S309-CAR-NK cells bind the Omicron variants in vitro and reduce SARS-CoV-2 viral loads in humanized ACE2-NSG mice.

Recent progress on chimeric antigen receptor (CAR)-NK cells has shown promising results in treating CD19-positive lymphoid tumors with minimal toxicities [including graft versus host disease (GvHD) and cytokine release syndrome (CRS) in clinical trials. Nevertheless, the use of CAR-NK cells in combating viral infections has not yet been fully explored. Previous studies have shown that CAR-NK cells expressing S309 single-chain fragment variable (scFv), hereinafter S309-CAR-NK cells, can bind to SARS-CoV-2 wildtype pseudotyped virus (PV) and effectively kill cells expressing wild-type spike protein in vitro. In this study, we further demonstrate that the S309-CAR-NK cells can bind to different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants in vitro. We also show that S309-CAR-NK cells reduce virus loads in the NOD/SCID gamma (NSG) mice expressing the human angiotensin-converting enzyme 2 (hACE2) receptor challenged with SARS-CoV-2 wild-type (strain USA/WA1/2020). Our study demonstrates the potential use of S309-CAR-NK cells for inhibiting infection by SARS-CoV-2 and for the potential treatment of COVID-19 patients unresponsive to otherwise currently available therapeutics. IMPORTANCE: Chimeric antigen receptor (CAR)-NK cells can be "off-the-shelf" products that treat various diseases, including cancer, infections, and autoimmune diseases. In this study, we engineered natural killer (NK) cells to express S309 single-chain fragment variable (scFv), to target the Spike protein of SARS-CoV-2, hereinafter S309-CAR-NK cells. Our study shows that S309-CAR-NK cells are effective against different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants. The S309-CAR-NK cells can (i) directly bind to SARS-CoV-2 pseudotyped virus (PV), (ii) competitively bind to SARS-CoV-2 PV with 293T cells expressing the human angiotensin-converting enzyme 2 (hACE2) receptor (293T-hACE2 cells), (iii) specifically target and lyse A549 cells expressing the spike protein, and (iv) significantly reduce the viral loads of SARS-CoV-2 wild-type (strain USA/WA1/2020) in the lungs of NOD/SCID gamma (NSG) mice expressing hACE2 (hACE2-NSG mice). Altogether, the current study demonstrates the potential use of S309-CAR-NK immunotherapy as an alternative treatment for COVID-19 patients.

Development of a novel human CD147 knock-in NSG mouse model to test SARS-CoV-2 viral infection.

BACKGROUND: An animal model that can mimic the SARS-CoV-2 infection in humans is critical to understanding the rapidly evolving SARS-CoV-2 virus and for development of prophylactic and therapeutic strategies to combat emerging mutants. Studies show that the spike proteins of SARS-CoV and SARS-CoV-2 bind to human angiotensin-converting enzyme 2 (hACE2, a well-recognized, functional receptor for SARS-CoV and SARS-CoV-2) to mediate viral entry. Several hACE2 transgenic (hACE2Tg) mouse models are being widely used, which are clearly invaluable. However, the hACE2Tg mouse model cannot fully explain: (1) low expression of ACE2 observed in human lung and heart, but lung or heart failure occurs frequently in severe COVID-19 patients; (2) low expression of ACE2 on immune cells, but lymphocytopenia occurs frequently in COVID-19 patients; and (3) hACE2Tg mice do not mimic the natural course of SARS-CoV-2 infection in humans. Moreover, one of most outstanding features of coronavirus infection is the diversity of receptor usage, which includes the newly proposed human CD147 (hCD147) as a possible co-receptor for SARS-CoV-2 entry. It is still debatable whether CD147 can serve as a functional receptor for SARS-CoV-2 infection or entry. RESULTS: Here we successfully generated a hCD147 knock-in mouse model (hCD147KI) in the NOD-scid IL2Rgammanull (NSG) background. In this hCD147KI-NSG mouse model, the hCD147 genetic sequence was placed downstream of the endogenous mouse promoter for mouse CD147 (mCD147), which creates an in vivo model that may better recapitulate physiological expression of hCD147 proteins at the molecular level compared to the existing and well-studied K18-hACE2-B6 (JAX) model. In addition, the hCD147KI-NSG mouse model allows further study of SARS-CoV-2 in the immunodeficiency condition which may assist our understanding of this virus in the context of high-risk populations in immunosuppressed states. Our data show (1) the human CD147 protein is expressed in various organs (including bronchiolar epithelial cells) in hCD147KI-NSG mice by immunohistochemical staining and flow cytometry; (2) hCD147KI-NSG mice are marginally sensitive to SARS-CoV-2 infection compared to WT-NSG littermates characterized by increased viral copies by qRT-PCR and moderate body weight decline compared to baseline; (3) a significant increase in leukocytes in the lungs of hCD147KI-NSG mice, compared to infected WT-NSG mice. CONCLUSIONS: hCD147KI-NSG mice are more sensitive to COVID-19 infection compared to WT-NSG mice. The hCD147KI-NSG mouse model can serve as an additional animal model for further interrogation whether CD147 serve as an independent functional receptor or accessory receptor for SARS-CoV-2 entry and immune responses.

