Antibody engineering Part 4 - unige.it · Antibody engineering - Part 4 ... Muyldermans S, Cambillau C, Wyns L. Recognition of antigens by single-domain antibody fragments: the

Antibody engineering - Part 4Alternative antibody formats: Nanobodies®

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HCAbs, camelid antibodies aka Nanobodies®

Muyldermans S. Single domain camel antibodies: current status. J Biotechnol. 2001 74(4):277-302

© 1993 Nature Publishing Group

Nature 1993 !Jun 3;363(6428):446-8.

HCAbs, VHH, nanobodies IgNARs Ig new antigen receptors

Flajnik MF1, Deschacht N, Muyldermans S. A case of convergence: why did a simple alternative to canonical antibodies arise in sharks and camels? PLoS Biol. 2011 Aug;9(8):e1001120. doi: 10.1371/journal.pbio.1001120.

Camelus dromedarius Dromedario

Camelus bactrianus Cammello

Lama glama Lama

Vicugna pacos Alpaca

Lama guanicoe Guanaco

Lama vicugna Vigogna

Muyldermans S, Cambillau C, Wyns L. Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. Trends Biochem Sci. 2001 Apr.;26(4):230–235.

Fv

Classical Ab


VHH!sdAb!

Nanobody©

Heavy-chain Ab


The heavy chains of the dromedary or llama HCAb are composed of a variable domain (VHH) immediately followed by a hinge, the CH2 and the CH3 domain


IgG1

IgG2

IgG3

Schematic representation of naturally occurring antibodies in sera of camelids

An extended loop implies a larger flexibility, and this is expected to be entropically counterproductive for binding. This issue is solved in camel VHHs by constraining the long loops with a disulfide bond.

Wesolowski J, Alzogaray V, Reyelt J, Unger M, Juarez K, Urrutia M, et al. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med. Microbiol. Immunol. 2009 Aug.;198(3):157–174.

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Flajnik MF1, Deschacht N, Muyldermans S. A case of convergence: why did a simple alternative to canonical antibodies arise in sharks and camels? PLoS Biol. 2011 Aug;9(8):e1001120. doi: 10.1371/journal.pbio.1001120.

Camelid VHHs locus organization

Shark IgNAR locus organization

( )S. Muyldermans ! Re"iews in Molecular Biotechnology 74 2001 277!302284

tution are expected to be silent at the structuralŽlevel Al-Lazikani et al., 1997; Chothia et al.,

.1992 . However, in contrast to the TTY codon forPhe, the TAY nucleotide triplet coding for Tyr isknown as a hotspot for somatic hypermutationŽ .Milstein et al., 1998 . In line with this, is theobservation that the amino acids at position 27and 29 become much more variable in VHHcompared to the corresponding amino acids in

Ž .VH domains Fig. 4 . Hence, the CDR1 region ofa VH that encompasses the amino acids 31!35Ž .Kabat et al., 1991 is extended in VHH to in-clude amino acids 27!35. The whole region has asolvent exposed location and the variability in thisregion is expected to have two consequences.First, the mutations are most likely involved inthe reshaping of the loop structure, and secondly,some of the amino acid mutations might provokesubtle surface modification that might improvethe VHH!antigen fit. The crystal structures ofthe VHH in complex with lysozyme and RNase Aconfirm these expectations. The loop conforma-tion in the CDR1 region adopts one of several

Ž .possible folds Decanniere et al., 2000 , and theamino acids 27 and 29 participate actively in therecognition process of RNase A and lysozyme,

Žrespectively Desmyter et al., 1996; Decanniere et.al., 1999 .

In summary, it is proposed that this frequentoccurrence of ‘off-sized’ CDR1 and CDR2 loopsand the somatic hypermutations in the CDR1region, leading to an extended hypervariable loopregion, will add to the diversity of the VHHdomains. This might explain how the VHHsingle-domains can exhibit such a broad antigen-binding repertoire in the absence of the VH!VLcombinatorial diversity.

