iGEM 2021 — Team NMU_China

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# Team:NMU China/Notebook

Combatting COVID-19

Naval Medical University CHINA

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## January

### - Administrative Work: Publicity: IGEM competition was advertised in our synthetic Biology class! Recruiting: We collected nearly 100 students who were interested in participating in the iGEM competition! First Training: Email etiquette and Synthetic Biology Second Training: Analysis of iGEM award conditions- Laboratory Work: Literature Reading and Learning: Understanding recent developments and research methods related to COVID-19.- Human Practice: Promotion of Synthetic Biology: We held a synthetic biology awareness session for freshman, sharing project content as well as overview and applications of synthetic biology from previous years, especially in the areas of disease treatment and diagnosis, food production, and environmental governance. HP Sharing: We read the iGEM integrated HP award conditions and shared HP's best cases.- Communication and Education: We introduced synthetic biology and iGEM to freshmen we participated in the data review and partial compilation of the synthetic biology study manual.

## February

### - Administrative Work: Homework: Read related literature and submitted a review. First Presentation: Analysis of Excellent wiki over the years Best wishes for the New Year! We prepared New Year gifts (cards and team USB flash disks) for each team member. - Laboratory Work: Learning theoretical knowledge related to experiment: plasmid construction, cell culture, lentivirus transfection, etc.- Human Practice: Expert Interview: On the treatment of COVID-19 patients, we interviewed Professor Li Chengzhong, Director of the Infection Division of Changhai Hospital. We initially established the theme "Treatment of COVID-19 patients, particularly for those severe cases with a high risk of hyperinflammation" and continued to seek possible treatment options.- Communication and Education: We participated in the data review and partial compilation of the synthetic biology study manual.

## March

### - Administrative Work: Third Training: Academic English writing standards- Laboratory Work: Brainstorm: Student members were encouraged to raise ideas about the project this year. After discussion with professors and instructors, the direction of this year's project was set as cell therapy for COVID-19. We began to do a lot of literature research and constantly improved our project design.- Human Practice: Expert Interview: After literature research, we interviewed the immunology professor Han Chaofeng and doctor Dong Yuchao from Naval Medical University. We understood the specific relation between infection and COVID-19. The professor suggested that we start with the CAR cell therapy which is a cynosure. And professor Han suggested that CAR components should enhance cells’ phagocytic capacity and decrease the release of cytokines. At the same time, doctor Dong stated that we need to design a cytokines’ sensor in order to seek proper time for therapy.- Communication and Education: Through intention investigation and application for courses, our team members facilitated the opening of synthetic biology elective course in our school. We participated in the data review and partial compilation of the synthetic biology study manual.

## April

### - Administrative Work: Second Presentation: Interpretation of champion projects in the past years Registered and paid the registration fee- Laboratory Work: IGEM safety and security rules learning- Human Practice: Expert Interview: Professor Han Chaofeng pointed out that our project are limited for only novel coronaviruses, and we’d better further consider the inflammatory response that mediated by cell fragments. We continued to optimize the experimental design. Also, he suggested that we should focus on bi-directional regulation so CAR-macrophages can improve immune response at the early stage of COVID-19 for recovery and decrease it later for safety. Establish Cooperation with Lanzhou University: We have identified long-term and in-depth cooperation with LZU in HP, experiments and model. “Virus Police” Seven-school Seminar: we gather teams from China in the past two years with the theme of novel coronavirus diagnosis and treatment to hold a seminar for seven schools, namely “Virus”. During this period, we received specific suggestions on the competition.- Communication and Education: We compiled four articles on antibody banking and CAR-T therapy, and displayed them on our WeChat official account.

## May

### - Administrative Work: Brainstorm: Determined the direction of the project- Laboratory Work: Basic Operation training: New team members stepped into the lab and began to learn experimental skill.- Human Practice: Public education: Due to the repeated outbreaks in the country, we mainly pushed out education of popular science. According to the results of the questionnaire survey, we first identified the topics: the application of synthetic immunity, the introduction of COVID-19, the treatment plan and principles of COVID-19 patients. Project Manual Production: Complete the production of the project manual, to provide assistance for the follow-up team to carry out publicity and education activities. Suggestions for the Rural Elderly in the Epidemic: We interviewed Dr. Zhang You, who supported Wuhan last year, to understand the inconvenience of the rural elderly living conditions under the conditions of the domestic epidemic. Then we wrote "Reflections and Suggestions on the Basic Life and Medical Security of The Elderly in Rural under the Epidemic", delivering them to the relevant departments and expecting to be answered.- Communication and Education: Questionnaire Survey: After establishing the project theme, we designed a questionnaire on the degree, method and content of the public's understanding of cellular immunotherapy. We collected 300 questionnaires and analyzed them to establish a final publicity plan.

## June

### - Administrative Work: CCiC (Conference of China iGEMer Community) registration Confirmed as presentation candidates- Laboratory Work: Carried out detailed project design and experimental feasibility analysis- Human Practice: Project promotion: We used multiple platforms for creative video production of synthetic biology, with videos posted by our team members.- Communication and Education: We made science popularization videos on synthetic biology and vaccination for Sars-Cov-2 and we uploaded them on the video website.

