the host cell culture (eg, from the host cell culture medium). do.
[52] The invention also provides a glycoprotein in an article of manufacture or kit that can be used for the purpose of treating a disease or disorder.
[53] Brief description of the drawings
[54] 1A is a schematic representation of native IgG and its enzymatic degradation to produce various antibody fragments. Disulfide bonds are indicated by double lines between the CH1 and CL domains and the two CH2 domains. V is the variable domain, C is the constant domain, L is the light chain and H is the heavy chain. 1B shows a fully processed or “mature” core carbohydrate structure 2100 attached to Asn297 of serum IgG, a mature core carbohydrate structure 2100 with a single galactose residue, and a core carbohydrate structure with two galactose residues and a bisected GlcNAc (3120). ) Is shown schematically. The number of GlcNAc, fucose, galactose and sialic acid residues is represented by the four-digit numbering system shown in this figure, respectively.
[55] Figure 2 illustrates the addition of oligosaccharides to Asn297 in the CH2 domain of IgG and processing them in cis, medial and trans Golgi bodies to produce a complex with a biantennary fully processed carbohydrate structure. Castanospermine inhibits the removal of glucose and mannose residues from natural oligosaccharides.
[56] 3 shows heavy chain (Fc) oligosaccharides found in antibodies expressed in CHO cells with normal fucose metabolism.
[57] Throughout further Figure Legends and Examples, the following symbols are used: "Hu4D5" is an abbreviation for humanized anti-HER2 4D5 antibody, Chinese Hamster Ovary is abbreviated as "CHO" and CHO cultured in 15 cm plate -DP12 cells are labeled "CHO-P", CHO-DP12 cells cultured in spinner flasks are labeled "CHO-S", human embryonic kidney 293 cells are abbreviated "HEK293", and "Lec 13" CHO cell line defective in fucose metabolism obtained from Pamela Stanley of Albert Einstein College of Medicine of Yeshiva University, Bronx, New York, and in the Fc region, S298A / Hu4D5 with E333A / K334A substitution is called “Hu4D5-AAA” and “E27” is an affinity matured / humanized anti-IgE antibody described in US Pat. No. 6,172,213 and E27 with S298A / E333A / K334A substitution in the Fc region Is represented by "E27-AAA" and peripheral blood Mononuclear cell antibody-dependent cell-mediated cytotoxicity is abbreviated as "PBMC ADCC".
[58] 4 shows the binding of Hu4D5 monomers to human FcγRI. Hu4D5 antibodies were expressed in CHO-S, HEK293 cells, CHO-P, or Lec13 CHO cells (two different batches).
[59] 5 shows the binding of Hu4D5 dimers to human FcγRIIB. Hu4D5 antibodies were expressed in CHO-S or Lec13 cells (three different batches).
[60] 6 shows binding of Hu4D5 dimer to human FcγRIIA (R131). Hu4D5 antibodies were expressed in CHO-S or Lec13 cells (three different batches).
[61] 7 shows the binding of Hu4D5 dimer to human FcγRIIA (H131). Hu4D5 antibodies were expressed in CHO-S or Lec13 cells (three different batches).
[62] FIG. 8 shows binding of Hu4D5 or Hu4D5-AAA dimers to human FcγRIIIA (V158) expressed in CHO-S or Lec13 cells (three and two batches, respectively).
[63] 9 shows binding to human FcγRIIIA (F158) of Hu4D5 dimers expressed in CHO-S or Lec13 cells (three different batches) or Hu4D5-AAA dimers expressed in Lec13 cells (two different batches).
[64] 10 depicts binding of anti-IgE (E27) dimers to human FcγRIIIA (V158). E27 expressed in HEK293 cells, CHO-P cells (two batches) or Lec13 cells (two batches) was tested in this assay.
[65] 11 depicts the binding of anti-IgE (E27) to human FcγRIIIA (F158). E27 expressed in HEK293 cells, CHO-P cells (two batches) or Lec13 cells (two batches) was tested in this assay.
[66] 12 depicts binding of anti-IgE (E27) hexamer and E27-AAA to FcγRIIIA (V158). Antibodies were expressed in CHO-P, Lec13 or HEK293 cells.
[67] FIG. 13 shows the binding of anti-IgE (E27) hexamer and E27-AAA to FcγRIIIA (F158). Antibodies were expressed in CHO-P, Lec13 or HEK293 cells.
[68] 14 shows binding of Hu4D5 to human FcRn expressed in CHO-P, CHO-S or Lec13 cells.
[69] 15 shows the binding of Hu4D5 and anti-CD20 (RITUXAN®) to human C1q. Hu4D5 was expressed in CHO-P or Lec13 cells (two batches). Rituxan® was expressed in CHO-P cells.
[70] Figure 16 shows binding of Hu4D5 or Rituxan® to human C1q. Hu4D5 used in this experiment was expressed in CHO-P, Lec13 (three different batches) or CHO-S cells. Rituxan® was expressed in CHO-P cells.
[71] 17 depicts PBMC ADCC of SKBR3 breast tumor cells (E: T 30: 1) using FcγRIII VF donors. Spontaneous ADCC compared to ADCC produced in Hu4D5 expressed in CHO-S or Lec13 cells.
[72] FIG. 18 shows PBMC ADCC of SKBR3 breast tumor cells (E: T 30: 1) using another FcγRIII VF donor. Spontaneous ADCC compared to ADCC produced in Hu4D5 expressed in CHO-S or Lec13 cells.
[73] 19 shows PBMC ADCC of SKBR3 breast tumor cells (E: T 30: 1) using FcγRIII FF donors. Spontaneous ADCC compared to ADCC produced in Hu4D5 expressed in CHO-S or Lec13 cells.
[74] 20 shows PBMC ADCC of SKBR3 breast tumor cells (E: T30: 1) using another FcγRIII FF donor. Spontaneous ADCC compared to ADCC produced in Hu4D5 expressed in CHO-S or Lec13 cells.
[75] 21 shows monocyte ADCC of SKBR3 breast tumor cells (E: T 10: 1) using FcγRIIA RR donors. Spontaneous ADCC compared to ADCC produced in Hu4D5 expressed in CHO-S or Lec13 cells (two different batches).
[76] 22 shows monocyte ADCC of SKBR3 breast tumor cells (E: T 10: 1) using FcγRIIA HH donors. Spontaneous ADCC compared to ADCC produced in Hu4D5 expressed in CHO-S or Lec13 cells.
[77] Figure 23 depicts alignment of native sequence IgG Fc regions. Native sequences human IgG Fc region sequences, humIgG1 (non-A and A allotypes) (SEQ ID NOs: 1 and 2, respectively) and humIgG2 (SEQ ID NO: 3), humIgG3 (SEQ ID NO: 4) and humIgG4 (SEQ ID NO: 5). The human IgG1 sequence is a non-A allotype and the difference between this sequence and the A allotype (at positions 356 and 358; EU numbering system) is shown below the human IgG1 sequence. Also shown are the native sequence murine IgG Fc region sequences murIgG1 (SEQ ID NO: 6), murIgG2A (SEQ ID NO: 7), murIgG2B (SEQ ID NO: 8), and murIgG3 (SEQ ID NO: 9).
[78] FIG. 24 depicts binding of Hu4D5 and Hu4D5-AAA to CD56 positive natural killer (NK) cells. Products tested were (1) FITC conjugated anti-human IgG, (2) Hu4D5 from CHO-S, (3) Hu4D5 expressed in Lec 13 cells, and (4) Hu4D5-AAA expressed in Lec 13 cells .
[79] 25 shows immunofluorescence staining of purified NK cells expressing FcγRIII (F / F) receptors.
[80] FIG. 26 provides a comparison of NK ADDC activity of Hu4D5 from CHO-S, Hu4D5 from Lec13 cells, Hu4D5-AAA from Lec13 cells, and Hu4D5 from HEK293 cells. Donor was FcγRIII (F / F).
[81] FIG. 27 repeats the experiment in FIG. 26 with different FcγRIII (F / F) donors.
[82] FIG. 28 depicts binding of anti-HER2 Hu4D5 monomers to CHO cell lines permanently-transfected with human FcγRIIIA α-chain and γ-chain (representative plot for one assay). Hu4D5 CHO-S, unpainted circle; Hu4D5 Lec13-D, unpainted square; Hu4D5 Lec13-E, unpainted diamond; Hu4D5 Lec13-F, unpainted triangle; Hu4D5 HEK293-AAA, painted circles; Hu4D5 Lec13-AAA-B, painted rectangles; Hu4D5 Lec13-AAA-C, painted diamonds.
[83] Detailed Description of the Preferred Embodiments
[84] I. Definition
[85] Throughout this specification and claims, the numbering of residues in an immunoglobulin heavy chain is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public health service, National Institutes of Health, Bethesda, MD (1991)]. "EU index in Kabat" refers to residue numbering of human IgG1 EU antibodies.
[86] Carbohydrate moieties of the present invention will be described according to the nomenclature commonly used for the description of oligosaccharides. A review of carbohydrate chemistry using this nomenclature can be found in Hubbard et al. Ann. Rev. Biochem. 50: 555-583 (1981). This nomenclature is for example Man; GlcNAc representing 2-N-acetylglucosamine; Gal representing galactose; Fuc representing fucose; And Glc, which represents glucose. Sialic acid is described by the abbreviation symbol NeuNAc for 5-N-acetylneuraminic acid, NeuNGc for 5-glycolylneuraminic acid.
[87] The term "glycosylation" refers to the attachment of oligosaccharides (carbohydrates containing two or more simple sugars linked together, such as from 2 to about 12 simple sugars linked together) to a glycoprotein. Oligosaccharide side chains are typically linked to the backbone of the glycoprotein via an N- or O-linking group. Oligosaccharides of the invention are generally attached to the CH2 domain of the Fc region like N-linked oligosaccharides.
[88] "N-linked glycosylation" means the attachment of a carbohydrate moiety to an asparagine residue in a glycoprotein chain. One skilled in the art recognizes, for example, that each of the murine IgG1, IgG2a, IgG2b and IgG3 and human IgG1, IgG2, IgG3, IgG4, IgA and IgD CH2 domains has a single site for glycosylation N-linked to amino acid residue 297 Kabat et al. Sequences of Proteins of Immunological Interest, 1991.
[89] A "glycoprotein" is a polypeptide with one or more oligosaccharide side chains attached.
[90] For the purposes of this application, the "mature core carbohydrate structure" is an Fc region generally consisting of the following carbohydrate structure GlcNAc (fucose) -GlcNAc-Man- (Man-GlcNAc) _{2, which} is typical of the biantennary oligosaccharides represented schematically below. Refers to the processed core carbohydrate structure attached to:
[91]
[92] The term specifically includes G-1 forms of core mature carbohydrate structures that lack β1,2 GlcNAc residues. Preferably, however, the core carbohydrate structure comprises both β1,2 GlcNAc residues. The mature core carbohydrate structures herein are generally not over-mannosylated.
[93] The mature core carbohydrate structure is generally attached to the Fc region of the glycoprotein via the N-linking group of the CH2 domain of the Fc region to Asn297.
[94] "Divided GlcNAc" is a GlcNAc residue attached to β1,4 mannose of mature core carbohydrate structure. Biparticulate GlcNAc can be enzymatically attached to the mature core carbohydrate structure by β (1,4) -N-acetylglucoamiminyltransferase III enzyme (GnTIII). CHO cells normally do not express GnTIII (Stanley et al. J. Biol. Chem. 261: 13370-13378 (1984)), but can be engineered to do so (Umana et al. Nature Biotech. 17: 176-180 ( 1999).
[95] Glycoproteins that are “essentially free” of one or more selected sugar groups (eg, bisected GlcNAc, one or more galactose residues, or one or more sialic acid residues) generally have about 90-100% glycoproteins in the composition as mature core carbohydrates. It is produced in host cells that are defective in attachment of the selected sugar group to the mature core carbohydrate structure such that there is no selected sugar group attached to the structure.
[96] "Glycosidase" is an enzyme involved in the biosynthesis of asparagine-linked (N-linked) glycoproteins. "Trimming" enzymes remove oligosaccharides, while "transferase" adds oligosaccharides. Examples of glycosidase include trimming glycosidase such as glucosidase I and glucosidase II; Trimming mannosidases such as rough endoplasmic reticulum mannosidase (rER mannosidase), mannosidase IA, mannosidase IB, and mannosidase II; And glycosyl transferases, for example, transferases such as β (1,4) -N-acetylglucoaminominyltransferase III (GnT III), Gal-transferase, sialic acid-transferase and fuc-transferase It includes.
[97] "Glycosidase inhibitor" means a compound or composition that reduces or prevents N-linked oligosaccharides processed by one or more glycosidase. Examples include nozirimycin, 1-deoxynojirimycin (dNM), N-methyl-1-deoxy-nojirimycin (N-dNM), castanospermine, bromoconduritol, 1-deoxymanno Zirimmycin (dMM), australine, MDL, lentiginosine, and swanissonin (Sw). Glycosidase inhibitors are described by Fuhrmann et al. Biochim. Biophys. Acta 825: 95-110 (1985); Kaushal and Elbein, Methods in Enzym. 230: 316-329 (1994) and Elbein, A. FASEB 5: 3055-3063 (1991).
[98] "Lec13" refers to a lectin-resistant Chinese hamster ovary (CHO) mutant cell line that exhibits defective fucose metabolism and thus has reduced ability to add fucose to complex carbohydrates. Cell lines are described in Ripka and Stanley, Somatic Cell & Molec. Gen. 12 (1): 51-62 (1986); And Ripka et al. Arch. Biochem. Biophys. 249 (2): 533-545 (1986) and available at Albert Einstein College of Medicine of Yeshiva University, Bronx, New York. Lec13 cells are thought to lack a transcript for GDP-D-mannose-4,6-dehydratase, a major enzyme for fucose metabolism. Ohyama et al. J. Biol. Chem. 273 (23): 14582-14587 (1988). GDP-D-mannose-4,6-dehydratase produces GDP-mannose-4-keto-6-D-deoxymannose from GDP-mannose, which is converted to GDP-L-fucose by the FX protein . The expression of the fucosylated oligosaccharides depends on the GDP-L-fucose donor substrate and the fucosyltransferase.
[99] A "fucosyltransferase" is an enzyme that adds one or more fucose to a glycoprotein. Examples include α1,6-fucosyltransferase, FucTI, FucTII, FucTIII, FucTIV, FucTV, FucTVI, and FucTVII. Porcine and human α1,6-fucosyltransferases are described in Uozumi et al. J. Biol. Chem. 271: 27810-27817 (1996) and Yanagidani et al. J. Biochem. 121: 626-632 (1997), respectively.
[100] "Sialyltransferase" is an enzyme that adds one or more sialic acid residues to a glycoprotein. α, 3 sialyltransferase can add sialic acid residues to galactose residues attached to mature core carbohydrate structures.
[101] "Galactotransferase" is an enzyme that adds one or more galactose residues to a glycoprotein. β1,4-galactosyltransferase may add galactose residues to the mature core carbohydrate structure.
[102] The term "Fc region-containing glycoprotein" means a glycoprotein such as an antibody or immunoadhesin comprising an Fc region.
[103] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, for example as shown in Figure 1A. An "Fc region" may be a native sequence Fc region or a variant Fc region. While the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined as affecting its carboxyl terminus from an amino acid residue at the Cys226 position, or from Pro230. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3, for example as shown in FIG. 1A.
[104] The "functional Fc region" has the "functional function" of the native sequence Fc region. Exemplary “agonist functions” include C1q binding; Complement dependent cytotoxicity; Fc receptor binding; Antibody-dependent cell-mediated cytotoxicity (ADCC); Phagocytosis; Down regulation of cell surface receptors (eg B cell receptor; BCR), and the like. Such agent function generally requires an Fc region associated with a binding domain (eg, an antibody variable domain), and can be assessed using, for example, various assays disclosed herein.
[105] "Native sequence Fc region" includes an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions are shown in FIG. 23, and the native sequence human IgG1 Fc regions (non-A and A allotypes); Native sequence human IgG2 Fc region; Native sequence human IgG3 Fc region; And native sequence human IgG4 Fc regions and naturally occurring variants thereof. The native sequence murine Fc region is shown in FIG. 23. Other examples of native sequence Fc regions include native sequence human IgA Fc regions and native sequence human IgD Fc regions.
[106] A “variant Fc region” includes an amino acid sequence that is different from the native sequence Fc region by one or more “amino acid modifications” as defined herein. Preferably, the variant Fc region is one or more amino acid substitutions, eg, about 1 to about 10 amino acid substitutions, preferably in the native sequence Fc region or the Fc region of the parent polypeptide as compared to the native sequence Fc region or the Fc region of the parent polypeptide. Preferably from about 1 to about 5 amino acid substitutions. The variant Fc regions herein are preferably at least about 80% homologous, most preferably at least about 90% homologous, more preferably at least about 95% at least a native sequence Fc region and / or an Fc region of the parent polypeptide Will have the same sex.
[107] "Homology" is defined as the percentage of identical residues in amino acid sequence variants after aligning the sequences and introducing gaps if necessary to obtain maximum percent homology. Methods and computer programs for alignment are known in the art. One such computer program is "Align 2" authored by Genentech, Inc., which was published in the U.S. Copyright Office Washington, DC 20559, December 10, 1991. Was submitted with.
[108] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. Preferred FcRs are native sequence human FcRs. Preferred FcRs also bind to IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibiting receptor”) having similar amino acid sequences that differ mainly in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see M. in Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). The Fc receptors herein are the two known, naturally occurring allotypes of human FcγRII, FcγRII (H131) and FcγRII (R131) (Clark et al. J. Immunol. 143: 1731-1734, determined by amino acid at position 131). (1989)), and naturally occurring allotypes of human FcγRIIIA. Human FcγRIIIA has naturally occurring allotypes at position 48 (Leu, His or Arg) and position 158 (Val or Phe). The FcγRIIIA (V158) allotype interacts better with human IgG than the FcγRIIIA (F158) allotype (Shields et al. J. Biol. Chem. 276: 6591-6604 (2001); Koene et al. Blood 90: 1109 -1114 (1997) and Wu et al. J. Clin. Invest. 100: 1059-1070 (1997)). FcRs are described 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). Other FcRs, including those to be identified in the future, are included in the term "FcR" herein. The term also includes neonatal receptor FcRn, which serves to deliver maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)).
[109] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to antibodies in which nonspecific cytotoxic cells expressing FcR (eg, natural killer (NK) cells, neutrophils and macrophages) are bound to target cells. The results refer to cell-mediated responses that cause lysis of target cells. The major cells for mediating ADCC, NK cells express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression in hematopoietic cells is described by Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991), page 464, Table 3.
[110] A "human agent cell" is a white blood cell that expresses one or more FcRs and performs an agent function. Preferably, these cells express one or more FcγRIII and perform ADCC agonist functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils, with PBMC and NK cells being preferred. Agonist cells can be isolated from natural sources such as blood or PBMCs.
[111] "Hinge region" is generally defined as affecting Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by positioning the first and last cysteine residues that form S-S bonds in the heavy chain at the same location.
[112] The "lower hinge region" of the Fc region is usually defined as the range of C-terminal residues immediately adjacent to the hinge region, ie 233 to 239 residues of the Fc region.
[113] The "CH2 domain" of the present invention is generally used herein to describe a CH2 domain having an attachment site to one or more N-linked oligosaccharides in Asn297. It is a feature of the glycoproteins of the invention that they contain or have been modified to contain one or more CH2 domains with N-linked oligosaccharides of the human IgG CH2 domain. The CH2 domain is preferably the CHγ2 domain of human IgG1. The human IgG CH2 domain is usually spanned from about 231 amino acids to about 340 amino acids in the Fc region, as expressed using the EU index for numbering of residues in an immunoglobulin heavy chain.
[114] “CH3 domain” encompasses the range of C-terminal residues for the CH2 domain in the Fc region (ie, amino acid residues about 341 to amino acid residues about 447 of IgG).
[115] The terms "amino acids" and "amino acids" refer to all naturally occurring alpha amino acids and analogs and derivatives thereof in both the D and L stereoisomeric forms. Analogs are usually defined as substituting atoms of amino acids with different atoms having similar properties. Derivatives are defined as amino acids with another molecule or atom attached to them. Derivatives will include, for example, acetylation of amino groups, amination of carboxyl groups, or oxidation of sulfur residues of two cysteine molecules to form cysteine.
[116] As used herein, "polypeptide" generally refers to peptides and proteins having more than about 10 amino acids. Polypeptides may be homologous to the host cell in which they are expressed, or may preferably be exogenous to the host cell to be used, such as chimeric, humanized or human antibodies produced by CHO cells, meaning that they are heterologous, ie foreign. have.
[117] The term “antibody” is used in its broadest sense and specifically refers to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and desired One antibody fragment that exhibits biological activity is included.
[118] “Antibody fragments”, defined for the purposes of the present invention, generally comprise a portion of a complete antibody, including the Fc region of an antibody or the antigen binding or variable region of a complete antibody. Examples of antibody fragments include straight antibodies; Single chain antibody molecules; And multispecific antibodies formed from antibody fragments.
[119] As used herein, the term "monoclonal antibody" refers to an antibody obtained from the same population of antibodies, except for possible naturally occurring mutations in which each antibody in the population may be present in small amounts. Monoclonal antibodies are very specific and are directed against a single antigenic site. Further, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies against different epitopes (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. The modifier "monoclonal" indicates the characteristics of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by a particular method. For example, monoclonal antibodies used in accordance with the present invention are prepared by the hybridoma method first described in Kohler et al., Nature 256: 495 (1975), or by recombinant DNA methods (e.g., US patents, 4,816,567). "Monoclonal antibodies" are described, for example, in Clackson et al., Nature 352: 624-628 (1991) and in Marks et al., J. Mol. Biol. 222: 581-597 (1991) may also be isolated from phage antibody libraries.
[120] Monoclonal antibodies herein specifically comprise a portion of the heavy and / or light chains that are identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the rest of the chain in another species. “Chimeric” antibodies (immunoglobulins) that are identical or homologous to corresponding sequences in antibodies that are derived or belong to other antibody classes or subclasses, and fragments of such antibodies as long as they exhibit the desired biological activity (US Pat. No. 4,816,567; And Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)).
[121] A “humanized” form of a non-human (eg murine) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are derived from residues from hypervariable regions of non-human species (donor antibodies), such as mice, rats, rabbits or non-human primates, where residues from the recipient's hypervariable regions have the desired specificity, affinity and ability. Replaced human immunoglobulin (receptor antibody). In some cases, Fv framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies have one or more, and typically two or more, in which all or substantially all hypervariable loops correspond to hypervariable loops of non-human immunoglobulins and all or substantially all FR regions are FR regions of human immunoglobulin sequences. Substantially all of the two variable domains. Humanized antibodies will also optionally include at least a portion of an immunoglobulin constant region (Fc), typically a constant region of human immunoglobulin. For further 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).
[122] “Human antibodies” have been prepared using techniques for making human antibodies disclosed herein and having amino acid sequences corresponding to the amino acid sequences of antibodies produced by humans. This definition of human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art. In one embodiment, human antibodies are selected from phage libraries that express human antibodies (Vaughan et al. Nature Biotechnology 14: 309-314 (1996): Sheets et al. PNAS (USA) 95: 6157-6162 (1998) ); Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991)). Human antibodies may also be prepared by introducing human immunoglobulin loci into transgenic animals, eg, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon loading, human antibody production is observed, which is closely similar to that observed in humans in all respects, including gene rearrangements, combinations, and antibody repertoires. This approach is described, for example, in US Pat. No. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016 and the following scientific publications: Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995). Alternatively, human antibodies can be prepared through immortalization of human B lymphocytes that produce antibodies to a target antigen, such B lymphocytes can be recovered from an individual or immunized in vitro. See, eg, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1): 86-95 (1991) and US Pat. No. 5,750,373.
[123] The term "hypervariable region" as used herein refers to an amino acid residue of an antibody that is responsible for antigen-binding. The hypervariable region may be referred to as the "complementarity determining region" or "CDR" (ie, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (in the heavy chain variable domain). H1), 50-65 (H2) and 95-102 (H3); Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD. (1991) ]) And / or "hypervariable loops" (ie, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1) in the heavy chain variable domain) , 53-55 (H2) and 96-101 (H3); Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). “Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.
[124] As used herein, the term “immune adhesin” refers to an antibody-like molecule that binds the “binding domain” (eg, receptor, ligand or enzyme) of a heterologous “adhesin” protein with an immunoglobulin constant domain. Structurally, immunoadhesin includes the fusion of an adhesin amino acid sequence (ie, “heterologous”) and an immunoglobulin constant domain sequence that has the desired binding specificity other than the antigen recognition and binding site (antigen binding site) of the antibody.
[125] As used herein, the term “ligand binding domain” refers to a region or derivative thereof that retains at least the qualitative ligand binding capacity of a natural cell-surface receptor or a corresponding natural receptor. In specific embodiments, the receptor is from a cell-surface polypeptide having an extracellular domain that is homologous to a member of an immunoglobulin supergene family. Other receptors that are not members of the immunoglobulin supergin family but specifically encompassed by this definition are those for cytokines and, in particular, receptors with tyrosine kinase activity (receptor tyrosine kinase), hematopoietic and neuronal growth factor receptor superfamily. Members, and cellular adhesin molecules such as (E-, L-, and P-) selectins.
[126] The term “receptor binding domain” refers to a region or derivative of such a natural ligand that retains the natural ligand to the receptor, or at least the qualitative receptor binding capacity of the corresponding natural ligand, including cellular adhesin molecules. This definition includes, among others, binding sequences from ligands to the receptor specifically.
[127] "Antibody-immunoadhesin chimera" includes molecules that bind one or more binding domains of an antibody to one or more immunoadhesin (as defined herein). Exemplary antibody-immune adhesin chimeras are bispecifics described by Berg et al., PNAS (USA) 88: 4723-4727 (1991) and Chaw et al., J. Immunol 153: 4268 (1994). CD4-IgG chimera.
[128] As used herein, the term "preparation" is used to define a glycoprotein or composition identified, separated and / or recovered as a component of its environment. Contaminant components of its environment are substances that interfere with diagnostic or therapeutic uses for glycoproteins or compositions such as unwanted or undesired glycoforms and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. have. The formulations of the present invention are substantially free of such contaminants. In a preferred embodiment, the glycoprotein formulation is (1) more than 95% by weight, most preferably more than 99% by weight of the antibody as measured by the Lowry method, and (2) using a spinning cup sequencer To be sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, or (3) to homogeneity levels by SDS-PAGE in reducing or non-reducing conditions using Coomassie Blue, preferably Silver Stain. will be.
[129] For the purposes of this application, a "pharmaceutical formulation" is one that is suitable and adapted for administration to a mammal, especially a human. Thus, the compositions can be used to treat diseases or disorders of mammals. In addition, the glycoprotein, the active ingredient in the composition, is subject to one or more purification or isolation steps such that contaminants that may interfere with the therapeutic use are separated therefrom. In general, pharmaceutical formulations include therapeutic glycoproteins and pharmaceutically acceptable carriers or diluents, examples of which are described below. The formulations are usually sterile and may be lyophilized.
[130] For the purposes herein, a "parental glycoprotein" is a glycoprotein having the same amino acid sequence and mature core carbohydrate structure as the glycoprotein variant of the invention, except that the fucose is attached to the mature core carbohydrate structure. For example, in a composition comprising maternal glycoproteins, about 50-100% or about 70-100% of the maternal glycoproteins comprise a mature core carbohydrate structure with fucose attached.
[131] Glycoprotein variants that bind to FcRs having “greater affinity” than the parent glycoproteins have substantially greater binding affinity than the parent glycoproteins when the amounts of the glycoprotein variants and the parent polypeptide are essentially the same in the binding assay. One or more of the above identified FcRs. For example, a glycoprotein variant with improved FcR binding affinity may exhibit about 5 to about 1000 times improved FcR binding affinity compared to the parent glycoprotein (eg, about 10 to about 500 times improved). Where FcR binding affinity is determined, for example, as disclosed in the Examples herein).
[132] Glycoprotein variants that "mediate antibody-dependent cell-mediated cytotoxicity (ADCC) glycoprotein variants more effectively in the presence of human effector cells" than the parent polypeptide are essentially dependent on the amount of glycoprotein variants and parent glycoproteins used in the assay. And when identical, it is substantially more effective at mediating ADCC in vitro or in vivo. Generally, such glycoprotein variants will be identified using the in vitro ADCC assays disclosed herein, but other assays or methods for measuring ADCC activity, such as methods in animal models and the like, are also contemplated. Preferred glycoprotein variants are about 1.5 to about 100 times more effective than the parent, for example about 2 to about 50 times more effective than the parent, for example in mediating ADCC in the in vitro assays disclosed herein.
[133] "Amino acid modification" refers to a change in the amino acid sequence of a predetermined amino acid sequence. Exemplary modifications include amino acid substitutions, insertions, and / or deletions. Preferred amino acid modifications herein are substitutions.
[134] Amino acid modification at a particular position, eg, an Fc region, refers to a substitution or deletion of a particular residue, or the insertion of one or more amino acid residues adjacent to a particular residue. Insertion "adjacent" to a particular residue means insertion within one or two residues thereof. Insertion can be N-terminus or C-terminus for a particular residue.
[135] "Amino acid substitution" means the replacement of one or more existing amino acid residues with another different "alternate" amino acid residue in a predetermined amino acid sequence. The replacement residue or residues may be “naturally occurring amino acid residues” (ie, those encoded by the genetic code) and may be selected from the group consisting of the following amino acids: alanine (Ala); Arginine (Arg); Asparagine (Asn); Aspartic acid (Asp); Cysteine (Cys); Glutamine (Gln); Glutamic acid (Glu); Glycine (Gly); Histidine (His); Isoleucine (Ile); Leucine (Leu); Lysine (Lys); Methionine (Met); Phenylalanine (Phe); Proline (Pro); Serine; Threonine (Thr); Tryptophan (Trp); Tyrosine (Tyr); And Val. Preferably, the replacement moiety is not cysteine. Substitution with one or more non-naturally occurring amino acid residues is included within the definition of amino acid substitutions herein. “Non-naturally occurring amino acid residues” refers to residues other than the naturally occurring amino acid residues listed above that can covalently bind adjacent amino acid residue (s) in a polypeptide chain. Examples of non-naturally occurring amino acid residues are norleucine, ornithine, norvaline, homoserine and Ellman et al. Meth. Enzym. 202: 301-336 (1991). To generate such non-naturally occurring amino acid residues, see Noren et al. Science 244: 182 (1989) and Ellman et al., Supra can be used. Briefly, this method involves chemically activating a suppressor tRNA with a non-naturally occurring amino acid residue followed by transcription and translation of the RNA in vitro.
[136] "Amino acid insertion" refers to the incorporation of one or more amino acids into a predetermined amino acid sequence. While the insertion will consist of the insertion of one or two amino acid residues, we contemplate larger “peptide insertions”, eg, from about 3 to about 5 or up to about 10 amino acid residues. The inserted residue (s) may be naturally occurring or non-naturally occurring as described above.
[137] "Amino acid deletion" refers to the removal of one or more amino acid residues in a predetermined amino acid sequence.
[138] "C1q" is a polypeptide comprising a binding site for the Fc region of an immunoglobulin. C1q, along with the two serine proteases C1r and C1s, form complex C1, which is the first component of the complement dependent cytotoxic (CDC) pathway. Human C1q can be purchased, for example, from Quidel, San Diego, Calif.
[139] "Treatment" refers to both therapeutic treatment and protective or preventative measures. Subjects in need of treatment include those already with the disorder as well as those for which the disorder is to be prevented.
[140] "Disorder" or "disease" herein is any disease that benefits from treatment with a glycoprotein. This includes chronic and acute disorders or diseases, including pathological conditions that make mammals prone to the disorder in question. In one embodiment, the disorder is cancer, autoimmune disease, anti-inflammatory disorders, infection or other disease, eg, goiter, in which removal of unwanted tissue or cells is desired. Preferred diseases or disorders to be treated herein are cancer or autoimmune diseases.
[141] The terms "cancer" and "of cancer" refer to or describe the physiological state in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer Bladder cancer, liver tumor, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vaginal cancer, thyroid cancer, liver carcinoma and various types of head and neck cancers.
[142] “B-cell malignancies” herein refers to low grade / follicular NHL, small lymphocyte (SL) NHL, medium grade / vesicular NHL, medium grade diffuse NHL, high grade immunoblast NHL, high grade non-cutting non-Hodgkin's lymphoma (NHL) including small cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS related lymphoma, and Waldenstrom megaglobulinemia; Leukemias including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia and chronic myeloid leukemia; And other hematologic malignancies. Such malignancies can be treated with antibodies to B-cell surface markers such as CD20.
