Natural Killer Cell Production Research Articles

A PSMA-targeted Tri-specific Killer Engager enhances NK cell cytotoxicity against prostate cancer.

Natural killer (NK) cell tumor infiltration is associated with good prognosis in patients with metastatic castration-resistant prostate cancer (mCRPC). NK cells recognize and kill targets by a process called natural cytotoxicity. We hypothesized that promoting an antigen-specific synapse with co-activation may enhance NK cell function in mCRPC. We describe a Tri-specific Killer Engager (TriKE) construct that engages with the activating receptor CD16 on NK cells, prostate-specific membrane antigen (PSMA) on mCRPC cells, and has an interleukin (IL)-15 moiety that is essential for NK cell survival, proliferation, and priming. We show that the PSMA TriKE specifically binds to PSMA-expressing cells and significantly enhances expansion, degranulation and cytokine production of NK cells derived from healthy donors or prostate cancer patients. Bystander killing of PSMA-negative was also achieved with PSMA TriKE treatment when co-cultured with PSMA-positive cells, suggesting potential PSMA TriKE benefit in controlling tumor antigen escape. When tested under physiologic conditions recapitulating the mCRPC tumor microenvironment (TME), NK cells treated with PSMA TriKE and prolonged exposure to hypoxia or MDSCs maintained their potent function while IL-15 treated NK cells showed greatly impaired cytotoxicity. Finally, in vivo testing of PSMA TriKE showed improved tumor control and survival of mice as compared to IL-15 and untreated control groups. In conclusion, PSMA TriKE demonstrates potential as a new therapy for advanced prostate cancer by providing additional signals to NK cells to maximize their anti-tumor potential in prostate cancer, especially in the setting of a hostile TME.

Phenotypic and transcriptomic profiling of induced pluripotent stem cell (iPSC)-derived NK cells and their cytotoxicity against cancers.

Adoptive immunotherapy using natural killer (NK) cells has attracted considerable interest in numerous clinical trials targeting both hematological and solid tumors. Traditionally, NK cells are primarily derived from either peripheral blood (PB) or umbilical cord blood (UCB). However, these methods can lead to variability and heterogeneity within the NK cell population. In contrast, induced pluripotent stem cell (iPSC)-derived NK (iNK) cells provide a more controlled and uniform cellular population, suitable for large-scale clinical applications. This makes iNK cells a promising option for developing "off-the-shelf" immunotherapeutic products. Nevertheless, current NK cell differentiation protocols, which rely on embryoid body (EB) cultures, are labor-intensive and susceptible to unwanted heterogeneity during differentiation. Here, we developed a more efficient approach for generating iNK cells by employing a monolayer and feeder-free differentiation protocol, alongside optimized culture media. The iNK cells were generated using a two-step in vitro monolayer feeder-free system following NK cell development. To evaluate their maturity, phenotypic analysis was performed using flow cytometry, comparing with PB-NK cells and the NK-92 cell line. Additionally, single-cell RNA sequencing was performed to examine their transcriptomic profiles. The cytotoxic activity of the iNK cells was evaluated by co-culturing with cholangiocarcinoma (CCA) and breast cancer (BCA) cell lines in both monolayer (2D) and tumor spheroid (3D) co-culture systems. We successfully differentiated iPSCs into mesoderm (ME), hematopoietic stem/progenitor cells (HSPCs), and NK cells. The resulting iNK cells exhibited typical NK cell markers such as CD45, CD56, and CD16, and expressed key functional proteins, including both activating and inhibitory receptors. Single-cell RNA sequencing confirmed that the transcriptomic profile of our iNK cells closely resembles that of PB-NK cells. Importantly, our iNK cells demonstrated strong cytotoxic abilities against various CCA and BCA cell lines, surpassing the NK-92 cell line in both monolayer cultures and tumor spheroid cultures. This study highlights the potential of iPSCs as an effective alternative cell source for generating NK cells. Using a two-step in vitro monolayer feeder-free system, we successfully generated iNK cells that not only expressed key NK cell markers and their receptors but also displayed a transcriptomic profile closely resembling PB-NK cells. Furthermore, iNK cells exhibited cytotoxicity against CCA and BCA cell lines comparable to that of PB-NK cells. This approach could pave the way for off-the-shelf NK cell products, potentially enhancing the effectiveness of adoptive NK cell therapy.

Antimicrobial activity of NK cells to Trypanosoma cruzi infected human primary Keratinocytes.