Development of a Novel Human CD147 Knock-in NSG Mouse Model to Test SARS-CoV-2 Viral Infection.

Background: An animal model that can mimic the SARS-CoV-2 infection in humans is critical to understanding the rapidly evolving SARS-CoV-2 virus and for development of prophylactic and therapeutic strategies to combat emerging mutants. Studies show that the spike proteins of SARS-CoV and SARS-CoV-2 bind to human angiotensin-converting enzyme 2 (hACE2, a well-recognized, functional receptor for SARS-CoV and SARS-CoV-2) to mediate viral entry. Several hACE2 transgenic (hACE2Tg) mouse models are being widely used, which are clearly invaluable. However, the hACE2Tg mouse model cannot fully explain: 1) low expression of ACE2 observed in human lung and heart, but lung or heart failure occurs frequently in severe COVID-19 patients; 2) low expression of ACE2 on immune cells, but lymphocytopenia occurs frequently in COVID-19 patients; and 3) hACE2Tg mice do not mimic the natural course of SARS-CoV-2 infection in humans. Moreover, one of most outstanding features of coronavirus infection is the diversity of receptor usage, which includes the newly proposed human CD147 (hCD147) as a possible co-receptor for SARS-CoV-2 entry. It is still debatable whether CD147 can serve as a functional receptor for SARS-CoV-2 infection or entry. Results: Here we successfully generated a hCD147 knock-in mouse model (hCD147KI) in the NOD- scid IL2Rgamma null (NSG) background. In this hCD147KI-NSG mouse model, the hCD147 genetic sequence was placed downstream of the endogenous mouse promoter for mouse CD147 (mCD147), which creates an in vivo model that may better recapitulate physiological expression of hCD147 proteins at the molecular level compared to the existing and well-studied K18-hACE2-B6 (JAX) model. In addition, the hCD147KI-NSG mouse model allows further study of SARS-CoV-2 in the immunodeficiency condition which may assist our understanding of this virus in the context of high-risk populations in immunosuppressed states. Our data show 1) the human CD147 protein is expressed in various organs (including bronchiolar epithelial cells) in hCD147KI-NSG mice by immunohistochemical staining and flow cytometry; 2) hCD147KI-NSG mice are marginally sensitive to SARS-CoV-2 infection compared to WT-NSG littermates characterized by increased viral copies by qRT-PCR and moderate body weight decline compared to baseline; 3) a significant increase in leukocytes in the lungs of hCD147KI-NSG mice, compared to infected WT-NSG mice. Conclusions: hCD147KI-NSG mice are more sensitive to COVID-19 infection compared to WT-NSG mice. The hCD147KI-NSG mouse model can serve as an additional animal model for further interrogation whether CD147 serve as an independent functional receptor or accessory receptor for SARS-CoV-2 entry and immune responses.

Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line.

