Imaging of colorectal carcinoma with radiolabeled antibodies.

A series of 14 monoclonal anti-carcinoembryonic antigen (CEA, Mr 180,000) antibodies (MAbs) that show a strong degree of selective reactivity for human colon carcinomas versus normal adult tissues were used to construct a serological map of the CEA molecule. The MAbs were generated using extracts of colon carcinomas as immunogen and are thus given a COL designation. None of the 14 COL-MAbs tested were reactive with purified non-specific cross-reacting antigen (NCA, Mr 55,000) from normal lung, although some showed reactivity to human granulocytes. All the COL-MAbs tested were reactive with normal fecal antigen-2 (NFA-2, Mr 170,000); however, many of the COL-MAbs demonstrated a higher affinity constant to CEA than to NFA-2. Cross-competition radioimmunoassays classified the 14 COL-MAbs into 5 groups. The chemical nature of the COL-binding domains was tested using chemically or enzymatically treated CEA; all reacted with periodate-treated CEA and deglycosylated CEA, indicating that the COL-reactive epitopes appear to be of a proteinaceous nature. Heat treatment, reduction, alkylation, pepsin digestion or pronase treatment of CEA, however, gave differential results with respect to COL binding. Antibody titration experiments were carried out to define differential reactivities to colorectal carcinoma versus NCA-containing granulocyte extracts; these results were compared with results obtained using several anti-CEA MAbs that have been used in clinical trials. Granulocyte binding and biochemical studies showed that the COL MAbs may distinguish as many as 7 to 10 CEA epitopes.

Serologic mapping and biochemical characterization of the carcinoembryonic antigen epitopes using fourteen distinct monoclonal antibodies.

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The carcinoembryonic antigen (CEA): Past, present, and future

The distribution of carcinoembryonic antigen (CEA) in colorectal cancer (CRC) differs from that in normal colorectal tissue, being found on all borders of the cell membrane and hence enabling access to intravenous antibody, making CEA a good target for antibody-based therapy. The distinctive anti-CEA antibody, PR1A3, binds only membrane-bound CEA. Humanised PR1A3 (hPR1A3) was assessed both in vitro cytotoxicity and binding assays with colorectal cancer cell lines expressing varying levels of CEA. Human peripheral blood mononuclear cells (PBMCs) and purified natural killer (NK) cells were used as effectors. The in vitro assays demonstrated hPR1A3 CEA-specific binding and antibody-dependent and CEA-specific killing of human colorectal cancer cell lines by human PBMCs. The effect increased with increasing concentration of antibody and surface CEA, and was lost by using the parent murine IgG1 PR1A3. Killing was also blocked by antibody to the Fc-γIIIA receptor. Purified human NK cells were effective at much lower effector:target ratios than unfractionated PBMCs, indicating that NK cells were the main mediators of hPR1A3-based CEA-specific killing. The results support the development of hPR1A3 for therapy of colorectal cancer.

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Targeted killing of colorectal cancer cell lines by a humanised IgG1 monoclonal antibody that binds to membrane-bound carcinoembryonic antigen

A new intraoperative approach to tumor localization using radiolabelled monoclonal antibody (MAb) B72.3 involves the use of a hand-held gamma-detecting probe (GDP) by the surgeon and, subsequently, the pathologist. We report here the use of 125j-labelled MAb B72.3 IgG and a GDP to localize primary and metastatic colorectal cancer in 31 patients. The patients were administered radiolabelled MAb i. v., and all underwent surgical exploration 5 to 35 days post-injection. In vivo localization of the MAb was evaluated using a GDP, with tumor and normal tissue counts being obtained. In each case, the subsequent tumor and normal tissue that were resected were analyzed in vitro for MAb localization; this was evaluated by calculating the radiolocalization index, i. e., the ratio of the injected dose per gram localized to tumor versus that of normal tissue. When the GDP was used intraoperatively, MAb B72.3 localized tumors in 68% (21/31) of the patients; the arbitrary criterion of tumor-to-normal tissue ratios higher than or equal to 2.0: I in vivo being taken as positive. Resected tumor radiolocalization indices ranged from 0.5 to 543, and 71% (22/31) of the patients studied had tumors with radiolocalization indices higher than or equal to 3. Of 50 carcinoma biopsies, 34 that were probe-positive were antigen-positive when B72.3 was used in immunoperoxidase assays, while 4 carcinoma biopsies that were probe-negative were also antigen-negative. Twelve of 50 biopsies were probe-negative and antigen-positive, but many of these lesions only contained a few antigen-positive cells; none of the 50 was probe-positive and antigen-negative. Tumors of all histologic grades localized injected MAb and, in general, higher in vivo probe ratios and radiolocalization indices were obtained from patients who underwent surgery 20 to 35 days following injection of the MAb. MAb B72.3 localized tumor in all sites to which colon carcinoma commonly metastasizes, including mesenteric and peri-aortic lymph nodes, liver, lung, and peri-rectal soft tissue. There was a strong statistical correlation (p = 0.001) between detecting MAb B72.3 localization to tumors using the GDP intraoperatively and subsequent in vitro analysis of cpm/g for tumor versus normal tissues. These studies thus further validate the use of 125I-labelled MAb B72.3 IgG and of a hand-held gamma probe for the intraoperative detection of carcinomatous lesions.

Intraoperative radioimmunolocalization of colorectal carcinoma with a hand-held gamma probe and MAb B72.3: Comparison of in vivo gamma probe counts with in vitro MAb radiolocalization

The localisation of tumour deposits by a 131 I labelled monoclonal antibody to carcinoembryonic antigen (CEA) has been evaluated in 24 patients with primary gastric, oesophageal and colorectal cancer and in 26 patients with clinically suspected recurrent gastric and colorectal cancer. Seventeen of 20 primary sites and 6/15 associated secondary sites were correctly identified by external scanning. Measurement of radiolabelled antibody in the resected specimens demonstrated a 2.6-3.3 fold increase in comparison with the surrounding normal tissue (P less than 0.01). The antibody scans were compared with computerised tomography (CT) in the detection of recurrent disease. The respective sensitivities and specificities for the two investigations were 61% and 33% for antibody scanning and 64% and 100% for CT. Assessment of the distribution of labelled antibody demonstrated rapid clearance with less than 2% detectable in serum samples at 24 h. The implications of these findings together with the mechanisms of excretion are discussed.

P Anderson

Papers

Localisation of gastrointestinal cancer with a 131 I labelled monoclonal antibody to CEA.