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Abstract Background Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging. Results We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals. Conclusions Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.

Abstract Background During the development of a therapeutic antibody, large numbers of monoclonal antibodies are required to screen for those that are best suited for the desired activity. Although the single cell-based immunoglobulin variable gene cloning technique is a powerful tool, the current methods remain an obstacle to the rapid production of large numbers of recombinant antibodies. Results We have developed a novel overlap extension polymerase chain reaction, the target-selective joint polymerase chain reaction (TS-jPCR), and applied it to the generation of linear immunoglobulin gene expression constructs. TS-jPCR is conducted using a PCR-amplified immunoglobulin variable gene and an immunoglobulin gene-selective cassette (Ig-cassette) that contains all essential elements for antibody expression and overlapping areas of immunoglobulin gene-specific homology. The TS-jPCR technique is simple and specific; the 3'-random nucleotide-tailed immunoglobulin variable gene fragment and the Ig-cassette are assembled into a linear immunoglobulin expression construct, even in the presence of nonspecifically amplified DNA. We also developed a robotic magnetic beads handling instrument for single cell-based cDNA synthesis to amplify immunoglobulin variable genes by rapid amplification of 5' cDNA ends PCR. Using these methods, we were able to produce recombinant monoclonal antibodies from large numbers of single plasma cells within four days. Conclusion Our system reduces the burden of antibody discovery and engineering by rapidly producing large numbers of recombinant monoclonal antibodies in a short period of time.

Abstract Background Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies. Results We developed a single-step cloning method, target-selective homologous recombination (TS-HR), in which PCR-amplified immunoglobulin variable genes were selectively inserted into vectors, even in the presence of nonspecifically amplified DNA. TS-HR utilizes Red/ET-mediated homologous recombination with a target-selective vector (TS-vector) with unique homology arms on its termini. Using TS-HR, immunoglobulin variable genes were cloned directly into expression vectors by co-transforming unpurified PCR products and the TS-vector into E. coli. Furthermore, the high cloning specificity of TS-HR allowed plasmids to be extracted from pools of transformed bacteria without screening single colonies for correct clones. We present a one-week protocol for the production of recombinant mouse monoclonal antibodies from large numbers of single plasma cells. Conclusion The time requirements and limitations of traditional cloning procedures for the production of recombinant immunoglobulins have been significantly reduced with the development of the TS-HR cloning technique.

Iβ6-N-acetylglucosaminyltransferase (IGnT) catalyzes the branching of poly-N-acetyllactosamine carbohydrate chains. In both humans and mice, three spliced forms of IGnT have been identified, and a common exon is present in all of them. We generated mice deficient in the common exon to understand the physiological function of poly-N-acetyllactosamine branching. IGnT activity was abolished in the stomach, kidney, bone marrow, and cerebellum of the deficient mice, while a low level of the activity persisted in the small intestine. Immunohistochemical analysis confirmed the loss of I antigen from the lung, stomach, and kidney. The deficient mice had reduced spontaneous locomotive activity. The number of peripheral blood lymphocytes was also reduced and renal function decreased in the deficient mice. Furthermore, in aged mice, vacuolization occurred in the kidney, and epidermoid cysts were frequently formed. However, cataracts did not develop earlier in the deficient mice. Decreased levels of lysosomal proteins, LAMP-2 and synaptotagmin VII, were found in the kidney of the deficient mice and correlated with renal abnormalities. [ABSTRACT FROM AUTHOR]

A novel form of murine β-1,6-N-acetylglucosaminyltransferase that forms branches in poly-N-acetyllactosamines (designated as IGnT B) was cloned based on sequence homology to the known IGnT (designated as IGnT A). When expressed as proteins, IGnT B showed higher specific activity than IGnT A. The C-terminal 1/4 of IGnT B was identical to that of IGnT A, while the rest of the predicted sequences showed 63% identity. Genomic analysis indicated that IGnT A and IGnT B were derived by alternative splicing; the unique portion was encoded by exon 1, and the common portion was encoded by exons 2 and 3. IGnT B showed an expression profile closely related to that of IGnT A and was strongly expressed in the liver, kidney and intestine, and moderately in the mammary gland, submaxially gland, embryonic stem cells, and embryonal carcinoma cells. The specificity of IGnT B examined using various substrates was indistinguishable from that of IGnT A, which is classified as the central acting IGnT (cIGnT). Thus, IGnT B acted on Galβ1–4GlcNAcβ1–3Galβ1–4Glc, but not on GlcNAcβ1–3Galβ1–4Glc. It formed branches in both of the internal galactosyl residues of Galβ1–4Glc-NAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4Glc, and prolonged incubation resulted in production of the di-branched oligosaccharide. Although addition of sialic acid to the terminal galactosyl residue did not abolish the acceptor activity, α2–6 sialylation was a preferred one as compared to α2–3 sialylation. [ABSTRACT FROM PUBLISHER]

