Allogeneic Natural Killer Cells Research Articles

Abstract LB072: Tumor-infiltrating lymphocytes (TIL) engineered with membrane-bound IL15 (cytoTIL15 cells) exhibit pharmacologically regulatable signal transduction in cis and trans

Abstract Introduction: We have previously shown the successful engineering of membrane-bound IL15 (mbIL15)-expressing tumor-infiltrating lymphocytes (TIL; cytoTIL15™ cells) from solid tumors such as melanoma, which are CD8+ enriched TIL exhibiting enhanced persistence and anti-tumor activity compared with unengineered TIL (SITC 2022, 2023; clinical candidate OBX-115: NCT05470283). The mbIL15 expressed by cytoTIL15 cells is regulated via a carbonic anhydrase 2 (CA2) drug-responsive domain, which allows expression to be induced upon delivery of the FDA-approved small molecule ligand acetazolamide (ACZ) and was designed to engage the IL2Rβ and IL2Rγ receptors in both cis and in trans. Using natural killer (NK) cells, which are potently responsive to soluble, trans-presented IL15, as an experimental model system, we examined the potential of mbIL15 on cytoTIL15 cells to potentiate activity in both cis and trans. Methods: cytoTIL15 cells and unengineered TIL were generated as described previously (SITC 2022, 2023). Cells were assessed for expression of mbIL15 and phosphorylation of downstream signaling molecules STAT5 and S6 by flow cytometry, and co-cultured with allogeneic healthy donor NK cells for 1-10 days with and without transwell inserts. Functional effects of cytoTIL15 cells were compared to those of soluble recombinant IL15. Supernatant was obtained from co-cultures for assessment of cytokine production. Results: cytoTIL15 cells demonstrated robust ACZ-dependent mbIL15 expression, and ACZ dosing regulated mbIL15 activity in cis by potentiating the phosphorylation of STAT5 (geoMFI with 0 vs. 25 µM ACZ, p=0.012) and S6 (geoMFI with 0 vs. 25 µM ACZ, p=0.016). Upon removal of ACZ, mbIL15 expression and pSTAT5 and pS6 returned to basal levels within 24 hours. mbIL15 expressed on the surface of cytoTIL15 cells could also activate cells in trans in an ACZ-dependent titratable manner. Specifically, co-culture of cytoTIL15 cells with NK cells led to an ACZ-dependent increase in pSTAT5 (geoMFI with 0 vs. 25 µM ACZ, p=0.038) in NK cells, which was not seen in co-cultures of unengineered TIL with NK cells. Additionally, NK cells showed a similar cytokine production profile when exposed to mbIL15 by cytoTIL15 cells as with soluble, trans-presented IL15. NK cell transactivation relied on direct cell contact with cytoTIL15 cells, as magnitudes of phosphorylation were decreased in co-cultures with NK and cytoTIL15 cells separated by a transwell membrane (pSTAT5 geoMFI p=0.008). Conclusions: cytoTIL15 cells demonstrate functional engagement of cytokine receptors via mbIL15 in both cis and in trans, including phosphorylation of STAT5 and S6. Taken together, these data highlight the potential for robust activity of cytoTIL15 cells through potentiation of ACZ-dependent mbIL15 activity in both cis and trans. Citation Format: Rachel Burga, Gauri Kulkarni, Zheng Ao, Dhruv K. Sethi, Jan ter Meulen, Michelle Ols. Tumor-infiltrating lymphocytes (TIL) engineered with membrane-bound IL15 (cytoTIL15 cells) exhibit pharmacologically regulatable signal transduction in cis and trans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB072.

Abstract 5236: Adoptive natural killer therapy is effective for synovial sarcoma cell lines

Abstract Despite the development of multimodal therapy consisting of surgery, radiotherapy, and chemotherapy, the prognosis of patients with metastatic or relapsed synovial sarcoma (SS) remains poor. A novel treatment option including immunotherapy is urgently required. Natural killer (NK) cells are innate immune cells that can recognize and kill tumor cells. The cytolytic activity of NK cells is determined by the balance of signals from both inhibitory and activating receptors expressed on cell surface. Activating receptors include NKp30, NKp44, NKp46, NKG2D, and DNAM-1. Furthermore, tumor cells with low HLA class I expression are potentially even more sensitive to NK-mediated cytotoxic killing due to a lack of inhibitory signals. We previously reported that HLA class I expression was down regulated in one third of patients with synovial sarcoma, suggesting that NK cell therapy may be effective for patients with SS. However, the efficacy of NK cells treatment in SS was not well established. In this study, we evaluated surface ligands for NK cell activation on SS cell lines and demonstrated the effectiveness of activated allogenic NK cells for SS cell lines in vitro.The surface expression of ligands for NK cell-activating receptors including NKp30, NKp44, NKp46, NKG2D, and DNAM-1 in three SS cell lines (SYO-I, HS-SY-II, and Yamato-SS) was evaluated using flow cytometry. Peripheral blood from two healthy donors were ficoll-separated to collect peripheral blood mononuclear cells (PBMCs). Then, PBMCs were stimulated with irradiated K562 cells expressing the NK-stimulatory molecules 4-1BB ligand and membrane-bound Interleukin 15 in the presence of human Interleukin-2 in RPMI1640 with 10% FBS. The cells were expanded for 14 days, and the expanded NK cells are CD3-CD56+ NK cells. These expanded cells are reproducible in their function, and we have utilized these cells for in vitro cytotoxicity assays. 2x10E4 of SS cell lines (SYO-I, HS-SY-II, and Yamato-SS) were seeded in a 96 well plates and cultured overnight. Then, allogenic NK cells or activated T cells as a control at various tumor effector ratio. The cells were co-cultured with activated T cells or NK cells in the presence of low dose interleukin2. Forty-eight hours later, cell viabilities were measured using WST-8 assay.Ligands for NKp44, NKp30, NK2GD, were expressed in all three SS cell lines. It suggested that SS cells upregulate activating receptor ligands for NK cells. In addition, cytotoxicity of allogenic NK cells for SS cell lines was significantly higher than that of activated T cells. To determine whether cytotoxic activity was effect of allogenic NK cells, CD107a mobilization assay using flowcytometry was performed. CD107a mobilization assay demonstrated degranulation of NK cells, leading us that NK cells had an antitumoral effect on SS cell lines in vitro. Based on these initial findings, we were able to demonstrate that allogenic NK cells may be a promising immune therapy for SS. Citation Format: Naoki Oike, Tomohiro Miyazaki, Yudai Murayama, Takashi Ariizumi, Akira Ogose, Minori Baba, Hiroyuki Kawashima, Chihaya Imai. Adoptive natural killer therapy is effective for synovial sarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5236.

