Agonist antibody to MuSK protects mice from MuSK myasthenia gravis.

Myasthenia gravis (MG) is a chronic and severe disease of the skeletal neuromuscular junction (NMJ) in which the effects of neurotransmitters are attenuated, leading to muscle weakness. In the most common forms of autoimmune MG, antibodies attack components of the postsynaptic membrane, including the acetylcholine receptor (AChR) or muscle-specific kinase (MuSK). MuSK, a master regulator of NMJ development, associates with the low-density lipoprotein-related receptor 4 (Lrp4) to form the signaling receptor for neuronal Agrin, a nerve-derived synaptic organizer. Pathogenic antibodies to MuSK interfere with binding between MuSK and Lrp4, inhibiting the differentiation and maintenance of the NMJ. MuSK MG can be debilitating and refractory to treatments that are effective for AChR MG. We show here that recombinant antibodies, derived from MuSK MG patients, cause severe neuromuscular disease in mice. The disease can be prevented by a MuSK agonist antibody, presented either prophylactically or after disease onset. These findings suggest a therapeutic alternative to generalized immunosuppression for treating MuSK MG by selectively and directly targeting the disease mechanism.

Serological Markers of Clinical Improvement in MuSK Myasthenia Gravis.

BACKGROUND AND OBJECTIVES: In this retrospective longitudinal study, we aimed at exploring the role of (a) MuSK-immunoglobulin G (IgG) levels, (b) predominant MuSK-IgG subclasses, and (c) antibody affinity as candidate biomarkers of severity and outcomes in MuSK-MG, using and comparing different antibody testing techniques. METHODS: Total MuSK-IgGs were quantified with radioimmunoassay (RIA), ELISA, flow cytometry, and cell-based assay (CBA) serial dilutions using HEK293 cells transfected with MuSK-eGFP. MuSK-IgG subclasses were measured by flow cytometry. SAffCon assay was used for determining MuSK-IgG affinity. RESULTS: Forty-three serum samples were obtained at different time points from 20 patients with MuSK-MG (median age at onset: 48 years, interquartile range = 27.5-72.5; women, 16/20), with 9 of 20 (45%) treated with rituximab. A strong correlation between MuSK-IgG levels measured by flow cytometry and RIA titers was found (rs = 0.74, 95% CI 0.41-0.89, p = 0.0003), as well as a moderate correlation between CBA end-point titers and RIA titers (rs = 0.47, 95% CI 0.01-0.77, p = 0.0414). A significant correlation was found between MuSK-IgG flow cytometry levels and disease severity (rs = 0.39, 95% CI 0.06-0.64, p = 0.0175; mixed-effects model estimate: 2.296e-06, std. error: 1.024e-06, t = 2.243, p = 0.032). In individual patients, clinical improvement was associated with decrease in MuSK-IgG levels, as measured by either flow cytometry or CBA end-point titration. In all samples, MuSK-IgG4 was the most frequent isotype (mean ± SD: 90.95% ± 13.89). A significant reduction of MuSK-IgG4 and, to a lesser extent, of MuSK-IgG2, was seen in patients with favorable clinical outcomes. A similar trend was confirmed in the subgroup of rituximab-treated patients. In a single patient, MuSK-IgG affinity increased during symptom exacerbation (KD values: 62 nM vs 0.6 nM) while total MuSK-IgG and IgG4 levels remained stable, suggesting that affinity maturation may be a driver of clinical worsening. DISCUSSION: Our data support the quantification of MuSK antibodies by flow cytometry. Through a multimodal investigational approach, we showed that total MuSK-IgG levels, MuSK-IgG4 and MuSK-IgG2 levels, and MuSK-IgG affinity may represent promising biomarkers of disease outcomes in MuSK-MG.

A bispecific antibody targeting EGFR and AXL delays resistance to osimertinib.

