Construction of stable membranal CMTM6-PD-L1 full-length complex to evaluate the PD-1/PD-L1-CMTM6 interaction and develop anti-tumor anti-CMTM6 nanobody

Article

Author: Gong, Li-Kun ; Yu, Xiao-Lu ; Geng, Yong ; Long, Yi-Ru ; Jia, Xiao-Min ; Chen, Run-Qiu

CKLF (chemokine-like factor)-MARVEL transmembrane domain containing protein 6 (CMTM6) is a novel regulator to maintain the stability of PD-L1. CMTM6 can colocalize and interact with PD-L1 on the recycling endosomes and cell membrane, preventing PD-L1 from lysosome-mediated degradation and proteasome-mediated degradation thus increasing the half-life of PD-L1 on the cell membrane. The difficulties in obtaining stable full-length PD-L1 and CMTM6 proteins hinder the research on their structures, function as well as related drug development. Using lauryl maltose neopentyl glycol (LMNG) as the optimized detergent and a cell membrane mimetic strategy, we assembled a stable membrane-bound full-length CMTM6-PD-L1 complex with amphipol A8-35. When the PD-1/PD-L1-CMTM6 interactions were analyzed, we found that CMTM6 greatly enhanced the binding and delayed the dissociation of PD-1/PD-L1, thus affecting immunosuppressive signaling and anti-apoptotic signaling. We then used the CMTM6-PD-L1 complex as immunogens to generate immune repertoires in camels, and identified a functional anti-CMTM6 nanobody, called 1A5. We demonstrated that the anti-CMTM6 nanobody greatly decreased T-cell immunosuppression and promoted apoptotic susceptibility of tumor cells in vitro, and mainly relied on the cytotoxic effect of CD8⁺ T-cells to exert tumor growth inhibitory effects in CT26 tumor-bearing mice. In conclusion, the stable membrane-bound full-length CMTM6-PD-L1 complex has been successfully used in studying PD-1/PD-L1-CMTM6 interactions and CMTM6-targeting drug development, suggesting CMTM6 as a novel tumor immunotherapy target.

01 Dec 2020·Applied Microbiology and BiotechnologyQ3 · ENGINEERING & TECHNOLOGY

Development of a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay for detecting canine distemper virus

Q3 · ENGINEERING & TECHNOLOGY

Article

Author: Liu, Baoyuan ; Zhang, Lu ; Zhou, En-Min ; Ji, Pinpin ; Zhang, Jingfei ; Zhang, Yuan ; Li, Yaning ; Zhao, Qin ; Zhao, Jiakai ; Sun, Yani ; Xu, Gang

AbstractCanine distemper virus (CDV) infection causes mass mortality in diverse carnivore species. For effective virus surveillance, rapid and sensitive assays are needed to detect CDV in field samples. In this study, after BABL/c mice were immunized with recombinant CDV-fusion (F) protein, monoclonal antibodies (mAbs) against recombinant CDV-F protein (designated 1A5, 1A6, and 7D5) were produced using traditional hybridoma cell technology. Next, capture antibody (1A6, 800 ng/well) and horseradish peroxidase (HRP)–conjugated detection antibody (HRP-7D5, 1:100, 500 ng/well) were used in a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay (ELISA) for CDV detection after optimization of both mAb amounts per well using a checkerboard titration test. Based on sandwich ELISA test results for 120 known CDV-negative samples, the cutoff value for a positive result was set to an OD450 nm value ≥ 0.196. As compared with test results obtained from commercial immune colloidal gold test strips, the low limits of detection for the two assays were revealed to be 100 TCID50 per 100 μL. In addition, the sandwich ELISA agreed 100% and 96.4% with commercial immune colloidal gold test strips when testing serum and stool samples. The sandwich ELISA assay provided statistically similar CDV detection. Thus, the sandwich ELISA developed here to detect CDV in fecal and serum samples provided good sensitivity, high specificity, and good reproducibility and should serve as an ideal method for large-scale surveillance of CDV infections in carnivores.Key points• Three CDV mAbs that recognized different epitopes and bound to virion were generated.• The sandwich ELISA based mAbs to detect CDV in fecal and serum samples was developed.• The sandwich ELISA is an ideal method for detecting CDV infections in the field.

01 Aug 2020·Veterinary MicrobiologyQ2 · AGRICULTURAL & FORESTRY SCIENCE

Identification of linear B cell epitopes on VP1 and VP2 proteins of Senecavirus A (SVA) using monoclonal antibodies

Q2 · AGRICULTURAL & FORESTRY SCIENCE

Article

Author: Bai, Juan ; Fan, Hui ; Zhu, Huixin ; Jiang, Ping ; Wang, Xianwei ; Li, Shihai ; Zhou, Erxuan ; Shi, Mengyu

Senecavirus A (SVA), previously called Seneca Valley virus, belongs to the family Picornaviridae, species Senecavirus A, in the Senecavirus genus, and can cause vesicular lesions in sows and acute death in piglets. In this study, recombinant VP1 and VP2 proteins were expressed in prokaryotic expression system and used to generate eight monoclonal antibodies (mAbs) against VP1 or VP2 protein. And all of the mAbs reacted specifically with SVA virus by both Western blot and indirect immunofluorescence assay (IFA). The resurts showed that all of the epitopes aganist these mAbs were B cell linear epitopes. To map the epitopes, both Western blot and indirect enzyme-linked immunosorbant assay (indirect ELISA) were performed. The epitope ²¹GELAAP²⁶ recognized by mAb 1G9, was likely to be a significant B cell epitope due to the high antigenic index and the fully exposure on the surface of the VP1. Other mAbs were recognized by VP2 protein. MAbs 1E7 and 8E8 recognized the same epitope at ¹²DRVITQT¹⁸, 1A5 recognized the epitope at ⁷¹WTKAVK⁷⁶, 1G2 recognized the epitope at ⁹⁸GGAFTA¹⁰³, 9D2 and 6B11 recognized the same epitope at ¹⁵⁰KSLQELN¹⁵⁶, and 7E4 recognized the epitope at ²⁴⁸YKEGAT²⁵³. Alignment of amino acids revealed that four epitopes were completely conserved among all SVA strains, including ²¹GELAAP²⁶, ⁷¹WTKAVK⁷⁶, ⁹⁸GGAFTA¹⁰³, and ²⁴⁸YKEGAT²⁵³. Interestingly, there were some amino acid mutations in ¹²DRVITQT¹⁸ and ¹⁵⁰KSLQELN¹⁵⁶, but no significant difference was detected on the reaction intensity between epitopes and the corresponding mAbs. This is the first report about the SVA epitopes, which will benefit to the study of viral pathogenic mechanism, vaccine design, as well as the establishment of detection methods.

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Indication	Highest Phase	Country/Location	Organization	Date
Neoplasms	Preclinical	CN	Shanghai Institute of Materia Medica Chinese Academy of Sci	22 Nov 2022

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