The development of a next generation NK cell engager platform by integrating a potency-reduced IL-15 variant to enhance antitumor activity

High dose IL-2 was the first modern immunotherapy to show complete responses in a subset of cancer patients. The liabilities of IL-2 prompted development of second-generation molecules, which signal through the IL-2Rβγ (“not-α” IL-2 and IL-15 variants). Although these molecules avoid vascular leak syndrome, their clinical efficacy appears to be suboptimal compared to high dose IL-2 [1]. Furthermore, not-α IL-2s show biased expansion of NK cells over CD8+ T cells in patients due to their high expression of IL-2Rβ on NK cells, which can act as a sink and contribute to toxicity, and these molecules do not eliminate IL-2Rβγ-mediated activation of Tregs [2, 3]. To maximize the therapeutic potential of IL-2, we developed AB248, a cis-targeted IL-2 fusion protein that selectively signals on CD8+ T-cells with limited activity on the highly IL-2/IL-15-sensitive NK cells and immunosuppressive Tregs. We have previously shown that selective expansion of CD8+ T cells over NK cells and Tregs is achieved in vivo using muAB248, AB248’s murine surrogate. Furthermore, a single dose of muAB248 elicits strong efficacy in multiple tumor models without body weight loss. Here, we detail the properties of AB248, a fusion of a human CD8-targeting antibody that selectively binds to CD8+ T cells over NK cells, and an IL-2 mutein with reduced affinity to IL-2Rα and IL-2Rβ. In STAT5 assays, AB248 showed approximately 1000-fold preference for the activation of CD8+ T cells over NK cells and Tregs. AB248 mediated a dose-dependent selective expansion of CD8 T cells over NK and Tregs in non-human primates. Furthermore, AB248 reproduced the expected in vitro gene signatures of an IL2Rβγ agonist, demonstrating that AB248 recapitulates native IL-2Rβγ signaling selectively on CD8+ T cells.We previously demonstrated in mice that while the efficacy of not-α IL-2 was mediated by CD8+ T cells and not NK cells, toxicity as measured by body weight loss was dependent upon NK cells but not CD8+ T cells. Here, we show that human NK cells may also drive IL-2βγ agonist-induced toxicities. Both IL-2 and not-α IL-2 induced IFN-γ secretion from human PBMCs, whereas AB248 did not. Strikingly, depletion of CD56+ NK cells eliminated IL-2-induced cytokine secretion, demonstrating that human NK cells are capable of spontaneously secreting IFN-γ in response to IL-2 signaling. In the context of TCR stimulation, AB248 induced robust secretion of effector cytokines from CD8+ T cells, but no cytokine secretion was seen in absence of TCR activation. Our data suggest that while AB248 can induce antigen-independent expansion of CD8+ T cells, it only induces effector cytokine secretion in the context of an additional activating signal via the TCR. Collectively, these data show that AB248 demonstrates an improved safety and efficacy profile as compared to not-α IL-2 and is a promising immuno-oncology therapeutic. Ref: 1. Overwijk, Ann. Rev. Med., 2020; 2. Janku, AACR, 2021; 3. Italiano, JCO, 2021 Citation Format: Kelly D. Moynihan, Danielle Pappas, Terrence Park, Wei Chen, Irene Ni, Paul Bessette, Henry Nguyen, David Liu, Mike Chin, Ruth Lan, Aaron Arvey, Ton N. Schumacher, Yik Andy Yeung, Ivana Djuretic. AB248 is a CD8+ T cell selective IL-2 designed for superior safety and anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3518.

BackgroundInnate immunity plays a vital role in cancer immunotherapy. Natural killer cell engager (NKCE) has garnered significant attention in recent years. Conventional NKCEs have shown limited anti-tumor activity as monotherapy...

Chimeric Antigen Receptor Modified Memory-like (CAR-ML) NK Cells Exhibit Potent Responses to NK-Resistant Tumors