Abstract 1333: Preclinical data supporting the Phase 1 trial design of SENTI-202, a next generation allogeneic logic-gated selective CAR NK cell therapy, engineered to overcome key limitations of first generation cell therapies in AML

Abstract First generation natural killer (NK) and chimeric antigen receptor (CAR) NK cell products are well-tolerated and have clinical activity (~20-60% CR) in patients with AML. CRs generally increase with the inclusion of CARs or cytokines, increased cell doses, and when Flu/Ara-C lymphodepletion (LD) is used for conditioning. Key clinical limitations have been low PK of the infused NK cells, short durability of the observed responses potentially due to immune evasion of leukemic stem cells (LSCs), and manufacturing challenges precluding higher or multiple doses. SENTI-202 is a next generation allogeneic logic-gated selective CAR NK cell therapy specifically designed to address these limitations and to augment endogenous NK anti-AML activity. SENTI-202 expresses a bivalent activating CAR (aCAR) that targets CD33 and FLT3 on bulk AML blasts and LSCs. As FLT3 is also expressed on healthy hematopoietic stem cells (HSCs), SENTI-202 is further engineered to express an inhibitory CAR that recognizes endomucin expressed on HSCs, thereby conferring protection from aCAR-mediated cytotoxicity even in the presence of FLT3/CD33. SENTI-202 expresses a calibrated-release IL15 that provides both auto- and paracrine cytokine support, mediating expansion, activation, and persistence of SENTI-202 and host immune cells. SENTI-202 demonstrated robust and specific killing of primary AML blasts, LSCs, and AML cell lines in vitro. SENTI-202 protected HSCs from CAR-mediated cytotoxicity while preserving their function. In vivo, SENTI-202 revealed robust efficacy that increased with higher E:T ratio and with 3 weekly vs 1 dose. Preclinically, 3 doses of SENTI-202 that were > 60-fold the planned starting clinical trial dose were well tolerated in acute and chronic toxicology studies with no CAR NK-related body weight, laboratory, or histopathology findings. SENTI-202 non-clinical PK revealed greater than dose proportional exposure, which was ~2-fold greater compared to non-engineered NK cells. Pretreatment of CD33/FLT3 negative AML cell lines with Ara-C resulted in upregulation of CD33 and FLT3 expression, sensitizing cells to robust SENTI-202-mediated killing, providing additional rationale for the use of Ara-C-based LD. In the presence of exogenous IL2, persistence, cytotoxicity, and serial killing activity of SENTI-202 were increased, supporting the use of low dose IL2 to further augment SENTI-202 clinical activity. Taken together, these results support the Phase 1 trial design of SENTI-202-101 in patients with R/R CD33 and/or FLT3 positive malignancies including AML, which uses Flu/Ara-C as LD followed by 3 weekly doses of SENTI-202 and includes the option of enrolling patients into cohorts that additionally receive low dose IL2 following SENTI-202 administration. Citation Format: Deepika Kaveri, Pearley Chinta, Elizabeth Leitner, Lawrence Naitmazi, Gozde Yucel, Mengxi Tian, Niran Almudhfar, Han Deng, Yongshuai Li, Alice Lam, Chen-Ting Lee, Enping Hong, Rochelle Emery, Brian Garrison, Aaron N. Nguyen, Kanya Rajangam. Preclinical data supporting the Phase 1 trial design of SENTI-202, a next generation allogeneic logic-gated selective CAR NK cell therapy, engineered to overcome key limitations of first generation cell therapies in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1333.

Abstract 2690: The role of PCNA tyrosine phosphorylation in evading innate immune surveillance

Abstract Cancer cells are continually exposed to intrinsic and extrinsic proliferative stresses that can send the genome stability awry. The aberrant DNA metabolism, evolving through deregulated replicative growth in cancer cells, is increasingly recognized as a major mechanism bridging the crosstalk between the immune microenvironment and tumor tissue, thereby either promoting or suppressing tumor progression. However, the triggering and regulation of these mechanisms remain largely unknown. Proliferating cell nuclear antigen (PCNA) and its homologs are the evolutionarily conserved central components of the DNA replication machinery. In eukaryotic cells, PCNA forms homotrimeric rings encircling the DNA double helix to coordinate the complex processes of DNA replication and damage repair, directed at least in part by differential post-translational modifications of the PCNA protein. In cancer cells, growth signal-stimulated phosphorylation at tyrosine 211 of PCNA (pY211-PCNA) is known to play an important function for active proliferation. Here, we demonstrate that inhibition of pY211 disrupts the processivity of replication forks. This disruption triggers single-stranded DNA (ssDNA) production catalyzed by the MRE11 nuclease, activating the cGAS-STING axis to launch an anti-tumor immune response by natural killer (NK) cells. We further show that the pY211 regulates site-specific post-translational modifications of MRE11, and loss of pY211 promotes the endonuclease mode of MRE11. This causes ssDNA generation in the cytosol, subsequently inducing type I interferon-activated cytotoxicity by NK cells and fostering an anti-tumor immunity that abrogates distant metastasis. Mechanistically, the pY211 determines the recruitment of the writer or eraser of the modification on MRE11, thus tuning the nucleolytic modes of MRE11 and, consequently, ssDNA generation. Therapeutic measures promoting MRE11-dependent ssDNA enhance the response of triple-negative breast cancer to cytotoxic killing mediated by endogenous or allogenic NK cells in mice. In breast cancer patients, the level of cytosolic ssDNA is inversely correlated with the expression of the eraser and pY211-PCNA, which is associated with poor overall survival in cancer patients. Our studies shed new light on the shaping of the tumor immune microenvironment and demonstrate the potential to develop therapeutic approaches that proactively leverage genomic instability and immune activation to target malignant tumors. Citation Format: Chuan-Chun Lee, Wan-Rong Wu, Yi-Chun Shen, Feng-Chi Chung, Yuan-Liang Wang, You-Zhe Lin, Chih-Hao Lu, Wei-Chung Cheng, Han Chang, Liang-Chih Liu, Ji-An Liang, Chang- Fang Chiu, Mien-Chie Hung, Shao-Chun Wang. The role of PCNA tyrosine phosphorylation in evading innate immune surveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2690.

