Obtaining parts

The structure of CAR and the kill-switch circuit:

2.Verification of the feasibility of our kill switch circuit

1)How to work:

Gal4-KRAB transcription inhibitor was constructed under the control of the NFAT-RE promoter. An inducible caspase-9 suicide gene was placed downstream of the Gal4-KRAB transcription inhibitor under the control of a combined UAS-SV40 promoter. Because the KRAB protein has been demonstrated to be capable of inhibiting all promoters within at least 3 kB and the forward construct would therefore inhibit the NFAT-RE promoter, 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.

When a CAR is activated, consequent NFAT will bind to the NFAT-RE promoter, thus generating Gal4-KRAB. Then Gal4-KRAB will inhibit the promoter UAS-PSV40, thus making the cell survive, while the rest will be induced apoptosis. We postulated that a particular antigen can activate a specific CAR, enabling that specific cell to survive and multiply under the selection pressure caused by the iCAS9 inducer; therefore, the specific CAR will automatically be enriched.

2)Test the kill-switch circuit:

1.Tested cells: NK-92 / NK92 CTX / NK92 TTZ

2.Condition: no stimulator

3.Method: co-culture experiment

4.Result: Data showed that AP1903 had no effect on the viability of NK-92 cells but caused apoptosis/necrosis of the engineered NK cells with no stimulatory cells. After 24h of stimulation with specific antigens, the engineered NK-92 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.

4.Verification of the coverage of the antigen library by the antibody library

In our project, we use the antibody library to fight against the antigen library. The effectiveness of the maneuver depends on the coverage to a large extent. In other words, as long as elements in the antigen library can be covered by the antibody library at a high percentage, we can achieve our goal that all tumor antigens, including neoantigens, can be recognized and eliminated by our CAR-NK92 library armed with the antibody library.

We use mathematical modeling to verify the coverage of the antigen library by the antibody library.

First, we should clarify that there is no absolute binding or non-binding relationship between antigen and antibody. In fact, antibody affinity reflects the ability of an antibody to react with a determinant cluster of an antigen or semi-antigen. It reflects the binding strength between the antibody and the antigen.

So here comes the question.

Q: How large the CAR library can cover the antigen library?

Known information: Extracted from healthy human bodies, the whole scFv library consists of 10^10 Kinds of ScFv.

Solution: We arranged and numbered these scFv from 1 to in the order of structural changes, which means that scFv with approximately close numbers have approximately similar affinity for a certain antigen. We randomly selected 5 numbers from the whole library species as the tumor antigen (each scFv corresponding to the number has the highest affinity to this antigen). Next, we then randomly select and generate a one-dimensional array of (or ) numbers from the full library. As mentioned before, each array represents our CAR library. Here, based on the results of the proliferation model, about 4-5 CAR-NK92 cells will exert targeted killing effects after CAR-NK92 library injection, so we chose 5 random antigens as antigenic mimics for in vivo tumors. We subtract each value in the array from the random number representing the antigen and take the minimum absolute value. We call this minimum absolute value the affinity gap.

Then the problem of setting the affinity gap threshold arises. CAR libraries of size have shown good coverage in the previous proliferation models. And after completing one hundred sets of pre-experiments for a CAR library of size we found that the average value of affinity gap for each antigen for a CAR library of this size is around 5600. Considering the discrete values and large variance that appeared in the pre-experiments, we finally set the threshold value of affinity gap at 10,000.

In each set of simulated experiments, if the affinity gap of all five antigens is less than 10,000, it means that the CAR library has effectively covered the tumor in vivo in this experiment. When all simulations are completed, the number of validly covered groups divided by the total number of simulated groups is the coverage rate of this size CAR library.

After running the simulated experimental program, we found that the coverage rate can approach 100 percent when the library size reaches a baseline between 10^6 and 10^7. However, the larger the CAR library capacity is, the higher the technical difficulty and cost of implementation. Furthermore, when the size reaches , 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 10^6 and 10^7 .

Safety concerns

Experimental safety

The Biosafety Law of the People's Republic of China was adopted at the 22nd Meeting of the Standing Committee of the 13th National People's Congress on October 17, 2020. The experiments of NMU-China iGEM were carried out under the premise of complying with relevant laws.

