Adoptive Transfer Of Natural Killer Cells Research Articles

Uncompromised NK cell activation is essential for virus-specific CTL activity during acute influenza virus infection

Natural killer (NK) cells are indispensable components of both the innate and adaptive immune response. However, their precise roles in the cross-talk between innate and adaptive immunity during influenza virus infection remain controversial. By comparing NK cell dynamics and activity under a sub-lethal dose and high dose of influenza virus infection, we showed that influenza virus PR8 directly infected NK cells during natural infection, which was consistent with our previous findings obtained from an in vitro investigation of human NK cells. The impairments in cytotoxicity and IFN-γ production by spleen NK cells following high-dose infection were accompanied by decreased virus-specific killing mediated by cytotoxic T lymphocytes (CTLs). Importantly, the weakened CTL activity could be reversed by adoptive transfer of spleen NK cells harvested from low-dose-infected mice but not healthy donors. Taken together, our data provide direct evidence supporting the contribution of NK cells to antiviral T-cell responses. This study also indicates that a novel NK-targeted immune evasion strategy is used by influenza virus to shrink both innate and adaptive immune responses.

Role of Recipient CD8+ T Cell Exhaustion in the Rejection of Adoptively Transferred Haploidentical NK Cells

Natural killer (NK) cells are cytotoxic innate lymphoid cells, which play a major role in tumor surveillance. We have tested the safety and efficacy of allogeneic NK cell adoptive transfer from heathy haploidentical donors and demonstrate that in vivo expansion and persistence of the adoptively transferred NK cells at Day 14 after infusion correlates with 30-50% remission in patients with refractory AML. However, the factors that influence successful persistence of donor-derived NK cells are unclear. We hypothesized that recipient T cells play a role in the rejection of allogeneic NK cells and a correlation could exist between persistence of donor-derived NK cells and exhaustion in recipient T cells. T cell exhaustion, a well-established state of T-cell dysfunction occurring in response to chronic and continuous antigen stimulation, is well-documented in human cancer, and characterized by progressive and hierarchical loss of effector functions including sustained up-regulation and co-expression of multiple inhibitory receptors such as PD-1 and Tim-3 and altered expression of key transcription factors including the gain of Eomes and T-bet. We used samples from a phase I/II trial of CD3/CD19 depleted, IL-15-activated, haploidentical donor NK cells delivered following conditioning with cyclophosphamide (50mg/kg) and fludarabine (35 mg/m2 x 3days) in adults with chemotherapy refractory AML. Patients received donor NK cells on Day 0 followed by 10 doses of recombinant human (rh) IL-15 (2 mcg/kg/day) manufactured by the NCI and delivered SQ on Days 1-5 and 8-12. A significant proportion of patients experienced donor NK cell expansion at Day 14 (expanders), but there were some that did not (non-expanders). Therapeutic benefit has only been noted among the expanders. We examined samples from a total of 10 patients with refractory AML, 5 expanders and 5 non-expanders, along with their 10 respective donors. Cryopreserved patient PBMCs were thawed and rested overnight in RPMI-1640 with 2% FBS. The cells were stained for viability, for surface markers using antibodies against CD3, CD8, CD56, PD-1, and Tim-3, intracellularly stained for Eomes and T-bet. We evaluated CD8+ T cell expression of PD-1 and Tim-3, in expanders and non-expanders, prior to chemotherapy and at Day 14. Paired donor T cells from the non-mobilized apheresis products served as controls. Prior to chemotherapy, both patient groups had equivalently elevated expression of both PD1 and Tim-3 on CD8+ T cells. However, at Day 14, the expanders had persistence of PD-1 and Tim-3 while expression on non-expander CD8+ T cells fell to donor level (Figure 1A). Furthermore, expanders had a significantly higher proportion of CD8+ T cells that either co-expressed PD-1 and Tim-3 (p=0.017) or had a PD-1high phenotype (p=0.032) at Day 14, both of which are suggestive of an exhausted state, as opposed to an activated one (Figure 1B,C). Next, we examined Eomes and T-bet expression in recipient T cells. While generally low among healthy T cell populations, as T cells become exhausted, they gain expression of these transcription factors. We looked specifically at the expression of these transcription factors in the recipient CD8+ T cell populations with the highest likelihood of being exhausted, i.e. those co-expressing PD-1 and Tim-3 or those with the PD-1high phenotype. Eomes expression in recipient PD-1high CD8+ T cells and in PD-1+Tim-3+ CD8+ T cells at Day 14 was significantly higher (p=0.01 and p=0.04, respectively) among expanders compared to non-expanders (Figure 2A,B). Likewise, T-bet expression was greater (p=0.004) among expanders in the PD-1high population (Figure 2A). There was no difference in the T-bet expression in PD-1+Tim-3+ CD8+ T cells between groups (Figure 2B). While all patients with refractory AML receiving NK cell adoptive transfer had an elevated percentage of CD8+ T cells with an exhausted phenotype prior to therapy, only patients with donor-derived NK cell expansion had persistence of the exhausted T cell phenotype at Day 14. Thus, T cell mediated rejection is a major obstacle to overcome for successful adoptive NK cell transfer which could in part be aided by a link between recipient T cell exhaustion and expansion of NK cells. This might further suggest that IL-15 reverses T cell exhaustion among those who failed to achieve donor-derived NK cell expansion. Disclosures Miller: Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