Activating EGFR (epidermal growth factor receptor) mutations can be inhibited by specific tyrosine kinase inhibitors (TKIs), which have changed the landscape of lung cancer therapy. However, due to secondary mutations and bypass receptors, such as AXL (AXL receptor tyrosine kinase), drug resistance eventually emerges in most patients treated with the first-, second-, or third-generation TKIs (e.g., osimertinib). To inhibit AXL and resistance to osimertinib, we compare two anti-AXL drugs, an antibody (mAb654) and a TKI (bemcentinib). While no pair of osimertinib and an anti-AXL drug is able to prevent relapses, triplets combining osimertinib, cetuximab (an anti-EGFR antibody), and either anti-AXL drug are initially effective. However, longer monitoring uncovers superiority of the mAb654-containing triplet, possibly due to induction of receptor endocytosis, activation of immune mechanisms, or disabling intrinsic mutators. Hence, we constructed a bispecific antibody that engages both AXL and EGFR. When combined with osimertinib, the bispecific antibody consistently inhibits tumor relapses, which warrants clinical trials.

Myasthenia gravis: the future is here.

Myasthenia gravis (MG) stands as a prototypical antibody-mediated autoimmune disease: it is dependent on T cells and characterized by the presence of autoantibodies targeting proteins located on the postsynaptic surface of skeletal muscle, known as the neuromuscular junction. Patients with MG exhibit a spectrum of weakness, ranging from limited ocular muscle involvement to life-threatening respiratory failure. Recent decades have witnessed substantial progress in understanding the underlying pathophysiology, leading to the delineation of distinct subcategories within MG, including MG linked to AChR or MuSK antibodies as well as age-based distinction, thymoma-associated, and immune checkpoint inhibitor-induced MG. This heightened understanding has paved the way for the development of more precise and targeted therapeutic interventions. Notably, the FDA has recently approved therapeutic inhibitors of complement and the IgG receptor FcRn, a testament to our improved comprehension of autoantibody effector mechanisms in MG. In this Review, we delve into the various subgroups of MG, stratified by age, autoantibody type, and histology of the thymus with neoplasms. Furthermore, we explore both current and potential emerging therapeutic strategies, shedding light on the evolving landscape of MG treatment.

Case report: Rapid clinical improvement in acute exacerbation of MuSK-MG with efgartigimod.

Myasthenia gravis with positive MuSK antibody often involves the bulbar muscles and is usually refractory to acetylcholinesterase inhibitors. For MuSK-MG patients who experience acute exacerbations and do not respond to conventional treatments, there is an urgent need to find more suitable treatment options. With the advent of biologic agents, efgartigimod has shown promising results in the treatment of MG. We report a 65-year-old MuSK-MG patient who presented with impaired eye movements initially, and the symptoms rapidly worsened within a week, affecting the limbs and neck muscles, and had difficulties in chewing and swallowing. Lymphoplasmapheresis did not achieve satisfactory results, but after a cycle of efgartigimod treatment, the patient's symptoms gradually improved and remained in a good clinical state for several months.

AXL-specific single domain antibodies show diagnostic potential and anti-tumor activity in Acute Myeloid Leukemia.

Rationale: AXL expression has been identified as a prognostic factor in acute myeloid leukemia (AML) and is detectable in approximately 50% of AML patients. In this study, we developed AXL-specific single domain antibodies (sdAbs), cross-reactive for both mouse and human AXL protein, to non-invasively image and treat AXL-expressing cancer cells. Methods: AXL-specific sdAbs were induced by immunizing an alpaca with mouse and human AXL proteins. SdAbs were characterized using ELISA, flow cytometry, surface plasmon resonance and the AlphaFold2 software. A lead compound was selected and labeled with 99mTc for evaluation as a diagnostic tool in mouse models of human (THP-1 cells) or mouse (C1498 cells) AML using SPECT/CT imaging. For therapeutic purposes, the lead compound was fused to a mouse IgG2a-Fc tail and in vitro functionality tests were performed including viability, apoptosis and proliferation assays in human AML cell lines and primary patient samples. Using these in vitro models, its anti-tumor effect was evaluated as a single agent, and in combination with standard of care agents venetoclax or cytarabine. Results: Based on its cell binding potential, cross-reactivity, nanomolar affinity and GAS6/AXL blocking capacity, we selected sdAb20 for further evaluation. Using SPECT/CT imaging, we observed tumor uptake of 99mTc-sdAb20 in mice with AXL-positive THP-1 or C1498 tumors. In THP-1 xenografts, an optimized protocol using pre-injection of cold sdAb20-Fc was required to maximize the tumor-to-background signal. Besides its diagnostic value, we observed a significant reduction in tumor cell proliferation and viability using sdAb20-Fc in vitro. Moreover, combining sdAb20-Fc and cytarabine synergistically induced apoptosis in human AML cell lines, while these effects were less clear when combined with venetoclax. Conclusions: Because of their diagnostic potential, sdAbs could be used to screen patients eligible for AXL-targeted therapy and to follow-up AXL expression during treatment and disease progression. When fused to an Fc-domain, sdAbs acquire additional therapeutic properties that can lead to a multidrug approach for the treatment of AXL-positive cancer patients.

