Our Project
1.Our Ultimate Goal
The ultimate goal of our project is to solve the problem of tumor heterogeneity.
2.Why our project is a “whack-a-mole” game?
For malignant tumors, the mainstream treatment methods are surgical resection, chemotherapy, radiotherapy, molecularly targeted therapy and adoptive cellular immunotherapy. For early-stage cancers, especially solid cancers, such as gastric cancer, lung cancer, etc., surgical resection is the gold standard of therapy. However, for advanced-stage cancers, resection may be no longer suitable due to the spread and metastasis of lesions.In these cases, Chemoradiotherapy, molecularly targeted therapy or other therapies should be applied. But all these maneuvers have a fatal drawback - tumor tolerance. In other words, we cannot eliminate all the tumor cells at one time, thus resulting in the recurrence of tumors.
The reason for this is that tumors are heterogeneous.
Scientists generally agree that the process of tumorigenesis and development can be regarded as a process of evolution: in most cases, the appearance of malignant tumors leads to Uncontrollable cell proliferation. At the same time, the genetic instability of tumor cells, the tumor microenvironment and other factors all provide plenty of opportunities for the generation of multiple mutants. They start from normal cells and end up with billions of malignant tumor cells, which have accumulated a large amount of mutations in the process. These mutations are the raw materials of tumor evolution. Tumor evolution result in dramatic differences on the expression of proteins, the proliferation rate and angiogenic potential between subspecies of tumor cells.
Due to the existence of tumor heterogeneity, the ever-emerging subspecies of tumor cells are just like the gophers. We try to hit them with the hammer over and over again, but it is often in vain. Gophers keep emerging from the earth, and the small hammer we have is no longer able to fight the huge number of gophers!
So here comes the question: How can we get rid of these stubborn gophers (tumor cells) completely?
That's right! We need a bigger, more powerful hammer!
The antibody library technology laid the foundation for the advent of the hammer: According to the clonal selection theory and antibody diversity, for each tumor antigen, there are always corresponding antibodies existing and able to bind to it. Antibody library technology is a technique that all antibody variable region genes of a certain species are cloned and expressed in plasmids or phages, and we can also using different antigens to select the corresponding antibody gene clones.
So, when we get this huge number of antibodies, we have a sledgehammer which can hit all the gophers simultaneously! This kind of treatment eliminates all the tumor cells and brings about a long-lasting immune response.
3.Why do we choose NK cells instead of the T cells which are commonly used as our chassis?
Now that we have the sledgehammer, we need to search for someone who can wield it. Our project combines the antibody library technology with CAR therapy. Compared to conventional treatments, CAR therapy is more lethal to tumor cells, and more accurate and longer-lasting, exerting powerful anti-tumor activity. Currently, CAR-T therapy has made great progress in the field of hematology oncology and T cells are the most commonly used chassis cells. However, using T cells as chassis has fatal drawbacks on safety and performs poorly in certain conditions, which are described as follows:
1.CRS (cytokine release syndrome)
2.poor results in solid tumors (due to lack of specific antigens, poor tumor infiltration, inhibition of tumor microenvironment )
3.off-target effect
Therefore, we need safer and more powerful immune cells to serve as our chassis. After literature review, we found that: NK cells are an important tumor-killing immune cell, and compared to CAR-T therapy, NK cells have many excellent properties as classis cells.
1. Allogeneic NK cells do not cause graft-versus-host reaction (GVHD), which has been confirmed in many clinical trials.
2. NK cells do not secrete inflammatory factors that cause CRS, such as IL-1 and IL-6.
3. In addition to CAR-mediated killing, CAR-NK cells can also identify and kill tumor cells in a missing-self way to improve the immunotherapeutic effect.
4. Allogeneic NK cells have a wide range of sources, including peripheral blood, NK cell lines, UCB (umbilical cord blood), iPSC (induced pluripotent stem cells), NK-92 and other cell lines. And T cells used for CAR-T mostly originate from the patients themselves or human healthy donors.
However, the proliferative rate of NK cells does not meet our requirements. Therefore, we put our eyes on NK-92 cells. Derived from tumors, NK-92 cells have a considerable proliferative rate. At the same time, NK-92 cells do not need to be extracted from the patients and are more convenient to be cultivated and genetically modified. The modified cells have the potential to be applied in homologous allogeneic transfer, promising to be a truly off-the-shelf product. This advantage further shows the superiority of NK-92 cells over T cells and other immune cells in this project.
4.How can we ensure that our CAR-NK cells are safe and effective?
As mentioned above, we plan to use NK-92 cell line as chassis. As a tumor-derived cell line, its powerful proliferation rate is a double-blade sword in our project. On the one hand, we want to take advantage of its rapid proliferation capacity; on the other hand, we are trying to avoid excessive proliferation in the human body, which is detrimental to therapeutic efficacy and the normal immune environment.
