General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold

Nanobodies, single-domain antigen-binding fragments of camelid-specific heavy-chain only antibodies offer special advantages in therapy over classic antibody fragments because of their smaller size, robustness, and preference to target unique epitopes. A Nanobody differs from a human heavy chain variable domain in about ten amino acids spread all over its surface, four hallmark Nanobody-specific amino acids in the framework-2 region (positions 42, 49, 50, and 52), and a longer third antigen-binding loop (H3) folding over this area. For therapeutic applications the camelid-specific amino acid sequences in the framework have to be mutated to their human heavy chain variable domain equivalent, i.e. humanized. We performed this humanization exercise with Nanobodies of the subfamily that represents close to 80% of all dromedary-derived Nanobodies and investigated the effects on antigen affinity, solubility, expression yield, and stability. It is demonstrated that the humanization of Nanobody-specific residues outside framework-2 are neutral to the Nanobody properties. Surprisingly, the Glu-49 → Gly and Arg-50 → Leu humanization of hallmark amino acids generates a single domain that is more stable though probably less soluble. The other framework-2 substitutions, Phe-42 → Val and Gly/Ala-52 → Trp, are detrimental for antigen affinity, due to a repositioning of the H3 loop as shown by their crystal structures. These insights were used to identify a soluble, stable, well expressed universal humanized Nanobody scaffold that allows grafts of antigen-binding loops from other Nanobodies with transfer of the antigen specificity and affinity. Nanobodies, single-domain antigen-binding fragments of camelid-specific heavy-chain only antibodies offer special advantages in therapy over classic antibody fragments because of their smaller size, robustness, and preference to target unique epitopes. A Nanobody differs from a human heavy chain variable domain in about ten amino acids spread all over its surface, four hallmark Nanobody-specific amino acids in the framework-2 region (positions 42, 49, 50, and 52), and a longer third antigen-binding loop (H3) folding over this area. For therapeutic applications the camelid-specific amino acid sequences in the framework have to be mutated to their human heavy chain variable domain equivalent, i.e. humanized. We performed this humanization exercise with Nanobodies of the subfamily that represents close to 80% of all dromedary-derived Nanobodies and investigated the effects on antigen affinity, solubility, expression yield, and stability. It is demonstrated that the humanization of Nanobody-specific residues outside framework-2 are neutral to the Nanobody properties. Surprisingly, the Glu-49 → Gly and Arg-50 → Leu humanization of hallmark amino acids generates a single domain that is more stable though probably less soluble. The other framework-2 substitutions, Phe-42 → Val and Gly/Ala-52 → Trp, are detrimental for antigen affinity, due to a repositioning of the H3 loop as shown by their crystal structures. These insights were used to identify a soluble, stable, well expressed universal humanized Nanobody scaffold that allows grafts of antigen-binding loops from other Nanobodies with transfer of the antigen specificity and affinity. Minimizing the size of antigen-binding entities from a multidomain protein such as a monoclonal antibody to a single-chain variable fragment or even a single domain has been one of the primary goals of antibody engineering. For drug therapy, these smaller formats can be beneficial in various aspects such as immunogenicity, biodistribution, renal clearance, serum half-life, tissue penetration, and target retention. However, the minimal sized antibody fragments need to retain sufficiently high antigen specificity and affinity, be expressed in high yields, and should have a low tendency to aggregate so as to maintain maximal potency and reduce possible risks of immunogenicity. Moreover functionality in adverse environments such as high concentrations of denaturant or elevated temperatures, and a concomitant increased shelf-life are additional assets. A significant proportion of the functional antibodies within species of the Camelidae are devoid of light chains. These immunoglobulins are referred to as heavy-chain antibodies (1Hamers-Casterman C. Atarhouch T. Muyldermans S. Robinson G. Hamers C. Songa E.B. Bendahman N. Hamers R. Nature.. 