Infection with the protozoan parasite Trypanosoma cruzi is causative for Chagas disease, which is a highly neglected tropical disease prevalent in Latin America. Humans are primary infected through vectorial transmission by blood-sucking triatomine bugs. The parasite enters the human host through mucous membranes or small skin lesions. Since keratinocytes are the predominant cell type in the epidermis, they play a critical role in detecting disruptions in homeostasis and aiding in pathogen elimination by the immune system in the human skin as alternative antigen-presenting cells. Interestingly, keratinocytes also act as a reservoir for T. cruzi, as the skin has been identified as a major site of persistent infection in mice with chronic Chagas disease. Moreover, there are reports of the emergence of T. cruzi amastigote nests in the skin of immunocompromised individuals who are experiencing reactivation of Chagas disease. This observation implies that the skin may serve as a site for persistent parasite presence during chronic human infection too and underscores the significance of investigating the interactions between T. cruzi and skin cells. Consequently, the primary objective of this study was to establish and characterize the infection kinetics in human primary epidermal keratinocytes (hPEK). Our investigation focused on surface molecules that either facilitated or hindered the activation of natural killer (NK) cells, which play a crucial role in controlling the infection. To simulate the in vivo situation in humans, an autologous co-culture model was developed to examine the interactions between T. cruzi infected keratinocytes and NK cells. We evaluated the degranulation, cytokine production, and cytotoxicity of NK cells in response to the infected keratinocytes. We observed a strong activation of NK cells by infected keratinocytes, despite minimal alterations in the expression of activating or inhibitory ligands on NK cell receptors. However, stimulation with recombinant interferon-gamma (IFN-γ), a cytokine known to be present in significant quantities during chronic T. cruzi infections in the host, resulted in a substantial upregulation of these ligands on primary keratinocytes. Overall, our findings suggest the crucial role of NK cells in controlling acute T. cruzi infection in the upper layer of the skin and shed light on keratinocytes as potential initial targets of infection.

Interim analysis of a phase I study using cryopreserved non-genetically modified allogeneic natural killer cells with enhanced cytotoxicity (SNK02) in patients with advanced solid tumors without lymphodepletion.

e14515 Background: Natural killer (NK) cells play a key role as the main effector cells toward cancer in innate immunity. A leading approach to boost NK cell-mediated anti-tumor activity is via the adoptive transfer of ex vivo activated NK cells. Current allogeneic donor-derived products require lymphodepletion to prevent immunologic rejection of donor cells by the recipient and ensure adequate cell delivery of the product cells to the target. However, lymphodepletion can negatively impact combination therapies where a robust T cell response is desired. SNK02 is a first-in-kind, cryopreserved allogeneic non-genetically modified NK cell product with significant anti-tumor cytotoxicity and over 90% expression of CD16, NKG2D, NKp46, and DNAM-1, that can be consistently produced on a large commercial scale. We hypothesized that higher doses of SNK02 (to overcome autodigestion) could be delivered frequently without the need for lymphodepletion and that it might demonstrate activity against solid tumors that have failed multiple prior standard-of-care treatment options. Methods: In this Phase 1 dose escalation study (NCT05990920), SNK02 was administered intravenously (IV) weekly for 8 consecutive weeks in patients with advanced solid tumors. The starting dose was 6 x 109 SNK02 cells. The primary endpoint was safety based on adverse events (AEs), vitals, laboratory tests, and physical exams. Results: As of Feb 1, 2023, 5 patients with advanced refractory solid tumors have been enrolled. Median age was 64 (range 44 – 71) and 3 were male. The subtypes were 1 leiomyosarcoma, 1 angiosarcoma, 1 endometrial adenocarcinoma, 1 undifferentiated pleomorphic sarcoma, and 1 colorectal adenocarcinoma. Four of five patients completed 8 cycles of SNK02. There was 1 death on study, which was deemed unrelated to the investigational product (IP). Out of the 36 doses administered through Cycle 8, there were 17 Grade 1, 3 Grade 2, and 1 Grade 3 adverse AEs related to IP. The Grade 3 AE of increased fatigue resolved after 1 day with no intervention required. Antibodies appeared to develop around cycle 5 and appeared to correlate with AEs. The best objective response of SD was demonstrated in 100% of patients that completed the 8 cycles. Conclusions: SNK02 was well tolerated as a monotherapy and appears to have some clinical activity against pretreated solid tumors despite the lack of lymphodepletion. SNK02 will continue to be studied as a monotherapy and in potential combination treatment regimens with monoclonal antibodies and immune checkpoint inhibitors. Clinical trial information: NCT05990920 .

