Fast on-rates of chimeric antigen receptors enhance the sensitivity to peptide MHC via antigen rebinding.

Chimeric antigen receptor (CAR) is a synthetic receptor that induces T cell-mediated lysis of abnormal cells. As cancer driver proteins are present at low levels on the cell surface, they can cause weak CAR reactivity, resulting in antigen sensitivity defects and consequently limited therapeutic efficacy. Although affinity maturation enhances the efficacy of CAR-T cell therapy, it causes off-target cross-reactions resulting in adverse effects. Preferentially expressed antigen in melanoma (PRAME) is an intracellular oncoprotein that is overexpressed in various tumors and restricted in normal tissues, except the testis. Therefore, PRAME could be an ideal target for cancer immunotherapy. In this study, we developed an experimental CAR system comprising six single-chain variable fragments that specifically recognizes the PRAMEp301/HLA-A∗24:02 complex. Cell-mediated cytotoxicity was demonstrated using a panel of CARs with a wide range of affinities (KD = 10-10-10-7 M) and affinity modulation. CAR-T cells with fast on-rates enhance antigen sensitivity by accelerating the killing rates of these cells. Alanine scanning data demonstrated the potential of genetically engineered CARs to reduce the risk of cross-reactivity, even among CARs with high affinities. Given the correlation between on-rates and dwell time that occurs in rebinding and cell-mediated cytotoxicity, it is proposed that CAR-binding characteristics, including on-rate, play a pivotal role in the lytic capacity of peptide-major histocompatibility complex-targeting CAR-T cells, thus facilitating the development of strategies whereby genetically engineered CARs target intracellular antigens in cancer cells to lyse the cells.

Vesicular Integral-Membrane Protein 36 Is Involved in the Selective Secretion of Fucosylated Proteins into Bile Duct-like Structures in HepG2 Cells.

Fucosylated proteins are widely used as biomarkers of cancer and inflammation. Fucosylated alpha-fetoprotein (AFP-L3) is a specific biomarker for hepatocellular carcinoma. We previously showed that increases in serum AFP-L3 levels depend on increased expression of fucosylation-regulatory genes and abnormal transport of fucosylated proteins in cancer cells. In normal hepatocytes, fucosylated proteins are selectively secreted in the bile duct but not blood. In cases of cancer cells without cellular polarity, this selective secretion system is destroyed. Here, we aimed to identify cargo proteins involved in the selective secretion of fucosylated proteins, such as AFP-L3, into bile duct-like structures in HepG2 hepatoma cells, which have cellular polarity like, in part, normal hepatocytes. α1-6 Fucosyltransferase (FUT8) is a key enzyme to synthesize core fucose and produce AFP-L3. Firstly, we knocked out the FUT8 gene in HepG2 cells and investigated the effects on the secretion of AFP-L3. AFP-L3 accumulated in bile duct-like structures in HepG2 cells, and this phenomenon was diminished by FUT8 knockout, suggesting that HepG2 cells have cargo proteins for AFP-L3. To identify cargo proteins involved in the secretion of fucosylated proteins in HepG2 cells, immunoprecipitation and the proteomic Strep-tag system experiments followed by mass spectrometry analyses were performed. As a result of proteomic analysis, seven kinds of lectin-like molecules were identified, and we selected vesicular integral membrane protein gene VIP36 as a candidate of the cargo protein that interacts with the α1-6 fucosylation (core fucose) on N-glycan according to bibliographical consideration. Expectedly, the knockout of the VIP36 gene in HepG2 cells suppressed the secretion of AFP-L3 and other fucosylated proteins, such as fucosylated alpha-1 antitrypsin, into bile duct-like structures. We propose that VIP36 could be a cargo protein involved in the apical secretion of fucosylated proteins in HepG2 cells.

Establishment and characterization of a fucosylated α-fetoprotein-specific monoclonal antibody: a potential application for clinical research.