Background and SignificanceBiological therapies harnessing the adaptive immune system have achieved a great success, given the clinical efficacy of CAR-T therapies and CD3 based T cell engagers in treating hematologic tumors, and the application of T cell immune checkpoint inhibitors in various indications. However, the challenges still exist due to the limitation of CAR-T therapies and T cell engagers in treating solid tumors. Side effects such as cytokine release storm and neurotoxicity remain a concern in the selection of CAR-T therapies and CD3-based T cell engagers as therapeutics. For PD1/L1-based immunotherapy, the relatively low objective response rate (ORR), short progression-free survival (PFS) of patients, and drug resistance and recurrence especially for solid malignancy, are the major clinical challenges. In contrast, innate effector cells such as NK cells and macrophages are naturally existing in human body as front line to defeat general pathogens and cancers，which will have a better safety profile. Also, NK cell activation is not limited by the antigen presentation of the MHC on the cell surface which makes it has broader anti-tumor effects. However, the innate effector cell based therapy is facing the limitation of low cell number, poor in vitro activation, and short effective duration in vivo.SunHo has generated a B7H3/NKp30 NK cell engager bispecific antibody which can directly activate and enrich NK cells to the TME when used alone or enhance the efficacy when used in combination NK cell therapy. NKp30 is a potent NK cell activation receptor with wide and persistent expression compared with other NK markers. B7H3 as a tumor associate antigen is also widely expressed on many kinds of tumor cells with great potential for multiple indications, especially for solid tumors.MethodsThree anti-NKp30 VHHs were identified from Alpaca immunized library. The VHHs were fused to the N-terminus or C-terminus of an anti-B7H3 mAb heavy chain with different IgG isotypes (IgG1 or IgG4). The binding activity to NK cells was evaluated by FACS. IFNγ level was detected in the NK cell activation assay. The NK cell mediated target cell killing was evaluated using either NK92MI-cd16a or Primary NK cells as effector cells. To evaluate the non-specific self-killing of NK cell without target cell, the B7H3/NKp30 candidates were incubated with NK92MI-cd16a and cell lysis percentage were calculated. NSG mice bearing Hs.746T tumors were used to evaluate the in vivo efficacy of B7H3/NKp30 candidates.ResultsThe B7H3/NKp30 candidates showed good binding activity and better activation to NK cells compared with benchmarks. Notably, the B7H3/NKp30 candidate IAN0982-VHH25 with NKp30 fused to the C-terminal with an IgG1 isotype showed lowest risk of non-specific NK cell killing. In the in vivo study, B7H3/NKp30 candidates in combination with 1x106 NK cells showed excellent anti-tumor activity with TGI over 95%. And we didn’t observe any significant change in body weight of the animals demonstrating good safety profile of the candidates. Moreover, even with lower NK cell amount (5x105), IAN0982-VHH25 showed great anti-tumor efficacy. We have also generated three mini pools of stably transfected cells for IAN0982-VHH25 for the commercial stable cell line development.ConclusionIAN0982-VHH25 is a first-in-class B7H3/NKp30 bispecific NK cell engager for cancer immunotherapy with excellent NK cell activation and in vivo anti-tumor efficacy, and also low risk of non-specific NK cell killing. It is expected to lift the limitations of current adaptive and innate effector cells-based therapies.

BackgroundEpidermal growth factor receptor (EGFR) is expressed at high levels across solid cancers. While EGFR antagonists have shown activity against EGFR-driven cancers, all are accompanied by significant toxicities, and tumors...

BackgroundNatural killer (NK) cells are emerging as a promising therapeutic option in cancer. To better understand how cancer cells evade NK cells, we studied interacting NK and blood cancer cells...

BackgroundPatients with acute myeloid leukemia (AML) are often older, which brings challenges of endurance and persistent efficacy of autologous chimeric antigen receptor (CAR)-T cell therapies. Allogenic CAR-natural killer (NK) cell therapies may offer reduced toxicities and enhanced anti-leukemic potential against AML. CD33 CAR-NK cells have been investigated for AML therapy. However, the fratricide-mediated lysis of CD33-expressing NK cells by CD33 CAR-NK cells limits the expansion and efficacy of CD33 CAR-NK cells. Mesothelin (MSLN), a tumor differentiation antigen, is highly expressed in a fraction of patients with AML, making it a promising target for AML therapy.MethodsWe designed a novel CD33-MSLN Loop CAR (Loop CAR) and evaluated its antitumor efficacy in human umbilical cord blood-derived NK (UCB-NK) cells and human pluripotent stem cell-derived NK (hPSC-iNK) cells. To further avoid fratricide caused by endogenous CD33 expression in NK cells, we established an hPSC-derived cell line via knockout of the CD33 gene (CD33KO) and engineered Loop CAR. We generated CD33KO-Loop CAR-iNK cells using an organoid induction approach. The efficacy of CD33KO-Loop CAR-iNK cells against tumor cells expressing CD33 and MSLN was investigated both in vitro and in AML xenograft mice.ResultsLoop CAR-NK cells exhibited superior cytotoxicity against dual-antigen-positive tumor cell lines and primary AML cells compared with CD33 CAR-NK and MSLN CAR-NK cells. Moreover, Loop CAR-NK cells showed upregulated signaling pathways related to NK cell activation and cytotoxic function. The loss of CD33 in iNK cells effectively avoided fratricide, improved expansion ability, and significantly enhanced CD33 and MSLN-mediated specific cytotoxicity of Loop CAR-iNK cells. Moreover, the CD33KO-Loop CAR-iNK cells demonstrated superior tumor-killing activity in AML xenograft mouse models and significantly prolonged mouse survival.ConclusionLoop CAR empowered both UCB-NK cells and hPSC-iNK cells with superior cytotoxicity against CD33+MSLN+ tumor cells. Genetic disruption of CD33 avoided fratricide and improved efficacy of Loop CAR-iNK cells against AML. This innovative strategy possesses unique advantages and translational potential for treating AML.