Abstract 1334: Evaluation of NK cell combination therapies in mouse models of cancer

Abstract Adoptive immunotherapy with natural killer (NK) cells has emerged as a promising innovative approach against hematological malignancies and solid tumors. To enhance the functional aspects of NK cells and to achieve stronger, sustained anti-tumor responses, combination strategies are under development. They are focused on optimization of NK cell potency, persistence and tumor targeting using monoclonal antibodies, multi-specific NK cell engagers and immune modulators like cytokines. SAR445419 is a universal off-the-shelf allogeneic human NK cell (hNK) product made using Sanofi’s proprietary platform with potential broad application across hematologic and solid tumor indications. We evaluated the therapeutic potential of these NK cells in combination with internal assets and standard of care treatments in preclinical mouse models. The studies were performed in immunodeficient NOG mouse strains transgenic for hIL-15, supporting long-term engraftment of functional hNK cells. We demonstrated the therapeutic advantage of NK cell combination with an anti-CD20 monoclonal antibody (mAb) in a disseminated lymphoma model. We also reported prolonged lifespan of leukemia-bearing animals treated with NK cells and pegenzileukin, a PEGylated non-alpha rhIL-2 variant engineered to preferentially bind to the IL-2 βγ receptors and promote NK cell function. The anti-tumor response correlated with pegenzileukin-induced increase in NK cell numbers in the blood. A combination of CD38KO NK cells with an anti-CD38 mAb was also investigated in a model of multiple myeloma (MM). FcγR-KO immunodeficient mice were used in these studies to limit the confounding contribution of residual murine immune cells engaging with the Fc-competent therapeutic antibody and to specifically detect hNK-cell mediated activity. The comparison of CD38KO versus WT NK cell numbers in the blood of animals treated with an anti-CD38 mAb showed a significant decrease in CD38-expressing NK cell count that was mitigated by the genetic deletion of CD38. Obtained results suggest that CD38KO NK cells are resistant to anti-CD38 mAb-mediated fratricide and provide a rationale for the combination of CD38KO NK cells with anti-an CD38 mAb against MM. Overall, these preclinical in vivo studies have a pivotal role in the evaluation of NK cell combination therapies against cancer and reveal their therapeutic potential. Citation Format: Katarzyna Franciszkiewicz, Pauline Rettman, Beatrice Gauthier, Laurence Noblet, Celine Nicolazzi, Andre Kunert, Rob Igarashi, Loreley Calvet, Valeria Fantin, Sukhvinder Sidhu. Evaluation of NK cell combination therapies in mouse models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1334.

Natural killer cell therapies.

Natural killer (NK) cells are lymphocytes of the innate immune system. A key feature of NK cells is their ability to recognize a wide range of cells in distress, particularly tumour cells and cells infected with viruses. They combine both direct effector functions against their cellular targets and participate in the generation, shaping and maintenance of a multicellular immune response. As our understanding has deepened, several therapeutic strategies focused on NK cells have been conceived and are currently in various stages of development, from preclinical investigations to clinical trials. Here we explore in detail the complexity of NK cell biology in humans and highlight the role of these cells in cancer immunity. We also analyse the harnessing of NK cell immunity through immune checkpoint inhibitors, NK cell engagers, and infusions of preactivated or genetically modified, autologous or allogeneic NK cell products.

Allogeneic NK cells induce the in vitro activation of monocyte-derived and conventional type-2 dendritic cells and trigger an inflammatory response under cancer-associated conditions.

Natural killer (NK) cells are innate lymphocytes capable to recognize and kill virus-infected and cancer cells. In the past years, the use of allogeneic NK cells as anti-cancer therapy gained interest due to their ability to induce graft-versus-cancer responses without causing graft-versus-host disease and multiple protocols have been developed to produce high numbers of activated NK cells. While the ability of these cells to mediate tumor kill has been extensively studied, less is known about their capacity to influence the activity of other immune cells that may contribute to a concerted anti-tumor response in the tumor microenvironment (TME). In this study, we analyzed how an allogeneic off-the-shelf cord blood stem cell-derived NK-cell product influenced the activation of dendritic cells (DC). Crosstalk between NK cells and healthy donor monocyte-derived DC (MoDC) resulted in the release of IFNγ and TNF, MoDC activation, and the release of the T-cell-recruiting chemokines CXCL9 and CXCL10. Moreover, in the presence of prostaglandin-E2, NK cell/MoDC crosstalk antagonized the detrimental effect of IL-10 on MoDC maturation leading to higher expression of multiple (co-)stimulatory markers. The NK cells also induced activation of conventional DC2 (cDC2) and CD8+ T cells, and the release of TNF, GM-CSF, and CXCL9/10 in peripheral blood mononuclear cells of patients with metastatic colorectal cancer. The activated phenotype of MoDC/cDC2 and the increased release of pro-inflammatory cytokines and T-cell-recruiting chemokines resulting from NK cell/DC crosstalk should contribute to a more inflamed TME and may thus enhance the efficacy of T-cell-based therapies.

High and selective cytotoxicity of ex vivo expanded allogeneic human natural killer cells from peripheral blood against bladder cancer: implications for natural killer cell instillation after transurethral resection of bladder tumor

BackgroundNon-muscle-invasive bladder cancer (NMIBC) is treated with transurethral resection of bladder tumor (TURBT) followed by intravesical instillation of chemotherapy or Bacillus Calmette–Guérin therapy. However, these treatments have a high recurrence rate and side effects, emphasizing the need for alternative instillations. Previously, we revealed that expanded allogeneic human natural killer (NK) cells from peripheral blood are a promising cellular therapy for prostate cancer. However, whether NK cells exhibit a similar killing effect in bladder cancer (BCa) remains unknown.MethodsExpansion, activation, and cryopreservation of allogeneic human NK cells obtained from peripheral blood were performed as we previously described. In vitro cytotoxicity was evaluated using the cell counting kit-8. The levels of perforin, granzyme B, interferon-γ, tumor necrosis factor-α, and chemokines (C-C-motif ligand [CCL]1, CCL2, CCL20, CCL3L1, and CCL4; C-X-C-motif ligand [CXCL]1, CXCL16, CXCL2, CXCL3, and CXCL8; and X-motif ligand 1 and 2) were determined using enzyme-linked immunosorbent assay. The expression of CD107a, major histocompatibility complex class I (MHC-I), MHC-I polypeptide-related sequences A and B (MICA/B), cytomegalovirus UL16-binding protein-2/5/6 (ULBP-2/5/6), B7-H6, CD56, CD69, CD25, killer cell Ig-like receptors (KIR)2DL1, KIRD3DL1, NKG2D, NKp30, NKp46, and CD16 of NK cells or BCa and normal urothelial cells were detected using flow cytometry. Cytotoxicity was evaluated using lactate dehydrogenase assay in patient-derived organoid models. BCa growth was monitored in vivo using calipers in male NOD-scid IL2rg−/− mice subcutaneously injected with 5637 and NK cells. Differential gene expressions were investigated using RNA sequence analysis. The chemotaxis of T cells was evaluated using transwell migration assays.ResultsWe revealed that the NK cells possess higher cytotoxicity against BCa lines with more production of cytokines than normal urothelial cells counterparts in vitro, demonstrated by upregulation of degranulation marker CD107a and increased interferon-γ secretion, by MICA/B/NKG2D and B7H6/NKp30-mediated activation. Furthermore, NK cells demonstrated antitumor effects against BCa in patient-derived organoids and BCa xenograft mouse models. NK cells secreted chemokines, including CCL1/2/20, to induce T-cell chemotaxis when encountering BCa cells.ConclusionsThe expanded NK cells exhibit potent cytotoxicity against BCa cells, with few toxic side effects on normal urothelial cells. In addition, NK cells recruit T cells by secreting a panel of chemokines, which supports the translational application of NK cell intravesical instillation after TURBT from bench to bedside for NMIBC treatment.