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, particularly CAR-NK cell, is one of the most promising 'off-the-shelf' development without severe life-threatening toxicity. However, the bottleneck for developing a successful CAR-NK therapy is achieving sufficient numbers of non-exhaustive, long-lived, 'off-the-shelf' CAR-NK cells from a third party. Here, we developed a new CAR-NK expansion method using an Epstein-Barr virus- (EBV) transformed B cell line expressing a genetically modified membrane form of interleukin-21 (IL-21). In this protocol, step-by-step procedures are provided to expand NK and CAR-NK cells from cord blood and peripheral blood, as well as solid organ tissues. This work will significantly enhance the clinical development of CAR-NK immunotherapy.

CAR-NK Cells Effectively Target SARS-CoV-2-Spike-Expressing Cell Lines In Vitro.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly contagious and presents a significant public health issue. Current therapies used to treat coronavirus disease 2019 (COVID-19) include monoclonal antibody cocktail, convalescent plasma, antivirals, immunomodulators, and anticoagulants. The vaccines from Pfizer and Moderna have recently been authorized for emergency use, which are invaluable for the prevention of SARS-CoV-2 infection. However, their long-term side effects are not yet documented, and populations with immunocompromised conditions (e.g., organ-transplantation and immunodeficient patients) may not be able to mount an effective immune response. In addition, there are concerns that wide-scale immunity to SARS-CoV-2 may introduce immune pressure that could select for escape mutants to the existing vaccines and monoclonal antibody therapies. Emerging evidence has shown that chimeric antigen receptor (CAR)- natural killer (NK) immunotherapy has potent antitumor response in hematologic cancers with minimal adverse effects in recent studies, however, the potentials of CAR-NK cells in treating COVID-19 has not yet been fully exploited. Here, we improve upon a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2 and its various mutants. CAR-NK cells were generated using the scFv domain of S309 (henceforward, S309-CAR-NK), a SARS-CoV and SARS-CoV-2 neutralizing antibody (NAbs) that targets the highly conserved region of SARS-CoV-2 spike (S) glycoprotein and is therefore more likely to recognize different variants of SARS-CoV-2 isolates. S309-CAR-NK cells can specifically bind to pseudotyped SARS-CoV-2 virus and its D614G, N501Y, and E484K mutants. Furthermore, S309-CAR-NK cells can specifically kill target cells expressing SARS-CoV-2 S protein in vitro and show superior killing activity and cytokine production, compared to that of the recently reported CR3022-CAR-NK cells. Thus, these results pave the way for generating 'off-the-shelf' S309-CAR-NK cells for treatment in high-risk individuals as well as provide an alternative strategy for patients unresponsive to current vaccines.

Deletion of ER-retention motif on SARS-CoV-2 spike protein reduces cell hybrid during cell-cell fusion.

BACKGROUND: The novel SARS-CoV-2 has quickly become a global pandemic since the first reported case in December 2019, with the virus infecting millions of people to date. The spike (S) protein of the SARS-CoV-2 virus plays a key role in binding to angiotensin-converting enzyme 2 (ACE2), a host cell receptor for SARS-CoV-2. S proteins that are expressed on the cell membrane can initiate receptor-dependent syncytia formation that is associated with extensive tissue damage. Formation of syncytia have been previously observed in cells infected with various other viruses (e.g., HIV, Ebola, Influenza, and Herpesviruses). However, this phenomenon is not well documented and the mechanisms regulating the formation of the syncytia by SARS-CoV-2 are not fully understood. RESULTS: In this study, we investigated the possibility that cell fusion events mediated by the S protein of SARS-CoV-2 and ACE2 interaction can occur in different human cell lines that mimic different tissue origins. These cell lines were transduced with either wild-type (WT-S) S protein or a mutated variant where the ER-retention motif was removed (Δ19-S), as well as human ACE2 expression vectors. Different co-culture combinations of spike-expressing 293T, A549, K562, and SK-Hep1 cells with hACE2-expressing cells revealed cell hybrid fusion. However, only certain cells expressing S protein can form syncytial structures as this phenomenon cannot be observed in all co-culture combinations. Thus, SARS-CoV-2 mediated cell-cell fusion represents a cell type-dependent process which might rely on a different set of parameters. Recently, the Δ19-S variant is being widely used to increase SARS-CoV-2 pseudovirus production for in vitro assays. Comparison of cell fusion occurring via Δ19-S expressing cells shows defective nuclear fusion and syncytia formation compared to WT-S. CONCLUSIONS: This distinction between the Δ19-S variant and WT-S protein may have downstream implications for studies that utilize pseudovirus-based entry assays. Additionally, this study suggest that spike protein expressed by vaccines may affect different ACE2-expressing host cells after SARS-CoV-2 vaccine administration. The long-term effects of these vaccines should be monitored carefully. Δ19-S mRNA may represent a safer mRNA vaccine design in the future.

