Team:SMMU-China/Design

Background:

The coronavirus disease 2019 (COVID-19) pandemic has caused a sudden significant increase in hospitalizations for pneumonia with multiorgan disease and has led to more than 300,000 deaths worldwide. COVID-19 is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel enveloped RNA betacoronavirus. SARS-CoV-2 infection may be asymptomatic or cause a wide spectrum of symptoms, ranging from mild symptoms of upper respiratory tract infection to life-threatening sepsis. Manifestations of COVID-19 include asymptomatic carriers and fulminant disease characterized by sepsis and acute respiratory failure. Approximately 5% of patients with COVID-19, including 20% of those hospitalized, experience severe symptoms necessitating intensive care. More than 75% of patients hospitalized with COVID-19 require supplemental oxygen. The case-fatality rate for COVID-19 varies markedly by age, ranging from 0.3 deaths per 1000 patients among patients aged 5 to 17 years to 304.9 deaths per 1000 patients among patients aged 85 years or older. Among patients hospitalized in the intensive care unit, the case fatality can reach 40%[1,2]. There is currently no human vaccine available for SARS-CoV-2, but approximately 17120 candidates are under development. In the development of an effective vaccine, a number of challenges must be overcome, such as technical barriers, the feasibility of large-scale production and regulation, legal barriers, the potential duration of immunity and thus the number of vaccine doses needed to confer immunity, and the antibody-dependent enhancement effect. Moreover, there is another complicated issues worth pondering: drug development for COVID-19, especially treatments for patients with severe or late-stage disease. Dexamethasone therapy was reported to reduce 28-day mortality in patients requiring supplemental oxygen compared with usual care (21.6% vs 24.6%; age-adjusted rate ratio, 0.83 [95% CI, 0.74-0.92])[3], and remdesivir was reported to accelerate the process of recovery (hospital discharge or no supplemental oxygen required) from 15 to 11 days[4]. In a randomized trial of 103 patients with COVID-19, convalescent plasma did not shorten the time of recovery[5]. Ongoing trials are testing antiviral therapies, immune modulators, and anticoagulants; however, there is no specific antiviral treatment recommended for COVID-19.

Chimeric antigen receptors (CARs) are synthetic receptors that redirect T cell activity towards specific targets[6]. A CAR construct includes antigen-recognition domains in the form of a single-chain variable fragment (scFv) or a binding receptor/ligand in the extracellular domains. A transmembrane domain provides the scaffold, signaling transduction area, and intracellular domains from the T cell receptor (TCR) and costimulatory molecules that trigger T cell activation[7]. Based on the longstanding interest in harnessing macrophages to combat tumor growth[8,9], human macrophages engineered with CARs have been developed and characterized for their antitumor potential. Macrophages, critical effectors of the innate immune system, are responsible for sensing and responding to microbial threats and promoting tissue repair. We therefore hypothesize that CAR macrophages can be used to combat SARS-CoV-2. However, the hyperinflammatory macrophage response, which has been found to be hazardous to the host, particularly in severe infections, including SARS-CoV-2. And cytokine release syndrome (CRS), which is also the most significant complication associated with CAR-T cell therapy, raises concerns regarding the safety of using CAR macrophages for virus clearance.

Our design:

In order to treat those severe COVID-19 patients and minimize the side effects, we developed the optimized CAR-Macrophage, namely, the ‘Silent-CAR’.

Firstly, we constructed chimeric antigen receptors based on recognition of the S protein and tested their ability to induce phagocytosis of SARS-CoV-2 virions. Then the CARs were combined with macrophages to build several types of CAR-Macrophages. After that, we tested the virus clearance rate of different CAR-Macrophages as well as testing their inflammatory factors releasing indicators. The certain type of CAR-Macrophage with high virus phagocytosis efficiency and low inflammatory factors release was selected. Finally, we verified that CAR structure wouldn’t facilitate the infection of CAR-Macrophages by SARS-CoV-2.

CAR structure:

The common CAR structure consists of 3 domains: the extracellular domain, the transmembrane domain and the intracellular domain. The extracellular domain targets at the distinctive S protein of SARS-CoV-2, the transmembrane domain permits the signal transduction and the intracellular domain activates the phagocytosis process. The extracellular domain is the single chain variable fragment of the viral structural spike (S) protein antibody. The S protein is chosen because of its important role in viral infection. SARS-CoV-2 binds to the angiotensin converting enzyme 2(ACE2) receptor. The type 2 transmemmal serine protease in host cells promotes viral absorption by cracking ACE2 and activating SARS-CoV-2 S protein, mediating the entry of SARS-CoV-2 into host cells[10,11]. The sequence encoding the scFv generated from CR3022 was chemically synthesized. The human CD8α molecule constitutes the transmembrane region. The intracellular domain is our main concern. We chose four CAR tags with the intracellular domains as follows: common gamma subunit of the Fc receptor (CARγ), MEGF10 (CARMEGF10), MERTK (CARMERTK) and CD3ζ(CARζ). Among them, MERTK was found to be the most promising one. It is a tyrosine kinase which has been reported to be in TAM family with TRYO3 and AXL. Its role is to promote macrophages, dendritic cells (DC) and other dedicated phagocytes to the rapid devouring of apoptotic cells (AC), which is one of the most common and effective signals of phagocytes to identify ACs. S protein and Gas6 have Gla domain on amino terminals, which makes them able to bind with phosphatidylserine (PtdSer). PtdSer is expressed on the surface of ACs, then, S protein and Gas6 bind and activate TAM receptors which expressed on the surface of phagocytes through their carboxyl terminals. Thereby bridging the phagocytes with ACs. In this correlation, the binding of Gas6 and S protein to the TAM receptor on macrophages activates the negative feedback loop, which inhibits the innate immune response induced by the TLR Toll-like receptor and type I interferon signaling pathway. Therefore, TAM receptors can be used to avoid further upregulation of pro-inflammatory cytokine levels[12]. Those fragments were subcloned into the pELNS vector. High-titer replication-defective lentiviruses were produced and concentrated. Lentiviral infection was used to stably express CAR constructs in THP-1 cells.

