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Biomedical subjects

L L Houston

Publications and source records attributed to L L Houston.

At least 19 recordsLinked to original sources
PubMed1992

In vivo efficacy of B43 (anti-CD19)-pokeweed antiviral protein immunotoxin against BCL-1 murine B-cell leukemia.

We show that a highly aggressive subclone of murine BCL-1 B-lineage leukemia expresses a single 2.4-kb transcript hybridizing to the human CD19 cDNA probe and reacts strongly with the anti-human CD19 monoclonal antibodies (MoAb) B43, B4, Leu-12, and J3-119. In contrast to their strong reactivity with anti-human CD19 MoAb, BCL-1 cells show no reactivity with MoAb directed against human CD22, CD72, HLA-DR, IgD, or IgM. Western blot analysis of BCL-1 whole cell lysates with the anti-human CD19 MoAb J3-119 showed a single 69-Kd protein band, which was not detected by the negative control MoAb G19.4 (anti-CD3). In contrast to BCL-1 cells, normal BALB/c splenocytes or mouse splenocyte/myeloma hybridoma cell lines did not (1) express any transcripts that hybridized to the human CD19 cDNA probe, (2) react with B43/anti-CD19 MoAb, or (3) express the 69-Kd protein that reacts with the anti-human CD19 MoAb J3-119. Murine BCL-1 B-cell leukemia thus provides a unique model of disseminated B-lineage leukemia to evaluate the antileukemic efficacy of anti-CD19 immunotoxins. This model was subsequently used to evaluate the in vivo homing ability, pharmacokinetics, and antileukemic efficacy of B43 MoAb conjugated to the plant hemitoxin pokeweed antiviral protein (PAP). B43-PAP immunotoxin (1) showed strong and antigen-specific reactivity with BCL-1 cells, (2) promptly penetrated the spleens of leukemic mice, (3) rapidly reduced the BCL-1 leukemia burden of leukemic mice and, most importantly, (4) improved survival. Finally, B43-PAP immunotoxin was more effective against BCL-1 leukemia than 700 cGy (LD100/30) total body irradiation (TBI) followed by syngeneic bone marrow transplantation (BMT).

Animals↗
PubMed1992

Inhibition of human subconjunctival fibroblast proliferation by immunotoxin.

The ability to target proliferating cells is important for agents used to modulate wound healing by decreasing the growth of fibroblasts. Proliferating cells are known to express increased numbers of transferrin receptors and have increased receptor turnover. 454A12 Mab-rRA, an immunotoxin containing anti-human transferrin receptor monoclonal antibody conjugated to recombinant ricin A chain, was shown to inhibit the proliferation of human subconjunctival fibroblasts in vitro. A dose-related reduction of cell counts was observed in proliferating cells. More than 90% inhibition was achieved with an immunotoxin concentration of 10 ng/ml per 20,000 cells plated. In contrast, confluent fibroblasts were markedly less sensitive to the immunotoxin at equivalent concentrations. Comparative experiments demonstrated that 5-fluorouracil has less specificity for proliferating cells, with significant death of confluent fibroblasts at high drug concentrations.

Antibodies, Monoclonal↗
PubMed1991

Elimination of clonogenic breast cancer cells from human bone marrow. A comparison of immunotoxin treatment with chemoimmunoseparation using 4-hydroperoxycyclophosphamide, monoclonal antibodies, and magnetic microspheres.