Potential role of IFN-α in COVID-19 patients and its underlying treatment options.

The coronavirus disease (COVID-19) caused by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly worldwide. Given that this contagious viral outbreak is still unfolding, it is urgent to understand the pathogenesis of SARS-CoV-2 infection and explore effective treatments to protect patients from developing a severe illness related to COVID-19. Recently, IFN-α has been considered a potential therapeutic strategy to treat COVID-19 disease, mainly because the innate immune system rapidly produces IFN-α as the first line of defense to combat viral infections. However, IFN-α can also play a role in immunoregulatory effects, causing pathogenic damage and uncontrolled inflammatory responses. There are 13 human IFN-α subtypes that bind to the same receptor and induce different interferon-stimulated gene (ISG) expression, regulating various antiviral and immunoregulatory effects. The varying degrees of inflammatory regulations may raise concerns about the possible side effects to enlarge the inflammatory responses, exacerbating the severity of infection. Thus, the analysis of various IFN-α subtype induction during SARS-CoV-2 infection is necessary in exploring the mechanism of COVID-19 pathogenesis. This review summarizes the current understanding of IFN-α in the pathogenesis of respiratory virus diseases and IFN-α based clinical intervention used in SARS-CoV-2 infection and other respiratory virus diseases. Besides, new ideas in selecting suitable IFN-α subtypes or combinations as drug candidates for viral infection treatment will also be discussed.Key Points• IFN-α plays an important role in anti-viral and immunoregulatory effects in COVID-19 patients caused by SARS-CoV-2.• The uncontrolled inflammation and disease severity correlated to the diversity of IFN-α subtype induction.• Selecting suitable IFN-α subtypes or combinations as drug candidates will be beneficial for the treatment of patients with COVID-19.

Development of a Novel Human CD147 Transgenic NSG Mouse Model to test SARS-CoV-2 Infection and Immune Responses.

An animal model that can mimic the SARS-CoV-2 infection in humans is critical to understanding the newly emerged, rapidly spreading SARS-CoV-2 and development of therapeutic strategies. Studies show that the spike (S) proteins of SARS-CoV (SARS-CoV-S-1-S) and SARS-CoV-2 (SARS-CoV-2-S) bind to human angiotensin-converting enzyme 2 (hACE2, a well-recognized, functional receptor for SARS-CoV and SARS-CoV-2) to mediate viral entry. Several hACE2 transgenic (hACE2Tg) mouse models are being widely used, which is clearly invaluable. However, the hACE2Tg mouse model cannot fully explain: 1) low expression of ACE2 observed in human lung and heart, but lung or heart failure occurs frequently in severe COVID-19 patients); 2) low expression of ACE2 on immune cells, but lymphocytopenia occurs frequently in COVID-19 patients; and 3) hACE2Tg mice do not develop strong clinical disease following SARS-CoV-2 infection in contrast to SARS-CoV-1. Moreover, one of most outstanding features of coronaviruses is the diversity of receptor usage, which includes the newly proposed human CD147 (hCD147) as a receptor for SARS-CoV-2-S. It is still debatable whether CD147 can serve as a functional receptor for SARS-CoV-2 infection or entry. Here we successfully generated a hCD147Tg mouse model in the NOD- scid IL2Rgamma null (NSG) background. In this hCD147Tg-NSG mouse model, the hCD147 genetic sequence was placed following the endogenous mouse promoter for mouse CD147 (mCD147), which creates an in vivo model that may better recapitulate physiological expression of CD147 proteins at the molecular level compared to the existing and well-studied K18-hACE2-B6 model. In addition, the hCD147Tg-NSG mouse model allows further study of SARS-CoV-2 in the immunodeficiency condition which may assist our understanding of this virus in the context of high-risk populations with immunosuppressed conditions. The hCD147Tg-NSG mouse mode can serve as an additional animal model for interrogate whether CD147 serve as an independent functional receptor or accessory receptor for SARS-CoV-2 entry and immune responses.

