RU2845361C1 - Modulation of enzyme and path with sulfhydryl compounds and derivatives thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and is an antibody to α-synuclein having monogalactosylated (G1) and digalactosylated (G2) glycans, wherein the antibody to α-synuclein has 17.2–48.0 (wt./wt.%) g1 and 3.1–15.0 (wt./wt.%) G2 to total glycan.

EFFECT: invention provides effective use of such antibody to α-synuclein for treating patients with Parkinson's disease.

10 cl, 12 dwg, 1 tbl, 8 ex

Description

AREA OF TECHNOLOGY

The present invention relates to proteins, in particular to antibodies, such as bispecific antibodies to CD20/CD3 and antibodies to α-synuclein, having monogalactosylated (G1) and digalactosylated (G2) glycans. More particularly, the present invention relates to engineering using galactosylation to produce proteins with improved therapeutic properties, including proteins with an increased titer. Furthermore, the invention relates to a cell culture medium and a mammalian cell, as well as methods of using said cell culture medium and said mammalian cell to produce said proteins. Furthermore, the present invention relates to the use of said antibodies as a medicament, for example, for the treatment of cancer, in particular B-cell-associated cancer, or Parkinson's disease.

LEVEL OF TECHNOLOGY

Many glycoproteins have been a staple of the biotechnology industry and have been used, in particular, for therapeutic purposes. Examples include erythropoietin (EPO), therapeutic monoclonal antibodies (therapeutic mAbs), tissue plasminogen activator (tPA), interferon-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), and human chorionic gonadotropin (hCG) (Dimming et al., Glycobiology 1: 115–130 (1991)). Thus, the oligosaccharide component of glycoproteins can influence their properties related to the efficacy of therapeutic glycoproteins, including, but not limited to, physical stability, resistance to protease attack, interaction with the immune system, pharmacokinetics, and specific biological activity. Such properties may depend not only on the presence or absence but also on the specific structures of the oligosaccharides.

Typically, immunoglobulins or antibodies in their native form are usually tetrameric glycoproteins consisting of two light and two heavy chains. Such immunoglobulins typically contain oligosaccharides at conserved positions in the heavy chain constant regions, which may differentially influence protein assembly, secretion, or functional activity (Boyd et al., (1995) Mol. Immunol. 32:1311-1318; Wittwer A., and Howard, S. C. (1990) Biochem. 29:4175-4180; Wright, A., and Morrison, S. L., Trends Biotech. 15:26-32 (1997)).

For example, increased galactosylation of antibodies may be functionally more anti-inflammatory, as described, for example, in the report by Karsten et al. (Nature Medicine 18.9 (2012) 1401–1406) in mice, where high galactosylation of IgG immune complexes promotes the association of Fcγ RIIB and Dectin-1, which blocks the pro-inflammatory effector functions of C5aR and CXCR226. Other reported effects of galactosylation on IgG molecules include alteration of physicochemical properties such as conformation and surface accessibility (Krapp et al., J. Mol. Biol. 325 (2003) 979–89; Mimura et al., Mol. Immunol. 37 (2000) 697–706). Fortunato and Colina (J. Phys. Chem. 118 (2014) 9844–9851) used explicit atomistic water molecular dynamics simulations to study the effects of galactosylation in the Fc domain of immunoglobulin G1. They suggested that glycosylation could be exploited as a route to improving the aggregation stability of monoclonal antibodies for therapeutic treatment.

Given the influence of different glycosylation levels and/or glycosylation patterns of glycoproteins on their properties, in particular those related to therapeutic efficacy, it is important to ensure that the glycosylation pattern of glycoproteins produced, in particular for clinical use, is uniform and thus at least to maintain the beneficial properties of antibodies.

However, typically, expression of recombinant glycoproteins in host cells results in variations in the oligosaccharide structures attached to a given glycosylation site, so that the produced glycoproteins exist as multiple glycoforms. Thus, it has been technically very challenging to precisely regulate and control the glycosylation level and/or glycosylation pattern in producer cells in vivo in a given therapeutic protein production process.

Over the past decades, various methods have been proposed and several process parameters have been investigated that can alter the glycosylation pattern of glycoproteins in host cells, including the introduction or overexpression of certain enzymes in host cells that are involved in the production of oligosaccharides (U.S. Patent No. 5,047,355, U.S. Patent No. 5,510,261), changes in oxygenation level, pH, purification schemes, etc. (Werner, R. and Noe, W. (1993), Drug Res. 43:1134-1139; Werner, R. and Noe, W. (1993), Drug Res. 43:1242-1249; Hayter et al, (1992) Biotech, and Bioeng. 39:327-335; Borys et al., (1994) Biotech and Bioeng. 43:505-514; Borys et al., (1993) Bio/technology 11:720-724; Hearing et al., (1989) J. Cell Biol. 108:339-353; Goochee et al., in Frontiers in Bioprocessing II, Todd et al., eds (1992) American Chemical Society pp.199-240; US Patent No. 5,096,816; Chotigeat, W, (1994) Cytotech. 15:217-221).

As noted above, the production of glycoproteins with a desired glycosylation level and/or glycosylation pattern is important to at least maintain and optionally optimize the beneficial properties of said antibodies, in particular properties related to their therapeutic efficacy.

The technical problem is solved by providing embodiments presented in this document below and described in the attached claims.

ESSENCE OF THE INVENTION

In recent years, much effort has been devoted to fundamentally understanding and engineering control of the protein galactosylation process in mammalian cells such as CHO cells. There are still several general strategies that can be used to influence the degree of protein glycosylation, where the effects are often cell type and product dependent (Hossler et al., Glycobiology 19(9) (2009) 936–949; Hossler, Genomics and Systems Biology of Mammalian Cell Culture 127 (2012) 187–219): 1) improving the activity of glycosyltransferases, 2) improving the availability and activity of nucleotide sugar transporters for nucleotide sugar transfer, 3) increasing the availability of nucleotide sugar substrates, and 4) decreasing glycosidase activity in extracellular glycan degradation. (Biotechnol Bioeng 96(3) (2007) 538–549) showed that addition of manganese, a preferred cofactor for both the oligosaccharyltransferase complex and β1,4-galactosyltransferase, to CHO cell cultures increased site occupancy and β1,4-galactosylation of rhEPO N-glycans in late culture. (Biotechnol Bioeng. 108(7) (2011) 1591–602) reported that the synergistic combination of uridine, MnCl ₂ , and galactose significantly increased mAb galactosylation relative to the total concentration of UMG supplied. Some approaches using overexpression and/or knockdown of glycosyltransferases and nucleotide sugar transporters have also been reported (Jeong et al., J Microbiol Biotechnol 18(12) (2008) 1945 1952; Weikert et al., Nat Biotechnol 17(11) (1999) 1116–1121). Nucleotide sugar precursor feeding has been reported as a possible strategy to control glycosylation of recombinant proteins (Wong et al., Biotechnology and Bioengineering 107.2 (2010) 321–336). In particular, addition of galactose, glucosamine, and N-acetylmannosamine to cell culture medium has been shown to increase intracellular nucleotide sugar levels. However, the effects of elevated intracellular nucleotide sugar levels on the expression of glycosylation genes have not been well studied. Moreover, increasing intracellular nucleotide sugar levels did not necessarily improve glycosylation of recombinant proteins. Some studies used a strategy to inhibit cellular glycosidases (Ngantung et al., Biotechnol Bioeng. 95(1) (2006) 106-19) to improve protein glycosylation. However, this approach worked only occasionally. Thus, there is still a need for basic research to understand how various external factors affect the process of intracellular galactosylation.

As described herein, antibody galactosylation depends on the concentration of UDP-galactose present in the cells, since these sugar molecules act as substrates required for galactosylation. Thus, it was found that increasing UDP-galactose content is associated with higher galactosylation and sialylation of antibody expressed in CHO cells. Different levels of UDP-galactose in cells can significantly affect glycan heterogeneity.

In this context, the UDP-glucose and UDP-galactose conversion pathway involving two identified enzymes, uridine diphosphate α-D-glucose epimerase (UDP-Glc-E) and UDP-α-D-glucose: α-D-galactose-1-phosphate uridyltransferase (UDP-Gal-T) was found to play a critical role in the UDP-glucose and UDP-galactose conversion pathway in mammalian cells such as CHO cells.

In this regard, UDP-Gal-T (EC 2.7.7.12) represents a special class of enzymes that can be regulated by several simple sulfhydryl molecules such as L-cysteine, glutathione, 2-mercaptoethanol and DTT. As early as 1966, Mayes and Hansen (Methods Enzymol. 9 (1966) 708-713) discovered that L-cysteine could be used to activate and stimulate the enzymatic activity of partially purified UDP-Gal-T from calf liver. In the following years, Mayes (Arch. Biochem. Biophys. 172 (1976) 715-720) carried out extensive studies with fully purified UDP-Gal-T from calf liver and further confirmed this activating function of L-cysteine. Similar studies have also been carried out with UDP-Gal-T from other organisms. Saito et al. (J. Biol. Chem. 242 (1967) 2362–2368) demonstrated that L-cysteine stimulated purified UDP-Gal-T from E. coli to its maximal activity. Chowdhury (Indian J. Biochem. Biophys. 16 (1979) 273–277) also confirmed this observation with purified UDP-GAL-T from E. coli.

Thus, by means of the present invention, it was found that it is possible to regulate and control the intracellular content of UDP-galactose and galactosylation of recombinant proteins such as antibodies during the production process using sulfhydryl compounds or derivatives thereof. For example, by means of the present invention, it was found that it is possible to regulate and control the processing of N-glycans of a recombinant monoclonal antibody during the production process using sulfhydryl compounds or derivatives thereof for successful and specific control of the cell culture process for fine-tuning the galactosylation of the antibody.

One of the features of the present invention is the use of simple sulfhydryl molecules or compounds such as L-cysteine to modulate the activity of UDP-Gal-T in vivo in mammalian cell lines such as CHO K1 and its derivative cell lines to produce a recombinant protein containing monogalactosylated (G1) or digalactosylated (G2) glycans. In other words, the present invention provides new approaches to regulating gluco- and galacto-configured UDP F-sugars in vivo, in particular by modulating the recently identified and reported dual enzyme pathway UDP-Gal-T and UDP-Glc-E in mammalian cells such as CHO K1 and its derivative cell lines to produce a recombinant protein having monogalactosylated (G1) or digalactosylated (G2) glycans. As shown in the examples provided herein, it has been demonstrated that by adjusting the relative concentrations of certain sulfhydryl compounds or derivatives thereof in the medium, galactosylation of various antibodies can be precisely controlled in a high-throughput, batch or fed-batch production process, with minimal impact on other glycoforms, other product quality characteristics, or cell culture productivity. Moreover, these data demonstrate that it is possible to precisely regulate and control a complex dynamic cellular process at the production scale to determine the molecular heterogeneity and biological activity of a recombinant antibody product. This provides insight into the key effector interactions underlying the knowledge-based design of a cell culture medium or nutrient composition to achieve a given level of antibody galactosylation while maximizing cell proliferation and productivity.

In particular, the present invention relates to a CD20/CD3 bispecific antibody comprising monogalactosylated (G1) and digalactosylated (G2) glycans, a CD20/CD3 bispecific antibody comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises a heavy chain variable domain (VH) comprising

(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and

(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; and

variable light chain (VL) domain containing

(d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25,

(d) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and

(e) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27;

wherein the second antigen-binding domain comprises a heavy chain variable domain (VH) comprising

(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34,

(b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and

(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; and

variable light chain (VL) domain containing

(d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37,

(d) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38, and

(e) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39; and

wherein the CD20/CD3 bispecific antibody has 19.0-29.0% (w/w) G1 and 1.3-2.8% (w/w) G2 per total glycan; preferably 20.0-28.0% (w/w) G1 and 1.4-2.7% (w/w) G2 per total glycan; more preferably 21.0-28.0% (w/w) G1 and 1.5-2.7% (w/w) G2 per total glycan, and most preferably 21.0-27.4% (w/w) G1 and 1.5-2.6% (w/w) G2 per total glycan.

Preferably, the CD20/CD3 bispecific antibody is an antibody in which

(a) the first antigen-binding domain comprises the VH sequence of SEQ ID NO: 28, and the second antigen-binding domain comprises the VH sequence of SEQ ID NO: 40;

(b) the first antigen-binding domain comprises the VL sequence of SEQ ID NO: 29 and the second antigen-binding domain comprises the VL sequence of SEQ ID NO: 41; or

(c) the first and second antigen-binding domains comprise a VH sequence as defined in (a) and a VL sequence as defined in (b).

More preferably, the CD20/CD3 bispecific antibody is an antibody in which

(a) the VH sequence of the first antigen-binding domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 28, and the VH sequence of the second antigen-binding domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 40;

(b) the VL sequence of the first antigen-binding domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 29, and the VL sequence of the second antigen-binding domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 41; or

(c) the bispecific antibody to CD20/CD3 comprises the VH sequence of the first and second antigen-binding domains defined in (a) and the VL sequence of the first and second antigen-binding domains defined in (b).

Even more preferably, the CD20/CD3 bispecific antibody is an antibody in which the CD20/CD3 bispecific antibody comprises

(a) a first heavy chain of SEQ ID NO: 46 and a second heavy chain of SEQ ID NO: 45,

(b) a first light chain of SEQ ID NO: 33 and a second light chain of SEQ ID NO: 44; or

(c) a first heavy chain and a second heavy chain as defined in (a), and a first light chain and a second light chain as defined in (b).

Most preferably, the CD20/CD3 bispecific antibody is an antibody in which the CD20/CD3 bispecific antibody comprises

(a) a first heavy chain of SEQ ID NO: 47 and a second heavy chain of SEQ ID NO: 45,

(b) a first light chain of SEQ ID NO: 33, a second light chain and a third light chain of SEQ ID NO: 44; or

(c) a first heavy chain and a second heavy chain as defined in (a), and a first light chain, a second light chain and a third light chain as defined in (b).

The present invention equally relates to an antibody to α-synuclein having monogalactosylated (G1) and digalactosylated (G2) glycans, wherein the antibody to α-synuclein comprises a heavy chain variable domain (VH) comprising

(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10,

(b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and

(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; and

variable light chain (VL) domain containing

(d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13,

(d) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and

(e) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and

wherein the anti-α-synuclein antibody has 17.2-48.0% (w/w) G1 and 3.1-15.0% (w/w) G2 per total glycan; preferably 25.4-48.0% (w/w) G1 and 3.5-15.0% (w/w) G2 per total glycan; preferably 27.2-47.0% G1 and 4.4-15.0% G2 per total glycan; preferably 40.0-46.0% (w/w) G1 and 8.4-15.0% (w/w) G2 per total glycan; more preferably 41.0-45.0% (w/w) G1 and 9.5-14.0% (w/w) G2 of total glycan, and most preferably 42.1-43.9% (w/w) G1 and 10.6-13.3% (w/w) G2 of total glycan.

Preferably, the antibody to α-synuclein comprises

(a) VH sequence SEQ ID NO: 16;

(b) the VL sequence SEQ ID NO: 17; or

(c) the VH sequence defined in (a) and the VL sequence defined in (b).

More preferably, the antibody to α-synuclein comprises

(a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16;

(b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17; or

(c) the VH sequence defined in (a) and the VL sequence defined in (b).

Even more preferably, the antibody to α-synuclein comprises the heavy chain of SEQ ID NO: 20 and the light chain of SEQ ID NO: 21.

Preferably, the monogalactosylated (G1) and digalactosylated (G2) glycans of the bispecific anti-CD20/CD3 antibody or anti-α-synuclein antibody as disclosed in the context of the invention are linked to N-acetylglucosamine.

Equally, the present invention relates to a cell culture medium for producing an antibody as disclosed in the context of the invention having monogalactosylated (G1) and digalactosylated (G2) glycans in a mammalian cell, wherein the cell culture medium comprises sulfhydryl group(s) of one or more sulfhydryl compounds at a concentration of greater than 4.0 mM and less than 10.0 mM and glucose at a concentration of at least greater than 3.0 g/L.

Preferably, the cell culture medium is a medium with a defined chemical composition, preferably a cell culture medium that does not contain serum, proteins and/or oligopeptides, more preferably a medium with a defined chemical composition, more preferably a cell culture medium that does not contain serum, proteins and oligopeptides.

Equally, the present invention relates to a mammalian cell producing a bispecific antibody to CD20/CD3 as disclosed in the context of the invention, having monogalactosylated (G1) and digalactosylated (G2) glycans, comprising a polynucleotide comprising a sequence having 80% identity to a polynucleotide encoding a first antigen-binding domain comprising a first and a second variable domain of a heavy chain (VH) as disclosed in the context of the invention, or comprising a sequence having 80% identity to a polynucleotide comprising a sequence encoding a second antigen-binding domain comprising a first and a second variable domain of a light chain (VL) as disclosed in the context of the invention, or

one or more vectors containing such polynucleotides,

wherein said cell is cultured in a cell culture medium as disclosed in the context of the invention.

Preferably, the mammalian cell further comprises a sequence having 80% identity with a polynucleotide encoding the first and second heavy chains as disclosed in the context of the invention, or comprising a sequence having 80% identity with a polynucleotide comprising a sequence encoding the first and second light chains as disclosed in the context of the invention, or

one or more vectors containing such polynucleotides,

wherein said cell is cultured in a cell culture medium as disclosed in the context of the invention.

The present invention equally relates to an antibody to α-synuclein having monogalactosylated (G1) and digalactosylated (G2) glycans, comprising a polynucleotide comprising a sequence having 80% identity with a polynucleotide encoding a heavy chain variable domain (VH) as described in the context of the invention, or a polynucleotide comprising a sequence having 80% identity with a polynucleotide encoding a light chain variable domain (VL) as disclosed in the context of the invention, or

one or more vectors containing such polynucleotides,

wherein said cell is cultured in a cell culture medium as disclosed in the context of the invention.

The present invention equally relates to a method for producing a CD20/CD3 bispecific antibody as disclosed in the context of the invention having monogalactosylated (G1) and digalactosylated (G2) glycans, said method comprising:

(a) culturing a mammalian cell as disclosed in the context of the invention in a cell culture medium as disclosed in the context of the invention, wherein the concentration of the sulfhydryl group(s) of one or more sulfhydryl compounds of at least greater than 4.0 and less than 10.0 mM and of glucose of at least greater than 3.0 g/L in the cell culture medium is maintained for at least 3, more preferably for at least 4 and even more preferably for at least 5 days,

(b) isolating said antibody.

Preferably, the concentration is maintained effective to produce a CD20/CD3 bispecific antibody having 19.0-29.0% (w/w) G1 and 1.3-2.8% (w/w) G2 per total glycan; preferably 20.0-28.0% (w/w) G1 and 1.4-2.7% (w/w) G2 per total glycan; more preferably 21.0-28.0% (w/w) G1 and 1.5-2.7% (w/w) G2 per total glycan, and most preferably 21.0-27.4% (w/w) G1 and 1.5-2.6% (w/w) G2 per total glycan.

Preferably, culturing the mammalian cell results in an increase in the titer of said antibody by at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60% and most preferably at least 80%.

relative to the titer when the mammalian cell is appropriately cultured without maintaining the sulfhydryl group(s) of one or more sulfhydryl compounds at a concentration of at least greater than 4.0 mM and less than 10.0 mM in the cell culture medium. As another example, culturing the mammalian cell results in an increase in the titer of said antibody of between 30 and 75% relative to the titer when the mammalian cell is appropriately cultured without maintaining the sulfhydryl group(s) of one or more sulfhydryl compounds at a concentration of at least greater than 4.0 mM and less than 10.0 mM in the cell culture medium.

The present invention equally relates to a method for producing an antibody to α-synuclein as disclosed in the context of the invention, having monogalactosylated (G1) and digalactosylated (G2) glycans, said method comprising:

(a) culturing a mammalian cell as disclosed in the context of the invention in a cell culture medium as disclosed in the context of the invention, wherein the concentration of the sulfhydryl group(s) of one or more sulfhydryl compounds of at least greater than 4.0 and less than 10.0 mM and of glucose of at least greater than 3.0 g/L in the cell culture medium is maintained for at least 3, more preferably for at least 4 and even more preferably for at least 5 days,

(b) isolating said antibody.

Preferably, the concentration is effective to produce an anti-α-synuclein antibody comprising 17.2-48.0% (w/w) G1 and 3.1-15.0% (w/w) G2 on a total glycan basis; 25.4-48.0% (w/w) G1 and 3.5-15.0% (w/w) G2 on a total glycan basis; preferably 27.2-47.0% G1 and 4.4-15.0% G2 on a total glycan basis; preferably 40.0-46.0% (w/w) G1 and 8.4-15.0% (w/w) G2 on a total glycan basis; more preferably 41.0-45.0% (w/w) G1 and 9.5-14.0% (w/w) G2 of total glycan, and most preferably 42.1-43.9% (w/w) G1 and 10.6-13.3% (w/w) G2 of total glycan.

Preferably, culturing the mammalian cell results in an increase in the titer of said antibody by at least 10%, preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80% and most preferably at least 100%.

relative to the titer in the corresponding culture of a mammalian cell without maintaining the sulfhydryl group(s) of one or more sulfhydryl compounds at a concentration of at least greater than 4.0 mM and less than 10.0 mM in the cell culture medium.

Preferably, culturing the mammalian cell results in the production of an antibody to α-synuclein having monogalactosylated (G1) and digalactosylated (G2) glycans characterized by

(i) increased binding of the target protein by at least 10%, preferably at least 20%, more preferably at least 35%, even more preferably at least 45%, and most preferably at least 50%;

(ii) increased neonatal Fc receptor (FcRn) binding by at least 10%, preferably at least 20%, more preferably at least 33%, more preferably at least 40%, and most preferably at least 45%; and/or

(iii) increased FcγRIIa binding by at least 10%, preferably at least 20%, more preferably at least 36%, even more preferably 45% and most preferably 50%,

with respect to non-monogalactosylated (G1) and non-digalactosylated (G2) forms of an antibody to α-synuclein upon appropriate culturing of a mammalian cell without maintaining the sulfhydryl group(s) of one or more sulfhydryl compounds at a concentration of at least greater than 4.0 mM and less than 10.0 mM in the cell culture medium.

The methods as disclosed in the context of the invention preferably comprise culturing a mammalian cell at an initial concentration of at least greater than 3.0 and less than 10.0 mM of the sulfhydryl group(s) of one or more sulfhydryl compounds. Preferably, the methods further comprise the step of pre-cultivating the mammalian cell in a cell culture medium prior to said culturing. Preferably, the concentration is maintained for at least 5 days, preferably for at least 7 days, more preferably for at least 10 days, even more preferably for at least 12 days and most preferably for at least 14 days.

Preferably, the sulfhydryl group(s) of one or more sulfhydryl compounds in the cell culture medium as disclosed in the context of the invention is/are contained in a reduced and/or oxidized form, more preferably the concentration of the reduced form of said sulfhydryl is in the range of more than 4.0 mM to less than 10.0 mM and/or the concentration of the oxidized form of said sulfhydryl is in the range of more than 2.0 mM to less than 5.0 mM.

Preferably, the methods as disclosed in the context of the invention further comprise collecting a bispecific CD20/CD3 antibody or an anti-α-synuclein antibody.

Preferably, the methods as disclosed in the context of the invention further comprise altering the level of monogalactosylated (G1) and digalactosylated (G2) glycans of the bispecific anti-CD20/CD3 antibody or the anti-α-synuclein antibody. Preferably, the methods further comprise formulating the bispecific anti-CD20/CD3 antibody or the anti-α-synuclein antibody into a medicinal product.

Preferably, the cell culture medium as disclosed in the context of the invention comprises at least more than 4.0 mM and equal to or less than 9.0 mM, preferably at least more than 4.0 mM and equal to or less than 8.0 mM, more preferably at least more than 4.0 mM and equal to or less than 7.0 mM and even more preferably at least more than 4.0 mM and equal to or less than 6.0 mM sulfhydryl group(s) of one or more sulfhydryl compounds.

Preferably, the cell culture medium as disclosed in the context of the invention comprises the sulfhydryl group(s) of one or more sulfhydryl compounds in a concentration of at least 5.0 mM and less than 10.0 mM, preferably at least 5.0 mM and equal to or less than 9.0 mM, more preferably at least 5.0 mM and equal to or less than 8.0 mM, even more preferably at least 5.0 mM and equal to or less than 7.0 mM and most preferably at least 5.0 mM and equal to or less than 6.0 mM.

Preferably, the cell culture medium as disclosed in the context of the invention comprises at least more than 2.0 g/l glucose, preferably at least more than 3.0 g/l glucose and more preferably at least more than 4.0 g/l glucose.

Preferably, the cell culture medium contains no more than 13.0 g/L glucose, preferably no more than 8.0 g/L, more preferably no more than 7.0 g/L, even more preferably no more than 6.0 g/L and most preferably no more than 5.0 g/L glucose.

Preferably, the cell culture medium contains from more than 2.0 g/L to no more than 13.0 g/L glucose, preferably from more than 2.0 g/L to no more than 8.0 g/L glucose, more preferably from more than 2.0 g/L to no more than 7.0 g/L glucose, even more preferably from more than 2.0 g/L to no more than 6.0 g/L glucose, and most preferably from more than 2.0 g/L to no more than 5.0 g/L glucose.

Preferably, the one or more sulfhydryl compound(s) of the cell culture medium as disclosed in the context of the invention is(are) selected from the group consisting of cysteine, cystine, succimer, methimazole, cysteamine, azathioprine, mercaptopurine, S-methylcysteine, selenocysteine, S-phosphocysteine, 4'-phosphopantetheine, butyrylthiocholine, carbocysteine, N-sulfocysteine, aletheine, acetylcysteine, dimercaprol, coenzyme M, sodium aurothiomalate, pantethine, bucillamine, methylselenocysteine, dimercaptosuccinic acid, acetylcysteine amide, thioglycolic acid, 2,3-dimercaptopropanol, O-methylmercaptoethanol, mercaptoacetic acid, β-mercaptopropionic acid, methyl mercaptan, S-methylmercaptoethanol, glutathione, glutathione derivatives and a combination thereof. More preferably, the one or more sulfhydryl compound(s) is(are) selected from the group consisting of cysteine, cystine and a combination thereof. Even more preferably, the one or more sulfhydryl compound(s) is(are) cysteine, and wherein the concentration of cysteine in the cell culture medium is greater than 4.0 mM and less than 10.0 mM, preferably the concentration of cysteine is at least 5.0 mM and equal to or less than 6.0 mM. Equally preferably, the one or more sulfhydryl compound(s) is cystine, and wherein the concentration of cystine in the cell culture medium is greater than 2.0 mM and less than 5.0 mM, preferably the concentration of cystine is at least 3.0 mM and equal to or less than 4.0 mM.

Preferably, the cultivation of a mammalian cell according to the method as disclosed in the context of the invention is carried out in a large-scale bioreactor, preferably in a bioreactor with a volume of 10,000 L.

The present invention equally relates to a CD20/CD3 bispecific antibody as disclosed in the context of the invention, having monogalactosylated (G1) and digalactosylated (G2) glycans obtained using a method or a mammalian cell as disclosed in the context of the invention.

The present invention equally relates to an antibody to α-synuclein as disclosed in the context of the invention, having monogalactosylated (G1) and digalactosylated (G2) glycans obtained using a method or a mammalian cell as disclosed in the context of the invention.

Preferably, the invention relates to a CD20/CD3 bispecific antibody as disclosed in the context of the invention, having monogalactosylated (G1) and digalactosylated (G2) glycans, for use as a medicament. More preferably, the CD20/CD3 bispecific antibody as disclosed in the context of the invention is for the treatment of patients with B-cell related cancers, preferably for use in the treatment of patients with chronic leukemia and lymphoma.

Equally preferably, the invention relates to an antibody to α-synuclein having monogalactosylated (G1) and digalactosylated (G2) glycans for use as a medicament. More preferably, the antibody to α-synuclein as disclosed in the context of the invention is for the treatment of patients with Parkinson's disease.

SYMBOLS OF FIGURES

Figure 1. Glucose conversion pathway for the formation of UDP-glucose and UDP-galactose in CHO K1M. EC 2.7.7.9: UTP: α-D-glucose-1-phosphate uridylyltransferase. UDP-Glc-E: Uridine diphosphate glucose epimerase.

Figure 2. Combined pathways for the interconversion of UDP-glucose and UDP-galactose into CHO K1M. EC 2.7.7.9: UTP: α-D-glucose-1-phosphate uridylyltransferase. UDP-Glc-E: Uridine diphosphate glucose epimerase. UDP-Gal-T: UDP-α-D-glucose: α-D-galactose-1-phosphate uridylyltransferase.

Figure 3. Modulation of the interconversion pathways of UDP-glucose and UDP-galactose to CHO K1M by sulfhydryl compounds. UDP-Glc-E: Uridine diphosphate glucose epimerase. UDP-Gal-T: UDP-α-D-glucose: α-D-galactose-1-phosphate uridylyltransferase.

Figure 4. Modulation of the interconversion pathways of UDP-glucose and UDP-galactose to CHO K1M with L-cystine in cell culture medium. UDP-Glc-E: Uridine diphosphate glucose epimerase. UDP-Gal-T: UDP-α-D-glucose: α-D-galactose-1-phosphate uridylyltransferase.

Figure 5. UDP-galactose is formed from UDP-glucose and is used in the Golgi apparatus for protein galactosylation. UDP-Glc-E: Uridine diphosphate glucose epimerase. UDP-Gal-T: UDP-α-D-glucose: α-D-galactose-1-phosphate uridylyltransferase. EC2.4.1.38: B-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase.

Figure 6A. Effect of different concentrations of L-cysteine on the G0 form of anti-α-synuclein antibody in CHO L965 cells cultured with 6 mM L-cysteine or 10 mM L-cysteine in the production medium. The percentage of G0 form of anti-α-synuclein antibody at the end of the 14-day production process is shown.

Figure 6B. Effect of different concentrations of L-cysteine on the G1 form of anti-α-synuclein antibody in CHO L965 cells cultured with 6 mM L-cysteine or 10 mM L-cysteine in the production medium. The percentage of G1 form of anti-α-synuclein antibody at the end of the 14-day production process is shown.

Figure 6B. Effect of different concentrations of L-cysteine on the G2 form of anti-α-synuclein antibody in CHO L965 cells cultured with 6 mM L-cysteine or 10 mM L-cysteine in the production medium. The percentage of G2 form of anti-α-synuclein antibody at the end of the 14-day production process is shown.

Figure 6D. Effect of different concentrations of L-cysteine on the titer of anti-α-synuclein antibody product in CHO L965 cells cultured with 6 mM L-cysteine or 10 mM L-cysteine in the production medium. The percentage of anti-α-synuclein antibody product titer at the end of the 14-day production process is shown.

