Antibody-mediated blockade for galectin-3 binding protein in tumor secretome abrogates PDAC metastasis.

The major challenges in pancreatic ductal adenocarcinoma (PDAC) management are local or distant metastasis and limited targeted therapeutics to prevent it. To identify a druggable target in tumor secretome and to explore its therapeutic intervention, we performed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis of tumors obtained from a patient-derived xenograft model of PDAC. Galectin-3 binding protein (Gal-3BP) is identified as a highly secreted protein, and its overexpression is further validated in multiple PDAC tumors and primary cells. Knockdown and exogenous treatment of Gal-3BP showed that it is required for PDAC cell proliferation, migration, and invasion. Mechanistically, we revealed that Gal-3BP enhances galectin-3-mediated epidermal growth factor receptor signaling, leading to increased cMyc and epithelial-mesenchymal transition. To explore the clinical impact of these findings, two antibody clones were developed, and they profoundly abrogated the metastasis of PDAC cells in vivo. Altogether, our data demonstrate that Gal-3BP is an important therapeutic target in PDAC, and we propose its blockade by antibody as a therapeutic option for suppressing PDAC metastasis.

A dominant negative variant of RAB5B disrupts maturation of surfactant protein B and surfactant protein C.

Pathogenic variants in surfactant proteins SP-B and SP-C cause surfactant deficiency and interstitial lung disease. Surfactant proteins are synthesized as precursors (proSP-B, proSP-C), trafficked, and processed via a vesicular-regulated secretion pathway; however, control of vesicular trafficking events is not fully understood. Through the Undiagnosed Diseases Network, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant in RAB5B was identified in the Ras/Rab GTPases family nucleotide binding domain, p.Asp136His. Functional studies were performed in Caenorhabditis elegans by knocking the proband variant into the conserved position (Asp135) of the ortholog, rab-5 Genetic analysis demonstrated that rab-5[Asp135His] is damaging, producing a strong dominant negative gene product. rab-5[Asp135His] heterozygotes were also defective in endocytosis and early endosome (EE) fusion. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in alveolar type II cells and that mature SP-B and SP-C were significantly reduced, while proSP-B and proSP-C were normal. Furthermore, staining normal lung showed colocalization of RAB5B and EEA1 with proSP-B and proSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, resulting in interstitial lung disease, and that RAB5B and EEs normally function in the surfactant secretion pathway. Together, the data suggest a noncanonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for a surfactant dysfunction disorder.

Direct and Indirect Chimeric Antigen Receptor T-Cell Imaging with PET/MRI in a Tumor Xenograft Model.

Background Chimeric antigen receptor (CAR) T cells are a promising cancer therapy; however, reliable and repeatable methods for tracking and monitoring CAR T cells in vivo remain underexplored. Purpose To investigate direct and indirect imaging strategies for tracking the biodistribution of CAR T cells and monitoring their therapeutic effect in target tumors. Materials and Methods CAR T cells co-expressing a tumor-targeting gene (anti-CD19 CAR) and a human somatostatin receptor subtype 2 (hSSTr2) reporter gene were generated from human peripheral blood mononuclear cells. After direct labeling with zirconium 89 (89Zr)-p-isothiocyanatobenzyl-desferrioxamine (DFO), CAR T cells were intravenously injected into immunodeficient mice with a CD19-positive and CD19-negative human tumor xenograft on the left and right flank, respectively. PET/MRI was used for direct in vivo imaging of 89Zr-DFO-labeled CAR T cells on days 0, 1, 3, and 7 and for indirect cell imaging with the radiolabeled somatostatin receptor-targeted ligand gallium 68 (68Ga)-DOTA-Tyr3-octreotide (DOTATOC) on days 6, 9, and 13. On day 13, mice were euthanized, and tissues and tumors were excised. Results The 89Zr-DFO-labeled CAR T cells were observed on PET/MRI scans in the liver and lungs of mice (n = 4) at all time points assessed. However, they were not visualized in CD19-positive or CD19-negative tumors, even on day 7. Serial 68Ga-DOTATOC PET/MRI showed CAR T cell accumulation in CD19-positive tumors but not in CD19-negative tumors from days 6 to 13. Notably, 68Ga-DOTATOC accumulation in CD19-positive tumors was highest on day 9 (mean percentage injected dose [%ID], 3.7% ± 1.0 [SD]) and decreased on day 13 (mean %ID, 2.6% ± 0.7) in parallel with a decrease in tumor volume (day 9: mean, 195 mm3 ± 27; day 13: mean, 127 mm3 ± 43) in the group with tumor growth inhibition. Enhanced immunohistochemistry staining of cluster of differentiation 3 (CD3) and hSSTr2 was also observed in excised CD19-positive tumor tissues. Conclusion Direct and indirect cell imaging with PET/MRI enabled in vivo tracking and monitoring of CAR T cells in an animal model. © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Bulte in this issue.

