Showing papers by "Serge Muyldermans published in 2011"

Specific Cell Targeting with Nanobody Conjugated Branched Gold Nanoparticles for Photothermal Therapy

Abstract: Branched gold nanoparticles are potential photothermal therapy agents because of their large absorption cross section in the near-infrared window. Upon laser irradiation they produce enough heat to destroy tumor cells. In this work, branched gold nanoparticles are biofunctionalized with nanobodies, the smallest fully functional antigen-binding fragments evolved from the variable domain, the VHH, of a camel heavy chain-only antibody. These nanobodies bind to the HER2 antigen which is highly expressed on breast and ovarian cancer cells. Flow cytometric analysis and dark field images of HER2 positive SKOV3 cells incubated with anti-HER2 conjugated branched gold nanoparticles show specific cell targeting. Laser irradiation studies reveal that HER2 positive SKOV3 cells exposed to the anti-HER2 targeted branched gold nanoparticles are destroyed after five minutes of laser treatment at 38 W/cm2 using a 690 nm continuous wave laser. Starting from a nanoparticle optical density of 4, cell death is observed, wherea...

Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer

Abstract: Accurate determination of tumor human epidermal growth factor receptor 2 (HER2)-status in breast cancer patients is possible via noninvasive imaging, provided adequate tracers are used. In this study, we describe the generation of a panel of 38 nanobodies, small HER2-binding fragments that are derived from heavy-chain-only antibodies raised in an immunized dromedary. In search of a lead compound, a subset of nanobodies was biochemically characterized in depth and preclinically tested for use as tracers for imaging of xenografted tumors. The selected compound, 2Rs15d, was found to be stable and to interact specifically with HER2 recombinant protein and HER2-expressing cells in ELISA, surface plasmon resonance, flow cytometry, and radioligand binding studies with low nanomolar affinities, and did not compete with anti-HER2 therapeutic antibodies trastuzumab and pertuzumab. Single-photon-emission computed tomography (SPECT) imaging quantification and biodistribution analyses showed that (99m)Tc-labeled 2Rs15d has a high tumor uptake in 2 HER2(+) tumor models, fast blood clearance, low accumulation in nontarget organs except kidneys, and high concomitant tumor-to-blood and tumor-to-muscle ratios at 1 h after intravenous injection. These values were dramatically lower for an irrelevant control (99m)Tc-nanobody and for (99m)Tc-2Rs15d targeting a HER2(-) tumor.

A Case Of Convergence: Why Did a Simple Alternative to Canonical Antibodies Arise in Sharks and Camels?

Abstract: The success of our adaptive humoral immune response, mediated by B cells, relies on its capacity to produce multiple antigen-recognition specificities against any possible invader. This antigen-recognition specificity is attributed to antibodies, which display remarkable flexibility and diversity. That diversity is generated by cutting and pasting of immunoglobulin (Ig) gene segments during B-cell development to generate functional variable (V) genes, which are selected from two separate regions of the genome to yield so-called “heavy” (H) and “light” (L) chains (Figure 1; see Section 1 in Text S1). Ig H and L chain V genes have been expanded in various vertebrate taxa to yield multiple families and subfamilies [1]. Given the obvious advantage of the diversity afforded by such multichain antibodies, it was extremely surprising to discover that some species—notably cartilaginous fish (holocephalins, e.g., chimeras, and elasmobranchs, e.g., sharks, skates, and rays) and camelids (e.g., camels and llamas)—developed functional H-chain-only antibodies (HCAbs) (Figures 1 and ​and2)2) [2],[3]. Loss of the combinatorial H-L diversification of heterotetrameric (H2L2) antibodies should result in a handicapped, less diversified antigen-binding repertoire in HCAbs; yet, contrary to expectations, high-affinity, antigen-reactive HCAbs can be elicited to all studied antigens [4],[5]. Even more peculiar, the HCAbs function alongside conventional H2L2 antibodies. What explains this remarkable evolutionary convergence of functional HCAbs in sharks and camels? What forces drove such distantly related species to produce antibodies with this simple structure? And, if they are so effective why is their prevalence not more widespread in evolution? Figure 1 Schematic of classical H2L2 and H2 homodimeric HCAbs. Figure 2 Simplified vertebrate phylogenetic tree emphasizing those taxa possessing single-domain Igs or TCRs. Occurrence of Functional Heavy Chain-Only Antibodies in Camelids and Shark The antigen-binding fragment of camelid HCAbs consists of a single domain known as a variable domain of the heavy chain of HCAbs (VHH) (Figure 1) [2]. It was demonstrated that HCAbs from dromedaries (one-humped camels) infected with trypanosomes are capable of associating tightly and specifically with these parasite antigens [2]. Subsequently, HCAbs were elicited to many different foreign antigens. HCAbs are present in all living species of the mammalian family Camelidae, but not in other ungulates (hoofed mammals). Approximately equal levels of HCAbs and H2L2 antibodies are present in camel blood [6]. The origin of camelid Ig genes encoding HCAbs is unambiguous. Phylogenetic analyses have demonstrated that the HCAb-dedicated γ genes were derived from γ genes coding for conventional antibodies. The genes emerged and diverged ∼25 million years ago [6],[7], after Tylopoda split from other mammals (60–80 million years ago) and before the camel and llama speciation (∼11 million years ago) (Figure 2) [6]. Also, the VHH germline genes descended from the classical variable region of a heavy-chain (VH) genes, and the diversity (D) and JH genes are even shared in the formation of the VH or VHH domains. Thus, the emergence of HCAbs was a relatively recent event in these species. In the nurse shark (Ginglymostoma cirratum), a novel secreted antigen receptor was reported and named the new antigen receptor (NAR) [3]. It too is composed of two covalently associated H-chains with no associated L-chains, but having six domains (Figure 1). Since the molecule shares several functional features with Ig isotypes, it was renamed IgNAR [8], and its N-terminal V domain is known as V-NAR. Later studies showed IgNAR to be induced by immunization with protein antigens with similar kinetics as in a typical IgM response [9]. Although the origin of shark IgNAR is poorly understood, IgNAR is found in all elasmobranchs and thus emerged at least 220 million years ago (Figure 2). However, the single-domain V is also found as a T-cell receptor (TCR) in all living cartilaginous fish (so-called NAR-TCR) [10],[11], showing that this immune system feature appeared at least 350 million years ago (Figure 2). In addition, V-NAR gene clusters actually are present in the TCR-δ loci of all cartilaginous fish and thus it remains uncertain whether they originated as a TCR, an Ig, or perhaps a common antigen receptor ancestor [10].

