Methods for in vitro glycoengineering of antibodies

Abstract

Herein is reported a method of producing an antibody comprising the step of forming an antibody-antibody light chain affinity ligand complex by applying a solution comprising an antibody to an immobilized antibody light chain affinity ligand wherein the antibody light chain affinity ligand is immobilized on a solid phase, the complex formed in the previous step is incubated with one or more enzymes to modify glycosylation of the antibody, thereby producing the antibody.

Description

[0001] This application is a divisional application of Chinese application No. 201780079057.7, whose application date is December 19, 2017 and whose invention name is “Method for in vitro glycoengineering of antibodies”.

[0002] The present invention belongs to the field of antibody engineering.Herein an in vitro glycoengineering method for glycosylation in the Fc region of antibodies is reported in more detail. Background Art

[0003] IgG is the most abundant antibody isotype, and IgG1 antibody is the subclass showing the most significant degree and effector function group (array). IgG1 antibody is the most commonly used antibody in immunotherapy, and ADCC and CDC are generally considered to be important. In the antibody structure, CH2 domain and IgG hinge region play a major role in the antibody effector function mediated by Fc. Each CH2 domain includes a conserved glycosylation site (according to the EU index number of Kabat) at the asparagine residue at about 297, wherein the polysaccharide moiety is covalently bound (Wright, A. and Morrison, SL, TIBTECH 15 (1997) 26-32). In mature IgG molecules, polysaccharides are buried between CH2 domains, affecting the tertiary structure of IgG molecules.

[0004] The glycans in the Fc region of antibodies are mainly highly heterogeneous complex biantennary structures. Although other non-conserved glycosylation sites may exist in the Fab region of antibodies, the effects of antibody glycosylation on its effector function have been attributed to Fc region glycosylation.

[0005] It is known that the N-linked glycans present in the Fc region of antibodies are essential for antibody-mediated effector functions such as ADCC (Lively, MR et al. Glycobiol. 8 (1995) 813-822; Jefferis R. et al. Immunol Rev. 163 (1998) 59-76). It has been shown that the composition of N-linked glycans affects the Fc region structure of IgG molecules, thereby changing antibody effector functions such as Fc-receptor binding, ADCC activity and CDC activity (Presta, L., Curr. Opin. Struct. Biol. 13 (2003) 519-525).

[0006] In IgG antibodies expressed in a recombinant expression system (e.g., by expression in a prokaryotic or eukaryotic host cell), the N-linked glycan structure is different between individual antibody molecules. Thus, antibodies produced in a recombinant expression system can be considered an "antibody population" (a term used further herein) in which the antibodies have identical amino acid sequences but exhibit heterogeneity in the N-linked glycan pattern of their Fc regions.

[0007] It is known that the composition of Fc region glycans is different between different host cell species used to express recombinant antibodies. Two commonly used host cell lines for recombinantly expressed antibodies are Chinese hamster ovary cells (CHO cells) and mouse myeloma cells (e.g., sp2 / 0, P3X63Ag8.653, NSO). CHO cells express recombinant antibodies that are essentially free of terminal sialic acid residues, while most of the glycan patterns are fucosylated. In contrast, the antibody population produced by mouse myeloma cells has up to 50% (relative frequency) of sialic acid residues but has fewer fucose residues.

[0008] Some terminal residues of the glycan structure are known to affect IgG effector functions. The presence of terminal fucose residues is known to contribute to reduced FcγRIIIa binding and reduced ADCC. Therefore, antibodies lacking terminal fucose residues ("afucosylated" antibodies) are associated with increased ADCC mediated by the antibody population. Although the effect of afucosylation on improved ADCC mediation is widely accepted in the art, the role of Fc region galactosylation in ADCC mediation is controversial. Some studies have shown that galactosylation has no effect on ADCC (Boyd, PN, et al. Mol Immunol. 32 (1995) 1311-1318; Hodoniczky, J., et al. Biotechnol. Prog. 21 (2005) 1644-1652; Raju, TS, Curr. Opin. Immunol. 20 (2008) 471-478); while other studies have reported that galactosylation of IgG increases FcγRIIIa binding (Houde, D., et al., Mol. Cell. Proteom. 9 (2010) 1716-1728; Kumpel, BM, et al., Hum. Antibod. Hybridom. 6 (1995) 82-88; Thomann, M., et al., Mol. Immunol. 73 (2016) 69-75).

[0009] Currently, the engineering of IgG molecules to improve antibody-mediated ADCC focuses on regulating the fucosylation of IgG molecules. Non-fucosylation of recombinantly expressed IgG can be achieved by expressing the antibody in genetically engineered host cells, for example, Lecl3 CHO cells that lack protein fucosylation or gene knockout cell lines, such as CHO cells with the α-1,6-fucosyltransferase (FUT8) gene knocked out.

[0010] However, antibodies produced by current expression systems (e.g., CHO cells) exhibit heterogeneous glycan patterns, resulting in different glycan species distribution variations within different batches of produced antibodies. Therefore, there is still a need to customize the effector functions of recombinant IgG antibodies, and in particular, there is a need to provide methods for improving ADCC mediated by therapeutic antibodies.

[0011] In WO2011 / 012297, a method for producing immunoglobulins or immunoglobulin fragments having a defined sugar structure is reported, which comprises the following steps: providing an affinity chromatography column eluate containing the immunoglobulin or the immunoglobulin fragment, incubating the affinity chromatography column eluate with (a1,3) galactosidase (EC3.2.1.22) of plant origin (e.g., from green coffee beans), applying the incubated affinity chromatography column eluate to a protein A chromatography material, and recovering the immunoglobulin or immunoglobulin fragment from the protein A chromatography material, thereby producing an immunoglobulin or immunoglobulin fragment having a defined sugar structure.

[0012] In WO 2015 / 123754, an enzymatic method for reconstructing a heterogeneous glycoform antibody sample bound by an affinity ligand into a substantially homogeneous single desired glycoform antibody sample is provided, the antibody sample is used for therapeutic purposes, and a kit for implementing the method is provided. The method for enzymatically changing the Fc region of an antibody bound by an affinity ligand from a heterogeneous glycoform to a substantially homogeneous single glycoform comprises: contacting the heterogeneous glycoform antibody bound by the affinity ligand with a reaction buffer designed for specific glycoform modification for a sufficient time, and under conditions that modify the glycoform of the Fc region to a substantially homogeneous single form; optionally adding one or more sugar nucleotides and / or cofactors; releasing the substantially homogeneous single glycoform antibody sample from the affinity ligand. The present invention also includes biopharmaceuticals for treating cancer and immune disorders, comprising enzymatically produced single glycoform mAbs and polyclonal antibodies, and compositions comprising single glycoform antibodies as biopharmaceuticals.

[0013] Galactose-engineered recombinant antibodies of the IgG1 isotype, methods for producing the antibodies and uses thereof are reported in WO 2016 / 037947. Summary of the invention

[0014] In one embodiment of a recombinantly produced monoclonal antibody, herein is reported a method for in vitro glycoengineering of an antibody, wherein the modification of glycosylation is performed while the antibody is bound to a solid phase comprising an affinity ligand for the antibody light chains. The method as reported herein is in particular an improved method for modifying immobilized antibodies.

[0015] In the method reported herein, a monoclonal antibody is bound to an affinity ligand, especially to an antibody light chain affinity ligand, for enzymatic modification and subsequent release as a monoclonal antibody preparation with a modified sugar structure. The modification can be carried out on an N-glycosylation site in a Fab fragment or in an Fc region. Surprisingly, it was found that antibodies bound to an antibody light chain affinity ligand can be effectively enzymatically modified, just as if the antibody were in solution. The method reported herein can be easily integrated into any antibody purification method, thereby providing a new, effective and cost-effective method for in vitro antibody glycan modification.

[0016] The method reported herein can be used for the modification of any monoclonal antibody without modifying the previous upstream production process steps. The method reported herein can be integrated into existing methods as a single in vitro modification and purification step. Intrinsically, there is no need to significantly change the existing antibody production cell lines, because the method as reported herein provides sugar structure modification during downstream processing.

[0017] Using the methods reported herein, enzymes used for antibody glycosylation modification can be removed from antibody preparations, resulting in improved preparations.

[0018] One aspect as reported herein is a method for the enzymatic preparation / production of an antibody with modified (substantially homogeneous) glycosylation at the N-glycosylation sites, wherein said antibody is bound to an antibody light chain affinity ligand during the enzymatic modification.

[0019] One aspect as reported herein is a method for enzymatically modifying glycosylation at N-glycosylation sites of an antibody to substantially homogeneous glycosylation, wherein the antibody is bound to an antibody light chain affinity ligand during the enzymatic modification.

[0020] In one embodiment of all aspects, the method comprises the following steps:

[0021] - incubating a monoclonal antibody having glycosylation at the N-glycosylation site to which the antibody light chain affinity ligand binds with one or more enzymes for a sufficient time and under conditions suitable to modify the glycosylation at the N-glycosylation site to a defined (substantially homogeneous) form (homoglycosylation).

[0022] In one embodiment of all aspects, the method comprises, before the incubation step, the step of

[0023] - allowing a monoclonal antibody having glycosylation at an N-glycosylation site to bind to an antibody light chain affinity ligand, and

[0024] Steps after the incubation step

[0025] - Release of antibodies with defined (substantially homogeneous) glycosylation at N-glycosylation sites from antibody light chain affinity ligands.

[0026] In one embodiment of all aspects, the method comprises the following steps in the following order

[0027] - applying a (buffered) solution comprising an antibody having glycosylation at an N-glycosylation site to an antibody light chain affinity ligand bound to a solid phase (antibody light chain affinity ligand chromatography material), whereby the antibody binds to the ligand (producing a ligand-bound antibody),

[0028] - optionally washing the solid phase with a buffer solution,

[0029] - Enzymatic modification of glycosylation at the N-glycosylation sites of antibodies by:

[0030] - applying a first (buffered) solution comprising a first glycosylation modifying enzyme (and a first activated sugar residue) for a sufficient time and under conditions suitable for enzymatic modification of the ligand-bound antibody, optionally washing the modified ligand-bound antibody, applying a second (buffered) solution comprising a second glycosylation modifying enzyme (and a second activated sugar) for a sufficient time and under conditions suitable for enzymatic modification of the modified ligand-bound antibody, optionally washing the ligand-bound antibody modified twice, or

[0031] - applying a first (buffered) solution comprising a first glycosylation modifying enzyme (and a first activated sugar) for a sufficient time and under conditions suitable for at least partially enzymatically modifying the ligand-bound antibody, and after a determined period of time, applying a second (buffered) solution comprising a second glycosylation modifying enzyme (and a second activated sugar) for a sufficient time and under conditions suitable for enzymatically modifying the modified ligand-bound antibody, and optionally washing the ligand-bound antibody modified twice, or

[0032] - applying a (buffered) solution comprising the first and second glycosylation modifying enzymes (and the first and second activated sugars) for a sufficient time and under conditions suitable for enzymatic modification of the ligand-bound antibody, optionally washing the modified ligand-bound antibody,

[0033] - Release of antibodies with defined glycosylation at N-glycosylation sites from antibody light chain affinity ligands.

[0034] The antibody used in the methods as reported herein may be any antibody or antibody fragment, including Fab fragments, single chain antibodies, multispecific antibodies and antibody fusions, as long as it contains an N-glycosylation site.

[0035] In one embodiment of all aspects the N-glycosylation site is in the Fab or in the Fc region.

[0036] Thus, in one embodiment of all aspects as reported herein the antibody is chosen from the group of antibodies consisting of an antibody Fab fragment, a full length antibody, a bivalent monospecific antibody, a bispecific antibody, a bivalent bispecific antibody, a trivalent bispecific antibody, a tetravalent bispecific antibody, a trivalent trispecific antibody and a tetravalent tetraspecific antibody.

[0037] In one embodiment, the antibody is a bivalent monospecific antibody.

[0038] In one embodiment the antibody is a bivalent or trivalent or tetravalent bispecific antibody.

[0039] In one embodiment, the antibody is a chimeric or humanized or human antibody.

[0040] In one embodiment, the antibody is a polyclonal antibody preparation.

[0041] In one embodiment, the antibody is a monoclonal antibody.

