Computational Mutagenesis of Antibody Fragments: Disentangling Side Chains from ΔΔG Predictions.

The development of highly potent antibodies and antibody fragments as binding agents holds significant implications in fields such as biosensing and biotherapeutics. Their binding strength is intricately linked to the arrangement and composition of residues at the binding interface. Computational techniques offer a robust means to predict the three-dimensional structure of these complexes and to assess the affinity changes resulting from mutations. Given the interdependence of structure and affinity prediction, our objective here is to disentangle their roles. We aim to evaluate independently six side-chain reconstruction methods and ten binding affinity estimation techniques. This evaluation was pivotal in predicting affinity alterations due to single mutations, a key step in computational affinity maturation protocols. Our analysis focuses on a data set comprising 27 distinct antibody/hen egg white lysozyme complexes, each with crystal structures and experimentally determined binding affinities. Using six different side-chain reconstruction methods, we transformed each structure into its corresponding mutant via in silico single-point mutations. Subsequently, these structures undergo minimization and molecular dynamics simulation. We therefore estimate ΔΔG values based on the original crystal structure, its energy-minimized form, and the ensuing molecular dynamics trajectories. Our research underscores the critical importance of selecting reliable side-chain reconstruction methods and conducting thorough molecular dynamics simulations to accurately predict the impact of mutations. In summary, our study demonstrates that the integration of conformational sampling and scoring is a potent approach to precisely characterizing mutation processes in single-point mutagenesis protocols and crucial for computational antibody design.

[Single chain antibody fragment display systems: a review].

Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.

Facile One-Step Generation of Camelid VHH and Avian scFv Libraries for Phage Display by Golden Gate Cloning.

Since its development in the 1980s, the Nobel Prize-awarded phage display technology has been one of the most commonly used in vitro selection technologies for the discovery of therapeutic and diagnostic antibodies. Besides the importance of selection strategy, one key component of the successful isolation of highly specific recombinant antibodies is the construction of high-quality phage display libraries. However, previous cloning protocols relied on a tedious multistep process with subsequent cloning steps for the introduction of first heavy and then light chain variable genetic antibody fragments (VH and VL). This resulted in reduced cloning efficiency, higher frequency of missing VH or VL sequences, as well as truncated antibody fragments. With the emergence of Golden Gate Cloning (GGC) for the generation of antibody libraries, the possibility of more facile library cloning has arisen. Here, we describe a streamlined one-step GGC strategy for the generation of camelid heavy chain only variable phage display libraries as well as the simultaneous introduction of heavy chain and light chain variable regions from the chicken into a scFv phage display vector.

Replica-exchange optimization of antibody fragments.

In the framework of the rational design of macromolecules capable of binding to a specific target for biosensing applications, we here further develop an evolutionary protocol designed to optimize the binding affinity of protein binders. In particular we focus on the optimization of the binding portion of small antibody fragments known as nanobodies (or VHH) and choose the hen egg white lysozyme (HEWL) as our target. By implementing a replica exchange scheme for this optimization, we show that an initial hit is not needed and similar solutions can be found by either optimizing an already known anti-HEWL VHH or a randomly selected binder (here a VHH selective towards another macromolecule). While we believe that exhaustive searches of the mutation space are most appropriate when only few key residues have to be optimized, in case a lead binder is not available the proposed evolutionary algorithm should be instead the method of choice.

Expression in Pichia pastoris of human antibody fragments that neutralize venoms of Mexican scorpions.

The methylotrophic yeast Pichia pastoris has been one of the most widely used organisms in recent years as an expression system for a wide variety of recombinant proteins with therapeutic potential. Its popularity as an alternative system to Escherichia coli is mainly due to the easy genetic manipulation and the ability to produce high levels of heterologous proteins, either intracellularly or extracellularly. Being a eukaryotic organism, P. pastoris carries out post-translational modifications that allow it to produce soluble and correctly folded recombinant proteins. This work, evaluated the expression capacity in P. pastoris of two single-chain variable fragments (scFvs) of human origin, 10FG2 and LR. These scFvs were previously obtained by directed evolution against scorpion venom toxins and are able to neutralize different toxins and venoms of Mexican species. The yield obtained in P. pastoris was higher than that obtained in bacterial periplasm (E. coli), and most importantly, biochemical and functional properties were not modified. These results confirm that P. pastoris yeast can be a good expression system for the production of antibody fragments of a new recombinant antivenom.