Comparison of NK cell count between 1% and 10% FBS culture media concentration by FACS analysis

Recently, there has been a growing research interest in the application of immunotherapy for cancer treatment. Various approaches have been developed, and one of these methods involves utilizing Natural Killer (NK) cells of the immune system to specifically target cancer cells. Research on this method necessitates the mass and stable production of NK cells through culturing. For NK cell culture, Fetal Bovine Serum (FBS) is widely used in the industry at concentrations ranging from 10% to 20%. In an effort to explore a more efficient culturing technique, this study aims to investigate the effect of 1% FBS on cell viability of NK cells in cell culture. Because NK cells are usually defined by cell surface molecules, the efficacy of using a lower FBS concentration was quantitatively measured using Fluorescence-Activated Cell Sorting (FACS). The cell counts of cells cultured in 1% FBS was compared to those of cells cultured in 10% FBS. The results showed that no significant difference was found between cell count of cells cultured in 1% and 10% FBS. Thus, 1% FBS concentration culturing method was not found to be inferior. Further research is needed on the effect of replicating cancer microenvironment on NK cell viability.

Cytokine Response of Natural Killer Cells to Hepatitis B Virus Infection Depends on Monocyte Co-Stimulation.

Hepatitis B virus (HBV) is a major driver of chronic hepatic inflammation, which regularly leads to liver cirrhosis or hepatocellular carcinoma. Immediate innate immune cell response is crucial for the rapid clearance of the infection. Here, natural killer (NK) cells play a pivotal role in direct cytotoxicity and the secretion of antiviral cytokines as well as regulatory function. The aim of this study was to further elucidate NK cell responses triggered by an HBV infection. Therefore, we optimized HBV in vitro models that reliably stimulate NK cells using hepatocyte-like HepG2 cells expressing the Na+-taurocholate co-transporting polypeptide (NTCP) and HepaRG cells. Immune cells were acquired from healthy platelet donors. Initially, HepG2-NTCP cells demonstrated higher viral replication compared to HepaRG cells. Co-cultures with immune cells revealed increased production of interferon-γ and tumor necrosis factor-α by NK cells, which was no longer evident in isolated NK cells. Likewise, the depletion of monocytes and spatial separation from target cells led to the absence of the antiviral cytokine production of NK cells. Eventually, the combined co-culture of isolated NK cells and monocytes led to a sufficient cytokine response of NK cells, which was also apparent when communication between the two immune cell subpopulations was restricted to soluble factors. In summary, our study demonstrates antiviral cytokine production by NK cells in response to HBV+ HepG2-NTCP cells, which is dependent on monocyte bystander activation.

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 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.

Therapeutic approaches to enhance natural killer cell cytotoxicity.

Enhancing the cytotoxicity of natural killer (NK) cells has emerged as a promising strategy in cancer immunotherapy, due to their pivotal role in immune surveillance and tumor clearance. This literature review provides a comprehensive overview of therapeutic approaches designed to augment NK cell cytotoxicity. We analyze a wide range of strategies, including cytokine-based treatment, monoclonal antibodies, and NK cell engagers, and discuss criteria that must be considered when selecting an NK cell product to combine with these strategies. Furthermore, we discuss the challenges and limitations associated with each therapeutic strategy, as well as the potential for combination therapies to maximize NK cell cytotoxicity while minimizing adverse effects. By exploring the wealth of research on this topic, this literature review aims to provide a comprehensive resource for researchers and clinicians seeking to develop and implement novel therapeutic strategies that harness the full potential of NK cells in the fight against cancer. Enhancing NK cell cytotoxicity holds great promise in the evolving landscape of immunotherapy, and this review serves as a roadmap for understanding the current state of the field and the future directions in NK cell-based therapies.

ITAM-based receptors in natural killer cells.

The ability of cells of the immune system to acquire features such as increased longevity and enhanced secondary responses was long thought to be restricted to cells of the adaptive immune system. Natural killer (NK) cells have challenged this notion by demonstrating that they can also gain adaptive features. This has been observed in both humans and mice during infection with cytomegalovirus (CMV). The generation of adaptive NK cells requires antigen-specific recognition of virally infected cells through stimulatory NK receptors. These receptors lack the ability to signal on their own and rather rely on adaptor molecules that contain ITAMs for driving signals. Here, we highlight our understanding of how these receptors influence the production of adaptive NK cells and propose areas in the field that merit further investigation.

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.

