Background
The six FDA approved autologous CAR-T cell therapies for hematological cancers have shown great effectiveness and potential of T cell-based immunotherapy in treating cancers that are resistant to standard chemotherapy and radiation therapy. However, critical manufacturing challenges still exist, including scale translation and transfer and the inherent process variability due to the inclusion of multiple manual operation steps. These challenges often lead to increased incurrence of contamination, operator errors and production failures. Consequently, many eligible patients cannot be treated. Attempts to automate cell production across all manufacturing steps have proved challenging with longer process time often required, resulting in longer vein-to-vein time that sometimes beyond what many eligible patients can wait.
Methods
To address these issues, Oncternal has developed a fully closed, robust and automated manufacturing process for ONCT-808, an autologous ROR1 CAR T cell product, using CliniMACS Prodigy. ONCT-808 cells are genetically modified via ex vivo transduction with lentivirus vector to express a CAR construct containing a ROR1-directed single chain variable fragment (scFv) derived from Oncternal’s clinical stage anti-ROR1 mAb zilovertamab. For process development, manufacturing was started with CD4+ and CD8+ cells enriched from frozen or fresh apheresis materials from healthy donors as well as a late-stage solid cancer patient and chronic lymphocytic leukemia patient. A number of critical parameters were evaluated during ONCT-808 manufacturing process development and verification, including seeding cell number, research vs GMP grade lentivirus vector, lentivirus MOI, culture medium exchange scale and frequency, presence or absence of human AB serum, sources of human AB serum and serum albumin, as well as manufacturing sites and environment.
Results
With the 8-day automated manufacturing process, greater than 3 billion T cells, including approximately 2 billion ROR1 CAR-positive T cells of mainly memory phenotypes were harvested from healthy donor apheresis. For manufacturing started with late-stage solid cancer patient and CLL patient apheresis, greater than 2 billion T cells, including approximately 1–2 billion ROR1 CAR-positive T cells were harvested. Analysis of ONCT-808 manufacturing from healthy donors showed that only a small fraction of the CAR T cells was terminally differentiated with fewer than 5 vector copies per transduced T cell, and undetected RCL. Combination of the automated closed process with optimization of release testing methodologies resulted in a vein-vein time for patient administration of 15–17 days.
Conclusions
A closed, high yield, robust and high-performance CAR T manufacturing process was developed and verified.