Control of yeast gene expression by transposable elements: maximum expression requires a functional Ty activator sequence and a defective Ty promoter.

Integration of a transposable element adjacent to a gene frequently results in an alteration in expression of the nearby gene. The purpose of our experiments was to identify cis-acting sequences within a yeast transposon (Ty) that are important for expression of the adjacent gene. The role of these sequences in Ty transcription was also analyzed in order to examine the relationship between Ty and adjacent gene expression. Three naturally occurring Ty elements located at the HIS4 locus were examined. These Ty elements differed by multiple sequence changes and had different effects on HIS4 expression. To determine which sequences were important to Ty and HIS4 expression, Ty::lacZ and Ty::HIS4::lacZ fusion genes were constructed and analyzed. Results of these experiments indicated that a sequence element is present in the Ty epsilon region that is necessary for HIS4 expression but which has only a modest effect on Ty transcription. Additionally, a mutation in the Ty promoter region decreased Ty transcription and increased HIS4 expression. The opposite effects of this mutation on Ty and adjacent gene expression were probably caused by promoter competition.

Chimeric murine-human antibodies directed against folate binding receptor are efficient mediators of ovarian carcinoma cell killing.

The MOv18 (gamma 1, kappa) and MOv19 (gamma 2a, kappa) murine monoclonal antibodies (MAbs) recognize different epitopes on the human folate binding receptor which is overexpressed on 90% of nonmucinous epithelial ovarian tumors. A chimeric murine-human (human gamma 1, kappa) version of both antibodies was constructed and expressed. The genes encoding the murine heavy and light chain variable regions of the MOv18 and MOv19 MAbs were cloned from the parental hybridomas, fused with genes encoding the human heavy (gamma 1) and light (kappa) chain constant regions, respectively, and expressed in the SP2/0 murine myeloma cell line. Using human peripheral blood mononuclear cells as effector cells and conditions that provide for maximum lysis (effector target = 50:1, saturating antibody concentration), the murine MOv18 MAb (IgG1) mediated variable levels of specific cytolysis of the target ovarian cancer cell line IGROV1. In contrast, the chimeric MOv18 MAb mediated higher and more consistent lysis even at a 10-100-fold lower antibody concentration. The murine MOv19 MAb (IgG2a) mediated specific lysis of IGROV1 cells, and the chimeric version of this antibody mediated an amount of lysis at least equal to that mediated by its murine counterpart. A comparison of the ED50 values obtained for the murine MOv19 and chimeric MOv19 antibodies indicates that the chimeric MOv19 MAb was 3 to 10 times more potent than the murine MOv19 antibody. In addition, the ED50 values obtained for the chimeric MOv18 and chimeric MOv19 MAbs were similar, indicating that these MAbs are equally potent. The level of maximal lysis obtained was dependent on the number of target molecules/cell; the same high level of lysis mediated by cMOv18, MOv19, and cMOv19 was observed with both IGROV1 and OvCA432 target cells. However, only low levels of lysis were obtained when the SW626 cell line, which expresses 1 x 10(4) folate binding protein sites/cell, was used as a target. An equimolar mixture of the chimeric MOv18 and MOv19 MAbs was no more effective in the mediation of lysis than an equivalent amount of either chimeric MAb alone. These data suggest that the folate binding receptor is expressed on IGROV1 cells at a density sufficient to provide for optimal levels of antibody-mediated lysis using a single chimeric antibody directed at the folate binding receptor.

Distribution of the folate receptor GP38 in normal and malignant cell lines and tissues.

