ReprintsOesk I 21612023 2:02:03 PM
`
`•
`
`Biology and clinical effects of natural killer cells in
`allogeneic transplantation
`Jonathan E. Benjamin, Saar Gill and Robert S. Negrin
`
`Division of Blood and Marrow Transplantation,
`Stanford University School of Medicine, Stanford,
`California, USA
`
`Correspondence to Robert S. Negrin, Division of Blood
`and Marrow Transplantation, Stanford University
`School ot Medicine, 300 Pasteur Drive H3249,
`Stanford, CA 94305-5627, USA
`Tel: + 1 650 723 0822; tax: + 1 650 725 8950;
`e-mail: negrs@stanford.edu
`
`Current Opinion in Oncology 2010, 22:130- 137
`
`Purpose of review
`Following allogeneic hematopoietic cell transplantation, donor-derived natural killer
`(NK) cells target recipient hematopoietic cells, result ing in an antileukemia effect and a
`lower incidence of graft rejection. NK cells do not mediate and may diminish graft versus
`host disease. Here we review the determinants of NK cell alloreactivity and their
`implications for adoptive NK cell therapy.
`Recent findings
`NK cell alloreactivity has been defined by the absence of recipient M HC class I ligands
`for donor inhibitory killer immunoglobulin-like receptor (KIR) receptors, as predicted by a
`number of algorithms. Recently, the role of activating NK receptors and their cognate
`ligands has received more attention. The beneficial clinical effect of NK-cell alloreactivity
`has not been uniformly demonstrated, likely reflecting differences in condit ioning
`regimens, graft components and posttransplant immune suppression. Investigations of
`NK cell phenotype and function after transplantation have helped demonstrate which
`NK cell subsets mediate the graft versus leukemia effect. These advances have
`proceeded in parallel with increasing facility in GMP-grade bulk purification and
`administration of NK cell preparations.
`Summary
`NK cells are a heterogeneous population of lymphocytes with diverse patterns of target(cid:173)
`cell recognition and effector function. Further clinical and functional correlations will
`help maximize 1heir potential for clinical benefit.
`
`Keywords
`adoptive immunotherapy, hematopoietic transplantation, natural killer cells
`
`Curr 0pin 0ncol 22:130-137
`0 2010 Wolters Kluwer Health I Lippincott Williams & Wilkins
`1040-8746
`
`Introduction
`Natural ki ller ( K) cells differentiate self from nonself
`by gauging the expression of M HC class I molecules on
`potential target cells. In the context of allogeneic he ma(cid:173)
`topoietic cell transplantation (HGT), particularly with
`HLA mismatched transplants, donor-derived K cells
`have been shown in some studies to influence the out(cid:173)
`come by a direct anti tumor effect as well as by mitigating
`graft versus host d isease (GVH D) and reducing the
`incidence of graft rejection. The optimal way to harness
`NK cell alloreactivity remains the subject of vigorous
`debate. In this article, we review the basic biology of NK
`cells in the context of recent clinical trials of H CT.
`
`sion and the presence or absence of the low affinity lgG
`receptor CD16. 'T'he CD56hrighcCD16" subset is enriched
`in lymphoid organs, secretes cytokines
`to help co(cid:173)
`ordinate adaptive immunity and is the major subtype
`recruited to sites of inflammation (including malignancy)
`[1,2]. In contrast, the CD56dimCD 16+ subset circulates in
`the peripheral blood and shows potent cytotoxicity [3].
`Several lines of evidence suggest that C D56hrighc K
`cells may differentiate into CD56°im K cells under
`some conditions [4,5]. K cells are reported to comprise
`5 - 25% of peripheral b lood lymphocytes, or approxi(cid:173)
`mately 100- 600cells/µl [6].