Interferon-epsilon is a novel regulator of NK cell responses in the uterus

The uterus is a unique mucosal site where immune responses are balanced to be permissive of a fetus, yet protective against infections. Regulation of natural killer (NK) cell responses in the uterus during infection is critical, yet no studies have identified uterine-specific factors that control NK cell responses in this immune-privileged site. We show that the constitutive expression of IFNε in the uterus plays a crucial role in promoting the accumulation, activation, and IFNγ production of NK cells in uterine tissue during Chlamydia infection. Uterine epithelial IFNε primes NK cell responses indirectly by increasing IL-15 production by local immune cells and directly by promoting the accumulation of a pre-pro-like NK cell progenitor population and activation of NK cells in the uterus. These findings demonstrate the unique features of this uterine-specific type I IFN and the mechanisms that underpin its major role in orchestrating innate immune cell protection against uterine infection.

A Scalable, Off-the-Shelf, and Cost-Effective NK Cell Manufacturing Platform for Developing NK Cell-Based Immunotherapy

Natural killer (NK) cells are innate lymphocytes that play a key role in the control of infection and malignancy. NK cells are potent killers of target cells and act in an MHC-independent manner, enabling their application as allogenic therapy for cancer. Human iPSCs are pluripotent with the ability to self-renew, thus providing an inexhaustible source for deriving NK cells. With a two-tiered cell bank system, we can manufacture NK cells with more homogeneity between batches. In principle, one single iPSC clone-derived master cell bank and working cell banks can support the life cycle of an affordable, off-the-shelf NK cell product. We developed a robust stepwise protocol for large-scale generation of high-purity NK cells from human iPSCs without the cell isolation procedure. First, human iPSCs formed embryoid bodies and then were differentiated into highly purified (>98.0%) CD34 + CD43 + hematopoietic stem and progenitor cells (HSPCs) using a combination of cytokines and small molecles such as BMP-4, VEGF, BFGF, SCF, FLT-3 and ascorbic acid. During this period, we observed up to an 180-fold cell increase in the number of HSPCs compared with human iPSCs. Second, these HSPCs were differentiated into NK cells with FLT3, SCF, IL-3, IL-7, and IL-15 etc., with cell number increased approximately 9000-fold compared with iPSCs. In the final step, iPSC-derived NK cells were further expanded up to 8000-fold which enable us to produce 1E12 NK cells from less than 1 million iPSCs. After expansion, iNK cells became more homogenous and exhibited a more mature phenotype. More than 99.0% of cells expressed both CD45 and CD56, and more than 70% expressed CD16. The expression of activating and co-stimulatory receptors (NKG2D, NKp46,NKp44, NKp30, CD244 and CD226) of NK cells was also high in these iNK cells. Intrestingly, the cells exhibited robust anti-tumor efficacy against K562 (chronic myelogenous leukemia), RPMI 8226 (multiple myeloma). Furthermore, the cryopreserved iNK cells demonstrated dramatic cytotoxicity to U-87 MG cells (malignant gliomas) in 2D culture and the tumor organoid, indicating that iNK cells derived from our protocol can be used as off-the-shelf therapy for both hematological and solid tumors. We also observed that these iNK cells had variable cytotoxicity in other tumors. For example, our iNK were not obviously cytotoxic to the NCI-H929 cell line, derived from a plasmacytoma myeloma patient and expressing BCMA at a high level. To improve the specific cytotoxicity of NK cells to NCI-H929 cells, we transduced a anti-BCMA CAR to the donor iPSC. Following differentiation, the CAR-transduced iNK cells showed approximately 6-fold cytotoxicity compared with WT iNK. (Figure 1) Collectively, our serum-free and strom cell-free NK cell generation produre from human iPSCs provides a scalable, consistent, off-the-shelf and cost-effective NK/CAR-NK cell manufacturing platform for clinical application and NK cell-based immunotherapies.

Engineered Natural Killer Cells Expressing Chimeric Ilt Receptors (CIR) Effectively Target HLA-G Positive AML Tumor Cells

