Genetically Engineered CLDN18.2 CAR-T Cells Expressing Synthetic PD1/CD28 Fusion Receptors Produced Using a Lentiviral Vector

This study aimed to develop synthetic Claudin18.2 (CLDN18.2) chimeric antigen receptor (CAR)-T (CAR-T) cells as a treatment for advanced gastric cancer using lentiviral vector genetic engineering technology that targets the CLDN18.2 antigen and simultaneously overcomes the immunosuppressive environment caused by programmed cell death protein 1 (PD-1). Synthetic CAR T cells are a promising approach in cancer immunotherapy but face many challenges in solid tumors. One of the major problems is immunosuppression caused by PD-1. CLDN18.2, a gastric-specific membrane protein, is considered a potential therapeutic target for gastric and other cancers. In our study, CLDN18.2 CAR was a second-generation CAR with inducible T-cell costimulatory (CD278), and CLDN18.2-PD1/CD28 CAR was a third-generation CAR, wherein the synthetic PD1/CD28 chimeric-switch receptor (CSR) was added to the second-generation CAR. In vitro, we detected the secretion levels of different cytokines and the killing ability of CAR-T cells. We found that the secretion of cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) secreted by three types of CAR-T cells was increased, and the killing ability against CLDN18.2-positive GC cells was enhanced. In vivo, we established a xenograft GC model and observed the antitumor effects and off-target toxicity of CAR-T cells. These results support that synthetic anti-CLDN18.2 CAR-T cells have antitumor effect and anti-CLDN18.2-PD1/CD28 CAR could provide a promising design strategy to improve the efficacy of CAR-T cells in advanced gastric cancer.