A retrospective multicenter study on clinical and serological parameters in patients with MuSK myasthenia gravis with and without general immunosuppression.

Introduction: Muscle-specific kinase (MuSK)- myasthenia gravis (MG) is caused by pathogenic autoantibodies against MuSK that correlate with disease severity and are predominantly of the IgG4 subclass. The first-line treatment for MuSK-MG is general immunosuppression with corticosteroids, but the effect of treatment on IgG4 and MuSK IgG4 levels has not been studied. Methods: We analyzed the clinical data and sera from 52 MuSK-MG patients (45 female, 7 male, median age 49 (range 17-79) years) from Italy, the Netherlands, Greece and Belgium, and 43 AChR-MG patients (22 female, 21 male, median age 63 (range 2-82) years) from Italy, receiving different types of immunosuppression, and sera from 46 age- and sex-matched non-disease controls (with no diagnosed diseases, 38 female, 8 male, median age 51.5 (range 20-68) years) from the Netherlands. We analyzed the disease severity (assessed by MGFA or QMG score), and measured concentrations of MuSK IgG4, MuSK IgG, total IgG4 and total IgG in the sera by ELISA, RIA and nephelometry. Results: We observed that MuSK-MG patients showed a robust clinical improvement and reduction of MuSK IgG after therapy, and that MuSK IgG4 concentrations, but not total IgG4 concentrations, correlated with clinical severity. MuSK IgG and MuSK IgG4 concentrations were reduced after immunosuppression in 4/5 individuals with before-after data, but data from non-linked patient samples showed no difference. Total serum IgG4 levels were within the normal range, with IgG4 levels above threshold (1.35g/L) in 1/52 MuSK-MG, 2/43 AChR-MG patients and 1/45 non-disease controls. MuSK-MG patients improved within the first four years after disease onset, but no further clinical improvement or reduction of MuSK IgG4 were observed four years later, and only 14/52 (26.92%) patients in total, of which 13 (93.3%) received general immunosuppression, reached clinical remission. Discussion: We conclude that MuSK-MG patients improve clinically with general immunosuppression but may require further treatment to reach remission. Longitudinal testing of individual patients may be clinically more useful than single measurements of MuSK IgG4. No significant differences in the serum IgG4 concentrations and IgG4/IgG ratio between AChR- and MuSK-MG patients were found during follow-up. Further studies with larger patient and control cohorts are necessary to validate the findings.

We are what we eat: macrophages and efferocytosis.

Liebold et al. recently revealed how the identity of dying cells drives distinct changes to the macrophages which engulf and clear them, a process known as efferocytosis. During infection with the helminth Schistosoma mansoni, liver macrophages recapitulate these phenotypes, mediated by Axl/MerTK receptors and regulating egg burdens.

[Muscle-Specific Receptor Tyrosine Kinase Antibody Positive Myasthenia Gravis].

