Showing papers in "Antibodies in 2019"

Antibody Structure and Function: The Basis for Engineering Therapeutics.

Abstract: Antibodies and antibody-derived macromolecules have established themselves as the mainstay in protein-based therapeutic molecules (biologics). Our knowledge of the structure–function relationships of antibodies provides a platform for protein engineering that has been exploited to generate a wide range of biologics for a host of therapeutic indications. In this review, our basic understanding of the antibody structure is described along with how that knowledge has leveraged the engineering of antibody and antibody-related therapeutics having the appropriate antigen affinity, effector function, and biophysical properties. The platforms examined include the development of antibodies, antibody fragments, bispecific antibody, and antibody fusion products, whose efficacy and manufacturability can be improved via humanization, affinity modulation, and stability enhancement. We also review the design and selection of binding arms, and avidity modulation. Different strategies of preparing bispecific and multispecific molecules for an array of therapeutic applications are included.

David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments.

Abstract: Since the licensing of the first monoclonal antibody therapy in 1986, monoclonal antibodies have become the largest class of biopharmaceuticals with over 80 antibodies currently approved for a variety of disease indications. The development of smaller, antigen binding antibody fragments, derived from conventional antibodies or produced recombinantly, has been growing at a fast pace. Antibody fragments can be used on their own or linked to other molecules to generate numerous possibilities for bispecific, multi-specific, multimeric, or multifunctional molecules, and to achieve a variety of biological effects. They offer several advantages over full-length monoclonal antibodies, particularly a lower cost of goods, and because of their small size they can penetrate tissues, access challenging epitopes, and have potentially reduced immunogenicity. In this review, we will discuss the structure, production, and mechanism of action of EMA/FDA-approved fragments and of those in clinical and pre-clinical development. We will also discuss current topics of interest surrounding the potential use of antibody fragments for intracellular targeting and blood-brain barrier (BBB) penetration.

Design and Production of Bispecific Antibodies.

Abstract: With the current biotherapeutic market dominated by antibody molecules, bispecific antibodies represent a key component of the next-generation of antibody therapy. Bispecific antibodies can target two different antigens at the same time, such as simultaneously binding tumor cell receptors and recruiting cytotoxic immune cells. Structural diversity has been fast-growing in the bispecific antibody field, creating a plethora of novel bispecific antibody scaffolds, which provide great functional variety. Two common formats of bispecific antibodies on the market are the single-chain variable fragment (scFv)-based (no Fc fragment) antibody and the full-length IgG-like asymmetric antibody. Unlike the conventional monoclonal antibodies, great production challenges with respect to the quantity, quality, and stability of bispecific antibodies have hampered their wider clinical application and acceptance. In this review, we focus on these two major bispecific types and describe recent advances in the design, production, and quality of these molecules, which will enable this important class of biologics to reach their therapeutic potential.

IgA: Structure, Function, and Developability.

Abstract: Immunoglobulin A (IgA) plays a key role in defending mucosal surfaces against attack by infectious microorganisms. Such sites present a major site of susceptibility due to their vast surface area and their constant exposure to ingested and inhaled material. The importance of IgA to effective immune defence is signalled by the fact that more IgA is produced than all the other immunoglobulin classes combined. Indeed, IgA is not just the most prevalent antibody class at mucosal sites, but is also present at significant concentrations in serum. The unique structural features of the IgA heavy chain allow IgA to polymerise, resulting in mainly dimeric forms, along with some higher polymers, in secretions. Both serum IgA, which is principally monomeric, and secretory forms of IgA are capable of neutralising and removing pathogens through a range of mechanisms, including triggering the IgA Fc receptor known as FcαRI or CD89 on phagocytes. The effectiveness of these elimination processes is highlighted by the fact that various pathogens have evolved mechanisms to thwart such IgA-mediated clearance. As the structure–function relationships governing the varied capabilities of this immunoglobulin class come into increasingly clear focus, and means to circumvent any inherent limitations are developed, IgA-based monoclonal antibodies are set to emerge as new and potent options in the therapeutic arena.

Bispecific T-Cell Redirection versus Chimeric Antigen Receptor (CAR)-T Cells as Approaches to Kill Cancer Cells.

