Targeting multidrug resistant tumor cells with a recombinant single-chain FV fragment directed to P-glycoprotein.

The MDR1 gene product P-glycoprotein (Pgp) plays a key role in multidrug resistance of cancer cells. Pgp is an ATP-driven efflux pump that extrudes a variety of dissimilar hydrophobic cytotoxic compounds. P-glycoprotein overexpression results in multidrug resistance (MDR) of tumor cell lines in vitro as well as in cancer patients. To selectively target and eliminate MDR tumor cells, we have isolated a monoclonal antibody that specifically reacts with the first extracellular loop of the human Pgp. We have cloned the variable domain genes of this antibody and assembled a functional single-chain Fv fragment capable of specifically targeting various Pgp-expressing MDR carcinoma cells lines. Targeting and specific elimination of Pgp-dependent MDR human cancer cells was achieved by constructing a single-chain immunotoxin in which the scFv fragment was fused to a truncated form of Pseudomonas exotoxin (PE38). We conclude that recombinant Fv-immunotoxins or other Fv-based molecules armed with potent cytotoxins represent an effective tool in targeted cancer therapy aimed at specific elimination of MDR tumor cell sub-populations. Recombinant antibody fragments targeting MDR proteins such as Pgp may be also used for intracellular expression and consequent phenotypic knockout of MDR.

Proteosome delivery of a protective 9B-antigen against Schistosoma mansoni.

We have previously characterized a stage specific, partially protective protein denoted 9B-antigen. This antigen is of 450 kDa in its native form but upon SDS-PAGE in reducing conditions it exhibits two subunits of 30 kDa and 45 kDa. The 9B-antigen is localized at the surface of schistosomula and persists at the surface of lung schistosomula. The 9B-antigen is also localized in internal organs of a vital function in the parasite such as flame cells and cytoplasmic tubes. Infected individuals or mice vaccinated with irradiated cercariae recognize the 9B-antigen. We have previously shown that when injected with complete Freunds adjuvant, the 9B-antigen can induce 40% protection against challenge infection. In this study we have used a more effective delivery system for this antigen. The 9B-antigen was coupled to proteosomes derived from meningoccocal outer membrane proteins. Vaccination of mice with this complex increased the protection level to 60%. Sera from these vaccinated mice induced high levels of complement mediated cytotoxicity of the parasite. Since the proteosomes are approved for human use, these results are promising towards the development of a vaccine against schistosomiasis.

Effective immunization of mice against cutaneous leishmaniasis using an intrinsically adjuvanted synthetic lipopeptide vaccine.

Two peptides representing predicted T-cell epitopes of gp63, a major surface glycoprotein of the parasite Leishmania major, were used in vaccines tested in a murine model of cutaneous leishmaniasis. Either subcutaneous or intraperitoneal immunization in saline with a peptide representing gp63 amino acids 467-482 (p467) significantly protected CBA mice against the development of severe cutaneous lesions only when the peptide was intrinsically adjuvanted by covalently adding a lauryl-cysteine moiety (LC-p467) to its amino terminus during synthesis. In marked contrast, administration of p467 alone, cysteinyl-p467 or gp63 protein in saline resulted in some disease exacerbation. Splenic cells of LC-p467 immunized mice stimulated in vitro with LC-p467 displayed strong proliferative responses and secretion of IL-2, IFN-tau and GM-CSF (but not IL-4 and IL-10) suggesting that immunization with the lipopeptide induced the TH1 type cytokine responses associated with cell-mediated immunity. The safety, efficacy, ease of production and standardization of such lipopeptide vaccines suggest that they have significant potential for the development of vaccines for humans against leishmaniasis or other parasitic or viral diseases that require cell-mediated immunity for protection.

Intranasal immunization of mice against influenza with synthetic peptides anchored to proteosomes.

Synthetic vaccines that are based on peptides representing immunogenic epitopes require a carrier molecule as well as an adjuvant in order to be effective. The choice of carriers or adjuvants approved for use in humans is very limited, and a considerable effort is devoted to develop new and efficient delivery systems. One of these vehicles utilizes preparations of outer membranes of meningococci, that form hydrophobic interactions, denoted proteosomes. Immunogenic proteins and peptides can be anchored to these proteosomes vesicles, which may serve as both carrier and adjuvant functions. In the present study we examined the ability of proteosomes to present epitopes of influenza, to elicit specific anti-influenza responses and to protect mice against viral challenge after intranasal immunization. Three influenza peptides were used--one corresponding to amino acid residues 91-108 of the haemagglutinin surface glycoprotein of H3 subtype, which comprises a B-cell epitope, and two from the internal nucleoprotein--a T-helper cell (Th) epitope (residues 55-69) and a cytotoxic T-lymphocyte (CTL) epitope (147-158). Mice were immunized intranasally (i.n.) with preparations containing each of the above epitopes, or various combinations thereof. The results obtained with this system demonstrate that influenza epitopes represented by synthetic peptides anchored to a proteosome carrier elicit both humoral and cellular specific immune responses, that can lead to partial protection of the mice from viral challenge. The importance of immunizing with vaccines containing both B- and T-cell peptide epitopes was emphasized by the demonstration that such vaccines elicited longer lasting immunity and led to more effective protection against influenza viral challenge.

