Immunotherapeutic potential of anti-human endogenous retrovirus-K envelope protein antibodies in targeting breast tumors.

BACKGROUND: The envelope (env) protein of the human endogenous retrovirus type K (HERV-K) family is commonly expressed on the surface of breast cancer cells. We assessed whether HERV-K env is a potential target for antibody-based immunotherapy of breast cancer. METHODS: We examined the expression of HERV-K env protein in various malignant (MDA-MB-231, MCF-7, SKBR3, MDA-MB-453, T47D, and ZR-75-1) and nonmalignant (MCF-10A and MCF-10AT) human breast cell lines by immunoblot, enzyme-linked immunosorbent assay, immunofluorescence staining, and flow cytometry. Anti-HERV-K env monoclonal antibodies (mAbs; 6H5, 4D1, 4E11, 6E11, and 4E6) were used to target expression of HERV-K, and antitumor effects were assessed by quantifying growth and apoptosis of breast cancer cells in vitro, and tumor growth in vivo in mice (n = 5 per group) bearing xenograft tumors. The mechanisms responsible for 6H5 mAb-mediated effects were investigated by microarray assays, flow cytometry, immunoblot, and immunofluorescence staining. The expression of HERV-K env protein was assessed in primary breast tumors (n = 223) by immunohistochemistry. All statistical tests were two-sided. RESULTS: The expression of HERV-K env protein in malignant breast cancer cell lines was substantially higher than nonmalignant breast cells. Anti-HERV-K-specific mAbs inhibited growth and induced apoptosis of breast cancer cells in vitro. Mice treated with 6H5 mAb showed statistically significantly reduced growth of xenograft tumors compared with mice treated with control immunoglobulin (control [mIgG] vs 6H5 mAb, for tumors originating from MDA-MB-231 cells, mean size = 1448.33 vs 475.44 mm(3); difference = 972.89 mm(3), 95% CI = 470.17 to 1475.61 mm(3); P < .001). Several proteins involved in the apoptotic signaling pathways were overexpressed in vitro in 6H5 mAb-treated malignant breast cells compared with mIgG-treated control. HERV-K expression was detected in 148 (66%) of 223 primary breast tumors, and a higher rate of lymph node metastasis was associated with HERV-K-positive compared with HERV-K-negative tumors (43% vs 23%, P = .003). CONCLUSION: Monoclonal antibodies against HERV-K env protein show potential as novel immunotherapeutic agents for breast cancer therapy.

Inhibitor design by wrapping packing defects in HIV-1 proteins.

Two viral proteins, HIV-1 protease and HIV-1 integrase, have been targeted for inhibitor design to prevent assembly and maturation of HIV-1 virions. The enzymatic mechanism of these proteins involves side-chain groups that serve as general acids or bases. Furthermore, catalytic activity requires that water be removed from the microenvironment surrounding the chemical reaction site or be constrained to serve as an activated nucleophile. Here, we identify previously unrecognized structural features that promote water removal from polar catalytic regions. Packing defects in the form of hydrogen bonds that are insufficiently dehydrated intramolecularly, named "dehydrons," are strategically placed in the structure to induce an anhydrous enzymatic pathway. Dehydrons become electrostatically enhanced and stabilized upon further desolvation. Thus, packing defects act synergistically with the polar active groups to enhance the enzymatic electrostatics. However, because dehydrons are sticky, they constitute targets for inhibitor design. We noticed that inhibitors attach to polar surfaces by further desolvating dehydrons, thus blocking the active sites or the sites involved in harnessing the substrate. The dehydrons are thus required for functional reasons, making them suitable targets. The differences in success when targeting HIV-1 protease, feline immunodeficiency virus protease, and HIV-1 integrase are rationalized in terms of the dehydron distribution, revealing possible improvements in the targeting strategy. Principles of design optimization are proposed to create an inhibitor that can be neutralized only at the expense of the loss of catalytic function. The possibility of using drugs that wrap dehydrons to block protein-protein associations is also discussed.