Infusion of Umbilical Cord Blood-Derived Natural Killer Cells in Immune-Deficient Mice for the Treatment of Resistant Acute Myeloid Leukaemia

Adoptive transfer of allogeneic natural killer (NK) cells represents a promising treatment approach against acute myeloid leukaemia (AML). Success of this NK cell immunotherapy is dependent on obtaining high numbers of functional NK cells that have the potential to survive in vivo. The use of umbilical cord blood (UCB) CD34+ cells as a source of allogenic NK cells is an interesting method that can generate a readily available, non-invasive, off the shelf cellular product.We developed a cytokine-based culture method for the generation of NK cell products derived from CD34+hematopoietic progenitor cells (HPC) isolated from fresh UCB units. Immuno-phenotyping of ex vivo expanded NK cells showed typical inhibitory and activating NK receptors except for CD16 and the KIR receptors.UCB-derived NK cells displayed good cytolytic activity against NK-sensitive K562 cells with a percentage of specific lysis varying from 30 to 50%. Cytolysis was directly correlated to CD94 expression since CD94-sorted NK cells were responsible for all the in vitro cytolytic function of differentiated NKs against K562 cells. There was an inconstant susceptibility of patient-derived primary AML cells to UCB-derived NK lysis in vitro with a specific lysis ranging from 0 to 25%.We further characterized UCB-derived NK cells by investigating their toxicity, biodistribution, and anti-leukemic potential in vivo. As adoptive transfer of NK cells is an attractive approach for treating refractory leukemia, immune deficient mice were engrafted with a patient derived AML strain resistant to NK-mediated lysis and doxorubicin.After successful engraftment and randomization, leukemic mice were injected with either UCB- derived NK cells or NK cells from healthy donors (NKhds) or doxorubicin, with one control group that didn't receive any treatment. Mice were sacrificed after 2 weeks of treatment and leukemia load along with NK distribution were evaluated by flow cytometry in the blood, bone marrow (BM) and spleen.There was no evidence of toxicity of UCB-derived or healthy donors NK cells in mice. Both types of cells were preferentially found in the blood and in the spleen, even though NKhds reached much higher levels than UCB-derived NKs.As for treatment efficacy, none of our treatment showed anti-leukemic potential based on the absence of decrease of leukemic cells in BM, blood, and spleen. In vivo microenvironment didn't overcome resistance of the patient derived AML cell to NK lysis or to doxorubicin.Remarkably, all of the UCB derived NK cells founded in vivo expressed the CD94 whereas not more than 20% of the injected cells were positive for this marker. Whether it was by in vivo selection or by in vivo differentiation must be investigated.Interestingly, a small cell population with CD56 and CD34 double staining was distinguished in UCB-derived NK and NK healthy donor treated leukemic mice suggesting in vivo interaction between leukemic and NK cells. Further characterization of this population may help to understand the molecular mechanism of leukemic recognition by NK cells and resistance of leukemic cells to cytolysis.In conclusion, UCB-derived NK generation is feasible. Investigation of the role of CD94 in these cells is needed, as cell sorting by CD94 selection in addition to the CD56 could be an interesting approach in the future to select highly functional expanded NK cells before therapeutic use. Furthermore, infusion of UCB-derived NK cells into immune-deficient mice is achievable and non-toxic. However, in vivo environment didn't overcome primary in vitro resistance of AML cells despite an established interaction. Additional elucidation of AML resistance mechanisms to NK lysis is mandatory before therapeutic application. DisclosuresNo relevant conflicts of interest to declare.

Targeting Ewing sarcoma with activated and GD2-specific chimeric antigen receptor-engineered human NK cells induces upregulation of immune-inhibitory HLA-G

ABSTRACTActivated and in vitro expanded natural killer (NK) cells have substantial cytotoxicity against many tumor cells, but their in vivo efficacy to eliminate solid cancers is limited. Here, we used chimeric antigen receptors (CARs) to enhance the activity of NK cells against Ewing sarcomas (EwS) in a tumor antigen-specific manner. Expression of CARs directed against the ganglioside antigen GD2 in activated NK cells increased their responses to GD2+ allogeneic EwS cells in vitro and overcame resistance of individual cell lines to NK cell lysis. Second-generation CARs with 4-1BB and 2B4 co-stimulatory signaling and third-generation CARs combining both co-stimulatory domains were all equally effective. By contrast, adoptive transfer of GD2-specific CAR gene-modified NK cells both by intratumoral and intraperitoneal delivery failed to eliminate GD2-expressing EwS xenografts. Histopathology review revealed upregulation of the immunosuppressive ligand HLA-G in tumor autopsies from mice treated with NK cells compared to untreated control mice. Supporting the relevance of this finding, in vitro co-incubation of NK cells with allogeneic EwS cells induced upregulation of the HLA-G receptor CD85j, and HLA-G1 expressed by EwS cells suppressed the activity of NK cells from three of five allogeneic donors against the tumor cells in vitro.We conclude that HLA-G is a candidate immune checkpoint in EwS where it can contribute to resistance to NK cell therapy. HLA-G deserves evaluation as a potential target for more effective immunotherapeutic combination regimens in this and other cancers.

