Minicircle DNA-Engineered CAR T Cells Suppressed Tumor Growth in Mice
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Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1535-7163.MCT-19-0204 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Minicircle DNA-Engineered CAR T Cells 2 Suppressed Tumor Growth in Mice 3 4 Jinsheng Han1 *, Fei Gao1 *, Songsong Geng3 *, Xueshuai Ye1,3, Tie Wang4, Pingping 5 Du3, Ziqi Cai3, Zexian Fu3,7, Zhilong Zhao3,5, Long Shi3,6, Qingxia Li2,3, Jianhui Cai1,2 † 6 7 1. Department of Surgery, Hebei Medical University, 361 West Zhongshan Road, 8 Shijiazhuang, Hebei, China, 050017. 9 2. Department of Surgery & Oncology, Hebei General Hospital, 348 West Heping 10 Road, Shijiazhuang, Hebei, China, 050051. 11 3. Hebei Engineering Technology Research Center for Cell Therapy, Hebei HOFOY 12 Biotech Corporation Ltd., 238 Changjiang Avenue, Shijiazhuang, Hebei, China, 13 050000. 14 4. Department of Surgery, Hebei Cangzhou Hospital of Integrated Traditional 15 Chinese Medicine and Western Medicine, 31 Huanghe road, Cangzhou, Hebei, 16 China, 061001. 17 5. Department of Surgery, the Third Affiliated Hospital of Jinzhou Medical University, 18 40 Section-Three of Songpo Road, Jinzhou, Liaoning, China, 121001. 19 6. Department of Oncology, The Second Hospital of Hebei Medical University, 215 20 West Heping Road, Shijiazhuang, Hebei, China, 050000. 21 7. Department of Surgery, Affiliated Hospital of Hebei Engineering University, 81 22 Congtai Road, Handan, Hebei, China, 056002. 23 * These three authors contributed equally to this work. 24 † Corresponding Author: Jianhui Cai ([email protected], +86 15130644888). 25 Running Title: Minicircle DNA PSCA-CAR T 26 Keywords: CAR T cells, chimeric antigen receptor T cells, minicircle DNA, Prostate 27 Stem Cell Antigen (PSCA), prostate cancer. 28 29 30 Conflict of Interest 31 The authors declare that there is no conflict of interest regarding the publication 32 of this paper. 33 34 1 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1535-7163.MCT-19-0204 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Abstract 2 Viral-based CAR T cell manufacturing has potential safety risks and relatively 3 high costs. The non-viral minicircle DNA (mcDNA) is safer for patients, cheaper to 4 produce, and may be a more suitable technique to generate CAR T cells. In this study, 5 we produced mcDNA-based CAR T cells specifically targeting PSCA (mcDNA-PSCA- 6 CAR T cells). Our results showed that mcDNA-PSCA-CAR T cells persisted in mouse 7 peripheral blood as long as 28 days and demonstrated more CAR T cell infiltration, 8 higher cytokine secretion levels, and better anti-tumor effects. Together, our results 9 suggest that mcDNA-CAR can be a safe and cost-effective platform to produce CAR T 10 cells. 11 12 13 Introduction 14 Recently, the Chimeric Antigen Receptor-engineered T (CAR T) 1,2 cell therapy 15 had successfully cured a few hematological cancers. Now, hundreds of new CAR T 16 therapies targeting blood and solid malignancies are being developed. However, current 17 clinical-grade CAR T cells are manufactured mainly using retroviral or lentiviral vectors 18 which have three problems: 1) potential insertional mutagenesis and oncogenesis3; 2) 19 risks of generating replication-competent viruses4; 3) high costs associated with 20 technical challenges in scaling up the viral production5. 2 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1535-7163.MCT-19-0204 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 On the contrary, non-viral methods, such as transposon and plasmid DNA, are 2 not yet widely used in producing CAR T cells. Transposons carrying a CAR cassette 3 (piggyBac or Sleeping Beauty) can integrate into the patients’ genome, and therefore a 4 long-lasting CAR expression can be achieved6-8. However, the transposon-based CAR 5 T production is complex and time-consuming, usually requiring purification and co- 6 transfection of two plasmids, as well as a co-culture with feeder artificial antigen 7 presenting cells or irradiated peripheral blood mononuclear cells6-8. In addition, plasmid 8 DNA (pDNA) is not suitable for producing therapeutic CAR T cells – because pDNA has 9 a transient in vivo transgene expression due to transcriptional silencing by the bacterial 10 DNA backbone9. 