Augmenting CAR T Cell Functions with LIGHT.

Chimeric antigen receptor (CAR) T-cell therapy has resulted in remarkable clinical success in the treatment of B-cell malignancies. However, its clinical efficacy in solid tumors is limited, primarily by target antigen heterogeneity. To overcome antigen heterogeneity, we developed CAR T cells that overexpress LIGHT, a ligand of both LTßR on cancer cells and HVEM on immune cells. LIGHT-expressing CAR T cells displayed both antigen-directed cytotoxicity mediated by the CAR and antigen-independent killing mediated through the interaction of LIGHT with LTßR on cancer cells. Moreover, CAR T cells expressing LIGHT had immunostimulatory properties that improved the cells' proliferation and cytolytic profile. These data indicate that LIGHT-expressing CAR T cells may provide a way to eliminate antigen-negative tumor cells to prevent antigen-negative disease relapse.

PEG-lipid-modified agonistic antibody against tumor necrosis factor receptor family elicits superior apoptosis-inducing activity against human carcinoma.

We have recently developed a novel PEG-lipid-modified antibody to enhance the induction of apoptosis by the agonistic antibody. The chemically modified TRA-8 antibody [anti-death receptor 5 (DR5) antibody] with PEG-lipid (DSPE-PEG) demonstrated significant cytotoxic activity in vitro without the need for crosslinking with a secondary antibody, which is typically required. We investigated the correlation between the PEG-lipid structure and the cytotoxic activity of the modified antibodies by varying the PEG length or lipid structure. However, when the DSPE-PEG-modified TRA-8 antibody was incubated with plasma, it lost its cytotoxic activity, likely due to degradation in the DSPE-PEG component. Nevertheless, by designing new PEG-lipids that are intended to be resistant to enzymatic degradation, we were able to prevent this degradation and restore the cytotoxic activity of the modified antibody. These findings provide valuable insights for the design of PEG-lipid-modified antibodies and suggest their potential effectiveness in enhancing cancer therapy.

Developing a membrane-proximal CD33-targeting CAR T cell.

BACKGROUND: CD33 is a tractable target in acute myeloid leukemia (AML) for chimeric antigen receptor (CAR) T cell therapy, but clinical success is lacking. METHODS: We developed 3P14HLh28Z, a novel CD33-directed CD28/CD3Z-based CAR T cell derived from a high-affinity binder obtained through membrane-proximal fragment immunization in humanized mice. RESULTS: We found that immunization exclusively with the membrane-proximal domain of CD33 is necessary for identification of membrane-proximal binders in humanized mice. Compared with clinically validated lintuzumab-based CAR T cells targeting distal CD33 epitopes, 3P14HLh28Z showed enhanced in vitro functionality as well as superior tumor control and increased overall survival in both low antigen density and clinically relevant patient-derived xenograft models. Increased activation and enhanced polyfunctionality led to enhanced efficacy. CONCLUSIONS: Showing for the first time that a membrane-proximal CAR is superior to a membrane-distal one in the setting of CD33 targeting, our results demonstrate the rationale for targeting membrane-proximal epitopes with high-affinity binders. We also demonstrate the importance of optimizing CAR T cells for functionality in settings of both low antigen density and clinically relevant patient-derived models.

Effects of Wearing Slippers While Obstacle Crossing in Healthy Young Adults: Strategy for Toe Clearance Enhancement and Prevention of Slipper Loss.

Wearing loose footwear, such as slippers, poses a risk factor for tripping. Previous studies have examined obstacle crossing to find strategies to avoid tripping. However, the effect of wearing slippers on the likelihood of tripping remains unclear. Therefore, this study aimed to determine whether wearing slippers while level walking and obstacle crossing affects kinematic characteristics and muscle activity. Sixteen healthy, young adults performed two tasks (a) while wearing slippers and (b) while barefoot: (1) level walking and (2) crossing a 10-cm obstacle. Toe clearance, joint angles, muscle activity, and cocontraction were measured for both the leading and trailing lower limbs. In the slipper-wearing condition, knee flexion and hip flexion angles were significantly increased in the swing phase for the leading limb (p < .001 and p < .001, respectively) and trailing limb (p < .001 and p = .004, respectively) compared with the barefoot condition. Tibialis anterior activity (p = .01) and muscle cocontraction of the tibialis anterior and the medial head of the gastrocnemius (p = .047) were significantly increased in the swing phase of the trailing limb for the slipper-wearing condition compared with the barefoot condition in the obstacle crossing task. Wearing slippers increased knee and hip flexion angles, and muscle cocontraction of the tibialis anterior and medial head of gastrocnemius increased during obstacle crossing. The results revealed that obstacle crossing while wearing slippers would require foot fixation adjustment in addition to increased knee and hip flexion to avoid toe collision.