`
`Effector functions : lytic machinery
`K cell cytotoxicity requires ta rget-cell recognition and
`Human natural killer immunophenotype
`the receipt of an activating signal in the absence of an
`inhibitory signal. Killing is mediated by several pathways;
`NK cells do not express a rearranged germline-antigen
`receptor and they are identified by expression ofCD56 in
`whereas immature N K cells rely on tumor necrosis(cid:173)
`the absence of GD3. Two subsets of mature K cells are
`related apoptosis-inducing ligand (TRAIL)-mediated
`recognized, defined by the brightness of CD56 expres-
`killing [7], mature K cells preferentially utilize the
`1040-8746 «, 2010 Wolters Kluwer Health I Lippincott W illiams & Wilkins
`
`DOI: 10.1097 /CCO.0b013e328335a559
`
`Copyright© Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
`
`UPenn Ex. 2056
`Miltenyi v. UPenn
`IPR2022-00855
`Page 130
`
`

`

`•
`
`ReprintsOesk I 21612023 2:02:03 PM
`
`granule-exocytosis pathway (requiring perforin and gran(cid:173)
`zymes) and the Fas- Fas ligand pathway (8]. In order to
`ach ieve the ir maximal cytotoxic potential in vivo, K
`cells must be activated, with evidence in mice that this
`step requires the rranspresentation ofIL-15 by dendritic
`cells [9]. The observed enhanced cytotoxic potential of
`activated cells is due in part to the translation of pre(cid:173)
`exist ing pools of granzyme B and perforin m RNA [10].
`
`Effector functions : cytokines
`Cytokine-producing NK cells link the innate and adap(cid:173)
`tive immune responses. l nterferon--y has protean effect~
`incl ud ing Th 1 polarization (11, 12], dendritic-cell matu(cid:173)
`ration and activation f 13], d irect antiviral effect f 14, 15], as
`well as various antiproliferative effects on transformed
`cells. TNFa en hances dendritic cell maturation [16] and
`also leads to increased IFN-y production f 17]. GM-CSF
`can stimulate phagocytosis by monocytes and contributes
`to dendritic-cell maturation (18].
`
`Natural killer receptors
`MHC class I molecules are critical determinants of K
`cell activity; NK cells effectively lyse cells lacking
`expression of some or all MHC molecules. Karre et al.
`f 19] recognized chat NK cytotoxiciry was abrogated if the
`effectors and targets shared MHC class I molecules. The
`'missing-self hypothesis correctly p redicted the exist(cid:173)
`ence of receptors for self-MHC that, when e ngaged,
`would in hibit cytotoxicity. In humans, inhibitory recep(cid:173)
`tors that recognize H L A-A, B, and C molecules belong to
`the killer immunoglobulin-like receptor (KIR) family
`[20,21]. Another inhibitory receptor, the C-type lectin
`NKG2A pairs with CD94 and recognizes the nonclassical
`MHC molecule H LA-E [22-24]. T he expression pat(cid:173)
`terns of inh ibitory receptors create a repertoire of K
`cells with nonoverlapping specificities.
`
`The human KIR gene cluster is located on chromosome
`19q13.4 and contains 14 KTR genes and 2 pseudogenes.
`Inhibitory receptors possess long cytoplasmic tails with
`immunoreceptor tyrosine-based inhibitory motifs (ITIM)
`that allow docking of tyrosine phosphatase molecules,
`whereas activating KIRs have short cytoplasmic tails chat
`enable pairing to adapter molecules with immunoreceptor
`tyrosine-based activating motifa (ITAM) [25]. The extra(cid:173)
`cell ular domains of the activating KIR share sequence
`similarity with the corresponding inhibitory KIR and
`may share HLA-binding specificities, though the ligand~
`for most activating KIRs remain unknown [26,27]. It is
`thought that inhibitory KIRs bind class I molecules with
`greater affinity than the correspondi ng activating KIR.
`
`KIR proteins recog nize allorypic motifs in the class I
`alpha helix, as described in Table 1. Two KIR haplotypes
`
`Effects of natural killer cells in transplantation Benjamin et al. 131
`
`Table 1 Inhibitory and activating receptors for HLA class I
`molecules
`
`Receptor
`
`Inhibitory
`KIR2DL2/3
`KIR2DL1
`KIR3DL1
`
`CD94/NKG2NE
`KIR3DL2
`KIR2DL4
`Activating
`KIR2DS4
`KIR2DS1
`KIR2DS2
`KIR2DS3
`KIR2DS5
`KIR3DS1
`KIR Haplotype
`A
`B
`
`Ligand
`
`HLA-C group1 (Cw1, Cw3)
`HLA-C group 2 (Cw2, Cw4)
`HLA-A and B with Bw4 motifs
`at position 77- 83
`HLA-E
`HLA-A3/A1 1
`HLA-G
`
`HLA-C2
`
`Inhibitory KIR and KIR2DS4
`Inhibitory KIR and combinations
`of activating KIR
`
`KIR genes are organized into diverse haplotypes, which have simplified
`into Groups A and B.
