Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells

NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy

Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations.

T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire

Gene expression in NK cells stimulated with mbIL15 or mbIL21 as assessed using the nCounter platform.

<p>Paired aliquots of purified NK cells from 4 donors were stimulated for one week with either Clone 4 (mbIL15) or Clone 9.mbIL21. Total RNA was purified and equal quantities hybridized for detection of expression of 96 genes. Gene expression was normalized to LDH (mean 6,076 copies detected), and the remaining data for each gene plotted as mean +/− SEM for each expansion condition. Genes with detection below background (set at ≤10 detected copies) were excluded as not biologically significant (grey box). The ten highest-expressed genes in addition to LDH are labeled, as are genes that are differentially expressed by >2 fold (red) or <0.5 fold (blue) in mbIL21-expanded cells. Differentially expressed genes were replotted (inset) for comparison, and two-tailed <i>t-</i>test applied.</p

Schema for NK cell manufacturing with aAPCs.

<p>Artificial antigen-presenting cells (aAPCs) were produced by genetic modification of K562 to express costimulatory molecules and membrane-bound cytokines. To expand NK cells <i>ex</i> vivo, unfractionated PBMC are stimulated weekly with irradiated PBMC, inducing rapid proliferation of NK cells and in some cases non-specific expansion of T cells. Contaminating T cells may be depleted, and the remaining purified NK cells may be stimulated weekly by the aAPCs as needed to obtain sufficient numbers. Expanded NK cells may be used directly or cryopreserved for future use.</p

Phenotype of NK cells expanded on aAPCs bearing membrane-bound cytokines.

<p>NK cells purified from PBMC were stimulated weekly with either Clone 4 (mbIL15) or Clone 9.mbIL21 for 3 weeks. Expression of NK cell receptors was determined by flow cytometry. A) Representative dot plots of NK cells expanded from the same donor on Clone 4 (mbIL15) or Clone 9.mbIL21. B) Component subpopulations of fresh NK cells or NK cells expanded on Clone 4 (mbIL15) or Clone 9.mbIL21 from 4 donors as determined by flow cytometry. Mean +/− SD is shown. P values are for 2-way repeated-measures ANOVA comparing against mbIL21-expanded NK cells with Bonferroni correction (all significant P values are indicated).</p