We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSVO-cat, in which the entire SV40 promoter region was removed and a unique HindIlI site was substituted for the insertion of other promoter sequences.As the number of putative mammalian promoter sequences isolated by recombinant techniques has increased over the past several years, the attention of many investigators has turned to ways in which the function of these sequences may be measured.

The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (pl85"m), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "hum " antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant do was constructed. Light-and heavy-chain variable regions were simultaneously humned in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonudleotides, respectively. The protooncogene HER2 encodes a protein tyrosine kinase (pl85HER2) that is homologous to the human epidermal growth factor receptor (1-3). Amplification and/or overexpression of HER2 is associated with multiple human malignancies and appears to be integrally involved in progression of 25-30%o of human breast and ovarian cancers (4, 5).Furthermore, the extent of amplification is inversely correlated with the observed median patient survival time (5). The murine monoclonal antibody mumAb4D5 (6), directed against the extracellular domain (ECD) of p185HER2, specifically inhibits the growth of tumor cell lines overexpressing p185HER2 in monolayer culture or in soft agar (7,8).mumAb4D5 also has the potential of enhancing tumor cell sensitivity to tumor necrosis factor (7,9). Thus, mumAb4D5 has potential for clinical intervention in carcinomas involving the overexpression of p185HER2.A major limitation in the clinical use of rodent mAbs is an anti-globulin response during therapy (10,11). A partial solution to this problem is to construct chimeric antibodies by coupling the rodent antigen-binding variable (V) domains to human constant (C) domains (12)(13)(14). The isotype of the human C domains may be varied to tailor the chimeric antibody for participation in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (15). Such chimeric antibody molecules are still =30% rodent in sequence and are capable of eliciting a significant anti-globulin response.Winter and coworkers (16-18) pioneered the "humanization" of antibody V domains by transplanting the complementarity determining regions (CDRs), which are the hypervariable loops involved in antigen binding, from rodent antibodies into human V domains. The validity of this approach is supported by the clinical efficacy of a humanized antibody specific for the CAMPATH-1 antigen with two non-Hodgkin lymphoma patients, one of whom had previously developed an anti-globulin response to the parental rat antibody (17,19). In some cases, transplanting hypervariable loops from rodent antibodies into human frameworks is sufficient to transfer high antigen binding affinity (16, 18), whereas in other cases it has been necessary to also replace one (17) or several (20) framework region (FR) residues. For a given antibody, a small number of FR residues are ...

We characterized the transcriptional activity of the long terminal repeat (LTR) of Rous sarcoma virus by constructing a recombinant plasmid, pRSVcat, in which bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol 3-0-acetyltransferase, EC 2.3.1.28) coding sequences are placed under LTR control. We find that the LTR directs relatively high levels of CAT synthesis within 48 hr after calcium phosphate-mediated introduction of this plasmid into CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, or mouse NIH/3T3 cells. The level of CAT synthesis is 3-fold higher in CV-1 cells and up to 10-fold higher in HeLa and mouse NIH/3T3 cells than after transfection with a related vector, pSV2cat, carrying CAT sequences under control of the simian virus 40 early promoter. We have shown, by primer extension, that the amounts of CAT-specific mRNAs encoded by pRSVcat and pSV2cat correlate with the levels of CAT enzyme activity. By both SI nuclease mapping and primer extension, we have demonstrated that the start site for RNA transcription within the LTR of pRSVcat corresponds to previous mapping data. We estimated transfection efficiencies by monitoring immunofluorescence induced by a rhodamine-labeled CAT antibody. Our results indicate that the Rous sarcoma virus LTR can direct synthesis of high levels of functional mRNA and has a wide expression range. The observed high transcriptional activity of the LTR is significant because it has been postulated that this LTR promotes activity of adjacent cellular oncogenes.

Tissue culture cells from several mammalian species, including three primate lines, were transfected with recombinant vectors carrying Escherichia coli xanthine-guanine phosphoribosyltransferase or Tn5 aminoglycoside phosphotransferase dominant selectable markers. Human HeLa and SV40-transformed xeroderma pigmentosum cells exhibited stable transformation frequencies of at least 10(-3) (0.1 percent). CV-1, an African green monkey kidney cell line, could be stably transformed with the exceptionally high frequency of 6 X 10(-2) (6 percent).

The simian virus (SV40) 72-base pair (bp) tandem repeated sequences have recently been shown to function as activators or enhancers of early viral transcription. A recombinant viral genome was recently constructed by inserting 72-bp tandem repeats from the Moloney murine sarcoma virus (MSV) in place of the 72-bp repeats of SV40. Although this genome replicates in monkey kidney cells, its rateof large tumor antigen expression and replication is considerably slower than that of wild-type SV40. In mouse cells, however, equivalent levels of large tumor antigen appear to be expressed from both wild-type and recombinant genomes, suggesting a relationship between the level of enhancer activity and the host cell. To confirm this observation, we have applied a sensitive quantitative assay for gene expression based on the conversion of chloramphenicol to its acetylated forms. The gene encoding the enzymatic function chloramphenicol acetyltransferase was inserted into two vectors in which the enhancer sequences from SV40 or MSV were placed adjacent to the early SV40 promoter. The SV40.tandem repeats appear to activate gene expression to significantly higher levels in monkey kidney cells, but-the MSV repeats are more active in two lines of mouse cells. These findings suggest that the tandem repeat elements may interact with host-specific molecules and, furthermore, may constitute one of the elements determining the host range of these eukaryotic viruses.Characterization of the nucleotide signals that constitute eukaryotic promoters is essential to an-understanding ofgene regulation. In addition to the Goldberg-Hogness sequence (or T-A-T-A box) which participates 'in the precise positioning of the 5' ends of RNA-molecules (1-3), upstream elements have been implicated in the activation or enhancement oftranscription for certain viral or eukaryotic genes by a number of studies (4)(5)(6)(7)(8)(9)(10)(11)(12). It was initially demonstrated that the simian virus 40 (SV40) 72-base-pair (bp) repeats activate early viral gene expression (5, 6). Further studies indicated that these sequences could also function when they were removed from their original location and placed at positions distant from the other promoter elements (8, 9). The tandem repeats also retained activity when inserted in an inverse orientation (9, 13). Other studies have indicated that the 72-bp repeats ofSV40 could increase the transformation efficiency of the herpes simplex virus thymidine kinase gene (10). An analogous fragment from the polyoma virus genome was shown to enhance the transcriptional activity of heterologous genes such as rabbit /3-globin gene (11).We have recently demonstrated that a retrovirus, the Moloney murine sarcoma virus (MSV), contains sequences in the long terminal repeats (LTRs) which can functionally replace the 72-bp repeats of SV40 (12). Preliminary data on large tumor antigen (T antigen) expression has suggested the possibility that the tandem repeats of SV40 and MSV activate gene expression in a host-specific manner.In th...