PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Supervisor

Raffaella De Maria

Phd thesis

Cytokine-Induced Killer Cells as immune cell-based therapy in the treatment of Canine Malignant Tumour

 

Scientific Background

Adoptive cell therapy has been a remarkable therapeutic strategy to combat human cancers in recent years, showing important results as antitumour methods. Of the available approaches regarding this subject, there is adoptive Natural Killer (NK) cell therapy, based on the induction and use of Cytokine-Induced Killer (CIK) cells. CIK cells consist of polyclonal ex vivo expanded NK-like T cells, capable of antigen and MHC-unrestricted antitumor activity (Todorovic et al., 2012).

When identifying ligands that are induced by stress, such as DNA damage or malignant transformation, NK cells carry strong cytotoxicity, and act through the release of cytolytic granules and cytotoxic cytokine. Also, their MHC-unrestricted antitumor activity allows them to bypass tumour immune escape mechanisms caused by MHC downregulation, assuring the attack of abnormal self-cells.

The advantages of an adoptive NK cell therapy encompass it not being associated with graft versus host disease (GvHD), allowing a safer option compared to T-cell based therapies, and also allogeneic transfer (Kisseberth and Lee, 2021). Furthermore, the complex manufacturing process of CAR (Chimeric Antigen Receptor) T cells increases costs and delays the start of the therapy, thus representing an obstacle for patients who, owing to rapidly progressing disease, are in critical need of treatment (Laskowski et al., 2022).

NK-cell based therapy has already shown outstanding rates in human cancer treatments, with its scope recently extending to solid tumours, besides previous success for immune cell tumours such as leukaemia and lymphomas. Concerning veterinary immunotherapy, NK cells have shown clinical efficiency in blood-based cancers, but still little improvement for solid tumours.

Dogs are the ideal candidates for those next steps because they share similar tumour biology with humans, which already have valuable literature for solid tumours treatment with CIK cells (Gingrich et al., 2021). Moreover, cross-species data have demonstrated similarities between dog NK cells to human NK cells, endorsing a great potential in canine studies of this unique and promising approach (Gingrich et al., 2021).

Melanomas are suitable candidates of solid tumours to be tested with this distinct technique, and have already been targeted by human researchers, who reported that NK cells show a good cytotoxic response against this type of tumour (Iaia et al. 2021). Therefore, canine patients could be the next ones to be studied, and benefit from a novel option of cancer treatment.

Goals

The aims of this project include the evaluation of expanding methods for canine NK cells and their activation into CIK cells, thus establishing future guidelines regarding NK-cell based therapy in solid tumours; the assessment of CIK cells persistence ex-vivo and anti-tumour response by their killing rate; to examine selectiveness and cytotoxicity of the CIKs in both allogenic and autogenic samples from canine solid tumours.

Methods

The acquisition of a high number of immune effectors is extremely important for the competence of the CIK-cells based immunotherapy. The classic protocol for the ex vivo expansion of CIK cells includes the culture of peripheral blood mononuclear cells (PBMC) and activation by specific cytokines (Mesiano et al., 2012). PBMCs are isolated from blood buffy coats of the patients by Ficoll separation, and are then stimulated by the addition of interferon-gamma (IFN-γ) (Iaia et al. 2021), interleukin 2 (IL-2) and microbeads with anti-CD28 and anti-CD3 antibodies (Rotolo et al. 2021). Expanded NK cells can be used immediately or cryopreserved for later use (Laskowski et al., 2022).

Tumoural cell lines can be grown in cultures with specific cell medium, 10% of Foetal Bovine Serum (FBS), 1% of antibiotics and 1% of L-glutamine. Anti-tumour response can be evaluated by allogenic and autogenic assessments of the CIK cells' potency towards tumoral tissue grown ex-vivo, such as CellTiter-Glo Luminescent Cell Viability Assay.

References

Gingrich A.A., Reiter T.E., Judge S.J., York D., Yanagisawa M., Razmara A., et al. 2021. Comparative Immunogenomics of Canine Natural Killer Cells as Immunotherapy Target. Front Immunol, 12.

Iaia, Ilenia, Loretta Gammaitoni, Giulia Cattaneo, Lidia Giraudo, Chiara Donini, Erika Fiorino, Luca Primo, et al. 2021. ‘Recruitment, Infiltration and Cytotoxicity of Hla-Independent Killer Lymphocytes in Three-Dimensional Melanoma Models’. Cancers 13 (10): 1–19. https://doi.org/10.3390/cancers13102302.

Kisseberth W.C. & Lee D.A. 2021. Adoptive Natural Killer Cell Immunotherapy for Canine Osteosarcoma. Front Vet Sci, 8.

Laskowski T.J., Biederstädt A. & Rezvani K. 2022. Natural killer cells in antitumour adoptive cell immunotherapy. Nat Rev Cancer, 22, 557–575.

Mesiano G., Todorovic M., Gammaitoni L., Leuci V., Giraudo Diego L., Carnevale-Schianca F., et al. 2012. Cytokine-induced killer (CIK) cells as feasible and effective adoptive immunotherapy for the treatment of solid tumors. Expert Opin Biol Ther, 12, 673–684.

Rotolo, Antonia, Matthew J. Atherton, Brian T. Kasper, Kumudhini P. Haran, and Nicola J. Mason. 2021. ‘Genetic Re-Direction of Canine Primary T Cells for Clinical Trial Use in Pet Dogs with Spontaneous Cancer’. STAR Protocols 2 (4): 100905. https://doi.org/10.1016/j.xpro.2021.100905.

Todorovic M., Mesiano G., Gammaitoni L., Leuci V., Giraudo Diego L., Cammarata C., et al. 2012. Ex Vivo Allogeneic Stimulation Significantly Improves Expansion of Cytokine-Induced Killer Cells Without Increasing Their Alloreactivity Across HLA Barriers. J Immunother, 35, 579–586.

 

Publications

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