[143] A "hormone independent" cancer is a cancer whose proliferation does not depend on the presence of hormones that bind to receptors expressed by the cells in the cancer. Such cancers do not cause clinical regression upon administration of pharmacological or surgical methods of reducing hormone concentrations in or near the tumor. Examples of hormone-independent cancers include androgen-independent prostate cancer, estrogen-independent breast cancer, endometrial cancer and ovarian cancer. Such cancers can start with hormone dependent tumors and transition from hormone-sensitive stages after anti-hormonal therapy to hormone-resistant tumors.
[144] An “autoimmune disease” herein is a non-malignant disease or disorder that occurs from or against an individual's own tissue. Examples of autoimmune diseases or disorders include inflammatory skin diseases including inflammatory reactions such as psoriasis and dermatitis (eg atopic dermatitis); Systemic sclerosis and sclerosis; Reactions associated with inflammatory bowel disease (eg Crohn's disease and ulcerative colitis); Dyspnea syndrome (including adult respiratory distress syndrome (ARDS)); dermatitis; Meningitis, encephalitis; Uveitis; Colitis; Glomerulonephritis; Allergic conditions such as eczema and asthma and other diseases and chronic inflammatory reactions involving T cell infiltration; Atherosclerosis; Leukocyte adhesion deficiency; Rheumatoid arthritis; Systemic lupus erythematosus (SLE); Diabetes (eg type 1 diabetes or insulin dependent diabetes); Multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Allergic encephalomyelitis; Thorgen syndrome; Childhood onset diabetes; And immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, typically found in tuberculosis, sarcoidosis, polymyositis, granulomatosis, vasculitis; Pernicious anemia (Addison's disease); Diseases associated with leukocyte leakage; Central nervous system (CNS) inflammatory disorders; Multiple organ damage syndrome; Hemolytic anemia (including but not limited to cryoglobulinemia or coombs positive anemia); Myasthenia gravis; Antigen-antibody complex mediated diseases; Anti-glomerular basement membrane disease; Antiphospholipid syndrome; Allergic nephritis; Grave's disease; Lambert-eaton dystonia syndrome; Bullous whey; pemphigus; Autoimmune polyendocrynopathy; Lighter's disease; Muscular stiffness syndrome; Behcet's disease; Giant cell arteritis; Immune complex nephritis; IgA nephropathy; IgM multiple neuropathy; Immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia and the like.
[145] "Inflammatory disorder" refers to a pathological condition in which inflammation occurs, typically due to neutrophil chemotaxis. Examples of such disorders include inflammatory dermatoses including psoriasis and atopic dermatitis; Systemic sclerosis and sclerosis; Reactions associated with inflammatory bowel disease (eg Crohn's disease and ulcerative colitis); Ischemic reperfusion disorders including myocardial ischemic diseases such as surgical tissue reperfusion injury, myocardial infarction, cardiac arrest, cardiac surgery reperfusion and stenosis after percutaneous coronary angioplasty, stroke, and abdominal extrathoracic aortic aneurysm; Secondary cerebral edema after stroke; Cranial trauma; Blood loss shock; choke; Adult respiratory distress syndrome; Acute lung injury; Behcet's disease; Dermatitis; Polymyositis; Multiple sclerosis; dermatitis; meningitis; encephalitis; Uveitis; Osteoarthritis; Lupus nephritis; Autoimmune diseases such as rheumatoid arthritis, Schorgen syndrome, vasculitis; Diseases associated with leukocyte leakage; Central nervous system (CNS) inflammatory disorders, sepsis or secondary multiple organ damage syndrome after trauma; Alcoholic hepatitis; Bacterial pneumonia; Antigen-antibody complex mediated diseases including glomerulonephritis; blood poisoning; Sarcoidosis; Immunopathological response to tissue / organ transplantation; Pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse pancreatitis, irritable pneumonia, idiopathic pulmonary fibrosis (IPF), and inflammation of the lung, including cystic fibrosis. Preferred indications include inflammatory bowel disease such as acute lung injury, adult respiratory distress syndrome, ischemic reperfusion (including traumatic tissue reperfusion injury, myocardial ischemia, and acute myocardial infarction), blood loss shock, asthma, bacterial pneumonia, and ulcerative colitis. Include. Autoimmune diseases can overlap with inflammatory disorders and vice versa.
[146] By “blocking an immune response” to an external antigen is meant reducing or preventing one or more immune-mediated responses resulting from exposure to the external antigen. For example, one can reduce the humoral response to foreign antigens by preventing or reducing the production of antibodies to antigens in mammals. Alternatively or additionally, inhibiting an individual subtype; "Settling" the removal of cells covered with alloantibody; Depletion of antigen-presenting cells may affect alloantigen presentation.
[147] "Exogenous antigen" means a molecule or molecules that are not endogenous or native to a mammal exposed to the antigen. Foreign antigens can elicit immune responses, such as humoral and / or T cell mediated responses in mammals. In general, foreign antigens cause the production of antibodies against them. Examples of foreign antigens contemplated herein include immunogenic therapeutic agents, for example proteins such as antibodies, in particular antibodies comprising non-human amino acid residues (eg rodents, chimeras / humanizations, and primatized). Antibodies); Toxin (optionally conjugated to a target molecule, such as an antibody, and the target molecule may also be immunogenic); Gene therapy viral vectors such as retroviruses and adenoviruses; Grafts; Infectious agents (eg bacteria and viruses); Alloantigen (ie occurs in some members of the same species but not in other members), for example blood type, human leukocyte antigen (HLA), platelet antigen, antigen expressed in transplanted organs, blood components, pregnancy (Rh) and hemophilia factors (eg, Factor VIII and Factor IX).
[148] For the purposes herein, a "tumor-associated antigen" is an antigen characterized by high expression in tumor cells compared to normal cells. Specific examples include ErbB receptors, B-cell surface markers, gangliosides GD2, GD3 and GM2 (Ragupathi G., Cancer Immunol. Immunother. 43: 152 (1996)); CD52 (Ginaldi et al., Leukemia Research 22: 185 (1998)); Prostate hepatocyte antigen (PSCA); And MAGE (Kirkin et al., APMIS 106: 665 (1998)).
[149] "Angiogenesis factor" is herein a molecule that stimulates angiogenesis. Examples include vascular endothelial growth factor (VEGF), basic or acidic fibroblast growth factor (FGF), and platelet-derived endothelial cell growth factor (PD-ECGF).
[150] "ErbB receptor" is a receptor protein tyrosine kinase belonging to the ErbB receptor family and includes the EGFR, ErbB2, ErbB3 and ErbB4 receptors and other members of this family to be identified in the future. ErbB receptors generally comprise an extracellular domain capable of binding an ErbB ligand; Lipophilic transmembrane domains; Conserved intracellular tyrosine kinase domains; And carboxyl-terminal signaling domains containing several tyrosine residues that can be phosphorylated.
[151] The terms "ErbB1", "epidermal growth factor receptor" and "EGFR" are used interchangeably herein and are described, for example, in Carpenter et al. Ann. Rev. Biochem. 56: 881-914 (1987), which refer to its naturally occurring mutant form (e.g., in Humphrey et al. PNAS (USA) 87: 4207-4211 (1990)). Deletion mutants EGFR). erbB1 refers to the gene encoding the EGFR protein product.
[152] The expressions "ErbB2" and "HER2" are used interchangeably herein and are described, for example, in Semba et al., PNAS (USA) 82: 6497-6501 (1985); And Yamamoto et al. Nature 319: 230-234 (1986)] (Genebank Accession No. X03363).
[153] Examples of antibodies that bind to HER2 are 4D5, 7C2, 7F3 and 2C4, and huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5 described in Table 3 of US Pat. No. 5,821,337, which is expressly incorporated by reference herein. Humanized variants thereof, including -5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8; And humanized 2C4 mutant Nos. 560, 561, 562, 568, 569, 570, 571, 574, or 56869 described in WO01 / 00245. 7C2 and 7F3 and humanized variants thereof are described in WO98 / 17797. Preferred antibodies are those comprising the heavy and light chain variable regions of huMAb4D5-8, or humanized 2C4 mutant 574.
[154] "Trastuzumab" (HERCEPTIN®) is a recombinant DNA-derived humanized antibody that binds with high affinity (Kd = 5 nM) to the extracellular domain of HER2 in cell-based assays. This antibody is an IgG1 antibody comprising the heavy and light chain variable regions of variant huMAb4D5-8 as described in Table 3 of US Pat. No. 5,821,337. This antibody is produced by CHO-DP12 cells.
[155] "ErbB3" and "HER3" are described, for example, in US Pat. Nos. 5,183,884 and 5,480,968 and Kraus et al. PNAS (USA) 86: 9193-9197 (1989).
[156] The terms "ErbB4" and "HER4" herein are described, for example, in European Patent Application Nos. 599,274; Plowman et al., Proc. Natl. Acad. Sci. USA, 90: 1746-1750 (1993); And receptor polypeptides as described in Plowman et al., Nature, 366: 473-475 (1993), which include, for example, those disclosed in WO99 / 19488, published April 22, 1999. Isomers are included.
[157] A “B cell surface marker” herein is an antigen expressed on the surface of a B cell that can be targeted with an antibody binding thereto. Exemplary B cell surface markers are CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD40, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82 , CD83, CDw84, CD85 and CD86 leukocyte surface markers. Particularly interesting B cell surface markers are preferentially expressed in B cells as compared to other non-B cell tissues in mammals and can be expressed in both precursor B cells and mature B cells. In one embodiment, the marker is the same as CD20 or CD19 found in B cells throughout the differentiation of the lineage from the hepatocellular state to just before the terminal differentiation into plasma cells. Preferred B cell surface markers herein are CD19, CD20, CD22 and CD40.
[158] The "CD20" antigen is a ˜35 kDa non-glycosylated phosphoprotein found on the surface of B cells from more than 90% peripheral blood or lymphoid organs. CD20 is expressed during early pre-B cell development and remains until plasma cell differentiation. CD20 is present in both normal and malignant B cells. Other names of CD20 in the literature include "B-lymphocyte-limiting antigen" and "Bp35". CD20 antigens are described, for example, in Clark et al. PNAS (USA) 82: 1766 (1985).
[159] Examples of antibodies that bind to the CD20 antigen include, but are not limited to, "C2B8" (US Pat. No. 5,736,137, which is expressly incorporated herein by reference), currently referred to as "rituximab"("RITUXAN®"); Yttrium- [90] -labeled 2B8 murine antibodies referred to as “Y2B8” (US Pat. No. 5,736,137, which is expressly incorporated herein by reference); Optionally labeled with ¹³¹ I ^"131 I-B1" antibody rat IgG2a generating a "B1" (BEXXAR ^TM) (U.S. Patent is incorporated herein expressly by reference No. 5595721 No.); Murine monoclonal antibody “IF5” (Press et al. Blood 69 (2): 584-591 (1987)); “Chimeric 2H7” antibodies (US Pat. No. 5,677,180, which is expressly incorporated herein by reference); And monoclonal antibodies L27, G28-2, 93-1B3, B-C1 or NU-B2 (Valentine et al., In: Leukocyte Typing III (McMichael, Ed.) Available from the International Leukocyte Typing Workshop. , p. 440, Oxford University Press (1987)).
[160] The term "rituximab" or "rituxan®" as used herein refers to a genetically engineered CD20 antigen and is referred to as "C2B8" in US Pat. No. 5,736,137, which is expressly incorporated herein by reference. By monoclonal antibody is meant. The antibody is an IgG ₁ kappa immunoglobulin containing murine light and single chain variable region sequences and human constant region sequences. Rituximab has a binding affinity for the CD20 antigen of about 8.0 nM. Rituximab is produced by CHO DG44 cells.
[161] The term "mammal" includes all animals classified as mammals, including humans, cattle, horses, dogs, and cats. In a preferred embodiment of the invention, the mammal is a human.
[162] "Growth inhibitor", as used herein, refers to a compound or composition that inhibits the growth of cells (eg, cancer cells) in vitro or in vivo. Thus, the growth inhibitory agent may be one that significantly reduces the percentage of cells on S. Examples of growth inhibitory agents include agents that block cell cycle progression (phases other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include vinca (vincristine and vinblastine), taxanes, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Agents that stop at G1 also lead to S-phase stops and include, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechloretamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information is provided in The Molecular Basis of Cancer, Mendelsohn and Israel, eds. (WB Saunders: Philadelphia, 1995), by Murakami et al., Especially on page 13 of Chapter 1 entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs."
[163] An example of a "growth inhibition" antibody is one that inhibits the growth of cells that bind to and express the antigen. Preferred growth inhibitory anti-HER2 antibodies exceed 20%, preferably greater than 50% (eg about 50%) growth of SK-BR-3 breast tumor cells in cell culture at antibody concentrations of about 0.5-30 μg / ml. 50% to about 100%), where growth inhibition was measured 6 days after exposure of SK-BR-3 cells to the antibody (see US Pat. No. 5,677,171, issued October 14,1997). Preferred growth inhibitory antibodies are huMAb4D5-8.
[164] An "induced cell death" antibody is one in which viable cells become non-viable cells. Here the cell expresses the antigen to which the antibody binds. In vitro cell death can be measured in the absence of complement and immune effector cells to distinguish cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Thus, assays for cell death can be performed using heat activated serum (ie in the absence of complement) and in the absence of immune effector cells. To determine if an antibody can induce cell death, either by propidium iodide (PI), trytan blue (see Moore et al. Cytotechnology 17: 1-11 (1995)) or by ingestion of 7AAD The loss of cell membrane fidelity evaluated can be assessed by comparison with untreated cells. Preferred cell-killing inducing antibodies are those that induce PI uptake in PI uptake assays in BT474 cells.
[165] "Apoptotic induction" antibodies are measured by binding of Annexin V, fragmentation of DNA, cell contraction, endoplasmic reticulum expansion, cell fragmentation, and / or formation of membrane vesicles (called an apoptotic body). To induce programmed cell death. The cell expresses the antigen to which the antibody binds. Preferably the cell is a tumor cell. Various methods are available for evaluating the events of cells associated with apoptosis. For example, phosphadidyl serine (PS) translocation can be measured by annexin binding; DNA laddering can be assessed through DNA laddering; Nucleus / chromosomal condensation with DNA fragmentation can be assessed by an increase in hypodiploid cells. Preferably, the antibody that induces apoptosis is about 2 to 50 times, preferably about 5 to 50 times, and most preferably about 10 to 50 times untreated cells in an annexin binding assay using BT474 cells. It induces 50 times of annexin binding.
[166] The term "therapeutically effective amount" means an amount of a drug effective for treating a disease or disorder in a mammal. In cancer, a therapeutically effective amount of drug reduces the number of cancer cells, reduces tumor size, inhibits (ie, slows to some extent and preferably stops) cancer cell infiltration into peripheral organs, and tumor metastasis (Ie, slow to some extent and preferably stop), inhibit tumor growth to some extent, and alleviate to some extent symptoms associated with one or more cancers. To the extent that the drug can prevent existing cancer cell growth and / or kill cancer cells, the drug can be cytostatic and / or cytotoxic. For cancer treatment, efficacy can be measured, for example, by evaluating time to cancer progression (TTP) and / or measuring response rate (RR).
[167] An "antigen-expressing cancer" is intended to include cells having a sufficient level of antigen on the surface of the cell so that the anti-antigen antibody can bind to it and have a therapeutic effect against the cancer.
[168] Cancers "characterized by overactivation" of a receptor are those in which the extent of receptor activation in cancer cells significantly exceeds the level of receptor activation in non-cancer cells of the same tissue type. This excessive activation may be due to overexpression of the receptor and / or beyond the normal levels of ligand available for activating the receptor in cancer cells. Such excessive activation may cause and / or be caused by a malignant state of cancer cells. In some embodiments, a diagnostic or prognostic analysis will be performed on cancer to determine if augmentation and / or overexpression of the receptor causing such overactivation of the receptor is occurring. Alternatively or in addition, a diagnostic or prognostic analysis can be performed on the cancer to determine if augmentation and / or overexpression of the ligand is occurring in the cancer causing the overactivation of the receptor. In this subset of cancers, overactivation of the receptor may be attributed to a self-regulating stimulatory pathway. ***
[169] Cancers that "overexpress" receptors have significantly higher levels of receptors such as HER2 on the cell surface as compared to noncancerous cells of the same tissue type. Such overexpression may be achieved by gene amplification or increased transcription or translation. May be caused. Receptor overexpression can be determined by diagnostic or prognostic analysis by evaluating increased receptor protein present on the cell surface (eg immunohistochemical analysis; IHC). Alternatively or additionally, the level of nucleic acid encoding a receptor in a cell can be determined, for example, by fluorescence in situ hybridization [FISH; WO98 / 45479, Oct. 1998, Southern blotting or polymerase chain reaction (PCR) techniques, such as real time quantitative PCR (RT-PCR), can be measured. Receptor overexpression can also be studied by measuring shed antigen (eg extracellular domain) in biological liquids such as serum [see, for example, US Patent No. 4,933,294 June 12, 1990; WO 91/05264, April 18, 1991; US Patent 5,401,638, March 28, 1995; And Sias et al. J. Immunol. Methods 132: 73-80 (1990). Various in vivo assays are available to those skilled in the art in addition to the above assays. For example, it is possible to expose cells in a patient's body to an antibody that is selectively detectable with a label, for example, a radioisotope, and the binding of the antibody in the patient's cell may, for example, be performed by external scanning or Or by analyzing a biopsy taken from a patient pre-exposed to the antibody.
[170] Tumors overexpressing ligands have significantly higher levels of these ligands compared to non-tumor cells of the same tissue type. Such overexpression can be caused by gene amplification or increased transcription or translation. Overexpression of the ligand assesses the ligand (or nucleic acid encoding it) levels in the patient, for example in tumor biopsies or various diagnostic assays such as IHC, FISH, Southern blotting, PCR or in vivo assays described above. Can be determined diagnostically.
[171] As used herein, the term “cytotoxic agent” refers to a substance that inhibits or inhibits cell function and / or causes cell destruction. The term refers to radioisotopes (eg, radioactive isotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and Lu), chemotherapy agents, toxins, It is intended to include enzymatically active toxins derived from bacteria, fungi, plants or animals, including, for example, small molecule toxins or fragments and variants.
[172] A "chemotherapeutic agent" is a chemical compound useful for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN ^™ ); Alkyl sulfonates such as busulfan, impprosulfan and pifosulfan; Aziridine such as benzodopa, carbocuone, meturedopa and uredopa; Ethyleneimines and methylamelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; Nitrogen mustards such as chlorambucil, chlornaphazine, colophosphamide, esturamustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, normovicin, fenste Lean, prednismustine, trophosphamide, uracil mustard; Nitrosureas such as carmustine, chlorozotocin, potemustine, lomustine, nimustine, rannimustine; Antibiotics, for example alaccinomycin, actinomycin, outramycin, azaserine, bleomycin, cocktinomycin, calicheamicin, carabicin, carminomycin, carcinophylline, chromomycin, dactinomycin , Daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin , Olibomycin, peplomycin, port pyromycin, puromycin, quelamycin, rhorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, ginostatin, zorubicin; Antimetabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogs such as denophtherine, methotrexate, pterophtherin, trimetrexate; Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmorpher, cytarabine, dideoxyuridine, doxyfluridine, enositabine, phloxuridine, 5-FU; Androgens such as calussterone, dromostanolone propionate, epithiostanol, mepitiostane, testosterone; Antiadrenal such as aminoglutetimide, mitotan, trilostane; Folic acid supplements such as proline acid; Aceglaton; Aldophosphamide glycosides; Aminolevulinic acid; Amsacrine; Vestravusyl; Bisantrene; Edatraxate; Depopamine; Demecolsin; Diajikuon; Elponnitine; Elftinium acetate; Etogluside; Gallium nitrate; Hydroxyurea; Lentinane; Rodidamine; Mitoguazone; Mitoxantrone; Fur mall; Nitracrine; Pentostatin; Penammet; Pyrarubicin; Grape filinic acid; 2-ethylhydrazide; Procarbazine; PSK ; Lakamic acid; Sizopyran; Spirogermanium; Tenuazone acid; Triazcuone; 2, 2 ', 2 "-trichlorotriethylamine;urethane;bindesin;dacarbazine;mannosemusin;mitobronitol;mitolactol;fibrobroman;psitocin; arabinoxide (" Ara-C ");Cyclophosphamide;thiotepa; taxanes such as paclitaxel (TAXOL , Bristol-Myers Squibb Onclology, Princeton, NJ) and doxtaxel (TAXOTERE , Rhone-Poulenc Rorer, Antony, France); Chlorambucil; Gemcitabine; 6-thioguanine; Mercaptopurine; Methotrexate; Platinum analogs such as cisplatin and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Navelvin; Novantron; Teniposide; Daunomycin; Aminopterin; Zeloda; Ibandronate; CPT-11; Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoic acid; Esperamicin; Capecitabine; And pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. This definition includes anti-hormonal agents that act to modulate or inhibit hormonal action on tumors, such as tamoxifen, raloxyphene, aromatase inhibitory 4 (5) -imidazole, 4-hydroxytamoxifen, trioxyphene, Antiestrogens such as keoxyphene, LY117018, onapristone and toremiphene; Anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; And pharmaceutically acceptable salts, acids or derivatives thereof.
[173] As used herein, an "EGFR labeled drug" is a therapeutic agent that binds to EGFR and, optionally, inhibits EGFR activation. Examples of such therapeutic agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL8509) [US Patent No. 4,943, 533, Mendelsohn et al . And variants thereof, for example chimeric 225 (C225) and reshaped human 225 (H225) [WO 96/40210, Imclone Systems Inc.]. Reference]; Antibodies that bind type II mutant EGFR. 5,212,290; Humanized and chimeric antibodies that bind EGFR [US Pat. 5,891,996; And human antibodies that bind EGFR [WO98 / 50433, Abgenix, see]. Anti-EGFR antibodies can be conjugated with a cytotoxic agent to produce an immunoconjugate (see eg EP659,439A2, Merck Patent GmbH). Examples of small molecules that bind to EGFR are ZD1839 (IRESSA (Astra Zeneca), CP-358774 or OSI-774 (TARCEVA ^™ ) (Genentech) and AG1478.
[174] "Cytocaine" is a generic term for a protein that acts on another cell as an intercellular mediator, released by one cell population. Examples of such cytokines are lymphokine, monocaine and typical polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; Parathyroid hormone; Thyroxine; insulin; Proinsulin; Relaxin; Prolylacin; Glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and progesterone (LH); Liver growth factor; Fibroblast growth factor; Prolactin; Placental lactogen; Tumor necrosis factor-α and -β; Mullerian inhibitors; Mouse gonadotropin related peptide; Inhibin; Activin; Vascular endothelial growth factor; Integrin; Thrombopoietin (TPO); Nerve growth factors such as NGF-β; Platelet growth factor; Transforming growth factors (TGF) such as TGF-α and TGF-β; Insulin-like growth factor-I and -II; Erythropoietin (EPO); Osteoinductive factor; Interferons such as interferon-α, -β and -γ; colony stimulating factor (CSF) such as macrophage-CSF (M-CSF); Granulocyte-macrophage-CSF (GM-CSF); And granulocyte-CSF (G-CSF); Interleukin (IL), for example IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 11, IL-12; Tumor necrosis factors such as TNF-α or TNF-β; And other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes biologically active equivalents of native sequence cytokines and proteins from natural sources or recombinant cell culture.
[175] As used herein, a “prodrug drug” is a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and is activated in a more active parent form by enzymes. Or a substance that can be converted. See, eg, Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (Ed.), Pp. 247-267, Humana Press (1985). Prodrugs of the invention include phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified precursor drugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally Substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted into more active cytotoxic free drugs But it is not limited thereto. Examples of cytotoxic drugs that can be induced in the form of prodrugs for use in the present invention include, but are not limited to, the chemotherapeutic agents described above.
[176] A “liposome” is a small vesicle composed of various types of lipids, phospholipids, and / or surfactants useful for delivering a drug (eg, the glycoprotein composition described herein and optionally a chemotherapeutic agent) to a mammal. to be. The components of the liposomes are usually arranged in a two-layered structure, similar to the lipid arrangement of a biological membrane.
[177] The term "package insert" refers to instructions normally included in a commercial therapeutic product package that includes information about indications, uses, dosages, administration, contraindications, and / or precautions regarding the use of the therapeutic product.
[178] An “isolated” nucleic acid molecule is a nucleic acid molecule that is typically identified and separated from one or more contaminant nucleic acid molecules that are bound together in a natural source of antibody nucleic acid. Isolated nucleic acid molecules are not in the form or condition found in nature. Thus, isolated nucleic acid molecules are distinguished from nucleic acid molecules present in natural cells. However, isolated nucleic acid molecules typically include nucleic acid molecules contained in cells expressing the antibody, for example, the nucleic acid molecules are present at different chromosomal positions from nucleic acids of natural cells.
[179] The expression "regulatory sequence" refers to a DNA sequence essential for the expression of a coding sequence operably linked in a particular host individual. For example, regulatory sequences suitable for prokaryotes include promoters, optionally operator sequences, and ribosomal binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
[180] Nucleic acids are “operably linked” when placed in a functional relationship with other nucleic acid sequences. For example, the DNA for a presequence or secretion leader is operably linked to the DNA for a polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to the coding sequence when it affects the transcription of the sequence; Ribosome binding sites are operably linked to coding sequences when placed to facilitate translation. In general, “operably linked” means that the DNA sequences to be linked are contiguous, and in the case of a secretory leader, contiguous and present in the reading phase. However, enhancers do not have to be contiguous. Linking is done by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.
[181] The expressions "cell", "cell line" and "cell culture" are used interchangeably herein and all such names include progeny. Thus, the terms “transformer” and “transformed cell” include primary subject cells and cultures derived therefrom regardless of the number of passages. It is also understood that not all progeny may be exactly the same in DNA content due to intentional or accidental mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. If a separate name is intended it will become apparent from the context.
[182] II.Form of practicing the present invention
[183] The present invention relates to a method for producing a substantially equivalent preparation of a glycoprotein comprising an Fc region comprising a mature coa carbohydrate lacking fucose, wherein 80-100% of the glycoprotein is attached to the glycoprotein Fc region in the composition. . In a preferred embodiment herein, the protein is an antibody or immunoadhesin. Glycoproteins include, for example, (a) the use of host cells that are engineered or mutated with reduced (or non-fucosylated) ability to fucose proteins expressed due to lack of fucose metabolism; (b) culturing the cells under conditions that interfere with or reduce fucosylation; (c) post-translational removal of fucose (eg, fucosidase enzyme); (d) post-translational addition of the desired carbohydrate (eg after recombinant expression of aglycosylated glycoprotein); (e) can be prepared by purifying glycoproteins to select non-fucosylated products. The present invention contemplates combining two or more of the above exemplary methods (a) to (e).
[184] Most preferably, the nucleic acid encoding the glycoprotein of interest is expressed in a host cell with reduced (or non-fucosylated) ability to fucosylate the expressed protein. Preferably, the host cell is substantially free of dehydrofolate reductase (DHFR) deficient Chinese hamster Ovari (CHO) cells, such as Lec13 CHO cells or glycoproteins produced by modification of the cells, for example. Unfamiliar CHO-K1, DUX-B11, CHO-DP12 or CHO-DG44 CHO host cells. Thus, the cells may have altered fucosyltransferase enzyme expression or activity, or other enzymes or substrates that add fucose to the N-linked oligosaccharides may have reduced activity and / or reduced levels in the host cell. have.
[185] The use of castanospermine that matures the core carbohydrate structure to inhibit or interfere with progression to inhibitors, for example mature carbohydrates, should generally be avoided. According to one preferred embodiment of the invention, about 80 to 100% of the glycoproteins in the composition recovered from the recombinant host cell producing the glycoprotein will have a core carbohydrate structure lacking fucose attached to the glycoprotein Fc region, Hereinafter referred to as "fucose-free glycoprotein composition". By "recovered" here is meant that the material obtained directly from the host cell culture does not go through a purification step to enhance the fucose-free glycoprotein.
[186] However, the present invention contemplates enhancing the amount of fucose-free glycoproteins with various techniques, for example, tablets using lectin substrates to remove the fucose-containing glycoproteins from the desired composition.
[187] It should be appreciated that the fucose-free glycoprotein amounts from the various batches that recombinantly produce glycoproteins may vary. For example, in the examples below, the total percentage of oligosaccharides without fucose attached to glycoproteins expressed by CHO-Lec13 cells ranged from 88% to 95%.
[188] Preferably about 90-99% of the glycoprotein in the composition comprises a mature core carbohydrate structure lacking fucose attached to the glycoprotein Fc region.
[189] Various types of carbohydrate structures can be present in the composition. For example, the carbohydrate attached to the glycoprotein can be represented by the following formula.
[190]
[191] here,
[192] M is mannose.
[193] GN is GlcNAc.
[194] X ₁ is a GlcNAc residue that is an optional dichotomy, with additional monosaccharides optionally attached to the bipartite GlcNAc.
[195] X ₂ is a preferred GlcNAc residue.
[196] X ₃ is an optional Gal residue, one Gal residue may be attached to each GN arm.
[197] X ₄ is an optional terminal sialic acid residue, with one or two sialic acid residues appended.
[198] Glycoproteins free of fucose exhibit improved binding to one or more FcγRIII receptors as compared to the same glycoprotein composition, although most of the glycoproteins in the composition (eg about 50-100% or about 70-100%). ) Has a fucose attached to a mature core carbohydrate structure (hereinafter "fucose-containing glycoprotein composition"). For example, a glycoprotein composition free of fucose may exhibit a 100-1000 fold improved binding to FcγRIII, such as FcγRIII (F158), as compared to the fucose-containing glycoprotein composition. It is believed that the F158 allotype is less effective at interacting with human IgG than V158, thus providing significant benefits in therapeutic aspects, particularly in patients expressing FcγRIII (F158). Moreover, herein the glycoprotein composition without fucose exhibits better ADCC activity, for example about 2-20 fold improved ADCC activity compared to its relative fucose containing glycoprotein composition.
[199] In addition to the mature coa carbohydrate structure free of fucose, additional oligosaccharides may be attached to the core carbohydrate structure. For example, bipartite GlcNAc may or may not be attached. For example, a GnTIII enzyme may be lacking in a host cell, thereby essentially free of biparticulate GlcNAc in the glycoprotein. Alternatively, glycoproteins can be expressed in host cells (eg Y0 host or engineered CHO cells) to which the bipartite GlcNAc is added. One or more (generally one or two) galactose may also be attached to the core carbohydrate structure. Finally, one or more terminal sialic acid residues (typically one or two) may be attached to and attached to the core carbohydrate structure, for example galactose residues.
[200] The compositions herein are prepared in preferred embodiments and intended for therapeutic use. Accordingly, preferred compositions are pharmaceutical preparations comprising the glycoprotein and the pharmaceutically acceptable carrier or diluent exemplified below. Such preparations are typically sterile and may be lyophilized.
[201] In a preferred embodiment of the invention, the glycoprotein is an antibody and an exemplary method of producing the antibody is described in more detail below. However, the glycoprotein may be any other glycoprotein including an Fc region, such as immune adhesin. Methods of making immune adhesin are described in more detail below.
[202] A. Fc region sequence variants
[203] In one embodiment of the invention, glycoprotein variants also include Fc region variants having an amino acid sequence that is different from the Fc region native sequence. When an Fc region variant has one or more amino acid substitutions, generally but not necessarily, combinations of amino acid substitutions in the same class achieve the desired result. Various classes of amino acid substitutions are described in the table below.
[204] Classes of Fc Region Variants class FcR binding properties Fc region substitution position 1A Reduced binding to all FcγR 238, 265, 269, 270, 297 *, 327, 329 1B Enhanced binding to both FcγRII and FcγRIII 239, 294, 295, 303, 338, 373, 376, 416, 435 2 Enhanced binding to both FcγRII and FcγRIII 256, 290, 312, 326, 330, 339, 378, 430 3 Enhanced binding to FcγRII and no effect on FcγRIII binding 255, 258, 267, 276, 280, 283, 285, 286, 305, 307, 309, 315, 320, 331, 337, 398 4 Enhanced Binding to FcγRII and Reduced Binding to FcγRIII 268, 272, 301, 322, 340 5 Reduced binding to FcγRII and no effect on FcγRIII binding 292, 324, 335, 414, 419, 438, 439 6 Reduced binding to FcγRII and enhanced binding to FcγRIII 298, 333 7 No effect on FcγRII binding and reduced binding to FcγRIII 248, 249, 252, 254, 278, 289, 293, 296, 338, 382, 388, 389, 434, 437 8 No effect on FcγRII binding and enhanced binding to FcγRIII 334, 360
