Generation of a Novel Mesothelin-Targeted Oncolytic Herpes Virus and Implemented Strategies for Manufacturing

Our plain-English summary of Generation of a Novel Mesothelin-Targeted Oncolytic Herpes Virus and Implemented Strategies for Manufacturing, by G. Froechlich, C. Gentile, L. Infante, et al (International Journal of Molecular Sciences, 2021).

The study

• Authors: G. Froechlich, C. Gentile, L. Infante, C. Caiazza, P. Pagano, S. Scatigna, G. Cotugno, A.M. D'Alise, A. Lahm, E. Scarselli, A. Nicosia, M. Mallardo, E. Sasso, N. Zambrano
• Journal: International Journal of Molecular Sciences
• Published: 2021;22(2)
• Cited by: 13 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: HER2-based retargeted viruses are in advanced phases of preclinical development of breast cancer models. Mesothelin (MSLN) is a cell-surface tumor antigen expressed in different subtypes of breast and non-breast cancer. Its recent identification as a marker of some triple-negative breast tumors renders it an attractive target, presently investigated in clinical trials employing antibody drug conjugates and CAR-T cells. The availability of MSLN-retargeted oncolytic viruses may complement the current immunotherapeutic panel of biological drugs against HER2-negative breast and non-breast tumors.

Methods: A fully virulent, tumor-targeted oncolytic Herpes simplex virus-1 (MSLN-THV) with a selectivity for mesothelin-expressing cancer cells was generated. Recombineering technology was used to replace an essential moiety of the viral glycoprotein D with antibody fragments derived from clinically validated MSLN monoclonal antibodies, and to allow IL12 cargo expression in infected cells. Panels of breast and female reproductive system cell lines were used to verify the oncolytic potential of the viral constructs. A platform for production of the retargeted viruses was developed in HEK 293 cells, providing stable expression of a suitable chimeric receptor.

Results: We demonstrated the selectivity of viral infection and cytotoxicity by MSLN-retargeted viruses in a panel of mesothelin-positive cancer cells, originating from breast and female reproductive system tumors. We also developed a second-generation oncolytic MSLN-THV, encoding IL12, to enhance the immunotherapeutic potential of the viral backbone. A non-tumor cell line expressing a chimeric MSLN/Nectin-1 receptor, de-sensitized from antiviral responses by genetic inactivation of the Stimulator of Interferon Genes (STING)-dependent pathway was engineered, to optimize viral yields.

Conclusions: Our proof-of-concept study proposes MSLN-retargeted herpesviruses as potential cancer immunotherapeutics for assessments in preclinical models of MSLN-positive tumors, complementing the available panel of oncolytic viruses to HER2-negative breast tumors.

In plain English

This laboratory study built and tested oncolytic Herpes simplex virus-1 constructs retargeted to mesothelin (MSLN), a tumour antigen the article describes as expressed in mesothelioma, ovarian and breast cancers. Recombineering replaced part of viral glycoprotein D (amino acids 6 to 38) of the wild-type recombinant virus R-LM55 with antibody fragments SD1, SS or SS1. The SD1 virus could not be recovered after the first infection passage, while THV_SS and THV_SS1 infected MSLN-positive cells; saturating anti-MSLN Amatuximab inhibited that infection, and with increasing antibody concentrations THV_SS1 infected more cells than THV_SS (p less than 0.05 between 0.1 and 50 micrograms per millilitre). Soluble mesothelin slightly reduced infection only at the highest concentrations. THV_SS1 killed MSLN-transduced HEK293 cells but left wild-type HEK293 cells 100% live. Across tumour cell lines, residual viability ranged from about 45% in CAL-120, HCC-1937 and SKOV3 to 80% in HeLa, with BT-549 and MDA-MB-231 almost unaffected. An IL12-armed virus was also made, and a STING knock-out HEK293 line expressing a non-secretable MSLN gave a 40-fold higher viral yield than SKOV3. The authors called this a proof-of-principle; no mesothelioma cells or animals were tested.

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 (in vitro) virus engineering and cell-culture study; described by the authors as a proof-of-concept/proof-of-principle study
• Who was studied: Human cell lines: HEK293 (MSLN-negative) and MSLN-transduced HEK293, plus ovarian (SKOV3, OVCAR3), cervical (HeLa) and triple-negative breast (CAL-120, BT-549, HCC1937, MDA-MB-231) tumour cell lines; no patients or animals
• Exposure: Infection with retargeted oncolytic HSV-1 constructs (THV_SD1, THV_SS, THV_SS1, THV_SS1-IL12) and parental wild-type R-LM55 or T-VEC, at multiplicities of infection from 0.001 to 10 pfu per cell
• Main results:
  • The SD1 single-domain construct did not give productive infection, whereas THV_SS and THV_SS1 were recovered
  • A saturating amount of the anti-MSLN antibody Amatuximab inhibited infection by THV_SS and THV_SS1, which the authors took as confirmation of retargeting to human mesothelin
  • In the assay with increasing Amatuximab concentrations, THV_SS1 infected more cells than THV_SS; the reported significance was p less than 0.05 over the 0.1 to 50 micrograms per millilitre range
  • Soluble mesothelin (SMRP) in conditioned SKOV3 medium reduced THV infection only at the highest concentrations, with no significant difference between THV_SS and THV_SS1
  • Cytotoxicity of THV_SS1 in HEK293 cells depended on MSLN expression, measured by Alamar Blue over 6 days
  • In the tumour cell line panel, killing by THV_SS1 tracked MSLN expression
  • BT-549 and MDA-MB-231 (low MSLN) were largely resistant to THV_SS1
  • The second-generation THV_SS1-IL12 virus produced the murine IL12 heterodimer in infected cells
  • STING knock-out in HEK293 cells increased yields of wild-type R-LM55 and of T-VEC
  • THV_SS1 production in the STING knock-out line expressing non-secretable MSLN exceeded that in SKOV3 cells
• The authors' conclusion: The authors concluded that their proof-of-principle study proposes mesothelin as a candidate antigen for retargeting oncolytic viruses, with the new viruses and the production cell platform intended for later preclinical testing in triple-negative breast cancer models
• Limitations the authors state:
  • The authors state the viruses have not yet been tested in animals; preclinical studies in immunocompetent, mesothelin-tolerant mice remain to be done
  • The authors note that cellular background, not only mesothelin levels, may affect susceptibility to infection

Topics

Medical terms in this study, explained in our Lexicon: Mesothelin, HER2, Clinical Trial, CAR-T Cell, Glycoprotein, Monoclonal Antibody.

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