Current Pharmaceutical Design - Volume 22, Issue 43, 2016

Background: Antibody phage display is highly dependent on the availability of antibody libraries. There are several forms of libraries depending mainly on the origin of the source materials. There are three major classes of libraries, mainly the naïve, immune and synthetic libraries. Methods: Immune antibody libraries are designed to isolate specific and high affinity antibodies against disease antigens. The pre-exposure of the host to an infection results in the production of a skewed population of antibodies against the particular infection. Results: This characteristic takes advantage of the in vivo editing machinery to generate bias and specific immune repertoire. The skewed but diverse repertoire of immune libraries has been adapted successfully in the generation of antibodies against a wide range of diseases. Conclusion: We envisage immune antibody libraries to play a greater role in the discovery of antibodies for diseases in the near future.

The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.

Background: The discovery of functional heavy chain-only antibodies devoid of light chains in sera of camelids and sharks in the early nineties provided access to the generation of minimal-sized, single-domain, in vivo affinity-matured, recombinant antigenbinding fragments, also known as Nanobodies. Methods: Recombinant DNA technology and adaptation of phage display vectors form the basis to construct large naïve, synthetic or medium sized immune libraries from where multiple Nanobodies have been retrieved. Alternative selection methods (i.e. bacterial display, bacterial two-hybrid, Cis-display and ribosome display) have also been developed to identify Nanobodies. The antigen affinity, stability, expression yields and structural details of the Nanobodies have been determined by standard technology. Nanobodies were subsequently engineered for higher stability and affinity, to have a sequence closer to that of human immunoglobulin domains, or to add designed effector functions. Results: Antigen specific Nanobodies recognizing with high affinity their cognate antigen were retrieved from various libraries. High expression yields are obtained from microorganisms, even when expressed in the cytoplasm. The purified Nanobodies are shown to possess beneficial biochemical and biophysical properties. The crystal structure of Nanobody::antigen complexes reveal the preference of Nanobodies for cavities on the antigen surface. Conclusion: Thanks to the properties described above, Nanobodies became a highly valued and versatile tool for biomolecular research. Moreover, numerous diagnostic and therapeutic Nanobody-based applications have been developed in the past decade.

Phage display technology has revolutionized the science of drug discovery by transforming the generation and manipulation of ligands, such as antibody fragments, enzymes, and peptides. The basis of this technology is the expression of recombinant proteins or peptides fused to a phage coat protein, and subsequent isolation of ligands based on a variety of catalytic, physicochemical/binding kinetic and/or biological characteristics. An incredible number of diagnostic and therapeutic domains have been successfully isolated using phage display technology. The variable domain of the New Antigen Receptors (VNAR) found in cartilaginous fish, is also amenable to phage display selection. Whilst not an antibody, VNARs are unquestionable the oldest (450 million years), and smallest antigen binding, single-domains so far identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established, enhancing our understanding of the evolutionary origins of humoral immunity and the unusual but divergent ancestry of the VNARs themselves. VNARs exhibit remarkable physicochemical properties, such as small size, stability in extreme conditions, solubility, molecular flexibility, high affinity and selectivity for target. The purpose of this review is to illustrate the important role phage display has played in the isolation and characterization of potent therapeutic and diagnostic VNAR domains.

Background: The complex multi-chain architecture of antibodies has spurred interest in smaller derivatives that retain specificity but can be more easily produced in bacteria. Domain antibodies consisting of single variable domains are the smallest antibody fragments and have been shown to possess enhanced ability to target epitopes that are difficult to access using multidomain antibodies. However, in contrast to natural camelid antibody domains, human variable domains typically suffer from low stability and high propensity to aggregate. Methods: This review summarizes strategies to improve the biophysical properties of heavy chain variable domains from human antibodies with an emphasis on aggregation resistance. Several protein engineering approaches have targeted antibody frameworks and complementarity determining regions to stabilize the native state and prevent aggregation of the denatured state. Conclusion: Recent findings enable the construction of highly diverse libraries enriched in aggregation-resistant variants that are expected to provide binders to diverse antigens. Engineered domain antibodies possess unique advantages in expression, epitope preference and flexibility of formatting over conventional immunoreagents and are a promising class of antibody fragments for biomedical development.