Autologous bone marrow transplantation (ABMT) may aid in the management of breast cancer, but is currently limited to patients without bone marrow metastases. In earlier studies, 5 logs of malignant clonogenic breast cancer cells could be eliminated from human bone marrow using a combination of chemoseparation with 4-hydroperoxycyclophosphamide (4-HC) and immunoseparation with monoclonal antibodies and magnetic microspheres. In this report the authors compare chemoimmunoseparation to treatment with immunotoxins for elimination of tumor cells from human bone marrow and for the preservation of normal precursors. Breast cancer cells from each of five cell lines were mixed with a tenfold excess of irradiated human bone marrow cells. Treatment with a combination of five immunotoxins reduced clonogenic tumor cell growth by 1.8 to 5.5 logs depending upon the cell line. With two of the five cell lines, clonogenic tumor cells were eliminated quantitatively. Using the CAMA-1 breast cancer cell line, treatment with multiple immunotoxins was compared with chemoimmunoseparation with 4-HC, a panel of five unconjugated monoclonal antibodies and magnetic microspheres. Chemoimmunoseparation eliminated 3.5 to 5.4 logs of malignant cells, while preserving 21% of Colony-forming unit-granulocyte-macrophage (CFU-GM) and 37% of burst-forming unit-erythrocyte (BFU-E). No clonogenic breast cancer cells could be detected. Immunotoxin treatment eliminated 2.2 to 5.4 logs of clonogenic breast cancer cells, but had no effect on the bone marrow precursors. In seven of ten experiments, however, clonogenic breast cancer cells remained after immunotoxin treatment. Consequently, treatment with 4-HC, multiple murine monoclonal antibodies and magnetic microspheres provided more consistent elimination of tumor cells than separation with immunotoxins, but was significantly more toxic for marrow precursors.

Antibodies, Monoclonal↗
PubMed1990

Use of immunotoxins in combination to inhibit clonogenic growth of human breast carcinoma cells.

Substantial heterogeneity has been observed in the expression of individual antigens within tumor cell populations. Immunotoxins which bind to different cell surface antigens might exert additive or synergistic cytotoxicity when used in combination to eliminate all clonogenic cells within a tumor. Immunotoxins have been prepared by conjugating recombinantly derived toxin A chain to different monoclonal reagents which recognize cell surface determinants of Mr 42,000 (317G5), 55,000 (260F9), and 200,000 (741F8). Each immunotoxin was evaluated for binding, internalization, and cytotoxicity with four breast cancer cell lines. Each of the three immunotoxins bound to the SKBr3 cell line and exerted antitumor activity in a limiting dilution clonogenic assay. Simultaneous treatment with two immunotoxins produced additive antitumor activity with each of the possible combinations. Additive binding could be demonstrated by immunofluorescent techniques, however, with only one of three combinations. With two of the three combinations, subpopulations of tumor cells could be identified which lacked one or the other antigenic determinant but not both. Consequently, log-additive antitumor activity was produced by immunotoxins in combination, and heterogeneity of antigenic targets may have contributed to the combined cytotoxicity in some but not all cases.

Antibodies, Monoclonal↗
PubMed1990

Antitransferrin receptor immunotoxin inhibits proliferating human retinal pigment epithelial cells.

Cultured human retinal pigment epithelial cells were exposed to an immunotoxin composed of a monoclonal antibody, 454A12, directed against transferrin receptors conjugated to a toxin, recombinant ricin A chain. Exposure of proliferating human retinal pigment epithelial cells to the immunotoxin (0.1 to 10,000 ng/mL) caused a statistically significant (P less than .0001) decrease in the number of cells. This inhibitory effect was induced after an exposure to the immunotoxin as short as 5 minutes and was maximal after 24 hours of exposure. The diminution in cell number was dose dependent over the range from 0.1 to 100 ng/mL. Monoclonal antibody alone, recombinant ricin A chain alone, or an irrelevant immunotoxin, MOP21C monoclonal antibody-recombinant ricin A, did not diminish the number of cells. There was a marked decrease in DNA synthesis measured by nuclear tritiated thymidine incorporation that accompanied the immunotoxin-mediated decrease in cell number. Viable cells remaining after exposure to the immunotoxin (0.1 to 10,000 ng/mL) were morphologically abnormal; typically the cells had elongated spindle-shaped processes and had lost their normal cuboidal appearance. In contrast, cell number was not decreased in confluent human retinal pigment epithelial cells after treatment with maximal doses of immunotoxin. Morphologic changes similar to those seen in proliferating cells were observed in confluent cells exposed to more than 100 ng/mL of immunotoxin. The effect of the immunotoxin was species specific because large doses of immunotoxin did not reduce the number of viable cells in proliferating or confluent pig retinal pigment epithelial cells or cause observable morphologic changes in this cell type. Our results indicate that the immunotoxin selectively inhibited proliferating retinal pigment epithelial cells by receptor-mediated internalization of the antitransferrin receptor monoclonal antibody-recombinant ricin A chain conjugate.

