Chimeric toxins targeted to the human immunodeficiency virus type 1 envelope glycoprotein augment the in vivo activity of combination antiretroviral therapy in thy/liv-SCID-Hu mice.

Highly active antiretroviral therapy (HAART), which combines multiple inhibitors of essential human immunodeficiency virus type 1 (HIV-1) enzymes, induces dramatic and sustained viral load reductions in many people infected with HIV-1. However, reservoirs of infected cells capable of producing replication-competent virus persist even after years of HAART, preventing elimination of infection. CD4-PE40 and 3B3(Fv)-PE38, chimeric toxins designed to target the HIV envelope (Env), represent a complementary class of agents that selectively kill productively infected cells. To investigate whether these Env-targeted toxins might serve as adjuncts to HAART for the elimination of infected cells, we tested their ability to augment HAART efficacy in vivo by using a thy/liv SCID-hu mouse model. CD4-PE40 and 3B3(Fv)-PE38 markedly enhanced the capacity of HAART to suppress acute HIV-1 infection and improved HAART-mediated viral load reduction in mice with established HIV-1 infection. These results represent the first demonstration of in vivo anti-HIV-1 efficacy for Env-targeted toxins and support their potential therapeutic utility in combination with HAART.

Regional treatment of epidermal growth factor receptor vIII-expressing neoplastic meningitis with a single-chain immunotoxin, MR-1.

The incidence of neoplastic meningitis is on the rise. Neoplastic meningitis can result from a direct seeding of the neuraxis by primary brain tumors or by hematogeneous spread of systemic solid tumors. A frequent genetic alteration in primary brain tumors such as gliomas is an in-frame deletion in the epidermal growth factor receptor (EGFR) gene EGFRvIII, which brings together what were normally distant polypeptide sequences in the intact receptor. A novel glycine is formed at the fusion junction, resulting in a unique and tumor-specific target. By using phage display, we have isolated a single-chain antibody specific for the EGFRvIII mutation and expressed it with a modified form of the Pseudomonas exotoxin to form the immunotoxin MR1scFvPE38KDEL (MR-1). The multiple dose toxicity and therapeutic efficacy of MR-1 immunotoxin were tested in an athymic rat model of neoplastic meningitis. The maximally tolerated doses in non-tumor-bearing rats were three doses of 3 microg each. For therapeutic studies, the target was a neoplastic meningitis induced by intrathecal inoculation of the EGFRvIII-expressing human glioma U87MG.deltaEGFR. A dose escalation study compared the survival of three equal doses of 1, 2, and 3 microg of MR-1 immunotoxin with saline or 3 microg of the control immunotoxin specific for the interleukin 2 receptor, anti-Tac. All animals treated with three doses of saline or 3 microg of anti-Tac died, with median survival of 7 and 10 days, respectively. There were 75% (six of eight) long-term survivors in the group treated with three doses of 1 microg and 57% (four of seven) long-term survivors in the groups treated with three doses of either 2 or 3 microg of MR-1 immunotoxin. None of the MR-1 immunotoxin-treated groups reached median survival by the termination of the study at 53 days. Therefore, median survival was estimated to be >53 days, resulting in an estimated increase in median survival of >657% compared with saline and 430% versus anti-Tac. Compartmental therapy with three doses of 2 microg of MR-1 immunotoxin is effective in the treatment of EGFRvIII-expressing neoplastic meningitis. This dose was found to have no clinical or histopathological effects on non-tumor-bearing animals. MR-1 immunotoxin is, therefore, considered specific and safe within its therapeutic window. Phase I clinical trials for tumors invading the intrathecal space that express the EGFRvIII target should be initiated.

Efficacy of compartmental administration of immunotoxin LMB-1 (B3-LysPE38) in a rat model of carcinomatous meningitis.

