Antibody Humanization Using Monovalent Phage Display

Antibody humanization often requires the replacement of key residues in the framework regions with corresponding residues from the parent non-human antibody. These changes are in addition to grafting of the antigen-binding loops. Although guided by molecular modeling, assessment of which framework changes are beneficial to antigen binding usually requires the analysis of many different antibody mutants. Here we describe a phage display method for optimizing the framework of humanized antibodies by random mutagenesis of important framework residues. We have applied this method to humanization of the anti-vascular endothelial growth factor murine monoclonal antibody A4.6.1. Affinity panning of a library of humanized A4.6.1 antibody mutants led to the selection of one variant with greater than 125-fold enhanced affinity for antigen relative to the initial humanized antibody with no framework changes. A single additional mutation gave a further 6-fold improvement in binding. The affinity of this variant, 9.3 nm, was only 6-fold weaker than that of a murine/human chimera of A4.6.1. This method provides a general means of rapidly selecting framework mutations that improve the binding of humanized antibodies to their cognate antigens and may prove an attractive alternative to current methods of framework optimization based on cycles of site-directed mutagenesis. Antibody humanization often requires the replacement of key residues in the framework regions with corresponding residues from the parent non-human antibody. These changes are in addition to grafting of the antigen-binding loops. Although guided by molecular modeling, assessment of which framework changes are beneficial to antigen binding usually requires the analysis of many different antibody mutants. Here we describe a phage display method for optimizing the framework of humanized antibodies by random mutagenesis of important framework residues. We have applied this method to humanization of the anti-vascular endothelial growth factor murine monoclonal antibody A4.6.1. Affinity panning of a library of humanized A4.6.1 antibody mutants led to the selection of one variant with greater than 125-fold enhanced affinity for antigen relative to the initial humanized antibody with no framework changes. A single additional mutation gave a further 6-fold improvement in binding. The affinity of this variant, 9.3 nm, was only 6-fold weaker than that of a murine/human chimera of A4.6.1. This method provides a general means of rapidly selecting framework mutations that improve the binding of humanized antibodies to their cognate antigens and may prove an attractive alternative to current methods of framework optimization based on cycles of site-directed mutagenesis. Monoclonal antibodies (mAbs) 1The abbreviations used are: mAb, monoclonal antibody; CDR, complementarity-determining region; VL, light chain variable region; VLκI, kappa light chain variable region subgroup I; VH, heavy chain variable region; VHIII, heavy chain variable region subgroup III; VEGF, vascular endothelial growth factor; MES, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline. 1The abbreviations used are: mAb, monoclonal antibody; CDR, complementarity-determining region; VL, light chain variable region; VLκI, kappa light chain variable region subgroup I; VH, heavy chain variable region; VHIII, heavy chain variable region subgroup III; VEGF, vascular endothelial growth factor; MES, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline. have enormous potential as therapeutic agents; however, most mAbs are derived from murine or other non-human sources, which severely limits their clinical efficacy. In addition to the immunogenicity of rodent mAbs when administered to humans (1Jaffers G.J. Fuller T.C. Cosimi A.B. Russell P.S. Winn H.J. Colvin R.B. Transplantation. 1986; 41: 572-578Crossref PubMed Scopus (182) Google Scholar, 2Shawler D.L. Bartholomew R.M. Smith L.M. Dillman R.O. J. Immunol. 1985; 135: 1530-1535PubMed Google Scholar, 3Miller R.A. Oseroff A.R. Stratte P.T. Levy R. Blood. 1983; 62: 988-995Crossref PubMed Google Scholar), further limitations arise from weak recruitment of effector function (4Riechmann L. Clark M. Waldmann H. Winter G. Nature. 