Elarekibep (PRS-060/AZD1402), a new class of inhaled Anticalin medicine targeting IL-4Ra for type 2 endotype asthma.

BACKGROUND: Type 2 endotype asthma is driven by IL-4 and IL-13 signaling via IL-4Ra, which is highly expressed on airway epithelium, airway smooth muscle, and immunocytes in the respiratory mucosa, suggesting potential advantages of an inhalable antagonist. Lipocalin 1 (Lcn1), a 16 kDa protein abundant in human periciliary fluid, has a robust drug-like structure well suited to protein engineering, but it has never been used to make an inhaled Anticalin protein therapeutic. OBJECTIVES: We sought to reengineer Lcn1 into an inhalable IL-4Ra antagonist and assess its pharmacodynamic/kinetic profile. METHODS: Lcn1 was systematically modified by directed protein mutagenesis yielding a high-affinity, slowly dissociating, long-acting full antagonist of IL-4Ra designated PRS-060 with properties analogous to dupilumab, competitively antagonizing IL-4Ra-dependent cell proliferation, mucus induction, and eotaxin expression in vitro. Because PRS-060 displayed exquisite specificity for human IL-4Ra, with no cross-reactivity to rodents or higher primates, we created a new triple-humanized mouse model substituting human IL-4Ra, IL-4, and IL-13 at their correct syntenic murine loci to model clinical dosing. RESULTS: Inhaled PRS-060 strongly suppressed acute allergic inflammation indexes in triple-humanized mice with a duration of action longer than its bulk clearance, suggesting that it may act locally in the lung. CONCLUSION: Lcn1 can be reengineered into the Anticalin antagonist PRS-060 (elarekibep), exemplifying a new class of inhaled topical, long-acting therapeutic drugs with the potential to treat type 2 endotype asthma.

A First-in-Human Study of cinrebafusp alfa, a HER2/4-1BB Bispecific Molecule, in Patients with HER2-Positive Advanced Solid Malignancies.

PURPOSE: 4-1BB (CD137) is a costimulatory immune receptor expressed on activated T cells, activated B cells, natural killer cells and tumor-infiltrating lymphocytes, making it a promising target for cancer immunotherapy. Cinrebafusp alfa, a monoclonal antibody-like bispecific protein targeting HER2 and 4-1BB, aims to localize 4-1BB activation to HER2-positive tumors. This study evaluated the safety, tolerability, and preliminary efficacy of cinrebafusp alfa in patients with previously treated HER2-positive malignancies. EXPERIMENTAL DESIGN: This was a multi-center dose escalation study involving patients with HER2-positive malignancies who had received prior treatment. The study assessed the safety and efficacy of cinrebafusp alfa across various dose levels. Patients were assigned to different cohorts, and antitumor responses were evaluated. The study aimed to determine the maximum tolerated dose (MTD) and to observe any clinical activity at different dose levels. RESULTS: Out of 40 evaluable patients in the 'active dose' efficacy cohorts, 5 showed an antitumor response, resulting in an overall response rate (ORR) of 12.5% and a disease control rate of 52.5%. Clinical activity was observed at the 8 mg/kg and 18 mg/kg dose levels, with confirmed objective response rates of 28.6% and 25.0%, respectively. Cinrebafusp alfa was safe and tolerable, with Grade ≤2 infusion-related reactions being the most frequent treatment-related adverse event. MTD was not reached during the study. CONCLUSION: Cinrebafusp alfa demonstrates promising activity in patients with HER2-positive malignancies who have progressed on prior HER2-targeting regimens. Its acceptable safety profile suggests it could be a treatment option for patients not responding to existing HER2-directed therapies.

The PD-L1/4-1BB Bispecific Antibody-Anticalin Fusion Protein PRS-344/S095012 Elicits Strong T-Cell Stimulation in a Tumor-Localized Manner.

