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Natural killer cell-based immunotherapeutic strategies
Cytotherapy (2005) Vol. 7, No. 1, 16 /22
Natural killer cell-based immunotherapeutic strategies H-G Klingemann Division of Hematology/Oncology, TUFTS /New England Medical Center, Boston, Massachusetts, USA
Not until recently have Natural killer (NK) cells stepped out of the shadow of T-cells to be considered for cellular therapy of malignant diseases. This evolution has been facilitated by the discovery of specific receptors on NK cells that interact with HLA molecules on target cells but also the discovery of specific activating receptors. Since NK cells represent only about 10% of the lymphocyte population in blood,
separation and enrichment are important steps if NK cells are to be used clinically. This is of particular consideration in the setting of allogeneic NK cell infusion where contaminating T-cells could potentially induce graft versus host disease. This review will describe the requirements for NK cells to recognize target cells, their ex vivo expansion and potential therapeutic applications.
Natural killer cells comprise a CD56 CD3 subpopulation of lymphocytes that is involved in responding to certain viruses, parasites, microbial pathogens and transformed cells by exhibiting cytotoxic functions and secreting a battery of cytokines [1 /5]. The history of using killer cells in an attempt to control cancer growth goes back to the mid-1980s, when the group of Rosenberg et al. [6] treated patients with advanced metastatic renal cell cancer and melanoma with mononuclear cells that were activated ex vivo with high doses of IL-2. The approach involved harvesting cells from cancer patients by leukapheresis and activation and expansion of these cells by ex vivo coculture with high doses of IL-2. The expanded cells were termed Lymphokine-activated killer (LAK) cells and were then re-infused into the patient supported by IL-2 administration aimed at maintaining the activity of the infused cells in vivo . Although this approach produced nearly 15 /20% partial and complete responses in initial trials, subsequent studies showed that a similar anti-tumor effect could be achieved with high dose IL-2 alone [7]. LAK cells generated in culture predominantly contain polyclonal T lymphocytes that proliferate with the high doses of IL-2 in the culture, and these T cells outgrow any NK cells with respect to numbers. With hindsight, we can now be critical of these studies but they still stand today as pioneering work that accelerated the field of cellular immunotherapy. Since
T-cell recognition of tumor cells depends on an intact MHC molecule, these polyclonal T cells cannot, in a predictable manner, interact with the malignant target. Expression of intact MHC molecules is variable, depending on the type of cancer [8,9]. It is also known that cytotoxic cells in patients with cancer can be functionally defective. For example the trans-membrane signaling receptor component CD3z of cytotoxic cells can be down-regulated [10]. Another example comprises patients with advanced CML in whom the NK cell function is down-regulated with advancing disease [11]. Taken together, these observations may explain why infusion of autologous NK cells, even when supported by IL-2 injections, showed no efficacy in patients with lymphoma or breast cancer receiving autologous NK-enriched cell preparations after stem cell transplant [12].
Pre-requisites for NK-cells to recognize malignant cells, become activated and execute the lethal hit As recently as a decade ago NK cells were considered immune cells with ill defined characteristics. However, the last few years have brought a flood of new research results with respect to recognition of target cells, NK receptors and their ligands (Figure 1 and Table 1) and their possible role in cancer surveillance [reviewed in 13/15]. NK cells will not be activated as long as the target cell expresses
Correspondence to: Hans-G Klingemann, MD, PhD, Tufts-New England Medical Center, 750 Washington Street, Boston, MA 02111, USA. – 2005 ISCT
DOI: 10.1080/14653240510018000
Natural killer cell-based immunotherapeutic strategies
CD94 Activating C-type lectin receptors (NKG2)
Inhibitory
C B A Activating
Killer cell immunoglobulin-like receptors (KIR)
Inhibitory
Natural cytotoxicity receptors (NCR) : p30 p44 p46
Figure 1. NK cells express three types of receptors. NKG2 conjugates to three different CD94 ligands.
‘self ’ MHC. Consequently an altered molecular structure of MHC, as can be in the case of malignant or virally infected cells, will activate the NK-cell killing machinery. Expression of self-MHC will activate inhibitory receptors on NK cells that belong to the family of killer cell immunoglobulin-like receptors (KIR) [16,17]. An increasing number of activating receptors is also being discovered on NK cells that, upon binding to ligands on tumor cells, will release perforin and granzyme from NK cells [18,19]. The killing of target cells is therefore the result of a balance of activating and inhibitory receptors. It is still unresolved whether inhibitory KIR signals recognizing class I MHC can override any effect of the naturally occurring activating receptors or receptors of the NKG2D family. Table 1. Some of the inhibitory and activating NK cells receptors and their ligands (listing is not necessarily complete)
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The advancing knowledge about the NK receptors has led to the concept of mismatching for KIR and their ligands (MHC) between recipients of allogeneic stem cell transplant and their respective donors, particularly if an additional anti-tumor effect is desired [20,21]. Such an anti-tumor effect of NK cells seems to be noticeable only if the allogeneic T lymphocytes have been silenced or depleted, either by ex vivo manipulation or, more commonly, in vivo depletion (such as ATG or Campath Ab), as T cells seem to overpower any detectable anti-tumor effect by allogeneic NK cells. It also appears from these published studies that myeloid malignancies are better targets for KIR mismatched NK cells than lymphoid malignancies. Initial results suggest that the NK cells are not fully activated after binding to the lymphoid blast [22,23]. As autologous NK cells will be inhibited by self-MHC Ag, most investigators believe that allogeneic NK cells, either enriched or expanded and activated with cytokines, will represent a better cell population for in vivo therapy than autologous NK cells. However, there is the potential risk of inducing GvHD in the recipient unless T cells in such a product are reliably removed beforehand. The treatment with NK cell infusions assumes that the infused cells will home to the tumor, as has been shown in murine studies with clonal NK cells [24]. The extravasation of NK cells and passage through the interstitial space to the tumor may rely on the production of metalloproteinases that can degrade the matrix surrounding tumor tissue [25,26].