1988; 332: 323-327Crossref PubMed Scopus (1272) Google Scholar, 5Junghans R.P. Waldmann T.A. Landolfi N.F. Avdalovic N.M. Schneider W.P. Queen C. Cancer Res. 1990; 50: 1495-1502PubMed Google Scholar) and rapid clearance from serum (6Hakimi J. Chizzonite R. Luke D.R. Familletti P.C. Bailon P. Kondas J.A. Pilson R.S. Lin P. Weber D.V. Spence C. Mondini L.J. Tsien W.H. Levin J.L. Gallati V.H. Korn L. Waldmann T.A. Queen C. Benjamin W.R. J. Immunol. 1991; 147: 1352-1359PubMed Google Scholar, 7Stephens S. Emtage S. Vetterlein O. Chaplin L. Bebbington C. Nesbitt A. Sopwith M. Athwal D. Novak C. Bodmer M. Immunology. 1995; 85: 668-674PubMed Google Scholar). As a means of circumventing these deficiences, the antigen-binding properties of murine mAbs can be conferred to human antibodies through a process known as antibody “humanization” (4Riechmann L. Clark M. Waldmann H. Winter G. Nature. 1988; 332: 323-327Crossref PubMed Scopus (1272) Google Scholar,8Jones P.T. Dear P.H. Foote J. Neuberger M.S. Winter G. Nature. 1986; 321: 522-525Crossref PubMed Scopus (1047) Google Scholar). A humanized antibody contains the amino acid sequences from the six complementarity-determining regions (CDRs) of the parent murine mAb, which are grafted onto a human antibody framework. For this reason, humanization of non-human antibodies is commonly referred to as CDR grafting. The low content of non-human sequence in humanized antibodies (∼5%) has proven effective in both reducing the immunogenicity and prolonging the serum half-life in humans (7Stephens S. Emtage S. Vetterlein O. Chaplin L. Bebbington C. Nesbitt A. Sopwith M. Athwal D. Novak C. Bodmer M. Immunology. 1995; 85: 668-674PubMed Google Scholar, 9Baselga J. Tripathy D. Mendelsohn J. Baughman S. Benz C.C. Dantis L. Sklarin N.T. Seidman A.D. Hudis C.A. Moore J. Rosen P.P. Twaddell T. Henderson I.C. Norton L. J. Clin. Oncol. 1996; 14: 737-744Crossref PubMed Scopus (1237) Google Scholar). Unfortunately, simple grafting of CDR sequences often yields humanized antibodies that bind antigen much more weakly than the parent murine mAb (4Riechmann L. Clark M. Waldmann H. Winter G. Nature. 1988; 332: 323-327Crossref PubMed Scopus (1272) Google Scholar, 10Queen C. Schneider W.P. Selick H.E. Payne P.W. Landolfi N.F. Duncan J.F. Avdalovic N.M. Levitt M. Junghans R.P. Waldmann T.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 10029-10033Crossref PubMed Scopus (544) Google Scholar, 11Kettleborough C.A. Saldanha J. Heath V.J. Morrison C.J. Bendig M.M. Protein Eng. 1991; 4: 773-783Crossref PubMed Scopus (174) Google Scholar, 12Tempest P.R. Bremner P. Lambert M. Taylor G. Furze J.M. Carr F.J. Harris W.J. Biotechnology. 1991; 9: 266-271Crossref PubMed Scopus (149) Google Scholar, 13Carter P. Presta L. Gorman C.M. Ridgway J.B. Henner D. Wong W.L. Rowland A.M. Kotts C. Carver M.E. Shepard H.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4285-4289Crossref PubMed Scopus (1527) Google Scholar, 14Presta L.G. Lahr S.J. Shields R.L. Porter J.P. Gorman C.M. Fendly B.M. Jardieu P.M. J. Immunol. 1993; 151: 2623-2632PubMed Google Scholar, 15Eigenbrot C. Gonzalez T. Mayeda J. Carter P. Werther W. Hotaling T. Fox J. Kessler J. Proteins. 1994; 18: 49-62Crossref PubMed Scopus (62) Google Scholar, 16Pulito V.L. Roberts V.A. Adair J.R. Rothermel A.L. Collins A.M. Varga S.S. Martocello C. Bodmer M. Jolliffe L.K. Zivin R.A. J. Immunol. 1996; 156: 2840-2850PubMed Google Scholar), and decreases in affinity of up to several hundredfold have been reported (13Carter P. Presta L. Gorman C.M. Ridgway J.B. Henner D. Wong W.L. Rowland A.M. Kotts C. Carver M.E. Shepard H.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4285-4289Crossref PubMed Scopus (1527) Google Scholar, 14Presta L.G. Lahr S.J. Shields R.L. Porter J.P. Gorman C.M. Fendly B.M. Jardieu P.M. J. Immunol. 1993; 151: 2623-2632PubMed Google Scholar, 15Eigenbrot C. Gonzalez T. Mayeda J. Carter P. Werther W. Hotaling T. Fox J. Kessler J. Proteins. 1994; 18: 49-62Crossref PubMed Scopus (62) Google Scholar). To restore high affinity, the antibody must be further engineered to fine tune the structure of the antigen-binding loops. This is achieved by replacing key residues in the framework regions of the antibody variable domains with the matching sequence from the parent murine antibody. These framework residues are usually involved in supporting the conformation of the CDR loops (17Chothia C. Lesk A.M. Tramontano A. Levitt M. Smith-Gill S.J. Air G. Sheriff S. Padlan E.A. Davies D. W.R. P.M. S. P.M. Nature. 