Prototypes developed within the CAR-NK project

• NK92 cells transduced with a vector co-expressing a CD64-2A CAR construct and interleukin 2 capable of producing IL2, thus becoming independent of the presence of interleukin
• NK92 cells transduced with a vector that co-expresses a CD64-2A CAR construct and interleukin 15 are able to produce IL15, thus becoming independent of the presence of exogenous interleukin.
• NK92 cells transduced with anti-CD19 CAR receptors
• K562 cells transduced to express CD19 antigen, for anti-CD19 CAR receptor testing
• The development of NK cells carrying universal CAR constructs that can mediate the cytotoxic response through monoclonal antibodies allows an increased level of flexibility of adoptive cell treatments.

Table 1. CAR constructs recognizing tumor antigens CD19, EGFR and PD-L1 containing different transmembrane domains and NK cell-specific co-stimulators

Map of a lentiviral vector indicating functional regions of the vector and relevant domains of the CAR construct

Cytotoxic activity of NK-92 cells transduced with an anti-CD19 CAR construct was assessed by determining cytolysis observed following contact of effector cells with fluorescently labeled tumor target cells. For this purpose, CD19- (U266) or CD19+ (NALM6) tumor cells in suspension were used as targets. Thus, it could be observed that the transduced cells specifically destroy, in a significantly increased proportion (**, p=0.0031), CD19+ NALM-6 tumor cells.

Cytotoxicity assays with untransduced NK-92 cells or transduced with anti-CD19 CAR against target cells expressing or not expressing the CD19 antigen. In the upper panel, CD19 antigen expression is determined by flow cytometry.

Table 2. CAR constructs with Fc receptor binding mode

These universal CARs are based on the use of the extracellular domain of the high-affinity receptor of the Fc constant domains of IgG immunoglobulins (CD64), and various transmembrane and intracytoplasmic modules. The use of these CARs based on the extracellular domain of high-affinity Fc receptors allows the "arming" of NK or T cells with various monoclonal antibodies or protein modules that recognize specific antigens, coupled to the Fc domain, giving these cells the flexibility to be personalized (used against various antigens). In addition, the specificity of these genetically modified cells can be optimized by conjugation with multiple antibodies, thereby creating cells with multivalent specificities. The use of a common backbone, CD64 or a common epitope tag (myc-tag), in the construction of these chimeric receptors allows, in addition, the easy detection of their expression on the surface of genetically modified cells.

To demonstrate the functionality of universal CAR receptors that can bind antibody molecules with therapeutic potential, we performed a cytotoxicity assay against Raji cells expressing CD20 and CD19 antigens with untransduced NK-92 cells or transduced with anti-CD19 CAR or CAR containing CD16(F158V) or CD64 high-affinity binding modules, in the absence or presence of therapeutic IgG1 anti-CD20 antibodies, Rituximab.

Determination of cytotoxic activity against Raji cells (CD19⁺CD20⁺) in the presence or absence of anti-CD20 antibodies of untransduced and transduced NK-92 cells with different CAR, anti-CD19 or universal constructs. The ratio of effector to target cells was 3:1.