11 To overcome this challenge, the minicircle DNA (mcDNA) was developed – two 12 recombination sites, attB and attP, were added to flank the bacterial DNA backbone so 13 that it can be removed through ΦC31 integrase-mediated recombinantion9. The mcDNA 14 vector, devoid of nearly all bacterial DNA, has significantly increased in vivo transgene 15 expression levels (10-200 folds more) and in vivo expression duration (from 4 to 7 16 weeks)9. To date, mcDNA has been used to engineer human pluripotent stem cells10. 17 In this study, we applied the mcDNA system to generate CAR T cells that 18 specifically targeting the Prostate Stem Cell Antigen (PSCA) molecule (mcDNA-PSCA- 19 CAR T cells). Our results demonstrated that mcDNA-PSCA-CAR can be easily 20 transfected and highly expressed in human T cells. We then performed in vitro and in 21 vivo assays and showed that mcDNA-PSCA-CAR T cells can specifically and effectively 22 attack PSCA-positive cancer cells. 23 3 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1535-7163.MCT-19-0204 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Materials and Methods 2 3 Animals. All animal experiments were conducted under the approval of Hebei Medical 4 University Animal Care and Use Committee, Hebei, China. All NOD/SCID (Non-Obese 5 Diabetic / Severe Combined Immuno-Deficiency) mice used in this study were healthy 6 males (6-8 weeks of age, weighing 200-250 g), which were randomly assigned to 7 experimental or control groups. 8 9 Cell lines. Human prostate cancer cell line RT4 and human bladder carcinoma cell line 10 PC-3M were purchased from and authenticated by the American Type Culture 11 Collection (ATCC). RT4 cells were cultured in McCoy’s 5A medium (ATCC, USA) 12 supplemented with 10% FBS (Fetal Bovine Serum, Gibco, USA) whereas PC-3M cells 13 were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS. These 14 cells were used when they were approximately passage 18-30. Cells were maintained 15 at 37°C in a humidified atmosphere containing 5% CO2. RT4 and PC-3M cells were 16 stained with a PE-labeled mouse anti-PSCA IgG1 (Santa Cruz, USA) and analyzed by 17 FACS for PSCA expression on the cell surface. For Mycoplasma testing of these cells, 18 both agar and broth methods were applied, and both cell lines were Mycoplasma- 19 negative up until Mar 20th, 2019. 20 4 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1535-7163.MCT-19-0204 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Construction of parental PSCA-CAR plasmid (pMC-Easy-PSCA-CAR). The amino 2 acid sequence of an anti-PSCA antibody’s single chain Fragment variable (scFv) was 3 previously published by Olafsen’s group11 and its nucleotide sequence obtained through 4 reverse genetics (see “Results”). The 3rd generation PSCA-CAR expression construct 5 (containing ≥ 2 co-stimulatory domains) was generated by fusing the anti-PSCA scFv 6 with the signaling domains of CD28, CD137 and CD3ζ. This PSCA-CAR construct 7 (1575 bp) was cloned into the pUC57 vector (4252 bp) and then cloned into the MC- 8 Easy™ (SBI, USA) parental minicircle plasmid MN511A-1 which contains a GFP 9 cassette (7064 bp) to form the parental minicircle PSCA-CAR plasmid (pMC-Easy- 10 PSCA-CAR, 8651 bp). 11 12 Generation of minicircle PSCA-CAR plasmid (mcDNA-PSCA-CAR). The bacterial E. 13 coli strain ZYCY10P3S2T (SBI, USA) was used to generate the minicircle PSCA-CAR 14 (mcDNA-PSCA-CAR). Transformed bacteria carrying pMC-Easy-PSCA-CAR was 15 cultured with LB medium containing 50 μg/mL kanamycin, with a rotation speed at 250 16 rpm at 30°C overnight. To assist the generation of mcDNA-PSCA-CAR, the expression 17 of ΦC31 integrase (recombinase) and SceI endonuclease (to degrade the bacterial 18 backbone) was induced by adding L-arabinose at an OD600 from 4 to 6. Minicircles 19 (mcDNA-PSCA-CAR) were produced from the parental minicircle plasmid (pMC-Easy- 20 PSCA-CAR) through ΦC31-mediated recombination between the ΦC321 attB and attP 21 sites on the parental plasmid (pMC-Easy-PSCA-CAR), resulting in separation of the 22 parental minicircle plasmid into the minicircle DNA (mcDNA-PSCA-CAR) and the 23 parental bacterial backbone. In addition, L-arabinose also induced SceI endonuclease 5 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1535-7163.MCT-19-0204 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 which degraded the bacterial backbone. After an additional 5 hr, bacteria cells were 2 harvested and mcDNA-PSCA-CAR purified using QIAGEN’s EndoFree Plasmid Maxi 3 Kit. For every 400 mL overnight culture, one 2500 column and 100 mL of buffers P1, P2 4 and P3 were used to ensure complete lysis and high yield of mcDNA-PSCA-CAR.