1993; 363: 446-448Google Scholar), and their antigen-binding fragment is reduced to a single domain (referred to as VHH or Nanobody), with a molecular size of only ∼15 kDa, which is smaller in comparison to single-chain variable fragment fragments (30 kDa), Fab fragments (60 kDa), and whole antibodies (150 kDa). All Nanobodies belong to the same sequence family, closely related to that of the human VH 3The abbreviations used are: VH, heavy chain variable domain; LDA, ligation during amplification protocol; GdmCl, guanidinium chloride; r.m.s.d., root mean square deviation; CDR, complementarity determining region.3The abbreviations used are: VH, heavy chain variable domain; LDA, ligation during amplification protocol; GdmCl, guanidinium chloride; r.m.s.d., root mean square deviation; CDR, complementarity determining region. of family III, although different subfamilies can be distinguished in dromedary based on the CDR2 length and the position of an additional cysteine in the CDR1 or the framework-2 (2Nguyen V.K. Hamers R. Wyns L. Muyldermans S. EMBO J.. 2000; 19: 921-930Google Scholar). Because extra cysteines are rare in llama VHH sequences, they cannot be used as a subfamily hallmark and alternative subfamily divisions had to be proposed for llama VHHs (3Achour I. Cavelier P. Tichit M. Bouchier C. Lafaye P. Rougeon F. J. Immunol... 2008; 181: 2001-2009Google Scholar, 4Harmsen M.M. Ruuls R.C. Nijman I.J. Niewold T.A. Frenken L.G. de Geus B. Mol. Immunol... 2000; 37: 579-590Google Scholar). Following immunization of a llama or dromedary, VHH genes can be easily cloned in a phagemid vector and antigen-specific VHHs can then be selected via phage display against virtually any antigen (5Arbabi Ghahroudi M. Desmyter A. Wyns L. Hamers R. Muyldermans S. FEBS Lett... 1997; 414: 521-526Google Scholar). Their small size, natural soluble behavior, and unique ability to target alternative epitopes make Nanobodies very attractive tools for tumor targeting, diagnostics, or even for in vivo therapy (6Baral T.N. Magez S. Stijlemans B. Conrath K. Vanhollebeke B. Pays E. Muyldermans S. De Baetselier P. Nat. Med... 2006; 12: 580-584Google Scholar, 7Cortez-Retamozo V. Backmann N. Senter P.D. Wernery U. De Baetselier P. Muyldermans S. Revets H. 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Wyns L. J. Scholar). Because antibody fragments a humanization to be as human is that Nanobodies from should a humanization It is in this to the of Nanobodies such humanization We investigated the humanization of a of Nanobodies more antibody fragments and their of antigen-binding Nanobodies, of were for this M. Conrath K. A. F. K. Frenken L.G. Muyldermans S. Wyns L. A. and M. M. A. J.M. J. Wyns L. Muyldermans S. Scholar). is the of the VHH subfamilies for of all dromedary antigen-specific Nanobodies (2Nguyen V.K. Hamers R. Wyns L. Muyldermans S. EMBO J.. 2000; 19: 921-930Google Scholar). We on the framework-2 because are to have the on solubility, and antigen of the We then the of the → on these antibody the residues in the framework and to a humanized Nanobody scaffold so as to the possible amino acid to a human VH It demonstrated that the the loops from a whole of Nanobodies with transfer of the antigen specificity of the loop D. M. R. E. M. A. Wyns L. Muyldermans S. Conrath K. J. Mol. Scholar). the loops of Nanobodies, and were on the framework to this framework can be used as a universal humanized Nanobody We and the antigen the and of humanized to different the of humanization on the scaffold of Nanobodies, the crystal of a humanized and in with its antigen and of the humanized on this a is proposed to a humanized of any Nanobody with maximal of and antigen-binding a universal humanized Nanobody scaffold that antigen-binding loops from Nanobodies, even from other subfamilies or were by the ligation during amplification Res... Scholar). were so as to or a The of the different by and by and of were The expression in the and of Nanobodies performed as M. M. A. J.M. J. Wyns L. Muyldermans S. Scholar). The of the by The protein the of as from their amino acid sequence E. A. C. I. A. Res... Scholar). concentrations from to of the and their were over a to which human and had been the to the All were performed a of in and and used for were with the of the on the of a with of the and The and were used to the were performed with a in the a protein of and A of of in by the from to a of and the as a of were with a of a and a performed J.M. A Scholar). The of the by of the protein and the to were and the the different were concentrations and for were with human or a of in in the were for with in with or and protein a antibody by an and as The of four different concentrations of by to the stability. a protein of and denaturant concentrations from to were by a in to the protein in the on an from to a of The of of as the of is the and is the Mol. Scholar). for were on the of a for the as for Nanobodies so M. Conrath K. A. F. K. Frenken L.G. Muyldermans S. Wyns L. A. Scholar, S. C. Conrath K. A. Scholar). this a performed to the and a as by and and M.M. Scholar). The of denaturant which of the protein is has been shown to be the the of and because can be and is to the Scholar). were to and J. 1993; and K. to the and can