Distinct features of B cell receptors in neuromyelitis optica spectrum disorder among CNS inflammatory demyelinating diseases.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) stands out among CNS inflammatory demyelinating diseases (CIDDs) due to its unique disease characteristics, including severe clinical attacks with extensive lesions and its association with systemic autoimmune diseases. We aimed to investigate whether characteristics of B cell receptors (BCRs) differ between NMOSD and other CIDDs using high-throughput sequencing. METHODS: From a prospective cohort, we recruited patients with CIDDs and categorized them based on the presence and type of autoantibodies: NMOSD with anti-aquaporin-4 antibodies, myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) with anti-myelin oligodendrocyte glycoprotein antibodies, double-seronegative demyelinating disease (DSN), and healthy controls (HCs). The BCR features, including isotype class, clonality, somatic hypermutation (SHM), and the third complementarity-determining region (CDR3) length, were analyzed and compared among the different disease groups. RESULTS: Blood samples from 33 patients with CIDDs (13 NMOSD, 12 MOGAD, and 8 DSN) and 34 HCs were investigated for BCR sequencing. Patients with NMOSD tended to have more activated BCR features compare to the other disease groups. They showed a lower proportion of unswitched isotypes (IgM and IgD) and a higher proportion of switched isotypes (IgG), increased clonality of BCRs, higher rates of SHM, and shorter lengths of CDR3. Notably, advanced age was identified as a clinical factor associated with these activated BCR features, including increased levels of clonality and SHM rates in the NMOSD group. Conversely, no such clinical factors were found to be associated with activated BCR features in the other CIDD groups. CONCLUSIONS: NMOSD patients, among those with CIDDs, displayed the most pronounced B cell activation, characterized by higher levels of isotype class switching, clonality, SHM rates, and shorter CDR3 lengths. These findings suggest that B cell-mediated humoral immune responses and characteristics in NMOSD patients are distinct from those observed in the other CIDDs, including MOGAD. Age was identified as a clinical factor associated with BCR activation specifically in NMOSD, implying the significance of persistent B cell activation attributed to anti-aquaporin-4 antibodies, even in the absence of clinical relapses throughout an individual's lifetime.

NFAT1 and NFκB regulates expression of the common γ-chain cytokine receptor in activated T cells.

INTRODUCTION: Cytokines of the common γ chain (γc) family are critical for the development, differentiation, and survival of T lineage cells. Cytokines play key roles in immunodeficiencies, autoimmune diseases, allergies, and cancer. Although γc is considered an assistant receptor to transmit cytokine signals and is an indispensable receptor in the immune system, its regulatory mechanism is not yet well understood. OBJECTIVE: This study focused on the molecular mechanisms that γc expression in T cells is regulated under T cell receptor (TCR) stimulation. METHODS: The γc expression in TCR-stimulated T cells was determined by flow cytometry, western blot and quantitative RT-PCR. The regulatory mechanism of γc expression in activated T cells was examined by promoter-luciferase assay and chromatin immunoprecipitation assays. NFAT1 and NFκB deficient cells generated using CRISPR-Cas9 and specific inhibitors were used to examine their role in regulation of γc expression. Specific binding motif was confirmed by γc promotor mutant cells generated using CRISPR-Cas9. IL-7TgγcTg mice were used to examine regulatory role of γc in cytokine signaling. RESULTS: We found that activated T cells significantly upregulated γc expression, wherein NFAT1 and NFκB were key in transcriptional upregulation via T cell receptor stimulation. Also, we identified the functional binding site of the γc promoter and the synergistic effect of NFAT1 and NFκB in the regulation of γc expression. Increased γc expression inhibited IL-7 signaling and rescued lymphoproliferative disorder in an IL-7Tg animal model, providing novel insights into T cell homeostasis. CONCLUSION: Our results indicate functional cooperation between NFAT1 and NFκB in upregulating γc expression in activated T cells. As γc expression also regulates γc cytokine responsiveness, our study suggests that γc expression should be considered as one of the regulators in γc cytokine signaling and the development of T cell immunotherapies. Video Abstract.