In some epithelial cells studied in vitro a membrane-bound folate receptor initiates the process for cell accumulation of 5-methyltetrahydrofolic acid. This receptor was found to be GP38, an overexpressed, glycosyl-phosphatidylinositol anchored glycoprotein, recognized by two monoclonal antibodies, designated MOv18 and MOv19. Using immunoblotting with MOv19, radioimmunoassay with MOv18 and 19, Northern blot analysis, and radioligand binding when possible, we describe the limited expression of the folate receptor in a large number of normal tissues from four autopsies. The immunoblot technique detected as little as 40 pg (approximately 1 fmol) of receptor protein. Choroid plexus consistently had the largest amount of folate receptor. Other tissues containing substantial amounts of receptor included lung, thyroid, and kidney. The liver, intestines, muscle, cerebellum, cerebrum, and spinal cord were immunologically nonreactive. Folate receptor gene expression determined by Northern blot analysis confirmed these observations. We also show that several malignant cell lines express significantly more receptor than normal epithelial cells or fibroblasts. Specifically, malignant cells bound greater than or equal to 20 pmol [3H]folate/10(6) cells, while normal epithelial cells and fibroblasts bound less than or equal to 1 pmol radioligand/10(6) cells. We also demonstrate that 4 of 6 brain tumors overexpress the folate receptor. These studies reveal the limited normal tissue distribution of the folate receptor, a cell surface protein which may be a useful immunological or pharmacological target for the development of selective cancer therapy.

Cellular localization of the folate receptor: potential role in drug toxicity and folate homeostasis.

In the past year, gp38, a glycosyl-phosphatidylinositol linked membrane protein that is overexpressed in some malignant tissues, has been shown to be the folate receptor. Using immunohistochemical techniques with the monoclonal antibody MOv19 against gp38, we evaluated the cellular localization of folate receptors in normal human tissues, which are potential target sites for drugs that utilize this uptake mechanism. The choroid plexus was intensely positive with staining limited to the epithelium, which in some foci had a distinct bilaminar pattern limited to the luminal and basal surfaces. The epithelium of the fallopian tube, uterus, and epididymis was highly immunoreactive. The acinar cells of the breast, submandibular salivary, and bronchial glands also showed intense staining as did the trophoblastic cells of the placenta. In the kidney reactivity was localized to the proximal tubules. Lung alveolar lining including type I and II pneumocytes stained intensely. Limited but focal reactivity was noted in the vas deferens, ovary, thyroid, and pancreas. This study in conjunction with previous work showing marked overexpression of folate receptor in some malignant cells suggests that the folate receptor may be an important target for diagnostic or therapeutic exploitation and indicates sites of potential drug toxicity.

Cloning of a tumor-associated antigen: MOv18 and MOv19 antibodies recognize a folate-binding protein.

Monoclonal antibodies MOv18 and MOv19, raised against a membrane preparation of an ovarian carcinoma surgical specimen, react with a surface antigen present on the majority of nonmucinous ovarian malignant tumors tested but not with normal adult tissue (S. Miotti, S. Canevari, S. Ménard, D. Mezzanzanica, G. Porro, S. M. Pupa, M. Regazzoni, E. Tagliabue, and M. I. Colnaghi, Int. J. Cancer, 39: 297-303, 1987). This surface antigen was purified as a soluble glycoprotein (molecular mass, 36-38 kDa) released from the cell surface of an ovarian carcinoma cell line (IGROV1) by digestion with Bacillus thuringiensis phospholipase C. Immunoblotting demonstrated that the purified protein reacted with MOv18 and MOv19 and that treatment of the purified preparation with N-glycanase resulted in a protein with a molecular mass of 27 kDa. The NH3-terminal amino acid sequence of the purified antigen was determined. This sequence is highly homologous to an internal stretch of 27 amino acids located near the NH3 terminus of human folate-binding protein. An oligonucleotide probe was synthesized and used to screen an IGROV1 ovarian carcinoma, lambda gt11 complementary DNA library to obtain three complementary DNA clones. The complete nucleotide sequence of one of these complementary DNA clones was determined. This sequence is nearly identical to that of a folate-binding protein clone obtained from the Caco-2 human carcinoma cell line. In addition, the nucleotide sequence of the 5'-untranslated region of the other two clones was determined. This region of all three clones was different. The product of the Caco-2 folate-binding protein clone expressed in Chinese hamster ovary cells was recognized by the MOv18 and MOv19 antibodies, confirming that the antigen and folate-binding protein are one and the same. Furthermore, a cell line that binds the MOv18 and MOv19 antibodies expressed increased levels of folate-binding protein mRNA compared with a cell line that does not bind these antibodies. These results indicate that the MOv18 and MOv19 monoclonal antibodies bind to at least one form of folate-binding protein and that this protein, which is evidently overexpressed in certain malignant tumors, may provide a suitable target for immunotherapy with these antibodies.

