Multifunctional Natural Killer Cell Engagers Targeting NKp46 Trigger Protective Tumor Immunity.

Over the last decade, various new therapies have been developed to promote anti-tumor immunity. Despite interesting clinical results in hematological malignancies, the development of bispecific killer-cell-engager antibody formats directed against tumor cells and stimulating anti-tumor T cell immunity has proved challenging, mostly due to toxicity problems. We report here the generation of trifunctional natural killer (NK) cell engagers (NKCEs), targeting two activating receptors, NKp46 and CD16, on NK cells and a tumor antigen on cancer cells. Trifunctional NKCEs were more potent in vitro than clinical therapeutic antibodies targeting the same tumor antigen. They had similar in vivo pharmacokinetics to full IgG antibodies and no off-target effects and efficiently controlled tumor growth in mouse models of solid and invasive tumors. Trifunctional NKCEs thus constitute a new generation of molecules for fighting cancer. VIDEO ABSTRACT.

Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells.

Checkpoint inhibitors have revolutionized cancer treatment. However, only a minority of patients respond to these immunotherapies. Here, we report that blocking the inhibitory NKG2A receptor enhances tumor immunity by promoting both natural killer (NK) and CD8+ T cell effector functions in mice and humans. Monalizumab, a humanized anti-NKG2A antibody, enhanced NK cell activity against various tumor cells and rescued CD8+ T cell function in combination with PD-x axis blockade. Monalizumab also stimulated NK cell activity against antibody-coated target cells. Interim results of a phase II trial of monalizumab plus cetuximab in previously treated squamous cell carcinoma of the head and neck showed a 31% objective response rate. Most common adverse events were fatigue (17%), pyrexia (13%), and headache (10%). NKG2A targeting with monalizumab is thus a novel checkpoint inhibitory mechanism promoting anti-tumor immunity by enhancing the activity of both T and NK cells, which may complement first-generation immunotherapies against cancer.

Antitumor immunity induced by antibody-based natural killer cell engager therapeutics armed with not-alpha IL-2 variant.

Harnessing innate immunity is emerging as a promising therapeutic approach in cancer. We report here the design of tetraspecific molecules engaging natural killer (NK) cell-activating receptors NKp46 and CD16a, the ß-chain of the interleukin-2 receptor (IL-2R), and a tumor-associated antigen (TAA). In vitro, these tetraspecific antibody-based natural killer cell engager therapeutics (ANKETs) induce a preferential activation and proliferation of NK cells, and the binding to the targeted TAA triggers NK cell cytotoxicity and cytokine and chemokine production. In vivo, tetraspecific ANKETs induce NK cell proliferation and their accumulation at the tumor bed, as well as the control of local and disseminated tumors. Treatment of non-human primates with CD20-directed tetraspecific ANKET leads to CD20+ circulating B cell depletion, with minimal systemic cytokine release and no sign of toxicity. Tetraspecific ANKETs, thus, constitute a technological platform for harnessing NK cells as next-generation cancer immunotherapies.

Targeting natural killer cells in solid tumors.

Natural killer (NK) cells are innate lymphoid cells endowed with cytolytic activity and a capacity to secrete cytokines and chemokines. Several lines of evidence suggest that NK cells play an important role in anti-tumor immunity. Some therapies against hematological malignacies make use of the immune properties of NK cells, such as their ability to kill residual leukemic blasts efficiently after conditioning during haploidentical hematopoietic stem cell transplantation. However, knowledge on NK cell infiltration and the status of NK cell responsiveness in solid tumors is limited so far. The pro-angiogenic role of the recently described NK cell-like type 1 innate lymphoid cells (ILC1s) and their phenotypic resemblance to NK cells are confounding factors that add a level of complexity, at least in mice. Here, we review the current knowledge on the presence and function of NK cells in solid tumors as well as the immunotherapeutic approaches designed to harness NK cell functions in these conditions, including those that aim to reinforce conventional anti-tumor therapies to increase the chances of successful treatment.

Targeting the Wnt Pathway and Cancer Stem Cells with Anti-progastrin Humanized Antibodies as a Potential Treatment for K-RAS-Mutated Colorectal Cancer.

Purpose: Patients with metastatic colorectal cancer suffer from disease relapse mainly due to cancer stem cells (CSC). Interestingly, they have an increased level of blood progastrin, a tumor-promoting peptide essential for the self-renewal of colon CSCs, which is also a direct ß-catenin/TCF4 target gene. In this study, we aimed to develop a novel targeted therapy to neutralize secreted progastrin to inhibit Wnt signaling, CSCs, and reduce relapses.Experimental Design: Antibodies (monoclonal and humanized) directed against progastrin were produced and selected for target specificity and affinity. After validation of their effectiveness on survival of colorectal cancer cell lines harboring B-RAF or K-RAS mutations, their efficacy was assessed in vitro and in vivo, alone or concomitantly with chemotherapy, on CSC self-renewal capacity, tumor recurrence, and Wnt signaling.Results: We show that anti-progastrin antibodies decrease self-renewal of CSCs both in vitro and in vivo, either alone or in combination with chemotherapy. Furthermore, migration and invasion of colorectal cancer cells are diminished; chemosensitivity is prolonged in SW620 and HT29 cells and posttreatment relapse is significantly delayed in T84 cells, xenografted nude mice. Finally, we show that the Wnt signaling activity in vitro is decreased, and, in transgenic mice developing Wnt-driven intestinal neoplasia, the tumor burden is alleviated, with an amplification of cell differentiation in the remaining tumors.Conclusions: Altogether, these data show that humanized anti-progastrin antibodies might represent a potential new treatment for K-RAS-mutated colorectal patients, for which there is a crucial unmet medical need. Clin Cancer Res; 23(17); 5267-80. ©2017 AACR.

Phosphoproteome analyses reveal specific implications of Hcls1, p21-activated kinase 1 and Ezrin in proliferation of a myeloid progenitor cell line downstream of wild-type and ITD mutant Fms-like tyrosine kinase 3 receptors.

The tyrosine kinase receptor Flt3 (Fms-like tyrosine kinase 3) is almost always expressed in AML (acute myeloid leukemia) cells, and constitutive activation of Flt3 by ITD (internal tandem duplication) mutations is one of the most common molecular alterations known in AML, especially monocytic AML. Furthermore, Flt3-ligand (FL) was shown as an in vitro growth factor for monocytic precursors, pointing to the important role of Flt3 in the regulation of monocyte/macrophage production. To get a relevant model for studying the molecular mechanisms underlying the physiopathological role of Flt3 on monocytic lineage development, we used the IL-3 dependent murine myeloid progenitors FDC-P1 cell line to generate cells stably co-expressing murine Fms (M-CSF receptor) and human Flt3. Wild type (WT)-Flt3 expressing cells could proliferate in an FL-dependent manner, whereas those expressing Flt3-ITD all survived IL-3 deprivation and showed autonomous proliferation, whereas both types of cells could differentiate to monocytic cells in response to M-CSF. Next, by combining phosphoprotein detection or purification, comparative 2D-PAGE and mass spectrometry sequencing, we sought for downstream mediators of Flt3-WT or Flt3-ITD in FD/Fms cell proliferation. Amongst the differentially expressed and/or phosphorylated proteins, 3 showed a specific implication in FD/Fms cell proliferation: Hcls1 and the Pak1/2 in FL-dependent proliferation of Flt3-WT expressing cells and Ezrin in autonomous proliferation of Flt3-ITD expressing cells.