Factors influencing mobility in community-dwelling older adults during the early COVID-19 pandemic: a cross-sectional study.

BACKGROUND: In older adults, mobility is important for maintaining their independence and quality of life, and it influences their physical, cognitive, and social health. This study aimed to identify the physical and psychosocial factors that affected the mobility of community-dwelling older adults, aged 65 years or older, who were socially isolated during the coronavirus disease 2019 (COVID-19) pandemic due to stay-at-home policies. METHODS: The participants in this study were 214 community-dwelling older adults in Korea, and a cross-sectional survey was conducted from December 2020 to January 2021. Variables included participants' general characteristics, mobility, sitting time, depression, social support, and cognitive function. RESULTS: Multiple linear regression analysis showed that the factors influencing older adults' mobility during the COVID-19 pandemic were depression (ß=-0.29, p < .001), age (65-74 years old) (ß = 0.19, p = .002), a lower level of education (ß=-0.17, p = .006), two or more comorbidities (ß=-0.18, p = .001), sitting time (ß=-0.17, p = .004), and the ability to drive a vehicle (ß = 0.14, p = .017). CONCLUSIONS: Home healthcare interventions are needed to limit psychosocial issues and improve mobility for older adults who had limited mobility during the COVID-19 pandemic.

The Effects of ICT-Based Interventions on Physical Mobility of Older Adults: A Systematic Literature Review and Meta-Analysis.

Systematic literature review and meta-analysis were conducted to integrate and analyze intervention studies dealing with the effects of information and communications technology- (ICT-) based interventions on the physical mobility of older adults in the community. The PubMed/MEDLINE, Embase, CINAHL, and Cochrane CENTRAL databases were searched for studies published from January 2000 to December 2022. We used the Risk of Bias 2 (RoB 2) tool to evaluate the quality of the randomized controlled studies in the systematic review. The meta-analysis was performed using a random-effects model. The model was used to calculate the standardized mean difference (SMD) and 95% confidence interval (CI) for both effect measures. I2 tests were used to measure the presence of heterogeneity. Thirty-seven randomized controlled trials were included (2,419 intervention participants), of which 23 were included in the meta-analysis. ICT interventions significantly improved Timed Up and Go (TUG) as a marker of physical mobility variable in older adults (SMD = -0.33, 95% CI: -0.57 to -0.10, p=0.005, I2 = 74.7%). A sensitivity analysis was performed on subgroups, and interventions were found to be effective in improving TUG in the exergame group (SMD = -0.40, 95% CI: -0.72 to -0.08, p < 0.001, I2 = 75.0%) and in the exergame with virtual reality (VR) group (SMD = -0.33, 95% CI: -1.01 to 0.35, p < 0.001, I2 = 91.0%) but both groups showed high heterogeneity. A meta-analysis was also performed on Short Physical Performance Battery (SPPB) but statistically significant results were not found (SMD = -0.19, 95% CI: -0.61 to 0.23, p=0.375, I2 = 87.7%). For the Berg Balance Scale (BBS), the post-intervention scores were significantly better than baseline (SMD = 1.52, 95% CI: 0.48 to 2.57, p=0.004, I2 = 93.5%). However, the number of studies included in the meta-analysis was small and heterogeneity was high, so follow-up studies are needed. This study confirmed that exergames, telecommunication, e-health, information applications, and robots were used as effective ICT-based interventions for improving the physical mobility of older adults. It is necessary to develop and apply more diverse ICT-based interventions that will prevent impairments of mobility and encourage older adults to live more independently, with a higher quality of life, based on extensive research on ICT-based interventions.

Development and evaluation of a problem-based learning simulation module for home-visit nursing.

OBJECTIVES: Although home-visit healthcare programs in Korea are expected to expand, providing hands-on experience to nursing students may be limited. This study aimed to develop and evaluate a problem-based learning (PBL) simulation module that reflects home-visit healthcare services provided by public health centers for pre-frail older adults. DESIGN AND SAMPLE: The simulation module, including PBL as prebriefing, was developed by the researchers and revised based on expert reviews. The module was evaluated using a mixed-method embedded one-group post-test-only design with focus group interviews (FGIs). Quantitative data (n = 29) were collected between April and June, 2021. FGIs (n = 10) were conducted twice in June 2021, and qualitative data were analyzed using an inductive content analysis approach. RESULTS: The average score of the Simulation Design Scale was 4.67 ± 0.36. The overall mean score of the Educational Practices Questionnaire was 4.75 ± 0.37. Three themes emerged from the FGIs: immersive learning experience, changes in perspective on nursing, and enhanced nursing competency. CONCLUSION: This PBL-based simulation module was evaluated as a systematic learning process in which nursing students could become self-directed learners, interacting and collaborating with colleagues, instructors, and environments. The module encourages them to practice home visit services.

