iGEM 2022 — Team nmu-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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Notebook

* [Overview](#pre)
* [January-March](#pro)
* [April](#ins)
* [May](#pg)
* [June](#ref)
* [July](#abc)
* [August](#abd)
* [September](#abe)
* [October](#abf)
* Go to the top

# Overview

We did lots of work in this year and recorded the work here.

![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p84-interview-notes.jpg)

# January-March

## Adrninistrative Work:

① We put out an open poster in the school to recruit members, and about 50 students became prospective iGEMers.

② Publicize the course of synthetic biology, and popularize the knowledge of synthetic biology while selecting the team members.

③ Explain iGEM rules and award requirements to prospective candidates, and let them understand the values that iGEM advocates.

④ Hold the first NMU-China 2022 plenary meeting, let everyone know each other, and understand everyone's strengths.

## Human Practice:

①To learn from old iGEMer, we searched and studied the scheme design and survey and interview methods of Best Human Practice in previous years.

② We conducted a social survey to understand the difficulties in cancer treatment, and summarized the public attitude towards cancer treatment.

③ By analyzing the collected data and information, it is confirmed that tumor heterogeneity is a difficult problem in tumor treatment. So we make it the core problem to be solved in the project.

## Education and Communication:

We introduce iGEM to students in the form of fun classes in the school.

## Laboratory Work:

A literature reading and study were conducted to understand the latest advances in the treatment of tumor heterogeneity and laboratory work.

# April

## Adrninistrative Work:

① We trained the team members' English writing ability, and trained how to build web pages and draw together.

② We learned the best projects of the past few years and summarized them.

③ After brainstorming, We proposed PlanA that express multiple CARs on the same cell to fight with tumor heterogeneity.

## Human Practice:

After the interviews with the doctors, we decided to take the chimeric antigen receptor technology in cell therapy as the basic treatment method, and put forward the improvement plan.

## Education and Communication:

After application, we opened an elective course of synthetic biology on campus.

## Laboratory Work:

①Team members were trained to learn plasmid construction, cell culture, lentivirus transfection and flow cytometry.

②Further understanding of cell experimental design was obtained through literature review

## Model Work:

Mathematical modeling was carried out according to Plan A, and the analysis showed that the optimal number of CAR species expressed by a single cell was 4-8, which was far from the 106 species we needed, so it was not feasible.

# May

## Adrninistrative Work:

① Apply for the team on the official website and pay the registration fee.

② We trained the basic methods of animation video production.

③ After brainstorming, NK-92 cells were registered as chassis cell after weighing the advantages and disadvantages.

④CCiC register

## Human Practice:

We interviewed Associate Professor Meng Guo from the Department of Immunology, Naval Medical University, and invited him to evaluate our project. He believed that when a single cell expresses multiple CARs, we should consider the ability of a single CAR to stimulate downstream signaling pathways. The expression of only a few CARs does not cause strong cluster effect, and the immune cells have poor ability to kill tumors. At the same time, he also proposed PlanB: whether different CARs can be expressed on multiple immune cells to achieve the killing effect.

## Education and Communication:

We have contributed to the revision of the introduction books of synthetic biology and participated in the review and compilation of the data of the Research Manual of synthetic biology

## Laboratory Work:

We preliminarily established a cell bank of 10CAR, and experimentally verified the specific killing ability of CAR-NK92 cells, the feasibility of suicide circuit and the dynamic proliferation ability of CAR-NK92 cell bank based on antigen repertory

## Model Work:

PlanB was fully discussed to determine the issues that need to be further demonstrated .We analyzed on the cell proliferation efficiency of CAR-NK92, CAR-NK92 signaling pathway, and preliminary models were established.

# June

## Adrninistrative Work:

Fill in the safety form, check in form.

## Human Practice:

① We had the first communication with ZJUintl-China where both sides introduced their own projects and put forward opinions and suggestions for each other's projects. We also established a long-term partnership, and they recommended UniProt webscraping for us.

② Associate Professor Guo Meng was interviewed again about the experiment of 10CAR library. He believed that our 10CAR library experiment is of guiding significance for the whole project, but 10CAR library is far from enough to deal with the heterogeneity of tumors. We need to expand the library capacity in a large number, and pay attention to negative selection in this process.

## Education and Communication:

We started to write educational resource package and discussed preliminary discussion of writing scheme, structure design and education audience.

## Laboratory Work:

We improved 10CAR related experiments and optimized the scheme, we also sorted out the experimental results and the were analyzed experimental data.

## Model Work:

We fully discussed AP1903 induced cell apoptosis and CAR-NK92 antibody library coverage issues then made preliminary modeling.

# July

## Adrninistrative Work:

①We started making promotional videos

②We started building the Wiki

③Submit track selection

## Human Practice:

①We had our first conversation with CHUK, who wanted to post our mathematical model on instagram to promote it.

②We had the second communication with ZJUintl-China, they invited us to participate in the preparation of synthetic biology altas, and put forward their own suggestions on the problem of high number of plasmids and low transduction efficiency.

## Education and Communication:

① Started the compilation of Wechat recommended readinbg menu.

② We interviewed stakeholders and improved the content of educational resource package.

## Model Work:

The four mathematical models were further improved, and the basic model was basically completed.

## Part work:

According to the experimental design requirements, we registered 13 new basic components.

# August

## Adrninistrative Work:

① Attend the 9th CCiC

② The safety work of all aspects of the project is fully discussed, and we wroted safety in Wiki.

## Human Practice:

①We completed cost analysis and market analysis of our project to establish the product form and its own advantages.

②We completed risk analysis to understand the risks that may be encountered, and to design the preplan to avoid the risks.

③We completed preliminary design of product development plan and future treatment process.

## Education and Communication:

① Use Blibli to promote the project and let more people know about Whack a Mole and synthetic biology

②Wechat recommended readinbg menu continued to push articles to publicize our project to the public

## Model Work:

According to the four basic models and experimental results, the cellular automata simulation experiment is programmed.

## Part work:

We also improved the BBa\_K3244013 and BBa\_K3755010 parts and updated the BBa\_K4421031 part.

