CAR T-cell-mediated delivery of bispecific innate immune cell engagers for neuroblastoma.

Novel chimeric antigen receptor (CAR) T-cell approaches are needed to improve therapeutic efficacy in solid tumors. High-risk neuroblastoma is an aggressive pediatric solid tumor that expresses cell-surface GPC2 and GD2 with a tumor microenvironment infiltrated by CD16a-expressing innate immune cells. Here we engineer T-cells to express a GPC2-directed CAR and simultaneously secrete a bispecific innate immune cell engager (BiCE) targeting both GD2 and CD16a. In vitro, GPC2.CAR-GD2.BiCE T-cells induce GPC2-dependent cytotoxicity and secrete GD2.BiCE that promotes GD2-dependent activation of antitumor innate immunity. In vivo, GPC2.CAR-GD2.BiCE T-cells locally deliver GD2.BiCE and increase intratumor retention of NK-cells. In mice bearing neuroblastoma patient-derived xenografts and reconstituted with human CD16a-expressing immune cells, GD2.BiCEs enhance GPC2.CAR antitumor efficacy. A CAR.BiCE strategy should be considered for tumor histologies where antigen escape limits CAR efficacy, especially for solid tumors like neuroblastoma that are infiltrated by innate immune cells.

Elp1 function in placode-derived neurons is critical for proper trigeminal ganglion development.

Background: The trigeminal nerve is the largest cranial nerve and functions in somatosensation. Cell bodies of this nerve are positioned in the trigeminal ganglion, which arises from the coalescence of neural crest and placode cells. While this dual cellular origin has been known for decades, the molecular mechanisms controlling trigeminal ganglion development remain obscure. We performed RNAsequencing on the forming chick trigeminal ganglion and identified Elongator acetyltransferase complex subunit 1 ( Elp1 ) for further study. Mutations in ELP1 cause familial dysautonomia (FD), a fatal disorder characterized by the presence of smaller trigeminal nerves and sensory deficits. While Elp1 has established roles in neurogenesis, its functions in placode cells during trigeminal gangliogenesis have not been investigated. Results: To this end, we used morpholinos to deplete Elp1 from chick trigeminal placode cells. Elp1 knockdown decreased trigeminal ganglion size and led to aberrant innervation of the eye by placode-derived neurons. Trigeminal nerve branches exhibited fewer axons, and abnormal interactions between placode-derived neurons and neural crest cells were observed. Conclusions: These findings reveal a new role for Elp1 in chick placode-derived neurons during trigeminal ganglion development. These results have potential high significance to provide new insights into trigeminal ganglion development and the etiology of FD. Bullet points: Elp1 is expressed in undifferentiated neural crest cells and placode-derived neurons contributing to the trigeminal ganglion.Elp1 knockdown in trigeminal placode cells reduces trigeminal ganglion size.Elp1 depletion from trigeminal placode cells leads to aberrant target tissue innervation and disrupts proper neural crest-placodal neuron interactions in the trigeminal ganglion. Grant sponsor and number: NIH R01DE024217 and NIH R03HD108480.

Human antibody VH domains targeting uPAR as candidate therapeutics for cancers.

The high expression of uPAR has been linked to tumor progression, invasion, and metastasis in several types of cancer. Such overexpression of uPAR makes it a potential target for immunotherapies across common cancers such as breast, colorectal, lung, ovarian cancer, and melanoma. In our study, two high-affinity and specific human VH domain antibody candidates, designed as clones 3 and 115, were isolated from a phage-displayed human VH antibody library. Domain-based bispecific T- cell engagers (DbTE) based on these two antibodies exhibited potent killing of uPAR-positive cancer cells. Thus, these two anti-uPAR domain antibodies are promising candidates for treating uPAR positive cancers.

Human Antibody VH Domains Targeting GPNMB and VCAM-1 as Candidate Therapeutics for Cancers.