The sialyl-Tn (sTn) antigen is a well known cancer-associated antigen, the expression of which is related to the prognosis of cancer patients. We aimed to isolate a human gene encoding an N-acetylgalactosamine α2,6-sialyltransferase which synthesizes sTn antigen, and to characterize the enzyme. Degenerate primers encoding sialyl motifs were used for the polymerase chain reaction to amplify complementary DNAs prepared from RNAs of human pyloric mucosae with intestinal metaplasia, which abundantly expressed sTn antigen, followed by screening of full-length cDNAs using the amplified DNA fragment as a probe. We isolated two human cDNA clones, long-form (2.46 kb) and short-form (2.23 kb) cDNAs. The former encodes an active enzyme with a predicted 600 amino acid sequence. The latter, a splice-variant of the long-form, encodes an inactive enzyme. HCT15 human colorectal cancer cells stably expressing the long-form cDNA expressed sTn epitopes on O-glycans. The long form cDNA was considered to encode a human homologue of chick ST6GalNAc I for the following reasons: (1) the putative amino acid sequence showed greater homology to that of chick ST6GalNAc I (55%) compared to other sialyltransferases, (2) it encodes the extraordinarily long stem region that is a typical feature of chick ST6GalNAc I, and (3) the substrate specificity was very similar to that of chick ST6GalNAc I. In situhybridization demonstrated that the localization of transcripts correlated well with that of sTn antigen in gastric cancer cells and Goblet cells in intestinal metaplastic glands. Thus, we determined that the long-form cDNA of the human ST6GalNAc I gene encodes the probable candidate for the human sTn synthase(s). [ABSTRACT FROM PUBLISHER]

Chondroitin 6‐sulfotransferase (C6ST) catalyzes the transfer of sulfate from 3′‐phosphoadenosine 5′‐phosphosulfate to position 6 of the N‐acetylgalactosamine residue of chondroitin. Using chick C6ST cDNA as a probe, we cloned the cDNA of mouse C6ST. The mouse enzyme was predicted to be composed of 472 amino acids, and exhibited 71% sequence identity with the chicken enzyme. The mouse and chicken catalytic domains exposed to the luminal side exhibited 81% identity, while the homology of the remaining regions was less. Transfection and expression of the mouse cDNA in COS‐7 cells yielded C6ST activity. Keratan sulfate sulfotransferase activity, which was simultaneously expressed, amounted to 3% of the C6ST activity, this value being significantly lower than that observed in the case of the chicken enzyme. Mouse C6ST mRNA was strongly expressed in the spleen, lung, and eye. In situ hybridization revealed that the transcript was localized in stromal cells in the marginal zone and red pulp of the spleen, and stromal cells in the bone marrow. Fluorescence in situ hybridization analysis revealed the gene is located in mouse chromosome 9. [ABSTRACT FROM PUBLISHER]

Intracellular tumor-associated antigens are targeted by antibodies known as T-cell receptor mimic antibodies (TCRm-Abs), which recognize T-cell epitopes with better stabilities and higher affinities than T-cell receptors. However, TCRm-Abs have been proven difficult to produce using conventional techniques. Here, we developed TCRm-Abs that recognize the survivin-2B-derived nonamer peptide, AYACNTSTL (SV2B80-88), presented on HLA-A*24 (SV2B80-88/HLA-A*24) from immunized mice by using a fluorescence-activated cell sorting-based antigen-specific plasma cells isolation method combined with a high-throughput single-cell-based immunoglobulin-gene-cloning technology. This approach yielded a remarkable efficiency in generating candidate antibody clones that recognize SV2B80-88/HLA-A*24. The screening of the antibody clones for their affinity and ability to bind key amino-acid residues within the target peptide revealed that one clone, #21-3, specifically recognized SV2B80-88/HLA-A*24 on T2 cells. The specificity of #21-3 was further established through survivin-2B-positive tumor cell lines that exogenously or endogenously express HLA-A*24. A bispecific T-cell engager comprised of #21-3 and anti-CD3 showed specific cytotoxicity towards cells bearing SV2B80-88/HLA-A*24 by recruiting and activating T-cells in vitro. The efficient development of TCRm-Ab overcomes the limitations that hamper antibody-based immunotherapeutic approaches and enables the targeting of intracellular tumor-associated antigens. [ABSTRACT FROM AUTHOR]