[205] Deglycosylated Version
[206] In addition to amino acid substitutions, the present invention contemplates otherwise modifying the parent Fc region amino acid sequence to produce Fc region variants with altered functional function.
[207] For example, one or more amino acid residues can be deleted in the Fc region to reduce binding to FcRs. Generally, one will delete one or more Fc region variants identified herein as achieving FcR binding to generate such Fc region variants. Generally, no more than one for about 10 Rc region residues will be deleted according to this embodiment of the invention. Wherein the Fc region comprising one or more amino acid deletions will maintain at least about 80%, preferably at least about 90% and most preferably at least about 95% of the parent Fc region of the human Fc region native sequence.
[208] It is also possible to produce Fc region variants with the addition of amino acids, which variants have altered functional function. For example, one or more amino acid residues may be introduced adjacent one (eg, one to two amino acid residues and generally up to 10 residues) to one or more Fc region positions identified as affecting FcR binding. "Near" means within one or two amino acid residues of the Fc region residues identified herein. Such Fc region residues may exhibit elevated or reduced FcR binding and / or ADCC activity. In order to generate such insertion variants, for example, to reasonably design Fc region variants with improved FcR binding capacity, the binding region of the FcR (eg the extracellular domain of the FcR of interest) and the amino acid are inserted Fc region [eg Deisenhofer, Biochemistry 20 (9): 2361-2370 (1981); and Burmeister et al ., Nature 342: 379-383, (1994). The insertion is generally made in the Fc region loop but not in the secondary structure (eg β-strand).
[209] Introducing appropriate amino acid sequence modifications in the parent Fc region to (a) mediate antibody dependent cell mediated cytotoxicity (ADCC) in the presence of human effector cells and / or (b) be more than a parent polypeptide. Good affinity can more effectively produce Fc region variants that bind to the Fc gamma receptor (FcγR). Such Fc region variants will generally comprise one or more amino acid modifications in the Fc region. Combining amino acid modifications is particularly preferred. For example, Fc region variants may include 2, 3, 4, 5, etc. substitutions at specific Fc region positions identified herein.
[210] Preferably the parent polypeptide Fc region is a human Fc region, for example a native sequence human Fc region human IgG1 (A and non-A allotypes), IgG2, IgG3 or IgG4 Fc region. The sequence is shown in FIG.
[211] In order to generate an Fc region with enhanced ADCC activity, the parent polypeptide preferably has ADCC activity already present, for example comprising a human IgG1 or human IgG3 Fc region. In one embodiment, variants with improved ADCC actually mediate ADCC more effectively than antibodies with native sequence IgG1 or IgG3 Fc regions and antigen binding regions of said variants. Preferably, the variant comprises or consists essentially of substituting two or three residues at the Fc regions 298, 333 and 334 positions. Most preferably, residues are substituted (eg, substituted with alanine residues) at positions 298, 333 and 334. Moreover, in order to generate Fc region variants with improved ADCC activity, Fc region variants with improved binding affinity for FcγRIII, which are considered to be important FcRs for mediating ADCC, can generally be engineered. For example, amino acid modifications (eg substitutions) to the parent Fc region at one or more of amino acid positions 256, 290, 298, 312, 326, 330, 333, 334, 360, 378 or 430 to generate the variants. ) Can be introduced. Said variant with improved binding affinity for FcγRIII further has a reduced binding affinity for FcγRII, in particular for affinity FcγRIIB receptors.
[212] Amino acid modifications are preferably introduced into the CH2 domain of the Fc region, as the experiment here indicates that the CH2 domain is important for FcR binding activity. Moreover, in contrast to the teaching of the techniques referenced above, the present specification contemplates introducing a modification to a portion other than the lower hinge region of the Fc region.
[213] Amino acid positions useful in modifications to generate IgG Fc region variants with altered Fc gamma receptor (FcγR) binding affinity or activity are Fc region amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, One or more of 416, 419, 430, 434, 435, 437, 438 or 439. Preferably, the parent Fc region used as a template to generate the variant comprises a human IgG Fc region. When residue 331 is substituted, the parent Fc region is preferably not human native sequence IgG3, or an Fc region variant comprising a substitution at position 331 is preferably an enhanced FcR binding to, for example, FcγRII. Indicates.
[214] Amino acid positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, to generate Fc region variants with reduced binding to FcγR 294, 295, 296, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, Amino acid modifications may be introduced at one or more of 438 or 439.
[215] Variants exhibiting reduced binding to FcγRI have those comprising an Fc region amino acid modification at one or more of amino acid positions 238, 265, 269, 270, 327 or 329.
[216] Variants exhibiting reduced binding to FcγRII include amino acid positions 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419 At least one of 435, 438 or 439.
[217] Fc region variants exhibiting reduced binding to FcγRIII are amino acid positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293, 294, 295, 296, 301, 303 At least one of 322, 327, 329, 338, 340, 373, 376, 382, 388, 389, 416, 434, 435 or 437.
[218] Variants with improved binding to one or more FcγRs can also be made. Such Fc region variants have amino acid positions 255, 256, 258, 267, 268, 272,276, 280, 283, 285, 286, 290, 298, 301, 305, 307, 309, 312, 315, 320, 322, Amino acid modification at one or more of 326, 330, 331, 333, 334, 337, 340, 360, 378, 398, or 430.
[219] For example, variants with improved FcγR binding activity may exhibit improved binding to FcγRIII and optionally further show reduced binding to FcγRII; For example, the variant may comprise amino acid modification at positions 298 and / or 333 of the Fc region.
[220] Variants with enhanced binding to FcγRII include amino acid positions 255, 256, 258, 267, 268, 272, 276, 280, 283, 285, 286, 290, 301, 305, 307, 309, 312, 315, 320 of the Fc region. At least one of 322, 326, 330, 331, 337, 340, 378, 398, or 430. Such variants may further exhibit reduced binding to FcγRIII. For example, an Fc region amino acid modification may be included at one or more of amino acid positions 268, 272, 298, 301, 322 or 340.
[221] While it is desirable to alter the binding to FcγR, Fc region variants with altered binding affinity for neoplastic receptors (FcRn) are also contemplated herein. Fc region variants with improved affinity for FcRn are expected to have longer serum half-lives, and the molecules are useful for methods of treatment of mammals, for example, treating chronic diseases or diseases, in which the administered polypeptide half-life is long. Will apply. Fc region variants with reduced FcRn binding affinity are expected to have shorter half-lives on the contrary, for example where the shortened circulation time is advantageous, for example in vivo diagnostic imaging or If left to circulate in the bloodstream for an extended period of time, it can be administered to mammals for polypeptides having toxic side effects and the like. Fc region variants with reduced FcRn binding affinity are not expected to cross the placenta and thus can be used to treat diseases or conditions in pregnant women. ***
[222] Fc region variants with altered binding affinity for FcRn are amino acid positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340 Containing an Fc region amino acid modification at one or more of 356, 360, 362, 376, 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 or 447 Have Representing reduced binding to FcRn generally results in an Fc region amino acid modification at one or more of amino acid positions 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435, 436, 439 or 447. Including; Enhancing binding to FcRn typically results in amino acid positions 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413 At least one of 424 or 434 will comprise an Fc region amino acid modification.
[223] Polypeptide variants prepared as such can often be further modified depending on the intended use of the polypeptide. Such modifications may involve further alteration of amino acid sequences (substitution, insertion and / or deletion of amino acid residues), fusion to heterologous polypeptides and / or covalent modifications. Such “additional modifications” can be made before, concurrently or after the amino acid modifications described above that result in Fc receptor binding and / or alteration of ADCC activity. In one embodiment, the Fc region modifications herein can be combined with the Fc region substitutions disclosed in the references mentioned in the "Related Art" herein.
[224] Alternatively or additionally, it may be useful to combine the amino acid modification with one or more additional amino acid modifications that alter the C1q binding and / or complement dependent cytotoxic function of the Fc region.
[225] Starting polypeptides of particular interest herein are those that typically bind C1q and exhibit complement dependent cytotoxicity (CDC). Additional amino acid substitutions described herein generally alter the ability of the initiating polypeptide to bind C1q and / or modify its complement dependent cytotoxic function, for example to reduce and preferably eliminate said agent function. . However, polypeptides are contemplated herein which include substitution at one or more of the positions described above having improved C1q binding and / or complement dependent cytotoxicity (CDC). For example, the starting polypeptide may not bind C1q and / or may not mediate CDC and may be modified according to the teachings herein, requiring such additional agent function. Moreover, polypeptides having pre-existing C1q binding activity, optionally further having the ability to mediate CDC, can be modified to enhance one or both of these activities.
[226] To generate an Fc region with altered C1q binding and / or complement dependent cytotoxic (CDC) function, the amino acid positions to be modified are generally selected from heavy chain positions 270, 322, 326, 327, 329, 331, 333 and 334. , Residue numbers in IgG heavy chains are described by Kabat et al., Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). In one embodiment, only one of the eight positions identified above is altered to produce a polypeptide variant region with altered C1q binding and / or complement dependent cytotoxic (CDC) function. Preferably only 279, 329 or 322 residues are altered in such cases. Alternatively, two or more are modified at the identified locations. If substitutions are combined, substitutions that generally enhance human C1q binding (eg residue positions 326, 327, 333 and 334) or substitutions that reduce human C1q binding (eg residue positions 270, 322, 329 and 331) ) Is combined. In the latter embodiment, all four positions (eg 270, 322, 329 and 331) may be substituted. Preferably, further substitutions at two, three or all positions at positions 326, 327, 333 or 334 are combined to optionally enhance human C1q binding and preferably enhanced ex vivo or with other Fc region substitutions. To produce polypeptides having CDC activity in vivo.
[227] Proline is conserved at human IgG position 329. The residue is preferably replaced with alanine, but it is contemplated that all other amino acids, such as serine, threonine, asparagine, glycine or valine, are substituted.
[228] Proline is conserved at human IgG1, IgG2 and IgG3 positions 331 but not at IgG4 (having serine residues at positions 331). Residue 331 is preferably replaced with alanine or another amino acid such as serine (for IgG regions other than IgG4), glycine or valine.
[229] Lysine 322 is conserved in human IgG, and this residue is preferably replaced by an alanine residue, but substitution with all other amino acid residues such as serine, threonine, glycine or valine is contemplated.
[230] D270 is conserved in human IgG and the residue may be replaced with other amino acid residues such as alanine, serine, threonine, glycine, valine or lysine.
[231] K326 is also conserved in human IgG. The residue may be substituted with, but not limited to, residues, valine, glutamic acid, alanine, glycine, aspartic acid, methionine or other residues, preferably tryptophan.
[232] Similarly, E333 is also conserved in human IgG. E333 is preferably replaced with an amino acid residue having a lower side chain volume, for example valine, glycine, alanine or serine, preferably serine.
[233] K334 is conserved in human IgG and can be substituted with other residues such as alanine or other residues.
[234] In human IgG1 and IgG3, residue 327 is alanine. In order to generate variants with enhanced C1q binding, the alanine may be substituted with other residues such as glycine. In IgG2 and IgG4, residue 327 is glycine and can be replaced with alanine (or other residues) to reduce C1q binding.
[235] As noted above, for example, an Fc region with altered agonist function can be designed by modifying C1q binding and / or FcR binding and thus altering CDC activity and / or ADCC activity. For example, one can generate Fc region variants with enhanced C1q binding and enhanced FcγRIII binding, for example with both enhanced ADCC activity and enhanced CDC activity. Alternatively, Fc region variants with reduced CDC activity and / or reduced ADCC activity can be engineered when agonist function is reduced or eliminated. In other embodiments, only one of the above activities can be increased and optionally also reduced the activity of the other, resulting in, for example, Fc region variants with enhanced ADCC activity but with reduced CDC activity and vice versa.
[236] For further amino acid sequence alterations, all cysteine residues that are not involved in maintaining the proper morphology of the polypeptide variant may also be substituted, generally with serine, thereby improving the oxidative stability of the molecule and preventing abnormal crosslinking. prevent.
[237] Other types of amino acid substitutions serve to alter the glycosylation mode of the polypeptide. This can be accomplished by deleting one or more hydrocarbon moieties found in the polypeptide and / or by adding one or more glycosylation sites that are not present in the polypeptide. Glycosylation of polypeptides is typically either N-linked or O-linked. N-bond indicates that the hydrocarbon moiety is attached to the side chain of the asparagine residue. The tripeptide of asparagine-X-serine and asparagine-X-threonine, where X is all amino acids except proline, is an enzyme recognition sequence with a hydrocarbon moiety attached to the asparagine side chain. Thus, each of these tripeptides in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars of N-acetylgalactosamine, galactose or xylose to hydroxyamino acid, most commonly serine or threonine, 5-hydroxyproline or 5-hydroxy Xylcin may also be used. Adding a glycosylation site to a polypeptide is conveniently accomplished by altering the amino acid sequence such that the amino acid contains one or more of the above-described tripeptide sequences (relative to the N-linked glycosylation site). It can also be altered by adding or substituting one or more serine or threonine residues (relative to the O-linked glycosylation site) to the original polypeptide sequence. Exemplary glycosylation variants have amino acid substitutions at the heavy chain Asn 297 residue.
[238] Moreover, one or more additional amino acid substitutions can be made to alter the class, subclass or allotype of the Fc region by generating an Fc region having an amino acid sequence that is more homologous to the different class, subclass or allotype of interest. For example, the murine Fc region can be altered to produce an amino acid sequence more homologous to the human Fc region, the human non-A allotype IgG1 Fc region can be modified to achieve a human A allotype IgG1 Fc region, and so forth. . In one embodiment, amino acid modifications herein that alter FcR binding and / or ADCC activity are made in the CH2 domain of the Fc region and the CH3 domain is deleted or replaced with another dimerization domain. Preferably, however, the CH3 domain is maintained (in addition to amino acid modifications that alter the function of the agents disclosed herein).
[239] Glycoproteins prepared as described above can often be further modified depending on the intended use of the glycoprotein. Such modifications may involve further amino acid sequence alterations (substitution, insertion and / or deletion of amino acid residues), fusion to heterologous polypeptides and / or covalent modifications.
[240] Another type of amanoic acid substitution serves to alter the glycosylation mode of glycoproteins. The glycosylation mutation may be in addition to the glycosylation variation for the fucose deficiency described herein and delete one or more carbohydrate moieties found in the glycoprotein, and / or one or more glycosylation sites not present in the glycoprotein. Can be achieved by adding Glycosylation of glycoproteins is typically either N-linked or O-linked. N-bond indicates that the hydrocarbon moiety is attached to the side chain of the asparagine residue. The tripeptide sequence of asparagine-X-serine and asparagine-X-threonine, where X is all amino acids except proline, is an enzyme recognition sequence with a hydrocarbon moiety attached to the asparagine side chain. Thus, each of the tripeptides in the glycoprotein is present, creating a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars of N-acetylgalactosamine, galactose or xylose to hydroxyamino acid, most commonly serine or threonine, 5-hydroxyproline or 5-hydroxy Xylcin may also be used. Adding a glycosylation site to a glycoprotein is conveniently accomplished by altering the amino acid sequence such that the amino acid contains one or more of the above-described tripeptide sequences (relative to the N-linked glycosylation site). Alterations can also be made by adding or substituting one or more serine or threonine residues to the original glycoprotein sequence (for O-linked glycosylation sites).
[241] B. Biological Activity Screening
[242] One or more assays can be performed to assess all changes in biological activity by comparing glycoprotein variants to the starting polypeptide.
[243] Preferably the glycoprotein variant maintains essentially the ability to bind antigen as compared to the non-variant polypeptide, eg, the binding performance is no less than about 20 times worse than the non- variant polypeptide, for example about 5 times worse. Not. The binding performance of polypeptide variants can be determined using, for example, fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA) techniques.
[244] The ability of glycoprotein variants to bind FcR can be assessed. When the FcR is a high affinity Fc receptor, e.g., FcγRI, FcRn, FcγRIIB or FcγRIIIA, the glycoprotein variant bound using an antigen that appropriates the monomeric glycoprotein variant in the reference ELISA format and specifically binds to the glycoprotein variant. Can be measured to determine binding (see Examples below). Other FcR binding assays for low affinity FcRs are described in WO00 / 42072 (Presta) and US Pat. 6,242,195B1.
[245] In order to analyze the ADCC activity of glycoprotein variants, ex vivo ADCC assays can be performed using a variety of agent: target ratios. Useful "effector cells" for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the protein glycoprotein variants is described in vivo, for example in Clynes et al. PNAS (USA) 95: 652-656 (1998).
[246] The ability of the variant to bind C1q and mediate complement dependent cytotoxicity (CDC) can be analyzed.
[247] To determine C1q binding, C1q binding ELISA can be performed. Briefly, assay plates can be coated overnight at 4 ° C. with glycoprotein variants or starting polypeptides (control) in coating buffer. The plate can then be washed and blocked. After washing, human C1q aliquots can be added to each well and incubated for 2 hours at room temperature. After further washing, the anti-C1q complement antibody conjugated with the peroxidase obtained in the sheep can be added to each well and incubated for 1 hour at room temperature. The plate can again be washed with 100 μl of substrate buffer containing wash buffer and OPD (O-phenylenediamine dihydrochloride (Sigma)). The oxidation reaction observed with yellow expression can be allowed to proceed for 30 minutes and stopped by addition of 100 μl of 4.5 NH ₂ SO ₄ . The absorbance can then be read at (492-405) nm.
[248] Exemplary monosaccharide variants show "significant reduction in C1q binding" in this assay. This indicates that about 100 μg / ml of the monoglycoprotein variant is reduced by at least about 50-fold in C1q binding compared to 100 μg / ml of the control antibody with non-mutant IgG1 Fc region. In the most preferred embodiment, the monosaccharide variants do not "bind to C1q", for example, about 100 μg / ml of monosaccharide variants show about a 100-fold reduction in C1q binding compared to 100 μg / ml of the control antibody. .
[249] Another exemplary variant is "having better binding affinity for human C1q than the parent polypeptide". Such molecules exhibit an improvement of at least about 2 times, preferably at least about 5 times, the human C1q binding compared to, for example, the parent polypeptide (eg IC ₅₀ values for both molecules). For example, human C1q binding is about 2 to 500 folds, preferably about 2 or about 5 to about 1000 fold improvement over the parent polypeptide.
[250] To analyze complement activity, complement dependent cytotoxicity (CDC) assays are described, for example, in Gazzano-Santoro et al ., J. Immunol. Methods 202: 163 (1996). Briefly, various concentrations of glycoprotein variants and human complement can be diluted in buffer. Cells expressing the antigen to which the glycoprotein variant binds can be diluted to ˜1 × 10 ⁶ cells / ml. A mixture of glycoprotein variants, diluted human complement and antigen expressing cells is added to a 96-well plate with flat bottomed tissue culture and incubated at 37 ° C. and 5% CO ₂ for 2 hours to facilitate complement mediated cell lysis. Can be. Thereafter, 50 μl of alamar blue (Accumed International) can be added to each well and incubated overnight at 37 ° C. Absorbance is stimulated at 530 nm and radiated at 590 nm and measured using a 96 well fluorometer. The results can be expressed in relative fluorescence units (RFU). Sample concentrations can be calculated from the reference curve and the percentage activity compared to the nonvariant polypeptide is reported for the glycoprotein of interest.
[251] Other exemplary variants also "do not activate complement". For example, 0.6 μg / ml of glycoprotein variant exhibits about 0-10% CDC activity in the assay compared to 0.6 μg / ml of the control antibody with non-mutated IgG1 Fc region. Preferably the variant appears to have no CDC activity in the CDC assay.
[252] Glycoproteins exhibit enhanced CDC compared to the parent polypeptide, for example between about 2 fold and about 100 fold in ex vivo or in vivo CDC activity (eg for each molecule compared). IC ₅₀ figures).
[253] Also contemplated herein are Fc region variants with altered binding affinity for angiogenic receptors (FcRn). Fc region variants with improved affinity for FcRn are expected to have longer serum half-lives, and the molecules are useful for methods of treatment in mammals, for example, in which it is desired that the administered glycoprotein half-life is long. Will be applied. Fc region variants with reduced FcRn binding affinity are expected to have shorter half-lives on the contrary, for example where the shortened circulation time is advantageous, for example in vivo diagnostic imaging or If left to circulate in the bloodstream for an extended period of time, it can be administered to mammals for polypeptides having toxic side effects and the like. Fc region variants with reduced FcRn binding affinity are not expected to cross the placenta and thus can be used to treat diseases or conditions in pregnant women.
[254] C. Antibody Preparation
[255] In a preferred embodiment of the invention, the glycoprotein modified according to the teachings herein is an antibody. Techniques for the production of antibodies are as follows:
[256] (i) Antibody Selection and Preparation
[257] If the glycoprotein is an antibody, it is against the antigen of interest. Preferably, the antigen is a biologically important glycoprotein, and the administration of the antibody to a mammal suffering from a disease or disorder can benefit therapeutically from the mammal. However, antibodies to nonpolypeptide antigens (eg, tumor-associated glycolipid antigens; see US Pat. No. 5,091,178) are also contemplated.
[258] If the antigen is a polypeptide, it may be a ligand such as a transmembrane molecule (eg, a receptor) or a growth factor. Exemplary antigens include molecules such as lenin; Growth hormones including human growth hormone and bovine growth hormone; Growth hormone releasing factor; Parathyroid hormone; Thyroid stimulating hormone; Lipoprotein; Alpha-1-antitrypsin; Insulin A-chain; Insulin B-chain; Proinsulin; Follicle stimulating hormone; Calcitonin; Progesterone; Glucagon; Coagulation factors such as factor VIIIC, factor IX, tissue factor (TF) and von Willebrands factor; Anticoagulant factors such as Protein C; Atrial sodium diuretic factor; Waste surfactants; Plasminogen activators such as urokinase or human urine or tissue plasminogen activators (t-PA); Bombesin; Thrombin; Hematopoietic growth factor; Tumor necrosis factor-alpha and -beta; Enkephalinase; RANTES (normally T-cells express and secrete and are regulated by activation); Human macrophage inflammatory protein (MIP-1-alpha); Serum albumin, eg human serum albumin; Muller-inhibiting component; Relaxin A-chain; Relaxin B-chain; Prolylacin; Mouse gonadotropin-associated peptide; Microbial proteins such as beta-lactam degrading enzymes; DNase; IgE; Cytotoxic T-lymphocyte related antigens (CTLA), for example CTLA-4; Inhibin; Activin; Vascular endothelial growth factor (VEGF); Receptors for hormones or growth factors; Protein A or D; Rheumatoid factor; Neurotropic factors such as bone-derived neurotropic factors (BDNF), neurotrophin-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6), or Nerve growth factors such as NGF-β; Platelet-derived growth factor (PDGF); Fibroblast growth factors such as aFGF and bFGF; Epidermal growth factor (EGF); Transforming growth factors (TGFs) such as TGF-alpha and TGF-beta including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; Tumor necrosis factor (TNF), for example TNF-α or TNF-β; Insulin-like growth factor-I and -II (IGF-I and IGF-II); des (1-3) -IGF-I (brain IGF-I), an insulin-like growth factor binding protein; CD proteins such as CD3, CD4, CD8, CD19, CD20, CD22 and CD40; Erythrocyte hematopoietic factor; Bone formation inducing factor; Immune toxins; Bone morphogenic protein (BMP); Interferons such as interferon-alpha, -beta and -gamma; Colony stimulating factors (CSFs) such as M-CSF, GM-CSF and G-CSF; Interleukins (ILs), for example IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 and IL-10; Superoxide dismutase; T-cell receptor; Surface membrane proteins; Decay promoting factor; Viral antigens, such as part of an AIDS envelope; Carrier protein; Homing receptor; Part of addressing; Regulatory proteins; Integrins such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; Tumor associated antigens such as HER2, HER3 or HER4 receptors; And fragments of any of the polypeptides listed above.
[259] Molecular targets for the antibodies encompassed by the invention include, by way of example, CD proteins such as CD3, CD4, CD8, CD19, CD20, CD22, CD34 and CD40; Members of the family ErbB receptor, for example EGF receptor, HER2, HER3 or HER4 receptor; Prostate stem cell antigen (PSCA); Cell attachment molecules such as αv / β3 integrins (eg, LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, α4 / β7 integrins, and α or β subunits thereof) Anti-CD11a, anti-CD18 or anti-CD11b antibodies); Growth factors such as VEGF; Tissue factor (TF); Tumor necrosis factor (TNF), for example TNF-α or TNF-β, alpha interferon (α-IFN); Interleukins such as IL-8; IgE; Blood type antigens; flk2 / flt3 receptor; Obesity (OB) receptor; mpl receptor; CTLA-4; Protein C and the like.
[260] Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens for antibody production. For transmembrane molecules such as receptors, fragments thereof (eg, extracellular domains of receptors) can be used as immunogens. Alternatively, cells expressing the transmembrane molecule can be used as the immunogen. The cell may be from a natural source (eg, a cancer cell line) or may be a cell transformed with recombinant technology to express a transmembrane molecule. Other antigens and forms thereof useful for preparing antibodies will be apparent to those skilled in the art.
[261] (ii) polyclonal antibodies
[262] Polyclonal antibodies are preferably produced by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the appropriate antigen and adjuvant into the animal. For example, difunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimide esters (linked with cysteine residues), N-hydroxysuccinimides (conjugated with lysine residues), glutaraldehyde, succinic anhydride , SOCl ₂ or R ¹ N═C═NR, where R and R ¹ are each different alkyl groups, proteins that are immunogenic in the species to be immunized with the appropriate antigen, for example keyhole limpet hemocyanin, KLH), serum albumin, bovine thyroid globulin or soybean trypsin inhibitor may be useful.
[263] Animals can combine 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and inject the solution into the dermis to inject the dermis into antigens, Immunized against an immunogenic conjugate or derivative. After one month, the animals are boosted by subcutaneous injection at various sites with 1/5 to 1/10 the original amount of peptide or conjugate in the Freund's complete adjuvant. After 7-14 days, the blood of the animals was drawn and the antibody titer of serum was measured. The animals were boosted until the titer reached plateau. Preferably, the animal is boosted with conjugates bound by the same antigen but with different proteins and / or different crosslinking agents. Conjugates can also be prepared in recombinant cell culture as protein fusions. In addition, flocculants such as alum are suitably used to enhance the immune response.
[264] (iii) monoclonal antibodies
[265] Monoclonal antibodies can be prepared by the hybridoma method first described in Kohler et al. , Nature , 256: 495 (1975), or by synthetic DNA method (US Pat. No. 4,816,567). Can be.
[266] In hybridomas, other suitable host animals, such as hamsters or macaque monkeys, produce or produce antibodies capable of producing antibodies capable of specifically binding to proteins used for immunization. Immunized as described. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cell lines with a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice , pp. 59-103 (Academic Press, 1986)).
[267] The hybridoma cells thus prepared are seeded and cultured in a suitable medium, which medium preferably comprises one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT) enzymes, the culture medium for hybridomas is typically hypoxanthine, aminopterin and thymidine (HAT Medium), and the substances inhibit the growth of HGPRT-deficient cells.
[268] Myeloma cells are preferably those that effectively fuse and sustain stable high concentrations of antibody production by selected antibody-producing cells and are sensitive to media such as HAT media. Among these, preferred myeloma cell lines are derived from MOPC-21 and MPC-11 mouse tumors available from Salk Institute Cell Distribution Center (San Diego, CA, USA), and SP-2 or American type. Rat myeloma cell line, such as X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, MD. Human myeloma and mouse-human heteromyeloma cell lines are also described for the production of human monoclonal antibodies (Kozbor , J. Immunol. , 133: 3001 (1984); and Brodeur et al. , Monoclonal Antibody Production Techniques and Applications , pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
[269] Medium in which hybridoma cells are grown is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is measured by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). do.
[270] After identifying hybridoma cells that produce antibodies with the specific specificity, affinity, and / or activity, the dilution process can be limited to subcloning and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice). , pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be cultured in vivo as ascites tumors of an animal.
[271] Monoclonal antibodies secreted by subclones can be prepared by conventional immunoglobulin purification methods, such as protein A-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography from media, ascites fluid or serum. By appropriate separation.
[272] DNA encoding a monoclonal antibody is readily isolated using conventional methods (e.g., using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody). And sequenced. Hybridoma cells function as a preferred source of the DNA. Once isolated, the DNA is placed in an expression vector, which is then placed into an E. coli cell, monkey COS cell, Chinese hamster ovary (CHO) cell or myeloma cell (if not otherwise producing an antibody protein). By transfecting into host cells, monoclonal antibodies can be synthesized in recombinant host cells. Recombinant production of antibodies is described in more detail below.
[273] In further embodiments, the antibody or antibody fragment can be isolated from an antibody phage library prepared using the techniques described in McCafferty et al., Nature , 348: 552-554 (1990). Clarkson et al. , Nature , 352: 624-628 (1991) and Marks et al ., J. Mol. Biol. , 222: 581-597 (1991) describe the isolation of each murine and human antibody using phage libraries. The following publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology , 10: 779-783 (1992)), and as a method for constructing very large phage libraries. Combination infection and in vivo recombination (Waterhouse et al., Nuc. Acids. Res. , 21: 2265-2266 (1993)). Thus, these techniques are viable alternatives to conventional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.
[274] DNA can also be used, for example, by replacing the coding sequences for human heavy and light chain constant domains with the homologous sequences of mice (US Pat. No. 4,816,567; and Morrison, et al., Proc. Natl Acad. Sci. USA , 81: 6851 (1984)), or by covalently linking an immunoglobulin coding sequence to all or a portion of a non-immunoglobulin polypeptide coding sequence.
[275] Typically, the non-immunoglobulin polypeptide may substitute for the constant domain of the antibody or for the variable domain of one antigen-binding site of the antibody, thereby allowing for one antigen-binding site and another antigen having specificity for the antigen. Chimeric bivalent antibodies can be generated that include other antigen-binding sites with specificity.
[276] (iv) humanized and human antibodies
[277] Humanized antibodies have one or more amino acid residues introduced from a non-human source. Such non-human amino acid residues are often referred to as "import" residues, typically obtained from an "import" variable domain. Humanization is substantially the same as Winter and its colleagues (Jones et al., Nature , 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science , 239: 1534-1536 (1988)), by replacing rodent CDRs or CDR sequences with the corresponding sequences of human antibodies. Thus, such “humanized” antibodies are substantially chimeric antibodies (US Pat. No. 4,816,567) in which fewer sites than the complete human variable domain have been substituted with the corresponding sequences of non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted with residues from analogous positions of rodent antibodies.