PURPOSE: While patients responding to checkpoint blockade often achieve remarkable clinical responses, there is still significant unmet need due to resistant or refractory tumors. A combination of checkpoint blockade with further T-cell stimulation mediated by 4-1BB agonism may increase response rates and durability of response. A bispecific molecule that blocks the programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis and localizes 4-1BB costimulation to a PD-L1-positive (PD-L1+) tumor microenvironment (TME) or tumor draining lymph nodes could maximize antitumor immunity and increase the therapeutic window beyond what has been reported for anti-4-1BB mAbs. EXPERIMENTAL DESIGN: We generated and characterized the PD-L1/4-1BB bispecific molecule PRS-344/S095012 for target binding and functional activity in multiple relevant in vitro assays. Transgenic mice expressing human 4-1BB were transplanted with human PD-L1-expressing murine MC38 cells to assess in vivo antitumoral activity. RESULTS: PRS-344/S095012 bound to its targets with high affinity and efficiently blocked the PD-1/PD-L1 pathway, and PRS-344/S095012-mediated 4-1BB costimulation was strictly PD-L1 dependent. We demonstrated a synergistic effect of both pathways on T-cell stimulation with the bispecific PRS-344/S095012 being more potent than the combination of mAbs. PRS-344/S095012 augmented CD4-positive (CD4+) and CD8-positive (CD8+) T-cell effector functions and enhanced antigen-specific T-cell stimulation. Finally, PRS-344/S095012 demonstrated strong antitumoral efficacy in an anti-PD-L1-resistant mouse model in which soluble 4-1BB was detected as an early marker for 4-1BB agonist activity. CONCLUSIONS: The PD-L1/4-1BB bispecific PRS-344/S095012 efficiently combines checkpoint blockade with a tumor-localized 4-1BB-mediated stimulation burst to antigen-specific T cells, more potent than the combination of mAbs, supporting the advancement of PRS-344/S095012 toward clinical development. See related commentary by Shu et al., p. 3182.

Antibody targeting of Cathepsin S induces antibody-dependent cellular cytotoxicity.

BACKGROUND: Proteolytic enzymes have been implicated in driving tumor progression by means of their cancer cell microenvironment activity where they promote proliferation, differentiation, apoptosis, migration, and invasion. Therapeutic strategies have focused on attenuating their activity using small molecule inhibitors, but the association of proteases with the cell surface during cancer progression opens up the possibility of targeting these using antibody dependent cellular cytotoxicity (ADCC). Cathepsin S is a lysosomal cysteine protease that promotes the growth and invasion of tumour and endothelial cells during cancer progression. Our analysis of colorectal cancer patient biopsies shows that cathepsin S associates with the cell membrane indicating a potential for ADCC targeting. RESULTS: Here we report the cell surface characterization of cathepsin S and the development of a humanized antibody (Fsn0503h) with immune effector function and a stable in vivo half-life of 274 hours. Cathepsin S is expressed on the surface of tumor cells representative of colorectal and pancreatic cancer (23%-79% positive expression). Furthermore the binding of Fsn0503h to surface associated cathepsin S results in natural killer (NK) cell targeted tumor killing. In a colorectal cancer model Fsn0503h elicits a 22% cytotoxic effect. CONCLUSIONS: This data highlights the potential to target cell surface associated enzymes, such as cathepsin S, as therapeutic targets using antibodies capable of elicitingADCC in tumor cells.

Antibody targeting of camptothecin-loaded PLGA nanoparticles to tumor cells.