Interleukin (IL)-15 superagonist mediates immunotoxicity by inducing hyperproliferation of activated NK cells and production of IFNγ

Abstract Interleukin (IL)-15 is currently undergoing clinical trials to assess its anti-tumor efficacy. IL-15 is essential for the generation, function and survival of natural killer (NK), natural killer T (NKT) and memory phenotype (MP) CD8+T cells. The combination of IL-15 with its soluble IL-15 receptor α (IL-15Rα) generates a complex termed IL-15 superagonist (IL-15 SA) that exhibits more profound biological activities on NK, NKT and MP CD8+T cells than IL-15 alone. Thus, IL-15 SA is a potentially more attractive therapeutic agent for use against advanced tumors. However, the safety of IL-15 SA administration in vivo is not clearly elucidated. This study was designed to evaluate toxic consequences of IL-15 SA in mice. Administration of 2 μg IL-15 SA for 4 days caused a time- and dose-dependent immune-toxicity, characterized by hypothermia, weight loss, liver damage and death. At 2ug, IL-15 SA expanded NK cells in spleens and livers compared to vehicle and significantly increased NK cell expression of CD69, NKG2D and NKp46 as well as production of IFNγ, granzyme B and perforin. IL-15 SA also prompted NK cell maturity with a preferential expansion of the pro-inflammatory (CD27+ CD11b+) NK subset. Mice depleted of NK cells by anti-asialoGM1or anti-NK1.1 were resistant to IL-15 SA-induced toxicity, whereas adoptive transfer of wild type NK cells to Rag2−/−γc−/− mice re-established IL-15 SA toxicity. While IL-15 SA expanded NKT and mCD8+ T cells, mice depleted of NKT or CD8+ T cells remained sensitive to IL-15 SA-mediated toxicity. In addition, IFNγ KO, but not TNF-α KO or perforin KO, mice were resistant to IL-15 SA-induced toxicity. In conclusion, high-dose IL-15 SA causes systemic immunotoxicity via induction of NK cell proliferation and activation.

Abstract P4-04-12: Enhancing the immunogenicity of breast cancer cells to stimulate innate immunity and augment the effects of cellular immunotherapy

Abstract A promising strategy for treating metastatic breast cancer is engaging the immune system to destroy disseminated breast cancer cells. However, breast cancer is a challenge for immunotherapy because of its inherent genetic heterogeneity and decreased immunogenicity. There is an unmet need to develop new, targeted therapies for breast cancer that stimulate the immune system and can be used in combination with checkpoint blockade or adoptive cell transfer to fight metastatic disease. One potential approach is to generate dying breast cancer cells that operate like a vaccine to stimulate a tumor-specific immune response. This is termed immunogenic cell death (ICD) and is characterized by a unique molecular signature, involving the release of molecules that attract and stimulate phagocytes like dendritic cells (DCs) and that could sensitize tumor cells to killing by natural killer (NK) cells. With the discovery of new ICD-inducing agents, interest in this approach is increasing. However, the current doses of drugs used to induce ICD may be too high to translate into clinically relevant regimens. To address these problems, our group developed a novel cytotoxic peptide, CT20p, and a nanotechnology-based platform to deliver and concentrate CT20p in breast tumors. We found that treatment of a murine xenograft model with nanomolar amounts of CT20p, encapsulated in nanoparticles formed with a novel hyperbranched polyester polymer (HBPE-NPs), resulted in regression of breast cancer tumors, and that dying breast cancer cells expressed markers characteristic of ICD, such as the pre-mortem exposure of the "eat me" signal, calreticulin (Crt). The intracellular target of CT20p is a protein called chaperonin-containing T-complex (CCT), which is essential for the folding of actin and tubulin and other critical proteins into their native forms. Disruption of CCT could cause endoplasmic reticulum (ER) stress through the accumulation of misfolded proteins. ER stress in turn initiates intracellular pathways that generate the danger signals associate with ICD. To this end, we observed that cancer cells treated with CT20p displayed alterations in PERK, a key mediator of the unfolded protein response (UPR) and translocation of Crt. As a result, CT20p-treated cancer cells were more readily phagocytosed. More importantly, NK cells more effectively killed cancer cells pre-treated with CT20p. This suggests that CT20p may stimulate NK cell cytotoxicity by inducing activating signals on cancer cells, which would greatly augment the curative effects of adoptive transfer of NK cells. In our experiments normal breast epithelial cells, macrophages or NK cells, themselves, were unaffected by CT20p treatment due reduced CCT. These studies indicate that a single molecule, CT20p, can do both the killing of cancer cells and activation of immune cells and can be combined with cellular immunotherapy (e.g. expanded NK cells) to provide a more complete protection from breast cancer recurrence and metastasis. Citation Format: Khaled AR, Limaye A, Bassiouni R, Showalter A, Oyer J, Pandey V, Igarashi R, Altomare DA, Copik AJ. Enhancing the immunogenicity of breast cancer cells to stimulate innate immunity and augment the effects of cellular immunotherapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-12.

Mouse Xenograft Model for Intraperitoneal Administration of NK Cell Immunotherapy for Ovarian Cancer.

