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

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

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

Blood ◽

10.1182/blood-2019-131947 ◽

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'>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.

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Genetically Reengineered K562 Cells Significantly Expand Cord Blood (CB) Natural Killer (NK) Cells Ex-Vivo Expanded with Increased NK Cell Activation and Cytolytic Activity Both In-Vitro and In-Vivo: Potential Use In Adoptive Cellular Immunotherapy (ACI)

Blood ◽

10.1182/blood.v116.21.3928.3928 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3928-3928

Author(s):

Michele Levin ◽

Janet Ayello ◽

Frances Zhao ◽

Andrew Stier ◽

Lauren Tiffen ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Granzyme B ◽

K562 Cells ◽

Cytolytic Activity ◽

Scid Mice ◽

Activation Marker

Abstract Abstract 3928 Background: NK cells play a role in reducing relapse in hematological malignancy following AlloSCT (Dunbar et al, Haematologica, 2008). NK cell limitations include lack of tumor recognition and/or limited numbers of viable and functional NK cells (Shereck/Cairo et al, Ped Bld Can, 2007). NK ACI provide safe and effective therapy against tumor relapse; yet NK cells are limited to specific cancer types and not all patients demonstrate optimal response (Ruggieri et al. Science, 2002; Ljunggren et al. Nat Rev Immuno, 2007). To circumvent these limitations, methods to expand and activate PBMNCs with genetically engineered K562 cells expressing membrane bound IL-15 and 41BB ligand (K562-mbIL15-41BBL [modK562]; Imai/Campana et al, Blood, 2005) have shown to significantly increase NK cells in number and maintain heterogeneous KIR expression (Fusaki/Campana et al BJH, 2009). We have shown that CB NK cells can be activated/expanded and exhibit enhanced cytolytic activity when cultured in a cytokines/antibody cocktail (Ayello/Cairo et al, BBMT, 2006; Exp Heme, 2009). Objective: To evaluate CBNK expansion, activation, cytolytic mechanism and function against Burkitt lymphoma (BL) tumor target and its influence on NK cell mediated in-vitro and in-vivo cytotoxicity in NOD-SCID mice following stimulation with modK562 cells (generously supplied by D.Campana, St Jude's Children's Hospital, Memphis, Tx). Methods: Following 100GY irradiation, modK562cells were incubated 1:1 with CBMNCs in RPMI+IL-2 (10IU/ml) for 7 days in 5%CO2, 37°C. NK activation marker (LAMP-1), perforin and granzyme B were determined by flow cytometry. Cytotoxicty was determined via europium assay at 20:1 E:T ratio with Ramos (BL) tumor targets (ATCC). The mammalian expression construct (ffLucZeo-pcDNA (generously supplied by L.Cooper, MD, PhD) was transfected to BL cells using lipofectin and selected by zeocin for stable transfection. Six week old NOD-SCID mice received 5×106 BL cells subcutaneously. Upon engraftment, xenografted NOD-SCID mice were divided in 5 groups: injected with PBS (control), BL only, 5×106 wildtype (WT) K562 expanded (E) CBNK cells, modK562 expanded (E) CB NK cells (5×106) and modK562 expanded (E) CBNK cells (5×107). Ex-vivo ECBNK cells were injected weekly for 5 weeks and xenografted NOD-SCID mice were monitored by volumetric measurement of tumor size (Tomayko/Reynolds, Can Chemother Pharmac, 1989), bioluminescent imaging (Inoue et al Exp Heme, 2007) and survival. The survival distribution for each group was estimated using the Fisher exact test. Results: On Day 0, NK cells (CD56+/3-) population was 3.9±1.3%. After 7 days, modK562 expanded CBNK cells was significantly increased compared to WTK562 and media alone (72±3.9 vs 43±5.9 vs 9±2.4%, p<0.01). This represented a 35-fold or 3374±385% increase of the input NK cell number. This was significantly increased compared to WTK562 (1771±300%, p<0.05). ModK562 ECBNK cells demonstrated increased perforin and granzyme B expression compared to WTK562 (42±1.5 vs 15±0.5%,p<0.001; 22±0.5 vs 11±0.3%,p<0.001, respectively). Cytotoxicity was against BL tumor targets was significantly increased (42±3 vs 18±2%,p<0.01), along with NK activation marker expression, CD107a (p<0.05). At 5 weeks, in-vivo studies demonstrated increased survival of NOD-SCID mice receiving both 5×106 and 5×107 modK562 ECBNK cells when compared to those with no treatment (p=0.05, p=0.0007, respectively). There was no difference in survival when comparing mice that received 5×106 vs 5×107 modK562 ECBNK cells (p=0.0894) at 5 weeks. Tumor volume of mice receiving either dose of modK562 ECBNK cells was significantly less than those receiving WTK562 ECBNK cells (1.92±0.57 and 0.37±0.05 vs 3.41±0.25, p=0.0096 and p=0.0001, respectively). Conclusions: CBMNCs stimulated and expanded with modK562 cells results in significant expansion of CBNK cells with enhanced in-vitro cytotoxicity, significant receptor expression of NK activation marker (LAMP-1), and perforin and granzyme B. Furthermore, modK562 ECBNK cells leads to increased survival and lower tumor burden of NOD-SCID mice xenografted with BL. Future directions include modK562 ECBNK cells to be genetically modified to express chimeric antigen receptor CD20 (MSCV-antiCD20-41BB-CD3 ζ) against CD20+ hematologic malignancies for future studies to evaluate whether targeting enhances in-vitro and in-vivo cytotoxicity. Disclosures: No relevant conflicts of interest to declare.