Antibody targeting of drug substances can improve the efficacy of the active molecule, improving distribution and concentration of the drug at the site of injury/disease. Encapsulation of drug substances into polymeric nanoparticles can also improve the therapeutic effects of such compounds by protecting the molecule until its action is required. In this current study, we have brought together these two rationales to develop a novel immuno-nanoparticle with improved therapeutic effect against colorectal tumor cells. This nanoparticle comprised a layer of peripheral antibodies (Ab) directed toward the Fas receptor (CD95/Apo-1) covalently attached to poly(lactide-co-glycolide) nanoparticles (NP) loaded with camptothecin. Variations in surface carboxyl density permitted up to 48.5 microg coupled Ab per mg of NP and analysis of nanoparticulate cores showed efficient camptothecin loading. Fluorescence visualization studies confirmed internalization of nanoconstructs into endocytic compartments of HCT116 cells, an effect not evident in NP without superficial Ab. Cytotoxicity studies were then carried out against HCT116 cells. After 72 h, camptothecin solution resulted in an IC 50 of 21.8 ng mL (-1). Ab-directed delivery of NP-encapsulated camptothecin was shown to be considerably more effective with an IC 50 of 0.37 ng mL (-1). Calculation of synergistic ratios for these nanoconstructs demonstrated synergy of pharmacological relevance. Indeed, the results in this paper suggest that the attachment of anti-Fas antibodies to camptothecin-loaded nanoparticles may result in a therapeutic strategy that could have potential in the treatment of tumors expressing death receptors.

Sustained plasma hepcidin suppression and iron elevation by Anticalin-derived hepcidin antagonist in cynomolgus monkey.

BACKGROUND AND PURPOSE: Anaemia of chronic disease (ACD) has been linked to iron-restricted erythropoiesis imposed by high circulating levels of hepcidin, a 25 amino acid hepatocyte-derived peptide that controls systemic iron homeostasis. Here, we report the engineering of the human lipocalin-derived, small protein-based anticalin PRS-080 hepcidin antagonist with high affinity and selectivity. EXPERIMENTAL APPROACH: Anticalin- and hepcidin-specific pharmacokinetic (PK)/pharmacodynamic modelling (PD) was used to design and select the suitable drug candidate based on t1/2 extension and duration of hepcidin suppression. The development of a novel free hepcidin assay enabled accurate analysis of bioactive hepcidin suppression and elucidation of the observed plasma iron levels after PRS-080-PEG30 administration in vivo. KEY RESULTS: PRS-080 had a hepcidin-binding affinity of 0.07 nM and, after coupling to 30 kD PEG (PRS-080-PEG30), a t1/2 of 43 h in cynomolgus monkeys. Dose-dependent iron mobilization and hepcidin suppression were observed after a single i.v. dose of PRS-080-PEG30 in cynomolgus monkeys. Importantly, in these animals, suppression of free hepcidin and subsequent plasma iron elevation were sustained during repeated s.c. dosing. After repeated dosing and followed by a treatment-free interval, all iron parameters returned to pre-dose values. CONCLUSIONS AND IMPLICATIONS: In conclusion, we developed a dose-dependent and safe approach for the direct suppression of hepcidin, resulting in prolonged iron mobilization to alleviate iron-restricted erythropoiesis that can address the root cause of ACD. PRS-080-PEG30 is currently in early clinical development.

New Cancer Immunotherapy Agents in Development: a report from an associated program of the 31stAnnual Meeting of the Society for Immunotherapy of Cancer, 2016.

This report is a summary of 'New Cancer Immunotherapy Agents in Development' program, which took place in association with the 31st Annual Meeting of the Society for Immunotherapy of Cancer (SITC), on November 9, 2016 in National Harbor, Maryland. Presenters gave brief overviews of emerging clinical and pre-clinical immune-based agents and combinations, before participating in an extended panel discussion with multidisciplinary leaders, including members of the FDA, leading academic institutions and industrial drug developers, to consider topics relevant to the future of cancer immunotherapy.

Annexin II cell surface and mRNA expression in human acute myeloid leukaemia cell lines.

