High log-scale expansion of functional human natural killer cells from umbilical cord blood CD34-positive cells for adoptive cancer immunotherapy

Abstract

Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56(+)CD3(-) NK cell products could be routinely generated from freshly selected CD34(+) UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34(+) UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56(+) NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34(+) cells for cancer immunotherapy.

Figure 1. Schematic diagram of the different culture methods used for the ex vivo generation of CD56⁺ NK cells from cytokine-expanded CD34⁺ UCB cells.

In Method I–III different combinations of cytokines were tested as described in detail in Materials and Methods starting with different numbers of initially seeded CD34⁺ cells. In Method I and II, NK cells were generated from freshly selected UCB donors and the culture duration was 5 weeks. In Method III, CD34⁺ cells were used from cryopreserved UCB donors and the culture period was 6 weeks. Finally, the diagram depicts which NK products were used and displayed as results in figures and supplemental material.

Figure 2. Ex vivo generation of CD56⁺ NK cells from cytokine-expanded CD34⁺ UCB cells.

CD34-enriched UCB cells were expanded for two weeks using three different media (H3000, Stemline I and Stemline II) and subsequently differentiated into NK cells for three additional weeks in the same basal medium using Method I (see Figure 1). Cell cultures were weekly analyzed for cell numbers and phenotype using FCM. (a) Representative example of antigen expression during the two-step culture period using H3000 medium. One week after the onset of the NK cell differentiation step 2 (i.e. after 3 weeks total culture duration) the CD56⁺CD161⁺CD94⁺ NK cell population increases and reaches high purity after 3 weeks of differentiation (i.e week 5). (b) Mean CD56⁺ cell frequency during the 5 week culture period for three different media, which have been tested in parallel experiments using 3–6 UCB donors. (c) Mean total CD56⁺ NK cell numbers after initial seeding of 1×10⁴ CD34⁺ UCB cells during 5 weeks of culture using Method I. Data represent a theoretical calculation based on the actual expansion rates of CD56⁺ cells. Total yield of CD56⁺ cells at each week was calculated by multiplying the expansion rate per week with the number of cultured cells. (d) Mean fold expansion of total cells after initial seeding of 1×10⁴ CD34⁺ UCB cells during 5 weeks of culture using Method I. Data represent a theoretical calculation based on the actual expansion rates of total cells.

Figure 3. Superior expansion of UCB-derived CD56⁺ NK cells using a novel clinical grade medium.

The new GBGM® medium was compared with two previously tested media in the NK cell generation system according to Method II (see Figure 1). Cell cultures were weekly analyzed for cell numbers and phenotype using FCM. (a) Fold expansion of total cells after initial seeding of 1×10⁴ CD34⁺ UCB cells was determined during 5 weeks of culture. Data represent the calculation based on the actual expansion rates of total cells and are displayed as mean ± SD of three different experiments. (b) CD56⁺ cell frequency during the differentiation stage for three different media, which have been tested in parallel experiments using three UCB donors. Data are depicted as mean ± SD. * represents a p-value of <0.05.

Figure 4. Phenotypical profile of ex vivo-generated NK cells using Method II with GBGM.

(a) Flow cytometric analysis of a representative NK cell product generated from CD34⁺ UCB progenitor cells. Cells at 5 weeks of culture were analyzed for expression of CD56, CD3, CD94 and CD16. (b) Expression of a repertoire of receptors important for regulating NK cell activity, including C-type lectin receptors, natural cytotoxicity receptors and cytokine receptors. Histograms show expression of the antigen of interest (black histogram) compared to the specific isotype control (grey histogram). (c) Acquisition of KIR⁺ NK cell subsets during ex vivo NK cell generation from expanded CD34⁺ UCB cells. KIR expression was determined at week 4 and 5 during the differentiation step by FCM.

Figure 5. Responsiveness of ex vivo-generated KIR⁺ and NKG2A⁺ NK cells to MHC class I-deficient target cells.

Ex vivo-generated NK cells using Method II with GBGM were incubated alone, or 18 hours with MHC class I-negative K562 or MHC class I-expressing KG1a cells at an E∶T ratio of 1∶1. Cells were then stained for CD56, CD3, KIR or NKG2A, and the degranulation antigen CD107a. (a) Degranulation of total CD56⁺CD3⁻ NK cells and KIR2DL2/DL3⁺ NK cell subset expanded for 5 weeks from CD34⁺ UCB cells. Density plots are gated on CD56⁺CD3⁻ NK cells and the histogram plots show the CD107a degranulation of the KIR2DL2/DL3⁺ NK cells. (b) Degranulation of total CD56⁺CD3⁻ NK cells and NKG2A⁺ NK cell subset expanded for 6 weeks from CD34⁺ UCB cells. Density plots are gated on CD56⁺CD3⁻ NK cells and the histogram plots show the CD107a degranulation of the NKG2A⁺ NK cells.

Figure 6. Functional activity of ex vivo-generated CD56⁺ NK cells using Method II with GBGM.

(a) Specific cytotoxicity of a CD56⁺ NK cell product (98% purity) against the myeloid leukemia cell lines K562 and KG1a. Specific lysis was determined after 4 and 24 hours of co-culture in a FCM-based cytotoxicity assay at an E∶T ratio of 2∶1. Data are displayed as mean ± SD of triplicate wells. (b) Degranulation of CD56⁺ NK cells was determined by FCM as the percentage of CD107a⁺ cells. Results are depicted as mean ± SD of triplicate wells. (c) IFNγ production was determined by ELISA and depicted as mean ± SD of triplicate measurements. (d) Specific cytotoxicity of another CD56⁺ NK cell product (95% purity) against the myeloid leukemia cell lines K562, Lama, Kasumi and KG1a, and the melanoma cell lines BLM and FM3. Specific lysis was determined after 18 hours of co-culture in a FCM-based cytotoxicity assay at an E∶T ratio of 1∶1. Data are displayed as mean ± SD of triplicate samples. (e) Degranulation of CD56⁺ NK cells was determined by FCM as the percentage of CD107a⁺ cells after overnight stimulation with different targets. Data are depicted as mean ± SD of triplicate samples. (f) IFNγ production was determined by ELISA and depicted as mean ± SD of triplicate measurements. (g) Specific cytotoxicity of a third CD56⁺ NK cell product (97% purity) against primary AML cells from 5 different patients. Specific lysis was determined after 24, 48 and 72 hours of co-culture in a FCM-based cytotoxicity assay at an E∶T ratio of 3∶1. Data are displayed as mean ± SD of triplicate samples.