Design of a novel switchable antibody display system in Pichia pastoris.

Yeast surface display (YSD) has been shown to represent a powerful tool in the field of antibody discovery and engineering as well as for selection of high producer clones. However, YSD is predominantly applied in Saccharomyces cerevisiae, whereas expression of heterologous proteins is generally favored in the non-canonical yeast Pichia pastoris (Komagataella phaffii). Establishment of surface display in P. pastoris would therefore enable antibody selection and expression in a single host. Here we describe the generation of a Pichia surface display (PSD) system based on antibody expression from episomal plasmids. By screening a diverse set of expression vectors using Design of Experiments (DoE), the effect of different genetic elements on the surface expression of antibody fragments was analyzed. Among the tested genetic elements, we found that the combination of P. pastoris formaldehyde dehydrogenase (FLD1) promoter, S. cerevisiae invertase 2 signal peptide (SUC2), and α-agglutinin cell wall protein (SAG1) including an autonomously replicating sequence of Kluyveromyces lactis (panARS) were contributing most strongly to higher display levels of three tested antibody fragments. Employing this combination resulted in the display of antibody fragments for up to 25% of cells. Despite significantly reduced expression levels in PSD compared to well-established YSD in S. cerevisiae, similar fractions of antigen binding single-chain variable fragments (scFvs) were observed (80% vs. 84%). In addition, plasmid stability assays and flow cytometric analysis demonstrated the efficient plasmid clearance of cells and associated loss of antibody fragment display after removal of selective pressure. KEY POINTS: • First report of antibody display in P. pastoris using episomal plasmids. • Identification of genetic elements conferring highest levels of antibody display. • Comparable antigen binding capacity of displayed scFvs for PSD compared to YSD.

Optically Responsive Protein Coating of DNA Origami for Triggered Antigen Targeting.

DNA nanostructures have emerged as modular building blocks in several research fields including biomedicine and nanofabrication. Their proneness to degradation in various environments has led to the development of a variety of nature-inspired protection strategies. Coating of DNA origami nanostructures with proteins can circumvent degradation and alter their properties. Here, we have used a single-chain variable antibody fragment and serum albumin to construct positively charged and stimuli-responsive protein-dendron conjugates, which were complexed with DNA origami through electrostatic interactions. Using a stepwise assembly approach, the coated nanostructures were studied for their interaction with the corresponding antigen in fluorescence-based immunoassays. The results suggest that the antibody-antigen interaction can be disturbed by the addition of the bulky serum albumin. However, this effect is fully reversible upon irradiation of the structures with an optical stimulus. This leads to a selective dissociation of the serum albumin from the nanostructure due to cleavage of a photolabile group integrated in the dendron structure, exposing the antibody fragment and enabling triggered binding to the antigen, demonstrating that serum albumin can be considered as an externally controlled "camouflaging" agent. The presented stimuli-responsive complexation approach is highly versatile regarding the choice of protein components and could, therefore, find use in DNA origami protection, targeting, and delivery as well as their spatiotemporal control.

A Recombinant Ig Fragment (IgCw-γεκ) Comprising the Cγ1-Cε2-4 and Cκ Domains Is an Alternative Reagent to Human IgE.

Human IgE is useful for immunological assays, such as sensitization of FcεRI-positive cells and IgE measurement. In this study, we report the development of a recombinant Ig fragment, designated IgCw-γεκ, as an alternative reagent to human IgE. IgCw-γεκ (∼130 kDa) comprises two hybrid constant H chain regions (Cγ1-Cε2-4, each ∼53 kDa) and two constant κ L chains (Cκ, each ∼12 kDa) and lacks a V domain. The presence of Cγ1 instead of Cε1 within the H chain increased the production yield and facilitated assembly of the H and L chains. IgCw-γεκ was produced in cultured human embryonic kidney 293F cells, with a yield of ∼27 mg/l. IgCw-γεκ bound to human FcεRIαRs expressed on the surface of rat basophilic leukemia-2H3 cells. A ß-hexosaminidase release assay revealed that the biological activity of IgCw-γεκ was comparable with that of IgE. The IgE concentration measured using IgCw-γεκ as a standard was similar to that measured using IgE as a standard. These results suggest that the IgCw-γεκ molecule retains the basic characteristics of IgE, but does not cross-react with Ags, making it an alternative to the IgE isotype references used in a variety of immunological assays.

