iGEM 2022 — Team zjuintl-china

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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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ENGINEERING

* [Overview](#Overview)
* [Research 1: Can we reverse the senescent phenotype through removing senescent cells?](#Research1)
* [Research 2: How to alleviate cytokine release syndrome (CRS) through the green small molecule switch?](#Research2)
* [Research 3: How to alleviate cytokine release syndrome (CRS) through the green small molecule switch?](#Research 3)

# Overview

Iterative design is a way of designing based on a cyclical process of developing, testing, evaluating, and enhancing items or methods, making modifications and enhancements according to the latest design iteration's test findings. This procedure seeks to eventually enhance the design's quality and utility. In our engineering section, we focused mostly on the engineering design cycle and how we created Ambrosia T. We completed three engineering cycles before obtaining a mature Ambrosia T cell. We initially created chimeric antigen receptor (CAR) T cells that specifically target senescent cells. Then, to minimize the most prevalent side effects of CAR-T immunotherapy, cytokine release syndrome (CRS), we developed our product by incorporating a caffeine-operated synthetic module (COSMO) and three IL-6-based negative feedback loops to control the activation and inactivation of CAR-T cells. Ambrosia T, a senolytic drug with a high degree of security and considerable potential to eliminate senescent cells and reverse senescent phenotypes, was finally developed.

# Ambrosia T development

# Research 1: Can we reverse the senescent phenotype through removing senescent cells?

Due to cellular senescence, ageing is characterised by a reduction in numerous physiological functions, accompanied by altered gene expression and a senescence-associated secretory phenotype (SASP) (Childs et al., 2017). The immune system detects and eliminates senescent cells. However, excessive stimulation of immune cells results in their fatigue, which is accompanied by a rise in senescent cell synthesis rate with age and immunological senescence (Lian et al., 2020). In prior research, elimination of senescent cells has been found to ameliorate ageing phenotypes and reverse ageing markers. It has been demonstrated that removing senescent fibroblasts alleviates liver fibrosis (Amor et al., 2020). Since the leading clinical causes of mortality in humans are now illnesses and organ failure, the elimination of senescent cells is an efficient method for combating ageing.

# Image 1: Can we remove the senescent cells through CAR-T immunotherapy?

At the first stage, we made our mind on taking advantages of the high accuracy of CAR-T cell therapy to target senescent cells by screening markers on the surface of senescent cells. CAR-T immunotherapy is highly effective by targeting cancer cells. CAR-T can specifically eliminate senescent cells since T-cell cytotoxic capabilities target all target cells. In prior research, urokinase-type plasminogen activator receptor (uPAR)-targeted CAR-T cells eliminated mouse liver senescent cells, reversing the ageing phenotype and showing that CAR-T has great potential in the field of anti-aging (Amor et al., 2020). However, uPAR is not widely expressed in senescent cells. Thus, we need to find a broader aging target for our CAR-T immunotherapy.

# Design 1: How can we let the CAR-T precisely remove the senescent cells?

We found dipeptidyl peptidase 4 (DPP4) as our potential senescent target. Previous studies showed that DPP4 selectively expressed on the surface of senescent, but not proliferating, human diploid fibroblasts (Kim et al., 2017), which suggested the elimination of DPP4 highly expressed cells can delay fibrosis and reverse senescence. Therefore, we found the anti-DPP4 scFv amino and DNA sequences and constructed anti-DPP4 CAR based on the 2rd-generation CAR structure, containing anti-DPP4 scFv, CD8 transmembrane domain, 4-1BB co-stimulator domain, and CD3-ζ domain. This can lead CAR-T cells to precisely target and remove the DPP4 highly expressed senescent cells. We also selected a strong promoter, EF-1a, to ensure the high level of CAR gene expression in T lymphocytes.

Anti-DPP4 scFv: [BBa\_K4175050](http://parts.igem.org/Part%3ABBa_K4175050)

CD8 hinge: [BBa\_K4175004](http://parts.igem.org/Part%3ABBa_K4175004)

CD8 transmembrane domain: [BBa\_K4175005](http://parts.igem.org/Part%3ABBa_K4175005)

4-1BB domain: [BBa\_K4175006](http://parts.igem.org/Part%3ABBa_K4175006)

CD3-ζ domain: [BBa\_K4175009](http://parts.igem.org/Part%3ABBa_K4175009)

# Build 1: How can we build it?

We first found the DNA sequence of the anti-DPP4 scFv from the patent, CN101282994A.

We first found the DNA sequence of the anti-DPP4 scFv from the patent, CN101282994A.
Other DNA sequences including the CD8 hinge, CD8 transmembrane domain, the 4-1BB DNA sequence and the CD3-ζ DNA sequence were obtained from the lab of our primary PI, HUANG He. Then all the sequences were arranged in the order, anti-DPP4 scFv, CD8 hinge, CD8 transmembrane, 4-1BB, and CD3-ζ (Figure 1). Considering that there was no way to obtain ready-made clone template, anti-DPP4 CAR sequence was artificially synthesized. In consideration of iGEM's official safety requirements, we did not use the traditional lentiviral transfection method for cell transfection, but instead used nucleofection.

