Periodic Reporting for period 1 - CHEMCELL (Chemical Engineering of Natural Killer Cells for Cancer Immunotherapy)
Reporting period: 2022-09-01 to 2024-02-29
Our immune system represents a powerful defense mechanism against various invaders and diseases. Immune cells employ intricate strategies to identify and eliminate abnormal cells while preserving our own cells. However, cancer cells often employ cunning tactics to evade this protective machinery, allowing them to thrive and multiply unchecked.
Immunotherapy, a promising treatment paradigm, seeks to harness the immune system's power to combat cancer by either enhancing its activity or by training immune cells. Among these approaches, adoptive cell transfer therapy, particularly in the form of chimeric antigen receptor (CAR) cells, has demonstrated remarkable success in treating specific cancer types.
Despite its efficacy, the production of genetically modified CAR cells poses significant challenges. The process is marred by inefficiency, time consumption, and high costs. Thus, there is a pressing need for novel methodologies that can expedite and streamline the production of modified immune cells, potentially revolutionizing therapy protocols.
This proposal endeavors to explore the feasibility of an innovative approach for generating chemically engineered immune cells and deploying them in immunotherapy. Leveraging a combination of metabolic engineering and biocompatible chemical labeling, we aim to develop antibody-modified natural killer (NK) cells for use in cancer-killing experiments.
Through comprehensive in vitro and in vivo assessments, we will conduct a thorough comparative analysis between our technology and traditional CAR cells. This evaluation will elucidate the respective advantages and limitations of each approach.
Furthermore, in collaboration with our Tech Transfer Office, we will try to introduce this groundbreaking technology to potential stakeholders. This may involve pursuing avenues such as patent licensing or establishing a spin-out company to further develop and commercialize the technology, thus advancing its translation into clinical practice.
Immunotherapy, a promising treatment paradigm, seeks to harness the immune system's power to combat cancer by either enhancing its activity or by training immune cells. Among these approaches, adoptive cell transfer therapy, particularly in the form of chimeric antigen receptor (CAR) cells, has demonstrated remarkable success in treating specific cancer types.
Despite its efficacy, the production of genetically modified CAR cells poses significant challenges. The process is marred by inefficiency, time consumption, and high costs. Thus, there is a pressing need for novel methodologies that can expedite and streamline the production of modified immune cells, potentially revolutionizing therapy protocols.
This proposal endeavors to explore the feasibility of an innovative approach for generating chemically engineered immune cells and deploying them in immunotherapy. Leveraging a combination of metabolic engineering and biocompatible chemical labeling, we aim to develop antibody-modified natural killer (NK) cells for use in cancer-killing experiments.
Through comprehensive in vitro and in vivo assessments, we will conduct a thorough comparative analysis between our technology and traditional CAR cells. This evaluation will elucidate the respective advantages and limitations of each approach.
Furthermore, in collaboration with our Tech Transfer Office, we will try to introduce this groundbreaking technology to potential stakeholders. This may involve pursuing avenues such as patent licensing or establishing a spin-out company to further develop and commercialize the technology, thus advancing its translation into clinical practice.
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.
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.
ChemCell provide faster, cheaper and safer way of producing immune cells for therapy. There are several aspects of our work that span beyond what is currently available. For example, our developed methodology represents a straightforward approach for attaching diverse functional moieties to cell surfaces, and that by using fast chemical reactions that do not require catalyst or an enzymes. It allows decoration of cellular surfaces with groups that are not present at cell surfaces naturally. Such artificial functional groups can endow cells with completely new properties and functions.
Our application of the methodology in cancer immunotherapy is also new, and represents novel opportunity to produce modified cells for therapeutic use.
Our work is also interesting regarding the underlying principles leading to enhanced cytotoxic effect of NK cells that we observe. Fundamentally, this opens up the question what kind of killing mechanism NK cells modified with targeting moieties using chemical tools use? How the sole targeting moiety and subsequent connection between target and effector cells triggers cytotoxic response? These and similar question may lead to new findings about how immunity works and which mechanisms are involved in immune responses. Answering these questions may lead to the development of novel therapeutic approaches.
Our application of the methodology in cancer immunotherapy is also new, and represents novel opportunity to produce modified cells for therapeutic use.
Our work is also interesting regarding the underlying principles leading to enhanced cytotoxic effect of NK cells that we observe. Fundamentally, this opens up the question what kind of killing mechanism NK cells modified with targeting moieties using chemical tools use? How the sole targeting moiety and subsequent connection between target and effector cells triggers cytotoxic response? These and similar question may lead to new findings about how immunity works and which mechanisms are involved in immune responses. Answering these questions may lead to the development of novel therapeutic approaches.