Background Human leukocyte antigen-G (HLA-G), the immune checkpoint of pregnancy, directs immunosuppression by binding to Ig-like transcript (ILT) receptors (ILT2 in lymphoid cells and ILT4 in myeloid cells) via immunoreceptor tyrosine-based inhibitory motif (ITIM) signaling domains. HLA-G is expressed in more than 50% of solid tumors and leukemias as a mechanism to evade immune attack. As a result, HLA-G is an excellent tumor-specific antigen since its expression in normal tissues is low except for cytotrophoblasts in the placenta where it protects the fetus from the mother's immune system. We present here a novel approach to target HLA-G-expressing Acute Myeloid Leukemia (AML) cells by utilizing the natural receptors of ILT2 and ILT4 coupled to positive intracellular signaling domains. Such chimeric ILT receptors, or CIRs drive activation of Natural Killer (NK) cells. Further, we have identified unique intracellular activation modules that function to robustly enhance cytotoxicity and expansion of NK cells upon engagement with tumor cells. Methods CD56 + NK cells were isolated from PBMCs, cultured with IL-15 and activated with a feeder-free cocktail of an immobilized cytokine and activating ligand. Activated NK cells were transduced with γ-retroviruses directing expression of CIR proteins (CIR.4-1BB.CD3ζ or CIR.X.Y where X and Y are candidate activation and coactivation moieties), soluble IL-15 and a ΔCD19 marker or CAR control constructs directed to HLA-G or CD33. After 8 days of expansion, CIR- or CAR-NK cells were cocultured with GFP ffluc-expressing AML target cell lines with or without transgenic HLA-G (G1, G2 and G5) isoforms. Cytotoxicity of CIR-NK cells was determined by reduction of GFP fluorescence in Incucyte or Celligo instruments or luciferase activity and supernatants were analyzed for IFN-γ and TNF-α release by ELISA. Transduced NK cells were labeled with nuclear-RFP for measurement of expansion in coculture. In comparative experiments, T cells from PBMCs were transduced to produce CIR-T cells by standard methods. Results CIR (ILT2 or ILT4.4-1BB.ζ) constructs were efficiently and stably expressed in T and NK cells (>80% ΔCD19 + 8- and 14-days post activation). Mock-transduced NK cells displayed innate killing activity against HLA-G negative AML cells (THP1 and KG1) that was not augmented by CIR constructs (mock = 6.16E6 ± 0.77E6 vs. CIR = 6.43E6 ± 0.37E6 in coculture against KG1 cells). In cocultures against THP1 and KG1 target cells expressing exogenous G1, G2 or G5 isoforms, CIR-NK cells or CIR-T cells displayed enhanced target cell killing in 2 and 7-day assays relative to mock-transduced effector cells (mock = 1.47E6 ± 0.49E6 vs. CIR = 0.57E6 ± 0.6 in coculture against KG1-HLA-G1). Further, while mock NK cells transduced with RFP alone could kill one-third of AML cells expressing endogenous HLA-G1 and G5 (Molm13 and Kasumi1), CIR-NK cells eliminated more than 90% of these AMLs and their activivation resulted in 4-fold higher IFN-γ secretion. Notably, robust cytotoxicity of CIR-NK cells was maintained even at 28 days post activation (mock = 36.95E6 ± 21.85E6 vs. CIR = 7.38E6 ± 1.92 in coculture against Kasumi1). To further enhance the functionality of the CIR constructs, a screen of novel costimulatory molecules to replace 4-1BB and/or CD3ζ resulted in several potent candidates ultilizing MyD88 signaling that greatly improved the cytotoxicity (> 80% tumor elimination), proliferation (> 2-fold expansion) and proinflammatory cytokine production (IFN-γ and TNF-α) of NK cells in cocultures. A subset is displayed in Figure 1. Conclusions The use of natural receptors ILT2 and ILT4 to target HLA-G takes advantage of the physiological pairing of the ILT receptors with the multiple immunosuppressive HLA-G isoforms thereby circumventing the possibility of antigen escape often observed in CAR-directed therapy. We demonstrated that engineered CIR-NK cells can convert HLA-G expression in AML from a potent inhibitor of leukocyte activity into a target for anti-tumor control.

Multimeric immunotherapeutic complexes activating natural killer cells towards HIV-1 cure

BackgroundCombination antiretroviral therapy (cART) has dramatically extended the life expectancy of people living with HIV-1 and improved their quality of life. There is nevertheless no cure for HIV-1 infection since HIV-1 persists in viral reservoirs of latently infected CD4+ T cells. cART does not eradicate HIV-1 reservoirs or restore cytotoxic natural killer (NK) cells which are dramatically reduced by HIV-1 infection, and express the checkpoint inhibitors NKG2A or KIR2DL upregulated after HIV-1 infection. Cytotoxic NK cells expressing the homing receptor CXCR5 were recently described as key subsets controlling viral replication.MethodsWe designed and evaluated the potency of “Natural killer activating Multimeric immunotherapeutic compleXes”, called as NaMiX, combining multimers of the IL-15/IL-15Rα complex with an anti-NKG2A or an anti-KIR single-chain fragment variable (scFv) to kill HIV-1 infected CD4+ T cells. The oligomerization domain of the C4 binding protein was used to associate the IL-15/IL-15Rα complex to the scFv of each checkpoint inhibitor as well as to multimerize each entity into a heptamer (α form) or a dimer (β form). Each α or β form was compared in different in vitro models using one-way ANOVA and post-hoc Tukey’s tests before evaluation in humanized NSG tg-huIL-15 mice having functional NK cells.ResultsAll NaMiX significantly enhanced the cytolytic activity of NK and CD8+ T cells against Raji tumour cells and HIV-1+ ACH-2 cells by increasing degranulation, release of granzyme B, perforin and IFN-γ. Targeting NKG2A had a stronger effect than targeting KIR2DL due to higher expression of NKG2A on NK cells. In viral inhibition assays, NaMiX initially increased viral replication of CD4+ T cells which was subsequently inhibited by cytotoxic NK cells. Importantly, anti-NKG2A NaMiX enhanced activation, cytotoxicity, IFN-γ production and CXCR5 expression of NK cells from HIV-1 positive individuals. In humanized NSG tg-huIL-15 mice, we confirmed enhanced activation, degranulation, cytotoxicity of NK cells, and killing of HIV-1 infected cells from mice injected with the anti-NKG2A.α NaMiX, as compared to control mice, as well as decreased total HIV-1 DNA in the lung.ConclusionsNK cell-mediated killing of HIV-1 infected cells by NaMiX represents a promising approach to support HIV-1 cure strategies.

