standardized-flow-cytometry-assay-using-dried-recombinant-antibody

Standardized Flow Cytometry Assay Using Dried Recombinant Antibody Panels for CAR T Cell Characterization

Flow cytometry is commonly used to assess critical quality attributes of Chimeric Antigen Receptor T (CAR T) cells, including viability, phenotype, and ...

Miltenyi Biotec

Oncogenic mutations in RAS genes, like KRAS or NRAS, trap Ras in the active state and cause myeloproliferative disorder and T cell leukemia (T-ALL) when induced in the bone marrow via Mx1CRE. The RAS exchange factor RASGRP1 is frequently overexpressed in T-ALL patients. In T-ALL cell lines overexpression of RASGRP1 increases flux through the RASGTP/RasGDP cycle. Here we expanded RASGRP1 expression surveys in pediatric T-ALL and generated a RoLoRiG mouse model crossed to Mx1CRE to determine the consequences of induced RASGRP1 overexpression in primary hematopoietic cells. RASGRP1-overexpressing, GFP-positive cells outcompeted wild type cells and dominated the peripheral blood compartment over time. RASGRP1 overexpression bestows gain-of-function colony formation properties to bone marrow progenitors in medium containing limited growth factors. RASGRP1 overexpression enhances baseline mTOR-S6 signaling in the bone marrow, but not in vitro cytokine-induced signals. In agreement with these mechanistic findings, hRASGRP1-ires-EGFP enhances fitness of stem- and progenitor- cells, but only in the context of native hematopoiesis. RASGRP1 overexpression is distinct from KRAS or NRAS, does not cause acute leukemia on its own, and leukemia virus insertion frequencies predict that RASGRP1 overexpression can effectively cooperate with lesions in many other genes to cause acute T-ALL.

Increased baseline RASGRP1 signals enhance stem cell fitness during native hematopoiesis.

Recent progress in chimeric antigen receptor (CAR) natural killer (NK) cell therapy has demonstrated their promising potential in cancer immunotherapy. However, most current CAR NK cell manufacturing processes utilize open systems with multiple manual steps, making it challenging to maintain consistent therapeutic quality and regulatory compliance for clinical applications. We specifically developed blood dendritic cell antigen 2 (BDCA2)-targeting CAR NK cells for treating blastic plasmacytoid dendritic cell neoplasm (BPDCN). Here, we present an automated, current good manufacturing practice (cGMP)-compliant Natural Killer Cell Transduction (NKCT) process for producing clinical-grade CAR NK cells on the CliniMACS Prodigy platform. This closed system integrates cell separation, activation, transduction, expansion, and harvest, thereby reducing contamination risks and ensuring cell product quality. The NKCT process achieved high transduction efficiency using baboon envelope pseudotyped lentiviral vectors (BaEV-LV) produced under cGMP conditions combined with Vectofusin-1, yielding CAR NK cells with high viability and purity. Both  and  studies demonstrated the potent antitumor activity of CliniMACS Prodigy-manufactured BDCA2 CAR NK cells, highlighting a promising treatment strategy for BPDCN. In summary, this automated NKCT process enables both centralized and decentralized CAR NK manufacturing and facilitates the efficient clinical translation of CAR NK cell therapies.

Article	Year
Increased baseline RASGRP1 signals enhance stem cell fitness during native hematopoiesis. Oncogene\| PubMed ID: 32989257	2020
Automated manufacturing of clinical-grade BDCA2 CAR NK cells in a closed system for the treatment of blastic plasmacytoid dendritic cell neoplasm. Frontiers in immunology\| PubMed ID: 41836419	2026

Marsilius Mues

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