## July

### - Administrative Work: Third presentation: Each group reported the latest work progress Promotion video Filled in the Check - in forms- Laboratory Work: Brainstorm and modify the design: Added IL-6 receptors to control the activation of Silent-CAR- Human Practice: Popular science adjustment: For large-scale outbreaks in many parts of the country, we adjusted the content and object of popular science. At the same time, we analyzed the reasons for the failure of prevention and control of many previous outbreaks, making recommendations to the relevant departments to help prevent disease. Expert Interview: We asked professor Guo Meng to evaluate the overall design of our project. He proposed that the existing IL-6 receptors need to be improved so that in the early stage of novel coronavirus infection, macrophages could initially convert to Kill CAR for an immune response, and in the late stage of infection, they could convert to Silent CAR to avoid further upregulation of inflammatory factors.- Communication and Education: We start to write our education package. We first conducted data review, research on education, proposal planning and collaboration discussion.

## August

### - Administrative Work: Formed a paper on the relationship between our synthetic biology curriculum and the iGEM competition Attended CCiC- Laboratory Work: Plasmid construction of the main experiment was carried out Completed CAR Macrophages construction- Human Practice: Team Building Management Sharing Conference: We asked National University of Defense Technology, which has won the iGEM Gold Award for many years, about the construction of the iGEM team at the military academy. Collaboration with NUDT-China: We achieved agreement with NUDT-China in the aspects of experimental collaboration, proposed some advice and received feedback so as to revise our project. Collaboration with High School: We provided technical guidance to four high school teams in China, who also raised questions about the failure of macrophage treatment when the viral load was too high. We used model to verify this problem. At the same time, we asked professor Yin from affiliated hospital to help them solve the problem of experimental model.- Communication and Education: The content of the education package began to be written. During this period, we conducted interviews and communications with stakeholders.

## September

### - Administrative Work: Made track selection on our Team Information Page- Laboratory Work: Finish the experiments of characterization SARS-CoV-2 Pseudovirus and Cell Infection Experiments Phagocytosis Assay Analysed inflammatory cytokines released by CAR macrophages- Human Practice: Hold a project sharing meeting with team Worldshaper-Shanghai. Helped the team contact the interviewee, Professor Yin you. Academic Evaluation: In order to evaluate the application of this therapy in real clinical environment, we interviewed Dr. Du Juan from the Hematology Department of Shanghai Changzheng Hospital since cell therapy is mostly used in the field of hematology at present. She gave us specific suggestions based on treatment cost and patient compliance. At the same time, she popularized the deficiency of current laws and regulations in relevant fields in real scenes, providing guidance for our investigation of laws and regulations in this field. Industrial Evaluation: In order to explore the development of this therapy in the commercial environment, we investigated cell therapy companies in Shanghai, drafted a business plan based on what we learned, and evaluated the development of the future commercialization of this therapy with Mr. Gao, director of investment Banking Division of Guosen Securities in Shanghai.- Communication and Education: The education package was put into use and feedback was obtained. We started to produce children’s book. We investigated related information of cellular immunotherapy, including policy, business and insurance.

## October

### - Administrative Work: Submitted team project title and abstract Completed safety form Finished wiki- Laboratory Work: An improved experiment was carried out- Human Practice: Completed the education package: We have completed the education package covering the content of synthetic biology in the fields of disease treatment, environmental management, etc. After the review of education experts, the volunteer teaching team of three school, including our school, Lanzhou University and Ocean University of China began to share synthetic biology education package resources to help children in poor areas receive scientific education.- Communication and Education: Education package application in progress. Children’s book collection started.

© Copyright **2021 iGEM NMU-China** . All Rights Reserved

**Phone:** +86 021-81870920
**Email:** iGEM@smmu.edu.cn

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# Team:NMU China/Engineering

Combatting COVID-19

Naval Medical University CHINA

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[Overview](#title1)
[Background Investigation and Information Collection](#title2)
[Design](#title3)
[Build and Test](#title4)
[Model, Learn and Improve](#title5)
[Reference](#title6)

![]( https://static.igem.org/mediawiki/2021/9/9d/T--NMU_China--es50.jpeg)

# Overview

We followed the engineering cycle for countless iterations over the course of our project. For clarity, we have condensed the iterations and dispersed our experience into the steps:
       1. Background Investigation and Information Collection
       2. Design
       3. Build and Test
       4. Model, Learn and Improve

![](	https://static.igem.org/mediawiki/2021/9/9d/T--NMU_China--eng2.jpeg)

# Background Investigation and Information Collection

Table of Contents

1. Overview
2. Macrophage and SARS-CoV-2
3. Macrophages, Cytokines, and Cytokine Storm
4. Macrophage Phenotype and Disease
5. Artificial Cell Therapy and Artificial Macrophage Therapy

Overview

Before designing our toggle macrophages, we conducted a thorough literature review on the important role of macrophages in response to COVID-19. According to literatures, Macrophages are divided into M1 (classically activated) and M2 (alternatively activated) macrophages, which are responsible for suppressing infection and regulating inflammation respectively. Through the analysis of the existing artificial cell therapies, we find the possibility in modifying the macrophages to alter their performances in phagocytosis and cytokine expression.
The findings of our literature review are summarized below.