Transforming CLL Management with Immunotherapy: Investigating the Potential of CAR T-Cells and Bispecific Antibodies

Immunotherapies, such as chimeric antigen receptor (CAR) T-cell therapy and bispecific antibodies or T-cell engagers, have revolutionized the treatment landscape for various B-cell malignancies, including B-acute lymphoblastic leukemia and many non-Hodgkin lymphomas. Despite their significant impact on these malignancies, their application in chronic lymphocytic leukemia (CLL) management is still largely under investigation. Although the initial success of CD19-directed CAR T-cell therapy was observed in three multiply relapsed CLL patients, with two of them surviving over 10 years without relapse, recent CAR T-cell therapy trials in CLL have shown reduced response rates compared to their efficacy in other B-cell malignancies. One of the challenges with using immunotherapy in CLL is the compromised T-cell fitness from persistent CLL-related antigenic stimulation, and an immunosuppressive tumor microenvironment (TME). These challenges underscore a critical gap in therapeutic options for CLL patients intolerant or resistant to current therapies, emphasizing the imperative role of effective immunotherapy. Encouragingly, innovative strategies are emerging to overcome these challenges. These include integrating synergistic agents like ibrutinib to enhance CAR T-cell function and persistence, and engineering newer CAR T-cell constructs targeting diverse antigens or employing dual-targeting approaches. Bispecific antibodies are an exciting "off-the-shelf" prospect for these patients, with their investigation in CLL currently entering the realm of clinical trials. Additionally, the development of allogeneic CAR T-cells and natural killer (NK) cells from healthy donors presents a promising solution to address the diminished T-cell fitness observed in CLL patients. This comprehensive review delves into the latest insights regarding the role of immunotherapy in CLL, the complex landscape of resistance mechanisms, and a spectrum of innovative approaches to surmount therapeutic challenges.

Optimisation of a primary human CAR-NK cell manufacturing pipeline.

Autologous chimeric antigen receptor (CAR) T-cell therapy of B-cell malignancies achieves long-term disease remission in a high fraction of patients and has triggered intense research into translating this successful approach into additional cancer types. However, the complex logistics involved in autologous CAR-T manufacturing, the compromised fitness of patient-derived Tcells, the high rates of serious toxicities and the overall cost involved with product manufacturing and hospitalisation have driven innovation to overcome such hurdles. One alternative approach is the use of allogeneic natural killer (NK) cells as a source for CAR-NK cell therapy. However, this source has traditionally faced numerous manufacturing challenges. To address this, we have developed an optimised expansion and transduction protocol for primary human NK cells primed for manufacturing scaling and clinical evaluation. We have performed an in-depth comparison of primary human NK cell sources as a starting material by characterising their phenotype, functionality, expansion potential and transduction efficiency at crucial timepoints of our CAR-NK manufacturing pipeline. We identified adult peripheral blood-derived NK cells to be the superior source for generating a CAR-NK cell product because of a higher maximum yield of CAR-expressing NK cells combined with potent natural, as well as CAR-mediated anti-tumor effector functions. Our optimised manufacturing pipeline dramatically improves lentiviral transduction efficiency of primary human NK cells. We conclude that the exponential expansion pre- and post-transduction and high on-target cytotoxicity make peripheral blood-derived NK cells a feasible and attractive CAR-NK cell product for clinical utility.

Interleukin-2 is required for NKp30-dependent NK cell cytotoxicity by preferentially regulating NKp30 expression.

Natural killer (NK) cells are key effectors in cancer immunosurveillance, eliminating a broad spectrum of cancer cells without major histocompatibility complex (MHC) specificity and graft-versus-host diseases (GvHD) risk. The use of allogeneic NK cell therapies from healthy donors has demonstrated favorable clinical efficacies in treating diverse cancers, particularly hematologic malignancies, but it requires cytokines such as IL-2 to primarily support NK cell persistence and expansion. However, the role of IL-2 in the regulation of activating receptors and the function of NK cells expanded for clinical trials is poorly understood and needs clarification for the full engagement of NK cells in cancer immunotherapy. Here, we demonstrated that IL-2 deprivation significantly impaired the cytotoxicity of primary expanded NK cells by preferentially downregulating NKp30 but not NKp46 despite their common adaptor requirement for expression and function. Using NK92 and IL-2-producing NK92MI cells, we observed that NKp30-mediated cytotoxicity against myeloid leukemia cells such as K562 and THP-1 cells expressing B7-H6, a ligand for NKp30, was severely impaired by IL-2 deprivation. Furthermore, IL-2 deficiency-mediated NK cell dysfunction was overcome by the ectopic overexpression of an immunostimulatory NKp30 isoform such as NKp30a or NKp30b. In particular, NKp30a overexpression in NK92 cells improved the clearance of THP-1 cells in vivo without IL-2 supplementation. Collectively, our results highlight the distinct role of IL-2 in the regulation of NKp30 compared to that of NKp46 and suggest NKp30 upregulation, as shown here by ectopic overexpression, as a viable modality to harness NK cells in cancer immunotherapy, possibly in combination with IL-2 immunocytokines.

Antitumor activity of genetically engineered NK-cells in non-hematological solid tumor: a comprehensive review.

