Single-Domain Antibody Fragments Derived from Heavy-Chain Antibodies: a Review

Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article

Single-domain antibody fragments derived from heavy-chain antibodies: a review

L. Eyer, K. Hruska

Veterinary Research Institute, Brno, Czech Republic

ABSTRACT: Single-domain antibody (sdAb) fragments derived from heavy-chain antibodies of camelids and cartilaginous fish represent a new generation of therapeutic agents and immunoreagents. Due to their unique characteristics, such as low molecular weight, high physical-chemical stability, good water solubility, and the ability to bind antigens inaccessible to conventional antibodies, they could potentially act as a substitute for conventional therapeutic drugs in the treatment of serious human diseases, and, moreover, could be broadly used in analyses and diagnostics. In this review article, an analysis of 826 publications oriented to heavy-chain antibodies and their sdAb fragments indexed ® in the Web of Science database since 1993 has been carried out. Attention has predominantly been paid to papers published from 2010 to June 2012. Key publications are presented in tables and are characterised by descriptive words, abstracts and references. The presented publications have been sorted according to seven basic criteria: review articles and monographs, heavy-chain antibodies of camelids and sharks, production of sdAb fragments using recombinant technology, characteristic properties of sdAb fragments, application of sdAb fragments in therapy, application of sdAb fragments in diagnostic and immunoanalytical methods and other prospective uses of sdAb fragments. This review article should highlight the typical properties of heavy-chain antibodies and sdAb fragments which differentiate them from conventional antibodies and other available recombinant fragments, and also emphasize their extremely broad application potential, mainly in human disease therapy. At the same time it allows an easy and rapid orientation in numerous publications written on this subject, and facilitates the search for the required data.

Keywords: single-domain antibody fragment; heavy-chain antibody; antigen-binding site; camelid; shark; therapy; recombinant technology

Contents Tables and figures

1. Introduction Table 1. Authors, countries, institutions and jour- 2. Review nals (Top 5) showing the highest publication 2.1. Methods of searching and publication analysis activities concerning heavy-chain antibodies 2.2. Review articles and monographs and single-domain antibody fragments (Web ® 2.3. Heavy-chain antibodies of camelids and sharks of Science , 826 papers from 1993 to June 2012) 2.4. Production of sdAb fragments using recom- Table 2. Important review articles and monographs binant technology concerning heavy-chain antibodies and single- 2.5. Characteristic properties of sdAb fragments domain antibody fragments 2.6. Application of sdAb fragments in therapy Table 3. Camelid and shark heavy-chain antibodies 2.7. Application of sdAb fragments in diagnostic Table 4. Production of single-domain antibody and immunoanalytical methods fragments using recombinant technology 2.8. Other prospective uses of sdAb fragments Table 5. Characteristic properties of single-domain 3. References antibody fragments

Supported by the Ministry of Education, Youth and Sports, Czech Republic (AdmireVet; Grant No. CZ 1.05/2.1.00/01.0006- ED 0006/01/01) and the Ministry of Agriculture of the Czech Republic (Grant No. MZE 0002716202). 439 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513

Table 6A. Application of single-domain antibody Table 6H. Application of single-domain antibody fragments in therapy: inhibition of enzymes, fragments in therapy: humanised single-domain toxins and other soluble proteins antibody fragments Table 6B. Application of single-domain antibody Table 7. Application of single-domain antibody frag- fragments in therapy: activity modulation of cell ments in diagnostic and immunoanalytic methods surface proteins Table 8. Other prospective uses of single-domain Table 6C. Application of single-domain antibody antibody fragments fragments in therapy: pathogen neutralisation Figure 1. The number of publications on heavy-chain Table 6D. Application of single-domain antibody antibodies and single-domain antibody frag- fragments in therapy: intracellular expression ments during the period from 1993 to June 2012 of single-domain antibody fragments Figure 2. Structure of antibodies. (a) Molecule of con- Table 6E. Application of single-domain antibody ventional antibody (down), recombinant fragments fragments in therapy: oral administration of (up); (b) molecule of camelid heavy-chain antibody single-domain antibody fragments (down), single-domain antibody fragment (up); (c) Table 6F. Application of single-domain antibody molecule of shark heavy-chain antibody (down), fragments in therapy: prevention of amyloid single-domain antibody fragment (up) plaque formation and protein aggregation Table 6G. Application of single-domain antibody fragments in therapy: multispecific and multifunctional constructs

1. Introduction show their numerous novel feasible applications in research and in clinical development. Publications, A surprising discovery was made in the 1990s: in which contain key information concerning heavy- addition to conventional antibodies, mammals of chain antibodies and their sdAb fragments, are in Camelidae family and also some cartilaginous fish this new designed review presented in tables and have evolved a distinctive type of antibody molecule are characterized with a few descriptive words, full composed entirely of two identical heavy chains or shortened abstracts and source references. The (Hamerscasterman et al. 1993; Greenberg et al. text in the tables contains several format imperfec- ® 1995). Because these antibodies are devoid of light tions, which exist in the Web of Science database chains, they are called heavy-chain antibodies. The and are caused by transmission and copying of data antigen-binding site of heavy-chain antibodies is between various information sources. The missing confined to one single domain referred to as the VHH format of lower and upper indexes (e.g. Ig(2) instead –6 (Variable domain of the Heavy chain of the Heavy- of Ig2 or 10(–6) instead of 10 ) can be given as a chain antibody) in Camelidae and VNAR (Variable typical example. Special characters, such as α, Å or domain of the shark New Antigen Receptor) in car- °C were transcribed as alpha, angstrom or degree C, tilaginous fish. These variable domains can be eas- respectively. The attention is predominantly focused ily expressed in bacteria, yeasts or in other hosts on papers published in the last three years, although as recombinant single-domain antibodies (sdAb), some older key papers had been also included. The which are the smallest available intact antigen-bind- presented publications have been classified ac- ing fragments. SdAb fragments derived from llama cording to seven basic criteria: review articles and and camel heavy-chain antibodies are often referred monographs, heavy-chain antibodies of camelids ® to as nanoantibodies or nanobodies . Due to their and sharks, production of sdAb fragments using biophysical and pharmaceutical properties, which recombinant technology, characteristic properties are conferred by their single-domain nature and of sdAb fragments, application of sdAb fragments in small molecule size, sdAb fragments offer a broad therapy, application of sdAb fragments in diagnos- application potential, especially as new immuno- tic and immunoanalytical methods and other pro- therapeutic drugs and also as efficient reagents in spective uses of sdAb fragments. This review article immunoanalytical and diagnostic methods. The aim should facilitate orientation in the above mentioned of this review is to highlight the unique features subjects and enable rapid and easy searching for of heavy-chain antibodies and sdAb fragments and particular data and information.

440 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article

2. Review (78.2%). Our analysis shows that 262 institutions from 47 countries are concerned with the subject of 2.1. Methods of searching and publication sdAb fragments derived from heavy-chain antibod- analysis ies. Authors, institutions, countries and scientific journals which show the highest publication activi- The publications were retrieved from the Web ties (Top 5) are listed in Table 1. ® of Science database using the following search profile: Topic = (nanobod* OR nanoantibod* OR “single-domain antibod*” OR “heavy-chain anti- 2.2. Review articles and monographs bod*” OR “heavy chain-only antibod*” OR VHH OR IgNAR OR “llama* antibod*” OR “camel* an- Eighteen review articles are listed in Table 2 and tibod*” OR “new antigen receptor”), Timespan = characterized by key words (left column), abstracts all years or 2010–2012, Citation databases = SCI- (in the middle), and references (right column). EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, These papers offer an overall review regarding all BKCI-S and BKCI-SSH, Chemical Databases = important topics, especially about the structure, CCR-EXPANDED and IC. properties, generation and evolution of heavy-chain Using the above mentioned search profile, a total antibodies in camelids and sharks (Conrath et al. of 826 publications were retrieved. The first paper 2003; Tillib 2011), engineering of sdAb fragments describing the occurrence of heavy-chain antibod- (Deffar et al. 2009), their production, structure and ies was published in 1993 by a Belgium research properties (Muyldermans et al. 2001; Harmsen and group. Since 2003, publication activity on the topic de Haard 2007), use of sdAb fragments as building has sharply increased (Figure 1). In the last three blocks for the construction of multivalent and mul- years, 297 papers were published. In 2012, sixty-four tispecific conjugates (Saerens et al. 2008), applica- papers have already been published at the time of tion of sdAb fragments in therapy (Van Bockstaele our manuscript preparation. The older publications et al. 2009; Wesolowski et al. 2009) and in im- are predominantly oriented to the elucidation of the munoanalytical and diagnostic methods (Huang structure and characteristics of heavy-chain anti- et al. 2010; de Marco 2011). All these topics are bodies and their antigen-binding sites. The latest discussed in detail also in original papers, which papers describe the possibilities of practical use of are presented in Tables 3 to 8. Recently, the new sdAb fragments, especially in medicine and therapy. monograph “Single Domain Antibodies” (Saerens ® The Web of Science utilities have been em- and Muyldermans 2012) has been launched with ployed for search results analysis. Of all the pub- a complete methodology and key protocols for the lished papers, original research articles prevail construction of sdAb libraries and for the selec-

160

140

120

100

0 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 Figure 1. The number of publications on heavy-chain antibodies and single-domain antibody fragments during the period from 1993 to June 2012

441 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 tion and expression of sdAb fragments and their has been focused also on the evolution of heavy- advanced derivatives. This publication hihlights the chain antibodies (Nguyen et al. 2002; Flajnik et al. broad application potential of sdAb fragments and 2011), their possible function in the immune system can be used as a practical guide to sdAb recombi- (Ferrari et al. 2007; Saccodossi et al. 2012), and nant technologies. molecular mechanisms of germline gene segment rearrangement (Nguyen et al. 2000). Some investi- gators have also used heavy-chain antibodies as a 2.3. Heavy-chain antibodies of camelids suitable alternative to conventional polyclonal an- and sharks tibodies, mainly for diagnostic purposes (Anderson and Goldman 2008; Torigoe et al. 2012). Twenty- Heavy-chain antibodies are homodimers of di- three papers (eleven of them published from 2010 sulphide-linked heavy chains (Figure 2) that occur to 2012) are presented in Table 3. naturally in the serum of camelids and cartilaginous fish. The total amount of heavy-chain antibodies of all serum immunoglobulins has been determined 2.4. Production of sdAb fragments using to be about 45% in llama species, while in camels recombinant technology it is about 75% (Hamerscasterman et al. 1993). The function of heavy-chain antibodies in the immune Due to their simple modular structure and a system has not yet been completely explained. Due single-gene nature, sdAb fragments are easily to the lack of light chains, the antigen-binding site produced in vitro as recombinant proteins. The of heavy-chain antibodies is formed by only three production of sdAb fragments is based on the complementary determining regions (CDRs), com- cloning of VHH or VNAR gene segments into pared to six CDRs in conventional antibodies. The phage display vectors, construction of large phage CDR3 region of the heavy-chain antibody, which libraries and selection of high-affinity binders us- usually plays a crucial role in an antibody-antigen ing a biopanning process. SdAb fragments are ef- interaction, can be formed by a long finger-like fectively expressed in different microbial hosts, of polypeptide loop. Therefore, the antigen-binding which Escherichia coli, Saccharomyces cerevisiae site of the heavy-chain antibodies often exhibits and Pichia pastoris are the most commonly used a convex shape and differs sharply from the con- (Makvandi-Nejad et al. 2011; Ezzine et al. 2012; cave, groove-shaped antigen-binding site of con- Gorlani et al. 2012). Bacterial species enable high- ventional antibodies. With regard to the shape of yield expression of sdAb fragments in the cytosol the antigen-binding site, heavy-chain antibodies or in periplasm, whereas yeast cells utilize secre- recognise preferably epitopes buried in clefts on tory pathways resulting in efficient disulfide bond protein surfaces, e.g. in enzyme active sites, which formation, N-glycosylation and secretion of the are usually less antigenic to conventional antibod- recombinant product into the growth medium. ies (Lauwereys et al. 1998; Decanniere et al. 1999; Recent papers also describe several alternative De Genst et al. 2006). The convex shaped antigen- strategies for sdAb fragment engineering and pro- binding site of heavy-chain antibodies is not very duction, such as yeast or ribosome display systems suitable for the binding of small molecules (hap- (Yau et al. 2003; Ryckaert et al. 2010), expression tens). Therefore, only a few papers focused on anti- of recombinant fragments in filamentous fungi hapten heavy-chain antibodies have been published (Joosten et al. 2005), in mammalian cells (Bazl et to this date (Spinelli et al. 2001; Ladenson et al. al. 2007) and in transgenic mice (Zou et al. 2005). 2006; Alvarez-Rueda et al. 2007). Other structural Mutagenesis and recombination of CDR regions features of heavy-chain antibodies, which differen- to improve the affinity and specificity of sdAb tiate them from conventional antibodies, include fragments have also been discussed (Swain et al. a higher proportion of hydrophilic amino acids in 2010; Fanning and Horn 2011). Current studies variable domains (VHH/VNAR), the presence of are focused on the development of new cloning disulfide bonds stabilizing the antigen-binding site, and expression strategies to produce humanised and an unusually high number of mutational hot fragments, multivalent constructs and fusions of spots responsible for structural variability of heavy- antibody fragments to other proteins (Saerens et al. chain antibodies (Harmsen and de Haard 2007). 2005; Vincke et al. 2009; Bell et al. 2010; Pollithy et In addition to structural studies, recent research al. 2011). Selected articles are presented in Table 4. 442 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article

Figure 2. Structure of antibodies. (a) Molecule of conventional antibody (down), recombinant fragments (up); (b) molecule of camelid heavy-chain antibody (down), single-domain antibody fragment (up); (c) molecule of shark heavy-chain antibody (down), single-domain antibody fragment (up)

2.5. Characteristic properties of sdAb neys can negatively affect their therapeutic activity. fragments Therefore, several strategies to prolong the half-life of sdAb fragments in serum have been described, Recombinant sdAb fragments are formed from e.g. conjugation of sdAb to another recombinant only one single polypeptide chain containing about fragment binding specifically to serum albumins or 120 amino acids. Due to their size (4 × 2.5 nm) immunoglobulins (Harmsen et al. 2005). SdAb frag- and molecular weight (12 kDa to 15 kDa), sdAb ments that bind to and are internalised by cerebro- fragments are the smallest available recombinant microvascular endothelial cells are able to cross the antibodies. Their small molecular size enables blood-brain barrier and transmigrate into tissues of sdAb to penetrate easily into tissues and inter- the central nerve system (Abulrob et al. 2005). Due to cellular spaces, but is, however, the cause of their their simple monomeric structure, sdAb fragments rapid clearance from the blood by renal filtration are conformationally very stable (Dumoulin et al. (Cortez-Retamozo et al. 2002). The rapid clear- 2002) and refold easily after heating to achieve their ance of sdAb fragments from the blood by kid- original native structure (Dolk et al. 2005b; Walper et 443 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 al. 2012). They show an increased resistance to high notherapy of gastrointestinal disorders using oral ad- pressure and to low pH (Dumoulin et al. 2002) and ministration (Harmsen et al. 2006; Vandenbroucke maintain their biological activity even in an environ- et al. 2010). Fourthly, the ability of sdAb fragments ment with a high concentration of proteases and to transmigrate across the blood-brain barrier and to detergents (Dolk et al. 2005a; Hussack et al. 2011a). prevent amyloid plaque formation could be utilized Owing to a higher content of hydrophilic amino in the diagnostics and therapy of neurodegenerative acids, sdAb fragments are characterised by good diseases (De Genst et al. 2010; Rutgers et al. 2011). solubility in water and limited agglutination ability Fifthly, a modular nature of sdAb fragments allows (Conrath et al. 2005). Another characteristic feature an easy engineering of multivalent or multispecific particularly concerns the affinity of sdAb fragments, formats that show an increased therapeutic potency which remains usually equivalent to that of the native compared with monovalent sdAb fragments. Recent heavy-chain antibodies. This is a substantial differ- research is focused also on the generation of bifunc- ence from other recombinant fragments, that show tional constructs by coupling of sdAb fragments with often lower affinity compared with their polyclonal enzymes and toxic substances for specific drug de- or monoclonal counterparts. Eight papers published livery to bacterial or tumour cells (Zhang et al. 2004; from 2010 to 2012 describing typical properties of Stone et al. 2007). Finally, the non-human origin of sdAb fragments are presented in Table 5. Eight older camelid and shark sdAb fragments could elicit their articles should also be considered. neutralisation by the human immune response and a decrease in their therapeutic effect. Therefore, a general strategy to produce humanised sdAb frag- 2.6. Application of sdAb fragments ments has been described as a promising way for in therapy lowering the potential risk of immunogenicity of therapeutic sdAb fragments (Vincke et al. 2009). The unique physicochemical and pharmacological More than half of the 826 analysed papers discuss properties of camelid and shark sdAb fragments give possible therapeutic applications of sdAb fragments. them a prospective use as new-generation thera- Key publications are presented in Tables 6A to H. peutic agents for the treatment of serious human diseases. At present, some sdAb-based drugs are in the stage of preclinical testing on animal models or 2.7. Application of sdAb fragments in vitro. Some other drugs have advanced to phase I in diagnostic and immunoanalytical or II of clinical testing in volunteers. The therapeutic methods effect of sdAb fragments is particularly based on the following principles: Firstly, a remarkable prefer- Their effective penetration into tissues makes sdAb ence of sdAb fragments for binding into clefts and fragments good candidates for the construction of cavities on protein surfaces offers the possibility to imaging probes used for in vivo monitoring of tu- develop selective therapeutics for efficient inhibition mours, metastatic lesions, amyloid fibrils, etc. Such of enzymes (Paalanen et al. 2011), neutralization of immuno-imaging probes are prepared by the labelling proteolytic toxins (Hussack et al. 2011b) and activity of sdAb fragments with short-lived isotopes, mainly modulation of cell surface proteins, such as recep- with 99mTc. In contrast to radiolabelled monoclonal tors, ion channels and leukocyte ecto-enzymes (Wei antibodies, small sdAb fragments show rapid antigen et al. 2011; Altintas et al. 2012) involved in cancer targeting and fast clearance from blood resulting in a and inflammatory diseases. Moreover, sdAb frag- contrast-enhanced imaging signal, reduced accumula- ments recognize also cryptic epitopes hidden deeply tion of labelled fragments in liver and lower radiation in the clefts of virus capsids and in surface envelopes burden. The main limitation of sdAb-based imaging of parasites (Stijlemans et al. 2004; Henderson et al. probes, however, is their high non-specific uptake in 2007; Forsman et al. 2008). Secondly, intracellular kidney and bladder (Vaneycken et al. 2010, 2011b). An expression of sdAb fragments as “intrabodies” is a innovative approach based on sdAb fragments fused potential strategy to target intracellular antigens, to fluorescent proteins and expressed in living cells as e.g. anti-apoptotic proteins, oncogenes or several “chromobodies” offers the possibility to trace intra- viral proteins (Vercruysse et al. 2010). Thirdly, the cellular antigens and to modulate protein function in high structural stability of sdAb fragments in harsh living cells (Schmidthals et al. 2010). Nanoantibodies conditions makes them ideally suited for the immu- can also be used in immunoassays and in biosensors 444 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article for the quantitative analysis of contaminants and tox- derived from camelid and shark heavy-chain an- ins in food and environmental samples (Conway et tibodies can also be employed as crystallography al. 2010; Dona et al. 2010; Kim et al. 2012) and also chaperones to stabilise the conformation of pro- for diagnostics of serious human diseases (De Marni teins during crystallisation trials (Abskharon et al. et al. 2012). Their small size and lower aggregation 2011). Immunoaffinity chromatography represents propensity enable immobilisation of extremely high another prospective field for sdAb fragment ap- numbers of nanoantibody molecules on the surface plication. SdAb fragments are highly resistant to of the well of microtitre plate or biosensor transducer, the effects of organic solvents used for the elution which leads to a substantial increase in the sensitivity of the captured ligands and for regeneration of im- of the immunodetection system. Moreover, highly munoaffinity columns (Franco et al. 2010). Recent stable sdAb fragments are resistant to denaturing con- applications, such as the development of transport ditions used during biochip regeneration. For details systems across epithelia (Iqbal et al. 2011), inacti- of new publications see Table 7. vation of phages infecting dairy bacterial cultures (Hultberg et al. 2007) and mimotope selection strategies (Simmons et al. 2008) show the extremely 2.8. Other prospective uses of sdAb broad application potential of sdAb fragments. Ten fragments recent publications discussing some alternative applications of sdAb fragments are listed in Table 8. Besides possible therapeutic, diagnostic and This table also includes two older papers which immunoanalytic applications, sdAb fragments make the review complete.

Table 1. Authors, countries, institutions and journals (Top 5) showing the highest publication activities con- ® cerning heavy-chain antibodies and single-domain antibody fragments (Web of Science , 826 papers from 1993 to June 2012)

Item Number of publications Authors (685 in total) Muyldermans S 96 Wyns L 32 Rahbarizadeh F 28 Conrath K 25 Tanha J 23 Verrips CT 23 Institutions (262 in total) Free University of Brussels/Flanders Institute for Biotechnology 188 Utrecht University 61 National Research Council Canada 47 Ablynx NV 32 Ghent University 23 Countries (47 in total) USA 200 Belgium 193 Netherlands 115 England 78 France 63 Journals (140 in total) Journal of Biological Chemistry 30 Journal of Molecular Biology 25 Molecular Immunology 24 Journal of Immunological Methods 22 PLoS ONE 19

445 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 - Wernery Wernery 2001 Conrath et et Conrath al. 2003 Muylder mans et al. 2009 - - - Camelid immunoglobulins differ from all other known antibodies and contradict all common theories on antibody diversity. It was Camelid all other known immunoglobulins differ from theories antibodiescommon all contradict and on antibody diversity. 75% of all serumimmunoglobulin G (IgG) molecules up to are proteins that demonstrated lacking chains. light IgG(2) and IgG(3), of heavy only consist which chains, a low molecular biodistribution their have improves which weight and allows a better tissue penetration. Of special importance long complementary is the determining region (CDR) loop inserts which active deep the into of an enzyme.site This binding property was only observedexperiments in to extraor gain structuralthe to out and data point dinary value of heavy antibodies chain as biochemical and pharmacological tools.absorption and The acquisition of adequate Pre-colostrum neonate, health serum essentialof the amounts the immunoglobulins of colostral are to IgG levels in camelids are - of 0.26 ± 0.23 mg/ml. Maximum IgG levels reached with concentra are a plateau after with concentrations 24 h and kept at low, IgG levels decline after 2–5 a successful above passive 10 mg/ml. indicate transfer. tions of 24.52 ± 8.8 mg/dl. IgG concentrations weeks and a marked increase is observed between systemhas neonate of the immune the one and two months, that indicating the testsavailablefor assessment of IgG serum are A numberstarted of different levels. mature. to Single radial immunodiffusion (SRID) specifically only method is the that testto of measuresfailure passivereliable It assay trans is a serum concentrations. IgG (FPT).fer FPT is factor a major in neonatal mortality in camelids, but very Therapeutic has adminis been little published so far. of colostrumtration will provide passive protection against infectious diseases a 2–3-week for period of risk, intravenous and the of 20–40 ml camelid plasmaFPT. administration combat helps to Antibodies composed are of jawed-vertebrates of paired heavy (L) polypeptide (H) and light sera the of camelids, chains. Surprisingly, and wobbegong shark nurse shark, and possiblyantibodiescontain L-chains. lack that ratfish In camelids,these Heavy-chain - anti bodies (HCAbs) gamma-isotypes, are functional and are binding. in antigen review In this we focus dedicated on the immunoglobu - HCAb encode the lin (Ig) genes that in Camelidae (camels, dromedaries and llamas), aboutorigin, their - and how these camel immu moglobulins evolved diversity. and diverse H-L combinatorial a large repertoire of the binding sites in absence and acquire of antigen It is well established that all camelids have unique antibodies established is all camelids well have blood. that circulating in their antibodiesIt Unlike species, from other these specialso-called heavy-chain antibodies devoid composed are and are chains of light of a heavy-chain homodimer.These antibodies (HCAbs) rearrangement and require dedicated expressed after are gamma-genes. a V-D-J constant An immune response is raised in these so-called heavy-chain antibodies following classical immunization protocols. These HCAbs are easily purified from serum,conventional the antigen-binding fragment interacts and to lesswith parts are antigenic that the of target antibodies. dromedary of the site antigen-binding the Since HCAb is comprised in one single as domain referred to variable domain of heavy of HCAb chain (VHH) or nanobody (Nb), we designed Nb repertoire a strategythe clone of an immunized to dromedary select and to Nbs the with specificityfor our antigens. target produced The monoclonalwell are Nbs in bacteria, are successfully have We developed very with high antigen affinity and and highly stable soluble,cognate and bind their specificity. Nbs research purposes, for recombinant as probe in biosensors, diagnose to infections, diseases treat and to cancer or trypa like - nosomosis. - fusion Failure of passiveFailure transfer Table 2. Important reviewTable articles and monographs concerning heavy-chain antibodies and single-domain antibody fragments Camelid immunoglobulin IgG concentration Single radial immunodif Evolution Jawed-vertebrates Sharks Immunoglobulin genes Heavy-chain antibody Nanobody

446 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article - - Tillib 2011 Tillib Deffar et al. 2009 Harmsen Harmsen and de 2007 Haard Muylder mans et al. 2001 Roovers et al. 2007 Van Bock Van etstaele al. 2009 - - - - - This short review provides an introduction to the rapidly developing field of generation and utilization of nanoantibodies”Thisshort“camel review and utilization developing of generation providesthe rapidly of field to an introduction “nanobody” “nanobodies”).“nanoantibody”or term(or The was to single-domain given variable fragments of special type- of anti classical (in addition to bodies exist types naturally that of antibodies) in blood of Camelidae family animals and in some- chon fishes.drichthyan of very existence The technology efficient of nanobody and some generation very characteristicfeatures useful usepromise their a big potential in immunobiotechnology for and medicine. Nanobodies antibody-derived are unique structural the and functional contain properties that therapeuticoccur proteins of naturally ring heavy-chain antibodies.Nanobody The technology was originally developedfollowingthe discovery (camels and camelidae that llamas) possess fully functional antibodies chains. lack light that These heavy-chain antibodies a singlecontain variable domain (VHH) cloned the and isolated domains (CH(2) VHHand two constant and CH(3)). domain is a perfectly Importantly, polypeptide stable har boring full capacity the original antigen-binding of the These heavy-chainnewly discovered antibody. VHHtheir unique domains with structural and functional properties basis form the of a new of therapeutic antibodies generation named were which Nanobodies. The properties paperaim of this the show is to of Nanobodies, clinical status. and their applications production their and expression, view propertiesantibodies, of their as conventional to compared production their in microorganisms, with a focus on yeasts, and therapeutic applications.their Camelids functional produce antibodies single devoid N-terminal the of which domain is chains fully of light of antigen capable binding. These single-domain antibody fragments (VHHsNanobodies) or several have advantagesfor biotechnological applica - Furthermore,they suitedfor well are tions.expressed They in microorganismsa high well and stability are solubility. and have construction molecules of larger and selection systems as such phage, yeast, This minireview or ribosome an over offers display. The antigen-binding site of antibodies site formedThe antigen-binding is the vertebrates combining variablefrom by domains of a heavychain (VH) and in antibodies sera their contain, (VL). in that fromthis chain camelsa light to and Ilamas an important are exception However, addition, a unique kind of antibody is formedthese that of by heavy site antibodies only The antigen-binding chains consists of one single as domain, referred to VHH. This articlereviewsto ken place ta at have th and structuralmutations adaptations the The VHH antigen- has a single-domainpotent VHHa of a sufficientreshape a VH pair variability, into of a VH-VL with retention pairs. VH-VL conventional binding capacityto inaccessible and providesepitopes with advantageare novel the of interacting that ing only a single Ig fold are called are ing only a single Ig fold Nanobodies. These antibodythem ideal make that fragments severalfeatures have remarkable cancer therapeutics. Particularlycandidates as simultaneously appealing to next-generation inhibit various ability is their crucial growth factor ligands receptors or their with a single molecule. In addition, they tip of filamen easy- on the are and express clone to opens phage, which tous possibility the select to Nanobodies for inducing particular biological effects.Nanobodies to have potential become important cancer therapeutics near in the future, displaying unequalled and unprecedented efficaciestreatment. in Over years, the antibodies been many have diseases, patients with life-threatening treat successfully to generated most notably of antibodies, first generation While the originating mice, causedfrom severecancer. inefficient, side effectsrelatively and were technological made it possible advances have obtain to fully antibodies human therapeutic use. for ‘Heavy-chain only’ antibodies been recently discoveredhave blood in the of camelids. Because units size, antigen-binding of their of these the antibodies compris Nanobodies derived therapeutic proteins are heavy-chain from the variable (V(H)H) in heavy-chain- occur naturally domains that only Ig molecules in camelidae. Thesethe smallest V(H)H domains are known antibody antigen-binding fragments.Nanobodies can be easily produced in prokaryotic or eukaryotic host organisms, unique biophysical and pharmacological and their character istics these render molecules ideal drug candidates for development.review This describesthe structural properties of nanobodies and focuses unique features, on their distinguishes which these molecules antibody from other formats and small-molecule drugs. of nanobodies therapeutic applications Possible discussed are anti- and data from phase I clinical trials ‘first-in-class’ of thenovel (Ablynx NV) ALX-0081 thrombotic agent presented. are

applications Nanobody technology Nanobodies Propeties Production Applications Selection systems Biotechnological Antigen-binding site Mutations pairVH-VL Novel epitopes Cancer therapeutics Structural properties Therapeuticapplication Clinical trials ALX-0081