Community Health Nursing During the COVID-19 Pandemic in Korea: Consequences, Challenges, and Directions.

PURPOSE: To explore the consequences, challenges, and future directions based on community health nurses' experiences during COVID-19. DESIGN: Qualitative study. Four focus group interviews were conducted with 27 community health nurses. FINDINGS: Three major themes emerged: 1) Coordination of roles and duties, 2) Identifying deterioration of patients' health and increasing demand for visits, and 3) Changing service delivery strategies: a testing ground for new services. CONCLUSIONS: Community health nurses were essential public healthcare providers during the pandemic. The findings are informative for nurses and policy makers who can develop and suggest different services in the post-COVID era.

An anti-Gn glycoprotein antibody from a convalescent patient potently inhibits the infection of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease localized to China, Japan, and Korea that is characterized by severe hemorrhage and a high fatality rate. Currently, no specific vaccine or treatment has been approved for this disease. To develop a therapeutic agent for SFTS, we isolated antibodies from a phage-displayed antibody library that was constructed from a patient who recovered from SFTS virus (SFTSV) infection. One antibody, designated as Ab10, was reactive to the Gn envelope glycoprotein of SFTSV and protected host cells and A129 mice from infection in both in vitro and in vivo experiments. Notably, Ab10 protected 80% of mice, even when injected 5 days after inoculation with a lethal dose of SFTSV. Using cross-linker assisted mass spectrometry and alanine scanning, we located the non-linear epitope of Ab10 on the Gn glycoprotein domain II and an unstructured stem region, suggesting that Ab10 may inhibit a conformational alteration that is critical for cell membrane fusion between the virus and host cell. Ab10 reacted to recombinant Gn glycoprotein in Gangwon/Korea/2012, HB28, and SD4 strains. Additionally, based on its epitope, we predict that Ab10 binds the Gn glycoprotein in 247 of 272 SFTSV isolates previously reported. Together, these data suggest that Ab10 has potential to be developed into a therapeutic agent that could protect against more than 90% of reported SFTSV isolates.

Bispecific anti-mPDGFRß x cotinine scFv-Cκ-scFv fusion protein and cotinine-duocarmycin can form antibody-drug conjugate-like complexes that exert cytotoxicity against mPDGFRß expressing cells.

Antibody selection for antibody-drug conjugates (ADCs) has traditionally depended on its internalization into the target cell, although ADC efficacy also relies on recycling of the receptor-ADC complex, endo-lysosomal trafficking, and subsequent linker/antibody proteolysis. In this study, we observed that a bispecific anti-murine platelet-derived growth factor receptor beta (mPDGFRß) x cotinine single-chain variable fragment (scFv)-kappa constant region (Cκ)-scFv fusion protein and cotinine-duocarmycin can form an ADC-like complex to induce cytotoxicity against mPDGFRß expressing cells. Multiple anti-mPDGFRß antibody candidates can be produced in this bispecific scFv-Cκ-scFv fusion protein format and tested for their ability to deliver cotinine-conjugated cytotoxic drugs, thus providing an improved approach for antibody selection in ADC development.

Efficient Selection of Antibodies Reactive to Homologous Epitopes on Human and Mouse Hepatocyte Growth Factors by Next-Generation Sequencing-Based Analysis of the B Cell Repertoire.

: YYB-101 is a humanized rabbit anti-human hepatocyte growth factor (HGF)-neutralizing antibody currently in clinical trial. To test the effect of HGF neutralization with antibody on anti-cancer T cell immunity, we generated surrogate antibodies that are reactive to the mouse homologue of the epitope targeted by YYB-101. First, we immunized a chicken with human HGF and monitored changes in the B cell repertoire by next-generation sequencing (NGS). We then extracted the VH gene repertoire from the NGS data, clustered it into components by sequence homology, and classified the components by the change in the number of unique VH sequences and the frequencies of the VH sequences within each component following immunization. Those changes should accompany the preferential proliferation and somatic hypermutation or gene conversion of B cells encoding HGF-reactive antibodies. One component showed significant increases in the number and frequencies of unique VH sequences and harbored genes encoding antibodies that were reactive to human HGF and competitive with YYB-101 for HGF binding. Some of the antibodies also reacted to mouse HGF. The selected VH sequences shared 98.3% identity and 98.9% amino acid similarity. It is therefore likely that the antibodies encoded by them all react to the epitope targeted by YYB-101.

