Novel tri-specific tribodies induce strong T cell activation and anti-tumor effects in vitro and in vivo.

BACKGROUND: Immunotherapy based on Bi-specific T Cell Engagers (TCE) represents one of the most attractive strategy to treat cancers resistant to conventional therapies. TCE are antibody-like proteins that simultaneously bind with one arm to a Tumor Associated Antigen (TAA) on cancer cells and with the other one to CD3 complex on a T-cell to form a TCR-independent immune synapse and circumvent Human Leucocyte Antigen restriction. Among them, the tribodies, such as Tb535H, a bi-specific molecule, made up of a Fab and a scFv domain both targeting 5T4 and another scFv targeting CD3, have demonstrated anti-tumor efficacy in preclinical studies. METHODS: Here, we generated five novel tri-specific and multi-functional tribodies, called 53X tribodies, composed of a 5T4 binding Fab arm and a CD3 binding scFv, but differently from the parental Tb535H, they contain an additional scFv derived from an antibody specific for an immune checkpoint, such as PD-1, PD-L1 or LAG-3. RESULTS: Compared with the parental Tb535H bi-specific T cell engager targeting 5T4, the novel 53X tribodies retained similar binding properties of Tb535H tribody, but showed enhanced anti-tumor potency due to the incorporation of the checkpoint inhibitory moiety. In particular, one of them, called 53L10, a tri-specific T cell engager targeting 5T4, CD3 and PD-L1, showed the most promising anti-tumor efficacy in vitro and led to complete tumor regression in vivo. CONCLUSIONS: The novel tribodies have the potential to become strong and safe therapeutic drugs, allowing to reduce also the cost of production as one single molecule contains three different specificities including the anti-TAA, anti-CD3 and anti-IC binding arms.

Successful treatment of a patient with Budd-Chiari syndrome with main hepatic vein obstruction with percutaneous transluminal angioplasty and stent placement.

A woman in her 40s presented at our department with abdominal fullness. Abdominal computed tomography showed hepatomegaly and ascites, and gastrointestinal endoscopy showed esophageal varices. A diagnosis of Budd-Chiari syndrome (BCS) was confirmed by percutaneous hepatic venography, which detected obstruction of the main hepatic vein. It was treated using percutaneous transluminal angioplasty and metallic stent placement. Rupture of the esophageal varices occurred 5 months later because of the occlusion of the stent lumen; however, she was successfully retreated with further stent placement.

Analysis of the fusion protein gene of Newcastle disease viruses isolated in Japan.

The complete nucleotide sequences of the fusion (F) protein gene of Newcastle disease viruses (NDV) isolated in Japan from 1930 to 2007 (45 strains total) were determined and genetically analyzed. In the deduced amino acid sequences of fusion protein, the 5 potential asparagine-linked glycosylation sites and 10 cysteine residues were all conserved in the NDV examined in this study. The major epitopes involved in virus neutralization are conserved in most of the NDV strains isolated in Japan except a few strains. By virus neutralization test, no major antigenic differences were observed among representative strains of each genotype in Japan. All chickens vaccinated with the B1 strain survived without clinical signs after challenge with 2 NDV strains isolated in Japan (velogenic strains, JP/Ibaraki/2000 and JP/Kagoshima/91), which possess amino acids substitutions involved in virus neutralization in the F protein gene.

A novel genotype of avian infectious bronchitis virus isolated in Japan in 2009.

Avian infectious bronchitis viruses (IBVs) isolated from commercial layer flocks kept in Ibaraki Prefecture in 2009 were genetically and serologically characterized. Reverse transcription-PCR coupled with direct nucleotide sequencing and GenBank BLAST database analysis of the hypervariable region of the S1 subunit of the virus spike gene showed that these isolates are genetically very different from the previously known IBV genotypes in Japan. Furthermore, none of the antisera used in this study neutralized the index isolate (JP/Ibaraki/168-1/2009) in virus neutralization tests. These results suggest that the isolates are a novel IBV genotype in Japan (designated JP-IV).

Genetic characterization of fowl adenoviruses isolated from chickens with hydropericardium syndrome in Japan.

We genetically characterized fowl adenoviruses (serotype 4 FAdV, FAdV-4) isolated from chickens with hydropericardium syndrome (HPS) in Japan by the polymerase chain reaction (PCR) method coupled with direct sequencing. Phylogenetic analysis based on the part of the hexon gene that included the L1 region revealed that all FAdV-4 isolates from chickens with HPS in Japan were identical and were distinguished completely from the cluster including FAdV strains from chickens with HPS in India and Pakistan. This suggested that FAdV-4 from the HPS chickens in India and Pakistan was derived from a common ancestor, but the origin of the FAdV-4 from the HPS chickens in Japan was completely different.

Identification of group I-III avian adenovirus by PCR coupled with direct sequencing of the hexon gene.

Polymerase chain reaction (PCR) coupled with direct sequencing of the product of the hexon gene was applied to avian adenoviruses (formerly group I-III). The expected sizes of DNA fragments were successfully amplified by PCR from all of the group I-III avian adenoviruses with our designed primers. The resulting PCR product contained diagnostically relevant hexon sequences that could be used to identify the group and type of avian adenovirus.

Genotyping of Newcastle disease viruses isolated from 2001 to 2007 in Japan.

Seventeen isolates of Newcastle disease virus (NDV) were obtained from various prefectures in Japan during the years 2001-2007 and were genotypically analyzed by the reverse transcriptase-polymerase chain reaction (RT-PCR) method coupled with direct sequencing. These NDV isolates were classified into three genetic groups that had been reported previously, namely, genotypes I, VI and VII. The isolate from an aigamo duck was classified into genotype I with isolates mainly from waterfowl. All isolates from pigeons were classified into genotype VI, the predominant genotype responsible for most Newcastle disease outbreaks in pigeons. The isolate from a pet bird was classified into genotype VI, distinct from the remaining viruses in genotype VI. All isolates from chickens were classified into genotype VII, the predominant genotype responsible for most Newcastle disease outbreaks in the East Asian countries. Among the isolates from chickens, isolates after 2002 were genetically most closely related with isolates in Korea. The single isolate from a wild cormorant was also classified into genotype VII, although it was different from the recent NDV epidemic strain in Japan.

Genetic diversity of avian infectious bronchitis viruses in Japan based on analysis of s2 glycoprotein gene.

To understand the genetic diversity of the S2 gene of infectious bronchitis viruses (IBV) isolated in Japan, we determined the nucleotide sequences of these IBVs using the reverse transcriptase polymerase chain reaction method coupled with direct sequencing. IBV isolated in Japan were classified into six different groups by phylogenetic analysis based on the S2 gene. However, the classification based on the S2 gene of IBV isolated in Japan was different for some of the strains from those obtained with our previous analysis of the S1 gene. This suggested that genetic recombination between the virus strains classified into different genetic groups had occurred in poultry, and that recombinant viruses might be epidemic in Japan.

Existence of avian infectious bronchitis virus with a European-prevalent 4/91 genotype in Japan.

Eight isolates of infectious bronchitis virus (IBV) were obtained from various prefectures in Japan during 2003-2007 and were genetically analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) coupled with direct sequencing. These IBV isolates were classified into three genetic groups, including two that have already been reported (JP-I and JP-III). The remaining group is related to the 4/91 (also known as 793/B) type, prevalent mainly in European countries, and has not been identified in Japan until now.