Cell Count↗
PubMed1990

Inhibition of hematopoietic progenitor colony growth by a monoclonal antibody against the transferrin receptor: comparison of unconjugated antibody with an immunotoxin containing recombinant ricin A chain.

We studied an immunotoxin consisting of recombinant ricin A chain (rRA) conjugated to 454A12 MoAb, a monoclonal antibody which recognizes an epitope on the human transferrin receptor, and compared the ability of 454A12 MoAb-rRA immunotoxin to inhibit the growth of erythroid burst-forming units (BFU-e) and myeloid colony-forming units (CFU-c) with unconjugated 454A12 MoAb. A significant reduction in BFU-e colony growth was observed at 0.001 microgram/ml of 454A12 MoAb-rRA versus 0.1 microgram/ml of unconjugated 454A12 MoAb (p = 0.005). Comparison of the effects of 454A12 MoAb-rRA and 454A12 MoAb on myeloid colony development gave markedly different results. Unconjugated antibody had no effect on CFU-c colony growth; in contrast, 0.01 microgram/ml of 454A12 MoAb-rRA reduced the number of colonies from 139 per 1 X 10(5) to 75 per 1 X 10(5) cells plated (p = 0.0005). No myeloid progenitor colonies developed at 0.1 microgram/ml of immunotoxin. These observations suggest that 454A12 MoAb-rRA inhibits growth by a potent, ricin A chain-mediated toxic effect on any proliferating cells expressing transferrin receptors, whereas the 454A12 MoAb exerts a selective inhibitory effect primarily on erythroid progenitors by perturbing the transferrin cycle. While growth factor receptors expressed on hematopoietic cells represent promising targets for immunotoxin therapy, our data indicate that an immunotoxin could inhibit cellular proliferation by a different mechanism than the corresponding unconjugated MoAb. Depending on the antibody used, these differences may be important in trials using immunotoxins for in vivo treatment or in vitro purging of malignant hematopoietic cells.

Antibodies, Monoclonal↗
PubMed1990

Selective inhibition of growing pigment epithelial cells by a receptor-directed immunotoxin.

An immunotoxin conjugate of a murine monoclonal antibody against human transferrin receptors and the A chain of ricin was examined for its potential to inhibit selectively the growth of retinal pigment epithelial (RPE) cells which grow in an uncontrolled manner in proliferative vitreoretinopathy. The probable efficacy of such an agent in vivo stems from the observation that actively proliferating cells possess many more transferrin receptors than normal quiescent cells. The authors showed in vitro that the immunoconjugate (454A12 MAB-rRA) inhibits protein synthesis in actively dividing RPE cells but has a smaller or no effect on protein synthesis by confluent, nondividing RPE cells. The effect was specific in that neither the free ricin A chain (rRA) nor the monoclonal antibody (454A12 MAB) alone has any inhibitory effect. Furthermore, the antibody competes with the immunotoxin and suppresses the latter's toxicity. This immunotoxin has applications for therapy in conditions in which the pathologic proliferation of RPE cells occurs.

Antibodies, Monoclonal↗
PubMed1990

Elimination of malignant clonogenic T cells from human bone marrow using chemoimmunoseparation with 2'-deoxycoformycin, deoxyadenosine and an immunotoxin.

