Modified antigen-binding of human antibodies with glycosylation variations of the light chains produced in sugar-limited human hybridoma cultures

Summary We have characterized the effects of serum andN-acetylglucosamine in a glucose-deprived condition on the glycosylation of antibody light chains, as well as the resulting biological properties of those antibodies. We have chosen for our investigation the human hybridoma lines producing monoclonal antibodies reactive to lung adenocarcinoma. Each antibody possess aN-glycosylated carbohydrate chain in the hypervariable region of the light chains. When the cell lines were grown in the absence of glucose, variant light chains with varying molecular masses were found to be secreted. Analysis of these light chains produced in a glucose-deprived condition revealed that the changed molecular-mass of the variant light chains is due to different glycosylation. Addition ofN-acetylglucosamine or fetal calf serum to the glucose-free medium led to the creation of other light chains that exhibit increased antigen binding activity.     

Generation of specificity-variant antibodies by alteration of carbohydrate in light chain of human monoclonal antibodies.

A hybridoma line, C5TN, produced human monoclonal antibody of which light chain had N-linked carbohydrate chain within the variable region. Some molecular-weight variants of light chain of the antibody were produced by C5TN variants resistant to cytotoxic effect of concanavalin A. The variant antibodies significantly altered the original cross-reactivity with antigens or lost the ability of antigen binding. The variants variously trimmed their carbohydrate chains by glycosidases, showed the changed reactivity or acquired the ability to bind for antigens. The carbohydrate-deficient antibodies from tunicamycin-treated C5TN and the variant clones behaved in a similar manner on antigen-binding reactivity. Furthermore, comparison of antibodies of which light chains have carbohydrate chains sensitive and resistant to some glycosidases showed that carbohydrate chain in variable region of light chain can influence their reactivity with antigen.     

Building high affinity human antibodies by altering the glycosylation on the light chain variable region in N-acetylglucosamine-supplemented hybridoma cultures

We attempted to improve antibody affinity by varying glycosylation on the light chain variable region. The human hybridoma line HB4C5 produces an antibody reactive to lung adenocarcinoma, which possess a N-glycosylated carbohydrate chain on the light chain hypervariable region. It has been shown that altering this carbohydrate structure can be accomplished by varying the level of N-acetylglucosamine in glucose free medium, a change in the carbohydrate chain could be induced which resulted in modifying antigen binding. By culturing the cells in media containing more than 20 mM N-acetylglucosamine, cells produced antibody with 10 fold improved affinity as compared with antibody produced in 20 mM glucose-containing medium. A newly induced light chain glycoform produced in the N-acetylglucosamine-containing medium was shown to be responsible for this antigen binding enhancement. Addition of glucose in the N-acetylglucosamine-containing media led to decreased antibody affinity and slightly inhibited production of a new light chain in a dose-dependent manner. Combination of 20 mM N-acetylglucosamine and 0.5 mM glucose gave a higher antibody production without the decrease of the antigen binding. These results indicate that optimization of N-glycosylation on the light chain, which leads to higher antigen binding, can be accomplished by adjusting a ratio of glucose and N-acetylglucosamine in the culture medium.     

Glucose-dependent glycosylation of secretory glycoprotein in mouse myeloma cells.

The mouse myeloma tumor, MOPC-46, produces a kappa-type immunoglobulin light chain that may be isolated from the urine of tumor-bearing animals. This protein possesses a single carbohydrate side chain, attached by glycosylamine linkage to asparagine residue 28. When viable single cell suspensions of the tumor are incubated in vitro in minimum essential medium containing sodium pyruvate as a source of carbon and energy, the major protein synthesized and secreted corresponds to a nonglycosylated form of the kappa light chain. However, when glucose or mannose are substituted for sodium pyruvate as a source of carbon, the immunoglobulin light chain is synthesized and secreted in the fully glycosylated, native form. The dependence of normal glycosylation of the protein on the presence of either glucose or mannose in the medium is relatively specific for these compounds since substitution with either fructose, galactose, glycerol, ribose, or N-acetylglucosamine was ineffective. The nonglycosylated protein produced in the presence of sodium pyruvate was characterized as nonglycosylated MOPC-46 light chain by immunoprecipitation and gel electrophoresis. An identical nonglycosylated protein was produced by tumor cells in the presence of glucose when the incubation mixtures contained tunicamycin.     

