CAR a number of hematologic and solid

CAR modified Natural killer cellsThe last several years have seen impressiveadvances in the engineering of immune cells as cancer therapy. Whereas chimericantigen receptors (CARs) have been used comprehensively to convey thespecificity of autologous T cells against hematological malignancies withremarkable clinical results, studies of CAR-modified natural killer cells havebeen mostly in preclinical phases.

NK cells for adoptive therapy can be derivedfrom several different sources which is explained in other parts. Allogeneic NKcells can be generated from the Peripheral blood of healthy donors or expandedfrom umbilical cord blood. Regardless of the source, there are several featuresof expanded, activated CB, or PB-derived NK cells that make them useful effectorsfor gene modification.with CAR-modified primary human NK cells canbe effector modified immune cells against a number of hematologic and solidtumor antigens, including CD19, CD20, GD2, and HER-2.

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While non-viralexpression techniques such as nucleofection or electroporation can producerobust CAR-mediated killing, the short-lived nature of these CAR moleculeswould likely dictate the need for repeated infusions in the clinical setting.( Engineering Natural Killer Cells for CancerImmunotherapy)NKG2D ReExpanded, activated NK cells generallyexpress a wide range of activating receptors, including CD16, NKG2D, and theNCRs (NKp44 and NKp46), in spite of donor-to-donor variability. These activatedNK cells are prepared with KIRs and are “licensed to kill.” in vivo expansionand persistence capacity of NK cells is clearly associated with antitumoractivity in trials involving hematologic malignancies such as AML. Moreover, exvivo expanded primary human NK cells produce a different storms of cytokinesmore than T cells, including interferon (IFN)-g, IL-3, and granulocytemacrophage colony-stimulating factor (GM CSF), which may be associated with alower risk of CRS(cytokine released syndrome)While normal NK cell counts are usuallydetected within the first month after alloSCT regardless of the graft source,several months are required to acquire the immunophenotypic and functionalcharacteristics of NK cells found in healthy donors. rebuilding NK cellsdisplay a more immature phenotype expressing the inhibitory natural killergroup two A (NKG2A) receptor at around 90% compared to around 50% in healthydonors 2,3. During the NK development and peripheral maturation, the CD56dimNK cells lose NKG2A expression but up-regulate the expression of the activatingNKG2C receptor, killer cell inhibitory immunoglobulin-like receptors (KIRs) andCD57.

The alloreactivity of NK cells is determined by various receptorsincluding the activating CD94/NKG2C and the inhibitory CD94/NKG2A receptors,which both recognize the non-classical human leukocyte antigen E (HLA-E). Herewe analyze the contribution of these receptors to NK cell alloreactivity in 26patients over the course of the first year after alloSCT due to acute myeloidleukemia, myelodysplastic syndrome and T cell Non-Hodgkin-Lymphoma. Our resultsshow that NK cells expressing the activating CD94/NKG2C receptor aresignificantly reduced in patients after alloSCT with severe acute and chronicgraft-versus-host disease (GvHD).

Moreover, the ratio of CD94/NKG2C to CD94/NKG2Awas reduced in patients with severe acute and chronic GvHD after receiving anHLA-mismatched graft. Collectively, these results provide evidence for thefirst time that CD94/NKG2C is involved in GvHD prevention.Moreover, the ratio of CD94/NKG2C to NKG2A/CD94was reduced in patients with acute or chronic GvHD after receiving anHLA-mismatched graft.

In conclusion, these results provide evidence that theCD94/NKG2C receptor is associated with alloreactivity of NK cells afteralloSCT, especially regarding acute or chronic GvHD prevention.( The Activating NKG2C Receptor IsSignificantly Reduced in NK Cells after Allogeneic Stem Cell Transplantation inPatients with Severe Graft-versus-Host Disease)Cytokines in improving NKIL2 and LAK cellsAt the University of Minnesota, reserachersfirst confirmed the use of low dose IL-2 daily to expand NK cells afterautologous HSCT in patients with non-Hodgkin lymphoma and breast cancer. Later,they activated autologous NK cells ex vivo with IL-2 for 24 hours, infused theminto patients and administered daily subcutaneous IL-2 .autologous NK cellstudies showed limited efficacy, they did yield important findings: 1).IL-2 canbe administered safely at daily or 3 times weekly intervals, 2) IL-2 can inducean increase in circulating cytotoxic lymphocytes with a disproportionateincrease in NK cells.In innovative studies at the NCI, Rosenbergand colleagues infused melanoma and renal cell carcinoma patients withautologous peripheral blood cells treated ex vivo with IL-2.