Bone marrow‐derived mesenchymal stem cells inhibit NK cell function via Tim‐3/galectin‐9 in multiple myeloma patients

Combination Therapy with Daratumumab and CAR-NK Targeting CS1 for Multiple Myeloma

Natural killer (NK) cells can spontaneously lyse certain virally infected and transformed cells. However, early in immune responses NK cells are further activated and recruited to tissue sites where they perform effector functions. This process is dependent on cytokines, but it is unclear if it is regulated by NK cell recognition of susceptible target cells. We show here that infiltration of activated NK cells into the peritoneal cavity in response to tumor cells is controlled by the tumor major histocompatibility complex (MHC) class I phenotype. Tumor cells lacking appropriate MHC class I expression induced NK cell infiltration, cytotoxic activation, and induction of transcription of interferon γ in NK cells. The induction of these responses was inhibited by restoration of tumor cell MHC class I expression. The NK cells responding to MHC class I–deficient tumor cells were ∼10 times as active as endogenous NK cells on a per cell basis. Although these effector cells showed a typical NK specificity in that they preferentially killed MHC class I–deficient cells, this specificity was even more distinct during induction of the intraperitoneal response. Observations are discussed in relation to a possible adaptive component of the NK response, i.e., recruitment/activation in response to challenges that only NK cells are able to neutralize.

BackgroundNatural killer (NK) cells hold great promise in treating diverse hematopoietic and solid tumors. Despite their availability from peripheral blood and cord blood, stem cell-derived NK cells offer an 'off-the-shelf' solution. Hematopoietic stem and progenitor cells (HSPCs) derived from cord blood pose no risk to the newborn or mother and are virtually ideal sources for NK cell differentiation.MethodsWe developed a modified protocol to differentiate HSPCs to NK cells under serum-free conditions using defined factors. The HSPC-derived NK (HSC-NK) cells could be expanded in a K562 feeder cell-dependent manner. Furthermore, using lentivirus transduction, chimeric antigen receptor (CAR)-modified HSPCs could be differentiated into NK cells, leading to the establishment of CAR-NK cells.ResultsThe efficiency of NK cell differentiation from HSPCs was increased through the simple modulation of osmotic pressure by the addition of sodium chloride or glucose. Furthermore, the hyperosmosis-primed HSC-NK cells exhibited enhanced proliferation capacity and maintained normal functional characteristics, including transcriptome and antitumor efficacy. The optimized protocol yielded approximately 1.8 million NK cells from a single CD34-positive cell within a 28-day cycle, which signifies more than a ten-fold increase in efficiency relative to the conventional methods. This optimized protocol was also suitable for generating CAR-NK cells with high yields compared to standard conditions.ConclusionsThe results of this study establish high osmotic pressure as a simple yet powerful adjustment that significantly enhances the efficiency and functionality of HSC-NK cells, including CAR-NK cells. This optimized protocol could lead to cost-effective, high-yield NK cell therapies, potentially revolutionizing cancer immunotherapy strategies.

