Properties, production, and applications of camelid single-domain antibody fragments

“Sequence analysis (Hamers-Casterman et al. 1993; Muyldermans et al. 1994; Vu et al. 1997; Harmsen et al. 2000) and elucidation of the crystal structure (Desmyter et al.”

“As compared to human VH domains, the VHH FRs show a high sequence homology of more than 80%, and their 3D structures can be superimposed (Muyldermans et al. 2001; Holliger and Hudson 2005).”

“Although isolated antibody heavy (Utsumi and Karush 1964) and light chains (Yoo et al. 1967) can retain antigenbinding specificity, their affinity and solubility is often reduced (Ward et al. 1989).”

“Although single-domain antibodies later were also identified in particular cartilaginous fish (Greenberg et al. 1995), most research on the biotechnological application of singledomain antibodies was done using camelids because of Appl Microbiol Biotechnol (2007) 77:13–22”

“Methods to isolate antigen-specific VHHs from immune ( Arbabi-Ghahroudi et al. 1997; Van der Linden et al. 2000a), nonimmune (Tanha et al. 2002; Yau et al. 2003; Verheesen et al. 2006), or semisynthetic (Goldman et al. 2006) libraries using phage, yeast, or ribosome display are now well established.”

“Because of their stability, VHHs are especially suited for intrabody production because this requires expression in the reducing environment of the cytoplasm (Gueorguieva et al. 2006; Rothbauer et al. 2006).”

“Occasionally, VHHs with affinity constants (KD) as low as 100 pM are isolated (Saerens et al. 2004; De Genst et al. 2006; Harmsen et al. 2006), which equals the affinity ceiling proposed for natural antibodies (Sundberg and Mariuzza 2002).”

“VHHs are especially suited for oral immunotherapy because of their resistance against extremes of pH and the capacity to bind to the target at high concentrations of chaotropic agents (Dumoulin et al. 2002, 2003).”

“This could rely on their high sequence homology to conventional VH domains and on their high stability because aggregation of proteins is known to increase immunogenicity (Hermeling et al. 2004).”

“For further reading on these topics, we refer to recently published reviews (Muyldermans 2001; Dufner et al. 2006).”

“Sequence analysis (Hamers-Casterman et al. 1993; Muyldermans et al. 1994; Vu et al. 1997; Harmsen et al. 2000) and elucidation of the crystal structure (Desmyter et al. 1996, 2001; Spinelli et al. 1996) has revealed several structural features of VHH domains.”

“However, this may now be feasible using P. pastoris strains with an engineered glycosylation machinery that are able to produce proteins with a specific human glycoform ( Hamilton et al. 2006 ).”

“Sequence analysis (Hamers-Casterman et al. 1993; Muyldermans et al. 1994; Vu et al. 1997; Harmsen et al. 2000) and elucidation of the crystal structure (Desmyter et al. 1996, 2001; Spinelli et al. 1996) has revealed several structural features of VHH domains.”

“VHHs can also recognize antigenic sites that are normally not recognized by conventional antibodies such as enzyme active sites (Lauwereys et al. 1998; De Genst et al. 2006) and conserved cryptic epitopes (Stijlemans et al. 2004).”

“Such molecules (diabodies, Fig. 1a) can be produced using conventional recombinant antibodies using linkers between the VH and VL domains of a specific length, although this often results in aggregation and reduced affinity because of mispairing of VH and VL domains ( Glockshuber et al. 1990; Whitlow et al. 1993).”

“Contrary to conventional antibodies, VHHs have been shown to remain functional at 90°C (Van der Linden et al. 1999) or after incubation at high temperatures (Van der Linden et al. 1999; Perez et al. 2001).”

“They contain an N-linked oligosaccharide attached to the second heavy-chain constant domain (CH2) that is essential for antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytolysis (CDC), and for retaining a long serum half-life.”