Deletion of ER-retention Motif on SARS-CoV-2 Spike Protein Reduces Cell Hybrid During Cell-cell Fusion.

The novel SARS-CoV-2 has quickly become a global pandemic since the first reported case in December 2019, with the virus infecting millions of people to date. The spike (S) protein of the SARS-CoV-2 virus plays a key role in binding to angiotensin-converting enzyme 2 (ACE2), a host cell receptor for SARS-CoV-2. S proteins that are expressed on the cell membrane can initiate receptor-dependent syncytia formation that is associated with extensive tissue damage. Formation of syncytia have been previously observed in cells infected with various other viruses (e.g., HIV, Ebola, Influenza, and Herpesviruses). However, this phenomenon is not well documented and the mechanisms regulating the formation of these syncytia by SARS-CoV-2 are not fully understood. In this study, we investigated the possibility that cell fusion events mediated by the S protein of SARS-CoV-2 and ACE2 interaction can occur in different human cell lines that mimic different tissue origins. These cell lines were stably transduced with either wild-type (WT-S) S protein or a mutated variant where the ER-retention motif was removed (Δ19-S), or human ACE2 vectors. Different co-culture combinations of spike-expressing 293T, A549, K562, and SK-Hep1 cells with hACE2-expressing cells revealed cell hybrid fusion. However, only certain cells expressing S protein can form syncytial structures as this phenomenon cannot be observed in all co-culture combinations. Thus, SARS-CoV-2 mediated cell-cell fusion represents a cell type-dependent process which might rely on a different set of parameters. Recently, the Δ19-S variant is being widely used to increase SARS-CoV-2 pseudovirus production for in vitro assays. Comparison of cell fusion occurring via Δ19-S expressing cells shows defective nuclear fusion and syncytia formation compared to WT-S. This distinction between the Δ19-S variant and WT-S protein may have downstream implications for studies that utilize pseudovirus-based entry assays. Additionally, this study suggest that spike protein expressed by vaccines may affect different ACE2-expressing host cells after SARS-CoV-2 vaccine administration. The long-term effects of these vaccines should be monitored carefully.

CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly contagious presenting a significant public health issue. Current therapies used to treat coronavirus disease 2019 (COVID-19) include monoclonal antibody cocktail, convalescent plasma, antivirals, immunomodulators, and anticoagulants, though the current therapeutic options remain limited and expensive. The vaccines from Pfizer and Moderna have recently been authorized for emergency use, which are invaluable for the prevention of SARS-CoV-2 infection. However, their long-term side effects are not yet to be documented, and populations with immunocompromised conditions (e.g., organ-transplantation and immunodeficient patients) may not be able to mount an effective immune response. In addition, there are concerns that wide-scale immunity to SARS-CoV-2 may introduce immune pressure that could select for escape mutants to the existing vaccines and monoclonal antibody therapies. Emerging evidence has shown that chimeric antigen receptor (CAR)- natural killer (NK) immunotherapy has potent antitumor response in hematologic cancers with minimal adverse effects in recent studies, however, the potentials of CAR-NK cells in preventing and treating severe cases of COVID-19 has not yet been fully exploited. Here, we improve upon a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2 and its D614G mutant. CAR-NK cells were generated using the scFv domain of S309 (henceforward, S309-CAR-NK), a SARS-CoV and SARS-CoV-2 neutralizing antibody that targets the highly conserved region of SARS-CoV-2 spike (S) glycoprotein, therefore would be more likely to recognize different variants of SARS-CoV-2 isolates. S309-CAR-NK cells can specifically bind to pseudotyped SARS-CoV-2 virus and its D614G mutant. Furthermore, S309-CAR-NK cells can specifically kill target cells expressing SARS-CoV-2 S protein in vitro and show superior killing activity and cytokine production, compared to that of the recently published CR3022-CAR-NK cells. Thus, these results pave the way for generating 'off-the-shelf' S309-CAR-NK cells for treatment in high-risk individuals as well as provide an alternative strategy for patients unresponsive to current vaccines.