Localization, mechanism and reduction of renal retention of technetium-99m labeled epidermal growth factor receptor-specific nanobody in mice.

Abstract: BACKGROUND: Nanobodies are single-domain antigen binding fragments derived from functional heavy-chain antibodies elicited in Camelidae. They are powerful probes for radioimmunoimaging, but their renal uptake is relatively high. In this study we have evaluated the role of megalin on the renal uptake of anti-EGFR (99m)Tc-7C12 nanobody and the potency of gelofusine and/or lysine to reduce renal uptake of (99m)Tc-7C12. METHODS: First we compared the renal uptake of (99m)Tc-7C12 in megalin-deficient and megalin-wild-type mice using pinhole SPECT/microCT and ex vivo analysis. The effect of gelofusine and lysine administration on renal accumulation of (99m)Tc-7C12 was analyzed in CD-1 mice divided into lysine preload at 30 min before tracer injection (LysPreload), LysPreload + gelofusine coadministration (LysPreload + GeloCoad), lysine coadministration (LysCoad), gelofusine coadministration (GeloCoad) and LysCoad + GeloCoad. The combined effect of gelofusine and lysine on tumor uptake was tested in mice xenografts. RESULTS: Renal uptake of (99m)Tc-7C12 was 44.22 +/- 3.46% lower in megalin-deficient compared with megalin-wild-type mice. In CD-1 mice, lysine preload had no effect on the renal retention whereas coinjection of lysine or gelofusine with the tracer resulted in 25.12 +/- 2.99 and 36.22 +/- 3.07% reduction, respectively. The combined effect of gelofusine and lysine was the most effective, namely a reduction of renal retention of 45.24 +/- 2.09%. Gelofusine and lysine coadministration improved tumor uptake. CONCLUSION: Megalin contributes to the renal accumulation of (99m)Tc-7C12. Gelofusine and lysine coinjection with the tracer reduces the renal uptake while tumor uptake is improved. Although this methodology allows for optimization of imaging protocol using nanobodies, further improvements are needed before using these molecules for radionuclide therapy.

High affinity nanobodies against the Trypanosome brucei VSG are potent trypanolytic agents that block endocytosis