Macrophage and SARS-CoV-2

A novel coronavirus, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which emerged in 2019, has been found to cause acute respiratory distress syndrome (ARDS) called coronavirus disease 2019 (COVID-19), with some patients developing a severe form of this disease leading to death. This virus is the third highly pathogenic coronavirus reported to date and has caused a worldwide outbreak of infection, a pandemic. Around 80% of individuals infected with SARS-CoV-2 appear to recover without antiviral treatments (Wu Z et al.2020), indicating that an adequate host immune response against the virus may be sufficient to cure the viral infection. In contrast, the other 20% of patients develop severe forms of this disease, suggesting that an inadequate immune response may predispose patients to poorer outcomes. **It has been reported that some strains invade the body in a dormant and a mitigatory way.**  At the beginning of the infection, SARS-CoV-2 can not only **suppress the innate immune response** by inhibition of interferon Ⅰ mediated inflammatory cells recruitment, but it can also escape the murder of acquired immune cells by lowering the expression of surface antigen. And the virus can consequently get mass propagation in alveolar epithelia cells without the worry of being destructed by the immune response.

![](https://static.igem.org/mediawiki/2021/8/8f/T--NMU_China--eng3.jpeg)

Understanding host immune responses against SARS-CoV-2 may be important in designing therapeutic strategies to fight viral infection. (Mallis P et al.2020) Macrophages, as an important component of the innate immune system, have unique effector functions as a first-line defense against SARS-CoV-2.

1. Macrophages participate in the innate immune response and play a critical role in the body’s defense against viral infections. They are powerful activators for triggering adaptive immune responses and killing viruses (Meidaninikjeh et al. 2021).

2. Naturally, they can transform into M1 type and M2 type, which is pro-inflammatory and anti-inflammatory, respectively. This characteristic of macrophages corresponds to our ideal therapy that can concurrently eradicate the viruses and balance the immune response.

3. Delayed IFN-I release is associated with inactivation of macrophages, which later causes delayed viral clearance and increased proinflammatory cytokine release.

**Therefore, we hypothesized that we could utilize macrophages in a synthetic way to combat SARS-CoV-2.**

![](https://static.igem.org/mediawiki/2021/e/ea/T--NMU_China--eng4.jpeg)

Macrophages, Cytokines, and Cytokine Storm

Cytokine, mainly produced by immune cells, is a kind of highly active small molecule polypeptide or protein. The network formed by the interactions among cytokines plays an essential role in immune response, inflammatory response, and cell proliferation and growth. (Yao Liteng et al.2020)
In the inflammatory response, a variety of cytokines exert their physiological functions and regulate the immune response when binding to their specific receptors. Some cytokines equipped with the capability of activating immune cells and promoting inflammatory response are called pro-inflammatory cytokines; while the other cytokines which could resist inflammation are called anti-inflammatory cytokines. When our body is invaded by pathogens, the pro-inflammatory cytokines produced by the immune system promote the progression of inflammation and elimination of the pathogens; and the anti-inflammatory cytokines inhibit the inflammatory response. The two sides work together, thus presenting a complex immune response.
It has been suggested that **pathophysiology of SARS-CoV-2 is related to elevated levels of inflammatory cytokines** such as IL-2, IL-6, IL-7, GSCF, IP10, MCP1, MIP1A, and TNF-α, an event which is known as “cytokine storm” (Mehta P et al.2020; Law HK et al.2005). After the infiltration of SARS-CoV-2 to the host cells, infected patients would go through three-phase immunopathology—non-severe stage, replication cycle and prolonged lung damage. It is right in the third stage that **“cytokine storm” has a high association with disease severity and mortality** (Mallis P et al.2020). We therefore concluded that **the key to preventing the progression of COVID-19 is to regulate the expression of cytokines.**  And the macrophage is the center of the cytokine storm to some extent (Chau AS et al.2021).

![](https://static.igem.org/mediawiki/2021/d/d5/T--NMU_China--eng5.png)

Picture from: Chau AS, Weber AG, Maria NI, Narain S, Liu A, Hajizadeh N, Malhotra P, Bloom O, Marder G, Kaplan B. The Longitudinal Immune Response to Coronavirus Disease 2019: Chasing the Cytokine Storm. Arthritis Rheumatol. 2021 Jan;73(1):23-35. doi: 10.1002/art.41526. Epub 2020 Dec 1. PMID: 32929876.

Macrophage Phenotype and Disease

Macrophages are distinguished to M1 (classically activated) and M2 (alternatively activated) macrophages (Yagi H et al.2010). M1 macrophages are responsible for pathogen phagocytosis and presentation of antigen epitopes to dendritic cells (DCs). In this process, a set of inflammatory cytokines are produced by M1 macrophages, such as TNF-α, ΙL-1α/β, IL-6, and IL-12, thus activating and promoting Th1 responses. On the other hand, M2 macrophages promote Th2 responses and are considered as immunosuppressive cells. These cells exhibit low expression of inflammatory cytokines and high production of anti-inflammatory IL-10, which is associated with tissue remodeling, wound repair, and clearance of apoptotic cells (Shapouri-Moghaddam A et al.2018).