then be from in very only the of Nanobody loops from loop Nanobodies and were to the scaffold of by The sequence of loop from the loop Nanobody by one and one the and the the sequences to the framework residues of the The were as with D. M. R. E. M. A. Wyns L. Muyldermans S. Conrath K. J. Mol. Scholar). The Nanobody of on with and the with as Nanobody with and fragments were cloned in the expression vector M. M. A. J.M. J. Wyns L. Muyldermans S. Scholar). The expression of is to the of the humanized Nanobody of for the of were The to were reduced the of 1997; Scholar). with the based D. Scholar, D. 2004; Scholar), which the of all VHH in the The framework region of crystal of with the loops from D. M. R. E. M. A. Wyns L. Muyldermans S. Conrath K. J. Mol. Scholar, A. Ghahroudi F. Hamers R. Muyldermans S. Wyns L. Nat. used as the of and with P.D. P. J. M. T. D. were with A. C. Scholar). were used the and the target and are in The and were and with the the very to reduce the by the or to different in different were and for and of of unique in a for the humanized of were of the to a of and as for the The same used for molecular and as for the to the same as for the that were The same to the humanized for which the were on The used as the for molecular and All of and are in the of Nanobodies, and with specificity for human and the of have been selected to the of the VHH hallmark amino acid residues in framework-2 on expression yield, affinity, and stability. The for this because of its therapeutic because an human that and M. Desmyter A. K. D. G. A. J. Muyldermans S. Wyns L. C. A. Robinson Nature.. Scholar). The high of M. M. A. J.M. J. Wyns L. Muyldermans S. and the of its framework to the antigen specificity loops from Nanobodies of the VHH D. M. R. E. M. A. Wyns L. Muyldermans S. Conrath K. J. Mol. Scholar), a as universal Nanobody The Nanobodies were mutated by the framework-2 hallmark amino acid 42, 49, 50, and to the amino acids in human VH i.e. and These referred to as and were expressed in the of E. and by and humanized in framework-2 are with a single for the amino acid 42, 49, 50, and these are they to the human sequence that The of protein to of for and The expression of the framework-2 humanized were of for and of for probably due to the or to are as by However, a in from the is for of the humanized with the for VH of antibodies K. C. Stijlemans B. J. K. Wyns L. Muyldermans S. R. J. Mol. Scholar, S. C. Conrath K. A. Scholar, H. P. C. J. 2008; Scholar). the has a tendency to The of the framework-2 region of and on the antigen-binding by an in the for with for and for with for and and in the of by and of the of VHH hallmark residues of humanized in framework-2 are with a to the residues 42, 49, 50, and The human hallmark residues these are in to humanized in framework-2 are with a to the residues 42, 49, 50, and The human hallmark residues these are in humanized in framework-2 are with a to the residues 42, 49, 50, and The human hallmark residues these are in J. 1993; Scholar, K. in a and in the of by and of the humanization of and of based on the to J. 1993; Scholar, K. in a of → on and of that in the with the domain and that is in VH domain is of the Nanobodies this to the is to a more M.M. de Scholar). → the domain more and shown to have an on the expression and of a VH domain F. M. A. P. I. J. D. J. J. Mol. 2004; Scholar). the of the amino acid position on the expression and functionality of and of an position of to a of the expression yield, a expression is for the same in the with an position a in The of of had been demonstrated to be for of the third antigen-binding loop D. M. R. E. M. A. Wyns L. Muyldermans S. Conrath K. J. Mol. Scholar). on loop to be of the Nanobody hallmark residues in because a in is the to of 42, 49, 50, and on of the on is for the of to the of mutated on the of The residues and are and mutated so that different and all the possible 42, 49, 50, and The humanized of were to the of with one for the an expression of of a on the of of the on the the of humanized of The and Gly to position is a to in the and The concomitant in is even more a Val is position to the that is for high antigen probably in with the position in were the residues and The of the different in framework-2 on the of by determining the of of all from and The is for all because the are from of the The are in the for of this size J.M. Scholar). A single a of the Nanobodies The Glu-49 → Gly and Arg-50 → Leu in a in protein Gly position and and a of of to Val position by these For all the different of a single with a proportion of the the from to with for the and that the a well of Nanobodies and human VH single-domain antibodies M. Conrath K. A. F. K. Frenken L.G. Muyldermans S. Wyns L. A. Scholar, S. C. Conrath K. A. Scholar, H. P. C. J. 2008; Scholar). However, this with a reduced antigen-binding of of the of of