Preclinical Evaluation of Human Placental-Derived Allogeneic CD19 CAR-T Cells Against B Cell Malignancies

Abstract

Celularity, Inc. is developing a CD19 CAR-T Cell therapy using an allogeneic platform derived from postpartum human placental cells. T cells isolated from placenta/ umbilical cord blood and genetically modified to express CD19 chimeric antigen receptor (CAR), termed Placental-derived (P-) CD19 CAR T cells, are in development for the treatment of B cell malignancies. Unlike adult peripheral blood mononuclear cell (PBMC)-derived T cells, P-T cells are mostly naïve (CD45RA+) and can be readily expanded while maintaining an earlier differentiation phenotype such as greater expression of naïve/ memory markers, lower expression of effector/ exhaustion markers, allowing for greater proliferative potential of these cells ex vivo. These cells are also known to have greater immune tolerance to HLA mismatch and display impaired allogeneic activation, contributing to lower incidences of severe graft-verse-host disease (GvHD) (Barker, et. al. Blood, 2001; Chen, et al. Biology of Blood and Marrow Transplantation, 2006), making them an attractive cell population for use as an allogeneic, adoptive cell therapy. A robust process for the isolation, transduction, and expansion of placental-derived T cells to generate "off-the-shelf" allogeneic P-CD19 CAR T cells was developed. Twenty-One day expanded, non-modified P-T cells (N=3) were compared to adult PBMCs for their allo-reactivity in a Xenogeneic GvHD model in NCG mice. P-T cells did not induce xeno-GvHD whereas PBMCs did, as evidenced by significant weight loss and death of all mice (N=5) by Day 28 post infusion. Despite expanded P-T cells demonstrating lack of in vivo GvHD, current manufacture of P-CD19 CAR T cells does include a CRISPR-mediated T-cell receptor a constant (TRAC) knockout (KO) step as an additional risk-mitigation strategy to circumvent any potential GvHD stemming from expression of endogenous T cell receptor. CD19 CAR transduction using a retrovirus provided by Sorrento Therapeutics, Inc., followed by TRAC knockout with CRISPR results in both high efficiency of CD19 CAR expression (~30% CD19 Fc+) and TCR KO (>96% CD3-/ TCR a/b-). In vitro, the functional activity of P-CD19 CAR-TRAC KO T cells against CD19+ Burkitt's Lymphoma (Daudi) and Acute lymphoblastic Leukemia (NALM6) cell lines was assessed in cytotoxicity and cytokine release assays. P-CD19 CAR T cells specifically lyse CD19+ Daudi/ Nalm6 targets in both 4-hour endpoint FACS and ACEA kinetic cytotoxicity assays, and in most cases at levels equivalent to or greater than PBMC-derived CD19 CAR T cells. When P-CD19 CAR T cells were co-cultured with CD19+ Daudi/ Nalm6 target cells for 24-hours, they secreted pro-inflammatory cytokines and effector proteins in an antigen-specific manner. In vivo, the anti-tumor activity of P-CD19 CAR T cells was assessed using a disseminated lymphoma xenograft model in NSG mice. Luciferase expressing Daudi cells (3×106) were intravenously (IV) injected on Day 0, followed by IV injection of P-CD19 CAR T cells (14×106) on Day 7. Bioluminescence Imaging (BLI) and survival were used as primary study endpoints. P- CD19 CAR T cells were well tolerated and safe. P-CD19 CAR T cells significantly reduced tumor burden, and improved survival. Four weeks after treatment, the vehicle group had a 100% mortality rate, while all animals from P-CD19 CAR T-treated group (N=5) remained alive without clinical symptoms including weight loss or changes in their fur. In summary, Celularity has defined a robust process for the generation and expansion of CD19 CAR T cells from human placenta. These cells exhibit potent anti-tumor activity both in vitro and in vivo with little evidence of acute GvHD induction, highlighting their potential as an allogeneic, adoptive cell therapeutic agent. Future in vivo GvHD studies will include assessment of both CD19 CAR and TRAC KO genetically modified P-T cells. Disclosures Karasiewicz: Celgene: Equity Ownership; Celularity, Inc.: Employment, Equity Ownership, Patents & Royalties: Patent Inventor. He:Celularity Inc: Employment. Ng:Celularity, Inc.: Employment. Tess:Celularity, Inc.: Employment. Ling:Celularity Inc: Employment. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Ji:Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.

Umbilical cord blood derived cellular therapy: advances in clinical development

“…First, like other allogeneic CAR T-cell therapies, solutions need be developed to tackle GvH and HvG effects. Although TCR knockout has been effective to reduce GvH in PB-derived ( 52 ) and CB-derived ( 53 ) CAR T-cells, it is possible to avoid genome editing, as mentioned above, by obtaining an HLA-matched CB unit from a limited and highly selected CB bank as the cell source for CAR T-cells ( 49 ). However, proof-of-concept clinical studies are lacking at this point.…”

Lentiviral Vectors for T Cell Engineering: Clinical Applications, Bioprocessing and Future Perspectives

“…Due to all these limitations, increasing interest has been generated around the allogeneic approach in a one-size-fits-all model. In this case, the material would originate from a universal source such as genetically modified donor T cells, umbilical cord blood (UCB), placenta, immortalised cell lines, or induced pluripotent stem cell (iPSC)-derived T cells, opening the possibility to large scale production [ 124 , 125 , 126 , 127 , 128 ]. At present, only the genetically modified donor T cell and immortalised cell line approaches have progressed to the clinical stage, as the stem cell-derived approaches still require further pre-clinical optimisation [ 125 , 127 ].…”

Optimizing the Clinical Impact of CAR-T Cell Therapy in B-Cell Acute Lymphoblastic Leukemia: Looking Back While Moving Forward

“…Recently, researchers have demonstrated that T cells isolated from postpartum human placenta/umbilical cord blood can be used for the generation of CD19-redirected CAR-Ts, termed P-CD19-redirected CAR-Ts (146). P-CD19-redirected CAR-Ts are mainly naïve (CD45RA + ) T cells and retain their naïve/memory marker expression and harbor a lower expression of effector/ exhaustion markers as compared to peripheral blood mononuclear cell-derived CAR-Ts (146). Moreover, these cells also have superior immune tolerance to HLA mismatch and exhibit weakened allogeneic activation resulting in a lower GvHD occurrence possibility (146).…”

“…P-CD19-redirected CAR-Ts are mainly naïve (CD45RA + ) T cells and retain their naïve/memory marker expression and harbor a lower expression of effector/ exhaustion markers as compared to peripheral blood mononuclear cell-derived CAR-Ts (146). Moreover, these cells also have superior immune tolerance to HLA mismatch and exhibit weakened allogeneic activation resulting in a lower GvHD occurrence possibility (146). Researchers have suggested that these features of P-CD19-redirected CAR-Ts make them a promising candidate for universal allogeneic CAR-T therapy (146).…”

“…Moreover, these cells also have superior immune tolerance to HLA mismatch and exhibit weakened allogeneic activation resulting in a lower GvHD occurrence possibility (146). Researchers have suggested that these features of P-CD19-redirected CAR-Ts make them a promising candidate for universal allogeneic CAR-T therapy (146). It is worth mentioning that the endogenous TCR expression of these T cells can be abrogated using the CRISPR genome-editing technique to minimize the risk of endogenous TCR-mediated GvHD (146).…”