Overview
Our CAR-NK92 library is a group of CAR-NK92 loading with several scFvs on the surface and a "target-missing" suicide circuit was built inside. When killing tumor cells, it works as follows: CAR-NK92 cells that recognize tumor antigens are allowed to proliferate due to the activation of the Zap70-NFAT pathway while the rest are induced apoptosis. The survival cells become the dominant population, enriching around the tumor and killing tumor cells until the corresponding antigens completely disappear.
We endowed our CAR-NK92 library with a synthetic system consisting of：
1. CAR library: recognize corresponding tumor antigen, and provide NFAT when successfully recognizing the antigen
2. Kill switch circuit:
①inducible casp9 section: induce suicide of CAR-NK92 controlled by exogenous AP1903
②Suicide repressor section: repress suicide, controlled by Zap70-NFAT pathway
More information about our circuits is detailed in the following parts.
In vitro experiments
To preliminarily verify the feasibility of this kill switch circuit, we first performed cellular experiments, which verified the specific killing effect of CAR-NK92 cells and cell dynamic growth with the kill switch circuit. Detailed design and related information will be mentioned below and more information about experiments can be found in our experiment part.
Classic cell: the NK-92 cell lines
After literature review, we found that: NK cells are important tumor-killing immune cells, and compared to CAR-T therapy, NK cells have many excellent properties as chassis cells.
1. Allogeneic NK cells do not cause graft-versus-host reaction (GVHD), which has been confirmed in many clinical trials.
2. NK cells do not secrete inflammatory factors that cause CRS, such as IL-1 and IL-6.
3. In addition to CAR-mediated killing, CAR-NK cells can also identify and kill tumor cells in a missing-self way to improve the immunotherapeutic effect.
4. Allogeneic NK cells have a wide range of sources, including peripheral blood, NK cell lines, UCB (umbilical cord blood), iPSC (induced pluripotent stem cells), NK-92, and other cell lines. And T cells used for CAR-T mostly originate from the patients themselves or human healthy donors.
However, the proliferative rate of NK cells does not meet our requirements. Therefore, we put our eyes on NK-92 cells. Derived from tumors, NK-92 cells have a considerable proliferative rate. At the same time, NK-92 cells do not need to be extracted from the patients and are more convenient to be cultivated and genetically modified. The modified cells have the potential to be applied in homologous allogeneic transfer, promising to be a truly off-the-shelf product. This advantage further shows the superiority of NK-92 cells over T cells and other immune cells in this project.
Target cell: MCF-7 cells and the derivative cells
Target cell: MCF-7 cells and the derivative cells
1.MCF-7 cells (EGFR- and HER2-negative cells)
2.MCF-7 EGFR cells (a derivative engineered to express EGFR)
3.MCF-7 HER2 cells (a derivative engineered to express HER2)
Module 1 CAR repertoire
Antibody libraries can be constructed based on different sources, such as immunizedanimals or naturally immunized or infected humans, or naive immune systems, which can be derived from nonimmune natural or computational and synthetic sources.
In vitro experiments, the MCF-7 cells and the derivative cells were characterized, which have been used in our previous study. A small library of 10 CARs comprising antigen-specific scFvs was constructed, termed as the CAR-NK92CTX-TTZ library. And the sequences encoding the scFv antibodies were generated from cetuximab(CTX), trastuzumab(TTZ), CH65, 9.8B, 2F5, 7D11, 8D6, omalizumab, TE33, and R10. The sequence information can be found in the Protein Data Bank.
The constitution of CAR
1.The extracellular domain: single chain variable fragment (scFv) library containing 10 scFvs which recognize corresponding antigens respectively
2.The transmembrane domain: CD8 protein allowing signal transduction
3.The intracellular domain: CD28 and CD3ζ for signal transduction
4.Myc tag is a fluorescent protein gene for subsequent detection of CAR transduction
Module2 inducible casp9 section of the kill switch circuit
For the purpose of rapid proliferation, the infinite proliferation capacity of NK92 cells is appropriately preserved. However, unrestrained proliferation can lead to a very large quantity of each CAR-NK92, which is extremely detrimental to both ensuring a normal immune environment and maintaining killing capacity. To ensure safety, we introduce a kill switch circuit and iCASP9 serves as a suicide gene controlled by exogenous AP1903.
