Development of a Novel Anti-CD19 Chimeric Antigen Receptor: A Paradigm for an Affordable CAR T Cell Production at Academic Institutions

Abstract

Genetically modifying autologous T cells to express an anti-CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials (CTs). Making this treatment available to our patients prompted us to develop a novel CART19 based on our own anti-CD19 antibody (A3B1), followed by CD8 hinge and transmembrane region, 4-1BB- and CD3z-signaling domains. We show that A3B1 CAR T cells are highly cytotoxic and specific against CD19+ cells in vitro, inducing secretion of pro-inflammatory cytokines and CAR T cell proliferation. In vivo, A3B1 CAR T cells are able to fully control disease progression in an NOD.Cg-Prkdc ^scid Il2rd ^tm1Wjl /SzJ (NSG) xenograph B-ALL mouse model. Based on the pre-clinical data, we conclude that our CART19 is clearly functional against CD19+ cells, to a level similar to other CAR19s currently being used in the clinic. Concurrently, we describe the implementation of our CAR T cell production system, using lentiviral vector and CliniMACS Prodigy, within a medium-sized academic institution. The results of the validation phase show our system is robust and reproducible, while maintaining a low cost that is affordable for academic institutions. Our model can serve as a paradigm for similar institutions, and it may help to make CAR T cell treatment available to all patients.

Keywords: 4-1BB; CD19; T cell; chimeric antigen receptor; immunotherapy; leukemia; lymphoma; preclinical studies.

Figure 1

ARI-0001 Anti-tumor Activity In Vitro (A) Diagram of A3B1 CAR19 construct. (B) Cytotoxicity assay of CART19 cells versus NALM6 cells at the 16-hr time point. Percent target surviving cells, relative to untreated, is shown (mean of 3 experiments ± SEM). Panels on the right show representative flow cytometry plots at E:T ratio = 1:8. (C) CAR19 T cell proliferation in vitro measured by CFSE assay at the 96-hr time point. Panels on the left show representative flow cytometry images. Panel on the right shows quantification of the proliferation index (PI). Mean of 4 experiments ± SEM is shown. (D) Cytokine production (IFNγ, TNF-α, and IL-10) of CART19 cells in co-culture with NALM6 cells at the 16-hr time point, measured by ELISA. Mean of 3 experiments ± SEM is shown. *Statistical significance, p < 0.05; n.s., not statistically significant.

Figure 2

ARI-0001 Anti-tumor Activity In Vivo (A) Upper panel shows a timeline of experimental design. Lower panels show bioluminescence images showing disease progression at different days. Animals indicated by a pound sign were sacrificed at day 16 due to advanced disease progression. The rest of the animals were sacrificed at day 17. (B) Detection of tumor (CD19+) cells in the bone marrow of mice shown in (A) (mean ± SD). (C) Detection of tumor (CD19+) cells in blood.

Figure 3

Comparison of Anti-tumor Activity of A3B1- and FMC63-based CAR T Cells (A) Detection of chimeric antigen receptor (CAR) expression by western blot. (B) Cytotoxicity assay of CART19 cells versus NALM6 cells after 4 hr of co-culture. Percent target surviving cells, relative to untreated, is shown (mean of 3 experiments ± SEM). (C) Upper panel shows a timeline of experimental design (*Im, bioluminescent image; *Bl, blood sample). Lower panels show bioluminescence images showing disease progression at different days. (D) Detection of tumor (GFP+) cells and CD3+ cells in blood by flow cytometry. Control, control mice (no tumor or T cells were injected).

Figure 4

Diagram Depicting the Main Steps of Large-Scale CAR19 Lentivirus Production as Currently Established at Hospital Clínic de Barcelona

Figure 5

Results of 3 Validation Processes of ARI-0001 Cell Production Using Healthy Donors (A) Total cell number at different time points. (B) Percentage of CAR19-expressing cells at different time points.