Preclinical assessment of PM21-particle expanded natural killer cells for ovarian cancer treatment.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 127-127
Author(s):  
Jeremiah Oyer ◽  
Sarah B. Gitto ◽  
Deborah Altomare ◽  
Dean A. Lee ◽  
Alicja Copik
Keyword(s):  
Ovarian Cancer ◽  
Cell Therapy ◽  
Nk Cells ◽  
Cell Expansion ◽  
Nk Cell ◽  
Ex Vivo ◽  
Feeder Cell ◽  
Nsg Mice ◽  
Nk Cell Therapy

127 Background: Ovarian cancer has high recurrence rate and could benefit from immunotherapy with NK cells. A necessity for NK cell therapy is an efficient way to generate high doses of NK cells. The best method currently used in clinical trials is ex vivo NK cell expansion by co-culture with K562 CML cells, modified to express 41-BBL and membrane bound IL21 (K562.mb21). However feeder cell based methods are limited to ex vivo co-culture, difficult to disseminate, and not allowed in many jurisdictions. To overcome these limitations and to further improve NK cell therapy, we developed a feeder cell free particle based method for NK cell stimulation. These particles (PM21) are nano-scale, made from cell membranes of K562.mb21 cells, and efficiently stimulate NK cell expansion (mean 825 fold in 14 days, range 163–2216, n = 13). Methods: PM21 particles were prepared from K562.mb21 cells with a procedure developed by our group. NK cells were expanded by culturing CD3 depleted PBMCs with PM21 particles or by co-culture with K562.mb21 cells for 14 days as previously described. NSG mice ( ≥ 8 per group) were implanted ip with 1 x 106 SKOV3 ovarian tumor cells, seeded 8 days, and then treated with vehicle or NK cells expanded with PM21 or K562.mb21 cells (two doses of 10 x 106, injected 6 days apart), with or without in vivo administration of PM21 particles (600 µg, 3x weekly), and IL2 (25 KU, 3x weekly), all delivered ip. Survival analysis was performed with log rank (Mantel-Cox) test. Results: Treatment of SKOV3 engrafted NSG mice with NK cells, expanded with K562.mb21 cells or with PM21 particles, allowed significant ( < 0.0001) 10 day increase in survival compared to untreated animals that succumbed on average 21 days after start of treatment. Administration of ip PM21 particles enhanced survival by 5 days (p = 0.056) over no in vivo PM21 groups. Conclusions: NK cells prepared with PM21 particles or with K562.mb21 cells are equivalent in anti-SKOV3 efficacy and in vivo application of PM21 particles provides further benefit. Clinical translation is underway and clinical trials are being planned. PM21 particles can be the next step in development of NK cell therapy for enhancing both efficacy and dissemination of NK cell therapeutics for ovarian cancer.

Assessment of antitumor function of NK cells expanded with exosomes from K562.mb21 cells.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 132-132 ◽  
Author(s):  
Jeremiah Oyer ◽  
Sarah B. Gitto ◽  
Sara Khederzadeh ◽  
Kari Shaver ◽  
Dean A. Lee ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Expansion ◽  
Nk Cell ◽  
Ex Vivo ◽  
K562 Cells ◽  
In Vitro Cytotoxicity ◽  
Feeder Cell ◽  
Nsg Mice

132 Background: NK cells can kill malignant cells to provide innate immunity against tumors. Due to their low abundance in blood, a focus is to expand NK cells ex vivo having enhanced anti-tumor cytotoxicity to be used as a treatment. Our group has pioneered a cell-free method using plasma membrane (PM) particles derived from K562 cells expressing 41BBL and membrane-bound IL-21 (K562.mb21) which were developed for NK cell expansion. Compared to feeder cell based methods for NK cell expansion, PM21-particles improve safety and allow for potential wide-spread dissemination, and also allows direct in vivo use. Exosomes, vesicles naturally secreted by cells, may yet be another novel feeder cell free way for NK cell expansion and may have further advantageous therapeutic dimensions. Methods: EX21-exosomes and PM21-particles were prepared from K562.mb21 cells and characterized by Nanosight and Western blot analysis. CD3-depleted PBMCs were cultured with EX21 for 14 days, NK cell amounts were monitored and media changed every 2-3 days. In vitro cytotoxicity against K562 cells were comparatively assessed for EX21-NK cells and PM21-NK cells. In vivo anti-tumor efficacy of EX21- and PM21-NK cells was assessed in NSG mice implanted ip with SKOV3_luc ovarian tumor cells (1 x 106 cells seeded for 4 days). SKOV3-bearing mice were treated with vehicle, or two doses of EX21-NK cells or PM21-NK cells (1 x 107, in 5 day intervals), and with or without in vivo administration of EX21 (10 µg, 3x/week) or PM21-particles (600 µg, 3x/week). All groups were injected ip with IL-2 (10 KU, 3x/week). Survival analysis was performed with a Log-rank (Mantel-Cox) test. Results: NK cells cultured with EX21 expanded 530 fold (344-710) over 14 days compared to 735 fold (667-802) in presence of PM21-particles. Treatment of SKOV3 engrafted NSG mice with NK cells, expanded with either EX21 or with PM21, allowed significant ( < 0.0001) increase in survival compared to untreated animals (41-44 vs 29 days post treatment). Ip delivery of EX21 to SKOV3 bearing mice had no effect on survival in either untreated control or EX21-NK cell treated groups. Conclusions: EX21 efficiently expands NK cells and EX21-NK cells have equal anti-tumor effect as PM21-NK cells, both in vitro and in vivo.

