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NKG2D is an activating receptor expressed by natural killer (NK) cells and other cytotoxic lymphocytes that plays a pivotal role in the elimination of neoplastic cells through recognition of different stress-induced cell surface ligands (NKG2DL). To employ this mechanism for cancer immunotherapy, we generated NKG2D-engaging bispecific antibodies that selectively redirect immune effector cells to cancer cells expressing the tumor-associated antigen ErbB2 (HER2). NKG2D-specific single chain fragment variable (scFv) antibodies cross-reactive toward the human and murine receptors were derived by consecutive immunization of chicken with the human and murine antigens, followed by stringent screening of a yeast surface display immune library. Four distinct species cross-reactive (sc) scFv domains were selected, and reformatted into a bispecific engager format by linking them via an IgG4 Fc domain to a second scFv fragment specific for ErbB2. The resulting molecules (termed scNKAB-ErbB2) were expressed as disulfide-linked homodimers, and demonstrated efficient binding to ErbB2-positive cancer cells as well as NKG2D-expressing primary human and murine lymphocytes, and NK-92 cells engineered with chimeric antigen receptors derived from human and murine NKG2D (termed hNKAR and mNKAR). Two of the scNKAB-ErbB2 molecules were found to compete with the natural NKG2D ligand MICA, while the other two engagers interacted with an epitope outside of the ligand binding site. Nevertheless, all four tested scNKAB-ErbB2 antibodies were similarly effective in redirecting the cytotoxic activity of primary human and murine lymphocytes as well as hNKAR-NK-92 and mNKAR-NK-92 cells to ErbB2-expressing targets, suggesting that further development of these species cross-reactive engager molecules for cancer immunotherapy is warranted. [ABSTRACT FROM AUTHOR]

Innate lymphocytes comprise cytotoxic natural killer (NK) cells and tissue‐resident innate lymphoid cells (ILC) that are subgrouped according to their cytokine profiles into group 1 ILC (ILC1), ILC2, and ILC3. However, cell surface receptors unambiguously defining or specifically activating such ILC subsets are scarcely known. Here, we report on the physiologic expression of the human activating C‐type lectin‐like receptor (CTLR) NKp65, a high‐affinity receptor for the CTLR keratinocyte‐associated C‐type lectin (KACL). Tracking rare NKp65 transcripts in human blood, we identify ILC3 to selectively express NKp65. NKp65 expression not only demarcates "bona fide" ILC3 from likewise RORγt‐expressing ILC precursors and lymphoid tissue inducer cells but also from mature NK cells which acquire the NKp65‐relative NKp80 during a Notch‐dependent differentiation from NKp65+ precursor cells. Hence, ILC3 and NK cells mutually exclusively and interdependently express the genetically coupled sibling receptors NKp65 and NKp80. Much alike NKp80, NKp65 promotes cytotoxicity by innate lymphocytes which may become relevant during pathophysiological reprogramming of ILC3. Altogether, we report the selective expression of the activating immunoreceptor NKp65 by ILC3 demarcating ILC3 from mature NK cells and endowing ILC3 with a dedicated immunosensor for the epidermal immune barrier. [ABSTRACT FROM AUTHOR]

NKG2D is an activating receptor of natural killer cells that recognizes stress-induced ligands (NKG2DL) expressed by many tumor cells. Nevertheless, NKG2DL downregulation or shedding can still allow cancer cells to evade immune surveillance. Here, we used lentiviral gene transfer to engineer clinically usable NK-92 cells with a chimeric antigen receptor (NKAR) which contains the extracellular domain of NKG2D for target recognition, or an NKAR, together with the IL-15 superagonist RD-IL15, and combined these effector cells with recombinant NKG2D-interacting bispecific engagers that simultaneously recognize the tumor-associated antigens epidermal growth factor receptor (EGFR) or ErbB2 (HER2). Applied individually, in in vitro cell-killing assays, these NKAB-EGFR and NKAB-ErbB2 antibodies specifically redirected NKAR-NK-92 and NKAR_RD-IL15-NK-92 cells to glioblastoma and other cancer cells with elevated EGFR or ErbB2 levels. However, in mixed glioblastoma cell cultures, used as a model for heterogeneous target antigen expression, NKAR-NK cells only lysed the EGFR- or ErbB2-expressing subpopulations in the presence of one of the NKAB molecules. This was circumvented by applying NKAB-EGFR and NKAB-ErbB2 together, resulting in effective antitumor activity similar to that against glioblastoma cells expressing both target antigens. Our results demonstrate that combining NK cells carrying an activating NKAR receptor with bispecific NKAB antibodies allows for flexible targeting, which can enhance tumor-antigen-specific cytotoxicity and prevent immune escape. [ABSTRACT FROM AUTHOR]