[278] The selection of human variable domains of both light and heavy chains used in the preparation of humanized antibodies is of great importance for reducing antigenicity. According to the so-called "best-fit" method, the sequences of rodent antibody variable domains are screened against the entire library of known human variable domain sequences. The human sequence closest to the rodent variable domain is accepted as the human framework (FR) for humanized antibodies (Sims et al., J. Immunol. , 151: 2296 (1993); Chothia et al., J. Mol. Biol. , 196: 901 (1987)). Another method uses a specific framework site derived from the consensus sequence of a particular light or heavy chain subgroup of all human antibodies. The same framework can be used for many different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA , 89: 4285 (1992); Presta et al., J. Immunol., 151: 2623 (1993)).
[279] It is also important to humanize antibodies while maintaining high affinity for antigens and other beneficial biological properties. To achieve this object, according to a preferred method, humanized antibodies are prepared by methods of analyzing parental sequences and various conceptual humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are commercially available and are familiar to those skilled in the art. Computer programs can also be used to depict and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of the display enables analysis of the possible role of residues in the function of candidate immunoglobulin sequences, ie, analysis of residues that affect the ability of a candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined with it and the import sequence from the recipient, so that certain antibody properties, such as an increase in affinity for the target antigen (s), can be achieved. Typically, CDR residues are directly and most substantially related to affecting antigen binding.
[280] Alternatively, transgenic animals (eg mice) can now be produced by immunization capable of producing the entire repertoire of human antibodies without the production of endogenous immunoglobulins. For example, if the antibody heavy chain linkage region (J _H ) gene of chimeric and germline mutant mice is homozygous for deletion, endogenous antibody production is described as completely inhibited. Delivering an array of human germline immunoglobulin genes to said germline mutant mice can result in the production of human antibodies against antigen administration. See, eg, 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); And Duchosal et al. Nature 355: 258 (1992). Human antibodies can also be derived from phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581- 597 (1991); Vaughan et al. Nature Biotech 14: 309 (1996)).
[281] (v) multispecific antibodies
[282] Multispecific antibodies have specificity for two or more different antigens. The molecule will normally only bind two antigens (ie bispecific antibodies, BsAbs), but as used herein the expression includes antibodies with additional specificity, such as trispecific antibodies. As examples of BsAbs, those having one arm against tumor cell antigens and the other arm against cytotoxicity promoting molecules, such as anti-FcγRI / anti-CD15, anti-p185 ^HER2 / FcγRIII (CD16), anti-CD3 Anti-malignant B-cells (1D10), anti-CD3 / anti-p185 ^HER2 , anti-CD3 / anti-p97, anti-CD3 / anti-renal cell carcinoma, anti-CD3 / anti-OVCAR-3, anti- CD3 / L-D1 (anti-colon carcinoma), anti-CD3 / anti-melanin cell promoting hormone analog, anti-EGF receptor / anti-CD3, anti-CD3 / anti-CAMA1, anti-CD3 / anti-CD19, anti -CD3 / MoV18, anti-neuronal cell adhesion molecule (NCAM) / anti-CD3, anti-folate binding protein (FBP) / anti-CD3, anti-pan carcinoma associated antigen (AMOC-31) / anti-CD3 ; BsAbs with one arm that specifically binds a tumor antigen and one arm that binds toxins, for example anti-saporin / anti-Id-1, anti-CD22 / anti-saporin, anti-CD7 / Anti-saporin, anti-CD38 / anti-saporin, anti-CEA / anti-lysine A chain, anti-interferon-α (IFN-α) / anti-hybridoma idiotype, anti-CEA / anti- Vinca alkaloids; BsAbs to convert enzyme-activated prodrugs such as anti-CD30 / anti-alkaline phosphatase (catalyzes the conversion of mitomycin phosphate prodrug to mitomycin alcohol); BsAbs that can be used as fibrin solubilizers, for example anti-fibrin / anti-tissue plasminogen activator (tPA), anti-fibrin / anti-urokinase type plasminogen activator (uPA); BsAbs for targeting immune complexes to cell surface receptors, eg anti-low density lipoprotein (LDL) / anti-Fc receptors (eg FcγRI, FcγRII or FcγRIII); BsAbs for use in the treatment of infectious diseases such as anti-CD3 / anti-simple herpes virus (HSV), anti-T-cell receptor: CD3 complex / anti-influenza, anti-FcγR / anti-HIV; BsAbs for tumor detection in vitro or in vivo, for example anti-CEA / anti-EOTUBE, anti-CEA / anti-DPTA, anti-p185 ^HER2 / anti-hapten; BsAbs as a vaccine adjuvant; And BsAbs, for example anti-rabbit IgG / anti-ferritin, anti-horse radish peroxidase (HRP) / anti-hormone, anti-somatostatin / anti-substance P, anti-HRP / anti-FITC as diagnostic means. Anti-CEA / anti-β-galactosidase. Examples of triple specific antibodies include anti-CD3 / anti-CD4 / anti-CD37, anti-CD3 / anti-CD5 / anti-CD37 and anti-CD3 / anti-CD8 / anti-CD37. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg, F (ab ') ₂ bispecific antibodies). Bispecific antibodies are described in Segal et al. J. Immunol. Methods 248: 1-6 (2001).
[283] Methods of making bispecific antibodies are known in the art. Typical production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Milstein et al., Nature , 305: 537- 539 (1983). Because of the random organization of immunoglobulin heavy and light chains, the hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, only one of which has an accurate bispecific structure. Purification of precise molecules, usually by affinity chromatography steps, is quite cumbersome and the product yield is low. Similar methods are described in WO 93/08829 and in Traunecker et al. , EMBO J., 10: 3655-3659 (1991).
[284] According to another approach, antibody variable domains (antibody-antigen binding sites) with predetermined binding specificities are fused to immunoglobulin constant domain sequences. Preferably, it is fused with an immunoglobulin heavy chain constant domain, C _H 2 and C _H 3 region comprising at least a portion of a hinge. It is preferred to have a first heavy chain constant region (C _H 1) comprising the position necessary for light chain binding, present in one or more fusions. The immunoglobulin heavy chain fusion, and, if necessary, the DNA encoding the immunoglobulin light chain, are inserted into a separate expression vector and coatfected into the appropriate host individual. This provides greater flexibility in controlling the mutual ratios of the three polypeptide fragments in embodiments, when unequal proportions of the three polypeptide chains used in the construction provide optimal yields. However, if high yields are obtained due to the expression of two or more polypeptide chains in the same proportion, or if the ratio is not particularly important, the coding sequences of two or three all polypeptide chains can be inserted into a single expression vector.
[285] In a preferred embodiment of this approach, the bispecific antibody consists of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. . Since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides an easy way of separation, the asymmetric structure has been found to facilitate the separation of certain bispecific compounds from unwanted immunoglobulin chain combinations. This approach is disclosed in WO 94/04690. For further details on the production of bispecific antibodies, see, eg, Suresh et al., Methods in Enymology, 121: 210 (1986). According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the proportion of heterodimers recovered from recombinant cell culture. Preferred interfaces include at least a portion of the C _H 3 domain of the antibody constant domains. In this method, at least one small amino acid side chain at the interface of the first antibody molecule is substituted with a larger side chain (eg tyrosine or tryptophan). Subjective “cavities” of the same or similar size to the large side chains are produced by replacing large amino acid side chains with smaller side chains (eg alanine or threonine) on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other undesired end products such as homodimers.
[286] Bispecific antibodies include crosslinked or “heteroconjugate” antibodies. For example, in heterologous conjugates, one of the antibodies may be coupled with avidin and the other with biotin. Such antibodies have been proposed, for example, for targeting immune system cells to unwanted cells (US Pat. No. 4,676,980), and for the treatment of HIV infection (WO 91/00360, WO 92/20373 and EP 03089). Heteroconjugate antibodies may be prepared using any conventional crosslinking method. Suitable crosslinkers are known in the art and are disclosed in US Pat. No. 4,676,980 with a number of crosslinking techniques.
[287] Antibodies having two or more valences are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60 (1991).
[288] (vi) multivalent antibodies
[289] Multivalent antibodies can be taken up (and / or catabolized) faster than divalent antibodies by cells expressing the antigen to which the antibody binds. Antibodies of the invention may be multivalent antibodies (different from the IgM group) having three or more antigen binding sites (e.g., tetravalent antibodies), which are readily facilitated by recombinant expression of nucleic acids encoding polypeptide chains of the antibody. Can be produced. Multivalent antibodies may comprise a dimerization domain and three or more antigen binding sites. Preferred dimerization domains comprise (or consist of) an Fc moiety or a hinge moiety. In this scenario, the antibody may comprise an Fc region and three or more antigen binding site amino-terminus for the Fc region. Preferred multivalent antibodies herein comprise (or consist of) three to about eight, but preferably four antigen binding sites. Multivalent antibodies comprise one or more polypeptide chains (and preferably two polypeptide chains), wherein the polypeptide chain (s) comprise two or more variable domains. For example, the polypeptide chain (s) may comprise VD1- (X1) _n -VD2- (X2) _n -Fc, where VD1 is the first variable domain, VD2 is the second variable domain, and Fc is One polypeptide chain of the Fc region, X 1 and X 2 are amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain (s) can comprise a VH-CH1-flexible linker-VH-CH1-Fc site chain, or a VH-CH1-VH-CH1-Fc site chain. The multivalent antibody herein preferably further comprises two or more (preferably four) light chain variable domain polypeptides. The multivalent antibodies herein may comprise, for example, about 2 to about 8 light chain variable domain polypeptides. Light chain variable domain polypeptides contemplated herein include light chain variable domains and optionally further comprise a CL domain. Multivalent antibodies are described in WO 01/00238 and WO 00/44788.
[290] (vii) affinity enhanced antibodies
[291] The antibody herein may be an affinity enhanced antibody in which one or more hypervariable site residues of a parent antibody (eg, a humanized or human antibody) are substituted. Typically, the resultant variant (s) selected for further improvement may have improved biological properties compared to the parent antibody from which it was produced. A convenient way to generate such substitutional variants involves affinity maturation using phage display. In short, various hypervariable site positions (eg, positions 6-7) are mutated to produce all possible amino substitutions at each position. The antibody variants thus produced are fusions with the gene III product of M13 packaged within each particle and are displayed in a monovalent fashion from filamentous phage particles. Phage-displayed variants are then screened for their biological activity (eg, binding affinity). Alanine scanning mutagenesis can be performed to identify candidate hypervariable site positions for modification to identify hypervariable site residues that significantly contribute to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the point of contact between the antibody and antigen. Such contact residues and adjacent residues are candidates for substitution according to the techniques described herein. Once the variants are generated, a panel of variants is screened as described herein, and antibodies with good properties in one or more appropriate assays can be selected for further improvement.
[292] (viii) immunoconjugates
[293] The invention also relates to the treatment with an immunoconjugate comprising a glycoprotein conjugated to an anticancer agent, for example a cytotoxic or growth inhibitory agent.
[294] Chemotherapeutic agents useful for the production of such immunoconjugates are described above.
[295] Conjugates of antibodies and one or more small molecule toxins such as calicheamicin, maytansinoids, trichothene and CC1065, and derivatives of these toxins having toxin activity are also contemplated herein do.
[296] In one preferred embodiment, the glycoproteins of the invention are conjugated to one or more maytansinoid molecules.
[297] Glycoprotein-maytansinoid conjugates can be prepared by chemically binding a glycoprotein (eg, an antibody) to a maytansinoid molecule without significantly reducing the biological activity of the glycoprotein or maytansinoid molecule. Can be. Although even one toxin / antibody molecule is expected to enhance cytotoxicity compared to the use of naked antibodies, the conjugation of an average of 3-4 maytansinoid molecules per antibody molecule adversely affects the function or solubility of the antibody. It has been shown to be effective in enhancing the cytotoxicity of target cells without reaching. Maytansinoids are known in the art and can be synthesized or isolated by known techniques from natural sources. Suitable maytansinoids are disclosed, for example, in US Pat. No. 5,208,020. Preferred maytansinoids are maytansinol and maytansinol analogues, eg, various maytansinol esters, modified in different positions or in aromatic rings of maytansinol molecules. For the preparation of antibody-maytansinoid conjugates, see, eg, US Pat. No. 5,208,020 or EP 0 425 235 B1 and Chari et al., Cancer Research 52: 127-131 (1992). There are a number of linking groups known in the art, including those disclosed. The linking groups include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups as disclosed in the aforementioned patents, with disulfide groups and thioether groups being preferred. Conjugates of antibodies and maytansinoids include a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- ( N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), difunctional derivatives of imidoesters (e.g., dimethyl adimidate HCL), active esters (e.g., disuccin Imidyl suverate), aldehydes (eg glutaraldehyde), bis-azido compounds (eg bis (p-amidobenzoyl) hexanediamine), bis-diazonium derivatives, (eg Bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (eg toluene 2,6-diisocyanate) and bis-active fluorine compounds (eg 1,5-difluoro-2, 4-dinitrobenzene). Particularly preferred coupling agents are N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) to provide disulfide bonds (Carlsson et al., Biochem. J. 173: 723-737 ( 1978)) and N-succinimidyl-4- (2-pyridylthio) pentanoate (SPP). The linker may be linked to the maytansinoid molecule at various positions depending on the type of binding. For example, ester bonds can be formed by reaction with hydroxyl groups using conventional coupling techniques. The reaction can take place at the C-3 position with hydroxyl groups, the C-14 position modified with hydroxymethyl, the C-15 position modified with hydroxyl groups and the C-20 position with hydroxyl groups. In a preferred embodiment, the bond is formed at the C-3 position of maytansinol or a maytansinol analogue.
[298] Other immunoconjugates of interest include glycoproteins conjugated to one or more caliciamycin molecules. Antibiotics with Caliciamycin can cleave double stranded DNA at concentrations below picomolar. Conjugation of Caliciamycin with US Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 See American Cyanamid Company. Structural analogs of caliciamycin that can be used are γ ₁ ^I , α ₂ ^I , α ₃ ^I , N-acetyl-γ ₁ ^I , PSAG and θ ^I ₁ (Hinman et al. Cancer Research 53: 3336-3342 (1993 ), Lode et al. Cancer Research 58: 2925-2928 (1998) and the American patents of the above American cyanamides). Another anti-tumor agent to which the glycoprotein can be conjugated is QFA, which is an antifolate. Caliciamycin and QFA both have intracellular site of action and do not readily cross the plasma membrane. Thus, cellular uptake of the drug via antibody mediated uptake significantly enhances its cytotoxic effects.
[299] Other anti-tumor agents that may be conjugated to the glycoproteins of the present invention are collectively described in BCNU, streptozocin, vincristine and 5-fluorouracil, US Pat. Nos. 5,053,394 and 5,770,710, in combination with the LL-E33288 complex. Together with a family of substances known as esperamicin (US Pat. No. 5,877,296).
[300] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotocin A chain (derived from Pseudomonas aeruginosa ), lysine A chain, abrin A chain, modesin A chain, alpha- sarsine , Aleurites fordii protein, diantine protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), Momordica Karan Thia inhibitors, curcin, crotin, sapaonaria opininalis inhibitors, gelonin, mitogeline, restrictocin, phenomycin, enomycin and tricortesense. See, for example, WO 93/21232 published October 28, 1993.
[301] The invention also relates to an immunoconjugate formed between a glycoprotein and a compound having a nucleolytic activity (eg ribonuclease or DNA endonuclease, eg deoxyribonuclease; DNase). .
[302] For selective destruction of the drug, the antibody may contain highly radioactive atoms. Various radioisotopes are available for the preparation of radioconjugated antibodies. Examples include radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu. When the conjugate is used for diagnosis, it is a radio label for scintogram imaging studies, for example tc ^99m or I ¹²³ , or spin labels for nuclear magnetic resonance (NMR) images (also known as magnetic resonance images, mri). Iodine-123 and iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[303] Radioactive or other labels may be introduced into the conjugate in a known manner. For example, peptides may be synthesized by chemical amino acid synthesis using biosynthesized or suitable amino acid precursors (eg, fluorine-19 instead of hydrogen). tc^99mOr I¹²³, Re¹⁸⁶, Re¹⁸⁸And In¹¹¹Labels such as may be bound by cysteine residues in a peptide. Yttrium-90 may be bound by lysine residues. IODOGEN Method (Frakeret al, Biochem. Biophys. Res. Commu.80: 49-57 (1978) may be used to introduce iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.
[304] Conjugates of glycoproteins and cytotoxic agents include a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), difunctional derivatives of imidoesters (e.g. dimethyl adimimidate HCL), active esters (e.g. disuccin Imidyl suverate), aldehydes (eg, glutaraldehyde), bis-azido compounds (eg, bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (eg, bis -(p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (eg toluene 2,6-diisocyanate) and bis-active fluorine compounds (eg 1,5-difluoro-2,4 -Dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a “cleavable linker” that facilitates release of the cytotoxic drug into the cell. For example, acid labile linkers, peptidase-sensitive linkers, light labile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Research 52: 127-131 (1992); US Pat. No. 5,208,020) Can be used.
[305] Alternatively, fusion proteins comprising glycoproteins and cytotoxic agents can be prepared, for example, by recombinant techniques or peptide synthesis. The length of the DNA may include regions encoding each of the two portions of the conjugate that are adjacent to each other or separated into regions encoding linker peptides that do not disrupt the desired properties of the conjugate.
[306] In another embodiment, the antibody is administered to a patient after the antibody-receptor conjugate is administered to remove unbound conjugates from circulation using a scavenger and conjugated to a cytotoxic agent (eg, a radionucleotide). It may be conjugated to a "receptor" (eg, streptavidin) for use in tumor pre-targeting, which is administered "ligand" (eg, avidin).
[307] (ix) antibody dependent enzyme mediated prodrug therapy (ADEPT)
[308] Antibodies of the invention can also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme that converts the prodrug (eg, peptide chemotherapeutic agent, see WO 81/01145) into an active anticancer agent. See, for example, WO 88/07378 and US Pat. No. 4,975,278.
[309] Enzymatic components of immunoconjugates useful for ADEPT include any enzyme that can act in a way to convert the prodrug into a more active cytotoxic form on the prodrug.
[310] Enzymes useful in the methods of the invention include alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; Arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; Cytosine deamino enzymes useful for converting non-toxic 5-fluorocytosine into an anticancer agent, 5-fluorouracil; Proteolytic enzymes useful for converting peptide-containing prodrugs into free drugs, such as serratia proteolytic enzymes, thermolysin, subtilisin, carboxypeptidase, and cathepsin (eg, Cathepsin B and L); D-alanylcarboxypeptidase useful for converting prodrugs containing D-amino acid substituents; Carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; β-lactam degrading enzymes useful for converting drugs derivatized with β-lactams into free drugs; And penicillin amidase, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized with phenoxyacetyl or phenylacetyl groups, respectively, on amine nitrogen to free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes", can be used to convert the prodrugs of the invention to free active drugs (eg, Massey, Nature 328). : 457-458 (1987). Antibody-Abzyme conjugates can be prepared as described herein for delivery of Abzyme to tumor cell populations.
[311] The enzymes of the present invention can be covalently linked to an antibody using techniques known in the art, for example using the heterobifunctional crosslinking agents described above. Alternatively, a fusion protein comprising at least an antigen binding site in an antibody of the invention, linked to one or more functionally active moieties of an enzyme of the invention, may be constructed by recombinant DNA techniques known in the art (eg, See, for example, Neuberger et al., Nature , 312: 604-608 (1984).
[312] (x) other glycoprotein modifications
[313] Other modifications of glycoproteins are also contemplated herein. For example, glycoproteins can be bound to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol. Antibodies can also be used, for example, in microcapsules prepared by coacervation techniques or interfacial polymerization (eg, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively), colloidal drugs It can be trapped in a delivery system (eg liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences , 16th edition, Oslo, A., Ed., (1980).
[314] Glycoproteins disclosed herein may also be formulated as immunoliposomes. A "liposome" is a vesicle composed of various forms of lipids, phospholipids, and / or surfactants useful for delivering a drug to a mammal. The components of the liposomes are typically arranged in the form of a bilayer similar to the lipid arrangement of the biofilm. Liposomes containing antibodies are known in the art, such as in Epstein et al., Proc. Natl. Acad. Sci. USA , 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci . USA, 77: 4030 (1980); U.S. Patents 4,485,045 and 4,544,545; And WO97 / 38731 (October 23, 1997 publication). Liposomes with increased circulation time are described in US Pat. No. 5,013,556.
[315] Particularly useful liposomes can be prepared by reverse phase evaporation using lipid compositions comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes extruded through a filter of defined pore size produce liposomes with the desired diameter. Fab ′ fragments of antibodies of the invention are described by Martin et al. J. Biol. Chem . 257: 286-288 (1982) may be conjugated to liposomes. Chemotherapeutic agents may optionally be contained in liposomes. Gabizon et al. J. National Cancer Inst . 81 (19) 1484 (1989).
[316] (xi) exemplary antibodies
[317] Preferred antibodies within the scope of the invention include those comprising the amino acid sequence of the following antibody:
[318] anti-HER2 antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285-4289 (1992), U.S. Patent No. 5), including antibodies comprising heavy and light chain variable regions of huMAb 4D5-8 5,725,856);
[319] Anti-CD20 antibodies, eg, chimeric anti-CD20 “C2B8” (RITUXAN) such as in US Pat. No. 5,736,137 ), Chimeric or humanized variants of 2H7 antibodies, such as in US Pat. No. 5,721,108, B1 or Tositumomab, BEXXAR );
[320] Anti-IL-8 (St John et al., Chest, 103: 932 (1993) and International Publication WO 95/23865);
[321] Anti-VEGF antibodies, including humanized and / or affinity enhanced anti-VEGF antibodies, eg, humanized anti-VEGF antibodies huA4.6.1 AVASTIN Kim et al., Growth Factors , 7: 53-64 (1992), WO 96/30046, and WO 98/45331 (October 15, 1998);
[322] Anti-PSCA antibodies (WO 01/40309);
[323] Anti-CD40 antibodies (WO 00/75348) comprising S2C6 and humanized variants thereof;
[324] Anti-CD11a (US Pat. No. 5,622,700, WO 98/23761, Steppe et al., Transplant Intl. 4: 3-7 (1991), and Hourmant et al., Transplantation 58: 377-380 (1994));
[325] Anti-IgE (Presta et al., J. Immunol. 151: 2623-2632 (1993) and International Publication WO 95/19181; US Pat. No. 5,714,338 (published Feb. 3, 1998) or US Pat. No. 5,091,313 (1992). 2. 25. published), WO 93/04173 (published March 4, 1993) or WO 99/01556 (published Jan. 14, 1999), US Pat. No. 5,714,338);
[326] Anti-CD18 (as in US Pat. No. 5,622,700 issued April 22, 1997) or WO 97/26912 (published July 31, 1997);
[327] Anti-Apo-2 receptor antibody (WO 98/51793 published Nov. 19, 1998);
[328] cA2 (REMICADE ), Anti-TNF-α antibodies including CDP571 and MAK-195 (US Pat. No. 5,672,347 issued September 30, 1997) Lorenz et al. J. Immunol . 156 (4): 1646-1653 (1996) And Dhainaut et al. Crit.Care Med. 23 (9): 1461-1469 (1995));
[329] Anti-tissue factor (TF) (European Patent 0 420 937 B1 (Patent on Nov. 9, 1994));
[330] Anti-human α ₄ -β ₇ integrin (WO 98/06248 published on Feb. 19, 1998); ***
[331] Anti-EGFR (chimeric or humanized 225 antibody, eg, as described in WO 96/40210 published December 19, 1996);
[332] Anti-CD3 antibodies, such as OKT3 (US Pat. No. 4,515,893, registered on May 7, 1985);
[333] Anti-CD25 or anti-tac antibody, eg, CHI-621 (SIMULECT ) And (ZENAPAX (See US Patent No. 5,693,762, registered December 2, 1997);
[334] Anti-CD4 antibodies, eg, cM-7412 antibody (Choy et al., Arthritis Rheum 39 (1): 52-56 (1996));
[335] Anti-CD52 antibodies such as CAMAPT-1H (Riechmann et al., Nature 332: 323-337 (1998));
[336] Anti-Fc receptor antibodies, eg, M22 antibody against FcγRI (Graziano et al. J. Immunol. 155 (10): 4996-5002 (1995));
[337] Anti-cancer embryo antigen (CEA) antibodies such as hMN-14 from Sharkey et al. Cancer Res. 55 (23 Suppl: 5935s-5945s (1995));
[338] antibodies against breast epithelial cells, including huBrE-3, hu-Mc3 and CHL6 (Cerianietal. Cancer Res. 55 (23): 5852s-5856s (1995); and Richman et al. Cancer Res. 55 (23 Supp): 5916s-5920s (1995));
[339] Antibodies against colon carcinoma cells, eg, C242 (Litton et al. Eur J. Immunol. 26 (1): 1-9 (1996));
[340] Anti-CD38 antibodies, for example AT 13/5 (Ellis et al. J. Immuunol. 155 (2): 925-937 (1995));
[341] Anti-CD33 antibodies such as Hu M195 (Jurcic et al. Cancer Res 55 (23 Suppl): 5908s-5910s (1995)) and CMA-676 or CDP771;
[342] Anti-CD22 antibodies such as LL2 or LymphoCide (Juweid et al. Cancer Res 55 (23 Suppl): 5899s-5907s (1995);
[343] Anti-EpCAM antibodies, eg, 17-1A (PANOREX );
[344] Anti-GpIIb / IIIa antibodies, for example, abciximab or c7E3 Fab (REOPRO );
[345] Anti-RSV antibodies, eg, MEDI-493 (SYNAGIS) );
[346] Anti-CMA antibodies, eg, PROTOVIR ;
[347] Anti-HIV antibodies, eg, PRO542;
[348] Anti-infective antibodies, eg, anti-Hep B antibody OSTAVIR ;
[349] Anti-CA 125 antibody, OvaRex;
[350] Anti-idiotype GD3 epitope antibody BEC2;
[351] Anti-αvβ3 Antibody VITAXIN ;
[352] Anti-human kidney cell carcinoma antibodies such as ch-G250; ING-1;
[353] Anti-human 17-1 antibody (3622W94);
[354] Anti-human colorectal tumor antibody (A33);
[355] Anti-human melanoma antibody R24 against GD3 gangliosides;
[356] Anti-human squamous cell carcinoma (SF-25); And
[357] Anti-human leukocyte antigen (HLA) antibodies such as Smart ID10 and anti-HLA DR antibody Oncolym (Lym-1);
[358] Glycoproteins of interest herein are preferably antibodies, and other Fc regions have been devised that include glycoproteins that can be modified according to the methods described herein. One example of such a molecule is immunoadhesin.
[359] D. Immune Adhesin Preparation
[360] The simplest and most direct immunoadhesin design is to bind the binding domain (s) of an adhesin (eg, the extracellular domain of the receptor (ECD)) with the Fc region of an immunoglobulin heavy chain. Typically, when preparing the immunoadhesin of the present invention, the binding domain of adhesin is fused at the C-terminus to a nucleic acid encoding the N-terminus of the constant domain sequence of the immunoglobulin. However, N-terminal fusions are also possible.
[361] Generally, in such fusions, the encoded chimeric polypeptide has one or more functionally active hinges, C _H 2 and C _H 3 domains of the constant region of the immunoglobulin heavy chain. Fusion may also be made at the C-terminus of the Fc portion of the constant domain, or directly at the N-terminus or light chain of the C _H 1 of the heavy chain. The exact location where the fusion is made is not important; Specific positions are known and can be selected to optimize the biological activity, secretion or binding properties of the immunoadhesin.
[362] In a preferred embodiment, the adhesin sequence is fused to the N-terminus of the Fc region of immunoglobulin G ₁ (IgG ₁ ). It is possible to fuse the entire heavy chain constant region to the adhesin sequence. However, more preferably, the sequence starts at the hinge region immediately preceding the papain cleavage site that chemically determines IgG Fc (ie, residue 216, such that the first residue of the heavy chain constant region is 114) or otherwise. Similar sites of immunoglobulins are used in fusions. In a particularly preferred embodiment, the adhesin amino acid sequence is fused to the (a) hinge region and C _H 2 and C _H 3 or (b) C _H 1, hinge, C _H 2 and C _H 3 domains of the IgG heavy chain.
[363] In the case of bispecific immunoadhesin, the immunoadhesin is assembled into a multimer, in particular into a heterodimer or heterotetramer. In general, these assembled immunoglobulins have a known unit structure. The basic four chain structural units are in the form of IgG, IgD and IgE. Four chain units are repeated in high molecular weight immunoglobulins. IgM generally exists as a pentamer where four basic monomers are assembled into disulfide bonds. IgA globulin and sometimes IgG globulin may also be present in the multimer form in serum. In the case of a multimer, each of the four units may be the same or different.
[364] Various exemplary assembled immunoadhesin that are within the scope of the present application are outlined below:
[365] (a) AC _L -AC _L ;
[366] (b) AC _H- (AC _H , AC _L -AC _H , AC _L -V _H C _H , or V _L C _L -AC _H );
[367] (c) AC _L -AC _H- (AC _L -AC _H , AC _L -V _H C _H , V _L C _L -AC _H , or V _L C _L -V _H C _H );
[368] (d) AC _L -V _H C _H- (AC _H , AC _L -V _H C _H , or V _L C _L -AC _H );
[369] (e) V _L C _L -AC _H- (AC _L -V _H C _H , or V _L C _L -AC _H ); And
[370] (f) (AY) _n- (V _L C _L -V _H C _H ) ₂ ,
[371] Wherein each A represents the same or different adhesin amino acid sequence;
[372] V _L is an immunoglobulin light chain variable domain;
[373] V _H is an immunoglobulin heavy chain variable domain;
[374] C _L is an immunoglobulin light chain constant domain;
[375] C _H is an immunoglobulin heavy chain constant domain;
[376] n is an integer greater than 1;
[377] Y represents the residue of a covalent crosslinker.
[378] For simplicity, the structures show only key features, no conjugation (J) or other domains of immunoglobulins, and no disulfide bonds. However, if such domains require binding activity, they will be found to be among the general positions they occupy among immunoglobulin molecules.
[379] Alternatively, the adhesin sequence is inserted between the immunoglobulin heavy and light chain sequences to obtain an immunoglobulin comprising a chimeric heavy chain. In this embodiment, the adhesin sequence is fused to the 3 'end of the immunoglobulin heavy chain of each arm of the immunoglobulin, between the hinge and the CH ₂ domain or between the C _H 2 and C _H 3 domains. Similar structures are described in Hoogenboom, et al., Mol. Immunol. 28: 1027-1037 (1991).
[380] While the presence of an immunoglobulin light chain is not required for the immunoadhesin of the present invention, the immunoglobulin light chain may be covalently linked to or directly fused to an adhesin-immunoglobulin heavy chain fusion polypeptide. In the former case, the DNA encoding the immunoglobulin light chain is generally co-expressed with the DNA encoding the adhesin-immunoglobulin heavy chain fusion protein. Upon secretion, the hybrid heavy and light chains are covalently linked to produce an immunoglobulin-like structure comprising two disulfide linked immunoglobulin heavy chain-light chain pairs. Suitable methods for the preparation of such structures are disclosed, for example, in US Pat. No. 4,816,567, registered on March 28, 1989.
[381] The cDNA sequence encoding the adhesin moiety is readily prepared by fusion in frame to the immunoadhesin immunoglobulin cDNA sequence. However, fusion to genomic immunoglobin fragments can also be used (eg, Aruffo et al., Cell 61: 1303-1313 (1990); and Stamenkovic et al., Cell 66: 1133-1144 (1991)). The latter type of fusion requires the presence of Ig regulatory sequences for expression. Based on published sequences of cDNA libraries derived from spleen or peripheral blood lymphocytes, hybridization or polymerase chain reaction (PCR) techniques can be used to isolate cDNA encoding IgG heavy chain constant regions. The cDNA encoding “adhesin” and the immunoglobulin portion of immunoadhesin are repeatedly introduced into plasmid vectors that direct effective expression in selected host cells.
[382] E. Vectors, Host Cells and Recombinant Methods
[383] The invention also provides isolated nucleic acids encoding the glycoproteins described herein, vectors and host cells comprising the nucleic acids, and recombinant techniques for the production of glycoproteins.
[384] For recombinant production of glycoproteins, nucleic acids encoding them are isolated and inserted into replicable vectors for further cloning (amplification of DNA) or expression. DNA encoding glycoproteins is readily isolated and sequenced using conventional methods (eg, using oligonucleotide probes that can specifically bind to a gene encoding a glycoprotein). Many vectors are available. Vector components generally include, but are not limited to, one or more of signal sequences, origins of replication, one or more marker genes, enhancer factors, promoters, and transcription termination sequences.
[385] (i) signal sequence components
