A study on influence of different signal peptides on anti-tumor effect of chimeric antigen receptor (CAR) T cells

doi:10.19401/j.cnki.1007-3639.2022.02.006

Abstract

Abstract:

Background and purpose: Signal peptide (SP) is a short peptide chain at the N-terminal of precursor protein, which can regulate the folding and transfer of precursor protein and plays an important role in protein secretion. In recent years, significant breakthroughs have been made in the treatment of leukemia with CD19-targeted chimeric antigen receptor (CAR) T cells, and many achievements have been reported in the intracellular domain modification of CAR structure, while the N-terminal SP of scFv presents less progress. The purpose of this study was to investigate the CD19-CAR expression of four different SP on the surface of T cells and their effect on the killing of CD19⁺ target cells. Methods: The CAR vectors containing four different SP (SP1, SP2, SP3, SP4) targeting CD19 antigen were constructed by gene synthesis and molecular cloning technology, and then packaged into lentivirus. The obtained lentivirus was transfected into T cells. The transfection efficiency of the cells was detected by flow cytometry, the killing effect of the cells on target cells was detected by calcein release assay, and the secretion levels of IFN-γ and TNF-α were detected by ELISA. Results: The recombinant lentiviral plasmids with four different SP were successfully constructed, and the four packaged lentiviruses were transduced into T cells. The results showed that 20.76%, 22.29%, 31.57% and 38.42% of T cells expressed CD19-CAR (named as SP1-CD19, SP2-CD19, SP3-CD19 and SP4-CD19 cells, respectively), respectively. The killing effect of SP4-CD19 on CD19^- tumor cells was significantly higher compared with SP1-CD19, SP2-CD19 and SP3-CD19 cells (P<0.01), and the secretion levels of IFN-γ and TNF-α in SP4-CD19 cells were significantly higher than those in SP1-CD19, SP2-CD19 and SP3-CD19 cells when the effect-target ratio was 10 to 1 for 24 h (P<0.05). There was no significant difference in the killing effect of CAR-T on CD19 negative cells K562 among four different SP (P>0.05). Conclusion: The transfection efficiency and killing effect of SP4-CD19 cells on CD19⁺ tumor cells were significantly higher compared with SP1-CD19, SP2-CD19 and SP3-CD19 cells, which laid a scientific foundation for the optimization and efficient clinical application of CAR-T.

Key words: Signal peptide, Chimeric antigen receptor, CD19-targeted chimeric antigen receptor T cells, Anti-tumor in vitro, Cytokines

CLC Number:

R730.51

LI Fan, ZHANG Qinxing, TONG Xiangwen, TIAN Gaohui, GU Lixing, XU Yao. A study on influence of different signal peptides on anti-tumor effect of chimeric antigen receptor (CAR) T cells[J]. China Oncology, 2022, 32(2): 142-151.