`
`have been defined, A and B [28] . The A haplotype has
`five inhibitory genes (2D L 1, 2D L3, 3DL1, 3DL2, 3DL3)
`and one activating gene (KIR2DS4). The B haplotypes
`are less uniform in gene content and have a greater
`number of activating receptors. H LA genes on chromo(cid:173)
`some 6 and KIR genes on chromosome 19 assort inde(cid:173)
`pendently, meaning that KIR genotype, rather than HLA
`type, is the major determ inant of KI R expression pattern.
`KIR expression is stochastic, generating a n K cell
`com partment with individual members expressing one
`or more inhibitory receptors. NK cells that express only
`inhibitory KlRs for absent class I molecules are detect(cid:173)
`able bu t rather tha n being autoreactive, as would be
`predicted by the missing-self model, such cells are hy por(cid:173)
`esponsive. T he licensing theory of K cell maturation
`suggests that engagement of an inhibitory KIR by the
`cognate class I molecule is necessary to acquire effector
`function [29,30]. Another model proposes that the K
`cells become functionally anergic without self-inhibitory
`signals f3 t].
`
`Licensing notwithstanding, failure to engage an inhibi(cid:173)
`tory KI R is insufficient to elicit an K at.tack. Rather, a
`cell-surface derived activating sig nal is necessary. In
`addition to activating KI R molecules, a nu mber of acti(cid:173)
`vating K cell receptors have now been characterized
`(see Table 2).
`
`Predictors of alloreactivity
`According to the missing-self model, mismatch between
`donor KI R and recipient. MHC class I molecule expres(cid:173)
`s ion predicts the existence of a subset of donor NK cells
`that are not inhibited and are thought to be alloreactive.
`
`Copyright© Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
`
`UPenn Ex. 2056
`Miltenyi v. UPenn
`IPR2022-00855
`Page 131
`
`

`

`132 Transplantation
`
`ReprintsOesk I 21612023 2:02:03 PM
`
`•
`
`Table 2 Some activating NK cell receptors
`
`Receptor
`
`Receptor expression
`
`Ligands
`
`Ligand expression
`
`NKG2D
`
`All NK cells, some T cells
`
`MICA, MICB, ULBP1-5
`
`Leukemia, carcinoma cells; induced by viral infection and ionizing
`radiation (DNA damage response)
`
`NKp30
`NKp44
`NKp46
`DNAM1
`
`All NK cells
`Activated NK cells only
`All NK cells
`All NK cells
`
`B7-H6, BAT3
`?
`Viral hemagglutin n
`CD112, CD155
`
`NK, natural killer.
`
`The Perugia group brought renewed atcention to K cell
`alloreacriviry in their pioneering stud ies of haploidentical
`transplantation. Recipients received myeloablative che(cid:173)
`moradiotherapy and ATG. In order to avoid GVHD ,
`donor grafts were T -cell depleted and
`'megadose'
`GD34+ (e.g. 107 /kg) cells were infused in order to over(cid:173)
`come the resistance co engraftment observed with T -cell
`depleted transplants [32].
`o posttransplant immune
`suppression was used. Under these conditions, KIR
`ligand mismatching in the GVH d irection (observed in
`30% of unselected haploidentical related donors) bene(cid:173)
`fited patients with myeloid leukemias [33,34]. Patients
`receiving KIR ligand mismatched transplants had better
`overall survival, improved rates of engraftment and a
`reduced incidence of GVHD. Protection against GVHD
`has been explored in a mouse model, where it was found
`that donor NK cell alloreactivity was linked to the
`depletion of recipient antigen-presenting cells [34].
