Team:NMU China/Implementation
Overview
Want to know Who are our proposed end users? Target patients
Want to know How we envision others using your project? Using Method
Want to know How we implement our project in the real world? Implement
Want to know What are the safety aspects need to be considered? Safety Aspect
Want to know What other challenges need to be considered? Other Challenges
Using Envision
Theoretically, all the COVID-19 patients can be treated with Toggle Macrophages! As a "smart" therapy, Toggle Macrophages can both reduce the viral load through phagocytosis and balance the immune system via phenotypic transformation.
CARMERTK macrophage (Ms) phagocytose the virus while not upregulating the inflammatory response. Coronavirus disease 2019 (COVID-19) patients, especially severe cases, are our target groups. The ‘severe’ here means that those patients have a severe inflammation, accompanied by high virion load. And our product is most suitable for late-stage patients with increased levels of the following cytokines: IL-6, IL-8, TNF-α, IFN-γ and IL-10. (Fu Wenyan, Lei Changhai, Ma Zetong et al., 2021)
Most importantly, because CAR extracellular domain can be redesigned, Toggle macrophage can treat not only COVID-19, but all patients who may develop immune system disorders, especially cytokine storm in the future.
Regarding the using method, we have created the following flow chart to help you visualize the possible future clinical applications of Toggle Macrophages. The specific treatment process is as follows:
1.Patients are informed of the possible risks of treatment and sign informed consent forms.
2.Peripheral blood of patients is collected to detect the initial il-6 concentration (I0) in vivo, assess the physical condition of patients, and determine the range of IL-6 control ([L, U]).
3.Determine the number of engineered cells.
4.Peripheral blood mononuclear cell (PBMC) is extracted from Peripheral blood of patients/volunteers, primary human macrophage are cultured, and Toggle macrophages are produced.
5.Return n units of engineered cells.
6.The concentration of IL-6 in patients is detected regularly, and if the concentration is within the control range ([L, U]), the treatment continues.
7.If symptoms do not improve or even worsen, or il-6 concentration exceeds the control range, AP1903 is administered and treatment is terminated.
（Flow chart of Toggle Macrophage therapy）
Implement
Shortly after the outbreak of COVID-19, our team decided to provide a new therapeutic method for COVID-19 patients with a synthetic biology perspective.
Through interviews with doctors who supported Wuhan, we learned that the effective treatment is currently lackingl for the severe cases and supportive treatment occupies in the main position.
Through literature review and expert interviews, we know that SARA-CoV-2 is mild in the early stage. However, it induces the production of a large number of cytokines, including IL-6, IL-8, TNF-α, IFN-γ and IL-10 in the later stage, and that the intensity of inflammatory response is related to the prognosis.
In conclusion, eliminating SARS-COV-2 and balancing the immune response are both important for late-stage COVID-19, which prompted us to start our experiment.
（interview with doctors who supported Wuhan）
At the beginning, we plan to genetically arm human macrophages with chimeric antigen receptors (CARs). The designed synthetic antiviral macrophages, termed "Toggle Macrophages", have the phagocytic capacity specific for SARS-COV-2 and showed pro-inflammatory/anti-inflammatory phenotypic transformation according to the level of local IL-6 concentration. We have demonstrated its feasibility through in vitro experiments. (More details in Engineering Success )
We further verified the feasibility of "Toggle Macrophages" in vivo through modeling, so as to guide clinicians to grasp the number of returned cells. Through this model, when we input the serum IL-6 concentration I0 of patients and the IL-6 concentration level we want to control ([L, U]), we can obtain the number of engineered macrophages n that is most suitable for transfusion. In this context, "best fit" refers to the fastest and most stable control of IL-6 concentration within the range of clinical requirements. （More details in Model ）
Application Example of model
After the completion of the experiment, we visited clinicians to evaluate if CAR macrophages have clinical application prospects.
Professor Han reminded us that our reprogramed macrophages can be theoretically employed in the late stage of severe cases. However, further clinical trials are needed, which is still a long way to go.