Figure 6D. Effect of different concentrations of L-cysteine on cell growth (IVCD) of a culture of CHO L965 cells producing an antibody to α-synuclein with 6 mM L-cysteine or 10 mM L-cysteine in the production medium. The percentage of cell growth (IVCD) is shown at the end of the 14-day production process.

Figure 7A. Effect of different concentrations of L-cysteine on the G0 form of the bispecific anti-CD20/CD3 antibody in CHO T104 cells cultured with 5 mM L-cysteine or 10 mM L-cysteine in the production medium. The percentage of the G0 form of the bispecific anti-CD20/CD3 antibody at the end of the 14-day production process is shown.

Figure 7B. Effect of different concentrations of L-cysteine on the G1 form of the bispecific anti-CD20/CD3 antibody in CHO T104 cells cultured with 5 mM L-cysteine or 10 mM L-cysteine in the production medium. The percentage of the G1 form of the bispecific anti-CD20/CD3 antibody at the end of the 14-day production process is shown.

Figure 7B. Effect of different concentrations of L-cysteine on the titer of the bispecific CD20/CD3 antibody product in CHO T104 cells with 5 mM L-cysteine or 10 mM L-cysteine in the production medium. The percentage of the bispecific CD20/CD3 antibody product titer at the end of the 14-day production process is shown.

Figure 7D. Effect of different concentrations of L-cysteine on cell growth (IVCD) of a culture of CHO T104 cells producing bispecific antibody to CD20/CD3 with 5 mM L-cysteine or 10 mM L-cysteine in the production medium. The percentage of cell growth (IVCD) is shown at the end of the 14-day production process.

Figure 8A. Effect of different concentrations of L-cysteine on the G0 form of the anti-CD20/CD3 bispecific antibody in CHO T104 cells cultured with 5 mM L-cysteine or 10 mM L-cysteine in the production medium. The percentage of the G0 form of the anti-CD20/CD3 bispecific antibody at the end of the 14-day production process is shown.

Figure 8B. Effect of different concentrations of L-cysteine on the G1 form of the bispecific anti-CD20/CD3 antibody in CHO T104 cells cultured with 5 mM L-cysteine or 10 mM L-cysteine in the production medium. The percentage of the G1 form of the bispecific anti-CD20/CD3 antibody at the end of the 14-day production process is shown.

Figure 8B. Effect of different concentrations of L-cysteine on the titer of the bispecific CD20/CD3 antibody product in CHO T104 cells cultured with 5 mM L-cysteine or 10 mM L-cysteine in the production medium. The percentage of the bispecific CD20/CD3 antibody product titer at the end of the 14-day production process is shown.

Figure 8D. Effect of different concentrations of L-cysteine on cell growth (IVCD) of a culture of CHO T104 cells producing bispecific antibody to CD20/CD3 with 5 mM L-cysteine or 10 mM L-cysteine in the production medium. The percentage of cell growth (IVCD) is shown at the end of the 14-day production process.

Figure 9A. Effect of different concentrations of L-cystine on the G0 form of anti-α-synuclein antibody in CHO L967 cells cultured with 2 mM L-cystine or 4 mM L-cystine. The percentage of G0 form of anti-α-synuclein antibody at the end of the 14-day production process is shown.

Figure 9B. Effect of different concentrations of L-cystine on the G1 form of anti-α-synuclein antibody in CHO L967 cells cultured with 2 mM L-cystine or 4 mM L-cystine in the production medium. The percentage of G1 form of anti-α-synuclein antibody at the end of the 14-day production process is shown.

Figure 9B. Effect of different concentrations of L-cystine on the G2 form of anti-α-synuclein antibody in CHO L967 cells cultured with 2 mM L-cystine or 4 mM L-cystine in the production medium. The percentage of G2 form of anti-α-synuclein antibody at the end of the 14-day production process is shown.

Figure 9D. Effect of different concentrations of L-cystine on the titer of anti-α-synuclein antibody product in CHO L967 cells cultured with 2 mM L-cystine or 4 mM L-cystine in the production medium. The percentage of anti-α-synuclein antibody product titer at the end of the 14-day production process is shown.

Figure 9D. Effect of different concentrations of L-cystine on cell growth (IVCD) of a culture of CHO L967 cells producing an antibody to α-synuclein with 2 mM L-cystine or 4 mM L-cystine in the production medium. The percentage of cell growth (IVCD) is shown at the end of the 14-day production process.

Figure 10A. Effect of different concentrations of L-cystine on the G0 form of anti-α-synuclein antibody in CHO L971 cells cultured with 2 mM L-cystine or 4 mM L-cystine in the production medium. The percentage of G0 form of anti-α-synuclein antibody at the end of the 14-day production process is shown.

Figure 10B. Effect of different concentrations of L-cystine on the G1 form of anti-α-synuclein antibody in CHO L971 cells cultured with 2 mM L-cystine or 4 mM L-cystine in the production medium. The percentage of G1 form of anti-α-synuclein antibody at the end of the 14-day production process is shown.

Figure 10B. Effect of different concentrations of L-cystine on the G2 form of anti-α-synuclein antibody in CHO L971 cells cultured with 2 mM L-cystine or 4 mM L-cystine in the production medium. The percentage of G2 form of anti-α-synuclein antibody at the end of the 14-day production process is shown.

Figure 10D. Effect of different concentrations of L-cystine on the titer of anti-α-synuclein antibody product in CHO L971 cells cultured with 2 mM L-cystine or 4 mM L-cystine in the production medium. The percentage of anti-α-synuclein antibody product titer at the end of the 14-day production process is shown.

Figure 10D. Effect of different concentrations of L-cystine on cell growth (IVCD) of a culture of CHO L971 cells producing an antibody to α-synuclein with 2 mM L-cystine or 4 mM L-cystine in the production medium. The percentage of cell growth (IVCD) is shown at the end of the 14-day production process.

Figure 11A. Effect of different concentrations of E-cystine/E-cysteine on the G1 form of anti-α-synuclein antibody in CHO L971 cells with 6 mM L-cysteine or 3 mM L-cystine in the production medium. The percentage of G1 form of anti-α-synuclein antibody at the end of the 14-day production process is shown.

Figure 11B. Effect of different concentrations of L-cystine/L-cysteine on the G2 form of anti-α-synuclein antibody in CHO L971 cells with 6 mM L-cysteine or 3 mM L-cystine in the production medium. The percentage of G2 form of anti-α-synuclein antibody at the end of the 14-day production process is shown.

Figure 11B. Effect of different L-cystine/L-cysteine concentrations on the relative level of anti-α-synuclein FcRn binding in CHO L971 cells with 6 mM L-cysteine or 3 mM L-cystine in the production medium. The percentage of relative anti-α-synuclein FcRn binding at the end of the 14-day production process is shown.

Figure 11D. Effect of different L-cystine/L-cysteine concentrations on the relative binding of Fcy-RIIa (H131) anti-α-synuclein antibody in CHO L971 cells cultured with 6 mM L-cysteine or 3 mM L-cystine in the production medium. The percentage of relative binding of Fcy-RIIa (H131) anti-α-synuclein antibody at the end of the 14-day production process is shown.

Figure 11D. Effect of different L-cystine/L-cysteine concentrations on the relative target binding of an anti-α-synuclein antibody in CHO L971 cells with 6 mM L-cysteine or 3 mM L-cystine in the production medium. The percentage of relative target binding at the end of the 14-day production process is shown.

Figure 12. Schematic representation of glycosylation sites in the Fc region (No. 1 Fc glycosylation), binding sites for FcγIIa and FcRn effectors (No. 2 FcγIIa binding and FcRn binding), the antigen binding site in the Fab region (4 Fab binding), and C-terminal modifications (No. 5 C-terminal alteration) of the antibody.

DETAILED DESCRIPTION OF THE ESSENCE OF THE INVENTION

Recombinant proteins having different glycosylation patterns, especially galactosylation patterns, are usually obtained by enzymatic production process using mammalian expression systems. Post-translational glycosylation, particularly galactosylation, of proteins is required for important physicochemical properties and functions such as protein solubility, stability, clearance, immunogenicity and immune effector functions. In this regard, differences in glycosylation patterns, especially galactosylation patterns of recombinantly produced proteins, have recently received much attention in the scientific community as recombinant proteins produced as potential prophylactic and therapeutic agents approach clinical practice. The oligosaccharide side chains of glycoproteins can influence protein function (Wittwer and Howard, Biochem. 29 (1990) 4175-4180) and the intramolecular interactions between glycoprotein moieties leading to conformation and three-dimensional surface presentation of the protein (Hart, Curr. Op. Cell Biol., 4 (1992) 1017-1023: Goochee, et al., Bio/Technology 9 (1991) 1347-1355; Parekh, Curr, Op. Struct, Biol. 1 (1991) 750-754). For example, the galactosylation status of a recombinant protein is regulated by different enzymes, and differences in the function of one or more of these enzymes can have a significant effect on the galactosylation status of the recombinant protein. In this context, multiple effects of modulation of the UDP-sugar enzymatic pathway have been demonstrated in recombinant mammalian cells, such as CHO cells, and include, but are not limited to, effects on cell growth, recombinant protein productivity, and/or protein quality, in particular on the galactosylation level. Additional effects on the biological functions of recombinant mAbs have been demonstrated. It is therefore important to maintain the galactosylation pattern of recombinant proteins, particularly proteins intended for use as therapeutics.

It is known that the quality of recombinant proteins, in particular therapeutic proteins such as antibodies, depends on various parameters such as the concentration of various nutrients present in the medium, since such nutrients, for example sugar molecules, act as precursors of the pool of sugar nucleotides inside the cells required for glycosylation. Different levels of sugar nucleotide concentration in cells can have a significant impact on the heterogeneity of antibodies, which is especially important in batch or fed-batch processes, where a constant product quality is necessary to achieve a predetermined clinical outcome.

Thus, one of the main objectives of the present invention is to use sulfhydryl compounds such as cysteine or cystine to regulate the intracellular concentration of UDP-glucose and UDP-galactose in mammalian cells in vivo, such as CHO K1 and its derived cell lines, thereby improving the quality of the final product for predetermined clinical applications.

In this context, the present invention relates to a glycoprotein, in particular a recombinant glycoprotein having monogalactosylated (G1) and digalactosylated (G2) glycans, as well as to means and methods for producing said glycoprotein. Therefore, a glycoprotein as described herein and in the context of the invention may be, for example, a therapeutic glycoprotein, such as a recombinant glycoprotein. Thus, as disclosed herein and used in the context of the invention, a glycoprotein, such as a recombinant protein, is a protein that can be linked to one or more glycans. As disclosed herein and illustrated in the accompanying examples, a protein linked to one or more glycans refers to a protein having monogalactosylated (G1) or digalactosylated (G2) glycans. A protein having one or more glycans may have multiple sites to which the glycan can be linked. A person skilled in the art knows potential sites where a glycan can be linked to a protein, as disclosed herein and in the context of the invention. For example, a protein can comprise at least one, more preferably at least two galactosylated glycans, wherein the galactosylated glycans can be linked to N-acetylglucosamine. Such glycans can be either monogalactosylated (G1) or digalactosylated (G2). As described herein, the level of monogalactosylated (G1) or digalactosylated (G2) protein can be measured by means and methods known to a person skilled in the art, and as shown in the accompanying examples.

As disclosed herein and in the context of the invention, the recombinant protein can be a therapeutic protein. Such a therapeutic protein can be, for example, an antibody, but is not limited to it. Therefore, said glycoprotein, for example, an antibody, can be used as a drug. Therapeutic proteins can include, but are not limited to, therapeutic proteins for use in the treatment of B-cell proliferative disorders such as non-Hodgkin's lymphoma and chronic lymphocytic leukemia, Parkinson's disease and related disorders. In one specific embodiment, the recombinant protein can be an antibody. For example, the antibody to be produced by a mammalian cell is an antibody intended for use in therapy or an antibody used as a potential drug for the development of a drug for use in therapy. In one embodiment, the recombinant protein can be an antibody to α-synuclein or a bispecific antibody to CD20/CD3.

Said glycoprotein, for example, an antibody, can be produced by a mammalian cell that contains a polynucleotide encoding said glycoprotein. For example, mammalian cells as described herein and in the context of the invention can be cultured in a large-scale bioreactor, such as a 10,000 L bioreactor. Chinese hamster ovary (CHO) cells are the most commonly used eukaryotic hosts for producing recombinant therapeutic protein.

As described herein and in the context of the invention, the glycoprotein can be an antibody, such as a bispecific antibody, for example, an anti-CD20/CD3 antibody. Said antibody can be expressed on a vector comprising one or more polynucleotides encoding said antibody. As another example, the antibody can be an anti-α-synuclein antibody and said antibody can be cloned into a suitable vector, such as, but not limited to, either LoxP.SV40.Puro.CMVi.FseI nbe or the Loxfas.puro.CMV.2L.vl expression vector. CHO-K1M TI host cells were transfected with this polycistronic plasmid to generate stable eukaryotic cell lines. As used herein, the term "vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., that is capable of replication when linked to appropriate control elements and that can effect the transfer of gene sequences between cells. Therefore, the term includes cloning and expression vehicles, as well as viral vectors. For example, one or more of the vectors described herein can be polycistronic vectors. As used herein, the term "polycistronic" refers to mRNA encoding more than one polypeptide chain. As one specific example, more than one vector can be used, wherein a first vector can express a first light chain and a second heavy chain, and a second vector can express a second light chain and a first heavy chain. One skilled in the art will know how to construct vectors suitable for expressing an antibody as described herein and in the context of the invention. Additionally, to generate recombinant cell lines, the host cell may be co-transfected with one or more vectors, wherein said vectors may contain a selection marker such as the dihydrofolate reductase (DHFR) gene. For example, expression of the murine DHFR gene is driven by the simian virus 40 (SV40) early promoter and is terminated by the SV40 polyadenylation signal (SV40 poly A).

Furthermore, a cell culture medium as described herein and in the context of the invention can be used to produce said glycoprotein, such as antibodies, wherein the cell culture medium comprises sulfhydryl group(s) of one or more sulfhydryl compounds at a concentration of greater than 4.0 mM and less than 10.0 mM and glucose at a concentration of at least greater than 3.0 g/L. The cell culture medium can be a medium with a defined chemical composition, preferably a cell culture medium that does not contain serum, proteins and/or oligopeptides. For example, conventional cell culture media with a defined chemical composition can contain up to 100 components that can be grouped by energy sources, amino acids, vitamins, trace elements and inorganic salts, nucleic acid derivatives, fatty acids and lipids and some others.

As described herein, a mammalian cell producing a glycoprotein as described herein and illustrated in the accompanying examples can be cultured by a method comprising

(a) culturing a mammalian cell in a cell culture medium as described herein, wherein the sulfhydryl group(s) of one or more sulfhydryl compounds at a concentration of at least greater than 4.0 mM and less than 10.0 mM and at least greater than 3.0 g/L glucose in the cell culture medium is maintained for at least 3, more preferably at least 4, and even more preferably at least 5 days,

(b) isolation of the said glycoprotein.

Said culturing of a mammalian cell may result in an increase in the titer of said glycoprotein compared to the titer of a corresponding culturing of the mammalian cell without maintaining the sulfhydryl group(s) of one or more sulfhydryl compounds at a concentration of at least greater than 4.0 mM and less than 10.0 mM in the cell culture medium. Said culturing may further result in the formation of a glycoprotein having monogalactosylated (G1) and digalactosylated (G2) glycans as described herein and characterized by increased target protein binding, increased neonatal Fc receptor (FcRn) binding, and/or increased FcγRIIa binding compared to non-monogalactosylated (G1) and non-digalactosylated (G2) forms of the glycoprotein when the mammalian cell is appropriately cultured without maintaining the sulfhydryl group(s) of one or more sulfhydryl compounds at a concentration of at least greater than 4.0 mM and less than 10.0 mM in the cell culture medium. In particular, culturing the mammalian cell may comprise culturing at an initial concentration of at least greater than 3.0 and less than 10.0 mM of the sulfhydryl group(s) of one or more sulfhydryl compounds. The method may further comprise a step of pre-cultivating the mammalian cell in a cell culture medium prior to said culturing.

The cell culturing process disclosed in this document and in the context of the invention refers to a cell culturing process that begins with the inoculation of cells into a cell culturing medium and may end with the collection of cells from the cell culturing medium. Said inoculation refers to day 1 of the cell culturing process, and the collection of cells from the cell culturing medium may be, for example, but not limited to, on day 12, 13 or 14 of cell culturing.

Therefore, a person skilled in the art knows how to select a suitable duration of the cell culturing process based on generally known knowledge. This is also illustrated by the attached examples. The entire cell culturing process disclosed herein and used in the context of the invention may include a growth phase and a production phase. As described herein and used in the context of the invention, the concentration of the sulfhydryl group(s) of one or more sulfhydryl compounds is maintained for at least 5 days, preferably for at least 7 days, more preferably for at least 10 days, even more preferably for at least 12 days and most preferably for at least 14 days during the cell culturing process.

The method as disclosed herein and in the context of the invention may further comprise a step of pre-cultivating the mammalian cells in a cell culture medium. Said pre-cultivating step may comprise culturing the cell in a cell culture medium comprising from 4.0 mM to 10.0 mM of a sulfhydryl group(s) of one or more sulfhydryl compounds as disclosed herein and in the context of the invention, or may not comprise a sulfhydryl group(s) of one or more sulfhydryl compounds at the specified concentration in the cell culture medium. In particular, the cell culture medium in the pre-cultivating step may either comprise a medium used to inoculate the bioreactor with a mammalian cell, or may comprise a medium that does not comprise a medium used to inoculate the bioreactor with a mammalian cell as disclosed herein and in the context of the invention.

The sulfhydryl group(s) of one or more sulfhydryl compounds in the cell culture medium may be present in an oxidized form and/or in a reduced form. The said oxidized form means that the sulfhydryl group(s) of one or more sulfhydryl compounds are present in the cell culture medium in a "bound form", while the reduced form means that the sulfhydryl group(s) of one or more sulfhydryl compounds are present in a "free form", which means that the sulfhydryl group(s) comprise a free -SH group. The cell culture medium as disclosed herein and used in the context of the invention may contain from 4 mM to 10 mM of the sulfhydryl group(s) of one or more sulfhydryl compounds in their oxidized form and/or in a reduced form. For example, the one or more sulfhydryl compound(s) can be cysteine, cystine, succimer, methimazole, cysteamine, azathioprine, mercaptopurine, S-methylcysteine, selenocysteine, S-phosphocysteine, 4'-phosphopantetheine, butyrylthiocholine, carbocysteine, N-sulfocysteine, alethene, acetylcysteine, dimercaprol, coenzyme M, sodium aurothiomalate, pantethine, bucillamine, methylselenocysteine, dimercaptosuccinic acid, acetylcysteine amide, thioglycolic acid, 2,3-dimercaptopropanol, O-methylmercaptoethanol, mercaptoacetic acid, β-mercaptopropionic acid, methyl mercaptan, S-methylmercaptoethanol, glutathione, glutathione derivatives. For example, the sulfhydryl group of one or more sulfhydryl compounds in the cell culture medium as disclosed herein and used in the context of the invention can be cysteine. As another example, the one or more sulfhydryl compound(s) in the cell culture medium as disclosed herein and used in the context of the invention can be cystine. As yet another example, the one or more sulfhydryl compound(s) in the cell culture medium as disclosed herein and used in the context of the invention can be cystine and cysteine.

The method may further comprise measuring the level of monogalactosylated (G1) and digalactosylated (G2) glycans of a glycoprotein, such as an antibody, as described herein.

A person skilled in the art knows means and methods for measuring and determining said content of sulfhydryl group(s) of one or more sulfhydryl compounds in a cell culture medium. Such a concentration can be obtained by determining the amount of sulfhydryl compound(s) using mass spectrometry and calculating the amount of sulfhydryl group(s) present in said sulfhydryl compound(s). Additional means and methods for measuring and determining the concentration of sulfhydryl group(s) of one or more sulfhydryl compounds are within the general knowledge of a person skilled in the art.

The cell culture method or medium as described herein may comprise, for example, at least greater than 4.0 mM and equal to or less than 9.0 mM, preferably at least greater than 4.0 mM and equal to or less than 8.0 mM, more preferably at least greater than 4.0 mM and equal to or less than 7.0 mM, and even more preferably at least greater than 4.0 mM and equal to or less than 6.0 mM of sulfhydryl group(s) of one or more sulfhydryl compounds. As another example, the cell culture medium comprises the sulfhydryl group(s) of one or more sulfhydryl compounds at a concentration of at least 5.0 mM and less than 10.0 mM, preferably at least 5.0 mM and equal to or less than 9.0 mM, more preferably at least 5.0 mM and equal to or less than 8.0 mM, even more preferably at least 5.0 mM and equal to or less than 7.0 mM, and most preferably at least 5.0 mM and equal to or less than 6.0 mM.

As a specific example, the one or more sulfhydryl compound(s) is(are) cysteine, and the concentration of cysteine in the cell culture medium is more than 4.0 mM and less than 10.0 mM, preferably the concentration of cysteine is at least 5.0 mM and equal to or less than 6.0 mM. As another specific example, the one or more sulfhydryl compound(s) is(are) cystine, and the concentration of cystine in the cell culture medium is more than 2.0 mM and less than 5.0 mM, preferably the concentration of cystine is at least 3.0 mM and equal to or less than 4.0 mM.

In most chemically defined culture media for culturing mammalian cells, glucose is the most commonly used carbohydrate because it can be efficiently transported into cells (Wright et al., J Exp Biol 196 (1994) 197-212). Glucose is a simple monosaccharide. In addition to being used as an energy source, glucose can also be used as a building block for many other molecules and structures. To serve these purposes, the monosaccharide must first be "activated." This activation involves the addition of a nucleoside diphosphate group to the sugar, resulting in the formation of a nucleotide sugar. For example, in CHO cells, the vast majority of nucleotide sugars are synthesized in well-characterized glucose reactions. As shown in Figure 1, D-glucose is converted to D-glucose-1-phosphate. Activation of D-glucose-1-phosphate is accomplished by UTP-glucose-1-phosphate uridylyltransferase (EC 2.7.7.9) to yield a cytoplasmic pool of UDP-glucose (Turnquist and Hansen, The Enzymes, 3rd. Ed. (Boyer, P. D., ed.) 8 (1973) 51-71; Chang et al., Eur. J. Biochem. 236 (1996) 723-728).

Therefore, the cell culture medium as described herein and used in the context of the invention may, for example, comprise at least more than 3.0 g/L glucose, and more preferably at least more than 4.0 g/L glucose. Additionally or alternatively, the cell culture medium comprises, for example, no more than 13.0 g/L glucose, preferably no more than 8.0 g/L, more preferably no more than 7.0 g/L, even more preferably no more than 6.0 g/L and most preferably no more than 5.0 g/L glucose. As a specific example, the cell culture medium contains from more than 3.0 g/L to no more than 13 g/L glucose, preferably from more than 3.0 g/L to no more than 8.0 g/L glucose, more preferably from more than 3.0 g/L to no more than 7.0 g/L glucose, even more preferably from more than 3.0 g/L to no more than 6.0 g/L glucose, and most preferably from more than 3.0 g/L to no more than 5.0 g/L glucose.

In particular, the cell culture medium for inoculating and culturing the cells as disclosed herein and in the context of the invention may comprise from 3.0 mM to 10.0 mM sulfhydryl group(s) of one or more sulfhydryl compounds, wherein the sulfhydryl compounds may be cysteine and/or cystine. For example, the cells producing the recombinant protein may be CHO cells producing an antibody to α-synuclein or a bispecific antibody to CD20/CD3 and may be cultured in a cell culture medium containing 6 mM cysteine. As another example, the cells producing the recombinant protein can be CHO cells producing an anti-α-synuclein antibody or an anti-CD20/CD3 bispecific antibody and can be cultured in a cell culture medium containing 5 mM cysteine. As yet another example, the cells producing the recombinant protein can be CHO cells producing an anti-α-synuclein antibody or an anti-CD20/CD3 bispecific antibody and can be cultured in a cell culture medium containing 4 mM cystine. As yet another example, the cells producing the recombinant protein can be CHO cells producing an anti-α-synuclein antibody or an anti-CD20/CD3 bispecific antibody and can be cultured in a cell culture medium containing cystine and cysteine, such as a combination of cystine and cysteine at a concentration of 4.0 mM to 5.0 mM.

As disclosed herein and in the context of the invention, the cells can be cultured in a cell culture medium containing at least more than 3 g/L of glucose. Said glucose can be present during inoculation of the cell with the cell culture medium as described herein and in the context of the invention, or can be added during the cell culture process, for example between days 4 and 14 of the cell culture process. In one embodiment, glucose can be present during the production phase of the cell culture process.

As shown in the accompanying examples, the cells can be cultured in the presence of 6 mM cysteine and in the presence of at least more than 3 g/L, namely equal to or more than 4 g/L glucose. As also shown in the accompanying examples, the cells can be cultured in the presence of 5 mM cysteine and in the presence of at least more than 3 g/L, namely equal to or more than 4 g/L glucose.

As shown in the examples, glucose can be added to the cell culture medium between the 4th and 14th day of the cell culture process. In other words, as shown in the examples, the cell culture medium can contain at least more than 3 g/L of glucose from the time of adding glucose to the cell culture medium, for example, between the 4th and 14th day. Glucose can be added to the cell culture medium as described herein and used in the context of the invention in a fed-batch or perfusion process, but the process by which glucose is added to said cell culture medium is not limited to this. It should be understood that the concentration of glucose present in the cell culture medium refers to a concentration that does not interfere with cell growth and/or recombinant protein production by the cell. In other words, a glucose concentration suitable for maintaining and/or increasing cell growth and/or recombinant protein production by the cell is selected.

As shown in the accompanying examples, said antibody to α-synuclein or bispecific antibody to CD20-CD3 can be produced in a CHO cell line, such as the CHO K1M cell line. It should be understood that the recombinant protein as disclosed herein and in the context of the invention can be produced in any mammalian cell line suitable for producing said recombinant protein. In this context, the person skilled in the art is familiar with such suitable mammalian cells and knows how to select such a mammalian cell for producing the recombinant protein as described herein and in the context of the invention. Said cells producing a recombinant protein, such as an antibody to α-synuclein or bispecific antibody to CD20-CD3, can include, but are not limited to, CHO cells, Vero cells, BHK cells, COS cells and HEK293/293T cells. Mammalian cells can be cultured in a chemically defined cell culture medium. Cell culture media used in the context of the invention are disclosed herein. In this regard, suitable commercially available chemically defined cell culture media, such as but not limited to DMEM, are known to those skilled in the art for use in the context of the invention. As shown in the accompanying examples, a serum-free, chemically defined medium can be used to culture mammalian cells. In one embodiment, the cell culture medium is a chemically defined medium, preferably a serum-, protein- and/or oligopeptide-free cell culture medium.

As disclosed herein and illustrated by the attached examples, mammalian cells, such as CHO cells, can be cultured, for example, for up to 14 days in a culture medium containing from 4.0 mM to 10 mM sulfhydryl group(s) of one or more sulfhydryl compounds, such as cysteine and/or cystine, and at least greater than 3 g/L glucose.

The method may further comprise collecting a glycoprotein, such as an antibody, as described herein. It should be understood that the cells may be collected from the cell culture medium during or at the end of the production phase. Accordingly, one skilled in the art may select an appropriate time to collect the cells from the cell culture medium, as also shown in the accompanying examples. The method may further comprise formulating a glycoprotein, such as an antibody, as described herein, into a medicinal product.

All aspects described below in the context of the cell culture medium used in the method of the present invention also apply to the cell culture medium used in the context of the present invention. Furthermore, the present invention relates to recombinant proteins having the desired content of galactosylated glycans, wherein the recombinant protein may comprise at least monogalactosylated, more preferably digalactosylated glycans. Preferably, the galactosylated glycans are linked to N-acetylglucosamine. All aspects described below in the context of the recombinant protein obtained by the method of the present invention also apply to the recombinant protein provided in the context of the present invention. The present invention also provides recombinant proteins obtained by the method of the present invention and are disclosed below.

In this context, it has been found in the present invention that sulfhydryl compound(s) and derivatives thereof, such as L-cysteine and/or cystine, can be used to activate UDP-α-D-glucose: α-D-galactose-1-phosphate uridylyltransferase (EC 2.7.7.12). Therefore, UDP-α-D-glucose: α-D-galactose-1-phosphate uridylyltransferase can be effectively activated. Furthermore, the sulfhydryl compound(s) can be used to modulate the UDP-sugar pathway, in particular in vivo. For example, L-cystine, the oxidized dimeric form of L-cysteine, can be used to modulate the UDP-sugar pathway, in particular in vivo. In particular, the UDP-sugar pathway, particularly in vivo, can be modulated by adding sulfhydryl compounds and their derivatives, such as cystine and/or cysteine, to increase cell growth, recombinant protein productivity and/or protein quality in the body, particularly by increasing the galactosylation level of recombinant protein by mammalian cell culture.

The term "oxidation state" refers to the extent to which the oxidizable sulfhydryl group(s) of one or more sulfhydryl compounds undergo(s) oxidation in a cell culture medium. For example, if a sulfhydryl compound contains one sulfhydryl group that is oxidized by forming a disulfide bridge with the sulfhydryl group of another (same or different) sulfhydryl compound, then an increase in the mass of said sulfhydryl compound indicates oxidation of the sulfhydryl group. The oxidation state can be measured using metrics known to those skilled in the art of protein and peptide chemistry, including, but not limited to, analysis of the number of oxidized residues, mass spectral peak intensity, integrated mass spectral area, and the like. In some embodiments of any of the aspects provided herein, the oxidation state is indicated as a percentage, where 0% refers to no oxidation and 100% refers to complete oxidation of the potentially oxidizable sulfhydryl group(s) of one or more sulfhydryl compounds in the medium. The term "potentially oxidizable sulfhydryl group" and the like refers to the total amount of sulfhydryl group(s) of one or more sulfhydryl compounds in the medium that can undergo oxidation, such as by forming a disulfide bridge.

The terms "unoxidized sulfhydryl group" and the like refer to the amount of sulfhydryl group(s) of one or more sulfhydryl compounds in a medium that has not been oxidized. An increase in the mass of a sulfhydryl compound compared to the mass of the same sulfhydryl compound that is not oxidized reflects the amount of oxidized sulfhydryl group(s) in the sulfhydryl compound. An increase in the amount of sulfhydryl compound(s) having oxidized sulfhydryl group(s) compared to the amount of sulfhydryl compound(s) not having oxidized sulfhydryl group(s) reflects the degree of oxidation. The oxidation state of the sulfhydryl group(s) of one or more sulfhydryl compounds is then determined based on the total amount of sulfhydryl group(s) of one or more sulfhydryl compounds having one or more oxidized sulfhydryl group(s) and the total amount of sulfhydryl group(s) of one or more sulfhydryl compounds in the cell culture medium.