[0042] In one embodiment of all aspects as reported herein the antibody (preparation) is of human IgG class.In one embodiment the antibody is of human IgG1 or IgG4 subclass.

[0043] In one embodiment of all aspects as reported herein the determined glycosylation is a glycosylation selected from the group consisting of a G2 glycoform, a G0 glycoform, an M3 glycoform, an S2 glycoform, an A2B glycoform, an A2BG2 glycoform and an S1 glycoform.

[0044] In one embodiment of all aspects as reported herein the defined glycosylation is a glycosylation selected from the group consisting of galactose as terminal sugar, GlcNAc as terminal sugar, mannose as terminal sugar and sialic acid as terminal sugar.

[0045] In one embodiment, the antibody is a recombinantly produced antibody.

[0046] One aspect as reported herein is an antibody produced using the method as reported herein.

[0047] One aspect as reported herein is a pharmaceutical formulation comprising an antibody with defined glycosylation produced by the method as reported herein.

[0048] Another aspect of the invention is a method for recombinantly producing an antibody with defined glycosylation at an N-glycosylation site, comprising the steps of:

[0049] a) recombinantly producing the antibody (of IgG1 isotype) in (mammalian or CHO) cells comprising a nucleic acid encoding the antibody, to obtain the antibody having glycosylation at the N-glycosylation site,

[0050] b) isolating (recovering and optionally purifying) antibodies having heterogeneous glycosylation at N-glycosylation sites,

[0051] c) enzymatically modifying an antibody having glycosylation at an N-glycosylation site using a galactosyltransferase and / or a sialyltransferase to obtain an antibody having defined glycosylation at an N-glycosylation site comprising a relative amount of at least 70% of a digalactosylated antibody (G2F glycoform) at the N-glycosylation site (wherein 100% corresponds to the amount of G0F, G1F and G2F glycoforms), followed by separating the modified antibody from the one or more enzymes,

[0052] d) optionally purifying the modified antibody by one or more chromatography steps,

[0053] This generates antibodies with defined glycosylation at the N-glycosylation sites.

[0054] In one embodiment of all aspects as reported herein the first glycosylation modifying enzyme is a galactosyltransferase.

[0055] In one embodiment of all aspects as reported herein the first glycosylation modifying enzyme is a galactosyltransferase and the second glycosylation modifying enzyme is a sialyltransferase.

[0056] In one embodiment, the galactosyltransferase is β4GalT1.

[0057] In one embodiment, the sialyltransferase is ST6.

[0058] In one embodiment, the sialyltransferase is ST6Gal1 or ST6Gal2.

[0059] In one embodiment, the (first) buffer solution comprises UDP-Gal.

[0060] In one embodiment, the (second) buffer solution comprises CMP-NANA.

[0061] In one embodiment, the incubation is at room temperature (20-25°C, preferably about 22°C).

[0062] In one embodiment, the incubation is at 25°C.

[0063] In one embodiment, the incubation is at 37°C.

[0064] In one embodiment, the incubation lasts from 7 to 48 hours.

[0065] In one embodiment of all aspects as reported herein, the solution comprises a chromatographically purified antibody, the (first) glycosylation modifying enzyme is GalT1, and the incubation with the (first) glycosylation modifying enzyme is for 24 hours at 20-27° C. or at 37° C. In one embodiment, the incubation is at room temperature (about 22° C.).

[0066] In one embodiment, the solution comprises a chromatographically purified antibody, the (second) glycosylation modifying enzyme is ST6, and the incubation with the (second) glycosylation modifying enzyme is for 24 hours at 20-27° C. or 37° C. In one embodiment, the incubation is at room temperature (about 22° C.).

[0067] In one embodiment, the solution is a buffered, cell-free culture supernatant containing an antibody, the first glycosylation modification enzyme is GalT1, the second glycosylation modification enzyme is ST6, which is added 6 to 24 hours after the first glycosylation modification enzyme, preferably 24 hours later, and the total incubation time is 24 hours to 48 hours, preferably 30 hours at 20-27° C. or 37° C. In one embodiment, the incubation is carried out at room temperature (about 22° C.).

[0068] One aspect as reported herein is a method for producing an antibody or a fragment thereof comprising the following steps in the following order:

[0069] - providing a cell comprising a nucleic acid encoding an antibody or a fragment thereof comprising at least one antibody light chain,

[0070] - culturing the cells under conditions suitable for the expression of an antibody or a fragment thereof having glycosylation at an N-glycosylation site, (the fragment comprising at least a light chain that can be specifically bound by an antibody light chain affinity chromatography material used in one of the following steps),

[0071] - recovering the antibody or fragment thereof from the cells or culture medium,

[0072] - applying a solution comprising the antibody or fragment thereof to an antibody light chain affinity chromatography column under conditions suitable for binding of the antibody or fragment thereof to the affinity chromatography material,

[0073] - modifying the glycosylation of an antibody or a fragment thereof at an N-glycosylation site using the method as reported herein, and

[0074] - recovering the modified antibody or fragment thereof having defined glycosylation at the N-glycosylation site from the affinity chromatography material,

[0075] Thereby producing antibodies.

[0076] In one embodiment, the method comprises the following steps as the final step:

[0077] - Purification of the modified antibody or fragment thereof using one to three chromatography steps.

[0078] In one embodiment of all aspects the N-glycosylation site is in the Fab or in the Fc region.

[0079] Detailed Description of the Invention

[0080] definition

[0081] As used herein, the amino acid positions of all constant regions and domains of heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), referred to herein as "numbered according to Kabat". Specifically, the Kabat numbering system of Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pages 647-660) is used for the light chain constant domains CL of kappa and lambda isotypes. Specifically, the Kabat EU index numbering system (see pages 661-723) is used for constant heavy chain domains (CH1, hinge, CH2 and CH3, further illustrated herein by being referred to as "numbered according to the Kabat EU index").

[0082] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. Likewise, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" are used interchangeably.

[0083] It is well known to those skilled in the art that an amino acid sequence (e.g., an amino acid sequence of a polypeptide) is converted to a corresponding nucleic acid sequence encoding the amino acid sequence. Therefore, a nucleic acid is characterized by its nucleic acid sequence consisting of individual nucleotides, and also by the amino acid sequence of a polypeptide encoded therefrom.

[0084] The term "about" means a range of + / - 20% of the value thereafter. In one embodiment, the term "about" means a range of + / - 10% of the value thereafter. In one embodiment, the term "about" means a range of + / - 5% of the value thereafter.

[0085] The term "antibody" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0086] The term "antibody-dependent cellular cytotoxicity (ADCC)" is a function mediated by Fc receptor binding, and refers to the lysis of target cells by the antibodies reported herein in the presence of effector cells. In one embodiment, in the presence of effector cells such as freshly isolated PBMCs (peripheral blood mononuclear cells) or effector cells purified from buffy coats (such as monocytes or NK (natural killer) cells), by treating erythrocytes expressing CD19 (e.g., K562 cells expressing recombinant human CD19) preparations with antibodies reported herein, ADCC is measured. Target cells are labeled with Cr-51 and subsequently incubated with antibodies. Labeled cells are incubated with effector cells, and the Cr-51 released by the supernatant is analyzed. Controls include incubation of target endothelial cells with effector cells, but without antibodies. The ability of antibodies to induce the initial step of mediating ADCC is studied by measuring its binding to cells expressing Fcγ receptors (such as cells or NK cells (essentially expressing FcγRIIIA) expressing FcγRI and / or FcγRIIA). In a preferred embodiment, binding to FcγR on NK cells is measured.

[0087] "Antibody fragment" refers to a molecule that is not an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0088] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0089] The "type" of an antibody refers to the type of constant domain or constant region possessed by the antibody heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0090] The term "complement dependent cytotoxicity (CDC)" refers to cell lysis induced by the antibodies reported herein in the presence of complement. In one embodiment, CDC is measured by treating human endothelial cells expressing CD19 with the antibodies reported herein in the presence of complement. In one embodiment, the cells are labeled with calcein. If the antibody induces 20% or more target cell lysis at a concentration of 30 μg / ml, CDC is present. Binding to complement factor C1q can be measured in ELISA. In this assay, in principle, ELISA plates are coated with antibodies of a concentration range, to which purified human C1q or human serum is added. C1q binding is detected by an antibody to C1q and then by a peroxidase-labeled conjugate. The detection of binding (maximum binding Bmax) is measured as a peroxidase substrate. Optical density of (2,2'-azino-di-[3-ethylbenzothiazoline-6-sulfonate]) at 405 nm (OD405).

[0091] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody type. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0092] Fc receptor binding-dependent effector functions can be mediated by the interaction of the Fc region of an antibody with an Fc receptor (FcR), which is a specialized cell surface receptor on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate the removal of antibody-coated pathogens by phagocytosis of immune complexes, as well as the lysis of erythrocytes and various other cellular targets (e.g., tumor cells) coated with the corresponding antibodies by antibody-dependent cell-mediated cytotoxicity (ADCC) (see, e.g., Van de Winkel, JG and Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined according to their specificity for immunoglobulin isotypes: the Fc receptors of IgG antibodies are called FcγRs. Fc receptor binding is described in, e.g., Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492; Capel, PJ, et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J. Lab. Clin. Med. 126 (1995) 330-341; and Gessner, JE, et al., Ann. Hematol. 76 (1998) 231-248.

[0093] Cross-linking of receptors (FcγRs) on the Fc region of IgG antibodies triggers a variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity and release of inflammatory mediators, as well as regulation of immune complex clearance and antibody production. In humans, three classes of FcγRs have been characterized, which are:

[0094] - FcγRI (CD64) binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils. Modification of the Fc region of IgG at at least one of amino acid residues E233-G236, P238, D265, N297, A327 and P329 (numbering according to the EU index of Kabat) reduces binding to FcγRI. Substitution of IgG2 residues at positions 233-236 with IgG1 and IgG4 reduces binding to FcγRI by 10 3 times, and eliminated the response of human monocytes to antibody-sensitized erythrocytes (Armour, KL, et al., Eur. J. Immunol. 29 (1999) 2613-2624).

[0095] -FcγRII (CD32) binds complexed IgG with moderate to low affinity and is widely expressed. This receptor can be divided into two subtypes, FcγRIIA and FcγRIIB. FcγRIIA is present on many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and appears to be able to activate the killing process. FcγRIIB appears to play a role in the inhibition process, and is present on B cells, macrophages, mast cells, and eosinophils. On B cells, it appears to play a role in inhibiting further production of immunoglobulins and isotype conversion to, for example, the IgE class. On macrophages, FcγRIIB inhibits phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, type B may help inhibit the activation of these cells by binding of IgE to their independent receptors. For example, reduced binding of FcγRIIA was found for antibodies comprising an IgG Fc region with a mutation in at least one of amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (numbering according to the EU index of Kabat).

[0096] -FcγRIII (CD16) binds IgG with medium to low affinity, and it exists in two types. FcγRIIIA is present on NK cells, macrophages, eosinophils, and some monocytes and T cells, and mediates ADCC. FcγRIIIB is highly expressed on neutrophils. For example, it was found that for antibodies with mutated IgG Fc regions on at least one of amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376 (according to the EU index numbering of Kabat), the binding of FcγRIIIA is reduced.

[0097] The location of the binding site on human IgG1 for Fc receptors, the above mutation sites and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, RL, et al. J. Biol. Chem. 276 (2001) 6591-6604.

[0098] As used herein, the term "Fc receptor" refers to an activating receptor characterized by the presence of a cytoplasmic ITAM sequence associated with the receptor (see, e.g., Ravetch, JV and Bolland, S., Annu. Rev. Immunol. 19 (2001) 275-290). These receptors are FcγRI, FcγRIIA and FcγRIIIA. The term "does not bind to FcγR" means that at an antibody concentration of 10 μg / ml, the binding of the antibodies reported herein to NK cells is 10% or less of the binding of the anti-OX40L antibody LC.001 as reported in WO2006 / 029879.

[0099] Although IgG4 showed reduced FcR binding, antibodies of other IgG subclasses showed strong binding. However, Pro238, Asp265, Asp270, Asn297 (Fc carbohydrate deletion), Pro329 and 234, 235, 236 and 237Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435 residues also reduced FcR binding if altered (Shields, RL, et al. J. Biol. Chem. 276 (2001) 6591-6604; Lund, J., et al., FASEB J. 9 (1995) 115-119; Morgan, A., et al., Immunology 86 (1995) 319-324; and EP 0 307 434).