As we have mentioned in iGEM2021, AP1903 is an excellent candidate for this inducible kill switch (Clackson, et al., 1998), as AP1903 has no other biologic effects in vivo(Straathof, K. C., et al.,2016). Remodeled dimers such as AP1903 are ideal reagents for controlling the activities of cells that have been modified by gene therapy procedures, without interference from endogenous FKBP(Clackson, et al., 1998).
The constitution of the inducible casp9 circuit
1.Upstream elements: Gal4-KRAB, generated from suicide repressor circuits when recognition happens, repressing the promoter of the iCASP9
2.Promoter: 5*UAS-Prosv40
3.iCASP9: inducible casp9, placed downstream of the Gal4-KRAB transcription inhibitor under the control of a combined UAS-SV40 promoter
4.2A peptide: allow the fusion peptide chain to be separated into two peptide chains
5.GFP: green fluorescent protein
Detailed information about our elements
Gal4-KRAB: (Actually, we have mentioned it in the wiki of our last project)
It is a mammalian synthetic transcription factor based on the Gal4 DNA binding domain (DBD)(Pengue et al. 1994) and KRAB transcription repression domain. Gal4-KRAB contains three core domains from N-terminal to C-terminal: GAL4 DNA binding domain, nuclear location sequence (NLS), and KRAB transcription repression domain(Morsut et al. 2016). And a (G4S) linker was added between DBD and NLS for providing region flexibility(Witzgall et al. 1994; Chen, Zaro, and Shen 2013). GAL4 DBD is capable of binding to specific DNA sequences and KRAB repressing the expression of the downstream gene, so we used Gal4-KRAB as a transcription repression factor to inhibit the activation of downstream synthetic promoter 5*UAS-Prosv40.
1.KRAB（Kruppel-associated box）: KRAB is the N-terminal reserve area of Kruppel-associated protein, Kid-1, which can reversibly inhibit gene expression by forming heterochromatin via recruiting various histone mediation factors(Wang et al. 1997). The KRAB protein has been demonstrated to be capable of inhibiting all promoters within at least 3 kB(Deuschle, Meyer, and Thiesen 1995). Strong transcriptional repression was observed when the KRAB domain was bound both at near or kilobase distances from the start site of transcription.
2.Gal4: Gal4 is a yeast transcriptional activator consisting of 881 amino acids. The DNA binding activity of Gal4 is located in the 74 amino acids in the N terminus (Keegan et al. 1986). The transcriptional activation function of Gal4 is mapped in two regions (residues 148–196 and 768–881) (Ma & Ptashne 1987). Gal4 binds to its specific recognition sequence UAS (upstream activating sequence) and activates the transcription of target genes. It has been demonstrated that the Gal4-UAS system can operate not only in yeast but also in various animal cells(Fischer et al. 1988).
iCASP9: (also mentioned in our last wiki)
Caspases are very important regulators of apoptosis induced by apoptosis stimuli (Stennicke and Salvesen, 1998). iCasp9 can be activated by AP1903 which has proven safe at the required dose for optimum deletional effect(Iuliucci, J. D., et al.,2001). Caspase9 will subsequently activate downstream effector caspases, such as caspase3, and ultimately induce apoptosis.
Module3 Suicide repressor section of the kill switch circuit
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.
The constitution of suicide repressor circuits
1.Upstream elements: NFAT, generated from Zap70-NFAT pathway when recognition happens, binding with the promoter Pro6*NFAT-RE
2.Promoter: Pro6*NFAT-RE
3.Gal4-KRAB
Note: We should pay special attention to the fact that because 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 the opposite ends and at a long distance.