A point mutation in the heavy chain complementarity-determining region 3 (HCDR3) significantly enhances the specificity of an anti-ROS1 antibody.

The proto-oncogene tyrosine kinase ROS1 plays a key role in carcinogenesis through gene rearrangement to form a fusion protein with other genes, in which the C-terminal intracellular region of ROS1 participates. The possibility of wild type ROS1 overexpression through epigenetic regulation has been proposed. Here, we generated an antibody, 3B20, reactive to the N-terminal region of ROS1 to use it for the detection of wild type ROS1 in cancerous tissues. Using immunoblot and immunoprecipitation analyses, we found that 3B20 also reacted with heat shock proteins (Hsp)70s. Using homology searching, ROS1 and Hsp70s were found to share an identical amino acid sequence: DLGT. Using alanine mutagenesis of ROS1, the epitope was found to harbor this sequence. To modify the idiotope with the aim of selecting more specific antibodies, we introduced random mutations into the heavy chain complementarity-determining region 3 and successfully generated an antibody clone, 3B20-G1K, with a point mutation that only reacted with ROS1 in enzyme-linked immunosorbent assays, and in immunoblot and immunoprecipitation analysis. In immunohistochemical analysis using 3B20-G1K, ROS1 was found to be absent in normal lung tissues and was overexpressed in a case of lung adenocarcinoma.

Establishment of a mammalian expression system for recombinant [-2]proPSA and a specific antibody against the truncated leader peptide.

A truncated precursor form of prostate-specific antigen (PSA), [-2]proPSA, is a well-known biomarker for prostate cancer. To develop a biomarker assay, highly purified [-2]proPSA is required as a standard reference and for generation of a specific antibody. In this study, we generated an efficient mammalian expression system for producing a recombinant [-2]proPSA-human kappa constant domain (Cκ ) fusion protein. N-terminal amino acid sequencing using Edman degradation demonstrated that over 95% of the recombinant protein produced is [-2]proPSA, thereby showing for the first time that recombinant [-2]proPSA can be produced as a major fraction. We also generated a recombinant chicken antibody specific to [-2]proPSA but not cross-reactive to recombinant [-7]proPSA-Cκ , [-5]proPSA-Cκ , and PSA purified from human seminal fluid in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Also, the recombinant chicken antibody reacted to recombinant [-2]proPSA protein bound to an anti-PSA antibody coated on the micrometer plate in a sandwich ELISA. All of these results suggest that the N-terminus of the [-2]proPSA-Cκ fusion protein resides on the exterior of the protein, thus allowing exposure to the antibody.

Antibodies to the DNA-directed RNA polymerase II subunit RPB1 occur with highest frequency in centenarians.

BACKGROUND: Recently, phage display technology has made it possible to define the circulating repertoire of humoral immunity. This study was designed to define the circulating antibodies specific to centenarians. RESULTS: We used a phage-displayed combinatorial peptide library to screen for peptides (YSATLRY and YSPTLFY) that preferentially react with the IgG fraction of centenarians aged 100-105 years. Centenarian sera binds to YSATLRY and YSPTLFY with higher frequency than that of healthy volunteers aged 60-79 years or healthy volunteers younger than or equal to 43 years of age. We prepared polyclonal antibodies to YSATLRY from human sera to immunoprecipitate the native antigen, which was identified as the carboxy-terminal domain (CTD) of DNA-directed RNA polymerase II subunit RPB1. The RBP1 CTD contains multiple YSPTSPS repeats, which are significantly homologous to YSATLRY and YSPTLFY. The immunoprecipitated RPB1 had significantly slower mobility than did RPB1 in cell lysates, and the polyclonal antibodies reacted with CTD peptide, depending on the phosphorylation pattern. Therefore, it appears that the polyclonal antibodies preferentially bind to highly phosphorylated RPB1. We also confirmed that human monoclonal antibodies reactive to both YSATLRY and YSPTLFY bound to the phosphorylated YSPTSPS motif. CONCLUSIONS: This study showed that centenarians possess IgG antibodies that are reactive to YSATLRY and YSPTLFY, mimicking the phosphorylated form of the YSPTSPS motif (CTD of RPB1), at a much higher frequency than that of the average population.