Ex vivo separation and expansion of NK cells Receptor
Ligand
Activating NK cell receptors 2B4 CD48 NKp44 Influenza/unknown NKp30 NKp46 Influenza/unknown CD16 IgG NKG2D MICA, MICB NKp80 ? DNAM CD112/CD155 Inhibitory NK cell receptors ILT2 MHC-A, B, G KIR3DL2 MHC-A KIR3DL1 MHC-B KIR2DL4 MHC-A, B, G KIR2DL1,2,3 MHC-C CD94 MHC-C
The objective of ex vivo selection of NK cells is to remove potentially unwanted cells (i.e. T cells) but also to be able to expand and activate the NK cells in a defined culture environment free of potentially inhibitory cytokines or competitor cells. To achieve selection of NK cells, different methods can be applied, yielding different purities of the cell product. In order to use the cell product for infusion into the patient, good manufacturing practice (GMP) conditions have to be followed that restrict the choices to enrich NK cells from blood. Only the magnetic bead selection process by Miltenyi (CliniMACS, Auburn, CA, USA) currently has a drug master file with the FDA and can be used for IND-sponsored clinical trials. Cells are labeled with a MAb that is coupled to magnetic microbeads and removed when they pass through a magnet. Depending on the purity of NK cells that one
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wants to achieve, the process can involve one or two steps. The decision about purity also depends on whether autologous or allogeneic cells are desired. If autologous NK cells are infused, any residual contamination with CD3 cells may be acceptable. However, in the allogeneic setting, T cells need to be removed below the threshold number known to induce transfusion-induced GvHD (3 / 104/kg body weight). When CD3 cells are removed by immunomagnetic selection, the flow through consists of NK cells (about 30%), with the remainder being B lymphocytes and monocytes [27]. The CD3 cell contamination is generally B/1%, and technically such a product is not expected to cause GvHD in an allogeneic recipient. To enrich further for CD56 NK cells after CD3 depletion, the flow through is positively selected using an anti-CD56 Ab coupled to immunomagnetic microbeads using again the CliniMACS system. These enriched cells can be infused without IL-2 activation, after overnight culture in high dose IL-2 or after being expanded in IL-2 for 2 /3 weeks. The purity of NK cells after this additional positive selection step is excellent, with more than 99% of cells expressing CD56 [27]. Our group has separated CD56 cells from mononuclear cells in a single-step procedure [28]. These cells, containing about 70% CD56 CD3 cells, could be expanded in a 2-week culture under GMP conditions with X-Vivo 10 serum-free medium, supplemented with 5% human serum and recombinant IL-2. Media change and IL-2 replacement occurred every 3 /4 days. Of note is that there was a lag period of about 1 week before proliferation and expansion of NK cells was seen. Similar observations have been reported by other groups, and even the presence of a feeder layer does not seem to shorten or alter this lag time [29 /31]. There is substantial donor-to-donor variability regarding the yield of expanded NK cells after the 2week culture. As NK T lymphocytes (CD56 CD3 ) were not removed for this protocol, they also showed substantial proliferation in the 2-week period. NK cells are major effector cells in Ab-dependent cellmediated cytotoxicity (ADCC), which occurs when the Fc portion of an Ab binds to the Fc receptor on NK cells (CD16). Although monocytes also express a Fc receptor, it has lower affinity for the Fc portion of the Ab. Clinical trials in patients with follicular lymphoma receiving MAb therapy with rituximab have suggested a lower relapse rate and improved survival if patients’ cells express the high-
affinity Fc receptor F158 compared with those who express the low affinity receptor V158, in which one amino acid has been exchanged [32,33]. Although the relationship between high-affinity receptor and lower relapse rate could not be confirmed for other B-cell malignancies such as CLL [34], initial trials combining rituximab with IL-2 to activate and expand the pool of NK cells available for ADCC are ongoing [35]. If these observations of enhanced efficacy in the presence of ‘more’ effector cells are confirmed, then consideration could be given to provide ex vivo-expanded NK cells to the patient.
The case for allogeneic NK cell therapy Recent data from the stem cell transplant group in Perugia, Italy, have generated some enthusiasm with regard to the potential contribution of allogeneic NK cells to mediate GvL effects [20,21]. These results suggested that recipient /donor mismatching for KIR receptors and their ligands (i.e. HLA-B and HLA-C) conveys a disease-free survival advantage and lower relapse rate in myeloid leukemias. This effect occurred in the absence of donor T lymphocytes, which had been removed prior to transplant. Although not all follow-up studies have been able to confirm these initial observations [36,37], several studies are now underway to better understand the GvL contribution of ‘KIR ligand mismatching’ in T-cell depleted stem cell grafts. Data from murine models also suggested a benefit of allogeneic over autologous NK cell administration [38]. In addition, inhibitory receptor blockade in murine transplants enhanced anti-leukemia activity of NK cells [39]. We have observed that myeloma cells separated from BM of affected patients are not lysed by autologous NK cells but only by allogeneic (Klingemann, unpublished).