The product wasenriched with NK cells and named “lymphocyte activated killer” (LAK) cells.High dose IL-2 was administered to patients after LAK infusions to promotetheir in vivo persistence and activity. In a subsequent trial, the NCI groupadoptively transferred in vitro expanded autologous tumor-infiltratinglymphocytes (TILs) to 20 patients with metastatic melanoma. Objective responseswere observed in 11 patients. Given the limited persistence of the transferredtumor-specific T-cells in vivo, a second course of TIL cells was infusedfollowing lympho-depleting chemotherapy combining high dose cyclophosphamidewith fludarabine 88. Objective responses were observed with TILs in patientswith melanoma.

These and other studies have contributed important newknowledge: 1) high-dose IL-2 used in vivo with the goal of activating NK cellshas significant but manageable toxicity owing to severe capillary leaksyndrome, whereas low-dose subcutaneous IL-2 was well tolerated, 2)lymphodepleting chemotherapy using high-dose cyclophosphamide and fludarabinefacilitated in vivo expansion of autologous adoptively transferred cytotoxic Tlymphocytes and lead to enhanced efficacy,3) chemotherapy induces lymphopenia,changes the competitive balance between transferred lymphocytes and endogenouslymphocytes, changes the cytokine milieu and depletes inhibitory cellpopulations (T regulatory cells Tregs) 8, 89–91.IL7,15Optimizing the proliferation of NK cellsmainly happened by the cytokine IL-15. As serum levels of both IL-15 and IL-7increases, this depletion allows for inundating levels on the surface of NKcells and CD8 T cells. Both are populations required for optimal tumorclearance.

It remains to be presented in humans how proliferation can promote along-lived population of NK cells. While the non-myeloblative conditioningregimen results in serum increases of IL-15 and IL-7, the response is limitedand the levels rapidly decrease after 1 week. Because of side effects andexpansion of Tregs that accompanies systemic IL-2 therapy, alternativecytokines have been sought to effectively expand lymphocytes in vivo.

The mostrecent advance in allogeneic NK cell therapy for AML includes an exogenousIL-15 currently being tested in Phase 1 dose escalation trials at the Universityof Minnesota (see and search NCT01385423). Patients withrefractory AML are treated with lymphodepleting chemotherapy, allogeneic NKcells and daily infusion of IL-15 for 10 days. An IL-15 dose has beenidentified for further study.IL10Researchers reported that very highexpression of IL-10 in a pattern that reflects the ‘proliferation-inducedconditioning’ observed within murine NK cells and which acts to suppressadaptive immunity. Importantly, higher numbers of NK cells at 14 days aftertransplant are associated with a reduced risk of acute GVHD.

MSC and NK therapyBone-marrow-derived MSCs (BM-MSCs) caninhibit NK cell proliferation, cytotoxicity, and cytokine production bysecreting IDO1, TGFb, HLA-G, and PGE2 (Casado et al., 2013; Krampera et al.,2006; Rasmusson et al., 2003; Spaggiari et al., 2008). However, they can alsobe lysed by activated NK cells, depending on their expression of activating NKreceptor ligands, including MHC class I polypeptide-related sequence (MICA, B),UL16 binding proteins (ULBPs), CD112, and CD155.

Mesenchymal Stem Cells (MSCs) showspleiotropic utilities factors with immunosuppressive activity involved incancer progression. We observed that T cell derived MSCs were more powerfullyimmunosuppressive than NK-MSCs and affected both NK function and phenotype byCD56 expression. T-MSCs shifted NK cells toward the CD56dim phenotype anddifferentially modulated CD56bright/dim subset functions. However MSCs affectedboth degranulation and activating receptor expression in the CD56dim subset,they mainly inhibited interferon-gama production in the CD56bright subset. Pharmacological inhibition ofprostaglandin E2 (PGE2) synthesis and, in some MSCs, interleukin-6 (IL-6)activity restored NK function, whereas NK cell stimulation by PGE2 alonemirrored T-MSC-mediated immunosuppression. Our observations provide insightinto how stromal responses to cancer reduce NK cell activity in cancerprogression.