[0100] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, or from Ala 231 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present.

[0101] The antibodies reported herein comprise an Fc region, in one embodiment, an Fc region derived from human origin. In one embodiment, the Fc region comprises all parts of a human constant region. The Fc region of an antibody is directly involved in complement activation, C1q binding, C3 activation and Fc receptor binding. Although the effect of an antibody on the complement system depends on certain conditions, binding to C1q is caused by a defined binding site in the Fc region. Such binding sites are known in the prior art and are described, for example, in Lukas, TJ, et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and Cebra, JJ, Mol. Immunol. 16 (1979) 907-917; Burton, DR, et al., Nature 288 (1980) 338-344; Thommesen, JE, et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, EE, et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75 (2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324; and EP 0 307 434. Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbered according to the EU index of Kabat). Antibodies of subclasses IgG1, IgG2 and IgG3 typically show complement activation, C1q binding and C3 activation, while IgG4 does not activate the complement system, does not bind C1q and does not activate C3. "Fc region of an antibody" is a term well known to those skilled in the art and is defined based on papain cleavage of an antibody. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region is a human IgG4 subclass comprising mutations S228P and / or L235E and / or P329G (numbered according to the EU index of Kabat). In one embodiment the Fc region is of human IgGl subclass comprising the mutations L234A and L235A and optionally P329G (numbering according to EU index of Kabat).

[0102] The term "wild-type Fc region" refers to an amino acid sequence that is identical to the amino acid sequence of a naturally occurring Fc region. Wild-type human Fc regions include native human IgG1 Fc regions (non-A and A allotypes), native human IgG2 Fc regions, native human IgG3 Fc regions, and native human IgG4 Fc regions, as well as naturally occurring variants thereof. Wild-type Fc regions are represented in SEQ ID NO: 01 (IgG1, Caucasian allotype), SEQ ID NO: 02 (IgG1, African American allotype), SEQ ID NO: 03 (IgG2), SEQ ID NO: 04 (IgG3), and SEQ ID NO: 05 (IgG4).

[0103] Variant (human) Fc regions are defined by the amino acid mutations they contain. Thus, for example, the term P329G represents a variant Fc region having a proline to glycine mutation at amino acid position 329 relative to the parent (wild-type) Fc region (numbered according to the EU index of Kabat). The identity of the wild-type amino acid may be unspecified, in which case the aforementioned variant is referred to as 329G.

[0104] The polypeptide chain of the wild-type human Fc region of the IgG1 subclass has the following amino acid sequence, which starts at a cysteine ​​residue at position 227 and ends at a glycine residue at position 446:

[0105]

[0106] The polypeptide chain of the variant human Fc region of the IgG1 subclass with the mutations T366S, L368A and Y407V has the following amino acid sequence:

[0107]

[0108] The polypeptide chain of the variant human Fc region of the IgG1 subclass with the mutation T366W has the following amino acid sequence:

[0109]

[0110] The polypeptide chain of the variant human Fc region of the IgG1 subclass with the mutations L234A and L235A has the following amino acid sequence:

[0111]

[0112] The polypeptide chain of the variant human Fc region of the IgG1 subclass with the mutations L234A, L235A, T366S, L368A and Y407V has the following amino acid sequence:

[0113]

[0114] The polypeptide chain of the variant human Fc region of the IgG1 subclass with the mutations L234A, L235A and T366W has the following amino acid sequence:

[0115]

[0116] The polypeptide chain of the variant human Fc region of the IgG1 subclass with the mutations L234A, L235A and P329G has the following amino acid sequence:

[0117]

[0118] The polypeptide chain of the variant human Fc region of the IgG1 subclass with the mutations L234A, L235A, P329G, T366S, L368A and Y407V has the following amino acid sequence:

[0119]

[0120] The polypeptide chain of the variant human Fc region of the IgG1 subclass with the mutations L234A, L235A, P329G and T366W has the following amino acid sequence:

[0121]

[0122] The polypeptide chain of the variant human Fc region of the IgG1 subclass with the mutations L234A, L235A, P329G, Y349C, T366S, L368A and Y407V has the following amino acid sequence:

[0123]

[0124] The polypeptide chain of the variant human Fc region of the IgG1 subclass with the mutations L234A, L235A, P329G, S354C and T366W has the following amino acid sequence:

[0125]

[0126] The polypeptide chain of the variant human Fc region of the IgG1 subclass with the mutations L234A, L235A, P329G, S354C, T366S, L368A and Y407V has the following amino acid sequence:

[0127]

[0128] The polypeptide chain of the variant human Fc region of the IgG1 subclass with the mutations L234A, L235A, P329G, Y349C and T366W has the following amino acid sequence:

[0129]

[0130] The polypeptide chain of the variant human Fc region of the IgG1 subclass with mutations I253A, H310A and H435A has the following amino acid sequence:

[0131]

[0132] The polypeptide chain of the variant human Fc region of the IgG1 subclass with the mutations H310A, H433A and Y436A has the following amino acid sequence:

[0133]

[0134] The polypeptide chain of the variant human Fc region of the IgG1 subclass with the mutations M252Y, S254T and T256E has the following amino acid sequence:

[0135]

[0136] The polypeptide chain of the wild-type human Fc region of the IgG4 subclass has the following amino acid sequence:

[0137]

[0138] The polypeptide chain of the variant human Fc region of the IgG4 subclass with the mutations S228P and L235E has the following amino acid sequence:

[0139]

[0140] The polypeptide chain of the variant human Fc region of the IgG4 subclass with the mutations S228P, L235E and P329G has the following amino acid sequence:

[0141]

[0142] The polypeptide chain of the variant human Fc region of the IgG4 subclass with the mutations S228P, L235E, P329G, T366S, L368A and Y407V has the following amino acid sequence:

[0143]

[0144] The polypeptide chain of the variant human Fc region of the IgG4 subclass with the mutations S228P, L235E, P329G and T366W has the following amino acid sequence:

[0145]

[0146] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0147] The term "glycan" means a polysaccharide or an oligosaccharide. Glycan is also used herein to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide, or proteoglycan. Glycans are generally composed only of monosaccharides with beta-glycosidic bonds between the monosaccharides. Glycans may be homopolymers or heteropolymers of monosaccharide residues and may be straight-chain or branched.

[0148] The term "glycosyltransferase" refers to an enzyme capable of transferring a monosaccharide moiety from a sugar nucleotide to an acceptor molecule (such as a sugar molecule in an oligosaccharide). Examples of such glycosyltransferases include, but are not limited to, galactosyltransferases and sialyltransferases.

[0149] The term "hinge region" refers to a portion of an antibody heavy chain polypeptide that connects the CH1 domain and the CH2 domain in a wild-type antibody heavy chain, e.g., from about position 216 to about position 230 according to the EU numbering system of Kabat, or from about position 226 to about position 230 according to the EU numbering system of Kabat. Hinge regions of other IgG subclasses can be determined by aligning the hinge region cysteine ​​residues with the IgG1 subclass sequence.

[0150] The hinge region is usually a dimer molecule consisting of two polypeptides with the same amino acid sequence. The hinge region usually contains about 25 amino acid residues and is flexible, allowing the associated target binding sites to move independently. The hinge region can be subdivided into three domains: upper, middle and lower hinge domains (see, e.g., Roux, et al., J. Immunol. 161 (1998) 4083).

[0151] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of non-human antibodies, and all or substantially all of the FRs correspond to those of human antibodies. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0152] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that comprises a segment of amino acid residues that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or a segment of amino acid residues that form structurally defined loops ("hypervariable loops"), and / or a segment of amino acid residues that contain antigen contact residues ("antigen contacts"). Typically, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3).

[0153] HVR includes

[0154] (a) hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3) (Chothia, C. and Lesk, AM, J. Mol. Biol. 196 (1987) 901-917);

[0155] (b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242);

[0156] (c) antigenic contacts present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and

[0157] (d) a combination of (a), (b) and / or (c), comprising amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0158] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0159] An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

[0160] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0161] The term "light chain" refers to the shorter polypeptide chain of a natural IgG antibody. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, called kappa (κ) and lamba (λ), see SEQ ID NO: 27 for the human kappa light chain constant domain and SEQ ID NO: 28 for the human lambda light chain constant domain.

[0162] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody group, that is, each antibody forming a group is identical and / or binds to the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or mutations produced in the production process of monoclonal antibody preparations, and these variants are usually present in a small amount. Contrary to the polyclonal antibody preparations that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" represents that the feature of an antibody is obtained from a substantially homogeneous antibody group, and should not be construed as requiring antibody to be produced by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0163] "Natural antibody" refers to a naturally occurring immunoglobulin molecule with different structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, which is composed of two identical light chains and two identical heavy chains connected by disulfide bonds. From N-terminal to C-terminal, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3), wherein the hinge region is located between the first and second constant domains. Similarly, from N-terminal to C-terminal, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the antibody light chain can be assigned to one of the two types called kappa (κ) and lamba (λ).

[0164] The term "N-linked oligosaccharide" refers to an oligosaccharide that is linked to the peptide backbone on an asparagine amino acid residue via an asparagine-N-acetylglucosamine bond. N-linked oligosaccharides are also referred to as "N-glycans". All N-linked oligosaccharides have a common pentasaccharide core of Man3GlcNAc2. They differ in the presence of peripheral sugars such as N-acetylglucosamine, galactose, N-acetylgalactosamine, fucose, and sialic acid and the number of side chains (also referred to as antennae). Optionally, the structure may also contain a core fucose molecule and / or a xylose molecule.

[0165] The term "O-linked oligosaccharide" refers to an oligosaccharide attached to the peptide backbone at a threonine or serine amino acid residue.

[0166] The term "sialic acid" refers to any member of a family of nine-carbon carboxylated sugars. The most common member of the sialic acid family is N-acetylneuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactosylnonylpyran-1-one acid (commonly abbreviated Neu5Ac, NeuAc, or NANA). The second member of the family is N-glycolylneuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl group of NeuAc is hydroxylated. The third sialic acid family member is 2-keto-3-deoxy-nonylketo acid (nonulosonic acid) (KDN) (Nadano et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265: 21811-21819 (1990)). Also included are 9-substituted sialic acids, such as 9-O-C1-C6 acyl-NeuSAc, such as 9-O-lactyl-Neu5Ac or 9-O-acetyl-NeuSAc, 9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxyNeu5Ac. For a review of the sialic acid family, see, for example, Varki, Glycobiol. 2 (1992) 25-40; Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed. (Springer-Verlag, New York (1992)). The synthesis and use of sialic acid compounds in sialylation methods are reported in WO 92 / 16640, the entire contents of which are incorporated herein.

[0167] With respect to antibodies, the term "substantially" means that the corresponding product (antibody) has a monoglycosylation state, whether or not the state includes glycosylation at a single site or multiple sites. Typically, an antibody is substantially pure when the antibody accounts for at least 60% of the weight of the antibody in the formulation. For example, the antibody in the formulation is at least about 75% of the weight of the desired antibody, in certain embodiments at least about 80%, in certain embodiments about 85%, in certain embodiments at least about 90%, in certain embodiments at least about 95%, 96%, 97%, 98%, and most preferably at least about 99%.

[0168] The term "glycosylation state" refers to a specific or desired glycosylation pattern of an antibody. A "glycoform" is an antibody comprising a specific glycosylation state. Such glycosylation patterns include, for example, one or more sugars attached at position N-297 (according to Kabat numbering) in the Fc region of an antibody, wherein the sugars are naturally, recombinantly, synthetically or semisynthetically produced. Glycosylation patterns can be determined by many methods known in the art. For example, methods for analyzing carbohydrates on proteins are reported in US2006 / 0057638 and US2006 / 0127950 (the disclosures of which are incorporated herein by reference in their entirety).

[0169] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in the binding of an antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt, TJ et al. Kuby Immunology, 6th edition, WH Freeman and Co., NY (2007), p. 91). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using VH or VL domains from antibodies that bind to an antigen to screen libraries that complement VL or VH domains, respectively. See, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

[0170] The term "N-glycosylation site" refers to an amino acid residue within the N-glycosylation site consensus sequence that is connected or can be connected to a glycan. Typically, an N-linked glycan is connected to the amide nitrogen atom of the asparagine (Asn, N) side chain. The N-glycosylation site consensus sequence is Asn-X-Ser / Thr, where X can be any amino acid residue except proline. The term "N-linked glycosylation" refers to the result of a sugar molecule oligosaccharide (expressed as a glycan) being connected to, for example, the amide nitrogen atom of asparagine.