Reportedly, patients with muscle-specific kinase (MuSK) antibody-positive myasthenia gravis (MG) account for approximately 3.0% of all patients with MG in Japan. Compared with patients who have acetylcholine receptor antibody-positive MG, those with MuSK antibody-positive MG show young-onset disease with female predominance, a low rate of ocular involvement (5.9%), and greater severity of dysphagia. The aforementioned types of MG are indistinguishable based on clinical symptoms and electrophysiological tests, and measurement of MuSK antibodies is essential for diagnosis. Thymectomy and complement inhibitors are not indicated for treatment, and acetylcholinesterase inhibitors, steroids, immunosuppressants, plasma exchange, intravenous immunoglobulin therapy, and neonatal Fc receptor inhibitors are used.

Immunological role of Gas6/TAM signaling in hemostasis and thrombosis.

The immune system is an emerging regulator of hemostasis and thrombosis. The concept of immunothrombosis redefines the relationship between coagulation and immunomodulation, and the Gas6/Tyro3-Axl-MerTK (TAM) signaling pathway builds the bridge across them. During coagulation, Gas6/TAM signaling pathway not only activates platelets, but also promotes thrombosis through endothelial cells and vascular smooth muscle cells involved in inflammatory responses. Thrombosis appears to be a common result of a Gas6/TAM signaling pathway-mediated immune dysregulation. TAM TK and its ligands have been found to be involved in coagulation through the PI3K/AKT or JAK/STAT pathway in various systemic diseases, providing new perspectives in the understanding of immunothrombosis. Gas6/TAM signaling pathway serves as a breakthrough target for novel therapeutic strategies to improve disease management. Many preclinical and clinical studies of TAM receptor inhibitors are in process, confirming the pivotal role of Gas6/TAM signaling pathway in immunothrombosis. Therapeutics targeting the TAM receptor show potential both in anticoagulation management and immunotherapy. Here, we review the immunological functions of the Gas6/TAM signaling pathway in coagulation and its multiple mechanisms in diseases identified to date, and discuss the new clinical strategies that may generated by these roles.

Serum AXL is a potential molecular marker for predicting COVID-19 progression.

Background: The severity, symptoms, and outcome of COVID-19 is thought to be closely linked to how the virus enters host cells. This process involves the key roles of angiotensin-converting enzyme 2 (ACE2) and the Tyrosine protein kinase receptor UFO (AXL) receptors. However, there is limited research on the circulating levels of ACE2 and AXL and their implications in COVID-19. Methods: A control group of 71 uninfected individuals was also included in the study. According to the Guidance for Corona Virus Disease 2019 (10th edition), a cohort of 358 COVID-19 patients were categorized into non-severe and severe cases. Serum ACE2/AXL levels in COVID-19 patients were detected by enzyme-linked immunosorbent assay (ELISA) at different time points post-COVID-19 infection, including days 0-7, 8-15, 31-179 and >180 days. Serum SARS-CoV-2 IgG/IgM antibodies in COVID-19 patients at the same intervals were assessed by using an iFlash 3000 Chemiluminescence Immunoassay Analyzer. The receiver operating characteristic (ROC) curves were used to assess the diagnostic value of the biological markers, and the association between laboratory parameters and illness progression were explored. Results: Compared with the uninfected group, the levels of ACE2 and AXL in the COVID-19 group were decreased, and the SARS-COV-2 IgG level was increased. AXL (AUC = 0.774) demonstrated a stronger predictive ability for COVID-19 than ACE2. In the first week after infection, only the level of AXL was statistically different between severe group and non-severe group. After first week, the levels of ACE2 and AXL were different in two groups. Moreover, in severe COVID-19 cases, the serum ACE2, AXL, and SARS-COV-2 IgM levels reached a peak during days 8-15 before declining, whereas serum SARS-COV-2 IgG levels continued to rise, reaching a peak at day 31-180 days before decreasing. In addition, the AXL level continued to decrease and the SARS-COV-2 IgG level continued to increase in the infected group after 180 days compared to the uninfected group. Conclusions: The levels of serum ACE2 and AXL correlate with COVID-19 severity. However, AXL can also provide early warning of clinical deterioration in the first week after infection. AXL appears to be a superior potential molecular marker for predicting COVID-19 progression.