Abstract: The concepts for T-cell redirecting bispecific antibodies (TRBAs) and chimeric antigen receptor (CAR)-T cells are both at least 30 years old but both platforms are just now coming into age. Two TRBAs and two CAR-T cell products have been approved by major regulatory agencies within the last ten years for the treatment of hematological cancers and an additional 53 TRBAs and 246 CAR cell constructs are in clinical trials today. Two major groups of TRBAs include small, short-half-life bispecific antibodies that include bispecific T-cell engagers (BiTE®s) which require continuous dosing and larger, mostly IgG-like bispecific antibodies with extended pharmacokinetics that can be dosed infrequently. Most CAR-T cells today are autologous, although significant strides are being made to develop off-the-shelf, allogeneic CAR-based products. CAR-Ts form a cytolytic synapse with target cells that is very different from the classical immune synapse both physically and mechanistically, whereas the TRBA-induced synapse is similar to the classic immune synapse. Both TRBAs and CAR-T cells are highly efficacious in clinical trials but both also present safety concerns, particularly with cytokine release syndrome and neurotoxicity. New formats and dosing paradigms for TRBAs and CAR-T cells are being developed in efforts to maximize efficacy and minimize toxicity, as well as to optimize use with both solid and hematologic tumors, both of which present significant challenges such as target heterogeneity and the immunosuppressive tumor microenvironment.

The Ligands for Human IgG and Their Effector Functions

Abstract: Activation of the humoral immune system is initiated when antibodies recognize an antigen and trigger effector functions through the interaction with Fc engaging molecules. The most abundant immunoglobulin isotype in serum is Immunoglobulin G (IgG), which is involved in many humoral immune responses, strongly interacting with effector molecules. The IgG subclass, allotype, and glycosylation pattern, among other factors, determine the interaction strength of the IgG-Fc domain with these Fc engaging molecules, and thereby the potential strength of their effector potential. The molecules responsible for the effector phase include the classical IgG-Fc receptors (FcγR), the neonatal Fc-receptor (FcRn), the Tripartite motif-containing protein 21 (TRIM21), the first component of the classical complement cascade (C1), and possibly, the Fc-receptor-like receptors (FcRL4/5). Here we provide an overview of the interactions of IgG with effector molecules and discuss how natural variation on the antibody and effector molecule side shapes the biological activities of antibodies. The increasing knowledge on the Fc-mediated effector functions of antibodies drives the development of better therapeutic antibodies for cancer immunotherapy or treatment of autoimmune diseases.

Phage Display Libraries for Antibody Therapeutic Discovery and Development

Abstract: Phage display technology has played a key role in the remarkable progress of discovering and optimizing antibodies for diverse applications, particularly antibody-based drugs. This technology was initially developed by George Smith in the mid-1980s and applied by John McCafferty and Gregory Winter to antibody engineering at the beginning of 1990s. Here, we compare nine phage display antibody libraries published in the last decade, which represent the state of the art in the discovery and development of therapeutic antibodies using phage display. We first discuss the quality of the libraries and the diverse types of antibody repertoires used as substrates to build the libraries, i.e., naive, synthetic, and semisynthetic. Second, we review the performance of the libraries in terms of the number of positive clones per panning, hit rate, affinity, and developability of the selected antibodies. Finally, we highlight current opportunities and challenges pertaining to phage display platforms and related display technologies.

Targeted Nanobody-Based Molecular Tracers for Nuclear Imaging and Image-Guided Surgery

Abstract: Molecular imaging is paving the way towards noninvasive detection, staging, and treatment follow-up of diseases such as cancer and inflammation-related conditions. Monoclonal antibodies have long been one of the staples of molecular imaging tracer design, although their long blood circulation and high nonspecific background limits their applicability. Nanobodies, unique antibody-binding fragments derived from camelid heavy-chain antibodies, have excellent properties for molecular imaging as they are able to specifically find their target early after injection, with little to no nonspecific background. Nanobody-based tracers using either nuclear or fluorescent labels have been heavily investigated preclinically and are currently making their way into the clinic. In this review, we will discuss different important factors in nanobody-tracer design, as well as the current state of the art regarding their application for nuclear and fluorescent imaging purposes. Furthermore, we will discuss how nanobodies can also be exploited for molecular therapy applications such as targeted radionuclide therapy and photodynamic therapy.

