Patent Publication Number: US-2019194349-A1

Title: Antibodies selective for cells presenting erbb2 at high density

Description:
RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 13/992,051, filed on Jun. 19, 2013, which is a national stage application of PCT International Application No. PCT/CA2011/050747, filed Dec. 2, 2011, which claims priority to U.S. Provisional Application Ser. No. 61/419,983, filed Dec. 6, 2010, each of which is incorporated by reference herein in its entirety. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to antibodies having therapeutic and diagnostic utility. More particularly, the present invention relates to antibodies that bind selectively to cells that present erbB2 at abnormally high density. The antibodies are useful therapeutically and diagnostically in the fields of oncology and other diseases. 
     BACKGROUND TO THE INVENTION 
     Drugs for the treatment of cancer and other diseases have a so-called “therapeutic window”. In the case of cancer, the therapeutic window defines the drug dosage that can kill cancer cells preferentially to normal cells, thereby establishing a safety range for the use of the drug. The therapeutic window for conventional chemotherapeutics is narrow with, in many cases, significant adverse effects coinciding with marginal slowing of tumour growth. Targeted treatments that spare normal cells are urgently needed. 
     Therapeutic antibodies form a newer class of cancer therapies that specifically target an antigen presented on the surface of cancer cells. When the target surface protein is unique to the cancer cell, adverse antibody effects on normal cells can be avoided. However, for the majority of antigens, target expression is not restricted completely to tumour cells, with some normal cells also expressing the antigen. In these cases, the antibody may have an effect on normal cells as well as tumor cells, leading to “on-target, off-tissue” adverse events. In the case of the ErbB2 antigen, because it is present on the surface of normal cells in cardiac tissue as well as on breast cancer cells, the clinical use of erbB2-targeting therapeutics is associated with adverse events that include cardiac toxicity. The incidence and degree of adverse events is considerably increased when given in combination with chemotherapeutics, particularly anthracyclines. 
     Considering the efficacy of anti-erbB2 therapies in treating patients that overexpress erbB2, the risk associated with cardiac toxicity is currently considered acceptable when managed properly. The risk of anti-erbB2 therapy-associated cardiac toxicity can be reduced by avoiding co-administration with anthracyclines, or by administering anti-erbB2 therapy and chemotherapeutics consecutively. 
     Currently, routine LVEF monitoring is performed to assess cardiac function in patients that are prescribed anti-erbB2 therapies, especially when given in combination with anthracyclines. This is a time-consuming and expensive process which requires patient compliance. 
     Efforts to improve upon erbB2 antibodies are aimed at generating antibodies having even greater affinity for the target antigen. For instance, U.S. Pat. No. 7,435,797 issued Oct. 14, 2008 describes a variety of trastuzumab analogs in which amino acid substitution is used to further increase target affinity. Substitutions are made at sites within different complementarity determining regions of trastuzumab. 
     It would be desirable to provide an erbB2 antibody that is useful to treat subjects presenting with erbB2 over-expressing disease cells, while avoiding significant interaction with cardiac and other normal cells that also present the erbB2 antigen. 
     It is an object of the present invention to provide therapeutic antibodies, and fragments and conjugates thereof that bind effectively to a given target only when that target is presented at a relatively higher density characteristic of a disease state. 
     It is a further object of the present invention to provide such antibodies, fragments and conjugates in pharmaceutical compositions, particularly for therapeutic and diagnostic use. 
     It is a further object of the present invention to provide a method useful, in a subject in need thereof, to control the growth of disease cells that present erbB2 at a density greater than normal erbB2 density, while avoiding or minimizing adverse effects on normal cells. 
     SUMMARY OF THE INVENTION 
     In one aspect, the present invention provides an isolated, erbB2 antibody or bivalent fragment thereof that binds preferentially to target cells that present erbB2 at a density above a normal erbB2 density. Cells that present erbB2 at a density greater than normal erbB2 density are disease cells, including cancer cells such as breast cancer cells, that over-express the her-2 gene, and manifest on their surface a greater number of erbB2 proteins than cells that express the her-2 gene at normal levels. 
     The antibodies of the present invention, and their bivalent fragments, display a preference for binding to disease cells having the higher erbB2 density, and show reduced and desirably minimal or negligible binding to normal cells having a normal erbB2 density. 
     The present antibodies and their bivalent binding fragments thus are well suited for use in reducing or eradicating high density erbB2 disease cells while minimizing or avoiding effects on normal cells, thereby reducing adverse events in subjects receiving erbB2 antibody therapy. 
     In one aspect, the erbB2 antibody comprises a heavy chain and a light chain, each chain having a constant region and a variable region, each variable region comprising framework regions and complementarity determining regions (CDRs), wherein the CDRs have an amino acid sequence set forth below: 
                            For the heavy chain:           (SEQ ID No. 1)                             CDR1   GFNIKDTYIH                                     (SEQ ID No. 2)                             CDR2   RIYPTNGY 57 TR 59 YADSVKG                                     (SEQ ID No. 3)                             CDR3   WGGDGFYAMDY                                     For the light chain:           (SEQ ID No. 4)                             CDR1   RASQDVN 30 TAVA                                     (SEQ ID No. 5)                             CDR2   SASF 53 LYS                                     (SEQ ID No. 6)                             CDR3   QQHY 92 TTPPT            
wherein at least one of Y57, R59, N30, F53, and Y92 is replaced by a substituting amino acid that reduces the erbB2 binding affinity of said antibody. In embodiments, the substituting amino acid(s) are selected to confer on the antibody a binding affinity (KD) for erbB2 that is about 10 fold or more weaker than the erbB2 binding affinity of trastuzumab.
 
     In embodiments, the present invention provides an ErbB2 antibody comprising a heavy chain and a light chain, each chain having a constant region and a variable region, wherein the light chain variable region comprises the sequence of SEQ ID No. 7 and the heavy chain variable region comprises the sequence of SEQ ID No. 8, wherein at least one of Y57, R59, N30, F53, and Y92 is replaced by a substituting amino acid that reduces the erbB2 binding affinity of said antibody. 
     In other embodiments, the substituting amino acid is selected to reduce erbB2 binding affinity of the antibody or bivalent fragment to a level that substantially eliminates binding to cells presenting erbB2 at a normal erbB2 density, and retains effective binding at targeted disease cells that present erbB2 at a greater density relative to normal cell erbB2 density. 
     In still other embodiments, the antibody or bivalent fragment is a variant of trastuzumab having one or more substitutions at the residues identified herein. 
     In another of its aspects, the present invention provides conjugates, i.e., immunoconjugates, comprising an antibody or bivalent fragment thereof according to the present invention and, conjugated therewith, an agent useful to treat or diagnose cells presenting erbB2 at a density characteristic of disease cells. 
     In a further aspect, the present invention provides medically useful compositions comprising an antibody, bivalent fragment thereof or immunoconjugate thereof according to the present invention, in combination with a medically acceptable carrier, such as a pharmaceutically acceptable carrier or a diagnostically useful carrier. 
     In a related aspect, the present invention provides a method for treating a subject having disease cells that present erbB2 at a density greater than the erbB2 density on normal cells, comprising the step of administering to the subject an effective amount of an antibody, bivalent fragment thereof, or an immunoconjugate of the present invention. 
     Subjects so treated will manifest adverse events that are fewer in number and/or severity given the reduced affinity of the present antibodies for normal cells and tissue. 
     These and other aspects of the present invention are now described in greater detail with reference to the accompanying drawings, in which: 
    
    
     