The productive recombination product ofVHH!D!J minigenes is expected to be expressed

Žas part of a "-chain Tonegawa, 1983; Rajewsky,.1993 . However, the co-expression of VHH with

the " constant gene is difficult to prove. At thegene level, it is difficult to amplify the VHH-"sequences using RT-PCR, so that the few clonesthat are obtained might result from an artefactual

Ž .PCR event e.g. PCR cross-over . At the proteinlevel, the production in dromedaries or llamas ofIgM-like molecules containing a VHH domain

Ž .Fig. 4. Variability plot of dromedary VH top and VHHŽ .bottom from amino acid 22!41. The white bars are frame-

Ž .work residues. The CDR1 as defined by Kabat et al. 1991 areshown in grey, while the extended hypervariable region out-side the conventional CDR1, and unique for camelid VHH, isin dark grey. The variability at each position is calculated asthe number of different amino acids occurring at that positiondivided by the frequency of occurrence of the most frequentamino acid at that position. Both databases were obtainedfrom shotgun cloning of VH and VHH cDNA sequences, andsome 20 clones of each were taken into account. The mostfrequent amino acid at each position for this database is given.

but without light chains remains elusive. It isdifficult to envisage that VHH-" chains contain-ing a CH1 domain could be secreted since chap-erone proteins recognising the CH1 domain needto be replaced by the light chain before secretionfrom the endoplasmic reticulum can proceed. Un-der the assumption that the constant domain ofthe light chain replaces the BiP chaperone andassociates with the CH1 domain of the " poly-

Ž .peptide Henderschot, 1987 , then the VL domainstill cannot associate with a VHH domain due tothe reshaped framework-two region and interfer-ence with the long CDR3 of the VHH. In con-trast, there is no indication for the generation of" polypeptide chains without CH1 domains, al-though such IgM-like molecules with a VHH,devoid of CH1 and light chains, could theoreti-cally be secreted from the endoplasmic reticulum.


tution are expected to be silent at the structuralŽlevel Al-Lazikani et al., 1997; Chothia et al.,

.1992 . However, in contrast to the TTY codon forPhe, the TAY nucleotide triplet coding for Tyr isknown as a hotspot for somatic hypermutationŽ .Milstein et al., 1998 . In line with this, is theobservation that the amino acids at position 27and 29 become much more variable in VHHcompared to the corresponding amino acids in

Ž .VH domains Fig. 4 . Hence, the CDR1 region ofa VH that encompasses the amino acids 31!35Ž .Kabat et al., 1991 is extended in VHH to in-clude amino acids 27!35. The whole region has asolvent exposed location and the variability in thisregion is expected to have two consequences.First, the mutations are most likely involved inthe reshaping of the loop structure, and secondly,some of the amino acid mutations might provokesubtle surface modification that might improvethe VHH!antigen fit. The crystal structures ofthe VHH in complex with lysozyme and RNase Aconfirm these expectations. The loop conforma-tion in the CDR1 region adopts one of several

Ž .possible folds Decanniere et al., 2000 , and theamino acids 27 and 29 participate actively in therecognition process of RNase A and lysozyme,

Žrespectively Desmyter et al., 1996; Decanniere et.al., 1999 .

In summary, it is proposed that this frequentoccurrence of ‘off-sized’ CDR1 and CDR2 loopsand the somatic hypermutations in the CDR1region, leading to an extended hypervariable loopregion, will add to the diversity of the VHHdomains. This might explain how the VHHsingle-domains can exhibit such a broad antigen-binding repertoire in the absence of the VH!VLcombinatorial diversity.

The productive recombination product ofVHH!D!J minigenes is expected to be expressed

Žas part of a "-chain Tonegawa, 1983; Rajewsky,.1993 . However, the co-expression of VHH with

the " constant gene is difficult to prove. At thegene level, it is difficult to amplify the VHH-"sequences using RT-PCR, so that the few clonesthat are obtained might result from an artefactual

Ž .PCR event e.g. PCR cross-over . At the proteinlevel, the production in dromedaries or llamas ofIgM-like molecules containing a VHH domain

Ž .Fig. 4. Variability plot of dromedary VH top and VHHŽ .bottom from amino acid 22!41. The white bars are frame-

Ž .work residues. The CDR1 as defined by Kabat et al. 1991 areshown in grey, while the extended hypervariable region out-side the conventional CDR1, and unique for camelid VHH, isin dark grey. The variability at each position is calculated asthe number of different amino acids occurring at that positiondivided by the frequency of occurrence of the most frequentamino acid at that position. Both databases were obtainedfrom shotgun cloning of VH and VHH cDNA sequences, andsome 20 clones of each were taken into account. The mostfrequent amino acid at each position for this database is given.

but without light chains remains elusive. It isdifficult to envisage that VHH-" chains contain-ing a CH1 domain could be secreted since chap-erone proteins recognising the CH1 domain needto be replaced by the light chain before secretionfrom the endoplasmic reticulum can proceed. Un-der the assumption that the constant domain ofthe light chain replaces the BiP chaperone andassociates with the CH1 domain of the " poly-

Ž .peptide Henderschot, 1987 , then the VL domainstill cannot associate with a VHH domain due tothe reshaped framework-two region and interfer-ence with the long CDR3 of the VHH. In con-trast, there is no indication for the generation of" polypeptide chains without CH1 domains, al-though such IgM-like molecules with a VHH,devoid of CH1 and light chains, could theoreti-cally be secreted from the endoplasmic reticulum.