LMB-1 (B3-LysPE38) is an immunotoxin composed of the tumor-reactive monoclonal antibody B3 and a genetically engineered form of Pseudomonas exotoxin. Monoclonal antibody B3 reacts with a carbohydrate epitope that is found on a number of solid tumors (e.g., breast, ovarian, and lung carcinomas) that frequently invade the intrathecal space, causing neoplastic meningitis. The Pseudomonas exotoxin has been engineered to remove the binding domain to eliminate nonspecific binding. A model of human neoplastic meningitis using rats bearing the human epidermoid carcinoma A431 was used for therapeutic studies of immunotoxin LMB-1. Therapy was initiated 3 days after injection of the tumor cells, which was one third of the median survival time of untreated rats. A single intrathecal injection of 40 microgram increased median survival from 9 days with saline injection to 16 days (78%, P < 0.001), and a single dose of 200 microgram increased median survival to 25 days (188%, P < 0. 001). Three doses of 40 or 200 microgram given on days 3, 6, and 8 significantly increased the median survival of 9.5 days associated with saline injection to 40.5 days (326% increase) and 33.0 days (247% increase), respectively, with two long-term survivors (191-day survival) in each treatment group. LMB-1 had no therapeutic effect on the treatment of two B3 antigen-negative neoplastic meningitis models. Treatment of the antigen-positive A431 neoplastic meningitis with B3 alone or a nonspecific monoclonal, MOPC, coupled to the engineered Pseudomonas exotoxin produced no survival effects. Nontumor-bearing athymic rats showed no toxicity with a single dose of either 40 microgram or 200 microgram, or 3 doses of 40 microgram. However, when they were given three doses of 200 microgram, these rats showed weight loss and loss of neurological function, and two of eight animals died. These studies indicate that, in the range of the most therapeutically effective dosage, the immunotoxin LMB-1 is tolerated in the intrathecal space and should be considered for human intrathecal trials.

Intrathecal administration of single-chain immunotoxin, LMB-7 [B3(Fv)-PE38], produces cures of carcinomatous meningitis in a rat model.

LMB-7 [B3(Fv)-PE38] is a single-chain immunotoxin constructed from the murine monoclonal antibody B3 and a truncated from of Pseudomonas exotoxin PE38. Antibody B3 recognizes a carbohydrate epitope found on solid tumors that frequently invade the intrathecal space and cause neoplastic meningitis. We tested the therapeutic value of intrathecally administered LMB-7 by using a model of human neoplastic meningitis in athymic rats. This model is representative of a clinical situation in that antibody B3 cross-reacts with a number of normal tissues that can be used to monitor potential systemic toxicity. Treatment was begun 3 days after A431 tumor implantation. Without treatment, the animals median survival was 10 days. Intrathecal administration of 10 micrograms of LMB-7 in 40 microliters on days 3, 5, and 7 produced 4 of 10 and 8 of 10 long-term survivors (> 170 days) in two experiments. Of the long-term survivors, 2 of 4 and 7 of 8 survivors had no microscopic evidence of tumor and were considered histologic cures. Lack of significant toxicity in the effective dose range and specificity make LMB-7 an excellent candidate for intrathecal treatment of neoplastic meningitis in humans.

Recombinant immunotoxins: new therapeutic agents for cancer treatment.

We have developed new agents for the treatment of cancer by genetically modifying Pseudomonas exotoxin. We have deleted the cell-binding region of Pseudomonas exotoxin and replaced it with various growth factors or the combining regions of antibodies in a single chain form. These new recombinant molecules are called recombinant toxins. Several different types of recombinant toxins have been produced. B3(Fv)-PE38KDEL is a recombinant toxin that kills many different adenocarcinomas and epidermoid carcinomas. The molecule is now undergoing preclinical development.

The multichain interleukin-2 receptor: a target for immunotherapy.

Activation of resting T-lymphocytes induces synthesis of interleukin-2 (IL-2) and expression of cell surface receptors for this lymphokine. In contrast to resting normal T-cells that do not express high-affinity IL-2 receptors (IL-2R), abnormal T-cells of patients with leukemia-lymphoma, certain autoimmune disorders, and individuals rejecting allografts express this receptor. Exploiting this difference in receptor expression, antibodies to the IL-2 receptor have been used effectively to treat patients with leukemia and lymphoma. One approach is to use monoclonal antibodies produced in mice; the disadvantage is that they are highly immunogenic. In an effort to reduce the immunogenicity of the mouse monoclonal antibodies, monoclonal-antibody-mediated therapy has been revolutionized by generating humanized antibodies produced by genetic engineering in which the molecule is human except for the antigen-combining regions, which are retained from the mouse. Further, to increase its cytotoxic effectiveness, the monoclonal antibody has been armed with toxins or radionuclides. Alternatively, IL-2 itself has been linked to a toxin to kill IL-2 receptor-bearing cells. Thus, IL-2 receptor-directed therapy provides a new method for treating certain neoplastic diseases and autoimmune disorders and for preventing allograft rejection.