Professor Dong believed that our project is theoretically feasible, but further animal experiments were still needed.
Professor Du reminded that polarization state is difficult to maintain in vivo and potential safety problems.
In short, the experts affirmed the theoretical feasibility of our project, but they all pointed out that our project still had a long way to go before clinical application.
（More details in Human Practice）
(More details in Human Practice )
（Interview with Professor Dong by Zhang Tiantian）
We interviewed several pharmaceutical representatives of biopharmaceutical companies, and all of them fully expressed their interest in our project and believed that our project has investment value. However, it requires us to further verify the safety of our program in humans.
In addition, I also investigated the commercial prospects of cellular immunotherapy in China and drew the following conclusions:
1. The commercial market for cellular immunotherapy is vast but brutal
CAR T plays a major role in cellular immunotherapy, and has been commercially used, including Fosun Kitt's Aquilunxel injection (Yicuda) and Minigolunxel Injection (Benoda), while CAR-M is still in the laboratory research stage.
Since the commercialization of Alkilunxel injection, the market size of CAR-T therapy in China will grow from 200 million YUAN in 2021 to 5.3 billion yuan, according to the analysis of Frost Sullivan, an internationally renowned consulting firm. At present, dozens of domestic enterprises have commenced CAR-T therapy, and many have entered the clinic.
However, the road to commercialization of CAR T is expected to be difficult due to the high cost of CAR T therapies, the concentration of drug targets and serious homogeneous competition.
2. The cost of CAR macrophage therapy is expected to be high
A dose of aquilunsai injection (Yi Kaida) from Fosun Kitt costs 1.2 million. Relmacabtagene autoleucel from JW Therapeutics has yet to be priced, with an estimated price of 1.3 million yuan.
The U.S. Food and Drug Administration (FDA) has currently approved five CAR-T therapies, including Novartis' Kymriah, Gilead Kitt's Yescarta and Tecartus, and Bristol-Myers Squibb's Breyanzi and AbecMA. The products are priced between $373,000 and $475,000 (2.48 million to 3.06 million yuan).
The cost of CAR-T is high because it is a personalized cell therapy. CAR T cells from each patient need to be produced in separate batches. The second reason is that the preparation process requires strict quality control and quality testing, and the whole process takes one month. The CAR-M therapy preparation process is broadly similar to CAR T, so we expect CAR macrophage therapy to be very expensive.
CAR macrophages belong to cellular immunotherapy. In order to understand the application environment of CAR macrophages in China, we conducted research on relevant laws and regulations.
Through data review, we found that China is actively promoting cellular immunotherapy. CAR T plays a major role in cellular immunotherapy, and has been commercially used, including Fosun Kitt's Aquilunxel injection (Yicuda) and Minigolunxel Injection (Benoda), while CAR-M is still in the laboratory research stage.
China has issued a number of relevant policies on cellular immunotherapy. For example, in the reply to the Proposal on Promoting the High-quality Development of China's Cell Industry issued by the National Health Commission in September 2020, it was clearly proposed to "jointly promote the development of cell industry". On February 9, 2021, the Drug Audit Center of the National Medical Products Administration organized and formulated the Technical Guidelines for Clinical Trials of Cellular Immunotherapy Products (Trial). In short, China is actively improving relevant laws and regulations to promote the clinical trials of cell therapy.
As for the medical insurance that people are most concerned about, neither of the two CAR-T models currently on the market in China has entered the basic medical insurance, and the high cost of about 1.2 million yuan needs to be fully borne by patients. Similarly, we do not believe that CAR-M therapy, even if successful, will enter the healthcare system in the near future.
（reply to the Proposal on Promoting the High-quality Development of China's Cell Industry issued by the National Health Commission）
According to National Health Commission of China Novel Coronavirus Laboratory Biosafety Guidelines (2nd edition), animal infection experiments should be carried out in the biosafety level-III laboratory. Our team's laboratory is only level-II, so relevant animal experiments cannot be carried out temporarily. If there is an opportunity to use a BSL-3 laboratory, we will seriously consider whether to carry out relevant animal experiments.
