AllergoOncology - the impact of allergy in oncology: EAACI position paper.

Th2 immunity and allergic immune surveillance play critical roles in host responses to pathogens, parasites and allergens. Numerous studies have reported significant links between Th2 responses and cancer, including insights into the functions of IgE antibodies and associated effector cells in both antitumour immune surveillance and therapy. The interdisciplinary field of AllergoOncology was given Task Force status by the European Academy of Allergy and Clinical Immunology in 2014. Affiliated expert groups focus on the interface between allergic responses and cancer, applied to immune surveillance, immunomodulation and the functions of IgE-mediated immune responses against cancer, to derive novel insights into more effective treatments. Coincident with rapid expansion in clinical application of cancer immunotherapies, here we review the current state-of-the-art and future translational opportunities, as well as challenges in this relatively new field. Recent developments include improved understanding of Th2 antibodies, intratumoral innate allergy effector cells and mediators, IgE-mediated tumour antigen cross-presentation by dendritic cells, as well as immunotherapeutic strategies such as vaccines and recombinant antibodies, and finally, the management of allergy in daily clinical oncology. Shedding light on the crosstalk between allergic response and cancer is paving the way for new avenues of treatment.

AllergoOncology: the role of IgE-mediated allergy in cancer.

Epidemiological studies have suggested inverse associations between allergic diseases and malignancies. As a proof of concept for the capability of immunoglobulin E (IgE) to destruct tumor cells, several experimental strategies have evolved to specifically target this antibody class towards relevant tumor antigens. It could be demonstrated that IgE antibodies specific to overexpressed tumor antigens have been superior to any other immunoglobulin class with respect to antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) reactions. In an alternative approach, IgE nonspecifically attached to tumor cells proved to be a powerful adjuvant establishing tumor-specific immune memory. Active Th2 immunity could also be achieved by applying an oral immunization regimen using mimotopes, i.e. epitope mimics of tumor antigens. The induced IgE antibodies could be cross-linked by live tumor cells leading to tumoricidic mediator release. Thus, IgE antibodies may not only act in natural tumor surveillance, but could possibly also be exploited for tumor control in active and passive immunotherapy settings. Thereby, eosinophils, mast cells and macrophages can be armed with the cytophilic IgE and become potent anti-tumor effectors, able to trace viable tumor cells in the tissues. It is strongly suggested that the evolving new field AllergoOncology will give new insights into the role of IgE-mediated allergy in malignancies, possibly opening new avenues for tumor therapy.

Induction of murine mucosal CCR5-reactive antibodies as an anti-human immunodeficiency virus strategy.

The genital mucosa is the main site of initial human immunodeficiency virus type 1 (HIV-1) contact with its host. In spite of repeated sexual exposure, some individuals remain seronegative, and a small fraction of them produce immunoglobulin G (IgG) and IgA autoantibodies directed against CCR5, which is probably the cause of the CCR5-minus phenotype observed in the peripheral blood mononuclear cells of these subjects. These antibodies recognize the 89-to-102 extracellular loop of CCR5 in its native conformation. The aim of this study was to induce infection-preventing mucosal anti-CCR5 autoantibodies in individuals at high risk of HIV infection. Thus, we generated chimeric immunogens containing the relevant CCR5 peptide in the context of the capsid protein of Flock House virus, a presentation system in which it is possible to engineer conformationally constrained peptide in a highly immunogenic form. Administered in mice via the systemic or mucosal route, the immunogens elicited anti-CCR5 IgG and IgA (in sera and vaginal fluids). Analogous to exposed seronegative individuals, mice producing anti-CCR5 autoantibodies express significantly reduced levels of CCR5 on the surfaces of CD4+ cells from peripheral blood and vaginal washes. In vitro studies have shown that murine IgG and IgA (i) specifically bind human and mouse CD4+ lymphocytes and the CCR5-transfected U87 cell line, (ii) down-regulate CCR5 expression of CD4+ cells from both humans and untreated mice, (iii) inhibit Mip-1beta chemotaxis of CD4+ CCR5+ lymphocytes, and (iv) neutralize HIV R5 strains. These data suggest that immune strategies aimed at generating anti-CCR5 antibodies at the level of the genital mucosa might be feasible and represent a strategy to induce mucosal HIV-protective immunity.

