Eight-Day Point of Care CAR T-Cell Manufacturing on Clinimacs Prodigy from Healthy Donors As a Proof-of-Concept Study
Authors: Hankey Kim G. , Luetkens Tim , Avila Stephanie , McLenithan John , Braxton John , Underwood Karen , Saba Clarissa , Lapidus Rena G. , Lufti Forat , Margiotta Philip , Yared Jean A. , Hardy Nancy M. , Abramowski-Mock Ulrike , Mittelstaet Joerg , Dudek Ron , Fan Xiaoxuan , Atanackovic Djordje , Rapoport Aaron P. , Dahiya Saurabh 
Published: November 2021

Abstract
Introduction
Chimeric Antigen Receptor (CAR) T-cell therapy has emerged as a powerful immunotherapy for various forms of cancer, especially hematologic malignancies. However, several factors limit use of CAR T-cells to a wider number of patients. Long manufacturing time (usually 3-4 weeks with standard of care products) poses a big challenge in treating these chemorefractory patients in a timely fashion. Thus, we evaluated the feasibility of a fresh in and fresh out, short, eight-day manufacturing process performed locally to expedite CAR T-cell drug product delivery. Herein we report the results of two experimental runs using this modified short eight-day culture process.
Methods
We used the CliniMACS Prodigy® closed manufacturing system and modified the 12-day T Cell Transduction (TCT) activity matrix protocol to produce anti-CD19 CAR T-cells in eight days. Normal donor mononuclear cells were collected by leukapheresis and enriched for CD4 and CD8 cells by immunomagnetic bead selection in three stages. Enriched T-cells were activated with MACS GMP T Cell TransACT and cultured at 37°C with 5% CO 2 for 16-24 hours in media supplemented with 12.5mcg/L each of IL-7 and IL-15, and 3% heat-inactivated human AB serum. On day 1 of the process, activated T-cells were transduced with lentiviral vector encoding the anti-CD19 CAR (Lentigen, LTG1563) at a multiplicity of infection (MOI) of 7-10. On day 3, the cells were washed twice and the media volume adjusted to feed the expanding cells. The culture was again fed on day 5 by exchanging half the volume of spent media with fresh supplemented media. Media supplemented with cytokines alone was used for the remaining four washes on day 6, 7 and 8. Transduction efficiency and T-cell subset frequencies were assessed by flow cytometry on the MACSQuant-10 and CAR-T Express Mode package on days 3, 6 and 8. Subsequently, we performed ELISPOT assay for CAR T-cell potency testing and in-vivo efficacy testing in NSG mice bearing Raji B cell lymphoma.
Results
Refer to Table 1 for details on cell populations of interest for experiment number 1 and 2. The total number of CD3 T-Cells increased from 97% on day 0 to &gt;99.5% on the harvest day (day 8). CD3 T-cells expanded 11.6- and 34.2-fold on day 8 when compared to day 0. Transduction efficiency of ~40% was observed in both experimental runs. Final CD19 CAR T-cells numbers ranged from 9.3-13.3 x 10e8 with viability of CD3+ cells &gt;93% for both the runs. Day 3 of the culture is an important day since a clinical decision to proceed with lymphodepletion must be made to facilitate the fresh in and fresh out approach. Here we observed reliable transduction of T-cells on day 3 with an average efficiency of 15.9%. Day 3 data reliably provided information to proceed with lymphodepletion.
A total of 100,000 CD19 CAR T-cells produced in experiment #1 were exposed to beads coated with CD19 protein, BCMA control protein, or T cell-activating beads coated with anti-CD3 and anti-CD28 antibodies in an ELISPOT plate. Spots in figure 1 represents individual CAR T-cells producing IFN-gamma. This novel ELISPOT assay shows high IFN-gamma by CD19 CAR T-cells in response to the target antigen or unspecific stimulation using CD3/CD28 beads.
Subsequently, NSG mice received injections of 5x10e5 Raji B cell lymphoma cells stably expressing luciferase into the tail vein. One week later, 4 mice per group received individual i.v. injections of 4x10e6 CD19 CAR T-cells, 0.3x10e6 CD19 CAR T-cells, 4x10e6 mock-transduced CAR T-cells, or media. Survival curves in figure 2 represent survival of the mice after receiving the treatment with best survival seen with 4x10e6 dose.
Conclusions
In these experimental runs, we were able to generate CD19 CAR+ T-cells in a short eight-day manufacturing process. The final product characteristics (viability, transduction efficiency and doses) were comparable to clinical formulations. Further, point-of-care potency assay suggests high IFN-gamma production and elimination of CD19 tumor in the in vivo murine model. The point-of-care CAR T-cell production allows for shorter vein-to-vein time and offers dramatic reduction in the product cost. Lastly, the novel potency assay via ELISPOT testing allows for rapid and visual functional analysis of the CAR T-cell product.
Figure 1 Figure 1.

Disclosures
Hardy: Kite/Gilead: Membership on an entity's Board of Directors or advisory committees; American Gene Technologies, International: Membership on an entity's Board of Directors or advisory committees; InCyte: Membership on an entity's Board of Directors or advisory committees. Abramowski-Mock: Miltenyi Biotec: Current Employment. Mittelstaet: Miltenyi Biotec: Current Employment. Dudek: Miltenyi Biotec: Current Employment.