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

Frontiers in Pharmacology ◽

10.3389/fphar.2021.733075 ◽

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 < 0.05), and the tumor was associated with improved infiltration of NK cells in the NK-anti-PD-1 pretreated group (*p < 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.

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Novel Approach for NK Cell Therapy for Cancer

Blood ◽

10.1182/blood.v124.21.3836.3836 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3836-3836 ◽

Cited By ~ 2

Author(s):

Reshmi Parameswaran ◽

David N. Wald ◽

Marcos De Lima ◽

Dean A. Lee ◽

Stephen Moreton

Keyword(s):

Cell Therapy ◽

Nk Cells ◽

Nk Cell ◽

Model Systems ◽

Target Cells ◽

Adhesion Assay ◽

Mouse Bone Marrow ◽

Clinical Potential ◽

Gsk3 Inhibitor ◽

Nk Cell Therapy

Abstract Novel therapeutic approaches are urgently needed for many malignancies such as Acute Myeloid Leukemia (AML). We have developed a new therapeutic strategy based upon NK cell immunotherapy that exhibits high clinical potential based upon cell and animal studies. While the harnessing of NK cells for cellular therapy against malignancies has been a topic of interest for several decades, our approach overcomes a major hurdle of insufficient NK cell cytotoxic activity. We have identified that targeting the kinase GSK3 through pharmacologic and genetic approaches leads to the hyperactivation of human blood derived NK cells and a significant improvement in efficacy as compared to traditionally used activated NK cells or chemotherapy in our mouse AML model systems. Importantly this GSK3 inhibition can be achieved through a short ex-vivo incubation of NK cells with a GSK3 inhibitor paving the way for a rapid implementation into a clinical trial. Utilizing both in vitro studies with AML cell lines (ex. OCI-AML3 and HL-60)) and primary human AML cells we observe approximately a 50% increase in efficacy with GSK3 inhibited NK cells as compared to untreated NK cells. Further, we demonstrate significant efficacy of GSK3 inhibited NK cells in a mouse model of circulating human AML. After 4 weekly injections of human NK cells, there is a 50% greater reduction in human AML cells present in the mouse bone marrow with GSK3 inhibited NK cells as compared to vehicle treated NK cells. Besides efficacy studies, our work has led a model of how GSK3-inhibition enhances NK cell activity as depicted in figure 1. GSK3 inhibition leads to a dramatic increase in adhesion of NK cells to target cells as demonstrated by a flow cytometric adhesion assay (49% vs 83% after 20 min incubation) as well as live cell imaging. Consistent with the increased adhesion, GSK3 inhibited NK cells as well as target cells (after co-incubation) exhibit increased expression of essential NK cell-target adhesion molecules including L-selectin (on NK cells) and ICAM (on target cells). The induction of ICAM on target cells is due to a marked induction in TNFa production from the NK cells upon incubation with target cells (>7 fold increase in TNFa production). TNFa neutralization impairs the NK activity of the GSK3 inhibited NK cells (~30%) but not vehicle treated cells. Finally, GSK3 inhibition also leads to changes in the NK cells that enhance activity such as increased expression of granzyme and perforin and secretion of IFNg. Overall, our work has a revealed a novel strategy for NK cell therapy that holds high clinical potential. Figure 1. Model of how GSK3 inhibition leads to hyperactive NK cells. GSK3I - GSK3 inhibitor Figure 1. Model of how GSK3 inhibition leads to hyperactive NK cells. GSK3I - GSK3 inhibitor Disclosures No relevant conflicts of interest to declare.