Body representation underlies response of proprioceptive acuity to repetitive peripheral magnetic stimulation.

Proprioceptive acuity is of great significance in basic research exploring a possible neural mechanism of fine motor control and in neurorehabilitation practice promoting motor function recovery of limb-disabled people. Moreover, body representation relies on the integration of multiple somatic sensations, including proprioception that is mainly generated in muscles and tendons of human joints. This study aimed to examine two hypotheses: First, different extension positions of wrist joint have different proprioceptive acuities, which might indicate different body representations of wrist joint in the brain. Second, repetitive peripheral magnetic stimulation (rPMS) applied peripherally to the forearm radial nerve and extensors could change proprioceptive acuity at the wrist joint. Thirty-five healthy participants were recruited then randomly divided into the real stimulation group (n = 15) and the sham stimulation group (n = 20). The participants' non-dominant side wrist joint position sense was tested at six extension positions within the physiological joint motion range (i.e., 10°, 20°, 30°, 40°, 50°, 60°) both before stimulation and after stimulation. Results showed that proprioceptive bias (arithmetic difference of target position and replicated position) among six extension positions could be divided into lower-extension position (i.e., 10°, 20°, 30°) and higher-extension position (i.e., 40°, 50°, 60°). One session rPMS could influence proprioceptive bias in lower-extension position but not in higher-extension position. However, proprioceptive precision (standard deviation within lower-extension position and higher-extension position) was not influenced. To conclude, proprioceptive bias may vary between different wrist extension positions due to different hand postures being related to changes in body representation, and different functions relating to proprioceptive bias and proprioceptive precision may underlie two aspects of body representation.

Human cytomegalovirus expands a CD8+ T cell population with loss of BCL11B expression and gain of NK cell identity.

CD8+ T cells not only are critical mediators of adaptive immunity but also may exhibit innate-like properties such as surface expression of NKG2C, an activating receptor typically associated with natural killer (NK) cells. We demonstrate that, similar to NK cells, NKG2C+TCRαß+CD8+ T cells are associated with prior human cytomegalovirus (HCMV) exposure. In addition to expressing several NK cell markers such as CD56 and KIR, NKG2C+CD8+ T cells are oligoclonal and do not up-regulate PD-1 even in response to persistent activation. Furthermore, we found that NKG2C+CD8+ T cells from some individuals exhibited strong effector function against leukemia cells and HCMV-infected fibroblasts, which was dictated by both NKG2C and TCR specificity. Transcriptomic analysis revealed that the transcription factor BCL11B, a regulator of T cell developmental fate, is down-regulated in NKG2C+CD8+ T cells when compared with conventional NKG2C−CD8+ T cells. BCL11B deletion in conventional CD8+ T cells resulted in the emergence of a similar innate-like CD56+CD94+DAP12+NKG2C+CD45RA+CCR7−PD-1−/low T cell population with activity against HLA-E+ targets. On the basis of their intrinsic capacity to recognize diseased cells coupled with lack of PD-1 induction, NKG2C+CD8+ T cells represent a lymphocyte population that resides at the boundary between innate and adaptive immunity, presenting an attractive alternative for cellular therapy, including CAR T cell­based therapies.

Effects of Lifestyle Changes on the Mental Health of Healthcare Workers with Different Sense of Coherence Levels in the Era of COVID-19 Pandemic.

