Overview
Target patients: Who are our proposed end users?
Therapy procedure: How do we envision others using the project?
Implementation: How would we implement the project in the real world?
Safety aspects: What are the safety aspects we would need to consider?
Other challenges: What other challenges would we need to consider?
Future prospect
Target patients
Cancer patients, especially those with solid tumors, are our main target patients. As we have described, our CAR-NK92 library is a novel therapy based on chimeric antigen receptor technology and antibody library technology, inspired by the cloning selection theory. Not only would it avoid side effects like CRS and GVHD, but also tackle the tumor heterogeneity problem. Originated from the immune system of healthy human bodies, our CAR library is diverse enough to fight against almost all kinds of tumor antigen libraries, even a rapidly changeable one.
Actually, due to the Astonishing diversity of our CAR-NK92 library, the target patients can be anyone who suffers because of foreign antigens or neoantigens in theory. We sincerely hope our project can contribute to Human Health, especially in the fight against tumors.
Therapy procedure
A general therapy procedure is shown as follows and we also make a flow chart to help you have a better understanding of it.
1. Draw 200 copies of peripheral blood from healthy volunteers.
2. Extract mRNAs encoding antibodies in B cells and use RT-PCR and SOE-PCR to form a scFv cDNA library.
3. Select leukocytes from volunteers on a large scale and obtain their HLA antigens as material for negative selection.
4. After negative selection, reserve the negative ones to form the final library that can be put into use.
5. Before the patient is treated, confirm that the scFv library has no affinity to the patient's HLA antigen.
6. Construct CAR-NK92 cells carrying the scFv library and infuse them into the patient for treatment.
What calls for special attention is that our CAR-NK92 library therapy can also be personalized as long as we perform a negative selection for each patient and construct a personalized negative library.
Implementation
Step1 previous investigation
We administered a questionnaire to the general public, especially patients and patient’s families, focusing on the problems encountered during tumor treatment. We revealed that cancer recurrence is widely concerned and is one of the most troubling problems for patients.
Step2 doctor consultation
We consulted Dr.Wang at Shanghai Xinhua Hospital and learned that one of the most tricky challenges to overcome in tumor treatment is tumor heterogeneity, which accounts for tumor recurrence and the limited effectiveness of current cellular therapy and monoclonal antibody therapy. We became interested and tried to tackle this problem.
Q: What is the core challenge of tumor treatment today?
Dr.Wang: In clinical practice, tumor recurrence is one of the core difficulties in tumor treatment, quite troublesome for patients. Tumor heterogeneity accounts for recurrence to a large extent, also responsible for the limited effectiveness of current cellular therapy and monoclonal antibody therapy.
Step3 cellular experiments
We performed cellular experiments, which verified the specific killing effect of CAR-NK92 cells and cell dynamic growth with the kill switch circuit. (Detailed information in Engineering Success and experiments)
Step4 expert inquiry
To further improve the project, we interviewed Professor Guo, who affirmed our current progress and pointed out that CARs shouldn’t be pre-defined, and we should improve them to deal with unpredictable neoantigens. So we modeled to simulate and visualize the condition and got positive results.
Step5 modeling
In modeling, we did the following work: (Detailed information in MODEL)
1.We demonstrated the limitations on the number and types of CARs that can be expressed in a single cell. Therefore, we choose to express multiple CARs on a large number of immune cells.
2.We tried to simulate expressing multiple CARs on a large number of immune cells and we built three models:
1)CAR-NK92 suicide gene pathway feasibility model
2)AP1903 pro-apoptosis model
3)CAR-NK92 proliferation model
3.We verified the coverage of the antigen library by the antibody library.
4.We finally performed cellular automata visualization simulation.
Step6 communication with other teams(throughout the project)
1)CUPK invited Professor Jingying Zhou from CUPK to evaluate our project.
2)ZJUintl-China and we helped each other on project optimization.
3)ZJUintl-China pointed out that when we modeled to assure that CAR-NK library injection would not damage normal cells, we should screened out not only highly expressed genes, but membrane-located ones in cancer cells to be specific targets. With their help, we used UniProt webscraping and addressed the problem.
4)We cooperated with six teams, CPU-China, TJUSLS-China, HiZJU-China, ZJU-China, ZJUT-China and ZJUintl-China to make the atlas of synthetic biology.