`
`Receptor-ligand
`The Ruggeri model assumes that K cells expressing
`inhibitory KIR for ligands absent on both donor and
`recipient are nonalloreactive. T he licensing model pre(cid:173)
`d icts that developing K cells that fail to receive a signal
`th rough an inhibitory receptor are hyporesponsive to
`activating stimuli. However, licensing can he bypassed
`in a p roinflammatory e nvironment [29], as is seen follow(cid:173)
`ing myeloablative conditioning. Le u ng et al. [35] argues
`that alloreactiviry may he predicted by considering only
`the expression of donor-inhibitory KI R (as determined by
`flow cytomerry) and recipient ligand, thus eliminating
`donor ligand from the equation. In a n analysis of 36
`pediatric patients receiving T -cell depleted haploiden(cid:173)
`tical transplants, the receptor-ligand model was a better
`predictor of leukemia relapse than the ligand-ligand
`mode l.
`
`Missing killer immunoglobulin receptor ligand
`The missing ligand model is a n extensio n and sim plifica(cid:173)
`tion of the receptor-ligand model. Regardless of HLA
`expression, most individuals express the full complement
`of inhibitory KIR. An individual who is homozygous for
`KTR ligand epitopes (e.g. H LA-Ct or H LA-Bw4) would
`he predicted to have a subset of NK cells expressing an
`inhibitory KI R for the absent ligand (KI R- L). Hsu et al.
`[36] analyzed the impact of missing ligand in 1770
`
`Melanoma, carcinoma, neuroblastoma
`
`myeloahlative, T -cell replete transplants from unrelated
`donors. Recipients were grouped accord ing to homozyg(cid:173)
`osity of HLA-C group t, HLA-C group 2, or HLA-Bw6.
`Patients homozygous for KTR epitopes who received
`transplants from HLA mismatched donors had a lower
`rate of relapse than heterozygotes. T his effect was greater
`among patients with AM L than those with CM L or ALL.
`However, among pa tients receiving HLA-matched unre(cid:173)
`lated donor transplants, homozygosity of these epitopes
`was associated with a sligh tly higher risk of relapse. In a
`similar analysis Miller et al. [37] analyzed 2062 unrelated
`donor transplants with myeloid malignancies. Of these
`patients, 70% were m issing one or more KIR-L. For
`patients with early stage myeloid malignancies, that is
`AML in C Rl , early stage MDS, or early chronic phase
`CML, absence of KIR- L was protective against relapse,
`hut this effect was lost in more advanced stage disease.
`Onlike the data of H su, this effect was seen in H L A
`matched and mismatched transplants alike. In a further
`analysis of patients receiving haploidentical transplants
`for myeloid malignancies, Ruggeri et al. [38] compared
`the predictive value of the missing ligand model with the
`original mismatched ligand model, finding the latter co he
`more informative.
`
`Activating killer immunoglobulin receptor
`Increased numbers of activating KTR molecules have
`correlated with increased propensity to autoimmune d is(cid:173)
`orders as well as to resisrance to CMV reactivat.ion in
`kidney-transplant recipie n ts [39] . In the setting of allo(cid:173)
`HCT, higher n umbers of activati ng Kl R genes has been
`correlated with increased frequency of acute and chronic
`GVHD. In a multivariate analysis of 448 unrelated trans(cid:173)
`plants for AM L, Cooley et al. [40•] found that presence of
`a donor, KIR-B haplotype, wh ich contains a higher nu m(cid:173)
`ber of activating KTR genes than the A haplotype, pre(cid:173)
`dicted improved relapse free and overall survival in which
`there was no KIR ligand mismatch. This benefit was not
`observed in patients with KIR ligand mismatched trans(cid:173)
`plants. An interesting recent study showed that in the
`setting of TCD haploidentical
`transplantation with
`KTR-L mismatch, K cells co-expressing the activating
`KTRZDSl with inhihicory KTR2 D L2/3 or KG2A were
`able to kill recipient leukemia blasts, highlighting that in
`some settings, recognition by activat ing KIR is able to
`overcome inhibitory signals [41 ].
`
`Copyright© Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
`
`UPenn Ex. 2056
`Miltenyi v. UPenn
`IPR2022-00855
`Page 132
`
`

`

`•
`
`ReprintsOesk I 21612023 2:02:03 PM
`
`Clinical outcomes
`The porential benefits of K cell alloreactivity have been
`explored in cohorts of patienrn receiving mismatched
`unrelated donor transplants, often with d iscordant con(cid:173)
`clusions. These results have been reviewed p reviously
`f42 - 4Sl.