Introduction
The long-standing dream of curing incurable diseases is gradually being realized, starting with a cure for blood cancer using chimeric antigen receptor (CAR)-T (CAR-T) gene therapy (June & Sadelain, 2018).Gene therapy, a field of synthetic biology, is a cutting-edge technology that corrects or treats diseases by synthesizing therapeutic genes and introducing them into the human body using delivery systems.In the field of CAR-T gene therapy, the United States was the first to conduct clinical trials in 1990 and began to announce clinical results in earnest in 2006.The United States and China are developing the most new drugs, leading clinical trials worldwide, followed by the United Kingdom and Germany (Maus & June, 2016).
Vectors used in gene therapy approved by the US Food and Drug Administration include viral and nonviral vectors.Viral vectors include lentiviruses, adenoviruses, retroviruses, adeno-associated viruses, and vaccinia viruses.Nonviral vectors include chemical delivery methods, such as plasmids, gold nanoparticles, lipid nanoparticles, and N-acetylgalactosamine, and physical delivery methods, such as microinjection and electroporation (Gill & June, 2015;Zhou et al., 2016).Viral vectors are commonly used in gene therapy development.Among them, self-inactivating lentiviral vectors, the safest third-generation lentiviruses, have recently been used in many clinical trials.It introduces four types of plasmid DNAs into production cells: one type of transfer plasmid DNA, two types of packaging plasmid DNAs, and one type of envelope plasmid DNA (VSV-G) (Milone & O'Doherty, 2018).Recently, as a more convenient method, there has been a trend to commercially use integrated, stable producer cell lines containing the above four plasmid DNAs without the need to separately produce transient lentiviral vectors containing each of these plasmids.
Genetically synthetic CAR-T cell therapy, which is attracting attention as a next-generation immunotherapy, is an ex vivo gene therapy developed by transducing lentiviral vectors containing transgenes into mature T cells.CAR-T cells are engineered to be specific for an antigen that is expressed on tumor cells but not on healthy cells (Srivastava & Riddell, 2015).Novartis' Kymriah, the first CAR-T cell treatment, was developed to efficiently destroy cancer cells by introducing a receptor gene into T cells that recognizes the CD-19 antigen expressed on the cancer cells of patients with B-cell malignancies (Gill & June, 2015;Maus & June, 2016).Since the US Food and Drug Administration approved Kymriah in 2017, numerous studies have been conducted on CAR-T treatments (Mohanty et al., 2019).To date, the following six CAR-T gene therapies have been approved only in the field of incurable blood cancer, including CD19 and B-cell maturation antigen (BCMA) targets (Mazinani & Rahbarizadeh, 2022): Kymriah™ (B-cell acute lymphoblastic leukemia [ALL] and B-cell non-Hodgkin lymphoma [NHL]), Yescarta™ (NHL and follicular lymphoma), Tecartus™ (Mantle cell lymphoma and ALL), Breyanzy™ (NHL), Abecma™ (multiple myeloma), and Carvykte™ (multiple myeloma).
CLDN18.2 is transiently expressed in gastric epithelial cells and is rarely expressed in other normal tissues.However, CLDN18.2 expression is abnormally elevated in several cancerous tissues (Niimi et al., 2001).CLDN18.2 is expressed in gastric, esophageal, pancreatic, lung, ovarian, and bile duct cancers and in various other tumors.Accordingly, CLDN18.2 is considered a very promising target for treating CLDN18.2-positivetumors such as gastric and pancreatic cancers and is also recognized as a very challenging target for developing safe and effective transformed CAR-T cells (Morin, 2005).CLDN18.2-specificCAR-T cells are a promising treatment strategy for CLDN18.2-positivetumors.However, further efforts are required to increase the efficacy of solid tumor treatment and reduce the side effects of CLDN18.2-specificCAR-T cell therapy (Jiang et al., 2019;Shi et al., 2023).
Existing CAR-T cells have a limitation in that their activation in vivo cannot be controlled.CAR-T cells commonly cause fatal cytokine release syndrome (Rafiq et al., 2020;Xu et al., 2018).Additionally, the interaction between programmed cell death protein 1 (PD-1) expressed on activated T cells and programmed cell death ligand 1 (PD-L1) expressed on tumor cells may lead to the failure of T-cell therapy (Abate-Daga et al., 2014;Wei et al., 2019).In other words, T-cell activity is inhibited by the binding of the immune checkpoint proteins PD-1 and PD-L1, making it difficult to predict the efficacy of cancer treatments (Lei et al., 2020;Liu et al., 2016;Wei et al., 2019).Therefore, to overcome the problems associated with existing CAR-T cells, synthetic switch molecules must be developed to change the signal between T and target cells (Lorenzini et al., 2023;Yu et al., 2019).In addition to the development of these switch molecules, the development of CAR-T cell therapy with improved efficacy and safety that expands the treatment indications for solid cancers in addition to blood cancer is required (Liang et al., 2021;Weng et al., 2018;Xu et al., 2021).Moreover, researchers have developed one to four generations of CAR structures, of which the second-generation CAR has the structure of CD3ζ (T cell activation signal I) and costimulatory molecule CD28 or 4-1BB (T-cell activation signal II), which is most commonly used in the recent studies (Poorebrahim et al., 2019;Zuccolotto et al., 2021).We used inducible T-cell costimulatory (ICOS) (CD278) instead of the existing CD28 or 4-1BB for applying to solid tumors.In addition, by attaching synthetic PD1/CD28 to the CAR structure using an internal ribosome entry site (Li & Wang, 2020;Mazinani & Rahbarizadeh, 2022), we developed a CAR-T-cell therapy that overcomes the inhibition of T-cell activity by immune checkpoint proteins.
In this study, we designed three types of anti-CLDN18.2CAR-T cells expressing an antigen-binding domain that specifically binds to CLDN18.2 and a switch molecule containing the extracellular domain of the PD-1 receptor and the intracellular domain of the CD28 receptor.CAR-T cells were produced using lentiviral vectors, and their functions and effects were analyzed.

Designing of CAR-PD1/CD28 Constructs and Subcloning into Lentiviral Vector
Anti-Claudin18.2CAR constructs were designed to include an antigen-binding domain, transmembrane domain, costimulatory domain, and chimeric-switch receptor (PD1/CD28).The DNA sequences for each domain and region in the anti-Claudin18.2CAR constructs were chemically synthesized by BIONICS.The synthesized DNAs for CT-001 and CT-002 were digested with the restriction enzymes XbaI and XhoI and subcloned into the CARBioV1-MCS vector, which was modified from pLenti-EF1a-Backbone (NG) (Addgene).
The EZ-FusionTM HT Cloning kit (Enzynomics) was used to subclone the CT-017 construct.The plasmid DNAs were assessed using electrophoresis, and the entire inserted DNA sequence was confirmed by sequencing analysis.Three plasmids carrying different combinations or sequences of anti-Claudin18.2CAR construct elements were obtained and named pCT-001, pCT-017, and pCT-002.

Lentivirus Production
Lentivirus carrying anti-CLDN18.2CAR constructs were produced using the LV-MAX™ Lentiviral System (A35684, Thermo Fisher).Viral Production cells (Gibco) were transfected with the target CAR plasmid and three types of packaging plasmids according to the manufacturer's instructions.At 48 h after the transfection, the culture medium was centrifuged at 1,300 × g for 15 min and then filtered through a 0.45-µm filter.The supernatant was transferred to a fresh tube to harvest the lentivirus.A Lenti-X concentrator (Takara, Cat# 631232) was used to concentrate the harvested lentiviruses.The supernatant with the concentrator was incubated at 4 °C for 24 h and centrifuged at 1,500 × g for 45 min.The produced lentivirus was aliquoted and stored at − 80 °C until use.
To investigate the titer of the produced lentivirus, HEK293 cells were transduced with the diluted lentivirus and incubated for 48 h.After transduction, the supernatant of the cell culture was harvested and the lentivirus titer was determined using flow cytometry.

