Exploitation of alloreactive NK cells in adoptive immunotherapy of cancer

Exploitation of alloreactive NK cells in adoptive immunotherapy of cancer Loredana Ruggeri, Antonella Mancusi, Marusca Capanni, Massimo F Martelli and Andrea Velardi NK cells are primed to kill by several activating receptors. Killing of autologous cells is prevented as NK cells co-express inhibitory receptors for self-MHC class I molecules. Human NK cells discriminate between different allelic forms of MHC molecules via killer cell immunoglobulin-like receptors (KIRs), which are clonally distributed, and each cell in the repertoire bears at least one receptor that is specific for self-MHC class I molecules. Consequently, when faced with mismatched allogeneic targets, NK cells in the repertoire will sense the missing expression of self-MHC class I alleles and will mediate alloreactions. Recent studies in murine transplant models and data from mismatched haematopoietic transplant trials demonstrate MHC class I mismatches, which generate an alloreactive NK-cell response in the graft-versus-host direction, eradicate leukaemia, improve engraftment and protect against T-cell-mediated graft-versus-host disease.

transplant because of delays due to the donor search and bone marrow harvesting. Virtually every patient, however, has a family member who is identical for one HLA haplotype and fully mismatched for the other (‘haploidentical’), and who could immediately serve as donor. Successful haploidentical transplantation is achieved when large numbers of haematopoietic stem cells are transplanted in order to overcome rejection [3–7]. These grafts have to be extensively depleted of T cells to prevent GvHD. This review focuses on the role of alloreactive natural killer (NK) cells in adoptive immunotherapy of leukaemia in the context of allogeneic haematopoietic transplantation.

NK cells and ‘missing self’ recognition Addresses Division of Haematology and Clinical Immunology, Department of Clinical and Experimental Medicine, University of Perugia, Policlinico Monteluce, 06122 Perugia, Italy Corresponding author: Velardi, Andrea ([email protected])

Current Opinion in Immunology 2005, 17:211–217 This review comes from a themed issue on Tumour immunology Edited by Rienk Offringa

0952-7915/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2005.01.007

Introduction Allogeneic haematopoietic cell transplantation cures leukaemia through alloreactions mediated by donor T cells in the graft that promote engraftment, eradicate leukaemia and reconstitute immunity [1,2]. Unfortunately, they also mediate graft-versus-host disease (GvHD). Together with the immunosuppression needed to prevent or treat it, GvHD underlies the major reasons for transplant failures: infection and neoplastic relapse. Although the matching of donor and recipient human leukocyte antigens (HLAs) is crucial to minimize the risk of rejection and GvHD, only 60% of patients have matched sibling or unrelated donors [3] and fewer than this make it to www.sciencedirect.com

NK cells are primed to kill by signals delivered through several different activating receptors [8]. The seminal discovery of NK-cell regulation by MHC class I on target cells was made by Klas Ka¨rre, who developed the ‘missing-self’ recognition hypothesis [9–13]. This work subsequently led to the identification of NK receptors for MHC class I molecules. NK killing of autologous cells is prevented as NK cells co-express clonally distributed receptors for self-MHC class I molecules [14,15,16– 18,19]. When faced with mismatched allogeneic targets, NK cells sense the missing expression of self-MHC class I molecules and mediate alloreactions (‘missing self’ recognition). In humans, NK cell alloreactions can occur because some inhibitory receptors, the killer-cell immunoglobulin-like receptors (KIRs), discriminate between groups of HLA class I molecules (Table 1). KIR2DL1 is the receptor for HLA-C group 2 alleles; KIR2DL2/3 receptors are specific for HLA-C group 1 alleles and KIR3DL1 is the receptor for HLA-Bw4 alleles. NK cell alloreactions are generated between individuals who are mismatched for HLA-C allele groups and/or the HLABw4 group (Figure 1). For example, individuals who express group 2 HLA-C alleles and possess NK cells that express the KIR specific for Group 2 HLA-C alleles (KIR2DL1) are alloreactive against cells from individuals who do not express Group 2 HLA-C alleles (who are homozygous for Group 1 HLA-C alleles). Individuals who express Group 1 HLA-C alleles possess NK cells with KIR-specific for Group 1 HLA-C alleles (KIR2DL2 and/ or KIR2DL3) and are alloreactive against cells from individuals who do not express Group 1 HLA-C alleles (who are homozygous for Group 2 HLA-C alleles). Current Opinion in Immunology 2005, 17:211–217