“…FMD virus VP1 Monovalent PEG Harmsen et al. 2007 Sepsis N. meningitidis LPS Monovalent None El Khattabi et al. 2006 Cancer – CEA Monovalent β-Lactamase Cortez-Retamozo et al. 2004 Cancer – EGF receptor Bivalent Anti-albumin VHH Roovers et al. 2007 Rheumatoid arthritis – TNFα Bivalent…”

“However, antigen binding by the C-terminal VHH in such fusions can be compromised (Conrath et al. 2001b) presumably because of steric hindrance by the N-terminal VHH.”

“Therefore, VHHs have been targeted to normally long-lived serum proteins such as albumin (Coppieters et al. 2006; Roovers et al. 2007) or immunoglobulin (Harmsen et al. 2005a) using bispecific VHHs recognizing these serum proteins in addition to the therapeutic target, resulting in half-lives that…”

“Sequence analysis (Hamers-Casterman et al. 1993; Muyldermans et al. 1994; Vu et al. 1997; Harmsen et al. 2000) and elucidation of the crystal structure (Desmyter et al.”

“The use of mixtures of a limited number of monoclonal antibodies (oligoclonal antibodies) is advantageous over single monoclonal antibodies for particular applications, such as toxin neutralization (Nowakowski et al. 2002).”

“As compared to human VH domains, the VHH FRs show a high sequence homology of more than 80%, and their 3D structures can be superimposed (Muyldermans et al. 2001; Holliger and Hudson 2005).”

“1a) can be produced using conventional recombinant antibodies using linkers between the VH and VL domains of a specific length, although this often results in aggregation and reduced affinity because of mispairing of VH and VL domains (Glockshuber et al. 1990; Whitlow et al. 1993).”

“However, antigen binding by the C-terminal VHH in such fusions can be compromised (Conrath et al. 2001b) presumably because of steric hindrance by the N-terminal VHH.”

“Firstly, the N-terminal part of CDR1 is more variable (Vu et al. 1997; Harmsen et al. 2000; Nguyen et al. 2000).”

“As a result, high-affinity antigenbinding VHHs can be isolated by directly screening a limited number of clones from immune libraries without prior selection using display technologies (Frenken et al. 2000; Harmsen et al. 2005b).”

“VHHs can also recognize antigenic sites that are normally not recognized by conventional antibodies such as enzyme active sites (Lauwereys et al. 1998; De Genst et al. 2006) and conserved cryptic epitopes ( Stijlemans et al. 2004 ).”

“For further reading on these topics, we refer to recently published reviews (Muyldermans 2001; Dufner et al. 2006).”

“For their use in targeting drugs across the blood–brain barrier (BBB) into the brain, VHHs were selected that transmigrate the human BBB in an in vitro model and accumulate in the brain after intravenous injection into mice (Muruganandam et al. 2002).”

“Until now, multiple injections of VHHs have not shown any immunogenicity in mice, as assessed by the presence of specific antibodies, T cell proliferation, or cytokine levels (Cortez-Retamozo et al. 2002; Coppieters et al. 2006).”

“However, the paired N-terminal variable domains of heavy (VH) and light (VL) chains are sufficient for antigen binding (Sundberg and Mariuzza 2002).”

“Similar to conventional VH domains, VHHs contain four framework regions (FRs) that form the core structure of the immunoglobulin domain and three complementarity-determining regions (CDRs) that are involved in antigen binding.”

“If necessary, technologies developed to decrease immunogenicity of mouse monoclonal antibodies (Presta 2006) could also be applied to VHHs.”

“Furthermore, it could complicate their therapeutic use because the addition of yeast-specific high-mannose oligosaccharides results in a high immunogenicity and decreased serum half-life because of binding to specific mannose receptors on cells of the reticulo-endothelial system (Sethuraman and Stadheim 2006).”

