Christopher H. Thompson

Monoclonal antibodies reactive with mucin expressed in breast cancer

Three murine monoclonal antibodies (BC 1, BC2 and BC3) were developed against human milk fat globule membrane (HMFGM). By immunoperoxidase staining, it was found that the antigenic determinants had a predominant distribution in breast cancer tissue. In addition, the antibodies reacted preferentially with mucin derived from human milk rather than that derived from the breast cancer cell line ZR75; they also recognized polymorphic high molecular weight components (MW ≥ 230 000) in serum and in human milk fat globule membrane. Thus the antibodies appear to react with a component of the family of mucins found in breast cancer and human milk and it appears likely that at least part of each epitope is protein in nature. Antibodies BC1, BC2 and BC3 recognized related but not identical epitopes, and they appear to be co‐expressed on the same molecules as 3E1·2‐defined antigen (mammary serum antigen, MSA) which is also a member of the family of breast cancer‐related mucin. However, the 3E1 2 epitope is distinct and non‐cross‐reactive with those described for BC1, BC2 and BC3. The BC2 and BC3 defined epitopes were examined for their value in serum assays. Immunoassay was developed with a combination of two antibodies, using antibody BC3 for antigen capture and antibody BC2 or 3E1·2 for antigen detection and gave reasonable sensitivity (~85%) and specificity (~95%) in such serum tests for breast cancer. In a limited study, these tests appeared to complement the MSA test in the detection of breast cancer.

Heterogeneity of the human transferrin receptor and use of anti-transferrin receptor antibodies to detect tumours in vivo.

The human transferrin receptor (TFR) which is present on dividing cells, many tumours and erthyroid precursors is readily identified using specific monoclonal antibodies. A new anti‐human TFR monoclonal antibody, HuLy‐m9, is described and its distribution on cell lines, normal and tumour tissue was examined and compared with two other anti‐TFR monoclonal antibodies, namely, OKT9 and 5E9. The three antibodies were shown to recognise different epitopes on the surface of the TFR and have different reactivities with in vitro cell lines. Peptide map analyses of the TFR recognised by each monoclonal antibody from the same cell line were identical; however, differences were observed between cell lines. 131I‐radiolabelled HuLy‐m9 was used to successfully localise a nasopharyngeal carcinoma in vivo.

Production of monoclonal antibodies to serum antigens in colorectal carcinoma

Two monoclonal antibodies (mAb) to colorectal cancer were produced using a novel immunization technique. This involved immunizing mice with whole serum obtained from patients with cancer of the colon (three with metastatic disease) and resulted in antibodies which were reactive with colonic tumor tissue by immunoperoxidase testing. Two mAbs (O-1, I-1) were isolated which were non-reactive with normal tissue and with tissues obtained from subjects with benign disease but were reactive with 34/50 (formalin-fixed) colon carcinoma specimens. Further testing on cell lines and other malignant tumors suggested both mAbs detect carcinoembryonic antigen (CEA) as their reactivities were similar to a known anti-CEA antibody, and they reacted with CEA in a solid-phase radioimmunoassay. The two mAbs were found to react with the same or closely associated epitopes on CEA by competitive tests. As the anti-CEA antibodies were made to serum (rather than tissue) CEA, it was possible that unique, highly specific mAb has been produced, particularly as there was a selective reaction of the mAbs for malignant but not normal tissues. A serum test with mAb I-1 was developed which detected raised serum CEA levels in 3/24 patients with benign colonic lesions, 7/19 patients with pancreatic cancer, 5/25 patients with colonic cancer but not in 20 normal individuals. There was direct correlation between these results and a commercially available CEA test kit.

Hu Ly-M3--a human leukocyte antigen.

A monoclonal antibody, 5–4.8, was produced against human peripheral blood lymphocytes and it appears to be leukocyte-specific in that it reacts with a common determinant (called Hu Ly-m3) present on the peripheral blood T, B and null lymphocytes of 40 individuals. The antibody also reacts with thymocytes, spleen cells, and bone marrow cells (30%) and weakly with granulocytes and platelets—but not with heart, liver, or kidney. Affinity to lentil-lectin and molecular weight analysis demonstrated that Hu Ly-m3 is a glycoprotein consisting of a single chain of 47,000 daltons which is not HLA because it is not present on all cells; because it is present on the surface of the phenotypically HLA-Daudi cell line; and because soluble HLA antigens did not inhibit the binding of the 5—4.8 antibody.

