Combinatory therapeutic approaches for haematological and solid tumors with Cytokine-Induced Killer (CIK) cells

In the last decade adoptive cell therapy (ACT) achieved extraordinary results in antitumor treatments, especially against haematological malignancies, even if with some limitations - such as - safety, high costs of manufacturing, and restricted cell sources. In this regard, Cytokine-Induced Killer cells are achieving considerable clinical relevance, due to the low risk of acute graft versus host disease (GvHD) in both autologous and allogeneic settings, their feasibility, and the limited costs of production. CIK cells are a heterogeneous population of T lymphocytes, expanded and activated ex vivo from peripheral blood mononuclear cells (PBMCs) or cord blood, with the addition of recombinant human interferon-γ (rhIFN- γ), anti-CD3 monoclonal antibody (mAb), and recombinant human interleukin-2 (rhIL-2). During the expansion, CIK cells acquire the expression of CD56 and present several functional and phenotypical properties of both T and Natural Killer (NK) cells. Importantly, CIK cells do not require any genetic engineering or antigen-specific stimuli to recognize tumor cells and exert a potent major histocompatibility complex (MHC)-unrestricted antitumor activity. In 2016, our group showed that CIK cells increase their ability to kill tumor cells by antibody-dependent cellular cytotoxicity (ADCC) due to the expression of the Fragment chain (Fc)-γ Receptor IIIa (CD16a). In this study, we assessed CIK cell cytotoxicity against CD20 + lymphoma patient-derived samples, both in vitro and in vivo, by the combination with two clinical grade anti-CD20 monoclonal antibodies (mAbs), Rituximab (RTX) or Obinutuzumab (Gazyva/Gazyvaro, OBI). Furthermore, to fulfil the huge amount of cell numbers required to achieve successful clinical responses, we optimized an efficient serum-free protocol for optimal expansion of CIK cells in gas-permeable rapid expansion (G-Rex®) devices from both healthy donors and B-cell malignant patients. G- Rex® devices allow the expansion of a high number of CIK cells, fully functional in terms of viability, phenotype, and lytic activity. Moreover, the addition of the bispecific T cell-engager (BiTe) Blinatumomab (CD3xCD19) to the cultures from B-cell lymphoma patients eliminated the residual tumor cells (CD19 + ), favouring the expansion of CIK cells, even if starting from blood samples containing very low percentages of CD3 + precursor cells. Next, we extended this efficient combinatorial strategy to solid cancers, focusing on Human epidermal growth factor receptor 2 (Her-2) - expressing breast cancers. We combined CIK cells with both the mAb Trastuzumab (TRS) or with the bispecific single chain Fragment variable (bsscFv) Her2xCD3 in a BiTE-like format. The synergistic effect exerted by CIK cells with the antibodies, especially with the Her2xCD3, resulted in a remarkable in vitro lytic activity against several Her-2 + cancer cell lines, even at very low effector/target ratio and a reduced dosage of the bsscFv, encouraging clinical perspectives of this approach for the ACT. To further gain insight into the trafficking and the infiltration of transferred CIK cells in solid tumors, we explored an agarose-embedded thick-tumor tissue slice assay combined with a high- resolution spinning disk confocal microscopy. This technique not only allows the tracking of labelled effector cells in the tumor tissue slice, but it can be useful also to explore the multicellular interactions taking place in the tumor microenvironment. Moreover, to study and characterize the tumor three-dimensional structure in terms of the stiffness of the extracellular matrix (ECM), we exploited the Second Harmonic Generation (SHG) microscopy which provides important information about collagen density and fibres organization.