SARS-CoV-2 pseudovirus:

The SARS-CoV-2 pseudovirus was constructed based on the spike genes of the strain Wuhan-Hu-1 (GenBank: MN908947) using published methods[13]. 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∗ΔG-VSV), in which the VSV-G gene was substituted with luciferase expression cassettes. The culture supernatants were harvested and filtered at 24 h postinfection. The SARS-CoV-2 pseudovirus could not be neutralized with anti-VSV-G antibodies, and no G∗ΔG-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 then were inoculated with pseudotyped virus. The transduction efficiency was quantified at 16 h posttransduction by measuring firefly luciferase activity. Generally speaking, pseudovirus mimics the characteristics of SARS-CoV-2. It is a replication-deficient VSV particle with coronavirus S protein, and is widely used in the laboratory to ensure safety.

Envision:

Still we find several aspects worthy of improving. For those experts who concerned about the safety of using VSV pseudotyped virus, we provide 2 alternatives: The first approach is to use lentivirus to carry S protein instead of using VSV. The second approach is to use GFP to mimic the viral antigen and construct with lentiviruses carrying GFP to evaluate the phagocytic property of CAR-Macrophages. Generally, those two approaches are safer, but their scientific reliability may be undermined as opposed to that of VSV pseudotypes. However, the pseudovirus we used in our project is somehow incomprehensive and simplified. Research showed that there is a new type of pseudovirus which contains a reporter gene that enabled researchers to easily quantify virus entry into angiotensin-converting enzyme 2 (ACE2)-expressing 293T cells[14]. Additionally, Cathepsin (Cat)B/L inhibitor E-64d could significantly block SARS-CoV-2 pseudovirus infection in 293T-ACE2 cells. We can follow the same idea to improve our pseudovirus and make it safer and easily monitored.

It should also be noted that our study used very simple infection models, therefore, the assays lack numerous physiological and pathological factors, such as IgG or complement-mediated immune complexes, that may interfere with the behavior of engineered cells. It is imperative for us to further engineered and developed synthetic receptors to achieve precise control as well as adding more systematic factors to fortify its authenticity.

Currently CARMERTK is the only CAR-Macrophage with significant viral clearance and barely no cytokine release. The function and application of CARMERTK is limited. We need more types of CAR-Macrophages which obtain the similar properties with CARMERTK. By the same token, more experiments concerning the optimal conditions of CAR-Macrophages remain to be conducted. It is still long way to go before the CAR-Macrophage therapy truly benefits those critical patients.

References:

1.Wiersinga W Joost., Rhodes Andrew., Cheng Allen C., Peacock Sharon J., Prescott Hallie C.(2020). Pathophysiology, Transmission, Diagnosis, and Treatment of Coronavirus Disease 2019 (COVID-19): A Review. JAMA, undefined(undefined), undefined. doi:10.1001/jama.2020.12839

2.Garg Shikha., Kim Lindsay., Whitaker Michael., O'Halloran Alissa., Cummings Charisse., Holstein Rachel., Prill Mila., Chai Shua J., Kirley Pam D., Alden Nisha B., Kawasaki Breanna., Yousey-Hindes Kimberly., Niccolai Linda., Anderson Evan J., Openo Kyle P., Weigel Andrew., Monroe Maya L., Ryan Patricia., Henderson Justin., Kim Sue., Como-Sabetti Kathy., Lynfield Ruth., Sosin Daniel., Torres Salina., Muse Alison., Bennett Nancy M., Billing Laurie., Sutton Melissa., West Nicole., Schaffner William., Talbot H Keipp., Aquino Clarissa., George Andrea., Budd Alicia., Brammer Lynnette., Langley Gayle., Hall Aron J., Fry Alicia.(2020). Hospitalization Rates and Characteristics of Patients Hospitalized with Laboratory-Confirmed Coronavirus Disease 2019 - COVID-NET, 14 States, March 1-30, 2020. MMWR Morb. Mortal. Wkly. Rep., 69(15), 458-464. doi:10.15585/mmwr.mm6915e3

