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     

Antigen binding of an ovomucoid-specific antibody is affected by a carbohydrate chain located on the light chain variable region

We cloned the variable regions of heavy and light chain genes of an anti-ovomucoid monoclonal antibody (MAb-OM21) produced by the mouse hybridoma cell line OM21. DNA sequence analysis showed that the light chain of the MAb-OM21 has only one potential N-glycosylation consensus sequence in the complementarity determining region 2 of the light chain. To find whether carbohydrate chains are located on the light chain, we assayed for the size of the light chain, after treatment with N-glycosidase, by western blotting, and also detection of the carbohydrate chains on the light chain was done using the lectin blot assay. A N-linked carbohydrate chain has been shown to bind to the light chain. To clarify the role of this carbohydrate chain in the light chain, we produced carbohydrate variant antibodies by N-deglycosylation using glycosidase or by expressing the antibody from different host cells. The N-deglycosylated variant antibody has greater antigen binding, and the antibody produced from the different host cells showed a reduced antigen binding activity and acquired the ability to react to ovalbumin. These results suggest that antigen binding of the ovomucoid specific antibody MAb-OM21 can be affected by the carbohydrate chain on the light chain variable region.     

A hydrophobic protein sequence can override a nuclear localization signal independently of protein context.

Simian virus 40 T antigen is specifically targeted to the nucleus by the signal Pro-Lys-Lys-128-Lys-Arg-Lys-Val. We have previously described the isolation of a simian virus 40 T-antigen mutant, 676FS, which retains a wild-type nuclear localization signal but fails to accumulate properly in the nucleus and interferes with the nuclear localization of heterologous proteins. Here we report that the hydrophobic carboxy-terminal sequence novel to 676FS T antigen overrides the nuclear localization signal if fused to other proteins, thereby anchoring the proteins in the cytoplasm. We discuss possible mechanisms by which missorting of such a fusion protein could interfere with the nuclear transport of heterologous proteins.     

Separation of three class II antigens from a homozygous human B cell line

Three class II molecules were isolated from a homozygous DRw6 human B lymphoblastoid cell line using the monoclonal antibodies L243 (L203), L227, LKT 111, and Genox 3.53. Two of the antigens appeared to employ the same heavy chain but expressed different light chains. The two light chains were separated after denaturation using L227 and LKT 111. One or both of these two molecules carried the DRw6 and MT2 determinants. The third class II antigen expressed the DC1 determinant. It was composed of a heavy and light chain different from the DR-like antigen subunits. The antibodies L243, L227, and LKT 111 did not preclear the cell lysate of the DC1 antigen recognized by Genox 3.53. However, a xenoanti-DR serum immunoprecipitated both the DR-like and the DC1 antigens. Thus, in total, one cell line can express at least two class II heavy chains and three class II light chains. This observation was not unique to this cell line.     

Characterization of humanized antibodies secreted by Aspergillus niger

Two different humanized immunoglobulin G1(kappa) antibodies and an Fab' fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex(6)GlcNAc(2) to Hex(15)GlcNAc(2). An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function.     

Contributions of immunoglobulin heavy and light chains to antibody specificity for lysozyme and two haptens

Chain recombination experiments with a set of structurally and/or functionally related antibodies were performed to assess the role of the heavy (H) and light (L) chains in determining antigen specificity. The results demonstrated that specificity for hen egg white lysozyme vs two haptens (dinitrophenyl or galactan) is H chain determined and for one set of proteins could be attributed specifically to the H3 region. In contrast to hapten vs lysozyme specificity, when reassociated molecules derived from structurally unrelated antibodies that bound nonoverlapping epitopes on lysozyme were tested, localization of binding to a particular epitope on lysozyme could be predominated by either H or L chains. Furthermore, in some cases, unique specificities distinct from those of either parental antibody were formed. Replacement of the native L chain with an isotypically homologous L chain was more likely to restore high affinity protein binding than was replacement of a less related L chain. When isotypically homologous L chains were compared in association with the same H chain, fine specificity profiles were sensitive to substitutions in as few as two residues that could be attributed to somatic mutation. These results demonstrate that both affinity and specificity derive from very subtle interactions between H and L chains and provide examples of how VH assembly, VL-VH pairing, and somatic mutation could contribute to development and maturation of the specificity repertoire.     

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.     

人源抗狂犬病毒单克隆抗体Fab段基因的获得和表达

运用噬菌体表面呈现（phage display）技术获得了人源抗狂犬病毒糖蛋白基因工程单克隆抗体Fab段基因及其表达。从狂犬病毒PM株Vero细胞疫苗免疫的人抗凝血中分离获得外周淋巴细胞,提取细胞总RNA,通过RTPCR方法,用一组人IgG Fab基因4特异性引物,从合成的cDNA中扩增了一组轻链和重链Fab段基因,将轻链和重链Fab段基因,将轻链和重链先后克隆入噬菌体载体pComb3,成功地建立了抗狂犬病毒抗原的方法,对此抗体库进行富积筛选表达,成功地获得了抗狂犬病毒的人源单抗Fab段基因及其在大肠杆菌中的有效表达,对其中一株单抗G10进行了较为系统的分析,发现它与一株鼠源中和性狂犬病毒糖蛋白特异性单抗存在竞争,证实该单抗能识别狂犬病毒糖蛋白,其序列资料分析表明,该单抗为一株新的抗狂犬病毒人源基因工程抗体。     

Rabbit variable kappa light chain regions: subgroups contain polypeptides encoded by multiple genes.

