Construction of a chimeric antibody with therapeutic potential for cancers which overexpress c-erbB-2.

We describe the chimerization of a monoclonal antibody directed against the c-erbB-2 protein using a novel PCR method for cloning immunoglobulin variable region genes. We also describe the characterization of the chimera and show its potential use for treating cancers which overexpress the c-erbB-2 protein. The genomic DNA fragments of heavy and light chain variable genes were cloned by PCR using uniquely designed primers which allowed for isolation of genes containing functional promoters, signal and coding sequences. The chimeric genes were then constructed by linking variable regions of murine genes to human constant gamma 1 and kappa genes. Expression of the chimeric immunoglobulin genes resulted in production of properly assembled chimeric antibody with improved biological properties.

An antigen immunologically related to the external domain of gp185 is shed from nude mouse tumors overexpressing the c-erbB-2 (HER-2/neu) oncogene.

An antigen, immunologically related to the external domain of the c-erbB-2 (HER-2/neu) protein, was found shed into the serum of nude mice bearing tumors that overexpress the c-erbB-2 protein (gp185). Utilizing paired combinations from a panel of monoclonal antibodies (TAbs 250-265), with specificity for extracellular epitopes of gp185, an immunoradiometric assay was developed to quantitate this shed antigen. The immunoradiometric assay detected membrane-bound and soluble gp185 as well as a soluble derivative corresponding in sequence to the extracellular domain of gp185 (designated gp75). This recombinantly expressed gp75 was immunoaffinity purified and used to generate a standard curve from which serum samples were quantitated. Increases in antigen levels measured in the sera of tumor-bearing nude mice correlated with both overexpression of the c-erbB-2 protein and increased tumor volume. Positive sera were obtained from mice given implants of NIH3T3 cells transfected with c-erbB-2 complementary DNA (NIH3T3t), or ovarian (SK-OV-3) or breast (MDA-MB-361) tumor cell lines overexpressing the c-erbB-2 protein. In mice bearing NIH3T3t tumors, increases in tumor volume from 80 to 9000 mm3 resulted in levels of shed antigen from 8 to greater than 1000 ng/ml gp75 equivalents. Sera from mice with c-erbB-2-negative tumors or tumors overexpressing the epidermal growth factor receptor were negative in the assay. This assay, and the quantitation of shed antigen levels, may have diagnostic or monitoring utility in cancers, such as breast and ovarian, in which the c-erbB-2 protein is overexpressed.

Human T-cell leukemia virus minus strand transcription in infected T-cells.

The plus strand of the Human T-cell Leukemia Virus RNA genome encodes proteins critical for infectivity, replication, and transformation. Thus far, gene products encoded on the minus strand of the HTLV-I provirus have not been found. We have identified several open reading frames, located between transcription start and poly (A) signal sequences on the minus strand of HTLV-I. The 3' terminus of HTLV-I was shown to promote test gene expression in either orientation. RNA blots probed with single stranded RNA complementary to the predicted transcribed region revealed 2.5 and 2.9 kilobase minus strand RNAs in HTLV-I infected T-cells but not in an uninfected control. The effect of mutations on minus strand gene products should also be considered when studying human retroviral protein function by mutational analysis.