Tumor resistance to anti-mesothelin CAR-T cells caused by binding to shed mesothelin is overcome by targeting a juxtamembrane epitope

Our plain-English summary of Tumor resistance to anti-mesothelin CAR-T cells caused by binding to shed mesothelin is overcome by targeting a juxtamembrane epitope, by X.F. Liu, M. Onda, J. Schlomer, et al (Proceedings of the National Academy of Sciences of the United States of America, 2024).

The study

• Authors: X.F. Liu, M. Onda, J. Schlomer, L. Bassel, S. Kozlov, C.H. Tai, Q. Zhou, W. Liu, H.E. Tsao, R. Hassan, M. Ho, I. Pastan
• Journal: Proceedings of the National Academy of Sciences of the United States of America
• Published: 2024;121(4):e2317283121
• Cited by: 32 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Despite many clinical trials, CAR-T cells are not yet approved for human solid tumor therapy. One popular target is mesothelin (MSLN) which is highly expressed on the surface of about 30% of cancers including mesothelioma and cancers of the ovary, pancreas, and lung. MSLN is shed by proteases that cleave near the C terminus, leaving a short peptide attached to the cell. Most anti-MSLN antibodies bind to shed MSLN, which can prevent their binding to target cells. To overcome this limitation, we developed an antibody (15B6) that binds next to the membrane at the protease-sensitive region, does not bind to shed MSLN, and makes CAR-T cells that have much higher anti-tumor activity than a CAR-T that binds to shed MSLN. We have now humanized the Fv (h15B6), so the CAR-T can be used to treat patients and show that h15B6 CAR-T produces complete regressions in a hard-to-treat pancreatic cancer patient derived xenograft model, whereas CAR-T targeting a shed epitope (SS1) have no anti-tumor activity. In these pancreatic cancers, the h15B6 CAR-T replicates and replaces the cancer cells, whereas there are no CAR-T cells in the tumors receiving SS1 CAR-T. To determine the mechanism accounting for high activity, we used an OVCAR-8 intraperitoneal model to show that poorly active SS1-CAR-T cells are bound to shed MSLN, whereas highly active h15B6 CAR-T do not contain bound MSLN enabling them to bind to and kill cancer cells.

In plain English

This was a laboratory and animal study of chimeric antigen receptor T (CAR-T) cells directed at mesothelin (MSLN), a protein the authors describe as expressed on mesothelioma and ovarian, pancreatic and lung cancers and shed from cells by proteases. The authors humanized the Fv of the mouse antibody 15B6, which binds close to the membrane, and compared h15B6 CAR-T cells with SS1 CAR-T cells, which bind a shed region. In cell-killing assays, killing was indistinguishable for the mouse and humanized CAR-T on OVCAR-8 (ovarian), KLM1 (pancreatic) and RH29 (mesothelioma) cells. In NSG mice, h15B6 CAR-T produced complete remissions in KLM1 subcutaneous tumors, in intraperitoneal OVCAR-8 tumors, and in a pancreatic cancer patient-derived xenograft model, where remissions were complete and durable in all 21 of 21 treated mice; h15B6 CAR-T had no activity against MSLN knockout tumors. SS1 CAR-T had no anti-tumor activity in the KLM1 and PDX models and was described as poorly active in the OVCAR-8 model. In mouse ascites, shed MSLN was bound to 34% (590/1,755) of SS1 CAR-T but under 1% (67/7,519) of h15B6 CAR-T. The authors concluded the data support clinical development of h15B6 CAR-T.

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 and animal experimental study: antibody humanization, in vitro cell-killing assays, mouse xenograft and patient-derived xenograft (PDX) tumor experiments, plus testing of CAR-T cells in patient ascites/pleural fluid
• Who was studied: Human cancer cell lines (OVCAR-8 ovarian, KLM1 pancreatic, RH29 mesothelioma, and an MSLN knockout line KLM1 E10); immunodeficient NSG mice bearing these tumors or a pancreatic ductal adenocarcinoma PDX (7 mice per group, experiment repeated 3 times); ascites or pleural fluid from 4 patients (one ovarian cancer, three mesothelioma) at the NIH
• Exposure: CAR-T cells made with the humanized 15B6 Fv (juxtamembrane MSLN epitope) or with the SS1 Fv (amino-terminal, shed epitope), given as a single intravenous dose; control saline or mock-transfected T cells
• Main results:
  • Binding affinities measured by mass photometry: mouse 15B6 Kd 23.5 nM, humanized 15B6 Kd 57.8 nM
  • In vitro killing by humanized and mouse 15B6 CAR-T was indistinguishable on three MSLN-expressing human cancer cell lines, including the mesothelioma line RH29
  • In mice with KLM1 pancreatic tumors, h15B6 CAR-T produced complete remissions while SS1 CAR-T had no anti-tumor activity
  • h15B6 CAR-T also produced complete remissions in OVCAR-8 tumors growing intraperitoneally; SS1 CAR-T were described in this model as poorly active rather than inactive
  • h15B6 CAR-T had no activity against KLM1 tumors with the MSLN gene knocked out, indicating MSLN dependence
  • In the pancreatic cancer PDX model, h15B6 CAR-T gave complete and durable remissions in all 21 of 21 treated mice; SS1 CAR-T had no anti-tumor activity
  • Across the mouse models overall, the authors characterized SS1 CAR-T as having little or no anti-tumor activity
  • In ascites of mice with OVCAR-8 tumors, shed MSLN was detected on 34% (590/1,755) of SS1 CAR-T cells versus less than 1% (67/7,519) of h15B6 CAR-T cells
  • After incubation with ascites or pleural fluid from four patients, 44 to 50% of SS1 CAR-T cells had bound MSLN versus 2 to 3% of h15B6 CAR-T cells
  • h15B6 CAR-T cells accumulated inside OVCAR-8 tumors on days 4, 7 and 10, while SS1 CAR-T cells were very few
  • In PDX tumors, cells stained positive for CD3, Ki-67 and DAPI, which the authors read as CAR-T replication inside the tumor
• The authors' conclusion: The authors concluded that the humanized 15B6 CAR-T, which does not bind shed MSLN, gives complete remissions and replicates within MSLN-expressing cancers, and that the data support its clinical development for testing in patients
• Limitations the authors state:
  • The authors stated that the extension of the mouse findings to people is an assumption

Topics

Medical terms in this study, explained in our Lexicon: Mesothelin, CAR-T Cell, Clinical Trial, Mesothelioma.

Related studies

• Development of Highly Effective Anti-Mesothelin hYP218 Chimeric Antigen Receptor T Cells With Increased Tumor Infiltration and Persistence for Treating Solid Tumors — S. Tomar, J. Zhang, et al. (2022)
• Affinity-tuned mesothelin CAR T cells demonstrate enhanced targeting specificity and reduced off-tumor toxicity — Y. Yang, Y. Vedvyas, et al. (2024)
• Efficacy and safety of novel multiple-chain DAP-CAR-T cells targeting mesothelin in ovarian cancer and mesothelioma: a single-arm, open-label and first-in-human study — T. Xu, T. Tian, et al. (2024)
• Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity in solid malignancies — G.L. Beatty, A.R. Haas, et al. (2014)
• Mesothelin-targeted CAR-T cell therapy for solid tumors — A. Klampatsa, V. Dimou, et al. (2021)
• Targeting a membrane-proximal epitope on mesothelin increases the tumoricidal activity of a bispecific antibody blocking CD47 on mesothelin-positive tumors — E. Hatterer, X. Chauchet, et al. (2020)

Browse more research in Proceedings of the National Academy of Sciences of the United States of America, or return to medical research.