Molecular engineering of antibodies for therapeutic and diagnostic purposes

During the past ten years, monoclonal antibodies (mAbs) have taken center stage in the field of targeted therapy and diagnosis. This increased interest in mAbs is due to their binding accuracy (affinity and specificity) together with the original molecular and structural rules that govern interactions with their cognate antigen. In addition, the effector properties of antibodies constitute a second major advantage associated with their clinical use. The development of molecular and structural engineering and more recently of in vitro evolution of antibodies has opened up new perspectives in the de novo design of antibodies more adapted to clinical and diagnostic use. Thus, efforts are regularly made by researchers to improve or modulate antibody recognition properties, to adapt their pharmacokinetics, engineer their stability, and control their immunogenicity. This review presents the latest molecular engineering results on mAbs with therapeutic and diagnostic applications.     

Augmentation of lenvatinib efficacy by topical treatment of miR-634 ointment in anaplastic thyroid cancer

Anaplastic thyroid cancer (ATC) is one of the most lethal types of human tumors. Lenvatinib can improve the disease control and prognosis in patients with ATC. However, there is an unmet need to develop a therapeutically safer and non-invasive strategy that improves the efficacy of lenvatinib for advanced ATC tumors, which grow larger close to the skin. We previously demonstrated that the topical application of an ointment incorporating tumor suppressive microRNA (TS-miR), miR-634, is a useful strategy as a TS-miR therapeutics. Here, we found that the overexpression of miR-634 synergistically increased lenvatinib-induced cytotoxicity by concurrently downregulating multiple genes related to cytoprotective processes, including ASCT2, a glutamine transporter, in ATC cell lines. Furthermore, the topical application of a miR-634 ointment on subcutaneous tumors effectively augmented the anti-tumor effects of lenvatinib in an ATC xenograft mouse model. Thus, we propose topical treatment of a miR-634 ointment as a rational strategy for improving lenvatinib-based therapy for ATC.     

Coordination property of poly(1-vinyl-2-methylimidazole)-heme complexes

Poly(1-vinyl-2-methylimidazole) and poly(1-vinyl-2-methylimidazole-co-1-vinylpyrrolidone) predominantly form five-coordinate heme complexes in aqueous solution. The apparent formation constants (K) of the heme complexes were estimated spectroscopically. The K values of the polymer-heme complexes were about 10² to 10³ times those of the corresponding monomeric ones. These large K values were canceled by adding poly(1-vinylpyrrolidone), alcohol, or dimethylformamide. The viscometric measurement of the polymer-heme solution showed that the polymer complex took a compact shape. These results indicate a hydrophobic interaction of heme with the polymer-ligand. Poly(1-vinyl-2-methylimidazole) could form the five-coordinate heme complex, even in the presence of a large amount of imidazole.     

Minor intron splicing is critical for survival of lethal prostate cancer

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Amine coupling versus biotin capture for the assessment of sulfonamide as ligands of hCA isoforms

This work was dedicated to the development of a reliable SPR method allowing the simultaneous and quick determination of the affinity and selectivity of designed sulfonamide derivatives for hCAIX and hCAXII versus hCAII, in order to provide an efficient tool to discover drugs for anticancer therapy of solid tumors. We performed for the first time a comparison of two immobilization approaches of hCA isoforms. First one relies on the use of an amine coupling strategy, using a CM7 chip to obtain higher immobilization levels than with a CM5 chip and consequently the affinity with an higher precision (CV% < 10%). The second corresponds to a capture of proteins on a streptavidin chip, named CAP chip, after optimization of biotinylation conditions (amine versus carboxyl coupling, biotin to protein ratio). Thanks to the amine coupling approach, only hCAII and hCAXII isoforms were efficiently biotinylated to reach relevant immobilization (3000 RU and 2700 RU, respectively) to perform affinity studies. For hCAIX, despite a successful biotinylation, capture on the CAP chip was a failure. Finally, concordance between affinities obtained for the three derivatives to CAs isozymes on both chips has allowed to valid the approaches for a further screening of new derivatives.     