Nanobody Engineering: Toward Next Generation Immunotherapies and Immunoimaging of Cancer

Abstract: In the last decade, cancer immunotherapies have produced impressive therapeutic results. However, the potency of immunotherapy is tightly linked to immune cell infiltration within the tumor and varies from patient to patient. Thus, it is becoming increasingly important to monitor and modulate the tumor immune infiltrate for an efficient diagnosis and therapy. Various bispecific approaches are being developed to favor immune cell infiltration through specific tumor targeting. The discovery of antibodies devoid of light chains in camelids has spurred the development of single domain antibodies (also called VHH or nanobody), allowing for an increased diversity of multispecific and/or multivalent formats of relatively small sizes endowed with high tissue penetration. The small size of nanobodies is also an asset leading to high contrasts for non-invasive imaging. The approval of the first therapeutic nanobody directed against the von Willebrand factor for the treatment of acquired thrombotic thrombocypenic purpura (Caplacizumab, Ablynx), is expected to bolster the rise of these innovative molecules. In this review, we discuss the latest advances in the development of nanobodies and nanobody-derived molecules for use in cancer immunotherapy and immunoimaging.

Macro- and Micro-Heterogeneity of Natural and Recombinant IgG Antibodies.

Abstract: Recombinant monoclonal antibodies (mAbs) intended for therapeutic usage are required to be thoroughly characterized, which has promoted an extensive effort towards the understanding of the structures and heterogeneity of this major class of molecules. Batch consistency and comparability are highly relevant to the successful pharmaceutical development of mAbs and related products. Small structural modifications that contribute to molecule variants (or proteoforms) differing in size, charge or hydrophobicity have been identified. These modifications may impact (or not) the stability, pharmacokinetics, and efficacy of mAbs. The presence of the same type of modifications as found in endogenous immunoglobulin G (IgG) can substantially lower the safety risks of mAbs. The knowledge of modifications is also critical to the ranking of critical quality attributes (CQAs) of the drug and define the Quality Target Product Profile (QTPP). This review provides a summary of the current understanding of post-translational and physico-chemical modifications identified in recombinant mAbs and endogenous IgGs at physiological conditions.

IgE Antibodies: From Structure to Function and Clinical Translation.

Abstract: Immunoglobulin E (IgE) antibodies are well known for their role in mediating allergic reactions, and their powerful effector functions activated through binding to Fc receptors FceRI and FceRII/CD23. Structural studies of IgE-Fc alone, and when bound to these receptors, surprisingly revealed not only an acutely bent Fc conformation, but also subtle allosteric communication between the two distant receptor-binding sites. The ability of IgE-Fc to undergo more extreme conformational changes emerged from structures of complexes with anti-IgE antibodies, including omalizumab, in clinical use for allergic disease; flexibility is clearly critical for IgE function, but may also be exploited by allosteric interference to inhibit IgE activity for therapeutic benefit. In contrast, the power of IgE may be harnessed to target cancer. Efforts to improve the effector functions of therapeutic antibodies for cancer have almost exclusively focussed on IgG1 and IgG4 subclasses, but IgE offers an extremely high affinity for FceRI receptors on immune effector cells known to infiltrate solid tumours. Furthermore, while tumour-resident inhibitory Fc receptors can modulate the effector functions of IgG antibodies, no inhibitory IgE Fc receptors are known to exist. The development of tumour antigen-specific IgE antibodies may therefore provide an improved immune functional profile and enhanced anti-cancer efficacy. We describe proof-of-concept studies of IgE immunotherapies against solid tumours, including a range of in vitro and in vivo evaluations of efficacy and mechanisms of action, as well as ex vivo and in vivo safety studies. The first anti-cancer IgE antibody, MOv18, the clinical translation of which we discuss herein, has now reached clinical testing, offering great potential to direct this novel therapeutic modality against many other tumour-specific antigens. This review highlights how our understanding of IgE structure and function underpins these exciting clinical developments.

Current advancements in addressing key challenges of therapeutic antibody design, manufacture, and formulation

Abstract: Therapeutic antibody technology heavily dominates the biologics market and continues to present as a significant industrial interest in developing novel and improved antibody treatment strategies. Many noteworthy advancements in the last decades have propelled the success of antibody development; however, there are still opportunities for improvement. In considering such interest to develop antibody therapies, this review summarizes the array of challenges and considerations faced in the design, manufacture, and formulation of therapeutic antibodies, such as stability, bioavailability and immunological engagement. We discuss the advancement of technologies that address these challenges, highlighting key antibody engineered formats that have been adapted. Furthermore, we examine the implication of novel formulation technologies such as nanocarrier delivery systems for the potential to formulate for pulmonary delivery. Finally, we comprehensively discuss developments in computational approaches for the strategic design of antibodies with modulated functions.