Detailed information about our elements
Zap70-NFAT pathway: ( ITAM - Zap70 - PLCγ1 - PIP2 - IP3 - Ca2+ - Ca2+/CaM - calcineurin - NFAT)
The early event of TCR activation is the phosphorylation of ITAMs on the cytoplasmic side of the TCR/CD3 complex by Lymphocyte-specific protein tyrosine kinase (Lck). Zap70 is recruited and activated, aggregating to the TCR/CD3 complex, thus initiating recruitment and phosphorylation of downstream proteins. SLP -76 is phosphorylated by Zap70 and promotes the recruitment of downstream proteins, including an inducible T cell kinase (Itk). PLCγ1, phosphorylated by Itk, leads to hydrolysis of PIP2, producing DAG and IP3. IP3 triggers Ca2+ release from the ER and entry of extracellular Ca2+ into the cell. Ca2+/CaM activates calcineurin, which promotes gene transcription via the transcription factor NFAT.
NFAT: NFAT proteins are best characterized as transcription factors that induce genes important in cellular processes. In the canonical pathway first elucidated in immune cells, NFAT is activated as a result of calcium flux released from endoplasmic reticulum stores and from the extracellular environment through the activation of store-operated channels in the plasma membrane.
In the basal state, NFAT is hyperphosphorylated in the cytoplasm. Subsequent to cell stimulation and calcium release, NFAT is dephosphorylated by the phosphatase calcineurin and translocates to the nucleus where it cooperates with other factors and co-activators to promote de novo gene transcription(Mancini, M., & Toker, 2009).
Modeling of in vivo experiments
In the cellular experiments, we got the expected results and verified the feasibility of the genetic circuit, which will be mentioned in our experiment part. We next simulated the tumor-killing effect of the therapy in vivo. Detailed information can be found in modeling.
Expected working mode
Stage1 (free proliferation phase): After the CAR-NK92 library injection but with no AP1903 added, CAR-NK92 cells proliferate freely but are monitored within the safe population.
Stage2 (selective enrichment phase): After regular addition of AP1903, CAR-NK92 cells proliferate freely depending on the presence or absence of CAR stimulation.
There are two conditions:
condition1
1. CAR-NK92 cells do not recognize the corresponding antigens.
2. Apoptosis is induced by iCASP9 after the addition of AP1903.
condition2
1. CAR-NK92 cells recognize the corresponding tumor antigens.
2. Zap70-NFAT pathway was activated.
3. NFAT binds to Pro6*NFAT-RE, initiating the synthesis of Gal4-KRAB.
4. Gal4-KRAB binds to 5*UAS-Prosv40 and inhibits the synthesis of iCASP9.
5. The apoptosis is inhibited or even blocked, and the CAR-NK92 cells continue to proliferate and enrich to kill tumor cells.
Stage3 (end of killing phase):
The tumor antigens in Stage2 are all eliminated and CAR-NK92 cells are all induced apoptosis as condition1.
The dynamic change of tumor heterogeneity leads to the alteration of dominant antigens and the appearance of new antigens, so the above process will occur cyclically until all tumor cells are eliminated.
How to therapy for humans and Our advantages
Despite the long way to go from experiment to clinic, we made a preliminary outlook on the future of this project.
Expected general process
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 a 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 off-the-shelf 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.
Note: 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 again by experiments before treatment.
How can we construct a scFv library
1.Blood samples from 200 nonimmunized, healthy volunteers were collected to construct a nonimmunized human single-chain variable fragment (scFv) library. B lymphocyte cDNA encoding a variable fragment was used to construct a phage display scFv library that consisted of ~ 1×10^8 individual colonies. The scFv gene corresponded to the size of the insert of more than 98% of the colonies.
2.To confirm the heterogeneity of the individual clones from the library, we sequenced 50 randomly selected clones, and each clone showed a distinct scFv sequence.
Advantages of our project
1.Cheaper: Because our library is an off-the-shelf one, the cost will be cheaper than CAR-T treatment.
2.Safer: Compared to CAR-T, CAR-NK cells do not bring CRS and GVHD.
3.More effective: Our CAR-NK92 cells can target all tumor antigens (Not only being given the CAR-mediated killing, but NK-92 cells also show stronger cytotoxicity against broad-spectrum tumor antigens.), avoiding tumor recurrence which brings about the trouble of repeated treatment.
Next work and new options
1.Synnotch replaces CAR as the provider of NFAT
2. Optimize Suicide repressor section and explore new ways:
①Use of siRNA (small interfering RNA)
②Use of antisense RNA
Reference
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