       REFERENCE TO THE FIGURES 
         FIG. 1  shows the in vitro effect of substitution on antibody affinity for erbB2; 
         FIG. 2A  is a graph showing binding of antibodies to cell surface erbB2 present on SkBr3 tumour cells at 0.1 and 1 ug/ml mAb, and compared to wt mAb (2-1 wt, set arbitrarily to 100%); 
         FIG. 2B  is a graph showing binding of antibodies to cell surface erbB2 present on BT474 tumour cells at 0.1 and 1 ug/ml mAb, and compared to wt mAb (2-1 wt, set arbitrarily to 100%); 
         FIG. 2C  is a graph showing binding of antibodies to cell surface erbB2 present on human cardiac myocytes (HCM) at 0.1 and 1 ug/ml mAb, and compared to wt mAb (2-1 wt, set arbitrarily to 100%); and 
         FIG. 3  is a graph representing binding selectivity of antibodies. The ratio of antibody binding to SKBr3 cells or BT474 cells (overexpressing erbB2) divided by their respective binding to normal HCM (at 0.1 mg/ml) was calculated and compared to that seen with 2-1 wild type antibody (set arbitrarily to 1). 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS 
     As used herein, the term “erbB2” refers to any protein that comprises the expressed and processed product of the her-2 gene, wherein the protein is designated as UniProtKB/Swiss-Prot P04626-1, including antibody-binding variants thereof. 
     The present invention relates to erbB2 antibodies and bivalent fragments thereof that display a preference for binding to disease cells presenting erbB2 at a density greater than normal cells. On cells that present erbB2, the normal density of erbB2 is generally less than about 10,000 erbB2 molecules per cell, and is usually less than about 1,000 erbB2 molecules per cell. ErbB2-presenting disease cells, on the other hand, present erbB2 at a density generally greater than 10,000 erbB2 molecules per cell, and usually greater than about 100,000 erbB2 molecules per cell. Generally, the erbB2 density is thus about 10 3  or less on normal cells, and about 10 5  or more on disease cells. The actual number of erbB2 molecules on any given cell can be determined by established methods, including the antibody based radiolabeled binding or flow cytometry binding to live cells herein exemplified. The binding avidity of the present antibodies is greater for the higher erbB2 density disease cells than for the lower erbB2 density normal cells. This greater avidity is revealed conveniently using techniques established for determining affinity constants for antibody-target interactions, also as exemplified herein. 
     In embodiments, the present erbB2 antibodies have a binding affinity for erbB2 that is about 10 fold or more weaker than the erbB2 binding affinity of trastuzumab. Desirably, the binding affinity of the antibody for erbB2 is about 15-fold, 20-fold, 25-fold, and preferably 30-fold or more weaker than the erbB2 binding affinity of trastuzumab. In absolute terms, and given an erbB2 binding affinity of about 0.03 nM for trastuzumab, the present antibodies incorporate amino acid substitution(s) that reduce their erbB2 binding affinity to about 0.1 nM and weaker, e.g., to an erbB2 binding affinity that is in the range from 0.1 nM to 100 nM, more desirably 0.5 nM to 100 nM, such as 0.7 nM to 100 nM, or 1 nM to 100 nM, or 1 nM to 75 nM, or 1 nM to 50 nM, or 1 nM to 25 nM, or 1 nM to 10 nM, or 1 nM to 5 nM. 
     In embodiments, the antibody is an intact antibody comprising features common to all natural antibodies, and thus comprises a heavy chain and a light chain, each chain having a constant region and a variable region, each variable region comprising framework regions (FRs) and complementarity determining regions (CDRs). In the alternative, the antibody is provided as a bivalent fragment, i.e., an antibody fragment comprising both “arms” of an intact antibody, joined through a linker that can be represented by the hinge region of the antibody or any equivalent. Such bivalent fragments include F(ab) 2  fragments and any other bivalent fragment that retains preference for high density erbB2. In particular embodiments, the bivalent fragment is a F(ab′) 2  fragment, generated for instance by papain-based digestion of the parent antibody using standard procedures for digestion and subsequent fragment isolation. In the alternative, the bivalent fragment can be a so-called single chain Fv (scFv), consisting of the variable light and variable heavy antibody domains joined by an amino acid linker, or a bivalent form of a so-called diabody prepared using a 5 amino acid linker such as SGGGG between the light and heavy chain variable domains and a C-terminal cysteine modification to GGC to give a final diabody product as VL-SGGG-VH-GGC. Still other bivalent fragments can be prepared by coupling the light and heavy chain variable domains through thioether linkages such as bis-maleimidomethyl ether (BMME), N,N′-p-phenylene dimaleimide (PDM and N,N′-bismaleimidohexane BMH), to stabilize the F(ab′)2 fragments. 
     In the intact antibody or bivalent fragment, the CDRs comprise or consist of the following amino acid sequences: 
                            For the heavy chain:           (SEQ ID No. 1)                             CDR 1   GFNIKDTYIH                                     (SEQ ID No. 2)                             CDR2   RIYPTNGY 57 TR 59 YADSVKG                                     (SEQ ID No. 3)                             CDR3   WGGDGFYAMDY                                     For the light chain:           (SEQ ID No. 4)                             CDR 1   RASQDVN 30 TAVA                                     (SEQ ID No. 5)                             CDR2   SASF 53 LYS                                     (SEQ ID No. 6)                             CDR3   QQHY 92 TTPPT            
wherein at least one of Y57, R59, N30, F53, and Y92 is replaced by a substituting amino acid that reduces the erbB2 binding affinity of said antibody or bivalent fragment.
 
     The substituting amino acids are most suitably genetically encoded amino acids that are selected desirably, but not essentially, from an amino acid class that is different from the amino acid class to which the parent amino acid belongs. For instance, in the case of Y57, suitable substituting amino acids are those that are not polar/neutral/large amino acids. The selection process can be conducted by applying computer aided tools that couple saturation virtual mutagenesis engines with algorithms for in silico scoring of binding affinities and/or association rates. Amino acid selections can also be made based on the following Table 1: 
     
       
         
           
               
               
               
               
               
               
             
               
                   
               
               
                   
                   
                   
                 Polarity 
                 Charge 
                   
               
               
                 Amino Acid 
                 3 letter 
                 1 letter 
                 (side chain) 
                 (pH 7.4) 
                 Size* 
               
               
                   
               
             
            
               
                 Alanine 
                 Ala 
                 A 
                 nonpolar 
                 neutral 
                 tiny 
               
               
                 Arginine 
                 Arg 
                 R 
                 polar 
                 positive 
                 large 
               
               
                 Asparagine 
                 Asn 
                 N 
                 polar 
                 neutral 
                 small 
               
               
                 Aspartic acid 
                 Asp 
                 D 
                 polar 
                 negative 
                 small 
               
               
                 Cysteine 
                 Cys 
                 C 
                 nonpolar 
                 neutral 
                 small 
               
               
                 Glutamic acid 
                 Glu 
                 E 
                 polar 
                 negative 
                 small 
               
               
                 Glutamine 
                 Gln 
                 Q 
                 polar 
                 neutral 
                 small 
               
               
                 Glycine 
                 Gly 
                 G 
                 nonpolar 
                 neutral 
                 tiny 
               
               
                 Histidine 
                 His 
                 H 
                 polar 
                 neutral (90%) 
                 large 
               
               
                 Isoleucine 
                 Ile 
                 I 
                 nonpolar 
                 neutral 
                 large 
               
               
                 Leucine 
                 Leu 
                 L 
                 nonpolar 
                 neutral 
                 large 
               
               
                 Lysine 
                 Lys 
                 K 
                 polar 
                 positive 
                 large 
               
               
                 Methionine 
                 Met 
                 M 
                 nonpolar 
                 neutral 
                 large 
               
               
                 Phenylalanine 
                 Phe 
                 F 
                 nonpolar 
                 neutral 
                 large 
               
               
                 Proline 
                 Pro 
                 P 
                 non-polar 
                 neutral 
                 small 
               
               
                 Serine 
                 Ser 
                 S 
                 polar 
                 neutral 
                 tiny 
               
               
                 Threonine 
                 Thr 
                 T 
                 polar 
                 neutral 
                 small 
               
               
                 Tryptophan 
                 Trp 
                 W 
                 nonpolar 
                 neutral 
                 bulky 
               
               
                 Tyrosine 
                 Tyr 
                 Y 
                 polar 
                 neutral 
                 large 
               
               
                 Valine 
                 Val 
                 V 
                 nonpolar 
                 neutral 
                 small 
               
               
                   
               
               
                 *based on volume in A 3 , where 50-100 is tiny, 100-150 is small, 150-200 is large and &gt;200 is bulky 
               
            
           
         
       
