Enhanced tumoricidal activity of PD-1 antibody-secreting c-Met CAR-T cells against pancreatic cancer cells

doi:10.12122/j.issn.1673-4254.2024.10.16

Abstract

Abstract:

Objective To construct c-Met CAR-T cells secreting PD-1 antibodies to reduce immune inhibitory effect of tumor cells and enhance the efficacy of CAR-T cell therapy against pancreatic cancer. Methods Kaplan-Meier Plotter, GEPIA, and Timer 2.0 bioinformatics databases were used to analyze c-Met expression in pancreatic cancer and its correlation with survival and immune infiltration status. In clinical samples of pancreatic cancer and pancreatic cancer Aspc-1 cells, c-Met and PD-L1 expressions were detected using immunohistochemistry or flow cytometry. Using gene editing technology, PD-1 secretory antibodies and HIS tags were linked to second-generation c-Met CAR molecules to construct PD-1/c-Met CAR plasmids, which were then packaged into lentiviruses for infection of activated T cells. The positive rate and cell subset distribution of CAR-T cells were analyzed with flow cytometry, and secretory PD-1 antibodies in cell supernatants were detected using Western blotting. The target cell killing efficiency and proliferative activity of the modified CAR-T cells were evaluated after activation, and cytokine secretion was analyzed using ELISA. Results The expression of c-Met was significantly higher in pancreatic cancer than in normal tissues, and its expression level was negatively correlated with the patients' survival and positively correlated with immune cell infiltration. The clinical samples of pancreatic cancer tissues expressed significantly higher levels of c-Met and PD-L1 than the adjacent tissues, and 90.7% and 57.7% of Aspc-1 cells were positive for c-Met and PD-L1, respectively. The constructed PD-1/c-Met CAR-T cells were capable of secreting PD-1 antibodies and showed a significantly higher killing efficiency against tumor cells than c-Met CAR-T cells at an effector-to-target ratio of 20:1, with also a higher proliferative activity after target cell stimulation and higher levels of IL-2 and TNF-α secretin. Conclusion PD-1/c-Met CAR-T cells have higher killing efficiency against pancreatic cancer cells with also higher proliferative activity than c-Met CAR-T cells.

Key words: chimeric antigen receptor T-cell therapy, c-Met, secretary PD-1 antibody, pancreatic cancer

Jingting MIN, Shang PENG, Nana DU, Ran AN, Xiangcheng ZHEN, Jiawei CAO, Chenhang ZHOU, Zhenghong LI. Enhanced tumoricidal activity of PD-1 antibody-secreting c-Met CAR-T cells against pancreatic cancer cells[J]. Journal of Southern Medical University, 2024, 44(10): 1976-1984.

Figures/Tables 8

Fig.1 Bioinformatics analysis of c-Met expression in pancreatic cancer and its clinical significance. A: Analysis of expression level of c-Met protein in PAAD tissues and adjacent normal tissues based on data from GEPIA database. *P<0.01 vs normal tissues. B: Expressions of MET, EGFR, MUC1 and GAS6 in esophageal cancer and normal tissues. C: Expression level of c-Met protein in PAAD patients in different clinicopathological stages. D: c-Met expression level in PAAD is negatively correlated with overall survival (OS). E: Expression level of c-Met in PAAD is negatively correlated with progression-free survival (PFS). F: High expression of c-Met is associated with low survival rate in stage 2 patients. G: High expression of c-Met is associated with poor survival in stage 3 patients. H: Expression of c-Met is positively correlated with CD8+ T cells, dendritic cells, NK cells and macrophages.

Fig.2 Expression of c-Met and PD-L1 protein in pancreatic cancer tissue samples detected by immunohistochemistry. A: c-Met immunohistochemical staining of clinical specimens of pancreatic cancer tissues (×400). B: PD-L1 immunohistochemical staining of pancreatic cancer tissues (×400). C: c-Met immunohistochemical staining of adjacent tissues (×400). D: PD-L1 immunohistochemical staining of adjacent tissues (× 400 ). E, F: Statistical chart of C-met and PD-L1 pathological scores.