![](https://static.igem.wiki/teams/4175/wiki/img/engineering/engineering1.png)

**Figure 1.**The overview of anti-DPP4 CAR structure.

# Test 1: Do we get we want?

After transfection, we used the flow cytometry to detect the expression of anti-DPP4 CAR. Then we co-cultured the anti-DPP4 CAR positive T lymphocytes with K562 transfected with DPP4 plasmid. The survival rate of DPP4 positive K562 cells were measured by flow cytometry 24h after the co-culture. The results satisfied our expected result.

![](https://static.igem.wiki/teams/4175/wiki/img/engineering/engineering2.png)

**Figure 2.**The results of anti-DPP4 CAR-T cells clearing senescent cells.

# Learn 1: What did we not know our design?

Current experiments were conducted only in vitro and shown that our engineered CAR T cells can eliminate the senescent cells with high DPP4 expression. However, its effect on senescent cells in vivo requires additional investigation. Although several studies indicate the high expression of the DPP4 gene in senescent cells (Kim et al., 2017), there are no in vivo experimental data that show the ability to reverse the senescent phenotype by removing these DPP4 senescent cells. Due to the risk of severe biosafety incidents resulting from in vivo trials at this point, we require more in vivo investigations in future to confirm the involvement of anti-DPP4 CAR T cells in reversing the ageing phenotype.

# Improve 1: Whether our product is manipulable and safe enough?

The first version of Ambrosia T just focused on its ability to clear senescent cells. However, the artificial controllability of Ambrosia T needs to be further improved. Therefore, we need to further explore other methods to build a switch to achieve regulation of CAR-T cells.

# Research 2: How to alleviate cytokine release syndrome (CRS) through the green small molecule switch?

During CAR-T therapy, the massive killing of target cells by CAR-Ts could result in substantial release of pro-inflammatory cytokines (Leclercq et al., 2022). Consequently, cytokine release syndrome (CRS) could be developed (Xiao et al., 2021). The symptoms of CRS range from mild to severe. As of severe CRS, symptoms such as high fever, hypotension, respiratory deficiency, and even multi-organ failure are presented, which could be life-threatening (Leclercq et al., 2022; Shimabukuro-Vornhagen et al., 2018). Therefore, we think it really worth considering how to ameliorate CRS when developed during the design of our product.

Biomarker screening in patients receiving CAR-T therapy showed that peak levels of interleukin-6 (IL-6), soluble IL-6 receptor, interferon-γ (IFN-γ) and soluble glycoprotein 130 (sgp130) are associated with the risk of severe CRS (Teachey et al., 2016). Also, IL-6 is considered holding a key role in CRS pathophysiology as it contributes to many key symptoms (Shimabukuro-Vornhagen et al., 2018). Meanwhile, IL-6 is not critical for the killing capacity of CAR-T cells (Barrett et al., 2016). Collectively, these properties of IL-6 made itself a potential target for alleviating CRS. Tocilizumab, a humanized monoclonal antibody for IL-6 receptor, has been shown to effectively treat severe or life-threatening CRS and has been approved by FDA (Shimabukuro-Vornhagen et al., 2018).

# Imagine 2: Can we implement an automatic switch within CAR-T cells to ameliorate CRS?

Monocyte and macrophage lineages are thought to be the main source of IL-6 during CRS (Norelli et al., 2018). The release of IL-6 following monocyte activation is mediated by damage-associated molecular patterns (DAMPs), which is the product of target cell pyroptosis after CAR-T cell killing (Xiao et al., 2021). Thus, if we can temporarily inhibit the cytotoxicity effect of CAR-T cells at the onset of CRS, the target cells will undergo less pyroptosis and release less DAMPs so that fewer monocytes will be activated. In this way, the serum level of IL-6 will decrease, resulting in the amelioration of CRS symptoms.

Before treating with tocilizumab, the patients need to be observed for some time to be diagnosed with CRS. This means that when receiving tocilizumab, the patients have already developed symptoms of CRS such as fever, fatigue and headache, which may make the patients uncomfortable. Also, treatment with tocilizumab requires additional cost. To alleviate the uncomfortableness during CAR-T therapy and reduce the cost, we wanted to implement an automatic switch within our CAR-T cells in such a way that CAR-T cells pause its cytotoxicity effect at the onset of CRS and get reactivated at the remission of CRS. Here we considered high concentration of IL-6 a plausible signal to downregulate the cytotoxic activity of CAR-T.

# Design 2: How to couple the IL-6 concentration with CAR-T cell activity?

![](https://static.igem.wiki/teams/4175/wiki/img/engineering/engineering3.png)

**Figure 3.**The illustration of how Toggle Macrophage works.

The project from Team NMU-China 2021, Toggle Macrophage, has enlightened us about how to design an automatic switch within CAR-T cells. In their project, they used macrophage as a tool to fight against virus and equipped it with CARs and IL-6 synthetic receptors. The macrophages are designed as such that when IL-6 level is low, there will be no signal transduced from the synthetic receptors and the macrophage expresses high level of CARγ. The macrophage remains in a pro-inflammatory state (M1 phenotype) at this time. However, when IL-6 level is high, IL-6R binds to IL-6 and recruits gp130. The TEV protease which fused with gp130 then cleaves the TEV cleavage site fused with IL-6R. The transcriptional inhibitor, Gal4KRAB, are subsequently released and enter the nucleus. Gal4KRAB inhibits the CARγ expression, which switches the macrophage into an anti-inflammatory state (M2 phenotype). However, implementing this kind of switches need to transduce at least two plasmids into the cells, which may have low efficiency. Thus, we decided to make a simplified switch for CAR-T in response to high level of IL-6.

