MaxCyte (United States)

Blood vessels show various COVID-19-related conditions including thrombosis and cytokine propagation. Existing in vitro blood vessel models cannot represent the consequent changes in the vascular structure or determine the initial infection site, making it difficult to evaluate how epithelial and endothelial tissues are damaged. Here, we developed a microphysiological system (MPS) that co-culture the bronchial organoids and the vascular bed to analyze infection site and interactions. In this system, virus-infected organoids caused damage in vascular structure. However, vasculature was not damaged or infected when the virus was directly introduced to vascular bed. The knockout of interferon-related genes and inhibition of the JAK/STAT pathway reduced the vascular damage, indicating the protective effect of interferon response suppression. The results demonstrate selective infection of bronchial epithelial cells and vascular damage by cytokines and also indicate the applicability of MPS to investigate how the infection influences vascular structure and functions.

The ubiquitous Epstein-Barr virus (EBV) is an oncogenic herpes virus associated with several human malignancies. EBV is an immune-evasive pathogen that promotes CD8+ T cell exhaustion and dysregulates CD4+ T cell functions. Burkitt lymphoma (BL) is frequently associated with EBV infections. Since BL relapses after conventional therapies are difficult to treat, we evaluated prospective off-the-shelf edited CAR-T cell therapies targeting CD19 or the EBV gp350 cell surface antigen. We used CRISPR/Cas9 gene editing methods to knock in (KI) the CD19CAR.CD28z or gp350CAR.CD28z into the T cell receptor (TCR) alpha chain (TRAC) locus. Applying upscaled methods with the ExPERT ATx® MaxCyte system, KI efficacy was ~20% of the total ~2 × 108 TCR-knocked-out (KO) generated cells. KOTCRKICAR-T cells were co-cultured in vitro with the gp350+CD19+ BL cell lines Daudi (infected with type 1 EBV) or with Jiyoye (harboring a lytic type 2 EBV). Both types of CAR-T cells showed cytotoxic effects against the BL lines in vitro. CD8+ KICAR-T cells showed higher persistency than CD4+ KICAR-T cells after in vitro co-culture with BL and upregulation of the activation/exhaustion markers PD-1, LAG-3, and TIM-3. Two preclinical in vivo xenograft models were set up with Nod.Rag.Gamma mice injected intravenously (i.v.) with 2 × 105 Daudi/fLuc-GFP or with Jiyoye/fLuc-GFP cells. Compared with the non-treated controls, mice challenged with BL and treated with CD19KICAR-T cells showed delayed lymphoma dissemination with lower EBV DNA load. Notably, for the Jiyoye/fLuc-GFP model, almost exclusively CD4+ CD19KICAR-T cells were detectable at the endpoint analyses in the bone marrow, with increased frequencies of regulatory T cells (Tregs) and TIM-3+CD4+ T cells. Administration of gp350KICAR-T cells to mice after Jiyoye/GFP-fLuc challenge did not inhibit BL growth in vivo but reduced the EBV DNA load in the bone marrow and promoted gp350 antigen escape. CD8+PD-1+LAG-3+ gp350KICAR-T cells were predominant in the bone marrow. The two types of KOTCRKICAR-T cells showed different therapeutic effects and in vivo dynamics. These findings reflect the complexities of the immune escape mechanisms of EBV, which may interfere with the CAR-T cell property and potency and should be taken into account for future clinical translation.

SARS-CoV-2 induces severe organ damage not only in the lung but also in the liver, heart, kidney, and intestine. It is known that COVID-19 severity correlates with liver dysfunction, but few studies have investigated the liver pathophysiology in COVID-19 patients. Here, we elucidated liver pathophysiology in COVID-19 patients using organs-on-a-chip technology and clinical analyses. First, we developed liver-on-a-chip (LoC) which recapitulating hepatic functions around the intrahepatic bile duct and blood vessel. We found that hepatic dysfunctions, but not hepatobiliary diseases, were strongly induced by SARS-CoV-2 infection. Next, we evaluated the therapeutic effects of COVID-19 drugs to inhibit viral replication and recover hepatic dysfunctions, and found that the combination of anti-viral and immunosuppressive drugs (Remdesivir and Baricitinib) is effective to treat hepatic dysfunctions caused by SARS-CoV-2 infection. Finally, we analyzed the sera obtained from COVID-19 patients, and revealed that COVID-19 patients, who were positive for serum viral RNA, are likely to become severe and develop hepatic dysfunctions, as compared with COVID-19 patients who were negative for serum viral RNA. We succeeded in modeling the liver pathophysiology of COVID-19 patients using LoC technology and clinical samples.