AFM13 in Combination with Allogeneic Natural Killer Cells (AB-101) in Relapsed or Refractory Hodgkin Lymphoma and CD30 + Peripheral T-Cell Lymphoma: A Phase 2 Study (LuminICE)

Background and Significance Limited treatment options are available for patients with relapsed or refractory (R/R) Hodgkin lymphoma (HL) and no standard-of-care therapy is established for R/R peripheral T-cell lymphoma (PTCL); novel therapies to improve outcomes in these patients are required. AFM13 is a tetravalent, bispecific innate cell engager that binds CD16A on natural killer (NK) cells and CD30 expressed (CD30 +) on HL and PTCL cells, enhancing NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). AFM13 monotherapy trials in patients with R/R HL and PTCL have shown promising clinical activity and a tolerable safety profile (Sasse et al. Blood 2020; Kim et al. Cancer Res 2023). Recently, a Phase 1/2 study of AFM13 in combination with cord blood (cb)-derived NK cells in patients with R/R CD30 + lymphomas treated at the recommended phase 2 dose (n=35) achieved an objective response rate (ORR) of 94% and a complete response (CR) rate of 71% (Nieto et al. Blood 2022, oral presentation). AB-101 is a non-genetically modified, allogeneic, cryopreserved, off-the-shelf, cb-derived NK cell product optimized for enhanced ADCC through selection for the KIR-B haplotype and the CD16 F158V polymorphism. AB-101 has demonstrated potent killing of tumor cell lines in vitro and in vivo and preliminary results of a Phase 1/2 trial of AB-101 alone and in combination with rituximab in patients with R/R B cell non-Hodgkin lymphoma demonstrated AB-101 is well tolerated (Khanal et al. J Clin Oncol 2023). Combining AFM13 with AB-101 has the potential to synergistically enhance and redirect antitumor immune responses to target HL and CD30 + PTCL cells. Study Design and Methods This Phase 2, open-label, multi-center, multi-cohort study (NCT05883449) aims to evaluate the efficacy and safety of AFM13 in combination with AB-101 in patients with R/R HL and certain R/R CD30 + PTCL subtypes. PTCL subtypes permitted are PTCL not-otherwise specified, angioimmunoblastic T-cell lymphoma, and ALK-positive and -negative anaplastic large cell lymphoma (ALCL). Patients aged ≥18 years are planned for enrolment and patients with R/R HL must have received at least two prior lines of therapy including prior combination chemotherapy, brentuximab vedotin (BV) and a checkpoint inhibitor. Patients with R/R PTCL must have confirmed CD30 expression of ≥1% by immunohistochemistry and have received at least one prior line of combination chemotherapy; patients with ALCL must have received or been intolerant to BV. Prior autologous or allogeneic hematopoietic stem cell transplant is permitted. Exclusion criteria include treatment with any anti-cancer agent ≤21 days prior to enrolment, continuing toxicity from a prior therapy, central nervous system involvement, or previous treatment with AFM13 or NK cells. The primary objective is to determine the ORR (complete and partial responses) by Independent Radiology Committee (IRC) based on PET-CT per Lugano classification. Secondary objectives include safety and immunogenicity, complete response rate, duration of response and progression free survival. Treatment will be given intravenously (IV) over 48-day cycles for up to 3 cycles. A run-in phase will assess two dose levels of AFM13 and AB-101 in 4 cohorts (Figure). A standard lymphodepletion regimen of fludarabine (30 mg/m 2/day) and cyclophosphamide (300 mg/m 2/day) will be administered IV from Day −5 to Day −3 at the start of each treatment cycle. Following this, AFM13 (200 mg or 300 mg once weekly) will be given, with AB-101 (dose level 1 or 2, see Figure) given 1 hour later per cycle. Patients will also receive 6 ×10 6 IU of IL-2 subcutaneously at least 1 hour after each AB-101 dose. Cohorts 1 and 2 will enrol in parallel; cohorts 3 and 4 will start only if cohorts 1 and 2 are cleared per protocol safety criteria. After cycle 1 has completed for each subject enrolled in all 4 cohorts, an analysis of the safety and clinical responses will be performed to determine the two dose levels to be evaluated in the main study. In addition, an exploratory cohort (cohort 5) will begin enrolment of patients with CD30 + PTCL. Disease and efficacy assessments will be conducted at screening and on Day 43 (± 3 days) of each cycle.