in with human has been M. Desmyter A. K. D. G. A. J. Muyldermans S. Wyns L. C. A. Robinson Nature.. The humanized of in with its antigen in a different crystal A and The antibody within the different crystal with an of for the the the of different crystal The a single for residues a of for all other this with the region the to be to crystal effects are in the CDR1 and CDR2 the loop is by the of position the Leu to which in against the chain of The of the for the of the antigen to the or the humanized is very and via the same a in the antigen position the have been for other antibody for which different are K. Desmyter A. D. Muyldermans S. Wyns L. J. Mol. Scholar). the of such in of which different are selected for by the crystal is that the are by of with only very to that that the high of heavy-chain antibodies in be due to a high tendency to the as this reduce the of the S. C. Wyns L. Scholar). by of the of from the Nanobody hallmark residues in other amino acids the VHH framework and the VH framework of a antibody various a single-domain antibody fragment with the possible to human sequences, humanized these residues the human as a G. G. J. Mol. to the VH family III, which the VHH and is well in P. EMBO J.. Scholar). The VH family in antibodies against a of different for this because of its high and the of its framework in D. M. R. E. M. A. Wyns L. Muyldermans S. Conrath K. J. Mol. Scholar). VHH framework residues outside framework-2 of were humanized to the sequence → → → → → → → → → → and → These residues are to have only on the Mol. Immunol... Scholar, J.M. U. S. Scholar), which is by the of and However, this the of because its from to for We demonstrated the of the humanization of framework-2 on the antigen-binding of on that this to the residues and only the residues and in framework-2 were to their human in and We then the of and and a in as well as the antigen-binding and are A and and The in even for the of for the for the and a more of the mutated in a longer on the the maximal humanization of the in a of the and to be to the spread the scaffold region of and is only to of of crystal of and humanized were to and A and The humanized of with in the are very for All mutated residues have their on the of the and a minimal of the The are from the with and for residues have in the to the in as a of that the are spread over the in in with the over the are for the and residues These to to and in be a of different and crystal The crystal of the on the single-domain human VH and from L. D. G. J. Mol. 2004; and H. P. C. J. 2008; Scholar), to the of the humanized sequences in the of the residues and a of the chain on all other amino acids had an on the of a stable, humanized Nanobody scaffold that allows of the antigen-binding loops of single-domain antibodies is an For to be a universal scaffold need to that its humanized framework as a universal for loop and for transfer of antigen We Nanobody by and their properties. is a of the dromedary the llama to the VHH to the of (3Achour I. Cavelier P. Tichit M. Bouchier C. Lafaye P. Rougeon F. J. Immunol... 2008; 181: 2001-2009Google Scholar). The Nanobody to the to transfer the antigen specificity and of a VHH to humanized dromedary VHH the of and on the framework of to and had significant on the expression and the of the and The of the with that of the in the of the for is by the a small in of the of the loops of It from the the to this of single-domain antibody fragments or Nanobodies have a high for and applications because of their small size, high antigen and and to their small size and the high of of their framework to the human VH framework of family III, Nanobodies are to a low M.M. de Scholar). for tumor therapy the is that the Nanobody to be humanized to a maximal on their expression affinity, solubility, and stability. The Nanobodies and human VH are the in the framework-2 region. of these a from a to a and are to be for the of the Nanobody J. L. FEBS Lett... Scholar, K. C. Stijlemans B. J. K. Wyns L. Muyldermans S. R. J. Mol. Scholar). human VH in only in with a and the of this domain a to the VH have a tendency to aggregate D. G. Nature.. Scholar). is to the of of these framework-2 on the of Nanobodies to the single-domain antibody to the of these VHH hallmark residues have on the of VH single to the of Nanobodies J. L. FEBS Lett... Scholar, F. M. A. P. I. J. D. J. J. Mol. 2004; Scholar, J. L. Scholar, J. P. F. H. J. Scholar). only as on human or VH shown to be due to framework and the tendency to and aggregate F. C. C. N. M. G. S. R. De R. M. 1997; Scholar, L. J. Mol. Scholar, S. T. D. J. Mol. 2000; Scholar). H. P. C. J. 2008; a molecular to a human VH a and single-domain antibody from a dromedary-derived Nanobody and a to this single-domain The of humanized Nanobodies over human is that VHH in the of a and as soluble, single outside the spread over the VHH