New Monoclonal Antibodies to Mesothelin Useful for Immunohistochemistry, Fluorescence-Activated Cell Sorting, Western Blotting, and ELISA

Abstract

Purpose: Mesothelin is a cell surface protein that is highly expressed in some malignant tumors, and is a promising target for immunotherapy. Recent data suggests that mesothelin is an adhesive protein and may have a role in the metastases of ovarian cancer. Although a few monoclonal antibodies (MAb) to mesothelin have been produced, they have limitations for the study of expression of native mesothelin because of their low affinity or reactivity only with denatured mesothelin protein. We have produced novel MAbs to mesothelin to help study mesothelin function and to develop improved diagnosis and immunotherapy of mesothelin-expressing tumors. Experimental Design: Mesothelin-deficient mice were immunized with plasmid cDNA encoding mesothelin, and boosted with a mesothelin-rabbit IgG Fc fusion protein prior to cell fusion. Hybridomas were screened by an ELISA using plates coated with mesothelin-Fc protein.Results: Seventeen hybridomas producing anti-mesothelin antibodies were established and shown to react with two epitopes on mesothelin. One group reacts with the same epitope as the low affinity antibody K1that was originally used to identify mesothelin. The other is a new group that reacts with a new epitope. One antibody from each group was chosen for further study and shown to react strongly on ELISA, on immunohistochemistry, and by fluorescence-activated cell sorting on living cells. Conclusion: Our two newly established MAbs, MN and MB, have different and useful properties compared with current antibodies used for the detection of mesothelin by immunohistochemistry, fluorescence-activated cell sorting, ELISA, and Western blotting.

Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes

“…Briefly, human sera were obtained under National Institutes of Health Institutional Review Board-approved protocols. Mesothelin-rFc was added to the ELISA plate (100 ng in 50 μL PBS/well) and incubated overnight at 4°C (22). After washing, SS1P (antimesothelin Fv-PE38, 100 ng in 50 μL blocking buffer/well) was added for 1 h and bound to the mesothelin.…”

Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

“…As a proof-of-concept, we chose to humanize the murine mAb MN, 33 specific for the cell surface glycoprotein mesothelin, 34,35 by screening mini-libraries consisting of humanized HCs and LCs. Humanized variable regions were designed by grafting of the MN HC and LC complementarity-determining regions (CDRs) into a series of 47 different frameworks for each immunoglobulin chain (Fig.…”

“…Parental anti-ROR1 mouse mAb 2A2 28 and rabbit mAb R11, 37 as well as anti-mesothelin mouse mAb MN, 60 were produced as chimeric, full-length IgG1 antibodies with human constant regions as follows: Variable region coding regions were produced by total gene synthesis (GenScript) using MNFGLRLIFLVLTLKGVQC as leader sequence, and were assembled with human IgH-g 1 and IgL-k constant regions in the expression vector pCB14b. This vector, a derivative of the episomal mammalian expression vector pCEP4 (Invitrogen), carries the Epstein-Barr virus (EBV) replication origin, encodes the EBV nuclear antigen (EBNA-1) to permit extrachromosomal replication, and contains a puromycin selection marker in place of the original hygromycin B resistance gene.…”

Expression of POTE protein in human testis detected by novel monoclonal antibodies

“…We used 4 vectors for expression of each paralog: pcDNA3 (Invitrogen, Carlsbad, CA) for full-length protein expression in mammalian cells, an Fc fusion vector derived from pSegTag2 (Invitrogen) to make POTE fragments (amino acid 1-130 of each paralog) as fusion proteins with rabbit IgG1 Fc portion in 293T cells [7], pGEX6P-3 (Amersham Biosciences, Piscataway, NJ) to make glutathione S-transferase (GST)-fusion proteins in E. coli, and pEGFP-N1 (Novagen, Madison, WI) to express each CRR as an EGFP-fusion protein. To make the cDNA inserts, the POTE paralog cDNAs were PCR-amplified using appropriate primers containing restriction enzyme sites from the original plasmid clones [2] (The GenBank accession numbers are: POTE-21, AY172978; POTE-2γC, AY462873; POTE-22, AY466021, POTE-2α, AY462871).…”

“…To harvest Fc-fusion proteins in the supernatants and to obtain POTE-expressing cells for Western blot, immunofluorescence or FACS analysis, 293T cells were transfected with each plasmid using Lipofectamine and Plus reagent (Invitrogen) as described previously [7].…”