Immunogenicity of Innovative and Biosimilar Monoclonal Antibodies

Abstract: The development of hybridoma technology for producing monoclonal antibodies (mAbs) by Kohler and Milstein (1975) counts as one of the major medical breakthroughs, opening up endless possibilities for research, diagnosis and for treatment of a whole variety of diseases. Therapeutic mAbs were introduced three decades ago. The first generation of therapeutic mAbs of murine origin showed high immunogenicity, which limited efficacy and was associated with severe infusion reactions. Subsequently chimeric, humanized, and fully human antibodies were introduced as therapeutics, these mAbs were considerably less immunogenic. Unexpectedly humanized mAbs generally show similar immunogenicity as chimeric antibodies; based on sequence homology chimeric mAbs are sometimes more "human" than humanized mAbs. With the introduction of the regulatory concept of similar biological medicines (biosimilars) a key concern is the similarity in terms of immunogenicity of these biosimilars with their originators. This review focuses briefly on the mechanisms of induction of immunogenicity by biopharmaceuticals, mAbs in particular, in relation to the target of the immune system.

Understanding Inter-Individual Variability in Monoclonal Antibody Disposition.

Abstract: Monoclonal antibodies (mAbs) are currently the largest and most dominant class of therapeutic proteins. Inter-individual variability has been observed for several mAbs; however, an understanding of the underlying mechanisms and factors contributing to inter-subject differences in mAb disposition is still lacking. In this review, we analyze the mechanisms of antibody disposition and the putative mechanistic determinants of inter-individual variability. Results from in vitro, preclinical, and clinical studies were reviewed evaluate the role of the neonatal Fc receptor and Fc gamma receptors (expression and polymorphism), target properties (expression, shedding, turnover, internalization, heterogeneity, polymorphism), and the influence of anti-drug antibodies. Particular attention is given to the influence of co-administered drugs and disease, and to the physiological relevance of covariates identified by population pharmacokinetic modeling, as determinants of variability in mAb pharmacokinetics.

IgG Charge: Practical and Biological Implications

Abstract: Practically, IgG charge can contribute significantly to thermodynamic nonideality, and hence to solubility and viscosity. Biologically, IgG charge isomers exhibit differences in clearance and potency. It has been known since the 1930s that all immunoglobulins carry a weak negative charge in physiological solvents. However, there has been no systematic exploration of this fundamental property. Accurate charge measurements have been made using membrane confined electrophoresis in two solvents (pH 5.0 and pH 7.4) on a panel of twelve mAb IgGs, as well as their F(ab')2 and Fc fragments. The following observations were made at pH 5.0: (1) the measured charge differs from the calculated charge by ~40 for the intact IgGs, and by ~20 for the Fcs; (2) the intact IgG charge depends on both Fv and Fc sequences, but does not equal the sum of the F(ab)'2 and Fc charge; (3) the Fc charge is consistent within a class. In phosphate buffered saline, pH 7.4: (1) the intact IgG charges ranged from 0 to -13; (2) the F(ab')2 fragments are nearly neutral for IgG1s and IgG2s, and about -5 for some of the IgG4s; (3) all Fc fragments are weakly anionic, with IgG1 < IgG2 < IgG4; (4) the charge on the intact IgGs does not equal the sum of the F(ab')2 and Fc charge. In no case is the calculated charge, based solely on H+ binding, remotely close to the measured charge. Some mAbs carried a charge in physiological salt that was outside the range observed for serum-purified human poly IgG. To best match physiological properties, a therapeutic mAb should have a measured charge that falls within the range observed for serum-derived human IgGs. A thermodynamically rigorous, concentration-dependent protein-protein interaction parameter is introduced. Based on readily measured properties, interaction curves may be generated to aid in the selection of proteins and solvent conditions. Example curves are provided.

Targeting the MHC Ligandome by Use of TCR-Like Antibodies.

