Phil Gold’s research while affiliated with Cancer Treatment Centre and other places

The antigen-induced leukocyte adherence inhibition response is directed to an organ-type specific neoantigen. Cancers of the colorectum, pancreas and stomach each expressed a unique organ-specific neoantigen. The relationship of the colon tumour antigen to the previously described carcinoembryonic antigen (CEA) of GIT cancers was examined by a tube blocking LAI assay and CEA radioimmunoassay. The colon tumour antigen was papain-solubilized from colon cancer membranes. An affinity column of horse anti-human β2-microglobulin bound the colon tumour antigen but did not bind CEA. In contrast, the colon tumour antigen failed to bind to an affinity column of antisera prepared to cell surface proteins that had failed to bind to the anti-human β2-microglobulin affinity column. Both the colon tumour antigen and CEA existed in the serum of patients with metastatic cancer. The colon tumour antigen co-isolated with the HDL fraction of serum by polyanion precipitation which suggested that the colon tumour antigen was lipoprotein in composition. By contrast, CEA was recovered in the non-lipoprotein fraction of serum. A proportion of the colon tumour antigen and CEA in the serum were eliminated into the urinary protein by filtration in the kidney. The results of the present study indicate that the colon tumour antigen epitope and the CEA epitope exist on separate molecules.

Carcinoembryonic antigen (CEA) was discovered in 1965 and defined as a tumor-specific antigen of the human digestive system (Gold and Freedman, 1965). The fact that CEA was absent from corresponding normal adult tissues, but was demonstrated in human embryonic and fetal digestive organs by the immunologic techniques employed in the initial observations, accounts for the designation given the molecule and the hypothesis that the reinitiation of its synthesis by bowel cancer cells in the adult is the result of a process variously termed derepressive dedifferentiation, retrodifferentiation, or antigenic reversion.

The blocking tube leukocyte adherence inhibition (LAI) assay was used to monitor the purification of human tumor-specific antigens (TSA's) from colon and breast cancer and malignant melanoma. The tumor antigens were specific for the organ from which the cancer arose and the histopathology of the cells of origin. Immunochemical studies revealed that TSA could be water solubilized from cancer cell membranes by limited papain digestion, and on Sephadex G-150 chromatography the majority of the TSA eluted in the molecular-weight range of 70,000 to 150,000. Affinity chromatography with anti-human β2-microglobulin (β2m) anti-serum indicated that the TSA's, like HLA molecules, contain a β2m subunit. The specificity of binding of the TSA to the anti-β2m immunoadsorbent affinity column and the immunologically specific abrogation of LAI activity were shown. Some patients with either colon adenomas or benign breast disease showed LAI activity to phosphate-buffered saline extracts of colon and breast cancer, respectively, but did not react to extracts of normal colon mucosa or breast tissue. Moreover, the LAI-reactive leukocytes were blocked by the papain-soluble TSA's from colon and breast cancer, respectively, purified by anti-β2m affinity chromatography. The present studies suggest that some 'benign' lesions express TSA's before the appearance of morphological evidence of cancer. It is not known, however, if the acquisition of a cell surface TSA is an irreversible step toward unrestrained growth and metastasis. In addition, the present studies indicate that the putative TSA's of cancers of different organs are similar in subunit structure, size, and genetic linkage and have some unexplained relationship to organ definition.

Carcinoembryonic antigen (CEA) was first detected by the immunization of rabbits with human colon cancer extract. The techniques of antiserum absorption and immunologic tolerance were employed in an attempt to render the resulting antisera tumor-specific. Using the procedures of precipitation and precipitin-inhibition in gel media, hemagglutination, passive cutaneous anaphylaxis and immunofluorescence, CEA was identified exclusively in all cancers of gastrointestinal origin and fetal digestive organs in the first two trimesters of gestation. The subsequent development of radioimmunoassays for CEA has raised the question of the presence of this material, in very low concentrations, in other normal and diseased (cancerous and noncancerous) tissue, but the problem of cross-reactivity within a family of closely related, but nonidentical, molecules remains to be resolved. CEA was initially purified by a sequence of steps involving extraction in perchloric acid, sieve chromatography, and preparative block electrophoresis. The molecule was characterized as a relatively heterogeneous acid glycoprotein with a molecular weight of approximately 200,000 and its carbohydrate (one-half to two-thirds of the molecule) and protein composition were determined. CEA was localized to the glycocalyx of the colon cancer cell and it was found that the CEA could be released from this site into the circulation of the cancer patient, where it could then be detected by means of radioimmunoassay. The clinical implications of this observation, as they have evolved over the past eight years, form the basis of the papers that constitute this supplement.

A method for the isolation of HLA antigen molecules from normal and cancerous solid human tissue is described. The method employs anti-β2-microglobulin (β2m) antiserum coupled to Sepharose beads as an immunosorbent affinity medium. The anti-β2m affinity chromatography procedure greatly purifies and selectively enriches HLA and any material that copurifies by affinity, with β2m and /or HLA molecules. The HLA isolated by this purification procedure was used to immunize rabbits. The antisera obtained were absorbed on β2m to remove all anti-β2m antibody activity. The use of such anti-HLA antisera in radioimmunoassays, immunoprecipitation studies, and F(ab′)2 blocking experiments demonstrated that these antisera are directed against a common HLA determinant present on the heavy (alloantigen-bearing) chain of all HLA molecules. The use of an identical procedure employing human tumor tissues has resulted in the isolation of HLA-like or HLA-associated tumor-specific antigens as demonstrated by the leukocyte adherence inhibition (LAI) assay.

A method for the purification of human alpha1-fetoprotein from the ascites fluid of a hepatoma-bearing patient is described that is capable of yielding large quantities of pure alpha1-fetoprotein within a relatively short period of time. The technique is based entirely on the physicochemical properties of the alpha1-fetoprotein molecule and uses sequential purification steps: ion-exchange chromatography on DEAE-Sephadex A-50, molecular-sieve chromatography on Sephadex G-200, negative-affinity chromatography on Sepharose-Blue Dextran, positivepaffinity chromatography on concanavalin A-Sepharose and, finally, molecular-sieve chromatography on Sephadex G-100. The efficiency of the entire procedure in its present form is 15% of the alpha1-fetoprotein activity of the starting preparation from ascites fluid. The purity of the final product was shown by polyacrylamide gel electrophoresis, radioimmunoelectrophoresis, and determinations of the NH2-terminal and COOH-terminal amino acid residues of the alphs1-fetoprotein isolated. Amino acid analysis of the final product revealed a composition very similar to those reported for alpha-fetoprotein preparations that have been previously isolated by the use of immunochemical technology.