# September

## Adrninistrative Work:

① Participate in academic communication conference

② Upload project promotion videos

③ Submit title and abstract

## Human Practice:

We communicated with Professor Zhou Jingying from CHUK. She believed that compared with CAR-T, which has a short half-life, we can effectively control the life span of NK cells by using suicide circuit to control cell apoptosis, so as to exert better killing effect. In addition, the professor also raised concerns about the safety of our project, thinking that even with the guarantee of suicide circuit, injection of NK-92 still has certain safety risks.

## Education and Communication:

① Go to middle school and present educational resource packs to middle school students in order to communicate our project and synthetic biology.

② We compiled synthetic biology altas, and explained to the public.

③ An educational resource package was written that systematically explained synthetic biology, synthetic immunology, Whack a Mole and iGEM.

## Model Work:

The above model and simulation experiments are optimized to further improve the modeling process and improve the credibility and accuracy.

# October

## Adrninistrative Work:

① Improve Wiki content

② Make the necessary preparations for the presentation

## Human Practice:

Upload relevant content to the Wiki

## Education and Communication:

A presentation of the project was given to freshmen and brochures were distributed.

## Model Work:

Submit the modeling to the Wiki and make the content more readable

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

Engineering success

* [Abstract](#cycle)

* [Cycle 1](#c1)

* [Cycle 2](#c2)

* [Cycle 3](#c3)

* [References](#ref)

* Go to the top

# Overview

![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p38-interview-notes.jpg)

**On this page, we will introduce the engineering design cycle of our entire project in chronological order. In our project, we improved and refined our design over three full engineering cycles, finally culminating in multi-targeted CAR-NK92 cell library that were invincible in “Whac-a-Mole” game.**

![](https://static.igem.wiki/teams/4421/wiki/engineering-p1-threecycles.png)

In the initial phase of the project,the first engineering cycle,we attempted to get a single immune cell which can express multiple CAR in response to multiple antigen targets due to tumor heterogeneity. However, with the continuous progress of our project, we finally rejected this plan through modeling and literature study.

In the second engineering cycle,we took the opposite approach and attempted to construct a population of CAR-NK cells expressing different types of CAR.At the same time, we did modeling and cell experiments to verify our ideas and achieve initial success.

In the third engineering cycle, we tried to use antibody library technology to further expand the variety of CAR carried by our CAR-NK cells to address unpredictable tumor targets.However, due to the serious pandemic situation in Shanghai, our wet experiment had to be canceled.But! through rigorous and substantial modeling, we still get complete results to confirm the feasibility of our design !

#### Next,we'll detailedly explain each step of our engineering cycles as follows.

## **Research 1:** *How can we deal with multi-target tumors?*

Nowadays,traditional treatment including surgery, chemotherapy, and radiotherapy are still recommended more often than novel treatment.However, these treatments not only bring serious side effects, but often fail to solve the metastasis and recurrence of the tumor.Tumor heterogeneity is a major cause of these problems.**How to solve the heterogeneity of tumor without bringing side effects to patients has become our research goal.**

So far there are no good effective means to deal with tumor heterogeneity. At this moment, an article regarding multi-target CAR-T therapy caught our attention (Han et al., 2019). In this paper, the authors constructed a CAR T cell that simultaneously carries three CAR and achieved good efficacy in experiments.

![](https://static.igem.wiki/teams/4421/wiki/arrow1.png)

## **Imagine 1:** *Can we build CAR immune cells which can target more antigens?*

Inspired by this paper, we imagine that can we build CAR immune cells which can target more antigens? At this point we thought the most direct way was to express multiple CARs on a single immune cell.In this way, as soon as any CAR binds to its antigen, the corresponding CAR immune cells concentrate near the antigen and kill the tumor cells that express it.

![](https://static.igem.wiki/teams/4421/wiki/engineering-p2-cellswithantigens.png)

![](https://static.igem.wiki/teams/4421/wiki/arrow1.png)

## **Design 1:** *Expressing multiple CARs on a single immune cell and making amplification.*

So we immediately started to design our circuit.The design of this idea is simple. We only need to transfer the scfv sequences of each antibody designed in advance into the same chassis cell to achieve the purpose of expressing multiple antibodies on one immune cell at the same time.When any CAR on a cell is stimulated by a tumor antigen, the stimulated immune cells proliferate and kill the tumor. In this way, the problem of multiple antigen targets caused by tumor heterogeneity is also solved.

(At this stage, the tentative chassis cells are T cells, as there are a number of clinical trials and applications of CAR T cells engineered from T cells.)

![](https://static.igem.wiki/teams/4421/wiki/engineering-p3-libraries.png)

![](https://static.igem.wiki/teams/4421/wiki/arrow1.png)

## **Build 1:** *Using modeling to test our design*

However, we are also concerned about the maximum number of foreign genes (CAR) that can be expressed simultaneously in a single cell.So before we started the cell experiment, we first tested it with modeling.

![](https://static.igem.wiki/teams/4421/wiki/engineering-p37-minzisuibian.png)

![](https://static.igem.wiki/teams/4421/wiki/arrow1.png)

## **Test 1:** *Modeling test results*

### **Modeling: Limitations on the number and types of CARs that can be expressed in a single cell.**

Studies have shown that multi-targeted CAR-T shows positive efficacy in the treatment of multiple malignancies and becomes an effective way to prevent antigen evasion, but this type of CAR-T cells can be recognized by multiple target antigens, leading to an excessive increase in the level of NFAT activation and over-activation of immune cells, which can easily over-kill normal cells expressing low target antigens. Through bioinformatics analysis, we derived the relationship between the number of types of CARs expressed by individual immune cells and the rate of antigen evasion and over-misfiring of normal cells. Taking colon cancer cells as an example, the number of CAR types should be controlled within 4-8 types.

Furthermore, research has shown that transfection of multiple CARs on a single cell is challenging and expression of multiple CARs on a single cell is prone to ligand non-dependent tonic signaling.