Sense of coherence (SOC) is a psychological factor that contributes to mental health maintenance under stressful environment. Likewise, level of SOC might affect mental health among healthcare workers during the COVID-19 pandemic differently. In this study, we investigated the relationships between lifestyle changes and mental health (General Health Questionnaire-12: GHQ-12) among different level of SOC (weak, moderate, or strong by SOC-13). The data of 898 healthcare workers from cross-sectional survey dataset were extracted and analyzed. As results, based on GHQ-12 score, 86.1% of 244 participants with weak SOC, 60.1% of 606 participants with moderate SOC, and 31.3% of 48 participants with strong SOC had poor mental health. Both SOC levels and lifestyle changes (except alcohol consumption) had significant main effects on the GHQ-12 score. Analysis on the association between lifestyle changes and mental health status stratified by SOC level reveled that among participants with weak SOC, those who increased their leisure and activity time had reduced odds of poor mental health than those who made no changes (OR: 0.08, CI: 0.01 to 0.64). Healthcare workers with weak SOC were at risk of poor mental health during the COVID-19 pandemic, and lifestyle changes may improve their mental health.

Anti-ORAI1 antibody DS-2741a, a specific CRAC channel blocker, shows ideal therapeutic profiles for allergic disease via suppression of aberrant T-cell and mast cell activation.

ORAI1 constitutes the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, which is responsible for store-operated calcium entry into lymphocytes. It is known that ORAI1 is essential for the activation of T cells and mast cells and is considered to be a potent therapeutic target for autoimmune and allergic diseases. Here, we obtained a new humanized antibody, DS-2741a, that inhibits ORAI1 function. DS-2741a bound to human-ORAI1 with high affinity and without cross-reactivity to rodent Orai1. DS-2741a demonstrated suppression of CRAC-mediated human and mouse T-cell activation and mast cell degranulation in human ORAI1 knock-in mice. Furthermore, DS-2741a ameliorated house dust mite antigen-induced dermatitis in the human ORAI1 knock-in mouse. Taken together, DS-2741a inhibited T-cell and mast cell functions, thus improving skin inflammation in animal models of atopic dermatitis and reinforcing the need for investigation of DS-2741a for the treatment of allergic diseases in a clinical setting.

Detection of Gas Drifting Near the Ground by Drone Hovering Over: Using Airflow Generated by Two Connected Quadcopters.

This paper describes the utilization of the downwashes of multicopters for gas-sensing applications. Multirotor drones are an attractive platform for sensing applications. Their high maneuverability enables swift scanning of a target area with onboard sensors. When equipped with a gas sensor and used for gas-sensing applications, however, the strong downwash produced by the rotors poses a problem. When a multicopter is hovering at a low altitude, gas puffs leaked from a gas source on the ground are all blown away. Here, we propose to use two multicopters connected by a rod or a string and place a gas sensor at the midpoint of the rod/string. The downwash generated by each multicopter spreads radially after it impinges on the ground. When two multicopters are connected, the airflows spreading radially along the ground from the two multicopters impinge at the center and are deflected in the upward direction. Gas puffs wafting near the ground surface between the two multicopters are carried by this upward airflow to the gas sensor. Experimental results are presented to show the soundness of the proposed method. The connected quadcopters hovering over an ethanol gas source was able to detect the gas even with a moderate cross-flow.

[Development of an In-house Couch Model to Improve Dose Attenuation Correction Accuracy for a Couch with Different Thickness in the Superior-inferior Direction].

As the couch used in external radiation therapy attenuate radiation by interaction, it is necessary to correct attenuation of radiation by inserting a couch model in the treatment planning systems. For a couch whose thickness is different in the superior-inferior direction, it is possible to perform dose calculations with an error within ±1% by using separate different couch models provided by vendors. However, it is difficult to correct attenuation correction accurately with a single couch model. In this study, we created an in-house couch model which can set couch shape and physical density in detail by acquiring CT images of actual couch. When we performed dose calculation by optimizing the physical densities of in-house and vendor couch, it was found that the difference between the measured and the calculated values can be significantly reduced by using in-house couch model. Additionally, by using in-house couch model, it is found that the dose attenuation can be corrected within ±1% for a couch whose thickness is different in the superior-inferior direction.