TRENDS in Biochem ical Sciences Vol.26 No.4 April 2001

http://tibs.trends.com

232 Review

share a high degree of identity (Fig. 2) and are mostsimilar (~80%) to the human VH of family III(Ref. 16), the most common human VH family interms of both expression and genome complexity17.The amino acids at positions that determine thetypical immunoglobulin fold18 are all well conservedin the VHH. However, four amino acids that areextremely well conserved in all VHs are constitutivelysubstituted in the VHH. These residues [Val37Phe(or Tyr), Gly44Glu (or Gln), Leu45Arg (or Cys) andTrp47Gly (or Ser, Leu, Phe) (Fig. 2)] discriminate theconventional VH from the heavy-chain specific VHH.

Three hypervariable regions can be clearlydistinguished in the VHH sequences, although theaverage variability of the remaining parts is increasedrelative to that in human or mouse VH (Ref. 19). Inaddition, the CDR3 is longer in VHH, on average, thanin VHs (17, 12 and 9 amino acids in dromedary VHHs,human VH and mouse VH, respectively16).

Dromedary HCAbs are generated from a limitednumber of diverse VHH germline segmentsThe unique, functional, heavy-chain IgG antibodiesoccur (to the best of our knowledge) exclusively withinthe Tylopoda. It is expected that their appearance mustbe paralleled by gene adaptations, and altered geneorganization and usage. Indeed, it seems that new anddedicated sets of immunoglobulin genes arose in thecommon ancestor of the camelids. Separate VH andVHH germline genes, probably residing within thesame locus, recombine with common D and JH genesegments to form a VH or VHH domain, respectively19.A limited number of VHH germline genes (~40) havebeen identified in the dromedary genome; this numberis approximately half that of functional human VHgenes20. However, the repertoire of the primary VHHdomains is apparently further diversified by activesomatic mutation mechanisms19.

The dromedary VHH germline genes encode a Cys residue in CDR1 (or in the framework region atposition 45). Cys residues at these positions are absent

in all VH germline genes including those of thedromedary19. In addition, a second Cys, in the VHHCDR3, is introduced exclusively during therecombination of the VHH–D–JH genes. Theseadditional Cys residues form an inter-loop disulphidebond that stabilizes the VHH domain21. Furthermore,this bond is expected to impose conformationalrestraints on the loop flexibility in the absence ofantigen so that the entropic penalty upon antigenbinding is minimized.

VHH structurePolymerase chain reaction and phage display areroutine techniques used to clone the antigen-bindingmodules (VH–VL pairs or VHHs; Figs 1c,d) fromantibodies and to select antigen-specific binders22. Ithas been shown repeatedly that the selected VHHfragments can be expressed extremely well as solubleproteins in bacteria and yeast23. Several of theserecombinant VHHs directed against haptens orvarious proteins were crystallized with or withouttheir antigen. This structural information confirmed

Review

Ti BS

10 20 30 abc 40 50 abcdef 60RR6-R2 QVQLQESGGGLVQAGGSLRLSCAASGRATSGHGHYGMGWFRQVPGKEREFVAAIRW-----SGKETWYKDSRR6-R9 QVQLQESGGGLVQAGESLKLSCAASGNTFSG---GFMGWYRQAPGKQRELVATIN------SRGITNYADFhCG-H14 QVQLQESGGGLVQAGGSLRLSCAASGRTGST---YDMGWFRQAPGKERESVAAINW-----DSARTYYASScAb-LYS3 DVQLQASGGGSVQAGGSLRLSCAASGYTIGP---YCMGWFRQAPGKEREGVAAINM-----GGGITYYADScAb-RN05 QVQLVESGGGLVQAGGSLRLSCAASGYAYTY---IYMGWFRQAPGKEREGVAAMDS-----GGGGTLYADScAb-CA05 QVQLVESGGGSVQAGGSLRLSCAASGYTVST---YCMGWFRQAPGKEREGVATIL-------GGSTYYGDSAMYL 07 QVQLVESGGGSVQAGGSLRLSCAASGYTFSS---YPMGWYRQAPGKECELVSRIF------SDGSANYAGSAMYL D10 DVQLVESGGGTVPAGGSLRLSCAASGNTLCT---YDMSWYRRAPGKGRDFVSGID------NDGTTTYVDSPot VH EVHLLESGGNLVQPGGSLRLSCAASGFTFNI---FVMSWVRQAPGKGLEWVSGVFG-----SGGNTDYADA

