The key developed methodology includes the preparation of modified immune cells, and natural killer (NK) cells in particular. The method consists of two steps: step one employs the metabolic machinery of sialic acid to achieve incorporation of a modified sialic acid into cellular glycoconjugates. Step two is based on chemical labeling of cell surfaces using biocompatible chemical reaction. The modified sialic acid employed in metabolic labeling bears an unnatural chemical reporter group, which is used for attachment of different targeting moieties in the second chemical modification step. In the next round, the modified NK cells are studied in vitro and in vivo for their ability to kill cancer cells. In the course of the last 18 month, we have been working on all these steps. The major achievements can be summarized as follows.
1) we elaborated the synthesis of the key metabolic precursor (TCO-Sia), which is the sialic acid bearing unnatural clickable trans-cyclooctene group. This work included thorough optimization of several synthetic steps and full characterization of each intermediate and product.
2) we have explored the conditions that lead to efficient incorporation of TCO-Sia in glycoconjugates of various cell lines, including NK cells. Time, dose and growing conditions were studied. This experiments revealed that incubation of the cells for two days using 0.5-1 mM concentration is optimal for majority of cell lines tested.
3) Modification of the metabolically labeled cells was optimized and compared to other methodology known from literature (labeling based on azide-alkyne chemistry). Our novel modification procedure revealed that lower amounts of the modification reagent is needed to achieve comparable level of modification. In other words, our method based on the TCO-Tetrazine chemistry seems to be much more efficient, which has practical consequences such as the use of lower amounts of regents, which is economically and ecologically viable. Importantly, our toxicity studies did not show any undesirable effects on cell viability or their phenotype.
4) The chemically modified NK cells were intensively studied in killing experiments. Among different cancer cell lines tested, our work mainly focused on the use of HG3 (a CD20 positive Human B-cell chronic lymphocytic leukemia line) as target cells. Using clinically approved Rituximab as the targeting moiety on NK cells, we performed numerous studies to obtain the best killing effect. Overall, using the optimized conditions, we observed clearly enhanced killing efficacy of NK cells modified with the antibody, vs unmodified cells.
5) Using the above-mentioned model (NK cells modified with Rituximab and HG3 cancer cell model), we performed in collaboration with The Czech Centre for Phenogenomics, first in vivo test using NSG mice. These experiments were approved by the respective authorities (Spisová značka AVCR-S 620/2023 SOV II, evidenční číslo: 064/PP/SOVII/2023). These experiments confirmed results obtained during in vitro testing that NK cells modified with our methodology are better at killing cancer cells. This was reflected by the lower percentage of cancer cells present in the animals and their prolonged survival.
6) Progress of the whole project was intensively consulted with the Tech transfer office at the Institute, who mediated numerous consultations with experts in biotech or immunology. Moreover, several discussions with founders were initiated, which include i&i Prague and Holecek family foundation. In addition, we started new collaboration with a research group of Dr. J. Fucikova, who is an expert in immunology and is also affiliated to a biotech company Sotio Biotech a.s.
7) There is a patent pending, which is currently entering the national phase in US (PCT/CZ2022/050058). There are two publications in preparation and promising data for additional publications were generated within the last couple of month.