212 Tumour immunology

Table 1 HLA-class I allele specificity of the main inhibitory KIR. KIR KIR2DL1 KIR2DL2/3 KIR3DL1

HLA-class I specificity Group 2 HLA-C alleles expressing Lys80 (such as, HLA-Cw2, -Cw4, -Cw5, -Cw6) Group 1 HLA-C alleles expressing Asn80 (such as HLA-Cw1, -Cw3, -Cw7, -Cw8) HLA-Bw4 alleles (e.g. HLA-B27)

Likewise, HLA-Bw4+ individuals expressing the Bw4specific KIR3DL1 receptor might possess NK cells that are alloreactive against Bw4– cells. The CD94–NKG2A inhibitory receptor complex is expressed primarily in NK cells that do not express an inhibitory KIR for self-HLA class I, so it fills the gaps in the KIR repertoire. Alloreactive NK cells are not found among CD94–NKG2A+ NK cells because HLA-E, the ligand for this receptor, is expressed on cells from all individuals [20].

The role of NK-cell alloreactivity in bone marrow transplantation NK cell alloreactions in haploidentical transplants were first observed in the hybrid resistance transplant model [21,22]. Parental bone marrow grafts are rejected by a subset of host F1 NK cells that is not equipped with the correct inhibitory receptor to recognize donor MHC class I alleles and is, therefore, activated to kill. These experiments illustrate that NK cell alloreactions in the hostversus-graft direction mediate rejection and play a major Figure 1

Cytolytic clones in 100 clones

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role in recognizing allogeneic cells in vivo. Moreover, as the hybrid recipient mouse tolerates skin and organ allografts, the model indicates that NK cell alloreactivity is restricted to lymphohaematopoietic targets. A recent report hypothesized that these in vivo effects of NK cell alloreactivity would hold true in the opposite direction (graft-versus-host, GvH) and could exert a graft-versus-leukaemia (GvL) effect [23,24]. H-2 heterozygous (d/b) F1 mice were used as donors of bone marrow and alloreactive NK cells, and homozygous b/b mice were used as recipients. The donor mouse has two major NK cell populations: one bears a receptor that recognizes, and is blocked by, the host MHC; the other expresses a receptor that does not recognize host MHC, and is activated to kill the recipient targets. Infusion of donor alloreactive NK cells, after mild host immune suppression, ablated the host lymphohaematopoietic cells, thus preventing rejection of the MHCmismatched bone marrow transplant. NK cells appeared to attack only the recipient haematopoietic cells because they did not cause GvHD. Therefore, as already observed in the hybrid mouse transplant model (see above), also in this case alloreactive NK cells are not apparently directed against normal tissues. Normal tissues, unlike lymphohaematopoietic lineage cells, might not express ligands for activating NK receptors. To date, only some ligands for mouse and human activating NK receptors have been identified, and the ligands for a major family of human activating NK receptors (the natural cytotoxicity receptors [NCRs]) are still unknown [12]. Their discovery and analysis of their tissue distribution should provide molecular evidence for the preferential engagement of NK cells by lymphohaematopoietic targets. Another noteworthy feature of alloreactive NK cells in transplantation is their killing of host-type dendritic cells which are known to initiate T-cell mediated GvHD by presenting host alloantigens to donor T cells [25]. Consequently, the infusion of donor versus recipient alloreactive NK cells prevented T-cell mediated GvHD [23] to such an extent that mice that were given alloreactive NK cells as part of the conditioning regimen were able to receive mismatched bone marrow grafts containing up to thirty-times the lethal dose of allogeneic T cells without clinical of histological evidence of GvHD.