Generation of Genetically Modified CAR-T Cells
Human peripheral blood mononuclear cells were purchased from Zen-Bio.T cells were activated by incubating CD3 T cells with CTS Dynabeads CD3/CD28 (Gibco) at a 1:1 ratio.Two days after activation, Claudin18.2CAR lentivirus was added at 3 multiplicity of infection to T cells in the presence of 8 μg/ml polybrene.At 24 h post-transduction, lentivirus was removed from the culture, and T cells were further cultured in CTS™ OpTmizer™ T Cell Expansion SFM (Gibco) supplemented with 5% human serum (Sigma) and 200 IU/ml interleukin 2 at 37 °C and 5% CO 2 .CAR expression levels were examined by staining with goat anti-mouse IgG F(ab')2 (Jackson ImmunoResearch) using flow cytometry at 4, 6, 8, and 11 days after transduction.

In Vivo Experiments
Five-week-old female NOG mice were purchased from COATECH Co., Ltd.To establish NCI-N87 xenograft mice, 5 × 10 6 cells of NCI-N87-CLDN18.2were subcutaneously injected into NOG mice.The size of tumors at the cancer cell-injection sites and the body weight of the mice were measured twice per week (Kusakawa et al., 2015).Mice were divided into four groups (NTD-T, CT-001, CT-002, and CT-017 CAR-T) at 23 days post-injection.NTD-T and anti-CLDN18.2CAR-T cells (CT-001, CT-002, and CT-017 CAR-T) were prepared at a concentration of 2 × 10 6 cells/100 µl in cold phosphate-buffered saline.CAR-T and NTD-T cells were injected intratumorally (IT) into mice, and seven days after the first IT injection, CAR-T and NTD-T cells were injected intravenously into mice via the tail vein.The anticancer efficacy of CAR-T cells in vivo was evaluated by measuring the changes in tumor volume and body weight of mice.A bioluminescence imaging system was used for the in vivo assessment of the anticancer activity of CAR-T cells using an in vivo imaging system (IVIS) Spectrum Imaging System (Perkin Elmer).At 7,12,20,23,27,and 33 days post CAR-T cell administration, the mice were anesthetized using isoflurane.Luciferin was injected intraperitoneally 10 min before imaging at 200 µl/mouse as a luminescent substrate.Images were acquired from the lateral position of the mice using the luminescence mode.The total flux value (unit: photons/s) was obtained and assessed from a region of interest of the same size from whole-body images.Data were analyzed using Living Image Version 4.7.2.20319.

Statistical Analysis
Data are presented as mean ± SD.Statistical significance was analyzed using post-hoc tests after one-way ANOVA.Statistical analyses were performed using GraphPad Prism 10 software.Statistical significance was defined as *, **, and *** for p-values < 0.05, < 0.01, and < 0.001, respectively.
We transduced T cells using lentivirus carrying synthetic anti-CLDN18.2CAR constructs to produce genetically engineered CAR-T cells targeting CLDN18.2.The CAR expression was examined using flow cytometry using FITC-labeled goat anti-mouse IgG F(ab')2.CT-001 CAR-T cells showed a CAR expression of 42.35%, CT-002 of 43.57%, and CT-017 of 57.93%.(Fig. 3A).We also assessed the proliferation rate of CAR-T cells.CT-001, CT-002, and CT-017 CAR-T cells and non-transduced T cells expanded approximately 200fold after 11 days of lentivirus transduction (Fig. 3B).
A cytokine release assay was performed to determine the amount of cytokines released from CLDN18.2 CAR-T cells in response to gastric cancer cells.IFN-γ and TNF-α were investigated from the supernatant of co-cultured CAR-T and cancer cells.CT-001 and CT-017 released significantly more IFN-γ and TNF-α in the presence of NCI-N87-C18.2,AGS-C18.2, and KATO-III-C18.2than non-transduced T cells.CT-002 CAR-T cells secreted more IFN-γ and TNF-α against AGS-C18.2 and KATO-III-C18.2than non-transduced T cells.However, in the presence of NCI-N87-C18.2,CT-002 CAR-T cells induced increased secretion of TNF-α but not IFN-γ compared with non-transduced T cells (Fig. 5B).These results suggest that CLDN18.2,CT-001, CT-002, and CT-017 CAR-T cells have anticancer activity against gastric cancer cells in vitro.CT-001 and CT-017 showed greater activity than CT-002.
Fig. 2 The subcloning of plasmids, pCT-001, pCT-002, and pCT-017.The DNA sequences of the self-designed CAR transgene were synthesized and subcloned into a third-generation lentiviral vector modified from the pLenti-EF1a-Backbone (NG).A Lentiviral plasmid maps of pCT-001, pCT-002, and pCT-017.For pCT-001 and pCT-002, the synthesized DNA fragments were subcloned into a lentiviral vector using XbaI and XhoI, while the EZ-FusionTM HT Cloning method was used to generate pCT-017.The total sizes of pCT-001, pCT-002, and pCT-017 are 10169, 10664, and 10293 bp, respectively.B Electrophoresis of plasmids, pCT-001, pCT-002, and pCT-017.Plasmids were prepared from each clone and examined using DNA electrophoresis.The insertions and sequences of the synthesized DNA fragments were confirmed by sequencing