The elevated expression of GPNMB and VCAM-1 has been observed in many cancers including breast cancer, melanoma, and prostate cancers. Such overexpression of GPNMB and VCAM-1 has been associated with poor prognosis and increased cancer metastasis. Thus, GPNMB and VCAM-1 are potential targets for immunotherapies across multiple cancers. In this study, two high-affinity specific human VH domain antibody candidates, 87 (GPNMB) and 1B2 (VCAM-1), were isolated from our in-house proprietary phage-displayed human VH antibody domain libraries. The avidity was increased after conversion to VH-Fc. Domain-based bispecific T-cell engagers (DbTE) based on these two antibodies combined with the anti-CD3ε OKT3 antibody exhibited potent killing against GPNMB and VCAM-1-positive cancer cells, respectively. Hence, these two domain antibodies are promising therapeutic candidates for cancers expressing GPNMB or VCAM-1.

Human antibodies targeting ENPP1 as candidate therapeutics for cancers.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is a type II transmembrane glycoprotein expressed in many tissues. High expression levels of ENPP1 have been observed in many cancer types such as lung cancer, ovarian cancer, and breast cancer. Such overexpression has been associated with poor prognosis in these diseases. Hence, ENPP1 is a potential target for immunotherapy across multiple cancers. Here, we isolated and characterized two high-affinity and specific anti-ENPP1 Fab antibody candidates, 17 and 3G12, from large phage-displayed human Fab libraries. After conversion to IgG1, the binding of both antibodies increased significantly due to avidity effects. Based on these antibodies, we generated antibody-drug conjugates (ADCs), IgG-based bispecific T-cell engagers (IbTEs), and CAR T-cells which all exhibited potent killing of ENPP1-expressing cells. Thus, these various antibody-derived modalities are promising therapeutic candidates for cancers expressing human ENPP1.

Neurogenin 2 and Neuronal Differentiation 1 Control Proper Development of the Chick Trigeminal Ganglion and Its Nerve Branches.

The trigeminal ganglion contains the cell bodies of sensory neurons comprising cranial nerve V, which relays information related to pain, touch, and temperature from the face and head to the brain. Like other cranial ganglia, the trigeminal ganglion is composed of neuronal derivatives of two critical embryonic cell types, neural crest and placode cells. Neurogenesis within the cranial ganglia is promoted by Neurogenin 2 (Neurog2), which is expressed in trigeminal placode cells and their neuronal derivatives, and transcriptionally activates neuronal differentiation genes such as Neuronal Differentiation 1 (NeuroD1). Little is known, however, about the role of Neurog2 and NeuroD1 during chick trigeminal gangliogenesis. To address this, we depleted Neurog2 and NeuroD1 from trigeminal placode cells with morpholinos and demonstrated that Neurog2 and NeuroD1 influence trigeminal ganglion development. While knockdown of both Neurog2 and NeuroD1 affected innervation of the eye, Neurog2 and NeuroD1 had opposite effects on ophthalmic nerve branch organization. Taken together, our results highlight, for the first time, functional roles for Neurog2 and NeuroD1 during chick trigeminal gangliogenesis. These studies shed new light on the molecular mechanisms underlying trigeminal ganglion formation and may also provide insight into general cranial gangliogenesis and diseases of the peripheral nervous system.

Potent and broad neutralization of SARS-CoV-2 variants of concern (VOCs) including omicron sub-lineages BA.1 and BA.2 by biparatopic human VH domains.

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics with high neutralization breadth. Here, we characterized a human VH domain, F6, which we generated by sequentially panning large phage-displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that broadly and potently neutralized VOCs including Omicron. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 variants including Omicron and highlight a vulnerable epitope within the spike that may be exploited to achieve broad protection against circulating variants.

Potent Neutralization of Omicron and other SARS-CoV-2 Variants of Concern by Biparatopic Human VH Domains.