Simple SummaryNatural killer (NK) cells are potent killers of tumor cells. Many tumors, including breast cancers, develop mechanisms to suppress anti-tumor immune responses, requiring the development of strategies to overcome suppression. Here, we tested a combination therapy that aims to (1) enhance NK cell activation and (2) reduce NK cell inhibition mediated by suppressive factors in tumors or in the tumor microenvironment. We cultured cell lines under hypoxia to mimic the tumor microenvironment or used patient-derived breast cancer cells that were primed by the patient’s tumor environment. Our results demonstrated that cytokine-activated NK cells remained active under hypoxia and that tumor-targeting antibodies enhanced the NK cell anti-breast cancer response. Moreover, we observed that NK cell suppression by inhibitory ligands on the tumor cells can be reduced by the selection of NK cell donors with NK receptors that are incompatible with these ligands. Collectively, we present two powerful strategies to enhance the NK cell responses against breast cancer.Natural killer (NK)-cell-based immunotherapies are an attractive treatment option for cancer. We previously showed that alloreactive mouse NK cells cured mice of 4T1 breast cancer. However, the tumor microenvironment can inhibit immune responses, and these suppressive factors must be overcome to unfold the NK cells’ full anti-tumor potential. Here, we investigated the combination of antibody-dependent cellular cytotoxicity (ADDC) and the selection of KIR-HLA-ligand mismatched NK cells to enhance NK cell anti-breast cancer responses in clinically relevant settings. Donor-derived and IL-2-activated NK cells were co-cultured with patient-derived breast cancer cells or cell lines MCF7 or SKBR3 together with the anti-HER2 antibody trastuzumab. NK cells mediated anti-breast cancer cytotoxicity under normoxic and hypoxic conditions. Under both conditions, trastuzumab vigorously enhanced NK cell degranulation (CD107a) against HER2-overexpressing SKBR3 cells, but we observed a discrepancy between highly degranulating NK cells and a rather modest increase in cytotoxicity of SKBR3. Against patient-derived breast cancer cells, the anti-tumor efficacy was rather limited, and HLA class I expression seemed to contribute to inhibited NK cell functionality. KIR-ligand-mismatched NK cells degranulated stronger compared to the matched NK cells, further highlighting the role of HLA. In summary, trastuzumab and KIR-ligand-mismatched NK cells could be two strategies to potently enhance NK cell responses to breast cancer.

BackgroundHead and neck squamous cell carcinomas (HNSCC) pose a significant challenge to currently available therapy. Despite the introduction of new treatment options like immune checkpoint inhibitors for advanced HNSCC, effective...

T cell engager (TCE) therapies targeting specific tumor associated antigens (TAAs) have shown clinical efficacy in multiple types of solid tumors. Indeed, TCE has been approved to treat melanoma and small cell lung cancer. Nevertheless, it remains necessary and challenging to improve the efficacy and safety profile of TCE to deliver benefits to more cancer patients. Recurrent and chemotherapy resistant ovarian cancer remains an urgent global unmet medical need. These is limited therapeutic options for this highly lethal and refractory disease. Recently, a MUC16 TCE demonstrated promising and durable clinical efficacy against heavily pretreated MUC16-high ovarian cancer, with acceptable safety profile. This underlines the potential to develop a MUC16 TCE with favorable safety profile and enhanced anti-tumor efficacy to possibly expand the benefits to MUC16-medium and MUC16-low ovarian cancer patients. MUC16 is a glycoprotein overexpressed in several types of solid tumors, especially in the majority of ovarian cancer. Here, we developed a 2+1 format MUC16 TCE, with improved tumor cell binding affinity, enhanced tumor cell killing potency, and MUC16-dependent T cell binding and activation. This MUC16 TCE targets membrane-proximal domains of MUC16 and achieves avidity-driven tumor cell binding in the presence of soluble CA125. Moreover, this MUC16 TCE elicited limited T cell binding and activation in the absence of MUC16, compared to benchmark molecule, to minimize off-target activity. This 2+1 format MUC16 TCE also showed favorable pharmacokinetics profile in mice. Functionally, systemic administration of this MUC16 TCE showed superior anti-tumor efficacy, with undetectable toxicity, in xenograft tumor models. Importantly, this MUC16 TCE demonstrated potent and synergistic anti-tumor efficacy in combination with clinically validated therapeutic strategies for ovarian cancer in pre-clinical tumor models. This provides rationale to develop combination therapies in future clinical trials. Indeed, combination of TCE with other therapies, e.g. ADC, chemotherapy, PD1/PDL1 mAb, are being tested clinically. Together, our pre-clinical data demonstrated that a 2+1 MUC16 TCE elicits potent anti-tumor efficacy and minimized off-target T cell activation, with potential to deliver benefits to ovarian cancer patients and patient with other MUC16-positive cancer. Citation Format: Jinyang Li, Yang Li, Nan Gao, Yi Wang, Mengqiang Di, Min Wu, Yiyi Li, Ran Xue, Shiyu Hao, Weiwei Wu, Xiguang Zhang, Li Li, Shuaixiang Zhou, Huizhong Xiong. A 2+1 format MUC16 targeting T cell engager induces MUC16-dependent T cell activity and superior anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3510.

siRNA Inactivation of the Inhibitory Receptor NKG2A Augments the Anti-Tumor Effects of Adoptively Transferred NK Cells In Tumor-Bearing Hosts