SIV gp41 binds to membranes both in the monomeric and trimeric states: consequences for the neuropathology and inhibition of HIV infection.

The viral envelope glycoprotein gp41 mediates membrane fusion in HIV/SIV infection. gp41 ectodomain (e-gp41, residues 27-149), which was shown to interact with phospholipid membranes, exists in an equilibrium between the monomeric and trimeric states. Here, we analyzed, by intrinsic Trp fluorescence and resonance energy transfer, whether SIV e-gp41-membrane interaction depends on the gp41 oligomeric state. We found that both gp41 monomers and trimers bind membranes, with the monomers' full binding being reached at substantially lower lipid to protein ratios. Furthermore, the different characteristics of the Trp fluorescence of monomers and trimers enabled us to detect binding of each form at concentrations at which both species were present. CD spectroscopy revealed that the secondary structure of gp41 monomers does not change upon membrane binding, suggesting that membrane-bound monomeric-gp41 is a possible target for DP-178, a potent peptide inhibitor of HIV infection. The consequences of the interaction between monomeric and trimeric gp41 with membranes in HIV/SIV infection, its inhibition, and its associated neuropathologies are discussed.

Experimental determination and calculations of redox potential descriptors of compounds directed against retroviral zinc fingers: Implications for rational drug design.

A diverse set of electrophilic compounds that react with cysteine thiolates in retroviral nucleocapsid (NC) proteins and abolish virus infectivity has been identified. Although different in chemical composition, these compounds are all oxidizing agents that lead to the ejection of Zn(II) ions bound to conserved structural motifs (zinc fingers) present in retroviral NC proteins. The reactivity of a congeneric series of aromatic disulfides toward the NC protein of the human immunodeficiency virus type 1 (HIV-1), NCp7, has been characterized by HPLC separation of starting reagents from reaction products. We calculated the absolute redox potentials of these compounds in the gas phase and in aqueous solvent, using a density functional theory method and a continuum solvation model. Pulsed polarography experiments were performed and showed a direct correlation between calculated and experimentally determined redox propensities. A dependence between protein reactivity and redox potential for a specific compound was shown: Reaction with NCp7 did not take place below a threshold value of redox potential. This relationship permits the distinction between active and nonactive compounds targeted against NCp7, and provides a theoretical basis for a scale of reactivity with retroviral zinc fingers. Our results indicate that electrophilic agents with adequate thiophilicity to react with retroviral NC fingers can now be designed using known or calculated electrochemical properties. This may assist in the design of antiretroviral compounds with greater specificity for NC protein. Such electrophilic agents can be used in retrovirus inactivation with the intent of preparing a whole-killed virus vaccine formulation that exhibits unaffected surface antigenic properties.

Structure of equine infectious anemia virus proteinase complexed with an inhibitor.

Equine infectious anemia virus (EIAV), the causative agent of infectious anemia in horses, is a member of the lentiviral family. The virus-encoded proteinase (PR) processes viral polyproteins into functional molecules during replication and it also cleaves viral nucleocapsid protein during infection. The X-ray structure of a complex of the 154G mutant of EIAV PR with the inhibitor HBY-793 was solved at 1.8 A resolution and refined to a crystallographic R-factor of 0.136. The molecule is a dimer in which the monomers are related by a crystallographic twofold axis. Although both the enzyme and the inhibitor are symmetric, the interactions between the central part of the inhibitor and the active site aspartates are asymmetric, and the inhibitor and the two flaps are partially disordered. The overall fold of EIAV PR is very similar to that of other retroviral proteinases. However, a novel feature of the EIAV PR structure is the appearance of the second alpha-helix in the monomer in a position predicted by the structural template for the family of aspartic proteinases. The parts of the EIAV PR with the highest resemblance to human immunodeficiency virus type 1 PR include the substrate-binding sites; thus, the differences in the specificity of both enzymes have to be explained by enzyme-ligand interactions at the periphery of the active site as well.

