Design and construction of a new human naïve single-chain fragment variable antibody library, IORISS1.

Human monoclonal antibodies are a powerful tool with increasingly successful exploitations and the single chain fragment variable format can be considered the building block for the implementation of more complex and effective antibody-based constructs. Phage display is one of the best and most efficient methods to isolate human antibodies selected from an efficient and variable phage display library. We report a method for the construction of a human naïve single-chain variable fragment library, termed IORISS1. Many different sets of oligonucleotide primers as well as optimized electroporation and ligation reactions were used to generate this library of 1.2×10(9) individual clones. The key difference is the diversity of variable gene templates, which was derived from only 15 non-immunized human donors. The method described here, was used to make a new human naïve single-chain fragment variable phage display library that represents a valuable source of diverse antibodies that can be used as research reagents or as a starting point for the development of therapeutics. Using biopanning, we determined the ability of IORISS1 to yield antibodies. The results we obtained suggest that, by using an optimized protocol, an efficient phage antibody library can be generated.

Process development of a human recombinant diabody expressed in E. coli: engagement of CD99-induced apoptosis for target therapy in Ewing's sarcoma.

Ewing's sarcoma (EWS) is the second most common primary bone tumor in pediatric patients characterized by over expression of CD99. Current management consists in extensive chemotherapy in addition to surgical resection and/or radiation. Recent improvements in treatment are still overshadowed by severe side effects such as toxicity and risk of secondary malignancies; therefore, more effective strategies are urgently needed. The goal of this work was to develop a rapid, inexpensive, and "up-scalable" process of a novel human bivalent single-chain fragment variable diabody (C7 dAbd) directed against CD99, as a new therapeutic approach for EWS. We first investigated different Escherichia coli constructs of C7 dAbd in small-scale studies. Starting from 60 % soluble fraction, we obtained a yield of 25 mg C7 dAbd per liter of bacterial culture with the construct containing pelB signal sequence. In contrast, a low recovery of C7 dAbd was achieved starting from periplasmic inclusion bodies. In order to maximize the yield of C7 dAbd, large-scale fermentation was optimized. We obtained from 75 % soluble fraction 35 mg C7 dAbd per L of cell culture grown in a synthetic media containing 3 g/L of vegetable peptone and 1 g/L of yeast extract. Furthermore, we demonstrated the better efficacy of the cell lysis by homogenization versus periplasmic extraction, in reducing endotoxin level of the C7 dAbd. For gram-scale purification, a direct aligned two-step chromatography cascade based on binding selectivity was developed. Finally, we recovered C7 dAbd with low residual process-related impurities, excellent reactivity, and apoptotic ability against EWS cells.

The human antibody fragment DIATHIS1 specific for CEACAM1 enhances natural killer cell cytotoxicity against melanoma cell lines in vitro.

Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1 malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1 melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1 melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro-expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell-mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin.

CD99 triggering in Ewing sarcoma delivers a lethal signal through p53 pathway reactivation and cooperates with doxorubicin.

PURPOSE: The paucity of new drugs for the treatment of Ewing sarcoma (EWS) limits the cure of these patients. CD99 has a strong membranous expression in EWS cells and, being also necessary for tumor survival, is a suitable target to aim at. In this article, we described a novel human monospecific bivalent single-chain fragment variable diabody (dAbd C7) directed against CD99 of potential clinical application. EXPERIMENTAL DESIGN: In vitro and in vivo evaluation of cell death and of the molecular mechanisms triggered by anti-CD99 agents were performed alone or in combination with doxorubicin to demonstrate efficacy and selectivity of the new dAbd C7. RESULTS: The dAbd C7 induced rapid and massive EWS cell death through Mdm2 degradation and p53 reactivation. Mdm2 overexpression as well as silencing of p53 in p53wt EWS cells decreased CD99-induced EWS cell death, whereas treatment with nutlin-3 enhanced it. Furthermore, cell death was associated with induction of p21, bax, and mitochondrial depolarization together with substantial inhibition of tumor cell proliferation. Combined treatment of anti-CD99 dAbd C7 with doxorubicin was additive both in vitro and in vivo against EWS xenografts. Normal mesenchymal stem cells showed no p53 activation and were resistant to cell death, unless transformed by EWS-FLI, the oncogenic driver of EWS. CONCLUSIONS: These results indicate that dAbd C7 is a suitable candidate tool to target CD99 in patients with EWS able to spare normal stem cells from death as it needs an aberrant genetic context for the efficient delivery of CD99-triggered cell death.