Abstract: The African trypanosome Trypanosoma brucei, which persists within the bloodstream of the mammalian host, has evolved potent mechanisms for immune evasion. Specifically, antigenic variation of the variant-specific surface glycoprotein (VSG) and a highly active endocytosis and recycling of the surface coat efficiently delay killing mediated by anti-VSG antibodies. Consequently, conventional VSG-specific intact immunoglobulins are non-trypanocidal in the absence of complement. In sharp contrast, monovalent antigen-binding fragments, including 15 kDa nanobodies (Nb) derived from camelid heavy-chain antibodies (HCAbs) recognizing variant-specific VSG epitopes, efficiently lyse trypanosomes both in vitro and in vivo. This Nb-mediated lysis is preceded by very rapid immobilisation of the parasites, massive enlargement of the flagellar pocket and major blockade of endocytosis. This is accompanied by severe metabolic perturbations reflected by reduced intracellular ATP-levels and loss of mitochondrial membrane potential, culminating in cell death. Modification of anti-VSG Nbs through site-directed mutagenesis and by reconstitution into HCAbs, combined with unveiling of trypanolytic activity from intact immunoglobulins by papain proteolysis, demonstrates that the trypanolytic activity of Nbs and Fabs requires low molecular weight, monovalency and high affinity. We propose that the generation of low molecular weight VSG-specific trypanolytic nanobodies that impede endocytosis offers a new opportunity for developing novel trypanosomiasis therapeutics. In addition, these data suggest that the antigen-binding domain of an anti-microbial antibody harbours biological functionality that is latent in the intact immunoglobulin and is revealed only upon release of the antigen-binding fragment.

Correlation Between Epidermal Growth Factor Receptor-Specific Nanobody Uptake and Tumor Burden: A Tool for Noninvasive Monitoring of Tumor Response to Therapy

Abstract: Purpose Nanobodies represent an interesting class of probes for the generic development of molecular imaging agents We studied the relationship between tumor uptake of the epidermal growth factor receptor (EGFR)-specific nanobody 99mTc-7C12 and tumor burden and evaluated the possibility of using this probe to monitor tumor response to erlotinib

Development of Cys38 knock-out and humanized version of NbAahII10 nanobody with improved neutralization of AahII Scorpion toxin

Abstract: During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim, causing serious medical problems. Nanobodies (Nbs), the recombinant single-domain antigen-binding fragments of camel-specific heavy-chain only antibodies, offer special advantages in therapy over classic antibody fragments due to their robustness and smaller size, matching the size of the scorpion toxins. Recently, a potent AahII scorpion toxin-neutralizing Nb was identified. However, this NbAahII10 contains a single Cys in its first antigen-binding loop, leading to Nb dimerization upon prolonged storage. In this work, we first investigate the efficacy of NbAahII10 variants in which this Cys was substituted by Ala, Ser or Thr. Second, the NbAahII10 Cys/Ser mutant displaying the best functional properties is subsequently humanized. It is demonstrated that the maximally humanized version of NbAahII10 Cys/Ser maintains its high affinity for the antigen without conceding much on expression yield and stability. More importantly, its neutralizing capacity is preserved as all mice survive injections of seven LD(50) and 50% of mice survived nine LD(50) of the scorpion toxin. Thus, this humanized Nb is the best candidate to develop a therapy in human against the most toxic venom compound of one of the most dangerous scorpions.

Generation of anti-infectome/anti-proteome nanobodies.

Abstract: The immunization of an animal with a whole proteome or the infection of an animal and the screening of the resulting antibody repertoire on either the same or different proteome(s) or the infecting agent(s), omits the laborious steps of recombinant protein expression and purification to obtain multiple antigen binders. This procedure allows the identification of antibodies that are specific to unique or common signatures of different proteomes without prior knowledge of these signatures.Nanobodies are the smallest (15 kDa, 2.2 nm diameter, 4 nm height) in vivo affinity-matured functional antigen-binding entities that are derived from camelid heavy-chain antibodies. Due to their small size, recognition of unique epitopes, high affinity, and easy tailoring, nanobodies are attractive affinity reagents for various applications, including diagnosis and therapy.We detail a protocol to generate, isolate, express, and purify anti-infectome/anti-proteome nanobodies.

Immunological Aspects of Scorpion Toxins: Current Status and Perspectives

Abstract: Significant progress has been made in immunological studies of scorpion toxins and several formats of antibodies directed against scorpion toxins have been reported. Some of these are commonly used in a specific treatment against envenoming; others are primarily used for immuno-biochemical characterizations. The preparation protocol of the antibody or its fragments can be substantially different from one laboratory to another, which complicates a direct comparison of the potency of the antivenom. The use of immune sera, the total immunoglobulin fraction or Fab and Fab'2 fragments as the therapeutic agent is widespread. A number of monoclonal antibodies have also been reported and used for engineering of Fv, ScFv or Fab fragments. Recently, a novel antibody format - known as nanobodies - derived from HCAbs of camelids and selected after phage display shows great potential to provide a more efficient therapy against scorpion envenoming. Subsequent bispecific derivatives have been designed and their pharmacokinetics have been studied. Distinct advantages and disadvantages have been attributed to these equine, murine or camelid antibodies and their derived fragments. Some fragments are easily amenable into next generation therapeutics after proper manufacturing and provide an ensured availability of the product to the medical community. Through examples, we will show how the comparison of the serotherapeutic effectiveness is compromised due to the absence of standardization, on the preparation of immunogens, production processes and / or nature of the products. We will report on recent advances in the field.