Expression of a human complementary DNA for the multidrug resistance gene in murine hematopoietic precursor cells with the use of retroviral gene transfer.

In multidrug resistance, cells become simultaneously resistant to anthracyclines, vinca alkaloids, epipodophyllotoxins, and certain other natural product cytotoxic drugs. Resistance results from synthesis of a multidrug transporter (P-glycoprotein) encoded by the MDR1 gene (also known as the PGY1 gene). In the present study, a retrovirus vector containing a complementary DNA for the human multidrug resistance gene HaMDR1/A was used to transfer the multidrug resistance phenotype to bone marrow cells of the DBA/2J mouse. A high proportion of transduced bone marrow cells showed resistance to both colchicine and vinblastine, as determined by in vitro colony formation of hematopoietic precursor cells. In addition, brief culturing of the cells in a cytotoxic drug following exposure to the retrovirus vector could be used to increase the proportion of bone marrow cell colonies that were resistant. These results may serve as a model for the generation and selection of bone marrow cells resistant to the toxic effects of chemotherapeutic agents in vivo.

Immunocytochemical localization of P170 at the plasma membrane of multidrug-resistant human cells.

P170 (P-glycoprotein) is a membrane protein found in high levels in multidrug-resistant cultured cell lines. We have localized this protein using monoclonal antibody MRK16 by immunofluorescence and electron microscopy in the multidrug-resistant human carcinoma cell line KB-C4. The P170 determinant recognized by antibody MRK16 was found on drug-resistant KB-C4 cells, but not on parental drug-sensitive KB-3-1 cells. The determinant was present on the external surface of the plasma membrane and on the luminal side of Golgi stack membranes. P170 was excluded from coated pits at the plasma membrane and absent from endocytic vesicles and lysosomes. This determinant was detected only in small amounts in the endoplasmic reticulum. The high protein concentration of P170 in the plasma membrane is consistent with a role of this protein as a drug efflux pump at the cell surface.

Increased vinblastine binding to membrane vesicles from multidrug-resistant KB cells.

Human KB carcinoma cells resistant to high levels of colchicine, vinblastine, vincristine, adriamycin, and actinomycin D exhibit reduced accumulation of these structurally unrelated chemotherapeutic agents (Akiyama, S.-I., Fojo, A., Hanover, J. A., Pastan, I., and Gottesman, M. M. (1985) Somatic Cell Mol. Genet. 11, 117-126; Fojo, A., Akiyama, S.-I., Gottesman, M. M., and Pastan, I. (1985) Cancer Res. 45, 3002-3007). To examine the mechanism of reduced drug accumulation in these cells, we measured [3H]vinblastine ([3H]VBL) binding to membrane vesicles made from drug-sensitive (KB-3-1), drug-resistant (KB-C4), and revertant (KB-R1) cells. Membrane vesicles from KB-C4 cells bound up to 8-fold more [3H]VBL than vesicles from the parental KB-3-1 or revertant KB-R1 cell lines. No difference in binding of [3H]dexamethasone, to which the cells are equally sensitive, was observed. The difference in [3H]VBL binding by vesicles from resistant and sensitive cells was eliminated by the addition of 10 micrograms/ml verapamil, which is known to reverse the multidrug-resistance phenotype. Drug binding by KB-C4 vesicles was osmotically insensitive, temperature-dependent, and trypsin-sensitive. Binding of [3H]VBL by KB-C4 vesicles was inhibited by vinblastine, vincristine, and daunomycin (in decreasing order). Dexamethasone at 100 microM, colchicine at 100 microM, and actinomycin D at 100 microM did not significantly inhibit [3H]VBL accumulation. No significant differences in tubulin content were detected among vesicles from sensitive and resistant cells. These data demonstrate that membrane vesicles from multiply drug-resistant cells bind increased amounts of vinblastine.