[Bispecific antibodies: An old story with a bright future with CAR-T cells!] / Les anticorps bispécifiques : une vieille histoire pleine d'avenir avec les CAR-T !

CAR-T cells originate from two different approaches, cellular immunotherapy based on tumor immunosurveillance by T lymphocytes, combined with molecular engineering of bispecific antibodies and antibody fragments. The latter makes it possible to retarget immune effector cytotoxic cells (such as NK cells and T lymphocytes) to tumor cells through the binding to tumor-associated antigens. We present herein the history of bispecific antibodies, highlighting how such antibodies played a major role in CAR-T cell development. We will first evoke how antibody engineering led to the construction of various bispecific formats, in particular using the single chain Fv fragment (scFv) which has been used as the initial building block to generate chimeric bi-, tri- or multifunctional molecules. We will also describe how bispecific antibodies, either full IgG or as scFv or F(ab')2 format, directed against Fcγ receptors or CD3ɛ and against tumor-associated antigens, induce a potent anti-tumor cytotoxicity following the recruitment and activation of immune effector cells, including CD3+ T lymphocytes. These anti-tumor effects have been translated into the clinics, especially to treat malignant hemopathies. At last, recently generated bispecific CAR-T cells suggest that the embrace between cell therapy and bispecific antibodies is not over and that we are yet to witness further discoveries enabling these cells to be even more efficient.

Competitive SPR using an intracellular anti-LMO2 antibody identifies novel LMO2-interacting compounds.

The use of intracellular antibodies as templates to derive surrogate compounds is an important objective because intracellular antibodies can be employed initially for target validation in pre-clinical assays and subsequently employed in compound library screens. LMO2 is a T cell oncogenic protein activated in the majority of T cell acute leukaemias. We have used an inhibitory intracellular antibody fragment as a competitor in a small molecule library screen using competitive surface plasmon resonance (cSPR) to identify compounds that bind to LMO2. We selected four compounds that bind to LMO2 but not when the anti-LMO2 intracellular antibody fragment is bound to it. These findings further illustrate the value of intracellular antibodies in the initial stages of drug discovery campaigns and more generally antibodies, or antibody fragments, can be the starting point for chemical compound development as surrogates of the antibody combining site.

Periplasmic expression of SpyTagged antibody fragments enables rapid modular antibody assembly.

Antibodies are essential tools in research and diagnostics. Although antibody fragments typically obtained from in vitro selection can be rapidly produced in bacteria, the generation of full-length antibodies or the modification of antibodies with probes is time and labor intensive. Protein ligation such as SpyTag technology could covalently attach domains and labels to antibody fragments equipped with a SpyTag. However, we found that the established periplasmic expression of antibody fragments in E. coli led to quantitative cleavage of the SpyTag by the proteases Tsp and OmpT. Here we report successful periplasmic expression of SpyTagged Fab fragments and demonstrate the coupling to separately prepared SpyCatcher modules. We used this modular toolbox of SpyCatcher proteins to generate reagents for a variety of immunoassays and measured their performance in comparison with traditional reagents. Furthermore, we demonstrate surface immobilization, high-throughput screening of antibody libraries, and rapid prototyping of antibodies based on modular antibody assembly.

Molecular determinants and mechanism for antibody cocktail preventing SARS-CoV-2 escape.

Antibody cocktails represent a promising approach to prevent SARS-CoV-2 escape. The determinants for selecting antibody combinations and the mechanism that antibody cocktails prevent viral escape remain unclear. We compared the critical residues in the receptor-binding domain (RBD) used by multiple neutralizing antibodies and cocktails and identified a combination of two antibodies CoV2-06 and CoV2-14 for preventing viral escape. The two antibodies simultaneously bind to non-overlapping epitopes and independently compete for receptor binding. SARS-CoV-2 rapidly escapes from individual antibodies by generating resistant mutations in vitro, but it doesn't escape from the cocktail due to stronger mutational constraints on RBD-ACE2 interaction and RBD protein folding requirements. We also identified a conserved neutralizing epitope shared between SARS-CoV-2 and SARS-CoV for antibody CoV2-12. Treatments with CoV2-06 and CoV2-14 individually and in combination confer protection in mice. These findings provide insights for rational selection and mechanistic understanding of antibody cocktails as candidates for treating COVID-19.