Lethally irradiated normal strains of mice radioprotected with SCID bone marrow develop sensitivity to low doses of staphylococcal enterotoxin B.

Normal strains of mice are rendered sensitive to small amounts (3-10 micrograms) of staphylococcal enterotoxin B (SEB) by transplanting bone marrow cells of SCID donor mice to lethally irradiated recipients. Four to 12 weeks post-transplantation, SEB induces 56-100% lethality. Transplantation of normal mouse bone marrow cells, either alone or with the SCID mouse selected bone marrow cells, does not confer SEB sensitivity. These data imply that either irradiation ablates certain cell population(s), that confer resistance to SEB in normal mice (populations that are absent in the SCID donor mice) or that the donor cells selectively repopulate recipients with SEB-sensitive cells. This model will help elucidate the cells, cytokines and the SEB peptide fragments responsible for SEB toxicity and will be useful in identifying promising vaccine candidates and in developing preventive medicines to protect against this potent toxin.

Indirect immunotargeting of cis-Pt to human epidermoid carcinoma KB using the avidin-biotin system.

Cis-diamminedichloroplatinum (II) (cis-Pt) complexed to a carboxymethyl dextran-avidin conjugate was targeted to biotin-monoclonal antibody 108 (b-MAb 108). This MAb recognizes the extracellular domain of the epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma (KB) cells over-expressing EGF-R. Cis-Pt-carboxymethyl-dextran-avidin (Pt-dex-Av) containing 60-90 M cis-Pt/M avidin was administered 24 hr following b-MAb108 containing 3-5 M biotin/M MAb. This treatment was potentially more effective in suppressing the growth of established KB tumor xenografts, or in inhibiting the development of lung metastases in nude mice, than free MAb 108, free drug or MAb 108 followed by drug. Replacing b-MAb 108 by unbiotinylated antibody or by b-MAb of a different specificity also yielded lower suppressive effects. The sequential administration of Pt-dex-Av following b-MAb was more effective than introduction of the Pt-dex-Av when already complexed to b-MAb 108. The results presented in this preliminary investigation suggest that Pt-dex-Av is specifically removed from the circulation by b-MAb 108 concentrated at the tumor site.

Inhibition of human tumor growth in nude mice by a conjugate of doxorubicin with monoclonal antibodies to epidermal growth factor receptor.

Monoclonal antibodies that recognize the extracellular domain of the epidermal growth factor receptor (mAb108) were conjugated with doxorubicin through a dextran bridge. Several antibody-drug conjugates, containing different amounts of doxorubicin, retained binding capacity to human epidermoid carcinoma (KB) cells overexpressing epidermal growth factor receptors. Slight decrease in drug cytotoxicity was seen in in vitro tests, as determined either by inhibition of thymidine incorporation into cells or by reduction in number and size of KB-cell colonies. Yet, when tested in vivo against KB tumor xenografted into nude mice, the anti-epidermal growth factor-receptor drug conjugates with high drug-substitution levels were significantly more effective than free doxorubicin, antibody alone, mixture of dextran-doxorubicin and antibody, or drug conjugated with irrelevant antibody. When the labile covalent bonds linking antibody to dextran bridge were stabilized by reduction, the therapeutic efficacy of the conjugate was markedly decreased. These results show that antibodies against the extracellular domain of the epidermal growth factor can deliver doxorubicin specifically and efficiently to tumor sites that express high receptor levels exerting a specific antitumor effect.

Cytotoxic activity of daunorubicin or vindesin conjugated to a monoclonal antibody on cultured MCF-7 breast carcinoma cells.

Conjugates were constructed between daunorubicin or vindesin and a monoclonal antibody to human milk fat globule membrane associated antigen. This antibody recognizes a high molecular weight glycoprotein present at the cell surface of human normal and tumour epithelial cells; after specific binding to plasma membrane of cultured MCF-7 human breast carcinoma cells, it is endocytosed and gains access to lysosomes, wherein it is broken down (Aboud-Pirak et al., Cancer Res 48: 3188-3196, 1988). Covalent linkage of daunorubicin (through a succinylated tetrapeptide arm) or of vindesin (through a hemisuccinate arm) yields conjugates with maximal molar ratios (drug molecule/specific IgG under monomeric form, i.e. unaggregated) or 2.0 and 4.5 respectively. The conjugate with daunorubicin inhibits the binding of the 3H labelled antibody to MCF-7 cells as efficiently as the native unconjugated antibody, whereas the conjugate with vindesin inhibits it only by 56%. Both conjugates are entirely stable in plasma and serum; after 24 hr incubation at pH 4.8 in the presence of rat liver lysosomal enzymes, 60 and 33% of daunorubicin and vindesin respectively are released from the conjugates. Adherent non-confluent cultures of cells recognized (MCF-7) or not (Hep-G2, human hepatocarcinoma cells) by the antibody were incubated from 1 hr to 6 days with different concentrations of daunorubicin or vindesin, free or conjugated to the specific or to a control monoclonal antibody. LD50, defined as the drug concentration required to reach 50% of the amount of cell associated protein obtained in the absence of drug were determined at the end of 6 days continuous incubation or after shorter incubation followed by reincubation in drug free medium up to 6 days. Both cell lines are almost equally susceptible to the free drugs. The conjugate between daunorubicin and the antibody appears inactive, even at saturating concentrations of antibody. This could result from the extrusion out of the cells of daunorubicin molecules released from the conjugate, impairing the drug to reach the intracellular concentration required for cytotoxicity. In contrast, conjugation of vindesin to the specific but not to a control antibody restricts the activity of the drug to cells selectively recognized by the specific antibody. However, even after corrections for the loss of immunoreactivity and for the incomplete release of vindesin from the conjugate, cytotoxicity is achieved at higher concentrations or requires longer exposure to the conjugated than to the free drug.(ABSTRACT TRUNCATED AT 400 WORDS)