447 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 - - Vanland schoot et al. 2011 Weso lowski et al. 2009 de Marco de Marco 2011 Huang et et Huang al. 2010 and intracellular imaging.and intracellular In 1989, a new type of antibody was thedromedaries identified, first in sera of alsoother later and in all species the of Camelidae it is still unclearToday what These antibodies constant chain and alsoheavy the first a light domain. contain lack do not family. evolutionarythe advantage heavy of such antibodies chain-only be. could In sharp isolated contrast,of the broad applicability the variable domains (VHH) antigen-binding was recognized, rapidly especially development of therapeutic the proteins, for called Nanobodies. we summarize Here characteristics first the unique of some of features VHHs. and Thesenext be will describedin NanobodiesHepatitisexperimental Btherapeutic of applications virus, viruses: of different against different context the HIV, influenza virus, RespiratorySyncytial Poliovirus, virus,the diagnostic Next, applica Rotavirus,- Rabies virus, and PERVs. FMDV, virus X), as as virus,well an industrial (lytiction of VHHs application lactococcal (Vaccinia virus Marburg 936 Tulip and plant phage) will be described. described In addition, the monovalent Nanobodies that data show can possess unique characteristics not observed antibodies. with conventional bivalent, The straightforwardmultivalent, into multispecificformatting and/or Nanobod - ies allowed tailoring molecules potency for and cross-reactivity against viral targets with high sequence diversity. Antibodies important are tools experimental research for and medical applications. Most antibodies composed are of two heavy these to In addition isconcave. which usually site or flat chains. antigen-binding and two light the to Both contribute chains antibodies,conventional llamas, camelids, other and sharks also antibodies produce composed only of heavy chains. The antigen- of thesebinding site unusual heavy antibodies chain is (hcAbs) formed only by a single domain, designated VHH in camelid hcAbs and VNAR hcAbs. in shark VHH and VNAR easily are produced as proteins, recombinant designated single domain antibodies (sdAbs) or nanobodies. The regionCDR3 these of sdAbs possessesextensions extraordinary the form to long fingerlike capacity cavities on antigens, into e.g., can extend that activecrevice the site of enzymes. Other advantageous of nanobodies features and good tissue penetration we review capacity, in vivo. Here refolding thermal stability, small size, their highinclude solubility, resultsthe of several proof-of-principle recent perspective open exciting the studies that of using modulating sdAbs for immune and microbes.functions targeting toxins and for The discovery hasthe single-domaintheir potentials antibody’s use of stimulated in an increasing variety of fields. rapid The of articles describingaccumulation new and further applications developments of established has approaches made it, therefore, necessary previous update the to reviews withsummary a new complete and more topic. of the Conclusions: Beside necessary the task of updating, analyses work this in detail aspects some applicative single-domain been antibodies of the have overseen that as co-crystallization past, in the as such efficacy their chaperones, chromatography, controllers, protein in affinity aggregation enzyme activity tuners, specificities and the single-domain fragments.the unconventional of With the advent of new advent the antibody engineeringWith technologies, antibodies been have conventional minimized smaller antibody into formats. Small size is an important advantageand future diagnostic current for development. Nanobodies smallest among the (Ablynx) are known antibody antigen-binding fragments, derived and are heavy-chain from the serum only antibodies in the occur of naturally that Camelidae. evolution, Endowed by natural these unique biophysical, biochemical Nanobodies exhibit and pharmacological inherently potential as moleculescharacteristics. in drug excellent development, their In addition to Nanobodies possess very functional attractive properties diagnostic development for aid in their tools. that of several we present applications available Here of currently examples in vivo chromatography, immunoaffinity Nanobodies of immunosensorfor field cancer, the to

modulating chromatography applications Therapeuticapplications Viruses VHH VNAR Immune function Toxin/microbe targeting Toxin/microbe Immunosensor Immunoaffinity Immuno-imaging Biotechnological

448 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article

Vaneycken Vaneycken et al. 2011a Holliger Holliger and Hudson 2005 Saerens et Saerens et al. 2008 Romer et Romer et al. 2011 - biopharma - Immuno-imaging is a developing technology studying disease aims at that in patients using imaging techniques as such positron with radiolabeled in combination immunoglobulin emission derived tomography targeting probes. Nanobodies smallest the are antibody-fragmentsantigen-binding fast and show and specific in targeting vivo. Theseinvestigationas probes under are currently therapeutics nanobodies but preclinical that studies indicate of magic also could generation bullets become immuno- next for the imaging. imaging can Initial that data be show performed as early as use radio- the one hour post-injection of short-lived enabling isotopes. These unique properties friendly imaging and safe patient should enable protocols. This review focuses the on current of radiolabeledstatus nanobodies as targeting probes immuno-imaging. for ceuticals with superior biodistribution and blood properties. clearance With 18 monoclonal antibody 100 in clinical trials, products than and more (mAb) market on the currently engiWith it is clear- that neered antibodies of age as come biopharmaceuticals. have In fact, by 2008, engineered antibodies for predicted are account to > 30% of all revenues biotechnology in the market. antibody Smaller recombinant fragmentsclassic monovalent example, (for antibody scFv)) and engineered fragments (Fab, variants (diabodies, triabodies, minibodies and single-domain antibodies) are now emerging as credible alternatives. These fragmentsretain the specificity targeting mAbs but of whole canproduced be more economically and possess unique and superior other properties a range of diagnostic for and therapeutic applications. Antibody fragments been and multispecific multivalent have reagents, into forged therapeutic linkedto (such payloads as radionuclides, toxins, enzymes, liposomesantibody and viruses)single have domains and engineered enhanced for therapeuticRecently, efficacy. been engineered and selected as targeting reagents cavities against hitherto immunosilent in enzymes, receptors and infectious agents. Single-domainrepertoire antibodiesthe expand significantly of antibody-based anticipated to are reagents againstthe vast being biomarkers discovered proteomics.range of novel through is all potential tremendous for there As review this show, aims to antibody fragments as either diagnostic robust in biosensors), reagentsexample or as (for in vivo nonimmunogenic Antibodies are large and complex molecules,Antibodies and complex large are and the antigen with parts two identical the bind independently onto of each other that therapeuticthe value of antibodies,To improve protein-engineering partthird molecule effector dictates of the the that function(s). it wassingle- reduced moietya single-domainendeavors the sizeunit. to antigen-binding the of the that demonstrated Occasionally, derivatives effectdomain antigen-binding their of antibodies on target. antagonistic) own – on their The – an agonistic can (or have biochemical with other properties in combination as such high solubilitysmall size and high specific and strict monomeric behavior, antibodies single-domain antigen, make cognate the harnessed for ideal to ity and affinity conjugates design man-made novel with innovative effector functionsclassical outsidereach of the antibodies. The rapid and ongoing discoveryThe rapid of new disease leadshuman healthcareparadigm and to a dramatic biomarkers change in related basis the a trulyconstitutes for personalized medicine. Molecular imaging enables early detection and classificationhuman diseases of biochemical the and physiologicaland provides properties valuable optimized, therapies. data for By now, of antibody target-oriented scaffoldsderivatives protein or alternative can be engineeredthe detectionfor of a wide range of structures.target The successful of these reagentsapplication in animals, xenograftmolecu utility for modelstheir in preclinical- and cells research demonstrate clearly lar imaging. Despite these perspectives, promising only a few used antibodies are proteins yetmolecular and recombinant for imag - medicine.ing in human Especially high needoff safety demands the effectsextensive and the target humans eliminate require to in research and development efforts.

antibody fragments reagent efficacy Immuno-imaging Man-made conjugate Molecular imaging therapies Target-oriented Antibody derivative Alternative protein Multivalent/multispecific Single antibody domains Enhanced therapeutic Smaller recombinant

449 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 Saerens Saerens - and Muyl dermans 2012 Koide 2009 Koide - - - tion, and cumbersome engineering retarded or hampered regularly streamlined their development in some applications. Consequently, these fragments. antigen-binding of their becamemAbs minimize size to the and complexity focus Eventually, the attempts of many efforts productionconnectsrecombinant or a led the scFv (where Fab synthetic of smaller antigenbinding fragmentslinker to such as variablethe domains of heavy i.e., VH chain, and VL), and light and even sdAbs (single domain antibodies derived VH). mostly of the first the setAlthough of offeredsdAbs significant advantages, they suffered multiple also shortcomings, have from which all of been remediated engineering. while scientists by elegant designing, were engineering, Interestingly, ideal the a serendipi - sdAb, and shaping discoverytous a similar engineering showed that occurred camelids, in the on, it was cartilagi already in nature and later that found - nous fish antibodies performed exercise the even on in earlier their bloodevolution. Thesehave in animals antibody functional isotype composed chains, lack light of heavyclassical the – only that in addition to chains antibodies two heavy containing and two light chains. These heavy-chain antibodies (HCAbs)recognize the antigen via a single V-NAR.variablereferredto domain, as VHH or The can smallest isfragment be the intactlow cost. antigen-binding that at producedVHH The recombinantly proper or V-NAR valuable ties high of man-made sdAbs, affinity and antigen, specificity VHHs,their for cognate solubility including and stability, and V-NARs small size and strict opportunities. monomeric many behavior offer result,As a severalhave companies Aus spin-off foundedbeen in tralia, Netherlands, Belgium, successfully introduced in a wide these England, and Scotlandproteins - range of applica that Germany, a special cover tions to editors). by the therapeutics (Preface clinic need in the in research or even next-generation produce to of the contributions: Titles I OVERVIEW OF SINGLE DOMAINPART ANTIBODIES Whole Monoclonal Antibodies Single Domain to From Antibodies: Think Small HeavyIntroduction to Chain Antibodies and Derived Nanobodies Overview and Discovery of IgNARs and Generation of VNARs II SINGLE DOMAINPART ANTIBODY LIBRARY CONSTRUCTION VH Repertoire and Vκ Domain Synthetic Antibodies Largeof Human of the Creation and Highly Functional Library of a Naïve Preparation of Camelid Single Domain Antibodies III SELECTION OF SINGLE DOMAINPART ANTIBODIES Selection of Single Display Domain by Phage AntibodiesNative Conformation Specific in Their to Antigens Camelidae of Naive LibrariesSemiautomated Panning and Selection of Single-Domain Antibodies Antigens Against Peptide Screening for Single Domain Antibodies Pichia Surface Display: A Tool Selection Intracellular Method One-Step Hybrid: A Versatile Bacterial Two Antibody (IAC) Capture Intracellular Methods or Single Domain Antibodies Inside Cells: Interactions Selection Single A “One Domain with Antibody Protein–Protein Interfering for of Functional Fragments System Plasmid” Mammalian Two-Hybrid http://www.springer.com/biomed/immunology/book/978-1-61779-967-9 hybridomatechnologythe The development of the created possibilityto obtain unlimited amounts of monoclonal antibodies (mAb) with high specificitymAbs in applications. introduce a wide range of target to any and antibody-based for of and affinity Examples drugs in therapeutic settings and antibody-basedthe bulky size of mAbs, probescostly produc in diagnostics However, infinite. are The preparation of diffractionThe preparation quality A major X-ray crystals the remains in macromolecular bottleneck crystallizacrystallography. - is an auxiliarytion chaperone as such fragments protein, of monoclonal antibodies, and increases binds to that crystallization the mask counterproductive surfaces of a target moleculeheterogeneity, probability chaperones reduce conformational of interest. Such surfaces predisposedwhile extending forming crystal to contacts, and provide phasing Crystallization information. chaperones gen - technologies usingerated recombinant emerged asinherent have superior increase and eliminate alternatives that throughput the associatedlimitations with antibody production by animal immunizationhybridoma technology. and the

chaperone heterogeneity monograph X-ray crystallographyX-ray Crystallization Recently published Conformational Conformational Single Domain Antibodies

450 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article Cell-Free Selection Compartmentalization Cell-Free of Domain Antibodies by In Vitro Conditions Selection Application of VHHs Under Single-Domain Toxin-Specific Antibodies Isolation and Characterization of Clostridium difficile Selection of VHH Recognize Antibody That Aβ Libraries Fragments Immune Depositions Different Using Complex IV EXPRESSION OF SINGLE DOMAINPART ANTIBODIES AND DERIVATIVES Expression of Single-Domain Antibodies in Bacterial Systems cerevisiaeExpression of VHHs in Saccharomyces Expression of ChimericStable Heavy Chain Antibodies in CHO Cells Production of Camel-Like Antibodies in Plants V IMPROVEMENT OF SINGLE AND APPLICATIONS PART DOMAIN ANTIBODIES Heavy (VH)Selecting Domains and Purifying Variable Human Autonomous Solubility Engineering and Stability VH Domains of Human of Proteolytic Engineering In Silico Through Stability Improvement Selection VH Single Domains of Human with Biophysical Improved Properties Display by Phage of Single Domain AntibodyImprovement by Disulfide Stability Bond Introduction Characterization of Single-Domain Antibodies with an Engineered Disulfide Bond Yeast Surface Display of Single-DomainAntibodiesMaturation Affinity by of Single-Domain Display Multivalent Antibodies Methods Determining for and of Long of AlbudAbs PK Parameters Serum the Technology Half-Life Drugs AlbudAb the Made Using Peptide Binding and Histone-Tail Interactions Specific Protein–Protein Study Protein to Nanotraps Fluorescent A Practical Guide Nanobodies 99mTc: with Site-Specific of His-Tagged Labeling Nanobody-Based Immunoprecipitation Chromatin Tags of VHHs Application-Specific to Expression Plasmids Fusion the Enable User-Friendly Histochemistry of Single-Domain AntibodiesApplication in Tumor VI CASE STUDIES PART Nanobodies as Formation Structural Probes and Fibril Misfolding of Protein Molecular Nanobodies: Imaging Using A Case Study Case Lamin-Chromobody on LiveApoptosis-Assay Using Cell Study Analysis Cell-Lines High-Content for

451 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 - - Hamer scaster man et al. 1993 Green - berg et al. 1995 Govaert et al. 2012 - - antibody maturation process favoringantibody VHH disulfide domains with maturation an interloop bond. Random association of VL and VH repertoires contributes considerably to antibody diversity. The diversity and the affinity affinity Random diversity and the association The of VL antibody and VH to repertoires diversity. considerably contributes increased then are by hypermutation located in B cells in germinaldisease, case in the centres. Except - of ‘heavy natu chain’ rally occurring heavy-chain antibodies not been have described, binding has antigen although been sepa for demonstrated - heavyrated of material amounts of IgG-like or cloned chains of considerable VHpresence the domains. we investigate Here serumM(r) 100K in the camel (Camelus of the dromedarius). molecules These composed are of heavy-chain dimers and are devoid chains,repertoire, of light question calls antigen-binding the a finding but nevertheless into that an extensive have camel. Camel heavy-chain in the chains of light role IgGs lack CH1, in one IgG class which be might structurally replaced by of all camelids. hinge. Heavy-chainan extended IgGs a feature are findings These open new perspectives engineering in the of antibodies. Immunoglobulin and T-cell receptor (TCR) molecules are central to the adaptive immune system, Sequence immune receptor (TCR) adaptive the molecules to conservation, central are Immunoglobulin and T-cell similarities in domain structure, and usage signal of similar recombination sequences machinery and recombination indicate was there a time during probably evolutionthat modern-day the receptor diverged when an ancestral to immunoglobulin not been Other moleculesand TCR(1–3). rearrangement have undergo described that intermediates in vertebrates, nor have report isolationbeen the of botha new here these features gene families,- member have identifiedimmuno of the that We globulin superfamily shark, nurse from the one variable Ginglymostoma contains domains which and five cirratum, constant asand is found a dimer in serum. Analyses sequence of complementarydiversity within variable extensive DNA clones show domains, is by both which generated diversification rearrangement and somatic mechanisms. Our results rear suggest that ranging loci arisen distinct have from during immunoglobulin and TCR evolution. The antigen-binding fragment antigen-binding of functionalThe heavy antibodies chain (HCAbs) in camelids comprises a single domain, named variablethe domain of heavy of HCAbs chain (VHH). VHH harborsframe The amino acid substitutions in the - remarkable substitutions The functions partner. chain of a light absence domain that in the an antigen-binding region-2 generate work to VHH the soluble, to but decrease more character hence hydrophilic, provide a more domain. of the stability intrinsic the functional the of dromedary of an additional hallmark role we investigate Here VHHs, disulfide bond extra i.e. the between loops. antigen-binding firstthe and third After cysteines substituting the cystine forming interloop this by all 20 amino acids, we selected and characterized VHH Although domains can func several binding capacity. retain VHHs antigen that a net its disulfideadvantage. the First, of an interloop constitutes presence absence that bond,tion in the we demonstrate disulfide bond destabilization stabilizes the domain and counteracts framework the by the region-2 amino acids. hallmark Second, disulfide the bond dual rigidifiesThis loop, interaction. antigen-binding antigen leading a stronger long third to the beneficial effect in vivo the explains

repertoire Table 3. Camelid heavy-chain and shark antibodiesTable Heavy-chain dimer Camelus dromedarius Antigen-binding CH1 domain Antibody engineering Nurse shark New antigen receptor Sequence analysis Rearrangement Somatic diversification Amino acid substitution Disulphide bond Cystein

452 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article Sircar et Sircar et al. 2011 Rese - mann et al. 2010 Spinelli Spinelli et al. 2001 - - Camelids have a specialCamelids have type known of Ab, as heavy Abs, chain devoid are which of classical chains. classical light to Ab Relative Ag recognition diversity and binding affinities, higher immunogenicity, sta - Abs, comparable have camelid heavy (cAbs) Abs chain therapeutic scaffolds. alternate candidates makingpromising for them Rational and better manufacturability, bility and solubility, engineering therapeutic function improve to of cAbs requires knowledgeof of sequence and structural differences features the between amino acid sequences and classicalvariable cAbs of 27 cAb regions Abs. (V(H)H) study, In this aligned were with the respective regions of 54 classicalcAb V(H)H of detect identification CDRs. to Abs amino acidautomatic differences, enabling CDR analysis revealed H1 often the (and sometimes H2) adopts that not classifiable the diverse by conformations established canonical rules. classical longer than H3 is Also, much H3 loops, cAb the although structural common motifs it often and some contains - times a disulfide the H1. to Leveragingbond these observations, Carlo-based we Monte created a V(H)H structural cAb modeling CDRtool, the where H1 and H2 loops exhibited a median root-mean-square deviation of 3.1 and 1.5 angstrom, natives respec to tively. The protocol generated 8–12, 14–16, and 16–24 residue 8–12, 14–16, and 16–24 generated H3 loops The protocol a medianwith root-mean-square deviation of to natives tively. deviation The large the predicted of 5.7, 4.5, and 6.8 angstrom, respectively. loopschallenge in modeling long the such underscores loops. V(H)H homology cAb models can mechanisms providedevelopment of novel structural enable interaction to insights into therapeutic and biotechnological for Abs use. The primary structure of a 13.6 kDa single heavychain antibody camelid (V(H)H) was determined by matrix-assisted laser desorp - top-downtion ionization-time-of-flight/time-of-flight analysis.sequence majority the The of (MALDI-TOF/TOF) sequencewas obtained by mass spectrometric de novo sequencing, N-terminal with the 14 amino acid residues being determined using T(3)- sequencing and database The determined interrogation. sequence was tandem confirmed mass chromatography by liquid spec trometry (LC-MS/MS) analysis of a tryptic digest, also which provided high-energy induced collisionally dissociation (CID) data permitting clear assignment the 14 isobaric of three of the Leu/Ile residues. 11 Leu/Ile of the ambiguities be Five could resolved by homology comparisons with known V(H)H sequences. The monoisotopic molecularthe of weight was determined by ultrahigh- agreement be established analysis with to the and found 13 610.6066 Da, in excellent resolution orthogonal electrospray (ESI)-TOF primarythe entire our knowledge, that first time is this a molecular structure the with of a protein > 13 kDa weight sequence. To has been established by mass spectrometric top-down sequencing. Camelids, camels and llamas, heavy-chain produce systemto able a unique immune only antibodies. have VH domains Their (VHHs) smallest the binding units are produced systems,biotechnological for suitable by immune applications and therefore by these documented, recognition VHHs antigens well The is of protein while less rather heterologous expression. through with a copper- structure of a VHHis knownreported in complex interactions. recently aboutVHH/hapten the The X-ray azo-dye settledof VHHcontaining ability recognize the to by forming a cavity between haptens three complementarity- the determining regions (CDR). we report Here structures the of a VHH (VHH with an azo-dye, RR1, A52) free or complexed of CDR2, CDR3 involvement the illustrates and a frameworkwithout complex structure metalresidue in a of the ion. The in classical found antibodies, that to is comparable site combining a lateral although Such hapten. with the interaction lateral VL. of the absence in the

interactions Amino acid alignment CDR identification Conformation study Primary structure MALDI-TOF/TOF LC-MS/MS Sequencing VHH/hapten VHH/hapten Azo-dye structure X-ray

453 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 Laden - son et al. 2006 Alvarez- Rueda et al. 2007 Nguyen Nguyen et al. 1999 - - We have isolated have and characterized a caffeine-specific,We heavy-chain-onlyantibody of (V-HH) capable is fragment that from llama being utilized analyze to cold caffeine in hot and beverages. speciesCamelid (llama and were selectedcamel) for immunization because heavy-chain-only antibodies. heat-stable, make potential to of their Llamas and camelsimmunized were with caffeine linked keyhole to covalently limpet hemocyanin, antibody and recombinant techniques used were phage displayed create librar to ies of variable region fragments heavy-chain of the antibodies. Caffeine-specific VHH fragmentsto selected were ability their by caffeine/bovinebind to serumconfirmed albumin (BSA) and by a positive reaction in a caffeine enzyme-linkedimmunosorbent ELISA).these (caffeine assay One of VHH fragments (VSA2) wasexpressed recover shownto its and reactivas a soluble protein ity after exposure to temperatures up to 90 degrees up to temperatures ity after to exposure C. In addition, VSA2 wasat 70 degrees bind caffeine to able competition C. A caffeine ELISA was developedthe measurement for of in caffeine of coffee,beverages, caffeine obtained for concentrations and Coca-Cola Classic, and Diet agreed Coke (HPLC) with high well performance and literature determination liquid chromatography values. VSA2 showed minimal cross reactivity with structurally methylxanthines. related Single-domain antibodies (MTX) obtained specific were after to methotrexate immunization of one llama (Llama glama).Specific VHH domains (V-D-J-region) selected were by panning library from- an immune-llama The anti using technology. phage display body fragments specificto MTX purified were from coli (C41 strain)Escherichia periplasm by immobilized chroma - metal affinity level 10 mg/l. A single withband16 000 Da of around an expression around correspondingtography VHH to fragments was found blotting, while competition ELISA selective demonstrated soluble and Western binding to MTX. after analysis by SDS-PAGE Surface plasmon analysis VHH anti-MTX (SPR) resonance showed domains had affinities the nanomolar that range (29–515 in to the to be similar MTX-serum albumin conjugates. nM) to The genes encoding anti-MTX found by IMGT/V-QUEST VHH were previously reported IGHV llama and human germlineV-D and D-J genes. junctionrearrangements the The in seven anti-MTX CDR3 they sequences originated were that indicate from three distinct B cells. progenitor camelid Our that results demonstrate single-domain antibodies Furthermore, these to low molecular of high capable affinity binding haptens. are hydrosoluble weight VHHanti-MTX give new binding repertoire insights antigen of llama on how the single-domain antibody can provide combining of a VL. absence in the haptens sites to This type of single-domain antibodies murine advantages to offers compared recombinant antibodies in termsand sequence of production IGHV3 similarity human rate the to subgroup genes. The molecular occurringthe C(H)1 domain in naturally absencebasis of the heavy-chainfor antibodiesthe of camelids was assessed Camelus dromedariusby determining entire the gamma 2a heavy-chain gene. The organizationcon - the constant of gamma 2a camel heavy-chainstant gene obtained from a liver genomic library appears be to typical mammalian gamma of all other genes sequenced to case the switch, to the In contrast C(H)1, hinge, C(H)2, C(H)3, M1 and M2 exons. in mouse contains heavy and human chain It date. diseases, camel gamma structural the 2a gene shows no major defect, sequence and its is intact. equivalent C(H)1 exon However, analysis is has defective revealedpoint mutations, due to after C(H)1 exon, immediately splicing especially the site, the that the A(+1) transversion consensus signalG(+1) to seems loss splice the of the is responsible be is to concludedthe that detrimental.for It a closer analysis C(H)1 domain in camel gamma hinge 2a heavy-chain of the entire removal of the immunoglobulins. Additionally, suggests of transposons possible the exon genetic involvement in the variation of mammalian C gamma hinges. Caffeine Methylxanthines Heat-stability ELISA Methotrexate Immune-llama library Anti-MTX VHH genes Hydrosoluble hapten C(H)1 domain absence Sequence analysis Splicing

454 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article - Lauw ereys et al. 1998 Decan - et niere al. 1999 De Genst et al. 2006 Evidence is provided dromedaryEvidence that heavy-chain antibodies, chains, of light absence in vivo-matured in the a unique are of inhibitorysource antibodies. After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine of heavy-chainpancreatic alpha-amylase, amount it was antibodies, a considerable that demonstrated acting as true competitive inhibitors, bloodstream. enzyme’s with in the the circulate antibodiesdo not interact In contrast, apparently conventional the peripheral we illustrated that bloodactive Next site. lymphocytes variable one-step of the for suitable cloning are domain frag - By bio-panning, severalments vector. in a phage-display antigen-specific single-domain fragments readily isolated are both for enzymes. among those isolated that In addition we show fragments represented. When produced well activebinders are site as in Escherichia these protein activebinders appear coli, recombinant site be to enzyme potent - when tested inhibitors in chro of these site single-domain antibodies antigen-binding of the mogenic assays. composed low complexity of only three The loops becould valuable designing for smaller synthetic inhibitors. Background: Camelid serum fraction a large of functional contains heavy antibodies chain – homodimers of heavy chains without chains. light variable The domains of these heavy-chain antibodies (VHH) determining a long complementarity have loops antigen-binding region 3 variable of the absence of the (VL). the (CDR3) chains for light compensates loop that In the case part 24 amino acid long CDR3 VHHof the of the fragment loop cAb-Lys3, protrudes surface antigen-binding from the Results: A dromedary a competitive enzymeand inserts active-site the inhibitor. cleft of its into rendering cAb-Lys3 antigen, VHH with specificity bovine for RNase A, cAb-RN05, has a short CDR3 loop of 12 amino acids and is not a competitive enzyme has cAb-RN05-RNase structure of the been The A complex solvedVHH 2.8 angstrom. scaffold The at architecture inhibitor. VH (variable of a human hypervariableis antigen-binding closestructure that heavy of the to The chain). 1 loop (H1) of both differknown from the canonical The structures;cAb-RN05 and cAb-Lys3 in addition these H1 loops resemble each other. antibodies. interacts withCDR3 VL domain that in conventional face of the provides surface the and shields an antigen-binding Conclusions: of variable immunoglobulin VHHsfold common domains, adopt the loops antigen-binding but the deviate from define a new canonical predictedthe H1 loop the structure for of immunoglobulins, canonical structure. We with cAb-RN05 structures. as reference new This loopand cAb-Lys3 structure also might occur or mouse in human VH domains. Surprisingly, only two loops recognition; involved in antigen CDR2 are the does not participate. Nevertheless, binding occurs antigen the with nanomolar affinities interaction. antigen becausefor usageatoms of mainchain of a preferential Clefts on protein surfaces are avoided by antigen-combining sites of conventional antibodies,Clefts sites of conventional surfaces heavy-chain to avoided by antigen-combining on protein in contrast are antibodies (HCAbs) seem of camelids be that to by enzymes’ attracted pockets. substrate pronounced this for explanation The of HCAbspreference was fragments single investigated. Eight domain antigen-binding of HCAbs (VHH) with nanomolar affini - ties lysozyme for isolated were from three immunized dromedaries. Six VHHs of eight with compete small lysozyme inhibitors. of activebinders is also site VHH ratio within the found This pool derived from polyclonal HCAbs purified serum from the of crystal immunizedthe The dromedary. structures of six with lysozyme VHHs surfaces interaction in complex and their were sizes antibodies interface antibodies of VHH The thosesame antigen. and conventional with to the of conventional compared lysozymeto very are contacts. similarfrom of the comes the as as main difference number well and chemical the nature The formed paratope, predominantly H3 loop by the and interacting, presents of VHH shape convex a large that prolate compact structures, lysozyme withalthough different concave pocket. the substrate-binding a single domain antigen- into Therefore, hastargeting clefts dimeric site for a clear format surfaces.combining structural on antigenic advantage over a conventional

surfaces Inhibitory antibodies active site Enzyme’s Carbonic anhydrase Alpha-amylase Recombinant protein VHH Antigen-binding loops RNase A Enzyme inhibitor Canonical structures Clefts on protein paratopeConvex Antigen-combining site Lysozyme inhibitor Crystal structure

455 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 Honda et et Honda al. 2010 Smith et et Smith al. 2012 Fennell Fennell et al. 2010 domains. Despite th to 5 rd domain in the constant region, constant and one lacks domain in the 3 the rd these differences, the IgW isoforms clustered with IgWs of other of other cartilaginous the C regionsfishes and showed 47–89% amino acid theseisoformsclustered differences, the IgW IgWs with identities. mRNAs detected IgM and IgNAR were for in various tissues, mRNA was while IgW mainly detected in pancreas. The banded hounded also and IgNAR shark has as ascartilaginous IgM, IgW other well the isoform. fish unique IgW with The cartilaginous(chimeras, immune to mammalsan fish sharks, in which adaptive relative the oldest are rays) group and skates system founded upon immunoglobulins has been manuscript In this dogfish found. we characterize spiny immunoglobulins of the the (Squalus both molecular acanthias) the at levels. and expressed protein Despite in this of hundreds presence of IgM clusters the analysis of cDNA sequences and serum suggestsspecies protein However, serum the levels isotype of this low. comparatively are used IgM heavyforms in the two microheterogeneity preferentially and supportsin are clusters chains different proposal the that IgNAR isotype the also that found (monomer or pentamer) species in this isotype. of this exists We in a previously unknown- multi we identified a newisotypeformthe IgW of IgD orthologue),shark the leader (the in which is splicedmeric in serum. format Finally, resulting domain, in a moleculeconstant first directly the domain. to lacking an antigen-binding The shark antigen-binding V(NAR) domain has the potential to provide an attractive alternative to traditional biotherapeutics V(NAR) alternative attractive domain hasto provide antigen-binding the an shark The potential based on its small size, advantageous physiochemical properties, target clefts to and unusual ability in enzymes surface or cell molecules.the properties of The V(NAR)shares many the well-characterised of less single-domainmuch V(H)H but is camelid chose hen-egg-lysozyme-specific the Typeunderstood molecular I V(NAR) the 5A7 and used at level. We archetypalribosome a high level found of mutational sequence We space. entire the mutagenesis interrogate to with error-prone in combination display plasticity acrossV(NAR) domain, particularly the framework within the 2 and hypervariable region 2 regions. A number of resi - AID, S61R,dues combining important identified, for and a affinity were mutant for triple and G62R resulted in a K(D) of 460 pM hen eggover wild-type lysozyme, improvement a 20-fold highest 5A7, and the K(D) yet reported interactions. V(NAR)-antigen for Theserationalised findings were importance using structural the and indicate of residuesmodelling outside the classical- comple We also resi - located two solvent-exposed mentarity determining modulate affinity. regions contacts that antigen in making novel dues V(NAR) (G15 and G42), distant from retain paratope, the which function cysteine upon to potential to the mutation and have be as targeted exploited modification. sites for covalent extended to all knownOur findings 5A7 were NAR structures with using and a newly analysis V(NAR) generated an in-depth bioinformatic database. of sequence literature in the data available This study first time, the V(NAR)-specific for both allowed identify, usresidues, to hallmark V(alpha) overlapping and V(NAR)/Ig V(L)/TCR critical are structuralwhich each the of our designated and functional for V(NAR)- integrity single domain. Intriguingly, of the specific cDNA‘cold nurse shark hallmarks spots’sequences.natural align in precisely These previouslywith defined mutational findings will aid V(NAR) engineeringfuture towardsthe development studies of V(NAR) and optimisation single-domain proteins as viable biotherapeutics. In this study, cDNAs secreted encoding the immunoglobulin (Ig) heavy forms of the of IgM, IgNAR, study, chains In this cloned were and IgW IgM heavy the clones for encoded chains 569 and 570 amino acids, scyllium. whose- con bandedfrom Two the Triakis houndshark served (C) region showed those 47–70% amino acid to reported identities the IgNAR cartilaginousclonesfor in other Four fish. encoded 673–670 amino acids with conserved Ig-superfamily domains.region The IgNAR C showed - 56–69% amino acid identi ties those to so far reported. High-throughput sequencing revealed in most IgNAR sequences, of the that two variable the regions (CDR1 and CDR3) each possess a cysteine residue. Three types contained identified; one were Ig-superfamily of IgW domains cartilaginous in other are that fish, one lacksthe 3

mutagenesis Cartilaginous fish sequencecDNA analysis IgNAR IgW V(NAR) domain V(NAR) Ribosome display Error-prone plasticityMutational Structural modelling IgM IgNAR IgW Houndshark