Current Opinion in Immunology

Donor NK cell allospecificity for ‘missing self’ on recipient targets. In a large series of haploidentical donor–recipient pairs [19,24], direct functional assessment of the donor NK repertoire through the generation of large numbers of donor NK clones and cytotoxicity assays against recipient target cells showed high-frequency alloreactive NK clones when allogeneic recipient targets did not express the class I group (HLA-C1, or HLA-C2, or HLA-Bw4) present in the donor. No alloreactive NK clones (i.e. <1 in 200 clones in 20/20 donor-recipient pairs tested) were detected when allogeneic targets expressed the class I group(s) present in the donor (‘no missing self’). Current Opinion in Immunology 2005, 17:211–217

In vitro studies of human cells show alloreactive NK cells kill acute myeloid leukaemia (AML) and chronic myeloid leukaemia (CML) cells [26]. In a series of primary lymphohaematopoietic lineage tumour cells, including common phenotype acute lymphoblastic leukaemia (ALL), T-cell ALL, chronic lymphocytic leukaemia, nonHodgkin’s lymphoma and multiple myeloma, the only non-susceptible target was common ALL [26,27]. Resistance was associated with lack of leukocyte www.sciencedirect.com

Alloreactive NK cells in adoptive immunotherapy Ruggeri et al. 213

Donor versus recipient NK-cell alloreactivity in clinical haploidentical transplantation In HLA haplotype-mismatched haematopoietic transplantation with a potential for NK-mediated GvH reactions, engrafted stem cells give rise to an NK cell wave of donor origin that regenerates the same repertoire as the donor, including donor versus recipient alloreactive NK cells [26]. Donor versus recipient NK cell alloreactivity reduced the risk of relapse in 57 AML patients while improving engraftment and protecting against GvHD [23]. An updated analysis of 93 AML patients transplanted from haploidentical donors between 1993 and 2003 confirms that grafts from NK alloreactive donors enhance engraftment (rejection rate 10% in the absence of NK alloreactivity, in comparison to a rejection rate of 2% in its presence), appear to protect from GvHD (9% versus 3%), control leukaemia relapse (Figure 2) and improve disease-free survival (Figure 3; [27]). Survival after transplantation from NK alloreactive donors comFigure 2

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function-associated antigen-1 (LFA-1) expression. Alloreactive NK cells exert significant cytotoxicity against melanoma and renal cell carcinoma cell lines [28]. In a pre-clinical model, transfer of alloreactive NK cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice eradicated transplanted human AML [23].

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This graph shows Kaplan-Meier estimates of the probability of survival of 40 high-risk AML recipients (25 transplanted in remission, 15 in relapse) who received haematopoietic stem cell grafts from haploidentical donors who were able to mount donor-versus-recipient NK alloreactions (Allo NK). Also plotted are the Kaplan-Meier estimates of the probability of survival of 53 high-risk AML recipients (26 transplanted in remission, 27 in relapse) who received haematopoietic stem cell grafts from haploidentical donors who were unable to mount donor-versus-recipient NK alloreactions (Non-allo NK). The probability of survival was 12% for the 53 patients transplanted from non-NK alloreactive donors versus 55% (p<0.005) for the 40 patients transplanted from NK alloreactive donors. Thus, transplantation from an NK alloreactive donor is a strong independent factor predicting survival (transplantation from NK alloreactive versus non-NK alloreactive donor: hazard ratio = 0.44, 95% confidence interval = 0.25–0.77, p = 0.004), even when compared with disease status at transplant (remission versus relapse: hazard ratio = 0.47, 95% confidence interval = 0.28-0.73, p = 0.004).

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pares favourably with survival reported after matched unrelated-donor transplant in adult patients in the same risk category [1].

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In children with acute leukaemia, a recent study confirmed that transplantation with haematopoietic cells from haploidentical donors with potential for NK cell alloreactivity decreases the risk of relapse [29].

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This graph shows Kaplan-Meier estimates of the probability of leukaemia relapse of 40 high-risk AML recipients (25 transplanted in remission, 15 in relapse) who received haematopoietic stem cell grafts from haploidentical donors who were able to mount donor-versus-recipient NK alloreactions (Allo NK). Also plotted are the Kaplan-Meier estimates of the probability of leukaemia relapse of 53 high-risk AML recipients (26 transplanted in remission, 27 in relapse) who received haematopoietic stem cell grafts from haploidentical donors who were unable to mount donor-versus-recipient NK alloreactions (Non-allo NK). The probability of relapse was 68% for the 53 patients transplanted from non-NK alloreactive donors versus 15% (p<0.005) for the 40 patients transplanted from NK alloreactive donors. www.sciencedirect.com