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FLT3 OR CD33 NOT EMCN Logic Gated CAR-NK Cell Therapy (SENTI-202) for Precise Targeting of AML

Blood ◽

10.1182/blood-2021-154201 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2799-2799

Author(s):

Brian S. Garrison ◽

Han Deng ◽

Gozde Yucel ◽

Nicholas W. Frankel ◽

Marcela Guzman-Ayala ◽

...

Keyword(s):

Cell Therapy ◽

Nk Cells ◽

Nk Cell ◽

Target Cells ◽

Gene Circuits ◽

Gene Circuit ◽

Cell Therapies ◽

Single Target ◽

Or Gate ◽

Nk Cell Therapy

Abstract Background: While chimeric antigen receptor (CAR) cell therapies have provided extraordinary clinical responses in some hematological malignancies, developing effective CAR cell therapies for acute myeloid leukemia (AML) has been challenging due to: (a) the lack of a single target antigen robustly expressed across both AML leukemic stem cell (LSC) and immature leukemic blast cell subpopulations, and (b) the lack of truly AML-specific target antigens, since current targets are also expressed on healthy tissues and may result in off-tumor toxicity. Using logic gated gene circuits, we are engineering our SENTI-202 CAR-NK cell therapy to overcome these long-standing challenges to treating AML patients. Methods: To maximize clearance of AML tumor subpopulations and minimize off-tissue toxicities, we used a proprietary bioinformatics paired antigen discovery platform to identify the optimal combinations of AML tumor-associated and healthy tissue antigens to target using an OR and NOT logic gated CAR gene circuit approach. The SENTI-202 therapeutic candidate is a FLT3 OR CD33 NOT Endomucin (EMCN) gene circuit-enabled allogeneic CAR-NK cell, designed to broadly target FLT3 and/or CD33-expressing AML tumor cells (including both LSCs and blasts) but not healthy hematopoietic stem cells (HSCs). Results: First, for the OR GATE portion of the logic circuit we demonstrated that engineered primary human NK cells expressing activating CARs (aCARs) that recognize both FLT3 and CD33 outperformed more traditional single target CAR approaches with FLT3 (p<0.05) or CD33 (p<0.01), and exhibited >80% cytotoxicity and significant cytokine secretion (GrB, IFN-g, and TNF-a) against multiple leukemia cell lines in vitro, including MOLM13, THP1, and SEM. We successfully engineered FLT3 OR CD33 CAR-NK cells using both bicistronic and bivalent CAR configurations, where bicistronic CARs possess separate FLT3 and CD33 CARs linked via a 2A peptide, and bivalent CARs use a loop structure to connect FLT3 and CD33 scFvs within the same CAR. While both approaches demonstrated robust efficacy against AML cells, the bivalent approach enabled greater CAR expression and cytotoxicity (p<0.05). Importantly, our FLT3 OR CD33 CAR-NK cells demonstrated significant cytotoxicity against primary AML patient samples (p<0.01-0.001) and significantly reduced tumor burden and improved mouse survival in MOLM13 (p<0.05) and MV4-11 (p<0.01) xenograft AML models. We believe that our strategy of concurrently targeting FLT3 and CD33 will result in a more robust synergistic anti-tumor effect, leading to a more durable remission with decreased risk of relapse due to single antigen escape. Second, for the NOT GATE portion of the logic circuit to protect healthy HSCs, we developed NK and T cell inhibitory CARs (iCARs) consisting of an scFv against a healthy cell antigen, hinge and transmembrane domains, and functional intracellular domains derived from inhibitory co-receptors containing immunoreceptor tyrosine-based inhibitory motifs. In the case of SENTI-202, the iCAR scFv recognizes EMCN, a surface antigen expressed on up to 76% of healthy HSCs but not on AML cells. Using two different iCAR configurations, we demonstrated that FLT3 (CD28z) aCAR-NK cells engineered with an EMCN-specific iCAR protected up to 67% (iCAR#1, p<0.01) or 50% (iCAR#2, p<0.01) of FLT3+ EMCN+ cells from FLT3 aCAR-mediated cytotoxicity. Next, to replicate a clinical context more closely, we mixed FLT3+ EMCN- (AML-like) and FLT3+ EMCN+ (HSC-like) target cells and demonstrated that FLT3 NOT EMCN CAR-NK cells exhibit preferential killing of FLT3+ EMCN- target cells (p<0.0001), demonstrating that our NOT GATED gene circuit controls NK-mediated responses on a cell-by-cell basis. Conclusion: SENTI-202 is a novel NK cell product candidate to be engineered with both OR and NOT logic gated CAR gene circuits, wherein the OR gate is designed to increase AML LSC/blast tumor clearance (to prevent relapse), and the NOT gate is designed to protect healthy HSCs from off-tumor toxicity, enabling regeneration of a healthy hematopoietic system and mitigating the need for a bone marrow transplant. Beyond AML, OR and NOT logic gated CAR-NK cell therapy has applicability to other cancer-associated antigen targets that are potentially limited by antigen escape and/or off-tumor toxicity, increasing the potential for enhanced efficacy and reduced risk of undesirable side effects. Disclosures No relevant conflicts of interest to declare.