scholarly journals Small Molecule Screening Identifies Rho-Associate Protein Kinase (ROCK) As a Regulator of NK Cell Cytotoxicity Against Cancer

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3607-3607
Author(s):  
Grace Lee ◽  
Sheela Karunanithi ◽  
Zachary Jackson ◽  
David Wald
Keyword(s):  
Cell Therapy ◽  
Cytotoxic Activity ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Akt Activation ◽  
Nk Cell Cytotoxicity ◽  
Rock Inhibition ◽  
Nk Cell Therapy

NK cells are a subset of lymphocytes that directly recognize and lyse tumor cells without the limitation of antigen specific receptor recognition. In addition to behaving as cytotoxic effector cells, NK cells unlike T cells are not thought to elicit graft versus host disease. The combination of these characteristics makes NK cells a powerful tool for adoptive cell therapy. Despite the promise of NK cell therapy, key hurdles in achieving significant clinical efficacy include both generating sufficient numbers of highly tumoricidal NK cells and maintaining the cytotoxic activity of these cells in vivo despite the immunosuppressive tumor microenvironment. Our lab and others have developed several feeder cell line-based expansion modules that robustly stimulate the ex vivo proliferation of NK cells. However, strategies to enhance and sustain the activity of NK cells once administered in vivo are still limited. In order to identify strategies to enhance the cytotoxic activity of NK cells, we developed a high-throughput small molecule screen (Figure 1A) that involved a calcein-based cytotoxicity assay of ex vivo expanded and treated NK cells against ovarian cancer cells (OVCAR-3). 20,000 compounds were screened and the screen was found to be highly robust (Z'&gt;0.59). We identified 29 hits that led to at least a 25% increase in cytotoxicity as compared to DMSO control-treated NK cells. One of the most promising hits was the pan-ROCK inhibitor, Y-27632 that led to an 30% increase in NK killing of the OVCAR-3 cells. We validated that ROCK inhibition leads to enhanced NK cell cytotoxic activity using Y-27632 (Figure 1B) as well as other well-established ROCK inhibitors such as Fasudil using a flow cytometry based killing assay. Y-27632 increased NK cell cytotoxicity in a dose- and time- dependent manner. ROCK inhibition consistently led to ~10-25% increase in NK cell cytotoxic activity directed against a variety of ovarian (Figure 1C) and other solid tumor cell lines (Figure 1D). Interestingly, we found that the NK hyperactivation persists for up to 48hrs after washing off the drug that may enable ex vivo stimulation before NK cell infusion. Our preliminary results showed that ROCK inhibition activates PI3K-dependent Akt activation (Figure 1E). We hypothesize that ROCK inhibition restores Akt activation which may be critical for NK cell activating receptor pathways and our current investigations will test these hypotheses. ROCK inhibitors, such as Y-27632 and Fasudil have been utilized in both preclinical and clinical studies for a variety of diseases such as atherosclerosis, neurodegenerative disorders, and ocular diseases. However, the consequences of ROCK inhibition in NK cells has not been thoroughly investigated. Our work shows a promising novel strategy to significantly enhance NK cell therapy against cancer that has high translational potential. Disclosures No relevant conflicts of interest to declare.

scholarly journals 799 Neoantigen-cytokine-chemokine multifunctional natural killer cell engager for immunotherapy of solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A834-A834
Author(s):  
Xue Yao ◽  
Sandro Matosevic
Keyword(s):  
Tumor Microenvironment ◽  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Nk Cell ◽  
Killer Cell ◽  
Nk Cell Therapy