The Effects of Thymosin Alpha-1 on Macrophages: A Cytological and Anti-Inflammatory Study

Thymosin alpha 1 (Tα-1) is a naturally occurring substance synthesized by the thymus tissue, known to activate various immune system cells. It has been reported to increase the production of Natural Killer cells, CD4 and CD8 cells, and cytokines such as IL-2, IL-3, and IFNγ. Furthermore, it plays a vital role in regulating immunity, inflammation, and tolerance. Its effect on the immune system is exerted via its modulatory action on innate immune system cells, thereby functioning as an endogenous regulator for both inflammatory and adaptive immune systems. In this study, we sought to evaluate the cytological and anti-inflammatory effects of Tα-1 on RAW 264.7 cells. The cytological effects of Tα-1 were assessed using the MTT assay, with the IC50 value against RAW 264.7 cells determined to be 368.105 ug/ml. The morphological observations made at various concentrations of Tα-1 showed increased cytotoxicity and decreased cell density of RAW 264.7 cells with increasing test concentration. Furthermore, the anti-inflammatory effects of Tα-1 were evaluated by analyzing the nitric oxide (NO) production in RAW 264.7 cells. Treatment with the test items at concentrations ranging from 7.813 to 31.25 ug/ml showed a dose-dependent reduction in NO production compared with the control group. These findings suggest that Tα-1 may have the potential as an anti-inflammatory agent in the treatment of various diseases associated with inflammation.

IMMU-22. ALLOGENEIC NK CELLS SECRETING IL15 AND RESISTANT TO TGF-β Show Antitumor Activity Against Gliobastoma

Abstract INTRODUCTION High grade brain cancers, like most solid tumors, often relapse and are resistant to current therapies. Limited efficacy to cell therapy may be related to intratumoral heterogeneity and the suppressive tumor microenvironment. Hence, we aimed to modify allogeneic natural killer (NK) cells, which can recognize expressed tumor stress ligands, to resist TGF-β and secrete IL15. METHODS Cord blood-derived NK cells were expanded and modified to express IL15 and a dominant negative receptor (DNR) against TGF-β. NK cell product activity against the GBM cell line U87MG were evaluated in vitro and in vivo. The behavior of these cells were also analyzed ex vivo, to evaluate the impact (including potential toxicity) of increasing the potency of this allogeneic NK cell product. RESULTS While animals injected with NK cells secreting IL15 and expressing DNR successfully cleared tumors 6 days after T cell infusion, we did not observe significant differences in survival between the groups (no treatment control mice vs treated with mock-transduced NK cell vs DNR-transduced NK cells vs IL-15/DNR-transduced NK cells). Therefore, we sought to determine whether cytokine-related toxicity was responsible for death in the setting of tumor clearance. We compared no treatment control mice vs mice treated with mock-transduced or IL-15/DNR-transduced NK cell products. Elevated levels of IL-15 were detected in the plasma of mice treated with IL-15/DNR-transduced NK cells. These elevated IL-15 levels corresponded with NK cell persistence in the brain measured by ddPCR. However, no other cytokines were upregulated. CONCLUSIONS We observed improved tumor clearance when NK cells were both protected against immune suppressive TGF-β and were capable of secreting the stimulatory cytokine IL15. We are currently performing additional experiments to determine whether toxicity from IL15 secretion occurs. To potentially mitigate this toxicity we plan to engineer a suicide gene into these constructs.