Visualization of metastases from colon carcinoma using an lodine 131‐radiolabeled monoclonal antibody

A murine monoclonal antibody that reacts with human colonic cancer (250–30.6) was labeled with radioactive iodine (131I) and the antibody was injected intravenously into 15 patients with known metastases originating from carcinoma of the colon (10 cases), malignant melanoma (1), breast (1), pancreas (1), hepatocellular carcinoma (1), and adenocarcinoma of unknown origin (1). Of the patients with metastatic colon carcinoma, there were 19 known deposits as judged by the techniques of clinical examination, x‐rays, and scans obtained using sulpha‐colloid. Of these 19 deposits, 17 (90%) were found using the 131I‐labeled monoclonal antibody. In one case, the primary tumor, previously undiagnosed, was found. In only 1 of the 10 patients was tumor not found and this was due to the subsequent finding that the undifferentiated tumor did not react with antibody. Of the five patients who did not have carcinoma of the colon, three had negative scans, but two were positive. Thus, the technique of immunoscintography can readily detect both primary and metastatic tumors.

Detection of Breast Cancer Using the Monoclonal Antibody 3E-1.2

The monoclonal antibody 3E-1.2 was produced by immunizing mice with fresh tissue from a primary breast carcinoma (CaB). The antibody is of the IgM class, reacts with formalin fixed tissues and by immunoperoxidase testing, all breast cancers were found to be 3E-1.2+, but normal breast tissue was either weakly positive or totally non-reactive; as were most other normal tissues, the exceptions being some cell types in lung, kidney, endometrium and urinary bladder. The antibody has the strongest reactions with breast cancer. The antibody has been used for two major purposes described herein, a) the localization of breast cancer in draining axillary lymph nodes. This was performed by injecting radiolabelled monoclonal antibody into the interdigital space of the hand and scanning axillae 16 to 24 hours later. Using this technique lymph nodes containing tumor deposits can be detected when the nodes did not contain a palpable mass. b) A serum test has been developed which detects circulating antigens in the blood and can readily distinguish between normal subjects (n >2000) and those with breast cancer (n > 100); in normal subjects the serum levels are 105 ± 53 (I.S.D.) wherein those with breast cancer the levels are > 267 in 90%. The patients with CaB with a normal serum level (10%) were found to be in remission or undergoing chemotherapy.

Production and characterization of a new monoclonal antibody to colorectal carcinoma

This study describes a new murine monoclonal antibody (MoAb) 5C1 raised against human colorectal carcinoma, which gave a differential reaction on formalin‐fixed sections of the gastrointestinal tract. The MoAb 5C1 of immunoglobulin M (IgM) isotype reacted with both the cytoplasm and membrane of all normal colonic epithelia, and with all benign colonic polyps and all premalignant colonic lesions. However, there was a decreased expression of the 5C1 antigen in most cases of colonic malignancy and it was this feature that makes MoAb 5C1 unique. The distribution of the 5C1 epitope in normal gastrointestinal tract is limited to a few epithelial cells in the mid‐portion of the small intestine but this distribution increased progressively down the digestive tract until it was found on > 90% of normal epithelial cells (in membrane and cytoplasm) of the colon. In addition, the 5C1 epitope was present on mucin secreting cells from normal organs of the gastrointestinal, reproductive and pulmonary tract and benign and malignant tissues of the colon. On Western blots, MoAb 5C1 was found to detect a heterogeneous population of molecules with molecular weights >100 kDa with the strongest staining bands found between 230 and 300 kDa. MoAb 5C1 does not detect carcino‐embryonic antigens (CEA), human milk fat globules (HMFG), human lymphocyte antigens (HLA) or ABO blood group antigens. The combination of its presence in mucin secreting cells and its broad molecular weight bands suggest that the antigen detected is a mucin.

The definition of an MB related specificity by a monoclonal antibody

A number of monoclonal antibodies have been described that react with monomorphic and polymorphic Ia-like specificities on human B cells, but it is not clear whether these react with HLA-DR encoded molecules or with the products of other closely linked genes within the MHC, such as MB, MT, or DC 1 antigens. A monoclonal antibody is described herein (MC-26.1) which detects a new Ia-like specificity as shown by B-cell reactivity, chemistry, family studies, including recombinant family and coprecipitation studies. The latter studies showed that specificity detected by the MC-26.1 antibody coprecipitated with determinants detected by conventional anti-MB3 and MT3, but not with HLA-DR4 antisera. The determinant was polymorphic but not related to any of the known MB or MT specificities. However, cross-reactions were demonstrated by the variability of reaction on different individuals. Coprecipitation studies indicate that the antigen defined by MC-26.1 coprecipitates with MB and MT specificities suggesting that the antibody defines a new specificity on the MB3 and MT3 molecules.