Safe Lab Work

Strict laboratory safety training is necessary. After understanding IGEM safety rules, we invited our advisor Shi Hu to the laboratory to conduct safety education for all members. We required each member to strictly abide by the safety guidelines of the laboratory and the school.

Blood transfusion from volunteers (future prospect)

The source of our scfv library is peripheral blood from around 200 healthy volunteers. The procedure is similar to blood donation, harmless to the human body.

Negative selection (future prospect)

To construct an off-the-shelf library, we will conduct a negative selection and reserve the negative ones to form the final library. The material for negative selection is HLA antigens, also from volunteers, but in a much greater number. For convenience and safety, HLA antigens are extracted from leukocytes, which can also be acquired through blood drawing, harmless to the human body. Then a human HLA library will be constructed by HLA antigens from a large number of volunteers. We will knock out those scfvs which have a high affinity to the human HLA library and reserve the negative ones to form the final library. Theoretically, the final library is an off-the-shelf one as long as the quantity of HLA volunteers is large enough to roughly represent humankind.

The reason why we choose HLA antigens (future prospect)

The largest antigenic difference between individuals is HLA antigens, and cross-matching is always performed before the organ transplantation to prevent mutual attacking heterologous antigens (mainly HLA antigens) between individuals. This phenomenon can also be avoided with the negative selection of HLA antigens on a large scale in our project. And in order to strictly exclude the binding of the scFv library to the patient's HLA antigens, it will be confirmed by experiments before treatment.

NK-92 cell line

NK-92 MI cell line was purchased from American Type Culture Collection (ATCC) and identified by short tandem repeat sequence analysis. Clinical trials have shown that NK-92 infusion is safe even at high doses(Suck et al., 2016). Irradiation is usually needed before using the NK-92 cell line for safety concerns(Klingemann et al., 2016). Here we use a kill switch circuit controlled by exogenous AP1903 to serve as an alternative way to control the population.

AP1903

As a dimerizer agent, AP1903 has been widely used to serve as the exogenous inducer of a safety switch to improve the safety of CAR-T.(Amatya et al., 2021)A study has shown that AP1903 was safe and well tolerated when administered to healthy male volunteers at dose levels up to 1 mg/kg over a 2-hour infusion period.(Iuliucci et al., 2001) After performing literature review and modeling, we estimated the suitable concentration of AP1903 for the human body in our project is around 0.4-0.6 mg/kg, which is within the safety range.

Lentivirus carrier

Lentivirus packaging kit was purchased from Shanghai Ji kai Ji Yin Chemical Technology Co., LTD. Lentivirus vectors including pCDH-iCAS9-KRAB lentivirus vector and pCDH-scFv-CAR lentivirus vector were used to transfect NK-92 cells to get targeted role and has the suicide lines of NK cells. Studies have shown that lentivirus vector have important biological safety characteristics(Schambach et al., 2013), its application in the body is relatively safe. The potential risks from lentiviral vectors are dependent upon the nature of the exposure. Aerosol exposures through droplet transmission are another potential route of lentiviral vector exposure(Schlimgen et al., 2016). And the non-specific nature of transgene integration by the viral integration machinery carries an inherent risk for genotoxicity(Schenkwein et al., 2020). When operating lentiviral vector, we will use commercial Lentiviral vector production kit in a biosafety class 2 cabinet, strictly following its protocols and procedures. Lentivirus operations that do not involve animal testing in a biosafety cabinet (BSL-2 level) are considered sufficient by the UK ACGM guidelines.

CAR-NK92

Compared to CAR-T therapy, CAR-NK92 cells will not cause graft-versus-host reaction (GVHD) and secrete inflammatory factors that cause CRS. Also, as the NK-92 cell line can be purchased and cultured, CAR-NK92 therapy is more likely to be an off-the-shelf one than CAR-T.

In our cellular experiments, we have demonstrated some safety aspects of our CAR-NK92 cells.

1)Specific killing effect: CAR-NK92 cells only kill specific cells with corresponding antigens, harmless to other cells.

2)Feasibility of the kill switch circuit: CAR-NK92 cells with no targets can be induced Apoptosis by exogenous AP1903 to ensure safety, as we have mentioned above.

Pandemic Prevention and Control

It was still a special time when the COVID-19 is raging, so we stipulated that our members must wear masks when entering the laboratory and disinfect their hands before and after the experiment to avoid contracting COVID-19. We've been granted a safe, healthy environment on campus to conduct our experiments.