70 80 abc 90 100 abcdefghijklmnop 110RR6-R2 VKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAARPVRVDDISLPVGF--------DYWGQGTQVTVSSRR6-R9 VKGRFTISRDNAKKTVYLEMNSLEPEDTAVYYCYTHYFR------------------SYWGQGTQVTVSShCG-H14 VRGRFTISRDNAKKTVYLQMNSLKPEDTAVYTCGAGEGGTW----------------DSWGQGTQVTVSScAb-Lys3 VKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSScAb-RN05 VKGRFTISRDKGKNTVYLQMDSLKPEDTATYYCAAGGYELRDRTY------------GQWGQGTQVTVSScAb-CA05 VKGRFTISQDNAKNTVYLQMNSLKPEDTAIYYCAGSTVASTGWCSRLRPYDY-----HYRGQGTQVTVSSAMYL 07 VKGRFTISRDNAKNTAYLQMDSLKPEDTAVYYCAAGPGSGKLVVAGRTCYGP-----NYWGQGTQVTVSSAMYL D10 VKGRFTISQGNAKNTAYLQMDSLKPDDTAMYYCKPSLRYGLPGCPI-----------IPWGQGTQVTVSSPot VH VKGRFTITRDNSKNTLYLQMNSLRAEDTAIYYCAKHRVSYVLTGF------------DSWGQGTLVTVSS

Fig. 2. A lignment of VHH sequences w ith known crystallographic structure and of one humanvariable domain of heavy chain (VH) (Pot VH). The hypervariable regions are shown in red, green andblue. The VHH hallmark am ino acids are in pink, and the Cys residues involved in either anintradomain (C22 and C92) or an inter-loop disulphide bond are highlighted in yellow. The twosequences at the top are against a hapten (azo-dye Reactive Red, RR6); all other VHHs are directedagainst proteins. Abbreviations: A MYL, amylase; CA , carbonic anhydrase; hCG , human chorionicgonadotropin hormone; LYS, lysozyme; RN , RNase A .

Fig. 3. The im munog lobu lin fo ld of a VHH (R2–VHH)27. The scaffo ld isin yellow (arrows are β strands), and the CDRs 1, 2 and 3 are in red,green and b lue, respectively. The hydrophob ic cluster of Phe37, Phe47,Tyr91, Trp103 and the Phe100h (g on fig) at three am ino acids upstreamfrom Trp103 are shown in purp le. The VHH hallmark am ino acids Arg45and G lu44 at the ‘VL-side’ of the VHH domain are shown in cyan andred, respectively.


Alignment of VHH sequences with known crystallographic structure and of one human variable domain of heavy chain ( )

Med Microbiol Immunol (2009) 198:157–174 159

123

Molecular cloning and reformatting of camelid and shark sdAbs

sdAbs are usually generated by PCR cloning of the V-domainrepertoire from blood, lymph node, or spleen cDNAobtained from immunized animals into a phage displayvector, such as pHEN2 (Fig. 5). Antigen-speciWc sdAbs are

commonly selected by panning phage libraries on immobi-lized antigen, e.g., antigen coated onto the plastic surface ofa test tube, biotinylated antigens immobilized on Streptavi-din beads, or membrane proteins expressed on the surfaceof cells. Several labs have also constructed semi-syntheticlibraries by cassette-mutagenesis of the CDR regions. Thelatter oVers the advantage of selecting antibodies againsttoxic or diYcult to express antigens. However, sdAbsderived from such non-immune libraries often show loweraYnities for their antigen than sdAbs derived from animalsthat have received several immunizations [8–10]. The highaYnity of sdAbs from immune libraries is attributed to thenatural selection of variant sdAbs during clonal expansionof B-cells in the lymphoid organs of the immunized ani-mals. The aYnity of sdAbs from non-immune libraries canoften be improved by mimicking this strategy in vitro, i.e.,by site directed mutagenesis of the CDR regions and furtherrounds of panning on immobilized antigen under conditionsof increased stringency (higher temperature, high or lowsalt concentration, high or low pH, and low antigen concen-trations).

sdAbs derived from camelid and shark hcAbs are readilyexpressed in and puriWed from the E. coli periplasm atmuch higher expression levels than the correspondingdomains of conventional antibodies (Fig. 6). sdAbs gener-ally display high solubility and stability and can also bereadily produced in yeast, plant, and mammalian cells [8].

