Splenic lymphocytes of mice, immunized with membrane-enriched fractions ofmetastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulinsecreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts ofapparently normal human liver. As a result offurther screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a "pancarcinoma" reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.Numerous investigators have reported the existence ofantigens associated with human mammary tumors (1-9). These studies, all conducted with conventional hyperimmune polyclonal sera, however, were unfortunately hampered with regard to the heterogeneity of the antibody populations employed and the amount of specific immunoglobulin that could be generated. Since the advent ofhybridoma technology (10), monoclonal antibodies of predefined specificity and virtually unlimited quantity may now be generated against a variety of antigenic determinants present on normal or neoplastic cells. The rationale of the studies reported here was to utilize extracts ofhuman metastatic mammary tumor cells as immunogens in an attempt to generate and characterize monoclonal antibodies reactive with determinants that would be maintained on metastatic, as well as primary, human mammary carcinoma cells. Multiple assays using tumor cell extracts, tissue sections, and live cells in culture have been employed to reveal the diversity of the monoclonal antibodies generated.MATERIALS AND METHODS Immunizations. Membrane-enriched cell extracts were prepared from breast tumor metastases to the liver from two patients as well as from apparently normal liver as described (11) Hybridoma Methodology. Somatic cell hybrids wer...

We report here both the range and patterns of reactivity of an IgG1 monoclonal antibody, B72.3, prepared against human, metastatic mammary carcinoma cells. When the avidin-biotin complex (ABC) immunoperoxidase technique was used on tissue sections, monoclonal B72.3 reacted with 19 of 41 (46%) primary mammary carcinomas and 13 of 21 (62%) metastatic lesions, either in axillary lymph nodes or at distal sites. Variable concentrations of antigen, recognized by B72.3, were observed among mammary tumors, as well as among different cell populations of a given tumor mass. Several patterns of antigen distribution were observed: membrane, diffuse cytoplasmic, focal and marginal. No reactivity was observed to normal mammary epithelium, stroma, or lymphocytes of the breast, nor to any cell types in a variety of other normal human tissues, melanomas, and sarcomas. Reactivity with all of four colon carcinomas was also observed. Assay of serial sections of mammary carcinomas with B72.3 and a monoclonal antibody directed against carcinoembryonic antigen demonstrated that these antigens were both distinct and non-coordinately expressed.

While several murine monoclonal antibodies (MAbs) directed against carcinoma associated antigens have shown excellent tumor targeting properties in clinical trials, the use of radiolabeled MAbs for both diagnostic and therapeutic applications has been hindered by two factors: (a) the induction of host anti-immunoglobulin (Ig) responses and (b) slow plasma clearance of unbound radiolabeled MAb, resulting in bone marrow toxicity for therapeutic application, and long intervals between MAb administration and tumor detection for diagnostic applications. This report describes the development of the first recombinant Ig with properties designed to reduce or eliminate both of the above problems: a complementarity determining region (CDR)-grafted humanized (Hu) MAb with a CH2 domain deletion (delta CH2). The MAb chosen for engineering was CC49, which is directed against a pancarcinoma antigen designated TAG-72 that is expressed on the majority of colorectal, gastric, breast, ovarian, prostate, pancreatic and lung carcinomas. When characterized for antigen binding in solid phase competition radioimmunoassays, the HuCC49 delta CH2 MAb completely inhibited the binding of murine (mu) CC49 and HuCC49 for TAG-72. The relative affinity constants (Ka) of MAbs HuCC49 delta CH2, HuCC49 and muCC49 were 5.1 x 10(-9), 2.1 x 10(-9) and 2.3 x 10(-9), respectively. The plasma clearance of 131I-HuCC49 delta CH2 was significantly faster than that of intact 125I-HuCC49 after either i.v. or i.p. administration in athymic mice (p(2)0.05). Biodistribution studies in athymic mice bearing human colon carcinoma xenografts after i.v. or i.p. administration of 131I-HuCC49 delta CH2 and 125I-HuCC49 demonstrated the efficient tumor localization and substantially lower percent of the injected dose (%ID/g) of the HuCC49 delta CH2 in normal tissues. This is reflected in the significantly higher radiolocalization indices (%ID/g in tumor divided by %ID/g in normal tissue) observed with the HuCC49 delta CH2 for most normal tissues tested (p(2)0.05). The differential between the rate of plasma clearance of HuCC49 delta CH2 and HuCC49 was even more pronounced in SCID mice, which have been shown to be an appropriate model to study the metabolism of human IgG. These studies thus describe the development of a recombinant Ig molecule which, for the first time, combines 1) the properties of more rapid blood clearance than an intact humanized Ig molecule--without loss of antigen binding affinity--and 2) reduced potential for eliciting a human anti-murine antibody (HAMA) response in patients. These studies also demonstrate the potential utility of HuCC49 delta CH2 for i.p. as well as i.v. radioimmunodiagnosis and radioimmunotherapy in patients with TAG-72 positive tumors.

The major neoplastic transformation-inducing genes of human solid tumors are members of the ras oncogene family. We used an immunohistochemical assay to assess expression of both the unaltered and the mutated ras oncogene protein (p21) in normal and neoplastic prostatic cells. With the concentration of monoclonal antibody used in this study, epithelial and stromal cells from subjects with normal prostates and from 19 patients with benign prostatic hyperplasia were negative for p21 antigen. This antigen was detected in 2 of 6 prostates with Grade I carcinoma, 4 of 6 with Grade II, and all of 17 with higher grades. A semiquantitative immunohistochemical method demonstrated that expression of the p21 antigen in a carcinoma strongly correlated with nuclear anaplasia and was inversely related to the degree of glandular differentiation. However, markedly anaplastic tumors were often more heterogeneous in expression of p21 and contained areas of low staining for the antigen. Comparison of p21 antigen with tumor carcinoembryonic antigen and prostate-specific antigen demonstrated that ras p21 was the only phenotypic marker that correlated with histologic tumor grade. Thus, ras oncogene p21 may represent a new class of biologically relevant tumor markers and may be a useful adjunct to histopathologic examination in determining the prognosis of patients with prostate cancer.