IgEs targeted on tumor cells: therapeutic activity and potential in the design of tumor vaccines.

Surface-bound IgE play a central role in antiparasite immunity; to exploit IgE-driven immune mechanisms in tumor prevention and control, monoclonal IgEs of irrelevant specificity were loaded through biotin-avidin bridging onto tumor cells, either by systemic administration to tumor-bearing mice or pre-loading of tumor cells before inoculation. Here we show that systemic administration of biotinylated IgEs to mice bearing tumors pre-targeted with biotinylated antibodies and avidin significantly decreased tumor growth rate. In addition, as compared with IgG-loaded control cells, inoculation of suboptimal doses of IgE-loaded tumor cells suppressed tumor formation in a fraction of animals and induced protective host immunity by eliciting tumor-specific T-cell responses. Similarly, tumor vaccination experiments showed that irradiated tumor cells (IgE loaded by biotin-avidin bridging) conferred protective immunity at doses 100-fold lower than the corresponding control cells without IgE. Finally, in vivo depletion of eosinophils or T cells abrogated IgE-driven tumor growth inhibition. These results demonstrate that IgEs targeted on tumor cells not only possess a curative potential but also confer long-term antitumor immunity and that IgE-driven antitumor activity is not restricted to the activation of innate immunity effector mechanisms but also results from eosinophil-dependent priming of a T-cell-mediated adaptive immune response. This suggests a potential role for IgEs in the design of new cell-based tumor vaccines.

Adoptive immunotherapy by avidin-driven cytotoxic T lymphocyte-tumor bridging.

We have shown previously that T cells, tagged with biotinylated anti-CD3 antibody fragments, can exert avidin-dependent cytolytic activity on suitably biotinylated tumor cells in vitro. In this study, we demonstrate that avidin-driven CTL-tumor bridging in vivo leads to growth inhibition of murine tumors WEHI-164 fibrosarcoma and RMA lymphoma. The biodistribution of biotin-tagged 111In-labeled T cells demonstrated a selective avidin-dependent and time-dependent accumulation of radioactivity at tumor sites. The specificity of lymphocyte tumor localization was demonstrated by the concurrent time-dependent decrease of radioactivity in the blood and in all other organs. Furthermore, we documented a therapeutic effect of the adoptively transferred T cells, i.e., a significant delay of tumor growth at early stages. All of the experiments included a control group of mice, which received all of the reagents, except avidin. These avidin-minus mice showed no specific localization and no delay in tumor growth, indicating that avidin bridging was essential for T-cell activity at tumor sites.

HIV neutralizing IgA in exposed seronegative subjects recognise an epitope within the gp41 coiled-coil pocket.

Human immunodeficiency virus (HIV)-specific IgA can be detected in cervical secretions, saliva, and sera of HIV-infected and HIV-uninfected individuals with a known exposure to the virus. IgA from HIV-uninfected exposed seronegative individuals (ESN) neutralize in vitro primary strains of HIV-1. We analyzed the epitopes of HIV recognized by serum HIV-specific IgA of ESN individuals to identify the antigenic correlates of HIV neutralization in exposed-uninfected subjects, and to verify whether different epitopes would be recognized by HIV-specific IgA of ESN and of HIV-infected patients. Results confirmed that HIV-neutralizing IgA are detected in sera of ESN and showed that neutralization of primary HIV strains is mediated by the recognition of different epitopes in HIV-infected patients and ESN. Thus, whereas IgA of HIV+ individuals recognize epitopes expressed both within gp120 and gp41, IgA of ESN exclusively bind to gp41-expressed epitopes. Epitope mapping revealed that the epitope recognized by serum IgA of ESN on gp41 is restricted to aa 581-584 (LQAR) and corresponds to coiled coil pocket in the alpha helic region. In contrast, the epitope seen by IgA of HIV-infected patients on gp41 is identified by two regions; the first is contained within the cystein loop (aa 589-618), the second correspond to C terminal region in the extra membrane region of gp 41 (aa 642-673). Thus, we have identified and characterized the epitopes that mediate neutralization of HIV in individuals in whom infection does not occur despite multiple exposures to the virus. These results have important implications for the development of a new therapy against HIV infection.

