Cytokine-Induced Killer (CIK) cells share several functional and phenotypical properties of both T and natural killer (NK) cells. They represent an attractive approach for cell-based immunotherapy, as they do not require antigen-specific priming for tumor cell recognition, and can be rapidly expanded in vitro. Their relevant expression of FcγRIIIa (CD16a) can be exploited in combination with clinical-grade monoclonal antibodies (mAbs) to redirect their lytic activity in an antigen-specific manner. Here, we report the efficacy of this combined approach against triple negative breast cancer (TNBC), an aggressive tumor that still requires therapeutic options. Different primitive and metastatic TNBC cancer mouse models were established in NSG mice, either by implanting patient-derived TNBC samples or injecting MDA-MB-231 cells orthotopically or intravenously. The combined treatment consisted in the repeated intratumoral or intravenous injection of CIK cells and cetuximab. Tumor growth and metastasis were monitored by bioluminescence or immunohistochemistry, and survival was recorded. CIK cells plus cetuximab significantly restrained primitive tumor growth in mice, either in patient-derived tumor xenografts or MDA-MB-231 cell line models. Moreover, this approach almost completely abolished metastasis spreading and dramatically improved survival. The antigen-specific mAb favored tumor and metastasis tissue infiltration by CIK cells, and led to an enrichment of the CD16a + subset. Data highlight the potentiality of this novel immunotherapy strategy where a nonspecific cytotoxic cell population can be converted into tumor-specific effectors with clinical-grade antibodies, thus providing not only a therapeutic option for TNBC but also a valid alternative to more complex approaches based on chimeric antigen receptor-engineered cells.

Cytokine-Induced (CIK) cells represent an attractive approach for cell-based immunotherapy, as they show several advantages compared with other strategies. Here we describe an original serum-free protocol for CIK cell expansion that employs G-Rex devices and compare the resulting growth, viability, phenotypic profile and cytotoxic activity with conventional culture in tissue flasks. CIK cells were obtained from buffy coats, seeded in parallel in G-Rex and tissue flasks, and stimulated with clinical-grade IFN-g, anti-CD3 antibody and IL-2. G-Rex led to large numbers of CIK cells, with a minimal need for technical interventions, thus reducing the time and costs of culture manipulation. CIK cells generated in G-Rex showed a less differentiated phenotype, with a significantly higher expression of naive-associated markers such as CD62L, CD45RA and CCR7, which correlates with a remarkable expansion potential in culture and could lead to longer persistence and a more sustained anti-tumor response in vivo. The described procedure can be easily translated to large-scale production under Good Manufacturing Practice. Overall, this protocol has strong advantages over existing procedures, as it allows easier, time-saving and cost-effective production of CIK effector cells, fostering their clinical application.

BackgroundPatients affected by aggressive B-cell malignancies who are resistant to primary or salvage chemoimmunotherapy have an extremely poor prognosis and limited therapeutic options. Promising therapeutic success has been achieved with the infusion of CD19 chimeric antigen receptor-T cells, but several limits still restrain the administration to a limited proportion of patients. This unmet clinical need might be fulfilled by an adoptive immunotherapy approach that combines cytokine-induced killer (CIK) cells and monoclonal antibodies (mAb) to the CD20 antigen. Indeed, CIK cells are an effector population endowed with antitumor activity, which can be further improved and antigen-specifically redirected by clinical-grade mAb triggering antibody-dependent cell-mediated cytotoxicity.MethodsCIK cells were generated from peripheral blood of patients affected by different B-cell malignancies using a blinatumomab-based cell culture protocol. Effector cells were combined with the anti-CD20 mAb obinutuzumab and their therapeutic activity was assessed both in vitro and in vivo.ResultsCIK cells were successfully expanded in clinically relevant numbers, starting from small volumes of peripheral blood with extremely low CD3+ counts and high tumor burden. This relied on the addition of blinatumumab in culture, which leads to the simultaneous expansion of effector cells and the complete elimination of the neoplastic component. Moreover, CIK cells were highly cytotoxic in vitro against both B-cell tumor cell lines and autologous neoplastic targets, and had a significant therapeutic efficacy against a B-cell malignancy patient-derived xenograft on in vivo transfer.ConclusionsThe combination of an easily expandable CIK cell effector population with a mAb already in clinical use establishes a tumor antigen-specific redirection strategy that can be rapidly translated into clinical practice, providing an effective therapeutic alternative for B-cell malignancies without any need for genetic modifications. Additionally, the approach can be potentially applied to an extremely vast array of different tumors by simply substituting the targeting mAb.