     
     In embodiments, the heavy chain variable region of the antibody or bivalent fragment incorporates at least one substitution at Y57 or R59. In other embodiments, the heavy chain variable region incorporates substitutions at both Y57 and R59. In an alternative embodiment, the heavy chain variable region is wild type and incorporates no such substitutions, provided there is at least one substitution and optionally two substitutions in the light chain variable region. 
     In embodiments, Y57 is replaced by a substituting amino acid having a side chain that is nonpolar and/or a side chain that is non-neutral and/or a side chain that is not large. Desirably, Y57 is replaced by an amino acid selected from A, C, G, I, L, M, F, W and V; preferably from A, G, I, L and V; and more preferably from A, V, I and L. In a specific embodiment, Y57 is replaced by A57, thus yielding the substitution designated Y57A. 
     In other embodiments, R59 is replaced by a substituting amino acid having a side chain that is nonpolar and/or is charge neutral or negative and/or is not large. Desirably, R59 is replaced by an amino acid having a side chain that is charge neutral or negative, as well as polar, as well as small, and is selected desirably from D and E. In a specific embodiment, R59 is replaced by E59, thus yielding the substitution designated R59E. 
     In embodiments, the light chain variable region of the antibody or bivalent fragment incorporates at least one substitution at N30 or at F53 or at Y92. In other embodiments, when the heavy chain comprises at least one substitution, then the light chain variable region comprises at least two such substitutions, or all three such substitutions. In a specific embodiment, the light chain variable region incorporates substitutions at both F53 and Y92. In another specific embodiment, the light chain variable region incorporates substitution only at N30, only at F53 or only at Y92. In an alternative embodiment, the light chain variable region is wild type and incorporates no such substitutions, provided there is at least one substitution in the heavy chain variable region. 
     When substituted, N30 is replaced by a substituting amino acid having a side chain that is either nonpolar and/or is negative or positive in charge and/or may not be small. In embodiments, N30 is substituted by an amino acid that is not S, and is selected from R, D, E, or K. In a preferred embodiment, N30 is substituted by D, yielding the substitution designated N30D. 
     When substituted, F53 is replaced by a substituting amino acid having a side chain that is either polar and/or is charge positive or negative and/or is not large. In embodiments, F53 is replaced by R, N, D, E, Q, H, K, S, T or Y. In particular embodiments, F53 is replaced by N, Q, H, S, T or Y. In a preferred embodiment, F53 is replaced by N, yielding the substitution designated F53N. 
     When substituted, Y92 is replaced by a substituting amino acid having a side chain that is nonpolar and/or a side chain that is non-neutral and/or is not large. Desirably, Y92 is replaced by an amino acid selected from A, C, G, I, L, M, F, W and V; preferably from A, G, I, L and V; and more preferably from A, V, I and L. In a specific embodiment, Y92 is replaced by A92, thus yielding the substitution designated Y92A. 
     The antibody or bivalent fragment thereof comprises at least one substitution at a location noted above. The at least one substitution can occur in either the light chain variable region or the heavy chain variable region. In embodiments, and in the case where there is a single substitution that is only within the antibody light chain, that substitution is other than N30A, F53N, Y92A and Y92F. 
     In other embodiments, the antibody or binding fragment thereof comprises at least two such substitutions, either both in the light chain variable region, both in the heavy chain variable region, or at least one substitution in each of the light and heavy chain variable regions. In specific embodiments, the light chain variable region and the heavy chain variable region incorporate substitutions as follows: 
     
       
         
           
               
               
             
               
                   
                 TABLE 2 
               
             
            
               
                   
                   
               
               
                   
                 Heavy chain 
               
            
           
           
               
               
               
               
            
               
                 Light Chain 
                 Wild type 
                 Y57A 
                 R59E 
               
               
                   
               
               
                 Wild type 
                 — 
                 wt, Y57A 
                 wt, R59E 
               
               
                 N30D 
                 N30D, wt 
                 N30D, Y57A 
                 N30D, R59E 
               
               
                 F53N 
                 F53N, wt 
                 F53N, Y57A 
                 F53N, R59E 
               
               
                 Y92A 
                 Y92A, wt 
                 Y92A, Y57A 
                 Y92A, R59E 
               
               
                 F53N&amp;Y92A 
                 F53N&amp;Y92A, 
                 F53N&amp;Y92A, 
                 F53N&amp;Y92A, 
               
               
                   
                 wt 
                 Y57A 
                 R59E 
               
               
                   
               
            
           
         
       
     
     In preferred embodiments, the erbB2 antibody incorporates (1) a wild type heavy chain and light chain substitutions of both F53 and Y92, such as F53N and Y92A, or (2) a heavy chain substituted at Y57, such as Y57A, and a light chain substituted at N30, such as N30D. 
     In addition to the recited three CDRs present in each of the light and heavy chain variable regions, the heavy and light chains of the intact antibody comprise four intervening framework regions that present the CDRs in a conformation suitable for erbB2 binding, and constant regions that confer antibody effector function. The CDRs can be integrated into any suitable acceptor antibody, by grafting the present CDRs into the acceptor antibody, in accordance with practices and techniques well established for the production of chimeric, humanized and human antibodies. 
     Particularly suitable acceptor antibodies are antibodies already known to have erbB2 binding affinity. Such donor antibodies are most desirably of human origin, but they can also derive from acceptor antibodies of non-human origin, including mouse, rat, rabbit, goat, sheep, primate and the like. It will be appreciated that human antibody acceptor sequences different from those exemplified herein can be identified and used to accommodate the presently desired CDRs. This is achieved by modeling the structure of a preferred antibody using for instance the Swiss-Model [http://swissmodel.expasy.org/repository] or similar software and selecting, from among the numerous human antibody sequences available in public databases, a human acceptor antibody sequence that, with CDR sequences altered as herein preferred, approximates the same structural conformation as the preferred antibodies. In embodiments, the acceptor antibodies, and the resulting present antibodies, are of the IgG1 isotype, but they may also be IgG2 or IgG4. Moreover, the isotype of the antibody, as dictated by the constant region, can be manipulated to alter or eliminate the effector function of the resulting antibody. That is, the constant region of the present antibodies is either wild type human antibody constant region, or a variant thereof that incorporates amino acid modifications, i.e., amino acid additions, substitutions or deletions that alter the effector function of the constant region, such as to enhance serum half-life, reduce complement fixation, reduce antigen-dependent cellular cytotoxicity and improve antibody stability. 
     The number of amino acid modifications in the constant region is usually not more than 20, such as 1-10 e.g., 1-5 modifications, including conservative amino acid substitutions. 
     In embodiments, the half life of the antibody is improved by incorporating one more amino acid modification, usually in the form of amino acid substitutions, for instance at residue 252, e.g., to introduce Thr, at residue 254, e.g., to introduce Ser, and/or at residue 256 e.g., to introduce Phe. Still other modifications can be made to improve half-life, such as by altering the CH1 or CL region to introduce a salvage receptor motif, such as that found in the two loops of a CH2 domain of an Fc region of an IgG. Such alterations are described for instance in U.S. Pat. Nos. 5,869,046 and 6,121,022. 
     Altered C1q binding, or reduced complement dependent cytotoxicity, can be introduced by altering constant region amino acids at locations 329, 331 and 322, as described in U.S. Pat. No. 6,194,551. The ability of the antibody to fix complement can further be altered by introducing substitutions at positions 231 and 239 of the constant region, as described in WO94/029351. 
     The framework regions of the light and heavy chains of the present antibodies and fragments also desirably have the sequence of a human antibody variable region, but incorporating the CDRs herein specified. In embodiments, the heavy chain variable region is human IgG4 in origin. In specific embodiments, the heavy chain variable region is that of human IgG, such as the human IgG1 antibody variant having the sequence designated Genbank gi 2414502. Alternatively, and preferably, the heavy chain variable region is that of human IgG4 antibody species designated Genbank gi 2414502. 
     The framework regions of the heavy and light chains of the present antibodies may also incorporate amino acid modifications, i.e., amino acid deletions, additions or substitutions, which further improve upon the properties of the antibody or fragment, in accordance with techniques established for antibody humanization. Such framework modifications can be modeled on the framework regions of antibody sequences provided in public databases, and on framework regions of antibodies known to bind erbB2, such as those antibodies referenced in the background section hereof. Preferred framework substitutions are those which yield antibodies having a greater preference for binding erbB2 at the higher density associated with disease cells, relative to normal cells. 
     Framework modifications can also be made to reduce immunogenicity of the antibody or to reduce or remove T cell epitopes that reside therein, as described for instance by Carr et al in US2003/0153043. 
     In accordance with embodiments of the present invention, the heavy and light chain variable regions are modeled on the antibody trastuzumab, and comprise a light chain variable region of SEQ ID No.7, and/or a heavy chain variable region having SEQ ID No.8, as follows: 
     
       
         
           
               
            
               
                 Light chain variable region (VL): 
               
               
                 [SEQ ID No. 7] 
               
               
                 DIQMTQSPSSLSASVGDRVTITCRASQDVN 30 TAVAWYQQKPGKAPKLL 
               
               
                   
               
               
                 IYSASF 53 LYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHY 92 T 
               
               
                   
               
               
                 TPPTFGQGTKVEIK, 
               
               
                   
               
               
                 wherein N30, F53, and Y92 are as defined 
               
               
                   
               
               
                 hereinabove; 
               
               
                   
               
               
                 Heavy chain variable region (VH) 
               
               
                 [SEQ ID No. 8] 
               
               
                 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV 
               
               
                   
               
               
                 ARTYPTNGY 57 TR 59 YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY 
               
               
                   
               
               
                 YCSRWGGDGFYAMDYWGQGTLVTVSS; 
               
               
                   
               
               
                 wherein Y57 and R59 are as defined hereinabove. 
               