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Romedepsin (RM), a Histone Deacytelase Inhibitor (HDACi), Significantly Increases the Expression of NKG2D Ligands, MIC A/B, In Pediatric Leukemia/Lymphoma Cells (LL), Resulting In Enhanced In Vitro and In Vivo NK Cytotoxicity In NOD-SCID Mice: Translational Approach for Adoptive NK Cell Targeted Immunotherapy

Blood ◽

10.1182/blood.v116.21.4297.4297 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4297-4297

Author(s):

Prakash Satwani ◽

Aniket Saha ◽

Sejal Bavishi ◽

Frances Zhao ◽

Janet Ayello ◽

...

Keyword(s):

Cell Therapy ◽

Nk Cells ◽

Nk Cell ◽

Scid Mice ◽

Rank Test ◽

P Value ◽

Nkg2d Ligands ◽

Log Rank Test ◽

Nk Cell Therapy

Abstract Abstract 4297 Introduction: Natural killer (NK) cells recognize malignant cells through the tumor-associated expression of NKG2D ligands, including MIC A/B (Crewenka et al, Science, 1998). Tumor cells expressing ligands for NKG2D can become susceptible to NK cell killing despite normal MHC class I expression (Lanier LL, Nat Med, 2001). However, tumor cells may shed MIC A/B and escape immuno-surveillance. HDACi increases the expression of NKG2D ligands MIC A/B. Glycogen synthase kinase-3 (GSK-3), a constitutively active serine/threonine kinase with numerous functions including regulation of cellular differentiation, stress and apoptosis, is also an important regulatory enzyme in the expression of MIC A/B in response to romidepsin (RM) (Skov et al, Cancer Res, 2005). Objective: To determine the expression of MIC A/B in response to RM in leukemia and lymphoma cells (LL), its influence on NK cell mediated in vitro and in vivo cytotoxicity in NOD-SCID mice and to investigate the role of the GSK-3 pathway in the regulation of expression of MIC A/B in response to RM. Methods: LL cells (106/ml, RS 4:11 [MLL-ALL], Ramos [Burkitt's lymphoma]) were exposed to RM (10 ng/mL) (generously provided by Gloucester Pharmaceuticals) for 24 hours, followed by FACS staining with PE-conjugated anti-MIC A/B. Peripheral blood NK cells were isolated via magnetic separation followed by 12 hrs incubation with interleukin-2 (IL-2) [3000 IU/ml]. Cytotoxicity assays (europium assay) were performed at effector target (E:T) ratio of 5–10:1. RS4:11 and Ramos cells were also pre-treated for 1 hour with 100mM lithium chloride (LiCl), a potent inhibitor of GSK-3 activity. The mammalian expression construct (ffLucZeo-pcDNA [generously provided by Laurence Cooper, MD, PhD]) was transfected to RS4:11 and Ramos cells using lipofectin. The transfected cells were selected by zeocin to make stable transfection cells using lipofectin. Six week old NOD-SCID mice received 5×106 LL cells subcutaneously. Once LL engraftment was established in NOD-SCID mice, the xenografted animals were divided in various groups, 1) Control NOD-SCID mice were injected with PBS, 2) NOD-SCID mice with leukemia or lymphoma, 3) NOD-SCID mice with leukemia or lymphoma + NK cell therapy and 4) NOD-SCID mice with leukemia or lymphoma + RM + NK cell therapy. NOD-SCID xenografted mice in group 3 received weekly injections of purified IL-2 activated adult NK cells (5×106) for 6 weeks and mice in group 4 received weekly injections of RM (4.4mg/kg) followed by an infusion of IL-2 activated NK cells 24 hrs later. Xenografted NOD-SCID mice were monitored by volumetric measurement of tumor size, bioluminescent imaging (Inoue et al, Exp. Hem, 2007) and survival. The survival distribution for each of the groups of mice was estimated using Kaplan-Meier estimates. The log-rank test was used to compare the survival distributions between treatment groups. Results: MIC A/B expression significantly increased in response to RM ([RS4:11 0.2% vs. 82%, p< 0.0001] and [Ramos 0.57% vs. 67%, p=0.0003]). Enhanced expression of MIC A/B in response to RM was inhibited when LL cells are pre-treated with LiCl RS 4:11 [RM vs RM+LiCl] 82% vs. 5%, p<0.0001; Ramos [RM vs. RM+LiCl] 67% vs. 35%, p<0.0001). In vitro cytotoxicity assays revealed significant increases against both RS 4:11 and Ramos cells at E:T ratio of 5–10:1 (p<0.01). The median survival time for NOD-SCID mice with RS4:11 was 24 days and RS4:11 + NK cell therapy was 34 days and RS4:11 +RM + NK cell therapy was: 46.5 days, respectively; Log-rank test p-value = 0.003. Median survival time for mice with Ramos was 16 days, Ramos + NK cells was 29 days and Ramos + RM + NK cell therapy was 32 days, respectively; Log-rank test p-value <0.001 Background: Expression of MIC A/B in LL cells is significantly increased by RM leading to enhanced susceptibility for NKG2D- MIC A/B mediated cytotoxicity by NK cells. NK cells + RM leads to increase in survival of NOD-SCID mice xenograft with LL. Furthermore, up-regulation of MIC A/B in LL cells secondary to RM exposure is in part regulated by the GSK-3 signal transduction pathway. Disclosures: No relevant conflicts of interest to declare.

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Natural Killer Cell Therapy: A New Treatment Paradigm for Solid Tumors

Cancers ◽

10.3390/cancers11101534 ◽

2019 ◽

Vol 11 (10) ◽

pp. 1534 ◽

Cited By ~ 10

Author(s):

Sooyeon Oh ◽

Joo-Ho Lee ◽

KyuBum Kwack ◽

Sang-Woon Choi

Keyword(s):

