Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies.

Monoclonal antibodies (mAbs) are one of the most successful and versatile protein-based pharmaceutical products used to treat multiple pathological conditions. The remarkable specificity of mAbs and their affinity for biological targets has led to the implementation of mAbs in the therapeutic regime of oncogenic, chronic inflammatory, cardiovascular, and infectious diseases. Thus, the discovery of novel mAbs with defined functional activities is of crucial importance to expand our ability to address current and future clinical challenges. In vitro, antigen-driven affinity selection employing phage display biopanning is a commonly used technique to isolate mAbs. The success of biopanning is dependent on the quality and the presentation format of the antigen, which is critical when isolating mAbs against membrane protein targets. Here, we provide a comprehensive investigation of two established panning strategies, surface-tethering of a recombinant extracellular domain and cell-based biopanning, to examine the impact of antigen presentation on selection outcomes with regards to the isolation of positive mAbs with functional potential against a proof-of-concept type I cell surface receptor. Based on the higher sequence diversity of the resulting antibody repertoire, presentation of a type I membrane protein in soluble form was more advantageous over presentation in cell-based format. Our results will contribute to inform and guide future antibody discovery campaigns against cell surface proteins.

C5a receptors C5aR1 and C5aR2 mediate opposing pathologies in a mouse model of melanoma.

The canonical complement component 5a (C5a) receptor (C5aR) 1 has well-described roles in tumorigenesis but the contribution of the second receptor, C5aR2, is unclear. The present study demonstrates that B16.F0 melanoma cells express mRNA for both C5aR1 and C5aR2 and signal through ERK and p38 MAPKs in response to C5a. Despite this, C5a had no impact on melanoma cell proliferation or migration in vitro. In vivo studies demonstrated that the growth of B16.F0 melanoma tumors was increased in C5aR2-/- mice but reduced in C5aR1-/- mice and wild-type mice treated with a C5aR1 antagonist. Analysis of tumor-infiltrating leukocyte populations showed no significant differences between wild-type and C5aR2-/- mice. Conversely, percentages of myeloid-derived suppressor cells, macrophages, and regulatory T lymphocytes were lower in tumors from C5aR1-/- mice, whereas total (CD3+) T lymphocytes and CD4+ subsets were higher. Analysis of cytokine and chemokine levels also showed plasma IFN-γ was higher and tumor C-C motif chemokine ligand 2 was lower in the absence of C5aR1. The results suggest that C5aR1 signaling supports melanoma growth by promoting infiltration of immunosuppressive leukocyte populations into the tumor microenvironment, whereas C5aR2 has a more restricted but beneficial role in limiting tumor growth. Overall, these data support the potential of C5aR1-inhibitory therapies for melanoma.-Nabizadeh, J. A., Manthey, H. D., Panagides, N., Steyn, F. J., Lee, J. D., Li, X. X., Akhir, F. N. M., Chen, W., Boyle, G. M., Taylor, S. M., Woodruff, T. M., Rolfe, B. E. C5a receptors C5aR1 and C5aR2 mediate opposing pathologies in a mouse model of melanoma.

Proteomic and functional variation within black snake venoms (Elapidae: Pseudechis).

Pseudechis (black snakes) is an Australasian elapid snake genus that inhabits much of mainland Australia, with two representatives confined to Papua New Guinea. The present study is the first to analyse the venom of all 9 described Pseudechis species (plus one undescribed species) to investigate the evolution of venom composition and functional activity. Proteomic results demonstrated that the typical Pseudechis venom profile is dominated by phospholipase A2 toxins. Strong cytotoxicity was the dominant function for most species. P. porphyriacus, the most basal member of the genus, also exhibited the most divergent venom composition, being the only species with appreciable amounts of procoagulant toxins. The relatively high presence of factor Xa recovered in P. porphyriacus venom may be related to a predominantly amphibian diet. Results of this study provide important insights to guide future ecological and toxinological investigations.

Enter the Dragon: The Dynamic and Multifunctional Evolution of Anguimorpha Lizard Venoms.

While snake venoms have been the subject of intense study, comparatively little work has been done on lizard venoms. In this study, we have examined the structural and functional diversification of anguimorph lizard venoms and associated toxins, and related these results to dentition and predatory ecology. Venom composition was shown to be highly variable across the 20 species of Heloderma, Lanthanotus, and Varanus included in our study. While kallikrein enzymes were ubiquitous, they were also a particularly multifunctional toxin type, with differential activities on enzyme substrates and also ability to degrade alpha or beta chains of fibrinogen that reflects structural variability. Examination of other toxin types also revealed similar variability in their presence and activity levels. The high level of venom chemistry variation in varanid lizards compared to that of helodermatid lizards suggests that venom may be subject to different selection pressures in these two families. These results not only contribute to our understanding of venom evolution but also reveal anguimorph lizard venoms to be rich sources of novel bioactive molecules with potential as drug design and development lead compounds.

Venom Profiling of a Population of the Theraphosid Spider Phlogius crassipes Reveals Continuous Ontogenetic Changes from Juveniles through Adulthood.