![](https://static.igem.wiki/teams/4175/wiki/img/engineering/engineering4v2.png)

**Figure 4.**The illustration of our designing of negative feedback loop.
The IL-6\_scfv-Notch-Gal4KRAB acts by inhibiting CAR transcription to reduce the number of CAR on the cell membrane. The IL-6R-PD-1 acts by directly inhibiting the downstream pathway of CAR.

We think of three ways to create a negative feedback loop for CAR-T activation. In the first case, we created a synthetic Notch receptor, where IL-6-scFv is fused with Notch core domain and Gal4KRAB domain. The IL-6-scFv is found to effectively reduce the serum IL-6 level through binding mechanism when equipped on CAR-T cells (Tan et al., 2020). The Notch core domain contains two proteolytic cleavage sites. When the synthetic Notch binds to its ligands (which is IL-6 in this context), the Notch core domain will undergo two subsequent cleavage events (Morsut et al., 2016). As a result, the intracellular domain is released (which is Gal4KRAB in this context). We chose UAS-pSV40 as the promoter for CAR so that we hope the expression of CAR can be inhibited by high level of IL-6. (See Parts: BBa\_K4175008)

The timing of inhibition is critical in the context of CAR-T therapy as we do not want the CAR-T be inhibited before it sufficiently exerts its cytotoxic effect. In other words, the affinity is crucial. Therefore, in the second case, we swapped the extracellular domain of the synthetic Notch receptor above with extracellular part of IL-6R (aa 1-309). Because IL-6-scFv and IL-6R have different affinities, we wanted to see which synthetic Notch is better in the context of CAR-T therapy. (See parts: BBa\_K4175010)

In the third case, we fused extracellular part of IL-6R (aa 1-386) with intracellular part of PD-1 (aa 191-289). This receptor cannot inhibit the expression of CAR upon binding to IL-6, but instead directly downregulate the CAR activity by disturbing the downstream pathway (Sharpe and Pauken, 2018). (See parts: BBa\_K4175011)

# Build 2: How can we build it?

We constructed four major DNA fragments in the negative feedback loop, which were IL6R – Notch – Gal4KRAB [(BBa K4175010)](http://parts.igem.org/Part%3ABBa_K4175010), IL6-scFv – Notch – Gal4KRAB [(BBa K4175008)](http://parts.igem.org/Part%3ABBa_K4175008), IL6R – PD1 [(BBa K4175011)](http://parts.igem.org/Part%3ABBa_K4175011), and UAS-pSV40 – CAR19 [(BBa K4175012)](http://parts.igem.org/Part%3ABBa_K4175012). IL-6R sequence and PD-1 sequence were found on Ensembl. IL-6-scFv sequence was obtained from Lens (Application No: 201917040387) (Adrian and Dario, 2019). The sequence of Notch core domain was obtained from the published paper of Prof. Morsut (Morsut et al., 2016). Gal4KRAB [(BBa K2446037)](http://parts.igem.org/Part%3ABBa_K2446037) and UAS-pSV40 [(BBa K511003)](http://parts.igem.org/Part%3ABBa_K511003) sequence were obtained from parts posted by iGEM17\_Fudan and NMU\_China 2021, respectively. P2A part was obtained from [(BBa K1537016)](http://parts.igem.org/Part%3ABBa_K1537016) designed by iGEM14\_UESTC-GreenLife. Furthermore, our plasmid backbone, MND63, with CAR19 including CD8 transmembrane domain, CD8 hinge, 4-1BB domain, CD3-ζ domain, and mCherry was presented by our primary PI, He, Huang. Each sequence except for UAS-pSV40 promoter has been codon optimized for human T cells by GenScript. Each part was ligated according to the plasmid map below and entrusted to GenScript company for plasmid synthesis, using seamless cloning. Then we transfected Jurkat cells with these plasmids to create CAR-T cells with internal negative feedback loop (NFL).

![](https://static.igem.wiki/teams/4175/wiki/img/engineering/engineering5a.png)
![](https://static.igem.wiki/teams/4175/wiki/img/engineering/engineering5b.png)
![](https://static.igem.wiki/teams/4175/wiki/img/engineering/engineering5c.png)

**Figure 5.**The map of plasmid we transfected into different Jurkat cells.

# Test 2: What is the effect of our switches?

![](https://static.igem.wiki/teams/4175/wiki/img/results/negative-feedback.png)

**Figure 6.**Results of IL-6R-PD-1 (blue, Raji; orange, Raji cocultured with untransfected Jurkat; gray, Raji cocultured with transfected Jurkat). The unit of IL6 concentration is pg/ml.