Human tumor cell lines with EGF receptor gene amplification in the absence of aberrant sized mRNAs.

A survey of human tumor cell lines for increased PDGF or EGF receptors identified 5 lines which bind from 6 to 13 times more EGF than normal human fibroblasts. Immunoprecipitation analysis links the elevated binding activity to increased quantities of the EGF receptor protein. EGF receptor gene amplification was detected in 2 of the cell lines. No evidence for EGF receptor gene rearrangements was found at the level of DNA or RNA structure. The results suggest that elevated levels of EGF receptor can be associated with at least three distinct mechanisms. These include gene amplification accompanied by rearrangement, gene amplification without accompanied alteration of mRNA transcripts, and extensive expression without gene amplification.

Transit of receptors for epidermal growth factor and transferrin through clathrin-coated pits. Analysis of the kinetics of receptor entry.

Selective enrichment of clathrin-coated membranes by anticlathrin immunoadsorption was used to examine the internalization of receptor-ligand complexes through coated pits. Using Staphylococcus aureus-anticlathrin antibody and [35S]methionine-labeled KB cells, the kinetics of association of the epidermal growth factor (EGF-R) and transferrin receptors (TF-R) with coated membranes were directly examined. The accumulation of EGF-R in coated pits at the cell surface was dependent upon EGF binding. EGF-R then passed sequentially through a compartment which did not react with anticlathrin antibody and a second clathrin-coated compartment. The EGF-R was degraded in lysosomes with a half-life of approximately 41-55 min. The tumor promoter, 4 beta-phorbol 12-myristate 13-acetate, appears to mimic the action of EGF in inducing EGF-R accumulation in coated pits at the cell surface and receptor internalization. In contrast to the results with EGF-R, the TF-R was found in clathrin-coated membranes in the presence or absence of TF, and the concentration of TF-R in clathrin-coated membranes did not significantly change with time. The method presented should be of great utility for examining the biochemical changes that occur during the receptor-mediated endocytosis and sorting of ligands and receptors.

Characterization of a cyclic AMP-resistant Chinese hamster ovary cell mutant containing both wild-type and mutant species of type I regulatory subunit of cyclic AMP-dependent protein kinase.

We have characterized a cyclic AMP-resistant Chinese hamster ovary (CHO) cell mutant in which one of two major species of type I regulatory subunit (RI) of cyclic AMP-dependent protein kinase is altered. Wild-type CHO cell extracts contain two cyclic AMP-dependent protein kinase activities. As shown by DEAE-cellulose chromatography, there is a peak of type I protein kinase activity in mutant extracts, but the type II protein kinase activity is considerably reduced even though free type II regulatory subunit (RII) is present. The type I kinase from the mutant has an altered RI (RI*) whose KD for the binding of 8-N3[32P] cAMP (KD = 1.3 X 10(-5) M) is increased by more than 200-fold compared to RI from the wild-type enzyme (KD = 5.5 X 10(-8) M). No differences were found between the catalytic subunits from the wild-type and mutant type I kinases. A large portion of RI in mutant and wild-type extracts is present in the free form. The RI* derived from mutant type I protein kinase shows altered labeling by 8-N3[32P]cAMP (KD = 1.3 X 10(-5) M) whereas the free RI from the mutant is labeled normally by the photoaffinity label (KD = 7.2 X 10(-8) M), suggesting that the RI* which binds to the catalytic subunit is functionally different from the free form of RI. The decreased amount of type II kinase activity in the mutant appears to be due to competition of RI* with RII for binding to the catalytic subunit. Translation of mRNA from wild-type CHO cells results in the synthesis of two different charge forms of RI, providing biochemical confirmation of two different species of RI in CHO cells. Additional biochemical evidence based on isoelectric focusing behavior of 8-N3[32P]cAMP-labeled RI species and [35S]methionine-labeled RI from mutant and wild-type extracts confirms the charge heterogeneity of RI species in CHO cells. These genetic and biochemical data taken together are consistent with the conclusion that there are at least two different species of RI present in CHO cells and that one of these species is altered in the mutant analyzed in this work.

src kinase catalyzes the phosphorylation and activation of the insulin receptor kinase.