For example, determining the oxidation state of the sulfhydryl group(s) of one or more sulfhydryl compounds in a cell culture medium comprises the steps of a) determining the mass and amount of the one or more sulfhydryl compounds in the cell culture medium; b) comparing the mass determined for the one or more sulfhydryl compounds with the mass of the one or more sulfhydryl compounds that are not oxidized, wherein an increase in the mass of the one or more sulfhydryl compounds compared to the mass of the one or more sulfhydryl compounds that are not oxidized reflects the amount of oxidized sulfhydryl groups in the one or more sulfhydryl compounds; and c) determining the oxidation state of the one or more sulfhydryl compounds by the total amount of the one or more sulfhydryl compounds containing at least one oxidized sulfhydryl group and, thus, the total amount of the sulfhydryl group(s) of the one or more sulfhydryl compounds.

Mass spectrometry is used to determine the mass of target fragments. The terms "mass spectrometry", "MS", etc. refer to the methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio (i.e., "m/z"). The terms "mass" and "m/z" are used interchangeably in the context of mass spectrometric analysis results and, unless otherwise noted, all m/z values assume singly ionized species. The terms "major isotope mass" and "major isotope m/z" refer to the mass reported for a molecular ion, taking into account the mass of the most abundant (i.e., major) isotope of each element. Typically, one or more molecules of interest are ionized and the ions are then introduced into a mass spectrometer where a combination of magnetic and electric fields causes the ions to follow a path through space based on their mass ("m") and charge ("z"). See, for example, U.S. Patent No. 6,204,500, entitled "Mass Spectrometry From Surfaces," U.S. Patent No. 6,107,623, entitled "Methods and Apparatus for Tandem Mass Spectrometry), U.S. Patent No. 6,268,144, entitled "DNA Diagnostics Based On Mass Spectrometry), U.S. Patent No. 6,124,137, entitled "Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes), Wright et al., Prostate Cancer and Prostatic Diseases 2:264-76 (1999); and Merchant and Weinberger, Electrophoresis 21:1164-67 (2000), each of which is incorporated herein by reference in its entirety. The terms "integrated intensity," "integrated area of the mass spectrum," "integrated intensity of the mass spectrum," and the like are herein incorporated by reference. refer to the area under the mass spectrometric curve corresponding to the amount of molecular ion having a particular major isotope m/z, as is well known in the art.

For example, in a quadrupole or quadrupole ion trap, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between the electrodes, the amplitude of the radio frequency signal, and the m/z. The voltage and amplitude can be selected so that only ions with a certain m/z value move along the length of the quadrupole, while all other ions are rejected. Thus, quadrupole instruments can act as both a "mass filter" and a "mass detector" for ions introduced into the instrument.

Uridine diphosphate (UDP) sugars in mammalian cells

It has been established that uridine diphosphate (UDP) α-D-glucose epimerase (EC 5.1.3.2) and UDP-α-D-glucose: α-D-galactose-1-phosphate uridylyltransferase (EC 2.7.7.12) play a crucial role in the UDP-glucose and UDP-galactose conversion pathway in mammalian cells, such as CHO cells.

As used herein, the term "uridine diphosphate (UDP) α-D-glucose epimerase" and "UDP-Glc-E" are used interchangeably and refer to the enzyme, which is a homodimeric epimerase found in bacteria, fungi, plants and mammalian cells belonging to EC class 5.1.3.2. As shown in Example 1, the UDP-Glc-E cDNA in the CHO K1M cell line was amplified, sequenced and further analyzed.

As used herein, the terms "UDP-α-D-glucose:α-D-galactose-1-phosphate uridylyltransferase", "UDP-Gal-T" and "EC 2.7.7.12" are used interchangeably and refer to the enzyme that catalyzes the nucleotide exchange between uridine-5'-diphosphate-glucose (UDP-glucose) and galactose-1-phosphate (Gal-1-P) to form uridine-5'-diphosphate-galactose (UDP-galactose) and glucose-1-phosphate (Glc-1-P) by a reversible mechanism. As shown in Example 2, the UDP-Gal-T cDNA in the CHO K1M cell line was amplified, sequenced and further analyzed.

Uridine diphosphate glucose epimerase

The pathway for the synthesis of UDP-glucose and UDP-galactose is shown in Figure 1. Thus, the pathway for the interconversion of UDP-sugars during the formation of UDP-glucose from α-D-glucose and the subsequent formation of UDP-galactose from UDP-glucose by UDP-glucose epimerase (UDP-Glc-E: Uridine diphosphate glucose epimerase. EC 5.1.3.2) is shown.

UDP-α-D-glucose: α-D-galactose-1-phosphate uridylyltransferase

The UDP-galactose and UDP-glucose pathway was further expanded as shown in Figure 2. Therefore, the interconversion pathway of UDP-sugars in the formation of UDP-galactose from α-D-glucose and the subsequent conversion of UDP-galactose back to UDP-glucose by UDP-α-D-glucose: α-D-galactose-1-phosphate uridylyltransferase (UDP-Gal-T:UDP-α-D-glucose: α-D-galactose-1-phosphate uridylyltransferase EC 2.7.7.12) is shown.

Establishment of the UDP-glucose/galactose conversion pathway

As shown in Figure 2, UDP-Gal-T catalyzes the reversible conversion of UDP-glucose and UDP-galactose. However, Wagstaff et al. ((2015) Carbohydrate Research 404: 17–25) found that under certain circumstances (e.g., in the presence of excess glucose-1-P), UDP-Gal-T primarily performs the reverse conversion, converting most UDP-galactose to UDP-glucose. This mechanism may result in an intracellular shift in the glucose- and galacto-configured pool of UDP-sugars, resulting in decreased intracellular UDP-galactose concentrations and increased intracellular UDP-glucose. Based on these experimental results, this new UDP-glucose/UDP-galactose pathway can be more precisely characterized, such as shown in Figure 2, for this therapeutic protein production process using a chemically defined glucose excess medium platform.

A new approach to regulation of the UDP-glucose/UDP-galactose pathway in mammalian cells using sulfhydryl compounds

As shown in Figure 3, sulfhydryl compounds and derivatives thereof such as L-cysteine and/or L-cystine can be added to the cell culture medium to further activate UDP-Gal-T and further regulate the gluco- and galactoconfiguration of UDP-sugars in mammalian cells such as CHO K1 and its derivative cell lines. Accordingly, the inventors of the present invention have found that by adding one or more sulfhydryl compounds, recombinant proteins containing monogalactosylated or digalactosylated glycans can be produced. The term "sulfhydryl compound(s)" as used herein refers to inorganic or organic compounds that contain sulfur as an integral part of the molecule, preferably compounds containing the -SH radical; and/or compounds originally containing the -SH radical and covalently linked to each other under reducing conditions to form a disulfide bridge. The inventors of the present invention have found that one or more sulfhydryl compounds that increase viable cell density and/or product titer during the production of a recombinant protein produced by a mammalian cell culture have a directly correlating effect on the galactose content of the resulting recombinant protein. In this context, the present invention provides methods for controlling the degree of galactosylation of a recombinant protein produced by a mammalian cell culture. Following the methods presented herein, a person skilled in the art can determine precise process parameters that ensure control of the galactose content of a recombinant protein produced by a mammalian cell culture.

In one aspect in the context of the present invention, during the production of a recombinant protein produced by a mammalian cell culture, the galactose content of the recombinant protein may be increased. In another aspect of the present invention, a recombinant protein having monogalactosylated (G1) or digalactosylated (G2) glycans is an anti-CD20/CD3 bispecific antibody, wherein the anti-CD20/CD3 bispecific antibody comprises 19.0-29.0% (w/w) G1 and 1.3-2.8% (w/w) G2 per total glycan; preferably 20.0-28.0% (w/w) G1 and 1.4-2.7% (w/w) G2 per total glycan; more preferably 21.0-28.0% (w/w) G1 and 1.5-2.7% (w/w) G2 on a total glycan basis, and most preferably 21.0-27.4% (w/w) G1 and 1.5-2.6% (w/w) G2 on a total glycan basis.

In particular, a bispecific antibody to CD20/CD3 having monogalactosylated (G1) and digalactosylated (G2) glycans comprises a first antigen-binding domain that binds to CD3 and a second antigen-binding domain that binds to CD20. The bispecific antibody to CD20/CD3 may comprise a first antigen-binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), and a second antigen-binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL).

More specifically, a CD20/CD3 bispecific antibody having monogalactosylated (G1) and digalactosylated (G2) glycans comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises a heavy chain variable domain (VH) comprising

(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,

(b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and

(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; and

variable light chain (VL) domain containing

(d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25,

(d) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26, and

(e) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27;

wherein the second antigen-binding domain comprises a heavy chain variable domain (VH) comprising

(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34,

(b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and

(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; and

variable light chain (VL) domain containing

(d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37,

(d) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38, and

(e) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39; and

wherein the CD20/CD3 bispecific antibody has 19.0-29.0% (w/w) G1 and 1.3-2.8% (w/w) G2 per total glycan; preferably 20.0-28.0% (w/w) G1 and 1.4-2.7% (w/w) G2 per total glycan; more preferably 21.0-28.0% (w/w) G1 and 1.5-2.7% (w/w) G2 per total glycan, and most preferably 21.0-27.4% (w/w) G1 and 1.5-2.6% (w/w) G2 per total glycan.

Preferably, the CD20/CD3 bispecific antibody is an antibody in which

(a) the first antigen-binding domain comprises the VH sequence of SEQ ID NO: 28, and the second antigen-binding domain comprises the VH sequence of SEQ ID NO: 40;

(b) the first antigen-binding domain comprises the VL sequence of SEQ ID NO: 29 and the second antigen-binding domain comprises the VL sequence of SEQ ID NO: 41; or

(c) the first and second antigen-binding domains comprise a VH sequence as defined in (a) and a VL sequence as defined in (b).

More preferably, the CD20/CD3 bispecific antibody is an antibody in which

(a) the VH sequence of the first antigen-binding domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 28, and the VH sequence of the second antigen-binding domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 40;

(b) the VL sequence of the first antigen-binding domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 29, and the VL sequence of the second antigen-binding domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 41; or

(c) the bispecific antibody to CD20/CD3 comprises the VH sequence of the first and second antigen-binding domains defined in (a) and the VL sequence of the first and second antigen-binding domains defined in (b).

As disclosed herein and in the context of the invention, an anti-CD20/CD3 bispecific antibody is an antibody in which the anti-CD20/CD3 bispecific antibody may comprise a third antigen-binding domain. For example, an anti-CD20/CD3 bispecific antibody is an antibody in which the anti-CD20/CD3 bispecific antibody comprises a third antigen-binding domain that binds to CD20. Alternatively, an anti-CD20/CD3 bispecific antibody is an antibody in which the anti-CD20/CD3 bispecific antibody comprises a third antigen-binding domain that binds to CD3. As used herein, the terms "first," "second," "third," etc., with respect to the antigen-binding domains are used to facilitate distinction when more than one of each type of domain is present. Use of these terms does not imply a particular order or orientation unless expressly stated otherwise.

For example, a CD20/CD3 bispecific antibody may comprise a second antigen-binding domain that binds to CD3, a first and third antigen-binding domain that bind to CD20. As a more specific example, the third antigen-binding domain may be identical to the first antigen-binding domain (i.e., the first and third antigen-binding domains may comprise the same heavy and light chain amino acid sequences). Even more specifically, the first and third Fab molecules comprising the first and third antigen-binding domains may be identical and, further, may have the same domain arrangement (i.e., normal or cross-over).

As another example, a CD20/CD3 bispecific antibody may comprise a first antigen-binding domain that binds to CD3, a second and a third antigen-binding domain that bind to CD20. As a more specific example, the third antigen-binding domain may be identical to the second antigen-binding domain (i.e., the second and third antigen-binding domains may comprise the same heavy and light chain amino acid sequences). Even more specifically, the second and third Fab molecules comprising the second and third antigen-binding domains may be identical and, further, may have the same domain arrangement (i.e., normal or cross-over).

As a more specific example described herein, a CD20/CD3 bispecific antibody may comprise a third antigen-binding domain, wherein the third antigen-binding domain comprises a heavy chain variable domain (VH) comprising

(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34,

(b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and

(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; and

variable light chain (VL) domain containing

(d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37,

(d) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38, and

(e) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39; and

wherein the CD20/CD3 bispecific antibody has 19.0-29.0% (w/w) G1 and 1.3-2.8% (w/w) G2 per total glycan; preferably 20.0-28.0% (w/w) G1 and 1.4-2.7% (w/w) G2 per total glycan; more preferably 21.0-28.0% (w/w) G1 and 1.5-2.7% (w/w) G2 per total glycan, and most preferably 21.0-27.4% (w/w) G1 and 1.5-2.6% (w/w) G2 per total glycan.

As an even more specific example, the CD20/CD3 bispecific antibody is an antibody in which the third antigen-binding domain may comprise a VH sequence of SEQ ID NO: 40 and a VL sequence of SEQ ID NO: 41. Preferably, the CD20/CD3 bispecific antibody is an antibody in which the third antigen-binding domain may comprise a VH sequence of the third antigen-binding domain that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 40, and the VL sequence of the third antigen-binding domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 41.

The first antigen-binding domain of the bispecific antibody to CD20/CD3 may be a crossover Fab molecule in which the variable domains or constant domains of the heavy and light chain of a Fab are exchanged, and the second and third, if present, antigen-binding domains may be a standard Fab molecule. For example, the antigen-binding domain that binds to CD3 is a crossover Fab molecule in which the variable domains or constant domains of the heavy and light chain of a Fab are exchanged (i.e., substituted for each other). As another more specific example, the first antigen-binding fragment is a crossover Fab molecule, and each of the second and first antigen-binding fragments is a standard Fab molecule.

"Fab molecule" refers to a protein consisting of the VH and CH1 domain of the heavy chain ("Fab heavy chain") and the VL and CL domain of the light chain ("Fab light chain") of an immunoglobulin. By "fused" is meant that the components (e.g., the Fab molecule and the Fc domain subunit) are linked by peptide bonds, either directly or through one or more peptide linkers.

By "crossover" Fab molecule (also called "Crossfab") is meant a Fab molecule in which the variable domains or constant domains of the heavy and light chains of the Fab are exchanged (i.e. substituted for each other), i.e. a crossover Fab molecule comprises a peptide chain consisting of the light chain variable domain VL and the heavy chain constant domain 1 CHI (VL-CH1, in the N- to C-terminal direction), and a peptide chain consisting of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, in the N- to C-terminal direction). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain containing the heavy chain constant domain 1 CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. At the same time, in a crossover Fab molecule in which the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain containing the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

In contrast, a "standard" Fab molecule means that the Fab molecule has its natural format, i.e. it contains a heavy chain consisting of the variable and constant domains of the heavy chain (VH-CH1, in the direction from N- to C-terminus), and a light chain consisting of the variable and constant domains of the light chain (VL-CL, in the direction from N- to C-terminus).

A third Fab molecule may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second Fc domain subunit. As used herein, the term "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that includes the C-terminal constant regions of an immunoglobulin heavy chain that are capable of stable self-association. For example, the Fc domain subunit of IgG contains the CH2 and CH3 constant domains of IgG.

For example, each of the second and third Fab molecules is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. More particularly, the antibody consists essentially of a first, second and third Fab molecules, an Fc domain consisting of a first and a second subunit, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a second Fab molecule, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain (see also EP 3252078 A1, in particular Figures 1B, 1E, 1K and 1P in conjunction with paragraph [0338], which are incorporated herein by reference).

In other words, the CD20/CD3 bispecific antibody may comprise a third antigen-binding domain, wherein the first antigen-binding domain of the CD20/CD3 bispecific antibody is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain, the second antigen-binding domain of the CD20/CD3 bispecific antibody is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen-binding domain of the CD20/CD3 bispecific antibody is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

The second and third Fab molecules may be fused to the Fc domain directly or via a peptide linker. For example, each of the second and third Fab molecules is fused to the Fc domain via an immunoglobulin hinge region. In particular, the immunoglobulin hinge region is a human IgG1 hinge region, in particular when the Fc domain is a human IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be further fused to each other.

Alternatively, each of the first and third Fab molecules is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first Fab chain of the second Fab molecule. More particularly, the antibody consists essentially of a first, second and third Fab molecules, an Fc domain consisting of a first and a second subunit, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain (see also EP 3 252 078 A1, in particular Figures 1B, 1E, 1L and 1C in conjunction with paragraph [0339], which are incorporated herein by reference).

In other words, the CD20/CD3 bispecific antibody may comprise a third antigen-binding domain, wherein the second antigen-binding domain of the CD20/CD3 bispecific antibody is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, the first antigen-binding domain of the CD20/CD3 bispecific antibody is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen-binding domain of the CD20/CD3 bispecific antibody is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

The first and third Fab molecules may be fused to the Fc domain directly or via a peptide linker. For example, each of the first and third Fab molecules is fused to the Fc domain via an immunoglobulin hinge region. In particular, the immunoglobulin hinge region is a human IgG1 hinge region, in particular when the Fc domain is a human IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be further fused to each other.

As a further alternative, each of the first and second Fab molecules is fused at the C-terminus of a Fab heavy chain to the N-terminus of one of the Fc domain subunits, and a third Fab molecule is fused at the C-terminus of a Fab heavy chain to the N-terminus of a first Fab chain of a second Fab molecule. More particularly, the antibody consists essentially of the first, second and third Fab molecules, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the third Fab molecule is fused at the C-terminus of a Fab heavy chain to the N-terminus of a Fab heavy chain of the first Fab molecule, the first Fab molecule is fused at the C-terminus of a Fab heavy chain to the N-terminus of the first Fc domain subunit, and the second Fab molecule is fused at the C-terminus of a Fab heavy chain to the N-terminus of the second Fc domain subunit. In other words, the CD20/CD3 bispecific antibody may comprise a third antigen-binding domain, wherein the third antigen-binding domain of the CD20/CD3 bispecific antibody is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, the first antigen-binding domain of the CD20/CD3 bispecific antibody is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first Fc domain subunit, and the second antigen-binding domain of the CD20/CD3 bispecific antibody is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit.

The first and second Fab molecules can be fused to the Fc domain directly or via a peptide linker. For example, each of the first and second Fab molecules is fused to the Fc domain via an immunoglobulin hinge region. In particular, the immunoglobulin hinge region is a human IgG1 hinge region, in particular when the Fc domain is a human IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the third Fab molecule can be further fused to each other.

Additional configurations of the CD20/CD3 bispecific antibody used herein and in the context of the invention can be found, for example, in EP 3 252 078 A1, in particular Figure 1 and paragraphs [0335] to [0367], which are incorporated herein by reference.

More specifically, the CD20/CD3 bispecific antibody is an antibody in which the CD20/CD3 bispecific antibody may comprise

(a) a first heavy chain having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 47, and a second heavy chain having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 45;

(b) a first light chain having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 33, and a second and third light chain having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 44; or

(c) a first heavy chain and a second heavy chain as defined in (a), and a first light chain, a second light chain and a third light chain as defined in (b).

More specifically, an anti-CD20/CD3 bispecific antibody is an antibody in which the anti-CD20/CD3 bispecific antibody comprises

(a) a first heavy chain of SEQ ID NO: 47 and a second heavy chain of SEQ ID NO: 45;

(b) a first light chain of SEQ ID NO: 33, a second light chain and a third light chain of SEQ ID NO: 44; or

(c) a first heavy chain and a second heavy chain as defined in (a), and a first light chain, a second light chain and a third light chain as defined in (b).

In another aspect of the present invention, a recombinant protein having monogalactosylated (G1) or digalactosylated (G2) glycans is an anti-α-synuclein antibody, wherein the anti-α-synuclein antibody has 17.2-48.0% (w/w) G1 and 3.1-15.0% (w/w) G2 per total glycan; preferably 25.4-48.0% (w/w) G1 and 3.5-15.0% (w/w) G2 per total glycan; preferably 27.2-47.0% G1 and 4.4-15.0% G2 per total glycan; preferably 40.0-46.0% (w/w) G1 and 8.4-15.0% (w/w) G2 based on total glycan; more preferably 41.0-45.0% (w/w) G1 and 9.5-14.0% (w/w) G2 based on total glycan, and most preferably 42.1-43.9% (w/w) G1 and 10.6-13.3% (w/w) G2 based on total glycan.

In particular, an antibody to α-synuclein that has monogalactosylated (G1) and digalactosylated (G2) glycans contains a heavy chain variable domain (VH) containing

(a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10,

(b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and

(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; and

variable light chain (VL) domain containing

(d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13,

(d) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and

(e) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and

wherein the anti-α-synuclein antibody has 17.2-48.0% (w/w) G1 and 3.1-15.0% (w/w) G2 per total glycan; preferably 25.4-48.0% (w/w) G1 and 3.5-15.0% (w/w) G2 per total glycan; preferably 27.2-47.0% G1 and 4.4-15.0% G2 per total glycan; preferably 40.0-46.0% (w/w) G1 and 8.4-15.0% (w/w) G2 per total glycan; more preferably 41.0-45.0% (w/w) G1 and 9.5-14.0% (w/w) G2 of total glycan, and most preferably 42.1-43.9% (w/w) G1 and 10.6-13.3% (w/w) G2 of total glycan.

More specifically, the α-synuclein antibody contains

(a) VH sequence SEQ ID NO: 16;

(b) the VL sequence SEQ ID NO: 17; or

(c) the VH sequence defined in (a) and the VL sequence defined in (b).

More specifically, the α-synuclein antibody contains

(a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16;

(b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17; or

(c) the VH sequence defined in (a) and the VL sequence defined in (b).

Even more particularly, the α-synuclein antibody comprises a heavy chain having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 20 and a light chain having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 21.

More specifically, the α-synuclein antibody comprises a heavy chain of SEQ ID NO: 20 and a light chain of SEQ ID NO: 21.

"Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways that are known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of this document, percent (%) amino acid sequence identity values are obtained using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and the source code has been filed with the user documentation in the United States Copyright Office, Washington, D.C. 20559, where it is registered under U.S. Copyright Registration Number TXU510087. The ALIGN-2 program is freely available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are not variable. In cases where ALIGN-2 is used to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A with or compared to a given amino acid sequence B (which may alternatively be stated as a given amino acid sequence A that has or contains a certain % amino acid sequence identity with or compared to a given amino acid sequence B) is calculated as follows: 100 multiplied by the ratio X/Y, where X represents the number of amino acid residues scored by the ALIGN-2 sequence alignment program as identical matches in the program's alignment of A and B, and Y represents the total number of amino acid residues in B. It should be understood that if the length of the amino acid sequence of A is not equal to the length of the amino acid sequence of B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein were obtained as described in the preceding paragraph using the ALIGN-2 computer program.

By a nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference sequence according to the present invention, it is meant that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations for every 100 nucleotides of the reference nucleotide sequence. In other words, in order to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with other nucleotides, or a number of nucleotides representing up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. These changes in the reference sequence may be at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between these terminal positions, located individually between residues in the reference sequence or one or more contiguous groups in the reference sequence. In practice, the determination that any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of the present invention can be made in a conventional manner using known computer programs, such as those discussed above for polypeptides (e.g., ALIGN-2).

Preferably, the monogalactosylated (G1) and digalactosylated (G2) glycans of the bispecific anti-CD20/CD3 antibody or anti-α-synuclein antibody as disclosed in the context of the invention are linked to N-acetylglucosamine.

As used herein, the terms "cell culture medium" and "culture medium" are used interchangeably and refer to a nutrient solution used to grow mammalian cells that typically contains at least one component from one or more of the following categories: 1) an energy source, typically in the form of a carbohydrate such as glucose; 2) all of the essential amino acids and typically the core set of twenty amino acids plus cysteine; 3) vitamins and/or other organic compounds required in low concentrations; 4) free fatty acids; and 5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required in very low concentrations, typically in the micromolar range. Essential components such as growth factors for a particular cell line are easily determined empirically without unnecessary experimentation, as described, for example, in Mammalian Cell Culture (Mather, J.P. ed., Plenum Press, N.Y. [1984]), and Barnes and Sato. (1980) Cell. 22:649).

As disclosed herein and in the context of the invention, a cell culture medium for producing an antibody having monogalactosylated (G1) and digalactosylated (G2) glycans is a cell culture medium containing a concentration of more than 4.0 mM and less than 10.0 mM sulfhydryl group(s) of one or more sulfhydryl compounds and a concentration of at least more than 3.0 g/L glucose.

In a preferred aspect of the present invention, the cell culture medium as disclosed in the context of the invention comprises at least greater than 4.0 mM and equal to or less than 9.0 mM, preferably at least greater than 4.0 mM and equal to or less than 8.0 mM, more preferably at least greater than 4.0 mM and equal to or less than 7.0 mM, and even more preferably at least greater than 4.0 mM and equal to or less than 6.0 mM of the sulfhydryl group(s) of one or more sulfhydryl compounds.

In another preferred aspect, the cell culture medium as disclosed in the context of the invention comprises a concentration of at least 5.0 mM and less than 10.0 mM, preferably at least 5.0 mM and equal to or less than 9.0 mM, more preferably at least 5.0 mM and equal to or less than 8.0 mM, even more preferably at least 5.0 mM and equal to or less than 7.0 mM and most preferably at least 5.0 mM and equal to or less than 6.0 mM of the sulfhydryl group(s) of one or more sulfhydryl compounds.

Alternatively (e.g. in the case of cysteine and/or cystine) the cell culture medium comprises from greater than 0.7 g/L to equal to or less than 1.2 g/L, preferably from greater than 0.7 g/L to equal to or less than 1.1 g/L, more preferably from greater than 0.7 g/L to equal to or less than 1.0 g/L, more preferably from greater than 0.7 g/L to equal to or less than 0.9 g/L, even more preferably from greater than 0.7 g/L to 0.8 g/L of one or more sulfhydryl compounds.

The sulfhydryl group(s) of one or more sulfhydryl compounds in the cell culture medium as disclosed in the context of the invention are contained in a reduced and/or oxidized form. For example, the concentration of the reduced form of said sulfhydryl is in the range from more than 4.0 mM to less than 10.0 mM, and/or the concentration of the oxidized form of said sulfhydryl is in the range from more than 2.0 mM to less than 5.0 mM.

As disclosed herein and in the context of the invention, the one or more sulfhydryl compound(s) may be selected from the group consisting of cysteine, cystine, succimer, methimazole, cysteamine, azathioprine, mercaptopurine, S-methylcysteine, selenocysteine, S-phosphocysteine, 4'-phosphopantetheine, butyrylthiocholine, carbocysteine, N-sulfocysteine, aletheine, acetylcysteine, dimercaprol, coenzyme M, sodium aurothiomalate, pantethine, bucillamine, methylselenocysteine, dimercaptosuccinic acid, acetylcysteine amide, thioglycolic acid, 2,3-dimercaptopropanol, O-methylmercaptoethanol, mercaptoacetic acid, β-mercaptopropionic acid, methyl mercaptan, S-methyl mercaptoethanol, glutathione, glutathione derivatives and combinations thereof. Preferably, the one or more sulfhydryl compound(s) is(are) selected from the group consisting of cysteine, cystine and combinations thereof.

In particular, the one or more sulfhydryl compound(s) may be(are) selected from the group consisting of cysteine, cystine and a combination thereof. For example, the one or more sulfhydryl compound(s) is(are) cysteine, and the concentration of cysteine in the cell culture medium is greater than 4.0 mM and less than 10.0 mM, preferably the concentration of cysteine is at least 5.0 mM and equal to or less than 6.0 mM.

Alternatively (e.g. in the case of cysteine and/or cystine), the cell culture medium for producing a recombinant protein having monogalactosylated or digalactosylated glycans in a mammalian cell contains from 0.5 g/L to 1.2 g/L, preferably from 0.6 g/L to 1.2 g/L, of one or more sulfhydryl compounds.

As described herein and in the context of the invention, the cell culture medium may comprise at least more than 3.0 g/L glucose, and more preferably at least more than 4.0 g/L glucose. Alternatively, the cell culture medium may comprise no more than 13.0 g/L glucose, preferably 8.0 g/L, more preferably no more than 7.0 g/L, even more preferably no more than 6.0 g/L, and most preferably no more than 5.0 g/L glucose. In particular, the cell culture medium as described herein and in the context of the invention may comprise from more than 3.0 g/L to 13 g/L glucose, preferably from more than 3.0 g/L to at most 8.0 g/L glucose, more preferably from more than 3.0 g/L to at most 7.0 g/L glucose, even more preferably from more than 3.0 g/L to at most 6.0 g/L glucose, and most preferably from more than 3.0 g/L to at most 5.0 g/L glucose.

As will be appreciated by those skilled in the art, the minimal and nutrient media used to culture cells for the production of a recombinant protein, as well as other variables such as feeding schedule, growth rate, temperature, and oxygen levels, can affect the yield and quality of the expressed protein. Methods for optimizing these conditions are known to those skilled in the art; illustrative conditions are set forth in the examples provided herein. As described herein and in the context of the invention, the cell culture medium is a chemically defined medium, preferably a cell culture medium that is serum-, protein-, and/or oligopeptide-free.

Chemically defined media have been extensively developed and published in recent years, including those for culturing mammalian cells. All components of the defined media are well characterized, and such media do not contain complex additives such as serum and hydrolysates. Typically, these media include certain amounts of purified growth factors, proteins, lipoproteins and other substances that would otherwise be provided by serum or extract supplements. Such media have been created for the sole purpose of maintaining high-yield cell cultures. Some defined media may be called low-protein media or may be protein-free if the typical components of low-protein media, insulin and transferrin are not included. Otherwise, serum-free media can be used in the methods of the present invention. Such media typically do not contain serum or protein fractions, but may contain undefined components. Examples of commercially available culture media include Ham's F10 (Sigma), minimal essential medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM, Sigma), as well as chemically defined media and feed supplements sold by Life Technologies. Any such media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™), and glucose or an equivalent energy source. The required nutrients and growth factors for the medium, including their concentrations, for a particular cell line are determined empirically and without undue experimentation, as described, for example, in Mammalian Cell Culture, Mather (Plenum Press: NY 1984); Barnes and Sato, Cell 22 (1980) 649 or Mammalian Cell Biotechnology: A Practical Approach M. Butler (IRL Press, 1991). A suitable medium comprises a base medium component such as a DMEM/HA F12 based formulation with altered concentrations of certain components such as amino acids, salts, sugars and vitamins, and optionally containing glycine, hypoxanthine, thymidine, recombinant human insulin, hydrolyzed peptone such as PRIM ATONE HS™ or PRIMATONE RL™ (Sheffield, England) or equivalent, a cell protecting agent such as PLURONIC F68™ or equivalent pluronic polyol and GENTAMYCIN™.