After transfecting Jurkat cells with these plasmids and confirming the expression of synthetic receptors and CARs using flow cytometry, we co-cultured these CAR-T cells with leukemic cell line, Raji, and measured the cytotoxicity over time. In human, the normal concentration of IL-6 is maintained in a range of 0 ~ 2.9 pg/ml, and in most severe cases of CRS, the level can reach 1000 pg/ml. Here, we co-cultured CAR-T cells with or without NFL we designed in media containing different concentrations of IL-6 (0, 1, 10, 100, 1000 pg/ml). Unfortunately, we found that high concentration of IL-6 failed to inhibit the cytotoxicity effect in CAR-T cells equipped with IL-6-scFv-Notch-Gal4KRAB or IL-6R-Notch-Gal4KRAB. However, as for CAR-T cells transfected with IL-6R-PD1, we found that the CAR activity has been successfully inhibited at high level of IL-6.

# Learn 2: What is going on with our designed switches?

We unfortunately found that the IL-6-scFv-Notch-Gal4KRAB and IL-6R-Notch-Gal4KRAB did not work as we expected. There were some possible explanations for this. Morsut et al. reported that for synthetic Notch receptors, when the ligand is soluble other than presented on the cell membrane, the receptors cannot be activated. This may indicate that synthetic Notch is not suitable for responding to soluble IL-6 in our case. Furthermore, the transfection efficiency in our experiment is not high, and the use of bipromoter systems may cause compromised expression of the latter protein, CAR. Both of these may bias our results.

For future experiments, we think we should pay more attention to IL-6R-PD-1 system and further investigate its inhibitory effects in the context of fluctuating IL-6 levels as seen in a CRS patient. From this, we could also test if the inhibition is reversible or not. We should also try other transfection method to improve transfection efficiency to make our result more accurate and credible.

# Research 3: How to alleviate cytokine release syndrome (CRS) through the green small molecule switch?

The background about CRS has been described above. The main switch being studied now is by means of a small molecule switch, and Prof. Ye Haifeng recently published a study on regulating CAR-T cells by means of a resveratrol switch to avoid CRS (Yang et al., 2021). The switch regulates CAR expression (ON) and inhibition (OFF) in T cells by resveratrol, which leads to the control of CAR-T cell immunotherapy and improves the safety of tumor immunotherapy. Another study controlled the viability of CAR-T somatic cells in solid tumors by focused ultrasound, which could mitigate off-target effects and reduce the damage of CAR-T to all normal structures (Wu et al., 2021). Considering that anti-aging therapy does not need to be as fast as anti-cancer therapy, but needs to be continuous, stable and gentle, and considering the other effects of small molecule drugs on body metabolism, choosing common molecules would be more beneficial for public recognition as well as circumventing the adverse effects of drugs. A genetically encoded caffeine manipulation synthesis module (COSMO) serves as a powerful chemically induced dimerization (CID) system. COSMO is capable of chemically genetically manipulating biological processes via caffeine and its metabolites as well as caffeine-containing beverages including coffee, tea, soda, and energy drinks. This CID tool evolved from caffeine-resistant nanosomes through a cell-based high-throughput screen. Further rationalized engineering of COSMO leads to 34-217-fold enhancement in caffeine sensitivity (EC50 = 16.9) (Wang et al., 2021). We fused the protein to the chimeric antigen receptor in the hope that the protein could block the signal from the single-chain fragment variable while activating its downstream signal in the presence of caffeine.

![](https://static.igem.wiki/teams/4175/wiki/img/engineering/engineering7.png)

**Figure 7.**The illustration of our designing of CAR-COSMO. When caffeine is not present, the CD3ζ structural domain cannot be activated even in the presence of antigen-activated CAR. When caffeine was present without antigen, 4-1BB could not be activated. In both cases, CAR-T cannot be activated to complete the kill. When antigen and caffeine are both present, CAR-T cells are activated to clear target cells.

![](https://static.igem.wiki/teams/4175/wiki/img/engineering/engineering8.png)

**Figure 8.**The map of plasmid (CAR-COSMO) we transfected into different Jurkat cells.

# Test 3: What is the effect of our switches?

![](https://static.igem.wiki/teams/4175/wiki/img/results/cosmo.jpg)

The CAR-COSMO-M killing system showed good killing after the addition of coffee, but the CAR-COSMO modulation system did not seem to completely inhibit the killing of Raji cell lines by CAR-M. Also, the CAR-M system did not show killing in the absence of coffee, which is not as expected. Considering the effect of coffee color on the measured value of the zymograph, we believe that this result is in error with the real result, although the CAR-COSMO-M killing system exhibited good killing in the presence of caffeine. We will explore the removal of the pigment in caffeine or the use of flow-through assays to eliminate the effect of coffee color on the experimental results.

# Learn 3: What is going on with our designed switches?

We used an enzyme marker to measure the data, but did not take into account the effect of the color of the coffee itself on the data. In addition, we chose macrophages as the chassis cells, which were not in full contact with the target cells (Raji cell line suspension cells), which may lead to a less effective killing.

## References

Adrian, T.H.J., Dario, C., 2019. Neutralization Of Human Cytokines With Membrane-bound Anti-cytokine Non-signaling Binders Expressed In Immune Cells. US 2021/0017271 A1.

