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.

No abstract

As an organ generally discarded after a normal full-term birth, the placenta is one of the most studied organs from the cellular standpoint. The placenta contains large numbers of immune cells, stem cells, and stromal cells. These cell types spurred the field of regenerative medicine by catalyzing the establishment of cord blood banks and hematopoietic stem cell reconstitution in the treatment of many diseases including cancer. Previously, many scientific articles and reviews have focused on the production, phenotype, and functional characterization of bone marrow-derived mesenchymal stromal cells. In this chapter, the focus will be solely on the biology, phenotype, and functional characterization of placentaderived stromal cells. Modulation of the immune response, including T cell proliferation, dendritic cell maturation, and monocyte differentiation by placenta-derived stromal cells, will be discussed. This chapter will span in vitro functional analyses, animal models highlighting the in vitro data culminating in a summary of current clinical activity.

Introduction: Celularity, Inc. is developing a CD19 CAR-T cell therapy using an allogeneic platform derived from postpartum human placental cells. To augment the CD19 CAR expression and activity of Placental-derived T (P-T) cells, we have evaluated the use of various lentiviral (LV) vectors, as well as a retroviral (RV) CD19 CAR construct and assessed the effects of each virus on the phenotypic characteristics and activity of our cells. Fully humanized scFv CD19 constructs have been shown to be less immunogenic with improved persistence and efficacy in patients (Sommermeyer, et. al. Leukemia, 2017), so human (Hu) vs. mouse (Ms) scFv CD19 CAR constructs were also evaluated. Experimental Procedures: Isolated P-T cells were activated and transduced with various Ms and Hu scFv CD19 CAR viral vectors, including two Ms scFv LV sequences (containing 4-1BB costimulatory domains), three different Hu scFv LV sequences (two containing 4-1BB and one containing CD28 costimulatory domains), and one Ms scFv RV sequence (containing 4-1BB costimulatory domain). In vitro functional activity of these P-T CD19 CAR cells was assessed against CD19+ Burkitt's Lymphoma (Daudi) and Acute lymphoblastic Leukemia (NALM6) cell lines in cytotoxicity assays. In vivo anti-tumor activity of P-T Ms scFv CD19 CAR LV vs. RV cells was assessed using a disseminated lymphoma xenograft model in NSG mice. Results: All CD19 CAR constructs containing the 4-1BB costimulatory domain resulted in high transduction efficiency in P-T cells: Ms LV: 32-70% (n=4); Hu LV: 26-42% (n=2); Ms RV: 22-52% (n=6). P-T cells transduced with LV resulted in lower frequency of CD8+ T cells (9-42%), as compared to cells transduced with RV (46-59%), and consequently had lower CD8+ CD19 CAR+ (of CD3+) expression (LV: 2.8-6.4%; RV: 14-30%). In vitro, all CD19 CAR constructs specifically lysed CD19+ Daudi/ Nalm6 targets in cytotoxicity assays. In vivo, both P-T Ms scFv CD19 CAR LV and RV cells significantly reduced tumor burden and improved survival over the vehicle control. P-T CD19 CAR RV cells demonstrated superior anti-tumor activity over P-T CD19 CAR LV with 100% survival out to day 120, while the CD19 CAR LV treated group succumbed to 100% mortality by day 70. Summary: We have demonstrated the ability to generate P-T CD19 CAR cells using a variety of viral vectors containing the 4-1BB costimulatory domain. Transduction of P-T cells using Hu vs. Ms scFv CD19 CAR sequences resulted in comparable CD19 CAR expression and in vitro cytotoxic activity, whereas use of LV vs. RV had an impact on the phenotypic characteristic of the cells and resulted in differences in the in vivo tumor activity of the cells. Citation Format: Kathy Karasiewicz, Shuyang He, Kristina Tess, Weifang Ling, Kevin Jhun, Gunnar Kaufmann, Jerome B. Zeldis, Henry Ji, Robert Hariri, Xiaokui Zhang. Generation and characterization of human placental-derived CD19 CAR-T cells using viral vectors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2188.