In Experiments
The effectiveness of the anticancer effects following administration of anti-CLDN18.2CAR-T cells was evaluated using an IVIS (Karimi et al., 2014) (Fig. 6).To assess the anticancer activity of CAR-T cells, 23 days after establishing the cancer-inducing animal model, 2 × 10 6 CAR-T cells were injected IT, and after seven days, 4 × 10 6 CAR-T cells were injected intravenously into the mice.The bioluminescence intensity of cancer cells in mice injected with CT-001 or CT-017 CAR-T cells decreased over time and significantly decreased 27 days after injection (Fig. 6).In addition, the tumor volume was measured at 3-day intervals from the time of tumor occurrence to the end of the experiment using a Digimatic caliper (Fig. 7).The results showed that the tumor volumes in each mouse were reduced in the CAR-T celladministered group.Considering that there were no or changes, such as weight loss, in the non-transduced T-cell administration group, the untreated group, and the anti-CLDN18.2CAR-T cell administration group, it was determined that the anti-CLDN18.2CAR-T cell therapy had no in vivo toxicity (Fig. 7).The anticancer efficacy of anti-CLDN18.2CAR-T cell therapy was confirmed in vivo, and additional testing for clinical trials on patients will be possible.

Discussion
We produced synthetic CLDN18.2-PD1/CD28CAR-T cells to increase the antitumor efficacy of CLDN18.2CAR-T cell therapy and verified their efficacy and safety in gastric cancer through in vitro and in vivo experiments.We designed CAR constructs containing ICOS (CD278) Fig. 3 The characteristics of CLDN18.2CAR-T cells.A Expression of CAR on T cells.Activated T cells were transduced using lentivirus encoding synthetic CLDN18.2-CARconstructs, and CAR expression level was assessed using flow cytometry after staining with FITC-labeled goat anti-mouse IgG F(ab')2.B Proliferation curves of CAR-T cells and non-transduced T cells.In all cases, the cells expanded 200-fold 11 days post-transduction as a costimulatory and synthetic PD1/CD28 CSR to enhance anticancer activity of CAR-T cells.In addition, we expanded the indications for CAR-T cell therapy, which is mainly used to treat blood cancer, to solid cancers, designed and manufactured a new concept of CAR-T cell therapy capable of overcoming the inhibition of T-cell activity by immune checkpoint proteins, and confirmed its efficacy.Specifically, anti-CLDN18.2CAR-T cells expressing a binding domain and a switch molecule that specifically binds to the cancer antigen CLDN18.2 were prepared.
In addition, CAR-T cells expressing a fusion protein of the PD-1 receptor, a CLDN18.2-specificantigen-binding domain, and a switch molecule were prepared.These are new concepts for manufacturing CAR-T cells based on the mechanism of action of PD-1 present on the surface of T cells and PD-L1 expressed on cancer cells.CAR-T cells have the advantage of overcoming the inhibition of T-cell activity by immune checkpoint proteins and have increased selectivity and reactivity to specific cancer antigens.The manufactured CAR-T cells exhibited excellent killing ability, specifically against cancer cell lines in which the CLDN18.2gene was introduced.This result is consistent with a previous report showing that the PD-1 receptor is fixed to the cell membrane of CAR-T cells and prevents the inhibition of T-cell activity by binding to PD-L1 overexpressed on cancer cells (Li & Wang, 2020).We found that synthetic CLDN18.2-PD1/CD28CAR-T cells activated the proliferation and differentiation of immune cells (e.g., T cells) due to the intracellular signaling action of the CD28 receptor and increased the secretion of cytokines such as IFN-γ and TNF-α which have immune response regulation and antitumor activities (Chen et al., 2021;Lorenzini et al., 2023).CAR-T cells are expected to effectively treat solid cancers, such as stomach, esophageal, pancreatic, and lung cancers.