Early production of HIV-1 neutralising antibodies in patients following highly active antiretroviral treatment (HAART) during primary HIV infection.

We investigate the effects of highly active antiretroviral therapy (HAART) on humoral immune responses during a 24-month follow up of 15 HIV patients with acute primary HIV infection. The patients were divided into three groups on the basis of the therapeutic protocol they were following at the time of entry: a) five naive patients (untreated or treated with only ZDV or AZT); b) five patients following a triple combination of ZDV+ lamivudine (3TC)+ saquinovir (SQV); and c) five patients on a four-drug combination of ZDV+3TC+SQV+ ritonavir (RTV). The results show that the early introduction of HAART greatly reduces plasma viremia levels and restores the number of CD4 cells. A significant correlation was found between anti HIV neutralising activity and the four-drug, but not the three-drug combination. The reduction in infectivity was directed against viruses of different clades and associated with immunoglobulin fractions. Moreover, the neutralising antibodies in the HAART-treated patients appeared after two weeks of treatment and remained stable throughout the 24 months of follow up. The early appearance of neutralising antibodies represent an important component of immune responses during primary HIV infection, may contribute towards immune reconstitution in patients on HAART, and give further information that may be useful in developing new strategies designed to eradicate the disease.

CCR5-reactive antibodies in seronegative partners of HIV-seropositive individuals down-modulate surface CCR5 in vivo and neutralize the infectivity of R5 strains of HIV-1 In vitro.

Exposure to HIV does not necessarily result in infection. Because primary HIV infection is associated with CCR5-tropic HIV variants (R5), CCR5-specific Abs in the sera of HIV-seronegative, HIV-exposed individuals (ESN) might be associated with protection against infection. We analyzed sera from ESN, their HIV-infected sexual partners (HIV+), and healthy controls (USN) searching for CCR5-specific Abs, studying whether incubation of PBMC with sera could prevent macrophage inflammatory protein 1 beta (Mip1 beta) (natural ligand of CCR5) binding to CCR5. Results showed that Mip1 beta binding to CCR5 was not modified by sera of either 40 HIV+ or 45 USN but was greatly reduced by sera of 6/48 ESN. Binding inhibition was due to Abs reactive with CCR5. The CCR5-specific Abs neutralized the infectivity of primary HIV isolates obtained from the corresponding HIV+ partners and of R5-primary HIV strains, but not that of CXCR4-tropic or amphitropic HIV strains. Immunoadsorption on CCR5-transfected, but not on CXCR4-transfected, cells removed CCR5-specific and virus-neutralizing Abs. Epitope mapping on purified CCR5-specific Abs showed that these Abs recognize a conformational epitope in the first cysteine loop of CCR5 (aa 89-102). Affinity-purified anti-CCR5-peptide neutralized the infectivity of R5 strains of HIV-1. Anti-CCR5 Abs inhibited Mip1beta-induced chemotaxis of PBMC from healthy donors. PBMC from two ESN (with anti-CCR5 Abs) were CCR5-negative and could not be stimulated by Mip1beta in chemotaxis assays. These results contribute to clarifying the phenomenon of immunologic resistance to HIV and may have implications for the development of a protective vaccine.

Anti-cell antibodies in exposed seronegative individuals with HIV type 1-neutralizing activity.

Despite repeated exposures to HIV-1, some individuals remain seronegative. This study reports that sera from a fraction of exposed seronegative (ESN) subjects showed HIV-neutralizing activity; 5 of 17 ESN sera and none of 17 controls neutralized two different HIV-1 primary isolates (range of neutralizing titers: 1/20 to 1/60). The neutralizing activity was associated with the IgG fraction of 4 of 4 neutralizing ESN sera. Moreover, in 11 of 17 and 9 of 17 ESN sera (but none of the control sera) we found antibodies against HLA class I and CD4, respectively. One of the ESN sera (EU22) neutralized efficiently the primary virus derived from the seropositive partner and showed a good broadly cross-reactive neutralization. Immunoadsorption of two IgG fractions from EU19 and EU22 on peripheral blood mononuclear cells (PBMC) removed virus-neutralizing antibodies. The correlations between the ESN status and neutralizing activity (p<0.05), anti-HLA antibodies (p<0.0002), and anti-CD4 antibodies (p<0.001) were statistically significant. However, there was no statistically significant correlation between neutralizing activity and either anti-HLA or anti-CD4 antibodies. It can therefore be said that exposure to HIV-1 without seroconversion is, in some individuals, associated with HIV-neutralizing antibodies (not directed against viral antigens) and/or with anti-cell autoantibodies, which are possibly specific for cellular antigens involved in the infection/entry process.