More communication can be found in , Partnership, Inclusivity.
Safety concerns
Experimental safety
The Biosafety Law of the People's Republic of China was adopted at the 22nd Meeting of the Standing Committee of the 13th National People's Congress on October 17, 2020. The experiments of NMU-China iGEM were carried out under the premise of complying with relevant laws.
Safe Lab Work
Strict laboratory safety training is necessary. After understanding IGEM safety rules, we invited our advisor Shi Hu to the laboratory to conduct safety education for all members. We required each member to strictly abide by the safety guidelines of the laboratory and the school. We treat all biological materials, waste and equipment in strict accordance with BSL-2 requirements.
Blood transfusion from volunteers (future prospect)
The source of our scfv library is peripheral blood from around 200 healthy volunteers. The procedure is similar to blood donation, harmless to the human body.
Negative selection (future prospect)
To construct an off-the-shelf library, we will conduct a negative selection and reserve the negative ones to form the final library. The material for negative selection is HLA antigens, also from volunteers, but in a much greater number. For convenience and safety, HLA antigens are extracted from leukocytes, which can also be acquired through blood drawing, harmless to the human body. Then a human HLA library will be constructed by HLA antigens from a large number of volunteers. We will knock out those scfvs which have a high affinity to the human HLA library and reserve the negative ones to form the final library. Theoretically, the final library is an off-the-shelf one as long as the quantity of HLA volunteers is large enough to roughly represent humankind.
The reason why we choose HLA antigens (future prospect)
The largest antigenic difference between individuals is HLA antigens, and cross-matching is always performed before the organ transplantation to prevent mutual attacking heterologous antigens (mainly HLA antigens) between individuals. This phenomenon can also be avoided with the negative selection of HLA antigens on a large scale in our project. And in order to strictly exclude the binding of the scFv library to the patient's HLA antigens, it will be confirmed again by experiments before treatment.
NK-92 cell line
NK-92 MI cell line was purchased from American Type Culture Collection (ATCC) and identified by short tandem repeat sequence analysis. Clinical trials have shown that NK-92 infusion is safe even at high doses(Suck et al., 2016). Irradiation is usually needed before using the NK-92 cell line for safety concerns(Klingemann et al., 2016). Here we use a kill switch circuit controlled by exogenous AP1903 to serve as an alternative way to control the population for safety.
AP1903
As a dimerizer agent, AP1903 has been widely used to serve as the exogenous inducer of a safety switch to improve the safety of CAR-T(Amatya et al., 2021). A study has shown that AP1903 was safe and well tolerated when administered to healthy male volunteers at dose levels up to 1 mg/kg over a 2-hour infusion period(Iuliucci et al., 2001). After performing literature review and modeling, we estimated the suitable concentration of AP1903 for the human body in our project is around 0.4-0.6 mg/kg, which is within the safety range.
Lentivirus carrier
Lentivirus packaging kit was purchased from Shanghai Ji kai Ji Yin Chemical Technology Co., LTD. Lentivirus vectors including pCDH-iCAS9-KRAB lentivirus vector and pCDH-scFv-CAR lentivirus vector were used to transfect NK-92 cells to get targeted role and has the suicide lines of NK cells. Studies have shown that lentivirus vector have important biological safety characteristics(Schambach et al., 2013), its application in the body is relatively safe. The potential risks from lentiviral vectors are dependent upon the nature of the exposure. Aerosol exposures through droplet transmission are another potential route of lentiviral vector exposure(Schlimgen et al., 2016). And the non-specific nature of transgene integration by the viral integration machinery carries an inherent risk for genotoxicity(Schenkwein et al., 2020). When operating lentiviral vector, we will use commercial Lentiviral vector production kit in a biosafety class 2 cabinet, strictly following its protocols and procedures. Lentivirus operations that do not involve animal testing in a biosafety cabinet (BSL-2 level) are considered sufficient by the UK ACGM guidelines.
CAR-NK92
Compared to CAR-T therapy, CAR-NK92 cells will not cause graft-versus-host reaction (GVHD) and secrete inflammatory factors that cause CRS. Also, as the NK-92 cell line can be purchased and cultured, CAR-NK92 therapy is more likely to be an off-the-shelf one than CAR-T.