Low Glucose but Not Galactose Enhances Oxidative Mitochondrial Metabolism in C2C12 Myoblasts and Myotubes

Background

Substituting galactose for glucose in cell culture media has been suggested to enhance mitochondrial metabolism in a variety of cell lines. We studied the effects of carbohydrate availability on growth, differentiation and metabolism of C2C12 myoblasts and myotubes.

Methodology/Principal Findings

We measured growth rates, ability to differentiate, citrate synthase and respiratory chain activities and several parameters of mitochondrial respiration in C2C12 cells grown in media with varying carbohydrate availability (5 g/l glucose, 1 g/l glucose, 1 g/l galactose, and no added carbohydrates). C2C12 myoblasts grow more slowly without glucose irrespective of the presence of galactose, which is not consumed by the cells, and they fail to differentiate without glucose in the medium. Cells grown in a no-glucose medium (with or without galactose) have lower maximal respiration and spare respiratory capacity than cells grown in the presence of glucose. However, increasing glucose concentration above physiological levels decreases the achievable maximal respiration. C2C12 myotubes differentiated at a high glucose concentration showed higher dependency on oxidative respiration under basal conditions but had lower maximal and spare respiratory capacity when compared to cells differentiated under low glucose condition. Citrate synthase activity or mitochondrial yield were not significantly affected by changes in the available substrate concentration but a trend towards a higher respiratory chain activity was observed at reduced glucose levels.

Conclusions/Significance

Our results show that using galactose to increase oxidative metabolism may not be applicable to every cell line, and the changes in mitochondrial respiratory parameters associated with treating cells with galactose are mainly due to glucose deprivation. Moderate concentrations of glucose (1 g/l) in a growth medium are optimal for mitochondrial respiration in C2C12 cell line while supraphysiological concentrations of glucose cause mitochondrial dysfunction in C2C12 myoblasts and myotubes.     

High-level production and secretion of a mouse-human chimeric Fab fragment with specificity to human carcino embryonic antigen in Escherichia coli

A high-level secretion system for the production of mouse-human chimeric antibody 21B2 (MHC 21B2) Fab fragment specific for human carcino embryonic antigen (hCEA) in Escherichia coli has been constructed. The genes encoding a light chain and an Fd fragment (a variable region and the CH₁ domain of a heavy chain) of a mouse-human chimeric antibody were directly fused to the signal peptide of the E. coli ompF gene sequence. E. coli cells containing expression vectors in which each of the two genes are located downstream of a separate tac promoter were able to secrete the light chain and Fd fragment as two of their major cellular proteins. The signal peptides were efficiently removed from the primary products by post-translational processing, although they formed insoluble aggregates, possibly in the periplasm. In high-cell-density culture experiments using a jar fermentor, the amount of light chain and Fd fragment produced was at levels of up to 2.88 g/l and 1.28 g/l culture, respectively. By optimizing the conditions that encourage correct folding, formation of disulphide bonds, and association of the light chain with the Fd fragment, we have established a procedure that can purify, refold, and combine aggregated products to electrophoretically homogeneous Fab fragment with a yield of approximately 47%. Fab fragment produced in this manner shows essentially the same antigen-binding activity and specificity to hCEA as the parental mouse antibody 21B2 (MoAb 21B2). Correspondence to: T. Shibui     

The carbohydrate structures ofβ-galactosidase from human liver

Acid -galactosidase (EC3.2.1.23) was obtained from human liver in a pure monomeric state (M_r63 000). The carbohydrate content of the enzyme was established to be, 9% by weight; mannose,N-acetylglucosamine, galactose andN-acetylneuraminic acid were found to be the constituent monosaccharides. The carbohydrate structures of the enzyme were studied at the glycopeptide level by employing 500 MHz¹H-NMR spectroscopy, carbohydrate composition analysis and methylation analysis involving GLCMS. Based upon the intensities of relevant signals in the¹H-NMR spectrum, approximately 60% of the chains were found to be of theN-acetyllactosamine type, having the structure The rest appeared to be of the oligomannoside type (Man₅-₆GlcNAc₂Asn). The carbohydrate composition and methylation analysis results sustained these findings, although the calculation of the distribution based upon these techniques indicated a somewhat lower percentage ofN-acetyllactosamine type chains. There are approximately three oligosaccharide chains per molecule. These findings offer an explanation for the abnormal distribution of -galactosidase in tissues and cultured fibroblasts of patients with I-cell disease.     

