Recombinant Immunotoxin with T-cell Epitope Mutations That Greatly Reduce Immunogenicity for Treatment of Mesothelin-Expressing Tumors

Our plain-English summary of Recombinant Immunotoxin with T-cell Epitope Mutations That Greatly Reduce Immunogenicity for Treatment of Mesothelin-Expressing Tumors, by R. Mazor, J. Zhang, L. Xiang, et al (Molecular Cancer Therapeutics, 2015).

The study

• Authors: R. Mazor, J. Zhang, L. Xiang, S. Addissie, P. Awuah, R. Beers, R. Hassan, I. Pastan
• Journal: Molecular Cancer Therapeutics
• Published: 2015;14(12):2789-96
• Cited by: 33 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

SS1P is a recombinant immunotoxin (RIT) that targets mesothelin. It consists of an antimesothelin Fv fused to a portion of Pseudomonas exotoxin A. In clinical studies, it has produced dramatic responses in patients with advanced mesothelioma, when combined with immunosuppressive therapy so that several treatment cycles could be given. Otherwise its activity is limited by its immunogenicity. In this work, we describe the development and characterization of LMB-T20, a highly potent RIT targeted at mesothelin-expressing cancers with low immunogenicity due to removal of its eight T-cell epitopes. LMB-T20 was more active than SS1P when tested on four different mesothelin-expressing cell lines as well as on cells obtained from patients with mesothelioma. It also has potent antitumor activity in mice, and has reduced immunogenicity as measured by cytokine secretion assays. In conclusion, LMB-T20 is a favorable candidate for evaluation in clinical trials due to its reduced immunogenicity and excellent activity.

©2015 American Association for Cancer Research.

In plain English

This was a laboratory study describing a new recombinant immunotoxin, LMB-T20, made by introducing six point mutations (R427A, F443A, L477H, R494A, R505A and L552E) into SS1-LR-GGS, an anti-mesothelin Fv joined to a shortened Pseudomonas exotoxin. Compared with the parent immunotoxin SS1P, peripheral blood mononuclear cells from 10 donors showed what the authors called "an 81% reduction in the total response", with the counts given as 105 positive responses to SS1P and 25 responses to LMB-T20. When whole protein was used to stimulate cells from 12 donors, SS1P gave medians of 702 IFN-gamma and 226 IL-2 spot-forming cells per million, while LMB-T20 gave 163 and 91 and was not significantly above the no-protein control (10 and 19). LMB-T20 was cytotoxic to all five mesothelin-expressing cell lines tested and, on the lines expressing endogenous mesothelin, more cytotoxic than SS1P, as it also was on cells from four mesothelioma patients. In mice bearing A431/H9 tumours, 7 of 8 treated animals had complete responses lasting to day 60; in the pancreatic KLM1 model there was an 11-day growth delay, and in the patient-derived models the authors reported a decrease in tumour size in one and stabilization in the second. The authors concluded LMB-T20 warrants further pre-clinical development.