            
           
         
       
     
     In more specific and preferred embodiments, the entire light and heavy chains of the intact antibody are set out below as SEQ ID Nos. 9 and 10, respectively: 
     
       
         
           
               
            
               
                 Entire Light chain 
               
               
                 [SEQ ID No. 9] 
               
               
                 DIQMTQSPSSLSASVGDRVTITCRASQDVN 30 TAVAWYQQKPGKAPKLLI 
               
               
                   
               
               
                 YSASF 53 LYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHY 52 TT 
               
               
                   
               
               
                 PPTEGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE 
               
               
                   
               
               
                 AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY 
               
               
                   
               
               
                 ACEVTHQGLSSPVTKSFNRGEC; wherein N30, F53, and 
               
               
                   
               
               
                 Y92 are as defined hereinabove; 
               
               
                   
               
               
                 Entire Heavy chain 
               
               
                 [SEQ ID No. 10] 
               
               
                 EVQLVESGGGLVQPGGSLRLSCAASGENIKDTYIHWVRQAPGKGLEWVA 
               
               
                   
               
               
                 RIYPTNGY 57 TR 59 YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYY 
               
               
                   
               
               
                 CSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA 
               
               
                   
               
               
                 LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS 
               
               
                   
               
               
                 SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDTPPPCPRCPAPELLGGPS 
               
               
                   
               
               
                 VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK 
               
               
                   
               
               
                 TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
               
               
                   
               
               
                 KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ 
               
               
                   
               
               
                 PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH 
               
               
                   
               
               
                 YTQKSLSLSPGK; 
               
               
                   
               
               
                 wherein Y57 and R59 are as defined hereinabove. 
               
            
           
         
       