Abstract: Monoclonal antibodies (mAbs) are valuable as research reagents, in diagnosis and in therapy. Their high specificity, the ease in production, favorable biophysical properties and the opportunity to engineer different properties make mAbs a versatile class of biologics. mAbs targeting peptide–major histocompatibility molecule (pMHC) complexes are often referred to as “TCR-like” mAbs, as pMHC complexes are generally recognized by T-cell receptors (TCRs). Presentation of self- and non-self-derived peptide fragments on MHC molecules and subsequent activation of T cells dictate immune responses in health and disease. This includes responses to infectious agents or cancer but also aberrant responses against harmless self-peptides in autoimmune diseases. The ability of TCR-like mAbs to target specific peptides presented on MHC allows for their use to study peptide presentation or for diagnosis and therapy. This extends the scope of conventional mAbs, which are generally limited to cell-surface or soluble antigens. Herein, we review the strategies used to generate TCR-like mAbs and provide a structural comparison with the analogous TCR in pMHC binding. We further discuss their applications as research tools and therapeutic reagents in preclinical models as well as challenges and limitations associated with their use.

Multiplex LC-MS/MS Assays for Clinical Bioanalysis of MEDI4276, an Antibody-Drug Conjugate of Tubulysin Analogue Attached via Cleavable Linker to a Biparatopic Humanized Antibody against HER-2.

Abstract: Bioanalysis of complex biotherapeutics, such as antibody-drug conjugates (ADCs), is challenging and requires multiple assays to describe their pharmacokinetic (PK) profiles. To enable exposure-safety and exposure-efficacy analyses, as well as to understand the metabolism of ADC therapeutics, three bioanalytical methods are typically employed: Total Antibody, Antibody Conjugated Toxin or Total ADC and Unconjugated Toxin. MEDI4276 is an ADC comprised of biparatopic humanized antibody attached via a protease-cleavable peptide-based maleimidocaproyl linker to a tubulysin toxin (AZ13599185) with an approximate average drug-antibody ratio of 4. The conjugated payload of MEDI4276 can undergo ester hydrolysis to produce the conjugated payload AZ13687308, leading to the formation of MEDI1498 (de-acetylated MEDI4276). In this report, we describe the development, validation and application of three novel multiplex bioanalytical methods. The first ligand-binding liquid chromatography coupled with tandem mass spectrometry (LBA-LC-MS/MS) method was developed and validated for simultaneous measurement of total antibody and total ADC (antibody-conjugated AZ13599185) from MEDI4276. The second LBA-LC-MS/MS assay quantified total ADC (antibody-conjugated AZ13687308) from MEDI1498. The third multiplex LC-MS/MS assay was used for simultaneous quantification of unconjugated AZ13599185 and AZ13687308. Additional stability experiments confirmed that quantification of the released warhead in the presence of high concentrations of MEDI4276 was acceptable. Subsequently, the assays were employed in support of a first-in-human clinical trial (NCT02576548).

Single-Domain Antibodies as Therapeutic and Imaging Agents for the Treatment of CNS Diseases.

Abstract: Antibodies have become one of the most successful therapeutics for a number of oncology and inflammatory diseases. So far, central nervous system (CNS) indications have missed out on the antibody revolution, while they remain ‘hidden’ behind several hard to breach barriers. Among the various antibody modalities, single-domain antibodies (sdAbs) may hold the ‘key’ to unlocking the access of antibody therapies to CNS diseases. The unique structural features of sdAbs make them the smallest monomeric antibody fragments suitable for molecular targeting. These features are of particular importance when developing antibodies as modular building blocks for engineering CNS-targeting therapeutics and imaging agents. In this review, we first introduce the characteristic properties of sdAbs compared to traditional antibodies. We then present recent advances in the development of sdAbs as potential therapeutics across brain barriers, including their use for the delivery of biologics across the blood–brain and blood–cerebrospinal fluid (CSF) barriers, treatment of neurodegenerative diseases and molecular imaging of brain targets.

Therapeutic Monoclonal Antibodies Targeting Immune Checkpoints for the Treatment of Solid Tumors.

Abstract: Recently, modulation of immune checkpoints has risen to prominence as a means to treat a number of solid malignancies, given the durable response seen in many patients and improved side effect profile compared to conventional chemotherapeutic agents. Several classes of immune checkpoint modulators have been developed. Here, we review current monoclonal antibodies directed against immune checkpoints that are employed in practice today. We discuss the history, mechanism, indications, and clinical data for each class of therapies. Furthermore, we review the challenges to durable tumor responses that are seen in some patients and discuss possible interventions to circumvent these barriers.

