Vladimir Senyukov

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.

Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells

BACKGROUND T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR. METHODS T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma and acute lymphoblastic leukemia safely underwent hematopoietic stem cell transplantation (HSCT) and infusion of CAR T cells as adjuvant therapy in the autologous (n = 7) or allogeneic settings (n = 19). RESULTS SB-mediated genetic transposition and stimulation resulted in 2,200- to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Following autologous HSCT, the 30-month progression-free and overall survivals were 83% and 100%, respectively. After allogeneic HSCT, the respective 12-month rates were 53% and 63%. No acute or late toxicities and no exacerbation of graft-versus-host disease were observed. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients. CONCLUSIONS CD19-specific CAR T cells generated with SB and AaPC platforms were safe, and may provide additional cancer control as planned infusions after HSCT. These results support further clinical development of this nonviral gene therapy approach. TRIAL REGISTRATION Autologous, NCT00968760; allogeneic, NCT01497184; long-term follow-up, NCT01492036. FUNDING National Cancer Institute, private foundations, and institutional funds. Please see Acknowledgments for details.

Manufacture of Clinical-Grade CD19-Specific T Cells Stably Expressing Chimeric Antigen Receptor Using Sleeping Beauty System and Artificial Antigen Presenting Cells

Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼1010 T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion.

Expansion, purification, and functional assessment of human peripheral blood NK cells.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC). NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials. The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days. Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15), which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL15. Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells

The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia.

Transcription of the activating receptor NKG2D in natural killer cells is regulated by STAT3 tyrosine phosphorylation

Signal transducer and activator of transcription 3 (STAT3) is considered a negative regulator of inflammation, as inhibition of STAT3 signaling enhances antitumor immunity. However, STAT3 activation is a key oncogenic pathway in natural killer (NK)-lineage large granular lymphomas, and we recently reported enhanced proliferation and function of human NK cells activated with IL-21, which signals primarily through STAT3. These IL-21-expanded NK cells also have increased NKG2D expression, which led us to focus our investigation on whether STAT3 regulates NKG2D. In this study, we show that modulation of STAT3 phosphorylation with cytokines and small-molecule inhibitors correlates with NKG2D expression on human NK cells, leading to altered NK-cell degranulation. Moreover, NKG2D expression on murine NK cells having conditional STAT3 ablation is lower than on NK cells from wild-type mice, and human NK cells carrying dominant-negative STAT3 mutations have decreased baseline NKG2D expression and blunted responses to IL-10 and IL-21. Lastly, we show binding of STAT3 to a predicted STAT3 binding site upstream of the NKG2D gene, which is enhanced by IL-10 and IL-21 and decreased by STAT3 inhibition. Taken together, these data show that NKG2D expression in NK cells is regulated at the transcriptional level by STAT3, resulting in a functional NK cell defect in patients with STAT3 mutations.

Interferon gamma induces changes in Natural Killer (NK) cell ligand expression and alters NK cell-mediated lysis of pediatric cancer cell lines

Natural killer (NK) cells have therapeutic potential for cancer due to their capacity for targeting tumor cells without prior sensitization. Our laboratory has developed an NK cell expansion protocol that generates large quantities of NK cells for therapeutic infusion that secret 20 times the amount of interferon gamma (IFNγ) than resting NK cells. IFNγ can upregulate major histocompatibility complex (MHC)-class I, an inhibitory ligand for NK cells, but can also upregulate intercellular adhesion molecule 1 (ICAM-1) which promotes NK:target cell interaction for an efficient lysis. Due to the opposing effects reported for IFNγ on tumor sensitivity to NK cells, we evaluated a panel 22 tumor cell lines from the pediatric preclinical testing program corresponding to different tumor types. We determined the impact of IFNγ on their expression of NK cell activating and inhibitory ligands, death receptors, and adhesion molecules using mass cytometry. We also evaluated the effect of IFNγ on their sensitivity to NK cell-mediated lysis. Our results show upregulation of PD-L1, ICAM-1, MHC-class I, HLA-DR, CD95/FasR, and CD270/HVEM after IFNγ treatment, this upregulation is variable across different tumor types. We also observed a variable impact of IFNγ in NK cell-mediated lysis. For six of the cancer cell lines IFNγ resulted in increased resistance to NK cells, while for three of them it resulted in increased sensitivity. Modeling of the data suggests that the effect of IFNγ on NK cell-mediated tumor lysis is mostly dependent on changes in MHC-class I and ICAM-1 expression. For three of the cell lines with increased resistance, we observed higher upregulation of MHC-class I than ICAM-1. For the cell lines with increased sensitivity after IFNγ treatment, we observed upregulation of ICAM-1 exceeding MHC-class I upregulation. ICAM-1 upregulation resulted in increased conjugate formation between the NK cells and tumor cells, which can contribute to the increased sensitivity observed. However, the effects of MHC-class I and ICAM-1 are not readily predictable. Due to the high IFNγ secretion of NK cell infusion products, a better understanding of the NK ligands on tumor cells and how they are affected by IFNγ is essential to optimize NK cell immunotherapy.

Cathepsin G is broadly expressed in acute myeloid leukemia and is an effective immunotherapeutic target.

Cathepsin G is broadly expressed in acute myeloid leukemia and is an effective immunotherapeutic target