`
`Umbilical cord transplantation
`As with haploidentical transplantation, umbilical cord
`blood (llCB) transplants are frequently c haracterized
`by a high degree of HLA m ismatching. UCB has as it~
`advantage the relatively low risk of acute GVHD due to a
`lower number of mature donor T cells and thus an
`increased ability to use HLA mismatched units. Will(cid:173)
`emze et al. [4(, ] reviewed the impact of KIR ligand
`m ismatching in lJCB transplants for acute leukemia
`(lymphoid or myeloid)
`in first complete remission
`(C Rl ). Of 218 transplants, 10% were HLA mismatched
`and 47% had greater than one mismatch. Thirty-two
`percent of donor recipient pairs were KI R-ligand incom(cid:173)
`patible in the graft versus host direction. Patients with
`KIR-ligand incompatible donors had improved overall
`survival (57 vs. 40%) and decreased relapse (20 vs. 37%)
`when compared with chose without these incompatibil(cid:173)
`ities. As was seen in the Perugia studies, benefits of KIR
`ligand incompatibility were most striking among patients
`with AM L although lJCB recipients with ALL also had a
`trend toward improved leukemia-free survival. However,
`Brunstein et al. (47] failed to observe any benefit of KIR(cid:173)
`L mismatch in lSS recipients ofOCB after myeloablative
`conditioning. In fact, in 102 patients who had received
`UCB after nonmyeloablative cond itioning, KI R-L mis(cid:173)
`match was associated with an increased rate of acute
`GVHD and higher treatment-related mortality.
`
`Nonmyeloablative transplantation
`Nonmyeloablative conditioning is marked by a period of
`mixed chimerism in which recipient NK cells may exert
`antidonor effects. KIR-L mismatch in the host versus
`graft direction in 31 patients conditioned with fludarab ine
`and 2Gy TBI was fou nd to predict for increased risk of
`graft rejectio n and lower incidence of complete do nor
`chimerism (48•].
`
`The impact of NK cell a lloreactivi cy in the graft versus host
`d irection was studied in a series of 282 donor- recipient
`pairs treated with 2Gy of'TBI with or without fludarabine,
`where the majority (88%) received H L A-matched graft~
`(49•]. High donor K cell chimerism before day 100 wa~
`associated with low relapse rates. The risk of relapse wa~
`lower for patients expressing ligands for all donor KIR,
`though this did not reach scaristical significance. Of note, a
`lower risk of relapse was nor significantly associated with
`high donor T -cell chimerism. In contrast, the risk of acute
`GVH D (grade II-IV) was associated with high levels of
`
`Effects of natural killer cells in transplantation Benjamin et al. 133
`
`do nor T -cell chimerism, whereas K cell chimerism did
`not correlate with GVHD.
`
`Natural killer reconstitution posttransplant
`During the first few mo nths post HCT, K cells are the
`predominant circulating lymphoid cell subset with the
`potential to control disease relapse. NK cells are the first
`ly mphoid cells to repopulate, reaching normal numbers
`within 1 month regardless of donor type or patient age in
`adults [S0,S 1] and children [S2]. Similar findings are
`shown after
`lJCB
`transplantation
`in children
`fS3] .
`Although some studies have highlighted the ability of
`K cells to exert a potent an tileukemic effect and
`reduced relapse in high-risk hematologic malignancy,
`the question remains whether K cells reconstituting
`after HCT manifest an actual antileukemic effect. This
`issue has been investigated by evaluating the phenotype
`and cytotoxic potential of K cells re -isolated at early
`time-points after transpla nt.
`
`Phenotypic changes
`According to the Perugia KIR-mismatch model, poren(cid:173)
`tially a lloreactive K cells posc-HCT are cells chat
`express KIR for missing KIR-L molecules in the absence
`of the inhibitory receptor GD94/NKG2A. The Perugia
`data, however, show that the majority of NK cells in the
`first S months post-HCT do not belong to this subset [38].
`Similarly, other groups have found that early after both
`HLA-matched and haploidentical HCT (with or without
`T cell depletion), NK cells are predominantly CDS6hrighc,
`KG2A+ and KIR- , a find ing which has been described
`as consistent with an ' immature phenotype' fS4,ss •,
`S6,S7] and has been related to increased post-HCT levels
`of the homeostatic cytokine IL-15 [S6]. The remaining
`CDS6tlim cells, which normally express low levels of
`KG2A, u pregulate expression of this receptor post(cid:173)
`HCT while expression of CD16 as well as of KIR is
`lower than in healthy controls fSS",57]. A mature donor(cid:173)
`type KI R repertoire appears within 3 months- 3 years
`[3s,ss· ,s8J.