Acetylcholinesterase inhibitors are ineffective in MuSK-antibody positive myasthenia gravis: Results of a study on 202 patients.

BACKGROUND: Myasthenia gravis (MG) with MuSK antibodies (MuSK-MG) represents a distinct subtype with different responses to treatments compared to patients with AChR antibodies, especially in terms of tolerance to acetylcholinesterase inhibitors (AChEI). However, AChEI are often used as first line symptomatic treatment in MuSK-MG, despite reports that they are poorly tolerated, seldom effective or even deleterious. METHODS: We analyzed demographic, clinical and therapeutic responses and side-effects in the large cohort of 202 MuSK-MG patients cared for at the MG Clinic of Azienda Ospedaliero-Universitaria Pisana. RESULTS: 165 patients had received AChEI at first evaluation. Only 7/165 patients (4.2%) reported an initial clinical benefit. Conversely, 76.9% of patients reported at least one side effect, most commonly neuromuscular hyperexcitability (68.4%), gastrointestinal (53.9%) and neurovegetative (35.8%) disturbances. 56 (33.9%) patients reported a concomitant worsening of muscle weakness and twelve patients (7.3%) suffered a cholinergic crisis. According to these patients, the severity of cholinergic side effects was greater at higher doses of AChEI, but side effects occurred regardless of the dose administered and ceased once the drug was discontinued. CONCLUSIONS: This is the largest population of MuSK-MG patients reported for perceived responsiveness and tolerance to AChEI treatment. Our obervations strongly suggest avoiding this treatment in MuSK-MG.

Duodenal mucosa of untreated celiac disease patients has altered expression of the GAS6 and PROS1 and the negative regulator tyrosine kinase TAM receptors subfamily.

Celiac disease (CD) is an immune-driven disease characterized by tissue damage in the small intestine of genetically-susceptible individuals. We evaluated here a crucial immune regulatory pathway involving TYRO3, AXL, and MERTK (TAM) receptors and their ligands PROS1 and GAS6 in duodenal biopsies of controls and CD patients. We found increased GAS6 expression associated with downregulation of PROS1 and variable TAM receptors levels in duodenum tissue of CD patients. Interestingly, CD3+ lymphocytes, CD68+, CD11c+ myeloid and epithelial cells, showed differential expressions of TAM components comparing CD vs controls. Principal component analysis revealed a clear segregation of two groups of CD patients based on TAM components and IFN signaling. In vitro validation demonstrated that monocytes, T lymphocytes and epithelial cells upregulated TAM components in response to IFN stimulation. Our findings highlight a dysregulated TAM axis in CD related to IFN signaling and contribute to a deeper understanding of the pathophysiology of CD.

High PD-L1 Expression Correlates with an Immunosuppressive Tumour Immune Microenvironment and Worse Prognosis in ALK-Rearranged Non-Small Cell Lung Cancer.

High tumour programmed cell death-ligand 1 (PD-L1) expression is associated with poor progression-free survival (PFS) after tyrosine kinase inhibitor (TKI) therapy in ALK-rearranged non-small cell lung cancer (NSCLC). However, the characteristics of the tumour microenvironment (TME) and their prognostic values in ALK-rearranged NSCLC are unknown. Here, we collected tumour tissues from pretreated ALK-rearranged NSCLC patients, immunohistochemical staining was used to assess PD-L1 expression, and tumour-infiltrating immune cells were determined via multiplex immunofluorescence staining (mIF). Our data showed that the median values of PFS for the high PD-L1 group and low PD-L1 group who received ALK-TKI treatment were 4.4 and 16.4 months, respectively (p = 0.008). The median overall survival (OS) of the two groups was 24.0 months and not reached, respectively (p = 0.021). Via univariate and multivariate analyses, a high PD-L1 expression and a worse ECOG PS were determined to be independent prognostic factors of OS (HR = 3.35, 95% CI: 1.23-9.11, p = 0.018; HR = 6.42, 95% CI: 1.45-28.44, p = 0.014, respectively). In addition, the high PD-L1 group had increased Tregs and exhausted CD8+ T cells in both the tumour and stroma (all p < 0.05). High PD-L1 expression was an adverse predictive and prognostic biomarker for ALK-rearranged NSCLC. The characteristics of the TME in patients with high PD-L1 expression were shown to have an immunosuppressive status.