Cell Therapy ◽

Nk Cells ◽

Natural Killer ◽

Solid Tumors ◽

Nk Cell ◽

Ex Vivo ◽

Killer Cell ◽

Genetic Defects ◽

New Paradigm ◽

Nk Cell Therapy

In treatments of solid tumors, adoptive transfer of ex vivo expanded natural killer (NK) cells has dawned as a new paradigm. Compared with cytotoxic T lymphocytes, NK cells take a unique position targeting tumor cells that evade the host immune surveillance by down-regulating self-antigen presentation. Recent findings highlighted that NK cells can even target cancer stem cells. The efficacy of allogeneic NK cells has been widely investigated in the treatment of hematologic malignancies. In solid tumors, both autologous and allogeneic NK cells have demonstrated potential efficacy. In allogeneic NK cell therapy, the mismatch between the killer cell immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) can be harnessed to increase the antitumor activity. However, the allogeneic NK cells cause more adverse events and can be rejected by the host immune system after repeated injections. In this regard, the autologous NK cell therapy is safer. This article reviews the published results of clinical trials and discusses strategies to enhance the efficacy of the NK cell therapy. The difference in immunophenotype of the ex vivo expanded NK cells resulted from different culture methods may affect the final efficacy. Furthermore, currently available standard anticancer therapy, molecularly targeted agents, and checkpoint inhibitors may directly or indirectly enhance the efficacy of NK cell therapy. A recent study discovered that NK cell specific genetic defects are closely associated with the tumor immune microenvironment that determines clinical outcomes. This finding warrants future investigations to find the implication of NK cell specific genetic defects in cancer development and treatment, and NK cell deficiency syndrome should be revisited to enhance our understanding. Overall, it is clear that NK cell therapy is safe and promises a new paradigm for the treatment of solid tumors.

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Haploidentical Mbil-21 Ex Vivo Expanded NK Cells (FC21-NK) for Patients with Multiple Relapsed and Refractory Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-134858 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 11-12

Author(s):

Stefan O. Ciurea ◽

Jolie Schafer ◽

Piyanuch Kongtim ◽

Julianne Chen ◽

Doris Soebbing ◽

...

Keyword(s):