Recent advancements in genetic engineering have made it possible to modify Natural Killer (NK) cells to enhance their ability to fight against various cancers, including solid tumors. This comprehensive overview discusses the current status of genetically engineered chimeric antigen receptor NK-cell therapies and their potential for treating solid tumors. We explore the inherent characteristics of NK cells and their role in immune regulation and tumor surveillance. Moreover, we examine the strategies used to genetically engineer NK cells in terms of efficacy, safety profile, and potential clinical applications. Our investigation suggests CAR-NK cells can effectively target and regress non-hematological malignancies, demonstrating enhanced antitumor efficacy. This implies excellent promise for treating tumors using genetically modified NK cells. Notably, NK cells exhibit low graft versus host disease (GvHD) potential and rarely induce significant toxicities, making them an ideal platform for CAR engineering. The adoptive transfer of allogeneic NK cells into patients further emphasizes the versatility of NK cells for various applications. We also address challenges and limitations associated with the clinical translation of genetically engineered NK-cell therapies, such as off-target effects, immune escape mechanisms, and manufacturing scalability. We provide strategies to overcome these obstacles through combination therapies and delivery optimization. Overall, we believe this review contributes to advancing NK-cell-based immunotherapy as a promising approach for cancer treatment by elucidating the underlying mechanisms, evaluating preclinical and clinical evidence, and addressing remaining challenges.

The optimal management of relapsed and refractory Hodgkin lymphoma: post-brentuximab and checkpoint inhibitor failure.

The treatment landscape of classical Hodgkin lymphoma has changed dramatically over the past decade. Relapsed and refractory mainstay therapeutics such as brentuximab vedotin (BV) and checkpoint inhibitors (CPIs) are being moved to earlier lines of therapy. However, the treatment of patients who progress after BV and CPI remains a challenge. Allogeneic stem cell transplantation still plays an important role in this patient population as the only current treatment approach with curative potential. Unfortunately, not all patients are transplant candidates, and many will still relapse afterward. Cytotoxic chemotherapy and radiation may be used for symptom palliation or as a bridge to transplant. Targeted therapies, including the antibody drug conjugate, camidanlumab tesirine, and transcriptional agents such mammalian target of rapamycin and histone deacetylase inhibitors have shown some potential in patients with refractory disease. In addition, combination therapies with CPIs and novel agents may help overcome resistance to therapy. Clinical trials with cellular therapies, including chimeric antigen receptor T cells targeting CD30 and allogeneic natural killer cells combined with AFM13, a CD30/CD16a-bispecific antibody, have shown promising results. The availability of more therapeutic options for this patient population is eagerly awaited.

Nicotinamide-Expanded Allogeneic Natural Killer Cells with CD38 Deletion, Expressing an Enhanced CD38 Chimeric Antigen Receptor, Target Multiple Myeloma Cells.

Natural killer (NK) cells are a vital component of cancer immune surveillance. They provide a rapid and potent immune response, including direct cytotoxicity and mobilization of the immune system, without the need for antigen processing and presentation. NK cells may also be better tolerated than T cell therapy approaches and are susceptible to various gene manipulations. Therefore, NK cells have become the focus of extensive translational research. Gamida Cell's nicotinamide (NAM) platform for cultured NK cells provides an opportunity to enhance the therapeutic potential of NK cells. CD38 is an ectoenzyme ubiquitously expressed on the surface of various hematologic cells, including multiple myeloma (MM). It has been selected as a lead target for numerous monoclonal therapeutic antibodies against MM. Monoclonal antibodies target CD38, resulting in the lysis of MM plasma cells through various antibody-mediated mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis, significantly improving the outcomes of patients with relapsed or refractory MM. However, this therapeutic strategy has inherent limitations, such as the anti-CD38-induced depletion of CD38-expressing NK cells, thus hindering ADCC. We have developed genetically engineered NK cells tailored to treat MM, in which CD38 was knocked-out using CRISPR-Cas9 technology and an enhanced chimeric antigen receptor (CAR) targeting CD38 was introduced using mRNA electroporation. This combined genetic approach allows for an improved cytotoxic activity directed against CD38-expressing MM cells without self-inflicted NK-cell-mediated fratricide. Preliminary results show near-complete abolition of fratricide with a 24-fold reduction in self-lysis from 19% in mock-transfected and untreated NK cells to 0.8% of self-lysis in CD38 knock-out CAR NK cells. Furthermore, we have observed significant enhancements in CD38-mediated activity in vitro, resulting in increased lysis of MM target cell lines. CD38 knock-out CAR NK cells also demonstrated significantly higher levels of NK activation markers in co-cultures with both untreated and αCD38-treated MM cell lines. These NAM-cultured NK cells with the combined genetic approach of CD38 knockout and addition of CD38 CAR represent a promising immunotherapeutic tool to target MM.

Abstract A036: Development of a stirred-tank bioreactor scale-up approach to allogeneic natural killer cell culture

Abstract Natural killer (NK) cells are innate lymphocytes that play a critical role in the immune response, as they recognize and eliminate virus-infected, cancerous, and other abnormal cells in an antigen-independent manner. This unique method of recognition makes NK cells an attractive target for utilization as an immunotherapy, as studies have demonstrated they reduce both the induction of graft-versus-host disease and the risk of cytokine release syndrome compared to T-cell based therapies. Additionally, these advantages over canonical T-cell therapies make NK therapies attractive candidates for allogeneic, as opposed to autologous, products. Given their cost-effective scale-up potential, developing a stirred-tank bioreactor (STR)-based NK cell expansion platform to achieve clinical-level expansion is of significant interest, as it would reduce the cost of producing such therapies and improve patient accessibility. To test ideal culture conditions for STR expansion, NK cells were first isolated from healthy donor Leukopaks prior to cryopreservation. Frozen, isolated NK cells were then thawed, co-cultured with varying ratios of irradiated K562 cells and supplemental cytokines from Cytiva in a 6-well G-Rex or DASbox® STR. Finally, cells were harvested for downstream analytics, including identity and functional characterization by flow cytometry and potency assays. A fold expansion of greater than 100 was achieved once the ratio of irradiated K562 to NK cells was optimized. One of the primary challenges encountered was donor-to-donor variation, not only with respect to proliferative capacity, but NK functional phenotype and activity as well. Therefore, a small-scale donor screen was conducted and donors with similar proliferative and functional capabilities were selected for large-scale expansion. Future work will focus on optimizing NK cell transduction to enable genetic customization and scaling-up to a clinically-relevant STR. The successful small-scale NK expansion in an STR demonstrated here lays the groundwork for an expansion platform that enables the manufacturing of various NK-based therapeutics at clinical scale. Citation Format: Mathura Yogalingam, Rebecca Jagroop, Alireza Khalajzadeh, Thergiory Irrazabal, Melissa Perea, Pooja Pushparaj, Roxana Roshankhah, Paul Bowles, Michael Le. Development of a stirred-tank bioreactor scale-up approach to allogeneic natural killer cell culture [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A036.