the spectrum of MSC immunosuppressiveactivity in humans includes secretion of human leukocyte antigen (HLA-G),transforming growth factor b (TGFb), prostaglandin E2 (PGE2), tumor necrosisfactor alpha-inducible protein 6 (TNFAIP6/ TSG-6), heme oxygenase 1(HO-1/HMOX1), IL-10, IL-6, indoleamine 2,3-dioxygenase 1 (IDO1), hepatocytegrowth factor (HGF), and leukemia inhibitory factor (LIF) as well as programmeddeath ligand (PD-L1/2) and Fas ligand (FasL) signaling.The finding that MSCs could inhibit theexpression of activating receptors on the surface of NK cells was indicative ofa possible loss of cytotoxic activity known to involve engagement of causingreceptors. To assess a possible MSC-mediated inhibitory effect on the lyticpotential of NK cells, researchres achieved cytolytic assays in differentNK-cell populations from different donors were used as effectors aftershort-term culture with 100 U/mL IL-2 either in the presence or in the absenceof MSCs. MSCs were originally shown to have  strong inhibitory effect on T-cell activationand function. In recent years, inhibition also has been observed on dendriticcells (DCs),B cells,and NK cells.

In this framework, researchers informed thatMSCs can block the IL-2–induced proliferation of fresh peripheral blood NKcells. the use of MSCs may become a common approach in BM transplantation notonly for their possible beneficial effect on the engraftment of hematopoieticstem cells,but also for their immunosuppressive potential.On the other hand, NKcells have been shown to play a central role in the successful outcome ofhaploidentical BM transplantation to treat AML.NK cells derived from the HSCsof the donor can exert a direct GVL effect, provided they express KIRs that donot recognize one or more HLA class I alleles of the patient.

Recent studies reported that NK-MSCinteractions not only provided  strongMSC-mediated anti proliferative effect on NK cells but also verified thatIL-2–activated NK cells can powerfully kill both allogeneic and autologousMSCs.  Killing reflects the fact thatMSCs are characterized by low levels of HLA class I antigens and also expressseveral ligands recognized by activating NK receptors. In the present study, NK cells and MSCs werederived from different donors (because MSCs were obtained from the BM ofpediatric patients, from whom it was not possible to obtain sufficient numbersof fresh NK cells). Though, as mentioned above, the results of the interactionbetween NK cells and autologous or allogeneic MSCs were fuzzy. Consequently, itis believable that also in an autologous MSCs would inhibit NK-cell functionfor kill cancererous cells. These data should be taken into account indesigning novel protocols of adoptive immunotherapy in both MSCs and NK cellscan be infused into the patient to improve the clinical outcome of HSCT.

Actually,the adoptive transfer of activated NK cells could potentially kill MSCs ifthese are infused shortly before or simultaneously with NK cells. In addition,MSCs could inhibit NK-cell proliferation and function. In conclusion, ourpresent study clearly shows that in addition to inhibiting NK-cellproliferation, MSCs noticeably suppress major NK functions, such as cytolyticactivity and cytokine production. Moreover this could have negative effects onthe NK-mediated GVL, mainly in the haploidentical HSC transplantation setting.Nevertheless, it is obvious that more confirmation of the relevance of in vitrofindings will need suitable in vivo studies in animal modelsNK cell production under Good ManufacturingPractice (GMP) conditionsNK products has changed over the years. Giventhe safety of apheresis methods for the donor, we have replaced a 3-hourapheresis product with a 5-hour product depleted of T cells and B cells usingCD3 and CD19 beads.

GMP cell processing resulted in a significant reduction of Tcells in all products, decreasing to < 1% following CD3-depletion, yieldinga final T cell dose of <3 × 105 cells/kg. There was an average of 40-foldless T cells than NK cells. Monocytes (sometimes >50%) comprised the othermajor component of the final product. While monocytes express IL-15 receptoralpha important for trans-presentation of IL-15, we do not yet understand theircontribution to successful adoptive transfer.

Although 5-hour apheresis allowsfor enhanced NK cell doses up to 20 × 106 cells/kg, definitive studies need tobe done to determine if differences in dose have an effect. In using ex vivoexpanded products, up to 1 × 108 cells/kg have been infused without majortoxicities 102. Depletion of CD3 cells below 0.1% prevents transfer of T cellsleading to GVHD. Depletion of CD19+ B cells prevents passenger lymphocytesyndrome and autoimmune phenomena. We observed passenger lymphocyte syndrome in2 patients prior to B-depletion 103.