Immunomodulatory effects of NK cells Studies of haploidentical stem cell transplants in murine models have suggested that NK cells can facilitate engraftment and down-modulate GvHD [21]. It appears that the GvHD effect is accomplished by NK cells recognizing host DC that are suspect candidates for inducing a donor-derived allo-immune response leading to clinical symptoms of GvHD [40,41]. T-cell depletion of the BM generally enhances the risk of graft rejection both in matched and, even more so, mismatched and unrelated donor transplants. The murine
Natural killer cell-based immunotherapeutic strategies
experiments have shown that in T-cell depleted grafts deliberate mismatching for KIR may promote engraftment [41]. Both observations in the mouse need to be confirmed in humans.
Clinical trials with NK cells Initial studies in autologous stem cell transplant recipients infused cytotoxic cells that had been harvested by leukapheresis following 28 and 42 days of IL-2 therapy [12]. The harvested cells were then incubated with IL-2 and reinfused into the patient the next day. The most common infusion-related events were fevers and mild to moderate chills. Although the infusion product and the cells obtained from patients after cell infusion showed significant cytotoxicity against Raji targets, no anti-tumor effect in these advanced patients (diagnoses of breast cancer, lymphoma and CML) was observed. The same group has started to explore allogeneic NK cell infusions. Studies in cancer patients have demonstrated that allogeneic NK cell infusions up to a dose of 2 /107/kg are well tolerated [42]. Recipients were given CY (750 mg/m2) and i.v. steroids (1g/m2) a couple of days before the infusion of allogeneic NK cells to prevent their immediate rejection by donor T cells. Even for the highest dose in this phase I trial, a single 5-h 15-L donor leukapheresis was sufficient to obtain enough NK-cells. The ‘NK-cell product’ was prepared by immunomagnetic Ab depletion of CD3 lymphocytes using the CliniMACS system. Although the remaining cell fraction also contained B cells and monocytes, this one-step process yielded an enriched NK cell fraction (c. 40%). Before infusion into the patients, the cells were activated overnight with IL-2 (1000 U/mL) and the patients also received 14 days of daily s.c. IL-2 injections. A total of 17 allogeneic NK cell infusions was given as part of a dose-escalation phase I trial. Except for the occasional constitutional symptom, no infusion-related toxicities were observed. Importantly there was no transfusion-induced GvHD and no marrow suppression occurred. One of the key questions with respect to allogeneic cells is how long these infused cells persist and/or possibly could expand in the recipient. In the Minnesota study, the CY/steroid regimen was not able to suppress a recipient MLR against the donor cells. Consequently, molecular studies for donor HLA-A1 showed that 0.1% of donor cells were detectable in the recipient at 4 h and up to day 5 after infusion, but that they were no longer detectable by day 7,
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with no evidence of in vivo expansion. Conversely, in the group of patients that received the higher dose of priming with CY (60 mg/m2) plus fludarabine (25 mg/m2 /5 days), donor cells could be detected beyond 28 days. It appears that such a preparative regime facilitates the expansion of donor NK cells, at least in some patients. The preliminary results from Minnesota, presented at this year’s ASH meeting, show a benefit with respect to disease reduction in some AML patients [43]. Our group has experience with infusing a NK cell line, which consists of ‘pure’ IL-2-activated NK cells [44]. Preclinical studies in immunocompromised mice had confirmed some anti-tumor effect in melanoma and leukemia [45,46]. Activity was also shown for ex vivo purging of leukemia, lymphoma and CML [47,48]. The NK-92 line has been administered to more than 20 patients with advanced cancer [49,50]. These phase I safety date suggested that NK-92 cell infusions were well tolerated and, in some cases, an anti-tumor effect was observed. The advantage of an allogeneic cell line is that the cells can be grown continuously under GMP conditions [51]. NK-92 do not express KIR, but have a spectrum of activating receptors [52]. Uherek et al. [53] engineered NK-92 cells to express a single-chain Fv recognizing Her-2/neu and showed that previously resistant Her-2-positive target cells were killed by the modified NK-92 cells. The data presented by R. Handgretinger et al. (personal communication) at a recent workshop in Italy on haploidentical stem cell transplantation again underscores the potential importance of allogeneic NK cells. The group depleted the haplo-mismatched stem cell graft of CD3 cells and enriched it for NK cells by positive selection using the Miltenyi device. Patients received a median of 10.3 /107/kg CD56 cells (range 0.84 /28.8). Not only had the patients less GvHD than would be expected with this degree of mismatching, but also the outcome so far suggests that these patients have a lower relapse rate than historic controls. The Stanford group has developed a protocol to expand cytokine-activated killer (CIK) cells that represent cytotoxic NK T cells that are dual positive for CD3 and CD56 and are able to recognize target cells in an MHC non-restricted fashion [54,55]. CIK cells are generated in a 2 /3 week culture with anti-CD3 Ab, IFN-g and IL-2. Results from phase I trial in patients with Hodgkin’s disease suggested that the cell product was tolerated well [56].