Natural killer (NK) cells are an attractive cell population for immunotherapy. Adoptive transfer of NK cells has been tested in multiple clinical trials including acute myeloid leukemia (AML) and ovarian cancer, although limitations do exist especially for treatment of solid tumors. In order to overcome these limitations, mouse xenograft models are needed for evaluation of various NK cell populations, as well as routes of NK cell administration. Here, we describe the methods used for the establishment of an intraperitoneal (ip) ovarian cancer mouse xenograft model with ip delivery of NK cells. This model has been successfully employed with multiple ovarian cell lines and could be applied to other tumor models where the tumor's primary location is in the peritoneal cavity. It is also compatible with multiple routes of NK cell administration. Bioluminescent imaging for monitoring tumor formation and response provides for easy visualization of NK cell tumor inhibition. This xenograft model is superior to other models because the tumor is implanted into the same physiological space where ovarian cancer is found, which allows for improved mimicking of actual disease.

The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Introduction:A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo.Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system..Methods:Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors.An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund.Results:ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2).Discussion:Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. [Display omitted] [Display omitted] DisclosuresWald:Invenio Therapeutics: Equity Ownership.

A Phase I Study of Allogeneic Natural Killer Cell Therapy Generated from Cord Blood Hematopoietic Stem and Progenitor Cells in Elderly Acute Myeloid Leukemia Patients

IntroductionElderly acute myeloid leukemia (AML) patients have a poor prognosis due to high relapse rates following standard therapy. Natural Killer (NK) cell alloreactivity has found to control relapse in AML in the HLA-mismatched haploidentical allogeneic stem cell transplantation (allo-SCT) setting. Moreover, allogeneic NK cell infusions can induce complete remission (CR) inpatients with advanced AML. As a consequence, adoptive NK cell transfer may be a promising treatment for elderly AML patients, who are not eligible for allo-SCT. Most clinical studies exploited NK cell products enriched from leukapheresis of haploidentical donors containing low numbers of T cells that could have contributed to the observed therapeutic effects and potentially induced graft-versus-host disease (GVHD). Therefore, we have developed a GMP-compliant culture system for the generation of large batches of NK cells from umbilical cord blood (UCB)-derived CD34+ progenitor cells, without T cell contamination. Here, we report results of a phase I dose escalation study (Dutch Trial Register nr. NTR2818) to evaluate the feasibility, safety and toxicity of allogeneic UCB-NK cell infusion following an immunosuppressive preparative regimen in elderly AML patients. Secondary endpoints were NK cell lifespan and the effects on minimal residual disease (MRD).MethodsElderly AML patients not eligible for allo-SCT, and in morphologic CR after standard therapy, were given preparative chemotherapy consisting of Cyclophosphamide (Cy;900 mg/m2/day) and Fludarabine (Flu;30 mg/m2/day) on days -6 to -2. At day 0, UCB-NK cells at a dose of 3, 10 or up to 30x106/kg body weight were infused without IL-2 treatment to study if in vivo expansion could be obtained without IL-2 support. Patients were assessed for toxicity and GVHD. Donor chimerism was measured by Q-PCR for discriminating DNA polymorphisms. NK cell expansion and phenotype were analyzed by flow cytometry. MRD was evaluated by flow cytometry and molecular techniques.ResultsTwelve AML patients (68-76 years) have been included, all in morphologic CR after 2 to 3 standard chemotherapy courses (n=6), or 1 standard chemotherapy course followed by subsequent treatment with hypomethylating agents (azacitidine or decitabine) (n=6). Patients were treated with Cy/Flu and an escalating dose of partially HLA-matched UCB-NK cells. Four patients had good/intermediate risk, 4 poor risk and 4 very poor risk AML. To date, 9 patients received NK cell products containing a median of 74% highly activated CD56+ NK cells, with <1x104/kg CD3+ T cells and <3x105/kg CD19+ B cells. Remaining non-NK cells were CD14+ and/or CD15+ monocytic and myelocytic cells. Follow up did not show GVHD or toxicity attributed to the NK cells. As expected, preparative Cy/Flu induced a neutropenic period of 20 ± 16 days, but no severe infections were seen. A temporary repopulation and persistence of UCB-NK cells could be detected in peripheral blood between days 1 and 8 post-infusion, which was associated with increased IL-15 plasma levels observed in most patients. Interestingly, donor chimerism increased with higher doses of infused UCB-NK cells, and donor chimerism up to 3.5% was found in bone marrow (BM) at day 7/8. Further UCB-NK cell maturation in vivo was observed by acquisition of CD16 and KIRs, while expression of activating receptors was sustained. Of the 9 treated patients so far, 5 (56%) are still in CR after 43, 35, 31, 5 and 4 months, whereas 4 patients relapsed after 5, 6 (2 pts) and 15 months. Despite morphologic CR during azacitidine treatment, residual disease of 6-7% with a leukemia-associated phenotype could be detected by flow cytometry before NK cell infusion in BM of two patients. In both patients MRD was reduced to less than 0.05% at 90 days after UCB-NK cell therapy following Flu/Cy conditioning.ConclusionThese results show that GMP-compliant UCB-NK cell products containing up to 30x106 NK cells/kg body weight can be safely infused in non-transplant eligible AML patients following immunosuppressive chemotherapy. After infusion, UCB-NK cells repopulate, mature and migrate to BM without supporting IL-2 infusion. Since we observed reduction in MRD in patients on treatment with hypomethylating agents, this UCB-NK cell therapy may induce or sustain CR in elderly AML patients, and could serve as an alternative consolidation therapy for patients with refractory AML or provide bridge to allo-SCT. DisclosuresSpanholtz:Glycostem Therapeutics: Employment. Tordoir:Glycostem Therapeutics: Employment. Bohme:Glycostem Therapeutics: Employment. Kok:Glycostem Therapeutics: Employment.