[386] Glycoproteins of the invention are produced recombinantly as well as directly as fusion polypeptides with heterologous polypeptides. The heterologous polypeptide is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. Selected heterologous signal sequences are preferably recognized and processed by the host cell (ie cleaved by signal peptidase). For prokaryotic host cells that do not recognize and process native polypeptide signal sequences, the signal sequence may be, for example, a prokaryotic signal selected from among alkaline phosphatase, penicillase, lpp, or heat stable enterotoxin II leader groups. Replaced with a sequence. For yeast secretion, the natural signal sequence can be, for example, a yeast invertase leader, α-factor leader (including Saccharomyces and Kluyveromyces α-factor leader) or acid phosphatase Leader, poem. C. albicans glucoamylase leader, or leader described in WO 90/13646. In mammalian cell expression, a mammalian signal sequence can be used as well as a viral secretion leader, such as the herpes simplex gD signal.
[387] DNA for such precursor regions is bound in a reading frame to the DNA encoding the polypeptide.
[388] (ii) origin of replication component
[389] Expression and cloning vectors include nucleic acid sequences that allow the vector to replicate among one or more selected host cells. In general, in cloning vectors, these sequences allow the vector to replicate independently of the host chromosomal DNA and include origins of replication or autonomous replication sequences. Such sequences are known for various bacteria, yeasts and viruses. The origin of replication from plasmid pBR322 is suitable for most Gram-negative bacteria, 2 μplasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) clone vectors in mammalian cells. Useful to do In general, the origin of replication components are not required for mammalian expression vectors (the SV40 origin is generally the only one used because it contains an early promoter).
[390] (iii) selection gene components
[391] Expression and cloning vectors may contain a selection gene, also called a selectable marker. Typical selection genes are (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, (b) supplement nutritional deficiencies, or (c) obtain from complex media. It encodes a protein that supplies a number of important nutrients (eg, genes encoding D-alanine racemase for Bacilli ).
[392] One example of a screening scheme is to use drugs that inhibit the growth of host cells. These cells successfully transformed with heterologous genes produce proteins that confer drug resistance, and thus survive the selection. Examples of such dominant selection use neomycin, mycophenolic acid and hygromycin.
[393] Other examples of suitable selection markers for mammalian cells include polypeptide nucleic acids such as DHFR, thymidine kinase, metallothionein-I and -II, preferably primate metallothionein gene, adenosine deaminase And cells that can accept ornithine decarboxylase and the like.
[394] For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a culture medium containing methotrexide (Mtx), a competitive antagonist of DHFR. When wild type DHFR is used, a suitable host cell is a Chinese hamster ovary (CHO) cell line lacking DHFR activity.
[395] Alternatively, the host cell (especially a wild type host containing endogenous DHFR) is transformed with a DNA sequence encoding a polypeptide of wild type DHFR protein and other selection markers such as aminoglycosidase 3′-phosphate transferase (APH) Or cotransformed can be selected by culturing the cells in a medium containing a selection marker such as an aminoglycoside antibiotic, for example kanamycin, neomycin, or G418. See US Pat. No. 4,965,199.
[396] A suitable selection gene for use in yeast is the trp 1 gene present in yeast plasmid YRp7 (Stinchcomb et al., Nature , 282: 39 (1979)). The trp 1 gene provides a selection marker for yeast mutant strains lacking the ability to grow in tryptophan, eg, ATCC No.44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of trp 1 lesions in yeast host cells provides an effective environment for detecting transformants by growth in the absence of tryptophan. Similarly, Leu 2-deficient yeast strains (ATCC 20,622 or 38,626) can be considered as known plasmids containing the Leu 2 gene.
[397] In addition, a vector derived from 1.6 μm circular plasmid pKD1 can be used for transformation of Kluyveromyces yeast. Alternatively, the expression system for large-scale production of recombinant bovine chymosin K. K. lactis has been reported (Van den Berg, Bio / Technology, 8: 135 (1990)). Suitable multi-copy vectors for the secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed (Fleer et al., Bio / Technology, 9: 968-975 (1991)).
[398] (iv) promoter components
[399] Expression and cloning vectors generally contain a promoter recognized by the host organism, which is operably linked to the polypeptide nucleic acid. Suitable promoters for use in prokaryotic hosts include hybrid promoters such as the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, and tac promoters. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems will also contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the polypeptide.
[400] Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region about 25 to 30 bases upstream from where transcription begins. Another sequence found 70 to 80 bases upstream from the transcription start site of many genes is the CNCAAT region, where N can be any nucleotide. The 3 'end of most eukaryotic genes is an AATAAA sequence that can be a signal for adding a poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.
[401] Examples of promoter sequences suitable for use in yeast hosts include 3-phosphoglycerate kinases or other glycolysis enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate dicar Promoters for carboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutasase-pyruvate kinase, triosphosphate isomerase, phosphoglucose isomerase and glucokinase Include.
[402] Other yeast promoters, which are inducible promoters with the added benefit of controlling transcription by growth conditions, include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3- Promoter region for phosphate dehydrogenase and enzymes responsible for the use of maltose and galactose. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers are also advantageously used with yeast promoters.
[403] Polypeptide transcription from a vector in a mammalian host cell can be, for example, polyoma virus, poultry virus, adenovirus (eg adenovirus 2), bovine papilloma if the following promoters are compatible with the host cell system. Promoters derived from the genome of viruses, avian sarcoma viruses, cytomegaloviruses, retroviruses, hepatitis-B viruses, most preferably monkey virus 40 (SV40), and promoters from heterologous mammalian promoters such as actin Regulated by a promoter derived from a promoter or immunoglobulin promoter and a heat-shock promoter.
[404] Early and late promoters of the SV40 virus are readily available as SV40 restriction enzyme fragments that also contain the origin of replication of the SV40 virus. The earliest expressed promoter of human cytomegalovirus can be conveniently obtained as a HindIII E restriction enzyme fragment. A system for expressing DNA in mammalian hosts using bovine papilloma virus as a vector is disclosed in US Pat. No. 4,419,446. A modification of this system is described in US Pat. No. 4,601,978. See Reyes et al., Nature 297: 598-601 (1982) for the expression of human β-interferon cDNA in mouse cells under thymidine kinase promoter control from herpes simplex virus. Alternatively long terminal repeats of rous sarcoma virus can be used as promoters.
[405] (v) enhancer factor component
[406] Transcription of the DNA encoding the polypeptides of the invention by higher eukaryotes is often increased by inserting enhancer sequences into the vector. Many enhancer sequences are known from mammalian genes (globin, esterase, albumin, α-fetoprotein and insulin). In general, however, enhancers derived from eukaryotic viruses will be used. Examples include the SV40 enhancer (bp 100-270) present on the late side of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer and the adenovirus enhancer present on the late side of the origin of replication. For enhancer factors for activation of eukaryotic promoters, see Yanib, Nature 297: 17-18 (1982). The enhancer can be spliced into the vector at the 5 'or 3' position of the polypeptide coding sequence, but is preferably located at 5 'of the promoter.
[407] (vi) transcription terminal components
[408] In addition, expression vectors used in eukaryotic host cells (nucleated cells from yeasts, fungi, insects, plants, animals, humans, or other multicellular organisms) will contain the sequences necessary for termination of transcription and stabilization of mRNA. Such sequences can be obtained from the 5 'and sometimes 3' untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleic acid segments of transcribed and polyadenylated fragments in the untranslated portion of the mRNA encoding the polypeptide. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vectors described therein.
[409] (vii) Selection and transformation of host cells
[410] Suitable host cells for cloning and expressing DNA in the vectors herein are the prokaryotic, yeast, or higher eukaryotic cells described above. Suitable prokaryotic cells for this purpose are soothing bacteria such as Gram-negative or Gram-positive organisms, for example enterobacteria, eg Escherichia , for example E. coli . Coli (E.coli), Enterobacter (Enterobacter), control Winiah (Erwinia), keulrep when Ella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella), e.g., Salmonella tie pimi Solarium (Salmonella typhimurium), Serra Serratia , for example Serratia marcesacna , and Shigella , as well as Bacilli , for example B. B. subtilis and b. Fort Lee Senigallia miss (B. licheniformis) (for example, 1989. 4. disclosed in DD 266 710, published on the 12 non-Li Senigallia formate miss (B. licheniformis) 41P), for Pseudomonas (Pseudomona), for example, , Blood. To include Rouge labor (P. aeruginosa), and Streptomyces (Streptomyces). One desirable tooth. Coli cloning host Collie 294 (ATCC 31,446), but other strains such as E. coli. Collie B, Lee. Collie X1776 (ATCC 31,537) and E. coli. Collie W3110 (ATCC 27,325) is suitable. These examples are illustrative rather than limiting.
[411] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning and expression vectors for polypeptide-coding vectors. Saccharomyces cerevisiae or the common baker's yeast is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species and strains can be commonly used herein and are useful, examples of which include Schizosaccharomyces pombe ; Kluyveromyces hosts, eg K. K. lactis , K. K. fragilis (ATCC 12,424), K. K. bulgalicus (ATCC 16,045 ), K. K. wickeramii (ATCC 24,178), K. K. waltii (ATCC 56,500); K. drosophilarum (ATCC 36,906), K. thermotolerans and K. marxianus ; Yaroyi O (yarrowia) (EP 402 226 the call); Pichia pastoris (EP 183,070); Candida (Candida); Trichoderma reesia (EP 244,234); Neurospora crassa ; Schawamziomyces , for example Schwanniomyces occidentalis ; And filamentous fungi, eg, Neurospora , Penicillium , Tolypocladium , and Aspergillus hosts, eg, A. A. nidulans and A. nidulans A. niger
[412] Suitable host cells for the expression of glycated polypeptides are derived from multicellular organisms. Examples of invertebrate cells are plant and insect cells. Many baculovirus strains and variants and corresponding permissive insect host cells include Spodoptera frugiperda (caterpillars), A edes aegypti (mosquitoes), Ades It has been identified from hosts such as Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori . Numerous viral strains for transfection are publicly available, including, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV and the like. Viruses can be used as the viruses according to the invention, in particular for the transfection of Spodoptera frugiperda cells.
[413] Plant cell cultures of cotton, corn, potatoes, beans, petunias, tomatoes, and tobacco may also be used as hosts.
[414] However, interest in vertebrate cells has grown, and proliferation of vertebrate cells in culture (tissue culture) has become a common method. Examples of useful mammalian host cell lines include monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); Human embryonic kidney cell lines (293 or 293 cells, subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36: 59 (1977)); Baby hamster kidney cells (BHK, ATCC CCL 10); Chinese Hamster Ovary Cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); And monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); Human cervical carcinoma cells (HELA, ATCC CCL 2); Canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human liver cells (Hep G2, HB 8065); Mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC5 cells; FS4 cells; Mouse myeloma cells, eg, NSO (eg, RCB0213, Bebbington et al., Bio / Technology 10: 169 (1992)) and SP2 / 0 cells (eg, SP2 / 0-Agl4 cells, ATCC CRL) 1581); Rat myeloma cells, eg, YB2 / 0 cells (eg, YB2 / 3HL.P2.G11.16Ag.20 cells, ATCC CRL 1662); And human liver tumor cell line (Hep G2). Dihydrofolate reductase (DHFR) deficient CHP cells are preferred cell lines for practicing the present invention, CHO-K1, DUX-B11, CHO-DO12, CHO-DG44 (Urlaub et al., Somatic Cell and Molecular Genetics 12 : 555 (1986)), and Lec13 are exemplary CHO host cell lines. DUX-B11 cells are transfected with pSVEHIGNeo carrying cDNA for preproinsulin, resulting in CHO-DP12 clones. For CHO-K1 (ATCC CRL 61), DUX-B11 (Simonsen et al. PNAS (USA) 80: 2495-2499 (1983)), DG44 or CHO-DP12 host cells, they express the fucosylate protein among them. It can be modified to lack the ability to be.
[415] In addition, the present invention can be applied to hybrid horse cells. The term "hybridoma" refers to a hybrid cell line produced by the fusion of an immortal cell line of immunoglobulin origin with a cell that produces an antibody. The term includes the progeny of a heterohybrid myeloma fusion that is the result of a fusion that results in the fusion of human cells with a murine myeloma cell line and then with the plasma cells, resulting in what is commonly known as a trioma cell line. In addition, the term includes any immortalized hybrid cell line that produces antibodies, such as antibodies such as quadromas (eg, Milstein et al., Nature, 537: 3053 (1983)). . Hybrid cell lines can be of any species, including humans and mice.
[416] In a preferred embodiment, the mammalian cell is a non-hybridoma mammalian cell transformed with an exogenous isolated nucleic acid encoding a polypeptide of interest. An “exogenous nucleic acid” or “heterologous nucleic acid” means a nucleic acid sequence that is heterologous to a cell or a nucleic acid sequence that is homologous to a cell but is typically located within a host cell nucleic acid in which no nucleic acid is found.
[417] (viii) culture of host cells
[418] Conventional nutritional media suitably modified to transform host cells with the expression or cloning vectors for the production of polypeptides described above, induce promoters, select transformants, and amplify genes encoding the desired sequences. Incubate in the air.
[419] Host cells used to produce the polypeptides of the invention can be cultured in a variety of media. Commercially available media include, for example, Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma). Suitable for culturing cells. See also, Ham et al., Meth. Enzym. 58: 44 (1979), Barnes et al., Anal. Bochem. 102: 255 (1980), US Pat. Nos. 4,767,704; 4,657,866; 4,927,762; Any medium described in US Pat. No. 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or US Pat. No. 30,985) may be used as culture medium for host cells. These optional media may be used, if necessary, with hormones and / or other growth factors (eg, insulin, transferrin, or epidermal growth factor), salts (eg, sodium chloride, calcium, magnesium and phosphate), buffers (eg For example, HEPES), nucleotides (eg adenosine and thymidine), antibiotics (eg GENTAMYCIN ™ drugs), trace elements (defined as inorganic compounds typically present in the final concentration micromolar range), and glucose Or an equivalent energy source. Any other necessary supplements may be included at appropriate concentrations known to those of ordinary skill in the art. Culture conditions such as temperature, pH, etc. will be apparent to those skilled in the art as previously used in host cells selected for expression.
[420] All culture media generally provide one or more components derived from one or more of the following categories:
[421] 1) an energy source, generally a carbohydrate form such as glucose;
[422] 2) all essential amino acids, and 20 basic amino acid sets plus cystine;
[423] 3) vitamins and / or other organic compounds required at low concentrations;
[424] 4) free fatty acids; And
[425] 5) Trace Elements, where trace elements are defined as inorganic compounds or naturally occurring elements which are usually required in very low concentrations, generally in the micromolar range.
[426] The culture medium preferably lacks serum, for example, less than about 5%, preferably less than 1%, more preferably 0-0.1% of serum and other animal-derived proteins. However, they can be used if necessary. In a preferred embodiment of the invention, the cell culture medium comprises an excess of amino acids. Amino acids provided in excess may be selected from, for example, Asn, Asp, Gly, Ile, Leu, Lys, Met, Ser, Thr, Trp, Tyr and Val. Preferably, Asn, Asp, Lys, Met, Ser and Trp are provided in excess. For example, amino acids, vitamins, trace elements and other media components can be used at one to two times the range covered by EP 307,247 or UP 6,180,401. These two documents are incorporated herein by reference.
[427] For the cultivation of mammalian cells which can express the protein of interest and add the desired carbohydrate to a specific site, a number of culture conditions can be used with particular attention to the host cell being cultured. Suitable culture conditions for mammalian cells are well known in the art (Cleveland et al., J. Immunol. Methods 56: 221-234 (1983)) or can be readily determined by those skilled in the art (eg, Animal Cell Culture). ^{:. a Practical Approach 2 nd Ed} , can vary according to Rickwood, D. and Hames, BD, eds Oxford University Press, New York (1992)), selected a specific host cells.
[428] (ix) glycoprotein tablets
[429] When using recombinant techniques, glycoproteins can be produced intracellularly, in the intracellular space, or directly secreted into the medium. If glycoproteins are produced intracellularly, the first step is that specific fragments, host cells or lysed fragments are removed, for example by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992). A method for isolating antibodies secreted into the intracellular space of collie is described. In brief, the cell paste is thawed for at least 30 minutes in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonylfluoride (PMSF). Cell debris is removed by centrifugation. When glycoproteins are secreted into the medium, such as supernatants from the expression system are generally first concentrated using commercially available protein enrichment filters, such as Amicon or Millipore Pellicon ultrafiltration units. Protease inhibitors such as PMSF are included in any of these steps to prevent proteolysis, and antibiotics may be included to inhibit the growth of foreign contaminants.
[430] Glycoprotein components prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc region present in the glycoprotein. Protein A can be used to purify glycoproteins based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is preferred for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5: 1567-1575 (1986)). The matrix to which the affinity ligand attaches is most often agarose, but other matrices may be used. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than are achieved by agarose. If the glycoprotein comprises a C _H 3 domain, Bakerbond ABX ™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Fractionation on ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE ™, chromatography on an anion or cation exchange resin (eg polyaspartic acid column) depending on the glycoprotein recovered ), Chromatographic focusing, SDS-PAGE and ammonium sulfate precipitation are also available.
[431] In one embodiment, the glycoprotein can be purified by enriching the glycoprotein lacking fucose by removing the fucose-containing glycoprotein from the preparation using precipitation on the lectin substrate (eg, lectin affinity column). have.
[432] F. Analysis of Glycoproteins
[433] The complex carbohydrate moiety of the glycoprotein produced according to the method of the present invention can be readily analyzed by measuring the completion of the glycosylation reaction described above. Oligosaccharides are analyzed according to conventional techniques for carbohydrate analysis. Thus, for example, lectin blotting known in the art reveals the proportion of other sugars such as terminal mannose or galactose.
[434] Preferably, the carbohydrates are prepared using MALD1-TOF mass spectral analysis as described in Example 1 and Shields et al., J. Biol. Chem. 9 (2): 6591-6604 (2001).
[435] Several methods are known in the art for glycosylation analysis and are useful within the scope of the present invention. This method provides information on the identity and composition of the oligosaccharides attached to the peptide. Useful methods of carbohydrate analysis in the present invention include lectin chromatography; HPAEC-PAD using high pH anion exchange chromatography to separate oligosaccharides on a charge basis; NMR; Mass spectrum; HPLC; GPC; Monosaccharide composition analysis; It includes, but is not limited to, continuous enzyme cleavage.
[436] In addition, methods for secreting oligosaccharides are known. These methods
[437] 1) enzymatic methods, for example using fucosidase such as α-L-fucosidase to remove fucose;
[438] 2) removal using a harsh alkaline environment primarily to release O-linked structures; And
[439] 3) chemical methods using anhydrous hydrazine to release N- and O-linked oligosaccharides.
[440] Neutral and amino-sugars can be measured by combining high performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD Carbohydrate System, Dionex Corp.), for example, 20% (v / v) sugar. It can be released by hydrolysis at 100 ° C. for 6 hours in trifluoroacetic acid. The hydrolyzate is then lyophilized or dried using Speed-Vac (Savant Instruments). The residue is then dissolved in 1% sodium acetate trihydrate solution, see Annumula et al. Anal. Biochem. 195: 269-280 (1991)] are analyzed on HPLC-AS6.
[441] Salicylic acid is described in Yao et al. Anal Biochem. 179: 332-335 (1989)], which can be measured individually using the direct colorimetric method. In a preferred embodiment, tribarvaturic acid (TBA) from Warren, L. J. Biol Chem 238: (8) (1959) is used.
[442] Alternatively, immunoblot carbohydrate assays can be performed. According to this method, protein bound carbohydrates are described by Haselbeck and Hosel, in Haselbeck et al. Glycoconjugate J., 7: 63 (1990)) is detected using a commercial glycan detection system (Boehringer) based on the oxidative immunoblot method described.
[443] Analytical methods include those described for analysis of antibody bound oligosaccharides, which are described, for example, in Warmald et al., Biochem. 36: 1370-1380 (1997); Sheeley et al. Anal. Biochem. 247: 102-110 (1997) and Cant et al., Cytotechnology 15: 223-228 (1994) and references therein.
[444] G. Pharmaceutical Formulations
[445] Therapeutic preparations of glycoproteins are frozen by mixing glycoproteins of the desired purity with any physiologically acceptable carrier, excipient or stabilizer ( Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). It may be prepared in the form of a dried formulation or an aqueous solution. Suitable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; Antioxidants including ascorbic acid and methionine; Preservatives (e.g., 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 other 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 (eg, Zn-protein complexes); And / or nonionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG).
[446] In addition, the formulations herein contain one or more active compounds, preferably those with complementary activities, each of which does not adversely affect each other, as needed for the particular condition being treated. For example, the formulation may additionally contain other antibodies or chemotherapeutic agents. Such molecules are suitably present in admixture in amounts that are effective for the purpose intended.
[447] The active ingredient can also be enclosed in microcapsules, for example coacervation techniques or interfacial polymerization, for example hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) micro, respectively. Capsules may be enclosed in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
[448] The formulations used for in vivo administration must be sterile. This is readily accomplished through filtration through sterile filtration membranes.
[449] Sustained release formulations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing glycoproteins whose matrix is in the form of shaped articles, eg, films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinylalcohol), polyacted (UP 3,773,919), 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 are capable of releasing molecules for more than 100 days, certain hydrogels release proteins for shorter times If the encapsulated antibody stays in vivo for a long time, it may denature or aggregate as a result of exposure to moisture at 37 ° C., resulting in loss of biological activity and altered immunogenicity. Thus, rational schemes can be devised for stabilization, for example, if the aggregation mechanism is found to form intramolecular SS bonds via thio-disulfide exchange, the sulfhydryl residues are modified, lyophilized from acidic solution, Stabilization can be achieved by controlling the wet content, using appropriate additives, and developing specific polymer matrix compositions.
[450] The pharmaceutical composition may be lyophilized. Lyophilized antibody formulations are described in UP 6,267,958. Stable water soluble antibody preparations are described in UP 6,171,586 B1.
[451] H. Non-Therapeutic Uses of Glycoproteins
[452] Glycoproteins of the invention can be used in affinity tablet formulations. In this method, the glycoprotein is immobilized using a method known in the art in a solid phase such as Sephadex resin or filter paper. The immobilized glycoprotein is contacted with a sample containing the antigen to be purified and then the support is washed with a suitable solvent that substantially removes all material in the sample except the antigen to be purified that is bound to the immobilized glycoprotein. Finally, the support is washed with another suitable solvent that releases the antigen from the glycoprotein, eg glycine buffer (pH 5.0).
[453] Glycoproteins may also be useful for diagnostic assays, eg, for detecting expression of a desired antigen in a particular cell, tissue or serum.
[454] For diagnostic applications, glycoproteins are generally labeled with a detectable moiety. Multiple labels are available, and they can generally be grouped into the following categories:
[455] (a) a radioactive isotope, e.g., ³⁵ S, C ^{l4, l25} I, ³ H, and I. ^l3l glycoproteins are described in [Current Prctocols in Inmmunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, New York, Pubs. (1991)] can be labeled, for example, radioisotope activity can be measured using scintillation counting.
[456] (b) Fluorescent labels, such as rare earth chelates (Europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, densyl, lysamine, phycoerythrin and Texas red can be used. Fluorescent labels can be conjugated to glycoproteins using, for example, the techniques described in Current Protocols in Immunology , supra. Fluorescence can be quantified using a fluorometer.
[457] (c) A variety of enzyme-substrate labels can be used, UP 4,275,149 provides a review of some of them. In general, enzymes catalyze the chemical modification of chromogenic substrates, which can be measured using a variety of techniques. For example, enzymes catalyze the color change in a substrate, which can be measured using a spectrophotometer. Alternatively, the enzyme can modify the fluorescence or chemiluminescence of the substrate. Techniques for quantifying changes in fluorescence are described above. The chemiluminescent substrate is electrically excited by a chemical reaction and then emits light that can be measured (eg, using a chemiluminometer) or provides energy to the fluorescent receptor. Examples of enzymatic labels include luciferase (eg, firefly luciferase and bacterial luciferase; UP No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, horseradish Peroxidases such as oxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g. glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase) ), Heterocyclic oxidase (eg, uricase and xanthine oxidase), lactoperoxidase, microperoxidase and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym . (ed J. Langone & H. Van Vunakis), Academic press, New York, 73: 147-166 (1981).
[458] Examples of enzyme-substrate combinations are, for example,
[459] (i) horseradish peroxidase (HRPO) and hydrogen peroxidase as substrate, wherein the hydrogen peroxidase is a dye precursor (e.g., orthophenylene diamine (OPD) or 3,3 ', 5,5' -Oxidize tetramethyl benzidine hydrochloride (TMB));
[460] (ii) para-nitrophenyl phosphate as alkaline phosphatase (AP) and a chromogenic substrate;
[461] (iii) β-D-galacoxidase (β-D-Gal) and chromogenic substrates (eg p-nitrophenyl-β-D-galactoxydase) or fluorescent substrate 4-methyllumbeliferyl β-D-galactosidase.
[462] Many other enzyme-substrate combinations are available to those skilled in the art. For a general review of these, see UP 4,275,149 and 4,318,980.
[463] Sometimes the label is conjugated indirectly to glycoprotein. Those skilled in the art will recognize various techniques for accomplishing this. For example, glycoproteins may be conjugated with biotin, and any of the three broad ranges mentioned above may be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated in a non-indirect manner with the glycoprotein. Alternatively, to achieve nonindirect conjugation of the glycoprotein with the label, the glycoprotein is conjugated with a small heptene (eg, digoxin), and one or more of the different types of labels mentioned above are anti-heptene polypeptides ( For example, an anti-digoxin antibody). Thus, indirect conjugation of glycoproteins and labels can be achieved.
[464] In other embodiments of the present invention, glycoproteins do not need to be labeled and their presence can be detected using labeled antibodies that bind to the glycoprotein.
[465] Glycoproteins of the present invention can be used in any known assay method, eg, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).
[466] Glycoproteins can also be used in in vivo diagnostic assays. Glycoproteins are generally radionuclides (eg, ¹¹¹ In, ⁹⁹ Tc, ¹⁴ C, ¹³¹ I, ¹²⁵ I, ³ H, ³² P) so that antigens or cells expressing them can be localized using immunosynthetic imaging. Or ³⁵ S).
[467] I. In vivo use of glycoproteins
[468] Glycoproteins of the present invention can be used to treat patients suffering from or susceptible to diseases or diseases that would benefit from administration of a mammal, eg, a glycoprotein. There are many symptoms that can be treated with glycoproteins and cancer (eg, glycoproteins are tumor-associated antigens, B-cell surface antigens, eg, ErbB receptors such as CD20, HER2 receptors, vascular endothelial growth factors) Angiogenic factors such as VEGF); Allergic symptoms such as asthma (using anti-IgE antibodies); And LFA-1-mediated diseases (eg, when the glycoprotein is an anti-LFA-1 or anti-ICAM-1 antibody) and the like. For antibodies that bind to B-cell surface markers such as CD20, the indication of choice is to block B-cell cancer (eg non-Hodgkin's lymphoma), autoimmune disease or immune response to foreign antigens (WO 01/03734).
[469] If the antibody binds to the ErbB receptor, the disease is preferably a benign or malignant carcinoma characterized by an overexpression of ErbB-expressing cancer, eg, ErbB receptor. Such cancers include breast cancer, squamous cell lung cancer, small-cell lung cancer, non-samll cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, liver cancer, colon cancer, colorectal cancer, and uterus. Endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma and various forms of head and neck cancers.
[470] According to the teachings herein, glycoproteins having variant Fc regions with improved ADCC activity can be prepared. Such molecules may find application in the treatment of different diseases.
[471] For example, glycoproteins with enhanced ADCC activity can be used for the treatment of diseases or conditions that require the destruction or removal of tissue or foreign microorganisms. For example, glycoproteins include cancer; Autoimmune diseases, inflammatory diseases; Infections (eg, bacterial, viral, fungal or yeast infections); And other symptoms that require tissue removal (eg, goiter).
[472] Glycoproteins can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intraperineal, and intranasal, as well as by intralesional administration when needed for local immunosuppressive treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, glycoproteins are suitably administered by pulse infusion, especially with decreasing dose of glycoproteins. Depending in part on whether the administration is temporary or long term, preferably the administration is by injection, most preferably given by intravenous or subcutaneous injection.
[473] For the prevention or treatment of a disease, appropriate dosages of glycoproteins are determined by the type of disease being treated, the depth and route of the disease, whether the glycoprotein is administered for prophylactic or therapeutic purposes, prior treatment, the clinical history of the patient It will depend on your response to the glycoprotein and your doctor's discretion.
[474] Depending on the type and severity of the disease, for example, by one or more separate administrations or by continuous infusion, about 1 μg / kg to 15 mg / kg (eg, 0.1-20 mg / kg Glycoproteins) are the first candidates for administration to the patient. Typical daily dosages will range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations of several days or more, depending on the condition, treatment is continued until suppression of the desired disease symptom occurs. However, other dosing regimens would be useful. The progress of this therapy is easily monitored according to conventional techniques and assays. The examples herein demonstrate that lower doses of glycoproteins (eg, antibodies without fucose) can be administered as compared to fucose containing glycoproteins.
[475] Glycoprotein compositions should be formulated, formulated, and administered in a fashion consistent with best medical practice. Factors considered within this context include the particular disease being treated, the particular mammal being treated, the clinical symptoms of the individual patient, the cause of the disease, the site of delivery of the drug, the method of administration, the dosing schedule, and other factors known to medical practitioners. Include. The "therapeutically effective amount" of glycoprotein to be administered is determined by this consideration and is the minimum amount necessary to prevent, alleviate or treat a disease or condition. Glycoproteins need not be formulated with one or more drugs currently used to prevent or treat a disease in question, but are optionally formulated together. The effective amount of such other drugs depends on the amount of glycoprotein present in the formulation, the type of disease or treatment, and other factors discussed above. They are generally used in the same dose depending on the route of administration used above, or at about 1 to 99% of the dose used above.
[476] The therapeutic antibody composition is generally located in a container with a sterile access port, such as a vial with a lid that can be permeated into an intravenous solution bag or a needle for subcutaneous injection.