In addition, synthetic CLDN18.2-PD1/CD28CAR-T cells (CT-001) expressing an antigen-binding domain that specifically binds to CLDN18.2 and a switch molecule containing the extracellular domain of the PD-1 receptor and the intracellular domain of the CD28 receptor, have excellent cytotoxicity specifically against CLDN18.2positive cancer cells.This result is consistent with the results that CAR-T cell therapy containing the synthetic PD-1/ CD28 switch molecule can prevent the inhibition of T-cell activity due to the binding of PD-1 and PD-L1 and activate T-cell proliferation and differentiation by CD28; therefore, it has the advantage of increasing the complete remission rate for solid cancers, such as pancreatic and liver cancers, more effectively than conventional CAR-T cell therapy (Guo & Cui, 2020).
The corresponding mRNA CAR gene was synthesized using the same constitution as CT-001.The gene was cloned by inserting it into an in vitro transcription vector and an mRNA expression vector, and the cloned pDNA was linearized and then used as a template to perform mRNA in vitro transcription to produce mRNA CAR-T, which was introduced into T cells by electroporation.The cytotoxicity of mRNA CAR-T cells against cancer cell lines was verified using the Calcein AM assay (Invitrogen).In the case of Claudin18.2-CAR-Tcells transduced with mRNA, 98.76% CAR-T cells were produced.In addition, CLDN18.2-CAR-Tcells showed cytotoxicity against cancer lines expressing CLDN18.2, which was more than two-fold than that against cancer cell lines not expressing CLDN18.2.However, in animal experiments, the effect was relatively weak compared with that of CAR-T cells using viral vectors (data not shown).From these results, it was found that mRNA CAR-T cells are easy to manufacture but have stability problems, and further follow-up studies are needed to address these problems.
This study reports the results of research on synthetic CLDN 18.2 PD1/CD28 CAR-T cells for treating gastric cancer.Applying synthetic PD1/CD28 costimulatory receptors to CAR-T cells improves the efficacy of CAR-T cells in solid tumors.The efficacy and safety of synthetic CLDN 18.2 PD1/CD28 CAR-T cells in patients with advanced gastric cancer should be validated in future studies using patient-derived xenograft models and clinical trials.
In the future, the CAR-T cell technology can be used to develop cancer vaccines for recurrent pancreatic cancer and refractory malignant sarcoma along with diseaserelated neoantigen mRNA (Lin et al., 2022;Rojas et al., 2023).Currently, we are developing various genetically engineered CAR-T cells to enhance function using lentiviruses to treat incurable diseases, which is related to methods to improve the TME and overcome antigen heterogeneity  (Ghartey-Kwansah et al., 2018;Lynn et al., 2019;Rafiq et al., 2020;Xu et al., 2018).addition, the synthetic CAR-T cell technology we developed will establish the basic technology for the development of multifunctional CAR-T cells for the treatment of refractory and recurrent diseases and will further accelerate the development of new treatments.Considering that there were no changes, such as weight loss, in any of the groups, it was determined that the anti-CLDN18.2CAR-T cells used in this experiment had no in vivo toxicity Cytotoxicity (%) = (experimental − ef fector spontaneous − target spontaneous) ∕(target maximum − target spontaneous) × 100 2

Fig. 5 Fig. 6
Fig. 5 Cytotoxicity and cytokine release of CLDN18.2CAR-T cells in the presence of Claudin18.2-positivetumor cells.A Target cells were co-cultured with different effector cells at the indicated E:T ratios for 24 h.Cytotoxicity was quantified based on the LDH release.Data represent the mean ± SD (n = 3), and p-values were calculated

Fig. 7
Fig. 7The changes in tumor volume in mice after infusion of CAR-T cells.A Tumor volume.B Body weight.Vehicle: untreated group; CT-001, CT-001 CAR-T cell administration group; CT-002, CT-002 CAR-T cell administration group; CT-017, CT-017 CAR-T cell administration group.It was confirmed that cancer cells in the CAR-T cell administration groups were significantly reduced, which was consistent with the results of an anticancer efficacy test using BLI performed at 12, 20 and 33 days after the administration of CAR-T cells.Considering that there were no changes, such as weight loss, in any of the groups, it was determined that the anti-CLDN18.2CAR-T cells used in this experiment had no in vivo toxicity