Enhanced HIV infectivity and changes in GP120 conformation associated with viral incorporation of human leucocyte antigen class I molecules.

BACKGROUND: Assembly of human immunodeficiency virus type 1 (HIV-1) occurs at the level of the plasma membrane of the host cell. During this process HIV incorporates significant quantities of cell surface-derived molecules into its lipid bilayer including human leucocyte antigen (HLA) class I and II, intercellular adhesion molecule-1 and lymphocyte function antigen-1. Several studies indicate that virion-bound host-cell-derived molecules are functional and affect the biological properties of HIV-1. Virion-associated HLA class II and intercellular adhesion molecule-1 enhance the infectivity of T-cell line-adapted (TCLA) viruses. No role for virion-associated HLA class I molecules has yet been identified. OBJECTIVE: To investigate the role of HLA class I molecules in HIV replication and infectivity. METHODS: HLA class I negative human cells lines transfected with the HLA Cw4 gene were infected with different TCLA viruses as well as primary X4 isolates. The infectivity of HLA Cw4 positive and negative viruses was determined on indicator cell lines and on phytohaemagglutinin-activated peripheral blood mononuclear cells. An entry polymerase chain reaction assay was used to determine differences in entry-competence of Cw4 positive and negative viruses. The expression of selected gp120 epitopes on native Env molecules derived from Cw4 positive and negative viruses was determined by a monoclonal antibody-based enzyme-linked immunosorbent assay. Immunoprecipitation experiments were performed to investigate the presence of gp120/HLA Cw4 complexes. Neutralization assays determined the differences in susceptibility to neutralization between HLA Cw4 negative and positive viruses. RESULTS AND CONCLUSIONS: The infectivity of primary HIV-1 X4 isolates and of TCLA viruses is increased upon viral incorporation of HLA Cw4 molecules. This effect is associated with changes in viral envelope proteins conformation including an enhanced expression of the V3 loop of gp120, and of epitopes that are exposed upon CD4 binding. The gp120 conformational changes are consistent with the formation of a multimolecular complex between HLA class I and gp120/160. HLA Cw4 incorporation is also associated to a lower susceptibility to antibody neutralization. These findings have important implications for understanding the immune response to cryptic and conformational epitopes of the viral envelope.

Anti-CD4 antibodies in exposed seronegative adults and in newborns of HIV type 1-seropositive mothers: a follow-up study.

In this work, an ELISA for the quantitative determination of IgG anti-CD4 autoantibodies was validated and utilized in the follow-up of two cohorts of HIV-1-exposed seronegative subjects. A serum with an arbitrarily assigned concentration of 100,000 units/ml was used as a reference, and the detection limit, inter- and intraassay variability, and analytical recovery were calculated. The study subjects included adults sexually exposed to HIV-1-infected partners and the newborns of HIV-1+ mothers who seroreverted by 18 months of age. Some of these individuals were studied over an 18- to 24-month period. The detection limit of the assay was 2000 AU/ml. Intra- and interassay variability was, respectively, 3.92 and 3.90%. Analytical recovery in an assay in which a fixed amount of anti-CD4 antibodies was added to different samples was 98%. A proportion of adults (16 of 47, 34.0%) and babies (12 of 27, 44.4%) had significantly higher concentrations of anti-CD4 antibodies. Among them, 8 adults maintained the same concentration as that found in the first determination; on the other hand, 12 babies born to seronegative mothers showed a significant increase in the concentration of anti-CD4 antibodies during their first months of life. In conclusion, anti-CD4 antibodies can be measured using a validated ELISA. They represent a serologic trait that is quantitatively conserved in HIV-1-exposed seronegative adult individuals and is actively acquired by newborns to HIV+ mothers.