Fig.3 Flow cytometry for analysis of c-Met and PD-L1 protein expressions on Aspc-1 cell surface.

Fig.4 CAR-T cell design, construction and detection. A: CAR core structure diagram. B: Electrophoresis of BamHI digestion products of CAR plasmid. C: CAR-T transfection positive rate. D: CAR-T cell subset distribution. ***P<0.001 vs T cells.

Fig. 5 Immunoblotting of HIS-labeled PD-1 Scfv in the cell supernatant.

Fig.6 Changes in proliferative activity of the effector cells stimulated by the target cells. *P<0.01 vs T cells; #P<0.01 vs CD19 CAR-T; &P<0.01 vs c-Met CAR-T.

Fig.7 Efficiency of the effector cells for target cell killing in different effector-target ratios. *P<0.01 vs T cells; #P<0.01 vs CD19 CAR-T; &P<0.01 vs c-Met CAR-T.

Fig.8 CAR-T cell cytokine release determined using ELISA. #P<0.01 vs T cells; *P<0.01 vs CD19 CAR-T; &P<0.01 vs c-Met CAR-T group.

References 33

1	Karunakaran M, Barreto SG. Surgery for pancreatic cancer: current controversies and challenges[J]. Future Oncol, 2021, 17(36): 5135-62.
2	Klein AP. Pancreatic cancer epidemiology: understanding the role of lifestyle and inherited risk factors[J]. Nat Rev Gastroenterol Hepatol, 2021, 18(7): 493-502.
3	Cai J, Chen HD, Lu M, et al. Advances in the epidemiology of pancreatic cancer: trends, risk factors, screening, and prognosis[J]. Cancer Lett, 2021, 520: 1-11.
4	Kokkinos J, Ignacio RMC, Sharbeen G, et al. Targeting the undruggable in pancreatic cancer using nano-based gene silencing drugs[J]. Biomaterials, 2020, 240: 119742.
5	Zhao YR, Tang JJ, Jiang K, et al. Liquid biopsy in pancreatic cancer - Current perspective and future outlook[J]. Biochim Biophys Acta Rev Cancer, 2023, 1878(3): 188868.
6	Sterner RC, Sterner RM. CAR-T cell therapy: current limitations and potential strategies[J]. Blood Cancer J, 2021, 11(4): 69.
7	Maalej KM, Merhi M, Inchakalody VP, et al. CAR-cell therapy in the era of solid tumor treatment: current challenges and emerging therapeutic advances[J]. Mol Cancer, 2023, 22(1): 20.
8	Ma S, Li XC, Wang XY, et al. Current progress in CAR-T cell therapy for solid tumors[J]. Int J Biol Sci, 2019, 15(12): 2548-60.
9	Liu GN, Rui W, Zhao XQ, et al. Enhancing CAR-T cell efficacy in solid tumors by targeting the tumor microenvironment[J]. Cell Mol Immunol, 2021, 18(5): 1085-95.
10	Marofi F, Motavalli R, Safonov VA, et al. CAR T cells in solid tumors: challenges and opportunities[J]. Stem Cell Res Ther, 2021, 12(1): 81.
11	Zhu GH, Huang C, Qiu ZJ, et al. Expression and prognostic significance of CD151, c-Met, and integrin alpha3/alpha6 in pancreatic ductal adenocarcinoma[J]. Dig Dis Sci, 2011, 56(4): 1090-8.
12	Hage C, Rausch V, Giese N, et al. The novel c-Met inhibitor cabozantinib overcomes gemcitabine resistance and stem cell signaling in pancreatic cancer[J]. Cell Death Dis, 2013, 4(5): e627.
13	Bauer TW, Somcio RJ, Fan F, et al. Regulatory role of c-Met in insulin-like growth factor-I receptor-mediated migration and invasion of human pancreatic carcinoma cells[J]. Mol Cancer Ther, 2006, 5(7): 1676-82.
14	Hill KS, Gaziova I, Harrigal L, et al. Met receptor tyrosine kinase signaling induces secretion of the angiogenic chemokine interleukin-8/CXCL8 in pancreatic cancer[J]. PLoS One, 2012, 7(7): e40420.