Amor, C., Feucht, J., Leibold, J., Ho, Y.-J., Zhu, C., Alonso-Curbelo, D., Mansilla-Soto, J., Boyer, J.A., Li, X., Giavridis, T., Kulick, A., Houlihan, S., Peerschke, E., Friedman, S.L., Ponomarev, V., Piersigilli, A., Sadelain, M., Lowe, S.W., 2020. Senolytic CAR T cells reverse senescence-associated pathologies. Nature 583, 127–132. https://doi.org/10.1038/s41586-020-2403-9

Barrett, D.M., Singh, N., Hofmann, T.J., Gershenson, Z., Grupp, S.A., 2016. Interleukin 6 Is Not Made By Chimeric Antigen Receptor T Cells and Does Not Impact Their Function. Blood 128, 654. https://doi.org/10.1182/blood.V128.22.654.654

Childs, B.G., Gluscevic, M., Baker, D.J., Laberge, R.-M., Marquess, D., Dananberg, J., van Deursen, J.M., 2017. Senescent cells: an emerging target for diseases of ageing. Nat Rev Drug Discov 16, 718–735. https://doi.org/10.1038/nrd.2017.116

Kim, K.M., Noh, J.H., Bodogai, M., Martindale, J.L., Yang, X., Indig, F.E., Basu, S.K., Ohnuma, K., Morimoto, C., Johnson, P.F., Biragyn, A., Abdelmohsen, K., Gorospe, M., 2017. Identification of senescent cell surface targetable protein DPP4. Genes Dev 31, 1529–1534. https://doi.org/10.1101/gad.302570.117

Leclercq, G., Steinhoff, N., Haegel, H., De Marco, D., Bacac, M., Klein, C., 2022. Novel strategies for the mitigation of cytokine release syndrome induced by T cell engaging therapies with a focus on the use of kinase inhibitors. Oncoimmunology 11, 2083479. https://doi.org/10.1080/2162402X.2022.2083479

Lian, J., Yue, Y., Yu, W., Zhang, Y., 2020. Immunosenescence: a key player in cancer development. J Hematol Oncol 13, 151. https://doi.org/10.1186/s13045-020-00986-z

Morsut, L., Roybal, K.T., Xiong, X., Gordley, R.M., Coyle, S.M., Thomson, M., Lim, W.A., 2016. Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell 164, 780–791. https://doi.org/10.1016/j.cell.2016.01.012

Norelli, M., Camisa, B., Barbiera, G., Falcone, L., Purevdorj, A., Genua, M., Sanvito, F., Ponzoni, M., Doglioni, C., Cristofori, P., Traversari, C., Bordignon, C., Ciceri, F., Ostuni, R., Bonini, C., Casucci, M., Bondanza, A., 2018. Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells. Nat Med 24, 739–748. https://doi.org/10.1038/s41591-018-0036-4

Sharpe, A.H., Pauken, K.E., 2018. The diverse functions of the PD1 inhibitory pathway. Nat Rev Immunol 18, 153–167. https://doi.org/10.1038/nri.2017.108

Shimabukuro-Vornhagen, A., Gödel, P., Subklewe, M., Stemmler, H.J., Schlößer, H.A., Schlaak, M., Kochanek, M., Böll, B., von Bergwelt-Baildon, M.S., 2018. Cytokine release syndrome. J Immunother Cancer 6, 56. https://doi.org/10.1186/s40425-018-0343-9

Tan, A.H.J., Vinanica, N., Campana, D., 2020. Chimeric antigen receptor–T cells with cytokine neutralizing capacity. Blood Adv 4, 1419–1431. https://doi.org/10.1182/bloodadvances.2019001287

Teachey, D.T., Lacey, S.F., Shaw, P.A., Melenhorst, J.J., Maude, S.L., Frey, N., Pequignot, E., Gonzalez, V.E., Chen, F., Finklestein, J., Barrett, D.M., Weiss, S.L., Fitzgerald, J.C., Berg, R.A., Aplenc, R., Callahan, C., Rheingold, S.R., Zheng, Z., Rose-John, S., White, J.C., Nazimuddin, F., Wertheim, G., Levine, B.L., June, C.H., Porter, D.L., Grupp, S.A., 2016. Identification of Predictive Biomarkers for Cytokine Release Syndrome after Chimeric Antigen Receptor T-cell Therapy for Acute Lymphoblastic Leukemia. Cancer Discov 6, 664–679. https://doi.org/10.1158/2159-8290.CD-16-0040

Xiao, X., Huang, S., Chen, S., Wang, Y., Sun, Q., Xu, X., Li, Y., 2021. Mechanisms of cytokine release syndrome and neurotoxicity of CAR T-cell therapy and associated prevention and management strategies. J Exp Clin Cancer Res 40, 367. https://doi.org/10.1186/s13046-021-02148-6

Wang, T., He, L., Jing, J., Lan, T.H., Hong, T., Wang, F., Huang, Y., Ma, G., and Zhou, Y. (2021). Caffeine-Operated Synthetic Modules for Chemogenetic Control of Protein Activities by Life Style. Adv Sci (Weinh) 8, 2002148.