Role of CD4 and CCR5 levels in the susceptibility of primary macrophages to infection by CCR5-dependent HIV type 1 isolates.

Macrophages are a preferred target for sexually transmitted human immunodeficiency virus type 1 (HIV-1) isolates that use CCR5 as a coreceptor in combination with CD4. To assess whether the susceptibility of MDMs to infection by an R5 isolate was influenced by CD4 and/or CCR5 expression, levels of membrane CD4 or CCR5 transcripts at the time of infection and ID50 values 15 days postinfection were measured in cultures of primary macrophages infected with HIV-1(10005). To analyze the data, subjects were divided so as to maximize differences in the levels of CD4 or CCR5 expression between groups. Indeed, the difference in CD4 expression between the CD4high (MFI, 16.7 +/- 2.2) and CD4low (MFI, 6.7 +/- 0.7) groups attained high significance (p < 0.005). Of note, susceptibility to infection of MDMs isolated from CD4high donors was strikingly enhanced as compared with CD4low subjects, as shown by a fourfold increase in ID50 titers at day 15 postinfection (p < 0.002). In contrast, no significant difference in ID50 was apparent when the subjects were grouped according to the levels of CCR5 transcripts, even though CCR5 expression in the two groups differed significantly (p = 0.01). These results suggest that, regardless of variations among individuals, the intensity of CD4 expression in macrophages is such that CCR5 levels are above the threshold required for efficient HIV-1 infection. Consistent with this hypothesis, macrophages from three additional donors selected for high CD4 expression and low CCR5 transcripts were found to be highly susceptible to HIV-1 infection.

Human immunodeficiency virus (HIV)-specific IgA and HIV neutralizing activity in the serum of exposed seronegative partners of HIV-seropositive persons.

The presence and activity of human immunodeficiency virus (HIV)-specific antibodies were analyzed in the sera of 15 sexually exposed seronegative persons who had systemic HIV-specific cell-mediated immunity and IgA-mediated mucosal immunity and in their HIV-infected partners. The HIV-positive subjects had HIV-specific serum IgG and IgA; the seronegative persons had HIV-specific serum IgA in the absence of IgG. Testing of the seronegative persons 1 year after the interruption of at-risk sex showed that no IgG seroconversion had occurred and that HIV-specific IgA serum concentrations had declined. Serum from the HIV-exposed seronegative persons was analyzed for the ability to neutralize primary HIV-1 isolates. Neutralizing activity was detected in 5 of 15 sera and in 2 cases was retained by serum-purified IgA. Thus, the immunologic picture for resistance to HIV infection should include HIV-specific cell-mediated immunity as well as HIV-specific IgA-mediated mucosal and systemic immunity.

Tumor pretargeting with avidin improves the therapeutic index of biotinylated tumor necrosis factor alpha in mouse models.

The clinical use of tumor necrosis factor alpha (TNF) as an anticancer drug is limited to local or locoregional administration because of dose-limiting systemic toxicity. We investigated in animal models whether the therapeutic index of systemically administered human or murine TNF can be increased by tumor pretargeting strategies based on the biotin-avidin system. Pretargeting of s.c. mouse WEHI-164 fibrosarcoma and RMA lymphoma genetically engineered to express the Thy 1.1 antigen on the cell membrane was achieved by i.p. injection of a biotinylated anti-Thy 1.1 antibody and avidin. This pretreatment increased the antitumor activity of systemically administered biotin-TNF conjugates by at least 5-fold. In contrast, pretargeting did not increase the toxicity of biotin-TNF, as judged by animal survival and weight loss after treatment. Ex vivo analysis of tumor cells 24 h after treatment showed that biotin-TNF persisted for several hours on the surface of pretargeted tumors, but not when avidin was omitted. The potentiation of the antitumor effects was related primarily to indirect mechanisms, involving a host-mediated response. The results indicate that tumor pretargeting improves the antitumor activity of TNF. Tumor pretargeting with avidin, which is currently used to increase the uptake of radioactive-labeled biotin in patients, could represent a new strategy for improving the therapeutic index of TNF.