BackgroundThe effectiveness of natural killer (NK) cell-based immunotherapy against solid tumors is limited by the lack of specific antigens and the immunosuppressive tumor microenvironment (TME). Glioblastoma multiforme (GBM) is one such heavily immunosuppressive tumor that has been particularly hard to target and remains without a viable treatment. The development of novel approaches to enhance the efficacy of NK cells against GBM is urgently needed. NK cell engagers (NKCE) have been developed to enhance the efficacy of NK cell therapy.MethodsTo improve the clinical efficacy of NK cell therapy, we are developing a new generation of multi-specific killer engagers, which consists of a neoantigen-targeting moiety, together with cytokine and chemokine-producing domains. Neoantigens are new antigens formed specifically in tumor cells due to genome mutations, making them highly specific tools to target tumor cells. Our engager has been designed to target Wilms' tumor-1 (WT-1), a highly specific antigen overexpressed in GBM among other solid tumors. This is done through the generation of an scFv specific targeting the complex of WT-1126-134/HLA-A*02:01 on the surface of GBM. On the NK cell side, the engager is designed to target the activating receptor NKp46. Incorporation of the cytokine IL-15 within the engager supports the maturation, persistence, and expansion of NK cells in vivo while favoring their proliferation and survival in the tumor microenvironment. Additionally, our data indicated that the chemokine CXCL10 plays an important role in the infiltration of NK cells into GBM, however, GBM tumors produce low levels of this chemokine. Incorporation of a CXCL10-producing function into our engager supports intratumoral NK cell trafficking by promoting, through their synthetic production, increased levels of CXCL10 locally in the tumor microenvironment.ResultsCollectively, this has resulted in a novel multifunctional NK cell engager, combining neoantigen-cytokine-chemokine elements fused to an activating domain-specific to NK cells, and we have investigated its ability to support and enhance NK cell-mediated cytotoxicity against solid tumors in vitro and in vivo against patient-derived GBM models. The multi-specific engager shows both high tumor specificity, as well as the ability to overcome NK cell dysfunction encountered in the GBM TME.ConclusionsWe hypothesize that taking advantage of our multi-functional engager, NK cells will exhibit superior ex vivo expansion, infiltration, and antitumor activity in the treatment of GBM and other solid tumors.

Immunostimulatory Properties of the Human Ortholog of the GMCSF/IL2 Fusion Protein for Cell Based Therapy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4894-4894
Author(s):  
Claudia Penafuerte Graduate ◽  
Jacques Galipeau
Keyword(s):  
Fusion Protein ◽  
Nk Cells ◽  
Cell Expansion ◽  
Cell Activation ◽  
Nk Cell ◽  
Ex Vivo ◽  
Potential Candidate ◽  
Activation Markers ◽  
Cell Based Therapy

Abstract NK cells constitute a potential candidate for cancer cell therapy because they express a diverse array of inhibitory and activating receptors, which recognize and kill infected or tumor cells without prior immune sensitization. However, autologous NK cell mediated adoptive immunotherapy is restricted due to insufficient cytolytic activity of NK cells from patient with aggressive malignancies. In contrast, the infusion of alloreactive NK cells has shown more successful outcomes in the treatment of cancer, but this approach also presents difficulties such as the high doses of cytokines required to induce NK cell expansion ex vivo, which may also sensitize NK cells to apoptosis. Therefore, a critical issue for NK cell based therapy is the use of appropriate growth factors or cytokines that promote NK cell expansion and activation. We have previously shown that a murine GM-CSF/IL-2 fusion protein (aka GIFT2) displays novel antitumor properties in vivo compared to both cytokines in combination regarding tumor site recruitment of macrophages and significant functional NK cell infiltration [Stagg et al., Cancer Research (December 2004)]. In the present work, we found that human GIFT2 will lead to a substantial two fold proliferation of human blood-derived NK cells which is significantly (p<0.05) superior to either IL2 or GMCSF single cytokine treatment or both cytokines combined at equimolar concentration. In addition, we observed that GIFT2 leads to robust expression of NK-cell activation markers CD69 and CD107a. In conclusion, the human GIFT2 fusokine is a novel and potent tool for ex vivo expansion of activated NK cells which may be of use in cell-based immunotherapy of cancer.

