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Use of chimeric antigen receptors (CARs), as an immune cell therapy, has generated excellent clinical outcomes against hematologic tumors in recent years. Among them, the CAR-NK (natural killer) therapy has shown better efficacy, and less toxicity, than chimeric antigen receptor T-cell (CAR-T) therapy. In our phase II clinical trials, administering chimeric costimulatory converting receptor (CCCR)-NK92 cells on advanced non-small cell lung cancer patients proved efficacious in cell and animal experiments. However, we observed occurrence of cytokine release syndrome (CRS), a rare and unexpected side effect, never reported before during CAR-NK therapy. Here, we provide a detailed report of the patient's case, emphasize on the need to pay attention to CRS in NK cell therapy, and suggest improvements that will minimize potential toxicity. [ABSTRACT FROM AUTHOR]

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 LDhiCD19+, LDhiCD11b+, and LDhiLy6G+ 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 LDhiCCR7hi 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 LDhiCCR7hi 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. [ABSTRACT FROM AUTHOR]

Objectives: synNotch receptors can get different inputs about the outside environment into cells; however, synNotch system doesn't work for soluble ligands. This study aims to explore the activation efficiency of different ligand carriers on synNotch system. Methods: SynNotch-based contact-dependent activation system was constructed in 293T cells (293T-synNotch), then the synNotch receptor ligands eGFP proteins were provided by three different carriers: 400 nm Ni magnetic beads (Mag Ni-eGFP), 300 nm carboxyl modified Fe3O4 magnetic beads (Mag COOH-deGFP), and 4T1 cell (4T1-mGFP). Three eGFP carriers were incubated with 293T-synNotch, the activation efficiencies of these three eGFP carriers were evaluated by fluorescence microscopy. Results: The 293T-synNotch cell activated by Mag Ni-eGFP shows no observable red fluorescence; when Mag COOH-deGFP are incubated with 293T-synNotch, about 50‰ cells with red fluorescence appeared, also eGFP proteins have not dropped off from the Mag COOH-deGFP beads; 4T1-mGFP cells show the highest activation efficiency, about 21% synNotch cells are activated, when 4T1-mGFP and 293T-synNotch are incubated at high cell density. Conclusion: The 4T1-mGFP cells are the most effective ligands for synNotch receptor activation. [ABSTRACT FROM AUTHOR]

Based on our previous work on the PLGA nanoparticles modified with biotinylated chitosan (Bio-CS-PLGA NPs), we further studied the stability, toxicity, pharmacokinetics, andin vivoefficacy. The safety of NPs was studied through single-dose toxicity test in mice, and the result showed that NPs were well tolerated at the dose of 300 mg/kg. Compared with the free EPB group, the NPs group exhibited higher plasma drug concentration, longer half-life time. EPB-loaded NPs significantly inhibited the tumor growth compared to free EPB. All results suggested that Bio-CS-PLGA NPs were stable, safe, and showed a promising potential on targeted drug delivery. [ABSTRACT FROM AUTHOR]

Engineered natural killer (NK) cell-based therapies have been potentially broadly applicable and exhibited promising results in clinical trials, particularly in the fight against cancers. NK cell immunotherapy however always remains variable. One major obstacle is the inhibitory pathway including PD1/PDL1, providing tumor cells an escape mechanism from immunosurveillance. In this regard, we rationally designed a chimeric switch-receptor (CSR) PD1-DAP10-41BB, which comprising the ectodomain of PD1 fused to the co-stimulatory receptor DAP10 and 41BB. Therefore, by exchanging the transmembrane and cytoplasmic tail of PD1 with positive costimulatory molecules DAP10 and 41BB signaling domains, the negative PD1/PDL1 signal pathway was thus converted into a positive one. This CSR-expressing NK92 cells showed a typical parental NK92 phenotype and improved cytotoxicity against human lung cancer H1299 cells. Besides, the expression of CSR elicited a significant increase of effector molecules such as perforin and granzymes, which can induce apoptosis of H1299 cells. More importantly, in the solid tumor cell H1299-bearing mice model, the CSR-modified NK92 cells significantly inhibited tumor growth. Collectively, we demonstrated that expression of PD1-DAP10-41BB augmented NK92-cell activation and killing in vitro and in vivo , which provides a considerable avenue of using NK-tailored chimeric receptor engineered NK92 cells to treat a wide range of solid tumors. • A novel NK-tailored chimeric switch-receptor PD1-DAP10-41BB is rationally designed. • Modified NK92 cells mediate the clearance of H1299 cells by triggering apoptosis. • PD1-DAP10-41BB-expressing NK92 cells elicit in vivo tumor control of H1299 cells. [ABSTRACT FROM AUTHOR]