![](https://static.igem.org/mediawiki/2021/7/76/T--NMU_China--eng6.jpeg)

M1 and M2 macrophages have different functions and transcriptional profiles. They have unique abilities by destroying pathogens or repairing the inflammation-associated injury. It is known that M1/M2 macrophage balance polarization governs the fate of an organ in inflammation or injury. **When the infection or inflammation is severe enough to affect an organ, macrophages first exhibit the M1 phenotype to release TNF-α, IL-1β, IL-12, and IL-23 against the stimulus.** But, if M1 phase continues, it can cause tissue damage. Therefore, **M2 macrophages secrete high amounts of IL-10 and TGF-β to suppress the inflammation,**  contribute to tissue repair, remodeling, vasculogenesis, and retain homeostasis. (Shapouri-Moghaddam A et al.2018).
Based on this, we designed the toggle macrophages which could cover the whole progression of the infection. In the early stage, the macrophages would transform into M1 phenotype, increasing the expression of the cytokines to eliminate immunosuppressive as much as possible. When the infection deteriorates into the late stage, the toggle macrophages would transform into M2 phenotype, exhibiting low levels of cytokine expression to decrease its critical damage to the severely ill patients.
For more detailed information, please visit our [Design.](https://2021.igem.org/Team%3ANMU_China/Design "Click to go to the Design")

Artificial Cell Therapy and Artificial Macrophage Therapy

**5.1 CAR-T cell therapy**
In recent years, Chimeric antigen receptor (CAR)-engineered T cells (CAR-T cells) have yielded unprecedented efficacy in the treatment of tumor, especially B cell malignancies (Wang Z et al.2017).
**Chimeric antigen receptor (CAR)** is a modular fusion protein comprising extracellular target binding domain usually derived from the single-chain variable fragment (scFv) of antibody, spacer domain, transmembrane domain, and intracellular signaling domain containing CD3ζ linked with zero or one or two costimulatory molecules such as CD28, CD137, and CD134 (Eshhar Z et al.2008; Curran KJ et al.2012; Dai H et al.2016). T cells engineered to express CAR by gene transfer technology are capable of specifically recognizing their target antigen through the scFv binding domain, resulting in T cell activation in a major histocompatibility complex (MHC)-independent manner (Eshhar Z et al.1993).
However, on-target or off-target tumor toxicity resulting from the recognition of healthy tissues by CAR-T cells would cause severe and even life-threatening toxicities. What’s worse, the activation of T cells is always accompanied by the overproduction of cytokines, which suggests that the application of CAR-T cells in the treatment of COVID-19 is impractical. But at least, **it provides with an idea of constructing CAR.**
**5.2 CAR-Macrophage therapy**
As mentioned above, CAR-T cell therapy is not so effective in combating solid tumor. Given that macrophages are the innate immune cells with the highest infiltration rate in the tumor microenvironment, researchers are trying to use macrophages modified with CAR (CAR-M) against solid tumors.
CAR-M with intracellular domain of MEGF10 or FcRγ, promotes the phagocytic potential of target antigens. (Chen Y et al.2021)
So, we began to think about whether we could **modify macrophages with different CARs to alter the performance, such as a strong phagocytosis and a better control of cytokine expression.**

# Design

Table of Contents

1. CAR Receptors
2. IL-6 Sensor and Linked Circuit Design（more details in [Proof of Concept）](https://2021.igem.org/Team%3ANMU_China/Proof_Of_Concept "Click to go to the POF")

1.CAR Receptors

We engineered THP-1 cells with 5 kinds of CAR to enhance its recognizing ability and phagocytosis of SARS-CoV-2. They share similar constructions in extracellular domain and transmembrane domain, except for intracellular domain. Notably, the Myc tag is inserted for flow cytometer.

1. The extracellular domain: a single chain variable fragment (ScFv) which specifically recognizes the spike protein of SARS-CoV-2.

2. The transmembrane domain: CD8 protein allowing signal transduction.

3. The intracellular domain: a domain consists of different signal motifs which stem from the intracellular structure of various natural receptors of immune cells. These signal motifs can trigger robust phagocytosis. Noteworthy, we designed a **CAR without intracellular structure** as negative control.

4. Myctag is a fluorescent protein gene for subsequent detection of CAR macrophage transduction.

Why we chose these four signal motifs as candidates? According to research, MERTK is a tyrosine kinase which belongs to TAM family of RTKs. The activated TAM receptors can inhibit the TLR and type I IFN signaling pathways and thus may avoid further upregulation of proinflammatory cytokine levels (Todt, Hu, and Curtis 2004). In previous study, Megf10 and FcRγrobustly triggered engulfment of CAR-macrophages by being phosphorylated by Src family kinases, while CD3ζ by recruiting syk kinase (Morrissey et al. 2018).