Recombinantly expressed sdAbs display several advan-tages as compared to conventional antibodies and the singlechain variable fragments (scFv) derived from theV-domains of conventional antibodies. Their high thermalstability, high refolding capacity, and good tissue penetrationin vivo [3, 11, 12] make nanobodies ideally suited for vari-ous biotechnological and therapeutic applications. More-over, sdAbs can be readily cloned into various formats byfusion to other proteins or peptides, thereby tailoring theirutility for certain diagnostic and/or therapeutic applications(Fig. 7) [13]. For example, fusion to a Xuorescent proteinyields a Xuorescent probe (also designated chromobody or

Fig. 3 3D-structures of enzyme-inhibiting sdAbs derived from cameland shark hcAbs. Images were generated with the PyMOL program[123]. The three CDR loops are color-coded as in Fig. 2: CDR1 red,CDR2 green, CDR3 blue, and disulWde bonds are depicted in yellow.a Chicken lysozyme in complex with an inhibitory shark sdAb VNAR(pdb code 1t6v). The VNAR contacts the enzyme only with its CDR1and CDR3 regions, the latter extends into and blocks the active site cre-vice [124]. b Chicken lysozyme in complex with an inhibitory camelsdAb (pdb code 1mel). The CDR3 extends into and Wlls out the activesite crevice of the enzyme [5]. c Chicken lysozyme in complex with itssubstrate (pdb code 1lsz) [125]. d Chicken lysozyme in complex withthe VL and VH domains of a conventional mouse mAb (pdb code1mlc). The Xat interaction surface with the conventional antibody liesoutside of the active site crevice [126]

Fig. 4 Amino-acid sequence alignment of heavy chain variabledomains of conventional (VH), camelid (VHH), and shark (VNAR)antibodies. Amino acids are color-coded as in previous Wgures: CDR1red, CDR2 green, CDR3 blue, hydrophilic motif in FR2 pink, and cysteine

residues yellow. The sequences are derived from the anti-lysozyme VH(mAbVH), VHH (cAbLys3), and VNAR domains shown in Fig. 3a, b,and d. VHH s+16a is an enzyme-blocking sdAb derived from a llamaimmunized with the murine T-cell ecto-enzyme ART2.2 [29]

Wesolowski J, Alzogaray V, Reyelt J, Unger M, Juarez K, Urrutia M, et al. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med. Microbiol. Immunol. 2009 Aug.;198(3):157–174.

( )S. Muyldermans ! Re"iews in Molecular Biotechnology 74 2001 277!302 279

Ž . Ž .Fig. 1. Schematic illustration of the conventional top and heavy-chain IgG antibodies bottom present in camelid serum. TheŽ . Ž .entire light chain curved lines and CH1 domain black are absent in HCAb. The antigen-binding domains of conventional

Ž .antibodies obtained after proteolysis Fab or after cloning, and expression of the gene VH and VL fragments are shown. Asynthetic linker introduced between the VH and VL stabilises the VH!VL dimer and forms the scFv. The recombinant VHH, thevariable domain of a heavy chain of HCAb is obtained after cloning and expression. The VHH is the minimal intact antigen-bindingfragment that can be generated.

frequently lead to aggregation of scFv and areŽeasily degraded by proteolysis Whitlow et al.,

.1993 .Antigen-binding fragments comprising the sin-

Ž .gle variable domain VH of the conventionalheavy chains have also been generated in the pastŽ .Ward et al., 1989 . In contrast to the VL do-mains, such VH domains often retain theantigen-specificity of the parental antibody sincetheir CDR3 is the major contributor to antigenbinding. However, removing the VL domain froma Fv exposes a large hydrophobic surface of the

ŽVH to the solvent i.e. the former interaction site.with the VL so that the isolated VH molecules

become ‘sticky’ and are, therefore, difficult toproduce in a soluble form. Moreover, affinitydrops by one to three orders of magnitude com-

Ž .pared to a scFv Borrebaeck et al., 1992 . Conse-quently, it has not been possible to develop sin-gle-domain VH antibodies as a valid alternativeof monoclonal antibodies.