The Intervening Removable Affinity Tag (iRAT) System for the Production of Recombinant Antibody Fragments.

Fv and Fab antibody fragments are versatile co-crystallization partners that aid in the structural determination of otherwise "uncrystallizable" proteins, including human/mammalian membrane proteins. Accessible methods for the rapid and reliable production of recombinant antibody fragments have been long sought. In this chapter, we describe the concept and protocols of the intervening removable affinity tag (iRAT) system for the efficient production of Fv and Fab fragments in milligram quantities, which are sufficient for structural studies. As an extension of the iRAT system, we also provide a new method for the creation of genetically encoded fluorescent Fab fragments, which are potentially useful as molecular devices in various basic biomedical and clinical procedures, such as immunofluorescence cytometry, bioimaging, and immunodiagnosis.

Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments.

Antibodies and antibody-derived molecules are continuously developed as both therapeutic agents and key reagents for advanced diagnostic investigations. Their application in these fields has indeed greatly expanded the demand of these molecules and the need for their production in high yield and purity. While full-length antibodies require mammalian expression systems due to the occurrence of functionally and structurally important glycosylations, most antibody fragments and antibody-like molecules are non-glycosylated and can be more conveniently prepared in E. coli-based expression platforms. We propose here an updated survey of the most effective and appropriate methods of preparation of antibody fragments that exploit E. coli as an expression background and review the pros and cons of the different platforms available today. Around 250 references accompany and complete the review together with some lists of the most important new antibody-like molecules that are on the market or are being developed as new biotherapeutics or diagnostic agents.

Secretion of proteins and antibody fragments from transiently transfected endothelial progenitor cells.

In neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis, neuroinflammation can lead to blood-brain barrier (BBB) breakdown. After intravenous or intra-arterial injection into mice, endothelial progenitor cells (EPCs) home to the damaged BBB to promote neurovascular repair. Autologous EPCs transfected to express specific therapeutic proteins offer an innovative therapeutic option. Here, we demonstrate that EPC transfection by electroporation with plasmids encoding the reporter protein GFP or an anti-ß-amyloid antibody fragment (Fab) leads to secretion of each protein. We also demonstrate the secreted anti-ß-amyloid Fab protein functions in ß-amyloid aggregate solubilization.

Multimeric antibodies with increased valency surpassing functional affinity and potency thresholds using novel formats.

The success of therapeutic antibodies is largely attributed for their exquisite specificity, homogeneity, and functionality. There is, however, a need to engineer antibodies to extend and enhance their potency. One parameter is functional affinity augmentation, since antibodies matured in vivo have a natural affinity threshold. Generation of multivalent antibodies is one option capable of surpassing this affinity threshold through increased avidity. In this study, we present a novel platform consisting of an array of multivalent antibody formats, termed Quads, generated using the self-assembling tetramerization domain from p53. We demonstrate the versatility of this tetramerization domain by engineering anti-tumor necrosis factor (TNF) Quads that exhibit major increases in binding potency and in neutralizing TNF-mediated cytotoxicity compared to parental anti-TNF molecules. Further, Quads are amenable to fusion with different binding domains, allowing generation of novel multivalent monospecific and bispecific formats. Quads are thus a novel group of molecules that can be engineered to yield potential therapeutics with novel modalities and potencies.

Single Domain Antibody Fragments as New Tools for the Detection of Neuronal Tau Protein in Cells and in Mice Studies.

Tau is a neuronal protein linked to pathologies called tauopathies, including Alzheimer's disease. In Alzheimer's disease, tau aggregates into filaments, leading to the observation of intraneuronal fibrillary tangles. Molecular mechanisms resulting in tau aggregation and in tau pathology spreading through the brain regions are still not fully understood. New tools are thus needed to decipher tau pathways involved in the diseases. In this context, a family of novel single domain antibody fragments, or VHHs, directed against tau were generated and characterized. Among the selected VHHs obtained from screening of a synthetic library, a family of six VHHs shared the same CDR3 recognition loop and recognized the same epitope, located in the C-terminal domain of tau. Affinity parameters characterizing the tau/VHHs interaction were next evaluated using surface plasmon resonance spectroscopy. The equilibrium constants KD were in the micromolar range, but despite conservation of the CDR3 loop sequence, a range of affinities was observed for this VHH family. One of these VHHs, named F8-2, was additionally shown to bind tau upon expression in a neuronal cell line model. Optimization of VHH F8-2 by yeast two-hybrid allowed the generation of an optimized VHH family characterized by lower KD than that of the F8-2 wild-type counterpart, and recognizing the same epitope. The optimized VHHs can also be used as antibodies for detecting tau in transgenic mice brain tissues. These results validate the use of these VHHs for in vitro studies, but also their potential for in-cell expression and assays in mouse models, to explore the mechanisms underlying tau physiopathology.

Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces.

Antibodies developed for research and clinical applications may exhibit suboptimal stability, expressibility, or affinity. Existing optimization strategies focus on surface mutations, whereas natural affinity maturation also introduces mutations in the antibody core, simultaneously improving stability and affinity. To systematically map the mutational tolerance of an antibody variable fragment (Fv), we performed yeast display and applied deep mutational scanning to an anti-lysozyme antibody and found that many of the affinity-enhancing mutations clustered at the variable light-heavy chain interface, within the antibody core. Rosetta design combined enhancing mutations, yielding a variant with tenfold higher affinity and substantially improved stability. To make this approach broadly accessible, we developed AbLIFT, an automated web server that designs multipoint core mutations to improve contacts between specific Fv light and heavy chains (http://AbLIFT.weizmann.ac.il). We applied AbLIFT to two unrelated antibodies targeting the human antigens VEGF and QSOX1. Strikingly, the designs improved stability, affinity, and expression yields. The results provide proof-of-principle for bypassing laborious cycles of antibody engineering through automated computational affinity and stability design.

Fusion of thymosin alpha 1 with mutant IgG1 CH3 prolongs half-life and enhances antitumor effects in vivo.

Thymosin alpha 1 (Tα1) is an immunomodulatory polypeptide secreted from the thymus. Tα1 has a wide range of biological functions, such as immunomodulation and endocrine regulation. Tα1 also displays antiviral and antitumor activities. Tα1 has been successfully used in clinical adjuvant therapy for solid tumors to improve the immune response of patients undergoing chemotherapy and radiotherapy. However, the half-life of Tα1 in the body is short, so frequent administration is required to maintain efficacy. In order to improve the pharmacokinetic profile of Tα1, we linked the mutated CH3 (mCH3) fragment of IgG1 (human) to the C-terminus of Tα1 to produce a long-acting fusion protein, Tα1-mCH3. The half-life of Tα1-mCH3 (47 h) was substantially increased compared with that of the parent molecule Tα1 (3 h). In vivo studies indicated that mCH3 fusion retained the original biological activity of Tα1, and Tα1-mCH3 showed slightly better immunomodulatory effect than Ta1. In the 4 T1 and B16F10 tumor xenograft models, Tα1-mCH3 induced a greater abundance of CD4+ and CD8+ T-cells in tumor tissues compared with Ta1. Tα1-mCH3 exhibited better effect in promoting the production of IL-2 and IFN-γ compared with Tα1. Therefore, Tα1-mCH3 more efficiently inhibited the growth of 4 T1 and B16F10 tumors than Tα1. In conclusion, fusion with mCH3 is an attractive strategy to lengthen the half-life and increase the activity of Tα1.

Antibody Fragments Humanization: Beginning with the End in Mind.

Molecular engineering has made possible to reformat monoclonal antibodies into smaller antigen-binding structures like scFvs, diabodies, Fabs with new potential in vivo applications because they do not induce Fc-mediated functions. However, most of these molecules are from rodent origin. As a consequence, they are immunogenic and approval for administration to humans requires prior humanization. Today, there is no well-identified strategy to create recombinant humanized antibody V-domains that preserve the antigen-binding characteristics of the parental antibody associated with high stability and solubility. Here, we propose a strategy that consists in grafting CDRs onto properly chosen human antibody frameworks in order to reduce immunogenicity. A flowchart indicates the way to proceed in order to introduce an internal affinity purification tag while structural refinements are proposed to maintain antigen-binding characteristics. The best humanized candidates are identified through selection steps including in silico analysis, research scale production followed by early functional evaluation, purification assays, aggregation, and stability assessment.