`
`Expression of NKp46 as well as of the activating receptor
`KG2D appear to be increased during rhe first year pose
`HLA-marched non-TCD HC'T, whereas NKp44 a nd
`Kp30 do not appear to be u pregulated [S6].
`
`With regard to the expression of effector molecules,
`production of both perforin and IFN-y is increased among
`the C DS6hright cells in patients post HLA-matched HCT
`when compared with the same subset in normal donors
`[S6].
`
`Functional changes
`inconsistent results,
`Several grou ps, with somewhat
`have investigated the fu nctional consequences of the
`
`Copyright© Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
`
`UPenn Ex. 2056
`Miltenyi v. UPenn
`IPR2022-00855
`Page 133
`
`

`

`134 Transplantation
`
`ReprintsOesk I 21612023 2:02:03 PM
`
`•
`
`phenotypic changes described earlier. The Perugia group
`found that N K alloreactive cells (defined as N K cells of
`donor origin capable of k ill ing cryopreserve<l host PHA
`b lasts) are detectable in about half of patie nts 1 month
`afrer haploi<len tical HCT and are no longer detectable
`afrer 12 months [38]. Afrer haplo i<lentical HCT K cells
`have poor effector function against primary leukemia
`cells, a finding which has been related to the increased
`frequency of NKG2A-bearing NK cells, althoug h result.s
`of blocking studies of this inhibitory receptor have
`yielded conflicting results [54,55•]. Evaluation of cells
`that are N KG2A- and single-KI R+ (thus predicted to
`have alloreactive potential under the models described
`earlier) after haploi<lentical HGT has demonstrated that
`these cells are first detectable approximately 75 <lays after
`HCT, their frequency is highly variable and shows no
`correlation with predicted donor K cell alloreactivity
`and that their functional capacity was reduced [55•]. T his
`finding is somewhat out of keeping with the conclusions
`from the Perugia group and may be related to the lack of
`licensing of these cells, as they are repopulating in the
`absence of a n H LA ligand for their single KI R.
`
`Furthermore, a small study in 'TCD HLA-matche<l
`HCT has shown that K cells exhibiting KI R for nonself
`class I ligands (predicted to be alloreactive according to
`the ' missing ligand' model but nonlicense<l according
`to the licensing hypothesis) are capable of mounting
`a robust cytotoxic and cytokine response in the first
`months posttransplant when incubated with MHC class
`I deficient cell lines [59•]. The a uthors explain this
`finding by postulati ng that N K cell tolerance to host
`cells takes some time to develop, <luring which time
`patients may benefit from NK cell alloreactivity, although
`it may be that licensing requirements are bypassed in the
`inflammatory posttransplant environment.
`
`The p resence of T cells in the graft has been found to
`impair K-cell reconstitution and function posttrans(cid:173)
`plant [43,60]. This find ing may in part explain the
`importance of T -cell depletion for ensuring an opti mal
`NK-cell effect. In addition, peritransplant immunosup(cid:173)
`pression could impact K cell function, as exposure ofin(cid:173)
`viuo differentiated K cells to cyclosporine A has been
`shown to lead to preferential expansion of C D56+ KIR(cid:173)
`NK cells [61].
`
`Conclusions drawn from these studies must be tempered
`by the recognition that different NK cell functional
`assays may y ield d iscordant results. Most studies evaluate
`NK cells by their capacity to <legranulate (using the
`GD107a mobilization assay) or produce IF
`'Y after
`exposure to tumor cell lines known to be susceptible
`to K cell killing, a somewhat artificial experimental. In
`addition, studies using as targets patient PHA blasts
`rather than cryopreserve<l p rimary patient leukemia cells
`
`likewise may not be representative of the actual anti(cid:173)
`tumor potential.
`
`Natural killer adoptive therapy
`As follows from the earlier d iscussion, K cells repopu(cid:173)
`lating the host after transplantat ion may be functionally
`defective as a result of immaturity, immunosuppression,
`or other u nidentified factors. Thus, it has been proposed
`that the adoptive transfer of mature NK cells may be
`beneficial by providing both a cytolytic effect and an
`immunomo<lulatory effect, the latter by utilizing NK(cid:173)
`<len<lritic cell cross talk to promote K-cell activation and
`subsequent Thl p olarization [14].