     
     As noted, final selection of an antibody or binding fragment is made based on the binding preference displayed by the desired antibody or bivalent fragment for cells that present erbB2 at a density greater than normal. The target cells are thus disease cells presenting greater than normal erbB2 density, as a hallmark. Screening can be performed in vitro, as exemplified herein, using as reference cells a first disease cell known from analysis to present erbB2 at a density greater than normal, such as the human breast adenocarcinoma cell line SkBr3 ATCC HTB-30 (˜2.5M ErbB2/cell) or the human breast ductal carcinoma cell line BT474 ATCC HTB-20 (˜3M Her2/cell) and a second, normal cell known from analysis to present erbB2 at a normal density, such as normal human cardiac myocytes (˜20,000 ErbB2/cell). The choice of cardiac myocytes as the reference, normal cell is prudent, given that marketed ErbB2 antibodies, such as trastuzumab, are known to elicit cardiac side effects through their interaction with these cells. Any other human cell line that presents erbB2 at normal density can be used, in the alternative. 
     The cell-based assay can use flow cytometry with appropriate erbB2 antibody and labeled secondary antibody to report and measure binding affinity and avidity, as exemplified herein. In the alternative, selection of the desired antibody can be performed based on absolute binding affinities obtained for instance using surface plasmon resonance, also as exemplified herein. 
     For purposes of identifying disease cells that can be targeted by the present erbB2 antibodies and bivalent fragments, the commercial test known as HerceptTest® can conveniently be used. This is a semi-quantitative immunohistochemical assay for determination of her-2 protein overexpression in breast cancer tissues. Positive or negative results aid in the classification of abnormal cells/tissues and provide a basis for treatment with erbB2 antibody. 
     The antibodies and binding fragments thus are useful for both diagnostic purposes, including sample testing and in vivo imaging, and for therapeutic purposes to treat diseases in which erbB2 density is increased on disease cells. 
     For either purpose, the antibody or binding fragment can be conjugated to an appropriate agent, to form an immunoconjugate. Agents appropriate for treating disease include cytotoxic agents include chemotherapeutics and radiotherapeutics. For diagnostic purposes, appropriate agents are detectable labels that include radioisotopes, for whole body imaging, and radioisotopes, enzymes, fluorescent labels and the like for sample testing. 
     For therapy, the cytotoxin may be conjugated with the antibody or bivalent binding fragment through non-covalent interaction, but more desirably, are coupled by covalent linkage either directly or, more preferably, through a suitable linker. In a preferred embodiment, the conjugate comprises a cytotoxin and an antibody. Immunoconjugates of the antibody and cytotoxin are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate, iminothiolane, bifunctional derivatives of imidoesters such as dimethyl adipimidate HCL, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azido compounds such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates such as toluene 2,6-diisocyanate, and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Carbon-14-labeled 1-isothiocyanobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is a chelating agent suitable for conjugation of radio nucleotide to the antibody. 
     The cytotoxin component of the immunoconjugate can be a chemotherapeutic agent, a toxin such as an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof, or a small molecule toxin, or a radioactive isotope such as  212 Bi,  131 I,  131 In,  111 In,  90 Y, and  186 Re, or any other agent that acts to inhibit the growth or proliferation of a cancer cell. 
     Chemotherapeutic agents useful in the generation of such immunoconjugates include adriamycin, doxorubicin, epirubicin, 5-fluoroouracil, cytosine arabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e.g. paclitaxel, and docetaxel, toxotere, methotraxate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosgamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins, 5-FU, 6-thioguanine, 6-mercaptopurine, actinomycin D, VP-16, chlorambucil, melphalan, and other related nitrogen mustards. Also included are hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onapristone. Toxins and fragments thereof which can be used include diphtheria A chain, nonbonding active fragments of diphtheria toxin, cholera toxin, botulinus toxin, exotoxin A chain (from  Pseudomonas aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,  Aleurites fordii  proteins, dianthin proteins,  phytolaca Americana  proteins (PAPI, PAPII, and PAP-S),  momordica charantia  inhibitor, curcin, crotin,  sapaonaria, officinalis  inhibitor, gelonin, saporin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothcenes. Small molecule toxins include, for example, calicheamicins, maytansinoids, palytoxin and CC1065. 
     Pharmaceutical Compositions 
     Therapeutic formulations of the antibody, bivalent fragment or the conjugate are prepared for storage by mixing the antibody or conjugate having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington&#39;s Pharmaceutical Sciences, 16 th  edition, Osol, A. Ed. [1980]), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum, albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagines, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN, PLURONICS or polyethylene glycol (PEG). 
     The active ingredients to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. 
     Sustained-release preparations may be prepared. Suitable examples of sustained-release include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shapes articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate), polyactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate, and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37° C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S—S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. 
     Pharmaceutical Combinations 
     Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. 
     Other therapeutic regimens may be combined with the administration of the anti-cancer agents, e.g., antibodies or conjugates, of the instant invention. For example, the patient to be treated with such anti-cancer agents may also receive radiation therapy, such as external beam radiation. Alternatively, or in addition, a chemotherapeutic agent may be administered to the patient. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers&#39; instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M. C. Perry, Williams &amp; Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may precede, or follow administration or the anti-tumor agent, e.g., antibody, or may be given simultaneously therewith. The antibody may be combined with an anti-estrogen compound such as tamoxifen or an anti-progesterone such as onapristone (see, EP 616812) in dosages known for such molecules. 
     It may be desirable to also administer antibodies or conjugates against other tumor associated antigens, such as antibodies which bind to the EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF). Alternatively, or in addition, two or more antibodies binding that same or two or more different antigens disclosed herein may be co-administered to the patient. Sometimes it may be beneficial to also administer one or more cytokines to the patient. In a preferred embodiment, the antibodies herein are co-administered with a growth inhibitory agent. For example, the growth inhibitory agent may be administered first, followed by an antibody of the present invention. However, simultaneous administration or administration of the antibody of the present invention first is also contemplated. Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to combined action (synergy) of the growth inhibitory agent and the antibody herein. 
     Kits 
     In another embodiment of the invention, an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described herein is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). The label on, or associated with, the container indicates that the composition is used for treating a cancer condition. The article of manufacture may further compromise a second container compromising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer&#39;s solution and dextrose solution. It may further include other matters desirable from a commercial and use standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. 
     Dosing and Administration 
     An anti-cancer therapeutic according to the invention may be administered with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Any appropriate route of administration can be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration. 
     For the treatment of subjects presenting with cancer cells presenting erbB2 at greater density than normal cells, the appropriate dosage of an anti-tumor agent, e.g., an antibody, fragment or conjugate, will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventative or therapeutic purposes, previous therapy, the patients clinical history and response to the agent, and the discretion of the attending physician. The agent is suitably administered to the patient at one time or over a series of treatments. 
     For example, depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of antibody or conjugate is a candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. 
     It will thus be appreciated that an effective amount of the antibody, fragment or immunoconjugate is an amount effective alone or as part of a treatment regimen that retards or inhibits the growth or proliferation of disease cells presenting with higher than normal erbB2 density. 
     In embodiments, the present antibodies are administered by intravenous infusion, such as at an initial dose of 4 mg/kg over 90 minutes, then 2 mg/kg over 30 minutes, once weekly for 52 weeks, with follow up as required. 
     The antibody and bivalent fragments are useful in the treatment of a variety of cancers, to inhibit the growth or proliferation of cancer cells and tumours comprising them, including hematopoietic cell cancers and solid tumours. Conditions or disorders to be treated include benign or malignant tumors (e.g., renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, lung, vulva, and thyroid); hepatic carcinomas; sarcomas; glioblastomas; and various head and neck tumors; leukemias and lymphoid malignancies. In particular embodiments, the antibody or bivalent fragment are used in the treatment of such cancer cells that express high density erbB2, as determined by the screening assays herein described. In particular embodiments, the cancer cells are erbB2-presenting breast cancer cells. 
     It will be appreciated that subjects who could benefit from the present method include mammals including humans as well as livestock, and pets. 
     Screening for High Density erbB2 Cancer Cells 
     Antibodies and bivalent fragments thereof that bind selectively to the target antigen, e.g. erbB2, are used, in accordance with an aspect of the invention, to screen cancer cells to detect those which present the erbB2 antigen at high density. In a preferred embodiment, screening is applied to a sample of cancer cells taken from a subject that is a candidate for erbB2 antibody therapy. Subjects testing positive for cancer cells that present the erbB2 antigen at high density can then be scheduled for therapy with the present antibody or fragment, or an immunoconjugate thereof. Standard techniques, combined with the antibodies or other binding agents herein described can be used to screen cancer cells. 
     Desirably, the antibodies incorporate a detectable label. The label may be detectable by itself. (e.g., radio-isotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. Radionuclides that can serve as detectable labels include, for example, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, and Pd-109. 
     In situ detection of the binding to cancer cells bearing high density erbB2 can be performed, using the present antibody or fragment, by immunofluorescence or immunoelectron microscopy. For this purpose, a histological specimen is removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample. This procedure also allows for distribution of the erbB2 antigen to be examined within biopsied tumour tissue, to reveal only those sites at which the antigen is presented at a density higher than normal. It will be apparent for those skilled in the art that a wide variety of histological methods are readily available for in situ detection. 
     More particularly, erbB2 antibodies or binding fragments may be used to monitor the presence or absence of antibody reactivity in a biological sample (e.g., a tissue biopsy, a cell, or fluid) using standard detection assays. Immunological assays may involve direct detection, and are particularly suited for screening large amounts of samples for the presence of erbB2 positive cancer cells. For example, antibodies may be used in any standard immunoassay format (e.g., ELISA, Western blot, immunoprecipitation, flow cytometry or RIA assay) to measure complex formation. Any appropriate label which may be directly or indirectly visualized may be utilized in these detection assays including, without limitation, any radioactive, fluorescent, chromogenic (e.g., alkaline phosphatase or horseradish peroxidase), or chemiluminescent label, or hapten (for example, digoxigenin or biotin) which may be visualized using a labeled, hapten-specific antibody or other binding partner (e.g., avidin). Exemplary immunoassays are described, e.g., in Ausubel et al., supra, Harlow and Lane, Antibodies: A Laboratory Approach, Cold Spring Harbor Laboratory, New York (1988), and Moynagh and Schimmel, Nature 400:105, 1999. For example, using the antibodies described herein, high density erbB2 is readily detected at the cell surface using standard flow cytometry methods. Samples found to contain increased levels of labeled complex compared to appropriate control samples are taken as indicating the presence of high density erbB2, and are thus indicative of a cancer or other disease amenable to treatment with the present antibodies. 
     The present antibody is produced suitably by recombinant DNA means, as exemplified herein. For production, there is provided a DNA molecule that encodes the heavy chain of the present antibody, and a DNA molecule that encodes the light chain thereof. The DNA further encodes any suitable signal peptide suitable for expression of a secretable chain precursor that enables proper externalization with folding and disulfide formation to elaborate the desired antibody as a secreted, dimerized and processed protein. To this end, the present invention provides, in one embodiment, a polynucleotide comprising a sequence that encodes the variable region of the light chain of a presently preferred erbB2 antibody, as set out in SEQ ID No. 9, supra. Also provided, in another embodiment, is a polynucleotide comprising a sequence that encodes the variable region of the heavy chain of a presently preferred erbB2 antibody, as set out in SEQ ID No. 10, supra. 