Dynamic Views of the Fc Region of Immunoglobulin G Provided by Experimental and Computational Observations

Abstract: The Fc portion of immunoglobulin G (IgG) is a horseshoe-shaped homodimer, which interacts with various effector proteins, including Fcγ receptors (FcγRs). These interactions are critically dependent on the pair of N-glycans packed between the two CH2 domains. Fucosylation of these N-glycans negatively affects human IgG1-FcγRIIIa interaction. The IgG1-Fc crystal structures mostly exhibit asymmetric quaternary conformations with divergent orientations of CH2 with respect to CH3. We aimed to provide dynamic views of IgG1-Fc by performing long-timescale molecular dynamics (MD) simulations, which were experimentally validated by small-angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our simulation results indicated that the dynamic conformational ensembles of Fc encompass most of the previously reported crystal structures determined in both free and complex forms, although the major Fc conformers in solution exhibited almost symmetric, stouter quaternary structures, unlike the crystal structures. Furthermore, the MD simulations suggested that the N-glycans restrict the motional freedom of CH2 and endow quaternary-structure plasticity through multiple intramolecular interaction networks. Moreover, the fucosylation of these N-glycans restricts the conformational freedom of the proximal tyrosine residue of functional importance, thereby precluding its interaction with FcγRIIIa. The dynamic views of Fc will provide opportunities to control the IgG interactions for developing therapeutic antibodies.

A Rabbit Monoclonal Antibody against the Antiviral and Cancer Genomic DNA Mutating Enzyme APOBEC3B.

Abstract: The DNA cytosine deaminase APOBEC3B (A3B) is normally an antiviral factor in the innate immune response. However, A3B has been implicated in cancer mutagenesis, particularly in solid tumors of the bladder, breast, cervix, head/neck, and lung. Here, we report data on the generation and characterization of a rabbit monoclonal antibody (mAb) for human A3B. One mAb, 5210-87-13, demonstrates utility in multiple applications, including ELISA, immunoblot, immunofluorescence microscopy, and immunohistochemistry. In head-to-head tests with commercial reagents, 5210-87-13 was the only rabbit monoclonal suitable for detecting native A3B and for immunohistochemical quantification of A3B in tumor tissues. This novel mAb has the potential to enable a wide range of fundamental and clinical studies on A3B in human biology and disease.

Using Nanobodies to Study Protein Function in Developing Organisms

Abstract: Polyclonal and monoclonal antibodies have been invaluable tools to study proteins over the past decades. While indispensable for most biological studies including developmental biology, antibodies have been used mostly in fixed tissues or as binding reagents in the extracellular milieu. For functional studies and for clinical applications, antibodies have been functionalized by covalently fusing them to heterologous partners (i.e., chemicals, proteins or other moieties). Such functionalized antibodies have been less widely used in developmental biology studies. In the past few years, the discovery and application of small functional binding fragments derived from single-chain antibodies, so-called nanobodies, has resulted in novel approaches to study proteins during the development of multicellular animals in vivo. Expression of functionalized nanobody fusions from integrated transgenes allows manipulating proteins of interest in the extracellular and the intracellular milieu in a tissue- and time-dependent manner in an unprecedented manner. Here, we describe how nanobodies have been used in the field of developmental biology and look into the future to imagine how else nanobody-based reagents could be further developed to study the proteome in living organisms.

Ion Channel Targeting with Antibodies and Antibody Fragments for Cancer Diagnosis

Abstract: The antibody era has greatly impacted cancer management in recent decades. Indeed, antibodies are currently applied for both cancer diagnosis and therapy. For example, monoclonal antibodies are the main constituents of several in vitro diagnostics, which are applied at many levels of cancer diagnosis. Moreover, the great improvement provided by in vivo imaging, especially for early-stage cancer diagnosis, has traced the path for the development of a complete new class of antibodies, i.e., engineered antibody fragments. The latter embody the optimal characteristics (e.g., low renal retention, rapid clearance, and small size) which make them ideal for in vivo applications. Furthermore, the present review focuses on reviewing the main applications of antibodies and antibody fragments for solid cancer diagnosis, both in vitro and in vivo. Furthermore, we review the scientific evidence showing that ion channels represent an almost unexplored class of ideal targets for both in vitro and in vivo diagnostic purposes. In particular, we review the applications, in solid cancers, of monoclonal antibodies and engineered antibody fragments targeting the voltage-dependent ion channel Kv 11.1, also known as hERG1.