New chimeric anti-pancarcinoma monoclonal antibody with superior cytotoxicity-mediating potency.

The monoclonal antibodies (MAbs) 323/A3 and 17-1A both recognize a 40-kDa carcinoma-associated epithelial glycoprotein (EGP40). MAb 17-1A has been used in many therapeutic trials as an immunotherapeutic agent to combat advanced colorectal cancer, and about 5-10% overall responses have been observed. It has been shown that MAb 323/A3 has a higher affinity than 17-1A, which might be an advantageous feature for a therapeutic agent. In our immunohistological studies different reaction patterns of these two MAbs were observed, suggesting that MAb 323/A3 reacts more intensely with carcinoma cells than MAb 17-1A. This also suggests that MAb 323/A3 might be a more effective immunotherapeutic tool. Because chimerization may reduce the immunogenicity of the murine MAb 323/A3 and increase the interaction with human effector mechanisms, we developed a chimeric form of murine MAb 323/A3. MAb 323/A3 heavy and light chain variable genes were cloned and grafted onto human C gamma 1 and C kappa domains, respectively. A chimeric antibody-producing cell line was established by transfection of the chimeric constructs into a nonproducing myeloma cell. The chimeric and murine 323/A3 MAbs were evaluated for efficacy of inducing complement-mediated cytotoxicity (CMC) and mediating antibody-dependent cellular cytotoxicity against LS 180 cells derived from human colon carcinoma. Both forms were found to mediate similar levels of CMC in the presence of human complement; however, higher levels of lysis of target cells were observed with human peripheral blood lymphocytes when the chimeric 323/A3 was used. Chimeric 323/A3 mediated higher maximal cytotoxicity than chimeric 17-1A in both CMC and antibody-dependent cellular cytotoxicity assays and was equally active as chimeric 17-1A at 100- to 1000-fold lower concentrations. The superior reactivity of chimeric 323/A3 with EGP40 on carcinoma cells and its higher cytotoxicity-mediating capacity, compared to chimeric 17-1A, are important characteristics, which support further clinical studies with chimeric MAb 323/A3 in immunotherapy of carcinomas.

Isolation and biochemical characterization of the soluble and membrane forms of folate binding protein expressed in the ovarian carcinoma cell line IGROV1.

The human ovarian carcinoma cell line, IGROV1, produces two forms of folate binding protein (FBP), the membrane form that is anchored to the cell surface by a glycosylphosphatidylinositol tail and the soluble form that is shed into the tissue culture medium. Both forms are recognized by the monoclonal antibodies MOv18 and MOv19. Here we describe their purification and biochemical characterization. The purified soluble protein appeared as a single band with an apparent Mr of 36 kDa after SDS-PAGE, whereas the membrane form appeared as a single band with an apparent Mr of 38 kDa. The size difference between the two forms of FBP was confirmed by gel filtration of both the native and the N-glycanase-treated proteins. Both purified proteins had equal capacity to bind folic acid. The immunological cross-reactivity and the folic acid binding capability of the FBPs extracted from IGROV1 gave more evidence of the possible existence of a precursor-product relationship between them.

Selective cleavage of bcr-abl chimeric RNAs by a ribozyme targeted to non-contiguous sequences.