Azacitidine, Venetoclax and Allogeneic NK Cells in Newly Diagnosed Acute Myeloid Leukemia (ADVENT-AML): An Investigator-Initiated Multicenter Phase Ib Trial

BACKGROUND AND SIGNIFICANCE : Older/unfit patients with acute myeloid leukemia (AML) have limited treatment options. Outcomes in adverse-risk subgroups of AML are modest with CR/CRi rates of 45% to 60% and median overall survival (OS) of 5 to 12 months with current standard frontline therapy of hypomethylating agent with venetoclax (HMA-VEN).While allogeneic stem cell transplantation (allo-SCT) can significantly improve OS in AML, only 10 to 20% of older pts are able to undergo allo-SCT (Pratz et al. Blood 2019, Maiti et al. Blood 2022) Natural killer (NK) cells bridge the innate and adaptive immune system and play a major role in graft-vs-leukemia (GVL) effect responsible for the clinical benefit noted with allo-SCT. NK cell-based adoptive cellular therapies have shown good safety profiles and promising activity in AML with responses noted in 25% to 88% of patients. Furthermore, NK cells are less likely to induce graft-versus-host disease (GVHD), severe cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). We hypothesize that the combination of azacitidine, venetoclax and allogeneic NK cells will be safe and improve cure rates in AML compared to either Aza/Ven or NK cells alone. Improved clinical activity may be mediated through multiple synergistic mechanisms including reduction of disease burden by Aza/Ven, upregulation of epigenetically silenced NKG2D ligands, dual activation of extrinsic and intrinsic apoptotic cascades, mitochondrial priming of leukemia cells by venetoclax, and bypassing significant morbidity and mortality associated allo-SCT. Significance: This will be the first trial to evaluate 1) cellular therapy in the frontline setting in AML; 2) the synergy of venetoclax with adoptive NK cell therapy; and 3) Aza/Ven as a lymphodepletion strategy. The results will have significant impact on the development of cellular therapies for hematological and solid tumors. STUDY DESIGN AND METHODS: This investigator-initiated, multicenter, open-label, phase Ib study will evaluate the combination of azacitidine, venetoclax and ex vivo-expanded, off-the-shelf cryopreserved allogeneic NK cells in patients with AML (NCT05834244). Eligibility criteria: The dose escalation phase will enroll adults with relapsed or refractory AML or MDS/AML, and the dose expansion phase will enroll older/unfit pts with newly diagnosed adverse-risk AML. Eligible Pts will be ≥ 75 years, or ≥ 18 years with at least one protocol-defined comorbidity. Patients with favorable risk AML, poor end-organ function, needing immunosuppression, uncontrolled infection or CNS leukemia will be excluded. Trial design: The dose escalation phase will enroll a maximum of 12 patients at 2 dose levels to establish a recommended phase 2 dose (RP2D) using a BOIN design. The dose expansion will enroll up to 20 patients at the RP2D ( Fig. 1). Study treatment: Patients will receive Aza/Ven as standard. Two dose levels of NK cells including 0.5 x10 9 and 1 x10 9 flat dose on day 8 and 15 of the first 4 cycles will be evaluated. Subsequently, pts will continue Aza/Ven indefinitely. Objectives: The primary objective is to determine the safety of this novel triplet combination in terms of dose-limiting toxicities including acute GVHD, CRS or ICANS. Secondary objectives include overall response rate, CR/CRi by 4 cycles, rate of negative measurable residual disease, OS and relapse-free survival. Exploratory objectives include additional response and survival endpoints, the persistence of NK cells using SNP-NGS chimerism, kinetics of allorejection, remodeling of host immune microenvironment and impact on AML stem/progenitor cell hierarchies using single cell CyTOF to understand response/resistance mechanisms. Status: Trial approved by the FDA and institutional IRB. Activation planned for 2H 2023. Funding: Gateway for Cancer Research, MDACC PRIME Award, Chimeric Therapeutics, NCI Cancer Center Support Grant.

Natural Killer Cells Are Active Against Myeloma, Leukemia or Lymphoma Cells with Loss of Tumor Suppressor Genes

The therapeutic targeting of tumor cells with loss of function (LOF) for tumor suppressor genes (TSGs) is challenging across cancers, including hematologic neoplasias, because pharmacological mechanisms to restore the function(s) of such genes are not readily feasible, in contrast to e.g., inhibition of oncogenic drivers. We reasoned, however, that, although LOF for TSGs leads to de-repressed growth of neoplastic hematopoietic cells, it may not necessarily protect them from immune attack. We thus explored the hypothesis that CRISPR-based loss of function of TSGs in cells from multiple myeloma (MM), leukemias or lymphoma may still be associated with substantial response to immune effector cells such as NK cells, which have the advantage to kill tumor cells across HLA barriers. We have conducted CRISPR-based studies in 7 cell lines that represent different hematologic malignancies and different levels of sensitivity to natural killer (NK) cells, namely the B-cell lymphoma (SUDHL4), precursor B cell acute lymphoblastic leukemia (NALM6), multiple myeloma (MM1.S, LP1, KMS11), chronic myeloid leukemia (K562), and acute myeloid leukemia (MOLM14). In these genome-scale or focused CRISPR screens for LOF (CRISPR-based gene editing) or gain of function (GOF, CRISPR activation), the blood cancer lines were exposed to allogeneic donor-derived NK cells (vs. control cultures without NK cells). We evaluated the performance of genes known to represent recurrent TSGs based on genomic data of patient samples or cell lines; as well as candidate TSGs, based on results from genome-scale CRISPR gene editing screens (e.g., CERES or CHRONOS scores >0.4 in multiple DepMap releases and TPM>1 [RNA-seq]) in the same cell lines as the NK cell resistance screens. These analyses sought to identify any TSGs whose LOF may potentially alter the response of blood cancer cells to NK cells. We also evaluated genes identified as top recurrent TSGs in patient samples from MM and other hematologic neoplasias (e.g., based on prior genomic studies). On aggregate, we evaluated a collection of known and recurrent TSGs (e.g., PTEN, TP53, RB1, CDKN2C, CDKN1B, TENT5C/FAM46C) as well as other, previously underappreciated candidate genes with TSG properties in hematologic neoplasias (e.g., HIF1A, DEPDC5). Perturbation of none of these genes was identified to meet criteria for association with significant resistance to allogeneic donor-derived NK cells (e.g., log2FC>1.0, at least 3-4 sgRNAs with enrichment upon CRISPR KO or depletion with CRISPR activation, p-value <0.05, enrichment rank <100, based on rank aggregation algorithm) in any of the MM, leukemia or lymphoma cell lines examined in LOF or GOF CRISPR screens for NK cell resistance. In fact, for a limited set of cases (e.g., PTEN in KMS11 cells), KO of a TSG was associated with sensitization to NK cell treatment. To probe the mechanistic basis for this lack of effect of TSG loss, we examined the molecular sequelae of CRISPR-based KO for one of these recurrent TSGs, PTEN, by performing scRNAseq using the CROP-seq platform. Pools of MM1.S and LP1 cells expressing sgRNAs targeting select hits from our CRISPR screens and also PTEN were co-cultured with NK cells for 24 h or left untreated, followed by scRNA-seq and sgRNA detection, differential gene-expression analysis. We observed in these studies that the transcriptional changes induced in MM cells by KO of PTEN included limited, if any, changes in the expression of key genes involved in regulation of NK cell responses of tumor cells, e.g., ligands for activating or inhibitor receptors in NK cells, death receptors (e.g., TRAIL, Fas) or their downstream effectors/regulators or other molecules identified from our aforementioned genome-scale and focused CRISPR LOF and GOF studies. Notably, our in-house results from studies of NK exposure of cell lines from hematologic neoplasias are concordant with results for these TSGs in CRISPR screens of non-hematological cancer cells treated with cytotoxic T-cells (e.g., 4T1 or RENCA cells; GSE149933). Overall, our observations indicate that MM, leukemia or lymphoma cells that become deficient for diverse TSGs based on CRISPR-based gene editing are equally responsive to NK cells as their TSG-proficient counterparts. NK cell-based therapies may thus be a promising approach to target TSG-deficient hematologic malignancies for which specific pharmacological therapies are not currently available.