3014 Background: MCY-M11 is a mesothelin-targeting chimeric antigen receptor (CAR) therapy made by a non-viral, mRNA-based platform, for rapid ( < 1 day) CAR manufacturing. We are conducting a phase I dose escalation trial in ovarian cancer and malignant peritoneal mesothelioma (MPM) (NCT03608618). Methods: MCY-M11 are fresh, non-expanded, autologous peripheral blood mononuclear cells (PBMCs) transfected by flow electroporation with mRNA encoding a human anti-mesothelin CAR. Following a 3+3 design, patients are treated in dose level (DL) escalating cohorts (DL1 1.0 x 107, DL2 5.0 x 107, DL3 1.0 x 108, DL4 5.0 x 108 cells/dose), in one cycle of weekly x 3 doses, intraperitoneal (ip) without preconditioning chemotherapy. Results: By January 2020, CP-M11-101 study successfully completed DL1 and DL2 without safety concerns. Based on 11 patients treated in DL1, DL2 and DL3, ip infusion of MCY-M11 is safe and well tolerated. No infusion-related adverse events and no dose limiting toxicities (DLTs) have occurred. No neurotoxicity has been observed. Most reported treatment-related adverse events have been Grades 1-2 per NCI CTCAE. One patient in DL3 presented with G2 pericarditis, fever and transient neutropenia clinically assessed as related SAEs, that resolved without further complications. These events were assessed as on-target off-tumor effects and possibly G1 cytokine release syndrome (CRS). Two unrelated SAEs (G2 confusion in a patient in DL2; G3 enterocutaneous fistula in a patient in DL3) were reported. These 2 patients have been replaced as they did not complete the evaluation period (3 weekly infusions and the DLT 43 day follow up). There have been no treatment-related discontinuations or deaths. Three patients in DL2 showed stable disease (SD) by RECIST 1.1 at the end of the DLT period. Of them, 1 completed the study and did not participate in additional follow up, 1 remained in SD 6 months, and 1 remained in SD 2 months. In DL3, 1 patient remains in SD at 2 months, and evaluation is pending for the other 2 patients. Enrollment is ongoing. Conclusions: Feasibility of 1-day manufacturing of MCY-M11 for ip delivery is demonstrated. Treatment has been safe. Initial SD observed in DL2 and DL3 with one-cycle infusions is encouraging and supports exploration of additional strategies such as the addition of preconditioning chemotherapy and multiple cycles to increase efficacy. Clinical trial information: NCT03608618 .

Abstract Background: Chimeric antigen receptor CAR T cell therapy for solid tumors has not yet achieved the success seen in hematologic malignancies. Many challenges remain, including the type of antigen being targeted, the risk for on-target off-tumor toxicity, a frequent high degree of immune tolerance within an immune suppressive environment, together with autologous product manufacturing hurdles, which hinder timely delivery of treatment to the patient. Also, most current CAR T therapies are viral based, limiting the ability to administer repeated, longer-term treatments. MaxCyte has developed its first CAR product, MCY-M11, applying the CARMA™ non-viral, mRNA-based platform for rapid (less than 1 day) manufacturing with efficient transfection of a human scFv targeting mesothelin, using fresh non-expanded peripheral blood mononuclear cells for autologous cell therapy. Mesothelin is an attractive and relevant therapeutic target because of its high differential expression in many solid tumors with only low levels of expression in mesothelial cells of mostly non-critical normal tissues. The transient expression of the CAR transgene may be important in limiting potential excessive or unwanted toxicity, while allowing for multiple therapeutic infusions needed to trigger a potent and more effective host immune response. In an in vivo murine model, MCY-M11 cells were shown to have the ability to traffic to and inhibit growth of mesothelin-expressing ovarian cancer and improve survival, both in a dose-dependent manner and with multicycle administration. The first in human phase 1 study of MCY-M11 in patients with advanced ovarian cancer and peritoneal mesothelioma (NCT03608618) is described here. Methods: A standard 3+3 design phase 1 study exploring 4 ascending cell dose levels, in patients with relapsed/refractory adenocarcinoma of the ovary, fallopian tube, primary peritoneum, or peritoneal mesothelioma. A minimum of 15 patients will be enrolled to receive one cycle of 3 weekly doses (D1, D8, D15) of MCY-M11 administered intraperitoneally. The primary objective is to characterize safety (Incidence and severity of adverse events assessed per NCI CTCAE v5.0), tolerability, and feasibility of infusion of MCY-M11. Secondary objectives include assessment of antitumor activity (RECIST, irRECIST, and serum biomarkers), and correlates of CAR performance and immune activation. The study is being conducted at two centers, and the first patient was dosed in October 2018. Recruitment in dose level 1 cohort continues. Citation Format: Armin Ghobadi, Premal Thaker, David Weng, Julie Vanas, Irene Ekwede, Mira Pavlova, Robert Keefe, Michael Kuo, Claudio Dansky Ullmann, Raffit Hassan, Christina Annunziata. A phase 1 study of intraperitoneal MCY-M11 therapy for women with platinum resistant high grade serous adenocarcinoma of the ovary, primary peritoneum, or fallopian tube, or subjects with peritoneal mesothelioma with recurrence after prior chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT159.