Wu, Y., Liu, Y., Huang, Z., Wang, X., Jin, Z., Li, J., Limsakul, P., Zhu, L., Allen, M., Pan, Y., et al. (2021). Control of the activity of CAR-T cells within tumours via focused ultrasound. Nat Biomed Eng 5, 1336-1347.

Yang, L., Yin, J., Wu, J., Qiao, L., Zhao, E.M., Cai, F., and Ye, H. (2021). Engineering genetic devices for in vivo control of therapeutic T cell activity triggered by the dietary molecule resveratrol. Proc Natl Acad Sci U S A 118.

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DESIGN

* [Engineered CAR-T cells target DPP4 to clear senescent cells](#Engineered CAR-T cells target DPP4 to clear senescent cells)
* [Inducible Caffeine switches regulate the activation of CAR-T cells](#Inducible Caffeine switches regulate the activation of CAR-T cells)
* [IL-6 based negative feedback loop to shut down the CAR-T cells](#IL-6 based negative feedback loop to shut down the CAR-T cells)

# Engineered CAR-T cells target DPP4 to clear senescent cells

Our experiments are composed of three main parts: **anti-DPP4 CAR-T cells construction，IL-6 based negative feedback loop, and caffeine-based inducible switch.** To prove the feasibility of our designs, we also build a cell line which highly expressed the DPP4, the target of our CAR-T cells. All the proof methods are widely used and reliable experiments.

# Anti-DPP4 CAR-T cells construction

# Part 1: Construction of anti-DPP4 CAR and DPP4-control plasmid

From the NCBI, we got the DNA sequence of the human DPP4 sequence. The anti-DPP4 CAR scFv DNA sequence was got from the patent, CN101282994A. Other DNA sequences of the CAR including signal peptide, CD8 transmembrane domain, 4-1BB domain, and CD3-ζ domain was presented by our primary PI, He, Huang.

Then we assembled our CAR structure which was **anti-DPP4 CAR scFv – CD8 transmembrane domain – 4-1BB domain – CD3-ζ domain.** The leader sequence was added at the front of the CAR. The anti-DPP4 CAR scFv can recognize the DPP4 expressed on the senescent cells surface. Then it will activate the 4-1BB domain and CD3-ζ domain, which activates the CAR-T cells to proliferate and secrete the granzymes and perforins to kill the target cells.

We entrusted the production of our plasmids, DPP4 control plasmid and anti-DPP4 CAR plasmid to the GeneScript company. The plasmid PB-YEF63, which is presented by the He, Huang’s lab, was chosen as our plasmid backbone.

# Part 2: Transfection of plasmids

Both Jurkat and K562 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin solution and maintained at 5% CO2 in air incubator at 37°C.

The DPP4 control plasmid was transfected into the K562 cells through the Advanced DNA RNA Transfection Reagent. The DPP4 plasmid was mixed with the transfection reagent as a ratio of 1:1 and added to the K562 cell medium.

The anti-DPP4 CAR plasmid was transfected into the Jurkat through electrotransfection and Lonza 4D-Nucleofector kit was used.

1. Jurkat cells were precultured to a density of 3 \* 105 cells/ ml before Nucleofection.

2. The Supplement solution was premixed with Nucleofector Solution.

3. Jurkat cells were centrifuged at 90g for 10 min at room temperature and the supernatant was completely removed.

4. Then, the cells were resuspended in 4D-Nucleofector Solution.

5. Add the anti-DPP4 plasmid in the Jurkat cells suspension.

6. The mixtures were transferred into the Nucleocuvette Vessels.

7. The Vessels were then placed into the retainer of the 4D-Nucleofector X unit and the program CL120 was used.

8. The transfected Jurkat cells were incubated in the fresh medium at 37°C.

# Part 3: Detection of positive rate of transfected cells

After transfection, the transfected K562 and Jurkat were collected and examined the positive rate through flow cytometry.

1. 100μL transfected K562 and Jurkat suspensions were collected.

2. Transfected K562 and Jurkat were centrifuged at 600 g for 3 min and then the supernatant was discarded.

3. The precipitation of cells was washed by 300 μL PBS for twice and resuspended by 100 μL PBS.

4. Then the transfected K562 and Jurkat were incubated with anti-DPP4 monoclonal antibodies which conjugated with APC and anti-human IgG monoclonal antibodies which conjugated with FITC, respectively.

5. The positive rate was detected by the flow cytometry.

# Part 4: Examination of anti-DPP4 CAR-T cell cytotoxicity

The anti-DPP4 CAR transfected (CAR-T group) and non-transfected (control group) macrophages were co-cultured with transfected K562-DPP4 cells at a ratio of 1 : 5 in 96-wells plate. After 4h, 8h, 24h, and 48h of co-culture, we collected the cell suspension from CAR-T group and control group. The cells in both groups were incubated with anti-DPP4 monoclonal antibody which conjugated APC and the survival rate of the DPP4 positive K562 cells were detected by flow cytometry.

In addition, we repeated the experiment. We co-cultured 10^5 DPP4-K562 cells with and without 3\*10^5 anti-DPP4 CAR-T cells, respectively, and added the appropriate amount of culture medium. And three sets of replicate experiments were set up. After that 200 µl of the co-culture system (with the addition of 0.2 test DPP4 antibody) was counted by flow collection at 24h.