[477] Cancer patients treated with antibodies as antagonists described herein may also be subjected to radiation therapy. Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient. Such preparations and dosing schedules for chemotherapeutic agents may be used according to the manufacturer's instructions or as determined empirically by a skilled practitioner. In addition, the preparation and dosing schedules for such chemotherapy are described in Chemotherapy Service Ed. , M. C. Perry, Williams & Wilkins, Baltimore, MD (1992). The chemotherapeutic agent may occur prior to, or after, or concurrently with the antagonist. For cancer symptoms, it may be desirable to administer additional antibodies to tumor associated antigens or angiogenic factors, such as antibodies that bind to HER2 or vascular endothelial growth factor (VEGF). Alternatively, or in addition, one or more cytokines may be co-administered to the patient.
[478] The present invention further provides articles of manufacture and kits containing substances useful for the treatment of cancer, for example. The article of manufacture comprises a labeled container. Suitable containers include, for example, bottles, vials and test tubes. The container may be formed from various materials such as glass and plastic. The container holds a composition comprising a glycoprotein preparation described herein. The active drug in the composition is a specific glycoprotein. The label on the container indicates that the composition is used for the treatment or prophylaxis of a particular disease or condition, and they also indicate the use in vivo, for example as mentioned above.
[479] The kit of the present invention comprises a container as described above and a second container comprising a buffer solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, filters, needles, injections and package inserts with instructions for use.
[480] The invention will be more fully understood by reference to the following examples. However, these should not be construed as limiting the scope of the invention. All documents and patent citations mentioned herein are expressly incorporated by reference.
[1] The present invention relates to a composition comprising a glycoprotein having a modified glycosylation pattern. In particular, the present invention relates to a composition comprising a glycoprotein having an Fc region, wherein about 80-100% of the glycoprotein in the composition comprises a mature coa carbohydrate structure without fucose attached to the Fc region of the glycoprotein. .
[481] To assess the role of fucosylated oligosaccharides in the function of IgG, Lec13 cell line (Ripka et al. Arch. Biochem. Biophys . 249: 533-545 (1986) was used to express human IgG1. Although lacking the ability to add cos, in other respects it provides IgG with oligosaccharides similar to those found in normal CHO cell lines and derived from human serum The resulting IgG product is directed against human FcγR, human C1q, human FcRn. It was used to evaluate the effect of fucosylated carbohydrates on antibody agonist function, including ADCC using binding and human agonist cells.
[482] Example 1
[483] Binding to Human FcR
[484] CDNA constructs for stable cell lines : heavy and light chains of the humanized anti-HER2 antibody Hu4D5 (Carter et al., Proc. Natl. Aacad. Sci. USA , 89: 4285 (1992)), humanized, affinity matured -IgE antibody E27 (UP 6,172,213) and chimeric anti-CD20 antibody C2B8 (UP 5,736,137) were subcloned into the mammalian cell expression vector described above (Lucas et al. Nucl. Acid Res. 24,1774) . -1779 (1996)). Puromycin was used as a selection marker in DHFR (+) cells, such as Lec13 cells, and the DHFR locus was maintained for methoxtrexate amplification of stable cell lines.
[485] Transfection and Culture of Lec13 and Wild-type CHO Cells : The CHO cell line Pro-Lec13.6a (Lec13) was obtained from Professor Pamela Stanley of Albert Einstein Colors College of Yeshiva Medical School. Parent strains were pro- (proline nutrients) and Gat- (glycine, adenosine, thymidine nutrients). The CHO-DP12 cell line used for wild type antibodies was a derivative of the CHO-K1 cell line (ATCC # CCL-61), which was deficient in dihydrofolate reductase and reduced the need for insulin. Cell lines were transfected with cDNA using the Superfect method (Qiagen, Valencia, Calif.). Selection of Lec13 cells expressing the transfected antibody contained MEM alpha medium containing L-glutamine, ribonucleotide and deoxyribonucleoside using puromycin dihydrochloride, 10% inactivated FBS (GIBCO), Using 10 μg / ml of puromycin dihydrochloride (Calbiochem, San Diego, CA) in growth medium (GIBCO-BRL, Calbiochem, San Diego, Calif.) Supplemented with 10 mM HEPES and 1 × penicillin / streptomycin (GIBCO) It was performed by. CHO cells were Ham's F12 without GHT; No glycine, low glucose DMEM containing NaHCO ₃ , 5% FBS (GIBCO), 10 mM HEPES, 2 mM L-glutamine, 1X GHT (glycine, hypoxanthine, thymidine) and 1X penicillin / streptomycin Similarly selected from growth media supplemented with. Colonies formed within 2-3 weeks were collected for proliferation and protein expression. Cell collections were first seeded into plates at 3 × 10 ⁶ cells / 10 cm for small batch protein expression. When cells grew to 90-95% confluency, the cells were converted to serum-free medium and Cell supernatants were collected 3-5 days later, and tested by Fc IgG- and total IgG-ELISA to assess protein expression levels. Lec13 and CHO cells were seeded on plates at about 8 × 10 ⁶ cells / 15 cm one day before conversion to PS24 production medium supplemented with 10 mg / L recombinant human insulin and 1 mg / L trace elements.
[486] Protein Expression : Lec13 cells and CHO cells were maintained for 3-5 days in serum-free production medium. Supernatants were collected and centrifuged in 150 ml conical tubes to remove cells and debris. Protein inhibitors PMSF and aprotinin (Sigma, St. Louis, MO) were added and prior to immediate purification using protein G chromatography (Amersham Pharmacia Biotech, Piscataway, NJ)) on stirred cells using a MWCO30 filter Concentrated 5 times (Amicon, Beverly, MA). Buffers of all proteins were exchanged into phosphate-buffered saline (PBS) using Centripriep-30 concentrator (Amicon) and analyzed by SDS-polyacrylamide gel electrophoresis. Protein concentration was measured using A280 and confirmed using amino acid composition analysis. On average, Lec13 cells produced 10 μg mAb per 15 cm plate; Expression in control CHO cells for all antibodies was 4-5 fold higher than in Lec13 cells. Antibodies from CHO-DP12 grown on plates are denoted CHO-P.
[487] CHO-DP12 cells were grown in spinner flasks. Cells were seeded at 6 × 10 ⁵ cells / ml and grown at 37 ° C. for 2 days. On day 3 the temperature was changed to 33 ° C., and the pH was lowered to ˜6.5 and grown until growth reached 70%. Antibodies derived from CHO-DP12 cells grown in spinner flasks are shown as CHO-S.
[488] Matrix-supported laser desorption / ionization time-of-flight (MALDI-TOF) Mass spectrum analysis of arparagine-linked oligosaccharides: N-linked oligosaccharides are described in Papac et al., Glycobiology 8 , 445-454 (1998), was used to release from recombinant glycoproteins. Briefly, wells of 96-well PVDF-coated microtiter plates (Millipore, Bedford, Mass.) Were adjusted to 100 μl of methanol exiting the PVDF membrane by applying vacuum to a Millipore Multiscreen vacuum manifold. The adjusted PVDF membrane was washed with 3 × 250 μl of water. In all washing steps, the wells were drained completely by gentle vacuum to the manifold. The membrane was washed with reduction and carboxymethylation buffer (RCM) consisting of 6 M guanidine hydrochloride, 360 mM Tris, 2 mM EDTA, pH 8.6. Glycoprotein samples (50 μg) were added to each well, drained back through the PVDF membrane with slight vacuum, and the wells washed with 2 × 50 μl RCM buffer. The immobilized samples were reduced by adding 50 μl of 0.1 M dithiothreitol (DTT) solution to each well and the microtiter plate incubated at 37 ° C. for 1 hour. DTT was removed by vacuum and the wells were washed with 4 × 250 μl of water. Cysteine residues were carboxymethylated by adding 50 μl of a 0.1 M iodiacetic acid (IAA) solution freshly prepared in 1 M NaOH and diluted to 0.1 M using RCM buffer. Carboxymethylation was achieved by incubation for 30 minutes in the dark at ambient temperature. The plate was vacuumed to remove the IAA solution and the wells were washed with 4 × 250 μL of purified water. 100 μl of 1% PVP360 (polyvinylpyrrolidine 360,000 MW) (Sigma) solution was added and incubated at ambient temperature for 1 hour to block PVDF membrane. Light vacuum was applied to remove the PVP-360 solution and the wells were washed with 4 × 250 μl of water. PNGase F (New England Biolabs, Beverly, MA) digestion solution in 10 mM Tris acetate, pH 8.4, 25 μl of 25 Unit / ml solution was added to each well and the digestion proceeded at 37 ° C. for 3 hours. After digestion, the samples were transferred to 500 μl Eppendorf tubes and 2.5 μl 1.5M acetic acid solution was added to each sample. The acidified sample was incubated at ambient temperature for 3 hours to convert the oligosaccharides of glycosylamine to hydroxyl form. Prior to MALDI-TOF mass spectral analysis, the released oligosaccharides were packed into compacted reaction tubes (US Biochemical, Cleveland, OH) in a slurried 0.7-ml bed of cation exchange resin (AG50W-X8 resin in hydrogen form) (Bio-Rad , Hercules, CA).
[489] For MALDI-TOF mass spectrum analysis of the sample in positive mode, desalted oligosaccharides (0.5 μL aliquots) were added to 0.1 mg of 5-methoxysilicone in 1 ml of ethanol / 10 mM sodium chloride 1: 1 (v / v). 0.5 μl of 2,5-dihydroxybenzoic acid matrix (sDHB) prepared by dissolving 2 mg of 2,5-dihydroxybenzoic acid with acid was added to the stainless target. The sample / matrix mixture was dried in vacuo. For analysis in the negative mode, desalted N-linked oligosaccharides (0.5 μl aliquots) were prepared in 0.5 μl 2 ′, 4 ′ prepared in 1: 3 (v / v) acetonitrile / 13.3 mM ammonium citrate buffer, A stainless' target was added with 6'-trihydroxyacetophenone matrix (THAP). The sample / matrix mixture was vacuum dried and then allowed to absorb ambient moisture prior to analysis. The released oligosaccharides were analyzed by MALDI-TOG on a PerSeptive BioSystems Voyager-DE mass spectrometer. The mass spectrometer was operated at 20 kV using delayed extraction in positive or negative mode in a linear fashion. Data was obtained in data weighting mode (240 scans) to enhance the signal to noise using a laser power of 1300. The instrument was calibrated using a mixture of standard oligosaccharides and the data was evened using a 19 point Savitsky-Golay algorithm prior to mass assignment. Integration of the mass spectral data was achieved using the Casesar 7.0 data analysis software package (SciBridge Software). The results are summarized in the table below.
[490]
[491]
[492] a. All values were measured at A ₄₉₀ nm in the ratio A (variant) / A (standard). CHO-S represents IgG expressed by CHO cells in a spinner flask, CHO-P represents IgG expressed by CHO cells on a 15 cm plate, Lec13 represents IgG expressed by Lec13 cells on a plate, HEK293 Represents IgG expressed in human embryonic kidney 293 cells, AAA represents Ser298Ala / Glu333Ala / Lys334Ala IgG variants, and Lec13-S represents IgG expressed in Lec13 cells in spinner flasks (instead of plates). Letters in parentheses indicate lots of independently expressed IgG.
[493] b. [mAb] Hu4D5 dimer at 0.12 μg / ml
[494] c. % -Fuc is the percentage of total oligosaccharides without fucose,% Gal0,% Gal1,% Gal2 are without galatose residues covalently linked to the terminal mannose residues (mugalactosyl), with one (monogallacto) Thread) or two (digalactosyl). Numbers in parentheses are deviations from the mean for four independently expressed lots of Lec13-Hu4D5.
[495] d. Hu4D5 dimer at [mAb] = 3.33 μg / ml
[496] e. [mAb] Hu4D5 dimer at 1.11 μg / ml
[497] f. [mAb] Hu4D5 dimer at 0.12 μg / ml
[498] g. EmA dimer at [mAb] = 0.12 μg / ml
[499] h. EmA hexamer at [mAb] = 0.12 μg / ml
[500] Assays (ELISA-format and cell-based) for measuring the binding of IgG1 to FcγR and FcRn have already been described by Shields et al. J. Biol. Chem. 276: 6591-6604 (2001) and WO 00/42072 (Presta).
[501] Human FcγRI bound monomers Lec13-Hu4D5 IgG1 equivalent to the binding of Hu4D5-CHO-S and CHO-P (FIG. 4; Table 2). The presence of carbohydrates is required for FcγRI binding (Walker et al. Biochem. J. 259: 347-353 (1989)), but differences in fucose content (Lec13 vs. CHO) or galactose content (CHO-P vs. CHO-S) Regardless, the equivalent binding of IgG1 indicates that human FcγRI is not sensitive to the presence of these residues on carbohydrates. The effect of galactosylation on IgG binding to human FcγRI has already been studied (Wright et al. J. Immunol. 160: 3393-3402 (1998); Kumpel et al. Human Antibod. Hybridomas 5: 143-151 (1994) And Tsuchiya et al. J. Rheumatol. 16: 285-290 (1989)), a review of these data suggests that if galactosylation affects FcγRI binding, it is subtle and probably isotype dependent. (Wright et al. J. Immunol. 160: 3393-3402 (1998)).
[502] In contrast to monomeric binding of IgG1 to human FcγRI, binding assays for low-affinity human FcγRI (FcγRII, FcγRIII) require the formation of dimers (Hu4D5, HuE27) or hexamers (HuE27) to induce binding detection. . Human FcγRIIA has two known, naturally occurring allotypes, identified as amino acids at position 131 (Clark et al. J. Immunol. 143: 1731-1734 (1989)). Human FcγRIIA has a native allotype at position 48 (Leu, His or Arg) and position 158 (Val or Phe); FcγRIIIA (Val158) allotypes interact better with human IgG than FcγRIIIA (Phe158) allotypes (Shields et al. J. Biol. Chem. 276: 6591-6604 (2001); Koene et al. Blood 90: 1109). -1114 (1997); and Wu et al. J. Clin. Invest. 100: 1059-1070 (1997).
[503] Binding of Lecl3-Hu4D5 dimers to human FcγRIIB and human R131-polymorphic forms of FcγRIIA showed 1.8- and 1.6-fold improvement in binding, respectively, compared to CHO-Hu4D5 (FIGS. 5, 6; Table 2). In contrast, the lack of fucose does not affect the binding of the human FcγRIIA to the H131-polymorphic form (FIG. 7; Table 2). In contrast to no effect on FcγRIIA (H131), the similar binding of fucose-free IgG1 to both human FcγRIIA (R131) and FcγRIIB with arginine at position 131 was similarly enhanced at position FcγR 131. It is suggested that fucose interacts directly with the FcγR residue at position 131 or modifies the IgG1 form to have a subtle, negative effect on binding when arginine is present.
[504] Both polymorphic forms of FcγRIIIA show significantly improved binding to IgG1 without fucose. Binding of dimer Lec13-Hu4D5 to FcγRIIIA (V158) showed a 14-fold improvement over CHO-Hu4D5 (FIG. 8; Table 2), and binding to FcγRIIIA (V158) showed a 100-fold improvement (FIG. 9). In addition, Lec13-HuE27 dimers show enhanced binding to both polymorphic forms of FcγRIIIA (FIGS. 10, 11; Table 2).
[505] In previous studies on the effect of protein-sequence variants of human IgG1 on binding to human FcγR, a hexamer complex consisting of three anti-IgE E27 and three IgE was used (Shields et al. J. Biol. Chzem .276: 6591-6604 (2001)); Thus, these complexes are trimers in anti-IgE E27. In this study, the binding enhancement of S298A / E333A / K334A-IgGl variants to FcγRIIIA (F158) and FcγRIIIA (V158) was 1.5 to 2 and 1.1 times, respectively; The improvement may be minimal, but the effect on ADCC was significant (Shields et al. J. Biol. Clem. 276: 6591-6604 (2001)). In the current study, S298A / E333A / K334A-IgGl showed improved binding to both FcγRIIIA high dose forms, consistent with the values obtained in previous studies (FIG. 12, 13; Table 2); In addition, the hexamer complex of Lec13-HuE27 (natural IgG1) showed about two-fold enhanced binding to FcγRIIIA (F158) (Table 2). When analyzed with dimers, the S298A / E333A / K334A-IgGl variants with fucose showed 9 and 20-fold improvements in binding to FcγRIIIA (V158) and FcγRIIIA (F158), respectively; The same variant without fucose showed a 21-fold and 33-fold higher improvement in binding to the polymorphic form, respectively (Table 2). Thus, the absence of fucose may increase binding of native IgG1 to FcγRIIIA, as well as enhance binding of IgG1 Fc variants. Thus, protein and carbohydrate modifications are synergistic.
[506] For all forms of HuE27 (natural, S298A / E333A / K334A-IgGl, Lec13-derived), the enhancement of binding in the large complex (Hu27 trimer) was much smaller than that observed for the same mAb as the dimer complex . For example, binding of Lec13-HuE27 as a dimer showed about a 20-fold improvement, but only a 2-fold improvement for the large complex (Table 2). This suggests that as the size of the immune complex increases, the effect of total affinity begins to dominate.
[507] Enhanced binding of fucose-deficient IgG1 to FcγRIIA was confirmed by FcγRIIA (Phe158) full-length α-chain co-expressed with γ-chain on CHO cells (FIG. 28; Table 2). For the α-chain fusion protein alone in ELISA-format, the fucose deficiency was synergistic with the S298A / E333A / K334A-IgGl variant.
[508] In addition, the binding of native and fucose (-) IgG1 to murine FcγRII and FcγRIII was evaluated. Human IgG1 binds insufficiently to these receptors even with dimers, and no binding improvement seen in IgG1 without fucose was seen. Other receptors for IgG, neonatal Fc receptor (FcRn), are structurally unrelated to FcγR (Burmeister et al. Nature 372: 379-383 (1994); and Raghavan et al. Annu. Rev. Cell Dev. Biol. 12 : 181-220 (1996)), has been suggested to be involved in a number of biological processes, including the clearance of IgG (Ghetie et al. Annu. Rev. Immunol. 18: 739-766 (2000)). The binding of fucosylated or non-fucosylated IgG1 to FcRn was equivalent (FIG. 14). This is not surprising since aglycosylated IgG1 binds to this receptor similarly to glycosylated IgG1 (Bglyco Ab binds to FcRn).
[509] Lack of fucose in Asn297-bound carbohydrates resulted in significantly enhanced binding to human FcγRIIIA (both F158 and V158 polymorphic forms) in ELISA-format analysis. Enhanced binding to FcγRIIIA was further supported by the ability of fucose (-) IgG to boost cytotoxicity in ADCC assays using purified human PBMCs. Improved cytotoxicity was particularly evident at low concentrations of antibody, suggesting that therapeutic antibodies using ADCC can be given at low doses that give equivalent cell killing effects, such as high doses of fucosylated IgG.
[510] Less improvement in binding of fucose (-) IgG was observed for human FcγRIIA (R131) and FcγRIIB; There was no difference from that observed for human FcγRIIA (H131). The preceding two receptors have arginine at position 131, which is not limited to the following theory, but in fucosylated IgG the fucose residues interact directly (and obviously, negatively) or IgG with FcγRII residue 131 It has a subtle effect on the shape of, suggesting that it can lead to negative interactions. Although the improvement in the binding of FcγRIIA (R131) and FcγRIIB to fucose (-) IgG in ELISA-formatted assays is very small (~ 2 fold), ADCC analysis using monocytes also shows some enhanced cytotoxicity at low concentrations of antibodies. It was. Monocytes express FcγRI, FcγRIIA, FcγRIIB, and only a subset of monocytes express FcγRIIIA. Since binding to human FcγRI is equivalent to both fucosyl IgG and fucose (−) IgG, no improvement in ADCC will occur due to different interactions with FcγRI. Both FcγRIIA (R131 / R131) and FcγRIIA (H131 / H131) donor monocytes showed constant improvement in ADCC (FIGS. 21, 22), which is not limited to either theory, but (1) FcγRIIA was between the two polymorphic forms. Is not expressed at sufficient levels on monocytes to show a difference in (2) FcγRIIB is predominantly FcγR binding (thus equally affects both R131 / R131 and H131 / H131 monocytes), or (3) expresses FcγRIIA Suggests that a subset of monocytes is involved in enhanced ADCC.
[511] Comparison of carbohydrates found in Lec13-generated IgG and native IgG derived from CHO-generated IgG showed no appreciable difference in the degree of galactosylation, thus the result is only due to the presence / absence of fucose. Can be. However, for S298A / E333A / K334A IgGl variants, Lec13-generated IgG, HEL-generated IgG and DP12-generated IgG showed differences in galactosylation. However, the combination of protein-sequence variants and lack of fucose has been shown to be additive.
[512] Previous studies of protein-sequence variants of human IgG have found that alanine (and other) substitutions at some Fc positions can reduce or enhance binding to FcγR and also exhibit enhanced ADCC (Shields et. J. Biol. Chem. 276 (9): 6591-6604 (2001). Interestingly, some of these were not near the interaction interface found in the crystal structure of the human IgG Fc-human FcγRIIIA complex (Sondermann et al., Nature 406: 267-273 (2000)). For example, in the three alanine substituents of S298A / E333A / K334A used in this study, only S298 is at the interface of Fc-FcγRIIIA in the crystal structure. In addition, in the co-crystal structure, none of the fucose residues on the two Fc heavy chains interact with FcγRIIIA. Close examination of the crystal structure of human and rodent Fc or IgG shows that fucose can adopt various forms and exhibit high B-factors, which suggests a high degree of fluidity.
[513] Normal CHO and HEK293 cells add fucose to IgG oligosaccharides at high levels (97-98%). Serum IgG is also highly fucosylated.
[514] Example 2
[515] C1q and FeRn Bonds
[516] Binding of Clq to the antibody is the first step in a typical complement activity pathway (Makrides, SC Pharmacol. Rev. 50: 59-87 (1998)). The properties of carbohydrates on IgG affect the interaction with Clq (Wright et al. J. Immunol. 160: 3393-3402 (1998); Boyd et al . Molec. Immunol. 32: 131-11-1318 (1995); and Tsuchiya et al . J. Rheumatol. 16: 285-290 (1989)). Hu4D5 is RITUXAN, an anti-CD20 mouse / human chimeric IgG1 Without binding better to human C1q (FIG. 15, 16) (Idusogie et al. J. Immunol. 164: 4178-4184 (2000)), the lack of fucose affects the ability of Hu4D5 to interact with human C1q. (FIG. 15, 16). In addition, the presence or absence of fucose has not been shown to affect binding of IgG1 to FcRn.
[517] Example 3
[518] Antibody-dependent Cytotoxicity (ADCC)
[519] The effect of lack of fucose on ADCC was assessed using Lec13-Hu4D5 IgG1 on human breast cancer cell line SK-BR-3 (Hudziak et al . Mol. Cell Biol. 9: 1165-1172 (1989)). PBMCs from two FcγRIIIA (V158 / F158) donors and FcγRIIIA (F158 / F158) donors were used as effector cells at an effector: target ratio of 30: 1. In all donors, IgG1 without fucose showed a significant improvement in ADCC compared to IgG1 with fucose (FIGS. 17-20). In particular, for all donors, the improvement in cytotoxicity was more pronounced as the concentration of antibody decreased. This may be a reflection of an improvement in dimer binding than that observed in hexamers. That is, the fucose (-) variant may be one that requires fewer mAbs on the surface of the target cell to activate binding / activity of the effector cell.
[520] Human monocytes express FcγRI, FcγRIIA, FcγRIIB, and only a subset expresses FcγRIIIA. ADCC was purified with agonist: target ratios of 20: 1, and 5: 1, because the lack of fucose did not affect binding to FcγRI, but had a small effect on binding to FcγRIIA (R131) and FcγRIIB. Human monocytes were used as effector cells. Purification of monocytes is more difficult than purification of PBMCs, thus making ADCC analysis more difficult. As in PBMC ADCC, the effect was less pronounced and the ability of monocytes to kill target cells was reduced than PBMCs, but monocyte ADCC showed improved cytotoxicity against IgG1 (FIGS. 21-22).
[521] Example 4
[522] ADCC Activity of Fc Variant Antibodies
[523] The following experiments were compared: (1) Hu4D5 (Hu4D5 CHO-S) expressed in CHO cells, (2) Hu4D5 fucose deficient variants (Hu4D5 Lec13) expressed in Lec13 cells, (3) Hu4D5 expressed in 293 HEK cells Triple alanine Fc domain substitution variants (Hu4D5 HEK293-AAA) and (4) Hu4D5 fucose deficient triple alanine Fc domain substitution variants (Hu4D5-Lec13-AAA) expressed in Lec13 cells.
[524] ADCC Method : Natural killer (NK) cells purified from peripheral blood of two donors were purified by negative screening using magnetic beads (Miltenyi Biotech, Auburn, CA). Donors were selected as homozygotes for alleles expressing the F158 form (F / F158) of FcγR3 (CD16) exhibiting a low degree of binding phenotype to IgG (Shields et al., J. Biol. Chem. 276 : 6591-6604 (2001). HER2-overexpressing SKBR-3 breast carcinoma cells were assayed at 1 ng / ml for 45 minutes at 25 ° C. in assay medium (50:50 Hams F12: DMEM, containing 1% inactivated small sun serum and 10 mM Hepes buffer). After opsonization with each antibody, treatment was performed for 5 hours in a humidified 37 ° C., CO ₂ incubator with varying concentrations of NK cells at an agent to target ratio (E: T) of 10: 1 to 0.156. Cytotoxicity was measured using the release of lactic acid dehydrogenase (LDH) using a commercial kit (Roche Diagnostics, Indianapolis, IN).
[525] Indirect immunofluorescence staining method of NK cells: Purified NK cells were stained at 4 ° C. for 30 minutes using 2 μg / ml of each Hu4D4 variant (phosphate buffered saline, 0.1% bovine serum albumin, 0.01). Incubated in% sodium azide). Cells were washed three times and phycoerythrin conjugated mouse mab anti-human CD56 (Pharminogen, San Diego, Calif.) And FITC-F (ab ') ₂ goat anti-human IgG (F (ab') ₂ specific (Jakson Immunoresearch, West Grove, PA) for an additional 30 minutes incubation at 4 ° C. Cells were analyzed by two-color immunofluorescence staining on a FACScan ™ flow cytometer (BDBiosciences, San Jose, Calif.).
[526] CONCLUSIONS : In both donors (5356 and 7580), ADCC activity was increased at twice the E / T ratio of Hu4D5-Lec13-AAA form of Hu4D5 compared to Hu4D5-Lec13 (Tables 26 and 27). This suggests a synergistic improvement of FcγRIII binding / ADCC in fucose deficient triple alanine Fc variants. This increased binding was confirmed by indirect immunofluorescence staining of NK cells in donor 5365 in CD56 / CD16 expressing NK cells (see FIGS. 24 and 25).
[527] Although the present invention has been described in connection with specific embodiments thereof, it will be understood that further modifications are possible. This application is intended to cover any variations, uses, or applications of the present invention, which follow the principles of the present invention and include such developments from the disclosure of the invention within the scope known or customary in the art to which the invention pertains. And, to the essential features described, as described within the appended claims,
<110> Genentech, Inc.<120> Glycoprotein Compositions<130> P1877R1PCT<140> PCT / US02 / 33739<141> 2002-10-22<150> US 60 / 337,642<151> 2001-10-25<150> US 60 / 347,694<151> 2002-01-09<160> 9<170> KopatentIn 1.71<210> 1<211> 218<212> PRT<213> homo sapiens<400> 1Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro  1 5 10 15Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys             20 25 30Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp         35 40 45Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu     50 55 60Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 65 70 75 80His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn                 85 90 95Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly            100 105 110Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu        115 120 125Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr    130 135 140Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn145 150 155 160Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe                165 170 175Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn            180 185 190Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr        195 200 205Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys    210 215<210> 2<211> 218<212> PRT<213> homo sapiens<400> 2Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro  1 5 10 15Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys             20 25 30Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp         35 40 45Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu     50 55 60Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 65 70 75 80His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn                 85 90 95Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly            100 105 110Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu        115 120 125Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr    130 135 140Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn145 150 155 160Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe                165 170 175Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn            180 185 190Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr        195 200 205Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys    210 215<210> 3<211> 217<212> PRT<213> homo sapiens<400> 3Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys  1 5 10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val             20 25 30Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr         35 40 45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu     50 55 60Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His 65 70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys                 85 90 95Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln            100 105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met        115 120 125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro    130 135 140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu                165 170 175Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val            180 185 190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln        195 200 205Lys Ser Leu Ser Leu Ser Pro Gly Lys    210 215<210> 4<211> 218<212> PRT<213> homo sapiens<400> 4Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro  1 5 10 15Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys             20 25 30Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp         35 40 45Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu     50 55 60Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu 65 70 75 80His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn                 85 90 95Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly            100 105 110Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu        115 120 125Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr    130 135 140Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn145 150 155 160Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe                165 170 175Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn            180 185 190Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr        195 200 205Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys    210 215<210> 5<211> 218<212> PRT<213> homo sapiens<400> 5Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro  1 5 10 15Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys             20 25 30Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp         35 40 45Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu     50 55 60Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 65 70 75 80His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn                 85 90 95Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly            100 105 110Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu        115 120 125Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr    130 135 140Pro Ser Asp Ile Ala Val Glu Trp Glx Ser Asn Gly Gln Pro Glu Asn145 150 155 160Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe                165 170 175Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn            180 185 190Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr        195 200 205Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys    210 215<210> 6<211> 215<212> PRT<213> Mus musculus<400> 6Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys  1 5 10 15Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val             20 25 30Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp         35 40 45Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe     50 55 60Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp 65 70 75 80Cys Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe                 85 90 95Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys            100 105 110Ala Pro Gln Val Tyr Thr Ile Pro Pro Lys Glu Gln Met Ala Lys        115 120 125Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp    130 135 140Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys145 150 155 160Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser                165 170 175Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr            180 185 190Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser        195 200 205Leu Ser His Ser Pro Gly Lys    210 215<210> 7<211> 218<212> PRT<213> Mus musculus<400> 7Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro  1 5 10 15Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys             20 25 30Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp         35 40 45Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg     50 55 60Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln 65 70 75 80His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn                 85 90 95Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly            100 105 110Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu        115 120 125Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met    130 135 140Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu145 150 155 160Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe                165 170 175Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn            180 185 190Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr        195 200 205Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys    210 215<210> 8<211> 218<212> PRT<213> Mus musculus<400> 8Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro  1 5 10 15Asn Ile Lys Asp Val Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys             20 25 30Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp         35 40 45Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg     50 55 60Glu Asp Tyr Asn Ser Thr Ile Arg Val Val Ser His Leu Pro Ile Gln 65 70 75 80His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn                 85 90 95Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly            100 105 110Leu Val Arg Ala Pro Gln Val Tyr Thr Leu Pro Pro Pro Ala Glu Gln        115 120 125Leu Ser Arg Lys Asp Val Ser Leu Thr Cys Leu Val Val Gly Phe Asn    130 135 140Pro Gly Asp Ile Ser Val Glu Trp Thr Ser Asn Gly His Thr Glu Glu145 150 155 160Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe                165 170 175Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser Lys Trp Glu Lys Thr Asp            180 185 190Ser Phe Ser Cys Asn Val Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu        195 200 205Lys Lys Thr Ile Ser Arg Ser Pro Gly Lys    210 215<210> 9<211> 218<212> PRT<213> Mus musculus<400> 9Pro Pro Gly Asn Ile Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro  1 5 10 15Lys Pro Lys Asp Ala Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys             20 25 30Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val His Val Ser Trp         35 40 45Phe Val Asp Asn Lys Glu Val His Thr Ala Trp Thr Gln Pro Arg Glu     50 55 60Ala Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Ala Leu Pro Ile Gln 65 70 75 80His Gln Asp Trp Met Arg Gly Lys Glu Phe Lys Cys Lys Val Asn Asn                 85 90 95Lys Ala Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly            100 105 110Arg Ala Gln Thr Pro Gln Val Tyr Thr Ile Pro Pro Pro Arg Glu Gln        115 120 125Met Ser Lys Lys Lys Val Ser Leu Thr Cys Leu Val Thr Asn Phe Phe    130 135 140Ser Glu Ala Ile Ser Val Glu Trp Glu Arg Asn Gly Glu Leu Glu Gln145 150 155 160Asp Tyr Lys Asn Thr Pro Pro Ile Leu Asp Ser Asp Gly Thr Tyr Phe                165 170 175Leu Tyr Ser Lys Leu Thr Val Asp Thr Asp Ser Trp Leu Gln Gly Glu            180 185 190Ile Phe Thr Cys Ser Val Val His Glu Ala Leu His Asn His His Thr        195 200 205Gln Lys Asn Leu Ser Arg Ser Pro Gly Lys    210 215