Theraphosid spiders (tarantulas) are venomous arthropods found in most tropical and subtropical regions of the world. Tarantula venoms are a complex cocktail of toxins with potential use as pharmacological tools, drugs and bioinsecticides. Although numerous toxins have been isolated from tarantula venoms, little research has been carried out on the venom of Australian tarantulas. We therefore investigated the venom profile of the Australian theraphosid spider Phlogius crassipes and examined whether there are ontogenetic changes in venom composition. Spiders were divided into four ontogenic groups according to cephalothorax length, then the venom composition of each group was examined using gel electrophoresis and mass spectrometry. We found that the venom of P. crassipes changes continuously during development and throughout adulthood. Our data highlight the need to investigate the venom of organisms over the course of their lives to uncover and understand the changing functions of venom and the full range of toxins expressed. This in turn should lead to a deeper understanding of the organism's ecology and enhance the potential for biodiscovery.

How the Cobra Got Its Flesh-Eating Venom: Cytotoxicity as a Defensive Innovation and Its Co-Evolution with Hooding, Aposematic Marking, and Spitting.

The cytotoxicity of the venom of 25 species of Old World elapid snake was tested and compared with the morphological and behavioural adaptations of hooding and spitting. We determined that, contrary to previous assumptions, the venoms of spitting species are not consistently more cytotoxic than those of closely related non-spitting species. While this correlation between spitting and non-spitting was found among African cobras, it was not present among Asian cobras. On the other hand, a consistent positive correlation was observed between cytotoxicity and utilisation of the defensive hooding display that cobras are famous for. Hooding and spitting are widely regarded as defensive adaptations, but it has hitherto been uncertain whether cytotoxicity serves a defensive purpose or is somehow useful in prey subjugation. The results of this study suggest that cytotoxicity evolved primarily as a defensive innovation and that it has co-evolved twice alongside hooding behavior: once in the Hemachatus + Naja and again independently in the king cobras (Ophiophagus). There was a significant increase of cytotoxicity in the Asian Naja linked to the evolution of bold aposematic hood markings, reinforcing the link between hooding and the evolution of defensive cytotoxic venoms. In parallel, lineages with increased cytotoxicity but lacking bold hood patterns evolved aposematic markers in the form of high contrast body banding. The results also indicate that, secondary to the evolution of venom rich in cytotoxins, spitting has evolved three times independently: once within the African Naja, once within the Asian Naja, and once in the Hemachatus genus. The evolution of cytotoxic venom thus appears to facilitate the evolution of defensive spitting behaviour. In contrast, a secondary loss of cytotoxicity and reduction of the hood occurred in the water cobra Naja annulata, which possesses streamlined neurotoxic venom similar to that of other aquatic elapid snakes (e.g., hydrophiine sea snakes). The results of this study make an important contribution to our growing understanding of the selection pressures shaping the evolution of snake venom and its constituent toxins. The data also aid in elucidating the relationship between these selection pressures and the medical impact of human snakebite in the developing world, as cytotoxic cobras cause considerable morbidity including loss-of-function injuries that result in economic and social burdens in the tropics of Asia and sub-Saharan Africa.

Vintage venoms: proteomic and pharmacological stability of snake venoms stored for up to eight decades.

For over a century, venom samples from wild snakes have been collected and stored around the world. However, the quality of storage conditions for "vintage" venoms has rarely been assessed. The goal of this study was to determine whether such historical venom samples are still biochemically and pharmacologically viable for research purposes, or if new sample efforts are needed. In total, 52 samples spanning 5 genera and 13 species with regional variants of some species (e.g., 14 different populations of Notechis scutatus) were analysed by a combined proteomic and pharmacological approach to determine protein structural stability and bioactivity. When venoms were not exposed to air during storage, the proteomic results were virtually indistinguishable from that of fresh venom and bioactivity was equivalent or only slightly reduced. By contrast, a sample of Acanthophis antarcticus venom that was exposed to air (due to a loss of integrity of the rubber stopper) suffered significant degradation as evidenced by the proteomics profile. Interestingly, the neurotoxicity of this sample was nearly the same as fresh venom, indicating that degradation may have occurred in the free N- or C-terminus chains of the proteins, rather than at the tips of loops where the functional residues are located. These results suggest that these and other vintage venom collections may be of continuing value in toxin research. This is particularly important as many snake species worldwide are declining due to habitat destruction or modification. For some venoms (such as N. scutatus from Babel Island, Flinders Island, King Island and St. Francis Island) these were the first analyses ever conducted and these vintage samples may represent the only venom ever collected from these unique island forms of tiger snakes. Such vintage venoms may therefore represent the last remaining stocks of some local populations and thus are precious resources. These venoms also have significant historical value as the Oxyuranus venoms analysed include samples from the first coastal taipan (Oxyuranus scutellatus) collected for antivenom production (the snake that killed the collector Kevin Budden), as well as samples from the first Oxyuranus microlepidotus specimen collected after the species' rediscovery in 1976. These results demonstrate that with proper storage techniques, venom samples can retain structural and pharmacological stability. This article is part of a Special Issue entitled: Proteomics of non-model organisms.