Interestingly, haploidentical and matched (T-celldepleted) transplants from donors expressing a KIR gene for which neither donor nor recipient have an HLA ligand are also associated with lower leukaemia relapse rates [29,30], implying that such donors possess potentially autoreactive KIR-bearing NK cells in an anergic and/or regulated state, and they presumably become activated and exert a GvL effect upon transfer into the recipient. Although self-tolerant anergic NK cells have been described in mice [31–34], their existence in humans and regeneration as effector cells after transplant remain to be demonstrated. No informative results emerged when outcomes of transplants from donors expressing a Current Opinion in Immunology 2005, 17:211–217

214 Tumour immunology

KIR gene for which neither donor nor recipient had an HLA ligand were analyzed in 93 AML and 71 ALL adult haploidentical transplant recipients, and in 39 AML recipients of matched sibling T-cell-depleted transplants (L Ruggeri, A Mancusi et al., unpublished).

Donor versus recipient NK-cell alloreactivity in unrelated donor haematopoietic transplantation As approximately 50% of unrelated donor transplants are performed in the presence of one or more HLA allele mismatches, NK cell alloreactivity might be expected to occur. Results from retrospective studies addressing this issue are now available [35,36,37–41], but several show no advantage of transplantation from donors with the potential to exert NK cell alloreactivity [35,37,41]. Common to these reports is the lack of functional assessment of either donor versus recipient NK cell alloreactivity and heterogeneous transplant protocols which differ from the haploidentical. Patient populations, underlying diseases (including adult common phenotype ALL which is resistant to alloreactive NK killing, see above), conditioning regimens, graft composition and post-transplant immunosuppressive regimens are all different. Unrelated donor transplants generally use unmanipulated bone marrow harvests (or, less frequently, peripheral blood progenitors) which contain approximately four-log more T cells and up to one-log fewer stem cells than the ‘megadose’ of extensively T-cell-depleted stem cells in haploidentical grafts [6,23]. As unrelated donor transplants are not usually T-cell depleted, they depend on post-transplant immune suppression to help prevent and/or control GvHD. The relatively few transplanted stem cells combined with post-transplant immune suppression adversely affect NK cell maturation from their bone marrow precursors. Indeed, under these conditions a recent study demonstrated a very delayed and poor reconstitution of potentially alloreactive KIR-bearing NK cells [42]. Some studies have, however, documented an increased GvL effect in transplants from unrelated donors with the potential to exert NK cell alloreactivity [36,38–40]. Interestingly, one study demonstrated a dramatic survival advantage in patients who received antithymocyte globulins pre-transplant (which provided in vivo T-cell depletion) and a graft containing twoto threefold more nucleated cells than usual in unrelated donor transplants [36]. Prospective studies are needed to determine whether strategies that harness donor versus recipient NK cell alloreactivity (high doses of stem cells, T-cell depletion, no post-transplant immune suppression) in haploidentical transplantation can be implemented to improve outcome in unrelated donor transplants.

Guidelines for NK alloreactive donor selection The search for NK alloreactive donors, which can require extension beyond the immediate family, increases the Current Opinion in Immunology 2005, 17:211–217

chance of finding a ‘perfect mismatch’ from the random 30% to >60% (which is close to the maximum, bearing in mind that the HLA type of about 1/3 of the population makes them resistant to alloreactive NK killing). First, the recipient is HLA typed. Those who express class I alleles belonging to the three class I groups recognized by KIRs (HLA-C group 1, HLA-C group 2 and HLA-Bw4 alleles) will block all NK cells from every donor. Recipients who express one or two of these allele groups might find NK alloreactive donors. Donor HLA typing will identify the family member who does not express the class I group(s) expressed by the patient and has, therefore, the potential for NK alloreactivity. Not all inhibitory KIRs are present in 100% of the population. Although KIR2DL2 and/or KIR2DL3, the receptors for HLA-C group 1, are present in everyone, KIR2DL1, the receptor for HLA-C group 2 is found in 97% of individuals and KIR3DL1, receptor for HLA-Bw4 alleles, is found in 90% [27,43,44]. Donor KIR genotyping, therefore, ensures the donor possesses the relevant NK cells. In a large series of KIR ligand-mismatched donor– recipient pairs, direct functional assessment of the donor alloreactive NK repertoire through the generation of large numbers of donor NK clones and cytotoxicity assays against recipient target cells showed high-frequency alloreactive NK clones in HLA-C group mismatches (Figure 1; [23,27]). In HLA-Bw4 mismatches, even when the KIR3DL1 gene is present, NK repertoire studies showed alloreactive NK cells in 2/3 of individuals [27]. This might be because they occur in highly variable frequencies, or because allelic KIR3DL1 variants might not allow receptor expression at the cell membrane [45]. For HLA-Bw4 mismatches, therefore, direct assessment of the donor NK repertoire is necessary. Finally, in 20 donor–recipient pairs that were not KIR ligandmismatched in the GvH direction, no donor alloreactive NK clones were found (Figure 1).