15	Min JT, Long CR, Zhang L, et al. C-Met specific CAR-T cells as a targeted therapy for non-small cell lung cancer cell A549[J]. Bioengineered, 2022, 13(4): 9216-32.
16	闵静婷, 张露, 龙赤荣, 等. 靶向c-Met 的嵌合抗原受体Ｔ细胞制备及其对非小细胞肺癌的杀伤作用[J]. 中华肿瘤杂志, 2023, 45（4）: 322-9.
17	Darwish IA, Alahmad W, Vinoth R. Novel ultrasensitive automated kinetic exclusion assay for measurement of plasma levels of soluble PD-L1, the predictive and prognostic biomarker in cancer patients treated with immune checkpoint inhibitors[J]. Heliyon, 2024, 10(10): e31317.
18	Qiu JY, Cheng ZL, Jiang Z, et al. Immunomodulatory precision: a narrative review exploring the critical role of immune checkpoint inhibitors in cancer treatment[J]. Int J Mol Sci, 2024, 25(10): 5490.
19	Xu XY, Xie TX, Zhou MX, et al. Hsc70 promotes anti-tumor immunity by targeting PD-L1 for lysosomal degradation[J]. Nat Commun, 2024, 15(1): 4237.
20	Lemoine J, Ruella M, Houot R. Born to survive: how cancer cells resist CAR T cell therapy[J]. J Hematol Oncol, 2021, 14(1): 199.
21	Norberg SM, Hinrichs CS. Engineered Tcell therapy for viral and non-viral epithelial cancers[J]. Cancer Cell, 2023, 41(1): 58-69.
22	Kashiwada T, Takano R, Ando F, et al. Lysosomal degradation of PD-L1 is associated with immune-related adverse events during anti-PD-L1 immunotherapy in NSCLC patients[J]. Front Pharmacol, 2024, 15: 1384733.
23	Li CC, Zhang Z, Cai QF, et al. Peripheral CX3CR1⁺ T cells combined with PD-1 blockade therapy potentiates the anti-tumor efficacy for lung cancer[J]. Oncoimmunology, 2024, 13(1): 2355684.
24	Andreu-Saumell I, Rodriguez-Garcia A, Mühlgrabner V, et al. CAR affinity modulates the sensitivity of CAR-T cells to PD-1/PD-L1-mediated inhibition[J]. Nat Commun, 2024, 15(1): 3552.
25	赵琳, 王菁, 杨金龙, 等. 分泌PD-1单链抗体的MesoCAR-T细胞治疗非小细胞肺癌的体内外研究[J]. 复旦学报: 自然科学版, 2022, 61(4): 405-16.
26	Li YT, Sharma A, Schmidt-Wolf IGH. Evolving insights into the improvement of adoptive T-cell immunotherapy through PD-1/PD-L1 blockade in the clinical spectrum of lung cancer[J]. Mol Cancer, 2024, 23(1): 80.
27	Prosser ME, Brown CE, Shami AF, et al. Tumor PD-L1 co-stimulates primary human CD8⁺ cytotoxic T cells modified to express a PD1: CD28 chimeric receptor[J]. Mol Immunol, 2012, 51(3/4): 263-72.
28	Cherkassky L, Morello A, Villena-Vargas J, et al. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition[J]. J Clin Invest, 2016, 126(8): 3130-44.
29	Zhu HF, You YP, Shen ZM, et al. EGFRvIII-CAR-T cells with PD-1 knockout have improved anti-glioma activity[J]. Pathol Oncol Res, 2020, 26(4): 2135-41.
30	Li DZ, Qin J, Zhou T, et al. Bispecific GPC3/PD-1 CAR-T cells for the treatment of HCC[J]. Int J Oncol, 2023, 62(4): 53.
31	Chen J, Zhu TC, Jiang GM, et al. Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer[J]. Mol Cancer, 2023, 22(1): 131.
32	Metanat Y, Viktor P, Amajd A, et al. The paths toward non-viral CAR-T cell manufacturing: a comprehensive review of state-of-the-art methods[J]. Life Sci, 2024, 348: 122683.
33	Cappabianca D, Pham D, Forsberg MH, et al. Metabolic priming of GD2 TRAC-CAR Tcells during manufacturing promotes memory phenotypes while enhancing persistence[J]. Mol Ther Methods Clin Dev, 2024, 32(2): 101249.