Efficacy of antibodies to epidermal growth factor receptor against KB carcinoma in vitro and in nude mice.

Iodine-125-labeled monoclonal antibody 108.4 (108.4 mAb), raised against the extracellular domain of the epidermal growth factor (EGF) receptor, was shown to visualize sc xenografts of human oral epidermoid carcinoma (KB) cells in nude mice. In vitro, although EGF caused an increase in the number of KB cell colonies (150% at a concentration of 160 mM), the anti-EGF receptor antibodies reduced clone formation. At a concentration at which EGF caused a 50% increase in colony number, the addition of a 100-fold molar excess of 108.4 mAb resulted in a decrease in the number of cell colonies to 20% of the original value. Therefore, the effect of antibody on the KB tumor was studied in vivo in three different modes of tumor transplantation. Antitumor activity was demonstrated first by retardation (versus controls) of the growth of tumor cells as sc xenografts (P greater than .017), then by prolongation of the life span of animals with the ip form of the tumor (P less than .001), and finally on an experimental lung metastasis by a reduction in the number and size of tumors (P less than .05). When the anti-EGF receptor antibodies were added together with cisplatin, the antitumor effect was greatly enhanced, suggesting that the toxic activity of these agents is synergistic (P less than .007). The antitumor effect persisted when animals were treated with the F(ab)'2 fragment of the antibody, although it was less efficient. The Fab fragment of the antibody, whose ability to bind to the cell-associated receptor was completely conserved, did not affect the growth of the tumor. The activity manifested by the F(ab)'2 fragment of the anti-EGF receptor antibodies suggested that the antitumor effect was not due to immune mechanisms requiring the Fc portion of the antibody.

Binding and endocytosis of a monoclonal antibody to a high molecular weight human milk fat globule membrane-associated antigen by cultured MCF-7 breast carcinoma cells.

The aim of this study was to analyze whether a monoclonal antibody to human milk fat globule membrane-associated antigens, recognized specifically and homogeneously by human breast carcinoma cells but also by normal epithelial cells active in secretion, could be used to restrict the access of antitumoral drugs to cells exposing the epitope. The drug-antibody conjugate to be used is constructed by means of a covalent peptidic linkage stable in extracellular medium but hydrolyzed by lysomal enzymes after endocytosis of the drug-carrier conjugate. This monoclonal antibody specifically immunoprecipitates radioactive material from MCF-7 cells biosynthetically radiolabeled with galactose, glucosamine, palmitic acid, or acetic acid but not with mannose, leucine, or methionine. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol, the label migrates as two bands with apparent molecular weights of about 350,000 and 400,000. These bands disappear, or their molecular weight is affected, after treatment of the cells with cycloheximide or of cell lysates with trypsin, Pronase, or neuraminidase but not treatment of the immunoprecipitate with endoglycosidase F. This suggests that these antigens are glycoproteins with O-linked oligosaccharides containing sialic acid in the epitope. By analogy, they should be similar, if not identical, to those recognized by the monoclonal antibodies designated HMFG1 (H. Burchell, H. Durbin, and J. Taylor-Papadimitriou, J. Immunol., 131:508-513, 1983) and DF3 (H. Sekine, T. Ohno, and D.W. Kufe, J. Immunol., 135:3610-3615, 1985). Binding at 4 degrees C of the 3H-labeled antibody by MCF-7 cells indicates the specific attachment of about 1.2 X 10(6) IgG molecules per cells with a Kd of about 14 nM. At 37 degrees C, cells take up the 3H-labeled antibody in amounts much higher than the binding capacity. In addition to cell-associated material, labeled digestion products are released into the culture medium. Cell fractionation by differential centrifugation and isopycnic equilibration on sucrose gradient indicates that the bulk of cell-associated antibody is distributed like the marker enzyme of lysosomes. Although the total uptake of the antibody by the cells is unaffected by either 50 microM chloroquine or 3 micrograms/ml cycloheximide, the release of digestion products is completely inhibited by chloroquine. Antigen-antibody dissociation is pH dependent, since, respectively, 50 and 84% of membrane-bound antibody are released during washing at pH 4.6 and 4.1.(ABSTRACT TRUNCATED AT 400 WORDS)