Innate Cell Engager (ICE®) AFM13 Combined with Preactivated and Expanded (P+E) Cord Blood (CB)-Derived Natural Killer (NK) Cells for Patients with Refractory CD30-Positive Lymphomas: Final Results

CONCLUSIONS Pts with refractory Hodgkin (HL) and other CD30+ lymphomas have few effective therapies available and targeted NK cell immunotherapy is an active area of research. Transfer of non-targeted NK cells has shown limited clinical benefit as target recognition of cancer cells by NK cells constitutes a barrier. The innate cell engager AFM13 is a first-in-class CD30/CD16A bispecific antibody construct that induces selective killing of CD30+ tumor cells by engaging and activating NK cells. As a single agent, AFM13 has limited clinical activity against HL, likely due to the reduced effector function of autologous NK cells in these pts, which provides the rationale for combining AFM13 with allogeneic NK cells. Our prior preclinical work identified a promising combination of CB-derived NK cells that were first IL-12/IL-15/IL-18-preactivated followed by ex vivo expansion (P+E) with engineered K562 feeder cells expressing membrane-bound IL-21, 4-1BBL and CD48 and exogenous IL-2, and subsequently complexed with AFM13 just before infusion; these cytokine-induced memory-like NK cells presented chimeric antigen receptor (CAR)-NK-like features and increased in vitro and in vivo antitumor activity compared to either non-AFM13-precomplexed P+E NK cells or to AFM13 alone (Kerbauy et al, Clin Cancer Res 2021). Building on this work we wished to study this CB NK-cell therapy in pts with refractory CD30+ lymphomas. This investigator-initiated single-center phase I-II trial (NCT04074746) evaluated the safety and activity of P+E AFM13-precomplexed CB NK cells followed by systemic AFM13. Pts ages 15-75 with CD30+ lymphomas refractory to brentuximab vedotin were enrolled. The goals were to identify the recommended phase 2 dose (RP2D) of NK cells, estimate the overall (ORR) and complete response (CR), event-free (EFS) and overall survival (OS) rates, and study NK cell activation and persistence. Each treatment cycle consisted of fludarabine/cyclophosphamide (days −5 to −3) followed by infusion (day 0) of the AFM13-precomplexed CB NK cells, cultured for 14 days as described above, and three weekly IV infusions of AFM13 (200 mg, days 7, 14 and 21). Two cycles were planned for pts 1-19 and up to 4 cycles from patient 20 on. Response was evaluated on day 28 of each cycle. Enrollment proceeded at 3 dose levels (DL) of 10 6, 10 7 and 10 8 NK/Kg, respectively. Each patient received the same NK dose in all cycles. Between 09/20-12/22 we enrolled 42 pts (37 HL, 5 NHL), pretreated with a median 7 lines, at DL1 (N=3), DL2 (N=3) and DL3 (N=36), with 1 (N=2), 2 (N=21), 3 (N=3) or 4 cycles (N=16). All had active progressive disease at enrollment and no bridging therapy was given. The cords used for each of the 117 cycles were selected without consideration for HLA match, which was 0/6 (N=53), 1/6 (N=46), 2/6 (N=14), 3/6 (N=3) and 4/6 (N=1). The product infused consisted of >94% NK cells (expanded >2,700 fold), with only 0.02% T cells. The NK cells were 96% viable, 91% bound to AFM13, and 97.6% CD16+. The products were infused fresh. There was 1 grade 2 infusion-related reaction (IRR) in 115 infusions of AFM13-NK (0.8%) and 27 IRR infusion related reactions in 350 infusions of AFM13 (7.7%) (1 G3, 26 G2). We saw no cases of CRS, ICANS or GVHD of any grade. DL3 (10 8 NK/Kg) was established as the RP2D. The ORR and CR were 92.8% and 66.7%, respectively (94.4% and 72.2%, respectively, in 36 pts treated at the RP2D). Nine pts had a response consolidated with SCT (5 allo, 4 auto). At median follow-up of 14 (6-34) months, the EFS/OS rates are 31%/76%; median EFS/OS are 8 months/not reached. No relapses were associated with antigen loss. In the subset of pts planned for 4 cycles at RP2D, 15 of 23 pts remained event free at 6 mo (4 pts with and 11 without SCT consolidation), for 6-month EFS/OS rates of 65%/83%. Overall, 8 of the 9 pts (89%) receiving a consolidation SCT are event free at 10 to 34 months. AFM-bound CB NK cells were detected in blood from day 1 post infusion for up to 3 weeks. Donor NK levels followed similar patterns after each cycle, which argues against a sensitization after the first NK infusion. CyTOF studies showed increased expression of AFM13 and activation markers in donor NK cells. We confirmed trafficking of AFM13-bound donor NK cells to tumor sites shortly after infusion. CB-derived cytokine-induced memory-like NK cells precomplexed with AFM13 have excellent tolerability and activity for pts with heavily pretreated and refractory CD30+ lymphoma.

