short review

98    Natural killer cell based immunotherapy for eradication of minimal residual disease in patients 1 3

Abstract  Natural killer (NK) cells are cytotoxic against a variety of transformed and virus-infected cells. Cytotoxic-ity of NK cells is regulated through complex interactions between  receptors  expressed  on  NK  surface  and  their cognate  ligands  expressed  on  the  surface  of  target  cells. A large body of data supports the concept that NK are key players  of  immune  surveillance  against  cancer,  and  NK cell based immunotherapy has been proved to be feasible and  effective.  Allogeneic  NK  cell  infusion,  in  nontrans-plant  recipients  with  malignant  disorders,  is  associated with  a  modest  but  of  a  short-term  duration  antitumor effect.  The  major  limitation  of  allogeneic-NK  therapy  is the  inevitable  rejection  of  allogeneic  effectors  and  the termination of antitumor activity. Allogeneic NK cells are not  rejected  by  the  host  if  infused  after  stem  cell  trans-plantation  from  the  same  donor.  Although  the  existing data are limited, allogeneic-NK immunotherapy is much more effective if performed after haploidentical stem cell transplantation. Future trials should address this issue.

Keywords:  Natural  killers,  Immunotherapy,  Stem  cell transplantation

Introduction

Natural  killer  (NK)  cells,  first  recognized  in  1975,  share a common lymphoid progenitor with B and T cells and recent  data  suggest  that  they  represent  a  bridge  that functionally  links  innate  with  adaptive  immunity.  NK cells exert significant cytotoxic activity against tumor or virus  infected  cells  and  therefore  are  considered  as  key players  in  immune  surveillance  against  infections  and malignant transformation [1, 2].

Only a few cases of patients with selective deficiency of  NK  cells  have  been  reported  so  far  and  almost  all  of them  died  from  opportunistic  infections  during  child-hood  [3].  Cytotoxic  activity  of  NKs  is  regulated  through receptors  expressed  on  their  surface.  NK-receptors  are either activating, or  inhibitory, depending on  the  func-tional  effect  of  the  transmitted  signal  after  binding  of the  cognate  ligand.  Human  NK-receptors  are  classified in  4  groups:  (1)  Killer  immunoglobulin-like  Receptors (KIR),  (2)  Heterodimer  CD94/C-type  lectin  Receptors, (3) Leucocyte Ig-like Inhibitory Receptors (LIR), and (4) Natural  Cytotoxicity  Receptors  (NCR).  Cognate  ligands are expressed on the surface of  target cells either cons-titutively  (ligands  for  inhibitory  receptors)  or  after  a stress-induced  signal  like  infection  or  neoplastic  trans-formation  (ligands  for  activating  receptors)  [4].  The “decision  to  kill”  is  regulated  by  the  interplay  between activating  and  inhibitory  signals  received  throughout receptor-ligand interaction. Many aspects of NK cell life, regarding  development,  maturation,  “licensing  to  kill”, regulation of cytotoxicity, etc. have been the focus of pre-vious reviews [5].

The cytotoxic potential of NK cells is a promising tool in the fight against cancer and NK cell based immunot-herapy is briefly summarized below:

Autologous NK cell immunotherapy

Almost three decades ago, Rosenberg et al. explored the cytotoxic  potential  of  autologous  lymphokine  activated killer cells (LAK) in the treatment of patients with meta-static renal carcinoma and melanoma [6]. LAK cells were expanded  ex-vivo  from  peripheral  blood  mononuclear cells  after  culture  in  the  presence  of  interleukin-2  (IL-2).  The  cytotoxic  potential  of  LAK  cells  was  mostly  due to activated NK cells present in the inoculums. Following the initial encouraging results, many other groups explo-red  the  therapeutic  efficacy  of  autologous  LAK  and/or 

memo (2012) 5:98–100DOI 10.1007/s12254-012-0010-7

Natural killer cell based immunotherapy for eradication of minimal residual disease in patients with malignant disordersPanagiotis D. Tsirigotis, Nikolaos Papanikolaou

P. D. Tsirigotis, MD () · N. Papanikolaou, MD2nd Department of Internal Medicine, Hematology Unit,  ATTIKON General University Hospital, Medical School,  University of Athens, Athens, Greecee-mail: panagtsirigotis@gmail.com

Received: 7 March 2012 / Accepted: 16 April 2012 / Published online: 5 June 2012© Springer-Verlag Wien 2012

short review

Natural killer cell based immunotherapy for eradication of minimal residual disease in patients    991 3

NK cells in patients with metastatic solid tumors, and in conclusion the results were rather unsatisfactory [7].