“Fourth, replacement of hydrophobic residues of conventional VH domains normally interacting with CH1 increased scFv production in E. coli (Nieba et al. 1997), suggesting that the hydrophilic mutations that naturally occur at these positions in VHHs also contributes to their high expression level.”

“VHHs are especially suited for oral immunotherapy because of their resistance against extremes of pH and the capacity to bind to the target at high concentrations of chaotropic agents (Dumoulin et al. 2002, 2003).”

“Sequence analysis (Hamers-Casterman et al. 1993; Muyldermans et al. 1994; Vu et al. 1997; Harmsen et al. 2000) and elucidation of the crystal structure (Desmyter et al. 1996, 2001; Spinelli et al. 1996) has revealed several structural features of VHH domains.”

“Contrary to conventional antibodies, VHHs have been shown to remain functional at 90°C (Van der Linden et al. 1999) or after incubation at high temperatures (Van der Linden et al. 1999; Perez et al. 2001).”

“However, antibody fragments that lack the Fc with its N-linked oligosaccharide are preferably produced in microbial systems (Arbabi-Ghahroudi et al. 2005).”

“This high apparent stability is mainly attributed to their efficient refolding after chemical or thermal denaturation and to a lesser extent because of an increased resistance against denaturation (Perez et al. 2001; Dumoulin et al. 2002; Ewert et al. 2002).”

“Methods to isolate antigen-specific VHHs from immune (ArbabiGhahroudi et al. 1997; Van der Linden et al. 2000a), nonimmune (Tanha et al. 2002; Yau et al. 2003; Verheesen et al. 2006), or semisynthetic (Goldman et al. 2006) libraries using phage, yeast, or ribosome display are now well established.”

“The avidity of VHHs has also been strongly increased using genetic fusions to the B-subunits of an E. coli toxin that selfassembles into a homopentamer (Zhang et al. 2004), resulting in pentameric recombinant antibodies (“pentabodies,” Fig.”

“Sequence analysis (Hamers-Casterman et al. 1993; Muyldermans et al. 1994; Vu et al. 1997; Harmsen et al. 2000) and elucidation of the crystal structure (Desmyter et al. 1996, 2001; Spinelli et al. 1996) has revealed several structural features of VHH domains.”

“For example, sleeping sickness was successfully treated with VHHs that bind to a trypanosome coat protein and were fused to the apolipoprotein L-1 enzyme, resulting in trypanosome lysis (Baral et al. 2006).”

“Although isolated antibody heavy (Utsumi and Karush 1964) and light chains (Yoo et al.”

“…human VH fragment that contains several of the hallmark hydrophilic amino acid residues of VHHs was more stable than the original VH fragment (Davies and Riechmann 1995, 1996), whereas “decamelization” of a VHH to mimic a VH domain reduces its thermodynamic stability (Conrath et al. 2005).”

“Fourth, replacement of hydrophobic residues of conventional VH domains normally interacting with CH1 increased scFv production in E. coli (Nieba et al. 1997), suggesting that the hydrophilic mutations that naturally occur at these positions in VHHs also contributes to their high expression level.”

“Thus, diarrhea could also be prevented by lactobacilli that produce rotavirus-neutralizing VHHs fused to a cell surface anchor (Pant et al. 2006).”

“Sequence analysis (Hamers-Casterman et al. 1993; Muyldermans et al. 1994; Vu et al. 1997; Harmsen et al. 2000) and elucidation of the crystal structure (Desmyter et al. 1996, 2001; Spinelli et al. 1996) has revealed several structural features of VHH domains.”

“Occasionally, VHHs with affinity constants (KD) as low as 100 pM are isolated (Saerens et al. 2004; De Genst et al. 2006; Harmsen et al. 2006), which equals the affinity ceiling proposed for natural antibodies (Sundberg and Mariuzza 2002).”

“This contrasts with shark single-domain antibodies that have a vestigial CDR2 that does not contribute to antigen binding (Streltsov et al. 2004).”