In another aspect of the present invention, the cell culture medium is a chemically defined medium, preferably a serum-, protein- and/or oligopeptide-free cell culture medium; and wherein the cell culture medium comprises from 4.0 mM to 10.0 mM of the sulfhydryl group(s) of one or more sulfhydryl compounds. In another preferred aspect, the cell culture medium is a chemically defined medium, preferably a serum-, protein- and/or oligopeptide-free cell culture medium; and wherein the cell culture medium comprises at least greater than 4.0 mM and equal to or less than 9.0 mM, preferably at least greater than 4.0 mM and equal to or less than 8.0 mM, more preferably at least greater than 4.0 mM and equal to or less than 7.0 mM, and even more preferably at least greater than 4.0 mM and equal to or less than 6.0 mM of the sulfhydryl group(s) of one or more sulfhydryl compounds. In yet another preferred aspect of the present invention, the cell culture medium is a chemically defined medium, preferably a serum-, protein- and/or oligopeptide-free cell culture medium; and wherein the cell culture medium comprises at least 5.0 mM and less than 10.0 mM, preferably at least 5.0 mM and equal to or less than 9.0 mM, more preferably at least 5.0 mM and equal to or less than 8.0 mM, even more preferably at least 5.0 mM and equal to or less than 7.0 mM and most preferably at least 5.0 mM and equal to or less than 6.0 mM of the sulfhydryl group(s) of one or more sulfhydryl compounds.

The concentration of one or more sulfhydryl compounds calculated for commercial media as described above will change if these media are supplemented with complex ingredients such as serum or peptones. When the method of the invention includes adjusting the concentration of one or more sulfhydryl compounds in a culture medium by adding the sulfhydryl compound(s) to the medium, any sulfhydryl compound provided herein and suitable for inclusion in the culture medium can be used to produce the recombinant protein.

Figure 3 shows the modulation of the interconversion pathways of UDP-glucose and UDP-galactose into CHO K1 by sulfhydryl compound(s). As shown in Figure 3, by adding sulfhydryl compound(s) such as L-cysteine to stimulate UDP-Gal-T, more UDP-galactose can be converted into UDP-glucose and therefore less UDP-galactose remains in the cytoplasm. As shown in Figure 4, by adding L-cystine instead of L-cysteine in the cell culture medium, the effect of stimulating the enzymatic activity of UDP-Gal-T by L-cysteine can be weakened in the cytoplasm. On the other hand, increased L-cystine absorption may provide a more efficient redox system to regulate cell survival and improve the metabolic process of UDP-galactose.

Another aspect of the present invention is the use of simple sulfhydryl molecules such as L-cysteine and/or cystine to regulate in vivo the intracellular concentration of UDP-glucose and UDP-galactose in mammalian cells such as CHO K1 and its derivative cell lines. For example, L-cystine can be added to the culture medium to regulate in vivo the intracellular concentration of UDP-glucose and UDP-galactose in CHO K1. In this way, the quantity and quality of the final product can be controlled and improved for predetermined clinical applications.

Modulation of the UDP-glucose/UDP-galactose pathway to control product glycosylation and cell culture process

As shown in Figure 5, UDP-galactose acts as a galactosyl donor in a variety of galactosylation reactions catalyzed by glycosyltransferases. For example, UDP-α-D-galactose: N-acetyl-β-D-glucosaminylglycopeptide 4-β-galactosyltransferase (EC 2.4.1.38) catalyzes the formation of Galpl-4GlcNAc linkages by transferring the galactosyl group from UDP-galactose to GlcNAc (Qasba et al., Curr. Drug. Targets. 9 (2008) 292–309).

Increased cytoplasmic L-cysteine levels may stimulate the UDP-Gal-T enzyme to convert more UDP-galactose into UDP-glucose, resulting in a decreased cytoplasmic pool of UDP-galactose for subsequent galactosylation reaction. On the other hand, in the presence of L-cystine instead of L-cysteine in the cell culture medium, L-cystine is transported through the x _c ^- system into the cells. With increased L-cystine uptake, the stimulation of the UDP-Gal-T enzyme may be weakened, resulting in a balanced cytoplasmic pool of UDP-galactose for subsequent galactosylation reaction.

According to the present invention, the concentration of one or more sulfhydryl compounds is adjusted to affect the galactosylation pattern of the recombinant protein produced by the mammalian cell. Concentrations of one or more sulfhydryl compounds with a sulfhydryl group(s) in the range of about 4 mM to 10 mM can be used and modified according to the particular host cell being cultured and the desired galactosylation pattern of the resulting recombinant protein. To obtain a protein with the desired galactosylation profile, a concentration of one or more sulfhydryl compounds is selected that provides a constant, homogeneous galactosylation profile of the recombinant protein. To increase the degree of galactosylation of the recombinant protein, generally a lower concentration provides an increased degree of galactosylation while maintaining the viability of the mammalian host cell culture. Typically, concentrations of one or more sulfhydryl compounds, such as cysteine and/or cystine, with a sulfhydryl group(s) in the range of from about 4 mM to 10 mM, preferably from about 5 mM to 10 mM are used. More preferably, concentrations of from about at least 5.0 mM to equal to or less than 9.0 mM, more preferably from about at least 5.0 mM to equal to or less than 8.0 mM, even more preferably from about at least 5.0 mM to equal to or less than 7.0 mM, and most preferably from about at least 5.0 mM to equal to or less than 6.0 mM are used. Alternatively (e.g. in the case of cysteine and/or cystine), concentrations of from about at least 0.6 g/L to equal to or less than 1.1 g/L, more preferably from about at least 0.6 g/L to equal to or less than 1.0 g/L, more preferably from about at least 0.6 g/L to equal to or less than 0.9 g/L, even more preferably from about at least 0.6 g/L to equal to or less than 0.8 g/L and most preferably from about at least 0.6 g/L to equal to or less than 0.7 are used.

As used herein, the term "concentration" with respect to one or more sulfhydryl compound(s) refers to the total concentration of all sulfhydryl compounds contained in the culture medium. As used herein, the term "concentration" with respect to a sulfhydryl group refers to the total concentration of all sulfhydryl compounds contained in the culture medium. In this context, a sulfhydryl group refers to -SH (also referred to herein as "[-SH]") and may also refer to a sulfhydryl group that has undergone a reaction in which a hydrogen dissociates from a sulfur atom of the sulfhydryl group and said sulfur atom forms an S-S bond with another sulfhydryl group that has undergone said reaction. In other words, the term "sulfhydryl group" also includes sulfhydryl groups that have been linked, for example, by a disulfide bridge. The concentration may be a measured or measurable, or a calculated or calculated actual concentration of the sulfhydryl compound(s) in the medium surrounding the cells at a given time. Methods for measuring the concentration of this(these) sulfhydryl compound(s) in the medium are known in the art. Examples of such methods include PCI-MS (Agilent, Boblingen, Germany). Thus, the concentration also refers to the amount of sulfhydryl compound(s) contained in the culture medium surrounding the cells during the culture and is thus the actual concentration of the corresponding sulfhydryl compound(s) at a given time. This concentration can be determined analytically and is the result of, for example, the introduction of the sulfhydryl compound(s) into the culture (by weighing, transfer of cells and medium from preculture, introduction of impurities, leaching, etc.), release by cells (e.g. by cell death or by active secretion), uptake by cells, and other factors.

As used in the context of the present invention, a cell culture medium comprises an increased level of one or more sulfhydryl compounds compared to a cell culture medium that does not comprise an increased level of one or more sulfhydryl compounds.

In particular, the invention relates to a method for producing a glycoprotein, such as a recombinant protein or an antibody, in particular a bispecific antibody to CD20/CD3 or an antibody to α-synuclein, said method comprising

(a) culturing a mammalian cell as described herein in a cell culture medium as described herein, wherein a concentration of sulfhydryl group(s) of one or more sulfhydryl compounds of at least greater than 4.0 mM and less than 10.0 mM and glucose of at least greater than 3.0 g/L in the cell culture medium is maintained for at least 3, more preferably for at least 4, and even more preferably for at least 5 days,

(b) isolating said antibody.

In the context of the method of the present invention, the recombinant protein has an increased level of monogalactosylated or

digalactosylated glycans compared to the corresponding level of a recombinant protein obtained using a medium not containing an elevated level of one or more sulfhydryl compounds, as used in the context of the method of the present invention, the recombinant protein has an elevated level of monogalactosylated or digalactosylated glycans by at least 3%, more preferably by at least 5%, even more preferably by at least 10%. As used in the context of the method of the present invention, the titer of the produced recombinant protein is increased compared to the titer of the corresponding recombinant protein obtained using a medium not containing an elevated level of one or more sulfhydryl compounds. Preferably, the titer of the obtained recombinant protein is at least 2000 mg/l.

As used in the context of the method of the present invention, the viable cell density in the cell culture medium is increased compared to the corresponding viable cell density in a medium that does not contain an increased level of one or more sulfhydryl compounds. Preferably, the maximum viable cell density in the cell culture medium is at least about 120×10 ⁵ cells/mL.

For example, the recombinant protein may be a CD20/CD3 bispecific antibody having 19.0-29.0% (w/w) G1 and 1.3-2.8% (w/w) G2 per total glycan; preferably 20.0-28.0% (w/w) G1 and 1.4-2.7% (w/w) G2 per total glycan; more preferably 21.0-28.0% (w/w) G1 and 1.5-2.7% (w/w) G2 per total glycan, and most preferably 21.0-27.4% (w/w) G1 and 1.5-2.6% (w/w) G2 per total glycan. Said culturing of a mammalian cell may result in an increase in the titer of said antibody by at least 20%, preferably by at least 30%, more preferably by at least 40%, more preferably by at least 50%, even more preferably by at least 60% and most preferably by at least 80% relative to the titer in a corresponding cultivation of the mammalian cell without maintaining the sulfhydryl group(s) of one or more sulfhydryl compounds at a concentration of at least more than 4.0 and less than 10.0 mM in the cell culture medium.

As another example, the recombinant protein can be an anti-α-synuclein antibody having 17.2-48.0% (w/w) G1 and 3.1-15.0% (w/w) G2 per total glycan; 25.4-48.0% (w/w) G1 and 3.5-15.0% (w/w) G2 per total glycan; preferably 27.2-47.0% G1 and 4.4-15.0% G2 per total glycan; preferably 40.0-46.0% (w/w) G1 and 8.4-15.0% (w/w) G2 per total glycan; more preferably 41.0-45.0% (w/w) G1 and 9.5-14.0% (w/w) G2 of total glycan, and most preferably 42.1-43.9% (w/w) G1 and 10.6-13.3% (w/w) G2 of total glycan. Said culturing of a mammalian cell may result in an increase in the titer of said antibody by at least 10%, preferably by at least 20%, more preferably by at least 30%, more preferably by at least 40%, more preferably by at least 50%, more preferably by at least 60%, more preferably by at least 70%, even more preferably by at least 80% and most preferably by at least 100% relative to the titer in the corresponding culturing of the mammalian cell without maintaining the sulfhydryl group(s) of one or more sulfhydryl compounds at a concentration of at least more than 4.0 and less than 10.0 mM in the cell culture medium. As another example, said culturing of a mammalian cell may result in an increase in the titer of said antibody between 9.0 and 75% relative to the titer of a corresponding culturing of the mammalian cell without maintaining the sulfhydryl group(s) of one or more sulfhydryl compounds at a concentration of at least greater than 4.0 mM and less than 10.0 mM in the cell culture medium.

In addition, the said mammalian cell culture results in the production of an antibody to α-synuclein having monogalactosylated (G1) and digalactosylated (G2) glycans characterized by

(i) increased binding of the target protein by at least 10%, preferably at least 20%, more preferably at least 35%, even more preferably at least 45%, and most preferably at least 50%;

(ii) increased neonatal Fc receptor (FcRn) binding by at least 10%, preferably at least 20%, more preferably at least 33%, more preferably at least 40%, and most preferably at least 45%; and/or

(iii) increased FcγRIIa binding by at least 10%, preferably at least 20%, more preferably at least 36%, even more preferably 45% and most preferably 50%,

with respect to non-monogalactosylated (G1) and non-digalactosylated (G2) forms of an antibody to α-synuclein upon appropriate culturing of a mammalian cell without maintaining the sulfhydryl group(s) of one or more sulfhydryl compounds at a concentration of at least greater than 4.0 mM and less than 10.0 mM in the cell culture medium.

"Recombinant protein" or "recombinantly expressed protein" as used herein refers to a protein expressed from a host cell that has been manipulated for the purposes of such expression. The manipulation includes one or more genetic modifications, such as the introduction of one or more heterologous genes encoding the expressed protein. The heterologous gene may encode a protein that is normally expressed in the cell or is foreign to the host cell. Alternatively, the manipulation may involve increasing or decreasing the level of one or more endogenous genes. As used in the context of the present invention, a recombinant protein has monogalactosylated or digalactosylated glycans. As used herein, the term "glycan" of a recombinant protein refers to a glycoprotein. As used herein, the term "glycoprotein" generally refers to peptides and proteins having more than about ten amino acids and at least one oligosaccharide side chain. The glycoproteins may be homologous to the host cell or, preferably, they are heterologous, i.e. foreign to the host cell used, such as the human protein produced by the Chinese hamster ovary (CHO) cell. Preferably, mammalian glycoproteins (glycoproteins that were originally obtained from a mammalian organism) are used, more preferably those that are directly secreted into the medium. Examples of mammalian glycoproteins include molecules such as cytokines and their receptors, as well as chimeric proteins containing cytokines or their receptors, including, for example, tumor necrosis factor alpha and beta, their receptors and derivatives thereof; growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone: thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle-stimulating hormone; Calcitonin; Luteinizing hormone; Glucagon; Coagulation factors such as Factor VIIIC, tissue factor IX, and von Willebrand factor; Anticoagulant factors such as protein C; Atrial natriuretic factor; Pulmonary surfactant; Plasminogen activator such as urokinase or human urine or tissue-type plasminogen activator (t-PA); Bombesin; Thrombin; Hematopoietic growth factor; Enkephalinase; RANTES (regulated by activation of normally expressed and secreted T cells); Human macrophage inflammatory protein (MIP-1-alpha); Serum albumin such as human serum albumin; Muller inhibitory substance; Relaxin A chain; Relaxin B chain; Prorelaxin; Mouse gonadotropin-associated peptide; Microbial protein such as beta-lactamase; DNase; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors: integrin; protein A or D; rheumatoid factors; neurotrophic factor such as bone neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factor such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic protein (BMP); interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs) such as M-CSF, GM-CSF, and G-CSF; interleukins (ILs) such as IL-1 through IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; cleavage accelerating factor; viral antigen such as, for example, the AIDS envelope portion; transport proteins; homing receptors; addressins; regulatory proteins; antibodies; chimeric proteins such as immunoadhesins, and fragments of any of the foregoing polypeptides. "Galactosylated" as used herein in relation to glycoproteins refers to a glycoprotein containing one or more galactose residues, resulting in the formation of G1 and G2 glycostructures. For example, galactosylated recombinant proteins having monogalactosylated or digalactosylated glycans that contain one or more sialic acid residues, such as GlcNAc ₃ Man ₃ GlcNAc ₂ Gal, GlcNAc ₃ Man ₃ GlcNAc ₂ Gal ₂ , GlcNAc ₃ Man ₃ GlcNAc ₂ GalSiai, GlcNAc ₃ Man ₃ GlcNAc ₂ Gal ₂ Siai, and GlcNAc ₃ Man ₃ GlcNAc ₂ Gal ₂ Siai ₂ .

In one aspect used in the context of the present invention, the recombinant protein is an antibody. As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule or an immunologically active portion of an immunoglobulin molecule, i.e., a molecule that contains an antigen binding site, such as a Fab or F(ab') ₂ fragment, regardless of whether they are natural or partially or wholly synthetic. The term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies (including full-length antibodies that have an immunoglobulin Fc region or intact monoclonal antibodies), antibody compositions with polyepitopic specificity, polyclonal antibodies, multivalent antibodies (usually engineered to have three or more antigen-binding sites), multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, diabodies and single-chain molecules such as scFv molecules, and antibody fragments (e.g., Fab, F(ab')2 and Fv), so long as they exhibit the desired antigen-binding activity. "Multispecific antibodies" are monoclonal antibodies that have binding specificity for at least two different sites, i.e., different epitopes on different antigens or different epitopes on a single antigen. Multispecific antibodies can be produced as full-length antibodies or antibody fragments.

Techniques for generating multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), and knob-into-hole engineering (see, e.g., U.S. Patent No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be generated by engineering using the electrostatic interaction effect to create Fc-heterodimeric antibody molecules (see, e.g., WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); the use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547–1553 (1992) and WO 2011/034605); the use of common light chain technology to overcome the problem of light chain mispairing (see, e.g., WO 98/50431); the use of diabody technology to generate bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444–6448 (1993)); and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and the production of trispecific antibodies, as described in, e.g., Tutt et al. J. Immunol. 147: 60 (1991).

Included within the definition of an antibody are antibody conjugates, such as antibody-drug conjugates (ADCs) or antibodies conjugated to, for example, labeling elements. In addition, included within the definition of an antibody are antibodies that bind with sufficient affinity to a particular target protein such that the antibody can be used as a diagnostic and/or therapeutic agent for targeting the particular protein. For example, an antibody can be an α-synuclein antibody that is capable of binding α-synuclein with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent for targeting α-synuclein.

As another example, the antibody can be an anti-CD20/CD3 antibody that is capable of binding CD20-CD3 with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent for targeting CD20-CD3. In one aspect, the degree of binding of the antibody to an unrelated, non-target protein is less than about 10% of the binding of the antibody to the target, as measured, for example, by surface plasmon resonance (SPR). In certain aspects, an antibody that binds to a target has a dissociation constant (K _D ) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 ^-8 M or less, e.g., from 10 ^-8 M to 10 ^-13 M, e.g., from 10 ^-9 M to 10 ^-13 M). An antibody is said to "specifically bind" to a target when the antibody has a K D of 1 μM or less. In certain aspects, an antibody binds to an epitope of a target that is conserved across targets from different species. In some aspects, as used in the context of the present invention, the recombinant protein is a therapeutic protein. For example, when the recombinant protein is an antibody, the antibody can be a therapeutically effective antibody and can bind to any protein, including a member of the angiopoietin family, such as Ang1, Ang2, Ang3 and Ang4, and bispecific antibodies to a member of the angiopoietin family and, for example, VEGF, such as Ang2/VEGF; a member of the HER receptor family, such as HER1 (EGFR), HER2, HER3 and HER4; CD proteins, such as CD3, CD4, CD8, CD18, CD19, CD20, CD21, CD22, CD25, CD33, CD34, CD38, CD40, CD44 and CD52; cell adhesion molecules such as LFA-1, VLA04, ICAM-1, VCAM, and integrin, including their α- or β-subunits (e.g., anti-CD11α, anti-CD18, or anti-CD11β antibodies); growth factors such as vascular endothelial growth factor (VEGF); cytokine receptors such as thymic stromal lymphopoietin receptor (TSLP-R); IgE; blood group antigens; flk2/flt3 receptor; obesity receptor (OR), and protein C. Other illustrative proteins include growth hormone (GH), including human growth hormone (hGH) and bovine growth hormone (bGH); growth hormone releasing factor; parathyroid hormone, thyroid stimulating hormone; lipoproteins; insulin A chain; insulin B chain; proinsulin, follicle-stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC; tissue factor (TF); von Willebrand factor; atrial natriuretic factor; pulmonary surfactant; plasminogen activator such as urokinase or tissue-type plasminogen activator (t-PA), bombasin, thrombin, tumor necrosis factor-α and -β; enkephalinase; RANTES (regulated by activation of normally expressed and secreted T cells); human macrophage inflammatory protein-1-α; serum albumin such as human serum albumin (HSA); Müllerian inhibitory factor; relaxin A-chain, relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; DNase; inhibin; activin; receptors for hormones or growth factors; protein A or D; fibroblast activating protein (FAP); carcinoembryonic antigen (CEA); rheumatoid factors; neurotrophic factor such as bone-derived neurotrophic factor (BDNF); neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6) or nerve growth factor such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR); transforming growth factor (TGF) such as TGF-α and TGF-β, including TGF-1, TGF-2, TGF-3, TGF-4, or TGF-βδ; insulin-like growth factor-1 and -II (IGF-I and IGF-II); des(1-3)-IGF-1 (brain IGF-I); insulin-like growth factor binding proteins (IGFBP); erythropoietin (EPO); thrombopoietin (TPO); osteoinductive factors; immunotoxins; bone morphogenetic protein (BMP); interferon (interferon-α, -β, or -γ); colony-stimulating factors (CSF), e.g. M-CSF, GM-CSF, and G-CSF; interleukins (IL), e.g. IL-1 through IL-10 and IL-17; superoxide dismutase; T-cell receptors; BlyS (Br3) receptor; Br3-Fc immunoadhesin; Apo-2 receptor; Fc receptor; surface membrane proteins; decay accelerating factor (DAF); viral antigen, such as part of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; immunoadhesins; and biologically active fragments or variants of any of the above. Alternatively, the antibody may be an antibody directed against mammary epithelial cells or binding to colon carcinoma cells, antibodies to EpCAM, antibodies to Gp11b/IIIa, antibodies to RSV, antibodies to CMV, antibodies to HIV, antibodies to hepatitis, antibodies to CA 125, antibodies to human renal cell carcinoma, antibodies to human colorectal tumor, R24 antibodies to human melanoma directed against ganglioside GD3, antibodies to human squamous cell carcinoma, antibodies to human leukocyte antigen (HLA), antibodies to HLA DR.

As used herein, the terms "antigen" and "epitope" are used interchangeably and refer to a site (e.g., a continuous stretch of amino acids or a conformational configuration consisting of different regions of non-contiguous amino acids) on an antigen, either protein or non-protein, to which an antibody binding moiety binds to form an antibody binding moiety-antigen complex. An epitope is thus a region of an antigen that is bound by an antibody. Epitopes may be formed from continuous stretches of amino acids (linear epitope) or contain discontinuous amino acids (conformational epitope), such as those brought into spatial proximity by folding of the antigen, i.e., by tertiary folding of a protein antigen. Linear epitopes typically remain bound to the antibody after exposure of the protein antigen to denaturing agents, whereas conformational epitopes typically are destroyed by treatment with denaturing agents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.

In some embodiments, the epitope determinant comprises chemically active surface moieties of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and in some embodiments may have specific three-dimensional structural characteristics and/or specific charging characteristics. Suitable antigenic determinants may be found, for example, on the surface of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in serum, and/or in the extracellular matrix (ECM). Proteins useful herein may be any native form of proteins from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. Selection of antibodies that bind to a particular epitope (i.e., those that bind to the same epitope) can be accomplished using methods conventional in the art, such as, for example, but not limited to, alanine scanning, peptide blots (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage assays, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-competition assays (see Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY).

In a specific embodiment, the antigen is a human protein. When a specific protein is referred to herein, the term includes the "full-length" unprocessed protein, as well as any form of the protein that results from processing in the cell. The term also includes naturally occurring protein variants, such as splice variants or allelic variants. In other words, the term also includes an antibody that has a structure substantially similar to that of a native antibody, or that has heavy chains that contain an Fc region, as defined herein.

By "antigen-binding" is meant that the antigen-binding site portion of the antibody specifically binds to the antigen. In other words, the term "antigen-binding site" refers to the portion of the antibody that contains a region that specifically binds to and is complementary to part or all of the antigen. By "specific binding" is meant that the binding is selective for the antigen and can be separated from unwanted or non-specific interactions. The ability of an antigen-binding molecule to bind to a particular antigen can be determined using enzyme-linked immunosorbent assay (ELISA) or other techniques known to those skilled in the art, such as surface plasmon resonance (SPR) (with BIAcore analysis) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

The variable regions of the heavy and light chains of an immunoglobulin molecule contain the binding domain that interacts with an antigen. The constant regions of antibodies (Ab) can mediate binding of immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classical complement pathway. "Variable domain" (variable domain of the light chain (VL), variable domain of the heavy chain (VH)) as used herein refers to each of the pairs of light and heavy chains that is directly involved in binding of the antibody to an antigen. The variable domains of human light and heavy chains have the same general structure, and each domain contains four framework regions (FRs), the sequences of which are largely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated by using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880–887 (1993); Clarkson et al., Nature 352:624–628 (1991). As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of the variable domain of an antibody that are hypervariable in sequence and that determine antigen-binding specificity, such as "complementarity determining regions" ("CDRs"). Unless otherwise noted, CDRs are defined in accordance with Kabat et al., supra. One skilled in the art will recognize that assignment of CDRs may also be determined in accordance with Chotia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

The framework regions adopt a β-sheet conformation and the CDRs can form loops connecting the β-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form, together with the CDRs from the other chain, an antigen-binding site. The CDR3 regions of the heavy and light chains of an antibody play a particularly important role in the binding specificity/affinity of the antibodies of the invention and therefore constitute a further aspect of the invention. Unless otherwise indicated herein, the numbering of amino acid residues in the variable region or constant region follows the EU numbering system, also referred to as the EU index, described in Kabat et al., Sequences of Proteins of Immunological Interest, ^5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. "Framework region" or "FR" refers to residues of the variable domain other than those of the hypervariable region (HVR). The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences are typically found in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. With the exception of CDR1 in VH, CDRs generally contain amino acid residues that form hypervariable loops. CDRs also contain "specificity-determining residues" or "SDRs," which are residues that contact the antigen. SDRs are contained in regions of the CDRs called abbreviated-CDRs or a-CDRs. Examples of a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31–34 L1, 50–55 L2, 89–96 L3, 31–35B H1, 50–58 H2, and 95–102 H3. (See Almagro and Fransson, Front. Biosci. 13:1619–1633 (2008)).

As indicated above, the term "antibody" as used herein, unless otherwise indicated or clearly contradicted by context, includes a full-length antibody and antibody fragments that are antigen-binding fragments, i.e., retain the ability to specifically bind to an antigen. In other words, the term "fragment" as used herein refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigens to which an intact antibody binds. Examples of antibody fragments include, but are not limited to, (i) a Fab' or Fab fragment; diabodies, linear antibodies; single-chain antibody molecules (e.g., scFv and scFab), multispecific antibodies formed from antibody fragments, a monovalent fragment consisting of the L, VH, CL and CH1 domains, or a monovalent antibody as described in WO 2007059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting essentially of the V _H and C _H 1 domains; (iv) an Fv fragment consisting essentially of the V _L and V _H domains of one arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of the V _H domain, also called the antibody domain (Holt et al.; Trends Biotechnol. 2003 Nov; 21(11):484-90); (vi) camel antibodies or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5(1): 111-24); and (vii) an isolated complementarity determining region (CDR). Pepsin treatment produces an E(ab')2 fragment that has two antigen-binding sites (two Fab fragments) and part of the Fc region. For a discussion of Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and having an increased half-life in vivo, see U.S. Patent No. 5,869,046. "Diabodies" are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404097; WO 1993/01161; Hudson et al., Nat. Med. 9:129–134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444–6448 (1993). Moreover, although the two domains of the Fv fragment, V _L and V _H , are encoded by separate genes, they can be joined recombinantly by a synthetic linker to form a single protein chain in which the V _L and V _H regions are paired to form monovalent molecules (known as single-chain antibodies or single-chain Fv (scFv), see, e.g., Bird et al., Science 242, 423–426 (1988) and Huston et al., PNAS USA 85, 5879–5883 (1988)). Thus, a "single-chain variable fragment" or "scFv" is a fusion protein of the variable domains of the heavy (VH) and light chains (VL) of an antibody joined by a linker. In particular, the linker is a short polypeptide of 10-25 amino acids and is usually enriched in glycine for flexibility and serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant regions and the introduction of the linker. For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. "Single domain antibodies" are antibody fragments comprising all or a portion of a heavy chain variable domain or all or a portion of a light chain variable domain of an antibody. In certain aspects, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1). Antibody fragments can be produced by a variety of methods, including, but not limited to, proteolytic cleavage of an intact antibody, as well as recombinant production by recombinant host cells (e.g., E. coli), as described herein.

Such single chain antibodies are encompassed by the term "antibody" unless otherwise indicated or clearly indicated by context. Although such fragments are generally included within the definition of antibody, they are individually and collectively unique features of the present invention, exhibiting distinct biological properties and utility. These and other useful antibody fragments in the context of the present invention, as well as bispecific formats of such fragments, are discussed further herein. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

The term "full-length antibody" means an antibody consisting of two "full-length antibody heavy chains" and two "full-length antibody light chains". A "full-length antibody heavy chain" is a polypeptide consisting, in N-terminal to C-terminal direction, of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), which is abbreviated as VH-CH1-HR-CH2-CH3; and optionally an antibody heavy chain constant domain 4 (CH4) in the case of an antibody of the IgE subclass. Preferably, a "full-length antibody heavy chain" is a polypeptide consisting, in N-terminal to C-terminal direction, of VH, CH1, HR, CH2, and CH3. A "full-length antibody light chain" is a polypeptide consisting, in N-terminal to C-terminal direction, of a light chain variable domain (VL) of an antibody and a light chain constant domain (CL) of an antibody, abbreviated as VL-CL. The light chain constant domain (CL) of an antibody can be κ (kappa) or % (lambda). The two chains of a full-length antibody are linked together by interpolypeptide disulfide bonds between the CL domain and the CH1 domain and between the hinge regions of the heavy chains of the full-length antibody. 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 IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . In certain aspects, an antibody is of the IgG ₁ isotype. The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain contains. In certain aspects, the antibody is of the IgG ₁ isotype with the P329G, L234A, and L235A mutations to reduce the effector function of the Fc region. In other aspects, the antibody is of the IgG ₂ isotype. In certain aspects, the antibody is of the IgG ₄ isotype with the S228P mutation in the hinge region to improve the stability of the IgG ₄ antibody. The constant domains of the heavy chain corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

It should also be understood that the term antibody, unless otherwise specified, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides such as chimeric antibodies and humanized antibodies, and antibody fragments that retain the ability to specifically bind to an antigen (antigen-binding fragments) obtained by any known technique, such as enzymatic cleavage, peptide synthesis and recombinant techniques. The resulting antibody may be of any isotype. 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 another source or species.

For example, an antibody may be a monoclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical to and/or bind to the same epitope, with the exception of possible variant antibodies, such as those containing mutations that occur naturally or arise during the production of the monoclonal antibody preparation, such variants generally being present in small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the characteristic of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, hybridoma techniques, recombinant DNA techniques, phage display techniques, and techniques using transgenic animals containing all or part of the human immunoglobulin loci, such techniques and other exemplary techniques for producing monoclonal antibodies are described herein.