# Inducible Caffeine switches regulate the activation of CAR-T cells

# Materials

Raji cell line

Raw 264.7 cell line

CAR-COSMO plasmid

Zetalife advanced DNA RNA transfection reagent

# CAR-COSMO-M cells construction

# Part 1: Construction of CAR-COSMO plasmid

From the addgene, we got the DNA sequence of the COSMO sequence (Plasmid #169684). Then we assembled our CAR structure which was anti-CD19 scFv (FMC63) – CD8 transmembrane domain – 4-1BB domain – COSMO - CD3-ζ domain. The leader sequence was added at the front of the CAR. The anti-CD19 scFv can recognize the CD19 expressed on the Raji cells surface. Then it will activate the 4-1BB domain and CD3-ζ domain, which activates the CAR-T cells to proliferate and secrete the granzymes and perforins to kill the target cells.

# Part 2: Transfection of plasmids

Raji were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin solution and maintained at 5% CO2 in air incubator at 37°C. Raw 264.7 were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin solution and maintained at 5% CO2 in air incubator at 37°C. The CAR-COSMO plasmid was mixed with the transfection reagent (Zetalife Advanced DNA RNA transfection reagent) as a ratio of 1:1 and added to the Raw 264.7 cell medium.

# Part 3: Detection of positive rate of transfected cells

We first transfected plasmids into RAW 264.7 cell line on the first day using Zetalife Advanced DNA RNA transfection reagent. The transfection efficiency was measured by flow assay on the third day. After the transfection efficiency test (>90%), we co-cultured 3\*10^5 RAW 264.7 cells and 10^5 Raji cells in a total system of 200 µl. We set three concentrations (0, 1, 10 µl) depending on the amount of coffee (coffee was provided by ZJY Coffee). The number of Raji cells (Raji cells transfected with the luciferase gene) was detected by enzyme marker at 0h, 4h, and 24h after co-culture.

Experiments

(1) CAR-COSMO Plasmid Construction

(2) CAR-COSMO Plasmid transformation and maxi prep

(3) Plasmid transfection into RAW 264.7 cell line through Zetalife advanced DNA RNA transfection reagent

(4) Flow assay for transduction efficiency of Raw 264.7 cells

(5) Raji cell line killing assay and detection of killing effect by enzyme marker

# IL-6 based negative feedback loop to shut down the CAR-T cells

# Part 1: Construction of plasmids of negative feedback loop

Totally four major plasmid fragments were constructed in the negative feedback loop, which were IL6R – Notch – Gal4KRAB (BBa K4175010), IL6-scFv – Notch – Gal4KRAB (BBa K4175008), IL6R – PD1 (BBa K4175011), and UAS-pSV40 – CAR19 (BBa K4175012).

IL6R sequences (amino acids 1 to 309 for BBa K4175010, and amino acids 1 to 386 for BBa K4175011) and PD1 (amino acids 191 to 289) were optimized from Ensembl, while IL6 scFv sequence was optimized from the patent, 201917040387 (Tan Hong Ji and Campana, 2021). The ‘notch’ part was the minimal core transmembrane domain of the wild-type notch, quoted from the published paper of Prof. Morsut (Morsut et al., 2016). Gal4KRAB and UAS-pSV40 part were optimized from parts posted by NMU\_China 2021 (BBa\_K511003 and BBa\_K2446037). P2A part was optimized from BBa K1537016 designed by iGEM14\_UESTC-GreenLife. Furthermore, our plasmid backbone, MND63, with CAR19 including CD8 transmembrane domain, CD8 hinge, 4-1BB domain, and CD3-ζ domain was presented by our primary PI, He, Huang. Each part was ligated according to the plasmid map below and entrusted to GenScript company for plasmid synthesis, using seamless cloning.

# Part 2: Transfection and detection of synthetic proteins

1. Jurkat cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin solution and maintained at 5% CO2 in air incubator at 37°C. The way culturing K562 cells was the same way as that for Jurkat cells, except for culturing IMDM medium.

2. All plasmids were transfected into precultured Jurkat via electrotransfection, using Lonza 4D-Nucleofector kit. Detailed procedures were listed in Anti-DPP4 CAR-T cells construction.

3. After incubation of transfected Jurkat cells for some time, 100 μl of them were collected and examined the positive rate through flow cytometry. Also, detailed procedures were listed in Anti-DPP4 CAR-T cells construction.

# Part 3: Examination of negative feedback loop

1. The Raji cells were transfected with the luciferase gene previously. Transfected Jurkat cells were co-cultured with Raji cells in a 3:1 ratio in 96-well plates at 0 ng/ml, 0.001 ng/ml, 0.01 ng/ml, 0.1 ng/ml, and 1 ng/ml IL-6, respectively. Also, untransfected Jurkat cells were co-cultured with Raji cells in the same condition as transfected groups, treated as the control group.

2. After co-culture for 4h, 8h, 16h and 24h, each group were added with same dose of fluorescein, and the fluorescence intensity of Raji, which is proportional to the survival rate, was measured by microplate analyzer to calculate inhibitory effect.

