The natural killer cells have been generally

The last years have seen remarkable advancesin the engineering of immune cells as cancer therapy. Whereas chimeric antigenreceptors (CARs) have been used comprehensively to convey the specificity ofautologous T cells against hematological malignancies with remarkable clinicalresults, studies of CAR-modified natural killer cells have been generally inpreclinical phases12. NK cells for adoptive therapy can bederived from several different sources which is explained in other parts.

Allogeneic NK cells can be generated from the Peripheral blood of healthydonors or expanded from umbilical cord blood. Regardless of the source, thereare several features of expanded, activated CB, or PB-derived NK cells thatmake them useful effectors for 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-2345. While non-viral expression techniques suchas nucleofection or electroporation can produce robust CAR-mediated killing,the short-lived nature of these CAR molecules would likely dictate the need forrepeated infusions in the clinical setting. 67NKG2D 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 variability897. These activated NK cells are prepared withKIRs and are “licensed to kill.

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” in vivo expansion and persistence capacity ofNK cells is clearly associated with antitumor activity in trials involvinghematologic malignancies such as AML1011. Moreover, ex vivo expanded primary human NKcells produce a different storms of cytokines more than T cells, includinginterferon (IFN)-g, IL-3, and granulocyte macrophage colony-stimulating factor(GM CSF), which may be associated with a lower risk of CRS(cytokine releasedsyndrome)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 .

During the NK development and peripheral maturation, the CD56dim NKcells lose NKG2A expression but up-regulate the expression of the activatingNKG2C receptor, killer cell inhibitory immunoglobulin-like receptors (KIRs) andCD5712. The allo reactivity of NK cells is determinedby various receptors including the activating CD94/NKG2C and the inhibitoryCD94/NKG2A receptors, which both recognize the non-classical human leukocyteantigen E (HLA-E. studies  showed that NKcells expressing the activating CD94/NKG2C receptor are significantly reducedin patients after alloSCT with severe acute and chronic graft-versus-hostdisease (GvHD). Moreover, the ratio of CD94/NKG2C to CD94/NKG2A was reduced inpatients with severe acute and chronic GvHD after receiving an HLA-mismatchedgraft. Collectively, these results provide evidence for the first time thatCD94/NKG2C is involved in GvHD prevention13.

Cytokines in 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 cells14.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 patients with metastatic melanoma15. These and other studies have contributedimportant new knowledge: 1) high-dose IL-2 used in vivo with the goal ofactivating NK cells has significant but manageable toxicity owing to severecapillary leak syndrome, whereas low-dose subcutaneous IL-2 was well tolerated,2) lymphodepleting chemotherapy using high-dose cyclophosphamide andfludarabine facilitated in vivo expansion of autologous adoptively transferredcytotoxic T lymphocytes and lead to enhanced efficacy,3) chemotherapy induceslymphopenia, changes the competitive balance between transferred lymphocytesand endogenous lymphocytes, changes the cytokine milieu and depletes inhibitorycell populations (T regulatory cells Tregs)16.IL7,15Optimizing the proliferation of NK cellsmainly happened by the cytokine IL-1517. 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 week18. Because of side effects and expansion ofTregs that accompanies systemic IL-2 therapy, alternative cytokines have beensought to effectively expand lymphocytes in vivo. The most recent advance inallogeneic NK cell therapy for AML includes an exogenous IL-15 currently beingtested in Phase 1 dose escalation trials at the University of Minnesota (seeClinicalTrials.gov and search NCT01385423).

Patients with refractory AML aretreated with lymphodepleting chemotherapy, allogeneic NK cells and dailyinfusion of IL-15 for 10 days. An IL-15 dose has been identified for furtherstudy19.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 GVHD20.

MSC and NK therapyBone-marrow-derived MSCs (BM-MSCs) caninhibit NK cell proliferation, cytotoxicity, and cytokine production bysecreting IDO1, TGFb, HLA-G, and PGE2 2122.However, they can also be lysed by activatedNK cells, depending on their expression of activating NK receptor ligands,including MHC class I polypeptide-related sequence (MICA, B), UL16 bindingproteins (ULBPs), CD112, and CD15523.Mesenchymal Stem Cells (MSCs) showspleiotropic utilities factors with immunosuppressive activity involved incancer progression24. This is observed that T cell derived MSCswere more powerfully immunosuppressive than NK-MSCs and affected both NKfunction and phenotype by CD56 expression. T-MSCs shifted NK cells toward theCD56dim phenotype and differentially modulated CD56bright/dim subset functions.However MSCs affected both degranulation and activating receptor expression inthe CD56dim subset, they mainly inhibited interferon-gama  production in the CD56bright subset.Pharmacological inhibition of prostaglandin E2 (PGE2) synthesis and, in someMSCs, interleukin-6 (IL-6) activity restored NK function, whereas NK cellstimulation by PGE2 alone mirrored T-MSC-mediated immunosuppression.

Ourobservations provide insight into how stromal responses to cancer reduce NKcell activity in cancer progression22.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) signaling2526.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 fuzzy21. Consequently, it is believable that also inan autologous MSCs would inhibit NK-cell function for kill cancererous cells.These data should be taken into account in designing novel protocols ofadoptive immunotherapy in both MSCs and NK cells can be infused into thepatient to improve the clinical outcome of HSCT21.

NK cell production under Good ManufacturingPractice (GMP) conditionsNK products has changed over the years. Giventhe safety of apheresis methods for the donor, it has been replaced replaced a3-hour apheresis product with a 5-hour product depleted of T cells and B cellsusing CD3 and CD19 beads. GMP cell processing resulted in a significantreduction of T cells in all products, decreasing to < 1% followingCD3-depletion, yielding a final T cell dose of <3 × 105 cells/kg.