Trispecific Killer Engagers (TriKEs) that contain IL-15 to make NK cells antigen specific and to sustain their persistence and expansion

Natural killer (NK) cells are capable of immune surveillance mediated by a balance of activating and inhibitory receptors. We have shown that adoptive transfer of NK cells can induce complete remissions in patients with refractory AML when combined with lymphodepleting chemotherapy and IL-2 to stimulate survival and in vivo expansion of the NK cells. Using this approach, 30-50% of patients with refractory attain clinical remissions. We hypothesize that clinical benefit is limited by the lack of antigen specificity and because IL-2 induces regulatory T cells (Treg) that inhibit NK cell proliferation. We have developed trispecific killer engagers (TriKE) to overcome these limitations. We have previously shown that bispecific killer engagers (BiKEs) are capable of creating immunologic synapses between NK cells and CD33 antigens on AML and MDS targets leading to NK cell signaling through the highly potent CD16 (FcγRIII) receptor. We observed that although CD16 engagement leads to enhanced killing and cytokine production by NK cells, there is no effect on proliferation (designated 1633 in Figure, Panel B). Because IL-15 is the homeostatic factor for NK cell proliferation, survival, activation, and development and because unlike IL-2, it does not stimulate CD4+ CD25+ Treg, we developed a TriKE that includes a modified human IL-15 crosslinker sandwiched between single chain Fv against CD16 and CD33 (designated 161533). The final target gene was spliced into pET21d vector expression plasmid and transformed into the Escherichia coli and compared to a 1633 BiKE with the identical Fv regions as above. TriKEs were recovered from inclusion bodies, refolded, and purified. The addition of the IL-15 crosslinker to the molecule reduced its isoelectric point by two pH units creating more favorable conditions for purification and enhancing yield. Despite identical amounts of starting inclusion bodies, the final yield of 161533 TriKE was twice the yield of 1633 BiKE indicating more favorable purification dynamics. To establish the selectivity of the anti-CD33 binding moiety, BiKEs and TriKEs were analyzed using flow cytometry and both bound to CD16 and CD33 selectively without interference from the IL-15 crosslinker. To determine the capacity of BiKE and TriKE reagents to mediate NK cell killing, activity was compared in a 4-hour chromium release assay (representative of 3 separate experiments) against CD33+ HL60 targets (Figure Panel A) or CD33- HT29 (not shown). The highest specific activity is with the 161533 TriKE containing a modified IL-15 linker as control samples of anti-CD16 and anti-CD33 alone did not augment cytotoxicity while 1633 BiKE mediating killing was intermediate. CD33- HT29 was not killed. We next tested BiKE and TriKE in a proliferation assay using CSFE labeled primary NK cells. NCI produced recombinant human IL-15 was used as a positive control. The TriKE exhibited potent proliferation of primary NK cells compared to BiKE (Figure Panel B). As target cell induced NK cell production of cytokines is an important aspect of the anti-leukemia response, we compared the BiKE and TriKE for their ability to mediate cytokine production with primary NK cells and HL60 targets. TriKE induced greater target cell specific TNFα, INFγ, IL-6, MIP1α, GM-CSF and IL-8 compared to BiKE. We then tested the activity of the TriKE in a xenogeneic model of human NK cell adoptive transfer where NSG mice are given 0.75 million HL60-Luc cells followed by 250 cGy XRT ± 1 million NK cells 3 days later from a CD3 and CD19 depleted clinical product and daily IP injection of 50 mcg/kg TriKE. Significant anti-tumor activity without toxicity was seen in the NK and TriKE treated mice (P<0.002). In addition, the 161533 TriKE had potent in vivo IL-15 activity to support the in vivo persistence and expansion of NK cells while BiKE did not (data not shown). In summary, we have shown that a novel TriKE construct can mediate CD16 directed cytotoxicity against CD33 targets and this is specifically maintained when adding a modified IL-15 linker. The IL-15 activity is fully preserved in vitro and in vivo and unexpectedly enhances the purification product. This will allow us to redirect NK cells to malignant targets while providing cytokine stimulation with an easily exportable off-the-shelf non-gene therapy strategy using a drug that also maintains NK cell persistence, survival and in vivo expansion. Drug production is in progress for clinical testing in 2016. [Display omitted] DisclosuresMiller:Coronado: Speakers Bureau; BioSciences: Speakers Bureau; Celegene: Speakers Bureau.

Improving natural killer cell cancer immunotherapy.