     In more specific embodiments, the present invention provides a polynucleotide that encodes the entire light chain (SEQ ID No. 11) and the entire heavy chain (SEQ ID No.12) of a preferred erbB2 antibody of the present invention. These sequences are provided at the end of this disclosure. 
     It will be appreciated that polynucleotide equivalents also can be used, in which synonymous codons are replaced within the sequences provided, to produce the present antibodies. 
     In embodiments, there are also provided vectors that comprise polynucleotides that encode the heavy chain or the variable region thereof and that encode the light chain or the variable region thereof. To express the antibodies, the polynucleotides are incorporated operably within expression vectors, i.e., operatively linked to transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, cosmids, and the like. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy gene can be inserted into separate vectors. In a preferred embodiment, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). 
     A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed, as described above. In such vectors, splicing usually occurs between the splice donor site in the inserted J region, and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The recombinant expression vector can also encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein). 
     Polynucleotides encoding the heavy chain and/or the light chain, and vectors comprising these can be used for transformation of a suitable mammalian host cell. Methods for introduction of heterologous polynucleotides into mammalian calls include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei. In addition, polynucleotides may be introduced into mammalian cells by viral vectors. Mammalian cell lines useful as hosts for expression of the antibody-encoding polynucleotides include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chine hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as S19 cells, amphibian cells, bacterial cells, plant cells and fungal cells. When recombinant expression vectors encoding the heavy chain or antigen-binding portion thereof are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. 
     It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from each other. However, all antibodies encoded by the polynucleotides provided herein, or comprising the amino acid sequences provided herein are part of the instant invention. 
     Embodiments are now described in the following examples. 
     EXAMPLES 
     The structure of trastuzumab bound to erbB2 [Ref TS1] was used as starting point for mutant design. Mutations were introduced only in the CDR regions of the light and heavy chain. First, single-point mutations were generated and evaluated computationally. Virtual mutagenesis was carried out with optional conformational relaxation upon mutation by means of conformational sampling algorithms, such as Monte Carlo minimization [Ref TS2]. Prediction of antigen-antibody relative binding affinities between parent and mutant antibodies was carried out with binding affinity scoring functions, such as the solvated interaction energy (SIE) function [Ref TS3]. Prediction of relative antigen-antibody association rates (k on ) between parent and mutant antibodies was carried out with methods that evaluate long-range electrostatic interactions, such as HyPARE [Ref TS4]. Candidate single-point mutants were the assembled into multiple-point mutants and re-scored for relative binding affinity. 
     Multiple-point mutants were generated by combining single-point mutants between light and heavy chains to achieve the targeted change in affinity. A requirement was to use as few single-point mutants as possible and to maximize the number of generated assembled antibodies. Another desirable feature was to generate a pool of mutants with reduced affinities due to either increased dissociation rates (k −off ) or to decreased association rates (k −on ). Among suitable candidate single-point mutations, those targeting distinct locations within the antibody-antigen interface, preferably at its periphery, were given higher priority. 
     Preparation of Plasmids 
     All the cDNAs encoding the heavy and light chains of the antibodies were ordered from GeneArt (Regensburg Germany). The cDNAs were removed from the plasmid provided by GeneArt by digestion with HindIII and cloned into the HindIII site of plasmid pKCR5 previously dephosphorylated with calf intestinal phosphatase (NEB) to prevent recircularization. In pKCR5, transcription of the cDNA is under the control of the strong CR5 promoter, part of the cumate gene switch. The plasmid pKCR5 is available from the Biotechnology Research Institute, Montreal, Canada and is described by Mullick et al, BMC Biotechnol., 2006, 6:43. This 3.9 kb plasmid incorporates a HindIII in proper contex with the CR5 promoter and a rabbit b-globin polyA, together with a B-lactamase gene for selection, and colE1 and f1 origins of replication. For transfection of CHO cells, all plasmids were isolated from large culture of  E. coli  using the Plasmid Maxi kit (Qiagen Inc, Mississauga, ON) according to the manufacturer&#39;s recommendation. Briefly, 3 ml of LB medium containing 100 μg/ml ampicillin were inoculated with a single colony of  E. coli  and grown for 6 h at 37° C. with vigorous shaking (250 RPM). This preculture was then used to inoculate 250 ml of LB medium containing 100 μg/ml ampicillin. The culture was incubated overnight at 37° C. with vigorous shaking (250 RPM). The bacteria were pelleted by centrifugation at 6000×g, for 15 min, at 4° C. and the plasmid was isolated using the protocols, buffers and columns provided by the kit. The pure plasmids was resuspended in sterile 50 mM TRIS, pH 8 and quantified by measuring the optical density at 260 nm. 
     Cell Line (CHO-cTA; Clone 5F1) and Growth Conditions 
     The CHO-cTA cell line (Gaillet, B. et al,  Biotechnol. Prog.  23:200-209; Mullick, A., et al.  BMC Biotechnol.  2006, 6:43.) used for transient transfection is a Chinese Hamster Ovary cell line (CHO) adapted to grow in suspension and in protein-free medium. The cell line stably expresses the cumate transactivator (cTA) which activates transcription by binding to the CR5 promoter. The CHO-cTA are maintained in CD-CHO medium (Invitrogen, CDCHO 10743), supplemented with 4 mM glutamine, 50 μg/mL and dextran sulfate (Amersham Pharmacia Biotech) at 37° C. under an atmosphere of 5% CO 2 . When the cells reach a concentration of 1.0×10 6  cells/ml (on average three times a week) they are passaged by diluting them to a concentration of 5.0×10 4  cells/ml using fresh medium. 
     Transient Transfection of CHO-cTA 
     Before transfection, the cells were washed with PBS and resuspended at a concentration of 2.5×10 6  cell/ml in growth medium without dextran sulfate for 3 hrs in suspension culture. 50 ml of cells were transfected by adding slowly 2.5 ml of a CDCHO medium supplemented with 1 μg/ml of plasmid and 5 μg/ml. polyethylenimine (PEI Max; Polysciences). After 2 hrs, the cells were transferred at 30° C. The next days, 50 μg/mL of dextran sulfate was added to the cells and they were incubated at 30° C. for a total of 4 days. The supernatant was clarified by centrifugation and filtered through a 0.22 μM filter and transferred at −80° C. until further analysis. 
     Polyacrylamide Gel Electrophoresis (SDS-PAGE) 
     Known amounts of supernatant were resuspended into an equal volume of Laemmli 2× and heated at 95° C. for 5 min and chilled on ice. The samples were then separated on a polyacrylamide Novex 10% Tris-Glycine gel (Invitrogen Canada Inc., Burlington, ON). A standard curve was made by adding known amount of purified human IgG. The gel was then stained using a solution of Coomassie Fluor™-Orange (Molecular Probes, Eugene Oreg.) according to the manufacturer&#39;s recommendations. The signal was visualized and quantified using the Typhoon Scanner. 
     Western Blot Analysis 
     Known amounts of supernatant were separated on a SDS-PAGE as described above and then transferred onto a Hybond-N nitrocellulose membrane (Amersham Bioscience Corp., Baie d&#39;Urfée, QC) for 1 h at 275 mA. The membrane was blocked for 1 h in 0.15% Tween 20, 5% skimmed milk in PBS and incubated for 1 h with an anti-human IgG conjugated to Cy5 (Jackson, Cat#109-176-099). The signal was revealed by scanning with the Typhoon Trio+ (Amersham Biosciences, GE Healthcare). 
     ELISA 
     96 wells/plates were coated with 50 μl of AffiniPure Goat Anti-Human IgG, (H+L) (Jackson Immuno Research) and incubated overnight at 4° C. The wells were washed with PBS and incubated for 30 min at 37° C. with 100 μl of 1% BSA in PBS at 37° C. 25 μl of samples diluted with 1% BSA in PBS were added to the wells, which were incubated for 2 hrs at 37° C. The wells were washed with 0.05% Tween 20 in PBS and incubated with an alkaline Phosphatase-conjugated AffiniPure Goat Anti-Human IgG (H+L) (Jackson Immuno Research) for 1 hr at 37° C. The wells were washed with 0.05% Tween 20 in PBS, followed by PBS. The signal was revealed by incubation with PNPP for 30 min at 37° C. The signal intensity was measure at 405 nm. A standard curve was made using known amount of purified antibody (IgG1, kappa from myeloma plasma (Athens Research Technology). 
     Purification of Antibody 
     The supernatant was concentrated with a Amicon Ultra (Ultracel-50K) at 1500 rpm to a volume of 500 μl. The wild type and mutant antibodies were purified using the Nab spin kit Protein A mini column (Thermo Scientific) according to the manufacture&#39;s recommendations. The purified antibodies were then desalted and resuspended in PBS using the desalting column PD-10 (GE Healthcare). The antibodies were then concentrated by centrifugation on an Amica Ultra 100,000 MWCO membrane. The purified antibodies were quantified by reading the optical density at 280 nm using the Nanodrop spectrophotometer. The purified antibodies were kept frozen at −20° C. in 50% glycerol. 
     In Vitro Binding by Surface Plasmon Resonance 
     Kinetic and affinity analysis was carried out using a BioRad Proteon surface plasmon resonance instrument. The running buffer for all steps was 10 mM HEPES, 150 mM NaCl, 3.5 mM EDTA and 0.05% Tween20 at pH 7.4. An antibody capture sensorchip was prepared by injecting 6.5 ug/mL of anti-mouse Fc (Jackson Immunochemicals Inc.) in 10 mM sodium acetate pH 4.5 at flow rate 25 uL/minute over a GLM sensorchip (BioRad Inc.) that had previously been activated with a 1/10 dilution of sNHS/EDC (BioRad Inc.) until the surface was saturated (approximately 5000 RUs). This procedure was carried out in the analyte direction to ensure all of the interspots for referencing have immobilized anti-mouse Fc. Wild-type trastuzumab and variants was captured in the ligand direction by injecting 100 uL of 4% culture supernatants or purified samples in running buffer at flow rate of 25 uL/min until 400 to 800 resonance units have been captured. This was immediately followed by two pulses of running buffer in the analyte direction, 50 uL each at flow rate 100 uL/min to stabilize the baseline. Next, the simultaneous injection of 100 uL of five ErbB2 (eBiosciences Inc.) concentrations (3-fold dilutions of 30 nM or 20 nM ErbB2) and buffer blank at a flow rate of 50 uL/min with a 600 s dissociation was carried out to analyse the ErbB2—antibody interaction. Kinetic rate constants (on- and off-rates) and affinity constants were generated from the aligned and double referenced sensorgrams with the Langmuir binding model using BioRad Proteon Manager software v3.2. 
     Cell Culture 
     The SkBr3 and BT474 cell lines were obtained from ATCC. Cell lines were maintained in DMEM (source) containing 10% fetal bovine serum (Gibco). Primary adult human cardiac myocytes (HCM) were obtained from ScienCell (Catalog #6200) and cultured using manufacturer&#39;s recommended Cardiac Myocyte Medium. Generally cells were passaged once or twice a week and used within 4-6 weeks for all experiments. 
     Detection of Antibody Binding to Surface erbB2 Level by Flow Cytometry 
     Prior to analysis, cells were plated such that they were not more than 80% confluent on the day of analysis. Tumor cells overexpressing her-2(SkBr3, ˜2.5M Her2/cell or BT474, ˜3M Her2/cell) or normal (human cardiac myocytes, ˜20,000 Her2/cell) were washed in PBS and harvested by the addition of cell dissociation buffer (Sigma.). A cell suspension containing 2.5×10 5  in 500 μl corresponding cell culture media) was incubated with various concentrations (0.01-100 ug/ml) of anti-HER2 antibodies for 2 h at 4° C. (to prevent internalization). Following 1 wash with cell culture media, primary antibody was incubated with 2 ug Dylight 488 conjugated AffiniPure goat anti-human IgG Alexa 488 secondary antibody (Jackson ImmunoResearch 109-487-003) in 100 ul of media for 1 h at 4° C. Cells were then pelleted and stored on ice until ready to analyzed by flow cytometry. Prior to analysis, cell pellets were resuspended in 300-500 ul media and filtered through a 50 um nylon mesh filter to remove cell aggregates. Flow cytometry analyses were performed on 10,000 viable cells gated on forward scattering, side scattering parameters and propidium iodide dye exclusion using a BD LSRII flow Cytometer (Becton-Dickinson Biosciences, CA, USA) and a standard filter set using BD FACSDiva™ acquisition software, according to manufacturer&#39;s instructions. 
     Specific antibody binding was calculated as the mean fluorescent intensity of binding to each antibody after background level subtraction of the mean fluorescent intensity of binding in the absence of primary antibody (but containing detection antibody). For all experiments, specific antibody binding was compared relative to that of the wild type version of trastuzumab that was produced and purified in the same manner (HC/LC). To examine the binding selectivity of antibodies, the value of antibody binding to tumor (overexpressing ErbB2) was divided by the binding observed with normal human cardiac myocyte cells. This parameter, named the ratio of binding, was calculated and compared to that seen with wild type antibody (named 2-1 wt, set arbitrarily to 1). A commercial source of trastuzumab (Roche) was used as a benchmark for comparison purposes. 
     Results: 
     1. Production of ErbB2 Antibodies. 
     Eight cDNAs corresponding to the coding sequence of the ErbB2 antibodies (Table above) were synthesized (GeneArt). All the cDNAs were cloned into the HindIII site of pKCR5, an expression vector regulated by the cumate-switch (pKCR5 vector). 
     For each antibody, 50 ml of CHOcTA (expressing the cumate transactivator, cTA) were transfected with various combinations of heavy and light chain. Four days after transfection the supernatant was analyzed by SDS-PAGE (not shown), with quantifications made by Western Blot (Table 4) and by ELISA (Table 3). 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                   
                 Quantification 
                 Quantification 
               