![](https://static.igem.org/mediawiki/2021/2/2e/T--NMU_China--design3.png)

Figure1：Vector maps of tested CAR designs and schematics showing the structures of CARs

2.IL-6 Sensor and Linked Circuit Design

According to the in vitro experiments，CARγ macrophage (Mγ) can promote inflammation and CARMERTK-macrophage (Ms) can inhibit inflammation. We determined to integrate CARγ and CARMERTK into a single macrophage by our circuit design in order to construct the smart Toggle Macrophage which can respond to the changing microenvironment by switching its phenotype. The circuit we designed determines the function of Toggle Macrophage by regulating the CAR expression based on the IL-6 concentration in the microenvironment. The circuit is as followed：

![](https://static.igem.org/mediawiki/2021/c/c3/T--NMU_China--design2.1.jpeg)

(figure2: Circuit Design of Toggle Macrophage)

# Build and Test

1.Overview

We aimed to test the 4 kinds of CAR macrophages we constructed. To accomplish this, we conducted several experiments to test their **engulfment effect, phenotypic transformation and the capacity of viral load.** Scroll down to learn more.

2.Obtaining Parts

We designed 7 basic parts synthesized by GENEWIZ to synthesize 4 composite parts.

|  | Name | Type | Description |
| --- | --- | --- | --- |
| Tag | [BBa\_K4040005](http://parts.igem.org/Part%3ABBa_K4040005) | Tag | Myc tag |
| Extracellular domains | [BBa\_K4040019](http://parts.igem.org/Part%3ABBa_K4040019) | Coding | CR3022 scFv |
| Transmembrane domains | [BBa\_K4040004](http://parts.igem.org/Part%3ABBa_K4040004) | Protein\_Domain | CD8 hinge |
| [BBa\_K4040007](http://parts.igem.org/Part%3ABBa_K4040007) | Protein\_Domain | CD8TM |
| Intracellular domains | [BBa\_K4040000](http://parts.igem.org/Part%3ABBa_K4040000) | Protein\_Domain | Intracellular Domain of the MEGF10 Protein |
| [BBa\_K4040001](http://parts.igem.org/Part%3ABBa_K4040001) | Protein\_Domain | Intracellular Domain of the MERTK Protein |
| [BBa\_K4040002](http://parts.igem.org/Part%3ABBa_K4040002) | Protein\_Domain | FcRγ |
| [BBa\_K4040003](http://parts.igem.org/Part%3ABBa_K4040003) | Protein\_Domain | CD3 zeta chain |
| Composite part | [BBa\_K4040015](http://parts.igem.org/Part%3ABBa_K4040015) | Composite | CAR-MEGF10 |
| [BBa\_K4040016](http://parts.igem.org/Part%3ABBa_K4040016) | Composite | CARγ |
| [BBa\_K4040017](http://parts.igem.org/Part%3ABBa_K4040017) | Composite | CAR ζ |
| [BBa\_K4040018](http://parts.igem.org/Part%3ABBa_K4040018) | Composite | CAR-MERTK |