Blocking monocyte transmigration in in vitro system by a human antibody scFv anti-CD99. Efficient large scale purification from periplasmic inclusion bodies in E. coli expression system.

Migration of leukocytes into site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells, inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies and the absence of toxic reagents utilized for solubilization and refolding step of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting, we herein describe an efficient and large scale production of the antibody fragments expressed in E. coli as periplasmic insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signaling. This protocol can be useful for the successful purification of other monomeric scFvs which are expressed as periplasmic inclusion bodies in bacterial systems.

Isolation of a new human scFv antibody recognizing a cell surface binding site to CEACAM1. Large yield production, purification and characterization in E. coli expression system.

The CEACAM1 cell adhesion molecule has recently received considerable interest as a tumour target antigen since its re-expression often occurs in the advanced stages of multiple malignancies including malignant melanoma, non-small cell lung cancer and other types of solid tumors. In this study, we describe the expression-purification and characterization of the new single chain variable fragment (scFv) antibody named DIATHIS1, that recognizes the N-terminal IgV-like domain present in CEACAM1. Three validation batches show that the production process is robust and reproducible. The scFv DIATHIS1 is formulated as a naturally occurring mixture of monomer and dimer. The antibody is biophysically stable at low temperature (-80°C), different concentrations and remains biologically active for at least 24months. The thermal stability of scFv DIATHIS1 at 37°C shows important features for its activity in vivo. The dimer behaves as a reservoir converting slowly into monomer. The monomer and dimer forms of scFv DIATHIS1 were isolated and characterized, showing high reactivity for CEACAM1. This new composition of antibody could have advantageous pharmacokinetics parameters over conventional scFv for in vivo applications.

Raltegravir does not revert efflux activity of MDR1-P-glycoprotein in human MDR cells.

BACKGROUND: Raltegravir (Isentress®)(RALT) has demonstrated excellent efficacy in both treatment-experienced and naïve patients with HIV-1 infection, and is the first strand transfer integrase inhibitor to be approved for use in HIV infected adults worldwide. Since the in vivo efficacy of this class of antiviral drugs depends on their access to intracellular sites where HIV-1 replicates, we analyzed the biological effects induced by RALT on human MDR cell systems expressing multidrug transporter MDR1-P-glycoprotein (MDR1-Pgp). METHODS: Our study about RALT was performed by using a set of consolidated methodologies suitable for evaluating the MDR1-Pgp substrate nature of chemical and biological agents, namely: i) assay of drug efflux function; ii) analysis of MDR reversing capability by using cell proliferation assays; iii) monoclonal antibody UIC2 (mAb) shift test, as a sensitive assay to analyze conformational transition associated with MDR1-Pgp function; and iv) induction of MDR1-Pgp expression in MDR cell variant subjected to RALT exposure. RESULTS: Functional assays demonstrated that the presence of RALT does not remarkably interfere with the efflux mechanism of CEM-VBL100 and HL60 MDR cells. Accordingly, cell proliferation assays clearly indicated that RALT does not revert MDR phenotype in human MDR1-Pgp expressing cells. Furthermore, exposure of CEM-VBL10 cells to RALT does not induce MDR1-Pgp functional conformation intercepted by monoclonal antibody (mAb) UIC2 binding; nor does exposure to RALT increase the expression of this drug transporter in MDR1-Pgp expressing cells. CONCLUSIONS: No evidence of RALT interaction with human MDR1-Pgp was observed in the in vitro MDR cell systems used in the present investigation, this incorporating all sets of studies recommended by the FDA guidelines. Taken in aggregate, these data suggest that RALT may express its curative potential in all sites were HIV-1 penetrates, including the MDR1-Pgp protected blood/tissue barrier. Moreover RALT, evading MDR1-Pgp drug efflux function, would not interfere with pharmacokinetic profiles of co-administered MDR1-Pgp substrate antiretroviral drugs.

The biology of MDR1-P-glycoprotein (MDR1-Pgp) in designing functional antibody drug conjugates (ADCs): the experience of gemtuzumab ozogamicin.