Method for Catalytic Enantioselective Alkylation of Aldehydes Using Grignard Reagents as Alkyl Sources.

Alkyltitanium reagents, generated in situ from Grignard reagents and ClTi(O iPr)3, can be employed without further manipulation in the enantioselective alkylation of aldehyde by the catalysis of a chiral titanium complex derived from DTBP-H8-BINOL. The reaction is performed with good stoichiometry [1.5 equiv each of Grignard reagents and ClTi(O iPr)3] at a low catalyst loading (2 mol %), affording a variety of chiral secondary alcohols in high enantioselectivity and yields and, hence, realizing an asymmetric version of the Grignard reaction in an indirect manner.

Novel Immunoliposome Technology for Enhancing the Activity of the Agonistic Antibody against the Tumor Necrosis Factor Receptor Superfamily.

We have developed a technology for efficiently enhancing the anticancer apoptosis-inducing activity of agonistic antibodies against the tumor necrosis factor receptor (TNFR) superfamily by the formation of immunoliposomes. To induce apoptosis in cancer cells, agonistic antibodies to the TNFR superfamily normally need cross-linking by internal immune effector cells via the Fc region after binding to receptors on the cell membrane. To develop apoptosis-inducing antibodies that do not require the support of cross-linking by immune cells, we prepared immunoliposomes conjugated with TRA-8, an agonistic antibody against death receptor 5 (DR5), with various densities of antibody on the liposome surface, and evaluated their activities. The TRA-8 immunoliposomes exhibited apoptosis-inducing activity against various DR5-positive human carcinoma cells at a significantly lower concentration without cross-linking than that of the original TRA-8 and its natural ligand (TRAIL). The activity of the immunoliposomes was correlated with the density of antibodies on the surface. As the antibody component, not only the full-length antibody but also the Fab' fragment could be used, and the TRA-8 Fab' immunoliposomes also showed exceedingly high activity compared with the parental antibody, namely, TRA-8. Moreover, cytotoxicity of the TRA-8 full-length or Fab' immunoliposome against normal cells, such as human primary hepatocytes, was lower than that for TRAIL. Enhanced activity was also observed for immunoliposomes conjugated with other apoptosis-inducing antibodies against other receptors of the TNFR superfamily, such as death receptor 4 (DR4) and Fas. Thus, immunoliposomes are promising as a new modality that could exhibit significant activity at a low dose, for cost-effective application of an antibody fragment and with stable efficacy independent of the intratumoral environment of patients as a TNF superfamily agonistic therapy.

Elucidation of Distinct Roles of Guinea Pig CXCR1 and CXCR2 in Neutrophil Migration toward IL-8 and GROα by Specific Antibodies.

Chemokine receptors CXCR1 and CXCR2 are conserved between guinea pigs and humans, but the distinct role of each receptor in chemotactic responses of neutrophils against chemokine ligands has not been elucidated due in part to the lack of specific inhibitors against these receptors in guinea pigs. In this study, we investigated the roles of guinea pig CXCR1 and CXCR2 on neutrophils in chemotactic responses to guinea pig interleukin (IL)-8 and growth-regulated oncogene (GRO)α by using specific inhibitory antibodies against these receptors. Neutrophil migration induced by IL-8 was partially inhibited by either anti-CXCR1 antibody or anti-CXCR2 antibody. In addition, the migration was inhibited completely when both anti-CXCR1 and anti-CXCR2 antibodies were combined. On the other hand, neutrophil migration induced by GROα was not inhibited by anti-CXCR1 antibody while inhibited profoundly by anti-CXCR2 antibody. These results indicated that CXCR1 and CXCR2 mediated migration induced by the IL-8 synergistically and only CXCR2 mediated migration induced by GROα in guinea pig neutrophils. Our findings on ligand selectivity of CXCR1 and CXCR2 in guinea pigs are consistent with those in humans.

Generation and Characterization of Inhibitory Antibodies Specific to Guinea Pig CXCR1 and CXCR2.