Cell Therapy ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

Median Number ◽

Publicly Traded ◽

Advisory Committees ◽

Nk Cell Therapy

Background: Allogeneic stem-cell transplantation (alloSCT) remains the only curative treatment for patients with advanced AML. However, only a minority of these patients achieve disease control prior to transplantation. Natural Killer (NK) cells have potent anti-leukemic activity but are functionally deficient in AML. Adoptive NK-cell therapy using high-doses of functionally active NK-cells could overcome these limitations. We previously developed an ex vivo NK-cell expansion method based on K562 feeder cells modified to express membrane bound IL-21 (mbIL-21) and 4-1BB ligand, (FC21), which resulted in high numbers of hyperfunctional FC21-NK cells with enhanced cytotoxicity and cytokine production. Here we report outcomes of a phase I clinical trial designed to assess the safety, feasibility and maximum tolerated dose (MTD) of haploidentical FC21-NK cells for patients with relapse/refractory (R/R) AML at MD Anderson Cancer Center. Methods: Eligible patients were ≥18 years, KPS ≥70 with good organ function. Patients with relapsed AML after alloSCT were eligible if they had no active GVHD and did not require immunosuppression. Haploidentical donors were selected based on KIR characteristics, when multiple donors were available. Donor NK cells were expanded over 3 weeks and cryopreserved. Three dose levels between 106-108 cells/kg were planned. Patients received cytoreductive chemotherapy with fludarabine 30 mg/m2/day and cytarabine 2 g/m2/day for 5 days (4 days for age >60) and G-CSF (subsequently eliminated). 3-7 days after chemotherapy, patients received FC21-NK cell infusions 3 times per week, up to 6 infusions. Results: As of 4/14/2020, 15 patients were screened, 12 of whom were eligible and received the FC21-NK cells. Median age was 60 years (range 25-70); 6 (50%) had adverse cytogenetics, 8 (66.7%) had adverse ELN genetic risk, 6 (50%) had primary induction failure, 2 (16.7%) had CNS disease and 4 (33.3%) had secondary AML. Median number of prior treatment regimens was 5 (range 2-8), median blast count at enrollment was 47% (range 7-88). Median time from diagnosis to enrollment and to first NK-cell infusion was 16.6 (range 2.5-98.1) and 17.2 (range 3.1-98.6) months, respectively. Donor-recipient NK-cell alloreactivity was seen in 5 patients (41.7%). Median number of NK-cell infusion was 6 (range 3-6); 8 (66.7%) and 4 (33.3%) patients received NK-cell dose of 1 X106 and 1 X107 cells/kg, respectively. MTD was not reached. Seven patients had ANC recovery post-NK cell infusion with cumulative incidence (CI) of ANC recovery to 500/mm3 at 60 days of 58.3%. Eight patients (66.7%) achieved complete remission (CR) (N=4, 33.3%) or CR with incomplete hematologic recovery (CRi) (N=4, 33.3%) at 30 days post-NK cell infusion. One patient with CR had negative minimal residual disease (MRD). Five patients (41.7%) proceeded to haploidentical alloSCT from the same donor and were transplanted in CR/CRi, all but one with persistent MRD. With a median follow-up of 13 months (range 4.1-42.7), median OS and DFS were 17.6 and 3.3 months, and 28 and 20 months for patients receiving alloSCT, respectively. Other outcomes including 2-year OS, DFS, relapse and TRM are shown in Figure 1 and Table 1. No infusion related toxicity or cytokine release syndrome was observed. Two patients were evaluable for FC21-NK cell persistence with haplotype-specific