From the PR
Title: A Combined Strategy of CD70 CAR Co-expression with Membrane-bound IL-15 and CISH Knockout Results in Enhanced NK Cytotoxicity and Persistence
This study illustrates multiple approaches to modify NK cells to target CD70, an antigen highly expressed in haematological malignancies and solid tumours, including renal cell carcinoma. Optimal CD70 chimeric antigen receptor (CAR) candidates were identified using high throughput screening approaches. Preclinical results showed that a combined editing and engineering strategy to armour primary NK cells via co-expression of the CD70 CAR and a membrane bound form of IL-15 (mbIL-15), together with knockout of CISH and CD70 genes using the CRISPR/Cas9 system enhanced the persistence in culture and the cytotoxicity of the cells against multiple tumour cell lines. The knockout of CISH also supported the resistance of primary NK cells to suppressive elements active in the tumour microenvironment.
Title: CISH Gene-knockout Anti-CD70-CAR NK Cells Demonstrate Potent Anti-tumor Activity Against Solid Tumor Cell Lines and Provide Partial Resistance to Tumor Microenvironment Inhibition
Primary NK cells engineered with CD70 chimeric antigen receptor (CAR), membrane bound form of IL-15 (mbIL-15) and knockout of CISH and CD70 genes using the CRISPR/Cas9 system could be produced efficiently, demonstrating consistent knockout and transduction efficiency across donors. Memory like NK cell differentiation of the gene edited NK cells was achieved using a modified K562 stimulatory cell line expressing membrane-bound IL-15 and 4-1BBL with the addition of IL-12 and IL-18 during expansion. Various gene editing candidates were assessed; the knockout of CISH in particular enhanced not only tumour cell killing by CD70 CAR NK cells, but also their persistence in culture and resistance to suppressive molecules associated with the tumour microenvironment, such as TGFß and adenosine.
Title: Potentiating the Large-Scale Expansion and Engineering of Peripheral Blood-Derived CAR NK Cells for Off-the-Shelf Application
The study highlights novel methods for scaling the expansion of engineered NK cells to potentially supply a life cycle's worth of commercial off-the-shelf product from a single donor. The methods entail sequential pulses with a proprietary K562 stimulatory cell line in the presence of IL-2 and use of IL-12 and IL-18 to achieve differentiation into memory-like NK cells. When these methods were used to achieve cell expansion greater than even 2 billion-fold, CAR expression was increased on the NK cell surface, engineered cells with CISH gene knockout, CAR and mbIL-15 were preferentially enriched during expansion, and the chromosomal integrity of the cells was well maintained.
Title: KIR Haplotype Can Inform Donor Selection in the Production of Allogeneic Memory-Like CAR NK Cells for Clinical Application
Study findings suggest that the profile of activating and inhibitory KIR genes expressed by healthy donors' NK cells may serve as a future criterion for selecting donors whose NK cells can be engineered for enhanced cytotoxic activity. The optimal KIR profile may depend on the process used to generate NK cells. Engineered CD19 CAR NK cells whose expansion includes IL-12 and IL-18 added to a proprietary K562 stimulatory cell line containing mbIL-15 and 15-41BBL stimulatory cells showed enhanced potency and upregulation of NK memory associated cell surface markers and natural cytotoxicity markers. Finally, donor KIR haplotype correlated best with CAR NK activity in cells expanded in the presence of IL-12 and IL-18, showing that donor selection and expansion methods must be considered together for development of optimal CAR NK therapies.