Natural killer (NK) cells are innate lymphoid cells specialized to eliminate malignant cells via direct cytotoxicity and immunoregulatory cytokine production. As such, NK cells are ideal as cellular therapy for cancer patients, and several studies have provided proof of principle that adoptively transferred NK cells can induce remissions in patients with leukemia. A clear understanding of the mechanisms underlying NK cell antitumor responses, including target cell recognition, activation status, and negative regulatory signals will improve NK cellular therapy for cancer patients. Clinical studies have demonstrated the safety and preliminary efficacy of NK cell adoptive transfer, especially in hematologic malignancies. Various NK cell sources, isolation techniques, activation approaches, and ex-vivo expansion strategies are under investigation. New approaches have been developed and are being tested to optimize NK cell therapy, including ways to better target NK cells to malignant cells, increase their functional competence, facilitate expansion in patients, and limit inhibitory signals or cells. NK cells represent a promising cellular immunotherapy for the treatment of cancer. In addition to adoptive cellular therapy, adjunct treatments that optimize NK cell targeting and function will enhance their potency and broaden their potential use to many cancer types.

NK cell-based cancer immunotherapy: from basic biology to clinical application.

Natural killer (NK) cells, which recognize and kill target cells independent of antigen specificity and major histocompatibility complex (MHC) matching, play pivotal roles in immune defence against tumors. However, tumor cells often acquire the ability to escape NK cell-mediated immune surveillance. Thus, understanding mechanisms underlying regulation of NK cell phenotype and function within the tumor environment is instrumental for designing new approaches to improve the current cell-based immunotherapy. In this review, we elaborate the main biological features and molecular mechanisms of NK cells that pertain to regulation of NK cell-mediated anti-tumor activity. We further overview current clinical approaches regarding NK cell-based cancer therapy, including cytokine infusion, adoptive transfer of autologous or allogeneic NK cells, applications of chimeric antigen receptor (CAR)-expressing NK cells and adoptive transfer of memory-like NK cells. With these promising clinical outcomes and fuller understanding the basic questions raised in this review, we foresee that NK cell-based approaches may hold great potential for future cancer immunotherapy.

Low Dose Focused Ultrasound Induces Enhanced Tumor Accumulation of Natural Killer Cells

Natural killer (NK) cells play a vital antitumor role as part of the innate immune system. Efficacy of adoptive transfer of NK cells depends on their ability to recognize and target tumors. We investigated whether low dose focused ultrasound with microbubbles (ldbFUS) could facilitate the targeting and accumulation of NK cells in a mouse xenograft of human colorectal adenocarcinoma (carcinoembryonic antigen (CEA)-expressing LS-174T implanted in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice) in the presence of an anti-CEA immunocytokine (ICK), hT84.66/M5A-IL-2 (M5A-IL-2). Human NK cells were labeled with an FDA-approved ultra-small superparamagnetic iron oxide particle, ferumoxytol. Simultaneous with the intravenous injection of microbubbles, focused ultrasound was applied to the tumor. In vivo longitudinal magnetic resonance imaging (MRI) identified enhanced accumulation of NK cells in the ensonified tumor, which was validated by endpoint histology. Significant accumulation of NK cells was observed up to 24 hrs at the tumor site when ensonified with 0.50 MPa peak acoustic pressure ldbFUS, whereas tumors treated with at 0.25 MPa showed no detectable NK cell accumulation. These clinically translatable results show that ldbFUS of the tumor mass can potentiate tumor homing of NK cells that can be evaluated non-invasively using MRI.

The Aryl Hydrocarbon Receptor Antagonist StemRegenin1 Improves In Vitro Generation of Highly Functional Natural Killer Cells from CD34(+) Hematopoietic Stem and Progenitor Cells.

Early natural killer (NK)-cell repopulation after allogeneic stem cell transplantation (allo-SCT) has been associated with reduced relapse rates without an increased risk of graft-versus-host disease, indicating that donor NK cells have specific antileukemic activity. Therefore, adoptive transfer of donor NK cells is an attractive strategy to reduce relapse rates after allo-SCT. Since NK cells of donor origin will not be rejected, multiple NK-cell infusions could be administered in this setting. However, isolation of high numbers of functional NK cells from transplant donors is challenging. Hence, we developed a cytokine-based ex vivo culture protocol to generate high numbers of functional NK cells from granulocyte colony-stimulating factor (G-CSF)-mobilized CD34(+) hematopoietic stem and progenitor cells (HSPCs). In this study, we demonstrate that addition of aryl hydrocarbon receptor antagonist StemRegenin1 (SR1) to our culture protocol potently enhances expansion of CD34(+) HSPCs and induces expression of NK-cell-associated transcription factors promoting NK-cell differentiation. As a result, high numbers of NK cells with an active phenotype can be generated using this culture protocol. These SR1-generated NK cells exert efficient cytolytic activity and interferon-γ production toward acute myeloid leukemia and multiple myeloma cells. Importantly, we observed that NK-cell proliferation and function are not inhibited by cyclosporin A, an immunosuppressive drug often used after allo-SCT. These findings demonstrate that SR1 can be exploited to generate high numbers of functional NK cells from G-CSF-mobilized CD34(+) HSPCs, providing great promise for effective NK-cell-based immunotherapy after allo-SCT.