Two identical light chain variable regions were identified in anti-streptococcal Group A-variant antibodies elicited in litter-mate rabbits by hyperimmunization with vaccine. In addition, one rabbit produced two additional clonally restricted antibodies to this polysaccharide antigen. The partial amino acid sequence of the light chain of one of these antibodies was identical with the dominant antibody light chain sequence, while the light chain of the other antibody, also partially established, showed significant variations in the framework-associated regions with identical CDRI and II. Since all of these light chains were from a small subset of rabbit kappa light chain pools (b4 allotype) the data suggest, together with other light chains reported in the literature, that more than one copy of variable region genes are present in the germ-line per subgroup. Furthermore, framework associated amino acid substitutions are not random; this suggests the existence of some "ordered" mechanism for linked amino acid substitutions (presumably recombination). Furthermore one light chain can pair with more than one heavy chain to yield functional antibodies.     

Synthesis and self-assembly of a functional monoclonal antibody in transgenic Nicotiana tabacum

Immunoglobulin light and heavy chains are synthesized in mammalian cells as precursors containing a signal peptide. Processing and assembling result in formation of active antibodies. Chimeric genes have been made containing the coding sequence of the barley -amylase signal peptide which has been fused to cDNAs coding for either the mature light or the mature heavy chain of a monoclonal antibody. A plasmid was constructed linking both chimeric genes under the control of plant active promoters in an expression cassette. This DNA fragment was stably integrated into the genome of Nicotiana tabacum by Agrobacterium tumefaciens mediated gene transfer. Synthesis of light and heavy chains and assembly to antibodies was detected in transgenic tobacco tissue using specific secondary antibodies. By electron microscopic immunogold labeling, the presence of assembled antibody could be detected within the endoplasmic reticulum. Affinity chromatography indicated biological activity of the assembled immunoglobulin produced in plant cells. Unexpectedly, a significant amount of assembled antibodies was found within chloroplasts.     

Mutational analysis of arsonate binding by a CRIA+ antibody. VH and VL junctional diversity are essential for binding activity

To assess the impact of various heavy and light chain mutations on p-azophenylarsonate binding, murine antibodies have been produced in insect cells (SF9) utilizing a baculovirus expression system. When expressed in this system, an antibody composed of a canonical CRIA+ heavy and light chain can bind antigen and express idiotype indistinguishably from analogous hybridoma-derived antibodies. Antibodies comprised of either light chains mutant at the V-J junction or heavy chains mutant at the V-D junction were found to be incapable of binding arsonate. In addition, substitutions in the first and second complementarity determining regions of the heavy chain were shown to play a role in arsonate binding, most likely related to affinity maturation targeted at the carrier protein. These results confirm the obligatory role that junctional diversity plays in the generation of arsonate-specific antibodies, as well as extend our understanding of the role of other variable region amino acids in arsonate binding.     

Two-Dimensional nmr studies of the interactions between a peptide of cholera toxin and monoclonal antibodies

To increase our understanding of the molecular basis for antibody specificity and for the cross-reactivity of antipeptide antibodies with native proteins, it is important to study the three-dimensional structure of antibody complexes with their peptide antigens. For this purpose it may not be necessary to solve the structure of the whole antibody complex but rather to concentrate on elucidating the combining site structure, the interactions of the antibody with its antigen, and the bound peptide conformation. To extract the information about antibody–peptide interactions and intramolecular interactions in the bound ligand from the complicated and unresolved spectrum of the Fab–peptide complex (Fab: antibody fragment made of Fv—the antibody fragment composed of the variable regions of the light and heavy chains forming a single combining site for the antigen—the light chain, and the first heavy chain constant regions), an nmr methodology based on measurements of two-dimensional transferred nuclear Overhauser effect (NOE) difference spectra was developed. Using this methodology the interactions of three monoclonal antibodies with a cholera toxin peptide were studied. The observed interactions were assigned to the antibody protons involved by specific deuteration of aromatic amino acids and specific chain labeling, and by using a predicted model for the structure of the antibody combining site. The assigned NOE interactions were translated to restraints on interproton distances in the complex that were used to dock the peptide into calculated models for the antibodies combining sites. Comparison of the interactions of three antibodies against a cholera toxin peptide (CTP3). which differ in their cross-reactivity with the toxin, yields information about the size and conformation of antigenic determinants recognized by the antibodies, the structure of their combining sites, and relationships between antibodies' primary structure and their interactions with peptide antigens. © 1994 John Wiley & Sons, Inc.     

