Development of Highly Effective Anti-Mesothelin hYP218 Chimeric Antigen Receptor T Cells With Increased Tumor Infiltration and Persistence for Treating Solid Tumors

Our plain-English summary of Development of Highly Effective Anti-Mesothelin hYP218 Chimeric Antigen Receptor T Cells With Increased Tumor Infiltration and Persistence for Treating Solid Tumors, by S. Tomar, J. Zhang, M. Khanal, et al (Molecular Cancer Therapeutics, 2022).

The study

• Authors: S. Tomar, J. Zhang, M. Khanal, J. Hong, A. Venugopalan, Q. Jiang, M. Sengupta, M. Miettinen, N. Li, I. Pastan, M. Ho, R. Hassan
• Journal: Molecular Cancer Therapeutics
• Published: 2022;21(7):1195-1206
• Cited by: 45 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Mesothelin targeting CAR T cells have limited activity in patients. In this study, we sought to determine if efficacy of anti-mesothelin CAR T cells is dependent on the mesothelin epitopes that are recognized by them. To do so, we developed hYP218 (against membrane-proximal epitope) and SS1 (against membrane-distal epitope) CAR T cells. Their efficacy was assessed in vitro using mesothelin-positive tumor cell lines and in vivo in NSG mice with mesothelin-expressing ovarian cancer (OVCAR-8), pancreatic cancer (KLM-1), and mesothelioma patient-derived (NCI-Meso63) tumor xenografts. Persistence and tumor infiltration of CAR T cells was determined using flow cytometry. hYP218 CAR T cells killed cancer cells more efficiently than SS1 CAR T cells, with a two- to fourfold lower ET50 value (effector-to-target ratio for 50% killing of tumor cells). In mice with established tumors, single intravenous administration of hYP218 CAR T cells lead to improved tumor response and survival compared with SS1 CAR T cells, with complete regression of OVCAR-8 and NCI-Meso63 tumors. Compared with SS1 CAR T cells, there was increased peripheral blood expansion, persistence, and tumor infiltration of hYP218 CAR T cells in the KLM-1 tumor model. Persistence of hYP218 CAR T cells in treated mice led to antitumor immunity when rechallenged with KLM-1 tumor cells. Our results show that hYP218 CAR T cells, targeting mesothelin epitope close to cell membrane, are very effective against mesothelin-positive tumors and are associated with increased persistence and tumor infiltration. These results support its clinical development to treat patients with mesothelin-expressing cancers.

©2022 The Authors; Published by the American Association for Cancer Research.

In plain English

This preclinical study tested whether the mesothelin epitope targeted by chimeric antigen receptor (CAR) T cells affects their activity. The authors built second-generation CAR T cells from the humanized rabbit antibody hYP218 (membrane-proximal mesothelin epitope) and from SS1 (membrane-distal epitope); humanization raised antibody affinity from KD 0.2 nmol/L to 0.07 nmol/L. In vitro, CAR T cells from six healthy donors and from six patients with malignant mesothelioma were tested against mesothelin-expressing cell lines; cells from one healthy donor were selected for the mouse studies. hYP218 CAR T cells killed tumour cells at lower effector-to-target ratios and released more IFN-gamma, IL2 and TNF-alpha than SS1 CAR T cells. In NSG mice, single intravenous infusions of hYP218 CAR T cells caused complete regression of OVCAR-8 ovarian and NCI-Meso63 mesothelioma tumours, with longer survival than SS1 in the OVCAR-8 and KLM-1 models. In the KLM-1 model, hYP218 showed greater blood expansion and tumour infiltration; three treated mice rechallenged with KLM-1 cells had no tumour growth or significant increase in burden (four others were excluded for graft-versus-host disease). The authors concluded that the epitope recognized significantly affects CAR T-cell efficacy.