456 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article Streltsov Streltsov et al. 2005 Nguyen Nguyen et al. 2002 Ferrari et et Ferrari al. 2007 - - remains remains The newreceptor (IgNAR) antibodies antigen sharks disulphide from are chains,bonded dimers of two protein containing each one variable domains.constant and five Three typesType appearing 3 of IgNARhave variable domains early been discovered, with Type have 1 and 2 response.deter we Here, development and beingin shark overtaken affinity-matured antigen-driven by the Type 2 IgNAR variable the domain, and identified links disulphidemined that first structureoccurring bond of a naturally and the stabilizes CDR1 the and CDR3 loops. This disulphide bridge locks the to other contrast CDR3 loopin “upright”conformation in an Type 3 isotype basedto modelthe we sought antibody shark on structures, configuration is observed. lateral a more where Further, crystallographicTypethe 3-specific structureinto reported to combine pack residues This internal modelincr here. that indicates supports that immunoglobulin core CDRa compact the loopIs there regions, low-sequence despite apparent variability, and that sufficient plasticity the in antigen-binding surface. diverse CDR3 formconformationally to a loop The emergence in speciesCamelidae The emergence of functional antibodies devoidto chains (referred of light as heavy-chain antibodies or HCAbs) is an intriguing evolutionary event. Homodimeric HCAbs also been have documented in spotted (Cos5-Abs) and ratfish reveal evolutionary the history (NAR). of HCAbs, To shark nurse we evaluated and phenotypic phylogenetic the relationships antibodiesamong HCAbs across and conventional taxa and confirmed the current viewpointgroups of different have that HCAbs evolved three independentlylineages. in the least, At camelids, in the HCAbs result of resuscitation not the are genes. of dormant recent of more They antibodiesthe outcome derived are conventional the apparently the lineage, Camelidae within and are from changes occurringadaptive of heteromeric antibodies. compartment in the shared The structural properties of HCAbs across taxa It chain. of pairing absence light the evolution to similarrelated constraints due to explained by convergent therefore are innovative evolutionaryappears that led changes in Camelidae a new have to level binding repertoire of antigen and diversification properties. allowed antigen-receptor of novel have acquisition In 1993, a fraction of antibodies devoid (Abs) wasoccurring of L chain naturally Camelidae. found in the Theyto lack found were L chains, asheavy-chainconstant as first well they the domain (CHI) named were therefore “heavy-chain and Abs” (HCAbs). Subsequent studies focused functional, on the structural and biochemical properties variable of recombinant fragments (rVHHs) of HCAbs. epitopes, was with rVHHsan augmented“partially capacity interact It stated to have hidden” enzymes that like active sites, an increased and have has thermal and chemical aggression. to stability been Abs It suggestedthese unconventional that an evolutionary represent could to inhibit microbial enzymes,Abs conventional advantage, than beingthus efficient more and response protective a more immune exerting against pathogens.focuses work The present the immunobiological on role of HCAbs, animal models selected, were capacity inhibit microbialver in their enzymes. to a modelcommon comprising for Two (Lama Abs glama). without HCAbs and unconventional A tebrates with both (rabbits), conventional and a modelvertebrates for bacterialrecombinant was P-lactamase selected (CTX-M-2) immunization as microbial enzymatic the After conventional antigen. schedules, serum neither nor serum titers inhibitory capacityrabbits showed when significant and com - llamas differences were pared.could of system immune the adaptive “prepared” the a prioricamelids Thesebe results that that better indicate assumption respondto bacterial to enzymes because llama of HCAbs, presence of the different when the Furthermore, is not always accurate. antibody isotypes and subclasses purified, were it the inhibitory wasthat demonstrated total capacity of serum wasexclusively due these results IgG I. HCAbsto but instead they not only failed activated Altogether, inhibit CTX-M-2, its to enzymatic activity. hypotheses the rVHHs that from the indicate extrapolated properties need be to revised; of HCAbs real role the in vivo unknown, as as evolutionary well their cause.

typic relationships response inhibition New antigen receptor Disulphide bond Loop stabilization Crystallographic studies Antibody evolution Phylogenetic and pheno - Camelidae Sharks Adaptive immune Adaptive Microbial enzyme P-lactamase

457 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 - Sacco dossi et al. 2012 Nguyen Nguyen et al. 2000 Torigoe Torigoe et al. 2012 - Heavy antibodies chain (HCAbs), CH1 devoid serum and the in the chains present light domain, are of the of camelids. IgG(2) HCAbs;and IgG(3) are immunological the study properties to structure. In order whereas of llama IgG(1) has conventional the HCAbs, is known, little llamas date to from which (Lama glama) HCAbs cloned, cDNA were sequenced with and compared other mammalian Igs. The sequence analysis llamashowed that HCAbs cDNA organization mammalian Igsto other is similarthe and of conservedpresence gamma RIII gamma A, RI,and C1q in HCAbs Protein binding motifs Fc to G, observed. Fc were Protein In a previous work, tofix IgG ability isotypes their were for assayed different chromatography G purifiedProtein A and Protein by Bothcomplement. fix total serum to complement, IgG(1) and the whereas able in were IgG(2) and IgG(3) complement even fixed we this (anticomplementaryactivating work in activity). activityperformed of antigen absence the complement Therefore, of the IgG isotypes different the purifiedphysiological hemagglutina under - to induce conditions ability using their G-150 and Sephadex tion. Llamas immunized were with sheep red blood (RBC) cells isotypes different and the stroma purified were from sera. Whole HCAbs not, C1q bind - (IgG(2) + IgG(3)) could of the presence serum despite the activate complement; however, and IgG(1) could primarying motif in their fraction IgG(1), the sequence. corresponding hemagglutinating IgG(2) + IgG(3) did not display Unlike to might site the C1q binding that Our findings antigens and HCAbs suggestthat activity. different with cannot efficiently crosslink variable of the be domains. hindered proximity by the mline database was constructed segments from PCR-amplified of a single specimen THH/V-H of dromedarius.Camelus total A of 33 VHH and 39 VH unique sequences identified, genes, encoded were compari by 42 and 50 different - respectively. Sequence V(H)Hs evolved the son indicates that VH within the subgroup III, Nevertheless, VHH the germline segments highly diverse, are leading a broad structural to loops, repertoire antigen-binding of the Seven VHH subfamilies recognized, were five were of which confirmedto expressed be lit vivo. Comparisonrearrangedthe of germline and cDNA that sequences V(H)Hs are demonstrates processes, diversifiedmutation by somatic extensively hypervariable leadingto an additional region- nucle and a high of incidence otide insertions or deletions, Theseprocesses diversification are driven by hypermutation hotspots and recombination embedded VHHin the germlineregions the genes loops. affecting at the antigen-binding the structure of The antigen-binding site of the heavy-chain of camel site The antigen-binding antibodies devoidconsistschain of light a single variable domain (VHH) VHH repertoire, of the antigen-binding a ger evaluate extent the To diversity, combinatorial obviously lacksthat V-H-V-L the A novel monoclonalA novel anti-pan leukocyte human (HLA) antigen EMR8-5, class I heavy was antibody, chain could established. It detect HLA-A, in formalin-fixed -B, and -C antigens paraffin embedded tissues.immunohistochemicalthe By staining using variousEMR8-5 cancer tissues antibody, from 246 cases examinedHLA were for was HLA that It found class I expression. class I carcinoma, cell renal wasexpression decreased hepatocellular carcinoma, cancer, colon cases 42% of the in 20% to of lung cancer, carcinoma.and urothelial In contrast, 85% of breast cancer cases had loss of or decreased HLA 35 breast Of the class I expression. cancer cases had decreased that HLA class 33 (94%) also I heavy had decreased expression, chain beta2-microglobulin expression detected by immunohistochemical staining. was It suggestedHLA that class I down-regulation be might characteristic a common of breast cancer mostly caused down-regulation by the of beta2-microglobulin expression.

properties activity activity repertoire antigen Immunological Complement activating Anticomplementary Hemagglutination Antigen-binding Germline database Sequence comparison Evolution Somatic mutation Application leukocyteHuman Cancer

458 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article - Tayebi et et Tayebi al. 2010 Ander son and Goldman 2008 Although there is currently no effective treatment for prion no effective diseases, is currently there treatment Although significanthave advances been made in suppressing its progress, be to In order effective of PrP(C) PrP(Sc). using individuals antibodies in treating blockinto that conversion the that prion diseases,have antibodies be must of arresting capable disease in its stages. late requires This the development of antibod - effectiveies and for with for PrP(Sc) higher affinity systems antibodies translocation of across the blood to brain barrier order in of these An additional advantage replication. ability antibodies is the of protein site the at of inhibitor achieve high concentrations PrP-specific generated this end, we have To antibodies cytosol the by immunization access to (known as of affectedPrioV) cells. of camels with scrapieadsorbed murine material antibodies immunomagnetic to beads. a range of specificities display The PrioV with some fragment, recognizing specific others PrP (27–30) proteinase the K-resistant for PrP(C) number and a dual bind - with shown to bind PrP(C)was antibodies (PrioV3) the one of PrioV specificity, conformation Independenttheir PrP of ing specificity. cytosolin the of neuroblastoma antibodies cells. anti-PrP In marked contrast,produced in mouse conventional against similar anti-PrP crossneuronal plasma to the unable membrane and instead were formedtarget antigen cells. a ring the around The PrioV antibodies be to compartments of affected prove a valuable neutralization/clearance could tool the in intracellular for of PrP(Sc) of so-called treatment diseases. the wider for protein-misfolding potentiallyapplicability have neurons and could Llamas possess unique subclasses of antibodies pairing chains, made by the lack light are of two heavy that and thus chains. IgG was purified two llamasfrom had which been immunized trinitrobenzene-keyholewith limpet hemocyanin. IgG1 Conventional The effectiveness and heavy of IgG2 and IgG3 subclasses chain chain heavy fractionated were usingchromatography. affinity antibodies detection the for (TNT) of trinitrotoluene using immunoassay a competitive fluid array was evaluatedto compared and was heavy antibody that chain It found bound TNTboth llama IgG1 as the antibody. as well monoclonal a murine anti-TNT with antibodies,selectivity conventional similar to yetheavy the antibodies chain the of possessed The titer thermal greater stability. heavy antibodies chain however analytical was IgGl1; thus be the to best lower than assays found were 10-fold using demonstrated The TNT fluid analyzerLuminex competitivethe had a on antibody. immunoassay dynamic range llama IgG1 conventional the 10 µg/ml. Utilizingdynamic same two-stepfrom monoclonal 100 ng/ml to the similar the range of the to competitive format assay antibody 1 µg/ml). This method a broad range (1 ng/ml to was have to was found on TNT demonstrated extracts contaminated soil using bothmouse llama IgG1 and the the monoclonal validatingutility of method the analysis samples. for of held immunoassay Application Prion immunotherapy Scrapie PrP conformation Application Competitive fluid array Trinitrotoluene

459 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 - Mak vandi- Nejad et al. 2011 Ezzine et al. 2012 Gorlani et al. 2012 Three V(H)Hs against the model hapten, azoxystrobin (MW 403), were isolated (MW 403), were from azoxystrobin a hyper-immunized phage-displayed V(H)Hs againstmodel Three the hapten, library This V(H)H library. was constructed by isolating V(H)H-coding genes the lymphocytes from the collected from a Lama was glama bovine that to immunized conjugated serum with azoxystrobin albumin (BSA). Six of panning rounds were serum or rabbit albumin (RSA) enrich clones to performed ovalbumin either (OVA) to conjugated against azoxystrobin V(H)Hs specificcontaining After screening hapten. the 95 clones, to amino three V(H)Hs (A27, A72, and A85) with different acid sequences identified, were expressed in Escherichia in soluble format HB2151, and purified coli - using nickel-immo Competitive inhibition enzyme-linkedbilized immunosorbent (CI-ELISA) assay metal affinity chromatography. showed specific A27 and A85 were that while A72 was azoxystrobin to not. IC(50) values The 7.2 and of A27 and A85 V(H)Hs were our knowledge highest A85 is one of the affinity has yet V(H)Hs that been isolated- against a hydro To 2.0 µm, respectively. as such azoxystrobin. phobic hapten Using eukaryoticUsing hosts, enables not antibody This fragments secretory generally expressed the by targeting are to pathway. only efficient disulfide bond but also formation secretion of soluble and correctly folded goal, this product. a recombi - For vector nant was using constructed a single-domain produce antibodytoxin to (NbAahI’22) directed against AahI’scorpion yeast. methylotrophic the corresponding The complementary oxidase- DNA was pro alcohol of the cloned under control Using pastoris strain X-33. cell secretion α-factor in frame signalmoter Saccharomyces withtransferred the P. and then to to medium. culture in the Targeting blot, NbAahI’22 exclusively we detected recombinant of the expression the Western histidine secreted label,the the nanobody was easily purified on nickelnitrilotriacetic acidtested resin and then in enzyme- production the level NbAahI’22 in its of the new glycosylated Interestingly, form reached more linked immunosorbent assay. pastoris as a heterologous obtained sixfoldthan that in E. findings These coli. give evidence more utilization the for of P. system. expression Variable domains of llama heavy-chain antibodies (VHH)Variable becoming are tool a wide a potent for range of biotechnological and medical applications. Because easy typicalthey- of structural pro relatively features to are single-domain nature, of their in lower eukaryotes, duce set out therefore some molecules that but it is poor not uncommon have secretion We efficiency. identified five computationally key crucial are residues and secre that folding for - production the study yield of VHH.to We tion, and we validated importance their with site-directed systematic observation mutations. The all key residues that were localised V segment, J segment in the of the of VHH, ledimportance in proximity the study usof J segment to in secretion use the of specific that we found J segments Intriguingly, efficiency. in VHH influence strongly could production the yield. Sequence with experiments chaperones, analysis suggestedinteractions strongly and expression that especially J with the segment, a crucial are aspect production of the yield of VHH.

tography promoter signal cerevisiae E. coli Azoxystrobin Metal affinity chroma - ELISA Table 4. Production of single-domain antibodyTable fragments using recombinant technology Pichia pastoris AahI’ scorpion toxin Alcohol oxidase A-factor secretion Saccharomyces Secretion efficiency J segment

460 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article Pollithy Pollithy et al. 2011 - Korou zhdehy et al. 2011 Conrad Conrad et al. 2011 - - - persis was by genetic fusion accomplished magnetosome of RBP the to protein synthesis of MNP-immobilized nanobodies. Moreover, intracellular nanotraps can be generated to can nanotraps synthesis be to intracellular generated of MNP-immobilized nanobodies. Moreover, MamC in the magnetite-synthesizingMamC in the bacterium Magnetospirillum isolated gryphiswaldense.that magneto - demonstrate We MamC-RBP efficientlysomes recognize expressing in vitro and can antigen be and bind their used immunoprecipitation for of RFP-tagged partners of monomeric extracts. interaction coexpression and their from cell proteins that In addition, we show RFP (mRFP or its variant mCherry) recognition and MamC-RBP results and magnetosome in intracellular recruitment of RFP within living of a functional bacteria. expression nanobody intracellular targeted The a specific to bacterial opens compartment new possibilities in vivo for Numerous applications of conventional and biogenic magnetic nanoparticles as of conventional such applications in diagnostics, (MNPs), Numerous immunomagnetic separations, and magnetic labeling, cell immobilization require the of antibodies. is by chemical usually This accomplished has several disadvantages, however, which, asconjugation, such poor efficiency Here, need and the chemistry. coupling for we describe strategy a functional display a novel to camelid antibody fragment (nanobody) from an alpaca (Lama pacos) on surfacethe of bacterial biogenic magnetic nanoparticles (magnetosomes). Magnetosome-specific of a red fluores expression (RFP)-binding nanobody protein cent (RBP) in vivo bacterialmanipulate structures in live cells. Several facile plants the are and economic large-scale bioreactor for production of industrial and pharmaceutical- pro agents like and antibodies.teins selected as we have host because the plant Here, tobacco scale of large other production and many capability advantages as such safety and lower production greater sub - costs animal-based to when compared we have systems. study, In this cloned pBI121 using VHH phasmid gene into pCANTAB5E. The newconstruct was usedthe Agrobacteriumto transform strains C58GV3101 and LBA4404. Agrobacterium strain C58GV3101 showed a higher virulence on leaf disks tabacum of Nicotiana The developed plants then were tobacco of CaMV 35S promoter. by introducing VHH control gene under the (NC25). Transgenic VHH of the presence antibody genome was plant gene in the verified by PCR analysis and hybridizationSouthern experiments. Northern genesVHH blot analysis be the codingplants. showedfor could expressed that in tobacco Three lines of transgenic high expresses levels that plant of mRNA screened were in a further analysis. of expression VHH The wasthen observed in trans genic plants by ELISA specific using the antibody developed,resultsthe showed folds three than non-transgenic higher to five tobaccos. Tumour necrosis factor (TNF) is pro-inflammatory a major Tumour cytokinemultiple inflammatory involved in diseases. The detrimental activity of TNF can be blocked by various antagonists, therapeutics and commercial based upon principlebeen this have approved disease and psoriasis. of diseases treatment for arthritis, anti- improved rheumatoid including new, In a search for Crohn’s inflammatorytherapeutics designed we have antibody The a single-domain monoclonal antibody recognizes (V(H)H), which TNF. (TNF-V(H)H) is basedcomponent TNF was uponCamelidae which heavy-chain an anti-human linked an monoclonalto antibody, fusion of the protein ELP accumulation enhances that fusion TNF-V(H)H the demonstrate to elastin-like polypeptide (ELP). We anti- this plant-derived that first time the for we show study, this plants.during biomanufacturing in transgenic tobacco With antibody was TNF-V(H)H human biologically therapeutic of TNF-V(H)H-ELP application active fusion in vivo. protein Therefore, was tested in humanized and was TNF mice be shown to effective in preventing death caused by septicshock. The in vivo tence of the ELPylated of the tence antibody of non-ELPylated was that longer than 24 fold similar to TNF-V(H)H. gryphiswaldense system Magnetospirillum Magnetic nanoparticle Magnetosome Red fluorescein protein Tobacco expression expression Tobacco Agrobacterium Tumour necrosis factor Tumour Fusion Elastin-like polypeptide Tobacco

461 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 Swain et et Swain al. 2010 Yau et al. 2003 - - Single-domain antibodies specific (sdAb) serotype neurotoxin botulinum A (BoNT for selected A) were from an immune llama library phage display derived was from a llama that immunized with BoNT constructed The phage library A toxoid. and BoNTwas toxoid panned A complex using two methods: panningwith coated BoNT on plates (BoNT A toxoid ATd) and BoNT (BoNT Both and panning on microspheres panning coupledBoNT methods A toxin to A Tx). (BoNT A Td Td) Ac selected sequences, had identical be may as binders that material for effective panning on toxoided suggesting as that pan - ning on bead-immobilized isolating for specific toxin binders. isolated All of the binders tested observed were recognize to bead-immobilized BoNT in direct binding assays, and showed very A Tx cross-reactivity little BoNT towards other sero - types Sandwichincorporated assays protein. that selected and unrelated demonstrated elements and tracer as sdAb capture recognize to able were sdAb with soluble all of the virtually (“live”) BoNT and BoNTthat no cross-reactivity Tx A Tx Ac BoNTwith other serotypes. high isolated degree the did not exhibit The sdAb of thermal often stability associated with these reagents; after first the heating cycle most binding activity of the was lost, did refold portion but the that protein of the activityand recover antigen-binding showed only minimal loss on subsequent heating and cooling cycles. binding kinet The ics of selected binders, assessed by both an equilibrium fluid as assay as array well surface plasmon using (SPR) resonance dissociation gave material, high-affinity 1.6 × 10(–10)M. These range 2.2 × 10(–11) to constants (K(D)) in the toxoided bind - detection beneficial prove rapid reagentsthe ers may of BoNT developmentfor of recombinant the to A, particularly in field screening and monitoring applications. Picloram-specific variable fragments (V(HH)s) of heavychain antibodies (HCAbs) were selected a naive-llama libraryfrom using ribosome A cDNA library technology. display of V(HH)s was constructed from lymphocytes of a non-immunized llama and engi - transcripts, codons on the present no stop neered of ribo allow in vitro transcription to trimeric- With and translation. complexes somes, mRNAs and nascent peptides produced were selection, affinity for i.e. panning. threeAfter cycles of panning, seven differ three cycles another subfamily isolated. 1 were Following of selection, V(HH)s all belonging sevenent V-HH only two of the the to V(HH)s persisted. A comparison of these two sequences with known suggests point mutations sequences that literature in the been DNA pool have the may introduced into during PCR steps of library amplification construction, cloning.panning and/or Threethree mutations independent causing separate amino acidchangespoint the (nonsynonomous in accumulated mutations) same sequence and enriched binding advantages. selection these the changes suggesting throughout confer protocol, that Surface plasmon analysis (SPR) resonance was used determine to binding kinetics two clonesthe (3-1D2 and 3-1F6) representing of the setstwo different of determiningisolatedregioncomplementarity (CDR)3s. The Measured respectively. 3 and 254 µM, K(D)s were ribosomeresults that indicate technology display can be usedhapten-specific efficiently isolate to antibody (Ab) fragments a from selectednaive library the diversity to introduce pool facilitating and concurrently molecular thereby evolution. Ribosome display naive librarytechnology limited the diversity of a V-HH and provide for an unlimitedof affinity-matured source can compensate immunoactive reagents in vitro. translation Panning methodsPanning Botulinum neurotoxin Binding kinetics Picloram Ribosome display In vitro transcription/ mutation Point Binding kinetics Molecular evolution

462 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article Fennell Fennell et al. 2010 Ryckaert Ryckaert et al. 2010 Martin et al. 2011 Joosten Joosten et al. 2005 We chose hen-egg-lysozyme-specific the Type I V(NAR) 5A7 and used We archetypalribosome error- combination with in display a high levelplasticity found of mutational acrossV(NAR) the sequence We space. entire the mutagenesis interrogate prone to domain, particularly framework within the 2 and hypervariable region 2 regions. A number of residues important affin - for S61R,combining AID, identified, mutant and a tripleity were resulted and G62R for hen in a K(D) of 460 pM egg lysozyme,a over wild-type improvement 20-fold highest 5A7, and the K(D) yet reported interactions. V(NAR)-antigen for These findings were rationalised using structural importance the modelling and indicate of residues outside classical the determin - complementarity We also residueslocated (G15 and G42), two solvent-exposed modulate affinity. ing regions contacts that antigen in making novel V(NAR)distant from retain paratope, the which function cysteine be upon to potential as to the exploited mutation and have targeted modification.sites for covalent extended to all knownOur findings 5A7 were NAR structures with using an in-depth and a newly analysis V(NAR) generated bioinformatic database. of sequence literature in the data available allowed This study first time, the V(NAR)-specific for both identify, usresidues, to hallmark V(alpha) overlapping and V(NAR)/Ig V(L)/TCR which criticalare structural each the of our designated and functional for V(NAR)-specific integrity single domain. Intriguingly, of the cDNA spots’nurseshark sequences. hallmarks‘cold align in natural precisely These with previously findings defined mutational will aid future V(NAR) engineering development as studies towardsof V(NAR) the and optimisation single-domain proteins viable biotherapeutics. Yeast surface is for display an efficient tool and engineeringisolating We describeantibody the use Yeast fragments,Fab. both scFv and on Pichia pastoris display selection rapid of protein the of camelid antibodies for composed only of heavy (nanobodies) chains from a library derived from a Ilama immunized The library with Protein. Green Fluorescent of nanobody-coding sequences was fused cerevisiae C-terminal the part to and expressed in glycoengineered alpha-agglutinin Saccharomyces gene (SAG1) of the pastoris. A high efficiencyprotocol yielded transformation a library clones. of 5 × 10(7) P. clones the strongly About 80% of nanobodyexpressed the fusion. Nanobody-displaying enriched activated rapidly clonessorting were cell by fluorescence (FACS), and GFP-specific nanobody-displayingclones isolated were and equilibrium dissociation determined.constants (K(d)) This pastoris isolation enables the and engineering of antibody-derivedtechnology molecules libraries displaying for protein on P. in a eukaryoticrobust host. expression A series cassettes of expression mediate secretion which or surface of antibody display fragments was- chromo integrated stably in the some of Lactobacillus paracasei. L. paracasei producing surface-anchored variable domain of llama heavy (VHH) chain (ARP1) directed against rotavirus rotavirus to showed binding protection efficient and the mouse in rotavirus of model infection. We report and production of llama expression variable the heavy-chain antibodyWe fragments (V(HH)s) by Aspergillus awamori. encoding cloned V(HH)sFragments were Aspergillus in a suitable vector fragments and transformants expression secreting V-HH analysedwere integrated gene copy-numbers, for mRNA detected V(HH)s were levels production. and protein Functional in the medium,culture feasibility the indicating of producing type this in a fungal system. Secreted of protein expression V(HH)s were degradation,be could which partiallysubjected medium. culture prevented the addition of BSA (extracellular) to by the to

mutagenesis residues protein system Ribosome display Error-prone Lysozyme Solvent-exposed Yeast surface display Yeast Pichia pastoris Green fluorescent Surface display Expression cassettes Lactobacillus paracasei Rotavirus Fungal expression expression Fungal Aspergillus awamori

463 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 Bazl et al. 2007 Zou et al. 2005 Vincke Vincke et al. 2009 - There is an increasing interest in the application of nanobodiesapplication is an increasingthe There in and imaging. interest such therapy as of the field VHHstudypresent in a the In geneticallystable engineered line of Chinese ovary cell hamster (CHO) origin transfected using two sets vectors was of expression - con structed permit to in order cytoplasmic the of single domain antibody expression along with green protein and extracellular fluorescent (GFP) as reporterconstructsthe gene. The quality of examined restriction were the by both map as well as sequence analysis. The gene wastransfection expression further and protein examined by reverse transcription-polymerase The transfected reaction chain (RT-PCR). line obtained media containing cell stable than grown activity showed and the were fluorescent for more in 200 µg/ml hygromycin cells a period of 180 days. The production of fluorescent fusion was protein spectroscopy also well detected as microscopy, as by fluorescent by enzyme-linked immunosorbent (ELISA) assay analysis. This strategy production allowsfluobodiesrecombinant a rapid of involving VHH, can which be used in various experiments as such imaging and detection a primary in which labeled antibody is required. In mature B cells of mice and most mammals, of mice B cells release cellular is withoutIn mature of singleAbs L chains prevented H chain asso by H chain - ciation with Ig-specific chain in of µ-H the endoplasmic surfaceexpression in chaperons reticiulum. In precursorcells, B however, has L chain been and conventional of surrogate absence identified. the this,Despite Ag-specific singlechain Ig repertoires, H using separately, or, removal of H chain Abs, not produced.µ-, gamma-, are epsilon-, in conventional Moreover, found or alpha-H chains (kappa/lambda)L chain locus sequences core by gene targeting has development. prevented B cell In contrast, Abs H chain-only can Abs test whether H chain be produced in in Camelidae produced asare abundantly C(H)l domain. To H2 IgG without the affects development, expression cell how their B rearranged we introduced investigate a and to mice, dromedaryH gamma 2a mouse the germline. The dromedary into chain transgene wasexpressed occurringas a naturally Ag-specific disulphide-linked - development can be of singlewithout B cell L chains. H chains showed instigated which by expression that Lympho homodimer, BCRcyte from was the of L chain absence despite the proliferation accomplished development Endogenous and B cell complex. Ig not becould detected, and IgH/L transcription V(D)J recombination although - was crossingdrom the unaltered. Furthermore, edary devoid can with mice Ab facilitate mice B cell a H chain-only H chain of all C genes without demonstrated a doubt that development early independentasdevelopmental such at stages. of endogenous chain, µ- or delta-H Ig expression, Nanobodies, fragments single-domain antigen-binding of camelid-specific heavy-chain only antibodies offer special advantages over classic antibodyin therapy fragments because smaller size, of their target robustness,unique epitopes. to and preference A Nanobodyhuman heavy a differs chain from aboutvariable domain in ten amino acids spread all over its surface,four hallmark Nanobody-specificregion amino acidsthe loop in (positionsframework-2 antigen-binding third 42, 49, 50, and 52), a longer camelid-specific the therapeutic applications area. over this (H3) folding amino acid For sequencesthe to in framework be have heavy performed human variable their chain to mutated domain equivalent, with humanization this exercise i.e. humanized. We Nanobodies represents subfamily close 80% of all dromedary-derived of the that to Nanobodies effects and investigated the on humanization the Nanobody-specific of that It is demonstrated residues expression and stability. yield, solubility, affinity, antigen Nanobodyoutside neutralthe are to framework-2 properties. -> Leu Glu-49 -> Gly and Arg-50 humanization the of Surprisingly, less substitu - probably soluble. though stable is The other more framework-2 amino acids a singlehallmark domain that generates to a repositioning due the H3 of loopby shown as affinity, detrimental antigen are for and Gly/Ala-52 -> Trp, tions, -> Val Phe-42 crystaltheir structures. These insights used were to identify expressedhumanized a universal well soluble, stable, Nanobody scaf fold that allows grafts that fold loops of antigen-binding Nanobodies from other specificity antigen of the with transfer and affinity. cells germline nanobody scaffold Chinese hamster ovary Expression vectors Reporter gene GFP-fusion protein Expression in mouse Dromedary transgene Gamma 2a H chain Universal humanized

464 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article - Vaney cken et al. 2010 Fanning Fanning and Horn 2011 - - - experiments revealed specificity targeting of tumor imaging of tumors. Nanobodies type with a novel beneficial are protein antigen-binding of immunoglobulinlike, pharmacologic and pharmacoki - molecularnetic for properties ideally antigens are suited targeting cellular imaging to that or therapeutic purposes. However, because origin, camelid, nonhuman possible of their the immunogenicity of Nanobodies when used is clinic a concern. in the a new we present humanizedHere strategy quickly generate Nanobodies to molecular for imaging purposes. Methods: We genetically grafted loops antigen-binding the of NbCEA5, a Nanobody with specificity carci carcinoma- marker colon the for noembryonic (CEA), antigen framework the onto of a humanized Nanobody scaffold. scaffold This has been previously charac terized in our laboratory as Nanobody a stable can serve that loops as antigen-binding for a universal loop from donor acceptor Nanobodies and has been about 10 crucial at additionally mutated surface-exposed resemble sequence sites to the of human variable immunoglobulin domains. Nanobodies 3 recombinant The (NbCEA5, humanized scaffold, and humanized CEA5 graft) Nanobodies biochemically were producedcharacterizedwere in bacteria in vitro and purified. Unlabeled and (99m)Tc-labeled and tested as probes SPECT/CT for of xenografted tumors. Results: of loop-grafting success The was confirmed by comparing these Nanobodies capacity recognize their to for soluble CEA in enzyme-linked protein immunosorbent and by surface assay plasmon CEA-positive bind to and to resonance LS174T and CEA-transfected carcinoma cells colon but not untransfected Chi - nese ovary hamster in flow cells cytometry. Specificity of binding was confirmed by competition studies. All Nanobodies were and recognized both be soluble could and membrane-bound efficiently labeled CEAheat-stable, with (99m)Tc, in bind - protein Nanobodies in biodistribution experiments performed were injecteding studies. with intravenously (99m)Tc-labeled Finally, in vivo These LS174T using pinhole SPECT/micro-CT. mice tumor-bearing all Nanobodies, for clearance renal and rapid with low signals besides in all organs kidneys. the shows study Conclusion: This potencythe loop-grafting of antigen-binding humanized efficiently to generate Nanobodies retaintargeting capaci their - that ties noninvasive for in vivo While several VHHsbeen have is known anti-hapten little generated, underlying structural regarding the and thermody namic basis for hapten recognition. Here, an anti-methotrexate VHH VHH) (anti-MTX wasnamic basis recognition. an anti-methotrexate using generated hapten Here, grafting for methods determining regions three complementarity the (CDRs)whereby inserted were VHH an existing framework. onto Thermody namic analysis VHH anti-MTX of the CDR1–3 Graft revealed crystal while the a micromolar binding affinity, structure of the revealedcomplex a somewhat surprising CDR1 noncanonical under the involved MTX which loop. tunneling binding site Due of MTX CDR4, to close the a nonhypervariable proximity to loop, CDR4 the loop sequence was subsequently introduced into CDR1–3the graft, Crystal increase resulted1000-fold which in a dramatic binding affinity. in the structure analysis of both the CDR1–4 anti-MTX free graft and complex revealed CDR4 a significant plays in both role intermolecular contacts and binding VHH anti-MTX the possessed high toward affinity appear contribute to binding. that conformation site relatively Additionally, high specificity VHH capable domains are that demonstrating MTXcompounds aminopterin and folate, for over closely related of binding low-molecular ligands weight with high affinity reduced despite their and specificity, interface.