In our cellular experiments, we have demonstrated some safety aspects of our CAR-NK92 cells.
1)Specific killing effect: CAR-NK92 cells only kill specific cells with corresponding antigens, harmless to other cells.
2)Feasibility of the kill switch circuit: CAR-NK92 cells with no targets can be induced apoptosis by exogenous AP1903 to ensure safety, as we have mentioned above.
Pandemic Prevention and Control
It was still a special time when the COVID-19 is raging, so we stipulated that our members must wear masks when entering the laboratory and disinfect their hands before and after the experiment to avoid contracting COVID-19. We've been granted a safe, healthy environment on campus to conduct our experiments.
Other challenges
Genetic mutations (future prospect)
Although we designed a kill-switch circuit for NK92 cells for safety concerns, we still wonder if the genetic mutations would be troublesome.
The proliferation rate of NK92 is much higher than normal cells, so the possibility of mutation is correspondingly larger. If the kill-switch circuit fails to work due to the mutation, it will be tricky. To address this problem, we need to estimate the risk potential and design remedial measures or preventive measures in our future work.
The number of volunteers (future prospect)
As we have mentioned above in therapy procedure, if we want to construct an off-the-shelf library, we should select leukocytes from volunteers on a large scale and obtain their HLA antigens as material for negative selection.
In clinical practice, the rate of HLA matching for unrelated individuals is approximately 1 in 100,000. So the quantity of volunteers is at least 100,000 to meet our satisfaction, which may be hard to carry out.
For this issue, the personalized library based on individual negative selection is also feasible in our project to serve as the alternative solution.
Reference
1. Suck, G., Odendahl, M., Nowakowska, P., Seidl, C., Wels, W. S., Klingemann, H. G., & Tonn, T. (2016). NK-92: An “off-the-shelf therapeutic” for adoptive natural killer cell-based cancer immunotherapy. Cancer Immunology, Immunotherapy: CII, 65(4), 485–492. https://doi.org/10.1007/s00262-015-1761-x
2. 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
3. Amatya, C., Pegues, M. A., Lam, N., Vanasse, D., Geldres, C., Choi, S., Hewitt, S. M., Feldman, S. A., & Kochenderfer, J. N. (2021). Development of CAR T Cells Expressing a Suicide Gene Plus a Chimeric Antigen Receptor Targeting Signaling Lymphocytic-Activation Molecule F7. Molecular Therapy: The Journal of the American Society of Gene Therapy, 29(2), 702–717. https://doi.org/10.1016/j.ymthe.2020.10.008
4. Iuliucci, J. D., Oliver, S. D., Morley, S., Ward, C., Ward, J., Dalgarno, D., Clackson, T., & Berger, H. J. (2001). Intravenous safety and pharmacokinetics of a novel dimerizer drug, AP1903, in healthy volunteers. Journal of Clinical Pharmacology, 41(8), 870–879. https://doi.org/10.1177/00912700122010771
5. Schambach, A., Zychlinski, D., Ehrnstroem, B., & Baum, C. (2013). Biosafety features of lentiviral vectors. Human Gene Therapy, 24(2), 132–142. https://doi.org/10.1089/hum.2012.229
6. Schlimgen, R., Howard, J., Wooley, D., Thompson, M., Baden, L. R., Yang, O. O., Christiani, D. C., Mostoslavsky, G., Diamond, D. V., Duane, E. G., Byers, K., Winters, T., Gelfand, J. A., Fujimoto, G., Hudson, T. W., & Vyas, J. M. (2016). Risks Associated With Lentiviral Vector Exposures and Prevention Strategies. Journal of Occupational and Environmental Medicine, 58(12), 1159–1166. https://doi.org/10.1097/JOM.0000000000000879
7. Schenkwein, D., Afzal, S., Nousiainen, A., Schmidt, M., & Ylä-Herttuala, S. (2020). Efficient Nuclease-Directed Integration of Lentivirus Vectors into the Human Ribosomal DNA Locus. Molecular Therapy: The Journal of the American Society of Gene Therapy, 28(8), 1858–1875. https://doi.org/10.1016/j.ymthe.2020.05.019