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Prospects One of the research objectives around NK cells is how one can manipulate them to the extent that they can overcome any de-activating effects from the tumor and also recognize tumor targets more predictably (Table 2). Similar to target-specific T cells that have been shown to be effective in preventing and treating viral infections, we need to be able to engineer autologous NK cells such that they are tumor specific and are not slowed down by KIR activation through self-MHC. Studies with the NK-92 cells line have shown that this can be accomplished by genetically engineering cells that express a single chain Fv receptor [53]. In addition to Her2/neu, tumor Ag-specific single-chain variants of NK-92 have been generated and shown to kill target cells that were initially resistant to lysis by non-modified NK-92 [57; W. Wels, personal communication]. It appears that this manipulation can override any inhibitory receptor effects. Unfortunately at this point this approach requires that the NK cells are transfected with a genetic construct. As physical gene transfection methods (electroporation, gene gun, lipofection) are hampered by a low transfection efficacy of generally B/10% of cells, viral approaches are being pursued. The highest transfection rate can be achieved with a retroviral construct consisting of the gene for the tumor-specific Fv fragment that is engineered to the CD8 hinge region and the zeta chain of the T-cell receptor that transmits the signal into the NK cells where it is picked up by ZAP 70 (Figure 2). The use of autologous cells in this setting has the obvious advantage that they would not be rejected and no prior T-lymphocyte depletion would be required. In vitro studies have suggested that blockage of inhibitory KIR can further enhance NK-directed killing [39]. One could also consider devising ways of stimulating the natural activating receptors (p30, p44, p46) on NK cells. Of Table 2. Strategies to improve cellular therapy with NK cells Use of KIR epitope mismatched allogeneic NK cells either alone or as part of a stem cell transplant Blocking KIR /MHC class I Ag interaction Induction of activating receptors (cytokines?) Devising mechanisms to overcome resistance mechanisms developed by tumor cells Infusion of autologous NK cells genetically engineered to recognize specific tumor receptors Co-stimulation of NK cells through DC activation
Malignant cell Perforin/granzyme
scFv CD8 hinge region
NK cell
ζ-chain ZAP 70
Figure 2. Schematic diagram of tumor-specific NK cells. The cells are transfected with a genetic construct that codes for the sequence of single-chain (sc) Fv recognizing a known tumor Ag. The construct also consists of a CD3 hinge region and the signal transducing zchain of the CD3 receptor that activates ZAP 70, which ultimately releases the NK molecules perforin and granzyme [53].
note is that p44 is only expressed on NK cells that have been exposed to IL-2. Similarly, modulation of activating ligands for NK cells on malignant cells may improve the efficacy of autologous NK cells. One could also consider ways to interfere with the ability of some tumors to inactivate perforin and granzyme. Even if we succeed in optimizing NK-cell based therapy, it should not be expected that such a treatment by itself will make a significant impact in advanced cancer. The best target for NK cell therapy is minimal disease, circumstances that are present after stem cell transplant or chemotherapy. Further, as the immune system after cord blood transplant lacks a sufficient graft versus tumor effect than one could easily imagine a place for NK cell infusions in these recipients as well. Cellular therapy with NK cells, expanded, activated and manipulated, could therefore add a comprehensive treatment approach for malignancies.
Acknowledgements I would like to thank Dr Jeffrey Miller for critically reviewing the manuscript.
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37 Giebel S, Locatelli F, Lamparelli T et al . Survival advantage with KIR ligand incompatibility in hematopoietic stem cell transplantation from unrelated donors. Blood 2003;102:814 /9. 38 Zeis M, Uharek L, Glass B et al . Allogeneic MHC mismatched activated natural killer cells administered after bone marrow transplantation provide a strong graft-versus-leukemia effect. Br J Haematol 1997;96:757 /61. 39 Koh CY, Blazar BR, George T et al . Augmentation of antitumor effects by NK cell inhibitory receptor blockade in vitro and in vivo . Blood 2001;97:3132 /7. 40 Shlomchik WD, Couzens MS, Tang CB et al . Prevention of graft-versus-host disease by inactivation of host antigen presenting cells. Science 1999;285:412 /5. 41 Asai O, Longo D, Tian Z-G et al . Suppression of graft-versushost disease and amplification of graft-versus-tumor effects by activated natural killer cells after allogeneic bone marrow transplantation. J Clin Invest 1998;9:1835 /42. 42 Miller JS, Soignier Y, McNearney S et al . Adoptive immunotherapy of cancer using haploidentical natural killer (NK) cell infusions to overcome inhibitory receptor recognition of class I molecules. Blood 2003;102:125a. 43 Miller JS, Soignier Y, Panoskaltsis-Mortari A et al . Successful remission of poor prognosis AML after adoptive transfer and in vivo expansion of human haploidentical NK cells. Blood 2004. 44 Gong J, Maki G, Klingemann H-G. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 1994;8:652 /8. 45 Tam YK, Miyagawa B, Ho VC, Klingemann H-G. Immunotherapy of malignant melanoma in a SCID mouse model using the highly cytotoxic natural killer cell line NK-92. J Hematother 1999;8:281 /90. 46 Yan Y, Steinherz P, Klingemann H-G et al . Antileukemia activity of a natural killer cell line against human leukemia. Clin Cancer Res 1998;4:2859 /68. 47 Klingemann H-G, Wong E, Maki G. A cytotoxic NK-cell line (NK-92) for ex vivo purging of leukemia from blood. Biol Blood Marrow Transplant 1996;2:68 /75.
