Anticalin N- or C-Terminal on a Monoclonal Antibody Affects Both Production and In Vitro Functionality.

Bispecific antibodies (BsAbs) represent an important advance in innovative therapeutic strategies. Among the countless formats of BsAbs, fusion with molecules such as anticalins linked to a monoclonal antibody (mAb), represents an easy and low-cost way to obtain innovative molecules. We fused an anticalin against human fibronectin to a molecule biosimilar to trastuzumab (H0) or rituximab (R0), in four different positions, two on the N terminal region of heavy or light chains and two on the C terminal region. The eight BsAbs (H family (HF) 1 to 4 and R family (RF) 1 to 4) were produced and their affinity parameters and functional properties evaluated. The presence of anticalin did not change the glycosylation of the BsAb, shape or yield. The antigenic recognition of each BsAb family, Her2 for HF1 to 4 and CD20 for RF1 to 4, was slightly decreased (HF) or absent (RF) for the anticalin N-terminal in the light chain position. The anticalin recognition of FN was slightly decreased for the HF family, but a dramatic decrease was observed for RF members with lowest affinity for RF1. Moreover, functional properties of Abs, such as CD16 activation of NK, CD32-dependent phagocytosis and FcRn transcytosis, confirmed that this anticalin position leads to less efficient BsAbs, more so for RF than HF molecules. Nevertheless, all BsAbs demonstrated affinities for CD16, CD32 and FcRn, which suggests that more than affinity for FcRs is needed for a functioning antibody. Our strategy using anticalin and Abs allows for rapid generation of BsAbs, but as suggested by our results, some positions of anticalins on Abs result in less functionality.

Intra-Domain Cysteines (IDC), a New Strategy for the Development of Original Antibody Fragment-Drug Conjugates (FDCs).

Antibody-drug conjugates (ADCs) derived from a full immunoglobulin-G (IgG) are associated with suboptimal solid-tumor penetration and Fc-mediated toxicities. Antibody fragment-drug conjugates (FDCs) could be an alternative. Nevertheless, innovative solutions are needed to implant cysteines as conjugation sites in the single-chain fragment variable (scFv) format, which is the backbone from which many other antibody formats are built. In addition, the bioconjugation site has the utmost importance to optimize the safety and efficacy of bioconjugates. Our previous intra-tag cysteine (ITC) strategy consisted of introducing a bioconjugation motif at the C-terminal position of the 4D5.2 scFv, but this motif was subjected to proteolysis when the scFv was produced in CHO cells. Considering these data, using three intra-domain cysteine (IDC) strategies, several parameters were studied to assess the impact of different locations of a site-specific bioconjugation motif in the variable domains of an anti-HER2 scFv. In comparison to the ITC strategy, our new IDC strategy allowed us to identify new fragment-drug conjugates (FDCs) devoid of proteolysis and exhibiting enhanced stability profiles, better affinity, and better ability to kill selectively HER2-positive SK-BR-3 cells in vitro at picomolar concentrations. Thus, this work represents an important optimization step in the design of more complex and effective conjugates.

Targeting IgE polyadenylation signal with antisense oligonucleotides decreases IgE secretion and plasma cell viability.

BACKGROUND: Allergy regroups numerous complex and various diseases classified as IgE-dependent or non-IgE-dependent hypersensitivities. IgEs are expressed as membrane and secreted forms by B cells and plasma cells, respectively. In IgE-mediated hypersensitivity, IgE secretion and binding to the high-affinity IgE receptor FcεRI on effector cells are responsible for the onset of allergic symptoms; in contrast, surface IgE expression as a B-cell receptor is barely detectable. OBJECTIVE: Our aim was to test an innovative antisense approach to reducing IgE secretion. METHODS: We designed an antisense oligonucleotide (ASO) targeting the polyadenylation signal of human secreted IgE to redirect IgE transcript polyadenylation from the secreted form to the membrane form. ASO treatments were performed on B cells from transgenic mice expressing humanized IgE (InEps mice), as well as on human primary B cells and myeloma cells. In vivo ASO delivery was tested by using an InEps mouse model. RESULTS: We demonstrated that treatment with a morpholino ASO targeting the secreted IgE polyadenylation signal drastically decreased IgE secretion and inversely increased membrane IgE mRNA expression. In addition, ASO treatment induced apoptosis of IgE-expressing U266 myeloma cells, and RNA sequencing revealed attenuation of their plasma cell phenotype. Remarkably, systemic administration of an ASO coupled with Pip6a as an arginine-rich cell-penetrating peptide decreased IgE secretion in vivo. CONCLUSION: Altogether, this ASO strategy could be an effective way to decrease IgE secretion and allergic symptoms in patients with IgE-dependent allergies, and it could also promote allergen tolerance through apoptosis of IgE+ antibody-secreting cells.

Exploration and Modulation of Antibody Fragment Biophysical Properties by Replacing the Framework Region Sequences.

In order to increase the successful development of recombinant antibodies and fragments, it seems fundamental to enhance their expression and/or biophysical properties, such as the thermal, chemical, and pH stabilities. In this study, we employed a method bases on replacing the antibody framework region sequences, in order to promote more particularly single-chain Fragment variable (scFv) product quality. We provide evidence that mutations of the VH- C-C' loop might significantly improve the prokaryote production of well-folded and functional fragments with a production yield multiplied by 27 times. Additional mutations are accountable for an increase in the thermal (+19.6 °C) and chemical (+1.9 M) stabilities have also been identified. Furthermore, the hereby-produced fragments have shown to remain stable at a pH of 2.0, which avoids molecule functional and structural impairments during the purification process. Lastly, this study provides relevant information to the understanding of the relationship between the antibodies amino acid sequences and their respective biophysical properties.