INTRODUCTION: Acute promyelocytic leukaemia (APL) (M3) is associated with both a characteristic t(15;17) and severe bleeding diathesis caused by disseminated intravascular coagulation (DIC) and/or hyperfibrinolysis. It has been suggested that annexin II, a coreceptor for tissue plasminogen activator (t-PA) and plasminogen (PLG), is overexpressed on the surface of promyelocytes, leading to an increased fibrinolytic potential. MATERIALS AND METHODS: This study examined the level of annexin II cell surface and mRNA expression in a range of acute myeloid leukaemia (AML) cell lines. The evidence that annexin II levels are higher in APL would lend support to the hypothesis that the bleeding disorder seen in APL is caused by hyperfibrinolysis. RESULTS: Cell surface annexin II was found to be expressed at higher levels on NB4 (promyelocytic) cells than on either KG1a (early myeloid) or HL60 (myelocytic) cells. However, even higher levels were found on U937 and MM6 (histo-monocytic) and HEL (erythroid) cells (p<0.01). MM6 cells showed a threefold increase in annexin II mRNA compared to any of the other cell lines. CONCLUSIONS: These findings do not fully support the concept of the coagulopathy associated with APL being caused by hyperfibrinolysis alone. Further investigations are required to identify the significance of annexin II expression and regulation in leukaemia.

In vivo visualization of MET tumor expression and anticalin biodistribution with the MET-specific anticalin 89Zr-PRS-110 PET tracer.

UNLABELLED: Anticalins are a novel class of biopharmaceuticals, displaying highly desirable attributes as imaging agents. The anticalin PRS-110 was rationally engineered to target the oncogene MET with high affinity and specificity. The aim of this study was to visualize MET expression and analyze biodistribution of (89)Zr-labeled PRS-110 in human tumor-bearing mice. METHODS: (89)Zr-PRS-110 was generated. For biodistribution studies (96 h after injection of tracer) 10 µg of (89)Zr-PRS-110 (with 0-490 µg of unlabeled PRS-110) were injected into BALB/c mice bearing high MET-expressing H441 non-small cell lung cancer xenografts. Further characterization with PET imaging was performed at 6, 24, 48, and 96 h after injection of 50 µg of (89)Zr-PRS-110 into mice bearing H441, primary glioblastoma U87-MG (intermediate MET), or ovarian cancer A2780 (low MET) xenografts. Drug distribution was also analyzed ex vivo using fluorescently labeled PRS-110. RESULTS: Biodistribution analyses showed a dose-dependent tumor uptake of (89)Zr-PRS-110, with the highest fractional tumor uptake at 10 µg of (89)Zr-PRS-110, with no unlabeled PRS-110. Small-animal PET imaging supported by biodistribution data revealed specific tumor uptake of (89)Zr-PRS-110 in the MET-expressing H441 and U87-MG tumors whereas the MET-negative A2780 tumor model showed a lower uptake similar to a non-MET binder anticalin control. Tumor uptake increased up to 24 h after tracer injection and remained high, whereas uptake in other organs decreased over time. Ex vivo fluorescence revealed intracellular presence of PRS-110. CONCLUSION: (89)Zr-PRS-110 specifically accumulates in MET-expressing tumors in a receptor density-dependent manner. PET imaging provides real-time noninvasive information about PRS-110 distribution and tumor accumulation in preclinical models.

A highly potent and specific MET therapeutic protein antagonist with both ligand-dependent and ligand-independent activity.

Activation of the MET oncogenic pathway has been implicated in the development of aggressive cancers that are difficult to treat with current chemotherapies. This has led to an increased interest in developing novel therapies that target the MET pathway. However, most existing drug modalities are confounded by their inability to specifically target and/or antagonize this pathway. Anticalins, a novel class of monovalent small biologics, are hypothesized to be "fit for purpose" for developing highly specific and potent antagonists of cancer pathways. Here, we describe a monovalent full MET antagonist, PRS-110, displaying efficacy in both ligand-dependent and ligand-independent cancer models. PRS-110 specifically binds to MET with high affinity and blocks hepatocyte growth factor (HGF) interaction. Phosphorylation assays show that PRS-110 efficiently inhibits HGF-mediated signaling of MET receptor and has no agonistic activity. Confocal microscopy shows that PRS-110 results in the trafficking of MET to late endosomal/lysosomal compartments in the absence of HGF. In vivo administration of PRS-110 resulted in significant, dose-dependent tumor growth inhibition in ligand-dependent (U87-MG) and ligand-independent (Caki-1) xenograft models. Analysis of MET protein levels on xenograft biopsy samples show a significant reduction in total MET following therapy with PRS-110 supporting its ligand-independent mechanism of action. Taken together, these data indicate that the MET inhibitor PRS-110 has potentially broad anticancer activity that warrants evaluation in patients.