Autologous bone marrow transplantation may contribute to the treatment of several types of lymphoreticular malignancies. Recent studies have suggested that a combination of chemoseparation and immunoseparation may be more effective than either modality alone in eliminating malignant cells from human bone marrow. In this report an immunotoxin has been prepared by conjugating pokeweed antiviral protein (PAP) to the 3A1 murine monoclonal antibody that recognizes a 40 kD (CD7) determinant expressed by most T cell acute lymphoblastic leukemias and a majority of normal mature peripheral T cells. When HSB-2 T lymphoma cells were mixed with normal human bone marrow and incubated with 3A1-PAP and 100 microM chloroquine, approximately 3 logs of clonogenic T cells could be eliminated from a 20-fold excess of bone marrow. Treatment of cell mixtures with 2'deoxycoformycin (2'-dCF) and deoxyadenosine (dAdo) eliminated 2 logs of clonogenic tumor cells. The use of 3A1-PAP and chloroquine with dCF/dAdo was more effective than either single modality, eliminating up to 6 logs of HSB-2 tumor cells in optimal experiments. Anti-tumor activity of the combined treatment extended to T leukemia cells taken directly from patients. Although 3A1-PAP reduced CFU-GM by only 13% and BFU-E by 36%, the addition of 2'-dCF and dAdo was more toxic for normal marrow precursors, further reducing CFU-GM, GEMM and BFU-E as well as preventing recovery of CFU-GM in long-term bone marrow culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiviral Agents↗
PubMed1989

Cytotoxicity against human tumor cells mediated by the conjugate of anti-epidermal growth factor receptor monoclonal antibody to recombinant ricin A chain.

We have produced monoclonal antibodies against the epidermal growth factor (EGF) receptor which bind to the receptor with high affinity, compete with EGF for binding, block EGF-induced tyrosine kinase activity, and activate internalization and down-regulation of the receptor. These antibodies are cytostatic against cultured A431 cells at concentrations of 5-20 nM. In addition, they prevent the growth of A431 tumor xenografts in athymic mice. In the present experiments, we have attempted to improve the antitumor activity of monoclonal antibody 528 IgG2a against the EGF receptor by linking it to recombinant ricin A chain (rRA). The immunoconjugate (528 IgG-rRA) showed a potent cytotoxic effect on A431 cells in vitro. At a concentration of 10 pM, it inhibited the proliferation of cultured A431 cells by 50% and also inhibited protein synthesis in these cells by 50%. Proliferation was prevented and cell death occurred at 528 IgG-rRA concentrations of 60 pM or greater. Recombinant free ricin A chain was far less toxic. The cytotoxic effect of the immunoconjugate was neutralized by 528 IgG at concentrations 100-fold higher than 528 IgG-rRA. When the cytotoxic effect of 528 IgG-rRA was compared among several human cell lines expressing different numbers of EGF receptors, the capacity to inhibit both proliferation and protein synthesis generally correlated with the number of EGF receptors on the plasma membranes of these cells. Since 528 IgG-rRA is a very potent immunotoxin against A431 cells in culture, we designed experiments to test its in vivo antitumor activity against A431 xenografts in athymic mice. To measure the clearance of 528 IgG-rRA, 50 micrograms of immunotoxin were injected i.p. into athymic mice, blood was collected from the animals at regular intervals, and the level of immunotoxin in the serum was assayed by protein synthesis inhibition in cultured A431 cells. The blood level of active immunoconjugate reached a maximum 6 h after i.p. injection. The half-life of the absorption phase was 2.2 h, the half-life for elimination was 9.2 h, and blood levels which could be potentially cytotoxic were maintained for 48-72 h. We investigated a number of immunotoxin treatment schedules, including every other day for 4 days, based on these data. The results demonstrate that, while 528 IgG-rRA has higher in vivo antitumor activity than 528 IgG against A431 cell xenografts, this is accompanied by toxicity against the murine host.

Animals↗
PubMed1989

Recombinant ricin A chain conjugated to monoclonal antibodies: improved tumor cell inhibition in the presence of lysosomotropic compounds.

