Isolation of Monomeric Human VHS by a Phage Selection

Abstract

Human VH domains are promising molecules in applications involving antibodies, in particular, immunotherapy because of their human origin. However, they are, in general, prone to aggregation. Therefore, various strategies have been employed to acquire monomeric human VHs. We had previously discovered that filamentous phages displaying engineered monomeric VH domains gave rise to significantly larger plaques on bacterial lawns than phages displaying wild type VHs with aggregation tendencies. Using plaque size as the selection criterion and a phage-displayed naïve human VH library we identified 15 VHs that were monomeric. Additionally, the VHs demonstrated good expression yields, good refolding properties following thermal denaturation, resistance to aggregation during long incubation and to 15 VHs as good and the selection employed have with Human VH domains are promising molecules in applications involving antibodies, in particular, immunotherapy because of their human origin. However, they are, in general, prone to aggregation. Therefore, various strategies have been employed to acquire monomeric human VHs. We had previously discovered that filamentous phages displaying engineered monomeric VH domains gave rise to significantly larger plaques on bacterial lawns than phages displaying wild type VHs with aggregation tendencies. Using plaque size as the selection criterion and a phage-displayed naïve human VH library we identified 15 VHs that were monomeric. Additionally, the VHs demonstrated good expression yields, good refolding properties following thermal denaturation, resistance to aggregation during long incubation and to 15 VHs as good and the selection employed have with of in that of to as and their of of a of of of a of of are with their in of in of resistance to refolding expression and of library and of the properties of are in applications involving However, the of their in human immunotherapy because of the human VH are applications because they are to However, human VHs are and prone to a to VHs were previously to human VHs VHs properties of as expression and resistance to aggregation. on VHs as library as a promising of as as and in and in human have been employed to monomeric human VHs. on VHs and were to to various monomeric human VHs were human VH to of the a monomeric human VH discovered a human VH library a selection on and monomeric VHs human VH the that selection to of human VHs with we monomeric human VHs. We the of 15 VHs and a phage-displayed naïve human VH a selection that on plaque and are their following to long incubation and are in good in the as the of the VHs of and in of VH selection of with good properties the selection and of and human and Using the as VH with were in VH and were and as the in the of to VH the and that and VH and a VH library as of the and and were a library as and were expression expression of and were as were of the on a as and and refolding of were with the the of VHs to of a were on were of in the the the of the to of of were as were in and a of and were with the the of the VHs were incubation were to to and were in a to were and as and were to a and were were the of the and the of the of a in to of VH in were in a of and of of and the with in and to VH were in the were in a in the of were a size and the monomeric were of to were as that of and were a of were in and were to were on a a were with and the were and of were on a with a of and a of the the were and the were with the on a with a with were and were in the and to were the of the of human and human VH we that the phages displaying monomeric VHs larger plaques on bacterial lawns than phages displaying human VHs with aggregation tendencies. We to plaque size as a of VHs the human VH a library displaying human VHs with a size of and as plaques on the the library of plaques with on that the plaques to the the the wild type phages VH of the phages were with plaque because of the of the monomeric VHs in the human and the size of the the of monomeric to the library size to a and wild type with the library to a of human VHs the a of the library and and of than plaques that had VH size of the plaques that were in 15 VHs that to the and and have been previously in VHs had a with their in of the the the with a the VH with a of VH to with a of in with to the and were in VHs and and and and to were in the were in the 15 and and VHs in that they had a of a However, the in the in and the VHs had the the and had a have been to in VH and were and that and and in a of the human VHs on and plaque in the with are the and that with are in VHs the as were in in in with a and to expression to of of bacterial in with the of the of and the expression gave of and as to and that of the VHs expression in expression significantly than the in the VHs to in with of a and with previously with of the VHs to the of the human of bacterial and properties of the various VH molecules are as and with and and were to a of and were and were to and were to and were and were of bacterial properties of the various VH molecules are as and with and and were to a of and were and were in a aggregation of the human VHs in of their and VHs were to to a VHs gave a the a and were of the in a human VH a of of the a with a a VH of the gave that were significantly than the with VH in the of the and of the VHs were in the VH to and a of the VH of VH of