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References 28

[1]	BLOBEL G,, DOBBERSTEIN B.Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma[J]. J Cell Biol, 1975, 67(3): 835-851.
[2]	HEGDE R S,, BERNSTEIN H D.The surprising complexity of signal sequences[J]. Trends Biochem Sci, 2006, 31(10): 563-571.
[3]	PROMCHAI R,, VISESSANGUAN W,, LUXANANIL P.An efficient ABC transporter signal peptide directs heterologous protein secretion in food-grade hosts[J]. World J Microbiol Biotechnol, 2020, 36(10): 154.
[4]	HUANG Q,, PALMER T.Signal peptide hydrophobicity modulates interaction with the twin-arginine translocase[J]. mBio, 2017, 8(4): e00909-e00917.
[5]	MAHMUD H,, ISMAIL A,, ABDUL RAHIM R, et al.Enhanced secretion of cyclodextrin glucanotransferase (CGTase) by Lactococcus lactis using heterologous signal peptides and optimization of cultivation conditions[J]. J Biotechnol, 2019, 296: 22-31.
[6]	FERRARESE L,, TRAINOTTI L,, GATTOLIN S, et al.Secretion, purification and activity of two recombinant pepper endo-beta-1, 4-glucanases expressed in the yeast Pichia pastoris[J]. FEBS Lett, 1998, 422(1): 23-26.
[7]	DE PINHEIRO C G,, PEDROSA M D E O,, TEIXEIRA N C, et al. Optimization of canine interleukin-12 production using a baculovirus insect cell expression system[J]. BMC Res Notes, 2016, 9: 36.
[8]	DUAN G D,, DING L M,, WEI D S, et al.Screening endogenous signal peptides and protein folding factors to promote the secretory expression of heterologous proteins in Pichia pastoris[J]. J Biotechnol, 2019, 306: 193-202.
[9]	LIN J,, NEO S H,, HO S, et al.Impact of signal peptides on furin-2A mediated monoclonal antibody secretion in CHO cells[J]. Biotechnol J, 2017, 12(9): 2017, 12(9).
[10]	CHEADLE E J,, GORNALL H,, BALDAN V, et al.CAR T cells: driving the road from the laboratory to the clinic[J]. Immunol Rev, 2014, 257(1): 91-106.
[11]	SRIVASTAVA S,, RIDDELL S R.Engineering CAR-T cells: design concepts[J]. Trends Immunol, 2015, 36(8): 494-502.
[12]	JEONG D W,, LEE J H,, KIM K H, et al.A food-grade expression/secretion vector for lactococcus lactis that uses an alpha-galactosidase gene as a selection marker[J]. Food Microbiol, 2006, 23(5): 468-475.
[13]	MASOMIAN M,, JASNI A S,, RAHMAN R N Z R A, et al. Impact of signal peptide and transmembrane segments on expression and biochemical properties of a lipase from Bacillus sphaericus 205y[J]. J Biotechnol, 2017, 264: 51-62.
[14]	HOMBACH A,, WIECZARKOWIECZ A,, MARQUARDT T, et al.Tumor-specific T cell activation by recombinant immunoreceptors: CD3 zeta signaling and CD28 costimulation are simultaneously required for efficient IL-2 secretion and can be integrated into one combined CD28/CD3 zeta signaling receptor molecule[J]. J Immunol, 2001, 167(11): 6123-6131.
[15]	QIN H,, DONG Z Y,, WANG X L, et al. CAR T cells targeting BAFF-R can overcome CD19 antigen loss in B cell malignancies[J]. Sci Transl Med, 2019, 11(511): eaaw9414.
[16]	SINGH N,, FREY N V,, ENGELS B, et al.Antigen-independent activation enhances the efficacy of 4-1BB-costimulated CD22 CAR T cells[J]. Nat Med, 2021, 27(5): 842-850.
[17]	COOK J,, LITZOW M.Advances in supportive care for acute lymphoblastic leukemia[J]. Curr Hematol Malig Rep, 2020, 15(4): 276-293.
[18]	ROGOSIC S,, GHORASHIAN S.CAR-T cell therapy in paediatric acute lymphoblastic leukaemia-past, present and future[J]. Br J Haematol, 2020, 191(4): 617-626.
[19]	FOUSEK K,, WATANABE J,, JOSEPH S K, et al.CAR T-cells that target acute B-lineage leukemia irrespective of CD19 expression[J]. Leukemia, 2021, 35(1): 75-89.
[20]	KOBAYASHI K,, HASHIMOTO M,, HONKAKOSKI P, et al.Regulation of gene expression by CAR: an update[J]. Arch Toxicol, 2015, 89(7): 1045-1055.
[21]	LIN W Y,, WANG H H,, CHEN Y W, et al.Gene modified CAR-T cellular therapy for hematologic malignancies[J]. Int J Mol Sci, 2020, 21(22): E8655.
[22]	HONG M H,, CLUBB J D,, CHEN Y Y.Engineering CAR-T cells for next-generation cancer therapy[J]. Cancer Cell, 2020, 38(4): 473-488.
[23]	RAFIQ S,, HACKETT C S,, BRENTJENS R J.Engineering strategies to overcome the current roadblocks in CAR T cell therapy[J]. Nat Rev Clin Oncol, 2020, 17(3): 147-167.
[24]	KNAPPSKOG S,, RAVNEBERG H,, GJERDRUM C, et al.The level of synjournal and secretion of Gaussia princeps luciferase in transfected CHO cells is heavily dependent on the choice of signal peptide[J]. J Biotechnol, 2007, 128(4): 705-715.
[25]	LIU J,, O’KANE D J,, ESCHER A. Secretion of functional Renilla reniformis luciferase by mammalian cells[J]. Gene, 1997, 203(2): 141-148.
[26]	JANDA C Y,, LI J,, OUBRIDGE C, et al.Recognition of a signal peptide by the signal recognition particle[J]. Nature, 2010, 465(7297): 507-510.
[27]	SARAOGI I,, SHAN S O.Molecular mechanism of co-translational protein targeting by the signal recognition particle[J]. Traffic, 2011, 12(5): 535-542.
[28]	LIANG Y,, LIU H,, LU Z M, et al.CD19 CAR-T expressing PD-1/CD28 chimeric switch receptor as a salvage therapy for DLBCL patients treated with different CD19-directed CAR T-cell therapies[J]. J Hematol Oncol, 2021, 14(1): 26.

SP	Sequence	Source
SP1	ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (MALPVTALLLPLALLLHAARP)	Human CD8α
SP2	ATGCTGCTGCTGGTGACCAGCCTGCTTCTGTGCGAACTGCCCCACCCCGCCTTCCTGCTAATCCCC (MLLLVTSLLLCELPHPAFLLIP)	Human CSF2Rα
SP3	ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATGTCTAGAATGGCC (MDFQVQIFSFLLISASVIMSRMA)	Mouse light chain Kappa-K32 precursor
SP4	ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGT (MYRMQLLSCI ALSLALVTNS)	Human IL-2

Primer name	Primer sequence(5'-3')
SP2-forwards	GGATCTATTTCCGGTGAATTCGCCACCATGCTGCTGCTGGTGACC
SP2-reverse	CTGGGTCATCTGGATGTCCGGCCTGGCGGCGTGGA
SP3-forwards	GGATCTATTTCCGGTGAATTCGCCACCATGGATTTTCAGGTG
SP3--reverse	CTGGGTCATCTGGATGTCGGCCATTCTAGACATTAT
SP4-forwards	GGATCTATTTCCGGTGAATTCGCCACCATGTACAGGATGCAAC
SP4-reverse	CTGGGTCATCTGGATGTCACTGTTTGTGACAAGTG
CAR-forwards	GACATCCAGATGACCCAGAC
CAR-reverse	GGGAGGGAGAGGGGCGGATCCTTAGCGAGGGGGCAGGGCC