Background: Antibody phage display is a major technological platform for the generation of fully human antibodies for therapeutic purposes. The in vitro binder selection by phage display allows researchers to have more extensive control over binding parameters and facilitates the isolation of clinical candidate antibodies with desired binding and/or functional profiles. Methods: Since the invention of antibody phage display in late 1980s, significant technological advancements in the design, construction, and selection of the antibody libraries have been made, and several fully human antibodies generated by phage display are currently approved or in various clinical development stages. Results: In this review, the background and details of antibody phage display technology, and representative antibody libraries with natural or synthetic sequence diversity and different construction strategies are described. The generation, optimization, functional and biophysical properties, and preclinical and clinical developments of some of the phage display-derived therapeutic antibodies approved for use in patients or in late-stage clinical trials are also discussed. Conclusion: With evolving novel disease targets and therapeutic strategies, antibody phage display is expected to continue to play a central role in the development of the next generation of therapeutic antibodies.

Background: The identification of combinatorial antibodies against many different targets in oncology, autoimmune, inflammatory and infectious diseases has uncovered novel strategies to control and prevent diseases’ onset and progression, and represents the fastest growing market for the pharmaceutical industry. Phage Display has been successfully used in the identification of unknown targets, which combines shotgun approaches with high throughput selection schemes. Methods: This specific review covers many aspects of combinatorial phage display technology starting from antibody selection strategies to its redesign for application purposes. Emphasis is specifically directed to how these biotherapeutics function on specific targets with an interactome view, especially within complex diseases. Conclusion: Novel combinatorial antibodies will lead to improved interventions in cancer, autoimmune and infectious diseases; however, the very large genetic diversity associated with environmental variations highlight the importance of the personalized medicine using a system’s biology approach. Therefore, combined therapies are expected in the near future.

Infectious diseases in the skin represent a major group of pathologies that contribute annually for significant health economic expenses. According to WHO, Buruli Ulcer (BU) and Cutaneous Leishmaniasis (CL) are two neglected tropical diseases for which therapy remains inadequate. Topical delivery of therapeutics constitutes an advantageous alternative to treat infected skin lesions as it allows a direct treatment of affected areas avoiding unwanted systemic effects and reducing the drug dosage. However, effective topical delivery of antimicrobial agents is still a challenge. The emergence of difficult-to-treat skin mycobacteriosis such as BU or the increasing incidence of CL highlights the need for new antimicrobial agents and new delivery systems. In the present review problems related to mycobacterial and parasitic infected skin lesions will be particularly focused as well as new alternative strategies for their treatment. Currently, progresses and challenges in constructing nanocarrier platforms for delivering various antimicrobial drugs for infected skin lesions have been attempted. This article reviews the potential of these nanocarriers in the treatment of cutaneous infections, especially regarding mycobacteria and Leishmania caused skin lesions. Nanostructured biomaterials, such as lipid-based nanocarriers, have unique physicochemical properties enabling the topical application of antimicrobial drugs, thereby overcoming some of the limitations of conventional therapies. These nanosystems have been investigated for the topical delivery of well-known drugs or new therapeutic candidates. This approach is just the beginning of a stimulating nanotechnological area.

Suramab (SUM) is a new pharmaceutical combination made up of suramine (SUR) and bevacizumab (BVM), which showed a high synergistic effect when administered jointly. As the pharmaceutical vehicle, poloxamer aqueous dispersions were used since this system is able to maintain their fluidity at low temperatures (<15ºC) but which become gel in the corporal environment (>35ºC). In the present study we aimed at evaluating the effect of Poloxamer to prolong the effect of SUM. These formulations were characterized using rheological, biopharmaceutical (drug release) and morphological (SEM) technique. Corneal NV was induced in Sprague Dawley rats Corneal. At 15 days of follow up animals were sacrificed and perfused with black drawing ink. Digital photographs were taken and the area of neovascularisation (ANV) was calculated using the image programmed. The rheological behavior was influenced by the addition of drugs, resulting in a decrease in the gelation temperature (Tsol/gel). Both drugs were released from poloxamer gels by means of an anomalous mechanism. However, BVM was released faster than SUR, with their combination (SUM) to appearing to reduce delivery, probably due to interactions between the drugs or with the polymeric matrix. The in vivo studies showed that SUM-poloxamer gel was able to increase the corneal antiangiogenic effect compared to the SUM solution and BVM alone at 15 days of follow-up. Furthermore no injurious effects were observed in the histological tissue examination after drug administration. The presence of Poloxamer, known to modulate control release of biological agents, seems to have a favorable effect on SUM subconjunctival administered.