![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p50-interview-notes.jpg)

**The result of our model showed that expressing multiple CARS on a single immune cell,especially when the number of CAR types is large, is not feasible.**

![](https://static.igem.wiki/teams/4421/wiki/arrow1.png)

## **Learn 1:** *Expert advice and online literature study*

Then we consulted Dr.guo, director of immunology Department of Naval Medical University, about our ideas and modeling results.He shared with us his experience in CAR research : In general, the human immune system works in concert. If you want to express multiple CARs on a single immune cell,you have to consider the stimulation of individual CAR on downstream signaling pathways.In other words, if only one or two CARs on these membranes are activated by tumor antigens, they do not form a "cluster effect" that activates downstream signals less strongly, and the immune cells are less capable of killing tumor cells.And it's not clear how many side effects this design might have, given the combined suppression of the tumor microenvironment and other immune cells.

At the same time, he also put forward an opposite way of our design : **Can multiple CARs be expressed on many immune cells? And expressing only one CAR per immune cell?**

**At the same time, we searched online for literature on the feasibility of this design** : Although many research data shows, more targeted CAR - T the curative effect of treatment of various malignant tumors showed positive, become the effective means to prevent antigen to escape, but this type of CAR - T cells can be multiple target antigen recognition and high strength activation of T cells, easy to cause a cytokine storm, and the lower expression of normal cells of the target antigen excessive destruction, Therefore, further optimization in structural design and function is needed to effectively play the role of tumor killing under the guarantee of safety.In addition, in the development process of CAR T cells, the positive rate of CAR transfection is a required test item for the quality control of CAR T cells. However, for multi-target CAR T cells, the expression of 2-3 CARS needs to be detected. Compared with single CAR, the detection of multiple Cars is more difficult. The use of different secondary antibody staining, prone to non-specific background strong, two CAR difficult to distinguish and other problems.The above information further confirms the infeasibility of our first design.

![](https://static.igem.wiki/teams/4421/wiki/engineering-p38-photo.png)

[**Please see Human\_Practices for more details!**](https://static.igem.wiki/teams/4421/wiki/mod-p1.png)

![](https://static.igem.wiki/teams/4421/wiki/arrow1.png)

## **Improve 1:** *Try the opposite way to achieve our goal.*

After listening to Dr.guo's advice, we began to try the opposite way to solve the tumor heterogenity ---expressing multiple CARs on a large number of immune cells but each immune cell expresses only one kind of CAR.

![](https://static.igem.wiki/teams/4421/wiki/engineering-p39-cellwithantigen2.png)

![](https://static.igem.wiki/teams/4421/wiki/arrow2.png)

## **Research 2:** *How can we implement our new idea?*

### Expressing different kinds of CAR on a large number of immune cells is not an easy task.Through the meeting and discussion, we identified two important issues to be solved.

First,because the number of engineered immune cells we can transfuse to patients is limited, the number of immune cells expressing each CAR will be small.It is possible that even after the immune cells come into contact with the corresponding antigen, they are unable to proliferate due to insufficient cardinal number.

![](https://static.igem.wiki/teams/4421/wiki/engineering-p40-patient1.png)

Second, due to the characteristics of cellular immunotherapy, in order to ensure its safe and controllable in human body, we need to design an apoptotic pathway to control their expansion in vivo.

![](https://static.igem.wiki/teams/4421/wiki/arrow2.png)

## **Imagine 2:** *The selection of chassis cell and the effect of gene circuit*

### The selection of chassis cell and the effect of gene circuit are the key to solve the above two problems.

#### the selection of chassis cells :

In the course of cellular immunotherapy, the chassis cells commonly used are T cells.However, T cells are not the best candidates because their proliferative capacity is not very prominent, and T cells can cause severe GVHD and CRS if they accumulate in large numbers in the body.(Zhao et al., 2021)

So we looked at the T cell's neighbor, the NK cell.NK cells have a number of characteristics that T cells do not have: First, allogeneic NK cells did not induce GVHD; Secondly, NK cells do not secrete inflammatory cytokines; Thirdly, in addition to CAR targeted killing, CAR-NK cells can also nonspecifically recognize tumor cells to improve the effect of immunotherapy. Finally, allogeneic NK cells come from a wide range of sources, including peripheral blood, NK cell lines, cord blood, etc.(Valipour et al., 2019)

**These features not only make allogeneic NK cell transplantation safer, but also enable our product to become an off-the-shelf product, bypassing the personalized customization process of traditional CAR T products, thus effectively reducing costs !** [(Please see Cost analysis for more details!)](https://2022.igem.wiki/nmu-china/entrepreneurship)

**Finally, we selected the tumor-derived NK-92 cell line as our chassis cells because we needed our chassis cells to have strong proliferative capacity.** The NK92 cell line, derived from a patient with acute non-Hodgkin's lymphoma, has the advantage of being able to proliferate indefinitely.(Suck et al., 2016)

#### The effect of gene circuit :

Due to the strong proliferative capacity of the NK-92 cell line, we need to design a suicide circuit to ensure the safety of our product.

**So we imagined that the final effect of our gene circuit would be something like this : CAR-NK92 cells that recognize cancer cells survived and proliferated,while CAR-NK92 cells that did not recognize the antigen were induced to apoptosis.**

![](https://static.igem.wiki/teams/4421/wiki/arrow2.png)

## **Design 2:** *The design of gene circuit*

So we initially designed three interconnected genetic circuits

**CAR structure**

![](https://static.igem.wiki/teams/4421/wiki/design-p6-picture.jpg)

**Gal4-KRAB---UAS promoter inhibitory protein**

![](https://static.igem.wiki/teams/4421/wiki/yuansheji2.jpeg)

**iCASP9---Induced suicide switch**

![](https://static.igem.wiki/teams/4421/wiki/engineering-new.jpg)

![](https://static.igem.wiki/teams/4421/wiki/arrow2.png)

## **※ Engineering optimization**

Although the three gene circuits designed by us can theoretically operate independently of each other and do not interfere with each other, the success rate of transfection will be relatively low because the three lines need to be transduced by three independent plasmids.