Evaluating the effects of action learning on the research competency of undergraduate nursing students: A mixed-methods study.

BACKGROUND: Research skills in nursing are crucial for guiding evidence-based practice and enhancing health care. However, undergraduate nursing students often encounter challenges in skill development because of curriculum constraints that prioritize clinical education. Bridging this skill gap is imperative for preparing students for evidence-based practice and nursing scholarship. AIM: This study aimed to investigate the impact of incorporating action learning into undergraduate nursing research classes to improve the quality of nurse education. DESIGN: A mixed-methods approach was employed using pre- and post-online surveys for quantitative analysis and reflective journals for qualitative analysis. SETTINGS: The study was conducted at a college of nursing in Seoul, South Korea. PARTICIPANTS: A convenience sample of 19 fourth-year nursing students participated in the study. METHODS: Action learning-based nursing research classes were implemented over ten sessions, integrating lectures and team activities. Pre- and post-assessment data on communication skills, critical thinking tendencies, and problem-solving abilities were analyzed using paired t-tests. The qualitative analysis involved content analysis of individual and team reflective journals. RESULTS: Participants in action learning-based nursing research classes showed significant improvements in their communication skills (t = 3.46, p = 0.002), critical thinking tendencies (t = 3.80, p = 0.001), and problem-solving abilities (t = 1.82, p = 0.043). From the analysis of reflective journals four main themes were developed: organized team projects, dynamics of learning goal achievement, extended application of learning outcomes, and recommendations for better action learning-based classes. CONCLUSIONS: This study highlights action learning as an effective educational method that integrates theory and practice in nurse education, helping students prepare for their future roles as nursing professionals. The findings underscore the effectiveness of action learning in improving undergraduate nursing students' research competency and support the need for continued development of such pedagogical approaches.

CD19/CD22 bispecific chimeric antigen receptor­NK­92 cells are developed and evaluated.

Anti-CD19 chimeric antigen receptor (CAR)-T cells have improved the outcomes of patients with B cell leukemia and lymphoma. However, their applications and positive outcomes remain limited. CAR-T cells are currently restricted to autologous blood as their source and their use can lead to downregulation of CD19 expression along with complications such as graft-versus-host disease and cytokine release syndrome. The present study aimed to develop anti-CD19/CD22 bispecific CAR structures using an anti-CD22 monoclonal antibody clone from chickens and analyze them in natural killer (NK)-92 cells, a human NK cell line, in vitro and in vivo. Anti-CD19/CD22 CAR-NK-92 cell cytotoxicity was assessed by the survival of target cells and counted using flow cytometry. Anti-CD22/CD19 and loop-structured anti-CD19/CD22 bi-specific CAR-NK-92 cells showed improved efficacy against OCI-Ly7 cells, a human B cell lymphoma cell line, compared with other CAR structures. These results demonstrate the potential of anti-CD19/CD22 bispecific CAR-NK cells and suggested that optimizing CAR structures in NK cells can improve the efficacy of CAR therapy.

A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets.

Natural killer (NK) cells are immune effector cells with outstanding features for adoptive immunotherapy. Immune effector cells with chimeric antigen receptors (CARs) are promising targeted therapeutic agents for various diseases. Because tumor cells exhibit heterogeneous antigen expression and lose cell surface antigen expression during malignant progression, many CARs fixed against only one antigen have limited efficacy and are associated with tumor relapse. To expand the utility of CAR-NK cells, we designed a split and universal cotinine-CAR (Cot-CAR) system, comprising a Cot-conjugator and NK92 cells (α-Cot-NK92 cells) engineered with a CAR containing an anti-Cot-specific single-chain variable fragment and intracellular signaling domain. The efficacy of the Cot-CAR system was assessed in vitro using a cytolysis assay against various tumor cells, and its single- or multiple- utility potential was demonstrated using an in vivo lung metastasis model by injecting A549-Red-Fluc cells. The α-Cot-NK92 cells could switch targets, logically respond to multiple antigens, and tune cytolytic activation through the alteration of conjugators without re-engineering. Therefore the universal Cot-CAR system is useful for enhancing specificity and diversity of antigens, combating relapse, and controlling cytolytic activity. In conclusion, this universal Cot-CAR system reveals that multiple availability and controllability can be generated with a single, integrated system.