The TAM receptor tyrosine kinases Axl and Mer drive the maintenance of highly phagocytic macrophages.

Many apoptotic thymocytes are generated during the course of T cell selection in the thymus, yet the machinery through which these dead cells are recognized and phagocytically cleared is incompletely understood. We found that the TAM receptor tyrosine kinases Axl and Mer, which are co-expressed by a specialized set of phagocytic thymic macrophages, are essential components of this machinery. Mutant mice lacking Axl and Mer exhibited a marked accumulation of apoptotic cells during the time that autoreactive and nonreactive thymocytes normally die. Unexpectedly, these double mutants also displayed a profound deficit in the total number of highly phagocytic macrophages in the thymus, and concomitantly exhibited diminished expression of TIM-4, CD163, and other non-TAM phagocytic engulfment systems in the macrophages that remained. Importantly, these previously unrecognized deficits were not confined to the thymus, as they were also evident in the spleen and bone marrow. They had pleiotropic consequences for the double mutants, also previously unrecognized, which included dysregulation of hemoglobin turnover and iron metabolism leading to anemia.

How to manage MuSK antibody-positive myasthenic crisis during pregnancy?

Myasthenia gravis (MG) is an autoimmune disease that is characterised by the formation of antibodies against acetylcholine receptors in the postsynaptic membrane of the neuromuscular junction. The course of the disease cannot be predicted during pregnancy. A subtype of MG with positive muscle-specific receptor tyrosine kinase (anti-MuSK) antibodies exhibits more localised clinical characteristics and a poor response to treatment compared with the disease subtype that involves positivity for acetylcholine receptor antibodies. Myasthenic crisis is more frequently observed in anti-MuSK-positive myasthenia patients. Anti-MuSK-positive myasthenic crisis management is very difficult and a risky situation during pregnancy. The reported case was 30 years old, female, 9 weeks pregnant and musk antibody positive. She stopped her treatment without asking her doctor because she was planning pregnancy in the 6-month period before her hospitalization. She was intubated for a long time in the intensive care unit due to myasthenic crisis and was very resistant to treatment. During this period, her pregnancy was terminated due to fetal anomaly. Plasmapheresis, IVIg and immunosuppressive treatments were applied. Our patient was discharged after a period of about 10 weeks. We share our treatment management.

Immunological Aspects of AXL/GAS-6 in the Context of Human Liver Regeneration.

AXL and its corresponding ligand growth arrest-specific 6 (GAS-6) are critically involved in hepatic immunomodulation and regenerative processes. Pleiotropic inhibitory effects on innate inflammatory responses might essentially involve the shift of macrophage phenotype from a pro-inflammatory M1 to an anti-inflammatory M2. We aimed to assess the relevance of the AXL/GAS-6-pathway in human liver regeneration and, consequently, its association with clinical outcome after hepatic resection. Soluble AXL (sAXL) and GAS-6 levels were analyzed at preoperative and postoperative stages in 154 patients undergoing partial hepatectomy and correlated with clinical outcome. Perioperative dynamics of interleukin (IL)-6, soluble tyrosine-protein kinase MER (sMerTK), soluble CD163 (sCD163), and cytokeratin (CK) 18 were assessed to reflect pathophysiological processes. Preoperatively elevated sAXL and GAS-6 levels predicted postoperative liver dysfunction (area under the curve = 0.721 and 0.722; P < 0.005) and worse clinical outcome. These patients failed to respond with an immediate increase of sAXL and GAS-6 upon induction of liver regeneration. Abolished AXL pathway response resulted in a restricted increase of sCD163, suggesting a disrupted phenotypical switch to regeneratory M2 macrophages. No association with sMerTK was observed. Concomitantly, a distinct association of IL-6 levels with an absent increase of AXL/GAS-6 signaling indicated pronounced postoperative inflammation. This was further supported by increased intrahepatic secondary necrosis as reflected by CK18M65. sAXL and GAS-6 represent not only potent and easily accessible preoperative biomarkers for the postoperative outcome but also AXL/GAS-6 signaling might be of critical relevance in human liver regeneration. Refractory AXL/GAS-6 signaling, due to chronic overactivation/stimulation in the context of underlying liver disease, appears to abolish their immediate release following induction of liver regeneration, causing overwhelming immune activation, presumably via intrahepatic immune regulation.