aggregation of the and and with and the of the a of with a and with the the that the had monomeric the of the human VHs. the of a human VH in to that of a and a human VH in to monomeric of and in the are a of to of the of the VHs in of their resistance to and following long the of a are and in the human VHs in the to of during the to a that had a the VH the VHs following a incubation with on a of the VHs and that in the of the to a VH with the as the gave a significantly than the and in the to in of the human VHs in of their resistance to and following long incubation the of the and VH and to a VH the of and of the VH that the to a of the VH to the of the VH of the in the to of a the of the of the VH to that of VH were because they were and in to monomeric and VH of the the the of to various VHs were that had been VHs in to were in of and of VHs VHs gave that were to of VHs and as the that the VHs their following and were and the to were and with the VHs the VHs were and and are to the the VHs that of VHs their that the VHs have been in a during the incubation and their to during and in of the that the VHs were to a with the of the human VHs the the of the and VHs to a of the VH the because on the of the of to a of VH to in and VH of the VH to in that the of the in and and the and the were are and of and were as were VHs had in the of and were to the and were and had in the of and were had significantly and previously had of the of and to and and were and to as and human VH are on human VH are a promising of However, human VH are and the human VH we 15 monomeric human VHs with and a phage-displayed naïve human VH a selection that on plaque VHs and monomeric following of of have thermal refolding are in good in and properties VHs that the VHs as VHs that have good and thermal refolding the of to VH are to their to VHs applications because of their VH and in and and to library VHs the human VHs with properties were on that the monomeric human VHs in that VHs are in of that we have monomeric VHs with and the we had we selection to a of VHs with of in of the VHs because of in the VH to the of the VHs because of the of in the VHs and the that to in the of that to and in and the and in have a in VHs of library that the of the VHs on in that the of library the VHs with because on of the VHs in of and the of VH have been on to in to the in that a of on the of the of the of VHs to that are on VH a of in have a the in significantly to and been in library on are significantly library library the of the of VHs in of to with good and good a library of on a VH the of the VHs in of VH with and to to to human VH library the of VHs. the and in of the human VH of the selection of with properties have been to selection of a library in a of VH to aggregation VHs of selection involving and the the selection to a on the that properties their than However, with the of a selection than the of to with on the that are and are to are engineered in a that the of the to a library with a the phages displaying their and of However, and have and and the selection of in a the with properties are identified the that in selection a that a to that the are However, the of the on plaque a in of the of that and to the of the to the library size a that a of library on a as on a to to VHs of good properties a of VHs. However, to the a library that with with good the identified on the of plaque incubation plaque size and selection to the of and as human with the in the selection of a involving and the plaque size and VH expression that the of with expression expression of significantly of in that of to as and their of of a of of of a of of are with their in of in of resistance to refolding expression and of library and of the properties of are in applications involving However, the of their in human immunotherapy because of the human VH are applications because they are to However, human VHs are and prone to a to VHs were previously to human VHs VHs properties of as expression and resistance to aggregation. on VHs as library as a promising of of a of refolding of of a of refolding of as as and in and in human have been employed to monomeric human VHs. on VHs and were to to various monomeric human VHs were human VH to of the a monomeric human VH discovered a human VH library a selection on and monomeric VHs human VH the that selection to of human VHs with we monomeric human VHs. We the of 15 VHs and a phage-displayed naïve human VH a selection that on plaque and are their following to long incubation and are in good in the as the of the VHs of and in of VH selection of with good properties the selection and of and human and Using the as VH with were in VH and were and as the in the of to VH the and that and VH and a VH library as of the and and were a library as and were expression expression of and were as were of the on a as and and refolding of were with the the of VHs to of a were on were of in the the the of the to of of were as were in and a of and were with the the of the VHs were incubation were to to and were in a to were and as and were to a and were were the of the and the of the of a in to of VH in were in a of and of of and the with in and to VH were in the were in a in the of were a size and the monomeric were of to were as that of and were a of were in and were to were on a a were with and the were and of were on a with a of and a of the the were and the were with the on a with a with were and were