Terminal glycosylation and disease: Influence on cancer and cystic fibrosis

Terminal glycosylation has been a recurring theme of the laboratory. In cystic fibrosis (CF), decreased sialic acid and increased fucosyl residues in 1,3 position to antennary N -acetyl glucosamine is the CF glycosylation phenotype. The glycosylation phenotype is reversed by transfection of CF airway cells with wtCFTR. In neuronal cells, polymers of 2,8sialyl residues are prominent in oligodendrocytes and human neuroblastoma. These findings are discussed in relationship to early studies in our laboratories and those of other investigators. The potential extension of these concepts to future clinical therapeutics is presented.     

Analysis of erythropoietin glycoform produced by recombinant CHO cells using the Lectin-blotting technique

The glycosylation pattern of Erythropoietin (EPO), produced by recombinant CHO cells, was studied using the simple and rapid technique of ‘Lectin-blotting’. In this experiment we used three different kinds of lectins, MAA (Maackia amurensis agglutinine), RCA (Ricinus communis agglutinine), and DSA (Datura stramonium agglutinine), which bind to the terminal sialic acid, galactose, and the N-acetyllactosamine chain respectively. The lectin-blotting technique was used to analyze the carbohydrate structure of EPO produced in the presence of two physiologically active chemical compounds, ammonium and chloroquine. The effect of the ammonium ion on the glycosylation of EPO was studied because it accumulated in the medium mainly as a by-product of glutamine metabolism. Ammonium chloride significantly inhibited the sialylation of the terminal galactose residue at concentrations of 8 mM or more. Chloroquine, a potent inhibitor of glycosylation, inhibited terminal sialylation at concentrations of 100 and 200 μM, and at a concentration of 300 μM also inhibited N-acetyllactosamine chain synthesis.     

Asn-linked sugar chain structures of recombinant human thrombopoietin produced in Chinese hamster ovary cells

Human thrombopoietin (TPO) that regulates the numbers of megakaryocytes and platelets is a heavily N- and O-glycosylated glycoprotein hormone with partial homology to human erythropoietin (EPO). We prepared recombinant human TPO produced in Chinese hamster ovary (CHO) cells and analyzed the sugar chain structures quantitatively using 2-aminobenzamide labeling, sequential glycosidase digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS).We found bi-, tri- and tetraantennary complex-type sugar chains with one or two N-acetyllactosamine repeats, which are common to recombinant human EPO produced in CHO cells. On the other hand, there were triantennary sugar chains with one or two N-acetyllactosamine repeats that were specific to the recombinant human TPO, and their distributions of branch structures were also different. These results suggested that proximal protein structure should determine the branch structure of Asn-linked sugar chains in addition to the glycosyltransferases subset.     

Photosensitized Oxygenation Reactions of β-Thujaplicin (Hinokitiol): Formation of a New Carbon Skeleton,Tetracyclo[7.3.2.02,8.04,14]tetradecane

N-Linked oligosaccharides were elongated by glycosylation with mannose and galactose residues in the secretory pathway of Schizosaccharomyces pombe. The wild-type S. pombe cells were agglutinated by the additions of not only concanavalin A lectin, which is specific for mannose residues, but also PNA (from Arachis hypogaea) and RCA (Ricinus communis) lectins, which are specific for terminal galactose residues. By PNA-binding selection, we isolated an S. pombe mutant defective in protein glycosylation. The mutant cells, named gmsl, were not agglutinated by PNA or RCA. In contrast, agglutination of the gmsl cells by the addition of concanavalin A was markedly increased. Structural studies on N-linked oligosaccharides from gmsl mutant cells showed that the number of x-l,2-linked galactose residues wes markedly reduced, and unsubstituted x-l,6-linked polymannose outer chains were attached to the core oligosaccharides.     

The antigen-binding capacity of the peptide chains of horse antibodies

The antigen-binding capacity of the peptide chains of horse anti-(diphtheria toxin) has been studied by using (125)I-labelled toxoid and electrophoresis of antibody-antigen mixtures on cellulose acetate. The heavy chains retained about 20% of the activity of the whole antibody and the light chains less than 5%. Recombination of specific heavy and light chains gave about 60% recovery of activity and recombination of specific heavy chains and non-specific light chains about 40% recovery. It is suggested that these results favour the heavy chain as the major site of the antigen-binding activity.     