Inhibition of Cathepsin S by Fsn0503 enhances the efficacy of chemotherapy in colorectal carcinomas.

Cathepsin S is a lysosomal cysteine protease implicated in tumourigenesis with key roles in invasion and angiogenesis. We have previously shown that the specific inhibition of Cathepsin S using a monoclonal antibody (Fsn0503) blocks colorectal carcinoma tumour growth and angiogenesis in vivo. We investigated whether Cathepsin S expression levels were affected by chemotherapy in human cancer cell lines by RT-PCR. Using colorectal xenograft models, we examined the therapeutic benefit of Cathepsin S inhibition using Fsn0503 in combination with a metronomic dosing regimen of CPT-11. We analysed the effects of the combination therapy on tumour progression and on tumour vascularisation by immunohistochemical staining of tumours. Cathepsin S expression levels are upregulated in HCT116, LoVo, Colo205 cell lines and HUVECs after exposure to CPT-11 in vitro. The administration of Fsn0503 in combination with CPT-11 significantly attenuated tumour growth in comparison to CPT-11 alone in colorectal HCT116 xenograft models. Furthermore, analysis of tumour vascularisation revealed that this was also significantly disrupted by the combination treatment. These results show that the combination of Cathepsin S inhibition with CPT-11 enhances the therapeutic effect of the chemotherapy. This rationale may have clinical application in the treatment of colorectal cancer upon further evaluation.

Application of the Enfer chemiluminescent multiplex ELISA system for the detection of Mycobacterium bovis infection in goats.

A study was conducted to optimise a multiplex serological immunoassay for use in identification of goats infected with Mycobacterium bovis. To assess assay specificity, 31 goats with a history of being free from M. bovis infection were used. To determine assay sensitivity, 180 Single Intradermal Comparative Tuberculin test (SICTT) positive goats were recruited. Additionally, 286 SICTT negative goats classed as potentially exposed animals present in the same positive herds were also included in the study. The results of the assay demonstrated a specificity of 100%. The multiplex assay detected 57/60 SICTT (95.0%) positive animals in one M. bovis infected herd and 120/120 (100%) in a second herd. In a separate experiment, 28 M. caprae culture confirmed infected goats from Spain were assayed, of which 24 (85.7%) were found positive in the test. The results show that inclusion of an antibody based assay can improve the ability to identify M. bovis and M. caprae infected goats. With further development and validation the multiplex assay may prove to be a useful tool for control of M. bovis and M. caprae infection in goats.

Stable expression and purification of a functional processed Fab' fragment from a single nascent polypeptide in CHO cells expressing the mCAT-1 retroviral receptor.

Monoclonal antibodies and derivative formats such as Fab' fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab' fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab' fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab' fragment against the Fas receptor, routinely expressed in 1-2mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab' fragments.

Expression and purification of diagnostically sensitive mycobacterial (Mycobacterium bovis) antigens and profiling of their humoral immune response in a rabbit model.