Antibody-guided three-step therapy for high grade glioma with yttrium-90 biotin.

While the incidence of brain tumours seems to be increasing, median survival in patients with glioblastoma remains less than 1 year, despite improved diagnostic imaging and neurosurgical techniques, and innovations in treatment. We have developed an avidin-biotin pre-targeting approach for delivering therapeutic radionuclides to gliomas, using anti-tenascin monoclonal antibodies, which seems potentially effective for treating these tumours. We treated 48 eligible patients with histologically confirmed grade III or IV glioma and documented residual disease or recurrence after conventional treatment. Three-step radionuclide therapy was performed by intravenous administration of 35 mg/m2 of biotinylated anti-tenascin monoclonal antibody (1st step), followed 36 h later by 30 mg of avidin and 50 mg of streptavidin (2nd step), and 18-24 h later by 1-2 mg of yttrium-90-labelled biotin (3rd step). 90Y doses of 2.22-2.96 GBq/m2 were administered; maximum tolerated dose (MTD) was determined at 2.96 GBq/m2. Tumour mass reduction (>25%-100%), documented by computed tomography or magnetic resonance imaging, occurred in 12/48 patients (25%), with 8/48 having a duration of response of at least 12 months. At present, 12 patients are still in remission, comprising four with a complete response, two with a parital response, two with a minor response and four with stable disease. Median survival from 90Y treatment is 11 months for grade IV glioblastoma and 19 months for grade III anaplastic gliomas. Avidin-biotin based three-step radionuclide therapy is well tolerated at the dose of 2.2 GBq/m2, allowing the injection of 90Y-biotin without bone marrow transplantation. This new approach interferes with the progression of high-grade glioma and may produce tumour regression in patients no longer responsive to other therapies.

Biochemical characterization and crystal structure of a recombinant hen avidin and its acidic mutant expressed in Escherichia coli.

The mature hen avidin encoded by a synthetic cDNA was expressed in Escherichia coli in an insoluble form. After resolubilization, renaturation and purification, a recovery of about 20 mg/l cell culture was obtained. ELISA assays indicated no apparent differences in biotin binding between the natural and recombinant avidins. In addition, an acidic avidin mutant, bearing the substitutions Lys3-->Glu, Lys9--> Glu, Arg26-->Asp and Arg124-->Leu of four exposed basic residues, was produced. The protein, expressed and renatured as wild-type avidin, showed unaltered biotin-binding activity. The acidic pI (approximately 5.5) and lack of aggregation of the mutant allowed easy electrophoretic analysis under non-denaturing conditions of the protein alone and of its complexes with biotin, biotinylated transferrin or peroxidase. Analysis of the sera from sensitized subjects revealed that the avidin mutant has altered antigenicity. Both recombinant avidins were crystallized and the three-dimensional structures solved by molecular replacement and refined to 0.22 nm resolution. The three-dimensional structures of the two recombinant molecules, in the absence of biotin and of glycosylation, are fully comparable with those of the natural hen avidin previously reported.

CXCR4 is a functional coreceptor for infection of human macrophages by CXCR4-dependent primary HIV-1 isolates.

The identification of HIV-1 coreceptors has provided a molecular basis for the tropism of different HIV-1 strains. CXC chemokine receptor-4 (CXCR4) mediates the entry of both primary and T cell line-adapted (TCLA) syncytia-inducing strains. Although macrophages (M phi) express CXCR4, this coreceptor is assumed to be nonfunctional for HIV-1 infection. We addressed this apparent paradox by infecting human monocyte-derived M phi with primary and TCLA isolates that were rigorously characterized for coreceptor usage and by adding the natural CXCR4 ligand, stem cell differentiation factor-1, to specifically block CXCR4-mediated entry. Our results show that primary HIV-1 isolates that selectively use CXCR4 productively infected both normal and C-C chemokine receptor-5-null M phi. By contrast, M phi supported the entry of CXCR4-dependent TCLA strains with variable efficiency but were not productively infected. Thus, the tropism of HIV isolates results from complex virus/host cell interactions both at the entry and postentry levels.

