Fate control engagement augments NK cell responses in LV/hu-IL-12 transduced sarcoma.

INTRODUCTION: NK cells are an untapped resource for cancer therapy. Sarcomas transduced with lentiviruses to express human IL-12 are only cleared in mice bearing mature human NK cells. However, systemic inflammation limits IL-12 utilization. Fate control a.k.a. "suicide mechanisms" regulate unchecked systemic inflammation caused by cellular immunotherapies. Despite increasing utilization, there remains limited data on immune consequences or tumor-directed effects of fate control. OBJECTIVES: We sought to engage the mutant thymidylate kinase (mTMPK) metabolic fate control system to regulate systemic inflammation and assess the impact on NK cell effector functions. METHODS: Primary human sarcoma short-passage samples and cell lines were transduced with LV/hu-IL-12_mTMPK engineering expression of IL-12 and an AZT-associated fate control enzyme. We assessed transduced sarcoma responses to AZT engagement and subsequent modulation of NK cell functions as measured by inflammatory cytokine production and cytotoxicity. RESULTS: AZT administration to transduced (LV/hu-IL-12_mTMPK) short-passage primary human sarcomas and human Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma cell lines, abrogated the robust expression of human IL-12. Fate control activation elicited a specific dose-dependent cytotoxic effect measured by metabolic activity (WST-1) and cell death (Incucyte). NK effector functions of IFN-γ and cytotoxic granule release were significantly augmented despite IL-12 abrogation. This correlated with preferentially induced expression of NK cell activation ligands. CONCLUSIONS: mTMPK fate control engagement terminates transduced sarcoma IL-12 production and triggers cell death, but also augments an NK cell-mediated response coinciding with metabolic stress activating surface ligand induction. Fate control engagement could offer a novel immune activation method for NK cell-mediated cancer clearance.

Novel anti-CD30/CD3 bispecific antibodies activate human T cells and mediate potent anti-tumor activity.

CD30 is expressed on Hodgkin lymphomas (HL), many non-Hodgkin lymphomas (NHLs), and non-lymphoid malignancies in children and adults. Tumor expression, combined with restricted expression in healthy tissues, identifies CD30 as a promising immunotherapy target. An anti-CD30 antibody-drug conjugate (ADC) has been approved by the FDA for HL. While anti-CD30 ADCs and chimeric antigen receptors (CARs) have shown promise, their shortcomings and toxicities suggest that alternative treatments are needed. We developed novel anti-CD30 x anti-CD3 bispecific antibodies (biAbs) to coat activated patient T cells (ATCs) ex vivo prior to autologous re-infusions. Our goal is to harness the dual specificity of the biAb, the power of cellular therapy, and the safety of non-genetically modified autologous T cell infusions. We present a comprehensive characterization of the CD30 binding and tumor cell killing properties of these biAbs. Five unique murine monoclonal antibodies (mAbs) were generated against the extracellular domain of human CD30. Resultant anti-CD30 mAbs were purified and screened for binding specificity, affinity, and epitope recognition. Two lead mAb candidates with unique sequences and CD30 binding clusters that differ from the ADC in clinical use were identified. These mAbs were chemically conjugated with OKT3 (an anti-CD3 mAb). ATCs were armed and evaluated in vitro for binding, cytokine production, and cytotoxicity against tumor lines and then in vivo for tumor cell killing. Our lead mAb was subcloned to make a Master Cell Bank (MCB) and screened for binding against a library of human cell surface proteins. Only huCD30 was bound. These studies support a clinical trial in development employing ex vivo-loading of autologous T cells with this novel biAb.

Sarcoma IL-12 overexpression facilitates NK cell immunomodulation.

Interleukin-12 (IL-12) is an inflammatory cytokine that has demonstrated efficacy for cancer immunotherapy, but systemic administration has detrimental toxicities. Lentiviral transduction eliciting IL-12-producing human sarcoma for autologous reintroduction provides localized delivery for both innate and adaptive immune response augmentation. Sarcoma cell lines and primary human sarcoma samples were transduced with recombinant lentivirus engineering expression of human IL-12 (hu-IL-12). IL-12 expressing sarcomas were assessed in vitro and in vivo following implantation into humanized NSG and transgenic human IL-15 expressing (NSG.Tg(Hu-IL-15)) murine models. Lentiviral transduction (LV/hu-IL-12) of human osteosarcoma, Ewing sarcoma and rhabdomyosarcoma cell lines, as well as low-passage primary human sarcomas, engendered high-level expression of hu-IL-12. Hu-IL-12 demonstrated functional viability, eliciting specific NK cell-mediated interferon-γ (IFN-γ) release and cytotoxic growth restriction of spheroids in vitro. In orthotopic xenograft murine models, the LV/hu-IL-12 transduced human sarcoma produced detectable IL-12 and elicited an IFN-γ inflammatory immune response specific to mature human NK reconstitution in the NSG.Tg(Hu-IL-15) model while restricting tumor growth. We conclude that LV/hu-IL-12 transduction of sarcoma elicits a specific immune reaction and the humanized NSG.Tg(Hu-IL-15) xenograft, with mature human NK cells, can define in vivo anti-tumor effects and systemic toxicities. IL-12 immunomodulation through autologous tumor transduction and reintroduction merits exploration for sarcoma treatment.