[0171] Antibody glycosylation

[0172] Human antibodies are mainly glycosylated on the asparagine residue at about position 297 (Asn297) in the heavy chain CH2 domain or Fab region, with more or less fucosylated biantennary complex oligosaccharides (antibody amino acid residues are numbered according to Kabat, supra). The biantennary sugar structure can terminate with up to two consecutive galactose (Gal) residues in each arm. The arms are represented as (1,6) and (1,3) according to the glycosidic bond with the central mannose residue. The sugar structure represented as G0 does not contain galactose residues. The sugar structure represented as G1 contains one or more galactose residues in one arm. The sugar structure represented as G2 contains one or more galactose residues in each arm (Raju, TS, Bioprocess Int.1 (2003) 44-53). The human constant heavy chain region is reported in detail in Kabat, supra and Brueggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361; Love, TW et al., Methods Enzymol. 178 (1989) 515-527. CHO-type glycosylation of the antibody Fc region is described, for example, in Routier, FH, Glycoconjugate J. 14 (1997) 201-207.

[0173] The term "antibody" refers to and includes various forms of antibodies, such as human antibodies, humanized antibodies, chimeric antibodies or T cell antigen depleted antibodies (see, for example, WO98 / 33523, WO98 / 52976 and WO00 / 34317). In one embodiment, the antibody in the method reported herein is a human or humanized antibody. The genetic engineering of antibodies is described, for example, in Morrison, SL et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244; Riechmann, L. et al., Nature 332 (1988) 323-327; Neuberger, MS et al., Nature 314 (1985) 268-270; Lonberg, N., Nat. Biotechnol. 23 (2005) 1117-1125.

[0174] Antibodies generally comprise two so-called full-length light chain polypeptides (light chains) and two so-called full-length heavy chain polypeptides (heavy chains). Each of the full-length heavy and light chain polypeptides contains a variable domain (variable region) (usually the amino terminal portion of the full-length polypeptide chain), which comprises a binding region that interacts with an antigen. Each of the full-length heavy and light chain polypeptides comprises a constant region (usually the carboxyl terminal portion). The constant region of the full-length heavy chain mediates the binding of antibodies i) to cells (such as phagocytes) carrying Fcγ receptors (FcγR) or ii) to cells carrying neonatal Fc receptors (FcRn) (also known as Brambell receptors). It also mediates the binding to certain factors, including factors of the classical complement system, such as components (C1q). The variable domains of the light or heavy chains of the full-length antibody again comprise different segments, i.e., four framework regions (FR) and three hypervariable regions (CDR). A "full-length antibody heavy chain" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody hinge region, an antibody constant domain 2 (CH2), an antibody constant domain 3 (CH3), and optionally, in the case of subclass IgE antibodies, an antibody constant domain 4 (CH4) in the N-terminal to C-terminal direction. A "full-length antibody light chain" is a polypeptide consisting of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) in the N-terminal to C-terminal direction. The full-length antibody chains are linked together by inter-polypeptide disulfide bonds between the CL domain and the CH1 domain and between the hinge regions of the full-length antibody heavy chains.

[0175] In recent years, it has been reported that the glycosylation pattern of antibodies, i.e., the sugar composition and multiple linked sugar structures, has a strong influence on biological properties (see, e.g., Jefferis, R., Biotechnol. Prog. 21 (2005) 11-16). Antibodies produced by mammalian cells contain 2-3% oligosaccharides by mass (Taniguchi, T. et al., Biochem. 24 (1985) 5551-5557). For example, in G class antibodies (IgG) this is equivalent to 2.3 oligosaccharide residues in mouse IgG (Mizuochi, T. et al., Arch. Biochem. Biophys. 257 (1987) 387-394), and 2.8 oligosaccharide residues in human IgG (Parekh, RB et al., Nature 316 (1985) 452-457), of which usually two are located on Asn297 in the Fc region and the rest are in the variable region (Saba, JA et al., Anal. Biochem. 305 (2002) 16-31).

[0176] The term "sugar structure" used in this application refers to a single, defined N- or O-linked oligosaccharide on a specific amino acid residue. Thus, the term "antibody with a G1 sugar structure" refers to an antibody that comprises a biantennary oligosaccharide at an asparagine amino acid residue at about amino acid position 297 according to the Kabat numbering scheme or in the FAB region, the biantennary oligosaccharide comprising only one terminal galactose residue at the non-reducing end of the oligosaccharide. The term "oligosaccharide" used in this application refers to a polymeric sugar comprising two or more covalently linked monosaccharide units.

[0177] For the annotation of different N- or O-linked oligosaccharides in the present invention, the individual sugar residues are listed from the non-reducing end to the reducing end of the oligosaccharide molecule. The longest sugar chain is selected as the basic chain for representation. The reducing end of the N- or O-linked oligosaccharide is a monosaccharide residue that is directly bound to an amino acid of the antibody amino acid backbone, while the N- or O-linked oligosaccharide end at the opposite end of the reducing end of the basic chain is referred to as the non-reducing end.

[0178] All oligosaccharides are described herein as the name or abbreviation of the non-reducing sugar (i.e., Gal), followed by the configuration of the glycosidic bond (α or β), the ring bond (1 or 2), the ring position of the reducing sugar involved in the bond (2, 3, 4, 6, or 8), followed by the name or abbreviation of the reducing sugar (i.e., GlcNAc). Each sugar is preferably a pyranose. For a review of standard glycobiology nomenclature, see Essentials of Glycobiology Varki et al., eds., 1999, CSHL Press.

[0179] The term "defined sugar structure" as used herein refers to a sugar structure in which the monosaccharide residue at the non-reducing end of the sugar structure is of a specific type. The term "defined sugar structure" as used herein refers to a sugar structure in which the monosaccharide residue at the non-reducing end of the sugar structure is defined and is of a specific type.

[0180] Antibody purification

[0181] The term "affinity chromatography" used in this application means a chromatographic method using an "affinity chromatography material". In affinity chromatography, antibodies are separated based on their biological activity or chemical structure, and the separation depends on electrostatic interactions, hydrophobic bonds and / or hydrogen bonds formed with the chromatographic functional groups of the chromatographic material. In order to recover the specifically bound antibody from the affinity chromatography material, a competing ligand can be added, or the chromatographic conditions, such as the pH value, polarity or ionic strength of the buffer, can be changed. Exemplary "affinity chromatography materials" are metal chelate chromatographic materials, such as Ni(II)-NTA or Cu(II)-NTA, or antibody affinity chromatography materials, such as chromatographic materials comprising protein A or protein G covalently linked thereto, or enzyme-bound affinity chromatography materials, such as chromatographic materials comprising enzyme substrate analogs covalently linked thereto, enzyme cofactors or enzyme inhibitors as chromatographic functional groups, or lectin-bound chromatographic materials, such as chromatographic materials comprising polysaccharides covalently linked thereto, cell surface receptors, glycoproteins or intact cells as chromatographic functional groups.

[0182] In one embodiment, the antibody light chain affinity ligand uses a light chain constant domain specific capture reagent, for example, the reagent is specific for kappa or lambda constant light chain, depending on whether the antibody contains kappa or lambda light chain. Examples of such light chain constant domain specific capture reagents are, for example, KappaSelect TM and LambdaFabSelect TM (Available from GE Healthcare / BAC), which are based on a high rigidity agarose matrix that can achieve high flow rates and low back pressures on a large scale. These materials contain ligands that bind to the kappa or lambda light chain constant region, respectively (will not bind antibodies or fragments lacking the light chain constant region). Therefore, both are able to bind other target molecules containing light chain constant regions, such as IgG, IgA and IgM. The ligands are attached to the matrix by long hydrophilic spacer arms, making them easy to bind to the target molecule. It is based on the screening of single chain antibody fragments of human Igkappa or lambda.

[0183] The term "light chain" refers to the shorter polypeptide chain of a natural IgG antibody. Antibody light chains can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains, with the human kappa light chain constant domain being shown in SEQ ID NO: 27 and the human lamba light chain constant domain being shown in SEQ ID NO: 28.

[0184] The term "application" and its grammatical equivalents used in this application represent the partial steps of the purification method, wherein a solution containing a substance of interest is contacted with a stationary phase. The solution containing the substance of interest to be purified passes through a stationary phase, which provides an interaction between the stationary phase and the substance in the solution. Depending on the conditions, such as, for example, pH, conductivity, salt concentration, temperature and / or flow rate, some substances of the solution are combined with the stationary phase and removed from the solution. Other substances remain in the solution. The substances retained in the solution may be present in a flow-through liquid." Flow-through liquid" means a solution obtained after passing through a chromatographic device, which may be an application solution or a buffer containing the substance of interest, which is used to rinse the column or elute one or more substances combined with the stationary phase. After the purification step, the substance of interest can be recovered from the solution by methods familiar to those skilled in the art, such as, for example, precipitation, salting out, ultrafiltration, diafiltration, freeze drying, affinity chromatography or solvent volume reduction to obtain a substantially homogeneous form of the substance.

[0185] Antibodies or antibody fragments can be produced by recombinant methods, and their sugar structures can be modified in the methods reported herein. The method for recombinant production is well known in the prior art, and includes protein expression in eukaryotic cells, and then separating antibodies or antibody fragments and purifying to a pharmaceutically acceptable purity. In order to express antibodies or antibody fragments, hybridoma cells or eukaryotic cells are used, wherein one or more nucleic acids encoding antibodies or antibody fragments have been introduced. In one embodiment, eukaryotic cells are selected from CHO cells, NS0 cells, SP2 / 0 cells, HEK 293 cells, COS cells, PER.C6 cells, BHK cells, rabbit cells or sheep cells. In another embodiment, eukaryotic cells are selected from CHO cells, HEK cells or rabbit cells. After expression, antibodies or antibody fragments are recovered from cells (recovered from supernatant or cells after lysis). General methods for the recombinant production of antibodies are well known in the art and are reported in review articles such as Makrides, SC, Protein Expr. Purif. 17 (1999) 183-202; Geisse, S. et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, RJ, Mol. Biotechnol. 16 (2000) 151-160; Werner, RG, Drug Res. 48 (1998) 870-880.

[0186] Purification of antibodies to eliminate cellular components or other contaminants, such as other cellular nucleic acids or proteins, can be performed by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods well known in the art (see, e.g., Ausubel, FM, et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (2005)). Different methods are well established and widely used for protein purification, such as affinity chromatography using microproteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (carboxymethyl resins), anion exchange (aminoethyl resins) and mixed mode exchange), thiophilic adsorption (e.g. using β-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. using phenyl sepharose, aza-arenophilic resins or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. using Ni(II)- and Cu(II)-affinity materials), size exclusion chromatography and electrophoretic methods (e.g. gel electrophoresis, capillary electrophoresis), and combinations thereof, such as affinity chromatography using microproteins, cation exchange chromatography and anion exchange chromatography (see, e.g. Vijayalakshmi, MA, Appl. Biochem. Biotech. 75 (1998) 93-102). General chromatography methods and their use are known to those skilled in the art.See, e.g., Heftmann, E. (ed.), Chromatography, 5th ed., Part A: Fundamentals and Techniques, Elsevier Science Publishing Company, New York (1992); Deyl, Z. (ed.), Advanced Chromatographic and Electromigration Methods in Biosciences, Elsevier Science BV, Amsterdam, The Netherlands (1998); Poole, CF and Poole, SK, Chromatography Today, Elsevier Science Publishing Company, New York (1991); Scopes, Protein Purification: Principles and Practice (1982); Sambrook, J. et al. (eds.), Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); or Ausubel, FM et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (2005).

[0187] In order to purify antibodies or antibody fragments that have been produced by cell culture methods, a combination of different chromatographic steps can generally be used. Typically, (Protein A) affinity chromatography is followed by one or two additional separation steps. In one embodiment, the additional chromatographic steps are cation and anion exchange chromatography steps, or vice versa. The final purification step is a so-called "correction step" to remove trace impurities and contaminants, such as aggregated immunoglobulins, residual HCPs (host cell proteins), DNA (host cell nucleic acids), viruses or endotoxins. In one embodiment, the final purification step is an anion exchange chromatography in flow-through mode.