48 Maki G, Tam Y, Berkahn L, Klingemann H-G. Ex-vivo purging with NK-92 cells prior to autografting for chronic myelogenous leukemia. Bone Marrow Transplant 2003;31:1119 /25. 49 Arai S, Kindy K, Swearingen M et al . Phase I study of adoptive immunotherapy using the cytotoxic natural killer (NK) cell line, NK-92, for treatment of advanced renal cell carcinoma and malignant melanoma. Blood 2003;102:693a. 50 Tonn T, Becker S, Esser R et al . Cellular immunotherapy of malignancies using the clonal natural killer cell line NK-92. J Hematoth Stem Cell Res 2001;10:525 /44. 51 Tam Y, Martinson JA, Doligosa K, Klingemann H-G. Ex vivo expansion of the highly cytotoxic human NK-92 cell line under cGMP conditions for clinical adoptive cellular immunotherapy. Cytotherapy 2003;5:259 /72. 52 Maki G, Klingemann H-G, Martinson JA, Tam YK. Factors regulating the cytotoxic activity of the human natural killer cell line, NK-92. J Hematoth Stem Cell Res 2001;10:369 /83. 53 Uherek C, Tonn T, Becker S et al . Retargeting of NK-cell cytolytic activity to ErbB2 expressing cancer cells results in efficient and selective tumor cell destruction. Blood 2002;100: 1265 /73. 54 Schmidt-Wolf I, Lefterova P, Johnston V et al . Propagation of large numbers of T-cells with natural killer cell markers. Br J Haematol 1994;87:453 /8. 55 Veneris M, Baker J, Ediner M, Negrin RS. Studies of ex vivo activated and expanded CD8 NK T cells in humans and mice. J Clin Immunol 2002;22:131 /6. 56 Leemhuis T, Wells S, Horn P et al . Autologous cytokine induced killer cells for the treatment of relapsed Hodgkin’s disease and non-Hodgkin’s lymphoma. Blood 2000;96:839a. 57 Koehne G, Guo HF, Trivedi D et al . Redirecting NK-cell cytolytic activity to solid tumors using chimeric scFv receptor gene-modified adoptive immunotherapy. Proc Am Soc Clin Oncol 2003;22:175a.
Natural killer cell-based immunotherapeutic strategies H-G Klingemann Division of Hematology/Oncology, TUFTS /New England Medical Center, Boston, Massachusetts, USA
Not until recently have Natural killer (NK) cells stepped out of the shadow of T-cells to be considered for cellular therapy of malignant diseases. This evolution has been facilitated by the discovery of specific receptors on NK cells that interact with HLA molecules on target cells but also the discovery of specific activating receptors. Since NK cells represent only about 10% of the lymphocyte population in blood,
separation and enrichment are important steps if NK cells are to be used clinically. This is of particular consideration in the setting of allogeneic NK cell infusion where contaminating T-cells could potentially induce graft versus host disease. This review will describe the requirements for NK cells to recognize target cells, their ex vivo expansion and potential therapeutic applications.
Natural killer cells comprise a CD56 CD3 subpopulation of lymphocytes that is involved in responding to certain viruses, parasites, microbial pathogens and transformed cells by exhibiting cytotoxic functions and secreting a battery of cytokines [1 /5]. The history of using killer cells in an attempt to control cancer growth goes back to the mid-1980s, when the group of Rosenberg et al. [6] treated patients with advanced metastatic renal cell cancer and melanoma with mononuclear cells that were activated ex vivo with high doses of IL-2. The approach involved harvesting cells from cancer patients by leukapheresis and activation and expansion of these cells by ex vivo coculture with high doses of IL-2. The expanded cells were termed Lymphokine-activated killer (LAK) cells and were then re-infused into the patient supported by IL-2 administration aimed at maintaining the activity of the infused cells in vivo . Although this approach produced nearly 15 /20% partial and complete responses in initial trials, subsequent studies showed that a similar anti-tumor effect could be achieved with high dose IL-2 alone [7]. LAK cells generated in culture predominantly contain polyclonal T lymphocytes that proliferate with the high doses of IL-2 in the culture, and these T cells outgrow any NK cells with respect to numbers. With hindsight, we can now be critical of these studies but they still stand today as pioneering work that accelerated the field of cellular immunotherapy. Since
T-cell recognition of tumor cells depends on an intact MHC molecule, these polyclonal T cells cannot, in a predictable manner, interact with the malignant target. Expression of intact MHC molecules is variable, depending on the type of cancer [8,9]. It is also known that cytotoxic cells in patients with cancer can be functionally defective. For example the trans-membrane signaling receptor component CD3z of cytotoxic cells can be down-regulated [10]. Another example comprises patients with advanced CML in whom the NK cell function is down-regulated with advancing disease [11]. Taken together, these observations may explain why infusion of autologous NK cells, even when supported by IL-2 injections, showed no efficacy in patients with lymphoma or breast cancer receiving autologous NK-enriched cell preparations after stem cell transplant [12].
