Abstract
Antibody targeting has potential for selective delivery of cancer therapy. However, there is a wide variation in the degree of antibody localisation in individual patients with colorectal adenocarcinoma. Colorectal adenocarcinomas are composed of glandular structures separated from fibrovascular stroma by a basal lamina which may represent a significant barrier to extravasated antibody. Basement membrane-associated CEA epitopes may be more accessible to antibodies than those which are cytoplasmic or lumenal. We have investigated by immunohistochemistry and in vivo localisation, the extent to which distribution of antigen epitopes influences targeting. Two monoclonal antibodies (A5B7 and EA77) recognising non-overlapping CEA epitopes were reacted immunohistochemically with samples of 39 tumours. Intensity and site of reaction were assessed for basement membrane, cytoplasmic or lumenal surface association. 125I-labelled antibodies were injected into nude mice bearing LS174T tumour. Per cent injected activity per gram was measured in tumour and normal tissues, 24, 72 and 168 h later. Tissues reacted immunohistochemically for CEA were autoradiographed to assess the relationship of injected antibody to target antigen. Immunohistochemistry showed that A5B7 antibody favours basement membrane aspects of malignant glands; in contrast, EA77 concentrated generally on lumenal surfaces. In vivo localisation showed that per cent inj.act g-1 in tumour for A5B7 reached 36.5% at 24 h. EA77 localised to a lesser extent (9.1% at 24 h), despite a longer circulatory half-life. Autoradiography combined with immunohistochemistry showed A5B7 reacting with antigen close to vasculature after 24 h, slowly penetrating deeper parts of the tumour by 72 h. In contrast, EA77 was confined mainly to fibrovascular stroma, showing little labelling of antigen-positive tumour cells. Localisation differences between A5B7 and EA77 may partly be due to accessibility of epitopes on tumour cells.
Highlights
Stained sections were counterstained with haematoxylin only. Both A5B7 and EA.77 antibodies were reactive with the apical region of epithelial cells in the INFLUENCE OF EPITOPE SPECIFICITY ON IN VIVO TUMOUR LOCALISATION OF ANTI-CEA ANTIBODIES 309 upper third of the colonic crypt
Some CEA reactivity was observed at the lumenal surfaces of the middle and lower parts of the crypt, but the intensity of binding was weaker than that observed in the upper part of the crypt
Figure la (EA.77) and b and c (A5B7) are representative photomicrographs illustrating the different distributions associated with the two anti-CEA antibodies in sections of colonic adenocarcinoma
Summary
A5B7 and EA77 (anti-CEA antibodies) were obtained from the Cancer Research Campaign (CRC) Targeting Group, University Department of Clinical Oncology, Royal Free Hospital, London, UK. BA57 (Harwood et al, 1986) and EA. (Nap et al, 1992) are mouse monoclonal anti-CEA. IgG antibodies which react with non-overlapping epitopes on CEA. Antibody IDIO (obtained from the CRC Targeting Group) is directed against fetal microvillous membrane antigen and has been used clinically by Blair et al (1990). B72.3 antibody to TAG-72 antigen (Nuti et al, 1982) and an anti-colon carcinoma antigen antibody, A33 (Welt et al, 1990), were obtained from Celltech.
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