**So we started to think about whether we could combine the latter two gene circuits to achieve engineering optimization.**

**So we designed the following optimization scheme**

![](https://static.igem.wiki/teams/4421/wiki/suicide-circuit.jpg)

we generated the opposite construct, in which the two expression cassettes were cloned in such a manner that both promoters were at opposite ends and at a long distance.The expression of Gal4-Krab does not inhibit P-NFAT because the two promoters are respectively on the positive and negative strands of DNA.The number of plasmids we use is reduced by one and the success rate of transduction is greatly improved, reflecting our engineering optimization.

#### [**BBa\_K4421027**](http://parts.igem.org/Part%3ABBa_K4421027)

#### [**BBa\_K4421029**](http://parts.igem.org/Part%3ABBa_K4421029)

#### [**BBa\_K4421030**](http://parts.igem.org/Part%3ABBa_K4421030)

[**Please see Design for more details !**](https://2022.igem.wiki/nmu-china/design)

![](https://static.igem.wiki/teams/4421/wiki/arrow2.png)

## **Build 2:** *Building a small CAR-NK92 library*

Next, in order to verify the feasibility of our designed circuit, we buillt a CAR-NK92 library containing ten kinds of established CARs.

We used this part (BBa\_K4421003) to gain ten kinds of CAR.This part is a phage display scfv library which consists of ten kinds of individual colonies:BBa\_K4421011, BBa\_K4421012, BBa\_K4421013, BBa\_K4421014, BBa\_K4421015, BBa\_K4421016, BBa\_K4421017， BBa\_K4421018, BBa\_K4421019 and BBa\_K4421020.

![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p63-interview-notes.jpg)

![](https://static.igem.wiki/teams/4421/wiki/arrow2.png)

## **Test 2:** *Cell experiments and mathematical modeling*

### Cell experiments

#### 1.Verification of specific killing effect of CAR-NK92 cells

![](https://static.igem.wiki/teams/4421/wiki/experiments-verification1-specific-killing.jpg)

The specific killing effect of CAR-NK92 cells in response to tumour cells. The killing effect of CAR-NK92 and control cells against MCF-7 cells was assessed by fluorescent target array killing assays at the indicated E:T ratios. Data are presented as mean ± s.d. of five independent biological replicates.

The specific killing effect was confirmed using fluorescent target array killing assays with MCF-7 cells and derivative cells.

#### 2.Verification of the ability of AP1903 to cause apoptosis when the engineered NK cells are not stimulated

![](https://static.igem.wiki/teams/4421/wiki/1.jpg)

![](https://static.igem.wiki/teams/4421/wiki/2.jpg)

Cells were first stimulated with different MCF-7 cells or unstimulated for 24 or 72 h. The addition of 10 nM AP1903 to the cultures induced apoptosis/necrosis of the CAR-NK92 cells, as assessed by annexin-V staining in four independent experiments. Data are presented as mean ± s.d. \*P< 0.0001 compared with no AP1903, from two-way ANOVA followed by Bonferroni post-test.

Our data showed that AP1903 had no effect on the viability of NK-92 cells but caused apoptosis/necrosis of the engineered NK92 cells with no stimulatory cells. However, after 24h of stimulation with specific antigens, the engineered NK92 cells were notably affected by the reduced pro-apoptotic effect of AP1903, and both the EGFR-specific and HER2-specific CAR-NK92cells were resistant to AP1903 after antigen stimulation for 72h.

#### 3.Verification of the growth dynamics of logic-gated NK-92 cells

![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p58-interview-notes.jpg)

Growth dynamics of the CAR-NK92 cell library in the different co-culture methods. Frequencies of αEGFR and αHER2 CAR-NK92 cells were analysed before the addition of fresh target cells.

The gene circuit was effective in the cell-based CAR screening method and adding AP1903 was essential for the enrichment of CAR NK-92 cells.

### mathematical modeling

#### 1.CAR-NK92 suicide gene pathway feasibility model

Based on the original McKeithan kinetic proofreading model, our CAR activation model was developed by relaxing various constraints, including the range of dephosphorylation rate and the order of phosphorylation and dephosphorylation, to obtain a more consistent phosphorylation model for CAR. In terms of the results, the biological properties of CAR-NK92 cells in terms of sensitivity, specificity and time efficiency in the recognition of exogenous molecules were reflected more successfully and realistically. It has implications for predicting the activity of CAR-NK92 cell gene circuits in animal and clinical experiments.

![](https://static.igem.wiki/teams/4421/wiki/engineering-p20-mathmodelformula1.png)
![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p43-interview-notes.jpg)

#### 2.AP1903 pro-apoptosis model

Optimize the linear model equation and obtain a relatively complete effect change equation to describe the relationship between efficacy, time and concentration of AP1903.

![](https://static.igem.wiki/teams/4421/wiki/engineering-p23-mathmodelformula2.png)(the linear model equation)

![](https://static.igem.wiki/teams/4421/wiki/engineering-p24-mathmodelformula3.png)(a relatively complete effect change equation)

When the initial concentration was set at 0.05, 0.1, 0.5 and 1.0mg/kg and AP1903 was injected at an interval of 12h, the relationship of plasma pharmacodynamics with time was observed.

![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p41-interview-notes.jpg)

When the initial concentration was set at 0.05, 0.1, 0.5 and 1.0mg/kg and AP1903 was injected at an interval of 24h, the relationship of plasma pharmacodynamics with time was observed.

![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p42-interview-notes.jpg)

#### 3.CAR-NK92 proliferation model

Build Lotka-Volterra predator-prey model，importing logistic elements
![](https://static.igem.wiki/teams/4421/wiki/engineering-p42-mathmodeltable1.png)
![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p44-interview-notes.jpg)

(Picture indicates how their numbers change over time)

**In these experiments, we determined the kinetics of the genetic circuit in which NFAT was coupled with KRAB activation in engineered NK-92 cells after CAR stimulation.The effectiveness of the circuit was verified by both cell experiments and mathematical modeling**

![](https://static.igem.wiki/teams/4421/wiki/arrow2.png)

## **Learn 2:** *Reflection and Summary*

Through the above experiments and modeling verification, we preliminarily proved the feasibility of our design.

Our ultimate goal is to solve the problem of multiple antigen targets caused by tumor heterogeneity.To accomplish this, we also need to determine how many CARs are needed in our constructed CAR library to ensure that CAR-NK92 recognizes all tumor antigens (both native and mutated neoantigens).