Efficacy of anti-CD147 chimeric antigen receptors targeting hepatocellular carcinoma.

Chimeric antigen receptor (CAR) therapy is a promising immunotherapeutic strategy for treating multiple refractory blood cancers, but further advances are required for solid tumor CAR therapy. One challenge is identifying a safe and effective tumor antigen. Here, we devise a strategy for targeting hepatocellular carcinoma (HCC, one of the deadliest malignancies). We report that T and NK cells transduced with a CAR that recognizes the surface marker, CD147, also known as Basigin, can effectively kill various malignant HCC cell lines in vitro, and HCC tumors in xenograft and patient-derived xenograft mouse models. To minimize any on-target/off-tumor toxicity, we use logic-gated (log) GPC3-synNotch-inducible CD147-CAR to target HCC. LogCD147-CAR selectively kills dual antigen (GPC3+CD147+), but not single antigen (GPC3-CD147+) positive HCC cells and does not cause severe on-target/off-tumor toxicity in a human CD147 transgenic mouse model. In conclusion, these findings support the therapeutic potential of CD147-CAR-modified immune cells for HCC patients.

The Role of Immunological Synapse in Predicting the Efficacy of Chimeric Antigen Receptor (CAR) Immunotherapy.

Chimeric Antigen Receptor (CAR) immunotherapy utilizes genetically-engineered immune cells that express a unique cell surface receptor that combines tumor antigen specificity with immune cell activation. In recent clinical trials, the adoptive transfer of CAR-modified immune cells (including CAR-T and CAR-NK cells) into patients has been remarkably successful in treating multiple refractory blood cancers. To improve safety and efficacy, and expand potential applicability to other cancer types, CARs with different target specificities and sequence modifications are being developed and tested by many laboratories. Despite the overall progress in CAR immunotherapy, conventional tools to design and evaluate the efficacy and safety of CAR immunotherapies can be inaccurate, time-consuming, costly, and labor-intensive. Furthermore, existing tools cannot always determine how responsive individual patients will be to a particular CAR immunotherapy. Recent work in our laboratory suggests that the quality of the immunological synapse (IS) can accurately predict CAR-modified cell efficacy (and toxicity) that can correlate with clinical outcomes. Here we review current efforts to develop a Synapse Predicts Efficacy (SPE) system for easy, rapid and cost-effective evaluation of CAR-modified immune cell immunotherapy. Ultimately, we hypothesize the conceptual basis and clinical application of SPE will serve as an important parameter in evaluating CAR immunotherapy and significantly advance precision cancer immunotherapy. Video abstract Graphic abstract for manuscript CCAS-D-20-00136 by Liu, D., et al., 'The Role of Immunological Synapse in Predicting the Efficacy of Chimeric Antigen Receptor (CAR) Immunotherapy". The various branches of evaluating cancer immunotherapy metaphorically represented as a Rubik's cube. The development of a novel approach to predict the effectiveness of Chimeric Antigen Receptor (CAR)-modified cells by quantifying the quality of CAR IS will introduce a new parameter to the rapidly expanding field of cancer immunotherapy. Currently, no single parameter can predict the clinical outcome or efficacy of a specific type of CAR-modified cell. IS quality will serve as a quantifiable measure to evaluate CAR products and can be used in conjunction with other conventional parameters to form a composite clinical predictor. Much like a Rubik's cube has countless configurations, several methods and combinations of clinical metrics have arisen for evaluating the ability of a given immunotherapeutic strategy to treat cancer. The quality of IS depicting cancer immunotherapy is metaphorically expressed as a Rubik's cube. Each face/color represents one aspect of cancer therapy. Each grid in one face indicates one factor within that aspect of cancer therapy. For example, the green color represents the tumor microenvironment, and one out of the nine grids in the green color indicates suppressor cells (suppressors in green). Changes in one factor may completely alter the entire strategy of cancer therapy. However, the quality of IS (illuminated center red grid) makes the effectiveness of CAR immunotherapy predictable.

Efficacy of Targeting SARS-CoV-2 by CAR-NK Cells.