As another example, the antibody can be a humanized antibody. The term "humanized antibody" refers to antibodies in which the framework regions or "complementarity determining regions" (CDRs) have been modified to comprise CDRs of an immunoglobulin with different specificity than the parent immunoglobulin. In other words, the term encompasses a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody comprises substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDRs correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. 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, such as a non-human antibody, refers to an antibody that has been humanized. Methods for producing humanized antibodies include conventional recombinant DNA and gene transfection technologies and are well known in the art. See, for example, Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M.S., et al., Nature 314 (1985) 268-270.

As another example, the antibody may be a human antibody. The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. In other words, the term encompasses an amino acid sequence that corresponds to the sequence of an antibody produced by a human or a human cell, or obtained from a non-human source that utilizes human antibody repertoires or other human antibody encoding sequences. This definition of a human antibody explicitly excludes a humanized antibody that contains non-human antigen-binding residues. Human antibodies are well known in the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368–74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450–459 (2008). Human antibodies can be produced by introducing an immunogen into a transgenic animal modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen stimulation. Such animals typically contain all or part of the human immunoglobulin loci that replace the endogenous immunoglobulin loci or that are extrachromosomal or randomly integrated into the animal's chromosomes. Typically, such transgenic mice have inactivated the endogenous immunoglobulin loci. For a review of methods for producing human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117–1125 (2005). Also see, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe the XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes the HuMab® technology; U.S. Patent No. 7,041,870, which describes the K-M MOUSE® technology; and U.S. Patent Application Publication No. US 2007/0061900, which describes the VelociMouse® technology. Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with a different human constant region.

Human antibodies can be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies produced by human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of human IgM monoclonal antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (trioma technology) is described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

Human antibodies can also be produced by isolating variable domain sequences from phage display libraries of human origin. Such variable domain sequences can then be combined with the desired human constant domain. Methods for selecting human antibodies from antibody libraries are described below.

In one aspect used in the context of the present invention, the recombinant protein is an antibody to α-synuclein or a bispecific antibody directed against CD3 and CD20, also referred to as a CD20/CD3 bispecific antibody. The term "bispecific antibody" in the context of the present invention refers to an antibody having two different antigen-binding regions defined by different antibody sequences. Examples of bispecific antibody formats that can be used for these purposes include, but are not limited to, so-called "BiTE" (bispecific T-cell engaging activator) molecules, in which two scFv molecules are linked by a flexible linker (see, e.g., WO 2004/106381, WO 2005/061547, WO 2007/042261 and WO 2008/119567, Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299–305 (1996)) and their derivatives, such as tandem diabodies (“TandAb”; Kipriyanov et al., J Mol Biol 293, 41–56 (1999)); “DART” (dual affinity retargeting) molecules, which are based on the diabody format but have a C-terminal disulfide bridge for additional stabilization (Johnson et al., J Mol Biol 399, 436–449 (2010)), and the so-called triomabs, which are fully hybrid mouse/rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458–467 (2010)). Specific T-cell bispecific antibody formats included herein are described in WO 2013/026833, WO 2013/026839, WO 2016/020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

The terms "α-synuclein antibody", "anti-α-synuclein antibody", and "antibody that binds to α-synuclein" refer to an antibody that is capable of binding human alpha-synuclein with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting α-synuclein. α-synuclein is a protein that plays a central role in the control of dopaminergic neuronal function in the brain and is believed to play a critical role in the pathophysiology of Parkinson's disease (PD). For example, synucleinopathies, also known as Lewy body diseases (LBDs), are characterized by degeneration of the dopaminergic system, movement disorders, cognitive impairment, and the formation of Lewy bodies (LB) and/or Lewy neurites (McKeith et al., Neurology (1996) 47:1113–24), and can be treated with antibodies to α-synuclein. Such synucleinopathies include Parkinson's disease (including idiopathic Parkinson's disease), diffuse Lewy body disease (DLBD), also known as Lewy body dementia (DLB), Alzheimer's disease variant with Lewy bodies (LBV), combined Alzheimer's disease and Parkinson's disease, pure autonomic failure, and multiple system atrophy (MSA; eg, olivopontocerebellar atrophy, striatonigral degeneration, and Shy-Drager syndrome).

Alpha-synuclein is part of a large family of proteins including beta- and gamma-synuclein, and synoretin. Naturally occurring wild-type human alpha-synuclein is a 140-amino acid peptide with the following amino acid sequence:

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA (SEQ ID NO: 9)

(Ueda et al., Proc. Natl. Acad. Sci. USA (1993) 90:11282-6).; GenBank accession number: P37840). The protein has three recognizable domains: a KTKE repeat domain spanning amino acids 1-61, a NAC (non-amyloid component) domain extending from approximately amino acids 60-95, and a C-terminal acidic domain extending from approximately amino acids 98 to 140. Reference to alpha-synuclein or fragments thereof includes the naturally occurring human wild-type amino acid sequences identified above and human allelic variants thereof, particularly those associated with Lewy body disease (e.g., E46K, A30P, and A53T, where the first letter represents the amino acid in SEQ ID NO: 9, the number represents the position of the codon in SEQ ID NO: 9, and the second letter represents the amino acid in the allelic variant).

Alpha-synuclein is expressed in the normal state associated with synapses and is thought to play a role in neural plasticity, learning, and memory. Several studies have shown that alpha-synuclein plays a central role in the pathogenesis of PD. The protein can aggregate to form insoluble fibrils under pathological conditions. For example, synuclein accumulates in LB (Spillantini et al., Nature (1997) 388:839-40; Takeda et al., J. Pathol. (1998) 152:367-72; Wakabayashi et al., Neurosci. Lett. (1997) 239:45-8). Mutations in the alpha-synuclein gene cosegregate with rare familial forms of parkinsonism (Kruger et al., Nature Gen. (1998) 18:106-8; Polymeropoulos, et al., Science (1997) 276:2045-7). Overexpression of alpha-synuclein in transgenic mice (Masliah et al., Science (2000) 287:1265-9) and Drosophila (Feany et al., Nature (2000) 404:394-8) mimics some of the pathological aspects of Lewy body disease. In addition, it has been suggested that soluble synuclein oligomers may be neurotoxic (Conway KA, et al., Proc Natl Acad Sci USA (2000) 97:571-576; VollesMJ, Lansbury PT, Jr Biochemistry (2003) 42:7871-7878). The accumulation of alpha-synuclein with similar morphological and neurological changes in animal species and models as diverse as humans, mice, and flies suggests that this molecule contributes to the development of Lewy body disease.

For example, an antibody to alpha-synuclein can be an antibody designated 9E4 (prasinezumab), BIIB054, 1H7, 5C1, 6H7, 8A5, and N1-202.21D11, and related antibodies. Prasinezumab or 9E4 is also known as PRX002 and RG7935. As shown in the accompanying examples, CHO L965 cells (Example 3), CHO L967 cells (Example 6), and CHO L971 cells (Examples 7 and 8) produce prasinezumab. References to such antibodies can be found in the art, for example, 9E4 (prasinezumab) and related antibodies can be found in U.S. Patents 8,609,820; U.S. Patents 9,556,259; U.S. Patents 9,884,906; US 8,697,082; US 8,506,959; US 9,034,337; US 7,919,088; US 8,092,801; US 8,147,833; US 8,673,593; US 7,910,333 and US 7,674,599. References to BIIB054, also known as N1-202.12F4, and related antibodies can be found in, for example, US Patents 10,301,381; US 9,975,947; US 8,896,504; US 9,580,493 and US 8,940,276. References to 1H7 and related antibodies can be found in, for example, US Patents 7,910,333; US 8,790,644; US 9,234,031; US 9,217,030; US 9,670,273 and US 10,118,960. References to 5C1 and related antibodies can be found, for example, in US Patents 9,605,056; US 1,008,1674 and US 10,301,382. References to 6H7 and related antibodies can be found, for example, in US Patents 8,673,593 and US 7,910,333. References to 8A5 and related antibodies can be found, for example, in US Patents 8,673,593 and US 7,910,333. References to N1-202.21D11 and related antibodies can be found, for example, in US Patent 9,580,493.

In particular, patients with Parkinson's disease and related disorders can be treated with an antibody to α-synuclein. Indeed, in addition to α-synuclein being the major protein component of Lewy bodies (LBs), genetic studies have shown that certain point mutations and multiplications of the α-synuclein gene cause familial forms of PD. A large body of evidence suggests that α-synuclein pathology at dopaminergic synapses may underlie the occurrence of neuronal dysfunction and degeneration in the brain in PD (Bellucci, A., et al., Brain Res. 1432 (2012) 95-113).

The term "CD20" refers to human CD20 (UniProtKB/Swiss-No. P11836) and includes any variants, isoforms, and species homologues of CD20 that are naturally expressed by cells, including tumor cells, or expressed by cells transfected with the CD20 gene or cDNA. The CD20 molecule (also called human B-lymphocyte differentiation-restricted antigen or Bp35) is a hydrophobic transmembrane protein of approximately 35 kDa localized on pre-B and mature B lymphocytes (Valentine et al. (1989) J. Biol. Chem. 264(19): 11282–11287; and Einfield et al., (1988) EMBO J. 7(3): 711–717). CD20 is found on the surface of more than 90% of B cells in peripheral blood or lymphoid organs and is expressed during early pre-B cell development and remains until plasma cell differentiation. CD20 is present on both normal B cells and malignant B cells. In particular, CD20 is expressed on more than 90% of B-cell non-Hodgkin lymphomas (NHL) (Anderson et al. (1984) Blood 63(6): 1424–1433), but is not detected on hematopoietic stem cells, pro-B cells, normal plasma cells, or other normal tissues (Tedder et al. (1985) J. Immunol. 135(2):973–979).

The term "CD3" as used herein refers to the cluster of differentiation protein 3, which is part of the T-cell co-receptor protein complex and consists of four separate chains. CD3 is also found in humans as well as other species, and thus the term "CD3" may be used herein and is not limited to human CD3 unless the context so requires. In mammals, the complex contains the CD3y (gamma) chain (human CD3y chain UniProtKB/Swiss-Prot No. P09693 or cynomolgus macaque CD3y UniProtKB/Swiss-Prot No. Q95LI7), the CD36 (delta) chain (human CD36 UniProtKB/Swiss-Prot No. P04234 or cynomolgus macaque CD36 UniProtKB/Swiss-Prot No. Q95LI8), two CD3s (epsilon) chains (human CD3s UniProtKB/Swiss-Prot No. P07766; cynomolgus macaque CD3s UniProtKB/Swiss-Prot No. Q95LI5; or rhesus macaque CD3s UniProtKB/Swiss-Prot No. G7NCB9) and the zeta chain (human CD3C UniProtKB/Swiss-Prot No. P20963, cynomolgus monkey CD3ζ UniProtKB/Swiss-Prot No. Q09TK0). These chains bind to a molecule known as the T-cell receptor (TCR) and generate an activation signal in T lymphocytes. The TCR and CD3 molecules together constitute the TCR complex.

According to the present invention, mammalian cells are cultured to produce a recombinant protein having monogalactosylated or digalactosylated glycans. When selecting a host cell for producing a recombinant protein in the context of the present invention, the person skilled in the art understands that different host cells have different properties and/or specific mechanisms for translational and post-translational processing and modification of the expressed protein, such as, but not limited to, glycosylation and cleavage. In this context, the person skilled in the art knows how to select a suitable cell in the context of the present invention. In other words, the person skilled in the art knows which cell line should be selected to allow post-translational modifications. Alternatively, the host cell can be modified with the means necessary for specific post-translational modification for expressing a recombinant protein having monogalactosylated or digalactosylated glycans. Recombinant methods for producing recombinant proteins, such as antibodies, which can be obtained, for example, as described in U.S. Patent 4,816,567. For these methods, one or more isolated nucleic acids encoding an antibody are provided. In the case of a native antibody or a native antibody fragment, two nucleic acids are required, one for the light chain or fragment thereof and one for the heavy chain or fragment thereof. Such nucleic acid(s) encode(s) an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). These nucleic acids can be in the same expression vector or in different expression vectors.

In the case of a bispecific antibody with heterodimeric heavy chains, four nucleic acids are required, one for the first light chain, one for the first heavy chain comprising a polypeptide of the first heteromonomeric Fc region, one for the second light chain and one for the second heavy chain comprising a polypeptide of the second heteromonomeric Fc region. The four nucleic acids can be contained in one or more nucleic acid molecules or one or more expression vectors. Such nucleic acids encode an amino acid sequence comprising a first VL and/or an amino acid sequence comprising a first VH comprising a first heteromonomeric Fc region and/or an amino acid sequence comprising a second VL and/or an amino acid sequence comprising a second VH comprising a second heteromonomeric Fc region of the antibody (e.g., the first and/or second light and/or first and/or second heavy chains of the antibody). These nucleic acids may be present in one expression vector or in different expression vectors, usually these nucleic acids are located in two or three expression vectors, i.e. one vector may contain more than one of these nucleic acids. Examples of these bispecific antibodies are CrossMab (see, e.g., Schaefer, W. et al, PNAS, 108 (2011) 11187-1191). For example, one of the heteromonomeric heavy chains contains so-called "knob mutations" (T366W and optionally one of S354C or Y349C), and the other contains so-called "hole mutations" (T366S, L368A and Y407V and optionally Y349C or S354C) (see, e.g., Carter, P. et al., Immunotechnol. 2 (1996) 73) according to the EU index numbering.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include initially transformed cells and progeny derived therefrom, regardless of passage number. Progeny may not be completely identical to the parent cell in nucleic acid content and may contain mutations. Included herein are mutant progeny that have the same function or biological activity for which the initially transformed cells were screened or selected. Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For information on the expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste into a soluble fraction and further purified.

The terms "mammalian host cell," "mammalian host cell line," and "mammalian host cell culture" are used interchangeably and refer to cell lines derived from mammals that are capable of growth and survival when maintained in either monolayer or suspension culture in medium containing appropriate nutrients and growth factors. The necessary growth factors for a particular cell line are readily determined empirically without undue experimentation, as described, for example, in Mammalian Cell Culture (Mather, J.P. ed., Plenum Press, N.Y. [1984]), and Barnes and Sato. ((1980) Cell. 22:649). Typically, the cells are capable of expressing and secreting large amounts of the particular glycoprotein of interest into the culture medium. Examples of suitable mammalian host cells in the context of the present invention may include Chinese hamster ovary/-DHFR cells (CHO. Urlaub and Chasm. Proc. Natl. Acad. Sci. USA. 77:4216 [1980]); dp12.CHO cells (EP 307.247 published March 15, 1989); the SV40-transformed monkey kidney line CV1 (COS-7, ATCC CRL 1651); the human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 [1977]); newborn hamster kidney cells (BHK, ATCC CCL 10); Mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 [1980]); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); brown rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562. ATCC CCL51); TRI cells (Mather et al, Annals N.Y. Acad. Sci. 383:44-68 [1982]); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In a preferred aspect of the present invention, the mammalian cells are selected from the group consisting of CHO cells, Vero cells, BHK cells, COS cells and HEK293/293T cells, more preferably the mammalian cells are CHO cells.

More preferably, the mammalian host cell is a CHO cell, even more preferably a CHO cell deficient in dihydrofolate reductase (DHFR) activity. The mammalian cell used in the present invention may be selected or manipulated to produce a recombinant protein. The manipulation includes one or more genetic modifications, such as the introduction of one or more heterologous genes encoding an expressed protein. The heterologous gene may encode a protein that is normally expressed in the cell or is foreign to the host cell. Additionally or alternatively, the manipulation may involve increasing or decreasing the level of one or more endogenous genes. Often, the cells are manipulated to produce a recombinant protein, for example, by introducing a gene encoding the protein and/or by introducing control elements that regulate the expression of the gene encoding the protein of interest. Genes encoding recombinant proteins and/or control elements may be introduced into a host cell using vectors such as plasmids, phages or viral vectors. As used herein, the term "vector" refers to a nucleic acid molecule capable of increasing the copy number of another nucleic acid to which it is linked. This term includes a vector in the form of a self-replicating nucleic acid structure as well as a vector incorporated into the genome of the host cell into which it is introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

Some vectors are capable of autonomous replication in the host cell into which they are introduced, while other vectors can be integrated into the host cell genome and thus replicate along with the host genome. Various vectors are publicly available and the precise nature of the vectors is not essential to the present invention. Typically, the components of a vector include one or more signal sequences, an origin of replication, one or more marker genes, a promoter and a transcription termination sequence. Such components are described in WO 97/25428.

The "growth phase" of a cell culture refers to the period of exponential cell growth (log phase), when cells typically divide rapidly. During this phase, cells are cultured for a specific period of time, typically 1 to 4 days, and under conditions such that cell growth is maximized. The definition of the growth cycle of a host cell can be determined for a particular intended host cell without unnecessary experimentation. "The period of time and under conditions that maximize cell growth," etc., refer to those culture conditions that are determined to be optimal for cell growth and division for a particular cell line. During the growth phase, cells are cultured in a nutrient medium containing the necessary additives. In addition, culture conditions such as temperature, pH, dissolved oxygen (dO ₂ ), etc., are used for a particular host and will be apparent to one skilled in the art. Typically, the pH is adjusted with either an acid (e.g., _CO2 ) or a base (e.g., _Na2CO or NaOH). A suitable temperature range for culturing mammalian cells such as CHO cells is approximately 30 to 38°C, and a suitable _dO2 value is 5 to 90% of air saturation in a humidified, controlled atmosphere so that optimal growth is achieved for a particular type of cell line. For example, fed-batch cell culture conditions can be used since fed-batch culture conditions are designed to enhance the growth of mammalian cells during the growth phase of the cell culture. "Fed-batch culture" as used herein refers to a cell culture method in which additional components are added to the culture during or after the start of the culturing process. Fed-batch culture is typically stopped at some point and the cells and/or media components are harvested and optionally purified. Additionally, at a certain stage, the cells may be used for inoculation in a cell culture preparation phase or step. Alternatively, the preparation phase or step may be continuous with the inoculation or growth phase or step.

The "transition phase" of a cell culture refers to the period of time during which the culture conditions of the acquisition phase are in effect. During the transition phase, environmental factors such as cell culture temperature, osmolality of the medium, etc., shift from growth conditions to acquisition conditions.

The "production phase" of cell culture refers to the period of time during which cell growth has stabilized. During the production phase, logarithmic cell growth has ended and protein production is primary. During this period, the medium is typically supplemented to support continued protein production and to produce the desired glycoprotein product. For example, the cell culture medium is controlled during the cell culture production phase. According to the method of the present invention, one or more factors affecting the viable cell density and/or the titer of the mammalian host cell culture product are manipulated to achieve a certain galactosyl content in the expressed recombinant protein. "Titer" in this context refers to the total amount of recombinantly expressed glycoprotein produced by a mammalian cell culture in a given amount of medium volume. The titer is typically expressed in milligrams of glycoprotein per milliliter of medium. In particular, the factors that increase the galactosyl content of the recombinant protein are controlled in the cell culture production phase so that the resulting recombinant protein contains a specific amount of galactosyl. As used herein, the cell culture production phase is preceded by a cell culture transition phase that involves parameters for the cell culture production phase.

In the method of the present invention, the concentration of one or more sulfhydryl compounds and/or the concentration of glucose is adjusted at any or all growth or production phases of the fermentation process. For example, the concentration of one or more sulfhydryl compounds and/or glucose can be adjusted at the beginning of or during the growth phase and/or at the beginning of or during the production phase. In particular, the concentration of one or more sulfhydryl compounds and/or glucose can be adjusted at the beginning of growth and at the beginning of the production phase or at any or all phases of the beginning of growth during the growth phase, at the beginning of the production phase and during the production phase. In a preferred aspect of the present invention, one or more sulfhydryl compounds and/or glucose are added to the medium at the beginning of or during the production phase to create a concentration of one or more sulfhydryl compounds of about 4 mM to 10 mM sulfhydryl group(s) in the medium and/or at least more than 3.0 g/L glucose. In another preferred aspect of the present invention, the method further comprises an initial step of culturing the mammalian cells in the same medium without said one or more sulfhydryl compounds comprising from 4 mM to 10 mM sulfhydryl group(s) and/or at least more than 3.0 g/L glucose. In yet another aspect of the present invention, the method further comprises an initial step of culturing the mammalian cells in the same medium without said one or more sulfhydryl compounds comprising from 4 mM to 10 mM sulfhydryl group(s) and/or at least more than 3.0 g/L glucose, and one or more sulfhydryl compounds and/or glucose are added to the medium at the beginning of or during the production phase to create a concentration of one or more sulfhydryl compounds with about 4 mM to 10 mM sulfhydryl group(s) in the medium and/or at least more than 3.0 g/L glucose.

The concentration of one or more sulfhydryl compounds can be controlled by increasing or decreasing the concentration of the sulfhydryl compound(s) in the culture medium. In the present invention, when the concentration of the sulfhydryl compound(s) increases or decreases, the increase or decrease in concentration is related to the concentration of the sulfhydryl compound(s) in the medium in the culture phase immediately preceding the increase. Thus, if an increase in the concentration of, for example, cystine and/or cysteine in the medium at the beginning of the production phase is observed, this leads to an increase in the concentration of the sulfhydryl compound(s) compared to the concentration of the sulfhydryl compound(s) in the medium of the immediately preceding growth phase. Similarly, if the concentration of, for example, cystine and/or cysteine in the medium during the production phase is to increase, this is an increase in the concentration of that(their) sulfhydryl compound(s) compared to the concentration of that(their) sulfhydryl compound(s) in the medium of the immediately preceding part of the production phase. Similarly, if a decrease in the concentration of, for example, cystine and/or cysteine in the medium is to be observed at the beginning of or during any of the growth or production phases, this is a decrease in the concentration of that(their) sulfhydryl compound(s) in the medium of the immediately preceding culture phase.

In general, it is preferred that the concentrations of any one or more sulfhydryl compounds are adjusted in the cell culture medium, wherein the concentrations of all of these one or more sulfhydryl compounds are adjusted simultaneously. However, the method of the present invention also includes adjusting one or more sulfhydryl compounds by simultaneously adjusting the first sulfhydryl compound(s) and then the second, or vice versa. In particular, if the concentration of one or more sulfhydryl compounds is to be increased and the concentration of one or more sulfhydryl compounds is to be decreased, the adjustments upward and downward may occur simultaneously or at different times. In this case, it is preferred that the adjustments to increase the concentration of one or more sulfhydryl compounds and to decrease the concentration of one or more sulfhydryl compounds occur at the same time or in the same medium.

In the method of the present invention, the adjustment of the concentration of one or more sulfhydryl compounds can be achieved by any method suitable for the fermentation conditions used. The method by which the concentration of one or more sulfhydryl compounds is adjusted is not essential to the present invention, and appropriate methods are known in the art. Thus, the adjustment of the concentration of one or more sulfhydryl compounds can occur either by adding to the medium (in the case of increasing the concentration of one or more sulfhydryl compounds) in which the cells are cultured, or by transferring all or part of the cells (e.g. by splitting) into fresh medium containing the desired concentration of one or more sulfhydryl compounds. If necessary, a combination of these methods can be used.

Thus, the adjustment of the concentration of one or more sulfhydryl compounds may be continuous during all or part of the culturing period or may be discontinuous, for example, as a response to an estimated, calculated or measured concentration of one or more sulfhydryl compounds in the culturing medium. The invention defines adjusting the concentration of one or more sulfhydryl compounds within certain ranges. If an actual measurement or calculation of the concentration of each or all of the one or more sulfhydryl compounds during a certain culturing period, for example during a growth or production phase, indicates that the concentration of each or all of the one or more sulfhydryl compounds falls within the ranges specified herein, an adjustment of this concentration may nevertheless take place, provided that the resulting concentration of the one or more sulfhydryl compounds remains within the specified range. If necessary, known techniques can be used to measure the actual concentrations of one or more sulfhydryl compounds in the culturing medium before making adjustments.

Thus, if batch fermentation conditions are used, the increase in the concentration of the one or more sulfhydryl compounds can be achieved, for example, by seeding into fresh medium containing or supplemented with concentrations of the one or more sulfhydryl compounds that are increased compared to the existing culture medium, or by splitting the cells into a medium containing or supplemented with increased concentrations of the respective one or more sulfhydryl compounds compared to the existing medium. If fed-batch fermentation conditions are used, the increase in the concentration of the one or more sulfhydryl compounds can be achieved, for example, by seeding into fresh medium containing or supplemented with increased concentrations of the respective one or more of the respective sulfhydryl compound(s), providing one or more bolus or continuous feeds of the respective one or more sulfhydryl compounds to the culture medium; by determining the feeding rate based on cell counts or calculated according to known metabolic models, metabolic surrogate markers, etc., or by dividing the culture into a medium that contains or has been supplemented with increasing concentrations of the relevant one or more sulfhydryl compounds. If a bolus or continuous feed is added, it may contain other nutrients/components required by the culture in addition to the one or more sulfhydryl compound(s). If perfusion fermentation conditions are used, achieving increasing concentrations of one or more sulfhydryl compounds may be achieved, for example, by continuously or intermittently adding one or more sulfhydryl compounds to the reactor, either simultaneously or separately with other nutrients/components added to the perfusion culture.

If a reduction in the concentration of one or more sulfhydryl compounds is desired, this can be achieved by seeding the cells into fresh medium in which the concentration of one or more sulfhydryl compounds is reduced compared to the concentration of one or more sulfhydryl compounds in the medium of the immediately preceding culture phase.

Specific values for the reduced or increased concentrations of one or more sulfhydryl compounds are based on either actual measurements of one or more sulfhydryl compounds in the culture medium, or on theoretical concentrations, or on calculations of the concentration of one or more sulfhydryl compounds in the culture solution surrounding the cells. The practitioner will recognize that some concentration of one or more sulfhydryl compounds may be present, introduced, for example, by impurities and leaching, and will take this into account when calculating the reduced or increased concentration of one or more sulfhydryl compounds in accordance with the invention.

In one aspect in the context of the present invention, the concentration of one or more sulfhydryl compounds can be adjusted in the culture medium to increase galactosylation of glycans of the recombinant protein. In another aspect of the present invention, the concentration of one or more sulfhydryl compounds can be adjusted in the culture medium to increase the viable cell density, to increase the titer of the recombinant protein product and/or then again to increase galactosylation of glycans of the recombinant protein. For example, the concentration of one or more sulfhydryl compounds can be adjusted in the culture medium first to enhance growth and then again to increase galactosylation of glycans of the recombinant protein. As another example, the concentration of one or more sulfhydryl compounds can be adjusted in the culture medium to increase the viable cell density and then to increase galactosylation of glycans of the recombinant protein. As another example, the concentration of one or more sulfhydryl compounds can be adjusted in the culture medium to increase the titer of the recombinant protein product, and then again to increase galactosylation of the glycans of the recombinant protein. In another aspect of the present invention, the concentration of one or more sulfhydryl compounds can be adjusted in the culture medium to increase the density of viable cells, to increase the titer of the recombinant protein product, and then again to increase galactosylation of the glycans of the recombinant protein.

As used herein, the term "bioreactor" refers to any vessel used to grow a prokaryotic or eukaryotic cell culture, such as an animal cell culture (such as a mammalian cell culture). The bioreactor may be of any size as long as it is suitable for culturing cells, such as mammalian cells. Typically, the bioreactor has a volume of at least 30 ml and may have a volume of 1, 10, 100, 250, 500, 1000, 2500, 5000, 8000, 10000, 12000 liters or more, or any volume in between. The internal conditions of the bioreactor, including but not limited to pH and temperature, are typically controlled during the culturing period. The bioreactor may be composed of any material suitable for storing mammalian cell cultures suspended in a medium under the culturing conditions of the present invention, including glass, plastic or metal. As used herein, the term "production bioreactor" refers to the final bioreactor used to produce the polypeptide or protein of interest. The volume of a large-scale cell culture production bioreactor is typically greater than about 100 ml, typically at least about 10 liters, and may be 500, 1000, 2500, 5000, 8000, 10000, 12000 liters or more, or any volume in between. For example, mammalian cell culture is performed in a large-scale bioreactor, preferably a 10000 L bioreactor.

One skilled in the art will be aware of and able to select suitable bioreactors for use in practicing the present invention.

As used in a preferred aspect in the context of the present invention, the method further comprises collecting the recombinant protein produced by the mammalian cell. The collection of the expressed protein either during or at the end of the culturing period, preferably the production phase, can be achieved using methods known in the art. The protein can be obtained from the culturing medium as a secreted protein, although it can be obtained from host cell lysates by direct production without a secretory signal. If the protein is membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X 100) or its extracellular region can be released by enzymatic cleavage. The expressed protein can be isolated and/or purified, if necessary, using methods known in the art. An "isolated" protein, such as an antibody, is an antibody that has been separated from a component of its natural environment. In some aspects, a protein, e.g., an antibody, is purified to greater than 95% or 99% purity, as determined by, e.g., electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reversed phase HPLC) methods. For a review of methods for assessing the purity of antibodies, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

As used herein, the term "expression" or "expresses" are used interchangeably and refer to transcription and translation in a host cell. The level of expression of a recombinant protein in a host cell can be determined based on either the amount of the corresponding mRNA present in the cell or the amount of protein encoded by the corresponding gene. For example, mRNA transcribed from the product gene is desirably quantified by Northern hybridization (Sambrook et al. Molecular Cloning A Laboratory Manual (Cold Spring Harbor Laboratory Press. 1989)). The protein encoded by the gene product can be quantified either by assaying the biological activity of the protein or by assays that are independent of such activity, such as Western blotting or radioimmunoassay using antibodies that are reactive with the protein (Sambrook et al. Molecular Cloning A Laboratory Manual, pp 18 1- 18 88 (Cold Spring Harbor Laboratory Press. 1989)).

In particular, mammalian cells that express a recombinant protein containing monogalactosylated or digalactosylated glycans must express or be caused to express specific enzymes such that, under appropriate conditions as described herein, the appropriate post-translational modification occurs in vivo. Enzymes include those enzymes that are required for the addition and replenishment of N- and O-linked carbohydrates, such as the enzymes described by Hubbard and Ivan above for N-linked oligosaccharides. The enzymes optionally include oligosaccharyl transferase, alpha-glucosidase I, alpha-glucosidase II, ER alpha (1,2) mannosidase, Golgi alpha-mannodase I, N-acetylglucosaminyl transferase I, Golgi alpha-mannodase II, N-acetylglucosaminyl transferase II, alpha (1,6) fucosyl transferase, and β (1.4) galactosyl transferase. In addition, the host cell expresses the corresponding enzyme(s) that can be expected to add galactose at a specific position and bind as part of the host cell genome. Optionally, the host cell can express the corresponding enzyme(s), such as by transfecting the host cell DNA encoding the enzyme(s). Enzyme(s) such as those described above are expected to add galactose to a corresponding oligosaccharide structure such as GlcNAc. Suitable enzyme(s) in the context of the present invention include, but are not limited to, enzyme(s) that catalyze galactosylation and branching of N- and O-linked oligosaccharides.