VHH-Photosensitizer Conjugates for Targeted Photodynamic Therapy of Met-Overexpressing Tumor Cells.

Abstract: Photodynamic therapy (PDT) is an approach that kills (cancer) cells by the local production of toxic reactive oxygen species upon the local illumination of a photosensitizer (PS). The specificity of PDT has been further enhanced by the development of a new water-soluble PS and by the specific delivery of PS via conjugation to tumor-targeting antibodies. To improve tissue penetration and shorten photosensitivity, we have recently introduced nanobodies, also known as VHH (variable domains from the heavy chain of llama heavy chain antibodies), for targeted PDT of cancer cells overexpressing the epidermal growth factor receptor (EGFR). Overexpression and activation of another cancer-related receptor, the hepatocyte growth factor receptor (HGFR, c-Met or Met) is also involved in the progression and metastasis of a large variety of malignancies. In this study we evaluate whether anti-Met VHHs conjugated to PS can also serve as a biopharmaceutical for targeted PDT. VHHs targeting the SEMA (semaphorin-like) subdomain of Met were provided with a C-terminal tag that allowed both straightforward purification from yeast supernatant and directional conjugation to the PS IRDye700DX using maleimide chemistry. The generated anti-Met VHH-PS showed nanomolar binding affinity and, upon illumination, specifically killed MKN45 cells with nanomolar potency. This study shows that Met can also serve as a membrane target for targeted PDT.

Selection and Characterization of a Nanobody Biosensor of GTP-Bound RHO Activities.

Abstract: RHO (Ras HOmologous) GTPases are molecular switches that activate, in their state bound to Guanosine triphosphate (GTP), key signaling pathways, which involve actin cytoskeleton dynamics. Previously, we selected the nanobody RH12, from a synthetic phage display library, which binds the GTP-bound active conformation of RHOA (Ras Homologous family member A). However, when expressed as an intracellular antibody, its blocking effect on RHO signaling led to a loss of actin fibers, which in turn affected cell shape and cell survival. Here, in order to engineer an intracellular biosensor of RHOA-GTP activation, we screened the same phage nanobody library and identified another RHO-GTP selective intracellular nanobody, but with no apparent toxicity. The recombinant RH57 nanobody displays high affinity towards GTP-bound RHOA/B/C subgroup of small GTPases in vitro. Intracellular expression of the RH57 allowed selective co-precipitation with the GTP-bound state of the endogenous RHOA subfamily. When expressed as a fluorescent fusion protein, the chromobody GFP-RH57 was localized to the inner plasma membrane upon stimulation of the activation of endogenous RHO. Finally, the RH57 nanobody was used to establish a BRET-based biosensor (Bioluminescence Resonance Energy Transfer) of RHO activation. The dynamic range of the BRET signal could potentially offer new opportunities to develop cell-based screening of RHOA subfamily activation modulators.

Peptides for Infectious Diseases: From Probe Design to Diagnostic Microarrays.

Abstract: Peptides and peptidomimetics have attracted revived interest regarding their applications in chemical biology over the last few years. Their chemical versatility, synthetic accessibility and the ease of storage and management compared to full proteins have made peptides particularly interesting in diagnostic applications, where they proved to efficiently recapitulate the molecular recognition properties of larger protein antigens, and were proven to be able to capture antibodies circulating in the plasma and serum of patients previously exposed to bacterial or viral infections. Here, we describe the development, integration and application of strategies for computational prediction and design, advanced chemical synthesis, and diagnostic deployment in multiplexed assays of peptide-based materials which are able to bind antibodies of diagnostic as well as therapeutic interest. By presenting successful applications of such an integrated strategy, we argue that they will have an ever-increasing role in both basic and clinical realms of research, where important advances can be expected in the next few years.

Super Potent Bispecific Llama VHH Antibodies Neutralize HIV via a Combination of gp41 and gp120 Epitopes.

