Christian P. Kalberer

Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients.

BACKGROUND AIMS Alloreactive natural killer (NK) cells are potent effectors of innate anti-tumor defense. The introduction of NK cell-based immunotherapy to current treatment options in acute myeloid leukemia (AML) requires NK cell products with high anti-leukemic efficacy optimized for clinical use. METHODS We describe a good manufacturing practice (GMP)-compliant protocol of large-scale ex vivo expansion of alloreactive NK cells suitable for multiple donor lymphocyte infusions (NK-DLI) in AML. CliniMACS-purified NK cells were cultured in closed air-permeable culture bags with certified culture medium and components approved for human use [human serum, interleukin (IL)-2, IL-15 and anti-CD3 antibody] and with autologous irradiated feeder cells. RESULTS NK cells (6.0 ± 1.2 x 10(8)) were purified from leukaphereses (8.1 ± 0.8 L) of six healthy donors and cultured under GMP conditions. NK cell numbers increased 117.0 ± 20.0-fold in 19 days. To reduce the culture volume associated with expansion of bulk NK cells and to expand selectively the alloreactive NK cell subsets, GMP-certified cell sorting was introduced to obtain cells with single killer immunoglobulin-like receptor (KIR) specificities. The subsequent GMP-compliant expansion of single KIR+ cells was 268.3 ± 66.8-fold, with a contaminating T-cell content of only 0.006 ± 0.002%. The single KIR-expressing NK cells were cytotoxic against HLA-mismatched primary AML blasts in vitro and effectively reduced tumor cell load in vivo in NOD/SCID mice transplanted with human AML. CONCLUSIONS The approach to generating large numbers of GMP-grade alloreactive NK cells described here provides the basis for clinical efficacy trials of NK-DLI to complement and advance therapeutic strategies against human AML.

Clinical‐grade purification of natural killer cells in haploidentical hematopoietic stem cell transplantation

BACKGROUND: Because of a high risk of graft‐versus‐host disease (GVHD), donor lymphocyte infusions with unmodified lymphapheresis products are not used after haploidentical hematopoietic stem cell transplantation. Natural killer (NK) cells have antitumor activity and may consolidate engraftment without inducing GVHD. Production of NK cells under good manufacturing practice (GMP) conditions in a sufficient number is difficult.

Human acute myeloid leukemia CD34+CD38− stem cells are susceptible to allorecognition and lysis by single KIR-expressing natural killer cells

In this study, the authors have investigated the anti-leukemic action of alloreactive single KIR positive natural killer cells on CD34+CD38− AML cells, showing that the HDAC inhibitor valproic acid augments this activity. The concept of tumor immunosurveillance has raised prospects for natural killer cell-based immunotherapy of human cancer. The cure of acute myeloid leukemia may depend on eradication of leukemic stem cells, the self-renewing component of leukemia. Whether natural killer cells can recognize and lyse leukemic stem cells is not known. To develop strategies that effectively target acute myeloid leukemia-leukemic stem cells, we investigated anti-leukemic effects of human alloreactive single KIR+ natural killer cells. Natural killer effectors with KIR specificity mismatched with respect to HLA class I allotype of target cells effectively recognized acute myeloid leukemia-leukemic stem cells defined phenotypically as CD34+CD38−, while healthy bone marrow-derived CD34+CD38− hematopoietic stem cells were spared, as demonstrated by cytotoxicity and hematopoietic colony-forming assays. The HDAC inhibitor valproic acid increased the activating NKG2D ligand-dependent lysis of acute myeloid leukemia-CD34+CD38− leukemic stem cells. These results show that alloreactive natural killer cells have the potential to detect and target leukemic stem cells, and thus to improve the treatment outcome in acute myeloid leukemia.

Retroviral Vectors Aimed at the Gene Therapy of Human β‐Globin Gene Disordersa

Abstract: We are focusing on the development of complex retroviral vectors containing human β‐globin gene and β‐LCR for the gene therapy of sickle cell disease and β‐thalassemias. First generation vectors containing mutated splice‐sites to insure stability of proviral transfer enabled long‐term reconstitution in 10/12 transplanted mice for a least 8 months with high expression levels in 2 out of 3 mice analyzed (5% and 20% murine β). Transfer and expression were also achieved in secondary recipients (range: 3–11% murine β). Position independent expression was not observed. In an effort to increase the efficiency of gene transfer and obtain complete reconstitution of recipient mice with exclusively transduced cells while enriching for proviral integration into active chromatin regions, we have incorporated a cassette expressing CD24 or the green fluorescent protein (GFP). Stable transfer to murine bone marrow cells allowed efficient FACS‐sorting of pure populations of transduced cells. A family of vectors based on these principles and containing segments of γ‐ or δ‐globin genes were also designed for systematic analysis of their anti‐sickling properties.

Combination therapy for multidrug-resistant cytomegalovirus disease.

Multidrug‐resistant (MDR) cytomegalovirus (CMV) emerged after transient responses to ganciclovir, foscarnet, and cidofovir in a CMV‐seropositive recipient who underwent allogeneic hematopoietic stem cell transplantation from a CMV‐seronegative donor. Experimental treatments using leflunomide and artesunate failed. Re‐transplantation from a CMV‐seropositive donor supported by adoptive transfer of pp65‐specific T cells and maribavir was followed by lasting suppression. This case illustrates that successful MDR CMV therapy may require individualized multidisciplinary approaches.