An  alternative  strategy  is  the  production  of  cytoki-ne-induced killer (CIK) cells. Large number of CIK cells have been produced after expansion of peripheral blood mononuclear cells in the presence of interferon gamma (IFN-γ),  IL-2,  and  anti-CD3  monoclonal  antibody.  CIK-cells are a unique population of cytotoxic CD3 +CD56 + T lymphocytes that express the natural killer cell receptor NKG2D.  CIK  cells  express  significant  non-MHC  restric-ted cytotoxicity against a variety of malignant cell lines.

Previous  studies  showed  that  infusion  of  autologous CIK-cells in patients with hematological malignancies is safe and effective [8].

However  we  consider  that  the  therapeutic  potential of autologous NK cells should be further tested in future trials. Interesting in-vitro data should not be ignored. NK cells from a proportion of patients with AML in remission have shown significant in-vitro cytotoxic activity against autologous leukemic blasts collected at the time of dia-gnosis. More interestingly, the patients with in-vitro NK cytotoxicity enjoyed significantly prolonged disease-free survival as compared with patients without NK cytotoxi-city [9, 10].

Allogeneic NK cell immunotherapy

The therapeutic potential of NK cells has been explored in the setting of haploidentical stem cell transplantation (haplo-SCT).  Extensive  clinical  and  laboratory  research from the team from Perugia showed the beneficial effect of  KIR-alloreactivity  in  the  graft-versus-host  direction. KIR-alloreactivity  was  associated  with  decreased  rejec-tion, graft-versus-host disease (GVHD), and relapse rates [11].  Of  significant  importance  is  the  observation  that although allogeneic NK cells are highly cytotoxic against normal and malignant hematopoietic tissues, they do not exert any cytotoxicity against normal nonhematopoietic cells [12]. Clinical trials are summarized below:

Allogeneic NK cell immunotherapy outside the setting of SCT

The therapeutic efficacy and safety of allogeneic NK cells infusion  was  tested  by  Miller  et  al.  in  43  patients  with various  malignancies  including  solid  tumors  and  acute myelogenous  leukemia  (AML).  NK  cells  were  purified, with the use of immunomagnetic beads, from peripheral blood MNCs collected from haploidentical family donors. Following harvesting, the NK cells were cultured ex-vivo in  the  presence  of  IL-2.  Immunosuppressive  treatment was administered to all patients before infusion with the aim  to  delay  the  immediate  rejection  of  allogeneic  NK cells. A cohort of patients received a  low-intensity regi-men, while another cohort received a high-intensity (flu-darabine (Flu) 25 mg/m2 × 5 days plus cyclophosphamide (Cy)  60  mg/kg × 2  days),  immunosuppressive  regimen. 

Low-intensity  regimen  resulted  in  transient  short-term persistence (hours to few days) of allogeneic NK cells. On the  contrary,  in  patients  treated  with  the  high-intensity regimen,  allo-NK  cells  expanded  in  vivo  and  persisted for more than 28 days. Of importance is the observation that  significant  antitumor  efficacy  was  observed  only in the cohort of patients treated with the high-intensity regimen [13]. In a similar trial performed by Curti et al., 13 patients with high risk AML, received highly purified KIR-mismatched  NK  cells  from  haploidentical  donors after immunosuppression with Flu-Cy. NK infusion was not  associated  with  GVHD  or  any  other  toxicity,  while antileukemic activity was documented in a proportion of patients [14]. Interpretation of these data showed that in-vivo  long  term  persistence  and  expansion  of  allogeneic NK cells is required for therapeutic efficacy.