The novel EpCAM-targeting monoclonal antibody 3–17I linked to saporin is highly cytotoxic after photochemical internalization in breast,pancreas and colon cancer cell lines

The epithelial cell adhesion molecule (EpCAM) is expressed by a wide range of human carcinomas, making it an attractive diagnostic and therapeutic target in oncology. Its recent identification on cancer stem cells has raised further interest in its use for tumor targeting and therapy. Here, we present the characterization and therapeutic potential of 3–17I, a novel human EpCAM-targeting monoclonal antibody. Strong reaction of 3–17I was observed in all lung, colon, and breast human tumor biopsies evaluated. By flow cytometry and confocal fluorescence microscopy, we demonstrate that 3–17I specifically targets EpCAM-positive cell lines. We also show evidence for mAb-sequestration in endo-/lysosomes, suggesting internalization of 3–17I by receptor-mediated endocytosis. The ribosomal-inactivating toxin saporin was linked to 3–17I, creating the per se non-toxic immunotoxin 3–17I-saporin, a promising candidate for the drug delivery technology photochemical internalization (PCI). PCI is based on a light-controlled destruction of endolysosomal membranes and subsequent cytosolic release of the sequestered payload upon light exposure. EpCAM-positive human cancer cell lines MCF7 (breast), BxPC-3 (pancreas), WiDr (colon), and the EpCAM-negative COLO320DM (colon), were treated with 3–17I-saporin in combination with the clinically relevant photosensitizer TPCS2a (Amphinex), followed by exposure to light. No cytotoxicity was observed after treatment with 3–17I-saporin without light exposure. However, cell viability, proliferation and colony-forming capacity was strongly reduced in a light-dependent manner after PCI of 3–17I. Our results show that 3–17I is an excellent candidate for diagnosis of EpCAM-positive tumors and for development of clinically relevant antibody-drug conjugates, using PCI for the treatment of localized tumors.     

Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain

Advances in recombinant antibody technology and protein engineering have provided the opportunity to reduce antibodies to their smallest binding domain components and have concomitantly driven the requirement for devising strategies to increase serum half-life to optimise drug exposure, thereby increasing therapeutic efficacy. In this study, we adopted an immunization route to raise picomolar affinity shark immunoglobulin new antigen receptors (IgNARs) to target human serum albumin (HSA). From our model shark species, Squalus acanthias, a phage display library encompassing the variable binding domain of IgNAR (VNAR) was constructed, screened against target, and positive clones were characterized for affinity and specificity. N-terminal and C-terminal molecular fusions of our lead hit in complex with a naïve VNAR domain were expressed, purified and exhibited the retention of high affinity binding to HSA, but also cross-selectivity to mouse, rat and monkey serum albumin both in vitro and in vivo. Furthermore, the naïve VNAR had enhanced pharmacokinetic (PK) characteristics in both N- and C-terminal orientations and when tested as a three domain construct with naïve VNAR flanking the HSA binding domain at both the N and C termini. Molecules derived from this platform technology also demonstrated the potential for clinical utility by being available via the subcutaneous route of delivery. This study thus demonstrates the first in vivo functional efficacy of a VNAR binding domain with the ability to enhance PK properties and support delivery of multifunctional therapies.     

Cartilage stress-relaxation proceeds slower at higher compressive strains

Articular cartilage is the connective tissue which covers bone surfaces and deforms during in vivo activity. Previous research has investigated flow-dependent cartilage viscoelasticity, but relatively few studies have investigated flow-independent mechanisms. This study investigated polymer dynamics as an explanation for the molecular basis of flow-independent cartilage viscoelasticity. Polymer dynamics predicts that stress-relaxation will proceed more slowly at higher volumetric concentrations of polymer. Stress-relaxation tests were performed on cartilage samples after precompression to different strain levels. Precompression increases the volumetric concentration of cartilage biopolymers, and polymer dynamics predicts an increase in relaxation time constant. Stress-relaxation was slower for greater precompression. There was a significant correlation between the stress-relaxation time constant and cartilage volumetric concentration. Estimates of the flow-dependent timescale suggest that flow-dependent relaxation occurs on a longer timescale than presently observed. These results are consistent with polymer dynamics as a mechanism of cartilage viscoelasticity.