Abstract A65: Immune modulation of natural killer cell adoptive transfer for the treatment of Osteosarcoma lung metastastasis: Targeting the PD-1/ PDL-1 signaling pathway

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumor in children. Pulmonary metastasis is the major cause of mortality in OS patients with a 25-30 % 5-year survival rate. Therefore, development of novel targeted approaches like immunotherapy has been the interest of investigators. Previously, we have demonstrated that Liposomal muramyl tripeptipe phosphatidyl ethanolamine (L-MTP-PE) reduced the metastatic burden in animal models and caused an 8% improvement in the overall survival of patients with OS metastatic disease through macrophage activation. The adoptive transfer of mature natural killer (NK) cells activated ex vivo has exhibited anti-tumor efficacy in breast cancer and lymphoma models. It has been shown that NK cells exert cytotoxicity against OS cells in vitro. Aerosolized interleukin 2 (IL-2) administered to dogs with OS lung disease has demonstrated therapeutic benefit. Additionally, Guma S. R. et al. showed that aerosol IL-2 enhanced the efficacy of infused NK cells against OS lung metastasis in vivo. Aerosol IL-2 and NK cell combination therapy resulted in the regression of OS lung metastasis through enhanced tumor cell apoptosis. Moreover, the combination therapy significantly increased the overall survival of mice with OS lung metastases. However, the treatment is not curative as metastasis persists leading to relapse. Recent studies have demonstrated the immunosuppressive role of checkpoint inhibitors such as programmed death receptor 1 (PD-1) and its ligand, programmed death ligand 1 (PDL-1). Inhibition of the PD-1/PDL-1 pathway has shown immunotherapeutic benefit in lung and renal tumors. Involvement of this pathway in OS lung metastases is unknown. Therefore, based on our previous studies of aerosol IL-2, we hypothesize that aerosol IL-2 causes upregulation of PDL-1 expression on OS tumor cells, which in turn interacts with PD-1 receptor on NK cells, restricting NK cells' killing ability against OS. Our preliminary data demonstrates high PDL-1 expression in human OS cells and OS lung metastases from patients. Using a human OS mouse model, we further show that aerosol IL-2 enhances PDL-1 expression in OS lung metastases. PDL-1 expression on OS lung nodules was observed towards the tumor periphery. Hence, we propose that targeting the PD-1/ PDL-1 axis using anti-PD-1 antibody will enhance the efficacy of NK cell-directed therapy, in the presence or absence of IL-2, against OS lung metastasis and improve the overall survival. Citation Format: Pooja M. Dhupkar, Eugenie Kleinerman, Nancy Gordon. Immune modulation of natural killer cell adoptive transfer for the treatment of Osteosarcoma lung metastastasis: Targeting the PD-1/ PDL-1 signaling pathway. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A65.

A novel method to expand large numbers of CD56+ natural killer cells from a minute fraction of selectively accessed cryopreserved cord blood for immunotherapy after transplantation

Background aimsUmbilical cord blood transplantation (UCBT) is increasingly used to treat acute leukemias. UCB units are thawed and infused in their entirety at transplant, precluding later use as immunotherapy to prevent or treat leukemia relapse. MethodsWe developed a device that selectively thaws only 1 mL of the UCB unit, leaving the remaining UCB unit cryopreserved for subsequent transplantation. We also show that large numbers of CD56+ natural killer (NK) cells can be expanded from these 1-mL fractions of selectively accessed UCB. Immunomagnetic depletion of CD3+ cells of the 1-mL fraction was performed, and the cells were subsequently stimulated with irradiated Epstein-Barr virus–transformed lymphoblastoid cell lines (EBV-LCLs) and set to culture in media containing interleukin (IL)-2. ResultsWhen a 1:20 ratio of total nucleated cells to EBV-LCL feeder cells was used, day-21 and day-35 NK cell cultures initiated from 1 mL of UCB contained a median of 430 × 106 (range: 44–4321 × 106) and 6092 × 106 (range: 165–20947 × 106) CD3–CD56+ NK cells. These cells expressed high levels of CD161, LFA-1, CD69, NKG2D, NKp30, NKp44, NKp80 and NKp46. UCB-derived NK cells were highly cytotoxic against K562 leukemia cells, although cytotoxicity was slightly lower than in expanded PBMC-derived NK cells. ConclusionsWe have developed and optimized a strategy to selectively access a small fraction from cryopreserved UCB and show that large numbers of CD56+ cells can be expanded from this selectively accessed fraction. This strategy presents a method to explore whether early adoptive transfer of NK cells expanded from the same UCB unit used for transplantation can prevent leukemic relapse and decrease graft-versus-host disease after UCBT.