Cell-based immunotherapy continues to be a promising avenue for cancers that standard therapy has failed. Although the specificity, avidity, and efficacy of infused cells have improved, immunocytotherapy still faces substantial hurdles. To this end, we developed a structure-based rational design approach and constructed a novel Dual Targeting Chimeric Receptor (DTCR) PD1-DAP10/NKG2D comprising the truncated ectodomain of PD1 fused to a key co-stimulatory receptor DAP10, and subsequently harnessed the activating receptor NKG2D, which evaluated the capacity of solid tumor cell killing. Retroviral transduction of DTCR dramatically increased NK92 cell surface expression of PD1 and NKG2D, which boosted robust cytotoxicity against human gastric cell SGC-7901. Chimeric receptor DTCR stimulation elicited a significant increase of TNF-α and TRAIL, which can trigger apoptosis of SGC-7901 cells. More importantly, DTCR-NK92 cells had considerable antitumor activity in the solid tumor cell SGC-7901-bearing mice model. Collectively, we demonstrated that expression of DTCR markedly augmented the cytotoxic potential of NK92 cells against solid tumor cells, and this potentially promising treatment modality will facilitate clinical translation of potent NK-tailored chimeric receptor strategy for a generalized cellular therapy that may be conducive to treat a wide range of solid tumors. • A novel NK-tailored bispecific chimeric receptor PD1-DAP10/NKG2D is rationally designed with a structure-based approach. • Engineered NK92 cells can not only trigger apoptosis, but seemingly induce pyroptosis of SGC-7901 solid tumor cells. • Expressing PD1-DAP10/NKG2D mediates robust in vitro cytotoxicity and in vivo tumor control of NK92 cells. [ABSTRACT FROM AUTHOR]

Highlights • mVEGF165b as an immunization adjunct was able to induce high titers of anti-VEGF antibody. • High anti-VEGF antibody titers decrease immunosuppression by reducing the proportion of Tregs in the spleen or in peripheral circulation. • The decrease immunosuppression augments the immune response induced by peptide vaccine from Mucin-1. • mVEGF165b can be used as a potential adjunct with other tumor vaccines to increase anti-tumor efficacy and also promote the T cell infiltration into tumor. Abstract Compelling evidence has shown that blocking VEGF via monoclonal antibodies may be beneficial in that it not only inhibits tumor angiogenesis but also reduces immune suppression and promotes T cell infiltration into tumors. Herein, we determined whether our recently generated VEGF165b mutant could be used as a co-immunization adjunct to augment the peptide cancer-vaccine- induced immune response in a mouse model of breast cancer. When co-immunized mVEGF165b with the peptide-based cancer vaccine (MUC1, a T-cell epitope dominant peptide vaccine from Mucin1), the VEGF antibody titers increased approximately 600,000-fold in mice. Moreover, the anti-VEGF antibody also reduced the frequency of regulatory T cells (Tregs) in both preventive and therapeutic scenarios. Mechanistically, the decrease of the Tregs population was associated with a remarkably increased MUC-1-specific IFN-γ-producing CD8+ T cells and anti-MUC1 humoral response. Finally, this combination co-immunization produced a superior antitumor response and significantly prolonged survival of tumor-bearing mice. In conclusion, our findings suggest that mVEGF165b may be an ideal immunization adjunct to enhance the immune efficacy of peptide-based tumor vaccines by overcoming immune tolerance. [ABSTRACT FROM AUTHOR]