anti-HLA antibodies. FC21-NK cells were detected 5 and 6 weeks after the last FC21-NK cell infusion, respectively. A progressive decrease of the blast population with progressive expansion of the FC21-NK cell population after repeated NK-cell infusions was noted in samples collected from one pt (Figure 2). Persistence is also being evaluated by STR chimerism. Conclusions: Multiple infusions of FC21-NK cells yielded unprecedented outcomes with 66.7% of patients responding and approximately half proceeding to alloSCT in a heavily pre-treated, ultra-refractory, high-risk patient population. Responses were observed irrespective of dose. FC21-NK cell therapy was very well tolerated with no attributable AEs and were shown to persist for at least 5 weeks after infusion. These encouraging results warrant further clinical evaluation of FC21-NK cells in R/R AML patients. Disclosures Ciurea: Kiadis Pharma: Current equity holder in publicly-traded company, Research Funding. Schafer:Kiadis Pharma: Current Employment. Shpall:Zelluna: Membership on an entity's Board of Directors or advisory committees; Adaptimmune: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Magenta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Licensing Agreement. Konopleva:Calithera: Research Funding; Eli Lilly: Research Funding; Kisoji: Consultancy; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Forty-Seven: Consultancy, Research Funding; Sanofi: Research Funding; AstraZeneca: Research Funding; Agios: Research Funding; Ablynx: Research Funding; AbbVie: Consultancy, Research Funding; Ascentage: Research Funding; Rafael Pharmaceutical: Research Funding; Cellectis: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding. Lee:Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Champlin:Actinium: Consultancy; Johnson and Johnson: Consultancy; Omeros: Consultancy; DKMS America: Membership on an entity's Board of Directors or advisory committees; Cytonus: Consultancy; Genzyme: Speakers Bureau; Takeda: Patents & Royalties.

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Cryopreservation impairs cytotoxicity and migration of NK cells in 3-D tissue: Implications for cancer immunotherapy

10.1101/812172 ◽

2019 ◽

Cited By ~ 1

Author(s):

Christoph Mark ◽

Tina Czerwinski ◽

Susanne Roessner ◽

Astrid Mainka ◽

Franziska Hörsch ◽

...

Keyword(s):

Clinical Trials ◽

Cell Therapy ◽

Nk Cells ◽

Natural Killer ◽

Solid Tumors ◽

Cell Function ◽

Nk Cell ◽

Target Cells ◽

Collagen Gels ◽

Nk Cell Therapy

AbstractNatural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation for clinical use, the cells are cryopreserved to bridge the necessary evaluation time. While a degranulation assay confirms the ability of cryopreserved NK cells to kill target cells, we find a significant decrease of cytotoxicity after cryopreservation in a chromium release assay. We complement these standard assays with measurements of NK cell motility and cytotoxicity in 3-dimensional (3-D) collagen gels that serve as a substitute for connective tissue. We find a 5.6 fold decrease of cytotoxicity after cryopreservation and establish that this is mainly caused by a 6-fold decrease in the fraction of motile NK cells. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.SynopsisCryopreservation of natural killer (NK) cells dramatically impairs their motility and cytotoxicity in tissue. This finding may explain the persistent failure of clinical trials in which NK cell therapy is used for treating solid tumors.