Pre-requisites for NK-cells to recognize malignant cells, become activated and execute the lethal hit As recently as a decade ago NK cells were considered immune cells with ill defined characteristics. However, the last few years have brought a flood of new research results with respect to recognition of target cells, NK receptors and their ligands (Figure 1 and Table 1) and their possible role in cancer surveillance [reviewed in 13/15]. NK cells will not be activated as long as the target cell expresses
Correspondence to: Hans-G Klingemann, MD, PhD, Tufts-New England Medical Center, 750 Washington Street, Boston, MA 02111, USA. – 2005 ISCT
DOI: 10.1080/14653240510018000
Natural killer cell-based immunotherapeutic strategies
CD94 Activating C-type lectin receptors (NKG2)
Inhibitory
C B A Activating
Killer cell immunoglobulin-like receptors (KIR)
Inhibitory
Natural cytotoxicity receptors (NCR) : p30 p44 p46
Figure 1. NK cells express three types of receptors. NKG2 conjugates to three different CD94 ligands.
‘self ’ MHC. Consequently an altered molecular structure of MHC, as can be in the case of malignant or virally infected cells, will activate the NK-cell killing machinery. Expression of self-MHC will activate inhibitory receptors on NK cells that belong to the family of killer cell immunoglobulin-like receptors (KIR) [16,17]. An increasing number of activating receptors is also being discovered on NK cells that, upon binding to ligands on tumor cells, will release perforin and granzyme from NK cells [18,19]. The killing of target cells is therefore the result of a balance of activating and inhibitory receptors. It is still unresolved whether inhibitory KIR signals recognizing class I MHC can override any effect of the naturally occurring activating receptors or receptors of the NKG2D family. Table 1. Some of the inhibitory and activating NK cells receptors and their ligands (listing is not necessarily complete)
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The advancing knowledge about the NK receptors has led to the concept of mismatching for KIR and their ligands (MHC) between recipients of allogeneic stem cell transplant and their respective donors, particularly if an additional anti-tumor effect is desired [20,21]. Such an anti-tumor effect of NK cells seems to be noticeable only if the allogeneic T lymphocytes have been silenced or depleted, either by ex vivo manipulation or, more commonly, in vivo depletion (such as ATG or Campath Ab), as T cells seem to overpower any detectable anti-tumor effect by allogeneic NK cells. It also appears from these published studies that myeloid malignancies are better targets for KIR mismatched NK cells than lymphoid malignancies. Initial results suggest that the NK cells are not fully activated after binding to the lymphoid blast [22,23]. As autologous NK cells will be inhibited by self-MHC Ag, most investigators believe that allogeneic NK cells, either enriched or expanded and activated with cytokines, will represent a better cell population for in vivo therapy than autologous NK cells. However, there is the potential risk of inducing GvHD in the recipient unless T cells in such a product are reliably removed beforehand. The treatment with NK cell infusions assumes that the infused cells will home to the tumor, as has been shown in murine studies with clonal NK cells [24]. The extravasation of NK cells and passage through the interstitial space to the tumor may rely on the production of metalloproteinases that can degrade the matrix surrounding tumor tissue [25,26].
Ex vivo separation and expansion of NK cells Receptor
Ligand
Activating NK cell receptors 2B4 CD48 NKp44 Influenza/unknown NKp30 NKp46 Influenza/unknown CD16 IgG NKG2D MICA, MICB NKp80 ? DNAM CD112/CD155 Inhibitory NK cell receptors ILT2 MHC-A, B, G KIR3DL2 MHC-A KIR3DL1 MHC-B KIR2DL4 MHC-A, B, G KIR2DL1,2,3 MHC-C CD94 MHC-C
The objective of ex vivo selection of NK cells is to remove potentially unwanted cells (i.e. T cells) but also to be able to expand and activate the NK cells in a defined culture environment free of potentially inhibitory cytokines or competitor cells. To achieve selection of NK cells, different methods can be applied, yielding different purities of the cell product. In order to use the cell product for infusion into the patient, good manufacturing practice (GMP) conditions have to be followed that restrict the choices to enrich NK cells from blood. Only the magnetic bead selection process by Miltenyi (CliniMACS, Auburn, CA, USA) currently has a drug master file with the FDA and can be used for IND-sponsored clinical trials. Cells are labeled with a MAb that is coupled to magnetic microbeads and removed when they pass through a magnet. Depending on the purity of NK cells that one
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wants to achieve, the process can involve one or two steps. The decision about purity also depends on whether autologous or allogeneic cells are desired. If autologous NK cells are infused, any residual contamination with CD3 cells may be acceptable. However, in the allogeneic setting, T cells need to be removed below the threshold number known to induce transfusion-induced GvHD (3 / 104/kg body weight). When CD3 cells are removed by immunomagnetic selection, the flow through consists of NK cells (about 30%), with the remainder being B lymphocytes and monocytes [27]. The CD3 cell contamination is generally B/1%, and technically such a product is not expected to cause GvHD in an allogeneic recipient. To enrich further for CD56 NK cells after CD3 depletion, the flow through is positively selected using an anti-CD56 Ab coupled to immunomagnetic microbeads using again the CliniMACS system. These enriched cells can be infused without IL-2 activation, after overnight culture in high dose IL-2 or after being expanded in IL-2 for 2 /3 weeks. The purity of NK cells after this additional positive selection step is excellent, with more than 99% of cells expressing CD56 [27]. Our group has separated CD56 cells from mononuclear cells in a single-step procedure [28]. These cells, containing about 70% CD56 CD3 cells, could be expanded in a 2-week culture under GMP conditions with X-Vivo 10 serum-free medium, supplemented with 5% human serum and recombinant IL-2. Media change and IL-2 replacement occurred every 3 /4 days. Of note is that there was a lag period of about 1 week before proliferation and expansion of NK cells was seen. Similar observations have been reported by other groups, and even the presence of a feeder layer does not seem to shorten or alter this lag time [29 /31]. There is substantial donor-to-donor variability regarding the yield of expanded NK cells after the 2week culture. As NK T lymphocytes (CD56 CD3 ) were not removed for this protocol, they also showed substantial proliferation in the 2-week period. NK cells are major effector cells in Ab-dependent cellmediated cytotoxicity (ADCC), which occurs when the Fc portion of an Ab binds to the Fc receptor on NK cells (CD16). Although monocytes also express a Fc receptor, it has lower affinity for the Fc portion of the Ab. Clinical trials in patients with follicular lymphoma receiving MAb therapy with rituximab have suggested a lower relapse rate and improved survival if patients’ cells express the high-
affinity Fc receptor F158 compared with those who express the low affinity receptor V158, in which one amino acid has been exchanged [32,33]. Although the relationship between high-affinity receptor and lower relapse rate could not be confirmed for other B-cell malignancies such as CLL [34], initial trials combining rituximab with IL-2 to activate and expand the pool of NK cells available for ADCC are ongoing [35]. If these observations of enhanced efficacy in the presence of ‘more’ effector cells are confirmed, then consideration could be given to provide ex vivo-expanded NK cells to the patient.
