Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible SH2-containing (CIS) protein, a key negative regulator of interleukin (IL)-15 signaling, with fourth generation 'armored' chimeric antigen receptor (CAR-IL-15) engineering of cord blood (CB) derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell anti-tumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15 secreting armored CAR-NK cells by promoting their metabolic fitness and anti-tumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.

Background Advantages associated with the use of cord blood (CB) transplantation include the availability of cryopreserved units, ethnic diversity and lower incidence of graft-versus-host disease when compared to bone marrow or mobilized peripheral blood. However, poor engraftment remains a major obstacle. We and others have found that ex vivo fucosylation can enhance engraftment in murine models and thus ex vivo treatment of CB with fucosyltransferase (FT)-VI prior to transplantation is under clinical evaluation (NCT01471067). However, FT-VII appears to be more relevant to hematopoietic cells and may alter acceptor substrate diversity. In this study, we compare the ability of FT-VI and FT-VII to improve the rapidity, magnitude, multi-lineage and multi-tissue engraftment of human CB hematopoietic stem and progenitor cells (HSPC) in vivo. Methods CD34-selected CB HSPC were treated with recombinant FT-VI, FT-VI or mock control, then injected into immunodeficient mice and monitored for multi-lineage and multi-tissue engraftment. Results Both FT-VI and FT-VII fucosylated CB CD34+ cells in vitro, and both led to enhanced rates and magnitudes of engraftment when compared to untreated CB CD34+ cells in vivo. Engraftment following treatment with either fucosyltransferase was robust at multiple timepoints, and in multiple tissues, with similar multi-lineage potential. In contrast, only FT-VII was able to fucosylate T- and B-lymphocytes. Discussion While we found that FT-VI and FT-VII were similarly able to fucosylate and enhance the engraftment of CB CD34+ cells, differences in their ability to fucosylate lymphocytes cells may modulate graft-versus-tumor and/or graft-versus-host effects and may allow further optimization of CB transplantation.

Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, the efficacy of these cells is hindered by the use of glucocorticoids, often given to patients for the management of complications such as graft-versus-host disease. To address this limitation, we have developed a novel strategy for the rapid generation of good manufacturing practice (GMP)–grade glucocorticoid-resistant multivirus-specific T cells (VSTs) using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) gene-editing technology. We have shown that deleting the nuclear receptor subfamily 3 group C member 1 (NR3C1; the gene encoding for the glucocorticoid receptor) renders VSTs resistant to the lymphocytotoxic effect of glucocorticoids. NR3C1-knockout (KO) VSTs kill their targets and proliferate successfully in the presence of high doses of dexamethasone both in vitro and in vivo. Moreover, we developed a protocol for the rapid generation of GMP-grade NR3C1 KO VSTs with high on-target activity and minimal off-target editing. These genetically engineered VSTs promise to be a novel approach for the treatment of patients with life-threatening viral infections post-HSCT on glucocorticoid therapy.

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Autologous peripheral blood progenitor cell (PBPC) transplantation is the treatment of choice for selected myeloma patients. However, tumor cells contaminating the apheresis product are a potential source of relapse. Here we report a sequential purging strategy targeting mature and immature clonogenic myeloma cell populations in the autograft. Thawed PBPC products of myeloma patients were treated with rituximab to kill CD138þ B cells (highly clonogenic immature cells), and bortezomib to target CD138 þ cells (normal and differentiated myeloma plasma cells), followed by coculture with allogeneic mesenchymal stem cells (MSC) from normal donors. After 7 days of coculture, nonadherent cells were removed and cultured in the absence of MSC for an additional 7 days. Then, efficacy of purging (removal of CD138 À 20 þ and CD138 þ cells)was assessed by flow cytometry and PCR. We used our ex vivo purging strategy to treat frozen aphereses from 16 patients. CD138 þ and CD138 À 20 þ (19 þ ) cells present in the initial products were depleted more than 3 and 4 logs, respectively based on 10 6 flow-acquisition events, and to levels below the limit of detection by PCR. In contrast, total nucleated cell (TNC), CD34 þ cell, and colony-forming cell numbers were increased by approximately 12 to 20, 8-, and 23-fold, respectively. Overall, ex vivo treatment of apheresis products with rituximab, bortezomib, and coculture with normal donor MSC depleted mature and immature myeloma cells from clinical aphereses while expanding the normal hematopoietic progenitor cell compartment. Cancer Res; 71(14); 5040-9. Ó2011 AACR.