We noticed that serum of camels, dromedaries

and llamas contains a unique type of antibodiesŽdevoid of light chains Fig. 1, Hamers-Casterman

.et al., 1993 . The heavy chains of these so-calledŽ .heavy-chain antibodies HCAb have a lower MW

than their counterparts in conventional anti-bodies due to the absence of the first constantdomain, the CH1. Since the light chain is missing,the heavy-chain antibodies should bind their anti-gen by one single domain, the variable domain ofthe heavy immunoglobulin chain, referred to as

ŽVHH, to distinguish it from classic VH Muylder-.mans et al., 1994 . As such, the single-domain

VHH is the smallest available intact antigen-bind-Ž .ing fragment 15 kDa derived from a functional

immunoglobulin.The cloning of the VHH in phage display vec-

tors, selection of antigen binders by panning andexpression of selected VHH in bacteria offer anattractive alternative to obtain small molecularrecognition units. The VHH obtained from animmunised dromedary or llama have a number ofadvantages compared to the Fab, Fv or scFv de-


( )S. Muyldermans ! Re"iews in Molecular Biotechnology 74 2001 277!302 287

Ž . !Fig. 5. Schematic overview of strategies to clone and select the VHH genes from an immunised dromedary or llama . See text foradditional explanation.

be amplified towards their VH and VHH end.This primary PCR yields two kinds of fragments:one kind contains the CH1 exon and is derivedfrom the heavy chain of a conventional antibody,and a second kind of fragment lacking the CH1

exon is derived from the heavy chain of theHCAb. These PCR fragments are easily sepa-rated on agarose gel and the recovery of theshorter fragment eliminates the VH sequencesefficiently. A secondary PCR with nested primers

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Table 22˚Ž .Interaction surface area of CDR with antigen in A and the

Ž .contribution in % of each CDR is given for the four VHHcrystal structures solved in complex with their antigen

CDR3 K Surface CDR1 CDR2 CDR3D3˚Ž . Ž . Ž .AA nM A

cAb-Lys3 24 65 847 14.2% 15.4% 70.4%cAb-RN05 12 35 570 43.9% ! 56.1%cAb-CA05 19 72 619 8.0% 1.0% 91.0%R2 16 22 295 43.0% 33.6% 23.4%

proach. The peptides that contained the CDR3loop sequences retained considerable binding

Žaffinity to lysozyme C. Granier and D. Laune,.personal communication and were shown to be

competitive inhibitors. Thus it is concluded thatthe cavity binding propensity of the CDR3 ofVHH can, therefore, form the ideal lead to de-sign peptidomimetic-based drugs acting as en-zyme inhibitors, and probably also as receptoragonists or antagonists.

It is evident that the VHH that are retrieved

from a synthetic library, in which the CDR3 is theonly randomised region, will be even more effi-cient in the design of peptide mimetics, since onlythe CDR3 of the antigen binder is different fromthe non-binders in the library.

2.7.3. VHH as modular building blocks for manifoldconstructs

Expanding the applications of conventionalantibodies through the design of antibodies withmultiple functions requires extensive protein en-gineering skills. An abundance of applicationsbecomes available for bispecific antibodies orother immuno-fusions.

2.7.3.1. Bispecific and multi"alent antibodies. Biva-Ž .lent and bispecific antibodies Fig. 6 have many

practical applications in immuno-diagnosis andtherapy. Bivalency can allow antibodies to bind to

Žantigens with great avidity Rheinnecker et al.,.1996; Terskikh et al., 1997 . Bispecificity permits

the cross-linking of two antigens, for example in

Ž . Ž .Fig. 6. Bivalent left and bispecific right antigen-binding constructs formed by expression of tandemly cloned scFv or VHH,separated by a linker. Bispecific antigen-binding constructs are generated by cloning two conventional scFv of different specificities

Ž A B . Ž .spaced by a long linker scFv !scFv or by a shorter linker and scrambling the VH and VL domains diabodies . Also, the VHHof different specificities can be cloned and expressed as one polypeptide with a linker. We selected the upper structural hinge ofdromedary IgG2a in our VHH-based constructs.


• smaller size (15 kD) and a single-domain immunoglobulin fold

• high expression yields in bacteria and yeast and ease of purification

• generation of antigen-specific, high-affinity binders

• a higher thermo and chemical stability than corresponding Fv derivatives

• recognition of unique conformational epitopes with the dominant involvement of its long CDR3

• close homology to human VH fragments.

HCAbs pros

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Antibody engineering Part 4 - unige.it · Antibody engineering - Part 4 ... Muyldermans S, Cambillau C, Wyns L. Recognition of antigens by single-domain antibody fragments: the