Numerous culture conditions can be used to culture mammalian cells expressing the desired protein and capable of adding the desired carbohydrates at the desired position and linkage, with particular attention to the host cell being cultured. Suitable culture conditions for mammalian cells are well known in the art (J. Immunol. Methods (1983) 56:221-234) or can be readily determined by one skilled in the art (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood. D. and Hames. B.D., eds. Oxford University Press. New York (1992)) and vary according to the particular host cell selected.

The mammalian cell culture of the present invention is prepared in a medium suitable for the particular cell being cultured. "Medium," "cell culture medium," and "culture medium" are used interchangeably herein and refer to a solution containing nutrients that support the growth of mammalian cells. Typically, such solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for minimal growth and/or survival. Such a solution may also contain additional components that enhance growth and/or survival above a minimal rate, including, but not limited to, hormones and/or other growth factors, certain ions such as sodium, chloride, calcium, magnesium, and phosphate, buffers, vitamins, nucleosides or nucleotides, trace elements, amino acids, lipids, and/or glucose or another energy source. The medium is preferably formulated with a pH and salt concentration that is optimal for cell survival and proliferation.

Commercially available media such as Ham's F10 medium (Sigma), minimal essential medium ([MEM], (Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium ([DMEM], Sigma) are typical nutrient solutions. In addition, any of the media described in Ham and Wallace (1979) Meth. Enz., 58:44; Barnes and Sato (1980) Anal. Biochem., 102:255; U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 5,122,469, or 4,560,655; International Publication Nos. WO 90/03430; and WO 87/00195; the disclosures of all of which are incorporated herein by reference, can be used as nutrient media. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as Gentamycin™), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), lipids (such as linoleic or other fatty acids) and their suitable carriers, and glucose or an equivalent energy source. Any necessary additives may also be included at appropriate concentrations as known to those skilled in the art.

As used herein, the term "under conditions under which the cell expresses the recombinant protein" means conditions that are used to culture the cell expressing the polypeptide and that are known or can be determined by one skilled in the art. One skilled in the art will recognize that these conditions may vary depending on the type of cell being cultured and the type of recombinant protein being expressed. Typically, the cell is cultured at a temperature of, for example, 20°C to 40°C and for a period of time sufficient to efficiently produce the conjugate, for example, for 4 to 28 days, in a volume of 0.01 to 10' L.

Preferably, the mammalian host cell is a CHO cell, preferably a CHO cell lacking dihydrofolate reductase (DHFR) activity, and the suitable medium comprises a base medium component such as a DMEM/HAMF-12 based formulation (for the formulation of DMEM and HAM F12 media, see the formulations of the growth media in the Catalog of Cell Lines and Hybridomas of the American Type Culture Collection. Sixth Edition. 1988. pp. 346-349) with altered concentrations of certain components such as amino acids, salts, sugars and vitamins, and optionally containing glycine, hypoxanthine, thymidine, recombinant human insulin, hydrolyzed peptone, a cell protective agent such as Pluronic F68 or an equivalent pluronic polyol, gentamicin and trace elements.

According to the present invention, mammalian host cells are cultured to produce a reconstitutable recombinant protein having monogalactosylated or digalactosylated glycans. The total galactose content of the recombinant protein is controlled by controlling cell culture parameters that affect viable cell density, product titer, and/or galactose content in the mammalian cell. Factors affecting viable cell density and/or product titer are well known in the art and include, but are not limited to, factors affecting DNA copy number, RNA copy number, factors affecting RNA such as RNA stabilizing factors, medium nutrients and other additives, concentration of transcription enhancers, osmolality of culture medium, temperature and pH of cell culture, and the like. According to the present invention, adjusting these factors individually or in combination to increase viable cell density and/or product titer produces a recombinant protein containing monogalactosylated or digalactosylated glycans. Adjusting these factors individually or in combination to increase viable cell density and/or product titer results in a recombinant protein with increased galactose content.

The terms "cell density", "cell concentration", etc., as used herein, refer to the number, weight, mass, etc. of cells present in a given volume of medium. "Peak cell density", etc., refers to the maximum number of cells that can be achieved in a given volume of medium, and "desired peak cell density", etc., refers to the maximum number of cells that a practitioner desires to obtain (e.g., targets) in a given cell volume. Variations of such target value(s) will be understood by those skilled in the art, for example, a person skilled in the art may express the target value(s) in terms of a desired cell mass, and such target value(s) may be expressed in one or more suitable units of measurement (e.g., desired peak units of cell mass).

As used herein, the term "cell viability" refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations. As used herein, the term also refers to the proportion of cells that are alive at a given time relative to the total number of cells, alive and dead, in the culture at that time.

As used herein, the terms "culture" and "cell culture" refer to a cell population that is suspended in a cell culture medium under conditions suitable for the survival and/or growth of the cell population. As used herein, the terms may refer to a combination that includes a cell population (e.g., an animal cell culture) and the medium in which the population is suspended.

In one aspect, the method further comprises the step of pre-cultivating the mammalian cells in a cell culture medium. As suggested herein, the cells can be pre-cultivated to exponential growth phase in a suitable cell culture medium with or without sulfhydryl group(s) from one or more sulfhydryl compounds. As used herein, the term "pre-cultivating" or "performing a pre-cultivating" is used interchangeably and refers to a culturing step preceding the culturing of the cells in a second culturing step. For example, the cells can be grown in a first cell culture as a pre-cultivating step and then inoculated into a second cell culture, such as in a bioreactor, such as a production bioreactor. Pre-cultivating steps are known to one skilled in the art and it is known how to perform such pre-cultivating steps.

The term "total viable cell density" or "IVCD" as used herein refers to the average viable cell density during a culture multiplied by the amount of time the culture was conducted. Assuming that the amount of polypeptide and/or protein produced is proportional to the number of viable cells present during the culture, the integrated viable cell density is a useful tool for estimating the amount of polypeptide and/or protein produced during a culture.

The recombinant protein of the present invention can be obtained by culturing cells that express the recombinant protein under various cell culture conditions. In other words, the formation of biomass and the expression of protein from mammalian cells are achieved according to the method of the invention by culturing the cells using any fermentation method or cell culture system that is suitable for growing cells to form biomass and express proteins that can be used in the present invention. For example, the cells can be grown in batch cultures, fed-batch cultures, perfusion cultures or split cultures, where the culture is stopped after sufficient expression of the proteins occurs, after which the proteins are collected and, if necessary, purified.

For example, the cell culture of the present invention may employ a fed-batch culture procedure. In a fed-batch culture, mammalian cells may initially be supplied to a culture vessel, and additional cellular nutrients are supplied continuously or gradually to the culture during the culture, with or without periodic harvesting of the cells and/or product before the culture is terminated. A fed-batch culture may include, for example, a semi-continuous fed-batch culture, in which the entire culture (including cells and medium) is periodically removed and replaced with fresh medium. A fed-batch culture differs from a simple batch culture, in which all components for culturing the cells (including cells and all nutrients for the culture) are supplied to the culture vessel at the beginning of the culture process. Fed-batch culture may further differ from perfusion culture in that the supernatant is not removed from the culture vessel during the process (in perfusion culture, the cells are calibrated in culture, such as by sequential filtration, encapsulation, attachment to microcarriers, etc., and the culture medium is continuously or periodically introduced and removed from the culture vessel). Alternatively, cells may be grown in perfusion cultures, where the culture does not disappear, and new nutrients and components are periodically or continuously added to the culture, and the expressed glycoprotein is removed either periodically or continuously.

In addition, cell cultures can be propagated according to any scheme or procedure that may be suitable for a particular host cell and the intended choice of production plan. For example, reactors, temperatures and other conditions for fermentation of cell culture for the formation of biomass and the production of proteins, such as oxygen concentration and pH, are known in the art. Any conditions suitable for culturing a selected mammalian cell can be selected using information available in the art. Cultivation conditions, such as temperature, pH, etc., correspond to those typically used previously with the host cell selected for expression and are obvious to those skilled in the art. If desired, the temperature and/or pH and/or CO ₂ can be changed during culturing to increase the yield and/or increase the relative amount of protein of the desired quality.

In addition, in this context, the present invention contemplates a single-step or multi-step culturing procedure. In a single-step culturing, the host cells are inoculated into a culture medium, and the methods of the present invention are applied during a single production phase of the cell culture. Alternatively, a multi-step culture is contemplated. In a multi-step culture, the cells can be cultured in multiple steps or phases. For example, the cells can be grown in a first step or growth phase of the culture, where the cells, possibly removed from storage, are inoculated into a medium suitable for promoting growth and high viability. As used herein, the term "cell viability" refers to the ability of cells in a culture to survive under a given set of culture conditions or experimental variations. As used herein, the term also refers to the proportion of cells that are alive at a given time relative to the total number of cells, alive or dead, in the culture at that time. Cells can be maintained in the growth phase for a suitable period of time by adding fresh medium to the host cell culture.

For example, cell culture procedures for large-scale or small-scale protein production are potentially useful in the context of the present invention. Procedures including, but not limited to, a fluidized bed bioreactor, a hollow fiber bioreactor, roller bottle culture, or a stirred tank bioreactor may be used and operated alternatively in a batch mode, a fed-batch mode, and/or a perfusion mode. "Perfusion culture" as used herein refers to a cell culture method comprising growing cells in a basal inoculation medium and, when the cells reach a desired cell density, replacing the spent medium with fresh medium. Perfusion may comprise either continuous or intermittent perfusion and may comprise delivering at least one bolus to the cell culture. Perfusion culture may be followed by fed-batch culture. As used herein, the term "bioreactor" refers to any vessel used to grow a culture of mammalian cells. Typically, the bioreactor has a volume of at least 1 liter and may have a volume of 10, 100, 250, 500, 1000, 2500, 5000, 8000, 10000, 12000 liters or more, or any volume in between. The internal conditions of the bioreactor, including but not limited to pH, dissolved oxygen and temperature, are typically controlled during the culturing period. The bioreactor may be composed of any material suitable for storing mammalian cell cultures suspended in a medium under the culturing conditions of the present invention, including glass, plastic or metal. In a preferred aspect of the present invention, the culturing of mammalian cells is carried out in a bioreactor, more preferably in a large-scale bioreactor. In a more preferred aspect of the present invention, the mammalian cell is cultured in at least a 10,000 L bioreactor.

The Importance of Fc-Galactosylation of Antibodies

As shown in Figure 5, the Asn297-linked carbohydrate chain consists of a common biantennary glycan structure of four N-acetylglucosamine (GlcNAc) and three mannose residues with various additions of fucose, galactose, and sialic acid residues. These glycans are often named according to the number of biantennary terminal galactose residues, i.e., G0 (no galactose), G1 (one galactose), G2 (two galactoses), and according to the presence of the backbone fucose residue, i.e., G0 (no galactose), G1 (one galactose), G2 (two galactoses).

In a preferred aspect used in the context of the present invention, the recombinant protein is used as a medicament. Altered IgG galactosylation was first reported in rheumatoid arthritis (Parekh et al., Nature. 316 (1985) 452-457) and more recently in other autoimmune diseases such as psoriatic arthritis and ankylosing spondylitis (Martin et al., J Rheumatol 28 (2001) 1531-1536). Increased galactosylation has been observed during pregnancy and in patients with rheumatoid arthritis who experience pregnancy-induced remission (Bondt et al., J. Proteome. Res. 12 (2013) 4522-4531). This suggests that increased galactosylation of antibodies may be functionally more anti-inflammatory (Zauner et al., Mol. Cell Proteomics. 12 (2013) 856-865). Karsten et al. (Nature Medicine 18.9 (2012) 1401-1406) confirmed this anti-inflammatory property by showing in mice that high galactosylation of IgG immune complexes promotes the association of Fcγ RIIB and Dectin-1, which blocks the proinflammatory effector functions of C5aR and CXCR226. In another preferred aspect used in the context of the present invention, the recombinant protein is for the treatment of patients with B-cell proliferative disorders such as non-Hodgkin's lymphoma and chronic lymphocytic leukemia, or patients with Parkinson's disease and related disorders.

Another important aspect to consider when considering the functional impact of the terminal galactose is that it provides a basis for the addition of sialic acid, the outermost sugar moiety of the IgG-Fc glycan. Oligosaccharide analysis revealed that the lack of galactosylation of the protein was a potential cause for the reduction in sialic acid content.

Terminal galactose structures are known to have a significant impact on affinity for the Clq complex, and their removal results in decreased complement lysis activity (Hodoniczky, J. et al., Biotechnol. Progr. 21 (2005) 1644-1652). In particular, Wright and Morrison (1998) (J Immunol. 1998; 160:3393 3402) and Hodoniczky et al. (2005) found that the absence of galactose on mAb Fc oligosaccharides reduces the affinity between the Fc and the complement component C1q, thereby reducing CDC activity.

Rituximab (Rituxan® anti-CD20), first approved in 1997, is a chimeric monoclonal antibody produced in CHO cells for the treatment of non-Hodgkin lymphoma and other B-cell-associated diseases. Rituximab is glycosylated at the Fc, and Fc glycans are highly heterogeneous, primarily due to the variable presence of terminal galactose residues. The effect of rituximab terminal galactose residues on CDC activity is related to the involvement of such residues in rituximab binding to complement C1lq (Hodoniczky et al. 2005).

The presence or absence of galactose on IgG glycans correlates with the effector function of modified Fc in some but not all (Boyd et al., Mol Immunol. 32 (1995) 1311–1318; Wright and Morrison, J Immunol. 160 (1998) 3393–3402) IgG mAbs, suggesting that the observed effects may be partly antibody specific. Tsuchiya et al. (1989) found that agalacto IgG reduced binding to C1q and Fc receptors, and Boyd et al. found that agalacto Campathl (an anti-CD52 mAb) had reduced cell-mediated lysis (CML) but retained the ability to trigger ADCC. Thus, as used herein, the term “effector functions” refers to the biological activities mediated by the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., the B cell receptor) and B cell activation; see also, e.g., Thomann et al, PLoS One. 2015 Aug 12;10(8):e0134949. doi: 10.1371/journal.pone, 0134949. eCollection 2015.

As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the Fc region of a human IgG heavy chain by definition typically extends from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expressing a particular nucleic acid molecule encoding a full-length heavy chain may comprise a full-length heavy chain or may comprise a cleaved version of a full-length heavy chain (also referred to herein as a "cleaved version heavy chain"). This may occur when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the EU Kabat index). Therefore, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

In some aspects, if the recombinant protein is an antibody, the Fc domain of the antibody may comprise one or more alterations compared to the wild-type Fc domain. These Fc domains will, however, retain substantially the same characteristics necessary for therapeutic use as compared to their wild-type counterparts. For example, certain alterations may be made in the Fc region that may result in altered (i.e., improved or decreased) C1q binding and/or complement-dependent cytotoxicity (CDC), such as described in WO 99/51642. See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 for other examples of Fc region variants. WO 00/42072 (Presta) and WO 2004/056312 (Lowman) describe antibody variants with increased or decreased binding to FcR. The contents of these patent publications are expressly incorporated by reference herein. See also Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001). Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton et al.). The half-life of an antibody may depend on the structure of the Fc region of the antibody, which in turn affects the efficiency of binding of said Fc region to FcRn of the neonatal receptor. Thus, the half-life is increased by maintaining the binding of the Fc region to FcRn. Specifically, at approximately pH 6.0, this binding results in endosomal translocation of the FcRn-bound antibody to lysosomal degradation pathways, recycling it instead to the plasma membrane where IgG is re-released into the bloodstream at pH 7.4. Thus, this pathway achieves an increased half-life of IgG in the blood, which is necessary for prolonged exposure of the antibody to its target and increased potential therapeutic efficacy; see Saxena, Abhishek; Bai, Bingxin; Hou, Shin-Chen; Jiang, Lianlian; Ying, Tianlei; et al. Methods in molecular biology (Clifton, N.J.) 1827: 399-417. (2018); Spearman, Maureen; Dionne, Ben; Butler, Michael. Cell Engineering, Vol 7: Antibody Expression and Production 7: 251-292. SPRINGER. (2011), which are expressly incorporated herein by reference. These antibodies comprise an Fc domain with one or more substitutions that improve binding of the Fc region to FcRn. Polypeptide variants with altered amino acid sequences of the Fc region and increased or decreased C1q binding capacity are described in U.S. Patent No. 6,194,551 and International Publication No. WO 99/51642. See also Idusogie et al. J. Immunol. 164: 4178-4184 (2000). The contents of such are expressly incorporated herein by reference.

Other reports on the effects of galactosylation on IgG molecules include changes in physicochemical properties such as conformation and surface accessibility (Krapp et al., J. Mol. Biol. 325 (2003) 979–89; Mimura et al., Mol. Immunol. 37 (2000) 697–706). Fortunato and Colina (J. Phys. Chem. 118 (2014) 9844–9851) used explicit water atomistic molecular dynamics simulations to study the effects of galactosylation in the Fc domain of immunoglobulin G1. They suggested that glycosylation could be exploited as a route to improve the aggregation stability of monoclonal antibodies for therapeutic treatment. As used herein, the term "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to a clinical intervention to alter the natural course of a disease in an individual being treated, and may be performed either prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, reducing the intensity or transient relief of a painful condition, and causing remission or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or to slow the progression of a disease.

Current evidence suggests that galactose residues in antibodies influence certain functions of IgG and may be necessary for effective monitoring and control during galactosidation of these molecules.

As previously stated, galactosylation of antibodies is known to be dependent on the concentration of UDP-galactose present in the cells, as these sugar molecules act as substrates required for galactosylation. Increasing UDP-galactose levels is associated with higher galactosylation and sialylation of the antibody expressed in CHO cells. Different levels of UDP-galactose in cells may also have a significant impact on other types of glycosylation in other therapeutic proteins.

Modulation of UDP-galactose concentration for O-linked glycosylation

In general, in the recombinant proteins used herein, sugars can be attached either to the amide nitrogen atom in the asparagine side chain via an N-linkage or to the oxygen atom in the serine or threonine side chain via an O-linkage. O-linked glycosylation occurs by the addition of N-acetylgalactosamine to serine or threonine residues by the enzyme UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (EC2.4.1.41), followed by other carbohydrates such as UDP-galactose. Therefore, the intracellular concentration of UDP-galactose can influence O-linked glycosylation. Therefore, the novel approach described in this invention may be useful for modulating the intracellular level of UDP-galactose and for further controlling the level of galactosylation in the final protein product.

Modulation of UDP-galactose concentration for glycosylation of Fab antibody

The existence of IgG Fab glycans has been known for some time. Fab glycans are thought to be more accessible to glycosyltransferases, resulting in greater processing compared to Fc glycans, which are spatially localized on the internal side of the CH2 domains. Accordingly, the intracellular concentration of UDP-galactose may also influence the galactosylation process of IgG Fab. Therefore, the novel approach described in this invention may be used to modulate the intracellular level of UDP-galactose and to further control the level of galactosylation in the final protein product.

As used in the context of the present invention, the level of N-linked galactosylated glycans of the recombinant protein is increased. Thus, the method of the present invention increases the production of N-linked galactosylated glycans of the recombinant protein. In one preferred aspect of the present invention, the recombinant protein comprises at least monogalactosylated, more preferably digalactosylated glycans, wherein the galactosylated glycans are linked to N-acetylglucosamine. The linkages of galactose to their respective target in the recombinant protein and their effect on the function of the recombinant protein are further described in the following example.

The present invention also relates to a pharmaceutical composition comprising a recombinant protein. The terms "composition" and "pharmaceutical composition" are used interchangeably and are to be understood as defining pharmaceutical compositions whose individual components or ingredients are themselves pharmaceutically acceptable, i.e. if oral administration is envisaged, oral administration is acceptable, and if topical administration is envisaged, topical administration is acceptable, and also includes combinations thereof, i.e. if oral and topical administration is envisaged, oral and topical administration are acceptable. The term also refers to a preparation that is in a form that provides effectiveness of the biological activity of the active ingredient contained therein and that does not contain additional components that are unacceptably toxic to the subject to which the pharmaceutical composition is to be administered. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or pharmaceutical formulation, other than the active ingredient, that is non-toxic to the subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

The pharmaceutical composition will be formulated and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site of delivery of the pharmaceutical composition, the route of administration, the schedule of administration and other factors known to practitioners. Accordingly, an "effective amount" of a pharmaceutical composition for the purposes of this invention is determined in accordance with such considerations. One skilled in the art will recognize that an effective amount of a pharmaceutical composition administered to an individual will depend, among other things, on the nature of the compound. In this context, an "effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective at doses and for periods of time necessary to achieve the desired therapeutic or prophylactic result.

A therapeutic protein, such as an antibody of the invention (and any additional therapeutic agent) can be administered by any suitable route, including parenteral, intrapulmonary, intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any convenient route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic. Various dosing regimens are contemplated herein, including, but not limited to, single or multiple administrations at different time points, bolus administration, and pulse infusion.

The antibodies of the invention are formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the route of administration, the schedule of administration and other factors known to practitioners. The antibody is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the pharmaceutical composition, the type of disorder or treatment and other factors discussed above. The other agents are typically used at the same dosages and administered by the routes described herein or at dosages that are from about 1% to 99% of the dosages described herein or at any dosage and by any route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of a disease, the appropriate dose of an antibody of the invention (used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, and the discretion of the treating physician.

As used herein, the term "and/or" shall be understood as a specific disclosure of each of the two stated features or components, either together or without the other. For example, "A and/or B" shall be understood as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were set forth herein separately.

Various aspects and features of the invention described herein are further described by way of example below. Although the foregoing invention has been described in detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention.

All patents and non-patent references cited herein are hereby incorporated by reference in their entirety.

EXAMPLES

The cDNAs of uridine diphosphate (UDP) α-D-glucose epimerase (UDP_Clc-E, EC 5.1.3.2) and UDP-α-D-glucose:α-D-galactose-1-phosphate uridylyltransferase from CHO K1M cell line were cloned and sequenced, respectively. These two enzymes were found to play a critical role in the UDP-glucose and UDP-galactose conversion pathway in CHO cells.

Example 1. Amplification of uridine diphosphate glucose epimerase (UDP-Glc-E) cDNA and sequence analysis of the CHO K1M cell line

The enzyme uridine diphosphate (UDP)-glucose-4-epimerase (UDP_Clc-E, EC 5.1.3.2), also known as UDP-galactose-4-epimerase, is a homodimeric epimerase found in bacterial, fungal, plant, and mammalian cells. This enzyme catalyzes the reversible conversion of UDP-glucose to UDP-galactose.

Total cellular RNA was extracted from the CHO K1M cell line (WO 2009047007) using the Roche MagNA Pure LC RNA Isolation Kit - High Performance (product no. 03542394001) running on a Roche MagNA Pure LC 2.0 instrument (product no. 05197686001, Roche Diagnostics). Purified RNA concentration was measured using NanoVue (GE Healthcare Bio-Science AB) and stored at 70°C.

This purified cellular RNA was used for UDP Glc-E cDNA synthesis and targeted amplification. cDNA synthesis and amplification were performed using the Roche Transcriptor One-Step RT-PCR Kit (product no. 04655877001, Roche Diagnopstics GmbH) with two UDP Glc-E specific primers homologously designed according to the NCBI GenBank database.

This document uses two primers:

forward primer UDF_ClcE-E2-21 (SEQ ID NO.: 5):

5' ATGGCCGAGAAGGTGCTGGTC 3', and

Reverse primer UDF_GlcE-R21 (SEQ ID NO.: 6):

5' TTAGGCCTGTGCTCCAAAGCC 3'.

RT-PCR conditions:

Reverse transcription: 50°C 30 min.

Initial denaturation: 94°C 7 min.

PCR amplification:

Denaturation: 94°C 10 s

Annealing: 56°C 30 sec

Elongation: 68°C 60 s (60 s/kb)

Cycle: 10

Denaturation: 94°C 10 s

Annealing: 56°C 30 sec

Elongation: 68°C 1:30 + 5 s (+5 s/tbp)

Cycle: 25

Final elongation: 68°C 7 min.

The amplified PCR product was first purified using the Roche High Pure PCR Product Purification Kit (Product No. 11732668001, Roche Diagnopstics GmbH) and then subjected to direct sequencing. The cDNA sequence of UDP_Clc-E is shown in SEQ ID NO.: 1. This cDNA encodes a predicted protein of 348 amino acids. A derivative of the amino acid sequence of UDP-glucose 4-epimerase in CHO K1M is shown in SEQ ID NO.: 2.

The protein sequence encoded by UDP-Glc-E CHO K1M is 94.5% identical and 96.6% similar to human UDP-glucose-4-epimerase and is closely related to other bovine and mouse UDP-glucose-4-epimerases.

Example 2. UDP-α-D-glucose: α-β-galactose-1-phosphate uridylyltransferase (UDP-Gal-T) cDNA amplification and sequence analysis in the CHO K1M cell line

UDP-α-D-glucose:α-D-galactose-1-phosphate uridylyltransferase (UDP-Gal-T, EC2.7.7.12) catalyzes the nucleotide exchange between uridine-5'-diphosphate-glucose (UDP-glucose) and galactose-1-phosphate (Gal-1-P) to form uridine-5'-diphosphate-galactose (UDP-galactose) and glucose-1-phosphate (Glc-1-P) by a reversible mechanism.

Total cellular RNA was extracted from the CHO K1M cell line using the Roche MagNA Pure LC - High Performance RNA Isolation Kit (product no. 03542394001) running on a Roche MagNA Pure LC 2.0 instrument (product no. 05197686001, Roche Diagnostics). Purified RNA concentration was measured using NanoVue (GE Healthcare Bio-Science AB) and stored at -70°C.

This purified cellular RNA was used for UDP-Gal-T cDNA synthesis and targeted amplification. cDNA synthesis and amplification were performed using the Roche Transcriptor One-Step RT-PCR Kit (product no. 04655877001, Roche Diagnopstics GmbH) with two UDP-Gal-T specific primers homologously designed according to the NCBI GenBank database.

This document uses two primers:

forward primer UDF-Gal-T-E2-21 (SEQ ID NO.: 7):

5' ATGTCGCAAACGGAGATGAT 3' and

Reverse primer UDP-Gal-T-R18 (SEQ ID NO.: 8):

5' TCAAGCAACAGCTGCTGT 3'. RT-PCR conditions:

Reverse transcription: 50°C 30 min.

Initial denaturation: 94°C 7 min.

PCR amplification:

Denaturation: 94°C 10 s

Annealing: 56°C 30 sec

Elongation: 68°C 60 s (60 s/kb)

Cycle: 10

Denaturation: 94°C 10 s

Annealing: 56°C 30 sec

Elongation: 68°C 1:30 + 5 s (+5 s/tbp)

Cycle: 25

Final elongation: 68°C 7 min.

The amplified PCR product was first purified using the Roche High Pure PCR Product Purification Kit (Product No. 11732668001, Roche Diagnopstics GmbH) and then subjected to direct sequencing. The cDNA sequence of UDP-Gal-T is shown in SEQUENCE-3 (SEQ ID NO.: 3). This cDNA encodes a predicted protein of 379 amino acids. A derivative of the amino acid sequence of UDP-α-D-glucose:α-D-galactose-1-phosphate uridylyltransferase in CHO K1M is shown in SEQUENCE-4 (SEQ ID NO.: 4).

The protein sequence encoded by UDP-Gal-T CHO K1M is 89.4% identical and 94.7% similar to the sequence of human UDP-α-D-glucose:α-D-galactose-1-phosphate uridylyltransferase and is closely related to other UDP-α-D-glucose:α-D-galactose-1-phosphate uridylyltransferases of mouse and bovine origin.

Example 3. Modulation of an enzyme and pathway by L-cysteine in the CHO L965 cell line to produce a recombinant antibody to human α-synuclein

In this example, CHO K1M, a cell line derived from Chinese hamster ovary (CHO) cells, was used as the host cell line (WO 2009047007).

CHO K1M cells were engineered to express a monoclonal antibody to human α-synuclein (described in U.S. Patent 9,670,274 B2 and U.S. Patent 9,890,209 B9) that binds to monomeric or oligomeric human α-synuclein and are designated herein as the CHO L965 cell line.

Human α-synuclein can form fibrillar aggregates, and these aggregates are the main component of Lewy bodies and Lewy neurites. Recent work suggests that prefibrillar oligomers of alpha-synuclein may play a key role in the progression of Parkinson's disease (Luk et al., 2012).

Specific antibody L965 can specifically bind to extracellular α-synuclein and may be used to prevent cell-to-cell transfer of aggregates and progression of Parkinson's disease.

For the cell culture process, a custom-made, chemically defined serum-free version of the medium was used as the basal culture medium for L965 cells. After thawing, cells were passaged in this medium in the presence of 3 μg/ml blasticidin (Blasticidin, InvivoGen SAS, France) and 10 μg/ml puromycin (Puromycin-Solution, InvivoGen SAS, France, cat. no. ANT-PR) in shake flasks on a 3-4 day schedule. Passage conditions were 36.5°C, 7% _CO2 and 160 rpm for 125 ml and 500 ml flasks using a Kuhner Shaker X platform (Adolf Kuhner AG, Birsfelden, Basel, Switzerland).

For the inoculation process and the production process, another custom-made version of chemically defined serum-free medium was used as the basic medium for L965 cell expansion.

During the preparation of this production medium, glucose, glutamine, amino acids, trace elements (RTE1.2 solution, Gibco, UK; Cat. No. 043-90585H) and salts were also additionally added. In this case, the final concentration of L-cysteine (Merck Chemicals GmbH, Cat. No.: 1.02735.1000) in the medium was adjusted to 6 mM and 10 mM, respectively.

Precultured cells were used for inoculation in step N-2 at approximately 3.0× ¹⁰⁵ cells/mL and in step N-1 at approximately 5.0× ¹⁰⁵ cells/mL in parallel with prepared production media containing 6 mM or 10 mM L-cysteine, respectively, all without blasticidin and puromycin. Production bioreactors were inoculated at approximately 10.0× ¹⁰⁵ cells/mL in medium containing 6 mM or 10 mM L-cysteine, respectively. Cells were cultured in production bioreactors under fed-batch conditions with predetermined pH, dissolved oxygen, temperature, and nutrient feeding strategy. Typically, 2 L bioreactors with an initial culture volume of 1.2 L were used unless otherwise noted.