imaging antigen analysis Humanized nanobodies Loop-grafting In vivo Scaffold Carcinoembryonic Grafting Hapten Methotrexate Crystal structure

465 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513

Teh and and Teh Kava- nagh 2010 Bell et al. 2010 Saerens Saerens et al. 2005 Veggiani Veggiani and de Marco 2011 imaging and/or therapy of cancer, three types of sdAb-based of cancer, moleculesimaging and/or therapy and its ability to be and its to ability functional conserved of the absence in the disulfide chime - bond. All five Here we describeHere benthamiana, high-level in Nicotiana expression, egglysozyme white of three versions of an anti-hen (HEWL) nanobody original the include which VHH a codon-optimized from an immunized libraryderivative, and a codon- (cAbLys3), optimized hybrid nanobody nanobody grafted CDRs ‘universal’ the comprising an alternative framework. onto of cAbLys3 His6- and StrepII-tagged derivatives of each nanobody cytoplasm, in the targeted accumulation were for chloroplast and apoplast using pre-sequences.different apoplast,the When to targeted intact highfunctional level nanobodiesexceptionally at an accumulated great potential the of plants demonstrating as 30% total leaf(up to protein), a nanobody production system. In the search for better search for antibodyIn the in vivo formats for directed against epidermal growth factor receptor (EGER) constructed, were characterized and tested. Eleven isolated sdAbs were from library a phage display constructed repertoire sdAb from the of a llama immunized with a variant of EGFR. A pentameric was V2C-EG2 constructed or pentabody, sdAb, by fusing sdAbs, one of the a pentamerization EG2, to domain. A chimeric protein HCAb was (cHCAb), EG2-hFc, constructed by fusing fragment the EG2 crystallizable to IgG1. Whereas of human EG2 and (Fc) was and this accumulation, largely tumor localizedV2C-EG2 exhibited excellent injection, kidneys EG2-hFc mainly in the after i.v. of IgGs. itsthat attributed to long serum size The moderate is which comparable to 80 kDa)human (similar half life, and intact HCAbs make outperform a unique antibody may whole IgGs which Fc as format imaging and therapeutic reagents. Camel single-domain antibody fragments (VHHs) tools promising biotechnological are in numerous and medical applications. antibodies under which some conditions used are they so can are demanding that be metmostHowever, by only the VHHs. robust A universal frameworkrequiredthe offering propertiesfor (e.g.use in as probevarious in biosensors applications or on as intrabody, ismicro-arrays) highly valuable be is and might envisaged. further of VHHs therapy when employment We in human implemented the identified VHH framework of cAbBCII10 specificities of antigen as a - exchange useful complemen candidate, potential the by for tarity determining region (CDR) grafting. number of CDRH Duelarge the to loop structures on VHHs, present grafting this tech - nique was expected unpredictable. be Nonetheless, to rather plasticity the cAbBCII10 of the framework allows of successful transfer specificityantigen its donor from VHHs cAbBCII10 onto scaffold. The was chosen for essentially its high stability (47 kJ/mol, of level good level (5 mg/l in E. expression ) coli ras by grafting generated CDR-Hs, from donor VHHs belonging encompass 75% of all antigen-specific subfamily to 2 that VHHs, on frameworkthe of cAbBCII10 functional were and generally had an increased The grafting loops of thermodynamic CDR-H stability. from VHHs belonging subfamilies other to resulted in chimeras of reduced capacity. antigen-binding Camelidae single domain antibodies- con (VHHs) alternatives to suitable them render that structural and binding features have IgG antibodies.ventional VHHs usually antibody are other easier than fragments. as produce proteins to recombinant However, somebiotechnologicalfor of the theybeen which for have applications proposed, and assisted- as such immunochromatography amounts of purified large crystallography, antibodies whereas necessary, are some VHH-fusionscommon tags with such as GFP poorlyand SNAP are of Erv1p bacterial expressed in the co-expression periplasm. the sulfhydryl shown that we have Here oxidase resulted in an astonishing yield increase constructs of VHH-SNAP bacterial expressed in the cytoplasm.resultingrecom The - antibodiesbinant antibodies the also than stable were more produced same plasmid, using the but in wild-type bacteria. Using it was approach, this possible obtainof milligrams to tens of purified fusion antibodies using a basicprotocol. flask fermentation the described Therefore, methodrepresents amounts of single a domain antibodieslarge inexpensively valid to produce solution and we expect in vitro applications for it will beproduction the antibody for suitable of other fragments.

framework factor receptor oxidase Grafting Tobacco Lysozyme nanobody‘Universal’ Epidermal growth Pentabody Long serum half life fragment Fc Universal framework Grafting Chimera VHH-SNAP constructVHH-SNAP Erv1p sulfhydryl

466 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article Rutgers Rutgers et al. 2011 Abulrob et al. 2005 Caljon et al. 2012 - - imaging and as efficient amyloidosis local cerebral drug moietypatients targeting insuffering from injection, inflammation-induced damage to the blood-brain barrier, as in the late encephalitic stage encephalitic of trypanosothe late as in injection,the blood-brain inflammation-inducedto damage barrier, - port was cells brain endothelial of FC5 across polarized, human dependent, independent and temperature charge suggesting a process. co-localizedreceptor-mediated cells brain endothelial FC5 taken up by human but not with with caveolin-1 clathrin by immunochemistry and was detected in clathrin-enriched subcellular fractions by western blot. migration The transendothelial of FC5 was reduced of clathrin-mediated by inhibitors endocytosis, K+ depletion and chlorpromazine, but was insensitivecaveolae to inhibitors,nystatin or methyl-beta-cyclodextrin.Following filipin, internalization, FC5 was to targeted endosomes, early bypassed endosomes/lysosomeslate and remained intact after transcytosis. The transcytosis process was affect inhibited that by agents actin with wheatgermcytoskeleton cells agglu signaling- PI3-kinase.brain endothelial through of human or intracellular Pretreatment tinin, sialic acid, alpha(2,3)-neuraminidase recognizes agglutinin or Maackia that amurensis alpha(2,3)-, but not with Sambucus nigra recognizes agglutinin that alpha(2,6)-sialylgalactosyl residues,reduced significantly FC5 transcytosis. FC5 failedrecognizeto cells-derivedbrain endothelial lipids, it binds luminal alpha(2,3)-sialoglycoproteintriggers suggesting receptor which clathrin- that mediated endocytosis. receptor This may be a new putative for developing target brain-targeting drug delivery vectors. such as Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA). disease angiopathy as amyloid such Alzheimer’s (AD) and cerebral Antibodies transcytosis undergo against receptors that acrossblood-brain the barrier been (BBB) have used as vectors target to transmi - FC5, which discovered single domain antibody, a novel recently have drugs or therapeutic peptides brain. We the into BBB in vivo. in vitro and the The purpose cells endothelial thisgrates cerebral acrossstudy of wascharacterize human to mechanisms of FC5 endocytosis and transcytosis cells. acrossBBB and itsbrain endothelial the The trans receptor on human putative Previously selected beta amyloid recognizing heavy antibody chain fragments (VHH) affinity binders derived thefrom Camelid heavy antibody chain repertoire propensity crossblood-brain tested their to the were for barrier (BBB) using an established in vitro system. Of all testedBBB co-culture VHH, ni3A showed highest transmigration efficiency which is, in part, facilitated three by a amino acidmechanism substitutions in its the passage studies indicatedof transcellular N-terminal that domain. Additional of ni3A is by active transport. As recognize to VHH ability beta the ni3A combines amyloid crossBBB, it has and to the potential as a tool non-invasive for in vivo miasis, significantly increased SPECTthe efficiencyComplementing analyses of with the nanobody passage of through this barrier. microdialysis analysis improved intracerebral of brain disposition. clearis There value in assessing the blood-brain of penetration bar rier by monovalent nanobodies in models of CNS clearance inflammation. blood Our from alsodata rapid suggestthat hamper might of efficient targeting specific nanobodies the to implications:CNS. Conclusions and Nanobodies from the brainparenchyma enter can systemicthe circulation, especially blood-brain in pathological the where conditions barrier integrity is compromised. Experimental approach: We have assessed have blood-brain the barrierExperimental We permeability approach: of Nb_An33, a nanobody againstTrypanosoma the brucei brucei variant-specific surface glycoprotein (VSG). This analysis was the performedin rats were rats that and in in healthy stageencephalitic of African trypanosomiasis microdialysis, using intracerebral tomography emission single computed photon Tc-99m-labelled(SPECT) nanobodies of both methodologies. or a combination This of unlabelled enabledthe quantification and using, a sensitive VSG-based respectively, nanobody-detection ELISA, radioactivity measurement in collected microdialysates and SPECT image analysis. Key results:combined The read-out methodologies Nb_An33 showed that was detected the brain of healthy in rats following i.v.

cells efficiency opathy permeability VSG Blood-brain barrier Transcytosis Endocytosis Cerebral endothelial Putative receptor Beta-amyloid Transmigration diseaseAlzheimer’s angiCerebral amyloid - Table 5. Characteristic propertiesTable of single-domain antibody fragments Blood-brain barrier Trypanosoma brucei Surface glycoprotein

467 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 Hussack Hussack et al. 2011a Omidfar et al. 2007 - The extreme pH and protease-rich environment of the upper pH and protease-rich of tractextreme gastrointestinal environment obstacleThe is a major facing orally-administered therapeutics,protein antibodies. including engineering, protein Through several toxinClostridium A-specific difficile chain heavy antibody variable domains (V(H)Hs) expressed with were an additional disulfide and Ile78Cys bond by introducing Ala/Gly54Cys antibodies wild-typemutations. their to Mutant compared were counterparts with respect yield, non-aggregation expression to toxinstatus, for affinity A-neuprotease- A,toxin circular dichroism resistance, (CD) structuraland signatures, thermal stability, expressed,to found although be well to lower compared V(H)Hs were with mutant wild-typeyields The tralizingcoun - capacity. terparts, non-aggregating were monomers,toxin retained for low nM affinity majority A, albeit the showed somewhat reduced to compared affinity wild-type counterparts,toxin A neutralizationcell-based of incapable in and were vitro and Far-UV assays. near-UV CD spectroscopy showed shifts consistently in peak and selective intensity peak wild-type minima for V(H)H and mutant A significant increase overall CD the remained thermaltemperature midpoint the unfolding profile very in pairs; similar. however, was observed both neutral all mutants at and acidic gastrointestinal for major pH. Digestionproteases, V(H)Hs with the of the at biologically revealed relevant concentrations, a significant increase in pepsinmutantsresistancefor all and an increase chymot in rypsin majority of mutants. the resistance for V(H)H trypsin Mutant resistance was of wild-type that similar to V(H)Hs, although trypsinthe wasintroduction resistance of one V(H)H mutant the reduced. of a significantly disulfide in Therefore, second bond not only increases core neutral pH, as hydrophobic V(H)H thermal at stability the previously shown, but also represents a generic strategy increase to low pH and impart at protease V(H)H stability resistance, with only minor perturbations in target binding affinities. characteristicsThese are all the desirable for design protein-based of therapeutics. oral has been antibodyIt fragments, shown with that, conventional in contrast variable the domains of these heavy-chain antibodies VHH the (variable domain of heavy-chain antibody) functional are high or after study, temperatures. to present exposure at In the camel antibody was subcloned vector Ppiczc and expressed into in Pichia pastoris. ORBI-83 VHH antibody recognizes external the EGFR growth [EGF (epidermal mutant domain of the factor) receptor], EGFR VIII.tumour-specific Thisantigen is ligand-inde - active tyrosinependent, kinase a constitutively contains domain and has been be shown to malig - in a number present of human pastoris resulted in a significantly increased the of levelexpression of report from P. that, expression although here nancies. We VIIIanti-EGFR VHH antibodies with compared Escherichia, antibody this selectively bound EGFR VIII the to peptide and reacted stabil - denaturation Furthermore, thermal specificallythe immunoaffinity-purified with lungcancer. antigen from non-small-cell ity and CD spectra analysis Camelus of the bactrianus (Bactrian camel) VHH and heavy-chaintemperature antibodies different at pastoris be system may expression proved reversibility P. Our and binding activity the results afterthat heat denaturation. indicate reversibly to without of camel singleaggreuseful expressedmelt domain antibody- protein of the expression ability the and the for allowinggation, regain it to binding activity after heat denaturation. and extreme pH and extreme receptor Thermal stability Resistance to trypsin Clostridium difficile Toxin A Oral administration Heat denaturation Epidermal growth factor Pichia pastoris Malignancy

468 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article Dona et al. 2010 Graef et Graef et al. 2011 Dumou - lin et al. 2002 - Food intended for celiac patients’ consumption must be must using analyzedprolamins high patients’ consumption delectability celiac tests. of toxic presence for intended the Food for Thoughcommonly the most 60% used 2-mercaptoethanol (2-ME) and is extraction, ethanol for prolamins guanidiniumchlo - solvent recognition with antigen agents interfere ethanol and denaturing ride (GuHCl) can be added However, increase to recovery. protein antibodies used. are when conventional work, present In the a new method gliadins shown. for quantification is The method is based selectionon the of llama single domain antibody fragmentsconditions. under denaturing operate to Six able out of 28 VHH-phages obtained retained binding capacity their in 15% ethanol. Selected clones presented a long CDR3 regiontwo additional containing clones (named One of the VHH26) cysteines was be could responsiblefully higher stability. the that of presence operative for in the 15% ethanol, 0.5% 2-ME, ELISA and 0.5M GuHCl. Capture using VHH26 was detect to able gliadins shown in samples as negatives ELISA. or any by conventional of proteins determination thisfor quantitative new platform Therefore, strategyappears excellent as an agents, of denaturing presence in the compound, immunogenic when specificother recognition unitsrequired. high are stability with Background: Camelids and sharks possess a unique subclass of antibodies comprised of only heavy chains. bind - The antigen ing fragments of these unique antibodies can be cloned and expressed as single domain antibodiesthese ability of (sdAbs). The moleculessmall antigen-binding after achieve heating to original refold their to structure, as size, as diminutive well makes their diagnostic candidates for attractive them assays. Results: we describe Here isolation against the of an sdAb Staphyloccocus aureus (Kd = 75 pM) and highclone, A3, B (SEB).affinity The wasto have found good enterotoxin specificity for SEB, showing no cross D (SED), and Shiga toxin. A (SEA),reactivity molecules enterotoxin related Staphylococcal to as such enterotoxin Staphylococcal of 85 degrees after 95 degrees heating to refold to C and an ability high Tm had an extremely sdAb anti-SEB this Most remarkably, Tm determined by circularcontrastthe dichroism, wasto C.gradualwith found decrease sharp The observed fluores in intrinsic cence. We demonstrated the utility of this sdAb as a capture and detector as sdAb utility of this the demonstrated a capture molecule based in Luminex assays providing limits of We cence. extraordinarily a high A3 affinity and an detectionsdAb wasto have leastfound (LODs) of at 64 pg/ml. Conclusion: The anti-SEB and strong,resilience of heat combination This recover activity specific to after still refold heat denaturation. and could high Tm a good sdAb this binding make use for candidate in antibody-based detection technologies. toxin A variety of techniques, transformfluorescence, infrared high-pressure including spectroscopy, monitored by Fourier unfolding beencircular and surface have dichroism, plasmon equilibrium used spectroscopy, the resonance properties investigate folding to of six binders derived single-domain antigen from camelid heavy-chain antibodies with specificitiesfor lysozymes, beta-lactamases, and (guanidinium conditions denaturing chloride,provided urea, and pressure) complementary temperature, and a dye (RR6). Various independent methods characterizing for properties and unfolding stability the antibody of the all binders, fragments. complete With recovery chemical- inducedis biological fully unfolding of the that reversible. demonstrates activity Furthermore, after renaturation experiments followed by optical spectroscopicdenaturation antibody methods the and affinity that measurements fragments indicate unfoldedare cooperatively in a single transition. Thus, equilibrium unfolding/refolding proceeds two-state mechanismvia a simple significantly are (Nreversible states denatured populated. arrowThermally- and the U), native only the induced where denaturation, reversible, partial and the is not completely loss of binding capacity be might least due, at in part,however, incorrect to refolding fragments resistant long loopsto all the of the rather are (CDRs), recognition. responsible are antigen which for Most interestingly, = 60–80 degrees-C), stabilities T-m (DeltaG(H2O) high and display conformational = 30–60 kJ/ (apparent denaturation heat-induced high thermodynamicmol). Such has stability never been reported antibody functional fragment, any for conventional even when engi - camelid heavy-chain and higher of the stability reduced the neered size, considered. Hence, solubility, improved binders are antigen antibody fragments of special are biotechnological for interest and medical applications. refolding Denaturing agents Celiac patients Prolamin Gliadin ELISA B Enterotoxin Denatured protein Diagnostic assay Denaturing conditions Refolding Thermodynamic stability

469 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 Dolk et al. 2005b Walper et al. 2012 Dolk et al. 2005a In a previous study we have shown that llamaIn a previous VHH shown that antibody we have after study a heat shock antigen of 90 fragments bind their to able are degrees molecular the C, monoclonal murine the antibodies. to mechanism in contrast antibody: by which However, antigen detail in more structural the examine and thermodynamic To unclear. remains conditions occursinteraction under these extreme aspects was between and NMR study ITC, interaction the CD, initiated. study In this binding mechanism, of the the an extensive llama VHH-R2 fragment dye Reactive and its the antigen, Red-6 (RR6) has most beenthe of that clearly show The data explored. VHH-R2 population80 degrees at In contrast, C is in an unfolded CD conformation. spectrabetween complex the representing VHH-R2 80 denatured at the dye to addition of the VHH-R2 80 degrees dye remained same up to and the the C. Interestingly, degrees C yielded spectrum the These complex. results native of the suggest of denatured VHH-R2 by its an inducedrefolding conditions.showed under these This inducedrefolding extreme antigen some similarities the well established“induced fit” with “induced fit0148” the with main difference the However, mechanism temperature. ambient of antibody-antigen at interactions most VHH start of the the antigen mechanism molecules addition of the at of the isThe that in an unfolded are conformation. VHHs make formation useful complex in a wide stable and the variety conditions - under these capability of appli extreme refolding cations. Significant efforts to laboratorydevelop both field-based and detection of biological for anpotential array threats assays started with renewed continued anthrax attacks of 2001 and have before the urgencywell following. assays and meth - While numerous laboratoryods for suitable been are have that utilization, explored detectionrequirements for by complicated is often field in the functionality environments, facilities limited in austere where cold-chain thesefor exist. limitations In an effortto overcome assay Bacillus anthracis, most one of the recognizable threats, a series of single domain antibodies isolated (sdAbs) were from a phage librarydisplay prepared from immunized llamas.thermal and stability conducted was Characterization affinity, of target specificity, sixfor families sdAb isolated from of selection rounds againstbacterial the for all six spore.target The protein sdAb families was determined be to S-layer the EA1, protein is in both which present and bacterial vegetative cells spores. sdAbs examined All of the exhibited a high degree of specificitythe for target bacterium and its spore, ability affinitiesthe the nanomolarwith range, and in functional molecules into antigen-binding refold to following several and refolding. research This of thermal rounds denaturation and developing current assays capabilities into and biosensors. of these the integration potential sdAbsdemonstrates for and their As part feasibility the of research exploring of using antibody fragments growth inhibit the to in dandruff, of organisms implicated we isolated antibodyof Malasseziathat surface found protein fragments a cell bind to that furfur of shampoo. presence in the We of llamaphage display single-domain antibody fragments (VHHs) can very be to extended conditions, harsh as such presence the selected of shampoo nonionic and anionic surfactants. Malf1 of M. wall several containing protein cell the bind to VHHs that We a fungus in causing implicated dandruff.shampoo,furfur, of the presence to highthese in stability In addition alsoVHHs are stable arginine at contain conditions, VHHs stable to denaturing of the found asunder other were such Many high urea concentrations. position amino acidposition at native of the most 44 with lacked arginine 44. Replacement in the VHH stable arginine this that the unique properties of combination The of togetherappliedVHHs resulted further in a dramatic with increase stability. in the engineering and protein phage display is a powerful method obtaining for highly can VHHs stable be that used in a wide range of applications. Heat shock Conformation studies Denaturation Refolding Bacillus anthracis Thermal stability Denaturation Refolding Dandruff Malassezia furfur Shampoo Denaturing conditions Amino acid replacement

470 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article - Cortez- Reta et mozo al. 2002 Conrath Conrath et al. 2005 Harmsen et al. 2005 - residence times as times residence as 33 nM). Four pIgG-binding VHH2s administered pigs in vivo were to and showed extended a 100-fold 33 nM). Four Two camel single-domain fragments, cAb-Lys2 and cAb-Lys3, recognizing epitope camel single-domain fragments, an overlapping of lysozyme and cAb-Lys3, with a dissociation cAb-Lys2 Two target transgenic tumors to ability their investigated for were and a bivalent cAb-Lys3 of 2 nM and 6S nM, respectively, constant lysozymeexpressing membrane. Biodistribution on their studies revealed these monomeric non-immunogenic and bivalent that binders specificallycamel single-domain antigen lysozyme-expressingtumors and metastatictarget anti - lesions. of excess The body blood from was the eliminated is retention rapidly circulationobserved and no cAb in normal organs.to organ tumor The The degree (2.1–10.8) : 1 and (6.2–23.7) range, respectively. and specificity in the 2 and 8 h were at - cAb-ratios tumor reten of single-domain their univalent tion is camel antigen and the independentfrom affinity of fragmentsrecombinant their of the for the successfulmonomeric and specific (15 kDa)(33 kDa). demonstrates or bivalentThis format study in tumors vivo of targeting by camel single-domain fragments. open may perspectives It small their due to future use their vehicle, as for tumor-targeting size, soluble behaviour and because conventional with for less epitopes they and interact are antigenic non-immunogenic are that antibodies. namic stability and affinity for antigen. Comparison and affinity namic stability its the of crystal structurescAbAn33 and humanised of its derivative reveals the prevent VL domain that pairing,Tyr37 and Arg45 by VHH-specific exerted steric hindrance whereas Glu44 and Arg45 residues insolubilitydomain. of the key avoid are to elements does VHH2 basis provide the pIgG.could that of VHHs not bind to therapeutic This a control application for in to compared pigs. Heavy chain only antibodies of camelids bind their antigens with a single domain, the VHH, which acquired adaptations relative to Heavy only antibodies to chain with VHH, a singlerelative of camelidsantigens domain, the acquired adaptations bind their which CDR Additional loopclassical conformations, outside VHs canonical function the to of a VL absence loop in the partner. structures of VHs,combined The site. effects repertoire broaden the antigen-binding of the “former” of part the folds the over of that CDR3 amino acids, hydrophilic solubility more the of VHH to enhance domains and prevent mutations VL and framework-2 binding site VL pairing. a VHH cAbAn33, domain specific moietythe the of of trypanosomes,variantfor carbohydrate surface glycoprotein has a short CDR3 does loop former VL as the Trp47 that as binding site not cover the well instead a VH-specificof VHH-specific VH restores of a human mimic that Glu44Gly and Arg45Leu) to Gly47. Resurfacing its Tyr37Val, region framework-2 (mutations albeit In solution,humanised the with reduced VHH thermody behaves as VLthe binding capacity. a soluble, monomeric entity, The therapeutic parenteral application of llama single-domain application antibody therapeutic parenteral fragmentsThe (VHHs) is hampered small size, by their we describe Here resulting in a fast body. a method from the increase to elimination serum the of VHHs half-life in pigs by isolated 19 pIgG-binding VHHs from an immunized fusion VHH another porcine to binding to immunoglobulin G (pIgG). We Six VHHs genetically were fusedllama using phage display. model Escherichia binds to VHH to F4 fimbriae. K609 that coli All six yeast-produced genetic fusions of two VHH domains (VHH2s) functional were in ELISA and bound pIgG with high to affin - ity (1 –

immunoglobulin G vehicle glycoprotein Serum half-live Porcine E. coli F4 fimbriae Biodistribution studies Blood circulation retention Tumour Tumour-targeting Solubility CDR3 Hydrophilic amino acids Trypanosome surface

471 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 - Ander son et al. 2010 Altintas et al. 2012 - mal stability, which makes them attractive alternatives to conventional monoclonal antibodies. We created library a phage display monoclonal antibodies. conventional alternatives to attractive makes them which We mal stability, from llamas and selected immunized with ricin a number of single domain antibodies. toxoid selected Phage on ricin found were ricin intact molecule; either or the bind to A chain to no ricin identified. By binders were chain, we identified B chain panning on characterized ricinbinders and have the While theya poorerpreviously binding to B chain. the their have than affinity described A binders,chain it was they performed that found detection better dramatically reagentsthe as for capture of ricin, providing a limit of detection in enzyme linked specificity immunosorbent (ELISA) assay for belowricinthe highly 100 pg/mLversus and excellent also reevaluated previously the RCA isolatedrelated 10 000). We 120 (1 to antiricin single domain antibody binding kinetics using those closely to previously matched surface plasmon obtained K(d)s their and found resonance under equilibrium- binding condi tions measured flow Luminex using the cytometer. discoveryThe occurring of naturally heavy only antibodies chain further and their small recombinant development into ‘nanobodies’ in drug applications targeting. offers attractive we describe Here, properties the of nanobodies been have that developed epidermal target the to growth factor characteristics receptor (EGFR)these the and contrast to of heavy only chain antibodies.antibodies and conventional tumor-directed target for and is an attractive tumors EGFR is in many overexpressed drug targeting. Single expressed domain antibodies binding fragments recombinantly the are derived from heavy antibodies chain in camels found and llamas.properties desirable These many elements offer unique binding such their small as to 15 ther size (similar kDa) and receptor nance Nanobody properties Epidermal growth factor Drug targeting Binding kinetics Ricin toxoid ELISA Surface plasmon reso -

472 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article Paalanen Paalanen et al. 2011 Li et al. 2012 Oyen et al. 2011 - The 10 isozymes roles the same biologicalkinase on the protein of process, C (PKC) different family can have making isozyme specificfew only pharmacological analysiscompounds of moderate functionisozymewith crucial.Currently, specific effects exist hamperingisozymes. individualthus research into PKC regions binding The antigen of singlechain antibodies camelid (VHHs) isozyme successfully provide a solutionobtainingselectedcould for PKC we have specific and modulators. study, the present In characterized epsilon specific PKC VHH The shown antibodies VHHs were VHH two immune librariesfrom using phage display. epsilon in ELISA PKC the bind to five of VHHsPKC had an effect and immunoprecipitation studies. on exclusively to Strikingly, epsilon kinase activity in vitro. VHHs A10, C1 and D1 increased epsilon kinase PKC manner activity in a concentration-dependent (EC(50) values: 212–310 nM), whereas epsilon E6 and G8 inhibited activity PKC (IC(50) values: 103–233 nM). None of these To our knowledge,these antibodies theta. VHHs isozymes had an effect and PKC delta PKC the activity novel on the other PKC of first described the are epsilonFurthermore, specificthe development of PKC kinase. for a protein VHH activators and inhibitors research. modulators is PKC an important to contribution Screening of inhibitory antibodies Ab1 is a critical producing step for catalytic antibodies How anti-idiotypic in the approach. ever, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional enzyme of heterotetrameric surface conventional activeof the site of the site antigen-binding incompatible and the the ever, antibodies become limiting step. Because the camelid-derived nanobodies the possess bind to preferentially potential to the active of enzymes site small size their and long CDR3, due to developed antibodies produce we have to approach with a novel alliinase activities molecular the by exploiting mimicry of camel nanobodies. By screening camelid-derived the variable region heavyof the library cDNA phage display chain with alliinase, we obtained an inhibitory nanobody VHHA4 recognizes that the screening with VHHA4active Further site. same variable from the heavy domain of the of a heavy-chain chain antibody library led a higherabzymes of anti-idiotypic incidence to Ab2 with alliinase activities. abzymes, One of the VHHC10, showed the highest activity can be that inhibited by Ab1VHHA4 and alliinase penicillamine competitive inhibitor and significantly sup - results growth feasibilitypressed of alliin in vitro. presence highlight The the in the cell of producing B16 tumor the abzymes via anti-idiotypic nanobody approach. Although it hasAlthough been antibodies years known that many properties for display characteristic of allosteric effectors,the molecular mechanisms responsible these for effectsremain poorly understood. we describeHere, a single-domain antibody fragment (nano - reductase enzymebody) in the change function modulates dihydrofolate conformational protein (DHFR). that by constraining Nanobody 216 (Nb216) behaves as allosteric a potent of DHFR, inhibitor giving rise mixed to hyperbolic inhibition kinetics. The crystal with DHFR epitope reveals of the nanobody activeHalf structure the the site. of Nb216 in complex to that binds adjacent consistsMet20 loop. of residues flexible from the This ordinarily loop, which oscillates betweenoccluded closed and conforma - Nb216 tions duringthat catalysis,show we stopped assumes Nb216-bound Using in the occluded flow, the state. conformation Met20 loop the inhibits kineticretains that DHFR data occluded indicate by stabilising the Surprisingly, Met20 loop conformation. to turnover occur. slow to allow the Nb216-boundsubstrate flexibility in state conformational sufficient Table 6A. of single-domain Application antibodyTable fragments in therapy: inhibition of enzymes, and other soluble toxins proteins Protein kinase C Enzyme modulator Anti-idiotypic Alliinase Molecular mimicry Abzyme Allosteric effector Dihydrofolate reductase Inhibition kinetics Crystal structure