We also recognized that transfer ofEBV-transformed B cells leading to donor-derived posttransplantlymphoproliferative disorder could be prevented.Future perspectives6.1 Genetic modification and alternativesources of NK cell productsTo overcome restrictions of the donor-derivedNK cell therapies, several groups have investigated alternative donor sourcesincluding UCB, NK cell lines and pluripotent stem cells. If cryopreservationcan be optimized, the quick availability of an off-the-shelf product denotes asignificant step forward. Further advantages include the ability to performpreclinical testing and to select for donors based on favorable characteristicsincluding optimal KIR-genotype 131.6.2 UCB-derived NK cellsUCB progenitors provide a rich source ofhematopoietic progenitor cells and serve as an important in vitro system forstudying the development of human NK cells 132.

Clinically appropriate dosesof UCB-derived NK cells can be generated without the use of feeder cells incompare to NK cells derived peripheral blood 106, 133. NK cells generatedfrom UCB contain a mixture of immature and mature cells that produce cytokinesand show cytotoxicity 116. Development of functional NK cells (e.g. CD34isolation, in vitro expansion) takes up to 4 weeks and requires processing in aGMP facility. Studies are uncompleted and preliminary data is insufficient toassess comparative advantages.

Yoon, et. al, have tested an approach usingCD34+ cells from adult donors. Fourteen patients received donor-derived NKcells that were differentiated in vitro in the presence of stem cell factor,FLT3 ligand, IL7 and hydrocortisone (HDC), followed by IL-7, IL-15 and HDC. Theinfusions were given ~ 6–7 weeks after transplant in the outpatient setting134.

Infusions were tolerated and no toxicity was observed except occasionalalanine aminotransferase elevation (grade III) in two patients and developmentof grade II skin GVHD in one patient, although the concurrent discontinuation ofimmunosuppression suggests that the NK cells were not responsible.6.3 NK cell linesMany research teams have explored the use ofcell lines derived from malignant NK cell clones (i.e. NK-92, NKL, KYHG-1, YT,NKG). NK cell lines keep some level of direct cytotoxic function and usuallylack expression of inhibitory KIR. Because they can be grown in culture,genetic modification with different cytokine genes or chimeric antigenreceptors is easily accomplished.

Among the lines, NK-92 cells remain the most establishedand have been tested in clinical trials that include patients with renal cellcarcinoma and malignant melanoma 135. In a phase I dose escalation studytreating 12 patients, investigators reported only transient toxicities andstable disease in 33% of patients. However, the in vivo activity of the NK-92cells was difficult to establish 100.

Additional data are needed concerningthe in vivo persistence of these infused lines. Because of their amenability toex vivo manipulation, these cell lines may provide an important platform tofacilitate whole-body in vivo imaging of infused cells. Appropriate technologyremains to be developed.

6.4 NK cells derived from pluripotent stemcellsPluripotent stem cells are available anadditional source of NK cells. These include human embryonic stem cells (hESCs)and induced pluripotent stem cells (iPSCs) 131, 136. Novel methods of iPSCgeneration have approached 100% efficiency, thus bringing closer the day thathematopoietic-based therapies derived from these lines become available forclinical use. A defined method for producing NK cells from hESCs and iPSCsamenable to clinical translation has been recently established 137.

Byadapting a feeder-free differentiation system, mature and functional NK cellscan be generated in a system agreeable to clinical scale-up. Significantly, incontrast to UCB-CD34+ derived NK cells or NK cell lines, the iPSC-derived NKcells maintained high levels of KIR and CD16 expression. If KIR expression doesindeed dictate acquisition of final effector function, some of the relativeadvantages of using iPSC-derived NK cells for anti-cancer therapies areclarified.

Using this improved differentiation method, it is estimated that one6-well plate of hESCs or iPSCs could provide enough NK cells to treat severalpatients at the PB-NK doses currently used 89, 137. Other advantages contain:1) unlimited source of KIR-typed NK cells foradoptive immunotherapy, 2) high level of function in preclinicalanimal models 3) a platform genetically responsive tomodify the therapy based on the patient’s cancer via tumor-specific receptors(TCRs or CARs) 138. At the present, however, using iPSCs on apatient-specific basis is impossible.