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Donor-Derived Ex-Vivo Activated NK Cells in Management of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation in High-Risk Acute Leukemia

Blood ◽

10.1182/blood.v124.21.312.312 ◽

2014 ◽

Vol 124 (21) ◽

pp. 312-312

Author(s):

Xing-Yu Cao ◽

Tong Wu ◽

Bi-Ping Deng ◽

Rui-Juan Sun ◽

Yue Lu ◽

...

Keyword(s):

High Risk ◽

Cell Therapy ◽

Acute Leukemia ◽

Nk Cells ◽

Culture System ◽

Nk Cell ◽

Ex Vivo ◽

Leukemia Cells ◽

Original Culture ◽

Nk Cell Therapy

Abstract Introduction: Relapse remains the main cause of failure of hematopoietic stem cell transplantation (HSCT) in acute leukemia. NK cells have the property of killing leukemia cells without GVHD aggravation theoretically. Moreover, in some cases, leukemia cells may lost HLA-I and/HLA-II antigens which would result in poor response to the immunotherapy except NK-based adoptive effectors. Objective: In present study, the safety and efficacy of donor-derived ex-vivo activated NK cells in management of relapse after allogeneic HSCT in high-risk acute leukemia were examined. Patients and methods: Between July 2012 and July 2014, 29 patients with acute leukemia who received NK cell infusion after HSCT were analyzed retrospectively. Some cases failed to chemotherapy combined with donor lymphocyte infusion (DLI) before NK cell therapy. The diagnosis were ALL (10 cases), AML (18 cases) and mixed acute leukemia (1 case). All patients were high-risk leukemia. The disease status before transplant was CR1 in 8 cases, CR2 in 7, CR3 in 1 and non-remission in 13. The types of donor included identical sibling (5 cases), haploientical family member (21 cases) and unrelated donor (3 cases). The conditioning and GVHD prophylactic regimens were reported previously (Lu DP et al., Blood 2006; 107:3065). Minimal residual disease (MRD) was detected by either quantitative RT-PCR for fusion genes or flow cytometry or both. The expression of HLA-I and HLA-II antigens in leukemia cells was evaluated by flow cytometry. Donor-derived either peripheral blood stem cells or lymphocytes were cultured for 6 days using original culture system (AIM-V medium with IL-2, IL-12, IL-15 and IL-21) or modified culture system (SCGM medium with IL-2, IL-12, IL-15, IL-18 and IL-21). Escalated dosage of NK cells were infused starting with 1×105 cells/kg (recipient’s body weight) with or without IL-2 injection. Nine patients were in prevention group and 20 cases were in treatment group. The patients with hematologic relapse received NK cells 3 days later after chemotherapy. Results: Compared with our original culture system, the modified culture system enhanced approximately 10% to 20% of the purity and 4 to 8 fold in number of NK cells by day 6. Furthermore, our modified culture system elevated the expression of function phenotype including TRAIL, NKG2D and CD62L on NK cells in approximately 8 to 10 folds at day 6 and simultaneously stimulated higher level of IFN-γ. One to 4 NK cell infusions were given in each case with two week interval. Two of 29 cases developed mild skin GVHD. No transfusion-related side effects were noted. In prevention group, four of 9 cases remain complete remission, and the other 5 patients became MRD positive or relapse. In treatment group, seven of 20 cases have response to NK cell therapy, and two out of 7 cases who response to NK cells had failed to chemotherapy plus DLI before. Among 11 patients who had response to NK cells, eight of them are AML, and the remaining 3 patients are ALL. Higher response rate (10/23 cases) was seen with NK cell therapy by our modified culture system compared with the one (1/6 cases) by our original culture system. Conclusions: Our preliminary results have demonstrated that donor-derived ex-vivo activated NK cells are safe and effective modality in the management of relapse after allogeneic HSCT in high-risk acute leukemia even failed to chemotherapy combined with DLI. Optimal culture system has improved not only NK cell’s purity, number and function phenotype but also clinical efficacy. Disclosures No relevant conflicts of interest to declare.

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