MuSK not MNGIE: Atypical MuSK-antibody myasthenia presenting as a genetic disorder.

Myasthenia gravis is a treatable autoimmune disease caused by autoantibodies directed against membrane proteins at the neuromuscular junction. While acetylcholine receptor antibodies are most common, a minority of patients have antibodies directed against muscle-specific kinase (MuSK-antibody). Differentiating features often include subacute onset and rapid progression of bulbar, respiratory and neck extensor muscles, with sparing of distal appendicular muscles, most commonly in middle-aged females. Here we present an atypical presentation of MuSK-antibody myasthenic syndrome in a young male consisting of a gradual-onset, insidiously-progressive, non-fatigable and non-fluctuating ocular, bulbar and oesophageal weakness, with a normal frontalis single fibre EMG. This case clinically resembled a mitochondrial myopathy (Mitochondrial Neurogastrointestinal Encephalopathy-MNGIE) with a poor prognosis. Because of the atypical presentation, MuSK antibodies were identified very late in the disease course, at which point the patient responded very well to immunotherapy. We report an unusual presentation of an uncommon but treatable condition, illustrating significant phenotypic heterogeneity possible in MuSK-antibody myasthenic syndrome.

PTK7-Targeting CAR T-Cells for the Treatment of Lung Cancer and Other Malignancies.

In spite of impressive success in treating hematologic malignancies, adoptive therapy with chimeric antigen receptor modified T cells (CAR T) has not yet been effective in solid tumors, where identification of suitable tumor-specific antigens remains a major obstacle for CAR T-cell therapy due to the "on target off tumor" toxicity. Protein tyrosine kinase 7 (PTK7) is a member of the Wnt-related pseudokinases and identified as a highly expressed antigen enriched in cancer stem cells (CSCs) from multiple solid tumors, including but not limited to triple-negative breast cancer, non-small-cell lung cancer, and ovarian cancer, suggesting it may serve as a promising tumor-specific target for CAR T-cell therapy. In this study, we constructed three different PTK7-specific CAR (PTK7-CAR1/2/3), each comprising a humanized PTK7-specific single-chain variable fragment (scFv), hinge and transmembrane (TM) regions of the human CD8α molecule, 4-1BB intracellular co-stimulatory domain (BB-ICD), and CD3ζ intracellular domain (CD3ζ-ICD) sequence, and then prepared the CAR T cells by lentivirus-mediated transduction of human activated T cells accordingly, and we sequentially evaluated their antigen-specific recognition and killing activity in vitro and in vivo. T cells transduced with all three PTK7-CAR candidates exhibited antigen-specific cytokine production and potent cytotoxicity against naturally expressing PTK7-positive tumor cells of multiple cancer types without mediating cytotoxicity of a panel of normal primary human cells; meanwhile, in vitro recursive cytotoxicity assays demonstrated that only PTK7-CAR2 modified T cells retained effective through multiple rounds of tumor challenge. Using in vivo xenograft models of lung cancers with different expression levels of PTK7, systemic delivery of PTK7-CAR2 modified T cells significantly prevented tumor growth and prolonged overall survival of mice. Altogether, our results support PTK7 as a therapeutic target suitable for CAR T-cell therapy that could be applied for lung cancers and many other solid cancers with PTK7 overexpression.