Determination of the bifunctional properties of high molecular weight kininogen by studies with monoclonal antibodies directed to each of its chains

In normal human plasma two forms of kininogen exist, low molecular weight kininogen (LMWK) and high molecular weight kininogen (HMWK). When these proteins are cleaved they are found to have a common heavy chain and bradykinin, but each has a unique light chain. Monoclonal antibodies to the heavy and light chains of HMWK have been developed, and the effects of each on the function of this protein are defined. Initial studies showed that an antibody, C11C1, completely neutralized the coagulant activity of plasma HMWK whereas another antibody, 2B5, did not. On a competitive enzyme-linked immunosorbent assay (CELISA) the C11C1 antibody was consumed by kininogen antigen in normal plasma but not by kininogen antigen in HMWK-deficient plasma. On immunoblot, the C11C1 antibody recognized one kininogen protein in normal plasma and did not recognize any kininogen antigen in HMWK-deficient plasma. These combined studies indicated that the C11C1 antibody was directed to an epitope on the unique 46-kDa light chain of HMWK. In contrast, the 2B5 antibody on a CELISA was consumed by kininogen antigen in both normal plasma and HMWK-deficient plasma but not by total kininogen-deficient plasma. On immunoblot, the 2B5 antibody recognized both kininogens in normal plasma but only LMWK in HMWK-deficient plasma. These combined studies indicated that the 2B5 antibody was directed to the common 64-kDa heavy chain of the plasma kininogens. Utilizing direct binding studies or competition kinetic experiments, the 2B5 and C11C1 antibodies bound with high affinity (1.71 and 0.77 nM, respectively) to their antigenic determinants on the HMWK molecule. The 2B5 antibody did neutralize the ability of HMWK to inhibit platelet calpain. These studies with monoclonal antibodies directed to each of the HMWK chains indicate that HMWK is a bifunctional molecule that can serve as a cofactor for serine zymogen activation and an inhibitor of cysteine proteases.     

通过构建轻链二级库对人源抗TNF-α抗体进行亲和力成熟

通过构建轻链二级库的方法,对人源抗ＴＮＦ－α单抗Ｆａｂ进行轻链置换,并筛选出具有更高亲和力的人源抗ＴＮＦ－α单抗的Ｆａｂ。首先用ＲＴ－ＰＣＲ技术扩增正常人全套抗体轻链基因,并与已获得的人源抗ＴＮＦ－α单抗的重链基因配对,构建人源抗ＴＮＦ－α噬菌体抗体轻链二级库,然后筛选与ＴＮＦ－α具有更高亲和力的克隆,经过三轮的生物淘筛（ｂｉｏｐａｎｎｉｎｇ）,获得了比原来的人源抗ＴＮＦ－α单抗Ｆａｂ具有更高亲和     

Production of an anti-mouse MHC class II monoclonal antibody with biological activity in transgenic tobacco

To produce a monoclonal antibody specific to a mouse major histocompatibility complex (MHC) class II protein, we synthesized the complementary DNAs for the heavy and light chains of a monoclonal antibody by PCR amplification. These cDNAs were then introduced separately into tobacco plant cells. After performing Northern blot analysis to confirm the expression of each of the chain genes in the transformed plants, we constructed transgenic plants expressing both the heavy and light chains by sexual crossing. The expression of the heavy and light chain genes in the sexually crossed plant was confirmed by Northern and Western blot analyses, respectively. Fluorocytometric analysis showed that the plant-derived antibodies, which we purified using a protein G affinity column, bound specifically to target cells that expressed the cognate MHC class II molecules on their cell surfaces. The results of this study demonstrate that a monoclonal antibody against mouse MHC class II proteins can be expressed in transgenic plants. They also show the specific binding activity of plant-derived antibodies to cognate antigens.     

Phenotypic and genotypic characterization of monoclonal anti-digoxin antibodies

Eleven hybridoma cell lines secreting monoclonal anti-digoxin antibody have been produced. They are primarily gamma heavy chain and kappa light chain molecules. Affinity constants for digoxin range from 2 X 10(6) to 3.5 X 10(8) liters/mole. Fine specificity analysis using a series of digoxin congeners demonstrates that an unsaturated lactone ring attached to the aglycone at the C-17 position is necessary for hapten recognition. The impact of other changes in digoxin's structure on antibody binding were also studied. DNA hybridization analysis demonstrates that there are at least three different variable region gene arrangements used to produce the heavy chains of the different hybridoma antibodies. Correlations between antigen binding characteristics and antibody V-gene arrangements are demonstrable.     

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.     

Characterization of Humanized Antibodies Secreted by Aspergillus niger

Two different humanized immunoglobulin G1(κ) antibodies and an Fab′ fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex₆GlcNAc₂ to Hex₁₅GlcNAc₂. An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function.