Usually, NK-92 cells need to be irradiated before injection or using to diminish its proliferation rate. However, irradiation is not stable, and it also poses threat to safety. So we developed a kill switch circuit to serve as an alternative maneuver to ensure the safety and controllability in vivo.
Our design mainly consists of two main modules: CAR and kill switch circuit. If CAR-NK92 cells recognize the corresponding antigens, they initiate a series of downstream pathways and finally inhibit the expression of iCASP9. But if CAR-NK92 cells fail to recognize the antigens, the iCASP9 is synthesized with no inhibition, which dimerizes in the presence of AP1903, leading to apoptosis of the cells. Thus, CAR-NK92 cells that recognize tumor antigens continue to proliferate and kill tumor cells, while the rest will be induced apoptosis after we inject AP1903, thus ensuring the safety and efficacy of the treatment.
Our Inspiration
CAR-T therapy has made great progress in the field of hematology-oncology. However, relying on specific targets determines that it can never overcome the challenge from tumor heterogeneity. So how to develop a new strategy to tackle tumor heterogeneity became the focus of our project.
Through an article, we found that there were already relevant studies in the field of CAR-T dealing with tumor heterogeneity through a three-target approach(Schneider et al., 2021). Thus, we envisioned whether we could construct a group of CAR-immune cells that aim for more targets simultaneously.
We tested and ruled out the primary plan① to express multiple CARs on a single cell through modeling. So we took our plan② to emulate how the immune system works in the normal body by constructing multiple cells expressing different CARs to form a CAR-immune cells library.
Our Project Goals
Our project aims to develop a new immunotherapeutic strategy to tackle tumor heterogeneity so that tumor cells have no place to hide. Also, we point out a new direction in tumor therapy through the concept of antibody library proposed in our project. This is the original purpose of our consistent participation in the iGEM therapeutic track for years: to make a modest contribution to human health and to be worthy of the Hippocratic oath. In the course of our exploration, we have conducted following works:
1. Conduct a review of current tumor therapeutic strategies, especially chimeric antigen receptor therapies, and find that the key point lies in tumor heterogeneity.
2.Propose the idea of a single cell expressing multiple CARs and rule it out by modeling.
3.Propose the idea of different cells expressing different CARs.
4.Review the current selection of CAR chassis cells and determine NK92 as the most suitable one in our project.
5.Design a genetic circuit to achieve the goal that CAR-NK92 cells that recognize tumor antigens are allowed to proliferate while the rest are induced apoptosis, and determine the feasibility of the circuit through cellular experiments.
6. Simulate the in vivo anti-tumor effect of the 10^6 CAR-NK92 library by modeling.
7. Consult with experts in the field of immunology and synthetic biology to evaluate the feasibility of this project and receive suggestions for improvement.
8. Investigate the view of stakeholders about our project and determine the future prospects.
Reference
1. Feng, Y., Liu, X., Li, X., Zhou, Y., Song, Z., Zhang, J., Shi, B., & Wang, J. (2021). Novel BCMA-OR-CD38 tandem-dual chimeric antigen receptor T cells robustly control multiple myeloma. Oncoimmunology, 10(1), 1959102. https://doi.org/10.1080/2162402X.2021.1959102
2. Fedorov, V. D., Themeli, M., & Sadelain, M. (2013). PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses. Science Translational Medicine, 5(215), 215ra172. https://doi.org/10.1126/scitranslmed.3006597
3. Giordano-Attianese, G., Gainza, P., Gray-Gaillard, E., Cribioli, E., Shui, S., Kim, S., Kwak, M.-J., Vollers, S., Corria Osorio, A. D. J., Reichenbach, P., Bonet, J., Oh, B.-H., Irving, M., Coukos, G., & Correia, B. E. (2020). A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy. Nature Biotechnology, 38(4), 426–432. https://doi.org/10.1038/s41587-019-0403-9
4. Schneider, D., Xiong, Y., Wu, D., Hu, P., Alabanza, L., Steimle, B., Mahmud, H., Anthony-Gonda, K., Krueger, W., Zhu, Z., Dimitrov, D. S., Orentas, R. J., & Dropulić, B. (2021). Trispecific CD19-CD20-CD22-targeting duoCAR-T cells eliminate antigen-heterogeneous B cell tumors in preclinical models. Science Translational Medicine, 13(586), eabc6401. https://doi.org/10.1126/scitranslmed.abc6401
5. Klingemann, H., Boissel, L., & Toneguzzo, F. (2016). Natural Killer Cells for Immunotherapy – Advantages of the NK-92 Cell Line over Blood NK Cells. Frontiers in Immunology, 7, 91. https://doi.org/10.3389/fimmu.2016.00091
6. Burnet, F. M. (1970). The concept of immunological surveillance. Progress in Experimental Tumor Research, 13, 1–27. https://doi.org/10.1159/000386035