## References

Morsut, L., Roybal, K.T., Xiong, X., Gordley, R.M., Coyle, S.M., Thomson, M., and Lim, W.A. (2016). Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell 164, 780-791.

Tan Hong Ji, A., and Campana, D. (2021). Neutralization of Human Cytokines with Membrane-Bound Anti-Cytokine Non-Signaling Binders Expressed in Immune Cells (US: NAT UNIV SINGAPORE).

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Results

* [The results of anti-DPP4 CAR-T cells clearing senescent cells.](#The results of anti-DPP4 CAR-T cells clearing senescent cells.)
* [The results of Inducible Caffeine switches regulate the activation of CAR-T cells.](#The results of Inducible Caffeine switches regulate the activation of CAR-T cells. )
* [IL-6 based negative feedback loop to shut down the CAR-T cells](#IL-6 based negative feedback loop to shut down the CAR-T cells)

# The results of anti-DPP4 CAR-T cells clearing senescent cells.

![](https://static.igem.wiki/teams/4175/wiki/img/results/d4-1.jpg)

**Figure 1.** The results of anti-DPP4 CAR-T cells clearing senescent cells.

We co-cultured 10^5 DPP4-K562 cells with 10^5 anti-DPP4 CAR-T cells and 10^5 Jurkat cells, respectively, and added the appropriate amount of medium. And three sets of replicate experiments were set up. Afterwards 200 µl of the co-culture system (with the addition of 0.2 test DPP4 antibody) was counted by flow collection at 24h and 48h, respectively.

![](https://static.igem.wiki/teams/4175/wiki/img/results/dpp4-2.jpg)

**Figure 2.** The results of repeated experiments of anti-DPP4 CAR-T cells clearing senescent cells.

We co-cultured 10^5 DPP4-K562 cells with and without 3\*10^5 anti-DPP4 CAR-T cells, respectively, and added the appropriate amount of culture medium. And three sets of replicate experiments were set up. After that 200 µl of the co-culture system (with the addition of 0.2 test DPP4 antibody) was counted by flow collection at 24h.

As can be seen from the figure, our constructed CAR-T killing system exhibited a significant killing effect in the target cells. This indicates that our killing system was engineered successfully. We will further test the release of cytokines.

# The results of Inducible Caffeine switches regulate the activation of CAR-T cells.

![](https://static.igem.wiki/teams/4175/wiki/img/results/cosmo.jpg)

**Figure 3.** The results of Inducible Caffeine switches regulate the activation of CAR-T cells.

We first transfected plasmids into RAW 264.7 cell line on the first day using Zetalife Advanced DNA RNA transfection reagent. The transfection efficiency was measured by flow assay on the third day. After the transfection efficiency test (>90%), we co-cultured 3\*10^5 RAW 264.7 cells and 10^5 Raji cells in a total system of 200 µl. We set three concentrations (0, 1, 10 µl) depending on the amount of coffee (coffee was provided by ZJY Coffee). The number of Raji cells (Raji cells transfected with the luciferase gene) was detected by enzyme marker at 0h, 4h, and 24h after co-culture.

As the result, The CAR-COSMO-M killing system showed good lethality after the addition of caffeine, but the CAR-COSMO modulation system did not seem to completely inhibit the killing of Raji cell lines by CAR-M, which we believed is in error with the real result. After analysis, we thought that two biases in the experiment lead to erroneous experimental results. The first reason is that we used an enzymatic marker to measure the data but did not consider the effect of the color of the coffee itself on the data. In addition, we selected macrophages as chassis cells, which are not in full contact with the target cells, suspension cells of the Raji cell line. This may result in poor killing efficacy. Anyway, the result showed that the CAR-COSMO-M killing system exhibited good killing in the presence of caffeine, verifying the lethality of our product and the feasibility of the COSMO switch.

# IL-6 based negative feedback loop to shut down the CAR-T cells

![](https://static.igem.wiki/teams/4175/wiki/img/results/nfl-1.jpg)

**Figure 4.** Results of PD1 (blue, Raji; orange, Raji cocultured with untransfected Jurkat; gray, Raji cocultured with transfected Jurkat). The title of each panel represents IL6 concentration (pg/ml).

In humans, the concentration of IL6 is usually maintained at 0.00-2.90 pg/ml. With this in mind, we set up two sets of normal concentrations and three sets of abnormal concentrations to treat the cells that were then transferred. We found that the killing ability of Jurkat was enhanced after IL6 treatment, as shown in Figure 7. In the design concept of module1, we were expecting the expression of CAR to be inhibited at abnormal IL6 concentrations, thus reducing the side effects of killing; hence the problem with the system we designed. As the results of PD1 are shown in Figure 7, we can see that at high concentrations, PD1 successfully disrupts the signaling downstream of CAR-T, reducing the killing ability.

Although the experiment successfully proved that our system is problem-free, the activity of transfected Jurkat is not really high and the choice of dual promoter in the plot resulted in not high CAR expression, thus his killing is not very obvious. In addition, the concentration gradient in the normal group was set low and additional experimental groups should be set up in subsequent experiments.

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