权利要求:
Claims (41)
[1" claim-type="Currently amended] A composition comprising a glycoprotein having an Fc region, wherein about 80-100% of the glycoprotein comprises a mature core carbohydrate structure lacking a fucose attached to the Fc region of the glycoprotein.
[2" claim-type="Currently amended] The composition of claim 1, wherein the glycoprotein comprises an antibody.
[3" claim-type="Currently amended] The composition of claim 1, wherein the Fc region comprises a human IgG Fc region.
[4" claim-type="Currently amended] The composition of claim 3, wherein the human IgG Fc region comprises a human IgG1, IgG2, IgG3 or IgG4 Fc region.
[5" claim-type="Currently amended] The composition of claim 1, wherein the glycoprotein binds to FcγRIII.
[6" claim-type="Currently amended] The method of claim 5, wherein the glycoprotein binds to FcγRIII with better affinity than a glycoprotein with a mature core carbohydrate structure comprising a fucose attached to the Fc region of the glycoprotein, or more effectively antibody-dependent cell-mediated cytotoxicity ( ADCC) mediated composition.
[7" claim-type="Currently amended] The composition of claim 2, wherein the antibody is a chimeric, humanized or human antibody.
[8" claim-type="Currently amended] The composition of claim 2, wherein the antibody binds to an antigen selected from the group consisting of B-cell surface markers, ErbB receptors, tumor-associated antigens and angiogenic factors.
[9" claim-type="Currently amended] The composition of claim 2, wherein the antibody binds to CD20, HER2, vascular endothelial growth factor (VEGF), CD40 or prostate hepatocyte antigen (PSCA).
[10" claim-type="Currently amended] The composition of claim 9 wherein the antibody comprises a humanized anti-HER2 antibody, a chimeric anti-CD20 antibody and a humanized anti-VEGF antibody.
[11" claim-type="Currently amended] The composition of claim 1, wherein about 90-99% of the glycoprotein comprises a mature core carbohydrate structure lacking fucose attached to the Fc region of the glycoprotein.
[12" claim-type="Currently amended] The composition of claim 1, wherein the glycoprotein is produced by Chinese Hamster Ovary (CHO) cells.
[13" claim-type="Currently amended] The composition of claim 12, wherein the CHO cells are Lec13 cells.
[14" claim-type="Currently amended] The composition of claim 1, wherein the glycoprotein is substantially devoid of bisecting N-acetylglucosamine (GlcNAc) attached to the mature core carbohydrate structure.
[15" claim-type="Currently amended] The composition of claim 1, wherein the glycoprotein has a bipartite N-acetylglucosamine (GlcNAc) attached to a mature core carbohydrate structure.
[16" claim-type="Currently amended] The composition of claim 1, wherein the glycoprotein has one or more galactose residues attached to mature core carbohydrates.
[17" claim-type="Currently amended] The composition of claim 1, wherein the glycoprotein is substantially free of one or more galactose residues attached to a mature core carbohydrate structure.
[18" claim-type="Currently amended] The composition of claim 1, wherein the glycoprotein has one or more salicylic acid residues attached to a mature core carbohydrate structure.
[19" claim-type="Currently amended] The composition of claim 1, wherein the glycoprotein is substantially devoid of one or more salicylic acid residues attached to a mature core carbohydrate structure.
[20" claim-type="Currently amended] The composition of claim 1 which is a pharmaceutical formulation.
[21" claim-type="Currently amended] The composition of claim 20 further comprising a pharmaceutically acceptable carrier.
[22" claim-type="Currently amended] The composition of claim 1, which is sterile.
[23" claim-type="Currently amended] The composition of claim 1, wherein the composition is lyophilized.
[24" claim-type="Currently amended] The composition of claim 1, wherein the glycoprotein is an immunoadhesin.
[25" claim-type="Currently amended] A composition comprising a glycoprotein having an Fc region, wherein about 51-100% of the glycoprotein comprises a mature core carbohydrate structure lacking a fucose attached to the Fc region of the glycoprotein, wherein the Fc region is combined with the native sequence Fc region. A composition comprising different amino acid sequences.
[26" claim-type="Currently amended] The composition of claim 25, wherein the Fc region comprises an amino acid substitution at one or more of amino acid positions 256, 290, 313, 326, 330, 333, 334, 360, 378 or 430 using EU numbering for the Fc region residues. .
[27" claim-type="Currently amended] The composition of claim 26, wherein the Fc region comprises amino acid substitutions at two or three residues at positions 298, 333, and 334.
[28" claim-type="Currently amended] The composition of claim 27, wherein the Fc region comprises amino acid substitutions at positions 298, 333, and 334.
[29" claim-type="Currently amended] The composition of claim 28, wherein the substitution residues at positions 298, 333, and 334 are alanine.
[30" claim-type="Currently amended] Vessel;
A label on the container; And
An article of manufacture comprising the composition of claim 1 contained in said container.
[31" claim-type="Currently amended] 31. The article of manufacture of claim 30, wherein the label on the container indicates that the composition can be used to treat cancer, autoimmune diseases, inflammatory diseases, infections, or other conditions that require the removal of cells or tissues.
[32" claim-type="Currently amended] A method of treating a mammal, comprising administering the composition according to claim 1 to the mammal in an effective amount to treat a disease or condition in the mammal which may benefit from the treatment.
[33" claim-type="Currently amended] 33. The method of claim 32, wherein the mammal is a human.
[34" claim-type="Currently amended] The method of claim 33, wherein the human expresses FcγRIII (F158).
[35" claim-type="Currently amended] 33. The method of claim 32, wherein the disease or condition is selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases, infections, or other symptoms in need of removal of cells or tissues.
[36" claim-type="Currently amended] A host cell comprising a nucleic acid encoding a glycoprotein comprising an Fc region, wherein a mature core carbohydrate structure lacking fucose having about 80-100% of the glycoprotein produced by the host cell attached to the Fc region of the glycoprotein. The host cell comprising a.
[37" claim-type="Currently amended] The host cell of claim 36, which is a Chinese hamster ovary (CHO) cell.
[38" claim-type="Currently amended] A method for producing a glycoprotein comprising culturing the host cell of claim 36 so that the nucleic acid is expressed.
[39" claim-type="Currently amended] The method of claim 38, wherein the glycoprotein comprises recovered from the host cell culture.
[40" claim-type="Currently amended] 40. The method of claim 39 comprising conjugating glycoprotein to a heterologous molecule.
[41" claim-type="Currently amended] The method of claim 40, wherein the heterologous molecule is a cytotoxic drug or enzyme.