The temperature in the bioreactors was maintained at 36.5°C and the stirrer speed was set at approximately 223 rpm. A gas mixture containing air, _CO2 , and _O2 was supplied. The concentration of dissolved carbon dioxide ( _dCO2 ) was measured offline once per day. The concentration of dissolved oxygen (DO) was monitored in real time and adjusted to 35% by varying the partial pressure of oxygen in the gas mixture. The pH was maintained at a set point of pH 7.00 with a dead band of ±0.03 pH units by adding _CO2 or 1.0 M _NaHCO3 unless otherwise stated.

The nutrient medium included a combination of internal medium with glucose, glutamine, amino acids and salts. This nutrient medium was prepared in solution and added to the culture on the 3rd, 6th and 9th day, each in an amount of approximately 10 vol. % of the working culture volume.

An additional glucose nutrient solution was prepared and added to the culture for 4–14 days to maintain glucose concentrations at approximately ≥4 g/L.

Cell culture samples for production were withdrawn daily with a syringe for offline analysis. The production time was typically approximately 14 days. Cell concentration and viability were measured by trypan blue exclusion on a CEDEX instrument (Roche Diagnostics GmbH, Germany). Offline measurements were performed using a COBAS INTEGRA® 400 plus (Roche Diagnostics GmbH, Germany) to measure glucose, glutamine, glutamate, lactate, ammonium and product concentrations. Dissolved carbon dioxide was analyzed on a Cobas b221 analyzer (Roche Diagnostics Ltd. CH-6343 Rotkreuz, Switzerland). Osmolality was measured by freezing point depression on an Osmomat autoosmometer (Gonotec GmbH, Berlin, Germany).

At the end of the main culture production process, the cell culture fluid was collected by centrifugation. The supernatants were then subjected to small-scale protein-A purification of mAb. The glycosylation pattern of the purified mAb was analyzed using 2AB.

Figures 6A-6D show the effect of different concentrations of L-cysteine on the G0 form of anti-α-synuclein antibody L965, the G1 form of anti-α-synuclein antibody L965, the G2 form of anti-α-synuclein antibody L965, product titer, and cell growth (IVCD).

Figure 6A shows that at the end of the 14-day production process, the amount of the G0 form of the α-synuclein antibody L965 in the process with 6 mM L-cysteine in the production medium was approximately 10.4% lower than in the process with 10 mM L-cysteine in the production medium.

Figure 6B shows that at the end of the 14-day production process, the amount of the G1 form of the α-synuclein antibody L965 in the process with 6 mM L-cysteine in the production medium was approximately 13% higher than in the process with 10 mM L-cysteine in the production medium.

Figure 6B shows that at the end of the 14-day production process, the amount of the G2 form of the α-synuclein antibody L965 in the process with 6 mM L-cysteine in the production medium was approximately 5% higher than in the process with 10 mM L-cysteine in the production medium.

Figure 6G shows that at the end of the 14-day production process, the product titer in the process with 6 mM L-cysteine in the production medium was approximately 73% higher than in the process with 10 mM L-cysteine in the production medium.

Figure 6D shows that at the end of the 14-day production process, the cell growth in the process with 6 mM L-cysteine in the production medium was higher than in the process with 10 mM L-cysteine in the production medium.

Example 4. Modulation of the enzyme and pathway by L-cysteine in the CHO T104 cell line to produce recombinant bispecific antibodies to CD20/CD3

In this example, CHO K1M, a cell line derived from Chinese hamster ovary (CHO) cells, was used as the host cell line (WO 2009047007). CHO K1M cells were engineered to express a T cell-targeted CD20-CD3 bispecific monoclonal antibody, bsAB to CD20/CD3 (described in EP3252078A1), and are designated herein as the CHO T104 cell line.

BsAB to CD20/K-CD3 is a T-cell bispecific (TCB) antibody targeting CD20 expressed on B cells and the CD3 epsilon chain present on T cells. The mechanism of action of anti-CD20/CD3 bsAB involves simultaneous binding to CD20+ B cells and CD3+ T cells, resulting in T cell activation and T cell-mediated B cell killing. In the presence of circulating or tissue-resident CD20+ B cells, pharmacologically active doses induce T cell activation and associated cytokine release. Anti-CD20/CD3 bsAB may be used to treat disease, particularly B cell proliferative disorder, and to reduce adverse effects following administration of a T cell activating therapeutic agent. For example, patients with chronic lymphocytic leukemia may be treated with an anti-CD20/CD3 bispecific antibody.

For the cell culture process, a custom-made, chemically defined serum-free version of the medium was used as the basal culture medium for T104 cells. After thawing, cells were passaged in this medium in the presence of 250 nM methotrexate (MTX; Pfizer, Cat.# 13999031) in shake flasks for 3-4 days. Passage conditions were 36.5°C, 7% _CO2 , and 160 rpm for 125 ml and 500 ml flasks using a Kuhner Shaker X platform (Adolf Kuhner AG, Birsfelden, Basel, Switzerland).

For the inoculation process and the production process, another custom-made version of the chemically defined serum-free medium was used as the basic medium for the expansion of T104 cells. During the preparation of this production medium, glucose, glutamine, amino acids, trace elements (RTE1.0 solution, SAFC, Cat. No. CR40054-1000M SLBR5143V) and salts were also additionally added. In this case, the final concentration of L-cysteine (Merck Chemicals GmbH, Cat. No.: 1.02735.1000) in the medium was adjusted to 5 mM and 10 mM, respectively.

Precultured cells were used for inoculation in step N-2 at approximately 3.0× ¹⁰⁵ cells/mL and in step N-1 at approximately 5.0× ¹⁰⁵ cells/mL in parallel with the prepared production media containing 5 mM or 10 mM L-cysteine, respectively, all without MTX. Production bioreactors were inoculated at approximately 10.0× ¹⁰⁵ cells/mL in medium containing 5 mM or 10 mM L-cysteine, respectively. Cells were cultured in production bioreactors under fed-batch conditions with predetermined pH, dissolved oxygen, temperature, and nutrient feeding strategy. Typically, 2 L bioreactors with an initial culture volume of 1.2 L were used unless otherwise stated. Ambr-250 bioreactors with an initial culture volume of 200 ml were used.

The temperature in the bioreactors was maintained at 36.5°C and the stirrer speed was set at approximately 223 rpm. A gas mixture containing air, _CO2 , and _O2 was supplied. The concentration of dissolved carbon dioxide ( _dCO2 ) was measured offline once per day. The concentration of dissolved oxygen (DO) was monitored in real time and adjusted to 35% by varying the partial pressure of oxygen in the gas mixture. The pH was maintained at a target value of pH 7.00 with a dead zone of ±0.03 pH units by adding _CO2 or 1.0 M _NaHCO3 unless otherwise stated.

The nutrient medium included a combination of internal medium with glucose, glutamine, amino acids and salts. This nutrient medium was prepared in solution and added to the culture on the 3rd, 6th and 9th day, each in an amount of approximately 10 vol. % of the working culture volume.

An additional glucose nutrient solution was prepared and added to the culture for 4–14 days to maintain glucose concentrations at approximately ≥ 3 g/L.

Cell culture samples for production were withdrawn daily with a syringe for offline analysis. The production time was typically approximately 14 days. Cell concentration and viability were measured by trypan blue exclusion on a CEDEX instrument (Roche Diagnostics GmbH, Germany). Offline measurements were performed using a COBAS INTEGRA® 400 plus (Roche Diagnostics GmbH, Germany) to measure glucose, glutamine, glutamate, lactate, ammonium and product concentrations. Dissolved carbon dioxide was analyzed on a Cobas b221 analyzer (Roche Diagnostics Ltd. CH-6343 Rotkreuz, Switzerland). Osmolality was measured by freezing point depression on an Osmomat autoosmometer (Gonotec GmbH, Berlin, Germany).

At the end of the main culture production process, the cell culture fluid was collected by centrifugation. The supernatants were then subjected to small-scale protein-A purification of mAb. The glycosylation pattern of the purified mAb was analyzed using 2AB.

Figures 7A–7D show the effect of different concentrations of L-cysteine on the G0 form of bsAB to CD20/CD3, the G1 form of bsAB to CD20/CD3, cell growth (IVCD), and product titer.

Figure 7A shows that at the end of the 14-day production process, the amount of the G0 form of bsAB to CD20/CD3 in the process with 5 mM L-cysteine in the production medium was approximately 5.5% lower than in the process with 10 mM L-cysteine in the production medium.

Figure 7B shows that at the end of the 14-day production process, the amount of the G1 form of bsAB to CD20/CD3 in the process with 5 mM L-cysteine in the production medium was approximately 3.4% higher than in the process with 10 mM L-cysteine in the production medium.

Figure 7B shows that at the end of the 14-day production process, the product titer in the process with 5 mM L-cysteine in the production medium was approximately 66% higher than in the process with 10 mM L-cysteine in the production medium.

Figure 7G shows that at the end of the 14-day production process, the cell growth in the process with 5 mM L-cysteine in the production medium was higher than in the process with 10 mM L-cysteine in the production medium.

Example 5. Modulation of the enzyme and pathway by L-cysteine in the CHO T104 cell line producing a recombinant bispecific antibody to CD20/CD3 to improve the production process

In this example, CHO K1M, a cell line derived from Chinese hamster ovary (CHO) cells, was used as the host cell line (WO 2009047007). CHO K1M cells were engineered to express a T cell-targeted bispecific CD20/CD3 monoclonal antibody, bsAB to CD20/CD3 (described in EP 3252078 A1), and are designated herein as the T104 cell line.

BsAB to CD20/K-CD3 is a T-cell bispecific (TCB) antibody that targets CD20 expressed on B cells and the CD3 epsilon chain (CD3I I) present on T cells. The mechanism of action of bsAB to CD20/CD3 involves simultaneous binding to CD20+ B cells and CD3+ T cells, resulting in T-cell activation and T-cell-mediated B-cell killing. In the presence of CD20+ B cells, circulating or in tissues, pharmacologically active doses induce T-cell activation and associated cytokine release. BsAB to CD20/CD3 may be used to treat disease, particularly B-cell proliferative disorder, and to reduce adverse effects in response to a T-cell activating therapeutic agent.

For the cell culture process, a custom-made, chemically defined serum-free version of the medium was used as the basal culture medium for T104 cells. After thawing, cells were passaged in this medium in the presence of 250 nM methotrexate (MTX; Pfizer, Cat.# 13999031) in shake flasks for 3-4 days. Passage conditions were 36.5°C, 7% _CO2 , and 160 rpm for 125 ml and 500 ml flasks using a Kuhner Shaker X platform (Adolf Kuhner AG, Birsfelden, Basel, Switzerland).

For the inoculation process and the production process, another custom-made version of the chemically defined serum-free medium was used as the basal medium for the expansion of T104 cells. During the preparation of this production medium, glucose, glutamine, amino acids, trace elements (diluted RTE1.2 solution, Gibco, UK; Cat. No. 043-90585H) and salts were also additionally added. In this case, the final concentration of L-cysteine (Merck Chemicals GmbH, Cat. No.: 1.02735.1000) in the medium was adjusted to 5 mM and 10 mM, respectively.

Precultured cells were used for inoculation in step N-2 at approximately 3.0× ¹⁰⁵ cells/mL and in step N-1 at approximately 5.0× ¹⁰⁵ cells/mL in parallel with the prepared production media containing 5 mM or 10 mM L-cysteine, respectively, all without MTX. Production bioreactors were inoculated at approximately 10.0× ¹⁰⁵ cells/mL in medium containing 5 mM or 10 mM L-cysteine, respectively. Cells were cultured in production bioreactors under fed-batch conditions with predetermined pH, dissolved oxygen, temperature, and nutrient feeding strategy. 2-L bioreactors with an initial culture volume of 1.2 L were used unless otherwise stated. Unless otherwise stated, Ambr-250 bioreactors with an initial culture volume of 200 ml were used.

The temperature in the bioreactors was maintained at 36.5°C and the stirrer speed was set at approximately 223 rpm. A gas mixture containing air, _CO2 , and _O2 was supplied. The concentration of dissolved carbon dioxide ( _dCO2 ) was measured offline once per day. The concentration of dissolved oxygen (DO) was monitored in real time and adjusted to 35% by varying the partial pressure of oxygen in the gas mixture. The pH was maintained at a set point of pH 7.00 with a dead zone of ±0.03 pH units by adding _CO2 or 1.0 M _NaHCO3 unless otherwise stated.

The growth medium consisted of a combination of RF1.0 powder (SAFC, Cat. No. CR60112), glucose, glutamine, amino acids and salts. This growth medium was prepared in solution and added to the culture on days 3, 6 and 9, each at approximately 10% by volume of the working culture.

An additional glucose nutrient solution was prepared and added to the culture for 4–14 days to maintain glucose concentrations at approximately ≥ 3 g/L.

Cell culture samples for production were withdrawn daily with a syringe for offline analysis. The production time was typically approximately 14 days. Cell concentration and viability were measured by trypan blue exclusion on a CEDEX instrument (Roche Diagnostics GmbH, Germany). Offline measurements were performed using a COBAS INTEGRA® 400 plus (Roche Diagnostics GmbH, Germany) to measure glucose, glutamine, glutamate, lactate, ammonium and product concentrations. Dissolved carbon dioxide was analyzed on a Cobas b221 analyzer (Roche Diagnostics Ltd. CH-6343 Rotkreuz, Switzerland). Osmolality was measured by freezing point depression on an Osmomat autoosmometer (Gonotec GmbH, Berlin, Germany).

At the end of the main culture production process, the cell culture fluid was collected by centrifugation. The supernatants were then subjected to small-scale protein-A purification of mAb. The glycosylation pattern of the purified mAb was analyzed using 2AB.

Figures 8A–8D show the effect of different concentrations of L-cysteine on the G0 form of bsAB to CD20/CD3, the G1 form of bsAB to CD20/CD3, cell growth (IVCD), and product titer.

Figure 8A shows that at the end of the 14-day production process, the amount of the G0 form of bsAB to CD20/CD3 in the process with 5 mM L-cysteine in the production medium was approximately 3.8% lower than in the process with 10 mM L-cysteine in the production medium.

Figure 8B shows that at the end of the 14-day production process, the amount of the G1 form of bsAB to CD20/CD3 in the process with 5 mM L-cysteine in the production medium was approximately 2.5% higher than in the process with 10 mM L-cysteine in the production medium.

Figure 8B shows that at the end of the 14-day production process, the product titer in the process with 5 mM L-cysteine in the production medium was approximately 67% higher than in the process with 10 mM L-cysteine in the production medium.

Figure 8G shows that at the end of the 14-day production process, the cell growth in the process with 5 mM L-cysteine in the production medium was higher than in the process with 10 mM L-cysteine in the production medium.

Example 6. Modulation of the pathway by L-cystine in the CHO L967 cell line to produce a recombinant antibody to human α-synuclein

In this example, CHO K1M, a cell line derived from Chinese hamster ovary (CHO) cells, was used as a host cell line (WO2009047007). CHO K1M cells were engineered to express a monoclonal antibody to human α-synuclein (described in U.S. Patent 9,670,274 B2 and U.S. Patent 9,890,209 B9) that binds to monomeric or oligomeric human α-synuclein, and are designated herein as the L967 cell line.

Human α-synuclein can form fibrillar aggregates, and these aggregates are the main component of Lewy bodies and Lewy neurites. Recent work suggests that prefibrillar oligomers of alpha-synuclein may play a key role in the progression of Parkinson's disease (Luk et al., 2012). A specific antibody, L967, can specifically bind to extracellular α-synuclein and may be used to prevent cell-to-cell transfer of aggregates and the progression of Parkinson's disease.

For the cell culture process, a custom-made, chemically defined serum-free version of the medium was used as the basal culture medium for L967 cells. After thawing, cells were passaged in this medium in the presence of 5 μg/ml puromycin (Puromycin-Solution, InvivoGen SAS, France, Cat. No. ANT-PR) in shake flasks on a 3-4 day schedule. Passage conditions were 36.5°C, 7% _CO2 and 160 rpm for 125 ml and 500 ml flasks using a Kuhner Shaker X platform (Adolf Kuhner AG, Birsfelden, Basel, Switzerland).

For the inoculation process and the production process, another custom-made version of the chemically defined serum-free medium was used as the basal medium for the expansion of L967 cells. During the preparation of this production medium, glucose, glutamine, amino acids, trace elements (RTE1.2 diluted solution, Gibco, UK; Cat. No. 043-90585H) and salts were also additionally added. Before use, the final concentration of L-cystine (L-cystine disodium monohydrate; SAFC, Supplier No. RES1523C-A154X) in the medium was adjusted to 2 mM and 4 mM, respectively.

Precultured cells were used for inoculation in step N-2 at approximately 3.0× ¹⁰⁵ cells/mL and in step N-1 at approximately 5.0× ¹⁰⁵ cells/mL in parallel with the prepared production media containing 2 mM or 4 mM L-cystine, respectively. Production bioreactors were inoculated at approximately 10.0× ¹⁰⁵ cells/mL in medium containing 2 mM or 4 mM L-cystine, respectively. Cells were cultured in production bioreactors under fed-batch conditions with predetermined pH, dissolved oxygen, temperature, and nutrient feeding strategy. Typically, 2 L bioreactors with an initial culture volume of 1.2 L were used unless otherwise noted. Ambr-250 bioreactors with an initial culture volume of 200 ml were used.

The temperature in the bioreactors was maintained at 36.5°C and the stirrer speed was set at approximately 223 rpm. A gas mixture containing air, _CO2 , and _O2 was supplied. The concentration of dissolved carbon dioxide ( _dCO2 ) was measured offline once per day. The concentration of dissolved oxygen (DO) was monitored in real time and adjusted to 35% by varying the partial pressure of oxygen in the gas mixture. The pH was maintained at a target value of pH 7.00 with a dead zone of ±0.03 pH units by adding _CO2 or 1.0 M _NaHCO3 unless otherwise stated.

The growth medium included a combination of RF1.0 powder (SAFC, Cat. No. CR60112), glucose, glutamine, amino acids and salts. This growth medium was prepared in solution and added to the culture on days 3, 6 and 9, each at approximately 10% by volume of the working culture.

An additional glucose nutrient solution was prepared and added to the culture for 4–14 days to maintain glucose concentrations at approximately ≥ 4 g/L.

Cell culture samples for production were withdrawn daily with a syringe for offline analysis. The production time was typically approximately 14 days. Cell concentration and viability were measured by trypan blue exclusion on a CEDEX instrument (Roche Diagnostics GmbH, Germany). Offline measurements were performed using a COBAS INTEGRA® 400 plus (Roche Diagnostics GmbH, Germany) to measure glucose, glutamine, glutamate, lactate, ammonium and product concentrations. Dissolved carbon dioxide was analyzed on a Cobas b221 analyzer (Roche Diagnostics Ltd. CH-6343 Rotkreuz, Switzerland). Osmolality was measured by freezing point depression on an Osmomat autoosmometer (Gonotec GmbH, Berlin, Germany).

At the end of the main culture production process, the cell culture fluid was collected by centrifugation. The supernatants were then subjected to small-scale protein-A purification of mAb. The glycosylation pattern of the purified mAb was analyzed using 2AB.

Figures 9A-9D show the effect of different concentrations of L-cystine on the G0 form of anti-α-synuclein antibody L967, the G1 form of anti-α-synuclein antibody L967, the G2 form of anti-α-synuclein antibody L967, product titer, and cell growth (IVCD).

Figure 9A shows that at the end of the 14-day production process, the amount of the G0 form of the α-synuclein antibody L967 in the process with 2 mM L-cystine in the production medium was approximately 6.6% higher than in the process with 4 mM L-cystine in the production medium.

Figure 9B shows that at the end of the 14-day production process, the amount of the G1 form of the α-synuclein antibody L967 in the process with 2 mM L-cystine in the production medium was approximately 5.9% lower than in the process with 4 mM L-cystine in the production medium.

Figure 9B shows that at the end of the 14-day production process, the amount of the G2 form of the α-synuclein antibody L967 in the process with 2 mM L-cystine in the production medium was approximately 1.6% lower than in the process with 4 mM L-cystine in the production medium.

Figure 9G shows that at the end of the 14-day production process, the product titer in the process with 2 mM L-cystine in the production medium was approximately 9% lower than in the process with 4 mM L-cystine in the production medium.

Figure 9D shows that at the end of the 14-day production process, cell growth in the process with 2 mM L-cystine in the production medium was approximately 3.7% lower than in the process with 4 mM L-cystine in the production medium.

Example 7. Modulation of the pathway by L-cystine in the CHO L971 cell line to produce a recombinant antibody to human α-synuclein

In this example, CHO K1M, a cell line derived from Chinese hamster ovary (CHO) cells, was used as a host cell line (WO2009047007). CHO K1M cells were engineered to express a monoclonal antibody to human α-synuclein (described in U.S. Patent 9,670,274 B2 and U.S. Patent 9,890,209 B9) that binds to monomeric or oligomeric human α-synuclein, and are designated herein as the CHO L971 cell line.

Human α-synuclein can form fibrillar aggregates, and these aggregates are the main component of Lewy bodies and Lewy neurites. Recent work suggests that prefibrillar oligomers of alpha-synuclein may play a key role in the progression of Parkinson's disease (Luk et al., 2012). A specific antibody, L971, can specifically bind to extracellular α-synuclein and may be used to prevent cell-to-cell transfer of aggregates and the progression of Parkinson's disease.

For the cell culture process, a custom-made serum-free version of the chemical medium was used as the basal culture medium for L971 cells. After thawing, the cells were passaged in this medium in the presence of 5 μg/ml puromycin (Puromycin-Solution, InvivoGen SAS, France, Cat. No. ANT-PR) in shake flasks on a 3-4 day schedule. Passage conditions were 36.5°C, 7% _CO2 and 160 rpm for 125 ml and 500 ml flasks using a Kuhner Shaker X platform (Adolf Kuhner AG, Birsfelden, Basel, Switzerland).

For the inoculation process and the production process, a different custom-made version of the chemically defined serum-free medium was used as the basal medium for the expansion of L971 cells. During the preparation of this production medium, glucose, glutamine, amino acids, trace elements (RTE1.2 diluted solution, Gibco, UK; Cat. No. 043-90585H) and salts were also additionally added. Before use, the final concentration of L-cystine (L-cystine disodium monohydrate; SAFC, Supplier No. RES1523C-A154X) in the medium was adjusted to 2 mM and 4 mM, respectively.

Precultured cells were used for inoculation in step N-2 at approximately 3.0× ¹⁰⁵ cells/mL and in step N-1 at approximately 5.0× ¹⁰⁵ cells/mL in parallel with the prepared production media containing 2 mM or 4 mM L-cystine, respectively. Production bioreactors were inoculated at approximately 10.0× ¹⁰⁵ cells/mL in medium containing 2 mM or 4 mM L-cystine, respectively. Cells were cultured in production bioreactors under fed-batch conditions with predetermined pH, dissolved oxygen, temperature, and nutrient feeding strategy. Typically, 2 L bioreactors with an initial culture volume of 1.2 L were used unless otherwise noted. Ambr-250 bioreactors with an initial culture volume of 200 ml were used.

The temperature in the bioreactors was maintained at 36.5°C and the stirrer speed was set at approximately 223 rpm. A gas mixture containing air, _CO2 , and _O2 was supplied. The concentration of dissolved carbon dioxide ( _dCO2 ) was measured offline once per day. The concentration of dissolved oxygen (DO) was monitored in real time and adjusted to 35% by varying the partial pressure of oxygen in the gas mixture. The pH was maintained at a target value of pH 7.00 with a dead zone of ±0.03 pH units by adding _CO2 or 1.0 M _NaHCO3 unless otherwise stated.

The growth medium consisted of a combination of RF1.0 powder (SAFC, Cat. No. CR60112), glucose, glutamine, amino acids and salts. This growth medium was prepared in solution and added to the culture on days 3, 6 and 9, each at approximately 10% by volume of the working culture.

An additional glucose nutrient solution was prepared and added to the culture for 4–14 days to maintain glucose concentrations at approximately ≥ 4 g/L.

Cell culture samples for production were withdrawn daily with a syringe for offline analysis. The production time was typically approximately 14 days. Cell concentration and viability were measured by trypan blue exclusion on a CEDEX instrument (Roche Diagnostics GmbH, Germany). Offline measurements were performed using a COBAS INTEGRA® 400 plus (Roche Diagnostics GmbH, Germany) to measure glucose, glutamine, glutamate, lactate, ammonium and product concentrations. Dissolved carbon dioxide was analyzed on a Cobas b221 analyzer (Roche Diagnostics Ltd. CH-6343 Rotkreuz, Switzerland). Osmolality was measured by freezing point depression on an Osmomat autoosmometer (Gonotec GmbH, Berlin, Germany).

At the end of the main culture production process, the cell culture fluid was collected by centrifugation. The supernatants were then subjected to small-scale protein-A purification of mAb. The glycosylation pattern of the purified mAb was analyzed using 2AB.

Figures 10A to 10D show the effect of different concentrations of L-cystine on the G0 form of anti-α-synuclein antibody L971, the G1 form of anti-α-synuclein antibody L971, the G2 form of anti-α-synuclein antibody L971, product titer, and cell growth (IVCD).

Figure 10A shows that at the end of the 14-day production process, the amount of the G0 form of the α-synuclein antibody L971 in the process with 2 mM L-cystine in the production medium was approximately 3% higher than in the process with 4 mM L-cystine in the production medium.

Figure 10B shows that at the end of the 14-day production process, the amount of the G1 form of the α-synuclein antibody L971 in the process with 2 mM L-cystine in the production medium was approximately 2% lower than in the process with 4 mM L-cystine in the production medium.

Figure 10B shows that at the end of the 14-day production process, the amount of the G2 form of the α-synuclein antibody L971 in the process with 2 mM L-cystine in the production medium was comparable to that in the process with 4 mM L-cystine in the production medium.

Figure 10G shows that at the end of the 14-day production process, the product titer in the process with 2 mM L-cystine in the production medium was approximately 30% lower than in the process with 4 mM L-cystine in the production medium.

Figure 10D shows that at the end of the 14-day production process, cell growth in the process with 2 mM L-cysteine in the production medium was slightly comparable to that in the process with 4 mM L-cystine in the production medium.

Example 8. Functional comparison of recombinant human α-synuclein antibody L971 produced by L-cysteine or L-cystine processes

In this example, CHO K1M, a cell line derived from Chinese hamster ovary (CHO) cells, was used as a host cell line (WO2009047007). CHO K1M cells were engineered to express a monoclonal antibody to human α-synuclein (described in U.S. Patent 9,670,274 B2 and U.S. Patent 9,890,209 B9) that binds to monomeric or oligomeric human α-synuclein, and are designated herein as the CHO L971 cell line.

Human α-synuclein can form fibrillar aggregates, and these aggregates are the main component of Lewy bodies and Lewy neurites. Recent work suggests that prefibrillar oligomers of alpha-synuclein may play a key role in the progression of Parkinson's disease (Luk et al., 2012). A specific antibody, L971, can specifically bind to extracellular α-synuclein and may be used to prevent cell-to-cell transfer of aggregates and the progression of Parkinson's disease.

For the cell culture process, a custom-made serum-free version of the chemical medium was used as the basal culture medium for L965 cells. After thawing, the cells were passaged in this medium in the presence of 5 μg/ml puromycin (Puromycin-Solution, InvivoGen SAS, France, Cat. No. ANT-PR) in shake flasks on a 3-4 day schedule. Passage conditions were 36.5°C, 7% _CO2 and 160 rpm for 125 ml and 500 ml flasks using a Kuhner Shaker X platform (Adolf Kuhner AG, Birsfelden, Basel, Switzerland).

For the inoculation process and the production process, another custom-made version of chemically defined serum-free medium was used as the basic medium for L971 cell expansion.

During preparation of this production medium, glucose, glutamine, amino acids, trace elements (diluted RTE1.2 solution, Gibco, UK; Cat. No. 043-90585H) and salts were also added. Before use, the final concentration of L-cystine (L-cystine disodium monohydrate; SAFC, Cat. No. RES1523C-A154X) in the medium was adjusted to 3 mM in one part of the medium. For direct comparison, the final concentration of L-cysteine (Merck Chemicals GmbH, Cat. No.: 1.02735.1000) in the medium was adjusted to 6 mM in another part of the medium.

The precultured cells were used for inoculation in step N-2 at approximately 3.0× ¹⁰⁵ cells/mL and in step N-1 at approximately 5.0× ¹⁰⁵ cells/mL in parallel with the prepared production media containing a predetermined amount of L-cystine or L-cysteine, respectively. The production bioreactors were inoculated at approximately 10.0× ¹⁰⁵ cells/mL in the medium containing a predetermined amount of L-cystine (3 mM) or L-cysteine (6 mM), respectively. The cells were cultured in the production bioreactors under fed-batch conditions with a predetermined pH, dissolved oxygen, temperature, and nutrient feeding strategy. Typically, 2 L bioreactors with an initial culture volume of 1.2 L were used unless otherwise noted. Ambr-250 bioreactors with an initial culture volume of 200 ml were used.

The temperature in the bioreactors was maintained at 36.5°C and the stirrer speed was set at approximately 223 rpm. A gas mixture containing air, _CO2 and _O2 was supplied. The concentration of dissolved carbon dioxide ( _cdO2 ) was measured offline once per day. The concentration of dissolved oxygen (DO) was monitored in real time and adjusted to 35% by varying the partial pressure of oxygen in the gas mixture. The pH was maintained at a target value of pH 7.00 with a dead zone of ±0.03 pH units by adding _CO2 or 1.0 M _NaHCO3 unless otherwise stated.

The nutrient medium included a combination of internal medium with glucose, glutamine, amino acids and salts. This nutrient medium was prepared in solution and added to the culture on the 3rd, 6th and 9th day, each in an amount of approximately 10 vol. % of the working culture volume.

An additional glucose nutrient solution was prepared and added to the culture for 4–14 days to maintain glucose concentrations at approximately ≥ 4 g/L.

Cell culture samples for production were withdrawn daily with a syringe for offline analysis. The production time was typically approximately 14 days. Cell concentration and viability were measured by trypan blue exclusion on a CEDEX instrument (Roche Diagnostics GmbH, Germany). Offline measurements were performed using a COBAS INTEGRA® 400 plus (Roche Diagnostics GmbH, Germany) to measure glucose, glutamine, glutamate, lactate, ammonium and product concentrations. Dissolved carbon dioxide was analyzed on a Cobas b221 analyzer (Roche Diagnostics Ltd. CH-6343 Rotkreuz, Switzerland). Osmolality was measured by freezing point depression on an Osmomat autoosmometer (Gonotec GmbH, Berlin, Germany).

At the end of the main culture production process, the cell culture fluid was collected by centrifugation. The supernatants were then subjected to small-scale protein-A purification of mAb. The glycosylation pattern of the purified mAb was analyzed using 2AB.