3.Testing CAR receptor

**3.0 Method**
       **Cell Lines and Primary Human Cells**
               All cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA). The identities of the cell lines were verified by STR analysis, and the cell lines were confirmed to be mycoplasma free. 293 and Vero cells were maintained in DMEM supplemented with 10% fetal bovine serum, and THP-1 cells were maintained in RPMI medium supplemented with 10% fetal bovine serum. Cell culture media and supplements were obtained from Life Technologies, Inc.
       **Vector Construction**
               The sequence encoding the scFv generated from CR3022 was chemically synthesized. As shown in Figure 1, synthetic receptors contained the human CD8a signal peptide followed by the scFv linked in-frame to the hinge domain of the CD8a molecule, transmembrane region of the human CD8 molecule, and intracellular signaling domains of the FCER1G, MEGF10, MERTK or CD3z molecules. The cDNA sequences containing the various fusion constructs were cloned into a third-generation lentiviral vector in which the CMV promoter was replaced with the EF-1a promoter, the pELNS vector. (Carpenito C et al. 2009) High-titer replication-defective lentiviruses were produced and concentrated (Carpenito C et al.). Lentiviral infection was used to stably express CAR constructs in THP-1 cells.
       **FACS-Based Phagocytosis Assay**
               UTD or CAR-expressing THP-1 cells were cocultured with GFP+ 293T cells or GFP+ 293T-S (S+ ) target cells for 4 h at 37°C. The effector-to-target (E:T) ratio was 1:1, and 1 × 105 cells were used as both effector cells and target cells. After coculturing, the cells were harvested and stained with an antiCD11b APC-Cy7-conjugated antibody (M1/70, BioLegend) and analyzed by FACS using a FACSCalibur flow cytometer (BD Biosciences). The percentage of phagocytosis was calculated based on the percent of GFP+ events within the CD11b+ population. Data are represented as the mean ± standard error of quadruplicate wells.
       **Flow Cytometry**
               Cell-surface staining was performed for 45 min at 4°C and was analyzed using a FACSCalibur flow cytometer (BD Biosciences). A minimum of 1 × 104 events per sample were examined.
       **In Vitro Cytotoxicity Assay and Luciferase-Based Killing Assay**
               293T and 293T-S cells were used as targets in luciferase-based killing assays including control (UTD) or CAR macrophages. The effector-to-target (E:T) ratio was 10:1 for all the groups. Bioluminescence was measured using a Bio-Tek Synergy H1 microplate reader. The percent specific lysis was calculated on the basis of the experimental luciferase signal (total flux) relative to the signal of the target alone, using the following formula: %Specific Lysis = [(Sample signal —; Target alone signal)]/ [Background signal — Target alone signal)] × 100.
       **SARS-CoV-2 Pseudovirus and Cell Infection Experiments**
               The SARS-CoV-2 pseudovirus was constructed based on the spike genes of the strain Wuhan-Hu-1 (GenBank: MN908947) using published methods (15). The SARS-CoV-2 spike gene was chemically synthesized and cloned into a eukaryotic expression plasmid. 293T cells were first transfected with the S expression vector and then infected with a VSV pseudotyped virus (G∗DGVSV), in which the VSV-G gene was substituted with luciferase expression cassettes. The culture supernatants were harvested and filtered at 24 h post infection. The SARS-CoV-2 pseudovirus could not be neutralized by anti-VSV-G antibodies, and no G∗DG-VSV was mixed with the SARS-CoV-2 pseudovirus stock. For cell-based infection assays, target cells were grown in plates until they reached 50%–75% confluency and were then inoculated with pseudotyped virus. The transduction efficiency was quantified at 16 h post transduction by measuring firefly luciferase activity according to the manufacturer’s instructions (Promega).
       **Phagocytosis Assay**
               In all cases, SARS-CoV-2 S pseudotyped virions were pelleted (90 min at 14,000 rpm and 4°C), and after removal of the supernatant, the pellets were resuspended in RPMI medium and incubated with phagocytes (THP-1 cells or CAR macrophages) at 37°C for 1.5 h. After allowing time for phagocytosis, the cells were washed three times with PBS and incubated with Accutase (Innovative Cell Technologies) for 10 min at 37°C, followed by a final wash in Accutase. Intracellular staining for the S protein was performed for 60 min on ice after using a fixation/permeabilization kit (eBioscience) and then analyzed using a FACSCalibur flow cytometer (BD Biosciences). The phagocytic score was determined by gating the samples on events representing cells and was calculated as follows: Percent S protein positive × median fluorescence intensity (MFI).
       **Cytokine Analysis**
               Cytokine analysis was performed on supernatants derived from cultures given the indicated treatments using a human cytokine 10-plex panel (Thermo Scientific) per the manufacturer’s instructions, with the panel results read on a Luminex Analyzer.
       **Statistical Analysis**
               Unless otherwise specified, Student’s t test was used to evaluate the significance of differences between two groups, and ANOVA was used to evaluate differences among three or more groups. Differences between samples were considered statistically significant when P < 0.05.
**3.1 engulfment effect test**
       The phagocytic potential of human macrophage THP-1 cell lines expressing different CAR receptors or a truncated CAR receptor (CARΔ) lacking the intracellular domain was measured with a cell-based assay. The results are demonstrated in figure 1 and figure 2, both of which suggested that **CAR macrophages and control untransduced (UTD) macrophages did not show notable phagocytosis of 293 cells;**  however, **CARMEGF10, CARγ and CARζ cells phagocytosed Spike-bearing 293 cells in an S-specific manner.**

![](https://static.igem.org/mediawiki/2021/f/f8/T--NMU_China--eng7.png)

Figure 3. Phagocytosis of 293T cells

![](https://static.igem.org/mediawiki/2021/7/7d/T--NMU_China--eng8.png)

Figure 4. Phagocytosis of 293T-S cells

        Antibody-mediated phagocytosis and internalization of virions are important mechanisms of antiviral activity performed by macrophages against pathogens; however, using the phagocytosis assay developed for SARS-CoV-2, we observed low levels of phagocytic activity when UTD cells directly contacted virions. Phagocytic activity was not significantly increased when CAR△ cells rather than UTD macrophages were the phagocytes in the assay, suggesting that **the extracellular domain of the CAR alone is not sufficient to induce strong virion internalization. CARγ, CARMEGF10, and CARζ mediated similar significantly stronger levels of SARS-CoV-2 phagocytosis by THP-1 cells than CAR△（truncated CAR）.**

![](https://static.igem.org/mediawiki/2021/a/aa/T--NMU_China--eng9.png)