[0188] Methods reported in this paper

[0189] The carbohydrate structures of recombinantly produced antibodies or antibody fragments will be determined by the cell line and culture conditions used. Using traditional downstream processing techniques, it is not possible to selectively remove specific carbohydrate structures.

[0190] In more detail, recombinantly produced monoclonal antibodies usually contain a heterogeneous mixture of glycoforms at glycosylation sites. This glycosylation profile is influenced by different factors during the recombinant production process, such as enzyme activities present in the host cells and in the culture medium and the culture conditions.

[0191] It may be desirable to generate antibodies with universal or even predetermined glycosylation, such as, for example, to have therapeutic effects and the like.

[0192] The method reported herein provides an antibody with defined glycosylation at an N-glycosylation site (e.g., at an N-glycosylation site in a Fab region or an Fc region) by enzymatically modifying the glycan at the N-glycosylation site after harvesting the antibody from the culture, i.e., the antibody substantially contains a single glycoform linked to an Fc region glycosylation site (e.g., at Asn297 in the Fc region). Since the antibody is tightly bound to the antibody light chain affinity ligand, its glycosylation can be modified in a desired manner, and therefore, the method reported herein has the advantage that it can be easily incorporated into a standard operating procedure for purifying antibodies from culture supernatants. Because the antibody is bound to the antibody light chain affinity ligand, which can be further immobilized on a solid phase, among other things, the amount of enzyme used for the modification can be reduced compared to the amount required for modification in solution; in addition, the entire modification can be achieved in a single step.

[0193] The term "antibody with defined glycosylation" or "antibody with defined sugar structure" refers to a population of antibody molecules in which a limited number of different glycans are attached to a (predetermined) N-glycosylation site, e.g., Asn297 in the Fc region (numbered according to the EU index of Kabat). In one embodiment, one of the glycans accounts for 50% or more of the G0F, G1F, G2F, G1S1F, G2S1F, and G2S2F glycoforms, or 30% or more of the G0F, G1F, G2F, G1S1F, G2S1F, and G2S2F glycoforms.

[0194] As used herein, the term "substantially" means that 40% or more, in one embodiment 50% or more of the compounds have the same glycosylation, ie comprise the same glycan at the N-glycosylation site, such as Asn297 of the Fc region (according to Kabat numbering).

[0195] Using the methods reported herein, antibodies (regardless of the type and size of the antibodies) can be modified to contain a determined glycoform. More specifically, the glycosylation of N-glycosylation sites in the Fc region can be customized, for example, for the intended therapeutic application of the antibody. For example, the galactosylation of the antibody Fc region can be used to treat cancer. Further, for example, the sialylated antibody Fc region is a determined glycoform that can be used to treat autoimmune diseases. For different applications, it may be desirable to remove galactosylation and / or desialylation of the Fc region. Still in other embodiments, it is possible to achieve the generation of a hybrid structure with a GlcNAc core and a mannose residue, such as N-acetylglucosamine, GlcNAc; or mannose-N-acetylglucosamine-N-acetylglucosamine, Man-GlcNAc-GlcNAc. Any of the aforementioned substances can be produced using the methods reported herein, because any antibody and any sugar structure of the antibody can be modified stepwise with different glycosylases, by a series of repetitions of the methods reported herein, to produce the desired determined glycoform antibody.

[0196] For example, antibodies with the G2 glycoform can be produced from a heterogeneous population of monoclonal antibodies using the methods reported herein. Non-fucosylated heterogeneous antibodies that can be produced by glycoengineering methods can be converted to homogeneous G2-glycoforms using the same methods. In addition, differences in antibody galactosylation between batches can also be resolved by adjusting galactosylation to the desired level using the methods reported herein.

[0197] In brief, the method reported herein comprises the steps of applying a solution containing an antibody with glycosylation at an N-glycosylation site (e.g. in the Fc region) to an antibody light chain affinity ligand immobilized on a solid phase / support. The support comprises a column washed with a wash buffer and then with a reaction buffer solution, which is suitable for the corresponding desired enzymatic sugar structure modification on the column. The reaction buffer can be further optimized by adding a selected second enzyme, optional cofactors and optional sugar nucleotides. The column is then incubated at room temperature or at an elevated temperature of about 37°C. The column is then washed with a wash buffer and the modified monoclonal antibody with a determined glycoform is eluted from the solid support with an elution buffer. The eluted antibody can then be neutralized with a neutralization buffer.

[0198] The sugar nucleotides used in the reaction buffer are selected from UDP-Glc, UDP-Gal, UDP-GalNAc, UDP-GlcNAc, UDP-GlcUA, UDP-Xyl, GDP-Man, GDP-Fuc, CMP-NeuSAc, CMP-NeuSGc and combinations thereof. The concentration range used in the reaction buffer is about 0.5 mM to about 5 mM, in some aspects about 1 mM to about 1.5 mM. The cofactor used in the reaction buffer can be selected from Mn 2+ , Ca 2+Mg 2+ 、Na + , K + , α-lactalbumin, and combinations thereof. The cofactor concentration used in the reaction buffer may be in the range of about 2 mM to about 10 mM.

[0199] The antibody light chain affinity ligand fixed on the solid phase is retained in the column during purification and modification. The solid phase includes but is not limited to agarose, sepharose, polyacrylic acid, polystyrene and other synthetic polymers, which provide negligible non-target proteins and non-specific adsorption of modified enzymes. The affinity ligand is covalently bound to the solid phase by any covalent bonding in various chemical reactions with the solid phase, such as N-hydroxysuccinimide (NHS) esters, epoxides, aldehydes or cyanogen bromide and the solid phase. Such conjugation chemical reactions are well known in the art, such as Hermanson, GT, Bioconjugate Techniques, Academic Press (Amsterdam, the Netherlands, Ed. 2008) and Wong, S., Chemistry of Protein Conjugation and Cross Linking, CRC Press (Boca Raton, Fla., 1991) exemplified.

[0200] Washing buffer ensures that the high affinity between the antibody and the affinity ligand is maintained during the washing step. For example, a phosphate buffered saline solution (PBS) with a pH of about 7.2 can be used as a washing buffer, but those skilled in the art understand that pH can vary to a certain extent. Washing and reaction buffers ensure that the high affinity between the antibody and the affinity ligand is maintained while maintaining the activity of each enzyme. Washing and reaction buffers are used at a temperature of about 25°C to about 40°C and at any temperature therebetween. A temperature of about 37°C is generally used. For the high affinity of the antibody and light chain affinity ligand, the optimal pH range is about 6.0 to about 8.0. Within this pH range, the buffer overlaps with the optimal pH range of the affinity ligand that can be used in the method reported herein. These include but are not limited to TRIS buffer, BIS-TRIS buffer, MES buffer, BES buffer, MOPS buffer and HEPES buffer.

[0201] The wash conditions of the affinity column minimize non-specific binding, thereby affecting the enzyme reaction and thus the antibody modification. The washing conditions are such that they do not disrupt the binding between the antibody light chain affinity ligand and the target monoclonal antibody.

[0202] The enzymes suitable for the methods as reported herein can be selected from the following group according to the modification: mannosyl-glucosamine transferases (MGAT1, MGAT2 and MGAT3), galactosyltransferases (β4GalT1, β4GalT2, β4GalT3, β4GalT4, β4GalT5, β4GalT6, β4GalT7), sialyltransferases (ST6Gal1, ST6Gal2); mannosidases (α mannosidase-I, α mannosidase-II, α(1-2) mannosidase, α(1-6) mannosidase, α(1-2,3) mannosidase, α(1-6,3) mannosidase, α(1-2,3) mannosidase, α(1-3) mannosidase, α(1-4) mannosidase, α(1-5) mannosidase, α(1-6) mannosidase, α(1-5) mannosidase, α(1-6,3) mannosidase, α(1-4) mannosidase, α(1-5) mannosidase, α(1-6) mannosidase, α(1- -2,3,6) mannosidase); hexosaminidase (β-N-acetylglucosaminidase, β-N-acetylglucosaminidase, α-N-acetylglucosaminidase); galactosidase (β-galactosidase, β(1-4) galactosidase, α(1-3,6) galactosidase); sialidase (α(2-3,6,8) sialidase, α(2-3) sialidase), fucosidase (α-L-fucosidase, α(1-6) fucosidase, α(1-2) fucosidase, α(1-3,4) fucosidase, α(1-2,3,4) fucosidase) and any combination thereof.

[0203] The methods reported herein can be used to remove or add terminal sialic acid from galactose to produce, for example, antibodies with homogeneous G2 sugar structures in the Fc region. Thus, for example, a non-specific neuraminidase that removes sialic acid from any connection or a specific sialidase that adds the corresponding sialic acid can be used. The enzyme can be used in combination with a galactosyltransferase to simultaneously affect galactosylation and remove or add sialic acid. Thus, an antibody with a determined G2 glycoform (e.g., in the Fc region) can be obtained from an antibody with glycosylation that contains at least glycoforms G0, G1, G2, G1S1, and G2S2 (e.g., in the Fc region).

[0204] Modification of antibody glycosylation according to the methods reported herein can be performed using continuous incubation with the respective enzymes or semi-continuous incubation, wherein a first enzyme is added and after a period of time a second enzyme is added without removing the first enzyme, or incubation with both enzymes present simultaneously. Any of these schemes results in improved modification compared to modification entirely in solution reactions or with antibody modification immobilized on protein A.

[0205] The method as reported herein is exemplified in the following by providing an antibody with defined galactosylation and sialylation in the Fc region using the corresponding transferases.

[0206] On-column galactosylation

[0207] Purified IgG1 subclass humanized antibodies were applied to protein A affinity chromatography material and antibody light chain affinity ligand chromatography material (Kappa select from GE Healthcare). The bound antibodies were incubated on column with a buffer solution containing galactosyltransferase (GalT1) and UDP-GAL. The results are shown in the table below. It can be seen that when the antibody is combined with a column containing an antibody light chain affinity ligand, a higher amount of galactosylation is achieved.

[0208]

[0209] G0F = complex N-glycan with two terminal N-acetylglucosamine residues and fucose

[0210] G1F = complex N-glycan with one terminal N-acetylglucosamine residue and one terminal galactose residue and fucose

[0211] G2F = complex N-glycan with two terminal galactose residues and fucose

[0212] The purified humanized antibody (homogeneous G2F glycoform) with homogeneous glycosylation of IgG1 subclass in the Fc region is applied to protein A affinity chromatography material and antibody light chain affinity ligand chromatography material (Kappaselect from GE Healthcare). The bound antibody is incubated on the column with a buffer solution comprising sialyltransferase (ST6) and CMP-NANA. The results are shown in the table below. It can be seen that when the antibody is combined with the column comprising the antibody light chain affinity ligand, a higher amount of sialylation is achieved.

[0213]

[0214]

[0215] The presence or absence of alkaline phosphatase did not change the yield of the Protein A column (19% G2F, 56% G2S1F, 25% G2S2F). In solution, the following results were obtained:

[0216]

[0217] G2F = complex N-glycan with two terminal galactose residues and fucose

[0218] G2S1F = complex N-glycan with two terminal galactose residues (one of which is sialylated) and fucose

[0219] G2S2F = complex N-glycan with two terminal galactose residues (both sialylated) and fucose

[0220] Human antibodies of IgG4 subclass are applied to protein A affinity chromatography material and antibody light chain affinity ligand chromatography material (Kappa select from GE Healthcare). The bound antibodies are incubated on column with a buffer solution containing galactosyltransferase (GalT1) and UDP-GAL. The results are shown in the table below. It can be seen that when the antibody is combined with a column containing an antibody light chain affinity ligand, a higher amount of galactosylation is achieved.

[0221]

[0222] G0F = complex N-glycan with two terminal N-acetylglucosamine residues and fucose

[0223] G1F = complex N-glycan with one terminal N-acetylglucosamine residue and one terminal galactose residue and fucose

[0224] G2F = complex N-glycan with two terminal galactose residues and fucose

[0225] The human antibody of IgG4 subclass with homogeneous glycosylation (homogeneous G2F glycoform) in Fc region is applied to protein A affinity chromatography material and antibody light chain affinity ligand chromatography material (Kappa select from GE Healthcare). The bound antibody is incubated on post with a buffer solution comprising sialyltransferase (ST6) and CMP-NANA. The results are shown in the table below. It can be seen that when the antibody is combined with the post comprising the antibody light chain affinity ligand, a higher amount of sialylation is achieved.