in the and to were and human and Using the as VH with were in VH and were and as the in the of to VH the and that and VH and a VH library as of the and and were a library as and were expression expression of and were as were of the on a as and and refolding of were with the the of VHs to of a were on were of in the the the of the to of of were as were in and a of and were with the the of the VHs were incubation were to to and were in a to were and as and were to a and were were the of the and the of the of a in to of VH in were in a of and of of and the with in and to VH were in the were in a in the of were a size and the monomeric were of to were as that of and were a of were in and were to were on a a were with and the were and of were on a with a of and a of the the were and the were with the on a with a with were and were in the and to were the of the of human and human VH we that the phages displaying monomeric VHs larger plaques on bacterial lawns than phages displaying human VHs with aggregation tendencies. We to plaque size as a of VHs the human VH a library displaying human VHs with a size of and as plaques on the the library of plaques with on that the plaques to the the the wild type phages VH of the phages were with plaque because of the of the monomeric VHs in the human and the size of the the of monomeric to the library size to a and wild type with the library to a of human VHs the a of the library and and of than plaques that had VH size of the plaques that were in 15 VHs that to the and and have been previously in VHs had a with their in of the the the with a the VH with a of VH to with a of in with to the and were in VHs and and and and to were in the were in the 15 and and VHs in that they had a of a However, the in the in and the VHs had the the and had a have been to in VH and were and that and and in a VHs the as were in in in with a and to expression to of of bacterial in with the of the of and the expression gave of and as to and that of the VHs expression in expression significantly than the in the VHs to in with of a and with previously with of the VHs to the of the human of bacterial and properties of the various VH molecules are as and with and and were to a of and were and were to and were to and were and were of bacterial properties of the various VH molecules are as and with and and were to a of and were and were in a aggregation of the human VHs in of their and VHs were to to a VHs gave a the a and were of the in a human VH a of of the a with a a VH of the gave that were significantly than the with VH in the of the and of the VHs were in the VH to and a of the VH of VH of aggregation of the and and with and the of the a of with a and with the the that the had monomeric the of the human VHs. the of a human VH in to that of a and a human VH in to monomeric of and in the are a of to of the of the VHs in of their resistance to and following long the of a are and in the human VHs in the to of during the to a that had a the VH the VHs following a incubation with on a of the VHs and that in the of the to a VH with the as the gave a significantly than the and in the to in of the human VHs in of their resistance to and following long incubation the of the and VH and to a VH the of and of the VH that the to a of the VH to the of the VH of the in the to of a the of the of the VH to that of VH were because they were and in to monomeric and VH of the the the of to various VHs were that had been VHs in to were in of and of VHs VHs gave that were to of VHs and as the that the VHs their following and were and the to were and with the VHs the VHs were and and are to the the VHs that of VHs their that the VHs have been in a during the incubation and their to during and in of the that the VHs were to a with the of the human VHs the the of the and VHs to a of the VH the because on the of the of to a of VH to in and VH of the VH to in that the of the in and and the and the were are and of and were as were VHs had in the of and were to the and were and had in the of and were had significantly and previously had of the of and to and and were and to as and the of the of human and human VH we that the phages displaying monomeric VHs larger plaques on bacterial lawns than phages displaying human VHs with aggregation tendencies. We to plaque size as a of VHs the human VH a library displaying human VHs with a size of and as plaques on the the library of plaques with on that the plaques to the the the wild type phages VH of the phages were with plaque because of the of the monomeric VHs in the human and the size of the the of monomeric to the library size to a and wild type with the library to a of human VHs the a of the library and and of than plaques that had VH size of the plaques that were in 15 VHs that to the and and have been previously in VHs had a with their in of the the the with a the VH with a of VH to with a of in with to the and were in VHs and and and and to were in the were in the 15 and and VHs in that they had a of a However, the in the in and the VHs had the the and had a have been to in VH and were and that and VHs the as were in in in with a and to expression to of of bacterial in with the of the of and the expression gave of and as to and that of the VHs expression in expression significantly than the in the VHs to in with of a and with previously with of the VHs to the aggregation of the human VHs in of their and VHs were to to a VHs gave a the a