The case for allogeneic NK cell therapy Recent data from the stem cell transplant group in Perugia, Italy, have generated some enthusiasm with regard to the potential contribution of allogeneic NK cells to mediate GvL effects [20,21]. These results suggested that recipient /donor mismatching for KIR receptors and their ligands (i.e. HLA-B and HLA-C) conveys a disease-free survival advantage and lower relapse rate in myeloid leukemias. This effect occurred in the absence of donor T lymphocytes, which had been removed prior to transplant. Although not all follow-up studies have been able to confirm these initial observations [36,37], several studies are now underway to better understand the GvL contribution of ‘KIR ligand mismatching’ in T-cell depleted stem cell grafts. Data from murine models also suggested a benefit of allogeneic over autologous NK cell administration [38]. In addition, inhibitory receptor blockade in murine transplants enhanced anti-leukemia activity of NK cells [39]. We have observed that myeloma cells separated from BM of affected patients are not lysed by autologous NK cells but only by allogeneic (Klingemann, unpublished).
Immunomodulatory effects of NK cells Studies of haploidentical stem cell transplants in murine models have suggested that NK cells can facilitate engraftment and down-modulate GvHD [21]. It appears that the GvHD effect is accomplished by NK cells recognizing host DC that are suspect candidates for inducing a donor-derived allo-immune response leading to clinical symptoms of GvHD [40,41]. T-cell depletion of the BM generally enhances the risk of graft rejection both in matched and, even more so, mismatched and unrelated donor transplants. The murine
Natural killer cell-based immunotherapeutic strategies
experiments have shown that in T-cell depleted grafts deliberate mismatching for KIR may promote engraftment [41]. Both observations in the mouse need to be confirmed in humans.
Clinical trials with NK cells Initial studies in autologous stem cell transplant recipients infused cytotoxic cells that had been harvested by leukapheresis following 28 and 42 days of IL-2 therapy [12]. The harvested cells were then incubated with IL-2 and reinfused into the patient the next day. The most common infusion-related events were fevers and mild to moderate chills. Although the infusion product and the cells obtained from patients after cell infusion showed significant cytotoxicity against Raji targets, no anti-tumor effect in these advanced patients (diagnoses of breast cancer, lymphoma and CML) was observed. The same group has started to explore allogeneic NK cell infusions. Studies in cancer patients have demonstrated that allogeneic NK cell infusions up to a dose of 2 /107/kg are well tolerated [42]. Recipients were given CY (750 mg/m2) and i.v. steroids (1g/m2) a couple of days before the infusion of allogeneic NK cells to prevent their immediate rejection by donor T cells. Even for the highest dose in this phase I trial, a single 5-h 15-L donor leukapheresis was sufficient to obtain enough NK-cells. The ‘NK-cell product’ was prepared by immunomagnetic Ab depletion of CD3 lymphocytes using the CliniMACS system. Although the remaining cell fraction also contained B cells and monocytes, this one-step process yielded an enriched NK cell fraction (c. 40%). Before infusion into the patients, the cells were activated overnight with IL-2 (1000 U/mL) and the patients also received 14 days of daily s.c. IL-2 injections. A total of 17 allogeneic NK cell infusions was given as part of a dose-escalation phase I trial. Except for the occasional constitutional symptom, no infusion-related toxicities were observed. Importantly there was no transfusion-induced GvHD and no marrow suppression occurred. One of the key questions with respect to allogeneic cells is how long these infused cells persist and/or possibly could expand in the recipient. In the Minnesota study, the CY/steroid regimen was not able to suppress a recipient MLR against the donor cells. Consequently, molecular studies for donor HLA-A1 showed that 0.1% of donor cells were detectable in the recipient at 4 h and up to day 5 after infusion, but that they were no longer detectable by day 7,
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with no evidence of in vivo expansion. Conversely, in the group of patients that received the higher dose of priming with CY (60 mg/m2) plus fludarabine (25 mg/m2 /5 days), donor cells could be detected beyond 28 days. It appears that such a preparative regime facilitates the expansion of donor NK cells, at least in some patients. The preliminary results from Minnesota, presented at this year’s ASH meeting, show a benefit with respect to disease reduction in some AML patients [43]. Our group has experience with infusing a NK cell line, which consists of ‘pure’ IL-2-activated NK cells [44]. Preclinical studies in immunocompromised mice had confirmed some anti-tumor effect in melanoma and leukemia [45,46]. Activity was also shown for ex vivo purging of leukemia, lymphoma and CML [47,48]. The NK-92 line has been administered to more than 20 patients with advanced cancer [49,50]. These phase I safety date suggested that NK-92 cell infusions were well tolerated and, in some cases, an anti-tumor effect was observed. The advantage of an allogeneic cell line is that the cells can be grown continuously