473 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 Ratier et Ratier et al. 2008 Hussack Hussack et al. 2011b - - The sialic protective of Trypanosoma acidcruzi in the present surfaceThe coat mucin is added by a membrane anchored trans- a modified work, In this sialidase we analyzed is expressed gene family. from a large that single domain camelid sialidase (TcTS), antibodies produced against trans-sialidase. Llamas immunized were trans-sialidase with a recombinant and inhibitory single- domain antibody fragments obtained were selection, by phage display taking advantage of a screening strategy using an inhibi - single domain antibodies displaying trans-sialidase Four strong classic tion test instead of the inhibition activity binding assay. enzymeagainst recombinant the identified. same complementarity-determining were region the They 3 length share (17 resi - verydues) and have similar sequences. they derived is only likely resultthere indicates that Probably This from a unique clone. our surprise, this used immunization. for To one structural binding inhibitory solutiontight for antibodies againstTcTS the failed enzymatic inhibit the to activity in parasite present extracts. single TcTS, domain antibody inhibits recombinant the that Analysis of individual trans-sialidases recombinant enzymes showed that genes inhibited dif expressed were fromto different ferent extents (from 8 to 98%) by the llama antibodies. 98%) by the (from 8 to extents ferent key Amino acid changes at be positions to responsible likely the are for enzymes. recombinant among the in inhibition found differences and diverse of a large presence results These the suggest that trans-sialidase family be might required inhibitory prevent the to response against essential this enzyme- consti thus and might cruzi evade to system. host the immune strategy a novel of T. tute Clostridium difficile is a leading cause of nosocomial infection healthcare in North to America challenge and a considerable professionals in hospitals homes. and nursing Gram-positive The bacterium produces two high molecular exotoxins, weight virulence major the factors are which responsible C. for difficile-associated B (TcdB), disease and toxin and A (TcdA) toxin targetsare C. for difficile-associated single-domain antibody recombinant Here, fragments (V(H)Hs), disease which therapy. isolated were llama library phage display from an immune or TcdB, specifically receptor binding domains of TcdA cell target the recognize V(H)Hs (A4.2, A5.1, A20.1, and A26.8), all shown to and characterized. epitopes, conformational Four potent were neutralizingneutralizers cytopathic The of the potency effects A on fibroblast was in an vitro assay. cells of toxin further suggest enhanced administered same overall V(H)H concentration, when V(H)Hs were in paired the at or triplet combinations ing recognition of nonoverlapping TcdA epitopes. epitope experiments Biacore revealed mapping some synergistic that - com ing recognition TcdA of nonoverlapping consisted of V(H)Hs recognizingbinations epitopes, overlapping epitope mere factors than blocking other that an indication responsible increasedare the V(H)Hs neutralized for A by binding assays toxin neutralization.revealed Further TcdA-specific characteristics favorable as such high production With binding pocket carbohydrate the toxin. than of the sites other binding to systemic therapeutics so these attractive more as V(H)Hs are but are neutralization, stability, yield, and intrinsic toxin potent oral therapeutics destabilizing gastrointestinal in the tract. of the environment Sialidase Trypanosoma cruzi Clostridium difficile Nosocomial infection North America A/B Exotoxin Gastrointestinal tract

474 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article - Pooja Pooja and Ajit 2010 Hen drickx et al. 2011 Conrad Conrad et al. 2011 - - - persis dAb clones of LPS-immunized dAb Indian desert camel constructed 7 Objective: To isolate and characterize (S. Staphylococcus aureus hemolysin aureus) neutralizing library phage display front dAbs Objective: To of Indian desert camel. Methods: library display Phage of 5 × 10 in our laboratory was usedspecific selection for clones by technique. of Ag-specific of S.panning Enrichment exotoxin aureus of panning rounds wasclones in successive assessed clonesto aureus binding dAb S. by phage-ELISA Different and phage titration. expression The Ags expressed with were C-terminal 6 X His tag in E.chromatography. exotoxin and purified coli by Ni-chelate was and western hemolytic blotting. verified in activity The purifiedSDS-PAGE by cloneswere tested for inhibition of ‘hot-cold’ vitro. clones Resistance was thermal inactivation dAb to of the studied by observing effect 50 degrees the from of heat treatment C hemolytic activity 99 degreesto in vitro. Results: ‘hot-cold’ 30 min on the C for Several S. clones binding to exotoxins dAb aureus isolatedwere to 16 kDa similar in size and enriched and by three of panning. rounds approximately clones The were soluble dAb reacted with 6 X His tag specificmurine monoclonal antibody in westernaffinity His purified blot. dAb.6 X the Ni-chelate One of clones, inhibited S. beta-hemolysin aureus activity in vitro and resisted thermal inactivation 99 degrees upto C. Conclusions: An S. beta-hemolysinaureus neutralizing of possible clone dAb therapeutic potential has been isolated. inducedclot lysis. Results:Five nanobodies,t-PA activity effect test their and to on raisedtowards an activated TAFIa stableing the more described. are These A(147)-C(305)-I(325)-I(329)-Y(333)-Q(335)), nanobodies TAFIa activity, inhibit specifically (TAFIa mutant TAFIa range between 0.38- and > 16-fold IC(50)’s of nanobody molar excess over TAFIa, a 16-fold 99% at resulting in an inhibition of up to nanobodiesmolar lysis In vitrothe excess. clot experimentsof thrombomodulin absence - that in the profi (TM) exhibit demonstrate TM, one nanobodybrinolytic of the presence exhibits in effects. an antifibrinolyticeffect whereas other nanobodies the However, show a pronouncedprofibrinolytic and a concentrations. antifibrinolytic pattern atThis slight effect concentrations higher effect at low biphasic theirTAFIa and The nanobodiesthe active-siteregionto bind in found of were concentration. was highly and t-PA dependent on TM TAFIa inactivationrevealed the of a yet unknownoccurrence confor binding during time-dependent intermediate behavior differential fibrinolysis. usefulconstitute tools and transition. Conclusion: TheseTAFIa inhibitors to accelerate mational nanobodies very are potent may inhibition be TAFIa of reversed TM. The identification presence of the by profibrinolytic the Our effect that data also of demonstrate a new transition provides inactivationTAFIa. unstable of the new conformational conformational the insights into Background: Because has very activated activatable fibrinolysis thrombin inhibitor powerful(TAFIa) antifibrinolyticproperties, co- nanobodies aimed generate to specifically treatment. Objective: inhibit We t-PA enhances inhibitor and a TAFIa of t-PA administration necrosis factor (TNF) is pro-inflammatory a major Tumour cytokinemultiple inflammatory involved in diseases. The detrimental activity of TNF can be blocked by various antagonists, therapeutics and commercial based upon principlebeen this have approved disease and psoriasis. of diseases treatment for arthritis, anti- improved rheumatoid including new, In a search for Crohn’s inflammatorytherapeutics designed we have antibody The a single-domain monoclonal antibody recognizes (V(H)H), which TNF. (TNF-V(H)H) is basedcomponent TNF was uponCamelidae which heavy-chain an anti-human linked an monoclonalto antibody, fusion of the protein ELP accumulation enhances that fusion TNF-V(H)H the demonstrate to elastin-like polypeptide (ELP). We anti- this plant-derived that first time the for we show study, this plants.during biomanufacturing in transgenic tobacco With antibody was TNF-V(H)H human biologically therapeutic of TNF-V(H)H-ELP application active fusion in vivo. protein Therefore, was tested in humanized and was TNF mice be shown to effective in preventing death caused by septicshock. The in vivo tence of the ELPylated of the tence antibody of non-ELPylated was that longer than 24 fold similar to TNF-V(H)H. fibrinolysis inhibitor activity Thrombin activatable Clot lysis necrosis factor Tumour Fusion Elastin-like polypeptide Tobacco Staphylococcus aureus Hemolysin hemolytic ‘Hot-cold’

475 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 Delanote Delanote et al. 2010 Altintas Altintas et al. 2012 Behdani et al. 2012 Zabeau et al. 2012 - - L-plastin, a conserved malignancy modular is cell ectopically F-actin to bundling protein, and contributes cells expressed in tumor in part,and invasion. The underlying molecular because mechanismsremain unclear, involved specific of inhibitors L-plastinare used alpaca-derived recombinant lacking. L-plastin We single-domain antibodies (nanobodies) as effector of L-plastin function nanobodiesin cells.that show KeyWe compared findings interact L-plastinstrongly with were with down-regulation by RNAi. L-plastin A nanobodyselectively interacts that epitopes the by targeting discrete with conformational with nanomolar affinity. tandem ABDs in L-plastin inhibits equimolar F-actin green bundling at - completely pro ratios, fluorescent a control to in contrast This “knockout”invasion nanobody (GFP) nanobody. expressedtein and cells. when inhibitsinPC-3 filopodia motility, formation, L-plastin showed no significant RNA effect interference on filopodialintegrity marginally and only restrained properties the motile of cells. L-plastin nanobodies exposein filopodia- uniquely migration. a fundamental There protein this cell for and role formation these molecules functionsfore, ablate withoutgene expres of structural instrument a potent manipulating to represent proteins they can that sion. In addition, we show be instrumental in uncovering new obscured remain functions that by RNAi. of proteins The discovery occurring of naturally heavychain only antibodies‘nanobod their recombinant and small - further development into in drugapplications attractive targeting.ies’ offers we describeHere, the properties of nanobodies have beenthat developed to epidermaltarget the growth factor characteristics receptor (EGFR)these the and contrast to of heavy only antibodies chain and antibodies. drug conventional tumor-directed targeting. target for and is an attractive tumors EGFR is in many overexpressed Vascular endothelial growth factor receptor-2 (VEGFR2) growth endothelial factor receptor-2 is an importantVascular tumor-associated receptor and blockade VEGF of the recep - signalingtor can inhibition of neovascularization lead the to metastasis. and tumor Nanobodies smallest binding the intact antigen are fragments derived from heavy antibodies chain-only occurring in camelids. we describe Here, of a VEGFR2-specific identification the by FACS, demonstrate named 3VGR19, from dromedariesNanobody, high levels immunized line expressing of VEGFR2. with a cell We 3VGR19 Nanobodythat specifically binds the surfaceVEGFR2 on of 293KDR and HUVECsNanobodyFurthermore,the 3VGR19 cells. inhibits of capillary-likepotently formation structures. TheseNanobodiesthe of show potential data the blockadefor of VEGFR2 signal - ing and provide a basis cancer therapeutics. development of novel the for The adipocyte-derived cytokine leptin acts as a metabolicconnecting switch, metabolismthe to high-energybody’s consuming processes assuch reproduction responses. and immune evidence pathologies, suggests in human leptin Accumulating a role plays that as such we generated development of leptin the antagonists. for providing a rationale thus study, present In the diseasesautoimmune and cancer, and evaluated of neutralizing a panel nanobodies LR targeting the (leptin receptor). A nanobody comprises variable the domain of the three identifiedclasses of neutralizing occurring nano - naturally We single-chain antibodies in members found Camelidae of the family. bodies LR subdomains: targeting different the i.e. (cytokineCRH2 receptor homology and FNIII 2), Ig-like (fibronectin type III) domains. Only nanobodies a nanobody directed targets that Ig-like show the that could againstCRH2 the domain inhibited leptin binding. We with leptin-dependent interfered domain potently regulation of hypothalamic NPY (neuropeptide Y) As a consequence, expression. daily intraperitoneal injection increased body weight, body food liver size content, fat intake, and serum insulin levels. All of these characteris tics resemble phenotype the animals. of leptin and LR-deficient study support The resultspresent the of proposed the of activatedmodels it is possible that block to LRLR and demonstrate signalling complex, without affecting ligand binding. These nanobodies form new tools mechanisms the study of BBB (blood-brainto barrier) leptin transport effect and the of LR inhibition in disease models.

receptor growth factor receptor-2 L-plastin Malignancy F-actin bundling Filopodia 6B.of single-domain Application antibody fragmentsTable in therapy: activity modulation of cell surface proteins Nanobody properties Epidermal growth factor Drug targeting Vascular endothelial Vascular Neovascularization Metastasis Leptin receptor Subdomain Neuropeptide Y Blood-brain barrier

476 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article Baral et al. 2011 Jamnani Jamnani et al. 2012 Wei et al. et al. Wei 2011 - - Carcinoembryonic adhesion molecule cell related (CEACAM) antigen types in different cells. of 6 is over-expressed cancer In addi - tion, it has also been molecule this in some implicated in diverse infectious by an antibodyapplications diseases. have might Targeting models.tumor Single domain antibody is (sdAb) becoming very useful in antibody format engineering as potential tools treat for diseaseing acute and chronic as such both cancer for conditions diagnostic sdAbs with as as Generally, well therapeutic application. good Discoveryare affinity library. isolatedfrom an immune of a antigen would a newrequire target new immunization purified with against an sdAb CEACAM6 isolated, we have by phage display, withnM an affinity of 5 study, In this is which not always easy. antigen from a llama immunized with cancer cells. The antibody has good biophysical properties,the target expressing cells the to and it binds targeting in vivo. tumor could This be sdAb in vitro and shows an excellent proliferation it reduces cancer cells Furthermore, antigen. useful in diagnosis as of CEACAM6 as we envisage well it would be therapy feasible isolate good to sdAbs tumors. expressing Finally, this immunization associated method tumor could Therefore, be a general strategyagainst interesting other from library. this antigens each time. isolating withoutof interest for surface sdAbs immunizing against antigen animal with any the the Modern anti-HER2 antibody therapy tends to exploit a panel of different antibodies against different epitopes on the antigen. For For antibodiesantigen. epitopes of different a panel Modern the against on different exploit anti-HER2 antibody to tends therapy aim, nanobodiesthis very are striking targeting agents and can be easily produced cell-specific against any The membrane antigen. oligoclonal nanobodies can be used one functional block than to of escape more epitopegeneration and inhibit the on a target antigen 12 nanobody clones selected camel libraryvariants from study, an immune In this examined associated were for with cancer therapy. differ to between ability their tumor markers. These oligoclonal nanobodiesbreast targeted cells than individual each cancer better In epitope mapping,nanobody. several nanobodies overlapped epitope in the recognized by trastuzumab and somenon-over of the nanobodieslapping the oligoclonal affectnanobodies that could the binding of trastuzumabto HER2. study demonstrates This are potential therapeutic can tools be that used with trastuzumab assess to instead of, or in combination viability tumor during treatment. Influenza A virus poseshumans. to serious threat healthNeutralizing antibodiesconserved againstthe highly is channel M2 ion broad protection offer against to influenzathought A viruses. we Here, screened antibody single-domain synthetic Camel (VHH) isolated One of the VHHs,libraries protein. M2 ion channel against native M2-7A, specifically mem - cell to M2-expressed bound brane as as well influenza A virion, inhibitedreplication of amantadine-sensitiveboth resistant and influenza A viruses in and vitro, showed M2-7A blocking influxprotected activityM2 through fromfor proton a lethal influenza mice virusMoreover, challenge. These a ion channel. piecesneutralizing that the first time of antibodyfor collectivelyevidence demonstrate broadagainst M2 with specificityfor cross protection numberis achievable, against have potential a of and M2-7A may variants and subtypes of influenza A viruses. antigen related cell adhesion molecule Carcinoembryonic Cancer 12 nanobody clones Oligoclonal nanobodies Breast cancer cells HER2 Ion channel M2 Ion Influenza A

477 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 - Ibanez et al. 2011 Hultberg et al. 2011 Jah et nichen al. 2010 - ful strategy. Llama heavy antibody chain ful fragments strategy. (VHH) of Respiratory againsttrimeric the proteins envelope Syncytial Virus Rabies virus protein), and H5N1 Influenza 5) were selected (Hemagglutinin (Fusion (Glycoprotein) llama derivedfrom immune Neutralizing VHHlibraries recognizing by phage display. epitopesreceptorthe spikes the binding sites on affini - in different with threethe for all ties identified low nanomolar viruses range were in the by viral neutralization assays. By fusion of VHH with vari - 1500-fold lengths, linker able constructsneutralization multimeric improved made that potencies were RSV, for 4000-fold up to Rabiesfor virus Influenza for and 75-fold H5N1. The potenciesthe constructs of VHH similar orthan were better best performing monoclonal antibodies. The cross protection viral strainscapacity against different threewas improved for all also viruses,by both constructs.VHH) (two or three VHH) identical (two different multivalent and biparatopic combining a By VHH neutralizing RSV subtype A, but not subtype B with a poorly neutralizing VHH with for subtype high affinity construct B, a biparatopic was made with low nanomolar neutralizing potency against both VHH subtypes. anti-H5N1 neutralized both Influenza H5N1 Trivalent clade1 and 2 in a pseudotype and was assay very in neutralizing potent NIBRG-14 Influenza the H5N1 strain with IC(50) of nine constructs Bivalent and biparatopic against Rabies virus cross neutralizedpicomolar. bothGenotype 10 different 1 strains and Genotypemultimerization results 5. The that of show VHH fragmentsmultiple epitopes targeting on a viral trimeric protein spike is a powerful achieve to tool anti-viral “best-in-class” and broader neutralization for therapy capacity. Influenza A virus infections imposerecurrent a and global disease antivirals againstCurrent influenzaare not always burden. We assessedthe protective Nanobodies of monovalent and bivalent effective.potential (Ablynx)this againstchallenge with virus. TheseNanobodies derived were llamasfrom and Nanobodies H5N1 hemagglutinin.target of Intranasal administration effectively homologouscontrolled influenza A Nanobodiesvirus of Administration replication. reduced challenge strongly H5N1 before virus lungs in the replication and protected from morbidity and mortality mice after with H5N1 virus. a lethal challenge The bivalent Nanobody was leastNanobody at effectivethe monovalent more than virus 60-fold controlling Nano - replication. in In addition, bodyrevealed Epitope after reduced strongly mapping challenge therapy viral to death. and significantly replication time delayed VHH the Nanobody B in H5 hemagglutinin.that Because site antigenic binds to - Nanobodies pro to and simple small, stable, are they a promising,duce, are against therapeutic influenza. agent novel For efficient prevention efficient of viral infectionscross andprotection, For simultaneous multiple of viral targeting epitopes is a power The importantreceptors hasfamily of G protein-coupled so far not been targeted very monoclonal successfullyconventional with antibodies. we report Here isolation the and characterization of functional VHH-based immunoglobulin single variable domains highly selective monovalent nanobodies, nanobodies) (or Two againstchemokine receptor CXCR4. 238D2 and 238D4, were the counterselection and method. The selectivehighly obtained VHH- cell immunization, phage display, using whole a time-efficient based immunoglobulin single variable inhibited CXCR4-mediated domains competitively the signaling and antagonized the the two nanobodies showed Epitopethat effectmapping ligandto distinctthe chemoattractant bind of but CXCR4 CXCL12. loops.partially extracellular sites in the overlapping Short peptide linkage of 238D2 with 238D4 resulted in significantly increased the monovalent nanobodiesCXCR4 and picomolar foractivityantichemotacticaffinity in behaved assays. as Interestingly, neutral antagonists, whereasnanobodies biparatopic the actedactive CXCR4-N3.35A. as agonists constitutively inverse the at The CXCR4 biparatopic nanobodies the displayedactivity antiretroviral strong strains. against and dual-tropic T cell-tropic HIV-1 Moreover, nanobody effectively mobilized CD34-positive in cells stem cynomolgus monkeys.Thus, the nanobodymay be highly platform and selectivereceptor modulators.potent G protein-coupled extremely effectiveat generating

constructs administration Multimeric/bispecific Influenza HIV virus CXCR4 Epitope mapping Table 6C. of single-domain Application antibodyTable fragments in therapy: pathogen neutralisation Bivalent nanobody Influenza virus Intranasal

478 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article

Smolarek Smolarek et al. 2010 Aladin et Aladin et al. 2012 Strokap- pe et al. 2012 - Fy blood carried are group antigens Duffy by the chemokinesPlasmodiumreceptorfor receptorfor red (DARC), a cells antigen vivax health and diseases. in human broadly implicated VHHs, Recombinant or nanobodies, smallest bind - the intact antigen ing fragment derivative heavy from the antibodies chain-only in camelids, present prepared were from a dromedary immunized domain and selected binding. N-terminal DARC against extracellular for DARC A described VHH, CA52, does recognize native vivax invasion of erythrocytes inhibits on cells. P. and displacesboundDARC It DARC. interleukin-8 to The targeted epitope idealto develop hence diagnosDARC Fy6 epitope. well-defined the K(D) of overlaps CA52-DARC equilibrium is sub-nanomolar, tic or therapeutic compounds. by immobilized Immunocapture CA52 yielded highly purifiedDARC engineeredfrom cells. K562 This first report on a VHH with specificity exemplifies to VHHs’target,a for potentialities to red and to protein cell purify, blood functionmodulate the markers. of cellular Rotavirus main cause is the the developmentantiviral prod of viral of inexpensive - gastroenteritis Therefore, in young children. of rotavirusucts preventionPreviouslya recombinant disease and/or treatment the shown for that we have a priority. remains monovalent antibody fragment as (referred to or ARP1) Anti-Rotavirus Proteins derived from a heavy antibody chain of a llama immunised with rotavirus was neutralise to able rotavirus infection in a mouse we investigated model work system. present In the specificitythe and neutralising activity of two llama antibody fragments, ARP1rotavi adapted- and ARP3, culture cell against 13 rus strains of diverse genotypes. electron microscopy In addition, immunocapture (IEM) was performed determine to binding of adaptedARP1 strains. culture clinical isolates to and cell ARP1 neutralise and ARP3 to able a broad variety were of rotavirus sero - types/genotypes in vitro, and in addition, IEM showed specificto a binding adaptedvariety strains cell of as well as strains from clinical specimens. Theseresultsthese molecules indicatedthat could potentially be used- and/or immuno as immunoprophylactic therapeutic products of infection prevention of a broad range clinically and/ or treatment the relevant for rotavirus strains. Many of the neutralising of the antibodies,Many limited display isolated activities date, to againstglobally the most subtypes prevalent HIV-1 Variable domains of thoseA and C. subtypes Therefore, consideredto are be an important for antibody-based target therapy. llama heavy antibodies chain (VHH) some superior have properties with compared classical antibodies. we describe Therefore the derived of subtype of trimeric(Env), application from proteins HIV-1 forms of envelope A and B/C, a prolonged for immunization of two llamas. selected were Env with CD4 with of VHH,use A panel HIV-1 interfere binding to of panning. which results The of a variant including with a stabilized CD4-bindingbinding and competitionvarious assays (gp120(Ds2)), to state Env, cross-compe - tition experiments, analysis and neutralisation assays, maturation enabled classify us to selected the three VHHgroups. into The VHH mainly efficient of groupagainst I were viruses of subtype B’/C. A, C and The VHH of group II the broadly resemble neu - tralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. group, 2E7, had an even third of the A representative higher neutralization neutralizing breadth, 26 tested 21 out of the strains belonging A, evaluate of certain the to potency contribution amino acids the the to A/G, VHH B, B/C and C subtypes. of the To a small setconstructed. mutants were of the Surprisingly neutralisation improved potency yielded this with slightly one mutant against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). Thesewell-known findings the and ofthe stability VHH indicate of these microbicides.potential VHH application as anti-HIV-1 for chemokines microscopy Plasmodium vivax Fy blood group Duffy antigen receptor HIV virus Envelope protein CD4 receptor Rotavirus Gastroenteritis Immunocapture electron

479 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 - Stijle mans et al. 2011 Ratier et al. 2008 The African trypanosomeTrypanosoma brucei, which persiststhe bloodstream within the mammalian host, of has evolved potent evasion.mechanisms immune for the antigenic variation of variant-specific Specifically, surface glycoprotein (VSG) and a highly active endocytosisantibodies. con - and recycling killing delay anti-VSG mediated by efficiently surface of the coat Consequently, VSG-specificventional non-trypanocidal intact immunoglobulins are sharpcomplement.contrast, In absence of the in monova- fragments, antigen-binding lent 15 kDa including nanobodies (Nb) derived from camelid heavy-chain antibodies (HCAbs) recog - nizing variant-specific VSG epitopes, efficiently lyse trypanosomesboth in and in vitro vivo. This Nb-mediated lysis ispreceded by very parasites, of the immobilisation rapid flagellar of the massivepocket enlargement blockade and major of endocytosis. This ATP-levels and lossis by severe accompanied of mitochondrial metabolic mem - perturbations reflectedreduced by intracellular Nbssite-directedthrough mutagenesis Modificationreconstitution death. of anti-VSG and by brane potential,in cell culminating HCAbs,into of trypanolytic combined with unveiling activity from intact immunoglobulins by papain proteolysis, demonstrates trypanolytic the that propose the We that activityrequires low molecular of Nbs and Fabs weight, monovalency and high affinity. of low moleculargeneration VSG-specific weight trypanolytic nanobodies impedethat endocytosis a offers new opportunity for developing trypanosomiasis novel therapeutics.- domain of an anti-micro antigen-binding In addition, these the data suggest that bial antibody harbours biological intact immunoglobulin and is in the revealed functionality is latent that only upon release of the fragment.antigen-binding The sialic acid present in the protective surface mucin coat of TrypanosomaThe sialicthe protective of acidcoat in mucin present surfacecruzi is added by a membrane anchored trans- this a modified work, In sialidase we analyzedexpressed is gene that a family. large from single domain camelid sialidase (TcTS), antibodies produced against trans-sialidase. Llamas immunized were trans-sialidase with a recombinant and inhibitory single- domain antibody fragments obtained were selection, by phage display taking advantage of a screening strategy using an inhibition single domain antibodies displaying trans-sialidase Four strong classictest instead of the inhibition activity binding assay. against enzyme recombinant the complementarity-determining identified. They were the same region share 3 length (17 residues) and veryhave similar sequences.they derived is only one structural resultthere Thisthat likely indicates clone. Probably a unique from our surprise,- single this domain anti used immunization. for To binding inhibitorysolution tight for antibodies againstTcTS the failed enzymatic inhibit the to activity in parasite present extracts. Analysis of individual body TcTS, inhibits recombinant the that trans-sialidasesrecombinant to extents 8 (from enzymes showed that genes inhibitedto different were expressed from different llama antibodies.98%) by the key Amino acid changes at be in inhibition positions to responsible differences likely the are for enzymes. recombinant among the found These and diverseresults of a the presence trans-sialidase large suggestthat family might cruzibe required inhibitory prevent the to response strategy against a novel essential this of T. enzyme constitute thus and might evadeto system. host the immune glycoprotein lysis Sialidase Trypanosoma cruzi Trypanosoma brucei surface Variant-specific Nanobody-mediated

480 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article - - Stijle mans et al. 2004 Hender son et al. 2007 - Antigen variationAntigen is a successful defense system adopted by several infectious evade agents to response. host the immune The principle defense strategy of this African in the trypanosome paradigm a dense involves packing of variant surface glycoproteins (VSG) exposing only highly system, epitopes variable whereas immune the and immuno-dominant to conserved epitopes become molecules. large for inaccessible Reducing conserved, target the size the of binders that less-immunogenic, cryptic VSG epitopes forms an obvious these parasites. solutioncombat to This goal was achieved by introducing dromedaryHeavy-chain antibodies. only these unique antibodies that found recognize VSG epitopes multiple classes. to common We After of their phage display repertoire,antigen-binding we isolated a single domain antibody fragment with high specificityconservedthe for Asn-linked of VSG.carbohydrate labeledstainsflagellar In sharp to only the contrast that pocket concanavalin-A are where carbohydrates becauseaccessible of less dense VSG packing, surface of viable single domain binder stainsentire the parasites, the irrespective of VSGthe type small antibody expressed.corroboratesthe idea Thisthat fragments, antibody lectins but not larger conventional or fragments, epitope. target densetheir penetrate to the VSG The diagnostic to labeled able coat are this fluorescently of potential binder was direct, by the proven selective, and sensitive detection of parasites in blood smears.this binder a of The employment as molecular designed recognition trypanosomosis unit in immunotoxins for becomes therapy feasible as This well. was illustrated by specificthe trypanolysis induced by an antibody: beta-lactamase fusion activating a prodrug. morphisms within surface-exposed of these loops ability AMA1 limit the may induced antibodies protect to against all parasite genotypes. an AMA1-specific Using IgNAR we single-variable-domainperformed antibody, mutagenesistargeted and selection cocrystal present falciparurm strains. structures of two antibody-AMA1against We AMA1 reveal which from three P. complexes IgNAR CDR3extended loops surface residues cleft and contacting antigen penetrating conserved on the deep a hydrophobic into across parasite species. Comparison of a series of affinity-enhancing allowed mutations contributions dissectionrelative their of binding kinetics withto inhibition of erythrocyte and correlation invasion. These findingsprovide of into insightsmechanisms single-domain antibody binding, design enable of reagents and may targeting otherwise cryptic epitopes antigens. in pathogen Apical membrane antigen 1 (AMA1) membrane antigen Apical is essential invasion for of erythrocytes and hepatocytes by Plasmodium parasites and is a leading antibodies malarial AMA1 to vaccine conventional Although candidate. poly can invasion, prevent such extensive

glycoproteins epitopes fusion antigen 1 Antigen variation surface Variant Cryptic epitopes membrane Apical Cryptic/conserved Trypanosomosis therapy Antibody: beta-lactamase Plasmodium falciparurm polymorphismExtensive IgNAR

481 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 Moller et et Moller al. 2010 Paalanen Paalanen et al. 2011 McGoni - gal et al. 2009 - IRF-1 is a tumor suppressor protein that activates gene expression from a range of promoters in response spanning from viral a range of promoters stimuli activates to IRF-1 gene expression that suppressor is a tumor protein regulationinfection post-translational of IRF-1 been DNA damage.on the Studies to have hampered bio - by a lack of suitable technology phage display was usedchemical tools study, In this endogenous of targeting develop capable the protein. to a mono - nanobodyclonal nanobody of the C-terminal targeting the Mf1 domain (residues expression 301–325) of IRF-1. Intracellular transcriptional the activity that demonstrated of IRF-1 is constrainedMf1 domain as by the nanobody an increase binding gave in Mf1-directed from IRF-1-responsive nanobodies Furthermore, 8-fold. expression of up to revealed promoters have an unexpected IRF-1 is the degraded. which protein at function rate Thus, domain in limiting the this for the increase in IRF-1 transcriptional activity observed on nanobody In support binding isby a significantreduction accompanied the protein. of the half-life in of data obtained using nanobodies, C-terminal the (P325A) involving residue a singleof IRF-1 point mutation has been identified, resultswhich transcriptional in greater activity and a significant increase of degradation. The results the rate presented in here In addition, the support Mf1 domain in limiting both the IRF-1-dependent for a role of IRF-1 transcription turnover. rate and the activation for of downstream usingdata biologics. highlight a route genes IRF-1 in the suppressor tumor pathway The 10 isozymes roles the same biologicalkinase on the protein of process, C (PKC) different family can have making isozyme specificfew only pharmacological analysiscompounds of moderate functionisozymewith crucial.Currently, specific effects exist hamperingisozymes. individualthus research into PKC regions binding The antigen of singlechain antibodies camelid (VHHs) isozyme successfully provide a solutionobtainingselectedcould for PKC we have specific and modulators. study, the present In characterized epsilon specific PKC VHH The shown antibodies VHHs were VHH two immune librariesfrom using phage display. epsilon in ELISA PKC the bind to five of VHHs had an effecton and immunoprecipitation studies. exclusively to Strikingly, epsilon kinasePKC activity in vitro. VHHs A10, C1 and D1 increased epsilon kinase PKC activity in a concentration-dependent manner (EC(50) values: 212–310 nM), whereas epsilon E6 and G8 inhibited activity PKC (IC(50) values: 103–233 nM). None of To our knowledge,these theta.these VHHs isozymes had an effect and PKC delta PKC the activity novel on the other PKC of antibodies first described the are epsiFurthermore,- the development of PKC kinase. for a protein VHH activators and inhibitors lon specificresearch. modulators to PKC is an important contribution ticular, specificthe activity modulators of of Caspaseticular, could 3 be veryfor diseases therapies importantthe development of for such two V(H)Hs specificto Caspase study, In this 3 (VhhCasp31as neurodegeneration and VhhCasp32) and cancer. isolated were from a heavy antibody chain variable domain (V(H)H) library phage display apoptosis-modulating and tested their for effects. While VhhCasp31 was be to found antagonistic towards Caspase 3, VhhCasp32 was when expressed agonistic. as Furthermore, neuroblastoma cells, VhhCasp31 oxidative-stress-induced resistant to apoptosis, rendered cells intrabodies in SHSY-5Y whereas VhhCasp32 resulted in apoptosis. Thesethe development of for apoptosis V(H)H antagonist and agonist of could have potential respectively. therapeutics neurodegenerative for diseases and cancer, Apoptosis, or programmed is an essential death, process affecting cell homeostasis cell growth, development, of elimina - the and causedtion of damaged stress has death been or dangerous cell cells.by oxidative development Inappropriate in the implicated process death hand, a defect other cell On the in the and stroke. of neurodegenerative diseases as such Parkinson’s, Alzheimer’s, of apoptosis, main player the cancers. example, p53, is human defective of the in many For leads development of cancer. the to Bcl-2 from the proteins family and caspases. and anti-apoptotic is of pro-apoptotic regulatedApoptosis interplay by the In par diseases IRF-1 suppressor Tumor Mf1 domain activity Transcriptional Protein kinase C Enzyme modulator Table 6D. Application of single-domain Application 6D. antibody fragmentsTable in therapy: intracellular of single-domain expression antibody fragments Apoptosis 3 Caspase Neurodegenerative Cancer