[0226]

[0227] nd = not determined

[0228] The humanized antibody of IgG1 subclass with extra glycosylation site in Fab is applied to protein A affinity chromatography material and antibody light chain affinity ligand chromatography material (Kappa select from GE Healthcare). The bound antibody is incubated on post with a buffer solution comprising sialyltransferase (ST6) and CMP-NANA. The result is as shown in the table below. In this embodiment, the glycosylation of N-glycosylation site in Fab is modified. It can be seen that when antibody is combined with the post comprising antibody light chain affinity ligand, the reaction kinetics of improvement is achieved.

[0229]

[0230]

[0231] G2 = complex N-glycan with two terminal galactose residues

[0232] G2S1 = complex N-glycan with two terminal galactose residues, one of which is sialylated

[0233] G2S2 = complex N-glycan with two terminal galactose residues, both of which are sialylated

[0234] The cell-free culture supernatant of the humanized antibody comprising the IgG1 subclass is applied to protein A affinity chromatography material and antibody light chain affinity ligand chromatography material (Kappa select from GE Healthcare). The bound antibody is first incubated with a buffer solution comprising galactosyltransferase (GalT1) and UDP-GAL on the column, and then incubated with a buffer solution comprising sialyltransferase (ST6) and CMP-NANA. The results are shown in the table below. Sialyltransferase is added after incubation for 6 hours.

[0235] Protein A

[0236]

[0237] Kappa select

[0238]

[0239] The same experiment was repeated with purified bulk material.

[0240] Protein A

[0241]

[0242]

[0243] Kappa select

[0244]

[0245] Improved Kappa select method by adding sialyltransferase after 24 hours

[0246]

[0247] Antibodies used in the methods reported herein

[0248] Chimeric and humanized antibodies

[0249] In certain embodiments the antibody modified in the methods reported herein is a chimeric antibody.

[0250] Certain chimeric antibodies are described in, for example, US 4,816,567; and Morrison, SL et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit or non-human primate such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof as long as they bind to the antibody light chain affinity ligand used in the methods reported herein.

[0251] In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies include one or more variable domains, wherein HVR (e.g., CDR) (or a portion thereof) is derived from a non-human antibody, and FR (or a portion thereof) is derived from a human antibody sequence. Humanized antibodies optionally also include at least a portion of human constant regions. In some embodiments, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies derived from HVR residues), for example, to restore or improve antibody specificity or affinity.

[0252] Humanized antibodies and methods for their preparation are reviewed in, for example, Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, and further described in, for example, Riechmann, I. et al., Nature 332 (1988) 323-329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033; US 5,821,337, US 7,527,791, US 6,982,321, and US 7,087,409; Kashmiri, SV et al., Methods 36 (2005) 25-34 (describing specificity determining region (SDR) grafting); Padlan, EA, Mol. Immunol. 28 (1991) 489-498 (describing "resurfacing"); Dall'Acqua, WF et al., Methods 36 (2005) 43-60 (describing "FR shuffling"); and Osbourn, J. et al., Methods 36 (2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (describing the "guided selection" method of FR shuffling).

[0253] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims, MJ et al., J. Immunol. 151 (1993) 2296-2308); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, LG et al., J. Immunol. 151 (1993) 2623-2632); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regions derived from screening FR libraries (see, e.g., Baca, M. et al., J. Biol. Chem. 272 ​​(1997) 10678-10684 and Rosok, MJ et al., J. Biol. Chem. 271 (19969 22611-22618).

[0254] Human Antibodies

[0255] In certain embodiments the antibody modified in the methods reported herein is a human antibody.

[0256] Human antibodies can be produced using various techniques known in the art.Human antibodies are generally described in van Dijk, MA and van de Winkel, JG, Curr. Opin. Pharmacol. 5 (2001) 368-374 and Lonberg, N., Curr. Opin. Immunol. 20 (2008) 450-459.

[0257] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to an antigenic attack. These animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or is present outside the chromosome or randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125. See also, for example, US 6,075,181 and US 6,150,584, describing XENOMOUSETM technology; US 5,770,429, describing technology; US 7,041,870, describes KM Technology, US2007 / 0061900, describes Technology, and WO 2007 / 131676, describe immune reconstitution mice. For example, the human variable regions of intact antibodies produced by these animals can be further modified by combining with different human constant regions.

[0258] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described (see, e.g., Kozbor, D., J. Immunol. 133 (1984) 3001-3005; Brodeur, BR et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; and Boerner, P. et al., J. Immunol. 147 (1991) 86-95). Human antibodies produced by human B cell hybridoma technology are also described in Li, J. et al., Proc. Natl. Acad. Sci. USA 103 (2006) 3557-3562. Additional methods include those described in, for example, US 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, J., Xiandai Mianyixue 26 (2006) 265-268 (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers, HP and Brandlein, S., Histology and Histopathology 20 (2005) 927-937, and Vollmers, HP and Brandlein, S., Methods and Findings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

[0259] Human antibodies can also be produced by isolating the Fv clone variable domain sequence selected from the human phage display library. Such variable domain sequence can then be combined with the desired human constant domain. The technology of selecting human antibodies from the antibody library is described below.

[0260] Antibodies from the library

[0261] The modified antibodies in the methods reported herein can be isolated by screening combinatorial libraries for antibodies with one or more desired activities. For example, various methods are known in the art for generating phage display libraries and screening these libraries for antibodies with desired binding characteristics. These methods are reviewed in, for example, Hoogenboom, HR et al., Methods in Molecular Biology 178 (2001) 1-37, and further described in, for example, McCafferty, J. et al., Nature 348 (1990) 552-554; Clackson, T. et al., Nature 352 (1991) 624-628; Marks, JD et al., J. Mol. Biol. 222 (1992) 581-597; Marks, JD and Bradbury, A., Methods in Molecular Biology 248 (2003) 161-175; Sidhu, SS et al., J. Mol. Biol. 338 (2004) 299-310; Lee, CV et al., J. Mol. Biol. 340 (2004) 1073-1093; Fellouse, FA, Proc. Natl. Acad. Sci. USA 101 (2004) 12467-12472; and Lee, CV et al., J. Immunol. Methods 284 (2004) 119-132.

[0262] In some phage display methods, the libraries of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, and then the antigen-binding phage in the library is screened, as described in Winter, G. et al., Ann. Rev. Immunol. 12 (1994) 433-455. Phages usually display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immune sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, natural libraries can be cloned (e.g., from humans) to provide a single source of antibodies against a variety of non-self and self antigens without any immunization, as described in Griffiths, AD et al., EMBO J. 12 (1993) 725-734. Finally, natural libraries can also be prepared synthetically by cloning non-rearranged V gene segments from stem cells, using PCR primers containing random sequences to encode highly variable CDR3 regions and completing rearrangement in vitro, as described in Hoogenboom, HR and Winter, G., J. Mol. Biol. 227 (1992) 381-388. Patent publications describing human antibody phage libraries include, for example: US5,750,373 and US2005 / 0079574, US2005 / 0119455, US2005 / 0266000, US2007 / 0117126, US2007 / 0160598, US2007 / 0237764, US2007 / 0292936 and US2009 / 0002360.

[0263] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0264] Multispecific Antibodies

[0265] In certain embodiments, the antibody modified in the methods reported herein is a multispecific antibody, for example, a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. Fragments of multispecific (bispecific) antibodies are included as long as they bind to the antibody light chain affinity ligand used in the methods reported herein.

[0266] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, AC, Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and “knobs-into-holes” engineering (see, e.g., US 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic steering effects for preparing antibody Fc-heterodimer molecules (WO2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., US 4,676,980 and Brennan, M. et al., Science 229 (1985) 81-83); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny, SA et al., J. Immunol. 148 (1992) 1547-1553; using "diabody" technology to prepare bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fv (sFv) dimers (see, e.g., Gruber, M et al., J. Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies as described, e.g., in Tutt, A. et al., J. Immunol. 147 (1991) 60-69).

[0267] Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576).

[0268] The antibodies or fragments modified in the methods as reported herein also include "dual-acting Fabs" or "DAFs" (see for example US 2008 / 0069820).

[0269] The antibodies or fragments herein also include the multispecific antibodies described in WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO2010 / 112193, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792 and WO2010 / 145793.

[0270] Recombinant methods and compositions

[0271] Antibodies can be produced using recombinant methods and compositions, for example, as described in US 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibodies are provided.

[0272] In the case of natural antibodies or natural antibody fragments, two nucleic acids are required, one for light chain or its fragment, one for heavy chain or its fragment. Such nucleic acid encoding comprises the amino acid sequence of antibody VL and / or comprises the amino acid sequence of antibody VH (e.g., the light chain and / or heavy chain of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

[0273] In the case of a bispecific antibody with a heterodimeric heavy chain, four nucleic acids are required, one for the first light chain, one for the second light chain comprising the first heteromonomer Fc region polypeptide, one for the second light chain, and one for the second heavy chain comprising the second heteromonomer Fc region polypeptide. For example, one of the heterodimeric heavy chains comprises a so-called "knob mutation" (T366W and optionally one of S354C or Y349C), and the other comprises a so-called "hole mutation" (T366S, L368A and Y407V and optionally Y349C or S354C) (see, e.g., Carter, P. et al., Immunotechnol. 2 (1996) 73). Such nucleic acid encoding comprises an amino acid sequence comprising a first VL and / or an amino acid sequence comprising a first VH (including a first heteromonomer Fc region) and / or an amino acid sequence comprising a second VL and / or an amino acid sequence comprising a second VH (including a second heteromonomer Fc region of the antibody) (e.g., the first and / or second light chain and / or the first and / or second heavy chain of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors, and usually these nucleic acids are located on two or three expression vectors, i.e. one vector can contain more than one of these nucleic acids. Examples of these bispecific antibodies are CrossMab and T cell bispecific antibodies (see, e.g., Schaefer, W. et al. Proc. Natl. Acad. Sci. USA, 108 (2011) 11187-1191).

[0274] In one embodiment an isolated nucleic acid encoding an antibody for use in the methods as reported herein is provided.

[0275] In further embodiments, one or more vectors (eg, expression vectors) comprising such nucleic acids are provided.

[0276] In a further embodiment, a host cell comprising such nucleic acid is provided.

[0277] In one such embodiment, the host cell comprises (e.g., has been transformed with):

[0278] - In the case of natural antibodies or natural antibody fragments:

[0279] (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of an antibody, or

[0280] (2) A first vector and a second vector, wherein the first vector comprises a nucleic acid encoding an amino acid sequence comprising antibody VL, and the second vector comprises a nucleic acid encoding an amino acid sequence comprising antibody VH.

[0281] - In the case of bispecific antibodies with heterodimeric heavy chains:

[0282] (1) a first vector and a second vector, the first vector comprising a first pair of nucleic acids encoding amino acid sequences, wherein one of the amino acid sequences comprises a first VL of an antibody and the other comprises a first VH of the antibody, and the second vector comprising a second pair of nucleic acids encoding amino acid sequences, wherein one of the amino acid sequences comprises a second VL of the antibody and the other comprises a second VH of the antibody, or

[0283] (2) a first vector comprising a first nucleic acid encoding an amino acid sequence comprising one of the variable domains (preferably a light chain variable domain), a second vector comprising a pair of nucleic acids encoding amino acid sequences, one of the amino acid sequences comprising a light chain variable domain and the other comprising a first heavy chain variable domain, and a third vector comprising a pair of nucleic acids encoding amino acid sequences, one of the amino acid sequences comprising a light chain variable domain relative to the light chain variable domain in the second vector and the other of the amino acid sequences comprising a second heavy chain variable domain, or

[0284] (3) a first vector, a second vector, a third vector and a fourth vector, wherein the first vector comprises a nucleic acid encoding an amino acid sequence comprising a first VL of an antibody, the second vector comprises a nucleic acid encoding an amino acid sequence comprising a first VH of an antibody, the third vector comprises a nucleic acid encoding an amino acid sequence comprising a second VL of the antibody, and the fourth vector comprises a nucleic acid encoding an amino acid sequence comprising a second VH of the antibody.