Enterotoxin A production in Staphylococcus aureus: inhibition by glucose

In this study, we investigated the relationship between carbohydrate metabolism and repression of staphylococcus enterotoxin A (SEA) in Staphylococcus aureus 196E and a pleiotrophic mutant derived from strain 196E. The mutant, designated at strain 196E-MA, lacked a functional phosphoenolpyruvate phosphotransferase system (PTS). The mutant produced acid, under aerobic conditions, from only glucose and glycerol. The parent strain contained an active PTS, and aerobically produced acid from a large number of carbohydrates. Prior growth in glucose led to repression of SEA synthesis in the parent strain; addition to the casamino acids enterotoxin production medium (CAS) led to more severe repression of toxin synthesis. The repression was not related to pH decreases produced by glucose metabolism. When S. aureus 196E was grown in the absence of glucose, there was inhibition of toxin production as glucose level was increased in CAS. The inhibition was related to pH decrease and was unlike the repression observed with glucose-grown strain 196E. The inhibition of SEA synthesis in mutant strain 196E-MA was approximately the same in cells grown with or without glucose and was pH related. Repression of SEA synthesis similar to that seen with glucose-grown S. aureus 196E could not be demonstrated in the mutant. In addition, glucose-grown S. aureus 196E neither synthesized -galactosidase nor showed respiratory activity with certain tricarboxylic acid (TCA) cycle compounds. Glucose-grown strain 196E-MA, however, did not show supressed respiration of TCA cycle compounds; -galactosidase was not synthesized because the mutant lacked a functional PTS. Cyclic adenosine-3, 5-monophosphate did not reverse the repression by glucose of SEA or -galactosidase synthesis in glucose-grown S. aureus 196E. An active PTS appears to be necessary to demonstrate glucose (catabolite) repression in S. aureus.Abbreviations SEA staphylococcal enterotoxin A - SEB staphylococcal enterotoxin B - SEC staphylococcal enterotoxin C - PTS phosphoenolpyruvate phosphotransferase system - CAS casamino acids salts medium - TCA tricarboxylic acid cycle     

Studies on the regulation of the three enzymes of the leloir pathway in cultured mammalian cells. I. Effect of substitution of galactose for glucose as the sole hexose in the medium in human diploid cell strains and in a rat hepatoma line

In human diploid cell strains, the substitution of galactose for glucose as the sole hexose in the medium had no measurable effect on the specific activity of the cell protein for any of the three enzymes of the Leloir pathway. These enzymes are galactokinase, α-D-galactose-1-phosphate:UDP glucose uridylyl transferase and UDP galactose 4-epimerase. A cell strain from a patient with galactosemia had no detectable activity for the transferase. The substitution of galactose for glucose in the medium of these cells (which has been shown to cause the cells to accumulate galactose-1-phosphate) also failed to affect cellular activity for the three enzymes. Similarly, the three activities failed to respond to the substitution of galactose for glucose in cultures of a rat hepatoma line. Cells of this line have been shown by others to perform a number of the tissue-specific functions of liver. The failure of galactose to stimulate increased cellular activity for the three enzymes represents a striking difference between the behavior of these enzymes in human diploid cell strains and their behavior in E. coli.     

Influence of baculovirus-host cell interactions on complex N-linked glycosylation of a recombinant human protein

The conditions required for mammalian-type complex N-linked glycosylation of human proteins produced in insect cells with the baculovirus expression vector system were investigated. Marked alterations to N-linked glycosylation of human placental secreted alkaline phosphatase (SEAP) were observed with different baculovirus species, insect cell lines, and cell culture media. When a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) was used to produce SEAP in Trichoplusia ni (Tn-4h) cells cultured in serum-free medium, structural analyses indicated <1% hybrid and no complex oligosaccharides attached to SEAP, a typical result with the baculovirus expression vector system. However, when fetal bovine serum was added to the culture medium, 48 +/- 4% of the oligosaccharides were hybrid or complex (but asialylated) glycans. When a recombinant T. ni nucleopolyhedrovirus (TnSNPV) was similarly used to express SEAP in Tn-4h cells cultured in serum-containing medium, only 24 +/- 3% of the glycans contained terminal N-acetylglucosamine and/or galactose residues. In contrast, SEAP produced in Sf9 cells grown in serum-containing medium with AcMNPV contained <1% hybrid oligosaccharides and no complex oligosaccharides. The results illustrate that baculovirus type, host cell type, and the growth medium all have a strong influence on the glycosylation pathway in insect cells, resulting in significant alterations in structures and relative abundance of N-linked glycoforms. Although the addition of sialic acid residues to the SEAP glycans was not detected, possible approaches to obtain sialylated glycans are discussed.     