Through our previous bioinformatic analysis, we were able to determine the number of tumor-surface antigens that were upregulated in tumor cells.(The bioinformatic analysis results are listed below with heat maps, volcano maps, and tables.[Please see Model for more details !](https://2022.igem.wiki/nmu-china/model)) **The results show that there are more than a thousand antigens that may be highly expressed on the surface of tumor cell membranes (including many different tumor models).**

![](https://static.igem.wiki/teams/4421/wiki/engineering-p28-mathmodelfigure6.png)
![](https://static.igem.wiki/teams/4421/wiki/engineering-p29-mathmodelfigure7.png)
![](https://static.igem.wiki/teams/4421/wiki/engineering-p30-mathmodelfigure8.png)

**Therefore, only relying on the prespecified ten kinds of CAR in our previous cell experiments is far from enough to cope with the huge antigen repertoire.**Moreover, because of the uncertainty of tumor cell mutation, we can't predict what the new antigen will be like.

![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p65-interview-notes.jpg)

![](https://static.igem.wiki/teams/4421/wiki/arrow2.png)

## **Improve 2:** *How do we get a CAR library consisting of more CARs?*

We have just analyzed the type and quantity of tumor surface antigens by bioinformatics,but the key question for these surface antigens is how many CARs should be used to cover the antigen repertoire and How do we get a CAR library consisting of more CARs.

※In our project, we envision a giant hammer, our CAR library, that can cover all tumor antigen.**No matter what antigen the tumor cells produce, there will always be a CAR that recognizes the tumor antigen.Just like whack-a-mole, no matter where the moles come from, this giant hammer can always hit them.**

![](https://static.igem.wiki/teams/4421/wiki/engineering-p32-hammerwithqmark.png)

![](https://static.igem.wiki/teams/4421/wiki/arrow3.png)

## **Research 3:***Diversity of antibodies*

Then we further studied and learned about the knowledge related to antibodies.We learned that Burnett's clonal selection theory could explain the diversity of antibodies.

This theory holds that there are many clones of immunocompetent cells in animals, and the cells of different clones have different surface receptors and can complement each other with the corresponding antigenic determinants. Once an antigen enters the body and binds to the receptor of the corresponding clone, it selectively activates the clone, causing it to expand and produce a large number of antibodies (i.e., immunoglobulins), molecules of the same specificity as the receptor on the surface of the selected cell (Cohn et al., 2007).**All in all, the human body can produce at least billions of antibodies against foreign antigens (including tumor antigens).**

![](https://static.igem.wiki/teams/4421/wiki/engineering-p33-scfvlibrary.png)

![](https://static.igem.wiki/teams/4421/wiki/arrow3.png)

## **Imagine 3:** *Using a whole library of antibodies,is it possible?*

Therefore, following the clue of clonal selection theory, we conducted in-depth research on the concept of "antibody library".

The antibody library technique is actually a very old technology.In a narrow sense, the so-called antibody library technology is a technology that uses gene cloning technology to clone the full set of variable region genes of heavy and light chains of antibodies, and then recombine them into specific prokaryotic expression vectors, and then transform Escherichia coli to express functional antibody molecular fragments, and obtain specific variable region genes of antibodies through affinity screening. The antibody genes screened by antibody library technology will be used to construct and express genetically engineered antibodies.

At present, the main application of antibody library technology is to screen a variety of important antibodies from antibody library, such as membrane protein antigen, self antigen, virus antigen antibodies, for clinical treatment or to modify existing antigens to improve the affinity.

**But few people would have thought of using an entire library of antibodies without screening for specific antibodies.Therefore,we imagined that we could use the antibody library technology to combine the sequences of all the light and heavy chains of antibodies to make scfv, and then express them on NK-92 cells to make CAR-NK92, so as to truly realize the CAR library.In this way, both native tumor antigens and newly mutated antigens can be recognized by one of the CARs in the CAR-NK92 library.**

![](https://static.igem.wiki/teams/4421/wiki/arrow3.png)

## **Design 3:** *Antibody library Vs Antigen library*

Based on our previous modeling and data from the literature, it is generally believed that millions of kinds of CARs are sufficient to recognize all possible tumor antigens.

In terms of gene circuit design, we continue to use the circuit designed in the second cycle.

Although the antibody library technology has matured, the digital antibody library on the Internet has not yet been realized, so we would use the phage antibody library to make the CAR-NK92 library.We need to build a library with a million kinds of antibodies that we can use before we actually use them in clinical practice.

![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p66-interview-notes.jpg)

![](https://static.igem.wiki/teams/4421/wiki/arrow3.png)

## **Build and Test 3:** *Cellular automata-Visual dynamic simulation of the CAR-NK92 library competing with tumour cells*

At this stage, due to the impact of the epidemic in Shanghai and our concern for the safety of animal experiments, we cancelled the original experiments in vivo.

But we can still rely on modeling to prove the effectiveness of our CAR-NK92 library !

Our ultimate goal is to address the many neoantigens that come with tumor heterogeneity.Since the process of tumor mutation and proliferation is very similar to the principle of cellular automata, we decided to use cellular automata for modeling.So our team modeled the killing effect of 10^6 kinds of CAR-NK92 cells against tumors and their mutants.

![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p45-interview-notes.jpg)

We also draw a whole flow chart to illustrate this process
**The results showed that a very small number of tumor cells survived at the end in most simulations !**

![](https://static.igem.wiki/teams/4421/wiki/arrow3.png)

## **Learn 3:** *Is it enough for our design?*

Through the above design, experiment and proof, we have tentatively constructed the seemingly invincible CAR-NK92 library.Through meeting after meeting, we found that there was a fatal flaw in our project.**Just building a CAR-NK92 library that contained all the antibodies could not be used directly for therapy.**The reason is that the antibody library contains almost all kinds of antibodies, so there must also be antibodies that can bind with HLA antigens in normal human tissues.This can cause a severe allergic reaction, which can be very dangerous.