“…human VH fragment that contains several of the hallmark hydrophilic amino acid residues of VHHs was more stable than the original VH fragment (Davies and Riechmann 1995, 1996), whereas “decamelization” of a VHH to mimic a VH domain reduces its thermodynamic stability (Conrath et al. 2005).”

“Methods to isolate antigen-specific VHHs from immune (ArbabiGhahroudi et al. 1997; Van der Linden et al. 2000a), nonimmune (Tanha et al. 2002; Yau et al. 2003; Verheesen et al. 2006), or semisynthetic (Goldman et al. 2006) libraries using phage, yeast, or ribosome display are now well established.”

“Although VHHs are highly suited for applications that require a high stability, such as use in shampoo for the prevention of dandruff (Dolk et al. 2005), as capturing reagents in immunoaffinity purification (Verheesen et al. 2003), or use in biosensors (Pleschberger et al. 2004), we would like to…”

“However, by selection for proteolytic stability, a VHH could be isolated from the original library that was not degraded in vivo (Harmsen et al. 2006).”

“Methods to isolate antigen-specific VHHs from immune (Arbabi-Ghahroudi et al. 1997; Van der Linden et al. 2000a), nonimmune ( Tanha et al. 2002; Yau et al. 2003; Verheesen et al. 2006), or semisynthetic (Goldman et al. 2006) libraries using phage, yeast, or ribosome display are now well established.”

“Methods to isolate antigen-specific VHHs from immune (ArbabiGhahroudi et al. 1997; Van der Linden et al. 2000a), nonimmune (Tanha et al. 2002; Yau et al. 2003; Verheesen et al. 2006), or semisynthetic (Goldman et al. 2006) libraries using phage, yeast, or ribosome display are now well established.”

“…human VH fragment that contains several of the hallmark hydrophilic amino acid residues of VHHs was more stable than the original VH fragment (Davies and Riechmann 1995, 1996), whereas “decamelization” of a VHH to mimic a VH domain reduces its thermodynamic stability (Conrath et al. 2005).”

“VHHs that successfully prevented diarrhea caused by rotavirus in a mouse model were similarly selected for resistance against the acidic environment of the stomach ( Van der Vaart et al. 2006 ).”

“…stability, such as use in shampoo for the prevention of dandruff (Dolk et al. 2005), as capturing reagents in immunoaffinity purification (Verheesen et al. 2003), or use in biosensors (Pleschberger et al. 2004), we would like to focus on their therapeutic applications, which are more challenging.”

“In addition to these specific mutations, the packing of extended CDR3 loops against this former VL interface contributes to domain stability (Bond et al. 2003).”

“However, hapten- and peptide-binding VHHs have been successfully isolated using strong selection systems (Spinelli et al. 2000; Yau et al. 2003; Alvarez-Rueda et al. 2007; Harmsen et al. 2007).”

“First, the presence of a potential N-linked glycosylation site often increases production levels in yeast (Sagt et al. 2000).”

“VHH production by the favored yeast expression host Pichia pastoris was only recently described (Rahbarizadeh et al. 2006).”

“Furthermore, transgenic mice containing hybrid llama/ human antibody loci that contain llama V regions and human D, J, and C regions have recently been used to generate human heavy-chain antibodies in mice (Janssens et al. 2006).”

“As a result, high-affinity antigenbinding VHHs can be isolated by directly screening a limited number of clones from immune libraries without prior selection using display technologies (Frenken et al. 2000; Harmsen et al. 2005b).”

“However, in contrast to the full protection afforded by the hyperimmune serum, these VHHs poorly protected guinea pigs from FMD viral challenge infection, suggesting that Fc-mediated effector functions are required for efficient in vivo protection (Harmsen et al. 2007).”

“2000b), as has often been done for conventional antibody fragments (Dufner et al. 2006).”