`
`GMP grade production of N K cells from apheresis pro(cid:173)
`ducts for adoptive therapy is feasible, with reported
`y ields of up to 10 x 108/kg with recovery of approximately
`40- 50% [62- 65].
`
`In the most important study of adoptive K-cell therapy
`to <late, haploi<lentical K cells were given to 43 patients
`with advanced malignancies (AML, metastatic mela(cid:173)
`noma and metastatic renal carcinoma) together with
`IL-2. There was minimal hematologic and nonhemato(cid:173)
`logic toxicity, the latter mostly attributable to IL-2.
`Results from this study revealed the following conditions
`for persistence in the host of donor NK cells: a high-dose
`conditioning regimen is g iven to achieve sig nificant lym(cid:173)
`pho<lepletion; high levels ofIL- 15 levels are present; and
`the addition of nonmyeloablative <loses of TB I, cyclopho(cid:173)
`sphami<le and flu<larabine and infusion of CD34+ p ro(cid:173)
`genitor cells leads to better K cell expansion and higher
`AML remission rates [66,67]. There was no GVHD, but
`the protocol was complicated by fatal infections and
`EBV-PTLD; interestingly, no correlation was found
`between efficacy and predicted K alloreactivity accord(cid:173)
`ing to KIR ligand mismatch.
`
`Q uestio ns remain regarding the optimal purity of the
`product, as there is evidence from early studies that
`monocytes may be required for optimal NK-cell prolifer(cid:173)
`ation [68], and more recent studies show that monocyte(cid:173)
`<lerive<l <len<lritic cells promote N K cell effector function
`[69]. Similarly, whether the optimal source of NK cells
`should be from nonmobilize<l or G-CSF mobilized per(cid:173)
`ip heral blood collections needs to be addressed, due to
`data suggesting that N K cells expanded from G-CSF
`mobilized PBPC collections are functiona lly abnormal
`and possess reduced n umbers of K progenitors [70- 72].
`
`Facilitation of NK cell eng raftment will likely depend on
`suppression of the host immune system, eradication of
`'Tregs (wh ich may inhibit transferred N K cells) [73]
`presence of 'space' for homeostatic proliferation follow(cid:173)
`ing lympho<lepletion, and the presence of high levels of
`
`Copyright© Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
`
`UPenn Ex. 2056
`Miltenyi v. UPenn
`IPR2022-00855
`Page 134
`
`

`

`ReprintsOesk I 21612023 2:02:03 PM
`
`Effects of natural killer cells in transplantation Benjamin et al. 135
`
`•
`
`cytokines especially I L-1:5 [74,75]. The assertion that
`I L- 15 is critically required for in-vivo K cell expansion
`has heen recently challenged in murine models of infec(cid:173)
`tion [76].
`
`Receptor and ligand modulation
`Modulation of NK cell recognition of tumors may he
`ach ieved hy hlockade of inhihitory KIR as was recently
`demonstrated using a humanized antihody to KI RZDL-1,
`KIRZD L-2, and KI RZD L-3 as well as the activating
`receptors KIRZDS-1 and KIRZDS-2 [77]. A phase I trial
`of this agent is ongoing.
`
`Additional approaches to augment K cell act1v1ty
`incl ude the use of hispecific antihodies (such as for
`HE RZ/neu and CD16) [78), genetic modification of
`NK cells to express receptors for tumor-associated anti(cid:173)
`gens not normally part of the K-receptor ligand reper(cid:173)
`toire or enforced expression of signaling receptors [79,80].
`Other approaches include the indirect pharmacologic
`modulation of K receptor ligands on tumor cells hy
`compounds such as horcezomih [81 l or hisrone deacety(cid:173)
`lase inhihitors [82••1. It may he that several of these
`techniques may be required in order to realize the full
`potential of K cell adoptive therapy.