Third party iPSC-derived NK cells aresubject to immune rejection in the recipient. To circumvent this limitation,specific genetic modulation must be used to decrease immunoreactivity of theinfused cells 139. Recently, Schwartz have shown the use ofhESC-derived retinal pigmented epithelial cells to be safe and potentiallyeffective in treating patients with macular degeneration, thus providing proofof concept for this cell source type 140 6.5 Bi- and Tri-specific antibodiesimprovements in recombinant technology andantibody production have led to a new class of therapeutics which use eitherall, or part, of the antibody structure to mediate enhanced effector activityat the tumor site 120. These include the fusion of two (bi-specific) or three(tri-specific) portions of the fragment of antigen-binding (Fab) region of atraditional antibody.

These reagents keep a high level of antigen specificity,but are derived from a moderately small segment of DNA and therefore offer thesignificant flexibility of swapping different reagents. The reagents serve tocrosslink specific tumor antigens (e.g.

CD19, CD20, CD33) with a potentstimulator on the effector cell (e.g. CD3, CD16, TCR) 120, 141, 142. Themajor advantage of this technology is flexibility in selecting from a number ofimmune effector cells (CD16 on NK cells, CD3 on T cells) as well as from avariety of tumor antigens (CD19, EpCAM, Her2/neu, EGFR, CEA, CD33, EphA2, andMCSP). We have focused on a platform using bispecific killer engagers (BiKEs)constructed with a single-chain Fv against CD16 and a single-chain Fv against atumor-associated antigen 143–145. Using CD16 ×19 BiKEs and a trispecific CD16×19 ×22 (TriKE), we have shown that CD16 signaling is potent and delivers adifferent signal comparable with natural recognition of rituximab, especiallyin regard to cytokine production. Flexibility and ease of production areimportant advantages of the BiKE and TriKE platform. We have recently developeda CD16 × 33 BiKE to target myeloid malignancies (AML and myelodysplasticsyndrome).

One of the most remarkable properties of this drug is its potentsignaling. In refractory AML, we found that CD16 × 33 BiKE overcomes inhibitoryKIR signaling, leading to potent killing and production of cytokines by NKcells 144. Interestingly, ADAM17 inhibition enhances CD16 × 33 BiKE responsesagainst primary AML targets. These immunotherapeutic approaches will bedeveloped for clinical testing for hematologic malignancies and will allow forNK cell activation via CD16 while approximating NK cells in direct contact withtargeted tumor cells In contrast to other therapies aimed at redirecting immunecells, such as chimeric antigen receptor (CARs), the effect of bi-specificantibodies can be titrated while maintaining specificity. Thus, the likelihoodof persistent B cell aplasia, which occurs with CD19 CART cells, is reduced,decreasing the risk of lifelong hypogammaglobulinemia. One limitation of this therapeutic approach is the very short half-life of bi and tri–specific antibodies,which potentially limits trafficking to all tissues.

conclusionClinical applications of NK cells has beeninspired by recognition of their potent anticancer activity. The studiesdiscussed above provide a solid basis for development of future NK cell trialsfor cancer therapy while minimizing risks and toxicities (Figure 1). Importantquestions remain to be answered including, most urgently, determination ofminimum in vivo NK cell expansion needed for clinically effective anti-tumoractivity. At present, outcomes involving NK cell expansion interventions remainunpredictable. Furthermore, NK therapy for solid tumors is limited by uncertainhoming and domination by an immunosuppressive, tumorinduced microenvironmentwhich may interfere with immune responses.

To advance NK cell therapies, bothfurther study of basic NK biology as well as a better understanding ofinteractions with other immune cells will be required. NK cell productcharacteristics and effective cytokine cocktail proportions will likely varyfor different tumor types and patient populations. Targeting through CD16remains a powerful and attractive way to increase specificity, rivaling that ofgenetically modified T cells. Future clinical trials will be designed toexploit strategies to overcome the host immune barriers. Similarly, strategiesto explore ex vivo NK cell expansion from blood, lymphoid progenitors, orpluripotent progenitor cells are being tested. In HCT, prospective studies arecurrently evaluating donor NK cell immunogenetics.

Strategies to apply CMV-inducedshaping of the immune response to enhance NK cell function are in development.


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