               
                   
                 Heavy and light chains 
                 Western blot 
                 ELISA 
               
               
                   
                 used (Heavy_Light) 
                 (mg/L) 
                 (mg/L) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 1_2 
                 67.02 
                 86.46 
               
               
                   
                 2_3 
                 107.27 
                 96.79 
               
               
                   
                 2_4 
                 87.69 
                 58.08 
               
               
                   
                 2_5 
                 54.36 
                 95.51 
               
               
                   
                 2_6 
                 26.21 
                 22.99 
               
               
                   
                 7_1 
                 66.04 
                 76.44 
               
               
                   
                 7_3 
                 80.14 
                 81.98 
               
               
                   
                 7_4 
                 60.72 
                 118.02 
               
               
                   
                 7_5 
                 34.49 
                 52.12 
               
               
                   
                 7_6 
                 50.10 
                 46.45 
               
               
                   
                 8_1 
                 81.49 
                 66.98 
               
               
                   
                 8_3 
                 43.78 
                 46.45 
               
               
                   
                 8_4 
                 48.22 
                 45.98 
               
               
                   
                 8_5 
                 48.77 
                 61.47 
               
               
                   
                 8_6 
                 16.62 
                 29.56 
               
               
                   
                 K- 
                 0 
                 0 
               
               
                   
                   
               
               
                   
                 Where, for the light chain, and for the heavy chain 
               
               
                   
                 1 = wild type light chain 
               
               
                   
                 2 = wild type 
               
               
                   
                 3 = F53N 
               
               
                   
                 4 = Y92A 
               
               
                   
                 5 = F53N, Y92A 
               
               
                   
                 6 = N30D 
               
               
                   
                 7 = Y57A 
               
               
                   
                 8 = R59E 
               
            
           
         
       
     
     The wild type and 9 mutants were purified by chromatography using protein A. The purified proteins were quantified by OD 280  (Nanodrop) see Table 4. The purified antibodies were analyzed by non-denaturing and denaturing SDS-PAGE. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Quantification of the purified antibodies by OD 280  (Nanodrop) 
               
            
           
           
               
               
               
               
            
               
                   
                 Purified 
                   
                 Concentration 
               
               
                   
                 mutants 
                   
                 (ug/ml) 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 1_2 
                 Wild type 
                 580 
               
               
                   
                 2_3 
                 wt-HC + m1-Lc 
                 500 
               
               
                   
                 2_4 
                 wt-HC + m2-LC 
                 370 
               
               
                   
                 2_5 
                 wt-HC + m3-LC 
                 450 
               
               
                   
                 2_6 
                 wt-HC + -m4-LC 
                 270 
               
               
                   
                 7_1 
                 m5-HC + wt-LC 
                 500 
               
               
                   
                 7_3 
                 m5-HC + m1-LC 
                 540 
               
               
                   
                 7_4 
                 m5-HC + m2-LC 
                 520 
               
               
                   
                 7_5 
                 m5-HC + m3-LC 
                 380 
               
               
                   
                 7_6 
                 m5-HC + m4-LC 
                 310 
               
               
                   
                   
               
            
           
         
       
     
     Binding Affinity Determination by SPR 
     The 2- and 7-series trastuzumab variants had detectible activity at the 20 nM ErbB2 concentrations used. While the 2-series fit quite well to a Langmuir binding model (1:1), the 7-series variants showed complexity in their kinetics as seen by deviations from ideal behaviour in the dissociation phase. This is especially evident with variant 7-5 which shows a complex biphasic off-rate that fit poorly to the Langmuir binding model. The affinity of this variant would likely be lower than the 0.3 nM indicated in Table 5, as the modeled fit uses the slow dissociation phase only. Series 8, those with the heavy chain substitution R59E, did not show any detectible binding activity indicating this position is critical for binding activity of trastuzumab. The wild-type version (2-1) had identical binding behaviour to the commercial trastuzumab. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Kinetic parameters of ErbB2 - mAb variant 
               
               
                 binding modeled to a 1:1 binding fit 
               
            
           
           
               
               
               
               
               
            
               
                   
                 ka 1/Ms 
                 kd 1/s 
                 KD 
                 Chi2 RU 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Variant 7-1 
                 7.07E+05 
                 1.72E−04 
                 2.43E−10 
                 3.38 
               
               
                 Variant 7-3 
                 6.70E+05 
                 9.24E−04 
                 1.38E−09 
                 14.92 
               
               
                 Variant 7-4 
                 6.01E+05 
                 1.10E−03 
                 1.83E−09 
                 25.9 
               
               
                 Variant 7-5 
                 8.12E+05 
                 2.11E−04 
                 2.59E−10 
                 5.48 
               
               
                 Variant 7-6 
                 5.76E+05 
                 2.91E−03 
                 5.05E−09 
                 3.68 
               
               
                 Variant 2-3 
                 8.46E+05 
                 1.17E−04 
                 1.39E−10 
                 3.76 
               
               
                 Variant 2-4 
                 7.47E+05 
                 2.95E−04 
                 3.95E−10 
                 3.55 
               
               
                 Variant 2-5 
                 7.19E+05 
                 1.12E−03 
                 1.56E−09 
                 11.91 
               
               
                 Variant 2-6 
                 6.21E+05 
                 4.30E−04 
                 6.92E−10 
                 3.7 
               
               
                 Trastuzumab 
                 8.82E+05 
                 2.92E−05 
                 3.31E−11 
                 5.84 
               
               
                 Wild-type 2-1 
                 8.56E+05 
                 1.66E−05 
                 1.94E−11 
                 3.99 
               
               
                   
               
            
           
         
       
     