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 preclinical study: construction of a mutated recombinant immunotoxin, in vitro cytotoxicity on cell lines and patient-derived cells, human PBMC T-cell activation (ELISpot) assays, and mouse xenograft experiments
• Who was studied: Five mesothelin-expressing human cancer cell lines (A431/H9, KLM1, L55, MKN74, HAY); mesothelioma cells from four patients (ascites or pleural fluid); PBMC from 10 donors for epitope mapping and 12 donors for whole-protein stimulation; athymic nude/SCID mice bearing A431/H9, KLM1 and patient-derived (NCI-Meso16, NCI-Meso21) tumours; NCI, Bethesda, MD
• Exposure: Treatment with the recombinant immunotoxins LMB-T20, SS1P and SS1-LR-GGS (anti-mesothelin Fv fused to Pseudomonas exotoxin A fragments); in vitro 5 or 50 micrograms/ml for PBMC stimulation; in mice 5 mg/kg intravenously, with toxicity doses up to 20 mg/kg
• Main results:
  • In PBMC from 10 donors, the authors reported an 81% reduction in the total T-cell response to LMB-T20 compared with SS1P, with 105 positive responses to SS1P and 25 responses to LMB-T20; the article prints the p value as p>0.0001 in 2-way ANOVA
  • With whole-protein stimulation of PBMC from 12 donors, SS1P gave median 702 IFN-gamma and 226 IL-2 SFC per 1x10^6 cells versus 10 and 19 for no protein; LMB-T20 gave 163 and 91 and was not significantly above the no-protein control
  • No new T-cell epitopes appeared in LMB-T20 apart from three very minor responses
  • LMB-T20 was cytotoxic to all five mesothelin-expressing cell lines and almost as active as SS1-LR-GGS; both LMB-T20 and SS1-LR-GGS were much more cytotoxic than SS1P on the cell lines expressing endogenous mesothelin. The authors state LMB-T20 was 2-3 fold more active than SS1P on cancer cell lines
  • On mesothelioma cells from four patients, LMB-T20 had lower IC50 values than SS1P in all four samples, described as 3-20 fold more active
  • In A431/H9 xenografts (8 mice treated, 8 vehicle), tumour size decreased significantly on day 9 (p<0.01, Mann Whitney), 2/8 had complete response by day 9, and complete responses persisted in 7/8 until day 60
  • In the KLM1 pancreatic model, LMB-T20 produced a significant growth delay of 11 days (p=0.008, Wilcoxon matched rank test), with differences starting on day 9 (p<0.05)
  • In the NCI-Meso16 patient-derived tumours, mean tumour size on day 55 was 110 mm3 with LMB-T20 versus 168 mm3 with vehicle (p<0.03), with regrowth after day 65
  • In the NCI-Meso21 patient-derived tumours, the average tumour size on day 90 was 87 mm3 in LMB-T20 treated mice while vehicle-treated tumours grew to 120 mm3 (p<0.008)
  • In mouse toxicity testing, 5 mg/kg daily for 6 days was well tolerated; two doses of 15 mg/kg LMB-T20 caused 9% weight loss and 20 mg/kg killed 1 of 2 mice, while 10 mg/kg of LR-GGS caused severe weight loss in 2 of 2 mice
• The authors' conclusion: The authors concluded that LMB-T20 is a de-immunized immunotoxin with low immunogenicity and high cytotoxic and anti-tumour activity that, in their view, warrants further pre-clinical development and is a favourable candidate for clinical evaluation
• Limitations the authors state:
  • The authors state the reason for the increased activity of LMB-T20 was not determined
  • The authors note that combination studies with taxanes had not yet been carried out with the mutant immunotoxins
  • Two of the 12 PBMC samples were not repeated because of limited cells, and one sample was invalidated when the response did not repeat

Topics

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

Medical terms in this study, explained in our Lexicon: Mesothelin, Mesothelioma, Cytokine, Clinical Trial.

Related studies

• Anti-Mesothelin Recombinant Immunotoxin Therapy for Colorectal Cancer — A. Cerise, T.K. Bera, et al. (2019)
• Immunogenicity of therapeutic recombinant immunotoxins — R. Mazor, M. Onda, et al. (2016)
• 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)
• Microspheres targeted with a mesothelin antibody and loaded with doxorubicin reduce tumor volume of human mesotheliomas in xenografts — S.L. Macura, J.L. Steinbacher, et al. (2013)
• Evaluation of (111)In labeled antibodies for SPECT imaging of mesothelin expressing tumors — R. Misri, K. Saatchi, et al. (2011)
• A human single-domain antibody elicits potent antitumor activity by targeting an epitope in mesothelin close to the cancer cell surface — Z. Tang, M. Feng, et al. (2013)

Browse more research in Molecular Cancer Therapeutics, or return to medical research.