3.Abdolahi Nafiseh., Kaheh Effat., Golsha Roghieh., Khodabakhshi Behnaz., Norouzi Alireza., Khandashpoor Mahmoud., Besharat Sima., Tavassoli Samane., Livani Somayeh., Azimi Sadegh Ali., Gharib Mohammad Hadi., Peivandi Babak., Fazel Abdolreza., Shirzad-Aski Hesamaddin., Roshandel Gholamreza.(2020). Letter to the editor: efficacy of different methods of combination regimen administrations including dexamethasone, intravenous immunoglobulin, and interferon-beta to treat critically ill COVID-19 patients: a structured summary of a study protocol for a randomized controlled trial. Trials, 21(1), 549. doi:10.1186/s13063-020-04499-5

4.Beigel John H., Tomashek Kay M., Dodd Lori E.(2020). Remdesivir for the Treatment of Covid-19 - Preliminary Report. Reply. N. Engl. J. Med., 383(undefined), undefined. doi:10.1056/NEJMc2022236

5.Wang Wenling., Xu Yanli., Gao Ruqin., Lu Roujian., Han Kai., Wu Guizhen., Tan Wenjie.(2020). Detection of SARS-CoV-2 in Different Types of Clinical Specimens. JAMA, undefined(undefined), undefined. doi:10.1001/jama.2020.3786

6.Luo Feifei., Qian Jiawen., Yang Jiao., Deng Yuting., Zheng Xiujuan., Liu Jie., Chu Yiwei.(2016). Bifunctional αHER2/CD3 RNA-engineered CART-like human T cells specifically eliminate HER2(+) gastric cancer. Cell Res., 26(7), 850-3. doi:10.1038/cr.2016.81

7.Fesnak Andrew D., June Carl H., Levine Bruce L.(2016). Engineered T cells: the promise and challenges of cancer immunotherapy. Nat. Rev. Cancer, 16(9), 566-81. doi:10.1038/nrc.2016.97

8.Morrissey Meghan A., Williamson Adam P., Steinbach Adriana M., Roberts Edward W., Kern Nadja., Headley Mark B., Vale Ronald D.(2018). Chimeric antigen receptors that trigger phagocytosis. Elife, 7(undefined), undefined. doi:10.7554/eLife.36688

9.Klichinsky Michael., Ruella Marco., Shestova Olga., Lu Xueqing Maggie., Best Andrew., Zeeman Martha., Schmierer Maggie., Gabrusiewicz Konrad., Anderson Nicholas R., Petty Nicholas E., Cummins Katherine D., Shen Feng., Shan Xinhe., Veliz Kimberly., Blouch Kristin., Yashiro-Ohtani Yumi., Kenderian Saad S., Kim Miriam Y., O'Connor Roddy S., Wallace Stephen R., Kozlowski Miroslaw S., Marchione Dylan M., Shestov Maksim., Garcia Benjamin A., June Carl H., Gill Saar.(2020). Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat. Biotechnol., 38(8), 947-953. doi:10.1038/s41587-020-0462-y

10.Wiersinga W Joost., Prescott Hallie C.(2020). What Is COVID-19? JAMA, undefined(undefined), undefined. doi:10.1001/jama.2020.12984

11.Maier Barbara., Leader Andrew M., Chen Steven T., Tung Navpreet., Chang Christie., LeBerichel Jessica., Chudnovskiy Aleksey., Maskey Shrisha., Walker Laura., Finnigan John P., Kirkling Margaret E., Reizis Boris., Ghosh Sourav., D'Amore Natalie Roy., Bhardwaj Nina., Rothlin Carla V., Wolf Andrea., Flores Raja., Marron Thomas., Rahman Adeeb H., Kenigsberg Ephraim., Brown Brian D., Merad Miriam.(2020). Author Correction: A conserved dendritic-cell regulatory program limits antitumour immunity. Nature, 582(7813), E17. doi:10.1038/s41586-020-2326-5

12.Bhattacharyya Suchita., Zagórska Anna., Lew Erin D., Shrestha Bimmi., Rothlin Carla V., Naughton John., Diamond Michael S., Lemke Greg., Young John A T.(2013). Enveloped viruses disable innate immune responses in dendritic cells by direct activation of TAM receptors. Cell Host Microbe, 14(2), 136-47. doi:10.1016/j.chom.2013.07.005

13.Nie Jianhui., Li Qianqian., Wu Jiajing., Zhao Chenyan., Hao Huan., Liu Huan., Zhang Li., Nie Lingling., Qin Haiyang., Wang Meng., Lu Qiong., Li Xiaoyu., Sun Qiyu., Liu Junkai., Fan Changfa., Huang Weijin., Xu Miao., Wang Youchun.(2020). Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2. Emerg Microbes Infect, 9(1), 680-686. doi:10.1080/22221751.2020.1743767

14.Hu Jie., Gao Qingzhu., He Changlong., Huang Ailong., Tang Ni., Wang Kai.(2020). Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2. Genes Dis, undefined(undefined), undefined. doi:10.1016/j.gendis.2020.07.006