Dual B- and T-cell de-immunization of recombinant immunotoxin targeting mesothelin with high cytotoxic activity

Our plain-English summary of Dual B- and T-cell de-immunization of recombinant immunotoxin targeting mesothelin with high cytotoxic activity, by R. Mazor, M. Onda, D. Park, et al (Oncotarget, 2016).

The study

• Authors: R. Mazor, M. Onda, D. Park, S. Addissie, L. Xiang, J. Zhang, R. Hassan, I. Pastan
• Journal: Oncotarget
• Published: 2016;7(21):29916-26
• Cited by: 39 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Recombinant immunotoxins (RITs) are genetically engineered proteins being developed to treat cancer. They are composed of an Fv that targets a cancer antigen and a portion of a protein toxin. Their clinical success is limited by their immunogenicity. Our goal is to produce a new RIT that targets mesothelin and is non-immunogenic by combining mutations that decrease B- and T-cell epitopes. Starting with an immunotoxin that has B-cell epitopes suppressed, we added mutations step-wise that suppress T-cell epitopes. The final protein (LMB-T14) has greatly reduced antigenicity as assessed by binding to human anti-sera and a greatly decreased ability to activate helper T-cells evaluated in a T-cell activation assay. It is very cytotoxic to mesothelioma cells from patients, and to cancer cell lines. LMB-T14 produces complete remissions of a mesothelin expressing cancer (A431/H9) xenograft. The approach used here can be used to de-immunize other therapeutic foreign proteins.

In plain English

This was a laboratory (preclinical) study in which the authors built and tested a recombinant immunotoxin (RIT) against mesothelin, LMB-T14, made by starting from an immunotoxin with silenced human B-cell epitopes (SS1-LO10R) and adding, step by step, mutations that suppress T-cell epitopes, giving a protein with domain II largely deleted and 10 point mutations in domain III. Against A431/H9 cells, SS1-LO10R had an IC50 of 1.7 pM and LMB-T20 2.2 pM, and LMB-T14 kept high cytotoxic activity, a little less than both parents; on established cell lines (HAY, L55, KLM1) it was 3- to 5-fold less active than its parents but more active than SS1P. On early-passage cells from four mesothelioma patients, LMB-T14, LMB-T20 and LO10R were all more cytotoxic than SS1P (IC50s under 100 pM). Binding to sera from 19 patients fell (mean 16% of SS1P), and T-cell activation spots fell 61% for LMB-T14 and 81% for LMB-T20. In nude mice bearing A431/H9 tumours, 5/6 tumours per dose group were undetectable by day 16. Unexpectedly, 4/10 donors responded to a new T-cell epitope.

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 protein-engineering study with in vitro cytotoxicity, immunogenicity and antigenicity assays and mouse toxicity/xenograft experiments
• Who was studied: Early-passage mesothelioma cells from four patients (NCI-Meso16, 19, 21, 29), six mesothelin-expressing cell lines, PBMC from 10 naive donors (plus 19 donors for epitope mapping), sera from 19 patients with neutralizing antibodies, Swiss mice and athymic nude mice, at the National Cancer Institute, USA
• Exposure: Recombinant immunotoxins targeting mesothelin: SS1P, SS1-LO10R (six B-cell epitope mutations), LMB-T20 (six T-cell epitope mutations) and LMB-T14 (domain II deletion plus 10 point mutations in domain III)
• Main results:
  • On A431/H9 cells, SS1-LO10R had an IC50 of 1.7 pM and LMB-T20 an IC50 of 2.2 pM
  • LMB-T14 retained high cytotoxic activity on A431/H9 cells, somewhat lower than LMB-T20 and LO10R
  • On cells from the four mesothelioma patients, LMB-T14, LMB-T20 and LO10R were all more cytotoxic than SS1P, with IC50s below 100 pM
  • On three established cell lines, LMB-T14 was 3-5 fold less active than its parent immunotoxins but still more active than SS1P
  • Against sera from 19 patients with neutralizing antibodies, LMB-T14 showed reduced binding, mean 16% relative to SS1P
  • In the IL-2 ELISpot T-cell activation assay with PBMC from 10 naive donors, IL-2 spots fell 61% with LMB-T14 and 81% with LMB-T20
  • A new T-cell response to peptides 63-65 of LMB-T14 appeared in 4 of 10 donors, and in further testing in 7 of 19 donors
  • In Swiss mice, LMB-T14 caused no significant weight loss at 20 mg/kg while the same dose of LMB-T20 was toxic in 4/4 mice; 28 mg/kg LMB-T14 was toxic in 4/4 mice
  • In nude mice with A431/H9 xenografts treated with four doses of LMB-T14 (5 or 7 mg/kg), 5/6 tumours in each group were undetectable by day 16 and regressions persisted to day 30
• The authors' conclusion: The authors concluded that LMB-T14, carrying mutations suppressing both B- and T-cell epitopes, has greatly reduced immunogenicity while remaining highly cytotoxic, being well tolerated in mice and producing complete remissions of mesothelin-expressing xenografts; they state this is the first therapeutic protein designed with silencing of both B- and T-cell epitopes
• Limitations the authors state:
  • The mutations reduced but did not completely eliminate T-cell responses, and the B-cell mutations did not reduce binding of all tested human anti-sera
  • The authors could not determine whether the B- or the T-cell epitope matters more for de-immunization
  • Adding de-immunizing mutations traded off some cytotoxic activity on several cancer cell lines

Topics

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

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

Related studies

• Immunogenicity of therapeutic recombinant immunotoxins — R. Mazor, M. Onda, et al. (2016)
• Recombinant Immunotoxin with T-cell Epitope Mutations That Greatly Reduce Immunogenicity for Treatment of Mesothelin-Expressing Tumors — R. Mazor, J. Zhang, et al. (2015)
• Anti-Mesothelin Recombinant Immunotoxin Therapy for Colorectal Cancer — A. Cerise, T.K. Bera, et al. (2019)
• Mesothelin-Targeted Recombinant Immunotoxins for Solid Tumors — B.L. Hagerty, G.J. Pegna, et al. (2020)
• Modulating mesothelin shedding to improve therapy — I. Pastan, Y. Zhang (2012)
• Immunogenicity of Immunotoxins Containing Pseudomonas Exotoxin A: Causes, Consequences, and Mitigation — R. Mazor, I. Pastan (2020)

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