There wasan average of 40-fold less T cells than NK cells. Monocytes (sometimes >50%)comprised the other major component of the final product. While monocytesexpress IL-15 receptor alpha important for trans-presentation of IL-15, it doesnot yet understand their contribution to successful adoptive transfer. Although5-hour apheresis allows for enhanced NK cell doses up to 20 × 106 cells/kg,definitive studies need to be done to determine if differences in dose have aneffect. In using ex vivo expanded products, up to 1 × 108 cells/kg have beeninfused without major toxicities .

Depletion of CD3 cells below 0.1% preventstransfer of T cells leading to GVHD. Depletion of CD19+ B cells preventspassenger lymphocyte syndrome and autoimmune phenomena. We also recognized thattransfer of EBV-transformed B cells leading to donor-derived post transplantlymphoproliferative disorder could be prevented.Future perspectives1.

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-genotype276 .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.

Clinically appropriate doses ofUCB-derived NK cells can be generated without the use of feeder cells in compareto NK cells derived peripheral blood28 . NK cells generated from UCB contain amixture of immature and mature cells that produce cytokines and showcytotoxicity .Development of functional NK cells (e.g. CD34 isolation, in vitroexpansion) takes up to 4 weeks and requires processing in a GMP facility.

Studies are uncompleted and preliminary data is insufficient to assesscomparative advantages29. 3. NK cell linesMany research teams have explored the use ofcell lines derived from malignant NK cell clones ( NK-92, NKL, KYHG-1, YT, NKG)26. NK cell lines keep some level of directcytotoxic function and usually lack expression of inhibitory KIR. Because theycan be grown in culture, genetic modification with different cytokine genes orchimeric antigen receptors is easily accomplished. Among the lines, NK-92 cellsremain the most established and have been tested in clinical trials thatinclude patients with renal cell carcinoma and malignant hematologicalmalignancies. .

Because of their amenability to ex vivo manipulation, these celllines may provide an important platform to facilitate whole-body in vivoimaging of infused cells. Appropriate technology remains to be developed. 30 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). Novel methods of iPSC generationhave 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 31.

By adapting a feeder-free differentiationsystem, mature and functional NK cells can be generated in a system agreeableto clinical scale-up. Significantly, in contrast to UCB-CD34+ derived NK cellsor NK cell lines, the iPSC-derived NK cells maintained high levels of KIR andCD16 expression. If KIR expression does indeed dictate acquisition of finaleffector function, some of the relative advantages of using iPSC-derived NKcells for anti-cancer therapies are clarified. Using this improveddifferentiation method, it is estimated that one 6-well plate of hESCs or iPSCscould provide enough NK cells to treat several patients at the PB-NK dosescurrently used . 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)32. 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 cells6.5. 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. 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 reagents33. The reagents serve to crosslink specifictumor antigens (e.g.

CD19, CD20, CD33) with a potent stimulator on the effectorcell (e.g. CD3, CD16, TCR)34 . The major advantage of this technology isflexibility in selecting from a number of immune effector cells (CD16 on NKcells, CD3 on T cells) as well as from a variety of tumor antigens (CD19,EpCAM, Her2/neu, EGFR, CEA, CD33, EphA2, and MCSP)33.there have focused on a platform usingbispecific killer engagers (BiKEs) constructed with a single-chain Fv againstCD16 and a single-chain Fv against a tumor-associated antigen. Using CD16 ×19BiKEs and a trispecific CD16 ×19 ×22 (TriKE), it has been showed that CD16signaling is potent and delivers a different signal comparable with naturalrecognition of rituximab, especially in regard to cytokine production.Flexibility and ease of production are important advantages of the BiKE andTriKE platform35.

It s been recently developed a CD16 × 33BiKE to target myeloid malignancies (AML and myelodysplastic syndrome)5. One of the most remarkable properties ofthis drug is its potent signaling. In refractory AML, this is  found that CD16 × 33 BiKE overcomes inhibitoryKIR signaling, leading to potent killing and production of cytokines by NKcells36. Interestingly, ADAM17 inhibition enhancesCD16 × 33 BiKE responses against primary AML targets37. 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. One limitation  of this therapeutic approach is the veryshort half-life of bi and tri –specific antibodies,which potentially limitstrafficking to all tissues35.conclusionClinical applications of NK cells has beeninspired by recognition of their potent anticancer activity. The studiesdiscussed  providing  a solid basis for development of future NKcell trials for cancer therapy by minimizing risks and toxicities.

Importantquestions remain to be answered, most urgently, determination of minimum invivo NK cell expansion needed for effective anti-tumor activity in clinical. Atpresent, upshots involving NK cell expansion interventions remain capricious. Also,NK therapy for solid tumors is limited by uncertain homing and domination by animmunosuppressive, tumor induced microenvironment that may interfere withimmune responses.

To improve and progress NK cell therapies, both further studyof basic NK biology as well as a better understanding of interactions withother immune cells will be required. NK cell products characteristics andeffective cytokine cocktails with optimal proportions will probably differ fromdifferent tumor types and patients. Targeting CD16 remains an attractive way toincrease specificity, resembling of genetically modified T cells. Futureclinical trials will be designed to exploit strategies to overcome the hostimmune barriers. In the same way, strategies to discover ex vivo NK cell expansionfrom blood, lymphoid progenitors, or other sources are being tested. In Hematopoeticstem cell transplantation, future studies are evaluating donor NK cellimmunogenetics. 

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