The Lens culinaris agglutinin (LCA)-reactive fraction of α-fetoprotein (AFP-L3) is a well-known cancer biomarker for hepatocellular carcinoma (HCC) with very high specificity. Because LCA recognizes only bi-antennary N-glycans with a core fucose, some of fucosylated AFP in HCC patients may not be detected. Then glycan antibodies, which recognize both specific glycan and protein, are desired for glycobiology. Here, we successfully established a novel glycan antibody for fucosylated AFP and demonstrated its potential clinical application. After immunization with a fucosylated AFP peptide, positive screening was performed for fucosylated AFP peptides using solid-phase enzyme-linked immunosorbent assay (ELISA). The newly developed antibody was designated: fucosylated AFP-specific mAb (FasMab). Western blot analysis showed that FasMab reacted with AFP produced by HepG2 cells, but not with AFP produced by α-1,6-fucosyltransferase deficient HepG2 cells. The specific binding of FasMab to fucosylated AFP was confirmed with ELISA as well as western blot analysis. A preliminary high sensitivity chemiluminescence enzyme immunoassay kit showed increased levels of fucosylated AFP in the sera of patients with HCC, but not in the sera of normal patients, or patients with chronic liver diseases. Thus, the novel glycan antibody, FasMab, is a promising tool to study fucosylated AFP with clinical and basic research applications.

Characterization of glycoengineered anti-HER2 monoclonal antibodies produced by using a silkworm-baculovirus expression system.

Silkworm-baculovirus expression systems are efficient means for the production of recombinant proteins that provide high expression levels and post-translational modifications. Here, we characterized the stability, glycosylation pattern and antibody-dependent cell-mediated cytotoxicity activity of anti-HER2 monoclonal antibodies containing native or glycoengineered mammalian-like N-glycans that were produced by using a silkworm-baculovirus expression system. Compared with a monoclonal antibody produced by using a Chinese hamster ovary cell expression system, the glycoengineered monoclonal antibody had comparable thermal stability and a higher antibody-dependent cell-mediated cytotoxicity activity. These results suggest that silkworm-baculovirus expression systems are next-generation expression systems potentially useful for the cost-effective production of therapeutic antibodies.

A novel endonuclease that may be responsible for damaged DNA base repair in Pyrococcus furiosus.

DNA is constantly damaged by endogenous and environmental influences. Deaminated adenine (hypoxanthine) tends to pair with cytosine and leads to the A:T→G:C transition mutation during DNA replication. Endonuclease V (EndoV) hydrolyzes the second phosphodiester bond 3' from deoxyinosine in the DNA strand, and was considered to be responsible for hypoxanthine excision repair. However, the downstream pathway after EndoV cleavage remained unclear. The activity to cleave the phosphodiester bond 5' from deoxyinosine was detected in a Pyrococcus furiosus cell extract. The protein encoded by PF1551, obtained from the mass spectrometry analysis of the purified fraction, exhibited the corresponding cleavage activity. A putative homolog from Thermococcus kodakarensis (TK0887) showed the same activity. Further biochemical analyses revealed that the purified PF1551 and TK0887 proteins recognize uracil, xanthine and the AP site, in addition to hypoxanthine. We named this endonuclease Endonuclease Q (EndoQ), as it may be involved in damaged base repair in the Thermococcals of Archaea.

Biochemical characterization of endonuclease V from the hyperthermophilic archaeon, Pyrococcus furiosus.

Endonuclease V (Endo V) is a DNA repair enzyme that recognizes deoxyinosine and cleaves the second phosphodiester bond on the 3' side of the deaminated base lesion. A database search revealed the presence of homologous genes for Endo V in most archaeal species, but the absence in some methanogenic species. We cloned a gene encoding the sequence homologous to Escherichia coli Endo V from the genome of the hyperthermophilic euryarchaeon, Pyrococcus furiosus and purified gene product (PfuEndoV) to homogeneity. In vitro characterization showed that PfuEndoV possesses specific endonuclease activity for the deoxyinosine-containing DNA strand. The activity of the enzyme was maximal at 90°C. Stable complex formation between PfuEndoV and nicked DNA produced by the cleavage reaction was detected by gel mobility shift assays. The molecular mechanisms of the inosine repair pathway including Endo V in the archaeal cells are discussed. Interestingly, PfuEndoV cleaved inosine-containing RNA strands as well as DNA substrates. PfuEndoV may also be involved in RNA metabolism.