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Comparison of Anti-Trop2 Extracellular Domain Antibodies Generated Against Peptide and Protein Immunogens for Targeting Trop2-Positive Tumour Cells

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Abstract

Trophoblast antigen 2 (Trop2) is a transmembrane glycoprotein upregulated in multiple solid tumours. Trop2-based passive immunotherapies are in clinical trials, while Trop2 targeting CAR-T cell-based therapies are also reported. Information about its T- and B-cell epitopes is needed for it to be pursued as an active immunotherapeutic target. This study focused on identification of immunodominant epitopes in the Trop2 extracellular domain (ECD) that can mount an efficient anti-Trop2 antibody response. In silico analysis using various B-cell epitope prediction tools was carried out to identify linear and conformational B-cell epitopes in the ECD of Trop2. Three linear peptide immunogens were shortlisted and synthesized. Along with linear peptides, truncated Trop2 ECD that possesses combination of linear and conformational epitopes was also selected. Recombinant protein immunogen was produced in 293-F suspension culture system and affinity purified. Antisera against different immunogens were characterized by ELISA and Western blotting. Two anti-peptide antisera detected recombinant and ectopically expressed Trop2 protein; however, they were unable to recognize the endogenous Trop2 protein expressed by cancer cells. Antibodies against truncated Trop2 ECD could bind to the endogenous Trop2 expressed on the surface of cancer cells. In addition to their high avidity, these polyclonal anti-sera against truncated Trop2 protein also mediated antibody-dependent cell-mediated cytotoxicity (ADCC). In summary, our comparative analysis demonstrated the utility of truncated Trop2 ECD as a promising candidate to be pursued as an active immunotherapeutic molecule against Trop2-positive cancer cells.

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Data can be made available upon request to the corresponding author.

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Acknowledgements

We acknowledge the technical support provided by Dr. S. Mukherjee, Ms. N. Joshi, Ms. G. Shinde and Ms. S. Khavale from DNA sequencing and Flow Cytometry Core Facility of ICMR-NIRRCH.

Funding

This study received financial support from Indian Council of Medical Research (ICMR), New Delhi, India (RA/1281/07-2022). ICMR Senior Research Fellowship (RBMH/FW/2020/36) has been granted to author Ms. Pradnya Kamble.

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Authors and Affiliations

  1. Cellular and Structural Biology Division, ICMR-National Institute for Research in Reproductive and Child Health, Mumbai, 400012, India

    Pradnya R. Kamble, Bhalchandra Kulkarni, Madhulika Bajaj, Ananya A. Breed, Dhanashree Jagtap, Smita Mahale & Bhakti R. Pathak

  2. Cactus Communications, Mumbai, India

    Amisha Malaviya

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  1. Pradnya R. Kamble
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Contributions

PK, experimental planning and execution, data analysis and interpretation and manuscript writing original draft preparation and editing; BK, assistance in peptide synthesis, immunization and SPR analysis; AM, MB and AB, assistance in experimentation; DJ, data analysis and editing the manuscript; SM, planning and supervision; BP, conceptualization, design and planning, data analysis and interpretation, manuscript writing and reviewing and editing.

Corresponding author

Correspondence to Bhakti R. Pathak.

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All animal procedures were approved by the Institutional Animal Ethics Committee (Project no: 21/15) recognized by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

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Kamble, P.R., Kulkarni, B., Malaviya, A. et al. Comparison of Anti-Trop2 Extracellular Domain Antibodies Generated Against Peptide and Protein Immunogens for Targeting Trop2-Positive Tumour Cells. Appl Biochem Biotechnol (2023). https://doi.org/10.1007/s12010-023-04706-4

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