Dysregulation of natural killer cells’ receptor expression and cytotoxic function by hydroxychloroquine

Abstract Background and Aim: Natural killer (NK) cells are crucial players of the innate immune system, especially in the fight against tumors and infections. Besides, NK cells are known to contribute in inflammatory processes associated with multiple diseases. The drug hydroxychloroquine (HCQ) has been repurposed to be used for the treatment of malaria, autoimmune diseases, and COVID-19 infection. However, its direct mechanism of action on NK cells remains unknown. In this study, we aimed to examine the effects of HCQ on NK cell activity and function. Materials and Methods: The human NK cell line NK92 was cultured and treated with HCQ at 1, 5, and 10 μM for 24 and 48 hours. Then, NK92 cell viability, NKG2D and CD107a expression, as well as NK92 cell cytotoxicity were assessed. Also, IFN-γ production was measured by ELISA after HCQ treatment. Statistical analysis was performed where p<0.05 was considered statistically significant. Results: Upon treatment with different concentrations of HCQ for 48 hours, NK92 cell viability significantly decreased in a dose-dependent manner, with the lowest viability noted at 10 μM of HCQ. Additionally, NKG2D and CD107a expression were significantly reduced upon treatment with 5 and 10 μM of HCQ. Similarly, the cytotoxicity of NK92 cells against K562 cells was reduced at both 5:1 and 10:1, effector to target (E: T) cell ratios, when 10 μM HCQ was used. In contrast, IFN-γ levels were significantly increased upon the treatment with 1, 5, and 10 μM HCQ. Conclusion: This study highlights the effect of HCQ on activating receptor expression, cytotoxicity, and IFN-γ production of NK cells. Despite being an immunomodulatory drug, more studies are needed to further understand HCQ’s effects on NK cells and perhaps other effector cells. None

Abstract 2919: A novel method for clinical scale production of natural killer cells from clonal master induced pluripotent stem cells with CISH knockout for next generation, off-the-shelf cancer immunotherapy

Abstract Natural killer (NK) cells are innate immune cells that play a key role in in tumor immune surveillance. NK cells are self-tolerant to healthy cells and can kill tumor cells in both hematological and solid malignancies. Recent advances allow for the derivation of immune cells such as NK cells, from human induced pluripotent stem cells, that can be utilized for cancer immunotherapy. Recent clinical trials suggest that high doses of NK cells (approx. 0.5-5 x 109Jaya cells per dose) and multiple doses are both safe and likely necessary for clinical efficacy. Manufacturing large number of NK cells from a clonal master iPSC line provides a promising strategy to enable next generation, off-the-shelf, affordable, cancer immunotherapies. We have developed a novel method to produce high purity, clinical scale iNK cells and improved the process for the expansion of NK cells efficiently and consistently. This method does not require cell sorting step and the usage of murine derived stromal cells. Briefly, hematopoietic progenitor cells were induced using an improved spin embryoid body (EB) method and the hematopoietic progenitor cells were subsequently differentiated into mature iNK cells in the absence of cell sorting and independent of stroma cells support. Using this novel method, differentiated NK cells could be expanded more than 8,000-fold to enable us to potentially produce 1 x 1012 iNK cells starting from 1 x 106 undifferentiated iPSC. This cell production scale can supply hundreds of doses in clinic from one cGMP manufacturing campaign. iNK cells produced using this method display the typical phenotype of NK cell activating receptor including NKG2D, NKp44, NKp46, DNAM-1 etc. Importantly, these iNK cells demonstrate better anti-tumor activities and higher levels of IFNγ and TNFα, compared to primary peripheral blood-derived NK cells isolated from healthy donors. Moreover, this protocol has been adapted and optimized for clinical scale manufacturing of iNK cells from genetically engineered, clonal master iPSC cell line with knock out of CISH (CISH KO iNK), a key intracellular checkpoint of NK cell anti-tumor immunity. CISH KO iNK cells produced using this optimized method show significantly better anti-tumor activities against multiple liquid and solid tumor cell lines including K562, Raji, Daudi, SUP-B15, MOLT-4, SKOV-3, OVCAR-4, HCC-827, ZR-75, and BT-474 etc. in vitro and in vivo in an AML xenograft mouse model, compared with unmodified iNK cells. Overall, this novel cell production strategy paves the way for clinical trials using higher doses of iPSC-derived NK cells with increased potency, creating next-generation NK cell-based immunotherapies. Citation Format: Davide Bernareggi, Caryn Gonsalves, Max Schabla, Alejandra Gárate-Carrillo, Duygu Ozmadenci, Jaya Thangaraj, Qin Li, Leah Mitchell, Dan S. Kaufman, Robert Hollingsworth, Huang Zhu. A novel method for clinical scale production of natural killer cells from clonal master induced pluripotent stem cells with CISH knockout for next generation, off-the-shelf cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2919.