Experiments

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

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

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.

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.

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.

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.

You can visit Model page to get more information.

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.