1989; PubMed Scopus Google Scholar), framework residues may the antigen A.R. J. 1991; PubMed Scopus Google Scholar). and Lesk C. Lesk A.M. J. PubMed Scopus Google Scholar) the of framework residues to CDR and a of framework residues that can antigen binding was by Foote and Winter J. Winter G. J. 1992; PubMed Scopus Google Scholar). These a of residues that can to CDR Although antigen affinity from the of residues a humanized antibody to the corresponding parent murine this is the of immunogenicity by further of murine from a therapeutic is to framework changes to the that a high affinity humanized antibody. As in the (4Riechmann L. Clark M. Waldmann H. Winter G. Nature. 1988; 332: 323-327Crossref PubMed Scopus (1272) Google Scholar, 10Queen C. Schneider W.P. Selick H.E. Payne P.W. Landolfi N.F. Duncan J.F. Avdalovic N.M. Levitt M. Junghans R.P. Waldmann T.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 10029-10033Crossref PubMed Scopus (544) Google Scholar, 11Kettleborough C.A. Saldanha J. Heath V.J. Morrison C.J. Bendig M.M. Protein Eng. 1991; 4: 773-783Crossref PubMed Scopus (174) Google Scholar, 12Tempest P.R. Bremner P. Lambert M. Taylor G. Furze J.M. Carr F.J. Harris W.J. Biotechnology. 1991; 9: 266-271Crossref PubMed Scopus (149) Google Scholar, 13Carter P. Presta L. Gorman C.M. Ridgway J.B. Henner D. Wong W.L. Rowland A.M. Kotts C. Carver M.E. Shepard H.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4285-4289Crossref PubMed Scopus (1527) Google Scholar, 14Presta L.G. Lahr S.J. Shields R.L. Porter J.P. Gorman C.M. Fendly B.M. Jardieu P.M. J. Immunol. 1993; 151: 2623-2632PubMed Google Scholar, 15Eigenbrot C. Gonzalez T. Mayeda J. Carter P. Werther W. Hotaling T. Fox J. Kessler J. Proteins. 1994; 18: 49-62Crossref PubMed Scopus (62) Google Scholar, 16Pulito V.L. Roberts V.A. Adair J.R. Rothermel A.L. Collins A.M. Varga S.S. Martocello C. Bodmer M. Jolliffe L.K. Zivin R.A. J. Immunol. 1996; 156: 2840-2850PubMed Google Scholar), the of framework residues that antigen binding is the most and of antibody Although these framework residues are derived from the by Foote and Winter J. Winter G. J. 1992; PubMed Scopus Google Scholar), a of changes usually to these often from one humanized antibody to the a molecular of the humanized antibody can the of mutations must be by this is achieved by a of mutants with framework residues by their murine These are for antigen and mutations that affinity are a As a means of antibody a of different have been A method is to the human framework most in sequence to that of the murine antibody of C. Schneider W.P. Selick H.E. Payne P.W. Landolfi N.F. Duncan J.F. Avdalovic N.M. Levitt M. Junghans R.P. Waldmann T.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 10029-10033Crossref PubMed Scopus (544) Google Scholar, 11Kettleborough C.A. Saldanha J. Heath V.J. Morrison C.J. Bendig M.M. Protein Eng. 1991; 4: 773-783Crossref PubMed Scopus (174) Google Scholar, 12Tempest P.R. Bremner P. Lambert M. Taylor G. Furze J.M. Carr F.J. Harris W.J. Biotechnology. 1991; 9: 266-271Crossref PubMed Scopus (149) Google Scholar). In this the of the humanized and parent murine framework is and that a key framework to be be that a single framework can have a on antigen binding C.A. Saldanha J. Heath V.J. Morrison C.J. Bendig M.M. Protein Eng. 1991; 4: 773-783Crossref PubMed Scopus (174) Google Scholar). alternative humanization was by Padlan E.A. Immunol. 1991; PubMed Scopus Google than grafting the onto a human the murine variable domains are and framework residues are with human residues on the of the antibody as by to are for These residues are for antigen binding most to to potential the other residues are as the murine sequence these are most to the structure antigen of the antibody are of low immunogenicity are in from the Although this has been used to different murine mAbs C.A. S.J. Lambert J.M. A.R. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google S. M. R. Protein Eng. 1994; PubMed Scopus Google Scholar), to be these antibodies are residues can a as a by a P.M. J. Immunol. 1985; 135: Google Scholar). In to these other an alternative is to antibodies only a single human of the sequence of the parent murine antibody. This method has been used to a of murine antibodies (13Carter P. Presta L. Gorman C.M. Ridgway J.B. Henner D. Wong W.L. Rowland A.M. Kotts C. Carver M.E. Shepard H.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4285-4289Crossref PubMed Scopus (1527) Google Scholar, 14Presta L.G. Lahr S.J. Shields R.L. Porter J.P. Gorman C.M. Fendly B.M. Jardieu P.M. J. Immunol. 