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: Preclinical laboratory study: in vitro cytotoxicity and cytokine assays plus mouse xenograft experiments comparing two anti-mesothelin CAR T-cell constructs
• Who was studied: Human tumour cell lines (ovarian OVCAR-8, pancreatic KLM-1 and KLM-1 MSLN-knockout, lung H292, mesothelioma H226, NCI-Meso63, NCI-Meso29, A431/A431-H9, H1703), CAR T cells made from PBMCs of six healthy donors and of patients with mesothelioma, and female NSG mice bearing xenografts; NCI, Bethesda, Maryland
• Exposure: CAR T cells carrying scFvs from hYP218 (membrane-proximal mesothelin region III) or SS1 (membrane-distal region I), given as a single intravenous infusion of 1 x 10^7 cells in mice
• Main results:
  • Humanization increased mesothelin binding affinity of the antibody
  • CAR T cells were made from six healthy donors; donor 1-derived hYP218 and SS1 CAR T cells were selected for the further in vitro and in vivo experiments
  • In CAR T cells manufactured from PBMCs of six patients with malignant mesothelioma, hYP218 CAR T cells killed OVCAR-8 and KLM-1 cells better than SS1 CAR T cells (in vitro)
  • In vitro, hYP218 CAR T cells killed mesothelin-expressing tumour cells at lower effector-to-target ratios than SS1 CAR T cells (lower ET50)
  • Cytokine release (IFN-gamma, IL2, TNF-alpha) by stimulated hYP218 CAR T cells exceeded that of stimulated SS1 CAR T cells
  • In the OVCAR-8 ovarian model, hYP218 CAR T cells produced complete tumour regression within 2 weeks, while SS1 only delayed growth
  • In the KLM-1 pancreatic model at day 42, among five hYP218-treated mice: two with persistent complete regression, two with no significant increase in tumour burden, one with progression and euthanized
  • Survival in the OVCAR-8 model: P less than or equal to 0.0001 for hYP218 versus SS1 CAR T-cell-treated mice
  • Survival in the KLM-1 model: P less than or equal to 0.01 for hYP218 versus SS1 CAR T-cell-treated mice
  • In the mesothelioma patient-derived NCI-Meso63 subcutaneous model, hYP218-treated mice had complete tumour regression within 4 weeks and remained tumour free; SS1 reduced growth but not complete regression in all mice
  • In the KLM-1 model, circulating human T cells in a hYP218-treated mouse rose from 0.8% (day 2) to 8.5% (day 7)
  • Tumour infiltration by hYP218 CAR T cells in the KLM-1 model was greater than by SS1 CAR T cells, which the authors proposed as a mechanism for the better in vivo activity
  • hYP218 CAR T cells persisted in blood of all treated KLM-1 mice at day 35, at 14% to 61%
  • On rechallenge with KLM-1 cells, the two previously treated mice with prior complete regression had no tumour growth and the one with residual tumour had no significant increase, while age-matched untreated mice grew tumours
  • YP218 antibody staining of normal human tissues showed reactivity mainly in mesothelial cells and some epithelia, without binding to parenchymal organs
• The authors' conclusion: The authors concluded that the mesothelin epitope recognized by CAR T cells significantly affects their efficacy, and that hYP218 CAR T cells have properties (humanization, increased potency, increased persistence and tumour accumulation) that could be useful clinically, to be tested in an upcoming trial
• Limitations the authors state:
  • The authors state the xenograft studies used immunodeficient mice, and the tumour immune environment affects CAR T-cell efficacy
  • The authors state the mechanism behind expansion and persistence of hYP218 CAR T cells is unclear and needs further study
  • In the rechallenge experiment, four of seven treated mice were excluded because of graft-versus-host disease
  • The authors note possible side effects at higher doses in patients, needing evaluation in clinical trials

Topics

The National Library of Medicine indexes this study under subjects our site covers: Ovarian cancer.

Medical terms in this study, explained in our Lexicon: Mesothelin, Ovarian Cancer, Mesothelioma.

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• Chimeric antigen receptor T cells engineered to recognize the P329G-mutated Fc part of effector-silenced tumor antigen-targeting human IgG1 antibodies enable modular targeting of solid tumors — S. Stock, M.R. Benmebarek, et al. (2022)
• Mesothelin as a target for chimeric antigen receptor-modified T cells as anticancer therapy — M. O'Hara, C. Stashwick, et al. (2016)
• Tumor resistance to anti-mesothelin CAR-T cells caused by binding to shed mesothelin is overcome by targeting a juxtamembrane epitope — X.F. Liu, M. Onda, et al. (2024)
• Improvement in the function of self-activating chimeric antigen receptor by replacing the linker sequence — T. Kouro, D. Hoshino, et al. (2025)

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