Camelid antibodies, more than meets the eye

Abstract

Nowadays therapeutic antibodies are gaining ground To validate camelid conventional type antibodies as a source for therapeutic antibodies, their human homology in both conformation (shape) and sequence is analyzed. To thoroughly assess (germline) sequence homology, human immunoglobulin variable regions (V regions) counterparts are identified amongst camelid (alpaca (Lama pacos), llama (Lama glama), and camel (Camelis ferus)) species. As human homology controls, cynomolgous monkey and murine immunoglobulin V regions are used (positive and negative respectively). In addition, two earlier selected Lama glama Fab fragments are crystalized and aligned with their corresponding human family members. Results indicate high sequence/structural homology in camelid heavy chain variable domain regions and a sound structural resemblance to human Fab domains. This is however not seen for other “target” proteins, allowing for efficient immunization, and ultimately making camelids an unexpectedly advantageous therapeutic antibody platform with features not seen in other animal platforms. The momentum gained makes delving deeper into the technical aspect of camelid Fab fragment phage-display seem like an obvious step. Based on earlier observations like finding similar heavy/light chain families paired after selection we sought to employ these to design a quick and easy Fab fragment phage-display method for generating highly potent monoclonal antibodies (mAbs). To do so, a llama is immunized using a cytokine (IL-6) as a soluble target. Selection against IL-6 revealed a panel of Fab fragments that are analyzed for binding the IL-6:IL-6R interface to prevent ligand to receptor binding. The Fab fragment that had these characteristics was made up from a VH and Vλ fragment, these fragments were separately captured in a library and randomly brought together in a working phagemid library. We believe that this happened as this was an original pair originating from the immunized llama. Therefore, the isolated Vλ fragment was recombined with the VH library and surely members (affinity variants) of the VH clone were retrieved. The somatic mutations present within these affinity variants were made into a new VH library containing all possible combinations. When joined with their Vλ fragment to form once again full Fab fragments, phage display was set-up to select for low off-rate molecules resulting in a highly potent Fab fragment that in IgG1 format performed second to none when potency and affinity are considered as compared to the known antibodies in therapy. G-protein coupled receptors (GPCRs) or seven transmembrane receptors (7TMR) form a highly desired druggable target that at the time presented a challenge for antibody platforms. To be prepared, a literature investigation resulted in the needed insight to tackle upcoming problems likely to be encountered. Components like lipoparticles were identified as bona vide vehicles for immunizing llama’s and performing selections with. In addition, therapeutic antibodies raised against chemokines and their receptors (7TMR type) are discussed to get a feel for what has been done in the past and present. Following our survey CXCR4 is picked for being a multifaceted receptor that is druggable in both cancer and HIV-1 context. To set the bar high it was decided to aim for modulating CXCR4 receptor function distinguishing between CXCL12 (ligand) signaling and HIV-1 entry blockade using the smallest camelid derived antibody fragment known as nanobodies. After selection on both CXCR4 nanodiscs and lipoparticles a resulting repertoire of nanobodies is put through rigorous testing to see to what degree and by which concentration (IC50) biologic function of CXCR4 was interfered with. CXCR4 driven calcium flux, migration, cell morphology (impedance), receptor internalization were analyzed. The absence of a significant correlation between the performed assays was used to distinguish a set of nanobodies that were further characterized for exhibiting biased receptor modulation.