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同族专利:

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公开号 | 公开日

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EP1443961A4|2005-11-23|

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PL213948B1|2013-05-31|

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HU0600342A3|2011-03-28|

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HU0600342A2|2006-08-28|

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CN100423777C|2008-10-08|

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BR0213761A|2005-04-12|

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IL220142D0|2012-07-31|

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EP1443961A2|2004-08-11|

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ES2326964T3|2009-10-22|

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EP1443961B1|2009-05-06|

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US20110086050A1|2011-04-14|

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JP2010248228A|2010-11-04|

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CN1607960A|2005-04-20|

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WO2003035835A3|2003-10-16|

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JP2005532253A|2005-10-27|

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AT430580T|2009-05-15|

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CA2463879A1|2003-05-01|

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WO2003035835A2|2003-05-01|

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US20080095762A1|2008-04-24|

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US20030157108A1|2003-08-21|

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US20100255013A1|2010-10-07|

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DE60232265D1|2009-06-18|

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MXPA04003798A|2004-07-30|

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IL161412D0|2004-09-27|

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NZ532526A|2007-01-26|

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US20070020260A1|2007-01-25|

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PL372709A1|2005-07-25|

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HK1076388A1|2008-12-12|

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KR100988949B1|2010-10-20|

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DK1443961T3|2009-08-24|

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AU2002337935B2|2008-05-01|

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JP2014076997A|2014-05-01|

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CA2463879C|2012-12-04|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2001-10-25|Priority to US33764201P

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2001-10-25|Priority to US60/337,642

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2002-01-09|Priority to US34769402P

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2002-01-09|Priority to US60/347,694

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2002-10-22|Application filed by 제넨테크, 인크.

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2002-10-22|Priority to PCT/US2002/033739

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2004-07-23|Publication of KR20040066105A

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2010-02-04|First worldwide family litigation filed

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2010-10-20|Application granted

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2010-10-20|Publication of KR100988949B1

优先权:

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申请号 | 申请日 | 专利标题

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US33764201P| true| 2001-10-25|2001-10-25|

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US60/337,642|2001-10-25|

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US34769402P| true| 2002-01-09|2002-01-09|

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US60/347,694|2002-01-09|

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PCT/US2002/033739|WO2003035835A2|2001-10-25|2002-10-22|Glycoprotein compositions|