Orvacabtagene Autoleucel (orva-cel; JCARH125): A Fully Human BCMA-Targeted Second-Generation CAR T Cell Product Characterized By a Predominant Central Memory Phenotype with High in Vitro and In Vivo Proliferative Potential and Sustained In Vivo Persistence

Background: Orva-cel is an investigational B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T cell product genetically modified with a lentiviral vector to express a CAR construct with a unique fully human single-chain variable fragment, optimized spacer, and 4-1BB costimulatory and CD3ζ activation domains. Orva-cel is currently being evaluated for efficacy and safety in the ongoing phase 1/2 EVOLVE study (NCT03430011) in heavily pretreated patients with relapsed/refractory multiple myeloma. We characterized orva-cel drug products, manufactured using the process in place for the phase 2 portion of the study and intended for commercial manufacturing, for CAR⁺ T cell purity, phenotype, and function.

Methods: Immunophenotyping was performed by flow cytometry of both surface and intracellular markers, including CD3, CD4, CD8, CD45, CCR7, CD45RA, CD28, CD27, and active caspase 3. Cytokine production after challenge with BCMA⁺ target cells was assessed by intracellular cytokine staining and Luminex multiplex assay of secreted cytokines, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and granzyme B (GrB). CAR-mediated in vitro proliferative capacity was measured after anti-idiotypic antibody stimulation using the IncuCyte Live-Cell Analysis System (Sartorius, Göttingen, Germany). In vivo CAR⁺ T cell proliferation and persistence were assessed by quantitative polymerase chain reaction (qPCR).

Results: The orva-cel manufacturing process was designed to enable consistent production of highly pure CD3⁺ cell products (median frequency of CD3⁺ T cells, 99.96%; quartiles 1-3 interquartile range, 99.9%-100.0%; n = 81). Orva-cel drug products were characterized by high frequencies of less-differentiated CAR⁺ T cells, leading to a dominant central memory-like population (CCR7⁺CD45RA^- CAR⁺ T cells) and substantial frequencies of naïve-like cells (CCR7⁺CD45RA⁺ CAR⁺ T cells) (Figure).

When assayed for in vitro functional activity, orva-cel drug products showed robust antigen-specific cytokine and effector molecule production (IFN-γ, TNF-α, IL-2, and GrB) upon challenge with BCMA⁺ tumor cells, as well as vigorous proliferation in response to CAR stimulation.

Preliminary correlative analysis suggested that the early memory phenotype may be linked to increased CAR⁺ T cell proliferative capacity, as determined by in vitro experiments and in vivo PK parameters (ie, maximum CAR⁺ T cell concentration observed in the blood [C_max], time to C_max, area under the curve from Day 0 to 28 [AUC_0-28], and CAR⁺ T cell persistence at Month 3 and Month 6). Consistent with the early CAR T cell memory phenotype, qPCR analysis showed robust in vivo proliferation of CAR⁺ T cells after infusion, with a median C_max of 1.54 × 10⁵ transgene copies/µg DNA and median AUC_0-28 of 1.61 × 10⁶ transgene copies/µg DNA*day, as well as long-term in vivo persistence, with CAR⁺ T cells detected in 69% of patients at 6 months postinfusion.

Conclusions: The orva-cel manufacturing process results in drug products characterized by highly pure T cells, with high frequencies of early memory and polyfunctional CAR⁺ T cells. Orva-cel drug products showed robust antigen-specific degranulation, production of multiple cytokines, sustained in vitro and in vivo proliferation, and in vivo persistence.

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