probably in the possible single-domain a the of the Nanobody hallmark amino acid in framework-2 on the solubility, and loop of K. C. Stijlemans B. J. K. Wyns L. Muyldermans S. R. J. Mol. Scholar). However, this and to the rare VHH the of the dromedary-derived Nanobodies belong to the the loop of over the as for Nanobodies with longer loops A. K. Muyldermans S. Wyns L. J. Scholar, K. Desmyter A. M. Ghahroudi Muyldermans S. Wyns L. Scholar, E. K. K. Conrath K. R. J. Muyldermans S. Wyns L. U. S. 2006; Scholar). more to in to the Nanobodies with longer this the of the of residues and in framework-2 to their human on affinity, and of and of VHH Nanobodies of this VHH subfamily of all dromedary antigen-specific Nanobodies (2Nguyen V.K. Hamers R. Wyns L. Muyldermans S. EMBO J.. 2000; 19: 921-930Google Scholar). These to which residues in framework-2 can be humanized the functionality of these single-domain humanized the Nanobody scaffold to a more with maximal of its Nanobody beneficial properties. The in of and humanization of residues and 50, the position is mutated to the VH hallmark is a A on had been for although in this the in of residues and is less because the natural position is by a K. C. Stijlemans B. J. K. Wyns L. Muyldermans S. R. J. Mol. Scholar). These are with on in framework-2 to a in domain J. L. FEBS Lett... Scholar). The residues and are residues in the VHH which the of the and the domain more A is that the VHH domain in by with residues and to its the and of a VH sequence a domain humanization of the Glu-49 and Arg-50 of a VHH a variable the of the Nanobodies is by the Glu-49 → Gly and Arg-50 → Leu as from the single on a more on the demonstrated that other in the sequences of single-domain antibodies of Camelidae to this soluble and S. Frenken L. D. de L. T. C. C. M. Nat. Scholar, J. G. T. J. Scholar). the and to be neutral for the antigen-binding of the Nanobodies the of to the antigen-binding of and The for this is that the of a Gly by the chain of a of the loop as in the crystal with the of the in Nanobodies for of the third antigen-binding loop K. C. Stijlemans B. J. K. Wyns L. Muyldermans S. R. J. Mol. Scholar). that the VHH genes to of the over the However, the of be the of the domain as demonstrated by the and a Gly this position and of and for the with and for The of Phe-42 is less this by the expression yield, and although these effects to be to this single from with residues 49, 50, and VH the of and and the for the The of a in VHH is crystal that the chain of Phe-42 a by the and the of and the A. Ghahroudi F. Hamers R. Muyldermans S. Wyns L. Nat. Scholar). → the and for the by the → and → is in with the of the human VH Val position a of the L. J. Mol. Scholar). Phe-42 to be to maintain the and the of the Nanobody is by the expression and the of the with a of a Val position a the crystal be for the which possible for the because of low expression and The → the expression of A significant for the and the of the of for the expression of However, the of a this position in Nanobodies cannot be by because of its detrimental on the antigen of these Nanobodies, probably by the of the third antigen-binding in this of demonstrated that the Nanobody residues and have a on the of the antigen Surprisingly, the residues and the Nanobodies, their and antigen affinity. We on the framework to the human VH sequence as as The sequence from human III, as to the used VH in the natural of human antibodies R. Mol. Immunol... Scholar, M. Mol. Immunol... Scholar). is an and S. S. B. A. L. C. A. J. Mol. Scholar). The stable human domain to as and the additional residues outside framework-2 were mutated to their human in the framework of These the antigen-binding of the Nanobody a in expression and of the single because of the residues → → and → were as of llama VH fragments J. G. T. J. Scholar). However, the beneficial effects of the residues and were to for this in stability. for these the scaffold more on the its the transfer of antigen specificity of loop the framework of that this scaffold can be used as a universal humanized for loop All these to to a humanized of a Nanobody with maximal of and antigen-binding For in vivo Nanobody with an antigen specificity of that to be humanized to its antigen-binding loops universal humanized Nanobody scaffold a and of the Nanobody with of the antigen specificity and of the loop However, because framework residues be or in antigen by the loops in the for antigen the transfer of the the universal humanized Nanobody scaffold the to these the framework of the Nanobody should be to the of Nanobody-specific The humanization of residues in framework and have a minimal the of amino acids in framework-2 and to Gly and even the domain the of and The humanization of amino acids and in framework-2 is due to their in antigen and of that universal humanized Nanobody is an to a by its