• With the versatility, CRISPR/Cas9 has dominated the realm of genome editing. • Various challenges of CRISPR/Cas9 remain to be overcome for patients with cancer. • CRISPR/Cas9 is revolutionizing and opening a new era for cellular immunotherapies. Immunotherapy has become one of the most promising strategies in cancer therapies. Among the therapeutic alternatives, genetically engineered NK/T cell therapies have emerged as powerful and innovative therapeutic modalities for cancer patients with precise targeting and impressive efficacy. Nonetheless, this approach still faces multiple challenges, such as immunosuppressive tumor microenvironment, exhaustion of immune effector cells in tumors, off-target effects manufacturing complexity, and poor infiltration of effector cells, all of which need to be overcome for further utilization to cancers. Recently, CRISPR/Cas9 genome editing technology, with the goal of enhancing the efficacy and increasing the availability of engineered effector cell therapies, has shown considerable potential in the novel strategies and options to overcome these limitations. Here we review the current progress of the applications of CRISPR in cancer immunotherapy. Furthermore, we discuss issues related to the NK/T cell applications, gene delivery methods, efficiency, challenges, and implications of CRISPR/Cas9. [ABSTRACT FROM AUTHOR]

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 ., Competing Interests: The authors report no conflicts of interest regarding the publication of this article., (Copyright © 2022 Zhiyuan Niu et al.)

A novel Gram-stain-negative, rod-shaped, non-spore-forming, non-flagellated, strictly aerobic strain, designated A6024 T , was isolated from coastal seawater near Rizhao, PR China (119.61° E 35.47° N). The organism grew optimally at 28 °C, in pH 6.0-7.0 and in the presence of 3.0 % (w/v) NaCl. The strain required seawater or artificial seawater for growth and NaCl alone did not support growth. Strain A6024 T contained ubiquinone 10 as the sole respiratory quinone and C 18 : 1 ω 7 c (75.2 %) as the most abundant fatty acid. The predominant polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, one unidentified aminolipid and two unidentified lipids. The DNA G+C content of strain A6024 T was 59.9 mol%. The results of phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain was related most closely to Aliiroseovarius halocynthiae MA1-10 T , Aliiroseovarius pelagivivens GYSW-22 T and Aliiroseovarius crassostreae CV919-312 T with 98.3, 97.6 and 97.4 % sequence similarities, respectively. The calculated average nucleotide identity values and DNA-DNA hybridization values between strain A6024 T and the phylogenetically related Aliiroseovarius species were in the range 76.0-85.6 % and 19.6-29.4 %, respectively. On the basis of the results of polyphasic analyses, strain A6024 T represents a novel species of the genus Aliiroseovarius , for which the name Aliiroseovarius marinus sp. nov. is proposed. The type strain is A6024 T (=KCTC 72114 T =MCCC 1K03595 T ).

A novel bacterial strain, C3212 T , was isolated from a marine alga collected from the sea shore of Yantai, China. The strain was Gram-stain-negative, rod-shaped, aerobic, non-motile, and oxidase- and catalase-positive. Growth was observed at 8-37 °C (optimum, 28 °C), at pH 6.0-9.0 (optimum, pH 7.0) and in the presence of 1.0-7.0 % (w/v) NaCl (optimum, 4.0 %). The major respiratory quinone was ubiquinone-8 (Q-8). The polar lipids of strain C3212 T consisted of diphosphatidylglycerol (cardiolipin), phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminophospholipid, an unidentified phospholipid and an unidentified polar lipid. The major fatty acids were C 16 : 1 ω 6c and/or C 16 : 1 ω 7c , and C 18 : 1 ω 6c and/or C 18 : 1 ω 7c . The DNA G+C content of strain C3212 T was 44.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain was related most closely to Leucothrix pacifica XH122 T , Leucothrix arctica IMCC 9719 T and Leucothrix mucor DSM 2157 T with similarities of 98.0, 97.5 and 94.3 %, respectively. Estimated DNA-DNA hybridization values were 14.2, 20.7 and 13.9 % between strain C3212 T and L. pacifica XH122 T , L. arctica IMCC 9719 T and L. mucor DSM 2157 T , respectively. Phenotypic, phylogenetic and genomic analyses revealed that strain C3212 T represents a novel species of the genus Leucothrix , for which the name Leucothrix sargassi sp. nov. is proposed. The type strain is C3212 T (=MCCC 1K03600 T =KCTC 72121 T ).