CXCR1 and CXCR2 are chemokine receptors that have different selectivity of chemokine ligands, but the distinct role of each receptor is not clearly understood. This is due to the absence of specific inhibitors in guinea pigs, which are the appropriate species for investigation of CXCR1 and CXCR2 because of their functional similarity to humans. In this study, we generated and evaluated monoclonal antibodies that specifically bound to guinea pig CXCR1 (gpCXCR1) and guinea pig CXCR2 (gpCXCR2) for acquisition of specific inhibitors. To assess the activity of antibodies, we established CHO-K1 cells stably expressing either gpCXCR1 or gpCXCR2 (CHO/gpCXCR1 or CHO/gpCXCR2). CHO/gpCXCR1 showed migration in response to guinea pig interleukin (IL)-8, and CHO/gpCXCR2 showed migration in response to both guinea pig IL-8 and guinea pig growth-regulated oncogene α. The receptor selectivities of the chemokines of guinea pigs were the same as the human orthologs. The inhibitory activities of the anti-gpCXCR1 and anti-gpCXCR2 monoclonal antibodies on cell migration were observed in a concentration-dependent manner. In conclusion, we successfully obtained inhibitory antibodies specific to gpCXCR1 and gpCXCR2. These inhibitory antibodies will be useful to clarify the physiological roles of CXCR1 and CXCR2 in guinea pigs.

Connexin30.3 is expressed in mouse embryonic stem cells and is responsive to leukemia inhibitory factor.

The expression of 19 connexin (Cx) isoforms was observed in the mouse embryonic stem (ES) cell line, EB3. Their expression patterns could be classified into either pluripotent state-specific, differentiating stage-specific, or non-specific Cxs. We focused on Cx30.3 as typical of the first category. Cx30.3 was pluripotent state-specific and upregulated by leukemia inhibitory factor (LIF), a specific cytokine that maintains the pluripotent state of ES cell, via a Jak signaling pathway. Cx30.3 protein was localized to both the cell membrane and cytosol. The dynamic movement of Cx30.3 in the cell membrane was suggested by the imaging analysis by means of overexpressed Cx30.3-EGFP fusion protein. The cytosolic portion was postulated to be a ready-to-use Cx pool. The Cx30.3 expression level in ES cell colonies dramatically decreased immediately after their separation into single cells. It was suggested that mRNA for Cx30.3 and Cx30.3 protein might be decomposed more rapidly than mRNA for Cx43 and Cx43 protein, respectively. These indicate possible involvement of Cx30.3 in the rapid formation and/or decomposition of gap junctions; implying a functional relay between Cx30.3 and other systems such as adhesion proteins.

Molecular complex composed of ß-cyclodextrin-grafted Chitosan and pH-sensitive amphipathic peptide for enhancing cellular cholesterol efflux under acidic pH.

Excess of cholesterol in peripheral cells is known to lead to atherosclerosis. In this study, a molecular complex composed of ß-cyclodextrin-grafted chitosan (BCC) and cellular cholesterol efflux enhancing peptide (CEEP), synthesized by modifying pH sensitive amphipathic GALA peptide, is introduced with the eventual aim of treating atherosclerosis. BCC has a markedly enhanced ability to induce cholesterol efflux from cell membranes compared to ß-cyclodextrin, and the BCC-CEEP complex exhibited a 2-fold increase in cellular cholesterol efflux compared to BCC alone under weakly acidic conditions. Isothermal titration calorimetry and fluorescence spectroscopy measurements demonstrated that the random coil structure of CEEP at neutral pH converted to the α-helical structure at acidic pH, resulting in a three-order larger binding constant to BCC (K = 3.7 × 10(7) at pH 5.5) compared to that at pH 7.4 (K = 7.9 × 10(4)). Such high-affinity binding of CEEP to BCC at acidic pH leads to the formation of 100-nm-sized aggregate with positive surface charge, which would efficiently interact with cell membranes and induce cholesterol efflux. Since the cholesterol efflux ability of HDL is thought to be impaired under acidic environments in advanced atherosclerotic lesions, the BCC-CEEP complex might serve as a novel nanomaterial for treating atherosclerosis.