When a partially purified insulin receptor preparation immobilized on insulin-agarose is incubated with [gamma-32P]ATP, Mn2+, and Mg2+ ions, the receptor beta subunit becomes 32P-labeled. The 32P-labeling of the insulin receptor beta subunit is increased by 2-3-fold when src kinase is included in the phosphorylation reaction. In addition, the presence of src kinase results in the phosphorylation of a Mr = 125,000 species. The Mr = 93,000 receptor beta subunit and the Mr = 125,000 32P-labeled bands are absent when an insulin receptor-deficient sample, prepared by the inclusion of excess free insulin to inhibit the adsorption of the receptor to the insulin-agarose, is phosphorylated in the presence of the src kinase. These results indicate that the insulin receptor alpha and beta subunits are phosphorylated by the src kinase. The src kinase-catalyzed phosphorylation of the insulin receptor is not due to the activation of receptor autophosphorylation because a N-ethylmaleimide-treated receptor preparation devoid of receptor kinase activity is also phosphorylated by the src kinase. Conversely, the insulin receptor kinase does not catalyze phosphorylation of the active or N-ethylmaleimide-inactivated src kinase. Subsequent to src kinase-mediated tyrosine phosphorylation, the insulin receptor, either immobilized on insulin-agarose or in detergent extracts, exhibits a 2-fold increase in associated kinase activity using histone as substrate. src kinase mediates phosphorylation of predominantly tyrosine residues on both alpha and beta subunits of the insulin receptor. Tryptic peptide mapping of the 32P-labeled receptor alpha and beta subunits by high pressure liquid chromatography reveals that the src kinase-mediated phosphorylation sites on both receptor subunits exhibit elution profiles identical with those phosphorylated by the receptor kinase. Furthermore, the HPLC elution profile of the receptor auto- or src kinase-catalyzed phosphorylation sites on the receptor alpha subunit are also identical with that on the receptor beta subunit. These results indicate that: the src kinase catalyzes tyrosine phosphorylation of the insulin receptor alpha and beta subunits; and src kinase-catalyzed phosphorylation of insulin receptor can mimic the action of autophosphorylation to activate the insulin receptor kinase in vitro, although whether this occurs in intact cells remains to be determined.

Immunoprecipitation of insulin receptors from cultured human lymphocytes (IM-9 cells) by antibodies to pp60src.

The family of tyrosine-specific protein kinases includes proteins encoded by retroviral oncogenes as well as receptors for insulin and several growth factors. Antibodies to pp60src, the protein encoded by the src oncogene of Rous sarcoma virus (RSV), can specifically immunoprecipitate affinity-labeled insulin receptors from cultured human lymphocytes (IM-9 cells). This precipitation is specifically inhibited by the src gene product purified from RSV-transformed rat cells. These observations provide evidence that there is structural homology between the insulin receptors and pp60src.

Immunocytochemical localization in normal and transformed human cells in tissue culture using a monoclonal antibody to the src protein of the Harvey strain of murine sarcoma virus.

Using a rat monoclonal antibody directed against the p21 src protein of the Harvey strain of Murine Sarcoma Virus (MSV), we have examined the reactivity of human cells in tissue culture using immunofluorescence and electron microscopy. Qualitative results indicated that untransformed mouse and human fibroblastic cells have undetectable amounts of p21; these levels were greatly increased after transformation with Harvey MSV. A group of human tumor cell lines adapted to tissue culture were examined and almost all of the epithelial tumor lines showed significant localization with this antibody. Notable exceptions were two melanoma cell lines which were negative for p21 by immunofluorescence. When normal human epithelial cells derived from esophageal or foreskin epithelium were examined, the antibody showed significant reactivity with subconfluent growing cells. After the normal cells were allowed to become quiescent, the reactivity with this antibody decreased. All of the localization seen by fluorescence was in a distribution consistent with the previously demonstrated location of p21 scr on the inner aspect of the plasma membrane. Electron microscope localization showed labeling for this antigen on the inner surface of the plasma membrane in both transformed mouse cells and in the human tumor cell lines MCF-7 and HTB-2 (RT4). These results suggest that the amounts of p21-like proteins detectable in human epithelial tumor cells do not necessarily reflect their malignant potential, but may be related to their epithelial nature. The loss of detectable localization at quiescence suggests that p21 levels decrease when these epithelial cells stop growing, and raises the possibility that an analog of p21 may be used by these human epithelial cells to regulate cell growth.