Tumor targeting with biotinylated tumor necrosis factor alpha: structure-activity relationships and mechanism of action on avidin pretargeted tumor cells.

We have recently described a new strategy for targeting biotinylated tumor necrosis factor-alpha (TNF-alpha) to tumors, based on pretargeting with biotinylated antibodies and avidin. Here, we have analyzed the structure-activity relationships of several biotin-TNF-alpha conjugates and studied the mechanism of their interaction with avidin and TNF-alpha receptors on tumor cells. The study has been carried out using an in vitro model based on human melanoma Colo 38 cells and monoclonal antibody 225, an antibody against the high molecular weight melanoma-associated antigen. Immunochemical and cytotoxicity studies showed that biotin-TNF-alpha but not TNF-alpha persists for several hours on the surface of cells pretargeted with biotin-monoclonal antibody 225 and avidin and triggers cytolytic effects. Studies on the mechanism of action showed that biotin-TNF-alpha trimers can slowly dissociate from targeted cells in a bioactive form, through trimer-monomer-trimer transitions. Structure-activity relationship studies showed that nonbiotinylated subunits must be present in the biotin-TNF-alpha trimers for efficient release of bioactive TNF-alpha. Colo 38 cells targeted with biotin-TNF-alpha were able to kill mouse L-M cells in coculture experiments, indicating that the released TNF-alpha can interact also with TNF-alpha receptors expressed by bystander cells. In conclusion, the targeting complex works as a system that slowly releases bioactive TNF-alpha in the microenvironment of the targeted cell. This opens up the possibility that cells other than those reached by the targeting antibody (e.g., endothelial cells and local cells of the immune system) can be affected in vivo.

In vitro selection of HIV-1 TAR variants by the Tat protein.

Starting from a pool of 10(13) RNA sequences, we isolated a number of TAR RNA variants after nine rounds of selection by binding to recombinant Tat in vitro (SELEX procedure). Sequence analysis of part of the selected molecular species indicated that two TAR variants (clones A and B) were, respectively, represented five and four times. These two groups of sequences constituted approximately 25% of the total number of analyzed clones (9/34). As far as the primary and presumptive secondary structures of the wild-type TAR are concerned, the selected A and B variants showed an almost complete sequence conservation of the Tat-binding domain, but the configuration of this nucleotide region differed within the secondary structure. Despite this difference, as verified by gel retardation and filter binding assays, both the A and B variants bound Tat in vitro with an affinity that was very close to that of the wild-type TAR. Conversely, neither variant sustained Tat-mediated trans-activation in vivo when they replaced the wild-type TAR inside the long terminal repeat of HIV_1. Taken together, our results suggest that these TAR variants have lost the ability to bind cell factor(s) in vivo and may therefore represent useful decoys for the inhibition of HIV-1 replication.

Biochemical modifications of avidin improve pharmacokinetics and biodistribution, and reduce immunogenicity.

Pretargeting techniques using the avidin-biotin system have shown encouraging results in both diagnostic and therapeutic clinical trials. It has been shown that in cancer therapy the ideal agent to be used for pretargeting should have a plasma half-life longer than avidin and lower immunogenicity than streptavidin in order for these procedures to be applied safely and repeatedly in patients. We prepared a recombinant form of avidin with no carbohydrates and avidins, biochemically modified either by decreasing the positive charges with succinic anhydride or by linking polyethylene glycol (PEG) at three different molar ratios and evaluated their in vivo behaviour after i.p. administration in mice. The succinylation and PEGylation of avidin increased the plasma half-life proportionally to the degree of protein modification. The procedures, however, affected the biotin binding to some extent. The biodistribution studies showed that, for all six time points (ranging from 20 min to 18 h post-injection), the liver and kidney to blood ratios were lower for PEGylated avidins than native, recombinant and succinyl avidin. Recombinant and low PEGylated avidin evoked an immune response in all mice after at least three injections. Native, recombinant and succinyl avidins showed higher serum titres than PEGylated avidins. In conclusion, the conjugation of avidin to PEG chains (n = 7) originates a compound with a suitable blood clearance, low immunogenicity and concurrent low cross-reactivity with avidin.