Increased sMICA and TGFβ1 levels in HNSCC patients impair NKG2D-dependent functionality of activated NK cells

Disseminated head-and-neck squamous cell carcinoma (HNSCC) escapes immune surveillance and thus frequently manifests as fatal disease. Here, we report on the distribution of distinct immune cell subpopulations, natural killer (NK) cell cytotoxicity and tumor immune escape mechanisms (TIEMs) in 55 HNSCC patients, either at initial diagnosis or present with tumor relapse. Compared to healthy controls, the regulatory NK cells and the ratio of pro/anti-inflammatory cytokines were decreased in HNSCC patients, while soluble major histocompatibility complex Class I chain-related peptide A (sMICA) and transforming growth factor β1 (TGFβ1) plasma levels were markedly elevated. Increased sMICA and TGFβ1 concentrations correlated with tumor progression and staging characteristics in 7 follow-up HNSCC patients, with significantly elevated levels of both soluble factors from the time of initial diagnosis to that of relapse. Patient plasma containing elevated sMICA and TGFβ1 markedly impaired NKG2D-dependent cytotoxicity against HNSCC cells upon incubation with patient-derived and IL-2 activated NK cells vs. those derived from healthy donors. Decreased antitumor recognition was accompanied by reduced NKG2D expression on the NK cell surface and an enhanced caspase-3 activity. In-vitro blocking and neutralization experiments demonstrated a synergistic negative impact of sMICA and TGFβ1 on NK cell functionality. Although we previously showed the feasibility and safety of transfer of allogeneic donor NK cells in a prior clinical study encompassing various leukemia and tumor patients, our present results suggest the need for caution regarding the sole use of adoptive NK cell transfer. The presence of soluble NKG2D ligands in the plasma of HNSCC patients and the decreased NK cell cytotoxicity due to several factors, especially TGFβ1, indicates timely depletion of these immunosuppressing molecules may promote NK cell-based immunotherapy.

Insulin-like growth factor-1 receptor (IGF-1R) inhibition promotes expansion of human NK cells which maintain their potent antitumor activity against Ewing sarcoma cells.

Patients with primary metastatic or relapsed Ewing sarcomas (EwS) have a poor prognosis. While inhibitory insulin-like growth factor 1 receptor (IGF-1R)-specific antibodies have shown single agent activity in some patients with refractory disease, effective therapeutic targeting will rely on optimal combinations with conventional or innovative therapies. Specifically, combination of inhibitory IGF-1R antibodies with adoptive transfer of activated natural killer (NK) cells may have therapeutic benefit in EwS without adding toxicity. We investigated the in vitro effects of IGF-1R targeting on the immunological profile of EwS cells and on the survival and tumor targeting capacity of K-562-activated NK cells. IGF-1R inhibition reliably reduced EwS cell viability without affecting expression of immune-modulatory and MHC molecules. In NK cells, we observed a significant superior expansion following in vitro activation in the presence of IGF-1R-specific antibodies, while expression of differentiation markers and activating receptors remained unaffected. Activated NK cells coincubated with EwS cells showed potent degranulation responses unaffected by IGF-1R inhibition. These findings were reproducible in a stimulator cell-free NK cell expansion system, suggesting that direct effects of IGF-R1 antibodies on the IGF-R1 pathway in NK cells induce their activation and expansion. Activated human NK cells respond to IGF-1R inhibition with superior expansion kinetics while maintaining potent antitumor responses against EwS. Combination of adoptive NK cell transfer with IGF-1R targeting may be an efficient means to eliminate minimal residual disease after conventional therapy and thereby rescue patients at the highest risk of relapse.

Enhanced NK cell adoptive antitumor effects against breast cancer in vitro via blockade of the transforming growth factor-β signaling pathway.

Natural killer (NK) cells have great potential for improving cancer immunotherapy. Adoptive NK cell transfer, an adoptive immunotherapy, represents a promising nontoxic anticancer therapy. However, existing data indicate that tumor cells can effectively escape NK cell-mediated apoptosis through immunosuppressive effects in the tumor microenvironment, and the therapeutic activity of adoptive NK cell transfer is not as efficient as anticipated. Transforming growth factor-beta (TGF-β) is a potent immunosuppressant. Genetic and epigenetic events that occur during mammary tumorigenesis circumvent the tumor-suppressing activity of TGF-β, thereby permitting late-stage breast cancer cells to acquire an invasive and metastatic phenotype in response to TGF-β. To block the TGF-β signaling pathway, NK cells were genetically modified with a dominant-negative TGF-β type II receptor by optimizing electroporation using the Amaxa Nucleofector system. These genetically modified NK cells were insensitive to TGF-β and resisted the suppressive effect of TGF-β on MCF-7 breast cancer cells in vitro. Our results demonstrate that blocking the TGF-β signaling pathway to modulate the tumor microenvironment can improve the antitumor activity of adoptive NK cells in vitro, thereby providing a new rationale for the treatment of breast cancer.

Understanding of molecular mechanisms in natural killer cell therapy.

Cancer cells and the immune system are closely related and thus influence each other. Although immune cells can suppress cancer cell growth, cancer cells can evade immune cell attack via immune escape mechanisms. Natural killer (NK) cells kill cancer cells by secreting perforins and granzymes. Upon contact with cancer cells, NK cells form immune synapses to deliver the lethal hit. Mature NK cells are differentiated from hematopoietic stem cells in the bone marrow. They move to lymph nodes, where they are activated through interactions with dendritic cells. Interleukin-15 (IL-15) is a key molecule that activates mature NK cells. The adoptive transfer of NK cells to treat incurable cancer is an attractive approach. A certain number of activated NK cells are required for adoptive NK cell therapy. To prepare these NK cells, mature NK cells can be amplified to obtain sufficient numbers of NK cells. Alternatively, NK cells can be differentiated and amplified from hematopoietic stem cells. In addition, the selection of donors is important to achieve maximal efficacy. In this review, we discuss the overall procedures and strategies of NK cell therapy against cancer.