The incidence of bovine tuberculosis (bTB) is increasingly giving rise to large economic losses in the agricultural industry. The current methods used for detection and control of bTB (skin test and interferon-gamma) lack desired sensitivity and specificity. Therefore, the development of a rapid and reliable bTB serological based assay is urgently required. An antibody assay using combinations of strain-specific mycobacterial antigens could resolve both specificity and sensitivity issues. We analyzed the ability of a series of selected mycobacterial antigens to outline a humoral immune response in a rabbit model experimentally challenged with different mycobacterium. Antibodies specific for three antigens, MTB40, ESAT6 and CFP10, were present in serum 2 weeks post-challenge (early indicator), while two other antigens, Rv3870 and Rv1580c, could be detected from 8 to 11 weeks post-challenge. These selected mycobacterial antigens did not exhibit any cross-reactivity with avian PPD and only a very low positivity with bovine PPD. This data suggests that this panel of strain-specific mycobacterial antigens could be used for identification of Mycobacteriumbovis infection in serum samples. The combinatorial application of these antigens could form part of a serum field test which may assist the future diagnosis of TB.

Antibody targeting of cathepsin S inhibits angiogenesis and synergistically enhances anti-VEGF.

BACKGROUND: Angiogenesis is a key hallmark of tumourigenesis and its inhibition is a proven strategy for the development of novel anti-cancer therapeutics. An important aspect of early angiogenesis is the co-ordinated migration and invasion of endothelial cells through the hypoxic tumour tissue. Cathepsin S has been shown to play an important role in angiogenesis as has vascular endothelial growth factor (VEGF). We sought to assess the anti-angiogenic effect of Fsn0503, a novel cathepsin S inhibitory antibody, when combined with anti-VEGF on vascular development. METHODOLOGY/PRINCIPAL FINDINGS: Cathepsin S expression and secretion from endothelial cells was characterised using RT-PCR and western blotting. We further show that cathepsin S promotes pericellular hydrolysis of extracellular matrix components in the tumour microenvironment and facilitates endothelial invasion. The cathepsin S inhibitory antibody, Fsn0503, blocks extracellular proteolysis, inhibiting endothelial invasion and tube formation in cell-based assays. The anti-angiogenic effects of Fsn0503 were also shown in vivo where it significantly retarded the development of vasculature in human xenograft models. Furthermore, when Fsn0503 was combined with an anti-VEGF antibody, a synergistic inhibition of microvascular development was observed. CONCLUSIONS/SIGNIFICANCE: Taken together, this data demonstrates that the antibody-mediated targeting of cathepsin S represents a novel method of inhibiting angiogenesis. Furthermore, when used in combination with anti-VEGF therapies, Fsn0503 has the potential to significantly enhance current treatments of tumour neovascularisation and may also be of use in the treatment of other conditions associated with inappropriate angiogenesis.

A Novel High-Throughput Technique for Identifying Monoclonal Antibodies Capable of Death Receptor Induced Apoptosis.

The study of death receptor family induced apoptosis has gained momentum in recent years with the knowledge that therapeutic antibodies targeting DR4 and DR5 (death receptor's 4 and 5) have proved efficacious in multiple clinical trials. The therapeutic rationale is based on targeting and amplifying a tumour tissues normal cell death programme (apoptosis). While advances in the targeting of DR4 and DR5 have been successful the search for an agonistic antibody to another family member, the Fas receptor, has proven more elusive. This is partly due to the differing in vitro and in vivo characteristics of individual antibodies. In order to induce Fas targeted cell death an antibody must be capable of binding to and trimerising the receptor. It has been shown that antibodies capable of performing this function in vivo, with the assistance of tumour associated cells, do not always induce apoptosis in vitro. As a result the use of current methodologies to detect functional antibodies in vitro may have dismissed potential therapeutic candidates ('false negative'). Here we report a novel high throughput screening technique which artificially cross-links antibodies bound to the Fas receptor. By combining this process with Annexin-V and Prodidium Iodide (PI) staining we can select for antibodies which have the potential to induce apoptosis in vivo.

Antibody-mediated inhibition of cathepsin S blocks colorectal tumor invasion and angiogenesis.