A novel Gram-stain-negative, straight or slightly curved rod-shaped, non-spore-forming, non-flagellated, strictly aerobic strain, designated RZG4-3-1 T , 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 RZG4-3-1 T 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 RZG4-3-1 T were phosphatidylethanolamine, phosphatidylglycerol and one unidentified aminophospholipid. The DNA G+C content of strain RZG4-3-1 T was 40.1 mol%. Strain RZG4-3-1 T exhibited the highest 16S rRNA gene sequence similarity value (96.0 %) to Thalassotalea eurytherma JCM 18482 T . Phylogenetic analysis based on 16S rRNA gene sequences showed that strain RZG4-3-1 T belonged to the genus Thalassotalea. On the basis of polyphasic analyses, strain RZG4-3-1 T represents a novel species of the genus Thalassotalea, for which the name Thalassotalea atypica sp. nov. is proposed. The type strain is RZG4-3-1 T (=JCM 31894 T =KCTC 52745 T =MCCC 1K03276 T ). An emended description of Thalassotalea eurytherma is also provided.

Objective To establish a melanoma B16 cell line stably over-expressing the HER2 (ErbB2) gene and study its effect on the proliferation, migration and invasion of melanoma cells. Methods The recombinant plasmid pCMV3-ErbB2 was transfected into B16 melanoma cell line by cationic liposome. The positive clones were screened with hygromycin B. Real-time quantitative PCR (qRT-PCR) was utilized to determine the expression of ErbB2 mRNA. Immunofluorescence was used to evaluate the expression of ErbB2 protein. MTT assay was applied to detect the cell proliferation ability. The cell migration ability was examined by a scratch assay. Transwell TM assay was performed to determine the effect of HER2 over-expression on the capability of B16s invasion. Results The cell line over-expressing HER2 was successfully established as showed by qRT-PCR. Over-expression of HER2 remarkably enhanced the proliferation, invasion and migration of B16 cells. Conclusion Over-expression of HER2 can enhance the growth and invasion of B16 cells.

A novel Gram-stain-negative, rod-shaped, non-spore-forming, non-flagellated, strictly aerobic strain, designated RZW3-2T, was isolated from seawater from near the Yellow Sea coast of PR China (35.475° N 119.613° E). The organism grew optimally between 24 and 28 °C, at pH 6.0-7.0 and in the presence of 2-3 % (w/v) NaCl. The strain requires seawater or artificial seawater for growth and NaCl alone does not support growth. RZW3-2T contained iso-C15 : 0, C16 : 1ω7c and/or C16 : 1ω6c and anteiso-C15 : 0 as the dominant fatty acids. The respiratory quinone detected in RZW3-2T was menaquinone 6 (MK6). The polar lipids of RZW3-2T comprised phosphatidylethanolamine (PE), four unidentified phospholipids (PL), two unidentified aminolipids (AL) and one unknown lipid (L). The DNA G+C content of RZW3-2T was 30.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain was related most closely to Polaribacter reichenbachii KCTC 23969T, Polaribacter dokdonensis KCTC 12392T and Polaribacter irgensii CIP 106478T with 98.0, 97.8 and 97.0 % sequence similarities, respectively. The DNA-DNA hybridization values between RZW3-2T and its closest phylogenetic relatives, P. reichenbachii KCTC 23969T and P. dokdonensis KCTC 12392T, were 52.0±0.6 % and 49.8±1.21 % respectively. On the basis of polyphasic analyses, RZW3-2T represents a novel species of the genus Polaribacter, for which the name Polaribacter marinaquae sp. nov. is proposed. The type strain is RZW3-2T (=JCM 30825T=KCTC 42664T=MCCC 1K00696T).