SARS-CoV-2, which causes COVID-19 disease, is one of greatest global pandemics in history. No effective treatment is currently available for severe COVID-19 disease. One strategy for implementing cell-based immunity involves the use of chimeric antigen receptor (CAR) technology. Unlike CAR T cells, which need to be developed using primary T cells derived from COVID-19 patients with lymphopenia, clinical success of CAR NK cell immunotherapy is possible through the development of allogeneic, universal, and 'off-the-shelf' CAR-NK cells from a third party, which will significantly broaden the application and reduce costs. Here, we develop a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2. CAR-NK cells were generated using the scFv domain of CR3022 (henceforth, CR3022-CAR-NK), a broadly neutralizing antibody for SARS-CoV-1 and SARS-CoV-2. CR3022-CAR-NK cells can specifically bind to RBD of SARS-CoV-2 and pseudotyped SARS-CoV-2 S protein, and can be activated by pseudotyped SARS-CoV-2-S viral particles in vitro. Further, CR3022-CAR-NK cells can specifically kill pseudo-SARS-CoV-2 infected target cells. Thus, 'off-the-shelf' CR3022-CAR-NK cells may have the potential to treat patients with severe COVID-19 disease.

Superior Expansion and Cytotoxicity of Human Primary NK and CAR-NK Cells from Various Sources via Enriched Metabolic Pathways.

Clinical success of chimeric antigen receptor (CAR) T cell immunotherapy requires the engineering of autologous T cells, which limits the broader implementation of CAR cell therapy. The development of allogeneic and universal cell products will significantly broaden their application and reduce costs. Allogeneic natural killer (NK) cells can be used for universal CAR immunotherapy. Here, we develop an alternative approach for the rapid expansion of primary NK and CAR-NK cells with superior expansion capability and in vivo cytotoxicity from various sources (including peripheral blood, cord blood, and tumor tissue). We apply a human B-lymphoblastoid cell-line 721.221 (hereinafter, 221)-based artificial feeder cell system with membrane-bound interleukin 21 (mIL-21) to propagate NK and CAR-NK cells. The expansion capability, purity, and cytotoxicity of NK cells expanded with 221-mIL-21 feeder cells are superior to that of conventional K562-mIL-21 feeder cells. RNA sequencing (RNA-seq) data show that 221-mIL-21 feeder cell-expanded NK cells display a less differentiated, non-exhausted, limited fratricidal, memory-like phenotype correlated with enriched metabolic pathways, which explains underlying mechanisms. Thus, "off-the-shelf" NK and CAR-NK cells with superior functionalities and expansion using a genetically modified 221-mIL-21 feeder cell expansion system will greatly support clinical use of NK immunotherapy.

Superior semicircular canal dehiscence: Diagnosis and management.

The authors provide an update on the clinical manifestations, diagnosis and various approaches to the treatment of superior semicircular canal dehiscence (SSCD). SSCD is a rare condition where the bone overlying the superior semicircular canal thins or dehisces causing characteristic clinical findings. Since this was first reported in 1998 by Minor and colleagues, there has been much advancement made in terms of diagnosis and treatment. Signs and symptoms include a wide variation of both vestibular and auditory manifestations. Diagnosis made solely on clinical signs is difficult due to how varied the presentations can be and the overlap with other otologic pathologies. High-resolution CT temporal scans have been the standard in confirming superior semicircular canal dehiscence, however, MRI FIESTA scans have recently been used to image SSCD. Additionally, audiometry and vestibular evoked myogenic potential (VEMP) testing are useful screening tools. Currently, the middle fossa approach is the most common and standard surgical approach to repair SSCD. The transmastoid, endoscopic and transcanal or endaural approaches have also been recently utilized. Presently, there is no consensus as to the best approach, material or technique for repair of SSCD. As we learn more, newer and less invasive approaches and techniques are being used to treat SSCD. We present a comprehensive review of SSCD, including clinical symptoms and presentation, histopathology, diagnosis, treatment strategies and outcomes of intervention.

A National WestlawNext Database Analysis of Malpractice Litigation in Radiation Oncology.

Although litigation involving radiation oncologists was infrequent and most verdicts were in favor of defendants, many cases resulted from claims of excessive radiation, unnecessary radiation, and a failure to refer and/or order appropriate tests.