Background Adenoid Cystic Carcinoma (ACC) is a lethal malignancy of exocrine glands, characterized by the co-existence within tumor tissues of two distinct populations of cancer cells, phenotypically similar to the myoepithelial and ductal lineages of normal salivary epithelia. The developmental relationship linking these two cell-types, and their differential vulnerability to anti-tumor treatments, remain unknown. Methods Using single-cell RNA-sequencing (scRNA-seq), we identified cell-surface markers (CD49f, KIT) that enabled the differential purification of myoepithelial-like (CD49fhigh/KITneg) and ductal-like (CD49flow/KIT+) cells from patient-derived xenografts (PDX) of human ACCs. Using prospective xeno-transplantation experiments, we compared the tumor-initiating capacity of the two cell-types, and tested whether one could differentiate into the other. Finally, we searched for signaling pathways with differential activation between the two cell-types and tested their role as lineage-specific therapeutic targets. Results Myoepithelial-like cells displayed higher tumorigenicity than ductal-like cells and acted as their progenitors. Myoepithelial-like and ductal-like cells displayed differential expression of genes encoding for suppressors and activators of retinoic acid signaling, respectively. Agonists of retinoic acid receptor (RAR) or retinoid X receptor (RXR) signaling (ATRA, bexarotene) promoted myoepithelial-to-ductal differentiation, whereas suppression of RAR/RXR signaling with a dominant-negative RAR construct abrogated it. Inverse agonists of RAR/RXR signaling (BMS493, AGN193109) displayed selective toxicity against ductal-like cells, and in vivo anti-tumor activity against PDX models of ACC. Conclusions In human ACCs, myoepithelial-like cells act as progenitors of ductal-like cells, and myoepithelial-to-ductal differentiation is promoted by RAR/RXR signaling. Suppression of RAR/RXR signaling is lethal to ductal-like cells and represents a new therapeutic approach against human ACCs.

BackgroundCytokine-Induced Killer (CIK) cells share several functional and phenotypical properties of both T and natural killer (NK) cells, and represent an attractive approach for cell-based immunotherapy as they do not require antigen-specific priming for tumor cell recognition, and can be efficiently and rapidly expanded in vitro. We recently reported that CIK cells have a relevant expression of FcγRIIIa (CD16a), which can be exploited in combination with clinical-grade monoclonal antibodies (mAbs) to redirect their lytic activity in an antigen-specific manner. Here, we report the assessment and the efficacy of this combined approach against triple negative breast cancer (TNBC), an aggressive tumor that still requires reliable therapeutic options.Materials and methodsDifferent primitive and metastatic TNBC cancer mouse models were established in NSG mice, either by implanting patient-derived TNBC samples or MDA-MB-231 cells subcutaneously or orthotopically into the mammary fat pad, or by injecting MDA-MB-231 cells intravenously. The combined treatment consisted in the repeated intratumoral or intravenous injection of CIK cells and cetuximab, while the mAb alone or CIK cells plus Rituximab served as control treatments. Tumor growth and metastasis were monitored by bioluminescence or immunohistochemistry, and survival was recorded.ResultsCIK cells plus cetuximab significantly restrained primitive tumor growth in mice, either implanted with TNBC patient-derived tumor xenografts or injected with MDA-MB-231 TNBC cell line. Moreover, in both experimental and spontaneous metastatic models the combined approach almost completely abolished metastasis spreading and dramatically improved survival. The antigen-specific mAb favored tumor and metastasis tissue infiltration by CIK cells, and in particular led to an enrichment of the CD16a+ subset.ConclusionsData highlight the potentiality of a novel immunotherapy approach where a non-specific cytotoxic cell population can be converted into tumor-specific effectors with clinical-grade antibodies, thus providing not only a therapeutic option for TNBC but also a valid alternative to more complex approaches based on chimeric antigen receptor-engineered cells.Disclosure InformationR. Sommaggio: None. E. Cappuzzello: None. A. Dalla Pietà: None. P. Palmerini: None. A. Tosi: None. D. Carpanese: None. L. Nicolè: None. A. Rosato: None.