Ex Vivo Activated Natural Killer (NK) Cells from Myeloma Patients Kill Autologous Myeloma and Killing Is Enhanced by Elotuzumab

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3666-3666
Author(s):  
Tarun K. Garg ◽  
Susann Szmania ◽  
Jumei Shi ◽  
Katie Stone ◽  
Amberly Moreno-Bost ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Therapy ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
K562 Cells ◽  
Scid Mice ◽  
Target Cells ◽  
Recent Trial ◽  
Nk Cell Therapy

Abstract Immune-based therapies may improve outcome for multiple myeloma (MM) by eradicating chemo-resistant disease. Our recent trial utilizing IL2 activated, killer immunoglobulin-like receptor-ligand mismatched NK cell transfusions from haplo-identical donors yielded (n) CR in 50% of patients. Unfortunately, after NK cell therapy, 2/10 patients had progressive disease, and the median duration of response for the other 8/10 patients was only 105 days (range 58–593). This may have been due to an insufficient dose of alloreactive NK cells and early rejection. Furthermore, appropriate donors were identified for only 30% of otherwise eligible patients. We therefore investigated whether NK cells from MM patients could be expanded and activated to kill autologous MM. We then examined whether pre-treatment of MM cell targets with elotuzumab, a humanized antibody to the MM tumor antigen CS1, could further enhance NK cell-mediated lysis. PBMC from 5 MM patients were co-cultured for 14 days with irradiated K562 cells transfected with 4-1BBL and membrane bound IL15 in the presence of IL2 (300U/ml) as previously described (Imai et al, Blood2005;106:376–383). The degree of NK cell expansion, NK immunophenotype, and ability to kill MM (4 hour 51Cr release assays) were assessed. To determine the ability of ex vivo expanded NK cells to traffic to bone marrow, activated NK cells were injected into the tail vein of NK cell depleted NOD-SCID mice, which were then sacrificed after 48 hours. Flow cytometry for human CD45, CD3, and CD56 was performed on cells from blood, marrow and spleen. There was an average 64-fold expansion of NK cells (range: 8–200) after 2 weeks of co-culture with K562 transfectants. Expansion of T cells was not observed. The NK cell activating receptor NKG2D, and natural cytotoxicity receptors NKp30, NKp44, and NKp46 were up-regulated following the expansion. Expanded NK cells were able to kill autologous MM (E:T ratio 10:1, average 31%, range 22–41%), whereas resting NK cells did not. Pretreatment of autologous MM cells with elotuzumab increased the activated NK cell-mediated killing by 1.7-fold over target cells pretreated with an isotype control antibody. This level of killing was similar to that of the highly NK kill-sensitive cell line K562 (Figure). Autologous PHA blasts and CD34+ stem cells were not killed. Activated human NK cells were detectable in the bone marrow of NOD-SCID mice 48 hours after injection. Ex vivo activation of NK cells from MM patients with K562 transfectants can induce killing of autologous MM and produce large numbers of NK cells for potential therapy. The addition of elotuzumab to activated NK cell therapy enhances anti-MM effects by ADCC thus invoking an additional NK cell-mediated mechanism of MM killing. Importantly, ex vivo activated NK cells traffic to the bone marrow in mice. Autologous NK cell therapy eliminates the issues related to allo-donor availability and early NK cell rejection, and could provide an option for patients refractory to chemotherapy agents. Figure Figure

Influence Of DNA Methyltransferese Inhibitors On The Anti-Leukemic Effect Of Umbilical Cord Blood Derived NK Cells Against Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4496-4496
Author(s):  
Harry Dolstra ◽  
Jeannette Cany ◽  
Anniek B. van der Waart ◽  
Marleen Tordoir ◽  
Basav Nagaraj Hangalapura ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Nk Cell ◽  
Ex Vivo ◽  
Dnmt Inhibitors ◽  
Nsg Mice ◽  
Drug Concentrations