In vivo effects of thymostimulin treatment on monocyte polarization, dendritic cell clustering and serum p15E-like trans-membrane factors in operable head and neck squamous cell carcinoma patients.

Head and neck squamous cell carcinoma patients have been characterized by impairments in their cell-mediated immune system, particularly by decreased chemotactic function of monocytes and impairments in the function of the monocyte-derived dendritic cells (viz, a decreased capability to form cell "clusters"). These impairments are thought to be due to immunosuppressive factors of low molecular mass released by tumor, the so-called p15E-like factors. These suppressive effects of p15-like factors can be neutralized in vitro by thymic peptides, such as thymostimulin (TP1). In a randomized double-blind, placebo-controlled multicenter trial in the Netherlands, 41 patients with operable head and neck squamous cell carcinomas (HNSCC) were treated for 10 days prior to surgery with intramuscular TP1 in one of three dosages (0.5 mg/kg; 1.0 mg/kg or 2.0 mg/kg body weight) or treated with placebo. Assessment of monocyte chemotaxis, the capability of dendritic cells to form clusters and the presence of p15E-like low-molecular-mass factors (LMMFs) in serum was performed before TP1 treatment and on the day of surgery. Findings demonstrated that TP1 in a dose of 1.0 mg/kg and 2.0 mg/kg resulted in normalization of impaired monocyte chemotactic capability. Although the cluster capability of dendritic cells after TP1 treatment improved, values only reached statistical significance for the 0.5 mg/kg group. Serum p15E-like LMMF levels were not affected by TP1 treatment in any of the patient groups. Contrary to expectations we found no correlation between elevated immunosuppressive LMMFs and defective monocyte chemotaxis or cluster capability of dendritic cells. We conclude that treatment with TP1 can improve monocyte chemotaxis in HNSCC patients but an effect on the production of p15E-like factors by carcinoma cells could not be demonstrated.

Stability of dimeric retroviral proteases.

The determination of dimer stabilities for the retroviral proteases has proved more challenging than anticipated, but it is a tractable problem when careful attention is made to potential interferences. For investigations of retroviral proteases not yet characterized, the fundamentally rigorous sedimentation equilibrium and other biophysical techniques may yet provide useful Kd values. They are preferable to the indirect methods emphasized in this chapter but nevertheless should be coupled with basic considerations such as recovery of activity at the end of an experiment and the relevance of values obtained to other situations. In the likely event that nanomolar Kd values are encountered in new investigations, the assay techniques provide the most readily available methods for many laboratories. Because these methods are sensitive to anything that affects enzyme activity, the use of complementary methods to verify dimerization constants is imperative. Inactivating reactions not due to monomer formation should be explored, and the potential impact of those reactions on the constants being measured should be estimated. Most of the Kd and dimerization rate data available for retroviral proteases are obtained with the HIV-1 protease, with each investigator choosing methods and solvent conditions different from the others. The confusing diversity of results should be the impetus for a direct comparison of methods for the identification of the sources of differences. If more comprehensive and rigorous measures of the kinetics and thermodynamics of subunit aggregation are obtained, they might be coupled with the large volume of detailed structural data accumulating for this class of protein to provide insights into more general problems of protein-folding chemistry.

A TIBO derivative, R82913, is a potent inhibitor of HIV-1 reverse transcriptase with heteropolymer templates.

R82913, (+)-S-4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)- imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (a TIBO derivative), inhibited the replication of thirteen different strains of HIV-1 in CEM cells with a median IC50 of 0.15 microM. The concentration of compound that killed 50% of the cells was much higher (46 microM), indicating that R82913 has a high selectivity index. R82913 was 20-fold more potent than AZT-TP in the inhibition of HIV-1 reverse transcriptase in an assay using a naturally occurring template (ribosomal RNA) that more accurately resembles native viral RNA than a synthetic homopolymer. With this template, R82913 inhibited HIV-1 reverse transcriptase with an ID50 (0.01 microM) that is equal to, or lower than, the IC50 for this compound in all of our cell culture assays (0.01-0.65 microM). R82913 has no effect on the replication of HIV-2 in CEM cells and does not inhibit the reverse transcriptase from this virus.

