Pre-Clinical Evaluation of Allogeneic 'Off-the-Shelf' CAR-iNKT Cells Targeting CD19 (ALA-101)-Expressing Hematological Malignancies

Background

Arovella Therapeutics is a preclinical stage company developing allogeneic 'off-the-shelf' human invariant natural killer T (iNKT) cell-based therapies for hematological malignancies and solid tumors. iNKT cells are a unique and rare subset of lymphocytes that express an invariant T cell receptor (iTCR) and are restricted by the glycolipid-presenting, non-polymorphic CD1d. Unlike allogeneic conventional T cells that can cause acute graft versus host disease (aGVHD) following administration, iNKT cells exert a protective effect against aGVHD after allogeneic stem cell transplantation in humans. Early clinical studies suggest that non-engineered and CAR-engineered allogeneic iNKT cells are safe in humans and represent a promising therapeutic approach, bypassing the requirement to delete the endogenous TCR. Their inherent biological properties enable iNKT cells to mount a swift and robust antitumor immune response when engineered with a Chimeric Antigen Receptor (CAR). The use of iNKT cells from healthy donors has many advantages over allogeneic T cells and NK cells, including recognition of CD1d+ on tumor cells through the iTCR in addition to the CAR mediated recognition of a tumor specific antigen. Here, we report results from early preclinical studies assessing the functional profile of iNKT cells engineered to express an optimized CAR targeting CD19 (ALA-101) for allogeneic use.

Methods

iNKT cells were purified from the peripheral blood of healthy donors followed by lentiviral vector transduction (development construct) to express a CD19-targeting CAR. After transduction, cells were expanded to generate ALA-101. The CD19 CAR functionality of ALA-101 was compared against non-transduced (NT) iNKT cells or with CAR19-T cells transduced with the same lentiviral vector. Flow cytometry was used to assess the expression of the CAR and ALA-101 phenotypes and to determine CD19 and CD1d expression levels in the primary patient samples; marginal zone lymphoma (MZL) cells and two chronic lymphocytic leukemia (CLL) samples. In vitro cytotoxicity assays and intracellular cytokine staining were used to evaluate the CAR-dependent and -independent anti-tumor activities using B cell lymphoblastic leukemia (B-ALL) SEM cell line and primary tumor cells from patients. In vivo efficacy testing of ALA-101 is ongoing in a disseminated NSG mouse model of SEM cells expressing luciferase.

Results

The purity of iNKT cells following selection from peripheral blood mononuclear cells of healthy donors was greater than 99%. ALA-101 was optimized for CD19 CAR functionality using a pre-clinical lentiviral vector. Using multiple independent donors, we confirmed that iNKT cells engineered with lentiviral vector encoding CD19 CAR (ALA-101) maintained the surface CAR expression over 35 days and expressed variable frequencies of CD27, CD62L and CCR7. Compared to day 11 CAR19-T cells, day 17 and day 35 ALA-101 cells demonstrated higher cytotoxicity against the CD19+CD1d- B-ALL SEM cells; similarly, ALA-101 demonstrated higher cytotoxic activity against CD19+CD1d+ malignant B cells from patients with CLL and MZL. Of note, when tested at different time points, ALA-101 cells expanded for 17 and 35 days showed similar cytotoxicity against primary patient samples and SEM cells. Intracellular cytokine staining from tumor cell co-cultures demonstrated that the majority of ALA-101 expressed TNFα, Perforin and Granzyme B.

Conclusions

Here we report the preclinical activity of ALA-101, a novel allogeneic CAR19-iNKT cell product for the treatment of CD19+ B-cell malignancies. Our data shows the successful generation of polyfunctional allogeneic iNKT cells in terms of cytotoxicity and cytokine profile when engineered to express a CD19-targeting CAR. We have initiated the manufacturing of ALA-101 with the intention to enter clinic trials in CD19 positive malignancies. Taken together, the data indicates that iNKT cells are a potent allogeneic, 'off-the-shelf' cell therapy to treat a broad spectrum of hematological malignancies and solid tumors.

Ponnusamy:Arovella Therapeutics: Current equity holder in publicly-traded company. Armugam:Arovella Therapeutics: Current Employment. Buchanan:Arovella Therapeutics: Current Employment. Baker:Arovella Therapeutics: Current Employment, Membership on an entity's Board of Directors or advisory committees. Bharathan:Arovella Therapeutics: Current Employment. Karadimitris:Arovella Therapeutics: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties, Research Funding.