482 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article - Alzogaray Alzogaray et al. 2011 Ver cruysse et al. 2010 Serruys et al. 2010 Walsh et et Walsh al. 2011 - ADP-ribosylation of host cell proteins is a common mode of cell intoxication by pathogenic bacterial by pathogenic toxins. Antibodies intoxication mode ADP-ribosylation is induced a common of cell proteins of host cell by immunization with inactivatedprotection provide ADP-ribosylating efficient in toxins case of some toxins,secreted e.g., diph - ADP-ribosylatingsecreted other are as toxins, such toxin, theria Salmonella SpvB directly and pertussis from the toxins. However, cytosol vacuole the Salmonella-containing into via encoded of target SPI-2 cells the bacterial type III secretion system, and thus antibodies. conventional to inaccessible Small-moleculeare ADP-ribosylation with fraught potential side effects are inhibitors caused by inhibition of endogenous ADP-ribosyltransferases. we report Here, development of a single-domain the antibody from an immunized blocks llama that ADP-ribosylate capacity to the of 1 : 1. The single-domain of SpvB a molar antibody, actin ratio at effectively protectedthe cells from activitywhen expressedchimeric as cytotoxic of a translocation-competent an intrabody, also were protected cells against cytoskeletal induced Transfected by wild-type alterations toxin. C2IN-C/SpvB SpvB-expressing strains of Salmonella. This prooffor developing of principletoxins. the new way paves against intracellular antidotes The human immunodeficiencyThe virus, typelateexpression ofviral 1 mRNAs.(HIV-1)-encoded the isprotein for Rev essential Rev Rev on the response organizedforms of these a large viral complex protein-protein element multimeric mRNA species and trans ports cytoplasm, the to nucleus from the them CRM1-mediated the we report exploiting Here selection the machinery. of cellular blocks derived efficiently the that from assemblya nanobody, llama of heavy-chain multimers.Rev only antibody, The nanobody and specifically in cells replication inhibits HIV-1 suppressesthe Rev-dependent of partiallyexpression spliced and unspliced RNA.HIV-1 In HIV-susceptible cells, nanobody this has thus potential as an effective anti-HIV using genetic agent immunization located N-terminal in the alpha-helical multimerization strategies. was binding site Its mapped Rev to and Tyr-23 residues Lys-20 we observed nanobody, of this presence domain. In the of dimeric Rev an accumulation species, supporting a head-to-head/tail- to-tailthe oligomeric molecular results The that assembly of Rev indicate modelRev for follows an ordered stepwise assembly. process and identify a new epitope withinguide Rev HIV could development strategiesof novel the for that inhibitors. Six single-domain antibodies (VHHs) (HBcAg) of HBV antigen been core have targeting the and three generated of these bound HBcAg to strongly Thesethree of both subtype studiedVHHs were as intrabodies ayw and adw. directed nucleus towardsthe A speckled cytoplasmor the staining was of HBcAg line that co-transfected was cell of a hepatoma observed with HBV. in the cytoplasm transfected an increased of cells hepatitis VHH with accumulation nucleotropic intrabodies. intracellular Moreover, HBcAg (HBeAg) disappearanceB e antigen of intracellular signal and a complete observed were targeted with nuclear HBcAg- specific VHHs. These results suggest that HBcAg-specificnucleus targeted the VHHsaffect to and HBcAg andexpression HBeAg HBV-transfected trafficking inhepatocytes. The HepatitisThe B virusis processed protein precore the endoplasmic in reticulum secreted (ER) into (HBeAg), hepatitis B e antigen acts infection.which establish to chronic Downregulation as tolerogen an immune of secreted HBeAg clinical improve should as significantly lower patients who effectivelyrespondoutcome, serum have (IFN-alpha) treatments to current HBeAg levels. we describeHere, reagent, a novel a single variable domain (V(NAR)) immunoglobulin new shark receptor (IgNAR) of the antigen antibodies. V(NARS) size possess 14 kDa) (similar to and cryptic advantages in stability, epitope- recognition conven to compared tional antibodies. The V(NAR) domain displayed biologically nativeuseful and forrecombinant affinity HBeAg, andrecognised reduce secreted to assess protein precore therapeutic potential in targeting intracellular epitope. To a unique conformational HBeAg, V(NAR) was the engineered ER-targeted for in vitro delivery function to antibody as (intrabody). an intracellular In vitro hepatocytedata from HBV/precore lines effective demonstrated cell intrabodyregulation of precore/HBeAg. protein ADP-ribosylation Salmonella SpvB toxin HIV-1 virus HIV-1 Rev protein Replication Multimerization domain Hepatitis B virus HBcAg HBeAg Hepatitis B HBeAg IgNAR Intracellular precore

483 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 Hussack Hussack et al. 2011a Blanco- et Toribio al. 2010 Tremblay Tremblay et al. 2010 - rypsin majority of mutants. the resistance for V(H)H trypsin Mutant resistance was of wild-type that similar to V(H)Hs, although trypsinthe wasintroduction resistance of one V(H)H mutant the reduced. of a significantly disulfide in Therefore, second bond not only increases core neutral pH, as hydrophobic V(H)H thermal at stability the previously shown, but also represents a generic strategy increase to low pH and impart at protease V(H)H stability resistance, with only minor perturbations in target binding affinities. characteristicsThese are all the desirable for design protein-based of therapeutics. oral The extreme pH and protease-rich environment of the upper pH and protease-rich of tractextreme gastrointestinal environment obstacleThe is a major facing orally-administered therapeutics,protein antibodies. including engineering, protein Through several toxinClostridium A-specific difficile chain heavy antibody variable domains (V(H)Hs) expressed with were an additional disulfide and Ile78Cys bond by introducing Ala/Gly54Cys antibodies wild-typemutations. their to Mutant compared were counterparts with respect yield, non-aggregation expression to toxinstatus, for affinity A-neuprotease- A,toxin circular dichroism resistance, (CD) structuraland signatures, thermal stability, expressed,to found although be well to lower compared V(H)Hs were with mutant wild-typeyields The tralizingcoun - capacity. terparts, non-aggregating were monomers,toxin retained for low nM affinity majority A, albeit the showed somewhat reduced to compared affinity wild-type counterparts,toxin A neutralizationcell-based of incapable in and were vitro and Far-UV assays. near-UV CD spectroscopy showed shifts consistently in peak and selective intensity peak wild-type minima for V(H)H and mutant A significant increase overall CD the remained thermaltemperature midpoint the unfolding profile very in pairs; similar. however, was observed both neutral all mutants at and acidic gastrointestinal for major pH. Digestionproteases, V(H)Hs with the of the at biologically revealed relevant concentrations, a significant increase in pepsinmutantsresistancefor all and an increase chymot in Intracellular proteins have a great potential as have targets proteins therapeutic antibodies for Intracellular plasma but the (Abs) membrane prevents- these to antigens. fragmentsaccess Ab and IgGs selected are and engineered in E. microorganismbe and this coli may also an (sdAbs) single-domain can Ab be that engineered we demonstrate work In this be to delivery. ideal vector intracellular their for by E.injected cells bacteria human coli into carrying molecular syringes assembled by a type secretion III protein system (T3SS). The injectedthe in cytoplasm accumulate sdAbs HeLaat levels of cells ca. 10(5)–10(6) moleculestheir and cell functional - per Injectionity iscomplexes. isolation shown of sdAbs by the of sdAb-antigen does not require bacterial invasion transfer or the of genetic These material. results proof-of-principle are the for capacity of coli sdAbs bacteriaE. to directly intracellular deliver analytical for cells (intrabodies) and therapeutic human purposes. into Botulinum neurotoxins (BoNTs) function by delivering a protease to neuronal cells that cleave SNARE proteins and inactivate and inactivate SNARE function cleave proteins by deliveringthat a protease neuronal cells (BoNTs) to Botulinum neurotoxins exocytosis.neurotransmitter Small (14 kDa) binding domains specificthe proteasefor of BoNT serotypes A or B were selected from libraries of heavy only antibody chain domains (VHHs or nanobodies) cloned from immunized alpacas. Several VHHs bind BoNTthe proteases with high of affinity (K(D) inhibitors near 1 nM) and include BoNT/A potent protease activity 1 (K(i) near nM). The retainVHHs their binding specificity and inhibitoryexpressed functionscells when as mammalian within neuronal intrabodies. A VHH of BoNT/A inhibitor protease from cleavage following was SNAP25 protein protect to neuronal cell able with BoNT/Aintoxication These componentsVHHtherapeutic that results domains have potential of as holotoxin. demonstrate reversalagents for of botulism intoxication. Intracellular proteins E. coli Intracellular delivery Molecular syringe Botulinum neurotoxins Protease inhibitor 6E. of single-domain Application antibody fragmentsTable in therapy: oral administration of single-domain antibody fragments Thermal stability Trypsin pH Extreme Clostridium difficile Toxin A

484 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article - Hussack Hussack et al. 2011b - Vanden broucke et al. 2010 Harm sen et al. 2006 - Inflammatory bowel disease (IBD) is a chronic inflammatory gastrointestinal disorder. Systemic treatment of IBD patients with ofpatientsInflammatory IBDtreatment with Systemic diseasebowel inflammatory (IBD) chronic is a disorder. gastrointestinal anti-tumor necrosis factor (TNF)-alpha antibodies hasbe but several to proven a highly approach, promising drawbacks remain, side effectsincluding to cost and high administration of treatment.systemic related Lactococcus lactis was engineeredto secrete monovalent and bivalent(m) TNF-neutralizing murine Nanobodies as therapeutic proteins. derived These are therapeutic proteins from fragments antibodies. of heavy-chain camelid antibodies conventional than stable more and are L. lactis-secreted anti-mTNF in vitro. Daily of Nanobody-secreting oral administration Nanobodies neutralized mTNF L. lactis resulted in local delivery of anti- sulfate Nanobodiesreduced and significantlydextran sodium colon (DSS)-induced the in micechronic with inflammation at mTNF colitis. was approach In addition, this also established10 (IL10)(–/–) successful in improving mice. in interleukin enterocolitis L. Nanobodies lactis-secreted with systemic Salmonella infection In did not interfere IL10(–/–) mice. in colitic Finally, anti-mTNF report this conclusion, colitis, details of chronic secretion in situ involving a new treatment for of anti-mTNF therapeutic approach Nanobodies by orally administered L. lactis bacteria.these Therapeuticof application engineered bacteria could leadto eventually humans. of effectiveIBD in more management and safer single-domain antibody recombinant of the previously fragment oral application (VHH) that demonstrated K609, clone We directed against Escherichia F4 fimbriae,reduced coli coli-induced diarrhoeaE. in piglets, We have at high but only VHH doses. portion a large now shown orally applied that K609 VHH of the is proteolytically degraded selection Stringent stomach. in the proteolyticfor identified stability seven increasedVHHsto 138-fold 7- with after stability in incubation in vitro gastricBy fluid. we obtained clonesDNA shuffling four a with These increased furtherto 3-fold most by at 1.5- differed VHHs in vitro stability. VHH scattered over the were amino acidsequence with residuesten predicted and did not overlap from and K609 that each other protease cleavage sites.retained The mostclone, K922, stable 41% activity after incubation in gastric fluid and 90% in jejunal fluid. piglets of K922 to In addition, K922 Oral application to F4 fimbriae confirmedbound itshigher with proteolytic improved stability. affinity and inhibited fimbrialfor promising of thus a piglet at lower adhesion prevention K922 concentrations. candidate is VHH diarrhoea. our findingscould by genetic guide engineeringselection Furthermore, - and improvement anti recombinant of other body fragments oral use. for Clostridium difficile is a causeleading of nosocomial infectionprofes to in North challenge healthcare America considerable and a sionals in hospitals and nursing homes. The Gram-positive bacterium produces two high molecular weight exotoxins, toxin A (TcdA) sionals in hospitals homes. and nursing The Gram-positive A bacterium producestoxin two high(TcdA) molecularexotoxins, weight virulence major the factors are which responsible C. for difficile-associated disease B (TcdB), are and toxin targets for C. difficile- single-domain antibodyassociated recombinant Here, fragments (V(H)Hs), disease specifically which receptor therapy. cell the target isolated were llama library phage display from an immune V(H)Hs (A4.2, and characterized. Four or TcdB, binding domains of TcdA recognizeA5.1, A20.1, and A26.8), all shown to epitopes, conformational neutralizers potent were cytopathic of the effects toxin of The neutralizing potencyA on fibroblast in an cells vitro assay. was further enhancedadministered when V(H)Hs were in paired epitopes. Biacore suggesting recognition TcdA of nonoverlapping same overall V(H)H concentration, the at or triplet combinations epitope experiments revealed mapping some synergistic consisted that of V(H)Hs recognizing combinations epitopes, overlapping epitope mere factors than blocking other responsible increased that an indication are the for binding assays neutralization. Further With binding the toxin. than pocketcarbohydrate the V(H)Hs neutralized of to sites other A by binding toxin revealed TcdA-specific these attractive V(H)Hs are characteristicsfavorable as such neutralization, high production stability, yield,and intrinsic toxin potent systemic therapeutics so more as but are oral therapeutics destabilizing gastrointestinal in the tract. of the environment disease domain Inflammatory bowel factor Tumor-necrosis Lactococcus lactis Secretion F4 fimbriae E. coli Diarrhoea Proteolytic stability Gastric fluid Clostridium difficile Nosocomial infection North America A/B Exotoxin Cell receptor binding

485 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 - Pant et et Pant al. 2006 Rutgers Rutgers et al. 2011 Kas turiran - gan et al. 2010 Vuchelen Vuchelen et al. 2009 - - - - imaging and as efficient amyloidosislocalsuch ascerebral drug moietypatients targeting Alzheimer’s insuffering from Previously selected beta amyloid recognizing heavy antibody chain fragments (VHH) affinity binders derived thefrom Came lid heavy antibodychain repertoire propensity crossblood-brain tested their to the were for barrier (BBB) using an established in vitro BBB co- system. culture Of all tested VHH, ni3A showed highest transmigration efficiency which is, in part, facilitated three by a amino acid mechanismsubstitutions in its the passage studies indicatedof transcellular N-terminal that domain. Additional of ni3A is by active transport. As recognize to VHH ability beta the ni3A combines amyloid crossBBB, it has and to the potential as- a tool non-inva for sive in vivo (CAA).disease angiopathy amyloid (AD) and cerebral Deposition of beta-amyloid (A beta) is disease considered an important early event pathogenesis in the of Alzheimer’s (AD), and reduction of A beta levels be brain could A potentially a viable in the noninflammatoryclearto therapeutic approach. facilitate route ance and reduce toxicity of A beta and reduce toxicity degradeance is to peptide the using proteolytic nanobodies. a proteolytic that nanobody we show Here engineered A beta cleave to its at alpha-secretase has site potential therapeutic value. The derivedAsec-1A proteolytic nanobody, prevents aggregation catalytic of monomericfrom a parent A beta, antibody, chain light inhibits further aggregation of preformed A beta aggregates, and reduces A beta-induced cytotoxicity line. The nanobody neuroblastoma a human toward cell alsoreduces toxic precursor (APP) amyloid in a Chinese protein human ovary of the hamster ity induced by overexpression (CHO) line by cleaving cell alpha-secretase the proteolysisAPP at of APP and A beta precludes of A beta.with which catalytic formation site nanobod Targeted - potentially AD harmful where treating ies for represents therapeutic side effects approach a novel can he minimized. Nanobodies produced singleantibodies uniquely chain are are in Camelidae, e.g. that camels and llamas. Theythe desir have of small sizes < 14 kDa) and highto nM), making them ideal(Kd similar features affinitiesantigens (Mw able against as structural previously have nanobody- probes shown that biomedically amyloido for binding to relevant motifs both in vitro and in vivo. We lysozymegenic human variantsthe originat can disis effectivelytheir aggregation,the processthat of inhibit amyloid systemic ease. we report Here NMR assignments the termed of a new recognises which NbSyn2, nanobody, - C-terminusintrinsi the of disease.cally alpha-synuclein disordered human (aS), whose protein, aberrant self-association is in Parkinson’s implicated Background. Rotavirus-induced diarrhea poses a worldwide medical in causing substantial problem morbidity and mortality developed therefore recombinant in which a system passive for immunotherapy in developingamong children countries. We neutralizing express lactobacilli variable constitutively domain of llama heavy-chain (VHH) antibody fragments against rotavirus. Methods. VHH expressed in Lactobacillus were paracasei, in both secreted surface-anchored forms. and cell Electron microscopy vivo the in functioninvestigate To of VHH-expressing was binding efficacy used the investigate to of lactobacilli. VHH-expressing lactobacilli, a mouse pup model of rotavirus infection was used. Results. theantibody binding of Efficient VHH to fragments rotavirus was shown by enzyme-linked VHH fragments immunosorbent and scanning assay expressed electron by microscopy. lactobacilli conferred a significant reductionlactobacilli-producing in infectioncultures.cell in When administeredsurface- orally, expressed VHH markedly shortened disease and viral load in a mouse severity, duration, model of rotavirus-induced diarrhea when administered both fresh and in a freeze-dried basis lactobacilli form the form may of of a novel form. Conclusions. Transformed against rotavirus treatment infectionsprophylactic diarrheal and other diseases.

angiopathy gration efficiency Beta-amyloid diseaseAlzheimer’s Proteolytic nanobody Aggregation Amyloidogenic lysozyme Alpha-synuclein disease Parkinson’s Self-association Rotavirus Diarrhoea Developing countries Passive immunotherapy lactobacilliTransformed of single-domain Application antibody fragments 6F. in therapy: prevention plaque formation and protein of amyloid Table aggregation Beta-amyloid transmi - diseaseAlzheimer’s Cerebral amyloid

486 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article - Pant et et Pant al. 2006 Rutgers Rutgers et al. 2011 Kas turiran - gan et al. 2010 Vuchelen Vuchelen et al. 2009 De Genst et al. 2010 Impagli - azzo et al. 2010 - - - - - imaging and as efficient amyloidosislocalsuch ascerebral drug moietypatients targeting Alzheimer’s insuffering from Previously selected beta amyloid recognizing heavy antibody chain fragments (VHH) affinity binders derived thefrom Came lid heavy antibodychain repertoire propensity crossblood-brain tested their to the were for barrier (BBB) using an established in vitro BBB co- system. culture Of all tested VHH, ni3A showed highest transmigration efficiency which is, in part, facilitated three by a amino acid mechanismsubstitutions in its the passage studies indicatedof transcellular N-terminal that domain. Additional of ni3A is by active transport. As recognize to VHH ability beta the ni3A combines amyloid crossBBB, it has and to the potential as- a tool non-inva for sive in vivo (CAA).disease angiopathy amyloid (AD) and cerebral Deposition of beta-amyloid (A beta) is disease considered an important early event pathogenesis in the of Alzheimer’s (AD), and reduction of A beta levels be brain could A potentially a viable in the noninflammatoryclearto therapeutic approach. facilitate route ance and reduce toxicity of A beta and reduce toxicity degradeance is to peptide the using proteolytic nanobodies. a proteolytic that nanobody we show Here engineered A beta cleave to its at alpha-secretase has site potential therapeutic value. The derivedAsec-1A proteolytic nanobody, prevents aggregation catalytic of monomericfrom a parent A beta, antibody, chain light inhibits further aggregation of preformed A beta aggregates, and reduces A beta-induced cytotoxicity line. The nanobody neuroblastoma a human toward cell alsoreduces toxic precursor (APP) amyloid in a Chinese protein human ovary of the hamster ity induced by overexpression (CHO) line by cleaving cell alpha-secretase the proteolysisAPP at of APP and A beta precludes of A beta.with which catalytic formation site nanobod Targeted - potentially AD harmful where treating ies for represents therapeutic side effects approach a novel can he minimized. Nanobodies produced singleantibodies uniquely chain are are in Camelidae, e.g. that camels and llamas. Theythe desir have of small sizes < 14 kDa) and highto nM), making them ideal(Kd similar features affinitiesantigens (Mw able against as structural previously have nanobody- probes shown that biomedically amyloido for binding to relevant motifs both in vitro and in vivo. We lysozymegenic human variantsthe originat can disis effectivelytheir aggregation,the processthat of inhibit amyloid systemic ease. we report Here NMR assignments the termed of a new recognises which NbSyn2, nanobody, - C-terminusintrinsi the of disease.cally alpha-synuclein disordered human (aS), whose protein, aberrant self-association is in Parkinson’s implicated Background. Rotavirus-induced diarrhea poses a worldwide medical in causing substantial problem morbidity and mortality developed therefore recombinant in which a system passive for immunotherapy in developingamong children countries. We neutralizing express lactobacilli variable constitutively domain of llama heavy-chain (VHH) antibody fragments against rotavirus. Methods. VHH expressed in Lactobacillus were paracasei, in both secreted surface-anchored forms. and cell Electron microscopy vivo the in functioninvestigate To of VHH-expressing was binding efficacy used the investigate to of lactobacilli. VHH-expressing lactobacilli, a mouse pup model of rotavirus infection was used. Results. theantibody binding of Efficient VHH to fragments rotavirus was shown by enzyme-linked VHH fragments immunosorbent and scanning assay expressed electron by microscopy. lactobacilli conferred a significant reductionlactobacilli-producing in infectioncultures.cell in When administeredsurface- orally, expressed VHH markedly shortened disease and viral load in a mouse severity, duration, model of rotavirus-induced diarrhea when administered both fresh and in a freeze-dried basis lactobacilli form the form may of of a novel form. Conclusions. Transformed against rotavirus treatment infectionsprophylactic diarrheal and other diseases. The aggregationthe intrinsically of disordered alpha-synucleinformto associated fibrillaramyloid structures protein intimately is disease.with a variety The molecular of neurological mechanism disorders, of alpha-synuclein aggrega most Parkinson’s notably - is not yet understoodtion and toxicity detail, in any not least because paucity of structural of the study probes to which through a disordered behavior of such the we describe NbSyn2, system. Here, a single-domain involving camelid antibody, an investigation selected of its effects alpha-synuclein, techniques exploration by phage display bind to the the in to including aggregation on vitro of using show isothermal calorimetric under a variety methods binds protein NbSyn2 specificallythe of conditions. that We to mono - meric alpha-synuclein with nanomolar affinity and by means of NMR interacts four spectroscopy it the C-terminal residues that with confirmed finding is Thisof thedetermination a by latter protein. of the NbSyn2crystal of to structure bound encompassinga peptide nine C-terminalthe residues of alpha-synuclein. : alpha-synuclein The NbSyn2 is mediated interaction interac mainly by side-chain we believe moleculestions while water be a feature of NbSyn2, could of alpha-synuclein cross-link atoms to atoms main-chain the The aggregation of alpha-synucleinat physiologicalbehavior important generally. more in intrinsically disordered interactions protein morphology the pH, including resulting fibrillar of the structures, unaffectedremarkably presenceis and indeed of NbSyn2 the by we aggregated the binds NbSyn2 to strongly as assoluble that the forms well of alpha-synuclein.show to Theseresults sup - give strong C-terminal is the not directly regionport mechanism protein involved in the that of the conjecture of aggregation the and suggest to be could binding of NbSyn2 a useful of alpha-synucleinthat identification aggregation probe the for in and vitro possibly in vivo. Oculopharyngeal muscularis caused 1 (PABPN1) dystrophy nuclear in polyadenylate binding protein by small alanine expansions PABPN1 3F5 llama muscle. antibody in skeletal specifically accumulation resulting in itsprotein intranuclear the with interferes understandstructural the basis its for epitope recognition we mappedaggregation binding process the in vitro and in vivo. To 3F5 complementarity that show We complex. and provide a structural model3F5-PABPN1 of the of 3F5 with PABPN1 interface alpha-helix domain resides critical by involving residues previouslydetermining regions- a cavityPABPN1 in which create impli cated aggregation in the process.PABPN1 aggregation mechanismthe These and results increase may our understandingthe of therapeutic potential of 3F5.

angiopathy gration efficiency structures muscular dystrophy Beta-amyloid diseaseAlzheimer’s Proteolytic nanobody Aggregation Amyloidogenic lysozyme Alpha-synuclein disease Parkinson’s Self-association Rotavirus Diarrhoea Developing countries Passive immunotherapy lactobacilliTransformed of single-domain Application antibody fragments 6F. in therapy: prevention plaque formation and protein of amyloid Table aggregation Beta-amyloid transmi - diseaseAlzheimer’s Cerebral amyloid Aggregation Alpha-synuclein amyloid Fibrillar disease Parkinson’s Aggregation Oculopharyngeal Alanine expansion PABPN1

487 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 Ibanez et al. 2011 Zhang et al. 2004 Roovers et al. 2011 model of athymic mice bearing mice - model of athymic A431 xeno Influenza A virus infections imposerecurrent a and global disease antivirals againstCurrent influenzaare not always burden. We assessedthe protective Nanobodies of monovalent and bivalent effective.potential (Ablynx)this againstchallenge with virus. TheseNanobodies derived were llamasfrom and Nanobodies H5N1 hemagglutinin.target of Intranasal administration effectively homologouscontrolled influenza A Nanobodiesvirus of Administration replication. reduced challenge strongly H5N1 before virus lungs in the replication and protected from morbidity and mortality mice after with H5N1 virus. a lethal challenge The bivalent Nanobody was leastNanobody at effectivethe monovalent more than virus 60-fold controlling Nano - replication. in In addition, bodyrevealed Epitope after reduced strongly mapping challenge therapy viral to death. and significantly replication time delayed VHH the Nanobody B in H5 hemagglutinin.that Because site antigenic binds to - Nanobodies pro to and simple small, stable, are they a promising,duce, are against therapeutic influenza. agent novel We describe type a novel of moleculeWe single-domain in which antibodies (sdAbs) isolated from a naive llama- single domain anti body library reagents. linked an oligomerization high-avidity are antigen-binding to generate domain to is An sdAb fused the to self-assembles which form a homopentamer toxin, to or shiga-like and resultsB-subunit- of Escherichiain simul verotoxin, coli taneous Molecular pentamerizationfusion this sdAb modeling (PDB: 10JF), indicated that protein and introduction of avidity. hasto surface-presentedtermedfor binding structural specific of an the instance pentabody, sdAb flexibility In a antigen. for peptide pentamerization antigen, resultedincrease in a dramatic in functional for immobilized affinity antigen. Thepentabody was thermostability and proteaseexpressed in high yieldresistance. in E. This in a non-aggregated and exhibited excellent coli state, molecules immobilized antigen-binding technology to bind strongly meansnovel - rapid of generating that anti provides a relatively is expected in proteomics, sdAbs pentavalent general applicability willgen. It have that immunochemical staining, cancer diagnosis presented are multivalently. antigens in which applications and other The epidermal growth receptor factor (EGFR) has beenAt present,shown to be a for antibody-basedvalid target cancer therapy. several monoclonal anti-EGFR antibodies beencrystallography have successfully used, X-ray as such and matuzumab. cetuximab these antibodiesepitopes that datathe show on different bind to ecto-domain of EGFR,combinedthe for use providing a rationale of these previously two antibodyWe have reported specificities. the successful on isolation nanobod of antagonistic anti-EGFR - efficacy the we aimed improve to these of ies. molecules combining nanobodies by In our study, with specificities to similar both molecule. a single biparatopic into Carefully and matuzumab cetuximab designed phage nanobody selections resulted in two sets of nanobodies specificallyfor compete each did not that that cetuximabto EGFR and or blocked matuzumab the binding of either binding.others’ of nanobodies A combination from both nanobody epitope biparatopic the groups CONAN-1 was into shown to than monovalent or bivalent block (monospecific) EGFR activation efficiently nanobodies. more nano - this In addition, biparatopic in an vivo body Importantly, inhibited potently EGF-dependentproliferation. cell grafts, CONAN-1 inhibited outgrowth tumour with an almost similar potency fact despite the cetuximab, as that whole mAb the effector portion mediate immune CONAN-1 iscould devoidfunctions. that of an Fc using bivalent,therapy to Compared mono - specific nanobodies, CONAN-1 tumour wasrational in growth clearlydesign inhibition. Thesethe more potent results that show nanobody-basedof biparatopic therapeuticsyield lead potent anticancer molecules may further for development. receptor tion Table 6G. of single-domain Application antibody fragmentsTable in therapy: multispecific and multifunctional constructs Pentabody B-subunit Verotoxin E. coli Thermostability Protease resistance Biparatopic nanobody Epidermal growth factor Anticancer therapeutics Bivalent nanobody Influenza virus Intranasal administra-