[1]	Yan HUANG, Lulu QIN, Shaoxing GUAN, Yanping GUANG, Yuru WEI, Ailing CAO, Dongmei LI, Guining WEI, Qibiao SU. Therapeutic mechanism of aqueous extract of Semiliquidambar cathayensis Chang root for pancreatic cancer: the active components, therapeutic targets and pathways [J]. Journal of Southern Medical University, 2024, 44(7): 1336-1344.
[2]	SUN Jingjie, LU Peng, GUAN Shasha, LIU Songsong. Heterogeneity analysis of pancreatic cancer and identification of molecular subtypes of tumor cells based on CEACAM5, LGALS1 and CENPF gene expression [J]. Journal of Southern Medical University, 2023, 43(9): 1567-1576.
[3]	LIANG Lidu, ZHANG Haojie, LU Qian, ZHOU Chenjie, LI Shulong. Advanced Faster RCNN: a non-contrast CT-based algorithm for detecting pancreatic lesions in multiple disease stages [J]. Journal of Southern Medical University, 2023, 43(5): 755-763.
[4]	. Detection of chitinase 3-like 1 combined with other biomarkers for diagnosis of pancreatic cancer [J]. Journal of Southern Medical University, 2018, 38(04): 450-.
[5]	. Predictive value of c-met for long-term mortality in patients with esophageal squamous cell carcinoma [J]. Journal of Southern Medical University, 2016, 36(08): 1153-.
[6]	. Cabozantinib inhibits Listeria monocytogenes infection in mice [J]. Journal of Southern Medical University, 2016, 36(01): 56-.
[7]	. Proanthocyanidins inhibit pancreatic cancer AsPC-1 cell growth and migration through up-regulation of let-7a [J]. Journal of Southern Medical University, 2015, 35(08): 1110-.
[8]	. Effect of MSX2 interference on epithelial-mesenchymal transitions of pancreatic cancer cell line PANC-1 [J]. Journal of Southern Medical University, 2015, 35(02): 179-.
[9]	. Effect of high-intensity focused ultrasound combined with gemcitabine on subcutaneous pancreatic cancer in nude mice [J]. Journal of Southern Medical University, 2013, 33(12): 1713-.
[10]	. Effect of Notch1 signaling pathway activation on pancreatic cancer cell proliferation in vitro [J]. Journal of Southern Medical University, 2013, 33(10): 1494-.
[11]	. Thyroid hormone inhibits the growth of pancreatic cancer xenograft in nude mice [J]. Journal of Southern Medical University, 2013, 33(08): 1160-.
[12]	. Influence of album and placement time outside the incubator on viability of isolated cytokine-induced killer cells: a simple method for detection [J]. Journal of Southern Medical University, 2013, 33(07): 1078-.
[13]	HUA Zhu-ming, LI Zhen, LOU Dong-lan Department of Anesthesia, Guangdong Provincial People’s Hospital, Guangzhou 510010, China. Relation of nerve growth factor expression with perineural invasion and pain in pancreatic cancer [J]. Journal of Southern Medical University, 2006, 26(08): 1251-1253.
[14]	GUAN Hai-tao, XUE Xing-huan, WANG Xi-jing, LI Ang, QIN Zhao-yin Department of Oncologic Surgery, Department of General Surgery, Second Hospital of Xi’an Jiaotong University;MedicaI Research Center, Stomatological Hospital of Xi’an Jiaotong University, Xi’an 710004, China. siRNA targeted against survivin induces apoptosis of pancreatic cancer cells [J]. Journal of Southern Medical University, 2006, 26(02): 169-173.
[15]	ZONG Li-li^1,2, LI Ya-li², SONG San-tai¹, JIANG Ze-Fei¹, ZHAO Jun². Expression of hepatocyte growth factor and its receptor c-met gene in the endometrium of women with endometriosis [J]. Journal of Southern Medical University, 2004, 24(06): 619-622.