Under the guidance of our mentor, we realized that the solution to this problem was not as difficult as we thought.Using **phage display technology** can solve this problem.(Phage display technology is a biotechnology in which the DNA sequence of foreign protein or polypeptide is inserted into the appropriate position of the structural gene of phage coat protein, so that the foreign gene can be expressed with the expression of coat protein, and the foreign protein can be displayed on the surface of phage.)

The derived phage display antibody library technology can display several different antibodies simultaneously on the phage surface. As long as we use healthy human blood cells mixed with the phage display library, after several screening, we can get a phage antibody library that will not bind with healthy human HLA antigens.

Such a screening process, consistent with the body's own production of mature T cells, is called **"negative selection".**

![](https://static.igem.wiki/teams/4421/wiki/engineering-p36-negativeselection.png)

![](https://static.igem.wiki/teams/4421/wiki/arrow3.png)

## **Improve 3:** *Outlook on the future of this project*

#### Despite the long way to go from experiment to clinic, we designed a preliminary outlook on the future of this project.

1. Draw 200 copies of peripheral blood from healthy volunteers.

2. Extract mRNAs encoding antibodies in B cells and use RT-PCR and SOE-PCR to form an scFv cDNA library.

3. Select leukocytes from volunteers on a large scale and obtain their HLA antigens as material for negative selection.

4. After negative selection, reserve the negative ones to form the final library that can be put into use.

5. Before the patient is treated, confirm that the scFv library has no affinity to the patient's HLA antigen.

6. Construct CAR-NK92 cells carrying the scFv library and infuse them into the patient for treatment.

![](https://static.igem.wiki/teams/4421/wiki/engineering-p37-projectfuture.png)

![](https://static.igem.wiki/teams/4421/wiki/arrow3.png)

# Engineering Success !

---

## References

1. Han, X., Wang, Y., Wei, J., & Han, W. (2019). Multi-antigen-targeted chimeric antigen receptor T cells for cancer therapy. Journal of Hematology & Oncology, 12, 128. https://doi.org/10.1186/s13045-019-0813-7

2. Zhao, C., Zhang, Y., & Zheng, H. (2021). The Effects of Interferons on Allogeneic T Cell Response in GVHD: The Multifaced Biology and Epigenetic Regulations. Frontiers in Immunology, 12, 717540. https://doi.org/10.3389/fimmu.2021.717540

3. Valipour, B., Velaei, K., Abedelahi, A., Karimipour, M., Darabi, M., & Charoudeh, H. N. (2019). NK cells: An attractive candidate for cancer therapy. Journal of Cellular Physiology, 234(11), 19352–19365. https://doi.org/10.1002/jcp.28657

4. Suck, G., Odendahl, M., Nowakowska, P., Seidl, C., Wels, W. S., Klingemann, H. G., & Tonn, T. (2016). NK-92: An “off-the-shelf therapeutic” for adoptive natural killer cell-based cancer immunotherapy. Cancer Immunology, Immunotherapy: CII, 65(4), 485–492. https://doi.org/10.1007/s00262-015-1761-x

5. Cohn, M., Mitchison, N. A., Paul, W. E., Silverstein, A. M., Talmage, D. W., & Weigert, M. (2007). Reflections on the clonal-selection theory. Nature Reviews. Immunology, 7(10), 823–830. https://doi.org/10.1038/nri2177

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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Experiment

* [Abstract](#pro)
* [Experiments](#ins)
* Go to the top

# Abstract

![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p36-interview-notes.jpg)

We firstly verified the specific killing effect of CAR-NK92 cells. Through modeling we rejected the plan to express diverse CARs on a single NK92 cell, which led us to express a CAR library on a group of NK92 cells to make up a CAR-NK92 library. We then verified the feasibility of the kill switch circuit and the growth dynamics of logic-gated NK-92 cells according to antigens. Together with statistics we gathered from modeling, we can preliminarily demonstrate that our project is feasible and promising in tumor therapy, especially in addressing tumor heterogeneity.

Our modeling work is detailed in [MODELS](https://2022.igem.wiki/nmu-china/model).

# Experiments

## 1.verification of the specific killing effect of CAR-NK92 cells

### Material：

①Engineered cell: NK-92-CTX and NK-92-TTZ

②Target cell: MCF-7 cells (EGFR-HER2-), MCF-7 EGFR+ cells and MCF-7 HER2+ cells

### Method:

fluorescent target array killing assays

### Result:

![](https://static.igem.wiki/teams/4421/wiki/experiments-verification1-specific-killing.jpg)

**Figure 1** The specific killing effect of CAR-NK92 cells in response to tumour cells. The killing effect of CAR-NK92 and control cells against MCF-7 cells was assessed by fluorescent target array killing assays at the indicated E:T ratios. Data are presented as mean ± s.d. of five independent biological replicates.

NK-92 cells with no CAR performed no killing effect and NK-92-CTX or NK-92-TTZ had specific killing effect only when they encountered corresponding antigens, which verified the specific killing effect of CAR-NK92 cells.

## 2.verification of the feasibility of the kill switch circuit

### Material:

①Engineered cell: NK-92-CTX and NK-92-TTZ, engineered with kill switch circuit

②Target cell: MCF-7 cells (EGFR-HER2-), MCF-7 EGFR+ cells and MCF-7 HER2+ cells

### Method:

co-culture experiment

### Result:

![](https://static.igem.wiki/teams/4421/wiki/1.jpg)

![](https://static.igem.wiki/teams/4421/wiki/2.jpg)

**Figure 2** Cells were first stimulated with different MCF-7 cells or unstimulated for 24 or 72 h. The addition of 10 nM AP1903 to the cultures induced apoptosis/necrosis of the CAR-NK92 cells, as assessed by annexin-V staining in four independent experiments. Data are presented as mean ± s.d. \*P< 0.0001 compared with no AP1903, from two-way ANOVA followed by Bonferroni post-test.

Our data showed that AP1903 had no effect on the viability of NK-92 cells but caused apoptosis/necrosis of the engineered NK92 cells with no stimulatory cells. However, after 24h of stimulation with specific antigens, the engineered NK92 cells were notably affected by the reduced pro-apoptotic effect of AP1903, and both the EGFR-specific and HER2-specific CAR-NK92 cells were resistant to AP1903 after antigen stimulation for 72h.

