Mesothelin-targeted second generation CAR-T cells inhibit growth of mesothelin-expressing tumors in vivo

Our plain-English summary of Mesothelin-targeted second generation CAR-T cells inhibit growth of mesothelin-expressing tumors in vivo, by L. Ye, Y. Lou, L. Lu, et al (Experimental and Therapeutic Medicine, 2019).

The study

• Authors: L. Ye, Y. Lou, L. Lu, X. Fan
• Journal: Experimental and Therapeutic Medicine
• Published: 2019;17(1):739-747
• Cited by: 36 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Non-small cell lung cancer (NSCLC) and mesothelioma are renowned for being diagnosed at a late stage and poor prognosis. Although surgery, chemotherapy, and radiotherapy have yielded successful outcomes, the improvement on the survival rate of NSCLC and mesothelioma have been less marked. Recently, adoptive immunotherapy, particularly chimeric antigen receptor T (CAR-T) cell therapy demonstrated promise for improving the survival of acute lymphoblastic leukemia with minimum toxicity. However, its application in solid tumors still warrants in-depth investigations and multiple consistent trial results, particularly in eliminating 'off-tumor' toxicity. To explore CAR-T therapy in NSCLC and mesothelioma, second-generation CAR-T cells were constructed targeting mesothelin (MSLN), which is abundant in NSCLC and mesothelioma but is under expressed in normal tissues. The second-generation design incorporated co-stimulatory CD28 and 4-1BB signaling domains to enhance the proliferation. Following the successful analysis of CAR-T cells by flow cytometry, cytotoxicity experiments were performed using the LDH kit to verify the killing effect of CAR-T cells on target cells. Otherwise, the in vivo killing tumor activity of MSLN CAR-T cells was verified by constructing a mouse model using tumor-derived cells from patients to inoculate the mice. When the effector-to-target ratio is >0.5:1, CAR-T MSLN cells exhibited significantly higher ability to kill tumor cells than T cells. In in vivo experiments, mice whose tail vein was injected with CAR-T MSLN cells demonstrated significantly slower tumor growth. Without continuous administration, both groups became gradually synchronized in growth of tumor size, which suggests that the persistence of CAR-T cells is an important issue in preclinical studies.

In plain English

This was a laboratory study in which the authors built second-generation chimeric antigen receptor T (CAR-T) cells directed at mesothelin (MSLN), using a lentiviral vector carrying CD28 and 4-1BB co-stimulatory modules, and tested them against tumour cells and in mice. T cells came from 5 patients (4 male, 1 female; age 64.80±2.77) and one 45-year-old healthy female donor, sampled at Shanghai Chest Hospital in December 2015. Flow cytometry showed 94.20% of HeLa cells expressed MSLN and 5.77% were MSLN negative. In LDH cytotoxicity assays, killing by CAR-T cells exceeded that by control T cells once the effector-to-target ratio reached 0.5:1 (P<0.05), and against CHO-K1-MSLN cells 78% of cells were lysed at 40:1. Of 15 male NPG mice obtained, 14 were allocated to the experiment (7 per group, one mouse unused) after inoculation with non-small cell lung cancer tissue from 5 patients with high MSLN expression; mice infused with CAR-T cells had slower tumour growth, significant at PG-D31 (P=0.03), after which the groups' growth rates became synchronised without further injection.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study: construction of MSLN-targeted second-generation CAR-T cells, with ex vivo cytotoxicity assays and an in vivo mouse xenograft experiment
• Who was studied: T-cell donors: 5 patients and 1 healthy control (blood taken at Shanghai Chest Hospital, December 2015); target cells HeLa and CHO-K1-MSLN; 15 male NPG mice aged 3-4 weeks obtained, randomised to two groups of 7 (one mouse unused), inoculated with patient NSCLC tissue
• Exposure: Lentiviral pCAR-MSLN construct containing CD28 and 4-1BB co-stimulatory modules used to make CAR-T cells; mice received 8x10^6 CAR-T cells or control T cells by tail vein
• Main results:
  • MSLN expression on target cells: 94.20% of HeLa cells positive, 5.77% negative
  • Against HeLa cells, CAR-T cells from both the healthy donor and the patients killed more cells than their respective control T cells (all P<0.05)
  • Against CHO-K1-MSLN cells, significantly elevated targeting from a 0.5:1 effector-to-target ratio (P=0.04); at 40:1, 78% of cells lysed
  • In mice (7 per group), tumours in the CAR-T group grew more slowly, with a significant difference at PG-D31 (P=0.03), after which both groups' tumour growth rates became synchronised
• The authors' conclusion: The authors concluded that MSLN-targeted CAR-T cells could inhibit tumour growth both in vivo and ex vivo, but that a more sophisticated method is needed for continuous tumour suppression; they also stated that CAR-T cells from healthy individuals appeared more potent than those from patients at the cell level
• Limitations the authors state:
  • The authors state that the target cell line used, HeLa, is a cervical cancer line and a considerable confounding factor, and that a single cell line may not be strong enough evidence
  • The authors state that an in vivo cytotoxicity assay is needed to complement their in vitro assay
  • The authors state that use of the 293T packaging cell line is a further limitation because of its phenotype and unstable, heterogeneous karyotype
  • The authors state that the absence of a blank control (non-MSLN-expressing or non-tumour cells) meant the specificity of the MSLN CAR-T cells could not be examined

Topics

Medical terms in this study, explained in our Lexicon: Mesothelin, CAR-T Cell, Non-Small Cell Lung Cancer, Mesothelioma, Prognosis, Chemotherapy, Immunotherapy.

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