Recombinant ricin A chain was chemically linked to monoclonal antibodies directed toward human breast cancer cells, a human T-cell differentiation antigen, and mouse transferrin receptor. Three types of immunotoxins were prepared; in two of them the antibody was linked to recombinant ricin A chain by a disulfide bond and in the third, a nonreducible thioether bond was used. Immunotoxins containing a nonreducible linkage may have some advantage over conjugates containing a reducible linkage because of improved stability in vivo. Conjugation of recombinant ricin A chain through either the endogenous thiol group or through a derivatized amino group produced immunotoxins with comparable cytotoxicity. The thioether conjugate was 1000-fold less cytotoxic to target tumor cells than the respective disulfide-linked immunotoxin. However, addition of monensin, a monocarboxylic ionophore, greatly enhanced the cytotoxicity of the thioether-linked immunotoxin. Monensin increased the immunotoxin activity better than other lysosomotropic reagents that were tested. The increase in activity of recombinant ricin A chain-containing immunotoxins mediated by monensin argues against a role for contaminating ricin B chain in potentiation.

Antibodies, Monoclonal↗
PubMed1988

Heterogeneity of a murine B cell lymphoma. Isolation and characterization of idiotypic variants.

mAb directed toward the idiotype of the 38C13 murine B cell lymphoma can be used to treat and cure a high percentage of mice challenged previously with an otherwise lethal dose of tumor cells. Tumors developing in animals despite antibody therapy were examined by immunofluorescence and found to demonstrate either loss of surface Ig, or expression of an altered idiotype that no longer bound the antibody used for treatment. Further immunofluorescence analysis of the variant tumors revealed individual patterns of cross-reactivity with anti-38C13 idiotype mAb other than that used for therapy. The variant tumor cells were fused to myeloma cells and hybrids were isolated which secreted large quantities of the altered idiotype proteins. Polyclonal antibodies and mAb prepared against the mutant proteins demonstrated cross-reactivity with the original 38C13 protein and its other variants. But the variants and wild type cells could be distinguished from each other by their patterns of reactivity with the panels of anti-idiotype antibodies. Differences in apparent m.w. were demonstrated in the L chains of each of the mutant proteins. Southern blot analysis of the H chain locus of these mutants established that they were all clonally related; however, the L chain loci were grossly different. Thus, rare cells with alteration in their Ig L chain genes and expressed proteins can give rise to idiotype variants in this B cell tumor.

Animals↗
PubMed1985

Genomic cloning and characterization of a ricin gene from Ricinus communis.

A genomic clone that specifies a single polypeptide precursor for ricin, a toxic lectin of Ricinus communis (castor bean), was isolated, sequenced and Sl mapped. The gene encodes a 64 kDa precursor which contains, in the following order: a 24 or 35 amino acid signal peptide, the A chain, a 12 amino acid linker peptide, and the B chain. The 5'-end of the ricin mRNA maps approximately 35 bases upstream from the first methionine codon. Two putative TATA boxes and a possible CAAT box lie in the 5'-flanking region. Two possible polyadenylation signals were found in the 3' flanking region. No introns were found, which is typical of other lectin genes that have been sequenced. Southern blot analysis suggests that the castor bean genome contains approximately six ricin-like genes.

Amino Acid Sequence↗
PubMed1979

Effect of sulfhydryl reagents and protease inhibitors on sodium dodecyl sulfate-heat induced dissociation of Ricinus communis agglutinin.

Ricinus communis agglutinin dissociated to lower molecular weight forms when heated in sodium dodecyl sulfate in the absence of reducing agents, while ricin was little affected by such treatment. The data suggest that strong noncovalent bonds hold together two A-B heterodimers in the Ricinus communis agglutinin tetramer. Protease inhibitors such as diisopropylfluorophosphate, phenylmethansefulonyl fluoride, and EDTA, did not prevent the sodium dodecyl sulfate-heat induced dissociation; however, sulfhydryl specific reagents (N-ethylmaleimide, 5,5'-dithiobis (2-nitrobenzoic acid) and p-chloromercuribenzoate) were effective. Titration of the lectins in sodium dodecyl sulfate indicated that ricin contains one sulfhydryl and Ricinus communis agglutinin four sulfhydryl groups, none of which react in the presence of 8 M urea. The sulfhydryl groups that could be titrated in the intact proteins in sodium dodecyl sulfate were on the A chains.

Lectins↗