Results of Cynk-001-AML-001: A Phase I Multi-Dose Study Evaluating the Safety, Tolerability, and Persistence of Cynk-001 in Adults with De Novo or Secondary Acute Myeloid Leukemia in Morphologic Complete Remission with Minimal Residual Disease or Relapsed/Refractory AML

Introduction: Acute myeloid leukemia (AML) is a clinically and genetically heterogeneous disease often with suboptimal outcomes. While progress has been achieved in the treatment of AML, relapse remains a significant challenge. Novel therapeutic options aimed at attaining minimal residual disease (MRD) negative complete remission (CR) are expected to reduce the incidence of relapse and prolong survival. Evidence shows that natural killer (NK) cells can exhibit potent anti-leukemia activity against AML. CYNK-001 is a CD56+CD3- enriched, off-the-shelf, unmodified allogeneic NK cells expanded from placental CD34+ cells. Aim CYNK-001-AML-001 is a phase I multi-dose study evaluating the safety, tolerability, and persistence of CYNK-001 with or without recombinant human interleukin-2 (rhIL-2) in adults with de novo or secondary acute myeloid leukemia (AML) in morphologic complete remission (CR) with minimal residual disease (MRD) or relapsed/refractory (R/R) AML. Methods This Phase I, single-arm, dose escalation clinical trial (NCT04310592) enrolled patients 18-80 years with R/R AML or patient in MRD positive CR. Two lymphodepletion regimens were assessed. Patients in cohorts 1-3 underwent standard lymphodepletion with fludarabine (25 mg/m2/day) and cyclophosphamide (300 mg/m2/day) on days -5, -4 and -3. Subsequent cohorts received enhanced lymphodepletion with fludarabine (30 mg/m2/day) and cyclophosphamide (900 mg/m2/day) on days -6, -5, -4 and -3. Escalating doses of NK cells were administered following a standard 3+3 design, with 2 separate arms (MRD+ arm and R/R arm) starting with cohort 4. A starting dose of 600 million cells on days 0, 7, and 14 was escalated to a maximum dose of 1.8 billion cells on days 0, 7, 14 and 21. The primary objective of the study was to determine the maximum tolerated dose (MTD) or maximum planned dose (MPD) of CYNK-001 and to assess the safety of multiple infusions of CYNK-001 administered using a flat, non-weight-based dose as assessed by the frequency and severity of adverse events (AE). The secondary objective was to assess the clinical efficacy of CYNK-001 in AML subjects. Results 28 patients (17 with R/R AML and 11 with MRD-positive AML) were enrolled during dose escalation, and 27 received CYNK-001. The highest dose of NK cells administered was 1.8 billion cells X 4 in cohort 6b (patients with R/R AML). Treatment was well tolerated with no dose limiting toxicities (DLTs) even at the highest dose. No cases of neurotoxicity or GvHD were reported. Four cases of grade 2 CRS were reported but there were no cases of ≥ grade 3 CRS. Two incidences of grade 4 thrombocytopenia and one grade 4 decreased white blood cell count were noted in patients who had received enhanced lymphodepletion. In R/R AML patients treated with the highest dose of CYNK-001, 2 of 4 patients achieved an objective response of Morphologic Leukemia-Free State (MLFS) on Day 28. One of these patients has maintained a response to D120 and is continuing follow up. One of 3 patients with MRD +ve AML treated with 3 doses of 1.8 billion CYNK-001 cells per dose achieved MRD negativity until day 120 at which time a blast count of 1.6% was observed. Conclusion Treatment with CYNK-001 showed biological activity as evidenced by achievement of MLFS in R/R AML patients and eradication of AML MRD positivity in AML patients in morphologic CR. Therapy was generally well-tolerated, with no dose-limiting toxicities observed with even the highest dose of CYNK-001. This trial yielded important insights into the optimal approach to lymphodepletion and the potential clinical efficacy of allogeneic NK cells in R/R AML. Based on these results, this trial is now closed to further enrollment and future development activities will focus on next generation NK cell therapies edited to optimize persistence and efficacy and limit the requirement for enhanced lymphodepletion.

Engineering Superaffinity Antibody Dependent Cellular Cytotoxcity Receptors into iPSC-Derived NK Cells As Next-Generation Immunotherapies for Cancer

Engineered immune cells have provided an important new approach to treating cancer. Most of the progress so far has focused on autologous CAR-T cell therapy, although this approach has been met with several limitations including high-cost and complexity of manufacturing, donor-to-donor variability, and severe toxicities in some patients. Allogenic natural killer (NK) cell therapies have also been shown to mount potent responses against hematologic malignancies but are less likely to cause high-grade toxicities and can be mass produced for off-the-shelf usage. We have developed an induced pluripotent stem cell (iPSC) derived NK (iNK) platform amenable to engineering multiple features in immune cells to improve efficacy against both solid and liquid tumors. Our previous studies showed that knocking out the CISH gene, which encodes a key regulator of activation, in iNK cells significantly improves their anti-cancer activity, in vivo persistence, metabolic fitness, polyfunctional cytokine production, and resistance to cell exhaustion. In these CISH Knockout (KO) iNK cells, we have screened a library of CAR constructs containing domains from diverse immune cell signaling receptors including NK cell activating receptors, cytokine receptors, and integrins. Our screen identified SLNK12, a CAR signaling domains that performed better than both T cell CARs and previously reported NK cell CARs in the context of CISH KO iNK cells. In this study, we describe the development of iNK cells with enhanced antibody dependent cellular cytotoxicity (ADCC) activity. NK cells normally bind antibodies via their CD16A receptor, which has medium-to-low affinity for the antibody Fc domain and is also typically expressed at a low level in unmodified iNKs. We have constructed a recombinant Fc-receptor that combines a high-affinity Fc-binding domain (to improve antibody binding) with our NK cell optimized SLNK12 CAR signaling domain to enhance ADCC-mediated killing. When co-cultured with multiple NC cell resistant tumor lines such as BT-474 (breast cancer) or FaDu (squamous cell carcinoma), these SuperAffinity ADCC receptor CISH KO iNK cells possess potent anti-cancer activity in combination with several different therapeutic monoclonal antibodies, including anti-EGFR (Cetuximab) and anti-HER2 (Trastuzumab) amongst others (Figure 1). We have assessed efficacy in a variety of different 2D and 3D spheroid cytotoxicity assays, as well as cytokine release and CAR activation assays. In addition, we demonstrate that our SuperAffinity receptor mediates tumor cell killing at lower antibody concentrations than do CD16A variants. Furthermore, introduction of serum IgG inhibits, but does not stop ADCC killing for both our SuperAffinity ADCC receptor and the CD16A receptor. Taken together, our results show that engineering CISH KO iNK cells with NK-optimized CARs for enhanced ADCC improves anti-tumor activity against a variety of solid tumor target lines. Future work will focus on advancing the new cellular therapies into clinical development.