Natural killer (NK) cell-based immunotherapy is a promising adjuvant, relatively non-toxic therapy approach for AML. However, further improvement of NK cell-based therapy is needed to increase the clinical effect. In this regard, NK cells generated ex vivo from hematopoietic progenitor cells (HPC) may have significant clinical benefits over enriched NK cells from adult donors, including the ability to choose an appropriate killer-cell immunoglobuline-like receptor (KIR)-ligand or KIR B haplotype alloreactive donor, as well as the capacity to reach high therapeutic dosages. Previously, we reported a GMP-compliant, cytokine/heparin-based culture protocol for the ex vivo generation of highly active NK cells from CD34+ HPC isolated from cryopreserved umbilical cord blood (UCB) units. Expansion in closed, large-scale bioreactors yields a clinically relevant dose of NK cells with high purity and cytolytic activity against AML cells in vitro. Currently, a clinical phase I trial with these HPC-NK cells is ongoing in our hospital. Trafficking studies in NOD/SCID/IL2Rgnull (NSG) mice demonstrated that these HPC-NK cells migrate to the bone marrow (BM) as well as to lymphoid organs where in vivo expansion and maturation can take place. Analysis of the chemokine receptor expression profile of UCB-NK cells matched in vivo findings. Particularly, a firm proportion of UCB-NK cells functionally expressed CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, high expression of CXCR3 and CCR6 supported the capacity of UCB-NK cells to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis. Importantly, a single HPC-NK cell infusion combined with supportive IL-15 administration was shown to efficiently inhibit growth of K562 leukemia cells implanted in the femur of NSG mice, resulting in significant prolongation of mice survival. Furthermore, we investigated whether modulation by the DNA methyltransferase (DNMT) inhibitors Azacytidine (Aza) and Decitabine (Deci) could further potentiate the antileukemic effect of HPC-NK cells against AML cells. In concordance with previous reports, we observed a dose-dependent effect of Aza and Deci on the growth of the AML cell lines THP1 and KG1a. In subsequent NK cell killing assays, we used clinical relevant low drug concentrations to pre-treat AML cells that did not affect HPC-NK cell viability and cytolytic function. Interestingly, increased killing of pre-treated THP1 and KG1a cells by HPC-NK cells could be observed, which was correlated with an increase in the NKG2D ligand ULBP2, the DNAM-1 ligands CD112 and CD155 as well as TRAIL-R2. Notably, maintenance of low-dose DNMT inhibitors during the KG1a/NK co-culture resulted in pronounced AML growth inhibition. To examine the effect of DNMT inhibitors in vivo, THP1.LucGFP-bearing NSG mice were treated with increasing dose of both agents, which were administered according to current standard protocols applied in humans. Data indicated that treatment with Aza or Deci at dosage equivalent in human to 12.5 and 5 mg/m2 respectively was well tolerated with minimal and/or transient weight loss, and efficiently reduced the progression of THP-1.LucGFP cells in vivo. Currently, we explore whether HPC-NK cells and DNMT inhibitors can work together to combat AML in our xenograft models. These preclinical studies may provide a rationale to investigate the possible additive and/or synergistic anti-AML effects of adoptive HPC-NK cell transfer in combination with these DNMT inhibitors in AML patients. Disclosures: Tordoir: Glycostem Therapeutics: Employment. Spanholtz:Glycostem Therapeutics: Employment.

scholarly journals Adaptive NK Cell Therapy Modulated by Anti-PD-1 Antibody in Gastric Cancer Model

2021 ◽  
Vol 12 ◽  
Author(s):  
Shahrokh Abdolahi ◽  
Zeinab Ghazvinian ◽  
Samad Muhammadnejad ◽  
Mohammad Ahmadvand ◽  
Hamid Asadzadeh Aghdaei ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Therapy ◽  
Tumor Growth ◽  
Growth Inhibition ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Tumor Growth Inhibition ◽  
Cancer Model ◽  
Nk Cell Therapy

Recently, adaptive NK cell therapy has become a promising treatment but has limited efficacy as a monotherapy. The identification of immune checkpoint inhibitor (ICI) molecules has opened a new horizon of immunotherapy. Herein, we aimed to demonstrate the cytotoxic effects of a polytherapy consisting of ex vivo expanded IL-2-activated NK cells combined with human anti-PD-1 antibody as an important checkpoint molecule in a xenograft gastric cancer mouse model. EBV-LCL cell is used as a feeder to promote NK cell proliferation with a purity of 93.4%. Mice (NOG, female, 6–8 weeks old) with xenograft gastric tumors were treated with PBS, ex vivo IL-2-activated NK cells, IL-2-activated NK cell along with human anti-PD-1 (Nivolumab), and IL-2-activated pretreated NK cells with anti-PD-1 antibody. The cytotoxicity of ex vivo expanded NK cells against MKN-45 cells was assessed by a lactate dehydrogenase (LDH) assay. Tumor volume was evaluated for morphometric properties, and tumor-infiltrating NK cells were assessed by immunohistochemistry (IHC) and quantified by flow cytometry. Pathologic responses were considered by H and E staining. Ex vivo LDH evaluation showed the cytotoxic potential of treated NK cells against gastric cancer cell line. We indicated that the adoptive transfer of ex vivo IL-2-activated NK cells combined with anti-PD-1 resulted in tumor growth inhibition in a xenograft gastric cancer model. Mitotic count was significantly decreased (*p &lt; 0.05), and the tumor was associated with improved infiltration of NK cells in the NK-anti-PD-1 pretreated group (*p &lt; 0.05). In conclusion, the combination approach of activated NK cells and anti-PD-1 therapy results in tumor growth inhibition, accompanied by tumor immune cell infiltration in the gastric tumor model.