488 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article Hussack Hussack et al. 2009 Hultberg Hultberg et al. 2011 - Antibody engineering of binding agents against has of interest, generation virtually predomi allowed antigen rapid - the any for has been therapeutic applications. for less Considerably attention nantly development given of diagnostic the to reagents and bio - sensors using engineered we produced bispecific pentavalent antibodies. a novel antibody (i.e., decabody) Recently, by pentameriz - B subunit (VTB)ing two single-domain antibodies verotoxin the (sdAbs) through both and found fusion partners be to functional. engineered we have a similar approach, a bispecificUsing cellulose-pentamericconsisting of five and five sdAbs fusion protein find an optimal designbinding modulesformat, constructed (CBMs) we linked six via bispecific VTB.of consisting To pentamers CBMs,three different fusedaureus-specificStaphylococcusthe to human sdAb HVHP428, in both orientations. One bispecific an N-terminal CBM9 containing and C-terminal HVHP428, waspentamer, soluble, non-aggregating, and did not degrade upon 4 degrees at storage over six C for months. This molecule was dually functional cellulose-basedas it to bound well filters as asS. cells.aureus filters, When impregnated in cellulose the bispecific pentamer recognizedcells in a flow-through aureus S. detection ability of The pentamerizedformthe basis cellulose may to bind multivalent display of an immobilizationassay. CBMs for platform for sensing of of high-avidityfilters pathogens, binding reagents and environmental pollutants. on cellulosic biomarkers ful strategy. Llama heavy antibody chain ful fragments strategy. (VHH) of Respiratory againsttrimeric the proteins envelope Syncytial Virus Rabies virus protein), and H5N1 Influenza 5) were selected (Hemagglutinin (Fusion (Glycoprotein) llama derivedfrom immune Neutralizing VHHlibraries recognizing by phage display. epitopesreceptorthe spikes the binding sites on affini - in different with threethe for all ties identified low nanomolar viruses range were in the by viral neutralization assays. By fusion of VHH with vari - 1500-fold lengths, linker able constructsneutralization multimeric improved made that potencies were RSV, for 4000-fold up to Rabiesfor virus Influenza for and 75-fold H5N1. The potenciesthe constructs of VHH similar orthan were better best performing monoclonal antibodies. The cross protection viral strainscapacity against different threewas improved for all also viruses, both constructs.VHH) (two or three VHH) identical (two different by multivalent and biparatopic combining a By VHH neutralizing RSV subtype A, but not subtype B with a poorly neutralizing VHH with for subtype high affinity construct B, a biparatopic was made with low nanomolar neutralizing potency against both VHH subtypes. anti-H5N1 neutralized both Influenza Trivalent H5N1 clade1 and 2 in a pseudotype and was assay very in neutralizing potent NIBRG-14 Influenza the H5N1 strain with IC(50) of constructs Bivalent and biparatopic against Rabies virus cross neutralized9 picomolar. bothGenotype 10 different 1 strains and Genotypemultimerization results 5. The that of show VHH fragmentsmultiple epitopes targeting on a viral trimeric protein spike is a powerful achieve to tool anti-viral “best-in-class” and broader neutralization for therapy capacity. For efficient prevention efficient of viral infectionscross andprotection, For simultaneous multiple of viral targeting epitopes is a power

bispecific constructs Biosensors Decabody Cellulose filter S. aureus Multimeric and and Multimeric Influenza

489 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 Van de de Van Broek et al. 2011 Talelli et et Talelli al. 2011 Iri-Sofla Iri-Sofla et al. 2011 - - using a 690 nm continuous wave laser. laser. wave using a 690 nm continuous 2 Starting from a nanoparticleis observed, death optical density of 4, cell samples, whereas nanoparticles control the with anti-PSA nano - These death. resultsthis new cell suggestthat any type do not show bodies, of bioconjugated branched and laser only, nanoparticles only, gold nanoparticles effective are antigen-targetedtherapeutic for treatment. photothermal agents cancer Branched agents gold nanoparticles because therapy potential photothermal are absorption large of their cross section near- in the laser Upon irradiationinfrared they cells. destroy enoughtumor produce window. heat to work, In this branched gold nanoparticles are blofunctionalized with nanobodies, smallest the fully functional fragments antigen-binding evolved variable from the VHH, domain, the These nanobodiesof a camel heavy antibody. the expressed which is highly chain-only HER2to bind antigen on breast and ovarian cancer cells. Flow cytometric analysis Images field of and dark incubatedcells HER2 branched conjugated anti-HER2 gold with positiveSKOV3 nanoparticles specific show targeting.cell Laserreveal Irradiation studies exposed cells HER2that the anti-HER2 to tar positiveSKOV3 geted branched gold nanoparticles destroyed are at 38 W/cm of afterlaser five minutes treatment The aim of this study methacrylamide-lactate]this study The aim of wasglycol)-b-poly[N-(2-hydroxypropyl) to develop poly(ethylene (mPEG-b- p(HPMAm-Lac(n)) thermosensitive core-crosslinked biodegradable polymeric targeting, active for tumor suitable micelles by end. PEG this was functionalized growth (epidermal anti-EGFR factor the receptor) EGa1coupling nanobody surface. their To to with 3-(2-pyridyldithio)-propionate N-succinimidyl yield Micelles a PDP-PEG-b-p(HPMAm-Lac(n)) to (SPDP) block copolymer. composed of 80% mPEG-b-p(HPMAm-Lac(n)) and 20% PDP-PEG-b-p(HPMAm-Lac(n)) prepared were and lysozyme (as a model wasprotein) modifiedhydrochloride and subsequently deprotectedN-succinimidyl-S-acetylthioacetate, hydroxylamine with with and gel characterized permeationchromatography were conjugates surface.SDS-PAGE using The micellar micellar coupled the to (GPC). knowledge the Using obtained with EGa1 lysozyme the nanobody conjugation, was micelles coupledmPEG/PDP-PEG to was conjugation successful and the as by western demonstrated blot and dot analysis. Rhodamine labeled EGa1-micelles untar than (A431 and UM-SCC-14C) showed cancer cells substantially higher binding as as well uptake by EGFR over-expressing geted nanobody no binding of the rhodarnine was micelles labeled observed micelles. Interestingly, (3T3) EGFR negative to cells the nanobodythe binding of that is micelles This demonstrates of free nanobody. of an excess presence as in the as cells well to14C EGa1 In conclusion, decorated (mPEG/PDP-PEG)-b-(pHPMAm-Lac(n)) polymericindeed EGF with receptor. the by interaction highly systems promising active are for drugmicelles targeting. The crucialrole of T lymphocytes has in anti-tumor immunity strategies ledcan that the developmentand acti of novel target to - cells. against tumor T cells DNA technology Recombinant vate has been used non-MHC-restricted generate to chimeric antigen receptors (CARs). we constructed CAR Here, of recombinant a panel harbors anti-MUC1 nanobody the that signaling and the and co-signaling moieties (CD3 zeta/CD28)regions spacer derived with different repeatshuman IgG3 one or two with from the of gamma RII.PhiC31 integrasehinge sequence The hinge region or the of Fc recombina system the - if wasto investigate employed tion efficiency could chimeric be cell genes.recruited receptor expression of T for high and The nuclearstable of effect localization (CMVsignal and promoters (NLS) on efficacyintegraseCAG) and two different cell lines PhiC31 T of human in was The evaluated. of integrasepresence with NLS, in combination mediated increases 7.6 and 8.5 fold up to in CAR in ZCHN-attB and expression ZCHHN-attB cassette Our Jurkat integrated T cells, results highlytransduction and respectively. efficient showedthe stable of that integrase line by PhiC31 is usecell a feasible for in cancer immunotherapy. chimeric T cells modality anti-cancer generating for

particle conjugates conjugate receptor receptor Nanobody-gold nano - HER2 Photothermal therapy Epidermal growth factor Lysozyme Drug targeting Nanobody-micelle Anti-MUC1 nanobody Cancer immunotherapy Chimeric antigen

490 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article - Baral et al. 2006 Oliveira Oliveira et al. 2010 Cortez- Reta et mozo al. 2004 studies demonstrated that the conjugate had an excellent biodistribution profile had an excellent conjugate the that studies demonstrated effectsthis of on system EGFR internalization and downregulationNano - investigated. were We identified a nanobody tumor-associated human carcinoembryonic the for subnanomolar affinity with antigen.We This nanobody cloacae beta-lactamase,was Enterobacter to conjugated and its site-selective prodrug anticancer activation capacity was evalu- wasreadilyconjugate purified The ated. in high yields aggregation without or loss of constituents. functionalitythe of In vitro nanobody-enzymeexperiments the showed that effectively activatedreleasethe conjugate mustard of phenylenediaminethe from cephalosporin prodrug mustard nitrogen 7-(4-carboxybutanamido) cephalosporin surface the of carcinoembryonic at mustard LS174T cancer cells.antigen-expressing In vivo High systemic and increasing drug toxicity prevalence human of drug of Africantreatment resistance hampers efficient trypanoso - development of new Hence, highly miasis specific (HAT). trypanocidal drugshuman Normalis necessary. serum (NHS)contains apolipoprotein lyses L-I (apoL-I), which African trypanosomes resistantas forms such Trypanosoma brucei rhodesiense. except T. b. rhodesiense apoL-I neutralizing the expresses serum resistance-associated (SRA) endowing parasite this protein, ability with the has been engineered A truncated apoL-I (Tr-apoL-I) its by deleting SRA-interacting domain, infect and causeto humans HAT. with a single-domain antibody (nanobody) efficiently that Tr-apoL-I b. rhodesiense. we conjugated Here, makes it lyticwhich T. for targets conserved cryptic epitopes variant of the (VSG) surface glycoprotein of trypanosomes a new generate manmade type to of with engineered this resulted in clear and curative conjugate with trypanosomiasis potential for Treatment immunotoxin therapy. alleviating effects infectionschronic on acute and of mice with NHS-resistantboth and NHS-sensitive trypanosomes. and induced regressions and cures of established xenografts. tumor The easy and manufacturing generation of nanobody-yield activitybased nanobodies antitumor make potent with together their new for conjugates cancer vehicles promising generation therapeutics. The epidermal growth receptor factor (EGFR)recognized is a and monoclonal antibodiestherapy tumor for target (mAbs, e.g. been have cetuximab) developed inhibit receptor activation. to Besides reports blocking ligand (e.g. EGF) receptor, the binding to receptor internalization slow and degradation promote mAbs in lysosomes, shown that have i.e. downregulation. The efficacy of receptor downregulation was depend shown surface. to study, In this size recently formed cell on the of receptor clusters the at is platform presented, of nanobodiesa multivalent consisting recognizing ectodomain the of EGFR (EGa1) coupledPEG- to liposomes, in vitro and in vivo and the bodies smallest the functional are immunoglobulin fragments antigen-binding EGa1 and the nanobody has been described as an EGFR-antagonist. EGa1-liposomes surface, (EGa1-L) as 90% removal of EGFR inducedthan cell from a result a more the of recep - massive this sequestration internalization.tor Furthermore, of EGFR mediated by EGa1-L lead receptor degradation, to while no degradation was detected The downregulatory monovalent nanobody. with the reported capacity here wasto found be independ - epitope of the on EGFRent recognized nanobody-liposomes, the to grafted by the and exclusive nanobody, as single anti-EGFR variablechain fragments (scFv)EGa1-L coupledliposomes effect. to this induced a significant induce to unable were Importantly, receptor in vitro, of downregulation an effect mediatedcombination recep likely the and proliferation, by - cell inhibition of tumor antagonism. Alsotor in vivo, EGFR downregulation was observed injected intravenously with EGa1-L, of mice in tumors indicating blocks platform multivalent this receptor and simultaneously downregulation ligandthe that the induces binding to of EGFR.

conjugate antigen conjugate L-I therapy downregulation conjugate receptor Nanobody-β-lactamase Carcinoembryonic Cancer therapy Nanobody-apolipoprotein Trypanosomiasis Immunotoxin Nanobody-liposome Epidermal growth factor Receptor

491 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 Inoue et Inoue et al. 2011 Stone et et Stone al. 2007 Szynol et al. 2004

VHH is the binding domain of the IgG heavy chain. Some VHHs have an extremely long CDR3 that contributes to antigen binding. antigen to VHH long CDR3 contributes IgG heavy binding domain of the is the Some that chain. VHHsan extremely have of CDR3 binding ability studied antigen by grafting the a CDR3 a nonbinding VHH. VHH from an antigen-binding onto We cAb-CA05-(1RI8), the CDR3-grafted VHH, had an antigen-binding ability. To find a human scaffold protein acceptable for acceptable VHH human protein scaffold find a cAb-CA05-(1RI8), To CDR3-grafted the VHH, ability. had an antigen-binding CDR3 grafting, we focused conserved on the structure of VHH, especially N-terminal the and C-terminal amino acid residues of CDR3the origin residue of CDR1. loop structuressearched Cys were same orientation and the protein with Human the in PDB and ubiquitin was selected. affinity Ubi-(1RI8), the though relatively was CDR3-grafted the ability, ubiquitin, had antigen-binding cAb-CA05-(1RI8). to low compared thermodynamic The parameters of from Ubi-(1RI8)to HEWL binding different were experiments showedcAb-CA05-(1RI8). decreased exchange Hydrogen-deuterium CDR3 the around stability grafting region of Ubi-(1RI8), decreased the thermodynamiccon - explain in might We which differences and the properties. ability antigen-binding CDR3 of the orientation the sequencecluded of Ubi-(1RI8) that not be could reconstructed correctly. Bispecific antibodies unique opportunities present terms in of newfor engineered applications designing antibodies.However, ideal bispecific antibodies we describeHere challenge. remains a bispecific a novel antibody five single in which domain model antibodies fused (sdAbs) are B (VTB) via verotoxin sequenceN-terminus of the a linker the to subunit, a pentamerization domain, and five are sdAbs fused via the a linker sequenceto C-terminus.VTB Fifteen decavalent such bispecific molecules, termed decabodies, constructed were and characterized purpose the for of identifying an optimal decabody design. fifteen One of the molecules existed in a non-aggregated isolation decavalent with the of sdAbs desired form. with In conjunction the specificities libraries, phage display from non-immune decabody the strategy provides a meanshigh bispecific avidity of generating antibody reagents, with good physical properties, quickly. relatively Enzymes as such lactoperoxidase and glucose oxidase (GOx) used are as agents in oral care antimicrobial products. Their low specificities and substantiveness can reduced moleculesVariablecoupling of antimicrobial be domains covalent by to antibodies. derived(V-HH) from llama heavy-chain antibodies particularly are The antibodiescomposed an approach. suited such are for of heavy-chainsolely production dimers; of active fusion therefore, by using molecular proteins biology-based techniques is less and GOx was a fusion of V-HH consisting antibodies. production protein than by usecomplicated of conventional study, In this cerevisiae.constructed and expressed A llama by Saccharomyces was immunized with Streptococcus strain HG982. mutans Subse- B lymphocytes isolated fragments were and cDNA fragmentsquently, obtained were V-HH encoding by reverse the transcription- PCR. library After construction of a V-HH in Escherichia and screening coli library of the against group mutans streptococci and Streptococcus sanguinis fragments strains, with high specificities two V-HH we found for mutans strains.S. A GOx gene was genes S. and cloned cerevisiae into two V-HH linked the yeasts. to The yeasts expressed and secreted into proteins recombinant the V-HH growth the fragments medium. showedtestmutans The that of binding to oral S. bacteriatheir fusionthrough proteins had been specificallyconstructs.the one of the fusiontargeted protein of by fusionwas protein GOx-S120, concentration A low selectivelyalso to kill able S. of lactoperoxidase presence within mutans 20 min in the and potassium iodide. These findings demon - is potentially valuable selective in the killing fusion of target the GOx-V-HH bacteria protein as that such S.strate mutans.

oxidase conjugate Bispecific antibody Decabody Linker sequence B subunit Verotoxin Nanobody-glucose Streptococcus mutans Antimicrobial agents of single-domain 6H. Application antibody fragmentsTable in therapy: humanised single-domain antibody fragments VHH CDR3 scaffold proteinHuman Grafting Ubiquitin

492 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article - - Vincke Vincke et al. 2009 Vaney cken et al. 2010 Ben Abder et razek al. 2011 - - imaging of tumors. experiments revealed specificityNanobodies,for all clearance renal tumor and rapid targeting of low with Nanobodies, fragments single-domain antigen-binding of camelid-specific heavy-chain only antibodies offer special advantages over classic antibodyin therapy fragments because smaller size, of their target robustness,unique epitopes. to and preference A Nanobodyhuman heavy a differs chain from aboutvariable domain in ten amino acids spread all over its surface,four hallmark Nanobody-specificregion amino acidsthe loop in (positionsframework-2 antigen-binding third 42, 49, 50, and 52), a longer camelid-specific the therapeutic applications area. over this (H3) folding amino acid For sequencesthe to in framework be have heavy performed human variable their chain to mutated domain equivalent, with humanization this exercise i.e. humanized. We Nanobodies represents subfamily close 80% of all dromedary-derived of the that to Nanobodies effects and investigated the on humanization the Nanobody-specific of that It is demonstrated residues expression and stability. yield, solubility, affinity, antigen Nanobodyoutside neutralthe are to framework-2 properties. -> Leu Glu-49 -> Gly and Arg-50 humanization the of Surprisingly, less substitu - probably soluble. though stable is The other more framework-2 amino acids a singlehallmark domain that generates to a repositioning due the H3 of loopby shown as affinity, detrimental antigen are for and Gly/Ala-52 -> Trp, tions, -> Val Phe-42 crystaltheir structures. These insights used were to identify expressedhumanized a universal well soluble, stable, Nanobody scaf allows grafts that fold loops of antigen-binding Nanobodies from other specificity antigen of the with transfer and affinity. Nanobodies type with a novel beneficial are protein antigen-binding of immunoglobulinlike, pharmacologic and pharmacokinetic molecular for properties ideally antigens are suited targeting cellular imaging to that or therapeutic purposes. because of However, origin, camelid, nonhuman possibletheir the immunogenicity of Nanobodies we present when used Here is clinic a concern. in the a new humanized strategy quickly generate Nanobodies to molecular for imaging purposes. genetically grafted Methods: the We loopsantigen-binding of NbCEA5, a Nanobody with specificity colon carcinoembryonic marker the carcinoma for (CEA), antigen framework the onto of a humanized Nanobody scaffold. This scaffold has been previously characterized in our laboratory a as Nanobodystable can serve that loops as antigen-binding for a universal loop from donor Nanobodies acceptor and has been addi - about 10 crucial at tionally mutated surface-exposed resemble sequence variable sites to the of human immunoglobulin domains. Nanobodies recombinant The 3 (NbCEA5,humanized humanized scaffold, and CEA5 graft)produced were in bacteria and puri - Nanobodies biochemically were characterized tested in and vitro Unlabeledfied.as probesSPECT/CTfor and (99m)Tc-labeled of xenografted tumors. Results: of loop-grafting The success wasconfirmed these comparing Nanobodies by to capacity their for recognize soluble CEA in enzyme-linked protein immunosorbent and by surface assay plasmon CEA- bind to and to resonance positive LS174T and CEA-transfected carcinoma cells colon but not untransfected Chinese ovary hamster in flow cells cytometry. Specificity of binding wasconfirmed competitionstudies. by could Nanobodieslabeled All be efficientlywith were heat-stable, and recognized both solublebiodistribution and membrane-bound CEA experiments (99m)Tc, in binding studies. protein Finally, Nanobodies in LS174T using pinhole SPECT/ mice tumor-bearing performedwere injected with intravenously (99m)Tc-labeled These in vivo micro-CT. signals besides in all organs kidneys. the the shows Conclusion: This study potency loop-grafting of antigen-binding to efficiently humanized generate Nanobodies retaintargeting capacities their noninvasive that for in vivo pion toxin. Thus, humanizedcompound this venom pion toxin. the human againstmost Nb toxic is in to developbest therapy a candidate mostof one the dangerous scorpions. Recently, a potent AahII a potent Cys scorpion in this a single NbAahII10 toxin-neutralizingcontains nanobody wasRecently, However, identified. its loop, first antigen-binding leadingto Nb dimerization uponthis prolonged work, In storage. the efficacyinvestigate we first of NbAahII10 variants was Cys/Ser Cys this Second,the NbAahII10 displayingthe mutant in which substituted by Ala, Ser or Thr. best functional properties maximally the is subsequently humanized is that demonstrated humanized. version of NbAahII10 It its importantly, More expression and stability. yield on much conceding antigenCys/Ser its maintains the without for high affinity neutralizing capacity is preserved as survive all mice injections of seven survived LD(50) and 50 of mice nine LD(50) scor of the imaging nanobody scaffold antigen Universal humanized AahII scorpion toxin Humanized nanobody Humanized nanobodies Loop-grafting In vivo Scaffold Carcinoembryonic

493 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 - Broisat et et Broisat al. 2012 Gainkam et al. 2011 Sukh anova et al. 2012 - - - < 0.05 < 0.05 competition competition in apolipoprotein E-deficient (ApoE(–/–)) in apolipoprotein (ApoE(–/–)) E-deficient bioluminescence imagingbioluminescence was performed followed by pinhole single-photon- tomog emission computed We aimed to generate, radiolabel, and evaluate aimed generate, to anti-VCAM1 nanobodies noninvasive for detection lesions.We of atherosclerotic anti-VCAM1 nanobodies radiolabeled generated, were Methods with and Results: technetium-99m, and screened Ten in vitro VCAM1on mouse recombinant and human and in vivo cells and endothelial proteins mice. Anontargeting control nanobody control was Anontargeting mice. All nanobodies used specificity. in all experiments demonstrate displayed to nanomolar murine affinities for VCAM1. Flow cytometry cells murine indicated analyses and human umbilical vein endothelial human using VCAM1human cross-reactivity 6 of 10 nanobodies. for was cross-reactive The leadcompound cAbVCAM1-5 human VCAM1 for (4.95 ± 0.85), lesion-to-heartand exhibited high lesion-to-control (8.30 ± 1.11), and lesion-to-blood (4.32 ± 0.48) ( P ratios and immunohistochemistryexperiments. uptake in VCAM1-positive Autoradiography further confirmed cAbVCAM1-5 lesions. Tc-99m-labeled, anti-VCAM1 allowed nanobody noninvasive detection cAbVCAM1-5 of VCAM1 Conclusions:expression The the of nanobodies potential demonstrates this study and displayedTherefore, as a new mouse cross-reactivity. and human class of cardiovascular for radiotracers applications. versus control C57Bl/6J mice). Aortic arch atherosclerotic lesions of ApoE(–/–) mice were successfully identified were by single-photon mice lesions of ApoE(–/–) atherosclerotic Aortic arch C57Bl/6J mice). versus control binding specificity was by in demonstrated vivo imaging. tomography emission computed Tc-99m-cAbVCAM1-5 between studiedepidermal relationship uptake of the the tumor growth factor receptor (EGFR)-specificWe nanobody (99m) and evaluated burden and tumor possibility the response tumor of usingThe specific probe this monitor erlotinib. to to Tc-7C12 resulted in a progressive uptake treatment of (99m) reduction Erlotinib and tumor burden. burden with tumor of relation burden. tumor - Tumor with Quantifi uptake is correlated EGFR binds with to high affinity and specificity. (99m)Tc-7C12 Tc-7C12. tumors. response monitoring uptake istherapy for promising of EGFR-expressing of (99m)Tc-7C12 cation Common strategy diagnostics for dots with quantum (QDs) utilizes specificity the of monoclonal antibodiesfor targeting. (mAbs) not always well-suited are purpose this However for conjugates QD-mAbs because we engineered size. large Here, of their ultr asmall of QDs with 13-kDa nanoprobes single-domain oriented antibodies through conjugation (sdAbs) derived from llama IgG. capacity refolding. for tagged sdAbs were with QDs excellent Monomeric 12 timesand demonstrate sdAbsmAbs are smaller than an additional cysteinethrough allowedapproach This sdAb. to develop residueC terminal integrated withinof the us the sdAbs- specific sdAbs-QD conjugates to QD nanoprobes of sdAbs copies coupled four comprising with a QD in highly oriented manner. carcinoembryonic specificity (CEA) antigen of flow excellent demonstrated cytometry discrimination quantitative of CEA-positive immunohistochemical the labeling of biopsy was samples or and CEA-negative be to found comparable cells. tumor Moreover, even superior quality obtained the to with gold standard sdAbs-QD-oriented of anatomopathology practice. protocols conjugates as developed a new represent of ultrasmall generation diagnostic in high-throughput probes diagnostic applications for platforms. ity and affinity of (99m)Tc-7C12 was determinedcells.firefly expressing luciferase on A431 were Cells used tumor to evaluate ity and affinity of (99m)Tc-7C12 uptake in vitro evaluated and (99m)Tc-7C12 effecttumor burden using the imaging. on bioluminescence burden of erlotinib We as as well in vivo. In vivo binds specifically receptor the Erlotinib highto affinity (3.67 ± 0.59 nM). with (99m)Tc-7C12 tomography. raphy/micro-computed showed good cor uptake of (99m)Tc-7C12 Tumor manner. viabilityreduced in a concentration-dependent uptake and cell tumor

receptor antigen computed tomography imaging Tumour uptake Tumour burden Tumour Epidermal growth factor Bioluminescence Nanoprobe Quantum dots Flow cytometry Carcinoembryonic Biopsy samples Table 7. Application of single-domain 7. Application antibody fragmentsTable in diagnostic and immunoanalytic methods Noninvasive detection Technetium-99m plaquesAtherosclerotic Single-photon emission

494 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article

Schmidt- hals et al. 2010 Caussi - etnus al. 2012 Lafaye et al. 2009 - The understanding of cellular processes and their pathophysiological alterations requires comprehensive data on the abundance, abundance, the on data processesrequirescellular The understanding comprehensive of their and pathophysiological alterations components.distribution, cellular modification, of all the one hand, artificially and interaction On introduced fluorescent fusion aboutdistribution provide their information and dynamicsproteins in livingbut not about endogenous cells factors. other On the hand, antibodies can detect endogenous proteins, posttranslational modifications, components but mostly in cellular and other fixed and permeabilizedcells. we highlight a newHere technology based domain of heavy-chainthe antigen-binding on antibod - ies (VHH) stable fromVHH Camelidae. domains Thesecan extremely be produced in bacteria,coupled to matrices, and used for these VHH domains canaffinity purification proteome studies. and expressed be Alternatively, proteins and fused with fluorescent “chromobodies” canin living be cells.proteins called usedother antigen-binding to and These detectproteins fluorescent and trace in vivo. components Chromobodiescellular can, in principle, structure,posttranslational detect modifi including antigenic - any analysis. can be that in high-content qualitygathered of information and quantity the cations, expand dramatically and thereby Depending epitope on the chosen, chromobodies can also be used function modulate protein to in living cells. The use of genetic mutations to study protein protein functions to study in The paradigmuse central advancesmutations vivo Recent reverse of geneticis a of modern in biology. genetics and morpholinos as such widely used RNA are interference further to paradigm. this apply Nevertheless, systems such act level,depletion targetupstream proteins.we present existing proteic and protein depends of the of the Here turnover rate on the a genetically encoded method direct for and fast depletion (GFP) of target green protein fusions in any fluorescent deGradFP, eukaryotic genetic system. This method is universal becauserelies on an it evolutionarilyconserved highly eukaryotic function,the because is traceable, It GFP tag the can be knockout. used protein cases, the monitor In many to ubiquitin pathway. it is a ready-to- use solution, as stock collections GFP protein-trap being are in Drosophila generated melanogaster and in Danio rerio. Neurotoxic oligomers of amyloid beta oligomers of amyloid (A beta)Neurotoxic peptide been disease. have incriminated pathogenesis in the of Alzheimer’s Further issue of this has beenexploration all previously fact by the hampered that date this described to beta anti-A antibodies unable are (oligomers,the discriminateof to conformations peptide between protofibrils and fibrils).Here we describe different the the gen - camelid single-chain binding domains (VHHs) of novel eration recognizes that specifically low molecular-weight (MW) oligomers. to Three able recognize VHH Two were specificselec for A beta obtained were from an immunized alpaca library. phage display tively intraneuronal A beta intraneuronal tively oligomers: furthermore, V31-1, prevented one of them, A beta-induced and inhibited neurotoxicity the that mayrecognize epitopes for confirms their VHHs study potential fibril Thisand illustrates formation. non-conventional immunodiagnostic of diseases accumulation. protein due to tein fications deGradFP Green fluorescent pro - Protein knockout Alzheimer’s diseaseAlzheimer’s conformation Peptide betaAmyloid Neurotoxicity Chromobody Posttranslational modi -

495 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 - - Vaney cken et al. 2010 Vaney cken et al. 2011b tumor. – imaging of tumors. tumor models, tumor fast bloodorgans in nontarget low accumulation clearance, + experiments revealed specificityNanobodies,for all clearance renal tumor and rapid targeting of low with Nanobodies type with a novel beneficial are protein antigen-binding of immunoglobulinlike, pharmacologic and pharmacokinetic molecular for properties ideally antigens are suited targeting cellular imaging to that or therapeutic purposes. because of However, origin, camelid, nonhuman possibletheir the immunogenicity of Nanobodies we present when used Here is clinic a concern. in the a new humanized strategy quickly generate Nanobodies to molecular for imaging purposes. genetically grafted Methods: the We loopsantigen-binding of NbCEA5, a Nanobody with specificity colon carcinoembryonic marker the carcinoma for (CEA), antigen framework the onto of a humanized Nanobody scaffold. This scaffold has been previously characterized in our laboratory a as Nanobodystable can serve that loops as antigen-binding for a universal loop from donor Nanobodies acceptor and has been addi - about 10 crucial at tionally mutated surface-exposed resemble sequence variable sites to the of human immunoglobulin domains. Nanobodies recombinant The 3 (NbCEA5,humanized humanized scaffold, and CEA5 graft)produced were in bacteria and puri - Nanobodies biochemically were characterized tested in and vitro Unlabeledfied.as probesSPECT/CTfor and (99m)Tc-labeled of xenografted tumors. Results: of loop-grafting The success wasconfirmed these comparing Nanobodies by to capacity their for recognize soluble CEA in enzyme-linked protein immunosorbent and by surface assay plasmon CEA- bind to and to resonance positive LS174T and CEA-transfected carcinoma cells colon but not untransfected Chinese ovary hamster in flow cells cytometry. Specificity of binding wasconfirmed competitionstudies. by could Nanobodieslabeled All be efficientlywith were heat-stable, and recognized both solublebiodistribution and membrane-bound CEA experiments (99m)Tc, in binding studies. protein Finally, Nanobodies in LS174T using pinhole SPECT/ mice tumor-bearing performedwere injected with intravenously (99m)Tc-labeled These in vivo micro-CT. signals besides in all organs kidneys. the the shows Conclusion: This study potency loop-grafting of antigen-binding to efficiently humanized generate Nanobodies retaintargeting capacities their noninvasive that for in vivo Accurate determination of tumor human epidermal human growth of tumor determination factor receptor 2 (HER2)-statusAccurate in breast cancer patients is possible via noninvasive imaging, we describe of 38 nanobodies, of a panel provided generation used. the are adequate tracers study, In this small HER2-binding fragments derived are that from heavy-chain-only antibodies raised in an immunized In search dromedary. of a lead a subset compound, of nanobodies was biochemically characterized in depth and preclinically tested use for as tracers imagingfor of xenografted tumors. The selectedcompound, 2Rs15d, specificallywasto found to interact and be stable with HER2 in ELISA, cells and HER2-expressing protein recombinant surface plasmon flow resonance, and radioligandcytometry, binding studies with low nanomolar affinities,anti-HER2 compete therapeuticantibodies and did not with trastuzumab and pertuzumab. Single-photon-emission (SPECT) tomography computed imaging and biodistribution quantification analyses (99m) showed that 2Rs15d has uptake in 2 HER2 a high tumor Tc-labeled except kidneys, and high concomitant tumor-to-blood and tumor-to-muscle ratios at 1 h after intravenous injection. kidneys, 1 h after intravenous These at ratios except values tumor-to-blood and tumor-to-muscle and high concomitant targeting a HER2 (99m)Tc-2Rs15d and for (99m)Tc-nanobody an irrelevant lower for dramatically were control imaging antigen HER-2 Breast cancer Noninvasive imaging Humanized nanobodies Loop-grafting In vivo Scaffold Carcinoembryonic