The neonatal Fe receptor (FcRn) influences the pharmacokinetic (PK) profile of IgG antibodies through its ability to extract antibodies from the early endosome and recycle them back into the circulation. Interaction with FcRn is considered the most important factor in determining the pharmacokinetics of therapeutic IgG antibodies and is considered a surrogate for clearance (reviewed in Nimmerjahn and Ravetch 2008).

Fcγ receptors are mediators of effector functions on immune effector cells. Among the various human Fcγ receptors, Fcγ-RIIa is considered the dominant factor in mediating antibody-dependent phagocytosis (ADCP). Of the two most common Fcγ-RIIa allotypes, the form with a histidine at amino acid position 131 (H131) is often described as the higher affinity allotype. (Nimmerjahn and Ravetch 2008, Yamada et al. 2013)

In this paper, the relative binding of small-scale purified aSyn-L971-mAb to FcRn and Fcγlla (His 131) was determined using surface plasmon resonance (SPR). The relative binding of small-scale purified aSyn-L971-mAb to target was determined using ELISA.

Figures 11A-HE show a direct comparison of the aSyn-L971 antibody produced in cell culture with 3 mM L-cystine or 6 mM L-cysteine in cell culture medium, respectively.

Figure 11A shows that at the end of the 14-day production process, the amount of the G1 form of the α-synuclein antibody L971 in the process with 6 mM L-cysteine in the production medium was only slightly higher than in the process with 3 mM L-cysteine in the production medium.

Figure 11B shows that at the end of the 14-day production process, the amount of the G2 form of the α-synuclein antibody L971 in the process with 6 mM L-cysteine in the production medium was comparable to that in the process with 3 mM L-cystine in the production medium.

Figure 11B shows that at the end of the 14-day production process, anti-α-synuclein antibody L971 exhibited approximately 33% higher relative FcRn binding in the process with 6 mM L-cysteine in the production medium (approximately 118% relative level) compared to anti-α-synuclein antibody L971 in the process with 3 mM L-cystine in the production medium (approximately 85% relative level).

Figure 11D shows that at the end of the 14-day production process, anti-α-synuclein antibody L971 exhibited approximately 36% higher relative level of Fcγ-RIIa (H131) binding in the process with 6 mM L-cysteine in the production medium (approximately 113% relative level) compared to anti-α-synuclein antibody L971 in the process with 3 mM L-cystine in the production medium (approximately 77% relative level).

Figure 11D shows that at the end of the 14-day production process, anti-α-synuclein antibody L971 had approximately 35% higher relative target binding in the process with 6 mM L-cysteine in the production medium (approximately 118% relative level) compared to anti-α-synuclein antibody L971 in the process with 3 mM L-cystine in the production medium (approximately 83% relative level).

--->

SEQUENCE LIST

<110> F. Hoffmann-La Roche AG

Hoffmann-La Roche Inc.

<120> MODULATION OF ENZYME AND PATHWAY BY SULFHYDRYL COMPOUNDS AND THEIR

DERIVATIVES

<130> AC1883 PCT S3

<150> EP20 17 1356.7

<151> 2020-04-24

<160> 47

<170> BiSSAP 1.3.6

<210> 1

<211> 1047

<212> DNA

<213> Artificial sequence

<220>

<223> cDNA sequence of UDP-glucose-4-epimerase in CHO K1M

<220>

<221> CDS

<222> 1..1047

<223> /transl_table=1

<400> 1

atg gcc gag aag gtg ctg gtc aca ggc gga gct ggc tac att ggc agc 48

Met Ala Glu Lys Val Leu Val Thr Gly Gly Ala Gly Tyr Ile Gly Ser

1 5 10 15

cac acg gta ctg gag ctg ctg gag gca ggc tac gcc cct gtg gtc atc 96

His Thr Val Leu Glu Leu Leu Glu Ala Gly Tyr Ala Pro Val Val Ile

20 25 30

gac aac ttc cat aat gcc att cgt gga ggg gat tcc atg cct gag agc 144

Asp Asn Phe His Asn Ala Ile Arg Gly Gly Asp Ser Met Pro Glu Ser

35 40 45

ctg cgg cgg gtc cag gaa ctg aca ggc cgc tct gtg gag ttt gag gag 192

Leu Arg Arg Val Gln Glu Leu Thr Gly Arg Ser Val Glu Phe Glu Glu

50 55 60

atg gac atc ttg gac cag gca gcg cta cag cac ctc ttt aag aag cac 240

Met Asp Ile Leu Asp Gln Ala Ala Leu Gln His Leu Phe Lys Lys His

65 70 75 80

agc ttt aag gct gtc atc cac ttt gct ggg ctc aag gct gtg ggc gag 288

Ser Phe Lys Ala Val Ile His Phe Ala Gly Leu Lys Ala Val Gly Glu

85 90 95

tcg gtg cag aag cct ctg gat tat tat aga gtt aac cta aca ggg acc 336

Ser Val Gln Lys Pro Leu Asp Tyr Tyr Arg Val Asn Leu Thr Gly Thr

100 105 110

atc cag ctt ctg gag atc atg agg gcc cac ggg gtg aag aat ctc gtg 384

Ile Gln Leu Leu Glu Ile Met Arg Ala His Gly Val Lys Asn Leu Val

115 120 125

ttc agc agc tca gcc acc gtg tat ggg aat ccc cag tac ctg cct ctg 432

Phe Ser Ser Ser Ala Thr Val Tyr Gly Asn Pro Gln Tyr Leu Pro Leu

130 135 140

gat gag gcc cac ccc acc ggg ggt tgt acc aac ccc tat gga aag tcc 480

Asp Glu Ala His Pro Thr Gly Gly Cys Thr Asn Pro Tyr Gly Lys Ser

145 150 155 160

aag ttc ttc atc gag gag atg gtc cgg gac ctg tgc cgg gca gat tcg 528

Lys Phe Phe Ile Glu Glu Met Val Arg Asp Leu Cys Arg Ala Asp Ser

165 170 175

gcc tgg aac gca gtg ctg cta cgc tac ttc aat ccc acg ggt gcc cac 576

Ala Trp Asn Ala Val Leu Leu Arg Tyr Phe Asn Pro Thr Gly Ala His

180 185 190

gcc tct ggc cgc atc ggc gag gat ccc cag ggc gtc ccc aac aac ctc 624

Ala Ser Gly Arg Ile Gly Glu Asp Pro Gln Gly Val Pro Asn Asn Leu

195 200 205

atg ccc tat gtc tcc cag gtg gca att ggg cga cga gag gcc ctg aat 672

Met Pro Tyr Val Ser Gln Val Ala Ile Gly Arg Arg Glu Ala Leu Asn

210 215 220

gtc ttt ggt ggt gac tat gat aca gag gat ggc aca ggt gta agg gat 720

Val Phe Gly Gly Asp Tyr Asp Thr Glu Asp Gly Thr Gly Val Arg Asp

225 230 235 240

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Tyr Ile His Val Val Asp Leu Ala Lys Gly His Ile Ala Ala Leu Lys

245 250 255

aag ctg aag gag caa tgt ggt tgc cgg atc tac aac ctg ggc aca ggc 816

Lys Leu Lys Glu Gln Cys Gly Cys Arg Ile Tyr Asn Leu Gly Thr Gly

260 265 270

aca ggc tac tct gtc ctg cag atg gtc caa gca atg gag aag gct tca 864

Thr Gly Tyr Ser Val Leu Gln Met Val Gln Ala Met Glu Lys Ala Ser

275 280 285

ggg aag aag atc cca tac aag gtg gtg gca cgg cgg gaa ggt gac gtg 912

Gly Lys Lys Ile Pro Tyr Lys Val Val Ala Arg Arg Glu Gly Asp Val

290 295 300

gca gcc tgt tat gcc aac ccc agc ctg gcc cat gag gag ctg ggc tgg 960

Ala Ala Cys Tyr Ala Asn Pro Ser Leu Ala His Glu Glu Leu Gly Trp

305 310 315 320

aca gca gcc ttg ggg ctg gac agg atg tgt gaa gat cta tgg cgc tgg 1008

Thr Ala Ala Leu Gly Leu Asp Arg Met Cys Glu Asp Leu Trp Arg Trp

325 330 335

cag aag cag aac cct tca ggc ttt gga gca cag gcc taa 1047

Gln Lys Gln Asn Pro Ser Gly Phe Gly Ala Gln Ala

340 345

<210> 2

<211> 348

<212> Protein

<213> Artificial sequence

<220>

<223> [CDS]:1..1047 from SEQ ID NO 1

<400> 2

Met Ala Glu Lys Val Leu Val Thr Gly Gly Ala Gly Tyr Ile Gly Ser

1 5 10 15

His Thr Val Leu Glu Leu Leu Glu Ala Gly Tyr Ala Pro Val Val Ile

20 25 30

Asp Asn Phe His Asn Ala Ile Arg Gly Gly Asp Ser Met Pro Glu Ser

35 40 45

Leu Arg Arg Val Gln Glu Leu Thr Gly Arg Ser Val Glu Phe Glu Glu

50 55 60

Met Asp Ile Leu Asp Gln Ala Ala Leu Gln His Leu Phe Lys Lys His

65 70 75 80

Ser Phe Lys Ala Val Ile His Phe Ala Gly Leu Lys Ala Val Gly Glu

85 90 95

Ser Val Gln Lys Pro Leu Asp Tyr Tyr Arg Val Asn Leu Thr Gly Thr

100 105 110

Ile Gln Leu Leu Glu Ile Met Arg Ala His Gly Val Lys Asn Leu Val

115 120 125

Phe Ser Ser Ser Ala Thr Val Tyr Gly Asn Pro Gln Tyr Leu Pro Leu

130 135 140

Asp Glu Ala His Pro Thr Gly Gly Cys Thr Asn Pro Tyr Gly Lys Ser

145 150 155 160

Lys Phe Phe Ile Glu Glu Met Val Arg Asp Leu Cys Arg Ala Asp Ser

165 170 175

Ala Trp Asn Ala Val Leu Leu Arg Tyr Phe Asn Pro Thr Gly Ala His

180 185 190

Ala Ser Gly Arg Ile Gly Glu Asp Pro Gln Gly Val Pro Asn Asn Leu

195 200 205

Met Pro Tyr Val Ser Gln Val Ala Ile Gly Arg Arg Glu Ala Leu Asn

210 215 220

Val Phe Gly Gly Asp Tyr Asp Thr Glu Asp Gly Thr Gly Val Arg Asp

225 230 235 240

Tyr Ile His Val Val Asp Leu Ala Lys Gly His Ile Ala Ala Leu Lys

245 250 255

Lys Leu Lys Glu Gln Cys Gly Cys Arg Ile Tyr Asn Leu Gly Thr Gly

260 265 270

Thr Gly Tyr Ser Val Leu Gln Met Val Gln Ala Met Glu Lys Ala Ser

275 280 285

Gly Lys Lys Ile Pro Tyr Lys Val Val Ala Arg Arg Glu Gly Asp Val

290 295 300

Ala Ala Cys Tyr Ala Asn Pro Ser Leu Ala His Glu Glu Leu Gly Trp

305 310 315 320

Thr Ala Ala Leu Gly Leu Asp Arg Met Cys Glu Asp Leu Trp Arg Trp

325 330 335

Gln Lys Gln Asn Pro Ser Gly Phe Gly Ala Gln Ala

340 345

<210> 3

<211> 1140

<212> DNA

<213> Artificial sequence

<220>

<223> UDP-α-D-glucose: α-D-galactose-1-phosphate cDNA sequence

uridylyltransferase in CHO K1M

<220>

<221> CDS

<222> 1..1140

<223> /transl_table=1

<400> 3

atg tcg caa aac gga gat gat cct gag cag cgc cag cag gcg tca gag 48

Met Ser Gln Asn Gly Asp Asp Pro Glu Gln Arg Gln Gln Ala Ser Glu

1 5 10 15

gcg gac gcc atg gca gcg acc ttc cgg gcg agc gaa cac cag cat att 96

Ala Asp Ala Met Ala Ala Thr Phe Arg Ala Ser Glu His Gln His Ile

20 25 30

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Arg Tyr Asn Pro Leu Gln Asp Glu Trp Val Leu Val Ser Ala His Arg

35 40 45

atg aag cgg ccc tgg caa gga caa gtg gag ccc cag ctt ctg aag aca 192

Met Lys Arg Pro Trp Gln Gly Gln Val Glu Pro Gln Leu Leu Lys Thr

50 55 60

gtg ccc cgc tat gac cca ctc aac cct ctg tgt ccc ggg gcc aca cga 240

Val Pro Arg Tyr Asp Pro Leu Asn Pro Leu Cys Pro Gly Ala Thr Arg

65 70 75 80

gcc aat ggg gag gtg aat cca cac tat gac agc acc ttc ctg ttt gac 288

Ala Asn Gly Glu Val Asn Pro His Tyr Asp Ser Thr Phe Leu Phe Asp

85 90 95

aat gac ttc cca gct ctg cag ccc gat gct ccg gat cca gga ccc agt 336

Asn Asp Phe Pro Ala Leu Gln Pro Asp Ala Pro Asp Pro Gly Pro Ser

100 105 110

gac cat cct ctt ttt cga gca gag gcg gcc aga gga gtt tgt aag gtc 384

Asp His Pro Leu Phe Arg Ala Glu Ala Ala Arg Gly Val Cys Lys Val

115 120 125

atg tgc ttc cat ccc tgg tcg gat gtg aca ctg cca ctc atg tca gtc 432

Met Cys Phe His Pro Trp Ser Asp Val Thr Leu Pro Leu Met Ser Val

130 135 140

cct gag atc cga gct gtc atc gat gcc tgg gct tca gtc acc gag gat 480

Pro Glu Ile Arg Ala Val Ile Asp Ala Trp Ala Ser Val Thr Glu Asp

145 150 155 160

ctg ggt gcc cag tat cct tgg gtg cag atc ttt gaa aac aaa gga gcc 528

Leu Gly Ala Gln Tyr Pro Trp Val Gln Ile Phe Glu Asn Lys Gly Ala

165 170 175

atg atg ggc tgt tct aac ccc cac ccc cac tgc cag gtt tgg gcc agc 576

Met Met Gly Cys Ser Asn Pro His Pro His Cys Gln Val Trp Ala Ser

180 185 190

agt ttc ctg cca gat att gcc cag cgt gaa gtg cga tcc cag cag aac 624

Ser Phe Leu Pro Asp Ile Ala Gln Arg Glu Val Arg Ser Gln Gln Asn

195 200 205

tat cac agc cag cat gga gaa cct ctg tta ctg gaa tat ggc cgc caa 672

Tyr His Ser Gln His Gly Glu Pro Leu Leu Leu Glu Tyr Gly Arg Gln

210 215 220

gaa ctc ctc agg aag gaa cgt ctg gtc tta tct agt gag cac tgg cta 720

Glu Leu Leu Arg Lys Glu Arg Leu Val Leu Ser Ser Glu His Trp Leu

225 230 235 240

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Val Leu Val Pro Phe Trp Ala Val Trp Ala Phe Gln Thr Leu Leu Leu

245 250 255

ccc cgc cgg cat gta cgt cgg cta cct gag ctg acc cct gct gaa cgt 816

Pro Arg Arg His Val Arg Arg Leu Pro Glu Leu Thr Pro Ala Glu Arg

260 265 270

gat gat cta gcc tcc atc atg aag aag cta ttg acc aag tat gac aac 864

Asp Asp Leu Ala Ser Ile Met Lys Lys Leu Leu Thr Lys Tyr Asp Asn

275 280 285

cta ttt gag aca tct ttt ccc tac tcc atg ggc tgg cat ggg gct ccc 912

Leu Phe Glu Thr Ser Phe Pro Tyr Ser Met Gly Trp His Gly Ala Pro

290 295 300

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Thr Gly Leu Lys Thr Gly Ala Ala Tyr Asp His Trp Gln Leu His Ala

305 310 315 320

cat tac tat ccc cca ctc ctg cgc tct gct act gtc cgg aaa ttc atg 1008

His Tyr Tyr Pro Pro Leu Leu Arg Ser Ala Thr Val Arg Lys Phe Met

325 330 335

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Val Gly Tyr Glu Met Leu Ala His Gly His Arg Asp Leu Thr Pro Glu

340 345 350

cag gct gca gag aga cta agg gcg ctt cct gag gta cat tat tgc ctg 1104

Gln Ala Ala Glu Arg Leu Arg Ala Leu Pro Glu Val His Tyr Cys Leu

355 360 365

gca aag aaa gac aag gaa aca gca gct gtt gct tga 1140

Ala Lys Lys Asp Lys Glu Thr Ala Ala Val Ala

370 375

<210> 4

<211> 379

<212> Protein

<213> Artificial sequence

<220>

<223> [CDS]:1..1140 from SEQ ID NO 3

<400> 4

Met Ser Gln Asn Gly Asp Asp Pro Glu Gln Arg Gln Gln Ala Ser Glu

1 5 10 15

Ala Asp Ala Met Ala Ala Thr Phe Arg Ala Ser Glu His Gln His Ile

20 25 30

Arg Tyr Asn Pro Leu Gln Asp Glu Trp Val Leu Val Ser Ala His Arg

35 40 45

Met Lys Arg Pro Trp Gln Gly Gln Val Glu Pro Gln Leu Leu Lys Thr

50 55 60

Val Pro Arg Tyr Asp Pro Leu Asn Pro Leu Cys Pro Gly Ala Thr Arg

65 70 75 80

Ala Asn Gly Glu Val Asn Pro His Tyr Asp Ser Thr Phe Leu Phe Asp

85 90 95

Asn Asp Phe Pro Ala Leu Gln Pro Asp Ala Pro Asp Pro Gly Pro Ser

100 105 110

Asp His Pro Leu Phe Arg Ala Glu Ala Ala Arg Gly Val Cys Lys Val

115 120 125

Met Cys Phe His Pro Trp Ser Asp Val Thr Leu Pro Leu Met Ser Val

130 135 140

Pro Glu Ile Arg Ala Val Ile Asp Ala Trp Ala Ser Val Thr Glu Asp

145 150 155 160

Leu Gly Ala Gln Tyr Pro Trp Val Gln Ile Phe Glu Asn Lys Gly Ala

165 170 175

Met Met Gly Cys Ser Asn Pro His Pro His Cys Gln Val Trp Ala Ser

180 185 190

Ser Phe Leu Pro Asp Ile Ala Gln Arg Glu Val Arg Ser Gln Gln Asn

195 200 205

Tyr His Ser Gln His Gly Glu Pro Leu Leu Leu Glu Tyr Gly Arg Gln

210 215 220

Glu Leu Leu Arg Lys Glu Arg Leu Val Leu Ser Ser Glu His Trp Leu

225 230 235 240

Val Leu Val Pro Phe Trp Ala Val Trp Ala Phe Gln Thr Leu Leu Leu

245 250 255

Pro Arg Arg His Val Arg Arg Leu Pro Glu Leu Thr Pro Ala Glu Arg

260 265 270

Asp Asp Leu Ala Ser Ile Met Lys Lys Leu Leu Thr Lys Tyr Asp Asn

275 280 285

Leu Phe Glu Thr Ser Phe Pro Tyr Ser Met Gly Trp His Gly Ala Pro

290 295 300

Thr Gly Leu Lys Thr Gly Ala Ala Tyr Asp His Trp Gln Leu His Ala

305 310 315 320

His Tyr Tyr Pro Pro Leu Leu Arg Ser Ala Thr Val Arg Lys Phe Met

325 330 335

Val Gly Tyr Glu Met Leu Ala His Gly His Arg Asp Leu Thr Pro Glu

340 345 350

Gln Ala Ala Glu Arg Leu Arg Ala Leu Pro Glu Val His Tyr Cys Leu

355 360 365

Ala Lys Lys Asp Lys Glu Thr Ala Ala Val Ala

370 375

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> forward primer UDF_GlcE-F2-21

<400> 5

atggccgaga aggtgctggt from 21

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer UDF_GlcE-R21

<400> 6

ttaggcctgt gctccaaagc from 21

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> forward primer UDF-Gal-T-F2-21

<400> 7

atgtcgcaaa acggagatga t 21

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer UDP-Gal-T-R18

<400> 8

tcaagcaaca gctgctgt 18

<210> 9

<211> 140

<212> Protein

<213> Artificial sequence

<220>

<223> Synthesized

<400> 9

Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val

1 5 10 15

Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys

20 25 30

Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val

35 40 45

Val His Gly Val Ala Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr

50 55 60

Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys

65 70 75 80

Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys

85 90 95

Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile

100 105 110

Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro

115 120 125

Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

130 135 140

<210> 10

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> α-synuclein; heavy chain CDR1 (CDR-H1)

<400> 10

Asn Tyr Gly Met Ser

1 5

<210> 11

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> α-synuclein; heavy chain CDR2 (CDR-H2)

<400> 11

Ser Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Asn Val Lys

1 5 10 15

Gly

<210> 12

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> α-synuclein; CDR3 heavy chain (CDR-H3)

<400> 12

Gly Gly Ala Gly Ile Asp Tyr

1 5

<210> 13

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> α-synuclein; CDR1 light chain (CDR-L1)

<400> 13

Lys Ser Ile Gln Thr Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 14

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> α-synuclein; CDR2 light chain (CDR-L2)

<400> 14

Trp Ala Ser Ile Arg Lys Ser

1 5

<210> 15

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> α-synuclein; CDR3 light chain (CDR-L3)

<400> 15

Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr

1 5

<210> 16

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> α-synuclein; variable domain of the heavy chain (VH)

<400> 16

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Asn Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ala Gly Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 17

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> α-synuclein; variable domain of the light chain (VL)

<400> 17

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ile Gln Thr Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg Lys Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 18

<211> 328

<212> Protein

<213> Artificial sequence

<220>

<223> α-synuclein; heavy chain constant domain (CH)

<400> 18

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 19

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> α-synuclein; light chain constant domain (CL)

<400> 19

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 20

<211> 444

<212> Protein

<213> Artificial sequence

<220>

<223> α-synuclein; heavy chain (H)

<400> 20

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Asn Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ala Gly Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440

<210> 21

<211> 220

<212> Protein

<213> Artificial sequence

<220>

<223> α-synuclein; light chain (L)

<400> 21

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ile Gln Thr Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg Lys Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 22

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> CD3; heavy chain CDR1 (CDR-H1)

<400> 22

Thr Tyr Ala Met Asn

1 5

<210> 23

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> CD3; heavy chain CDR2 (CDR-H2)

<400> 23

Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser

1 5 10 15

Val Lys Gly

<210> 24

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> CD3; heavy chain CDR3 (CDR-H3)

<400> 24

His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr

1 5 10

<210> 25

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> CD3;CDR1 light chain (CDR-L1)

<400> 25

Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

1 5 10

<210> 26

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> CD3; CDR2 light chain (CDR-L2)

<400> 26

Gly Thr Asn Lys Arg Ala Pro

1 5

<210> 27

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CD3; CDR3 light chain (CDR-L3)

<400> 27

Ala Leu Trp Tyr Ser Asn Leu Trp Val

1 5

<210> 28

<211> 125

<212> Protein

<213> Artificial sequence

<220>

<223> CD3; variable domain of the heavy chain (VH)

<400> 28

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 29

<211> 109

<212> Protein

<213> Artificial sequence

<220>

<223> CD3; variable domain of light chain (VL)

<400> 29

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 30

<211> 101

<212> Protein

<213> Artificial sequence

<220>

<223> CD3; heavy chain constant domain (CH)

<400> 30

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys

100

<210> 31

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> CD3; light chain constant domain (CL)

<400> 31

Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 32

<211> 101

<212> Protein

<213> Artificial sequence

<220>

<223> CD3 heavy chain constant domain (CD3 CH)

<400> 32

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys

100

<210> 33

<211> 232

<212> Protein

<213> Artificial sequence

<220>

<223> CD3; light chain (L)

<400> 33

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 34

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> CD20; heavy chain CDR1 (CDR-H1)

<400> 34

Tyr Ser Trp Ile Asn

1 5

<210> 35

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CD20; heavy chain CDR2 (CDR-H2)

<400> 35

Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe Lys

1 5 10 15

Gly

<210> 36

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> CD20; heavy chain CDR3 (CDR-H3)

<400> 36

Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr

1 5 10

<210> 37

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> CD20; CDR1 light chain (CDR-L1)

<400> 37

Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr

1 5 10 15

<210> 38

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> CD20; CDR2 light chain (CDR-L2)

<400> 38

Gln Met Ser Asn Leu Val Ser

1 5

<210> 39

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CD20; CDR3 light chain (CDR-L3)

<400> 39

Ala Gln Asn Leu Glu Leu Pro Tyr Thr

1 5

<210> 40

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> CD20; variable domain of the heavy chain (VH)

<400> 40

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 41

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> CD20; variable domain of the light chain (VL)

<400> 41

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val

115

<210> 42

<211> 101

<212> Protein

<213> Artificial sequence

<220>

<223> CD20; heavy chain constant domain (CH)

<400> 42

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Glu

85 90 95

Lys Val Glu Pro Lys

100

<210> 43

<211> 104

<212> Protein

<213> Artificial sequence

<220>

<223> CD20; light chain constant domain (CL)

<400> 43

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

1 5 10 15

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

20 25 30

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

35 40 45

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

50 55 60

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

65 70 75 80

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

85 90 95

Lys Ser Phe Asn Arg Gly Glu Cys

100

<210> 44

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<223> CD20 light chain (CD20 VL-CL)

<400> 44

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg

115 120 125

Lys Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 45

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<223> CD20 heavy chain [CD20 VH-CHl(EE)-Fc (hollow, P329G LALA)]

<400> 45

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 46

<211> 439

<212> Protein

<213> Artificial sequence

<220>

<223> CD20-CD3 heavy chain [CD3 VL-CH1-Fc (knob, P329G LALA)]

<400> 46

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala

100 105 110

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

115 120 125

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

130 135 140

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

145 150 155 160

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

165 170 175

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

180 185 190

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

195 200 205

Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

210 215 220

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

225 230 235 240

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

245 250 255

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

260 265 270

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

275 280 285

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

290 295 300

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

305 310 315 320

Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

325 330 335

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu

340 345 350

Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro

355 360 365

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

370 375 380

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

385 390 395 400

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

405 410 415

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

420 425 430

Lys Ser Leu Ser Leu Ser Pro

435

<210> 47

<211> 673

<212> Protein

<213> Artificial sequence

<220>

<223> CD20 heavy chain [CD20 VH-CHl(EE)-CD3 VL-CH1-Fc (knob, P329G

[LALA]

<400> 47

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Gly

210 215 220

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln

225 230 235 240

Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys

245 250 255

Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val

260 265 270

Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn

275 280 285

Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly

290 295 300

Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala

305 310 315 320

Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly

325 330 335

Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser

340 345 350

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

355 360 365

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

370 375 380

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

385 390 395 400

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

405 410 415

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

420 425 430

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

435 440 445

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

450 455 460

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

465 470 475 480

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

485 490 495

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

500 505 510

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

515 520 525

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

530 535 540

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

545 550 555 560

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

565 570 575

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

580 585 590

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

595 600 605

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

610 615 620

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

625 630 635 640

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

645 650 655

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

660 665 670

Pro

<---

Claims (23)

1. An antibody to α-synuclein having monogalactosylated (G1) and digalactosylated (G2) glycans, wherein the antibody to α-synuclein comprises a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:10, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:11, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:12; and variable light chain (VL) domain containing (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:13, (d) CDR-L2 comprising the amino acid sequence of SEQ ID NO:14, and (e) CDR-L3 comprising the amino acid sequence of SEQ ID NO:15; and wherein the anti-α-synuclein antibody has 17.2-48.0% (w/w) G1 and 3.1-15.0% (w/w) G2 per total glycan; preferably 25.4-48.0% (w/w) G1 and 3.5-15.0% (w/w) G2 per total glycan; preferably 27.2-47.0% G1 and 4.4-15.0% G2 per total glycan; preferably 40.0-46.0% (w/w) G1 and 8.4-15.0% (w/w) G2 per total glycan; more preferably 41.0-45.0% (w/w) G1 and 9.5-14.0% (w/w) G2 of total glycan, and most preferably 42.1-43.9% (w/w) G1 and 10.6-13.3% (w/w) G2 of total glycan. 2. The antibody to α-synuclein according to claim 1, wherein the antibody to α-synuclein comprises (a) VH sequence SEQ ID NO:16; (b) the VL sequence SEQ ID NO:17; or (c) the VH sequence defined in (a) and the VL sequence defined in (b). 3. The antibody to α-synuclein according to claim 1 or 2, wherein the antibody to α-synuclein comprises the heavy chain of SEQ ID NO:20 and the light chain of SEQ ID NO:21. 4. A method for producing an antibody to α-synuclein according to any one of claims 1-3, having monogalactosylated (G1) and digalactosylated (G2) glycans, wherein said method comprises: (a) culturing a mammalian cell in a cell culture medium, wherein the concentration of the sulfhydryl group(s) of one or more sulfhydryl compounds is at least greater than 4.0 mM and less than 10.0 mM and of glucose is at least greater than 3.0 g/L in the cell culture medium is maintained for at least 3, more preferably at least 4, and even more preferably at least 5 days, (b) isolating said antibody. 5. The method according to claim 4, wherein the concentration is maintained for at least 5 days, preferably for at least 7 days, more preferably for at least 10 days, even more preferably for at least 12 days and most preferably for at least 14 days. 6. The method according to any one of claims 4 or 5, wherein the cell culture medium comprises sulfhydryl group(s) of one or more sulfhydryl compounds at a concentration of at least 5.0 mM and less than 10.0 mM, preferably at least 5.0 mM and equal to or less than 9.0 mM, more preferably at least 5.0 mM and equal to or less than 8.0 mM, even more preferably at least 5.0 mM and equal to or less than 7.0 mM and most preferably at least 5.0 mM and equal to or less than 6.0 mM. 7. The method according to any one of claims 4 to 6, wherein the cell culture medium contains from more than 3.0 g/L to no more than 13 g/L glucose, preferably from more than 3.0 g/L to no more than 8.0 g/L glucose, more preferably from more than 3.0 g/L to no more than 7.0 g/L glucose, even more preferably from more than 3.0 g/L to no more than 6.0 g/L glucose and most preferably from more than 3.0 g/L to no more than 5.0 g/L glucose. 8. The method according to any one of claims 4-7, wherein one or more sulfhydryl compounds are selected from the group consisting of cysteine, cystine, and a combination thereof. 9. The method according to any one of claims 4 to 8, wherein the cell culture medium is a medium with a specific chemical composition, preferably a cell culture medium that does not contain serum, proteins and/or oligopeptides. 10. Use of an antibody to α-synuclein according to any one of claims 1-3 for the treatment of patients with Parkinson's disease.