Treatment of Ex Vivo Expanded NK Cells with Daratumumab F(ab')2 Fragments Protects Adoptively Transferred NK Cells from Daratumumab-Mediated Killing and Augments Daratumumab-Induced Antibody Dependent Cellular Toxicity (ADCC) of Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4244-4244 ◽  
Author(s):  
Elena Cherkasova ◽  
Luis Espinoza ◽  
Ritesh Kotecha ◽  
Robert N. Reger ◽  
Maria Berg ◽  
...  
Keyword(s):  
Nk Cells ◽  
Adoptive Transfer ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Killing ◽  
Cellular Toxicity ◽  
Myeloma Cells ◽  
Nsg Mice

Abstract Daratumumab is a fully humanized monoclonal antibody (IgG1) that targets CD38 expressed on myeloma cells. Daratumumab kills myeloma cells through antibody dependent cellular toxicity (ADCC), compliment dependent cytotoxicity (CDC), and antibody dependent phagocytosis (ADCP). In early clinical trials, daratumumab has showed significant anti-myeloma activity in patients with treatment refractory disease. In vivo, daratumumab has been found to induce NK cell lymphopenia of unclear etiology. We found that NK cells isolated from the peripheral blood of healthy and cancer patients expressed variable surface levels of CD38 (Fig. 1A). Further, surface expression of CD38 increased substantially when NK cells underwent ex vivo cytokine activation by culturing cells overnight in IL-2 containing media or ex vivo expansion using irradiated EBV-LCL feeder cells (Fig. 1B). Remarkably, daratumumab induced apoptosis of expanded NK cells in a dose dependent manner, with substantial NK cell apoptosis occurring within 2 hours following in vitro exposure to daratumumab at a concentration of 1 and 10 ug/ml (Fig. 1C). Further, adoptive transfer of ex vivo expanded human NK cells into NSG mice that had been pre-treated with daratumumab showed daratumumab induced NK cell killing in vivo: the numbers of NK cells isolated from the lungs, blood, spleen and bone marrow of NSG mice 24 hours after infusion of expanded human NK cells was reduced by 90% in mice that were pretreated with 1 mg/kg of daratumumab i.p. compared to controls that had not received the antibody (Fig. 1D). In vitro experiments showed NK cell killing by daratumumab occurred as a consequence of ADCC and was dependent on NK cell CD16 expression; when CD56+ NK cells were sorted by FACS into CD16 positive and negative populations, only NK cells expressing CD16 were killed by daratumumab, with no effect on NK cell viability occurring in the CD16- NK cell. Further, we observed that NK cells obtained from donors who have high affinity FCgR3 as a consequence of a single nucleotide polymorphism in the FCGR3A gene resulting in an amino acid substitution at position 158 (F158V) in CD16 were more sensitive to daratumumab killing compared to NK cells isolated from donors carrying the low affinity CD16 polymorphism. Although NK cell counts and NK reduction in peripheral blood and bone marrow were not associated with daratumumab clinical response in myeloma studies, NK cells play an important role in mediating antitumor responses through ADCC following mAb therapy. In this regard, combining mAb therapy with adoptive transfer of ex vivo expanded NK cells could be utilized as a strategy to potentiate the antitumor effects of mAbs. To overcome daratumumab-mediated killing of adoptively transferred NK cells in daratumumab-treated patients, we blocked CD38 on the surface of NK cells by pretreating them with daratumumab F(ab')2 fragments. The F(ab')2 fragments that were generated using pepsin cleavage of daratumumab were confirmed to bind and block the CD38 epitope expressed on NK cells. Importantly, these F(ab')2 fragments remained bound to the surface of NK cells for at least 96 hours, did not induce NK cell apoptosis, protected NK cells from daratumumab-mediated NK cell killing, and bolstered their tumor cytotoxicity against daratumumab-treated myeloma targets. In vitro experiments showed NK cell tumor cytotoxicity vs myeloma cells in daratumumab-containing media was significantly higher by NK cells that had CD38 blocked with F(ab')2 fragments compared to unblocked controls (Fig. 1E). Importantly, pretreatment with daratumumab F(ab')2 fragments also protected human NK cells from daratumumab-mediated killing in vivo; expanded NK cells pretreated with F(ab')2 fragments prior to adoptive transfer into NSG mice that had been treated with daratumumab were detectable at significantly higher numbers in the blood compared to untreated NK cell controls (Fig. 1F). Conclusion: Expression of CD38 on activated NK cells makes them susceptible to killing by daratumumab, which could compromise the ability of adoptively transferred NK cells to bolster ADCC following treatment with this mAb. Pretreatment of ex vivo expanded NK cells with daratumumab F(ab')2 fragments protects cells from daratumumab-mediated killing, potentially offering a strategy to augment the anti-tumor effects of adoptively transferred NK cells in myeloma patients that have received daratumumab treatment. Disclosures No relevant conflicts of interest to declare.

