PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Supervisor

Raffaella De Maria

Phd thesis

In vitro assessment of Natural Killer-like T Cells as immune cell-based therapy in the treatment of Canine Malignant Melanoma

Scientific Background

In recent years, adoptive cell therapy^a has been a remarkable therapeutic strategy to fight cancer, showing important results in antitumoral research. Regarding this field, an emerging approach is adoptive Natural Killer (NK) cell therapy, based on the usage of ex vivo^b expanded NK-like T cells capable of antitumor activity.

Briefly, when identifying ligands induced by stress, such as malignant transformation, NK cells carry strong cytotoxicity by the release of cytokines¹. Also, their activity does not depend on the recognition of Major Histocompatibility Complex (MHC) molecules, that usually present antigens. This allows them to bypass tumour immune escape mechanisms caused by MHC downregulation, assuring the attack of abnormal self-cells¹.

Additionally, NK cell therapy has shown many results in human cancer treatments, with its scope recently extending to solid tumours², besides previous success for leukaemia and lymphomas. Concerning the veterinary field, NK cells have still show modest advance in solid tumours treatment³.

Dogs are the ideal candidates to be tested next because they share similar tumour biology with humans, which already contain valuable literature for cell-based therapies. Also, cross-species data have shown similarities between dog and human NK cells^3,4. Melanomas are a suitable choice of tumours to be evaluated, and have already been targeted by human researchers, who reported NK cells showing a good cytotoxic response against this type of tumour^5–9.

 

Goals

The aims of this project include the evaluation of expansion methods for canine T cells and their activation into Cytokine-Induced Killer (CIK) cells, that consist of polyclonal ex vivo expanded NK-like T cells. After that, CIK cells' persistence ex-vivo and anti-tumour response will be assessed by their killing rate with respect to control samples, including CIK-only and Target-only cell cultures. Finally, I plan to examine the selectiveness and cytotoxicity of the CIKs in 5 allogenic and 10 autogenic samples from Canine Malignant Melanomas. By that, it is intended to establish future guidelines regarding cell-based immunotherapy. 

 

Methods

The standard protocol for the ex vivo expansion of CIK cells includes the culture of peripheral blood mononuclear cells (PBMC) and activation by specific cytokines. The PBMCs are isolated from the blood of the patients by Ficoll separation. Then, the activation is driven by the addition of interferon-gamma (IFN-γ), interleukin 2 (IL-2) and microbeads with anti-CD28 and anti-CD3 antibodies¹⁰. This choice of cytokines serves to achieve a NK-like phenotype. Then, the expanded NK cells can be used as effectors immediately or cryopreserved for later use.

Tumoral cell lines will be grown in cultures with specific cell medium, 10% of Foetal Bovine Serum (FBS), 1% of antibiotics and 1% of L-glutamine, under incubation in 38^oC. After target cells growth, they are merged with different concentrations of effector cells, added to each well of a multi-well plate by serial dilution and co-cultured for three days. Then, anti-tumoral response will be evaluated by CellTiter-Glo Luminescent Cell Viability Assay and also Flow Cytometry, assessing CIK cells' potency towards tumoral tissue grown ex-vivo.

Statistical analysis and descriptive statistics will be performed in R-Studio environment. The target cells viability for each proportion of target cells per effector cells will be normalised to the values obtained from untreated tumoral cells that have not been incubated with the CIK cells. Additionally, the values from wells with CIK cells-only will be subtracted from the obtained values.

 

Expected Results

Solid tumours such as melanomas are still a challenge concerning immunotherapies, mostly because they exhibit a complex micro environment that intercepts immune activity. The success of this approach using NK-like T cells could be a breakthrough in that field, allowing a higher level of choices for possible cancer therapies, and developing a superior status of quality, efficiency, and safety in veterinary medicine.

Because in vitro cultures do not completely replicate the tumoral environment of a living body, this project still has limitations concerning its viability as a treatment. However, dogs demonstrate great similarity to human patients, who have shown encouraging results with reference to adoptive NK cell therapy. This envisages a favourable outcome for the presented project, which could endorse future clinical studies of CIK cell therapy on dogs affected by Malignant Melanoma.

 

a. Adoptive cell therapy: isolation of native immune cells to be expanded and then transferred to a patient as treatment.

b. Ex vivo samples: samples that have been collected from an organism and will be tested outside of it, maintaining similar conditions to the original tissue.

 

References

1. Laskowski, T. J., Biederstädt, A. & Rezvani, K. Natural killer cells in antitumour adoptive cell immunotherapy. Nat Rev Cancer 22, 557–575 (2022).
2. Mesiano, G. et al. Cytokine-induced killer (CIK) cells as feasible and effective adoptive immunotherapy for the treatment of solid tumors. Expert Opin Biol Ther 12, 673–684 (2012).
3. Gingrich, A. A. et al. Comparative Immunogenomics of Canine Natural Killer Cells as Immunotherapy Target. Front Immunol 12, (2021).
4. Gingrich, A. A., Modiano, J. F. & Canter, R. J. Characterization and Potential Applications of Dog Natural Killer Cells in Cancer Immunotherapy. J Clin Med 8, 1802 (2019).
5. Filin, I. Y. et al. Cell Immunotherapy against Melanoma: Clinical Trials Review. Int J Mol Sci 24, (2023).
6. Li, H. et al. Selective effect of cytokine-induced killer cells on survival of patients with early-stage melanoma. Cancer Immunology, Immunotherapy 66, 299–308 (2017).
7. Gammaitoni, L. et al. Cytokine-induced killer cells kill chemo-surviving melanoma cancer stem cells. Clinical Cancer Research 23, 2277–2288 (2017).
8. Kim, J. S. et al. Adoptive Cell Therapy of Melanoma with Cytokine-induced Killer Cells. Immune Netw 15, 58 (2015).
9. Iaia, I. et al. Recruitment, infiltration and cytotoxicity of hla-independent killer lymphocytes in three-dimensional melanoma models. Cancers (Basel) 13, 1–19 (2021).
10. Rotolo, A., Atherton, M. J., Kasper, B. T., Haran, K. P. & Mason, N. J. Genetic re-direction of canine primary T cells for clinical trial use in pet dogs with spontaneous cancer. STAR Protoc 2, (2021).

 

 

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