1993; 151: 2623-2632PubMed Google Scholar, 15Eigenbrot C. Gonzalez T. Mayeda J. Carter P. Werther W. Hotaling T. Fox J. Kessler J. Proteins. 1994; 18: 49-62Crossref PubMed Scopus (62) Google Shepard H.M. Presta L. P.C. M. Carter P. J. 1992; PubMed Scopus Google Scholar) a framework derived from sequences of the most human subgroup and subgroup E.A. H.M. C. of of of Scholar). of the most human potential immunogenicity of the humanized antibody and with one framework. In this framework has been to yields of antibody when in or an important for antibodies that are to for clinical on the from these and other antibody we have that the framework residues that most often antigen binding are derived from a of only residues. We have this the of a random mutagenesis to antibody this of framework residues and by display of the library of antibody on the of phage S. R. J.A. Proteins. 1990; PubMed Scopus Google Scholar, J.A. 1991; PubMed Scopus Google Scholar), framework sequences can be Here we describe the of this to the humanization of the murine antibody A4.6.1 J. 1992; PubMed Scopus Google Scholar, J. M. Nature. 1993; PubMed Scopus Google Scholar), an antibody that to vascular endothelial growth factor The murine mAb A4.6.1 binding and has been J. 1992; PubMed Scopus Google Scholar, J. M. Nature. 1993; PubMed Scopus Google Scholar). The variant of humanized was by site-directed mutagenesis a of (13Carter P. Presta L. Gorman C.M. Ridgway J.B. Henner D. Wong W.L. Rowland A.M. Kotts C. Carver M.E. Shepard H.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4285-4289Crossref PubMed Scopus (1527) Google Scholar) that for a human light chain and human heavy chain The A4.6.1 CDR sequences to the sequence of E.A. H.M. C. of of of Scholar), for which we to both sequence and C. Lesk A.M. J. PubMed Scopus Google Scholar) residues The sequence was the S. R. J.A. Proteins. 1990; PubMed Scopus Google Scholar, J.A. 1991; PubMed Scopus Google Scholar). This the initial humanized A4.6.1 with the of the heavy chain to the of the is in to a for display of L.J. M. Henner Biotechnology. 1991; 9: PubMed Scopus Google Scholar). and a and of an the antibody heavy chain This of both heavy chain or heavy of a was with or in of These of M. H. G. PubMed Scopus Google Scholar) and for The and for in of for and for The was applied to a and with of The was with of acid and with of and yields of by and by amino acid The humanized A4.6.1 library was by site-directed mutagenesis to the method of T.A. J. 1991; PubMed Scopus Google Scholar). A of and was for as the mutagenesis sequence is to E.A. H.M. C. of of of This was to by The for and and and To heavy chain and with a single from and by of or with of of with sequence to the of and the of was to of the and and the was to of for for to the and The on a and from the in The in and in The was of framework and VH, and was in was achieved by additional of the and in the light chain or the and was from of these with the these for of the light chain framework sequence and to the or a of and used to heavy chain framework of the The and The of was greater than the of sequences in the the humanized A4.6.1 in the in of and phage J. J. PubMed Scopus Google Scholar). from by with a and in of in was onto a The was and this in addition to an was with for and with of to with serum and was to the and the phage with of and with of of this was used to the of phage The phage from the for in the selection A of of selection was which and S. A.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). and for binding of humanized A4.6.1 to by R. A. L. J. Immunol. 1991; PubMed Scopus Google Scholar) on a was on the amino of humanized A4.6.1 was by of in PBS, the a of binding was from the by with of by a simple binding initial humanized A4.6.1 was in which the from A4.6.1 grafted onto a human framework. other residues in as the human of this variant to was weak that was on the relative affinity of other weakly binding humanized A4.6.1 the for binding of was This with an affinity of for a of the and domains from murine A4.6.1 and human binding of to was relative to the The of binding by variant the important of the framework in the CDR loops for antigen binding. on the from a of murine antibodies onto a human framework (13Carter P. Presta L. Gorman C.M. Ridgway J.B. Henner D. Wong W.L. Rowland A.M. Kotts C. Carver M.E. Shepard H.