scholarly journals Eomes and T-Bet Expression Are Required By Mature Primary Human NK Cells for Anti-Leukemia Responses In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 194-194
Author(s):  
Pamela Wong ◽  
Carly C. Neal ◽  
Lily Chang ◽  
Julia A Wagner ◽  
Melissa M. Berrien-Elliott ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
T Box ◽  
Nsg Mice ◽  
Healthy Donors ◽  
And Function

Abstract Natural Killer (NK) cells are innate lymphoid cells that respond to hematologic cancers via cytotoxicity (perforin/granzyme and death receptors) and cytokine/chemokine production, yet the molecular determinants underlying their proliferation, function, and persistence are poorly understood. There are promising reports of pre-clinical and clinical NK cell responses to leukemia and lymphoma, which represent a nascent cellular therapy for these blood cancers. The T-box transcription factors (TFs) Eomes and T-bet are expressed by NK cells throughout their lifespan, and are required for development as evidenced by NK cell loss in Eomes and T-bet deficient mice. However, the roles of these TFs in mature human NK cell molecular programs and functions remain unclear. We hypothesized Eomes and T-bet, which are the only T-box TFs expressed in NK cells, are critical regulators of NK cell homeostasis and functionality, and are necessary for proper mature NK cell responses. To address this, we utilized the CRISPR-Cas9 system to genetically delete both Eomes and T-bet in primary human NK cells isolated from healthy donors, and investigated their role beyond guiding NK cell development, specifically in the anti-leukemia response. Gene-editing of primary human NK cells has been technically challenging, thus most reports that modified NK cells were performed with cell lines, in vitro-differentiated, or highly expanded NK cells that likely do not reflect primary human NK cell biology. Here, we introduced Cas9 mRNA and sgRNA targeting T-bet and Eomes by electroporation into unexpanded primary human NK cells isolated from healthy donors using the MaxCyte GT system. We observed highly efficient reductions of Eomes and T-bet protein expression, quantified by flow cytometry (p &lt; 0.0001, Fig A-B) without viability differences between control (sgRNA targeting TRAC, an unexpressed locus in NK cells), and Eomes/T-bet double CRISPR-edited (DKO) cells after one week in vitro. To study Eomes and T-bet in NK cell anti-leukemia response, control or DKO primary human NK cells were engrafted into NSG mice, supported with human IL-15, and challenged with K562 leukemia cells. Utilizing bioluminescent imaging to visualize leukemia burden, we observed that NK cells lacking both TFs were unable to suppress leukemia growth in vivo. To understand the mechanism responsible for impaired leukemia control, we investigated in vivo persistence and proliferation, cytotoxic effector molecule expression, as well as ex vivo degranulation and cytokine production of DKO NK cells compared to control NK cells. DKO or control human NK cells were transferred into NSG mice and supported with human IL-15. After 2-3 weeks, significantly fewer (&lt;30%) DKO NK cells persisted compared to control NK cells: spleen (5-fold decrease, control 240e3±65e3 vs DKO 47e3±15e3 NK cells, p&lt;0.01, Figure C), blood (6-fold decrease, p&lt;0.01), and liver (4-fold decrease, p&lt;0.05). Using intracellular flow cytometry, double T-bet/Eomes CRISPR-edited NK cells that lacked both Eomes and T-bet protein after in vivo transfer were identified. A proliferative defect was evident in flow-gated DKO (62±6% undivided), compared to unedited (WT) NK cells (4±2% undivided) assessed by CellTrace Violet dilution (Figure D). In addition, there were marked reductions in granzyme B and perforin protein (p&lt;0.001) in flow-gated DKO NK cells compared to controls. To assess DKO NK cell functional capacity, we performed an ex vivo functional assay on NK cells from spleens of the NSG mice as effectors, and K562 targets or IL-12/15/18 stimulation for 6 hours. Degranulation to K562 targets was impaired (p&lt;0.05), and IFN-γ production was reduced (p&lt;0.0001) after cytokine stimulation in flow-gated DKO NK cells (Figure E). Thus, CRISPR-editing of unexpanded, primary human NK cells revealed that Eomes and T-bet are required by mature human NK cells for their function and homeostasis, distinct from their role in development. This is translationally relevant, as defects in proliferation and function of human DKO NK cells manifested markedly reduced response against human leukemia cells in vivo in xenografts. These findings expand our understanding of key molecular regulators of mature NK cell homeostasis and function, with the potential to provide new avenues to enhance NK cell therapy. Figure 1 Figure 1. Disclosures Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Foltz-Stringfellow: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Fehniger: HCW Biologics: Research Funding; Compass Therapeutics: Research Funding; Affimed: Research Funding; ImmunityBio: Research Funding; Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; Kiadis: Other; OrcaBio: Other; Indapta: Other.