In a subsequent trial and in an attempt to increase host immunosuppression, TBI 400 cGy was added to the initial Flu-Cy regimen. IL-2 was administered post infusion with the  aim  to  harness  NK  cell  expansion.  Due  to  concerns of  prolonged  aplasia,  a  CD34  positively  selected  blood graft  from  the  same  donor  was  given  to  all  patients.  In this trial, authors defined in vivo allo-NK cell expansion as the existence in PB of > 100 allogeneic-NK cells/ml at day  + 14  post  infusion.  Thirty-eight  patients  included  in the trial and allo-NK-expansion at day + 14 was observed in half of them. Successful NK cell expansion was statisti-cally associated with significantly increased probability of complete remission (84 vs 10 %, p > 0.0001) [15].

In  a  similar  trial,  Slavin  et  al.  explored  the  safety and  efficacy  of  allogeneic  activated  lymphocytes  (LAK) administered  in  40  patients  with  chemotherapy-resis-tant  metastatic  solid  tumors  and  high  risk  hematologi-cal malignancies. Thirty-two patients received LAK cells from  haploidentical  family  members  while  8  patients received cells from totally mismatched volunteer donors. No  T-cell  depletion  was  performed  prior  to  infusion. Low-intensity  immunosuppression  consisted  of  either Flu or low-dose Cy in order to prevent immediate rejec-tion  of  allogeneic  LAK  cells  was  administered  prior  to infusion. Self-limited acute GVHD was observed in only 1 out of 40 patients [16].

The major limitation of allo-NK infusions outside the setting of SCT is the inevitable immune rejection of allo-geneic cells resulting in a short-term antitumor effect.

Post-transplant NK cell immunotherapy

Slavin  et  al.  explored  the  safety  and  efficacy  of  post-transplant  administration  of  purified  allo-NK  cells  [17]. Nineteen  patients  with  chemo-refractory  hematological malignancies were included in this trial. Fifteen patients underwent SCT from haploidentical related donor, while 5 patients received a stem cell graft from a matched donor. Donor peripheral blood MNCs incubated in the presence of IL-2. Following initial ex-vivo expansion and activation in the presence of IL-2, NK cells were purified with the use of immunomagnetic beads. GVHD developed in 4 out of 

100    Natural killer cell based immunotherapy for eradication of minimal residual disease in patients

short review

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19 patients (1 grade 2; 1 grade 4; 1 limited chronic; 1 exten-sive chronic), and was due to high T-cell contamination.

Passweg et al. explored the safety and efficacy of infu-sion of purified allo-NK cells from the original donors in 5 patients after haplo-SCT. Infusions were well tolerated and none of the patients developed GVHD [18]. Koehl et al.  tested  the  efficacy  of  KIR-alloreactive  NK  cells  after haplo-SCT in 3 children with refractory acute leukemia. Similar to previous trials, no GVHD was observed [19].

Laport et al. explored the safety and efficacy of alloge-neic CIK infusions in patients with relapsed hematologic malignancies  after  allo-SCT.  Authors  observed  that  all-ogeneic CIK cells retain significant antileukemic activity, while the incidence of GVHD was surprisingly low. Infu-sion of allogeneic CIK should be tested in future trials as a method for eradication of minimal residual disease in high risk malignancies after allo-SCT [20].

Conclusions

The  major  limitation  of  allo-NK  infusion  outside  the context  of  SCT  is  the  short-term  antitumor  effect  due to inevitable rejection of allogeneic cells. Moreover, it is highly  possible  that  the  development  of  immunologic memory will prevent  the efficacy of  repeated  infusions. On the contrary, allo-NK infusions after haplo-SCT can-not be rejected by the host and might result in a long term antitumor reaction.

NK  cell  immunotherapy  is  the  focus  of  intensive research  and  further  improvements  are  anticipated  in the near future.

Take-home message

The  antitumor  effect  of  allogeneic  NK  cell  therapy  in nontransplant recipients is time-limited due to rejection of infused cells, and therefore it might be effective only in cases of minimal residual disease.

Allogeneic NK cell  therapy after allo-SCT is a promi-sing  strategy  since  it  induces  a  significant  graft  versus tumor effect without causing graft-versus-host disease.

Conflict of interestThe  authors  declare  that  there  is  no  actual  or  potential conflict of interest in relation to this article.

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