The roles of C-terminal helices of human apolipoprotein A-I in formation of high-density lipoprotein particles.

Apolipoprotein A-I (apoA-I) accepts cholesterol and phospholipids from ATP-binding cassette transporter A1 (ABCA1)-expressing cells to form high-density lipoprotein (HDL). Human apoA-I has two tertiary structural domains and the C-terminal domain (approximately amino acids 190-243) plays a key role in lipid binding. Although the high lipid affinity region of the C-terminal domain of apoA-I (residues 223-243) is essential for the HDL formation, the function of low lipid affinity region (residues 191-220) remains unclear. To evaluate the role of residues 191-220, we analyzed the structure, lipid binding properties, and HDL formation activity of Δ191-220 apoA-I, in comparison to wild-type and Δ223-243 apoA-I. Although deletion of residues 191-220 has a slight effect on the tertiary structure of apoA-I, the Δ191-220 variant showed intermediate behavior between wild-type and Δ223-243 regarding the formation of hydrophobic sites and lipid interaction through the C-terminal domain. Physicochemical analysis demonstrated that defective lipid binding of Δ191-220 apoA-I is due to the decreased ability to form α-helix structure which provides the energetic source for lipid binding. In addition, the ability to form HDL particles in vitro and induce cholesterol efflux from ABCA1-expressing cells of Δ191-220 apoA-I was also intermediate between wild-type and Δ223-243 apoA-I. These results suggest that despite possessing low lipid affinity, residues 191-220 play a role in enhancing the ability of apoA-I to bind to and solubilize lipids by forming α-helix upon lipid interaction. Our results demonstrate that the combination of low lipid affinity region and high lipid affinity region of apoA-I is required for efficient ABCA1-dependent HDL formation.

Construction of antibody/insulin receptor chimera for growth induction of mammalian cells.

The insulin receptor (IR) is expressed ubiquitously in various tissues, where insulin exerts various biological effects on the target cells, such as cellular metabolic changes, cell proliferation and differentiation. Therefore, mimicry of insulin signaling would be a promising strategy to realize artificial control of such cellular fates. In this study, we constructed an antibody/insulin receptor chimera that enables to utilize any antigen as the ligand in principle. We constructed chimeric receptors consisting of anti-fluorescein single chain Fv (scFv), the extracellular D2 domain of erythropoietin receptor and the transmembrane/intracellular domains of IR (scFv-IR; S-IR). The function of S-IR was evaluated in terms of growth signal transduction in murine pro-B Ba/F3 cells and murine fibroblast NIH/3T3 cells. S-IR exerted IL-3-independent cell growth in Ba/F3 cells, while NIH/3T3 cells expressing S-IR acquired growth advantage over parental NIH/3T3 cells in a low-serum condition. S-IR induced phosphorylation of S-IR itself and key signaling molecules downstream of IR. Although antigen-independent activation was significantly observed, S-IR enabled specific amplification of the gene-transduced cells.

Selection and growth regulation of genetically modified cells with hapten-specific antibody/receptor tyrosine kinase chimera.

Although receptor tyrosine kinases (RTKs) play a pivotal role in the development and maintaining the homeostasis of the body, overexpression or mutation of RTKs often induces tumorigenesis or metastasis. To mimic the function of RTKs, we developed two fusion receptors consisting of anti-fluorescein antibody single-chain Fv, extracellular D2 domain of erythropoietin receptor and transmembrane/intracellular domains of epidermal growth factor receptor or c-fms based on previously constructed antibody/cytokine receptor chimeras. The expression of these chimeric receptors in the hematopoietic cell line Ba/F3 and non-hematopoietic cell line NIH/3T3 resulted in the activation of receptors themselves, downstream signaling molecules and cell proliferation in response to fluorescein-conjugated BSA, leading to selective expansion of transduced cells up to almost 100%. These results indicate that the cognate antigen could activate the chimeric receptors even though the wild-type extracellular domains were switched to the antibody fragment. This is the first study to show that our antigen-mediated genetically modified cell amplification (AMEGA) system could be applied to non-hematopoietic cells by utilizing antibody/RTK chimeras.