Conventionally designed ribozymes may be unable to cleave RNA at sites which are inaccessible due to secondary structure. In addition, it may also be difficult to specifically target a conventionally designed ribozyme to some chimeric RNA molecules. Novel approaches for ribozyme targeting were developed by using the L6 bcr-abl fusion RNA as a model. Using one approach, we successfully directed ribozyme nucleation to a site on the bcr-abl RNA that is distant from the GUA cleavage site. These ribozymes bound to the L6 substrate RNA via an anchor sequence that was complementary to bcr sequences. The anchor was necessary for efficient cleavage as the anchor minus ribozyme, a conventionally designed ribozyme, was inefficient at catalyzing cleavage at this same site. The effect of anchor sequences on catalytic rates was determined for two of these ribozymes. Ribozymes generated by a second approach were designed to cleave at a CUU site in proximity to the bcr-abl junction. Both approaches have led to the development of a series of ribozymes specific for both the L6 and K28 bcr-abl chimeric RNAs, but not normal abl or bcr RNAs. The specificity of the ribozyme correlated in part with the ability of the ribozyme to bind substrate as demonstrated by gel shift analyses. Secondary structure predictions for the RNA substrate support the experimental results and may prove useful as a theoretical basis for the design of ribozymes.

Generation of chimeric bispecific G250/anti-CD3 monoclonal antibody, a tool to combat renal cell carcinoma.

The monoclonal antibody (MAb) G250 binds to a tumour-associated antigen, expressed in renal cell carcinoma (RCC), which has been demonstrated to be a suitable target for antibody-mediated immunotherapy. A bispecific antibody having both G250 and anti-CD3 specificity can cross-link G250 antigen-expressing RCC target cells with T cells and can mediate lysis of such targets. Therapy studies with murine antibodies are limited by immune responses to the antibodies injected (HAMA response), which can be decreased by using chimeric antibodies. We generated a chimeric bispecific G250/anti CD3 MAb by transfecting chimeric genes of heavy and light chains for both the G250 MAb and the anti-CD3 MAb into a myeloma cell line. Cytotoxicity assays revealed that the chimeric bispecific MAb was capable of mediating lysis of RCC cell lines by cloned human CD8+T cells or by IL-2-stimulated peripheral blood lymphocytes (PBLs). Lysis mediated by the MAb was specific for target cells that expressed the G250 antigen and was effective at concentrations as low as 0.01 microgram ml-1. The chimeric bispecific G250/anti-CD3 MAb produced may be an effective adjuvant to the currently used IL-2-based therapy of advanced renal cell arcinoma.

Apoptotic cell death induced by a mouse-human anti-APO-1 chimeric antibody leads to tumor regression.

The murine anti-APO-1 antibody (gamma 3, kappa) induces programmed cell death (apoptosis) following binding to the APO-1 antigen (m.w., 48 kDa) expressed, e.g., on activated or malignant lymphocytes. APO-1 expression on malignant cell lines and tissues suggested potential clinical utility supported by anti-APO-1-mediated tumor regression in a nude mouse model. A mouse-human anti-APO-1 chimeric antibody (gamma 3, kappa) with an affinity similar to that of the murine antibody was produced. Chimeric anti-APO-1 showed the same potential to inhibit growth of the SKW6.4 B-lymphoblastoid cell line as murine anti-APO-1. In addition, both the chimeric and murine anti-APO-1 antibodies were equally capable of mediating complete macroscopic tumor regression of a SKW6.4 xenotransplant in SCID mice by induction of apoptosis. Induction of apoptosis was the only mechanism for tumor regression because neither murine nor chimeric anti-APO-1 showed anti-tumor activity against solid H53 tumor (APO-1 antigen-positive, anti-APO-1-resistant) xenotransplants. Our results indicate that the chimeric anti-APO-1 antibody effectively induces apoptosis and suggest that chimeric anti-APO-1 should be evaluated for the treatment of malignant cells expressing the APO-1 antigen. However, chimeric anti-APO-I might only be used therapeutically when the antibody can be targeted specifically to tumor cells.