`
`Conclusion
`Successful utilization of adoptive K therapy may
`require a n availahle niche as defined either by lympho(cid:173)
`pen ia, selective host NK cell lymphopenia, high IL-15 or
`other homeostatic cytokine levels, or a comhination of
`these. Other open questions include the appropriate
`method for collection and activation, optimal cytokine
`type and schedule, and in-vivo or ex-vivo stimulation, and
`it will he important to ensu re that cells which have been
`activated for several days in vitro do not lose the ir pro(cid:173)
`liferative potential in v ivo, as has heen found for adop(cid:173)
`tively transferred T cells [83]. Although it is likely that
`apheresis products will represent the most easily acces(cid:173)
`sihle source of mature NK cells, it is important to keep in
`m ind that K cells enriched from G-CSF mohilized
`hlood exhibit reduced functional capacity compared with
`those from unstimulated peripheral hlood [70]. It may he
`that a particular suhset of K cells should he targeted for
`expansion for adoptive infusion, althoug h studies addres(cid:173)
`sing this particular point are presently lacking. Finally,
`the optimal methods to e nsure persistent expansion and
`recognition of target cells still await development, and
`perhaps will require a com hi nation of cytokines and K
`receptor ligand modulating agents.
`
`Acknowledgements
`This work was supported by the National Institutes of Health (R01-CA-
`125276) and the Hematology Society of Australia and New Zealand.
`
`We apologize to those whose studies were not cited due to space
`considerations.
`
`References and recommended reading
`Papers of particular interest, published within the annual period of review, have
`been highlighted as:
`of special interest
`•
`•• of outstanding interest
`Additional references related to this topic can also be found in the Current
`World Literature section in this issue (pp . 159-160).
`
`Dalbeth N, Gundle R, Davies RJ, et al. CD56bright NK cells are enriched at
`inflammatory sites and can engage with monocytes in a reciprocal program of
`activation. J lmmunol 2004; 173:6418-6426.
`
`2 Carrega P, Morandi B, Costa R, et al. Natural killer cells infiltrating human
`nonsmall-cell lung cancer are enriched in CD56 bright CD16(- ) cells and
`d isplay an impaired capability to kill tumor cells. Cancer 2008; 112:863-875.
`
`3 Gill S, O lson JA, Negrin RS. Natural killer cells in allogeneic transplantation:
`effect on engraftmen~ graft-versus-tumor, and graft-versus-host responses.
`Biol Blood Marrow Transplant 2009; 15:765-776.
`
`4
`
`Chan A, Hong DL, Atzberger A, et al. CD56bright human NK cells differentiate
`into C D56dim cells: role of contact with peripheral fibroblasts. J lmmunol
`2007; 179:89-94.
`5 Romagnani C, Juelke K, Falco M, et al. C D56brightCD16- killer lg-like
`receptor- NK cells display longer telomeres and acquire features of CD56dim
`NK cells upon activation. J lmmunol 2007; 178:4947-4955.
`
`6
`
`7
`
`8
`
`Jentsch-Ullrich K, Koenigsmann M, Mohren M, Franke A. Lymphocyte subsets'
`reference ranges in an age- and gender-balanced population of 1 00 healthy
`adults: a monocentric German study. Clin lmmunol 2005; 116:192-197.
`Zamai L, Ahmad M, Bennett IM, et al. Natural killer (NK) cell-mediated
`cytotosicity: differential use of TRAIL and Fas ligand by immature and mature
`primary human NK cells. J Exp Med 1998; 188:2375-2380.
`
`Freud AG, Yokohama A, Becknell B, et al. Evidence for discrete stages of
`human natural killer cell differentiation in vivo. J Exp Med 2006; 203:1033-
`1043.
`
`9
`
`Lucas M, Schachterle W , Oberle K, et al. Dendritic cells prime natural killer
`cells by trans-presenting interleukin 15. Immunity 2007; 26:503-517.
`1 0 Fehniger TA, Cai SF, Cao X, et al. Acquisition of murine NK cell cytoto,icrty
`requires the translation of a preexisting pool of granzyme B and perforin
`mRNAs. Immunity 2007; 26:798-81 1.
`
`11 Martin-Fontecha A, Thomsen LL, Brett S, et al. Induced recruitment of NK cells
`to lymph nodes provides IFN-gamma for T(H)1 priming. Nat lmmunol 2004;
`5:1260- 1265.
`
`12 Morandi B, Bougras G, Muller WA, et al. NK cells of human secondary
`lymphoid tissues enhance T ce