## 3.verification of the growth dynamics of logic-gated NK-92 cells according to antigens

While AP1093 extensively induces apoptosis, we want to ensure that CAR-NK92 cells that recognize the corresponding antigen, could continue to proliferate and enrich without interference. To achieve this goal, we introduced the suicide repressor section.

### Material:

①Engineered cell: a group of NK-92 cells engineered with 10 different kinds of CARs and the kill switch circuit, making up a 10-CAR-NK92 library.

The origin of 10 CARs: cetuximab, trastuzumab, CH65, 9.8B, 2F5, 7D11, 8D6, omalizumab, TE33, and R10

②Target cell: MCF-7 cells (EGFR-HER2-), MCF-7 EGFR+ cells and MCF-7 HER2+ cells

### Method:

co-culture experiment

### Result:

![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p58-interview-notes.jpg)

**Figure 3** Growth dynamics of the CAR-NK92 cell library in the different co-culture methods. Frequencies of αEGFR and αHER2 CAR-NK92 cells were analysed before the addition of fresh target cells.

Repeat stimulations with MCF-7 EGFR cells or MCF-7 HER2 cells resulted in no significant change in the CAR composition of the library co-cultured with either MCF-7 cells or antigen-positive MCF-7 derivatives without AP1903 treatment, while enrichment of NK-92 CTX and NK-92 TTZ cells was observed after AP1903 treatment in the presence of specific MCF-7 derivatives. These data suggest that the gene circuit was effective in the cell-based CAR screening method and adding AP1903 was essential for the enrichment of CAR NK-92 cells.

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

Result

* [Overview](#pro)
* [Experiments](#ins)* [Model](#mod)* [Product design and development plan](#pda)
    * [Product cost analysis](#pca)
    * [The process of future treatment](#pea)
    * Go to the top

# Overview

Tumor heterogeneity is a huge problem in cancer treatment. Our project aims to develop a highly targeted cell therapy with few side effects and relatively low price, which we believe will be achieved in the near future.

# Experiments

## 1.verification of the specific killing effect of CAR-NK92 cells

![](https://static.igem.wiki/teams/4421/wiki/experiments-verification1-specific-killing.jpg)

The specific killing effect of CAR-NK92 cells in response to tumour cells. The killing effect of CAR-NK92 and control cells against MCF-7 cells was assessed by fluorescent target array killing assays at the indicated E:T ratios. Data are presented as mean ± s.d. of five independent biological replicates.

NK-92 cells with no CAR performed no killing effect and NK-92-CTX or NK-92-TTZ have specific killing effect only when they encountered corresponding antigens, which verified the specific killing effect of CAR-NK92 cells

## 2.verification of the feasibility of the kill switch circuit

![](https://static.igem.wiki/teams/4421/wiki/1.jpg)

![](https://static.igem.wiki/teams/4421/wiki/2.jpg)

Cells were first stimulated with different MCF-7 cells or unstimulated for 24 or 72 h. The addition of 10 nM AP1903 to the cultures induced apoptosis/necrosis of the CAR-NK92 cells, as assessed by annexin-V staining in four independent experiments. Data are presented as mean ± s.d. \*P< 0.0001 compared with no AP1903, from two-way ANOVA followed by Bonferroni post-test.

Our data showed that AP1903 had no effect on the viability of NK-92 cells but caused apoptosis/necrosis of the engineered NK92 cells with no stimulatory cells. However, after 24h of stimulation with specific antigens, the engineered NK92 cells were notably affected by the reduced pro-apoptotic effect of AP1903, and both the EGFR-specific and HER2-specific CAR-NK92cells were resistant to AP1903 after antigen stimulation for 72h.

## 3.verification of the growth dynamics of logic-gated NK-92 cells according to antigens

While AP1093 extensively induces apoptosis, we want to ensure that CAR-NK92 cells that recognize the corresponding antigen, continue to proliferate and enrich without interference. To achieve this goal, we introduced the suicide repressor section.

![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p58-interview-notes.jpg)

Growth dynamics of the CAR-NK92 cell library in the different co-culture methods. Frequencies of αEGFR and αHER2 CAR-NK92 cells were analysed before the addition of fresh target cells.

Repeat stimulations with MCF-7 EGFR cells or MCF-7 HER2 cells resulted in no significant change in the CAR composition of the library co-cultured with either MCF-7 cells or antigen-positive MCF-7 derivatives without AP1903 treatment, while enrichment of NK-92 CTX and NK-92 TTZ cells was observed after AP1903 treatment in the presence of specific MCF-7 derivatives. These data suggest that the gene circuit was effective in the cell-based CAR screening method and adding AP1903 was essential for the enrichment of CAR NK-92 cells.

# Model

## 1.Modeling of number of CAR types

As the number of CARs increased, the antigen evasion rate is controlled to less than 10% when the total number of CARs reaches 4, and the trend of normal cell being killed is about 0.02 at this time. When the total number of CARs reached 8, the antigen evasion rate is still controlled below 10%, but the trend of normal cells being killed is around 0.1. The probability of being mistakenly injured exceed the range when CAR expression types are increased further up, so the ideal range of CARs expressed in a single immune cell is 4-8, which cannot satisfy the requirement of identifying a large number of tumor antigens.

## 2.Mathematical modeling of CAR-NK92 proliferation

Close-to-reality assumptions:the initial number of tumor cells in a local tissue of 300 × 300 units size is 300, and the initial injection volume of CAR-NK-92 is N ml. There are five types of CAR-NK-92 recognized by this tumor surface antigen, five of each, and a total of 25 are able to expand and actually complete the killing process, and the rest will apoptosis due to the modulation of AP1903. The maximum environmental accommodation of tumor cells in local tissues is 5 × 104.

![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p44-interview-notes.jpg)

Population size of tumor cells and CAR-NK-92 over time

During the initial phase, tumor cells expand heavily and quickly reach the environmental holding capacity. At around 70-80 days, CAR-NK-92 start to be recruited around the tumor tissue and some of them start to proliferate and perform the function of killing tumor cells. At around 140 days most of the tumor cells are cleared.