Conclusions Donor versus recipient NK cell alloreactivity derives from a mismatch between donor NK clones (carrying specific inhibitory receptors for self-MHC class I molecules) and MHC class I ligands on recipient cells [46,47,48]. When faced with mismatched allogeneic targets, these donor NK clones sense the missing expression of self-HLA class I alleles and mediate alloreactions. Transplantation from NK alloreactive haploidentical donors controls AML relapse, and improves engraftment without causing GvHD. The effectiveness of NK-cell alloreactivity, as revealed in the haploidentical transplant setting, has led to the establishment of specific criteria for donor selection which have enhanced survival rates of leukaemia patients. These results will encourage extending the www.sciencedirect.com

Alloreactive NK cells in adoptive immunotherapy Ruggeri et al. 215

use of mismatched transplants to more leukaemia patients without a matched donor. Other developments might include designing protocols that will facilitate exploitation of NK-cell alloreactivity in the setting of unrelated donor transplantation. Perspectives from murine models suggest that alloreactive NK cells might become part of the conditioning regimen, as they may potentiate the GvL effect of the transplant, help engraftment and protect from T-cell mediated GvHD. They might also be infused post-transplant to help prevent or control leukaemia relapse. Studies are currently seeking to apply donor alloreactive NK cells beyond the field of transplantation as adoptive immunotherapy for cancer. One can even envisage ways to overcome autologous NK-cell inhibition by self-HLA molecules to direct autologous NK cells against tumours, thus obviating the need for immune suppression and/or allogeneic transplantation.

Update A recent study of relapsing AML patients has demonstrated that the infusion of NK cells from haploidentical donors is safe. Cells expanded in vivo after lymphopenia was induced by high-dose cyclophosphamide and fludarabine. Interestingly, remissions were observed when potentially alloreactive KIR-ligand mismatched donors were used [49]. Another recent report shows that NK-cell overexpression of activating receptors overcomes the inhibitory signals delivered through receptors for HLA molecules and enhances NK killing of NK-resistant leukaemia targets [50]

Acknowledgements This review is based on studies supported by grants from the Italian Association for Cancer Research, the Italian Ministry of Further Education and the Italian Ministry of Health, by a Translational Research Grant from the Leukemia and Lymphoma Society, by the European Community ‘Allostem’ Project (Contract number: 503319) and by the National Institutes for Health of the USA (Project Number 1 PO1 CA 100265-01A1). AM is a student of the International PhD Program in Molecular Medicine, Vita-Salute San Raffaele University, Milan, Italy.