[0285] In one embodiment, the host cell is a eukaryotic cell, e.g., a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In one embodiment, a method for preparing an antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as described above under conditions suitable for expressing the antibody, optionally recovering the antibody from the host cell (or host cell culture medium), and modifying the glycosylation of the antibody using the methods reported herein.

[0286] In order to recombinantly produce antibodies, nucleic acids encoding antibodies such as those described above are separated and inserted into one or more vectors for further cloning and / or expression in host cells. These nucleic acids can be easily separated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can be specifically bound to the genes encoding the heavy and light chains of the antibodies) or produced by recombinant methods or obtained by chemical synthesis.

[0287] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector energy are not required. For expressing antibody fragments and polypeptides in bacteria, see, for example, US5,648,237, US 5,789,199 and US5,840,523 (also see Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes expression of antibody fragments in Escherichia coli. After expression, antibodies can be separated from the soluble fraction of bacterial cell paste and can be further purified.

[0288] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" so that antibodies with partially or fully human glycosylation patterns are produced. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0289] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0290] Plant cell cultures can also be used as hosts. See, for example, US5,959,177, US6,040,498, US6,420,548, US7,125,978 and US6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants). TM technology).

[0291] Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed by SV40; human embryonic kidney cell lines (293 or 293 cells, such as, for example, Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); small hamster kidney cells (BHK); mouse testicular support cells (TM4 cells, such as, for example, Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; rat liver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather, JP et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For certain mammalian host cell lines suitable for producing antibodies, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0292] Pharmaceutical preparations

[0293] Pharmaceutical formulations of antibodies modified by the methods reported herein are prepared by mixing the antibodies having the desired purity with one or more optional pharmaceutically acceptable carriers in the form of lyophilized formulations or aqueous solutions (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations used, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenolic alcohols, butyl alcohols or benzyl alcohols; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as poly(vinyl pyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US2005 / 0260186 and US2006 / 0104968. In some aspects, sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.

[0294] Exemplary lyophilized antibody formulations are described in US 6,267,958. Aqueous antibody formulations include those described in US 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0295] The formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

[0296] The active ingredient can be embedded in microcapsules, prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, respectively. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. (ed.) (1980).

[0297] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0298] Preparations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0299] Methods of treatment and compositions

[0300] Any antibody modified as described herein by any of the methods may be used in therapeutic methods.

[0301] In one aspect, antibodies modified as reported herein by any method are provided for use as medicines. In other aspects, antibodies modified as reported herein by any method are provided for the treatment of diseases. In certain embodiments, antibodies modified as reported herein by any method are provided for use in therapeutic methods. In certain embodiments, the present invention provides antibodies modified as reported herein by any method, which are used in methods for treating individuals with diseases, the method comprising administering an effective amount of antibodies modified as reported herein by any method to the individual. In such an embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual. In certain embodiments, the present invention provides antibodies modified as reported herein by any method for the treatment of individuals, the method comprising administering an effective antibody modified as reported herein by any method to the individual. "Individual" according to any of the above embodiments is preferably a person.

[0302] On the other hand, the invention provides the use of antibodies modified by any method as reported herein in the manufacture or preparation of medicines. In one embodiment, the medicine is used to treat a disease. In a further embodiment, the medicine is used in a method for treating a disease, the method comprising administering an effective amount of medicine to an individual suffering from the disease. In such an embodiment, the method further comprises administering an effective amount of at least one other therapeutic agent to the individual. In another embodiment, the medicine is used in a method for treating in an individual, the method comprising administering an effective amount of medicine to the individual. According to an "individual" of any of the above embodiments can be a person.

[0303] On the other hand, the invention provides methods for treating a disease. In one embodiment, the method comprises administering an effective amount of an antibody modified by any method as reported herein to an individual suffering from the disease. In such an embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual. According to any of the above embodiments, an "individual" can be a human.

[0304] On the other hand, the invention provides pharmaceutical preparations comprising any antibody modified by any method as reported herein, for example for use in any of the above-mentioned methods of treatment. In one embodiment, the pharmaceutical preparation comprises any antibody modified by any method as reported herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical preparation comprises any antibody modified by any method as reported herein and at least one additional therapeutic agent.

[0305] The antibodies of the invention can be used alone or in combination with other agents in therapy. For example, the antibodies of the invention can be co-administered with at least one additional therapeutic agent.

[0306] Such combination therapies described above include combined administration (wherein two or more therapeutic agents are included in the same or separate preparations) and separate administration, in which case the antibodies modified by any method as reported herein can be administered before, simultaneously and / or after the administration of one or more other therapeutic agents. In one embodiment, the administration of the antibodies modified by any method as reported herein and the administration of other therapeutic agents occur within about one month, or within about one week, two weeks or three weeks, or within about one, two, three, four, five or six days. The antibodies modified by any method as reported herein can also be used in combination with radiotherapy.

[0307] The antibody (and any other therapeutic agent) modified by any method as reported herein can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and can be used for local treatment, intralesional administration if necessary. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Can be administered by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, which depends in part on whether the administration is short-term or long-term. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations, push administration and pulse infusions at different time points.

[0308] As reported herein, any method modified antibodies will be formulated, dosed and applied in a manner consistent with good medical practice. Factors to be considered in this case include the specific illness being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the illness, the delivery site of the medicament, the method of administration, the administration regimen, and other factors known to the doctor. Not necessarily, but optionally, the antibody is formulated with one or more medicaments currently used to prevent or treat the illness in question. The effective amount of these other medicaments depends on the amount of the antibody present in the preparation, the type of illness or treatment, and other factors discussed above. These are generally used with the same dosage and route of administration as described herein, or with about 1% to 99% of the dosage described herein, or with any dosage and any route determined to be suitable by experience / clinical.

[0309] For the prevention or treatment of disease, the appropriate dosage of the antibody modified by any method as reported herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is used for prevention or treatment purposes, previous treatment, the patient's clinical history and reaction to the antibody, and the discretion of the attending physician. The antibody is suitable for administration to the patient once or in a series of treatments. Depending on the type and severity of the disease, the initial candidate dose administered to the patient can be about 1 μg / kg to 15 mg / kg (e.g., 0.5 mg / kg-10 mg / kg) antibody, for example, or by one or more separate administrations or by continuous infusion. According to the above factors, a typical daily dose can be about 1 μg / kg to 100 mg / kg or more. For repeated administration for several days or longer, depending on the condition, treatment is usually continued until the desired disease symptom suppression occurs. An exemplary dosage range of the antibody is about 0.05 mg / kg to about 10 mg / kg. Therefore, one or more dosages of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient. These dosages can be administered intermittently, for example, weekly or every three weeks (for example, so that the patient receives about 2 to about 20, or for example about 6 dosages of the antibody). An initial higher load dosage can be administered, followed by one or more lower dosages. However, other dosage regimens may be useful. The progress of the therapy can be easily monitored by conventional techniques and assays.

[0310] The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

[0311] The following examples are provided to aid the understanding of the present invention, the true scope of the invention being set forth in the appended claims. It is understood that modifications can be made in the procedures described without departing from the spirit of the invention. Example

[0312] Material

[0313] GalT reaction solution (5mM MnCl2, 10mM UDP-Gal, 100mM MES, 0.05mg / ml GalT, pH 6.5)

[0314] 153mg UDP-Gal (MW=610.27g / mol)

[0315] 32 mg MnCl2 (MW = 125.84 g / mol)

[0316] 460 μL GalT (c = 5.43 mg / mL; 10 μg / 2 mg antibody --> 10 μg in 300 μL = 0.033 mg / ml)

[0317] In 100 mM MES buffer pH 6.5

[0318] ST6 reaction solution (0.1mM ZnCl2, 200nM AP, 50mM MES, 1.7mg / ml CMP-NANA, 0.7mg / mlST6, pH 6.5)

[0319] 50 μL ZnCl (100 mM solution: 13.6 mg in 1 mL 50 mM MES)

[0320] 28 μL alkaline phosphatase (AP) (c = 20 mg / mL, MW = 56,000 g / mol)

[0321] 167mg CMP-NANA (1000μg / 2mg antibody-->1000μg pro 300μL=3.34mg / mL)

[0322] 6mL ST6 (c = 5.45mg / mL, target: 200μg (2mg AK) in 300μL = 0.67mg / mL)

[0323] In 50 mM MES buffer pH 6.5

[0324] Buffer:

[0325] Regeneration buffer 1 (0.1 M phosphoric acid)

[0326] Regeneration buffer 2 (3M guanidine hydrochloride)

[0327] Equilibration buffer (25 mM Tris, 25 mM NaCl, 5 mM EDTA, pH 7.1)

[0328] Wash buffer 1 (100 mM MES, pH 6.5): 21.3 mg MES in 1000 mL H2O, pH 6.5 (adjusted with 50% (w / v) NaOH)

[0329] Wash buffer 2 (1M Tris, pH 7.2)

[0330] Wash buffer 3 (50 mM MES, pH 6.5): 1:1 wash buffer 100 mM MES and distilled water

[0331] Elution buffer Kappa select (0.1 M glycine, pH 2.7): 750 mg glycine in 100 mL H2O, pH 2.7 (adjusted with 25% (w / v) HCl)

[0332] Elution buffer Protein A (25 mM sodium citrate, pH 2.8)

[0333] Example 1

[0334] On-column galactosylation of bulk material

[0335] Regenerate, equilibrate, and wash the Protein A or Kappa select column by applying 2 column volumes of Regeneration Buffer 1, 10 column volumes of Equilibration Buffer, and 4 column volumes of Wash Buffer 1.

[0336] Apply 2 mg IgG (bulk material) to the column

[0337] Wash with 10 column volumes of Wash Buffer 1

[0338] Apply 2 mL of galactosylation reaction solution (containing 0.033 mg / ml GalT) and allow 0.8 mL to flow through

[0339] Incubate at 25°C (2, 7 or 24 hours)

[0340] Wash with 8 column volumes of Wash Buffer 1

[0341] Elute with the corresponding elution buffer (2 column volumes for Protein A; 8 column volumes for Kappa select) and adjust the pH with 1 M Tris buffer (pH 9.0)

[0342] Example 2

[0343] On-column sialylation of IgG1 bulk material (Protein A)

[0344] Regenerate, equilibrate and wash the Protein A column by applying 2 column volumes of Regeneration Buffer 1, 10 column volumes of Equilibration Buffer and 10 column volumes of Wash Buffer 3

[0345] Apply 2 mg IgG (bulk material) to the column

[0346] · Apply 2 mL of sialylation reaction solution (3.3 mg / ml CMP-NANA, + / - AP) and allow 0.8 mL to flow through

[0347] Incubate at 37°C (2, 7, 24 or 48 hours) and 25°C (48 hours)

[0348] Wash with 4 column volumes of Wash Buffer 3

[0349] Elute with 2 column volumes of elution buffer Protein A (sodium citrate) and adjust the pH with 1 M Tris buffer (pH 9.0)

[0350] On-column sialylation of IgG1 bulk material (Kappa select)

[0351] Regenerate, equilibrate and wash the kappa Select column by applying 2 column volumes of equilibration buffer, 3 column volumes of regeneration buffer 2, 4 column volumes of equilibration buffer and 2 column volumes of wash buffer 3

[0352] Apply 2 mg IgG (bulk material) to the column

[0353] Wash with 3 column volumes of wash buffer 3

[0354] · Apply 2 mL of sialylation reaction solution (3.3 mg / ml CMP-NANA, + / - AP) and allow 0.8 mL to flow through

[0355] Incubate at 37°C (2, 7 and 24 hours) and 25°C (24 hours)

[0356] Wash with 3 column volumes of wash buffer 3

[0357] Elute Kappa select with 8 column volumes of elution buffer and adjust the pH with 1M Tris buffer (pH 9.0)

[0358] Example 3

[0359] Sequential galactosylation and sialylation of cell culture supernatants

[0360] - Regenerate and equilibrate the Protein A or Kappa Select column by applying 2 column volumes of Regeneration Buffer 1 and 10 column volumes of Equilibration Buffer.