Figure 5. The uptake of pseudotyped virions by UTD and other CAR macrophages

**3.2 Phenotypic test**
       Because the systemic cytokine profiles observed in patients with severe COVID-19 show similarities to those observed in patients with macrophage activation syndrome, culture supernatants from THP-1 cells with different CARs treated with virions were further analyzed in a multiplex cytokine assay (Figure5). Following SARS-CoV-2 treatment of THP-1 cells, we observed slightly increased secretion of the cytokines IL-6, IL-8 and TNF-a, but no discernable patterns could be confidently drawn for GM-CSF, IL-1b, IL-2, IL-4, IL-5, IL-8, IL-10, and IFN-g. CAR△ cells showed a cytokine profile similar to that of UTD macrophages. Notably, **we observed not only strongly increased induction of IL-6, IL-8 and TNF-a but also induction of IFN-g and IL-10 in SARS-CoV-2-treated CARγ and CARζ cells.**  However,  **for CARMERTK cells, we did not observe significant changes in cytokines.**

![](https://static.igem.org/mediawiki/2021/f/f1/T--NMU_China--eng10.png)

Figure 6. Cell lines were infected with the SARS-CoV-2 pseudotyped virus or mock infected. Cytokine levels in the supernatants were determined by a multiplex bead array.

**3.3 Viral load test**
       We further used a transwell-based coculture model to evaluate the protective role of CAR macrophages in SARS-CoV-2 infection (Figure 7). **All the CAR-expressing macrophages potently inhibited Vero E6 cell infection with the SARS-CoV-2-S pseudotyped virus.**  Interestingly, **CAR△ cells showed no protective effect in the infection assay,** although they had a similar capacity to bind to the S protein, suggesting that the intracellular signaling domain is necessary for virion clearance by CAR macrophages (figure 8).

![](	https://static.igem.org/mediawiki/2021/b/bb/T--NMU_China--eng11.png		)

Figure 7. Transwell coculture model

![](	https://static.igem.org/mediawiki/2021/3/3c/T--NMU_China--eng12.png	)

Figure 8. Pseudotype entry intodifferent cell cocultures

After testing, we found that although cytosolic domains from MERTK (CARMERTK) did not trigger antigen-specific cellular phagocytosis or killing effects, unlike those from MEGF10, FcRγ and CD3zeta did, these CARs all mediated similar SARS-CoV-2 clearance in vitro. Notably, we showed that CARMERTK macrophages reduced the virion load without upregulation of proinflammatory cytokine expression. These results suggest that CARMERTK drives an ‘immunologically silent’ scavenger effect in macrophages and paves the way for further investigation of CARs for the treatment of individuals with COVID-19, particularly those with severe cases at a high risk of hyperinflammation. While the other three CARs reduced the virion load with upregulation of proinflammatory cytokine expression.

# Model, Learn and Improve

We have expanded upon our Silver medal work for [Proposed Implementation](https://2021.igem.org/Team%3ANMU_China/Implementation "Click to go to the Proposed Implementation") and developed a [Proof of Concept](https://2021.igem.org/Team%3ANMU_China/Proof_Of_Concept "Click to go to the POF") for our project.