under GMP conditions [51]. NK-92 do not express KIR, but have a spectrum of activating receptors [52]. Uherek et al. [53] engineered NK-92 cells to express a single-chain Fv recognizing Her-2/neu and showed that previously resistant Her-2-positive target cells were killed by the modified NK-92 cells. The data presented by R. Handgretinger et al. (personal communication) at a recent workshop in Italy on haploidentical stem cell transplantation again underscores the potential importance of allogeneic NK cells. The group depleted the haplo-mismatched stem cell graft of CD3 cells and enriched it for NK cells by positive selection using the Miltenyi device. Patients received a median of 10.3 /107/kg CD56 cells (range 0.84 /28.8). Not only had the patients less GvHD than would be expected with this degree of mismatching, but also the outcome so far suggests that these patients have a lower relapse rate than historic controls. The Stanford group has developed a protocol to expand cytokine-activated killer (CIK) cells that represent cytotoxic NK T cells that are dual positive for CD3 and CD56 and are able to recognize target cells in an MHC non-restricted fashion [54,55]. CIK cells are generated in a 2 /3 week culture with anti-CD3 Ab, IFN-g and IL-2. Results from phase I trial in patients with Hodgkin’s disease suggested that the cell product was tolerated well [56].
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Prospects One of the research objectives around NK cells is how one can manipulate them to the extent that they can overcome any de-activating effects from the tumor and also recognize tumor targets more predictably (Table 2). Similar to target-specific T cells that have been shown to be effective in preventing and treating viral infections, we need to be able to engineer autologous NK cells such that they are tumor specific and are not slowed down by KIR activation through self-MHC. Studies with the NK-92 cells line have shown that this can be accomplished by genetically engineering cells that express a single chain Fv receptor [53]. In addition to Her2/neu, tumor Ag-specific single-chain variants of NK-92 have been generated and shown to kill target cells that were initially resistant to lysis by non-modified NK-92 [57; W. Wels, personal communication]. It appears that this manipulation can override any inhibitory receptor effects. Unfortunately at this point this approach requires that the NK cells are transfected with a genetic construct. As physical gene transfection methods (electroporation, gene gun, lipofection) are hampered by a low transfection efficacy of generally B/10% of cells, viral approaches are being pursued. The highest transfection rate can be achieved with a retroviral construct consisting of the gene for the tumor-specific Fv fragment that is engineered to the CD8 hinge region and the zeta chain of the T-cell receptor that transmits the signal into the NK cells where it is picked up by ZAP 70 (Figure 2). The use of autologous cells in this setting has the obvious advantage that they would not be rejected and no prior T-lymphocyte depletion would be required. In vitro studies have suggested that blockage of inhibitory KIR can further enhance NK-directed killing [39]. One could also consider devising ways of stimulating the natural activating receptors (p30, p44, p46) on NK cells. Of Table 2. Strategies to improve cellular therapy with NK cells Use of KIR epitope mismatched allogeneic NK cells either alone or as part of a stem cell transplant Blocking KIR /MHC class I Ag interaction Induction of activating receptors (cytokines?) Devising mechanisms to overcome resistance mechanisms developed by tumor cells Infusion of autologous NK cells genetically engineered to recognize specific tumor receptors Co-stimulation of NK cells through DC activation
Malignant cell Perforin/granzyme
scFv CD8 hinge region
NK cell
ζ-chain ZAP 70
Figure 2. Schematic diagram of tumor-specific NK cells. The cells are transfected with a genetic construct that codes for the sequence of single-chain (sc) Fv recognizing a known tumor Ag. The construct also consists of a CD3 hinge region and the signal transducing zchain of the CD3 receptor that activates ZAP 70, which ultimately releases the NK molecules perforin and granzyme [53].
note is that p44 is only expressed on NK cells that have been exposed to IL-2. Similarly, modulation of activating ligands for NK cells on malignant cells may improve the efficacy of autologous NK cells. One could also consider ways to interfere with the ability of some tumors to inactivate perforin and granzyme. Even if we succeed in optimizing NK-cell based therapy, it should not be expected that such a treatment by itself will make a significant impact in advanced cancer. The best target for NK cell therapy is minimal disease, circumstances that are present after stem cell transplant or chemotherapy. Further, as the immune system after cord blood transplant lacks a sufficient graft versus tumor effect than one could easily imagine a place for NK cell infusions in these recipients as well. Cellular therapy with NK cells, expanded, activated and manipulated, could therefore add a comprehensive treatment approach for malignancies.
Acknowledgements I would like to thank Dr Jeffrey Miller for critically reviewing the manuscript.
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