[0361] Apply 1 mg IgG (in supernatant) to the column

[0362] Wash with 10 column volumes of equilibration buffer, followed by 2 column volumes of wash buffer 2 and 6 column volumes of wash buffer 1

[0363] ·Apply 2 mL of galactosylation reaction solution and allow 0.8 mL to flow through

[0364] Incubate at 25°C for approximately 6 to 24 hours (to allow for sufficient galactosylation)

[0365] Wash with 8 column volumes of Wash Buffer 1, 10 column volumes of Equilibration Buffer, 2 column volumes of Wash Buffer 2, and 6 column volumes of Wash Buffer 3

[0366] ·Apply 2 mL of sialylation reaction solution and allow 0.8 mL to flow through

[0367] Incubation (e.g. 25°C for 2, 7 or 24 hours or even longer)

[0368] Wash with 8 column volumes of Wash Buffer 1

[0369] Elute with the corresponding elution buffer (2 column volumes for Protein A; 8 column volumes for Kappa select) and adjust the pH with 1 M Tris buffer (pH 9.0)

[0370] Example 4

[0371] Continuous galactosylation and sialylation of batch material

[0372] Regenerate, equilibrate, and wash the Protein A or Kappa Select column by applying 2 column volumes of Regeneration Buffer 1, 10 column volumes of Equilibration Buffer, and 4 column volumes of Wash Buffer 1.

[0373] Apply 1 mg IgG (bulk material) to the column

[0374] Wash with 10 column volumes of Wash Buffer 1

[0375] ·Apply 2 mL of galactosylation reaction solution and allow 0.8 mL to flow through

[0376] Incubate at 25°C for approximately 6 to 24 hours (to allow for sufficient galactosylation)

[0377] Wash with 8 column volumes of Wash Buffer 1, 10 column volumes of Equilibration Buffer, 2 column volumes of Wash Buffer 2, and 6 column volumes of Wash Buffer 3

[0378] ·Apply 2 mL of sialylation reaction solution and allow 0.8 mL to flow through

[0379] Incubation (e.g. at 25°C for 2, 7 or 24 hours, respectively, or even longer)

[0380] Wash with 8 column volumes of Wash Buffer 1

[0381] • Eluted with corresponding elution buffer (2 column volumes for Protein A; 8 column volumes for Kappa select) and adjusted the pH using 1 M Tris buffer (pH 9.0).

Claims

1. A method for producing an antibody, comprising the following steps: - forming an antibody-antibody light chain affinity ligand complex by applying a solution comprising the antibody to an immobilized antibody light chain affinity ligand, wherein the antibody light chain affinity ligand is specific for a kappa or lambda constant light chain and is immobilized on a solid phase, - incubating the complex formed in the previous step with one or more enzymes to modify the glycosylation of the antibody, Thereby producing antibodies.

2. The method for producing a glycosylated antibody according to claim 1, comprising the following steps: - forming an antibody-antibody light chain affinity ligand complex by applying a solution comprising the antibody to an immobilized antibody light chain affinity ligand, wherein the antibody light chain affinity ligand is specific for a kappa or lambda constant light chain and is immobilized on a solid phase, - incubating the complex formed in the previous step with one or more enzymes and one or more activated sugar residues to modify the glycosylation of the antibody, This results in the production of glycosylated antibodies.

3. The method according to any one of claims 1 to 2, comprising the following steps after the incubation step: - Recovering the modified antibody from the antibody light chain affinity ligand.

4. A method according to any one of claims 1 to 3, comprising the following steps in the following order: - applying a solution comprising an antibody having glycosylation at an N-glycosylation site to an antibody light chain affinity ligand bound to a solid phase, whereby the antibody binds to the ligand, - optionally washing the solid phase with a buffer solution, - Enzymatic modification of glycosylation at the N-glycosylation sites of the antibody by: - applying a first solution comprising a first glycosylation modifying enzyme for a sufficient time under conditions suitable for enzymatic modification of the ligand-binding antibody, optionally washing the modified ligand-binding antibody, applying a second solution comprising a second glycosylation modifying enzyme for a sufficient time under conditions suitable for enzymatic modification of the modified ligand-binding antibody, optionally washing the twice-modified ligand-binding antibody, or - applying a first solution comprising a first glycosylation modifying enzyme for a sufficient time and under conditions suitable for at least partially enzymatically modifying the ligand-bound antibody, and after a determined period of time, applying a second solution comprising a second glycosylation modifying enzyme for a sufficient time and under conditions suitable for enzymatically modifying the modified ligand-bound antibody, and optionally washing the ligand-bound antibody modified twice, or - applying a solution comprising a first and a second glycosylation modifying enzyme for a sufficient time and under conditions suitable for enzymatic modification of the ligand-bound antibody, and optionally washing the modified ligand-bound antibody, -releasing the antibody from the antibody light chain affinity ligand, thereby producing the antibody.

5. A method according to any one of claims 1 to 3, comprising the following steps in the following order: - applying a buffer solution containing an antibody having glycosylation at an N-glycosylation site to an antibody light chain affinity ligand bound to a solid phase, whereby the antibody is bound by the ligand, thereby producing a ligand-bound antibody, - optionally washing the solid phase with a buffer solution without desorbing the antibodies, - Enzymatic modification of glycosylation at the N-glycosylation sites of the antibody by: - applying a first buffer solution comprising a first glycosylation modifying enzyme and a first activated sugar residue under conditions sufficient for enzymatic modification of the ligand-bound antibody for a sufficient time, optionally washing the modified ligand-bound antibody, applying a second buffer solution comprising a second glycosylation modifying enzyme and a second activated sugar residue under conditions sufficient for enzymatic modification of the modified ligand-bound antibody for a sufficient time, optionally washing the modified ligand-bound antibody twice, or - applying a first buffer solution comprising a first glycosylation modifying enzyme and a first activated sugar for a sufficient time and under conditions sufficient to at least partially enzymatically modify the ligand-binding antibody, and after the specified time, applying a second buffer solution comprising a second glycosylation modifying enzyme and a second activated sugar under conditions sufficient to enzymatically modify the modified ligand-binding antibody, and optionally washing the modified ligand-binding antibody twice, or - applying a buffer solution comprising a first and a second glycosylation modifying enzyme and a first and a second activated sugar under conditions suitable for enzymatic modification of the ligand-binding antibody for a sufficient time, and optionally washing the modified ligand-binding antibody, - Release the antibody from the antibody light chain affinity ligand, thereby generating a glycosylated antibody.

6. The method according to any one of claims 1 to 5, wherein the antibody is selected from the group consisting of a full length antibody, a bivalent monospecific antibody, a bispecific antibody, a bivalent bispecific antibody, a trivalent bispecific antibody, a tetravalent bispecific antibody, a trivalent trispecific antibody, an antibody Fab fragment and a tetravalent tetraspecific antibody.

7. The method according to any one of claims 1 to 6, wherein the antibody is a bivalent or trivalent or tetravalent bispecific antibody or a Fab fragment.

8. The method according to any one of claims 1 to 7, wherein the antibody is a chimeric antibody or a humanized antibody or a human antibody. 9 . The method according to any one of claims 4 to 8 , wherein the first glycosylation modification enzyme is a galactosyltransferase, and the second glycosylation modification enzyme is a sialyltransferase.

10. The method according to any one of claims 1 to 9, wherein the solution comprises a chromatographically purified antibody, the (first) glycosylation modifying enzyme is GalT1, and the incubation with the (first) glycosylation modifying enzyme is at 37°C or room temperature for 24 hours. 11 . The method according to claim 1 , wherein the solution comprises a chromatographically purified antibody, the first glycosylation modifying enzyme is GalT1, and the incubation time with the first glycosylation modifying enzyme is 24 hours at 37° C. or room temperature.

12. The method according to any one of claims 1 to 11, wherein the solution comprises a chromatographically purified antibody, the (second) glycosylation modifying enzyme is ST6, and the incubation time with the (second) glycosylation modifying enzyme is 24 hours at 37°C or room temperature.

13. The method according to any one of claims 1 to 12, wherein the solution comprises a chromatographically purified antibody, the second glycosylation modification enzyme is ST6, and the incubation time with the second glycosylation modification enzyme is 24 hours at 37°C or room temperature.

14. The method according to any one of claims 1 to 13, wherein the solution is a buffered cell-free culture supernatant, the supernatant contains antibodies, the first glycosylation modification enzyme is GalT1, the second glycosylation modification enzyme is ST6, which is added 6 to 24 hours after the first glycosylation modification enzyme is added, and the total incubation time is 24 to 48 hours at 37°C or room temperature.

15. A method according to any one of claims 1 to 14, comprising the steps of: a) recombinantly producing the antibody or Fab fragment in a cell comprising a nucleic acid encoding the antibody or Fab fragment to obtain the antibody or Fab fragment having glycosylation at the N-glycosylation site, b) isolating the antibody or Fab fragment produced in step a) having heterogeneous glycosylation at the N-glycosylation site, c) enzymatically modifying an antibody or Fab fragment having heterogeneous glycosylation at an N-glycosylation site using a galactosyltransferase and a sialyltransferase to obtain a modified antibody, followed by separation of the modified antibody or Fab fragment from the enzyme, d) optionally purifying the modified antibody or Fab fragment by one or more chromatography steps, wherein the produced antibody or Fab fragment comprises a relative amount of at least 70% of digalactosylated antibody or Fab fragment at the N-glycosylation site (wherein 100% corresponds to the amount of G0, G1 and G2 glycoforms).

16. A method according to any one of claims 1 to 15, comprising the steps of: a) recombinantly producing an antibody or Fab fragment of the IgG1 isotype in a mammal or a CHO cell comprising a nucleic acid encoding the antibody or Fab fragment to obtain the antibody or Fab fragment having glycosylation at an N-glycosylation site, b) recovering and purifying the antibody or Fab fragment having heterogeneous glycosylation at the N-glycosylation site produced in step a), c) enzymatically modifying an antibody or Fab fragment having heterogeneous glycosylation at an N-glycosylation site with a galactosyltransferase and a sialyltransferase to obtain a modified antibody, followed by separation of the modified antibody or Fab fragment from the enzyme, d) optionally purifying the modified antibody or Fab fragment by one or more chromatography steps, wherein the produced antibody or Fab fragment comprises a relative amount of at least 70% of digalactosylated antibody or Fab fragment at the N-glycosylation site (wherein 100% corresponds to the amount of G0, G1 and G2 glycoforms).

17. The method according to any one of claims 1 to 16, wherein the N-glycosylation site is the Fab region N-glycosylation site or the Fc region N-glycosylation site at asparagine residue 297 (according to Kabat numbering).

18. A method for producing an antibody or a fragment thereof, comprising the following steps in the following order: - providing a cell comprising a nucleic acid encoding an antibody or a fragment thereof, said antibody or fragment thereof comprising at least an antibody light chain, - culturing the cells under conditions suitable for the expression of an antibody glycosylated at the N-glycosylation site or a fragment thereof comprising at least a light chain that can specifically bind to an antibody light chain affinity chromatography material used in one of the subsequent steps, - recovering the antibody or fragment thereof from the cells or culture medium, - applying a solution containing the antibody or fragment thereof to an antibody light chain affinity chromatography column under conditions suitable for binding of the antibody or fragment thereof to the affinity chromatography material, - modifying the glycosylation of an antibody or fragment thereof at an N-glycosylation site using the method as reported herein, and - recovering the modified antibody or fragment thereof having defined glycosylation at the N-glycosylation site from the affinity chromatography material, Thereby producing antibodies.

19. A pharmaceutical formulation comprising an antibody with defined glycosylation, wherein the antibody is selected from the group consisting of a G2 glycosylation form, a G0 glycosylation form, an M3 glycosylation form, an S2 glycosylation form, an A2B glycosylation form, an A2BG2 glycosylation form and an S1 glycosylation form.

20. A pharmaceutical formulation comprising an antibody with defined glycosylation selected from the group consisting of galactose as terminal sugar, GlcNAc as terminal sugar, mannose as terminal sugar and sialic acid as terminal sugar, wherein the antibody is embedded in hydroxymethylcellulose or gelatin microcapsules or poly-(methyl methacrylate) microcapsules prepared by coacervation technology or interfacial polymerization.

21. A pharmaceutical formulation according to claim 20, wherein the formulation is a sustained release formulation selected from semipermeable matrices of solid hydrophobic polymers containing the antibody, said matrix being in the form of shaped articles, e.g. films or microcapsules.

Images