Abstract Antibody drug conjugates (ADC’s) define an emerging class of targeted therapies for cancer, with over 60 ADC’s currently in clinical development. Although antibodies can exhibit exquisite target specificity, expression of the tumor target in normal tissue can limit the tolerated dose that one can achieve with an ADC. We designed two different affinity variant biparatopic antibodies that recognize distinct epitopes on EGFR and promote receptor clustering that leads to enhanced lysosomal trafficking. These ADC’s comprise two different strategies to target EGFR expressing tumors: a high affinity, potency driven approach and a lowered affinity, threshold driven approach. We show that both ADC’s bind to tumor cells, are rapidly internalized, and traffic to the lysosome, where the warhead is efficiently released by lysosomal hydrolases. In vitro, the high affinity ADC exhibited potent killing of cancer cell lines, with IC50’s in the double digit picomolar range in cells with relative EGFR densities as low as 25,000, while the low affinity ADC was most effective when the EGFR density was greater than 500,000. The high affinity ADC was active in patient derived xenograft (PDX) models derived from colorectal and lung cancer patients harboring clinically relevant oncogenic drivers, such as KRAS and BRAF mutations or EGFR aberrations. This ADC induced tumor stasis or regression, with minimally efficacious doses between 1 and 3 mg/kg, in multiple tumor models across a broad range of EGFR expression (IHC scores ranging from 1 to 3). In a cynomolgus monkey safety study, this ADC showed a manageable safety profile at 2 mg/kg, with skin effects as the dose limiting toxicity. The potency of the high affinity ADC may provide meaningful activity across a range of EGFR expression levels, at doses that are tolerable in the clinic. The lowered affinity ADC exhibited an improved safety profile compared to the high affinity ADC, with a highest non-severely toxic dose (HNSTD) of 6 mg/kg, presumably driven by a reduction in binding to normal tissues that express comparably lower EGFR levels relative to the tumor. This lowered affinity ADC showed a more restricted anti-tumor activity, causing regressions at a minimally efficacious dose between 3 and 5 mg/kg, in PDX models that expressed high levels of EGFR, as judged by IHC scores of 3. The lowered affinity ADC required higher EGFR expression for activity, but the improved tolerability may provide a selectivity threshold that discriminates tumor from normal tissue in patients whose tumors overexpress EGFR. Our findings support the concept that modulating antibody affinity can yield distinct ADC’s that represent different strategies for balancing safety and efficacy. The work presented here provides a conceptual framework for designing unique ADC candidates tailored to the particular expression profile and safety considerations around specific tumor associated targets. Citation Format: Frank I. Comer, Xinzhong Wang, Andy Q. Yuan, Vanessa Muniz-Medina, Agnieszka Sadowska, Lan Xu, Samuel Perry, Pamela Thompson, Ralph Minter, Robert Woods, Mary Jane Hinrichs, Molly Reed, Shameen Affif-Rider, Marlon Rebelatto, Charles Brown, Hui Feng, Rakesh Dixit, Steven Coats, Jane Osbourn. Strategies to Improve the Safety and Efficacy of Biparatopic Antibody Drug Conjugates [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A20.