The morphologic pathway of binding and internalization of epidermal growth factor in cultured cells. Studies on A431, KB, and 3T3 cells, using multiple methods of labelling.

We have prepared several electron and light microscopic labels of epidermal growth factor (EGF) to analyse the morphologic features of its binding and internalization by cultured cells. These include a ferritin conjugate of EGF, a covalent conjugate of EGF and horseradish peroxidase (EGF-HRP), a colloidal gold marker system using EGF-HRP as a primary antigen, and a covalent complex of EGF with rhodamine-labelled lactalbumin. All of the light and electron microscopic labels showed similar patterns of binding. EGF initially bound to diffusely distributed cell surface receptors at 4 degrees C. The EGF-receptor complexes clustered into clathrin-coated pits on the cell surface only when the temperature was raised to 37 degrees C. In KB and Swiss 3T3 cells, this was followed by rapid internationalization into receptosomes, compartmentalization into the Golgi system, clustering in the clathrin-coated regions of the Golgi, and finally delivery into lysosomes from the Golgi. This general pathway was seen in Swiss 3T3 cells which have a low number of EGF receptors, KB cells which have a moderate number of receptors and A431 cells that have a high number of receptors. However, the ruffling activity induced in A431 cells by EGF produced some internalization through macropinosomes, making the pathway of entry more difficult to evaluate. Double label experiments showed that EGF is internalized together with alpha 2-macroglobulin and adenovirus particles. These data clarify the route of entry of EGF in different cell types using multiple labels, and shows that it enters cells through the same coated pit entry pathway as most other ligands previously examined.

Ultrastructural immunocytochemical localization of calmodulin in cultured cells.

Using an antibody prepared against performic acid-treated calmodulin, we have localized calmodulin in cultured fibroblastic cells by immunofluorescence and immunoelectron microscopy. In interphase cells, calmodulin was found to be diffusely distributed throughout the cytosol. An increased amount of calmodulin was found in the pericentriolar region of interphase cells. No significant aggregation of calmodulin was found in association with microfilaments, peripheral cytoplasmic microtubules or clathrin-coated structures. Calmodulin was present in moderate amounts in microvilli, ruffles, and zeiotic blebs of the cell surface. In motitic cells, calmodulin was found concentrated in the pericentriolar region, and appeared to concentrate along radiating spindle microtubules proximal to the centrioles. Redistribution of calmodulin was seen between early and late telophase, in which the pericentriolar pattern of calmodulin in early telophase shifted to an aggregation on the intercellular bridge, with a large part of the midbody portion of the bridge being devoid of calmodulin. These results show that calmodulin is distributed throughout the cytosol, but is markedly concentrated in the region of the microtubule organizing center in interphase cells, as well as in elements of the mitotic spindle apparatus. This distribution suggests that calmodulin has a regulatory role in the organization and function of microtubules during interphase, as well as during mitosis.

A versatile method for the coupling of protein to DNA: synthesis of alpha 2-macroglobulin-DNA conjugates.

We describe a simple, general method to link proteins covalently to DNA. The method uses two reagents, N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine and 2-iminothiolane. The former reacts specifically with nonpaired quanine residues and upon reduction generates a free sulfhydryl group. The latter reacts with a protein to provide another sulfhydryl group which is subsequently conjugated to DNA by an intermolecular disulfide interchange reaction. Using this method alpha 2-macroglobulin was conjugated to plasmid DNA encoding the Herpes simplex virus-1 thymidine kinase gene or a DNA fragment containing the E. coli chloramphenicol acetyltransferase gene. Up to 20% of the total DNA was conjugated to alpha 2-macroglobulin and the alpha 2-macroglobulin-DNA conjugate had a protein/DNA molar ratio of approximately two. The whole reaction takes place under very mild conditions in aqueous solution. The structure of DNA appears not to be significantly affected by the chemical modification. This method may prove useful in ligand directed gene transfer studies.