Chimeric bispecific OC/TR monoclonal antibody mediates lysis of tumor cells expressing the folate-binding protein (MOv18) and displays decreased immunogenicity in patients.

The bispecific OC/TR monoclonal antibody (mAb) cross-links the CD3 molecule on T cells with the human folate-binding protein (FBP), which is highly expressed on nonmucinous ovarian carcinomas. Clinical trials of patients with ovarian carcinoma with the OC/TR mAb have shown some complete and partial responses. Most patients developed human anti-murine immunoglobulin antibodies (HAMA), which can inhibit OC/TR mAb-mediated lysis. We generated a chimeric version of the OC/TR mAb to decrease the immunogenicity of the OC/TR mAb and to allow more extended treatment schedules. Sp2/0 myeloma cells were transfected with chimeric heavy- and light-chain genes encoding the anti-CD3 mAb and the MOv18 mAb, respectively, which are reactive with FBP. The resulting cell line produced 80 micrograms/ml of total immunoglobulin G (IgG), of which 11.5% was the functionally active chimeric OC/TR mAb. Chimeric OC/TR F(ab')2 fragments mediated lysis of IGROV-1 ovarian carcinoma cells by human T cells at antibody concentrations of > or = 1 pg/ml. Specific lysis was still detectable at an effector-to-target cell ratio as low as 0.4. Two patients with ovarian carcinoma treated with F(ab')2 fragments of the murine OC/TR developed distinct HAMA titers, which were mainly anti-idiotypic and only partly directed against the murine antibody constant regions. However, of the two patients that were treated with the F(ab')2 fragments of the chimeric OC/TR mAb, only one developed a low transient HAMA response just above background level. In conclusion, the generation of chimeric OC/TR may allow more extended clinical studies of bispecific mAb-mediated immunotherapy of ovarian carcinoma.

Comparison of IgE and IgG antibody-dependent cytotoxicity in vitro and in a SCID mouse xenograft model of ovarian carcinoma.

Allergic reactions are mediated by IgE antibodies bound to high-affinity receptors on mast cells in peripheral tissues and are characterized by their immediacy and hypersensitivity. These properties could also be advantageous in immunotherapy against cancer growth in peripheral tissues. We have constructed chimeric IgE and IgG1 antibodies with murine V regions and human C regions corresponding to the MOv18 monoclonal antibody against the human ovarian tumor-associated antigen, folate binding protein. The antibodies exhibited the expected binding affinities for antigen and Fc receptors, and effector activities with human basophils and platelets in vitro. The protective activities of MOv18-IgE and MOv18-IgG1 were compared in a SCID mouse xenograft model of ovarian carcinoma. The beneficial effects of MOv18-IgE were greater and of longer duration than those of MOv18-IgG1. Our results suggest that the allergic reaction could be harnessed for the suppression of ovarian tumors.

First human trial of a DNA-based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response.

A DNA-based vaccine containing human immunodeficiency virus type 1 (HIV-1) env and rev genes was tested for safety and host immune response in 15 asymptomatic HIV-infected patients who were not using antiviral drugs and who had CD4+ lymphocyte counts of > or = 500 per microliter of blood. Successive groups received three doses of vaccine (30, 100, or 300 microg) at 10-week intervals in a dose-escalation trial. Vaccine administration induced no local or systemic reactions, and no laboratory abnormalities were detected. Specifically, no patient developed anti-DNA antibody or muscle enzyme elevations. No consistent change occurred in CD4 or CD8 lymphocyte counts or in plasma HIV concentration. Antibody against gp120 increased in individual patients in the 100- and 300-/microg groups. Some increases were noted in cytotoxic T lymphocyte activity against gp160-bearing targets and in lymphocyte proliferative activity. The safety and potential immunogenicity of an HIV-directed DNA-based vaccine was demonstrated, a finding that should encourage further studies.