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics that are effective against a variety of strains of the virus. Herein, we characterize a human V H domain, F6, which we generated by sequentially panning large phage displayed V H libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized V H domain, resulted in a construct (F6-ab8-Fc) that neutralized Omicron pseudoviruses with a half-maximal neutralizing concentration (IC 50 ) of 4.8 nM in vitro . Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 VOCs - including the recently emerged Omicron variant - and highlight a vulnerable epitope within the spike protein RBD that may be exploited to achieve broad protection against circulating variants.

Associations between routine coronary computed tomographic angiography and reduced unnecessary hospital admissions, length of stay, recidivism rates, and invasive coronary angiography in the emergency department triage of chest pain.

OBJECTIVES: This study was designed to assess the effects on resource utilization of routine coronary computed tomographic angiography (CCTA) in triaging chest pain patients in the emergency department (ED). BACKGROUND: The routine use of CCTA for ED evaluation of chest pain is feasible and safe. METHODS: We conducted a retrospective multivariate analysis of data from two risk-matched cohorts of 894 ED patients presenting with chest pain to assess the impact of CCTA versus standard evaluation on admissions rate, length of stay, major adverse cardiovascular event rates, recidivism rates, and downstream resource utilization. RESULTS: The overall admission rate was lower with CCTA (14% vs. 40%; p < 0.001). Standard evaluation was associated with a 5.5-fold greater risk for admission (odds ratio [OR]: 5.53; p < 0.001). Expected ED length of stay with standard evaluation was about 1.6 times longer (OR: 1.55; p < 0.001). There were no differences in the rates of death and acute myocardial infarction within 30 days of the index visit between the two groups. The likelihood of returning to the ED within 30 days for recurrent chest pain was 5 times greater with standard evaluation (OR: 5.06; p = 0.022). Standard evaluation was associated with a 7-fold greater likelihood of invasive coronary angiography without revascularization (OR: 7.17; p < 0.001), while neither group was significantly more likely to receive revascularization (OR: 2.06; p = 0.193). The median radiation dose with CCTA was 5.88 mSv (n = 1039; confidence interval: 5.2 to 6.4). CONCLUSIONS: The routine use of CCTA in ED evaluation of chest pain reduces healthcare resource utilization.

Shoulder impact response and injury due to lateral and oblique loading.

Little is known about the response of the shoulder complex due to lateral and oblique loading. Increasing this knowledge of shoulder response due to these types of loading could aid in improving the biofidelity of the shoulder mechanisms of anthropomorphic test devices (ATDs). The first objective of this study was to define force versus deflection corridors for the shoulder corresponding to both lateral and oblique loading. A second focus of the shoulder research was to study the differences in potential injury between oblique and lateral loading. These objectives were carried out by combining previously published lateral impact data from 24 tests along with 14 additional recently completed lateral and oblique tests. The newly completed tests utilized a pneumatic ram to impact the shoulder of approximately fiftieth percentile sized cadavers at the level of the glenohumeral joint with a constant speed of approximately 4.4 m/sec. Of the 14 tests, four of them were conducted lateral to the shoulder along the subject's y-axis, four of them were conducted 15 anterior to this axis, and six were conducted 30 anterior to the subject's y-axis. As in the previous testing, the first thoracic vertebrae and both shoulders of the subject were instrumented with tri-axial linear accelerometers on the sternum, clavicle, acromion process, and inferior angle of the scapula. The impacting mass was instrumented with an accelerometer and displacement transducer. In addition to this instrumentation, the tests were documented by high-speed digital imagery. Radiographs (x-rays), magnetic resonance images (MRIs), and autopsies were used to document injury to the subjects. The results from the tests revealed differences between the stiffness of the shoulder when loaded laterally to that when it is loaded obliquely. The shoulder was found to deflect twice as much medially when loaded obliquely then when it is loaded laterally. This can be attributed to the ability of the scapula to slide posteriorly around the thoracic cage. The ability of the shoulder to displace medially while simultaneously deflecting posteriorly in oblique impact is important to replicate in the ATDs because it results in the load being transmitted to the upper thoracic cage.