In clinical situation, tumor cells are often difficult to be detected when they first start to proliferate. As can be seen from the prediction diagram, when tumor cells first start to proliferate, it is difficult for CAR-NK92 to detect tumor cells and recruit around them due to the small number of tumor cells. When the tumor cells grow to a certain number, CAR-NK92 starts to initiate proliferation and killing mechanism.

Injected in the first 80 days, the killing effect is the best. The latest injection timing should be controlled on the 70th to 80th day, and the killing effect is basically the same when injected at this time point compared with the first day.

## 3.Modeling of CAR-NK92 signaling pathway

Based on the original McKeithan kinetic calibration model, our CAR activation model is developed by relaxing various constraints, including the range of dephosphorylation rate β and the order of phosphorylation and dephosphorylation, to obtain a more consistent phosphorylation model for CAR. In terms of the results, the biological properties of CAR-NK92 cells in terms of sensitivity, specificity and time efficiency in the recognition of exogenous molecules are reflected more successfully and realistically. It has implications for predicting the activity of CAR-NK92 cell gene circuits in animal and clinical experiments.

## 4.Modeling of AP1903-induced apoptosis of CAR-NK92

According to literature review, the optimal concentration range of AP1903 for injection in NSG mice is 2.5-5mg /kg.The initial injection time of AP1903 in the NSG mouse model is 0, and the interval injection time is 50.0106h(4 decimal places are reserved).This result is basically consistent with the data obtained by referring to relevant AP1903 literature,so the calculation formula is valid.

![](https://static.igem.wiki/teams/4421/wiki/model-ap1903-04.jpg)

Based on the equivalent dose conversion between mice and human models (data from FDA guidelines)

We attempt to further apply the formula to the prediction and guidance of clinical AP1903 injection. According to the equivalent dose conversion method,we obtain that the initial injection time of AP1903 in human body was 3.8147h, and the interval injection time is 4.8060h(4 decimal places are reserved).

## 5.Modeling of CAR antibody library coverage

We find that the coverage rate can approach 100 percent when the library size reaches 107 and 108. However, the larger the CAR library capacity is, the higher the technical difficulty and cost of implementation.

Furthermore, when the size reaches 107, there are about 20 effective scFv per tumor antigen, which is sufficient to ensure that a sufficient number of CAR-NK92s are stocked and perform tumor killing tasks. Therefore, a reasonable CAR bank size should be between 106 and 107.

## 6.Metacellular automata simulation

①We simulate the process of tumor cell removal by CAR-NK92 library using metacellular automata and successfully resolved the tumor heterogeneity.

②We figure out that NK92 with different CARs have different recognition ability to tumor cells:"Central Blossom" killing (one point of recognition and central point of breakthrough) and "Fireworks" killing ( multiple points of recognition and multiple points of breakthrough).

③We find the "protective circle" of tumor cells and the "killing limit" mechanism of CAR-NK92 are important factors for the remnants of tumor, which provide guidance for subsequent experiments.

**Video** Simulation process of metacellular automata

You can visit [Model](https://2022.igem.wiki/nmu-china/model) page to get more information.

# Product design and development plan

Our program has many advantages over conventional anticancer therapies and CAR-T cell therapies
(1)The application of NK-92 cells in chassis cells does not cause graft-versus-host disease, cytokine storm, etc., and has less side effects on the body.
(2)The application of antibody library makes it possible to combat tumor heterogeneity and reduce tumor recurrence.
(3)The tumor-associated antigen is used as an inhibitory signal of suicide circuit to determine the number of immune cells by the dynamic situation of the tumor.
(4)The extraction and recovery of immune cells enriched in patients can provide a new idea for screening tumor antigens
Considering patent factors, we do not set the ultimate goal of the product as a marketed drug, because patent and market factors will cause a significant increase in price. We can set the product as an information repository, and treat the sequence information library obtained from healthy individuals or NK cell library stored in the laboratory, when the patient is willing to accept treatment, we can directly apply the sequence information storage or cell library into treatment.
We designed two plans for our project, and we list their characteristics, advantages, and disadvantages in the follow chart.

![](https://static.igem.wiki/teams/4421/wiki/integrated-hp-p22-picture.jpg)

This is our initial idea. After that, we will listen to the opinions of all parties and improve the project. We have also made the improvement plan of the project

![](https://static.igem.wiki/teams/4421/wiki/policy-and-market-p12-picture.jpg)

# Product cost analysis

Compared with CAR-T, our product has the advantages of being more economical and easier to be accepted by the public. However, since there is no CAR-NK product on the market, it is necessary to demonstrate the cost of the product，it is also a suggest from ZJUtil iGEM. Since the project exists as a repository, the construction of sequence information database and other steps can be completed only with peripheral blood of normal people, which can be used as a common step in the treatment of all patients, embodied the characteristics of off the shelf, for individual patient treatment process, the step of cost is negligible. Defining the project in the form of the repository, can effectively avoid the patent, market and other factors lead to rising prices and uncertainty, so compared with drugs, the repository of cost analysis also has guiding significance for the final price.
To bring these highlights to fruition, we did the following planning for the course of the project, with specific timing to continue adjusting based on subsequent progress. Now, we are at the first phase and we have the opportunity to continue our project after the iGEM competition.

![](https://static.igem.wiki/teams/4421/wiki/policy-and-market-p6-cost-analysis.jpg)

# The process of future treatment

The treatment process of CAR-NK antibody library highlighted the characteristics of off the shelf. First, we extracted peripheral blood of healthy people to isolated and purified B cells, then we extracted total mRNA of B cells, and amplified scfv DNA sequences by RT-PCR and SOE-PCR. Then we used phage display sequence libraries and negative selection were performed to eliminate antibody sequences capable of binding to human normal tissue cells and construct a CAR-NK cell library. After cell culture, the CAR-NK antibody library was injected into the patient, and AP1903 was injected after a period of time. The patient's condition was closely observed, and various indicators were monitored. Follow-up treatment was performed according to the tumor killing.

![](https://static.igem.wiki/teams/4421/wiki/policy-and-market-p7-result.jpg)

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