Purification and biochemical characterization of recombinant simian immunodeficiency virus protease and comparison to human immunodeficiency virus type 1 protease.

Simian immunodeficiency virus protease (SIV-PR) was produced in Escherichia coli with a recombinant expression system in which the mature enzyme autoprocessed from a precursor form. Recombinant SIV and HIV-1 (human immunodeficiency virus, type 1) proteases were purified from bacterial cell lysates by use of sequential steps of ammonium sulfate precipitation and size-exclusion and ion-exchange chromatography. The amino acid composition, amino-terminal sequence, and molecular weight (monomer) of the recombinant SIV-PR were in accord with that of the 99 amino acid polypeptide predicted from the SIVMac-PR nucleotide sequence. The active form of SIV-PR was shown to be dimeric by gel filtration chromatography. Inhibition by pepstatin A, time-dependent inactivation by 1,2-epoxy-3-(4-nitrophenoxy)propane, and pH rate profiles using oligopeptide substrates demonstrated that SIV-PR behaves as an aspartic protease. Recombinant HIV-1 Pr55gag precursor was processed in vitro by SIV-PR and HIV-1 PR with indistinguishable proteolytic patterns upon NaDodSO4-polyacrylamide gel electrophoresis. Oligopeptide substrates for HIV-1 PR were found to be suitable substrates for recombinant SIV-PR with the exception of a peptide containing the site identified for p66/p51 cleavage (Phe*Tyr) within HIV-1 reverse transcriptase (RT). Several synthetic peptide analogue inhibitors of HIV-1 PR were also potent inhibitors of SIV-PR, indicating that SIV infection in macaques and rhesus monkeys should be useful models for the preclinical evaluation of acquired immunodeficiency syndrome (AIDS) therapeutics targeted towards the virally encoded HIV-1 protease.

Antibody response to reverse transcriptase in cats infected with feline immunodeficiency virus.

The antibody response in cats to feline immunodeficiency virus (FIV) reverse transcriptase (RT) was followed for 3 years. Eight of the nine cats used in this study produced reverse transcriptase-inhibiting (RTI) antibodies. Relative inhibitory means of 2.9%, 18.4%, 33%, and 47% were found 6, 12, 24, and 36 months, respectively, after infection with FIV. The enzyme activity was suppressed by greater than or equal to 78% with the use of 100 micrograms of FIV-associated IgG. The RTI antibodies were FIV-specific, as they did not inhibit other mammalian retroviral polymerases, including feline leukemia virus RT. An RT-inhibition assay with sera in the presence of protein A and immunoblot analysis showed that antibody binding to FIV RT protein p62 is independent of antibody ability to block enzyme activity. Viral RT released by detergent-treated virus was stable for more than 6 weeks at 4 degrees C, whereas its activity was reduced by 50% after 2 weeks at 37 degrees C. Because significant concentrations of RTI antibodies are detected only at 1 to 2 years after infection, they can be used to determine the approximate time of virus infection and as a marker for disease progression.

Comparison of the human immunodeficiency virus type 1 and 2 proteases by hybrid gene construction and trans-complementation.

To determine the cleavage specificity of the proteases of the type 1 and 2 human immunodeficiency viruses (HIV-1, HIV-2), we interchanged this domain of the polymerase (pol) genes and analyzed the maturation programs of the chimeric polyproteins in an Escherichia coli expression system. In both cases, release of reverse transcriptase and integrase was observed, together with the respective 10-kDa protease form resulting from autocatalysis, although the maturation proceeded less efficiently compared to the homologous system. In further experiments, the ability of both HIV-1 and HIV-2 proteases to release in vivo gag p24 from an in-frame fusion of the full length gag and protease precursors was analyzed. In either case, p24 was released, albeit with greater efficiency in the heterologous gene construction. In vitro mixed lysate experiments with the HIV-1 gag precursor furthermore demonstrate that both enzymes respond to the aspartyl proteinase inhibitor pepstatin A. Taken together, these results illustrate that although different cleavage recognition sequences exist for HIV-1 and -2, they are amenable to the proteases of both viruses, but additionally that subtle differences in the mode of action of both enzymes are observable.

