Production of IgG1-based bispecific antibody without extra cysteine residue via intein-mediated protein trans-splicing.

A major class of bispecific antibodies (BsAbs) utilizes heterodimeric Fc to produce the native immunoglobulin G (IgG) structure. Because appropriate pairing of heavy and light chains is required, the design of BsAbs produced through recombination or reassembly of two separately-expressed antigen-binding fragments is advantageous. One such method uses intein-mediated protein trans-splicing (IMPTS) to produce an IgG1-based structure. An extra Cys residue is incorporated as a consensus sequence for IMPTS in successful examples, but this may lead to potential destabilization or disturbance of the assay system. In this study, we designed a BsAb linked by IMPTS, without the extra Cys residue. A BsAb binding to both TNFR2 and CD30 was successfully produced. Cleaved side product formation was inevitable, but it was minimized under the optimized conditions. The fine-tuned design is suitable for the production of IgG-like BsAb with high symmetry between the two antigen-binding fragments that is advantageous for screening BsAbs

p300/CBP-associated factor (P/CAF) interacts with nuclear respiratory factor-1 to regulate the UDP-N-acetyl-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyltransferase-3 gene.

We demonstrated recently that expression of the UDP- N -acetyl-alpha-D-galactosamine: polypeptide N -acetylgalactosaminyltrans-ferase-3 (GalNAc-T3) gene is restricted to epithelial glands [Nomoto, Izumi, Ise, Kato, Takano, Nagatani, Shibao, Ohta, Imamura, Kuwano, Matsuo, Yamada, Itoh and Kohno (1999) Cancer Res. 59, 6214-6222]. In the present study, we show that sodium butyrate treatment of human breast cancer MCF-7 cells transcriptionally activates the GalNAc-T3 gene. Transient transfection of plasmids containing a reporter gene under the control of GalNAc-T3 indicated that several transcriptional elements are involved in response to sodium butyrate, with the nuclear respiratory factor-1 (NRF-1)-binding motif located between -88 and -77nt being the most important. Incubation of a labelled probe encompassing the NRF-1-binding motif with a nuclear extract of sodium butyrate-treated MCF-7 cells yielded a higher level of specific DNA-protein complex versus controls. Flag-tagged NRF-1 expressed in MCF-7 cells can bind to the NRF-1-binding motif of the GalNAc-T3 promoter. Nuclear content of NRF-1 remained constant in MCF-7 cells treated with or without sodium butyrate. Moreover, NRF-1 interacts with and is acetylated by p300/CBP-associated factor (P/CAF). Acetylation of NRF-1 enhances DNA binding. Co-transfection of the GalNAc-T3 reporter plasmid with either NRF-1 or P/CAF expression plasmid resulted in the activation of the GalNAc-T3 promoter. These results indicate a correlation between acetylation of NRF-1 by P/CAF and the butyrate-induced expression of the GalNAc-T3 gene. Additionally, induced expression of P/CAF may be a component of the adenocarcinoma differentiation process

Development of a 1:1-binding biparatopic anti-TNFR2 antagonist by reducing signaling activity through epitope selection

Abstract Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we develop BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of antibody variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulate the size of BpAb–TNFR2 immunocomplexes to result in controlled agonistic activities. Our series of results indicate that the relative positions of the two epitopes recognized by the BpAb are critical for controlling its signaling activity. One particular antagonist, Bp109-92, binds TNFR2 in a 1:1 manner without unwanted signal transduction, and its structural basis is determined using cryo-electron microscopy. This antagonist suppresses the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions

Physical interaction of tumour suppressor p53/p73 with CCAAT-binding transcription factor 2 (CTF2) and differential regulation of human high-mobility group 1 (HMG1) gene expression.

The CCAAT-binding transcription factor (CTF)/nuclear factor I (NF-I) group of cellular DNA-binding proteins recognizes the sequence GCCAAT and is implicated in eukaryotic transcription, as well as DNA replication. Molecular analysis of human CTF/NF-I cDNA clones revealed multiple mRNA species that contain alternative coding regions, apparently as a result of differential splicing. Expression and functional analysis established that individual gene products can bind to GCCAAT recognition sites and serve as both promoter-selective transcriptional activators and initiation factors for DNA replication. The interaction between CTF2 and p53/p73 was shown to modulate their ability to regulate transcription of their respective target genes. In the present paper, we report that p53 down-regulates the activity of the high mobility group 1 (HMG1) gene promoter, whereas p73alpha up-regulates the activity of this promoter. Furthermore, CTF2 transactivates p53-induced p21 promoter activity, but inhibits p73alpha-induced p21 promoter activity. Using deletion mutants, we found that the DNA-binding domains of both p53 and p73alpha are required for physical interaction with CTF2 via the regions between amino acid residues 161 and 223, and 228 and 312 respectively. CTF2 enhances the DNA-binding activity of p53 and inhibits the DNA-binding activity of p73alpha. These results provide novel information on the functional interplay between CTF2 and p53/p73 as important determinants of their function in cell proliferation, apoptosis, DNA repair and cisplatin resistance

FCRL1 on chronic lymphocytic leukemia, hairy cell leukemia, and B-cell non-Hodgkin lymphoma as a target of immunotoxins

FCRL1 (Fc receptor–like 1) is a cell-surface membrane protein belonging to FCRL family and is preferentially expressed on B cells. To evaluate FcRL1 as an immunotherapy target for B-cell malignancies, we prepared anti-FCRL1 mAbs without cross-reactivity to other FCRL family proteins and analyzed FCRL1 protein expression on malignant cells from patients and on B-cell lines. Frequent FCRL1 expression was observed by flow cytometry on 12 B-cell non-Hodgkin lymphoma (B-NHL) cell lines and many patient samples: 12 of 14 chronic lymphocytic leukemia (CLL), 7 of 7 follicular lymphoma (FL), 13 of 17 hairy cell leukemia (HCL), and 2 of 3 mantle cell lymphoma (MCL). Two recombinant immunotoxins, E3(Fv)-PE38 and E9(Fv)-PE38, were constructed. Both immunotoxins bound to FCRL1-positive cells with similar affinities (3.4 and 3.2 nM) and were cytotoxic to cell lines, but E9(Fv)-PE38 was 4- to 20-fold more cytotoxic than E3(Fv)-PE38. The concentrations that inhibited response by 50% (IC50s) of E9(Fv)-PE38 on 11 different FCRL1-positive cell lines ranged from 1.0 ng/mL to 90 ng/mL and correlated with the FCRL1 expression levels. Our results suggest that anti-FCRL1 immunotoxin E9(Fv)-PE38 exhibits remarkably specific cytotoxicity and merits further evaluation for the treatment of FCRL1-positive malignancies, including CLL, HCL, FL, MCL, and other B-NHL

Infective Endocarditis from Furuncle with Meningitis Complication Caused by Methicillin-resistant Staphylococcus aureus

Infective endocarditis (IE) may be acquired in the community as community-acquired (CA) IE or in the healthcare setting. In Japan, cases of CA-methicillin-resistant Staphylococcus aureus (MRSA) infection as skin infection have been increasing. CA-MRSA strains, including the USA300 clone, have higher pathogenicity and are more destructive to tissue than healthcare-associated MRSA strains because of the toxins they produce, including arginine-catabolic mobile element (ACME) and Panton-Valentine leukocidin (PVL). However, only a few IE cases induced by USA300 have been reported. We herein report a 64-year-old man who developed CA-IE from a furuncle caused by USA300 MRSA producing PVL and ACME, which resulted in complications of meningitis