A Novel Triple Umbilical Cord Blood Transplant (UCBT) Strategy To Promote NK Cell Immunotherapy (Unit 1) with a Fully Ablative Preparative Regimen Followed by a Double UCBT in Patients with Refractory AML.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3111-3111 ◽  
Author(s):  
Jeffrey S. Miller ◽  
Claudio G. Brunstein ◽  
Sarah Cooley ◽  
Michael R. Verneris ◽  
Angela Panoskaltsis-Mortari ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Expansion ◽  
Nk Cell ◽  
Ex Vivo ◽  
Treatment Options ◽  
Hematopoietic Stem ◽  
Preparative Regimen ◽  
Neutrophil Engraftment ◽  
Refractory Aml

Abstract Treatment options for refractory AML are usually ineffective. Previously, we tested adoptive transfer of haploidentical peripheral blood (PB) derived NK cells without transplantation and demonstrated correlation between in vivo NK cell expansion and those who achieved a complete remission. This therapy is limited by: the inability to expand NK cells in most patients, prolonged neutropenia in some patients and inconsistent efficacy. UCB, in contrast to adult PB, is rich in NK precursors with CD34+/CD7−, CD34+/CD7+ and CD34−/CD7+ phenotypes. We hypothesized that UCB-derived NK cells may show better in vivo expansion than adult derived NK cells after cytoreduction. Therefore, we tested our triple UCBT strategy in patients with refractory relapsed AML who were <45 years old, without active infection and eligible for myeloablative conditioning. The UCB NK product (unit 1) was CD3 depleted (using a CliniMacs system) and activated with IL-2 (1000U/ml for 16–20 hours). The UCB-derived NK cells (matched at 3 HLA loci and KIR-ligand mismatched when possible) were infused on day -12 after conditioning with cyclophosphamide 120 mg/kg, fludarabine 125 mg/m2 and TBI 1320 cGy on days -19 to -13. Subcutaneous IL-2 (10 MU) was was given on days -12, -10, -8, -6, -4 and -2 to facilitate in vivo NK cell expansion. On day 0, two UCB units (≥4/6 match) were transplanted for hematopoietic rescue and followed by mycophenolate mofetil and cyclosporine for GVHD prophylaxis. Compared to pre-treatment levels, endogenous IL-15 was markedly increased after the preparative regimen at the time of the NK UCB unit. The NK UCB units contained both precursor and mature NK cells. Three product samples were cultured for 28 days in limiting dilution on a murine stromal feeder, demonstrating cloning frequencies of 1:5, 1:9 and 1:12 infused UCB cells giving rise to NK progeny. Two of the 3 patients had partial chimerism derived from the NK product on day -1. Unexpectedly, these same two patients demonstrated prompt neutrophil engraftment on days 3 and 7 after hematopoietic stem cell rescue. In both instances, chimerism was achieved from the NK product. Of the non-NK and non-T cells in the NK UCB units from these 2 patients there were 9.6% and 5.3% CD34+/CD7− cells. In the third patient, the NK UCB unit had only 2% CD34+/CD7− cells and they did not contribute to neutrophil engraftment which occurred on day 36 after UCBT. Of note, 3/3 tolerated the NK infusion without toxicity and were leukemia-free at the time of engraftment. Two remain alive (one died of TRM) with one relapse before day 100. These data suggest UCB NK cells may be administered safely and, despite CD3 depletion and IL-2 activation (ex vivo and in vivo), provide long term engraftment potential that may dominate over unmanipulated UCB infused subsequently. In summary, UCB is a rich source of NK precursors capable of in vivo expansion which are potentially better suited than adult NK cells for use in treatment of patients with refractory AML.

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