Many animal trials of CAR-M have been completed (Chen et al., 2021), and no ethical or safety issues have been reported. Mouse studies such as Santoni et al were conducted in accordance with national guidelines for the humane treatment of animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pennsylvania (Santoni et al., 2021.) If we apply for animal experiment, we believe it can pass the examination.
Although CAR-M therapy has been verified by animal experiments, the safety and effectiveness of CAR-M in human body still need to be verified.
As of November 2020, two clinical trials based on CAR-M strategies have received FDA approval. CARISMA Therapeutics' candidate cT-0508 (Phase I clinical trial), along with Maxyte's McY-m11, is currently recruiting volunteers for phase I clinical trials. Since both trials are still ongoing, we do not yet know how to evaluate the in vivo safety of CAR-M. We are aware that our product is still far from clinical trials. We are united in our hope that one day our product will get through clinical trials and provide a new option for COVID-19 patients.
Safety Aspect
We promise that we will not take any biological samples out of our BSL-2 laboratory，and our operators have accepted safety training. (See safety page for more information.)
Reliable source and expansion are the necessary conditions for the clinical application of CAR-M, which can be prepared by PBMC or by iPSCs. In addition, unlike T cells, macrophage has a low risk of GVHD, which means that products can be made in advance for patients to use on demand. (Chen et al., 2021)
CRS represents the major problem of CAR T therapies. Giulia’s analysis has not revealed new adverse drug reactions. (Giulia et al., 2021) Due to the Immunoregulation capability of Toggle macrophage, the possibility of cytokine storm is small.
As for Toggle Macrophage, we have demonstrated some safety aspects in vitro experiment.
1. Do not harm normal cells（Ms）
The phagocytic potential of human macrophage THP-1 cell lines expressing different CAR receptors or a truncated CAR receptor (CAR△) lacking the intracellular domain was measured with a cell-based assay. Consistent with previous reports (Morrissey et al., 2018), CAR macrophages and control untransduced (UTD) macrophages did not show notable phagocytosis of 293 cells; however, CARMEGF10, CARγ and CARz cells but not CARMERTK, CARD, or UTD macrophages phagocytosed Spike-bearing 293 cells in an S-specific manner (Figure 1).
Figure 1
CAR-mediated macrophage phagocytosis was further confirmed by a luciferase-based killing assay, and our data showed that CARMEGF10, CARg and CARz cells eradicated S protein expressing 293T cells in an antigen-specific manner (Figure 2). Interestingly, CARMERTK and UTD macrophages showed-no difference in killing effect.
Figure 2
The above two experiments suggest that CARMERTK macrophages (Ms) do not phagocytose or lyse 293T cells expressing S protein (Mimicking infected normal cells) under high inflammatory conditions. This feature is beneficial for COVID-19 patients suffering from cytokine storms, considering that the release of lysosomal inflammatory substances after cell membrane destruction leads to a strong inflammatory response. Of course, this property brings with it the problem of not being able to eliminate the infected normal cells, which is also a safety threat to patients. Our interpretation is that suppression of the inflammatory response is more urgent than elimination of the virus when cytokine storms occur. After the cytokine storm subsides, Ms switches to Mγ to quickly eliminate the virus.
2. Would not be infected
Although there is currently no evidence that SARS-CoV-2 can infect THP-1 cells with or without IgGs (Banerjee et al., 2020), THP-1 cells have been shown to support antibody-mediated enhancement of SARS-CoV infection in previous studies (Jaume et al., 2021). We therefore sought to determine whether synthetic receptors facilitate the entry of SARS-COV-2 into macrophages as host cells, as the extracellular domain of the CAR constructs has the capacity to directly bind to the S protein. We employed VSV pseudovirus bearing SARS-2-S (Kleine-Weber et al., 2019) to study the cell entry of SARS-CoV-2. Our data showed that Vero E6 cells were susceptible to entry driven by SARS-S (Figure 3); however, no evidence of infection was detected in THP-1 cells with or without synthetic receptors. Synthetic receptors did not facilitate the entry of SARS-COV-2 into macrophages as host cells.