# Reference

Carpenito C, Milone MC, Hassan R, Simonet JC, Lakhal M, Suhoski MM, et al. Control of Large, Established Tumor Xenografts With Genetically Retargeted Human T Cells Containing CD28 and CD137 Domains. Proc Natl Acad Sci USA (2009) 106:3360–5. doi: 10.1073/pnas.0813101106
Nie J, Li Q, Wu J, Zhao C, Hao H, Liu H, et al. Establishment and Validation of a Pseudovirus Neutralization Assay for SARS-CoV-2. Emerg Microbes Infect (2020) 9:680–6. doi: 10.1080/22221751.2020.1743767
Wu Z, McGoogan JM. Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72 314 Cases From the Chinese Center for Disease Control and Prevention. JAMA 2020; 323: 1239-1242 [PMID: 32091533 DOI: 10.1001/jama.2020.2648]
Mallis P, Michalopoulos E, Chatzistamatiou T, Stavropoulos-Giokas C. Mesenchymal stromal cells as potential immunomodulatory players in severe acute respiratory distress syndrome induced by SARS-CoV-2 infection. World J Stem Cells. 2020 Aug 26;12(8):731-751. doi: 10.4252/wjsc.v12.i8.731. PMID: 32952855; PMCID: PMC7477656.
Merad M, Martin JC.Nat Rev Immunol. 2020 Jun;20(6):355-362. doi: 10.1038/s41577-020-0331-4. Epub 2020 May 6.
Xu Na, Lv Yingfeng. Research progress of macrophages in the immune microenvironment of lung cancer[J]. Journal of Clinical Oncology, 2021, 26(08): 756-761.
Sui Yang, Zhao Zhiyu, Wu Changjun. Research progress of tumor-associated macrophages in triple-negative breast cancer[J/OL]. Chinese Journal of Immunology: 1-11[2021-10-04]. http: // kns. cnki. net/ kcms/ detail/ 22.1126.r.20210624.1517.008.html.
Yao Liteng, Min Jianping, Su Haixiang. The mechanism of cytokines in new coronavirus pneumonia and their monitoring value[J]. Gansu Medicine, 2020, 39(02): 148-150+157.
Mehta P, McAuley DF, Brown M, Sanchez E, Tattersall RS, Manson JJ; HLH Across Speciality Collaboration, UK. COVID-19: consider cytokine storm syndromes and immunosuppression. Lancet 2020; 395: 1033-1034 [PMID: 32192578 DOI: 10.1016/S0140-6736(20)30628-0]
Law HK, Cheung CY, Ng HY, Sia SF, Chan YO, Luk W, Nicholls JM, Peiris JS, Lau YL. Chemokine upregulation in SARS-coronavirus-infected, monocyte-derived human dendritic cells. Blood 2005; 106: 2366- 2374 [PMID: 15860669 DOI: 10.1182/blood-2004-10-4166]
Mallis P, Michalopoulos E, Chatzistamatiou T, Stavropoulos-Giokas C. Mesenchymal stromal cells as potential immunomodulatory players in severe acute respiratory distress syndrome induced by SARS-CoV-2 infection. World J Stem Cells. 2020 Aug 26;12(8):731-751. doi: 10.4252/wjsc.v12.i8.731. PMID: 32952855; PMCID: PMC7477656.
Chau AS, Weber AG, Maria NI, Narain S, Liu A, Hajizadeh N, Malhotra P, Bloom O, Marder G, Kaplan B. The Longitudinal Immune Response to Coronavirus Disease 2019: Chasing the Cytokine Storm. Arthritis Rheumatol. 2021 Jan;73(1):23-35. doi: 10.1002/art.41526. Epub 2020 Dec 1. PMID: 32929876.
Yagi H, Soto-Gutierrez A, Parekkadan B, Kitagawa Y, Tompkins RG, Kobayashi N, Yarmush ML. Mesenchymal stem cells: Mechanisms of immunomodulation and homing. Cell Transplant 2010; 19: 667- 679 [PMID: 20525442 DOI: 10.3727/096368910X508762]
Shapouri-Moghaddam A, Mohammadian S, Vazini H, Taghadosi M, Esmaeili SA, Mardani F, Seifi B, Mohammadi A, Afshari JT, Sahebkar A. Macrophage plasticity, polarization, and function in health and disease. J Cell Physiol 2018; 233: 6425-6440 [PMID: 29319160 DOI: 10.1002/jcp.26429]
Shapouri-Moghaddam A, Mohammadian S, Vazini H, Taghadosi M, Esmaeili SA, Mardani F, Seifi B, Mohammadi A, Afshari JT, Sahebkar A. Macrophage plasticity, polarization, and function in health and disease. J Cell Physiol. 2018 Sep;233(9):6425-6440. doi: 10.1002/jcp.26429. Epub 2018 Mar 1. PMID: 29319160.
Wang Z, Wu Z, Liu Y, Han W. New development in CAR-T cell therapy. J Hematol Oncol. 2017 Feb 21;10(1):53. doi: 10.1186/s13045-017-0423-1. PMID: 28222796; PMCID: PMC5320663.
Eshhar Z. The T-body approach: redirecting T cells with antibody specificity. Handb Exp Pharmacol. 2008;181:329–42.
Curran KJ, Pegram HJ, Brentjens RJ. Chimeric antigen receptors for T cell immunotherapy: current understanding and future directions. J Gene Med. 2012;14(6):405–15.
Dai H, Wang Y, Lu X, Han W. Chimeric antigen receptors modified T-cells for cancer therapy. J Natl Cancer Inst. 2016;108(7):djv439.
Eshhar Z, Waks T, Gross G, Schindler DG. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proc Natl Acad Sci U S A. 1993;90(2):720–4
Chen Y, Yu Z, Tan X, Jiang H, Xu Z, Fang Y, Han D, Hong W, Wei W, Tu J. CAR-macrophage: A new immunotherapy candidate against solid tumors. Biomed Pharmacother. 2021 Jul;139:111605. doi: 10.1016/j.biopha.2021.111605. Epub 2021 Apr 23. PMID: 33901872.
Cao X. COVID-19: immunopathology and its implications for therapy. Nat Rev Immunol 2020; 20: 269-270 [PMID: 32273594 DOI: 10.1038/s41577-020-0308-3]
Ruan Q, Yang K, Wang W, Jiang L, Song J. Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan, China. Intensive Care Med 2020; 46: 846-848 [PMID: 32125452 DOI: 10.1007/s00134-020-05991-x]
Xu X, Han M, Li T, Sun W, Wang D, Fu B, Zhou Y, Zheng X, Yang Y, Li X, Zhang X, Pan A, Wei H. Effective treatment of severe COVID-19 patients with tocilizumab. Proc Natl Acad Sci USA 2020; 117: 10970-10975 [PMID: 32350134 DOI: 10.1073/pnas.2005615117]
Levine BL, Kessler DA, Rappel WJ. Gene Transfer in Humans Using a Conditionally Replicating Lentiviral Vector. Proc Natl Acad Sci (2006) 103:17372–7. doi: 10.1073/pnas.0608138103
Fu W, Lei C, Liu S, Cui Y, Wang C, Qian K, et al. CAR Exosomes Derived From Effector CAR-T Cells Have Potent Antitumour Effects and Low Toxicity. Nat Commun (2019) 10:1–12. doi: 10.1038/s41467-019-12321-3

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# Team:NMU China/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NMU China/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)