Abstract: Broad and potent neutralizing llama single domain antibodies (VHH) against HIV-1 targeting the CD4 binding site (CD4bs) have previously been isolated upon llama immunization. Here we describe the epitopes of three additional VHH groups selected from phage libraries. The 2E7 group binds to a new linear epitope in the first heptad repeat of gp41 that is only exposed in the fusion-intermediate conformation. The 1B5 group competes with co-receptor binding and the 1F10 group interacts with the crown of the gp120 V3 loop, occluded in native Env. We present biophysical and structural details on the 2E7 interaction with gp41. In order to further increase breadth and potency, we constructed bi-specific VHH. The combination of CD4bs VHH (J3/3E3) with 2E7 group VHH enhanced strain-specific neutralization with potencies up to 1400-fold higher than the mixture of the individual VHHs. Thus, these new bivalent VHH are potent new tools to develop therapeutic approaches or microbicide intervention.

A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells.

Abstract: Single-domain antibodies have emerged as highly versatile nanoprobes for advanced cellular imaging. For real-time visualization of endogenous antigens, fluorescently labelled nanobodies (chromobodies, CBs) are introduced as DNA-encoded expression constructs in living cells. Commonly, CB expression is driven from strong, constitutively active promoters. However, high expression levels are sometimes accompanied by misfolding and aggregation of those intracellular nanoprobes. Moreover, stable cell lines derived from random genomic insertion of CB-encoding transgenes bear the risk of disturbed cellular processes and inhomogeneous CB signal intensities due to gene positioning effects and epigenetic silencing. In this study we propose a strategy to generate optimized CB expressing cell lines. We demonstrate that expression as ubiquitin fusion increases the fraction of intracellularly functional CBs and identified the elongation factor 1α (EF1-α) promoter as highly suited for constitutive CB expression upon long-term cell line cultivation. Finally, we applied a CRISPR/Cas9-based gene editing approach for targeted insertion of CB expression constructs into the adeno-associated virus integration site 1 (AAVS1) safe harbour locus of human cells. Our results indicate that this combinatorial approach facilitates the generation of fully functional and stable CB cell lines for quantitative live-cell imaging of endogenous antigens.

Current Approaches and Future Perspectives for Nanobodies in Stroke Diagnostic and Therapy

Abstract: Antibody-based biologics are the corner stone of modern immunomodulatory therapy. Though highly effective in dampening systemic inflammatory processes, their large size and Fc-fragment mediated effects hamper crossing of the blood brain barrier (BBB). Nanobodies (Nbs) are single domain antibodies derived from llama or shark heavy-chain antibodies and represent a new generation of biologics. Due to their small size, they display excellent tissue penetration capacities and can be easily modified to adjust their vivo half-life for short-term diagnostic or long-term therapeutic purposes or to facilitate crossing of the BBB. Furthermore, owing to their characteristic binding mode, they are capable of antagonizing receptors involved in immune signaling and of neutralizing proinflammatory mediators, such as cytokines. These qualities combined make Nbs well-suited for down-modulating neuroinflammatory processes that occur in the context of brain ischemia. In this review, we summarize recent findings on Nbs in preclinical stroke models and how they can be used as diagnostic and therapeutic reagents. We further provide a perspective on the design of innovative Nb-based treatment protocols to complement and improve stroke therapy.

IMGT® and 30 Years of Immunoinformatics Insight in Antibody V and C Domain Structure and Function.

Abstract: At the 10th Human Genome Mapping (HGM10) Workshop, in New Haven, for the first time, immunoglobulin (IG) or antibody and T cell receptor (TR) variable (V), diversity (D), joining (J), and constant (C) genes were officially recognized as ‘genes’, as were the conventional genes. Under these HGM auspices, IMGT®, the international ImMunoGeneTics information system®, was created in June 1989 at Montpellier (University of Montpellier and CNRS). The creation of IMGT® marked the birth of immunoinformatics, a new science, at the interface between immunogenetics and bioinformatics. The accuracy and the consistency between genes and alleles, sequences, and three-dimensional (3D) structures are based on the IMGT Scientific chart rules generated from the IMGT-ONTOLOGY axioms and concepts: IMGT standardized keywords (IDENTIFICATION), IMGT gene and allele nomenclature (CLASSIFICATION), IMGT standardized labels (DESCRIPTION), IMGT unique numbering and IMGT Collier de Perles (NUMEROTATION). These concepts provide IMGT® immunoinformatics insights for antibody V and C domain structure and function, used for the standardized description in IMGT® web resources, databases and tools, immune repertoires analysis, single cell and/or high-throughput sequencing (HTS, NGS), antibody humanization, and antibody engineering in relation with effector properties.