In Vitro Nanobody Library Construction by Using Gene Designated-Region Pan-Editing Technology.

Camelid single-domain antibody fragments (nanobodies) are an emerging force in therapeutic biopharmaceuticals and clinical diagnostic reagents in recent years. Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting the large-scale application of nanobodies. In this study, we developed three kinds of gene designated-region pan-editing (GDP) technologies to introduce multiple mutations in complementarity-determining regions (CDRs) of nanobodies in vitro. Including the integration of G-quadruplex fragments in CDRs, which induces the spontaneous multiple mutations in CDRs; however, these mutant sequences are highly similar, resulting in a lack of sequences diversity in the CDRs. We also used CDR-targeting traditional gRNA-guided base-editors, which effectively diversify the CDRs. And most importantly, we developed the self-assembling gRNAs, which are generated by reprogrammed tracrRNA hijacking of endogenous mRNAs as crRNAs. Using base-editors guided by self-assembling gRNAs, we can realize the iteratively diversify the CDRs. And we believe the last GDP technology is highly promising in immunization-free nanobody library construction, and the full development of this novel nanobody discovery platform can realize the synthetic evolution of nanobodies in vitro.

Chimeric antigen receptor-modified macrophages trigger systemic anti-tumour immunity.

Preliminary results and emerging data have shown that lipid droplet high (LDhi ) immunosuppressive cells accumulate in tumour tissues. By tracking and phenotypic profiling of LDhi cells, we find that LDhi CD19+ , LDhi CD11b+ , and LDhi Ly6G+ immune cell populations appear in the spleen, thymus, and tumour tissues in a syngeneic tumour model. Using a contact-dependent reporter system, we discover a LDhi CCR7hi immunosuppressive cell population that migrates from tumour tissues to the spleen and thymus. Hence, we engineered a family of chimeric antigen receptor-modified macrophages (CAR-Ms) that direct macrophages to CCR7-positive cells and show that the cytosolic domain from Mer receptor tyrosine kinase (MerTK) triggers tumour cell cytotoxicity by the CAR-Ms. In vivo, CCR7-targeted CAR-Ms suppressed tumour growth and prolonged survival by preventing metastasis and by inducing systemic anti-tumour immunity through retarding the migration of LDhi CCR7hi immunosuppressive cells from tumour tissues to distal immune organs, indicating an important role for CCR7 in tumour cell-induced immune tolerance. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Characterization and overexpression of a glycosyl hydrolase family 16 ß-agarase Aga0917 from Pseudoalteromonas fuliginea YTW1-15-1.

A gene (aga0917) encoding a putative ß-agarase was identified from the genome of Pseudoalteromonas fuliginea YTW1-15-1. The nucleotide sequence analysis revealed that aga0917 had significant homology to the agarase genes of the GH16 family. aga0917 encodes a putative protein of 290 amino acids with an estimated molecular mass of 32.5 kDa, including a 21-amino acid signal peptide. A gene fragment encoding only the putative mature form of Aga0917 (269 amino acids) was overexpressed in Escherichia coli BL21 (DE3) pLysS as a 6 × histine-tagged fusion protein (rmAga0917). The Km, Vmax, and kcat for agarose of rmAga0917 were 39.6 mg/mL, 334 (U/mg) of protein, and 178 (1/s), respectively. According to the results of thin-layer chromatography and mass spectrometry analysis, the main end product from agarose with rmAga0917 was neoagarotetraose, in addition to a small amount of neoagarobiose. Notably, the recombinant protein rmAga0917 showed optimum activity at 60°C and retained approximately 100% agarolytic activity after being kept at 40°C for 1 h and 57% residual activity after incubation at 50°C for 1 h. The rmAga0917 exhibited maximum agarase activity at pH 6.0, and retained more than 80% of activity after incubation over a range of pH 4.0-9.0 for 1 h at 4°C.

Thalassotalea atypica sp. nov., isolated from seawater, and emended description of Thalassotalea eurytherma.

A novel Gram-stain-negative, straight or slightly curved rod-shaped, non-spore-forming, non-flagellated, strictly aerobic strain, designated RZG4-3-1T, was isolated from coastal seawater of Rizhao, China (119.625° E 35.517° N). The organism grew optimally at 24-28 °C, at pH 7.0 and in the presence of 2.0 % (w/v) NaCl. The strain required seawater or artificial seawater for growth, and NaCl alone did not support growth. Strain RZG4-3-1T contained ubiquinone 8 (Q-8) as the major respiratory quinone and contained C16 : 1ω7c and/or C16 : 1ω6c and C16 : 0 as the dominant fatty acids. The polar lipids of strain RZG4-3-1T were phosphatidylethanolamine, phosphatidylglycerol and one unidentified aminophospholipid. The DNA G+C content of strain RZG4-3-1T was 40.1 mol%. Strain RZG4-3-1T exhibited the highest 16S rRNA gene sequence similarity value (96.0 %) to Thalassotalea eurytherma JCM 18482T. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain RZG4-3-1T belonged to the genus Thalassotalea. On the basis of polyphasic analyses, strain RZG4-3-1T represents a novel species of the genus Thalassotalea, for which the name Thalassotalea atypica sp. nov. is proposed. The type strain is RZG4-3-1T (=JCM 31894T=KCTC 52745T=MCCC 1K03276T). An emended description of Thalassotalea eurytherma is also provided.