Development of Optimized Protocol for Generation of NK Cells Expressing Chimeric Antigen Receptors from Hematopoietic Stem Cells for Cancer Immunotherapy

Background: Natural Killer (NK) cells are innate immune cells that mediate cytotoxicity against tumor and virus-infected cells, and represent a very promising source for adoptive cellular approaches for cancer immunotherapy. Extensive research has been conducted, including clinical trials, attempting to harness their properties. Gene modification of NK cells can direct their specificity and enhance their function, but the efficiency of gene transfer in mature NK cells is very limited. We have previously published a protocol for generation of human NK cells from gene-modified hematopoietic stem cells (HSC) isolated from umbilical cord blood. Generation of NK cells from HSC provides the opportunity of generation of younger NK cells and expansion of specific gene-modified clones starting from a smaller number of previously isolated and cryopreserved initial cells, with added advantage of generation of multiple batches from the same donor. Chimeric antigen receptors (CAR) are engineered fusion proteins that combine the antigen specificity of antigen-binding moieties of monoclonal antibodies and intracellular activation motifs capable to activate immune cells. Preliminary evidence suggests that NK cells with specificity directed by second-generation CAR may have enhanced cytotoxicity. The goal is to develop a protocol with maximal generation of CAR-expressing NK cells from human HSC for clinical applications. We have evaluated the use of HSC isolated from G-CSF-mobilized apheresed peripheral blood mononuclear cells (PBSC), the elimination of serum in culture media and the elimination of feeder stroma.

Significance: Development of a protocol for clinical translation and large-scale good manufacturing practice (GMP) compatibility, maximally generating functional CAR-expressing NK cells. G-CSF mobilized peripheral blood stem cells (PBSC) were used because of availability of larger HSC numbers, increasing safety and efficacy. Changes in culture media based on available literature were evaluated to promote generation of larger number of cells. Human AB serum and serum free media were tested to determine the cell yield for large-scale GMP-compatible protocol.

Methods: A third-generation lentiviral vector co-delivering CD19-specific CAR and enhanced green fluorescent protein (EGFP) was used for gene modification of primary human PBSC. Gene-modified cells were then co-cultured with OP9-DL1 stromal cells over 35-40 days in six different culture media conditions for evaluation. Medium “A” was our previously published protocol and consisted of alpha-MEM enriched with 20% of fetal bovine serum and recombinant human cytokines SCF 5ng/mL, Flt3L 5ng/mL, IL-7 5ng/mL, and IL-15 10ng/mL. Medium “B” was AIM V enriched with 10% of human AB serum and cytokines SCF 5ng/mL, Flt3L 5ng/mL, IL-7 20ng/mL, and IL-15 50ng/mL. Medium “C” was similar to medium “B” excluding human AB serum. After 10 days of culture, IL-2 10ng/mL was added to all three media (“plus”) creating six different conditions. Flow cytometry was used for detection of EGFP expression and NK cell surface markers. Digital droplet PCR was used for analysis of number of integrated viral copies. Feeder-free culture conditions were developed with the addition of recombinant human IGF-1 100ng/mL to AIM V culture media enriched with SCF, Flt-3 and IL-15. 

Results: NK cell differentiation was achieved in all conditions. Feeder-free conditions seemed to present mature NK cells earlier (days 25-30) than stromal co-culture (days 35-45), but lower cell yield. As previously reported, PBSC had lower yields of NK differentiation as compared to umbilical cord blood, but higher concentrations of IL-7 and IL-15 rescued the differentiation. Total cell yields were 100-220-fold expansions, with highest counts recovered from conditions with higher IL-7 and IL-15. The removal of serum and addition of IL-2 did not seem to affect differentiation or proliferation. CD56+/CD16+/CD94+ NK cells were present in 10-40% of all CD56+ cells.    

Conclusions: Large-scale GMP-compatible generation of clinically-relevant numbers of gene-modified NK cells from HSC is feasible. Higher doses of cytokines IL-7 and IL-15 successfully increase the yield of NK cells from PBSC. Absence of serum did not decrease differentiation or proliferation. PBSC showed folds of expansion and NK cell differentiation comparable to those obtained from umbilical cord blood.