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36. Giebel S, Locatelli F, Lamparelli T, Velardi A, Davies S, Frumento G,  Maccario R, Bonetti F, Wojnar J, Martinetti M et al.: Survival advantage with KIR ligand incompatibility in hematopoietic stem cell transplantation from unrelated donors. Blood 2003, 102:814-819. The authors describe the survival advantage of KIR ligand incompatibility in the GvH direction in unrelated donor bone marrow transplantation for acute leukaemia. In this situation, a large bone marrow cell dose is transplanted and anti-thymocyte globulin is added to the conditioning regimen (to provide in vivo T cell depletion of the graft). 37. Lowe EJ, Turner V, Handgretinger R, Horwitz EM, Benaim E, Hale GA, Woodard P, Leung W: T-cell alloreactivity dominates natural killer cell alloreactivity in minimally T-cell-depleted HLA-non-identical paediatric bone marrow transplantation. Br J Haematol 2003, 123:323-326. 38. Morishima Y, Yabe T, Inoko H, Saji H, Juji T, Yamamoto K, Sasazuki T, Kodera Y: Clinical significance of killer Ig-like receptor (KIR) on acute GvHD, rejection and leukemia relapse in patients transplanted non-T cell depleted marrow from unrelated donors; roles of inhibitory KIR epitope matching and activating KIR genotype. Blood 2003, 102:526a. 39. Beelen DW, Ottinger H, Ferencik S, Elmaagacli AH, Peceny R, Grosse-Wilde H: Genotypic inhibitory killer immunoglobulin-like receptor ligand incompatibility enhances the long-term antileukemic effect of unmodified allogeneic hematopoietic stem cell transplantation in patients with myeloid leukemias. Blood 2004. DOI 10.1182/blood2004-04-1441. 40. Elmaagacli AH, Ottinger H, Koldehoff M, Peceny R, Trenschel R, Beelen DW: Reduced risk of molecular and haematological relapse in patients with CML after KIR-mismatched haematopoietic stem cell transplantation. Bone Marrow Transplant 2004, 33:S59. 41. Bornhauser M, Schwerdtfeger R, Martin H, Frank KH, Theuser C, Ehninger G: Role of KIR ligand incompatibility in hematopoietic stem cell transplantation using unrelated donors. Blood 2004, 103:2860-2861. 42. Shilling HG, McQueen KL, Cheng NW, Shizuru JA, Negrin RS,  Parham P: Reconstitution of NK cell receptor repertoire following HLA-matched hematopoietic cell transplantation. Blood 2003, 101:3730-3740. The authors show that unrelated donor transplants using unmanipulated bone marrow harvests (which contain approximately four-log more T cells and up to one-log fewer stem cells than the haploidentical grafts) [6,30] and post-transplant immune suppression to control GvHD, are associated with delayed reconstitution of potentially alloreactive, KIR-bearing NK cells. 43. Uhrberg M, Valiante NM, Shum BP, Shilling HG, LienertWeidenbach K, Corliss B, Tyan D, Lanier LL, Parham P: Human diversity in killer-cell inhibitory receptor genes. Immunity 1997, 7:753-763. 44. Shilling HG, Young N, Guethlein LA, Cheng NW, Gardiner CM, Tyan D, Parham P: Genetic control of human NK-cell repertoire. J Immunol 2002, 169:239-247. 45. Pando MJ, Gardiner CM, Gleimer M, McQueen KL, Parham P: The protein made from a common allele of KIR3DL1 (3DL*004) is poorly expressed at cell surfaces due to substitution at position 86 in Ig domain 0 and 182 in Ig domain 1. J Immunol 2003, 171:6640-6647. 46. Farag SS, Fehniger TA, Ruggeri L, Velardi A, Caligiuri M: Natural Killer cell receptors: new biology and insights into the graft versus leukemia effect. Blood 2002, 100:1935-1947. 47. Velardi A, Ruggeri L, Moretta A, Moretta L: NK-cells: a lesson from mismatched haematopoietic transplantation. Trends Immunol 2002, 23:438-444. 48. Parham P, McQueen KL: Alloreactive killer cells: hindrance and  help for haematopoietic transplants. Nat Rev Immunol 2003, 3:108-122. These three papers [46,47,48] review recent advances in NK cell biology and summarize genetic and functional evidence that, in human NK cells, the KIR inhibitory recognition of class I molecules regulates tolerance to self and alloreactivity, and impacts on the cure rate of leukaemia patients www.sciencedirect.com

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transplanted from donors who exhibit a mismatch that triggers donor versus recipient NK-cell alloreactivity. 49. Miller JS, Soignier Y, Panoskaltsis-Mortari A: Successful  adoptive transfer and in vivo expansion of human haploidentical NK cells in cancer patients. Blood 2005, DOI 10.1182/blood-2004-07-2974. This is the first report of safety and in vivo expansion of infused haploidentical NK cells in cancer patients.

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50. Imai C, Iwamoto S, Campana D: A new method for  propagating primary natural killer (NK) cells allows highly efficient expression of anti-CD19 chimeric receptors and generation of powerful cytotoxicity against NK resistant acute lymphoblastic leukemia cells. Blood 2004, 104:91a. These novel findings illustrate the potential of NK cell engineering for cancer therapy.

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