Nonphosphorylatable substrate analogs selectively block autophosphorylation and activation of the insulin receptor, epidermal growth factor receptor, and pp60v-src kinases.

The receptors for insulin and epidermal growth factor undergo tyrosine autophosphorylation in response to ligand stimulation, while pp60v-src is an unregulated tyrosine kinase. In this report we show that each of the kinases phosphorylates an exogenous peptide that corresponds to the insulin proreceptor sequence 1142-1153. When the kinases were pre-phosphorylated, saturable Michaelis-Menten kinetics were observed. However, when the kinases had not been pre-phosphorylated biphasic kinetics were observed; at progressively higher substrate concentrations (greater than Km) less substrate phosphorylation was seen. Furthermore, when the kinases had not been pre-phosphorylated kinase autophosphorylation was inhibited at high substrate concentrations. On this basis we postulated that the substrate inhibition of substrate phosphorylation resulted directly from substrate inhibition of kinase autophosphorylation. To test this we designed additional peptides to function specifically as inhibitors of the kinases. Each of the 3 tyrosine residues within the substrate sequence were replaced either by 4-methoxyphenylalanine or phenylalanine, residues structurally similar to tyrosine but unable to accept phosphoryl transfer. Both analogs inhibited insulin and epidermal growth factor receptor autophosphorylation, whereas only the Phe-substituted analog inhibited pp60v-src phosphorylation. These data suggest that autophosphorylation of tyrosine residues near the kinase active site is a generalized mechanism for tyrosine kinase activation and that activation can be selectively blocked by substrates and nonphosphorylatable analogs.

D-penicillamine inhibits transactivation of human immunodeficiency virus type-1 (HIV-1) LTR by transactivator protein.

D-Penicillamine, an amino acid analogue of cysteine, has been shown to inhibit the transactivation of HIV-1 LTR by the transactivator protein, tat protein. The transactivation was studied in Jurkat cells co-transfected with plasmids containing HIV-LTR sequences fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and HIV tat gene. The expression of CAT activity was a measure of transactivation of LTR by the tat protein. Incubation of transfected Jurkat cells with D-penicillamine led to inhibition of CAT activity. This inhibition was found to be concentration-dependent; more than 90% inhibition of chloramphenicol acetylation was seen in extracts prepared from cultures incubated with 40 micrograms/ml of D-penicillamine. Earlier experiments have shown that D-penicillamine at 40 micrograms/ml can completely inhibit HIV-1 (HTLV-III B) replication in H9 cells [(1986) Drug Res. 36, 184-186]. These results suggest that inhibition of transactivation may be the molecular mechanism involved in the inhibition of HIV-1 replication by D-penicillamine.

Potassium inhibition of transforming protein P85gag-mos and reversal of the transformed phenotype in 6m2 cells.

K+ at high concentrations (52-72 mM hypertonic KCl) has been reported to induce reverse transformation in the 6m2 cell, which is a clone of normal rat kidney cells (NRK) infected with a temperature-sensitive transformation virus. When exposed to high K+, 6m2 cells grown at the permissive temperature (33 degrees C) exhibit normal morphology and reduced soft agar growth, characteristics of cells grown at nonpermissive temperature (39 degrees C). In the current study, flattening of cells and rearrangement of surface microvilli were demonstrated by scanning electron microscopy to occur within 6 hr of exposure to high K+, similar to the effect of temperature shift to 39 degrees C. Exposure to K+ resulted in a 90% inhibition of P85gag-mos-associated serine kinase activity within 5 min, with a subsequent reduction of up to 75% of the synthesis of this protein. These alterations in the putative transforming protein were similar to those induced by temperature shift and were considered to be the basis for retrotransformation. The cell microtubular system and F-actin cables were affected more slowly by K+ than by a temperature shift to 39 degrees C. The former did not achieve the fine reticulum network seen in NRK cells until 72 hr later, but the latter remained aberrant. The effect on the enzyme might be mediated by alteration in phosphorylation, but the mechanism by which kinase inactivation induces retrotransformation is not yet known.

Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants.

We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a protein with an apparent molecular mass of 66 kDa that differs from human immunodeficiency virus (HIV) RNA-dependent DNA polymerase (deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase or reverse transcriptase, EC 2.7.7.49) only in that it has two additional amino-terminal amino acids. This protein is soluble in E. coli extracts, is active in reverse transcriptase assays, and shows inhibition profiles with dideoxy-TTP and dideoxy-GTP that are indistinguishable from the viral enzyme. The deletion of 23 amino-terminal or carboxyl-terminal amino acids or the insertion of 5 amino acids at position 143 substantially decreases the polymerizing activity of the HIV reverse transcriptase made in E. coli. The properties of a 51-kDa reverse transcriptase-related protein made in E. coli suggests that the p51 found in the virion probably does not have substantial polymerizing activity. The full-length HIV reverse transcriptase and the various mutant proteins produced in E. coli should be quite useful for structural and biochemical analyses as well as for the production of antibodies.

Amiloride inhibits the protein tyrosine kinases associated with the cellular and the transforming src-gene products.

Amiloride inhibits a protein tyrosine kinase from rat brain extracts. The kinase activity is characterized by an anti-serum (TBR-serum) which immunoprecipitates pp60c-src, the cellular counterpart of the transforming protein pp60v-src of Rous sarcoma virus. In immunocomplexes, TBR-IgG serves as an artificial but specific phosphate acceptor. The phosphate incorporation into TBR-IgG is a time- and temperature-dependent process. In the presence of amiloride the TBR-IgG phosphorylation is reduced. The drug does not influence the immunocomplexes formed by TBR-IgG and pp60src and no amiloride-activated protein tyrosine phosphatase can be detected in the immunocomplex system. Half-maximal inhibition of the tyrosine kinase occurs at 300 microM amiloride and is competitive with respect to ATP. Viral pp60src kinase of transformed cells is more sensitive to amiloride (IC50: 50-100 microM). Furthermore, normal cellular tyrosine kinases are to a lesser extent inhibited by amiloride as compared to the transforming viral pp60src kinase. These results may indicate different amiloride-sensitive forms of cellular pp60src kinases.

Inhibition of the tyrosine kinase activity of v-src, v-fgr, and v-yes gene products by a monoclonal antibody which binds both amino and carboxy peptide fragments of pp60v-src.

A monoclonal antibody, R2D2, raised to the src gene product of Rous sarcoma virus was found to inhibit the tyrosine protein kinase activity of pp60v-src in autophosphorylation reactions and in reactions involving exogenously added substrates, such as casein and histone. R2D2 also inhibited the enzymatic activity of two related viral transforming proteins, pp70gag-fgr and pp90gag-yes. The inhibitory ability of R2D2 was dependent upon immunoglobulin concentration and could be demonstrated in both immune complexes formed directly with R2D2 and preformed immune complexes to which R2D2 was added. Binding sites in both the amino-terminal 110 amino acid residues and the carboxy-terminal 240 amino acids of pp60v-src were identified for R2D2. These results indicate that at least part of the epitope recognized by R2D2 resides within a region of the src protein which is required for protein kinase activity. The localization of the R2D2 epitope to the amino- as well as to the carboxy-terminal portions of pp60v-src, together with results of studies analyzing the relative binding efficiencies of R2D2 to the intact protein and to V-8 proteolytic fragments of pp60v-src, are consistent with the view that the R2D2 epitope is conformational in nature and that it is assembled from residues contained within both N-terminal and C-terminal regions of the molecule.