PURPOSE: Cathepsin S is a cysteine protease that promotes the invasion of tumor and endothelial cells during cancer progression. Here we investigated the potential to target cathepsin S using an antagonistic antibody, Fsn0503, to block these tumorigenic effects. EXPERIMENTAL DESIGN: A panel of monoclonal antibodies was raised to human cathepsin S. The effects of a selected antibody were subsequently determined using invasion and proteolysis assays. Endothelial cell tube formation and aorta sprouting assays were done to examine antiangiogenic effects. In vivo effects were also evaluated using HCT116 xenograft studies. RESULTS: A selected cathepsin S antibody, Fsn0503, significantly blocked invasion of a range of tumor cell lines, most significantly HCT116 colorectal carcinoma cells, through inhibition of extracellular cathepsin S-mediated proteolysis. We subsequently found enhanced expression of cathepsin S in colorectal adenocarcinoma biopsies when compared with normal colon tissue. Moreover, Fsn0503 blocked endothelial cell capillary tube formation and aortic microvascular sprouting. We further showed that administration of Fsn0503 resulted in inhibition of tumor growth and neovascularization of HCT116 xenograft tumors. CONCLUSIONS: These results show that blocking the invasive and proangiogenic effects of cathepsin S with antibody inhibitors may have therapeutic utility upon further preclinical and clinical evaluation.

Multiplex immunoassay for serological diagnosis of Mycobacterium bovis infection in cattle.

Efforts to develop a better diagnostic assay for bovine tuberculosis have shown that the sensitivity and specificity of an assay can be improved by the use of two or more antigens. As reported here, we developed a multiplex chemiluminescent immunoassay that can simultaneously detect antibody activity to 25 antigens in a single well in a 96-well plate array format. The chemiluminescent signal is captured with a digital imaging system and analyzed with a macro program that tracks each serum for its pattern of antibody activity for Mycobacterium bovis antigens. The comparison of sera from 522 infected and 1,489 uninfected animals showed that a sensitivity of 93.1% and a specificity of 98.4% can be achieved with a combination of antigens. The assay system is rapid and can be automated for use in a centralized laboratory.

All-trans retinoic acid-induced downregulation of annexin II expression in myeloid leukaemia cell lines is not confined to acute promyelocytic leukaemia.

Most acute promyelocytic leukaemia (APL) patients suffer from disordered haemostasis. APL can be treated successfully in most instances by all-trans retinoic acid (ATRA) therapy, which induces endpoint maturation of the leukaemic promyelocytes with the characteristic t(15;17). Annexin II (AnII), a profibrinolytic protein, has been implicated in the bleeding manifestation seen in APL. Our group has shown previously that high levels of AnII are expressed on other acute myeloid leukaemia subtypes that are sometimes associated with disordered haemostasis, albeit less frequently than APL. This study examined the effects of ATRA on AnII expression and cell differentiation, on myeloid leukaemia cell lines to determine whether a regulatory influence on AnII may contribute to the return of haemostatic stability in APL following treatment. The results confirmed that AnII expression in the APL cell line (NB4) was significantly downregulated in response to ATRA (P < 0.01), with associated morphological and immunophenotypical evidence of myeloid differentiation. ATRA also downregulated AnII expression on other myeloid cell lines, albeit to a lesser extent than observed on NB4 cells. The results provide evidence that ATRA may resolve the hyperfibrinolysis in APL by downregulation of AnII expression.

Antibody conjugates and therapeutic strategies.

Immunotherapeutics represent the largest group of molecules currently in development as new drug entities. These versatile molecules are being investigated for the treatment of a range of pathological conditions including cancer, infectious and inflammatory diseases. Antibodies can be used to exert biological effects themselves or as delivery agents of conjugated drug molecules. Site-specific delivery of therapeutic agents has been an ultimate goal of the pharmaceutical industry in order to maximize drug action and minimize side effects. Antibodies have the potential to realize this objective and in this review we will examine some of the main strategies currently being employed for the development of these diverse therapeutic molecules.