Correspondence: Dr Evren Alici , Karolinska Institutet, Department of Medicine, Karolinska University Hospital Huddinge, Stockholm, Sweden. E-mail: evren.alici@ki.se

(Received 5 January 2011; accepted 4 February 2011)

LETTER TO THE EDITOR

Ex vivo expansion of natural killer cells: a question of function

TOLGA SUTLU 1 & EVREN ALICI 1,2

1 Department of Medicine, Karolinska Institutet, and 2 Hematology Center, Karolinska University Hospital Huddinge, Stockholm, Sweden

Key Words: cancer immunotherapy , ex vivo expansion , good manufacturing practice , natural killer cells

We have read, with great interest, the commentary by Maria Berg and Richard Childs (1) about our study on the expansion of clinical-grade natural killer (NK) cells (2), published in the December 2010 issue of Cytotherapy . In the commentary, the authors have presented very valid concerns regarding cancer immunotherapy using adoptive transfer of NK cells in general and more specifi cally the implications of the expansion method we have presented. Therefore, we feel that it is of great importance to clarify certain aspects and present our standpoint regarding such therapeutic approaches.

The NK cell expansion protocol that we have pre-sented is the result of a long-term effort to optimize a clinical-grade and feeder-free closed system for the generation of highly cytotoxic NK cells with anti-tumor activity. We initially developed this system using the same medium and culture components in a con-ventional cell culture fl ask-based system along with extensive characterization of phenotype and function in such expanded cells. The paper under discussion presents the fi nal steps of optimizing this protocol in a closed and automated bioreactor under good manu-facturing practice (GMP) conditions. We believe that many of the questions raised by the authors have their answers in our previous reports, and we would like to bring them to their attention in this response.

Regarding lineage-specifi c cytolytic function against autologous tumor targets, we have presented in a previous study (3) the analysis of cytotoxic activity by different subsets of the fi nal product. Our observations clearly show that the main anti-tumor activity against autologous tumor cells from multiple myeloma (MM) patients was carried out

not by T cells (CD3 � CD56 − ) or NK-like T cells (CD3 � CD56 � ) but by NK cells (CD3− CD56 � ) in the fi nal expansion product. This has been shown by analysis of CD107a expression after stimulation with autologous tumor targets, which is an indicator of degranulation-mediated cytotoxicity.

We fully agree with the comments that a higher NK cell purity in the fi nal cell product would be desirable in order to relate clinical effects better to NK cells. The comment that T-cell depletion and/or NK-cell selection would be required is well taken, and we are indeed working on cheaper GMP-compliant methods for depletion of T cells after expansion, as the current golden standard systems prove to be quite expensive for the intended cell dose. We believe this is just a technical issue that will soon be overcome.

Nevertheless, although the number of T cells in the fi nal product might seem to pose a risk of graft-versus-host disease (GvHD) if used in the allogeneic setting, previous research has convincingly shown (4 – 9) that T cells cultured more than 7 days under such conditions lose their alloreactive potential. Although the exact mechanism is unknown, various hypotheses, such as the deletion of alloreactive cells by activation-induced cell death, involvement of split anergy, loss of alloreactive precursors during expan-sion, and loss of naive cells or induction of suppres-sive cells, have been put forward (7,10,11).

In agreement with such observations, we have completed and published a phase I clinical trial infusing cells expanded under the same conditions (using fl asks) without any depletion and/or purifi ca-tion steps (12). The patients included in this study had undergone allogeneic stem cell transplantation

Cytotherapy, 2011; 13: 767–768

ISSN 1465-3249 print/ISSN 1477-2566 online © 2011 Informa HealthcareDOI: 10.3109/14653249.2011.563295

768 T. Sutlu & E. Alici

for colorectal carcinoma, hepatocellular carcinoma, renal cell carcinoma and B-cell chronic lymphocytic leukemia. The cells were given with or without subcu-taneous interleukin-2 (IL-2) infusions and, although a high number of T cells were infused, we did not observe GvHD or any other side-effects attributable to the infusion of the cell product.

Regarding yield versus purity, we believe that both are equally crucial. As mentioned above, the purity might be key, especially in early phase clinical trials, for directly relating clinical effects to NK cells. How-ever, the yield is no less important. We have previously demonstrated that infusion of syngeneic NK cells in MM-bearing mice has a dose-dependent effect on pro-longation of survival (13). Therefore, it is possible that the amount of cells that can possibly be infused is a key factor in seeing a clinical benefi t from such treat-ments. In line with this hypothesis, in future clinical trials we intend to improve the effector-versus-target cell ratio in vivo , by preceding the NK cell infusion with chemo- and/or radiotherapy to reduce the number of tumor targets and using multiple escalating doses of NK cell infusions for keeping an optimum balance between safety and effi cacy.

Another important factor is the phenotype of the cells in the fi nal product. It is evident that not every NK cell expansion protocol and not every different donor yields expanded cells with a similar phenotype. Factors such as the distribution of KIR-expressing populations and expression of other activating and inhibitory receptors may be of importance and need to be checked thoroughly. The level of CD16 expres-sion in the fi nal product should also be optimized, in order to make way for possible future use of expanded NK cells along with tumor-targeting antibody infu-sions. Our fi nal expanded product contains NK cells that almost uniformly express CD16 at higher levels than unstimulated NK cells (2,3), in contrast to other reports of differently expanded NK cells showing low levels of CD16 expression or a large CD16 – fraction (14 – 16).

Keeping in mind that such differences in NK cell phenotype as a result of the expansion method used might have a signifi cant effect on the clinical applica-bility and effi ciency, the ultimate answers lie in testing these cells in clinical trials. Such clinical experience is warranted to provide a conclusive answer to which approach will be the most suitable to induce a graft-versus-tumor effect with a minimum of adverse events. Some of these trials are already underway and, in due time, we hope to have a better insight into the dose, purity and, most important of all, the phenotype of effector cells that will have a positive clinical effect.

Declaration of interest: The authors report no con-fl icts of interest. The authors alone are responsible for the content and writing of the paper.

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