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4285-4289Crossref PubMed Scopus (1527) Google Scholar, 14Presta L.G. Lahr S.J. Shields R.L. Porter J.P. Gorman C.M. Fendly B.M. Jardieu P.M. J. Immunol. 1993; 151: 2623-2632PubMed Google Scholar, 15Eigenbrot C. Gonzalez T. Mayeda J. Carter P. Werther W. Hotaling T. Fox J. Kessler J. Proteins. 1994; 18: 49-62Crossref PubMed Scopus (62) Google Scholar, Shepard H.M. Presta L. P.C. M. Carter P. J. 1992; PubMed Scopus Google Scholar), we have that most framework as to antigen are to of the residues in and We that this as the for a to selecting framework we a to the humanization of mAb A4.6.1 by these key framework residues and the library of on the of of residues and in the light chain and and from the heavy chain to the selection of the murine human or sequences commonly in other human and murine that of these residues was to a the human or A4.6.1 framework of additional amino commonly in other human and murine framework sequences for the that additional may to the selection of binding heavy chain framework residues in a to the human and murine A4.6.1 framework and are in a to the antigen-binding The of and are in antibody and their potential in the conformation of and is known C.A. Saldanha J. Heath V.J. Morrison C.J. Bendig M.M. Protein Eng. 1991; 4: 773-783Crossref PubMed Scopus (174) Google Scholar, 13Carter P. Presta L. Gorman C.M. Ridgway J.B. Henner D. Wong W.L. Rowland A.M. Kotts C. Carver M.E. Shepard H.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4285-4289Crossref PubMed Scopus (1527) Google Scholar, Shepard H.M. Presta L. P.C. M. Carter P. J. 1992; PubMed Scopus Google Scholar, A. C. Lesk A.M. J. 1990; PubMed Scopus Google Scholar). the other the of and are to has been that these residues can antigen binding C. Gonzalez T. Mayeda J. Carter P. Werther W. Hotaling T. Fox J. Kessler J. Proteins. 1994; 18: 49-62Crossref PubMed Scopus (62) Google Scholar), to antigen in of their in sequence and we and that only of this be human or murine A4.6.1 residues and only to the and A4.6.1 We have and in antibody the and A4.6.1 sequences and have been as important for CDR conformation J. Winter G. J. 1992; PubMed Scopus Google Scholar), we to in the A of have been for the display of antibody on the of phage G. A.D. Immunol. 1994; PubMed Scopus Google Scholar). These the display of or single chain variable as to the or of We to a to that by L.J. M. Henner Biotechnology. 1991; 9: PubMed Scopus Google Scholar) in which a is as a This has single chain variable have no to the of which can selection of the to the of the a potential of that from the of of a on S. R. J.A. Proteins. 1990; PubMed Scopus Google Scholar, J.A. 1991; PubMed Scopus Google Scholar). A in the humanized A4.6.1 library was that residues for the and in the of that of of these framework can be achieved only through the of as the of the of mutagenesis decreases the of mutants that sequence derived from of the To this we library The was to different mutagenesis for of the framework This was simple to the of the light chain framework different was beneficial in that the for from the mutagenesis we from This of and with a single than The mutagenesis only onto different from the humanized A4.6.1 library based on binding to of as by for phage from a up to the of affinity one additional of to framework residues These in the of the the the framework in and and residues and as the human or murine A4.6.1 This that framework may from the the human and parent murine framework we have this by the mutants. and murine A4.6.1 antibodies are The of for sequence is in in the sequences of selection of the human framework sequence as in other amino acid sequences the and of these in addition to framework and the human sequence and the light chain sequence and bind when in a phage J.A. J. 1994; PubMed Scopus (62) Google Scholar). We have the of heavy or light chain sequence with other and J. A. and these are for on the of enhanced can often be by reducing the of cycles or by on and in and from by affinity yields of for these from to The affinity of of these for antigen was by on a of these binding that the the affinity for of the library was for the binding The for was nm, 125-fold than for which contains no framework changes The other weaker binding to VEGF, to a of for the for nm, was only weaker than that of and only one of this was This may have been to a of and as was the when the of this variant was