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A novel human monoclonal antibody specific to the A33 glycoprotein recognizes colorectal cancer and inhibits metastasis.

ABSTRACT: Colorectal cancer represents the second most common cause of cancer-related death. The human A33 transmembrane glycoprotein is a validated tumor-associated antigen, expressed in 95% of primary and metastatic colorectal cancers. Using phage display technology, we generated a human monoclonal antibody (termed A2) specific to human A33 and we compared its epitope and performance to those of previously described clinical-stage anti-human A33 antibodies. All antibodies recognized a similar immunodominant epitope, located in the V-domain of A33, as revealed by SPOT analysis. The A2 antibody homogenously stained samples of poorly, moderately, and well differentiated colon adenocarcinomas. All antibodies also exhibited an intense staining of healthy human colon sections. The A2 antibody, reformatted in murine IgG2a format, preferentially localized to A33-transfected CT26 murine colon adenocarcinomas in immunocompetent mice with a homogenous distribution within the tumor mass, while other antibodies exhibited a patchy uptake in neoplastic lesions. A2 efficiently induced killing of A33-expressing cells through antibody-dependent cell-mediated cytotoxicity in vitro and was able to inhibit the growth of A33-positive murine CT26 and C51 lung metastases in vivo. Anti-A33 antibodies may thus represent useful vehicles for the selective delivery of bioactive payloads to colorectal cancer, or may be used in IgG format in a setting of minimal residual disease.

SUBMITTER: Murer P

PROVIDER: S-EPMC6999842 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Monoclonal antibody (MAb) 667 is a neutralizing mouse monoclonal antibody recognizing the envelope glycoprotein (Env) of the ecotropic neurotropic murine retrovirus CasBrE but not that of other murine retroviruses. Since 667 can be used for preclinical studies of antiviral gene therapy as well as for studying the early events of retroviral infection, we have cloned its cDNAs and molecularly characterized it in detail. Spot technique-based experiments showed that 667 recognizes a linear epitope of 12 amino acids located in the variable region A of the receptor binding domain. Alanine scanning experiments showed that six amino acids within the epitope are critical for MAb binding. One of them, D(57), is not present in any other murine retroviral Env, which suggests a critical role for this residue in the selectivity of 667. MAb 667 heavy- and light-chain cDNAs were functionally characterized by transient transfection into Cos-7 cells. Enzyme-linked immunosorbent assays and Biacore studies showed that the specificities as well as the antigen-binding thermodynamic and kinetic properties of the recombinant 667 MAb (r667) produced by Cos-7 cells and those of the parental hybridoma-produced MAb (h667) were similar. However, h667 was shown to contain contaminating retroviral and/or retrovirus-like particles which interfere with both viral binding and neutralization experiments. These contaminants could successfully be removed by a stringent purification protocol. Importantly, this purified 667 could completely prevent retrovirus binding to target cells and was as efficient as the r667 MAb produced by transfected Cos-7 cells in neutralization assays. In conclusion, this study shows that the primary mechanism of virus neutralization by MAb 667 is the blocking of the retroviral receptor binding domain of CasBrE Env. In addition, the findings of this study constitute a warning against the direct use of hybridoma cell culture supernatants for studying the initial events of retroviral cell infection as well as for carrying out in vivo neutralization experiments and suggest that either recombinant antibodies or highly purified antibodies are preferable for these purposes.

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A novel human monoclonal antibody specific to the A33 glycoprotein recognizes colorectal cancer and inhibits metastasis.

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