Genetic modification of murine hematopoietic stem cells by retroviruses.

Among the currently available methods for gene transfer, recombinant murine retroviruses remain the best established method for achieving stable integration of a transgene with high efficiency. Pioneering work by a number of groups has demonstrated the feasibility of using this method for gene transfer to primitive, multipotential long-term repopulating hematopoietic stem cells (HSC) (1-4). In the case of the hematopoietic system, it is required that the introduced gene integrates into the genome of HSC in order to be expressed in multiple lineages over an extended period of time. However, HSC are found at low frequency, and are normally in a quiescent or slow cycling state. Both factors represent challenges to successful retroviral gene transfer. The former places a premium on high titer, and the latter dictates methods to trigger HSC cycling during the infection, since stable integration of murine retroviruses requires cell division of the target cell and breakdown of the nuclear membrane (5,6). In general, titers greater than 1 × 10(5) U/mL allow some degree of gene transfer for HSC, but 1 × 10(6) or higher are a reasonable goal for achieving useful efficiencies of at least 20%. For activation of HSC, most protocols invoke a combination of in vivo and in vitro stimulation. The former is most easily and routinely achieved by administration of cytotoxic agents like 5-fluorouracil (5-FU) 4 d prior to bone-marrow harvest. This procedure removes a large proportion of actively cycling, more differentiated cells, thus achieving a.

Comment: Shall Killer Cell Immunoglobulin-Like Receptor Genotyping be Incorporated in Donor Search Algorithms?

The article in Biology of Blood and Marrow Transplantation by Bachanova et al. [1] analyzed outcomes for 614 adult recipients of non–T cell–depleted transplants from unrelated donors for treatment of non-Hodgkin lymphoma over a 19-year period. Killer cell immunoglobulin-like receptor (KIR) genotyping was done on stored samples from the National MarrowDonor Program repository. Patients had various lymphoma types and most had advanced disease. Of the unrelated donors, 30% of donors were of KIR AA genotypes and the remaining donors had either 1 or 2 B haplotypes. Receiving stem cells from a donor with at least 1 KIR B haplotype was associated with a reduced relapse risk, with an effect size of 11% absolute reduction in relapse incidence. This was observed in 10/10 HLA–matched donorrecipient pairs only; after correction for confounders, the hazard ratio was .63. This effect was not observed in patients receiving a transplant from an HLA-mismatched donor. There appeared to be a “dose” effect, with higher activating KIR gene numbers associated with lower relapse rates than lower gene numbers in recipients of stem cells from a donor with at least 1 B haplotype. In allogeneic hematopoietic stem cell transplantation, immunological effects (ie, the graft-versus-malignancy effect) are considered to carry the main responsibility for maintaining patients in remission [2]. Although initially all graftversus-malignancy effects were ascribed to effector T cells, a contribution of natural killer (NK) cells has become more clear over time. Not astonishingly, initially, NK cell effects were observed in extensively T cell–depleted haploidentical transplantations, where the lack of engagement of inhibitory KIR receptors on NK cells was associated with reduced relapse rates [3]. Differential regulation of ligands for activating receptors in tumor cells impacts the sensitivity to NK cell lysis. Thus, the effects of NK cells are thought to be dependent on the integration of signals delivered by a complex array of inhibitory and activating receptors [4]. Most KIR genes are encoded on chromosome 19 and are summarized as belonging to 2 haplotypes: the A haplotype with only genes coding for inhibitory KIR receptor and the B haplotypes, associated with a variable number of activating KIR. It was, therefore logical to study whether a measurable antitumor effect could be observed in correlation with the KIR gene content of the donor. SuchNK cell–mediated graft-versusmalignancy effectswere foundmore frequently in patientswith acute myeloid leukemia (AML) than with other diseases. Myeloid leukemia is thought to be particularly sensitive to NK cell–mediated antimalignancy effects [5,6]. This paper is remarkable for showing similar effects in patients with nonHodgkin lymphoma. This is of particular importance, as the fact that such effects can be shown in disease other than AML strengthens the evidence for the postulated importance of activating KIR gene content in exerting graft-versus-malignancy effects. This study also bears witness to the great organizational capacity of the National Marrow Donor Program, where biobanking of donor samples allows to study questions as shown here. The question now is: are these data highly reliable and should these influence our donor selection algorithms? Interestingly, the observed effects were seen only in patients with 10/10 HLA–matched donors and not in donors with HLA mismatches. Such differences may be explained, however, by more important T cell effects in patients receiving stem cells from an HLA-mismatched donor. In a study by Cooley in patients with AML [5], it was donor centromeric or telomeric KIR B/B haplotypes that protected from relapse rather than KIR B/x; ie, a KIR haplotype homozygosity in donor NK cells was most protective against relapse. Is this due to gene dose Financial disclosure: See Acknowledgments on page 1540. * Correspondence and reprint requests: Jakob Passweg, MD, MS, EBMT Activity Survey Office, Division of Hematology, University Hospital Basel, CH-4031 Basel, Switzerland. E-mail address: jakob.passweg@usb.ch (J. Passweg). http://dx.doi.org/10.1016/j.bbmt.2016.06.021 1083-8791/© 2016 American Society for Blood andMarrow Transplantation. Biol Blood Marrow Transplant 22 (2016) 1539–1542