on and are and the improvement in antigen affinity the initial humanized variant, binding of to was weaker than that of a the murine A4.6.1 This that further optimization of the humanized framework be through additional the residues by Foote and Winter J. Winter G. J. 1992; PubMed Scopus Google Scholar), only residues and in the A4.6.1 human framework these in A molecular of the humanized A4.6.1 that the and the conformation of this amino acid is in most E.A. H.M. C. of of of Scholar) is in A4.6.1. a was this in the of of binding for this variant, a further 6-fold improvement in the for the of in antibody A4.6.1. The for was 6-fold that of the In to no improvement in the binding affinity of was for replacement of or with the corresponding from murine A4.6.1. A to antibody humanization has a the site-directed method in that a much of mutants can be with the rapidly from a phage display library based on relative antigen We have applied an to humanization of the murine mAb a antibody known to and as an therapeutic J. M. Nature. 1993; PubMed Scopus Google Scholar). A library of was and from this a of that than the CDR variant and The binding variant in the was the and of one additional mutation a humanized A4.6.1 variant with an affinity 6-fold that of the Although framework these to the in antigen binding. The in the relative that framework changes to antigen binding. As a 6-fold improvement in antigen affinity was with replacing with the corresponding amino acid in A4.6.1 of the improvement in affinity was to an in the that may a in the antibody structure a conformation more for antigen binding. mutations that antigen affinity to changes in the for binding. of and a improvement in affinity for of residues and with the A4.6.1 of to the A4.6.1 sequence on binding with than with the in both the A4.6.1 and human of other humanized A4.6.1 that the in a improvement in to antigen by this or to a in the conformation of variant has sequence relative to the has a This that these have on antigen binding. Although the of changes and with binding for other is that the more has on binding. Harris L.J. J. Lin H. J. 1996; PubMed Scopus Google Scholar) reported a to the humanization of murine mAb the method used in that from that in several key For we used the human framework that has been used in antibody (13Carter P. Presta L. Gorman C.M. Ridgway J.B. Henner D. Wong W.L. Rowland A.M. Kotts C. Carver M.E. Shepard H.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4285-4289Crossref PubMed Scopus (1527) Google Scholar, 14Presta L.G. Lahr S.J. Shields R.L. Porter J.P. Gorman C.M. Fendly B.M. Jardieu P.M. J. Immunol. 1993; 151: 2623-2632PubMed Google Scholar, 15Eigenbrot C. Gonzalez T. Mayeda J. Carter P. Werther W. Hotaling T. Fox J. Kessler J. Proteins. 1994; 18: 49-62Crossref PubMed Scopus (62) Google Scholar, Shepard H.M. Presta L. P.C. M. Carter P. J. 1992; PubMed Scopus Google Scholar), a human framework based on to the sequence of the parent murine we humanized the framework region for only framework residues that have been to be important to antigen binding. In humanized only the residues that in sequence the murine mAb and human the of residues the of humanization We have that phage display methods can be applied to the humanization of a murine monoclonal antibody. to the humanization of mAb A4.6.1 led to selection of a humanized variant that greater than 125-fold than the variant with no framework changes. additional mutation the of the phage was to improve the affinity to 6-fold that of murine A4.6.1. be however, that usually replacement in antibody the of this in most antibody E.A. H.M. C. of of of Scholar). this we that the general phage method usually for the selection of binding humanized the of this in with (13Carter P. Presta L. Gorman C.M. Ridgway J.B. Henner D. Wong W.L. Rowland A.M. Kotts C. Carver M.E. Shepard H.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4285-4289Crossref PubMed Scopus (1527) Google Scholar, 14Presta L.G. Lahr S.J. Shields R.L. Porter J.P. Gorman C.M. Fendly B.M. Jardieu P.M. J. Immunol. 1993; 151: 2623-2632PubMed Google Scholar, 15Eigenbrot C. Gonzalez T. Mayeda J. Carter P. Werther W. Hotaling T. Fox J. Kessler J. Proteins. 1994; 18: 49-62Crossref PubMed Scopus (62) Google Scholar, Shepard H.M. Presta L. P.C. M. Carter P. J. 1992; PubMed Scopus Google Scholar), the of a single human framework as a for humanized We for with the for for amino acid and the