Improvement of antigen binding ability of human antibodies by light chain shifting

Human HB4C5 hybridoma cells produce a lung cancer-specific IgM human monoclonal antibody (mAb). HB4C5 human mAb cross-reacts with Candida cytochrome c (Cyt c) and carboxypeptidase (Cpase). Concanavalin A (ConA)-resistant variants of HB4C5 cells loss the original light chain followed by expression of various new light chains at a high incidence (light chain shifting) (Tachibana et al., 1996). HTD8 cells, one of the ConA-resistant variant subclones of HB4C5 cells, undergo the active light chain shifting and produce various sublines, each of which stably secretes new mAb consisting of a new light chain and a HB4C5 heavy chain. The new mAb exhibits altered antigen binding ability from that of the original antibody. We could expect that HTD8 cells can be used as ‘a light chain stem cell line’ to improve antigen binding ability and specificity of established human mAbs. A BD9D12 IgG human mAb recognizes lung cancer cells and cross-reacts with cytokeratin 8. Introduction of the heavy chain gene of BD9D12 mAb into HTD8 cells resulted in establishment of various sublines which secreted various kinds of hybrid antibodies consisting of different light chains derived from HTD8 subclones which underwent light chain shifting and a common IgG heavy chain derived from BD9D12. These hybrid antibodies exhibited different or improved reactivities to Cyt, Cpase, cytokeratin 8 and various cancer cells from those of parental mAb, demonstrating that light chain shifting can be applied to improve the affinity and specificity of human mAb. This revised version was published online in June 2006 with corrections to the Cover Date.     

Immunogenicity and antigenicity of immunoglobulins: detection of human immunoglobulin light-chain carbohydrate, using concanavalin A

Concanavalin A binding to glycoprotein bands on nitrocellulose blots was used to detect mannose, sorbose, N-acetylgalactosamine and/or glucose residues on 100% (31/31) of human Bence Jones protein light chains, following sodium dodecyl sulphate-polyacrylamide gel electrophoresis. All (20/20) light chains form IgG myeloma proteins and light chains from a preparation of normal polyclonal human IgG were also bound by concanavalin A. The specificity of concanavalin A for glycoproteins was demonstrated by its binding to human Fc fragments and a human monoclonal anti-Rhesus D antibody (REG-A), but not to human albumin pFc' fragments and aglycosylated REG-A derived from cells grown in the presence of the glycosylation inhibitor tunicamycin. These results suggest that all Bence Jones proteins and light chains from myeloma IgG proteins contain mono- or oligosaccharides linked O-glycosidically to serine or threonine residues.     

Expression of antibodies using single‐open reading frame vector design and polyprotein processing from mammalian cells

We describe a novel polyprotein precursor‐based approach to express antibodies from mammalian cells. Rather than expressing heavy and light chain proteins from separate expression units, the antibody heavy and light chains are contained in one single‐open reading frame (sORF) separated by an intein gene fused in frame. Inside mammalian cells this ORF is transcribed into a single mRNA, and translated into one polypeptide. The antibody heavy and light chains are separated posttranslationally, assembled into the functional antibody molecule, and secreted into culture medium. It is demonstrated that Pol I intein from P. horikoshii mediates protein splicing and cleavage reactions in mammalian cells, in the context of antibody heavy and light chain amino acid sequences. To allow the separation of antibody heavy chain, light chain, and the intein, we investigated a number of intein mutations designed to inhibit intein‐mediated splicing but preserve cleavage reactions. We have also designed constructs in which the signal peptide downstream from intein has altered hydrophobicity. The use of some of these mutant constructs resulted in more efficient antibody secretion, highlighting areas that can be further explored in improving such an expression system. An antibody secreted using one of the sORF constructs was characterized. This antibody has correct N‐terminal sequences for both of its heavy and light chains, correct heavy and light chain MW as well as intact MW as measured by mass spectrometry. Its affinity to antigen, as measured by surface plasmon resonance (SPR), is indistinguishable from that of the same antibody produced using conventional method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009