BACKGROUND: The treatment of cancer remains a formidable challenge owing to the difficulties in differentiating tumor cells from healthy cells to ameliorate the disease without causing intolerable toxicity to patients. In addition, the emergence of MDR1-Pgp mediated multi-drug resistance (MDR) it is a biological phenomenon that inhibits the curative potential of chemotherapeutic treatments. One way to improve the selectivity of therapeutic molecules in tumors would be to target them on the tumor site, thereby sparing normal tissues. AIMS: In this overview, we will discuss the biological factors influencing the safety and efficacy of the humanized mAb hP67.6 linked to the potent cytotoxic drug calicheamicingamma1 (gemtuzumab ozogamicin) that target CD33 cell surface antigen expressed on AML cells. In addition, we highlight key aspects of MDR1-Pgp biology as a platform to understand its functional role in gemtuzumab ozogamicin immunotherapy which is tightly linked to an accurate assessment of the MDR status of AML cells. DISCUSSION: Several factors may affect the efficacy and safety of immunoconjugates. These include the common issues of chemical and antibody therapeutics such as specificity, heterogeneous target antigen expression and the complex pharmacokinetics profile of conveyed antibody. Further, the delivered drug may not be sufficient for providing therapeutic benefit, since the curative cytotoxic compound may be affected by intrinsic or acquired resistance of target cells. These and other potential problems, as well as the possible ways to overcome them will be discussed in this review by examining the biological factors involved in safety and efficacy of the first in class antibody drug conjugate (ADC) gentuzumab ozogamicin. Despite this set-back, the extensive recorded data and the lessons learned from gentuzumab ozogamicin recently withdrawn from the market for safety concerns helped to pave the way for next generations of clinically promising new ADCs which are currently investigated in clinical trials and two of them, Brentuximab vedotin, and Trastuzumab emtansine (T-DM1) have been recently approved for commercial distribution in US by Food and Drug Administration (FDA).

Targeting MDR1-P-glycoprotein (MDR1-Pgp) in immunochemotherapy of acute myeloid leukemia (AML).

BACKGROUND: Monoclonal antibodies represent the fastest growing sector of pharmaceutical biotechnology and a number of antibody-based biopharmaceuticals have been approved for cancer treatment. However, in many cases the antibodies used for the treatment of tumors offer only a modest survival benefit to cancer patients. AIMS: In the present review-article we intend to analyze: i) the curative regimen gemtuzumab ozogamicin (GO) -mediate characterized by the absence of cytotoxic drugs MDR1-Pgp substrates to overcome the mechanism of action of this multidrug transporter, ii) the safety and efficacy of MDR reversing strategy in AML outcome and, iii) chemical and biological MDR modulators playing a dual relevant medical role as a therapeutic and MDR reversing agents but not yet entered in the clinical setting of AML. Since the similar multidrug transporter protein MDR1-Pgp and its down modulation factors may affect safety and efficacy of already generated antibody drug conjugates (ADCs) a comprehensive overview of the most clinically representative immunoconjugates is reported. DISCUSSION: ADCs represent one of the most promising strategies to enhance the antitumor activity of antibodies. ADCs comprise an antibody (or an antibody fragment) conjugated to a cytotoxic drug via a chemical linker. The therapeutic concept of ADCs is to use an antibody as a vehicle to selectively delivering a cytotoxic drug specifically to a tumor cell, in most cases by means of binding to target cell surface antigen. As a consequence, ADCs have significant potential for enhancing the antitumor activity of "naked" antibodies and reducing the systemic toxicity of the conjugated drugs.

Clinical development of monoclonal antibody-based drugs in HIV and HCV diseases.

Today there are many licensed antiviral drugs, but the emergence of drug resistant strains sometimes invalidates the effects of the current therapies used in the treatment of infectious diseases. Compared to conventional antiviral drugs, monoclonal antibodies (mAbs) used as pharmacological molecules have particular physical characteristics and modes of action, and, therefore, they should be considered as a distinct therapeutic class. Despite being historically validated, antibodies may represent a novel tool for combatting infectious diseases. The current high cost of mAbs' production, storage and administration (by injection only) and the consequent obstacles to development are outweighed by mAbs' clinical advantages. These are related to a low toxicity combined with high specificity and versatility, which allows a specific antibody to mediate various biological effects, ranging from the virus neutralization mechanisms to the modulation of immune responses.This review briefly summarizes the recent technological advances in the field of immunoglobulin research, and the current status of mAb-based drugs in clinical trials for HIV and HCV diseases. For each clinical trial the available data are reported and the emerging conceptual problems of the employed mAbs are highlighted.This overview helps to give a clear picture of the efficacy and challenges of the mAbs in the field of these two infectious diseases which have such a global impact.