“Although such effector functions can be indirectly recruited using bispecific (conventional) antibody fragments binding to host immunoglobulin (Holliger et al. 1997), it may be more efficient to recruit these functions by fusing VHHs to host Fc domains.”

“…a high stability, such as use in shampoo for the prevention of dandruff (Dolk et al. 2005), as capturing reagents in immunoaffinity purification (Verheesen et al. 2003), or use in biosensors (Pleschberger et al. 2004), we would like to focus on their therapeutic applications, which are more…”

“Methods to isolate antigen-specific VHHs from immune (ArbabiGhahroudi et al. 1997; Van der Linden et al. 2000a), nonimmune (Tanha et al. 2002; Yau et al. 2003; Verheesen et al. 2006), or semisynthetic (Goldman et al. 2006) libraries using phage, yeast, or ribosome display are now well established.”

“As a result, high-affinity antigenbinding VHHs can be isolated by directly screening a limited number of clones from immune libraries without prior selection using display technologies (Frenken et al. 2000; Harmsen et al. 2005b).”

“2004), in vivo diagnosis using imaging, and treatment of snake bites (Harrison et al. 2006).”

“VHHs have also often been produced in the yeast Saccharomyces cerevisiae (Frenken et al. 2000; Thomassen et al. 2002; Van der Vaart 2002).”

“Finally, Bax-specific VHHs have been expressed in the cytoplasm, resulting in so-called intrabodies, to prevent oxidative-stress-induced apoptosis that is implicated in several neurodegenerative diseases (Gueorguieva et al. 2006).”

“Although isolated antibody heavy (Utsumi and Karush 1964) and light chains (Yoo et al. 1967) can retain antigenbinding specificity, their affinity and solubility is often reduced (Ward et al. 1989).”

“Third, the presence of unpaired C-terminal cysteines reduces expression levels (Simmons et al. 2006).”

“Methods to isolate antigen-specific VHHs from immune (ArbabiGhahroudi et al. 1997; Van der Linden et al. 2000a), nonimmune (Tanha et al. 2002; Yau et al. 2003; Verheesen et al. 2006), or semisynthetic (Goldman et al. 2006) libraries using phage, yeast, or ribosome display are now well established.”

“VHHs have often been produced in E. coli (ArbabiGhahroudi et al. 1997; Rahbarizadeh et al. 2005).”

“…Pant et al. 2006 Piglet diarrhea E. coli F4 fimbriae Monovalent None Harmsen et al. 2006 Caries S. mutans I/II adhesion Monovalent None Kruger et al. 2006 FMD FMD virus VP1 Monovalent PEG Harmsen et al. 2007 Sepsis N. meningitidis LPS Monovalent None El Khattabi et al. 2006 Cancer –…”

“Piglet diarrhea E. coli F4 fimbriae Monovalent None Harmsen et al. 2006 Caries S. mutans I/II adhesion Monovalent None Kruger et al. 2006 FMD FMD virus VP1 Monovalent PEG Harmsen et al. 2007 Sepsis N. meningitidis LPS Monovalent None El Khattabi et al. 2006”

“Production of such functional antibodies requires the correct glycosylation of the CH2 domain, which until recently could only be accomplished using higher eukaryotic cells (Nguyen et al. 2003) but not by microbial production.”

“In baker’s yeast, the specific VHH production rate is correlated with growth rate (Thomassen et al. 2005) and can be up to fivefold increased by growing on ethanol as the carbon source (Van de Laar et al.”

“There is only one example of VHH production in filamentous fungi, which resulted in limited proteolytic degradation of the secreted product (Joosten et al. 2005) because of the high levels of proteases secreted by filamentous fungi (Gerngross 2004).”

“Indeed, llamas immunized with clenbuterol developed conventional but not heavychain antibodies against this hapten (Lange et al. 2001).”

“An example of a successful application of PEGylation to nanobodies is the PEGylated nanobody neutralizing foot and mouth disease (FMD) virus [15].”