and were of the in a human VH a of of the a with a a VH of the gave that were significantly than the with VH in the of the and of the VHs were in the VH to and a of the VH of VH of aggregation of the and and with and the of the a of with a and with the the that the had monomeric the We the of the VHs in of their resistance to and following long the of a are and in the human VHs in the to of during the to a that had a the VH the VHs following a incubation with on a of the VHs and that in the of the to a VH with the as the gave a significantly than the and in the to in VHs in to were in of and of VHs VHs gave that were to of VHs and as the that the VHs their following and were and the to were and with the VHs the VHs were and and are to the the VHs that of VHs their that the VHs have been in a during the incubation and their to during and in of the that the VHs were to a with We the of the human VHs the the of the and VHs to a of the VH the because on the of the of to a of VH to in and VH of the VH to in that the of the in and and the and the were are and of and were as were VHs had in the of and were to the and were and had in the of and were had significantly and previously had of human VH are on human VH are a promising of However, human VH are and the human VH we 15 monomeric human VHs with and a phage-displayed naïve human VH a selection that on plaque VHs and monomeric following of of have thermal refolding are in good in and properties VHs that the VHs as VHs that have good and thermal refolding the of to VH are to their to VHs applications because of their VH and in and and to library VHs the human VHs with properties were on that the monomeric human VHs in that VHs are in of that we have monomeric VHs with and the we had we selection to a of VHs with of in of the VHs because of in the VH to the of the VHs because of the of in the VHs and the that to in the of that to and in and the and in have a in VHs of library that the of the VHs on in that the of library the VHs with because on of the VHs in of and the of VH have been on to in to the in that a of on the of the of the of VHs to that are on VH a of in have a the in significantly to and been in library on are significantly library library the of the of VHs in of to with good and good a library of on a VH the of the VHs in of VH with and to to to human VH library the of VHs. the and in of the human VH of the selection of with properties have been to selection of a library in a of VH to aggregation VHs of selection involving and the the selection to a on the that properties their than However, with the of a selection than the of to with on the that are and are to are engineered in a that the of the to a library with a the phages displaying their and of However, and have and and the selection of in a the with properties are identified the that in selection a that a to that the are However, the of the on plaque a in of the of that and to the of the to the library size a that a of library on a as on a to to VHs of good properties a of VHs. However, to the a library that with with good the identified on the of plaque incubation plaque size and selection to the of and as human with the in the selection of a involving and the plaque size and VH expression that the of with expression expression of significantly human VH are on human VH are a promising of However, human VH are and the human VH we 15 monomeric human VHs with and a phage-displayed naïve human VH a selection that on plaque VHs and monomeric following of of have thermal refolding are in good in and properties VHs that the VHs as VHs that have good and thermal refolding the of to VH are to their to VHs applications because of their VH and in and and to library VHs the human VHs with properties were on that the monomeric human VHs in that VHs are in of that we have monomeric VHs with and the we had we selection to a of VHs with of in of the VHs because of in the VH to the of the VHs because of the of in the VHs and the that to in the of that to and in and the and in have a in VHs of library that the of the VHs on in that the of library the VHs with because on of the VHs in of and the of VH have been on to in to the in that a of on the of the of the of VHs to that are on VH a of in have a the in significantly to and been in library on are significantly library library the of the of VHs in of to with good and good a library of on a VH the of the VHs in of VH with and to to to human VH library the of VHs. the and in of the human VH of the selection of with properties have been to selection of a library in a of VH to aggregation VHs of selection involving and the the selection to a on the that properties their than However, with the of a selection than the of to with on the that are and are to are engineered in a that the of the to a library with a the phages displaying their and of However, and have and and the selection of in a the with properties are identified the that in selection a that a to that the are However, the of the on plaque a in of the of that and to the of the to the library size a that a of library on a as on a to to VHs of good properties a of VHs. However, to the a library that with with good the identified on the of plaque incubation plaque size and selection to the of and as human with the in the selection of a involving and the plaque size and VH expression that the of with expression expression of significantly We and and and We are to and with