496 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article Kim et al. Kim et al. 2012 Swain et et Swain al. 2011 Conway et al. 2010 - - affinity maturation and typically have low affinity toward their targets. We expressed We five targets. and their have low affinity toward typicallymaturation affinity We expressed VHHs from an immunized alpaca and developedWe a VHH-based using immunoassay 3-phenoxybenzoic acid (3-PBA), metabolitea major insecticides of pyrethroid as a model system. A phage VHH library was constructed, and seven VHH clones selectedwere by competitive binding with 3-PBA. The best developed immunoassay these one of with VHHsshowed an IC50 of 1.4 ng/ml (limit of detection (LOD) = 0.1 ng/ml). These parameters were furtherthe phage by improved using borne VHH, IC50 50% in assay up to = 0.1 ng/ml and LOD 0.01 ng/ml. Both methanol and dimethylsulfoxide assays showedto a similar tolerance The assay 3-PBA,was highly specific4-hydroxy derivative, to 3-PBA and4-hydroxylated (150% cross reactivity) its buffer. with neg - ligible cross reactivity tested with other structural analogues, recovery and the from spiked ranged urine sample 112%. from 80 to a highly specificIn conclusion, and sensitive for 3-PBAVHH was developed using sequences immunizedfrom alpaca and phage technologydisplay antibody for selection. Naive librariesNaive of single domain antibodies target. isolation nearly any of binders to rapid (sdAbs) These enable binders, however, benefitslack the bestowed by in vivo low-affinity toxin binding sdAbs,low-affinity previously selected from a naive library derivedfrom variable regions of llama heavy chain-only antibodies, as fusions with a hyperactive Escherichia alkaline mutant phosphatase coli (AP) and examinedimpact on apparent the AP spontaneously dimerizesaffinity and utility. ineffectively solution, dimerizing the fusedsdAbs, the imparting of avidity in place interactions.lower affinity monomeric sdAb-AP The fusiontarget combines the recognition also domainwith a signal transduction useddomain, commonly in enzyme-linked immunosorbent assays (ELISAs). The sdAb-APfunctional the affinity of fusions, often increased by a factor of 10 over unfused sdAbs, utility as reagents and their in ELISAs tracer was giving improved, dramatically limits of detection tested. of 300 ng/ml or less, The concentrations whereas no discernible sdAbs parental gave toxin signal the at of sdAb-basedfusion AP of sdAbs implementation presentsfacilitate to selected the to a valuablerapidly immunoreagents route from naive libraries existing new toward or emerging threats. Background: There are currently 7 Background:known are currently There serotypes (BoNT) neurotoxin of botulinum classified upon non-cross reactivityof neutralizing immunoglobulins. cancross-reactivities Non-neutralizing exhibit immunoglobulins, between however, 2 or more serotypes, particularly mosaic forms, can which development hamper of highly the specific immunoassays, especially if based on polyclonal antisera. facile antibody we employ recombinant Here technology select subtractively to ligands 7 BoNT each of the to serotypes, resulting in populations with very high specificity their intended serotype.for Findings:Methods and A single llama was immunized with a cocktail library a phage display of 7 BoNT generate to of single domain antibodies toxoids VHH (sdAb, or nanobodies) with the selected were complex which of detecting on live toxins. capable Resulting and toxin were sdAb both toxin detectcombined to The mostbest able were in 100s-10ssensitive sdAb combinations of pg per in a liquid bead 50 µl sample array. identify to assay a heptaplex BoNT each of the and serotypes in orange juice carrot juice, to a lesserextent and milk in buffer cola. Several recognized sdAb anti-A(1) subtype showing A2 complex, that cross-reactivity within a serotype was evident. Many suggesting can sdAb be used complexes act as several as and toxin could of our sdAb for architec bothand tracer captor toxin probes BoNTtural indicate to neuro-2A cleavage in the oligomerisation. Six clones exhibited inhibition of SNAP-25 of 14 anti-A neutralizing some indicating assay had toxin sdAb capabilities. also were sdAb high after be shown to to exposure Many refoldable polyclonal to antisera, in contrast temperatures as monitored by circular Conclusions: dichroism. Our of molecularly panel flexible antibodies not only serve should as a good starting ruggedizing point for assays architec and inhibitors, intricate the but enable be to of BoNT probedtures extensively. more and complexes toxins

capability phosphatase fusion VHH-based immunoassay 3-phenoxybenzoic acid Pyrethroid insecticides Botulinum neurotoxin neutralizing Toxin High temperatures ELISA Toxin SdAb-alkaline

497 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 De Marni et al. 2012 Tabares- da Rosa et al. 2011 Goldman et al. 2010 - - The preparation of effective conventional antibody of effective microarraysThe preparation conventional depends of highmaterialavailability the the and on quality on spotting that show antibodycorrect of the accessibility active moieties immobilization following their support on the We slide. or epoxy slides (puri - bacteria expose antibodies surface recombinant directly that external on nanostructured-TiO(2) on their for preparing alternative sensitive and specificreliable fication-independent and microarray – PIM) is a simple for microarrays domains of single heavy-chain antibodies detection. (VHHs)antigen Variable against fibroblast growth receptor factor 1 (FGFR1) diluted in serum antigen used the were or BSA capture solution. to The FGFR1 detection was performed direct by either antigen labeling or using a sandwich system FGFR1 was in which first to itsbound antibody identifiedlabeled using a and successively FGF. In both cases signal the distribution within each spot was The signal-to-noise uniform and spotthe morphology of ratio regular. signal elevated was specificity and the antigen extremely the the for The LOD of was the system of system was proved statistically. calculated being dynamic 0.4 ng/ml and the range between 0.4 ng/ml and 10 μg/ml. The microarrays prepared with bacteriaexpos ing antibodies remain fully functional for at least 31 clays after spotting. We finally demonstrated that the method is suitable for the methoding antibodiesfor that is suitable finally demonstrated fully remain functional least at after for spotting. 31 clays We antigen-antibodyother pairsbe and expect it could easily that further adapted as to of scFv such display and IgG the applications antibodies autoantibody or the detection using protein. Using triclocarban antibodies,Using (TCC) conventional that as IgG1 fraction, we found a model reacted hapten, with with free a TCC the (IgG2 and IgG3, 497 370 ng/ml, affinity than did sdAbs respectively).(IC(50) 51.0 ng/ml) higher relative A VHH library and competitive selection of TCC was of binders, to able prepared, we were of phage with limiting concentrations and by elution isolate high-affinity clones, TCC an allowed with IC(50) (SPR), which competitive K(D) 0.98–1.37 nM for of a assay development the regardto the performanceserum of sdAb with represents= 3.5 ng/ml (11 nM). This improvement fraction, a 100-fold and it Despite modest the overall antihapten VHHs reported IC(50) betterthe attainedis antihapten with 100-fold than other far. thus sdAbs response in llamas, a small subpopulation of high-affinity can that VHHs is generated be isolated by careful design the of selection process. Previously, we selected specific were that sdAb Previously, for both ricin A (BoNT and librar phage display botulinum from complex toxin A) ies and evaluated of sdAb phage-displayed used were sdAb Here, solubly the expressed protein. as reporter reagents and compared using phage-displayed as that sdAb found reporter improved formats in immunoassay gave elements soluble, to unfused We sdAb. detection over using unfused, soluble reporters. sdAb In enzyme-linked immunosorbent assays (ELISAs), lowest the level of toxin detected both for ricin was and BoNT decreased complex A toxoid of magnitude using two orders phage-displayed by one to sdAb as reporter reagents. phage preserved of the Use discriminate to ricin ability the and RCA120 least by at a factor In an of 10 fold. numberreducethe of stepsthe effortto in assays, we directly labeled dye. Signal Cy-3 phage displaying fluorescent a sdAb with was decreased greatly dye-labeled usingphage followed the biotinylated by streptavidin-phycoerythrin. to phage compared In these use assays the of phage-displayed gives sdAb sensitive more detection soluble alone, however than sdAb directly dye labeling phage failedthe provide responses to of a similar magnitude. receptor 1 Competitive assay Triclocarban ELISA Ricin Botulinum A toxin fluorescentCy-3 dye Antibody microarrays Surface expression Fibroblast growth factor

498 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article - - Dona et al. 2010 Deck ers et al. 2009 Good et child al. 2011 - Food intended for celiac patients’ consumption must be must using analyzedprolamins high patients’ consumption delectability celiac tests. of toxic presence for intended the Food for Thoughcommonly the most 60% used 2-mercaptoethanol (2-ME) and is extraction, ethanol for prolamins guanidiniumchlo - solvent recognition with antigen agents interfere ethanol and denaturing ride (GuHCl) can be added However, increase to recovery. protein antibodies used. are when conventional work, present In the a new method gliadins shown. for quantification is The method is based selectionon the of llama single domain antibody fragmentsconditions. under denaturing operate to Six able out of 28 VHH-phages obtained retained binding capacity their in 15% ethanol. Selected clones presented a long CDR3 regiontwo additional containing clones (named One of the VHH26) cysteines was be could responsiblefully higher stability. the that of presence operative for in the 15% ethanol, 0.5% 2-ME, ELISA and 0.5 M GuHCl. Capture using VHH26 was detect to able gliadins shown in samples as negatives ELISA. or any by conventional of proteins determination thisfor quantitative new platform Therefore, strategyappears excellent as an agents, of denaturing presence in the compound, immunogenic when specificother recognition unitsrequired. high are stability with mission of infection. Specificto diagnose assays living cysts in pigs lacking. are The monodonal-basedantigen detection ELISAis - aimedto intro therefore, We, the hampertestusethisto hydatigena of screenTaenia genus-specific pigs. and cross-reactions with nanobodies,duce T. solium camelid-derivedcysticercosis, single-domain antibodiesto develop unambiguous specifictests. for solium-specific nano - T. and eight solium antigen Nanobodies cloned were following immunization of two dromedaries with T. bodiescharacteristics Their binding the diagnosisfor and selected were potential of after porcine phage display. were cysticercosis Trichinella spiralis and Taenia crassiceps or Taenia saginata,investigated. The nanobodies do not cross-reacthydatigena, T with categorizedwere epitope-binding four groups. into (Ts14), but The was protein target identified as 14 kDa diagnostic glycoprotein Nanobodies nanobodiesthe tested were in a sandwich alsoELISA same family. reacted of the with proteins cyst with other fluid, and one particular nanobody detected its serum cognate in a species-specific antigens inhibition ELISA. Consideringtheir benefi - cial production properties, and stability these highly specific nanobodiestool a promising to diagnose constitute after cysticercosis sensitivity of the further validation. and future assay improvement Taenia solium cysticercosis zoonosis is helminth a major in developing countries. hosts intermediate Pigs the are mediating Taenia trans Members of the genus EbolavirusMembers genus of the cause fulminating outbreaks of disease populations primate with a and non-human in human detection facilitate rapid samples, of these in clinical and environmental pathogens reagents robust 90%. To mortality up to rate of providingcapable sensitive and specific detectionrequired. antibodyrecombinant are this work In libraries generated were from (singlevariablemurine chain domain fragment; scFv) shark, and nurse Ginglymostoma (IgNAR cirratum V) hosts immunised with Zaire ebolavirus. This providesthe first recorded IgNAR V response nurse shark. the against aantigen in particulate Both IgNAR scFvV librariesmurine and shark panned were technology by phage display identify to useful antibodies generation the for specificity have scFvshown murine to were the of immunologicalto ebolavirus detectionZaire reagents.viral matrix protein Two viable Zaire capture (NP) isolated viral and to the ebolavirus nucleoprotein bind to shown IgNAR to V were with a high VP40. Two Assays developeddegree of sensitivity. with IgNAR V cross-reacted Reston ebolavirus, to Sudan ebolavirus and Bundibugyo ebola - virus. of IgNAR V showed neither reactivity ebolavirus. divoire DespiteCote to broad reactivity, this IgNAR V was substantially test. scFv murine and monoclonal than irreversible resistant IgG in a comparative The demonstrable to more thermal denaturation enhanced offer utility robustness IgNARas immunological V domains may of the detectionreagents biosensor in fieldable applica - usetions for outbreaks in tropical or subtropicalwhere countries of Ebolavirus haemorrhagic fever occur.

environmental samples Denaturing agents Celiac patients Prolamin Gliadin ELISA ELISA solium Taenia Cysticercosis Biosensor Ebolavirus Clinical and IgNAR Haemorrhagic fever

499 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 Hussack Hussack et al. 2009 Trilling Trilling et al. 2011 10) M. Conclusions/Significance: A broad set of different llama antibodies10) M. Conclusions/Significance: llama A broad set of different specific for 16 protein kDaheat shock Antibody engineering of binding agents against has of interest, generation virtually predomi allowed antigen rapid - the any for has been therapeutic applications. for less Considerably attention nantly development given of diagnostic the to reagents and bio - sensors using engineered we produced bispecific pentavalent antibodies. a novel antibody (i.e., decabody) Recently, by pentameriz - B subunit (VTB)ing two single-domain antibodies verotoxin the (sdAbs) through both and found fusion partners be to functional. engineered we have a similar approach, a bispecificUsing cellulose-pentamericconsisting of five and five sdAbs fusion protein find an optimal designbinding modulesformat, constructed (CBMs) we linked six via bispecific VTB.of consisting To pentamers CBMs,three different fusedaureus-specificStaphylococcusthe to human sdAb HVHP428, in both orientations. One bispecific an N-terminal CBM9 containing and C-terminal HVHP428, waspentamer, soluble, non-aggregating, and did not degrade upon degrees four at storage over six C for months. This molecule was dually functional cellulose-basedas it to bound well filters as asS. cells.aureus filters, When impregnated in cellulose the bispecific pentamer recognizedcells in a flow-through aureus S. detection ability of The pentamerizedformthe basis cellulose may to bind multivalent display of an immobilizationassay. CBMs for platform for sensing of of high-avidityfilters pathogens, binding reagents and environmental pollutants. on cellulosic biomarkers Background: antibodies Recombinant powerful are tools in engineering diagnostics. of novel Duesmall size the and stable to of llama antibody nature domains selected antibodies can serve as and sensitive M. a detectionassays for in multiplexed reagent tuberculosis. Methodology/Principal Findings: Antibodies tuberculosis recognition Mycobacterium for raised (M. tb) were in variable recombinant domains of heavy-chainAlpaca, antibodies (VHH) and, by phage display, M. tuberculosis binding to antigens selection phage display strategies followed: were one direct selection using isolated. semi-purifiedwere a Two and antigen, protein depletion strategy with lysates, cross-reaction avoid mycobacteria. aiming to other to Both panning methods selected a set of bind - determiningregions.ers withcomplementarity widely differing Selected recombinant producedVHHs were in shown coli and E. bind immobilizedto in direct lysate Enzymelinked Immunosorbent (ELISA) Assay tests by surface and soluble plasmon antigen analysis. (SPR) resonance All tested VHHs specific were tuberculosis-causingfor mycobacteriatuberculosis, (M. M. bovis) and recognizedexclusively (hsp). The highest 16 kDa an immunodominant heat shock protein affinity VHH had a dissociation constant (KD) of 4 × 10( – of M. tuberculosistuberculosis. in M. abundant is highly isThis and stable available. protein are Thedetect that protein thisVHH tuberculosis-causing of sensorapplied SPR in a robust identification for mycobacteria. strategies Biosensor Decabody Cellulose filters S. aureus SRP-sensor ELISA M. tuberculosis Phage display selection Heat shock protein

500 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article - ron et al. et ron al. 2011 Doman - ska et al. 2011 Abskha Park et et Park al. 2012 - Atomic-level structural investigation of the key conformational intermediates of amyloidogenesis remains a challenge. Here we we Here intermediates key a challenge. structuralof amyloidogenesis of the remains investigation conformational Atomic-level utility of nanobodies the and characterizedemonstrate trap intermediates to of beta 2-microglobulin (beta 2m) amyloidogenesis purpose, this For we selected crystallography. five single domain antibodies blockthe fibrillogenesis that by X-ray proteolytic of a fragmentamyloidogenic of beta 2m (Delta N6 beta 2m). The crystal structure of these N6 Delta one of with betacomplex 2m in nanobodies domain (Nb24) identifies swapping as a plausible mechanism of self-association In protein. this amyloidogenic of hinge loops-corresponding two extended heptapetide the swapped to the NHVTLSQ dimer, in isolation-are forms amyloid that a newunmasked into two-stranded and fold beta-sheet. antiparallel The beta-strandsthissheetto of self-associate prone are and stack perpendicular direction intermolecular the build large strands to to of the beta-sheets axis run the to parallel that of growing oligomers, mechanism providing an elongation by self-templated growth. Prion proteins (PrPs) are difficult to crystallize, probably due to their inherent flexibility. Several PrPs structures havePrPs structures Several difficult to are been solvedflexibility. (PrPs) Prion proteins crystallize, their inherent to due probably we Here crystallography. only three structures magneticsolvedby nuclear were (NMR) techniques; resonance by X-ray however, combined in-situ proteolysismicroseed with automated matrix screening (MMS)crystallize to PrP(C)-nanobody two different Nanobodies(Nb) complexes. single-domain antibodies are derived from heavy-chain-only antibodies of camelids. Initial crystal - lization screening proteolysis using with in situ conditions a nanobody of mouse prion thin (23–230) in complex (Nb_PrP_01) gave needle aggregates, Next, we of poorused were which these diffraction microcrystals quality. MMS. automated nucleantsfor as Good-quality crystals obtained were from mouse PrP (89–230)/Nb_PrP_01, belonged monoclinic the group space P 1 21 1, with to parameters a = 59.13, b 63.80, c 69.79 angstrom, β = 101.96° and diffractedresolutionto 2.1 angstrom using unit-cell - synchro PrP (90–231)/Nb_PrP_01 crystals Human radiation. parameters tron belonged monoclinic the group space C2, with to unit-cell a = 131.86, b 45.78, c 45.09 angstrom, β = 96.23° and diffractedresolution.to 1.5 angstrom combined This strategy benefits powerfrom the MMS of the technique without proteolysis.the drawbacks suffering the in situ of from It provedto be a successful strategy crystallize to crystallization the be for and could exploited PrP-nanobodies complexes antigenantibody difficult of other complexes. The parasite Trypanosomasleeping of Africa,The parasite agent the sickness brucei, causative across sub-Saharan dependsremarkable on a U-insertion/deletion called complex, 20 S multi-protein RNA edito editing the - process in its mitochondrion. A approximately some, is an essential machinery editing for pre-mRNA molecules majority encoding of mitochondrial the proteins. Editosomes proteins, six where interaction called OB-fold proteins of twelve a crucial A1-A6, play we report core Here, a common role. contain form a het surprisingly, A3 and A6 that structure proteins of two single-strandthe acid-binding from OB-folds interaction nucleic Crystal growth requirederodimer. assistance the nanobody of an anti-A3 as a crystallization anti-A3 this Unexpectedly, chaperone. nanobody both binds to A3(OB) and A6, despite40% amino acid only similar sequence to between identity of A3 and OB-folds the of presence attributed the This to mainly is heterodimerThe A3(OB)-A6 A6. buries homodimer. A6 the surfacethan more 35% area and of a dimer heterodimers observeda conserved the A3(OB)-A6 heterodimer, in loop Pro-rich of in A3(OB). The implications crystals,the the of core center’the editosome in resulting in a proposal OB-fold of the profound, architecture of a ‘five are the for editosome.

intermediates screening X-ray crystallographyX-ray Conformational Amyloidogenesis Beta 2-microglobulin Table 8. Other prospective usesTable of single-domain antibody fragments Crystal structure Editosome Trypanosoma brucei X-ray crystallographyX-ray Prion In situ proteolysis Microseed matrix

501 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 - Emmer son et al. 2011 et Talelli al. 2010 De Vooght et al. 2012

The polymericreceptorthe transport immunoglobulin (pIgR) ensures (dIgA) and - of dimericpentameric immu immunoglobulin A lungs and the intestines via the transcytosis. example of for layer noglobulin mucosal the M (pIgM) across to epithelia the day Per of luminal organs. mucosa the 5 gramsThisusefulcould prove of 2 to of dIgA into system pIgR mediates excretion human the use variable mucosa. we investigated the the of Here domain of camelid of compounds into excretion aiming at therapies for derived heavy only antibodies, chain also known as VHHs or Nanobodies, pIgR, human targeting the as a transport system across 1 VHHs receptor with high bind the to affinity (similar directedto that show able pIgR are againsthuman the cells.epithelial We ligand, dIgA. they natural with compete the nM) and that In a transcytosis and phage-bound both assay native VHH only able were get to across polarized pIgR gene in a basolateralVHHs the fashion. apical human MDCK to the that Indicating express that cells VHHs by making particles multivalent take translocatealong large acrosswe and to to epithelia able Furthermore, of cargo. are transport the system,by inducing recep probably enhance - compoundsand Caco-2 to both cell of the able in a MDCK-hpIgR were clustering.tor These results VHHsthat show can be used human pIgR transcytoticas a the carrier exploit to system and system the transport significantly enhance to able across compounds are monolayers. multivalent epithelial that Core-crosslinked thermosensitive and biodegradable cancer polymeric targeted actively EGFR-overexpressing were to micelles nanobody an anti-EGFR surface micelles. by conjugating on the of the cells A methacrylated an derivative containing doxorubicin acid was sensitivespacer hydrazone encapsulated micelles. during attached of the and subsequently core-crosslinking covalently Encapsulation efficiencyreleased DOXdoxorubicin any completely was 60% and at pH 5, while hardly (DOX) 24 h after free similartowards ovarian to showed micelles doxorubicin toxicity released carcinoma cells. pH 7.4. DOX-loaded at Background: Sodalis glossinidius, a gram-negative bacterial endosymbiont has tsetse of the been proposed fly, as a in potential vivo drug delivery vehicle to control trypanosomedrug deliveryapproach known control an to as vehicle paratransgenesis. parasite fly, this development in the Despite of S. glossinidiusinterest as organism in tsetse platform a paratransgenic flies,few effector moleculespotential have been identified none of theseso molecules far date and to been have successfully S. expressed bacterium. in this glossinidius Results: study, In this (Nanobody) targets conserved efficiently was Nb_An33, that a single transformeddomain antibody, express to cryptic epitopes variantof the (VSG) surface glycoprotein parasite of the Trypanosoma brucei. Next, we analyzed of two predicted capability the the leaderpelB that show secretionWe peptide delivery signals direct to extracellular of significant the levels of active Nb_An33. was successful in directing export the of fully functional periplasm the Nb_An33 to of S. glossinidius resulting in significant levels S. glossinidius pelBNb_An33 exhibited no significantreduction expressing terms in of fitness, release. Finally, of extracellular determined by in vitro growth kinetics, wild-type the to compared strain. Conclusions:of Thesethe first demonstration are data release of functional and extracellular trypanosome-interfering expression the Nanobodies in S. glossinidius. Sodalis Furthermore, released efficiently were not effector affected the protein strains that in their thatmaybe with suggesting competitive they growth, the of for the notion potential these reinforce data Collectively, tsetseendogenous of the midgut microbiota in the environment fly. S. glossinidius be to developed organism. platform a paratransgenic into

receptor Transport systems Transport Micelle EGFR Doxorubicin Encapsulation efficiency Drug delivery vehicle Sodalis glossinidius Paratransgenesis Trypanosoma brucei Transport systems Transport Immunoglobulin Carrier system Transcytosis Epithelial monolayer

502 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article Simmons Simmons et al. 2008 Iqbal et Iqbal et al. 2011 - ) constrained library have may 8 imaging of drug biodistribution may provide an alternative to “classical” pharmacoki- imaging of drug biodistribution “classical” to provide an alternative may selection of mimotopes from phage-displayed molecular libraries. ing the powering the of in vivo allowed selection of variants with even epitape favorable topologies more original in the present structure, than illustrat antigenic netics and biodistribution studies in that a contrast-enhanced imagingnetics signal serve and biodistribution could a contrast-enhanced of drug studies in that amount as or the for a surrogate brain-targeting the applications, it isbiologic of interest. necessary organ For the delivered to develop strategies to formulation we describe methods encapsulating a simultaneous for enable delivery drugthat agent and contrast chapter, acrossBBB. In this the drugs liposome into nanocarriers imaging with one or multiple modalities surface for of both imaging display the agent contrast single-domain antibodyand the undergoes transcytosis receptor-mediated that imaging signal acrossBBB. Contrast-enhanced the detected injection is the of drug proportional brain after formulation(s) amount in the delivered intravenous of such into the to brain parenchyma. This methodfor a prospective, allows elimination and accumulation, noninvasive of drug estimation delivery, brain. from the The development of imagingtherapeutic and agents against neuronal targets the is hampered of probesthe limited by access into nervouscentral system acrossblood the brain barrier (BBB). The experimental evaluation of drugthe brain in into penetration models procedures, often requires complex drug including radiolabeling, as as each animals well for in multiple determinations or time point.condition Prospective in vivo Mimotopes three-dimensional topology mimic the epitope, frequently of an antigen recognized and are by antibodies with affini - those to obtained originalties the comparable for and anti-idiotypic antibody-antigen antibodies Peptides interaction. two are classes topology we mimic the mimotopes of protein that (but not necessarily study, In this sequence) antigen. the parental of the these two classescombine by selecting mimotopes based on single domain IgNAR antibodies, long exceptionally display which CDR3 loop regions (analogous a constrained to peptide of an immunoglobulin framework library) with presentedcontext in the and supportingadjacent CDR1 loops. By screening an in vitro library phage-display of IgNAR variable domains (V(NAR)s) against monoclonal target antibody antigen the MAb5G8, we obtained potential mimotopes. four MAb5G8 targets a linear tripeptide signal flexible Plasmodium in the sequencethe of (AMA1),ApicalfalciparumMembrane Antigen-1 epitope (AYP) this or and similar detected motifs were CDR in the loops V(NAR)s. of all four The V(NAR)s,to demonstrated -7, -11, and -14, were 1-A-2, bind specificallythiscompetition to by paratope studies an artificialwith showedpeptide and all enhanced nM) affinities (3–46 (175 nM). Crystallographic antigen the parental showed the and 1-A-11 to that compared 1-A-7 proteins studies of recombinant SYP motifs exposed on these tip of the V(NAR)s the presented at CDR3 loops, ideally positioned structures within bulge-like to These loops, MAb5G8 with the contact make antibody. in particular loop and intra- were in 1-A-11, further stabilized by inter- disulphide bridges, bonds,of higher hydrogen rationalizeaffinity the electrostatic interactions,residue packing. and aromatic We CDR1 adjacent and framework by suggesting binding that to antigen parental residues the V(NAR)s contribute to compared the course though definitive support of otherinteractions this of through with antibody, CDR regions the hypothesis on will affinity, on co-crystallographic selection the rely of mimotopes (< 4 × 10 from a large studies. Alternatively, imaging Mimotope selection IgNAR Plasmodium falciparum Blood-brain barrier Drug biodistribution In vivo Liposome nanocarrier

503 Review Article Veterinarni Medicina, 57, 2012 (9): 439–513 - Hultberg Hultberg et al. 2007 Mur taugh et al. 2011 Franco et et Franco al. 2010 - - - ing their binding affinity, due to the incorporation of multiple the incorporationhistidine to of groups. due binding affinity, trendsing their were Several observed, such as histidine as development of pH switchas the proteins well will enhance increase which help of our understandingrole of the “hot-spots”, ionizable interfaces. residues be in protein and should general, and robust is of capable Overall, rapid, approach combinatorial the applications.producing highlynumber pH-sensitive affinity for a reagents of different protein tions typically rely on the rational introduction of ionizable groups in the protein interface. Such experiments introductiontypically of ionizabletions typically are Such rational on the interface. groups time protein rely in the the at affinity permissive pH. The underlying thermodynamicsproton-linkage dic of and oftenthe protein’s sacrifice intensive ionizable of multiple presence the groups, binding, that on protein a pK(a) change tate necessary undergo are which result in to testhypothesis, this histidine library combinatorial a novel highly pH-dependent was binding. developed To every where possible of histidine and wild-typecombination of a model anti-RNase interface residue is the sampled A single throughout domain VHH Antibodies coselected were antibody. high-affinity binding and pH-sensitivity for using an in dual-function vitro, selection strategy. resultingThe antibodiesretained near wild-type affinity yet became highly to small sensitive decreases in pH, drastically decreas Background: Bacteriophages infecting lactic acid bacteria (LAB) widely acknowledged are as main cause the of milk fermentation we describefailures. as as surface-expression secretion well the the study, In this of two functional llama heavy-chain antibody (RBP) lactococcal of the proteins fragments, receptor-binding (MCP)the capsidto major protein other the and the one binding to bacteriophage p2, by lactobacilli neutralise to in order lactococcal phages. Results: The antibody fragment is directedthat VHH5 was fused tag and expressed in a secreted a c-myc to form by a Lactobacillus strain. The fragmentagainst RBP, the that VHH2 was fused an E-tag to surface lactobacilli. and anchored on the of VHH2 of the Surface was expression MCP, the is binding to confirmed by flow cytometry binding the Efficient both theof VHH2 and anti-E-tag using an secretedantibody. VHH5 fragment was phage antigens the to shown in ELISA. Scanning electron lactobacilli VHH2 microscopy showed anchored that expressing at bind lactococcal to surface able their were phages. A neutralisation also assay the secreted confirmedthat the anchored VHH5 and VHH2 fragments prevented adsorption the of lactococcal host phages their cells. to Conclusion: Lactobacilli express to able were functional VHH fragments in both a secreted surface form and reduced and a cell phage infection of lactococcal cells. Lactobacilli llama heavy-chainexpressing antibody fragments limit phage infection. to way a novel represent switches, the development of protein is There in whosegrowing proteins interest are which function, such as binding a target molecule, can be triggers. modulated environmental through Effortsto engineer highly interac pH sensitive protein-protein This work demonstrates the feasibilitythe demonstrates This of work using a singlechromatographic domain antibody camelid for immunoaffinity seperation of small molecules. An anti-caffeine VHH antibody was produced determining by sequencesa complementanty grafting of the previously antibody generated an anti-RNase A antibody onto scaffold in followed expression by coli. Analysisthe binding E. of properties antibody of the by ELISA and fluorescence-basedthermalshift recognizes it showed that assays not only but caffeine, effect the investigation of Furtherenvironmental of also theobiomine theophylline, and paraxanthine, albeit with lower affinity. antibody pH, and ionic strength, on the conditions, using these methods i.e., temperature, provided useful about information be to used conditions applications.potential elation Immobilization in chromatographic a high VHH flow-through of the onto synthetic support resulted material In a stationary phase of separating capable caffeine and its metabolites.

proteins library chromatography Bacteriophage p2 Major capsid protein Receptor-binding Neutralisation assay Protein switches Combinatorial histidine pH-dependent binding Immunoaffinity Caffeine Grafting

504 Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article

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Yau KYF, Groves MAT, Li SH, Sheedy C, Lee H, Tanha tion of single-domain antibodies from phage libraries: A J, MacKenzie CR, Jermutus L, Hall JC (2003): Selection novel strategy for the rapid generation of high-avidity of hapten-specific single-domain antibodies from a antibody reagents. Journal of Molecular Biology 335, non-immunized llama ribosome display library. Jour- 49–56. nal of Immunological Methods 281, 161–175. Zou XG, Smith JA, Nguyen VK, Ren LM, Luyten K, Zabeau L, Verhee A, Catteeuw D, Faes L, Seeuws S, De- Muyldermans S, Bruggemann M (2005): Expression cruy T, Elewaut D, Peelman F, Tavernier J (2012): Se- of a dromedary heavy chain-only antibody and B cell lection of non-competitive leptin antagonists using a development in the mouse. Journal of Immunology random nanobody-based approach. Biochemical Jour- 175, 3769–3779. nal 441, 425–434. Zhang JB, Tanha J, Hirama T, Khieu NH, To R, Hong TS, Received: 2012–08–30 Stone E, Brisson JR, MacKenzie CR (2004): Pentameriza- Accepted after corrections: 2012–09–25

Corresponding Author: Mgr. Ludek Eyer, Ph.D., Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic Tel. +420 533 331 911, E-mail: [email protected]

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