Figure 3
Although Toggle Macrophage is considered safe in vivo, a final safety catch is still needed. This way, the Toggle Macrophage can be rapidly eliminated in vivo. An excellent candidate for this inducible kill switch is the AP1903 described by Clackson, et al., 1998, as AP1903 has no other biologic effects in vivo. (Straathof, K. C., et al.,2016) ICasp9 can be activated by AP1903 that has proven safe at the required dose for optimal eradicating effect. (Iuliucci, J. D., et al.,2001) (More details in Safety )
We have verified that Toggle Macrophage can regulate immune response in a bidirectional way by in vitro experiments. However, the environment in vivo is often complex. So we established a model to quantitatively analyze the cytokine levels that Toggle Macrophage can control. The model can tell us that the concentration range of IL-6 in patients can be greatly improved by importing a certain amount of macrophages, which greatly improves the safety of Toggle Macrophage.
Other Challenges
We have not been able to obtain live SARS-CoV-2 due to the domestic epidemic control. Since we could not acquire the live virus, we used replication-defective VSV Particles. Though pseudovirus bearing Coronavirus S proteins could reflect key aspects of host cell entry by SARS-CoV-2, it should be noted that our study used very simple infection models.
Similarly, the network of inflammatory responses in vivo is so complex that we cannot simulate the true inflammatory effects of CAR-M. Professor Dong reminded us that the practical anti-inflammatory efficiency of the CARMERTK macrophages is what really matters.
Professor Li warned us that the inflammation reaction is not only initialed by the virus, but also by the cell debris of the apoptotic alveolar epithelial cells. He suggested that we should pay more attention to the cell debris mediated pathway of inflammation activated by SARS-CoV-2.
In addition, since our project could not get out of the laboratory, we mentioned three challenges that we imagined we might encounter clinically.
At the early stage of novel coronavirus infection, the body starts mainly the nonspecific immune response. At this time, the phagocytes play a dominant role. But as the viral load gradually rises, the body mainly activates humoral immune response by producing antibodies to neutralize the virus. At this time, the macrophage is not the main force in eliminating the virus, and its anti-inflammatory effect by secreting inhibitory cytokines is limited.
Silent Macrophage can not only inhibit inflammation, but also promote scar tissue repair. The generation of lung scar can hinder lung ventilation and ventilation, resulting in impaired lung function.
Reference
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2.Fu Wenyan., Lei Changhai., Ma Zetong., Qian Kewen., Li Tian., Zhao Jian., Hu Shi.(2021). CAR Macrophages for SARS-CoV-2 Immunotherapy. Front Immunol, 12(undefined), 669103. doi:10.3389/fimmu.2021.669103
3.Morrissey, M. A., Williamson, A. P., Steinbach, A. M., Roberts, E. W., Kern, N., Headley, M. B., & Vale, R. D. (2018). Chimeric antigen receptors that trigger phagocytosis. eLife, 7. doi:10.7554/elife.36688
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8.Santoni, M. , et al. "Re: Human Chimeric Antigen Receptor Macrophages for Cancer Immunotherapy." European Urology (2021).
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10.Iuliucci, J. D. , et al. "Intravenous Safety and Pharmacokinetics of a Novel Dimerizer Drug, AP1903, in Healthy Volunteers." The Journal of Clinical Pharmacology 41.8(2001).
11.https://clinicaltrials.gov/ct2/show/study/NCT04660929?term=04660929&draw=2&rank=1
12.https://clinicaltrials.gov/ct2/show/record/NCT03608618?term=03608618&draw=2&rank=1