Simple Summary: The disialoganglioside GD2 is produced at high levels by neuroblastomas and other tumors of neuroectodermal origin, where its expression correlates with increased tumor progression and poor prognosis. Although established therapies with GD2-specific antibodies can be efficacious, more effective treatment options are needed for advanced and relapsed tumors. The aim of this work was the generation and functional evaluation of natural killer cells engineered with a GD2-specific chimeric antigen receptor (CAR) as a novel off-the-shelf adoptive immunotherapy approach. In preclinical in vitro models, these CAR-NK cells displayed high and selective cytotoxicity against GD2-expressing tumor cells. Moreover, GD2-CAR NK cells further modified to express an interleukin (IL)-15 superagonist displayed enhanced functionality also in the absence of exogenous cytokines and modulated proliferation and antitumor activity of surrounding innate immune cells and T lymphocytes. In contrast to T lymphocytes, natural killer (NK) cells do not require prior sensitization but are rapidly activated upon encountering virally infected or neoplastic cells. In addition, NK cells can be safely applied in an allogeneic setting, making them important effector cells for the development of off-the-shelf therapeutics for adoptive cancer immunotherapy. To further enhance their therapeutic potential, here, we engineered continuously expanding NK-92 cells as a clinically relevant model to express a humanized second-generation chimeric antigen receptor (CAR) with a composite CD28-CD3ζ signaling domain (hu14.18.28.z) that targets the disialoganglioside GD2, which is expressed at high levels by neuroblastoma cells and other tumors of neuroectodermal origin. In a separate approach, we fused an IL-15 superagonist (RD-IL15) to the GD2-CAR via a P2A processing site. Lentivirally transduced NK-92/hu14.18.28.z and NK-92/hu14.18.28.z_RD-IL15 cells both displayed high and stable CAR surface expression and specific cytotoxicity toward GD2-positive tumor cells. GD2-CAR NK cells carrying the RD-IL15 construct in addition expressed the IL-15 superagonist, resulting in self-enrichment and targeted cell killing in the absence of exogenous IL-2. Furthermore, co-culture with RD-IL15-secreting GD2-CAR NK cells markedly enhanced proliferation and cytotoxicity of bystander immune cells in a paracrine manner. Our results demonstrate that GD2-CAR NK cells co-expressing the IL-15 superagonist mediate potent direct and indirect antitumor effects, suggesting this strategy as a promising approach for the further development of functionally enhanced cellular therapeutics. [ABSTRACT FROM AUTHOR]

Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a high relapse rate and still limited therapeutic options. Natural killer (NK) cell-based immunotherapy has the potential to improve outcomes for patients with AML. Summary: Recent preclinical studies and early-stage clinical trials aim to enhance the intrinsic anti-leukemic properties of NK cells by selectively targeting AML cells with chimeric antigen receptors (CARs). Furthermore, NK and CAR-NK cells can be combined with other therapeutic modalities or engineered further to overcome the immunosuppressive microenvironment, and treatment resistance of AML blasts and leukemia-initiating cells (LIC). Key Messages: In this review, we summarize preclinical studies with cytokine-stimulated or genetically engineered NK cells derived from different cell sources for the treatment of AML and their translation into early-phase clinical trials. We also provide an overview of promising recent developments toward innovative NK cell-based therapies that may be implemented in the near future. [ABSTRACT FROM AUTHOR]

NKG2D is an activating receptor expressed by natural killer (NK) cells and other cytotoxic lymphocytes that plays a pivotal role in the elimination of neoplastic cells through recognition of different stress-induced cell surface ligands (NKG2DL). To employ this mechanism for cancer immunotherapy, we generated NKG2D-engaging bispecific antibodies that selectively redirect immune effector cells to cancer cells expressing the tumor-associated antigen ErbB2 (HER2). NKG2D-specific single chain fragment variable (scFv) antibodies cross-reactive toward the human and murine receptors were derived by consecutive immunization of chicken with the human and murine antigens, followed by stringent screening of a yeast surface display immune library. Four distinct species cross-reactive (sc) scFv domains were selected, and reformatted into a bispecific engager format by linking them via an IgG4 Fc domain to a second scFv fragment specific for ErbB2. The resulting molecules (termed scNKAB-ErbB2) were expressed as disulfide-linked homodimers, and demonstrated efficient binding to ErbB2-positive cancer cells as well as NKG2D-expressing primary human and murine lymphocytes, and NK-92 cells engineered with chimeric antigen receptors derived from human and murine NKG2D (termed hNKAR and mNKAR). Two of the scNKAB-ErbB2 molecules were found to compete with the natural NKG2D ligand MICA, while the other two engagers interacted with an epitope outside of the ligand binding site. Nevertheless, all four tested scNKAB-ErbB2 antibodies were similarly effective in redirecting the cytotoxic activity of primary human and murine lymphocytes as well as hNKAR-NK-92 and mNKAR-NK-92 cells to ErbB2-expressing targets, suggesting that further development of these species cross-reactive engager molecules for cancer immunotherapy is warranted., Competing Interests: KS, JH, AM, PO, HK and WW are named as inventors on patents and patent applications in the field of cancer immunotherapy owned by their respective academic institutions. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Pfeifer Serrahima, Schoenfeld, Kühnel, Harwardt, Macarrón Palacios, Prüfer, Kolaric, Oberoi, Kolmar and Wels.)