     Experimental testing of the designed Trastuzumab mutants using the SPR technique showed that the erbB2 binding affinity is weakened over 100-fold for 1 mutant, between 10-100-fold for 5 mutants, and below 10-fold for another 3 mutants, relative to the parent Trastuzumab antibody (see also  FIG. 1 ). 
     Evaluation of Antibody Binding to Tumor and Normal Cell Lines 
     Binding of the antibodies to various cell lines was also determined by indirect flow cytometry as described in the Materials and Methods. As shown in  FIG. 3 , these results demonstrate weaker binding of 2-5 and 7-6 antibody mutants to HCM cells compared to tumor cells (relative to binding observed for wt control and commercial benchmark). Also as shown in  FIG. 4 , this result clearly shows that some of the antibody mutants exhibit better binding to tumor relative to normal HEK cells (e.g. 2-5 exhibits 10-12×, 7-6 exhibits 6-8× more binding to tumor than normal). The pattern of binding specificity was similar amongst the tumor cell lines analyzed (SKBr3 or BT474) suggesting that the selectivity of binding is universally high for all tumor cells presenting erbB2 at a density greater than the normal erbB2 density (˜3 million receptors per cell or more). 
     Polynucleotides encoding the various mutant antibody chains are provided below. Substituted codons are italicized and in bold font, and HindIII sites are italicized: 
     
       
         
           
               
               
            
               
                 Light chain: WT 
                   
               
               
                 [SEQ ID No. 11] 
                   
               
               
                 GGTACC AAGCTT GCCACCATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGG 
                   
               
               
                   
               
               
                 ATCTCTGGCGCCTACGGCGACATCCAGATGACCCAGTCCCCCTCCTCCCTGTCTGCCTCC 
               
               
                   
               
               
                 GTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGGATGTGAACACCGCCGTGGCC 
               
               
                   
               
               
                 TGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTCCGCCTCCTTCCTG 
               
               
                   
               
               
                 TACTCCGGCGTGCCCTCCCGGTTCTCCGGCTCCAGATCCGGCACCGACTTCACCCTGACC 
               
               
                   
               
               
                 ATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC 
               
               
                   
               
               
                 CCTCCAACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCTCCTTCC 
               
               
                   
               
               
                 GTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCTGTCGTCTGC 
               
               
                   
               
               
                 CTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTG 
               
               
                   
               
               
                 CAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACTCTACCTACTCC 
               
               
                   
               
               
                 CTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGC 
               
               
                   
               
               
                 GAAGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGC 
               
               
                   
               
               
                 TGATGA AAGCTT GAGCTC 
               
               
                   
               
               
                 &gt;m1; Light chain: F53N (changed TTC for AAC) 
               
               
                 [SEQ ID No. 12] 
                   
               
               
                 GGTACC AAGCTT GCCACCATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGG 
                   
               
               
                   
               
               
                 ATCTCTGGCGCCTACGGCGACATCCAGATGACCCAGTCCCCCTCCTCCCTGTCTGCCTCC 
               
               
                   
               
               
                 GTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGGATGTGAACACCGCCGTGGCC 
               
               
                   
               
               
                 TGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTCCGCCTCC  CTG 
               
               
                   
               
               
                 TACTCCGGCGTGCCCTCCCGGTTCTCCGGCTCCAGATCCGGCACCGACTTCACCCTGACC 
               
               
                   
               
               
                 ATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC 
               
               
                   
               
               
                 CCTCCAACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCTCCTTCC 
               
               
                   
               
               
                 GTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCTGTCGTCTGC 
               
               
                   
               
               
                 CTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTG 
               
               
                   
               
               
                 CAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACTCTACCTACTCC 
               
               
                   
               
               
                 CTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGC 
               
               
                   
               
               
                 GAAGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGC 
               
               
                   
               
               
                 TGATGA AAGCTT GAGCTC 
               
               
                   
               
               
                 &gt;m2; Light chain: Y92A (changed TAC for GCC) 
               
               
                 [SEQ ID No. 13] 
                   
               
               
                 GGTACC AAGCTT GCCACCATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGG 
                   
               
               
                   
               
               
                 ATCTCTGGCGCCTACGGCGACATCCAGATGACCCAGTCCCCCTCCTCCCTGTCTGCCTCC 
               
               
                   
               
               
                 GTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGGATGTGAACACCGCCGTGGCC 
               
               
                   
               
               
                 TGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTCCGCCTCCTTCCTG 
               
               
                   
               
               
                 TACTCCGGCGTGCCCTCCCGGTTCTCCGGCTCCAGATCCGGCACCGACTTCACCCTGACC 
               
               
                   
               
               
                 ATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCAC  ACCACC 
               
               
                   
               
               
                 CCTCCAACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCTCCTTCC 
               
               
                   
               
               
                 GTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCTGTCGTCTGC 
               
               
                   
               
               
                 CTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTG 
               
               
                   
               
               
                 CAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACTCTACCTACTCC 
               
               
                   
               
               
                 CTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGC 
               
               
                   
               
               
                 GAAGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGC 
               
               
                   
               
               
                 TGATGA AAGCTT GAGCTC 
               
               
                   
               
               
                 &gt;m3; Light chain: F53N, Y92A (changed TTC for AAC and TAC 
               
               
                 for GCC) 
               
               
                 [SEQ ID No. 14] 
                   
               
               
                 GGTACC AAGCTT GCCACCATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGG 
                   
               
               
                   
               
               
                 ATCTCTGGCGCCTACGGCGACATCCAGATGACCCAGTCCCCCTCCTCCCTGTCTGCCTCC 
               
               
                   
               
               
                 GTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGGATGTGAACACCGCCGTGGCC 
               
               
                   
               
               
                 TGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTCCGCCTCC  CTG 
               
               
                   
               
               
                 TACTCCGGCGTGCCCTCCCGGTTCTCCGGCTCCAGATCCGGCACCGACTTCACCCTGACC 
               
               
                   
               
               
                 ATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCAC  ACCACC 
               
               
                   
               
               
                 CCTCCAACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCTCCTTCC 
               
               
                   
               
               
                 GTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCTGTCGTCTGC 
               
               
                   
               
               
                 CTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTG 
               
               
                   
               
               
                 CAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACTCTACCTACTCC 
               
               
                   
               
               
                 CTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGC 
               
               
                   
               
               
                 GAAGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGC 
               
               
                   
               
               
                 TGATGA AAGCTT GAGCTC 
               
               
                   
               
               
                 &gt;m4; Light chain: N30D (changed AAC for GAC) 
               
               
                 [SEQ ID No. 15] 
                   
               
               
                 GGTACC AAGCTT GCCACCATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGG 
                   
               
               
                   
               
               
                 ATCTCTGGCGCCTACGGCGACATCCAGATGACCCAGTCCCCCTCCTCCCTGTCTGCCTCC 
               
               
                   
               
               
                 GTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGGATGTG  ACCGCCGTGGCC 
               
               
                   
               
               
                 TGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTCCGCCTCCTTCCTG 
               
               
                   
               
               
                 TACTCCGGCGTGCCCTCCCGGTTCTCCGGCTCCAGATCCGGCACCGACTTCACCCTGACC 
               
               
                   
               
               
                 ATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACC 
               
               
                   
               
               
                 CCTCCAACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCTCCTTCC 
               
               
                   
               
               
                 GTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCCTCTGTCGTCTGC 
               
               
                   
               
               
                 CTGCTGAACAACTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTG 
               
               
                   
               
               
                 CAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACTCTACCTACTCC 
               
               
                   
               
               
                 CTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGC 
               
               
                   
               
               
                 GAAGTGACCCACCAGGGCCTGTCCAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGC 
               
               
                   
               
               
                 TGATGA AAGCTT GAGCTC 
               
               
                   
               
               
                 Heavy chain: WT 
               
               
                 [SEQ ID No. 16] 
                   
               
               
                 TTAATT AAGCTT GCCACCATGGACTGGACCTGGCGGATCCTGTTTCTGGTGGCCGCTGCT 
                   
               
               
                   
               
               
                 ACCGGCACACACGCCGAGGTGCAGCTGGTGGAGTCTGGCGGAGGACTGGTGCAGCCTGGC 
               
               
                   
               
               
                 GGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACACCTACATCCAC 
               
               
                   
               
               
                 TGGGTCCGGCAGGCTCCAGGCAAGGGACTGGAATGGGTGGCCCGGATCTACCCTACCAAC 
               
               
                   
               
               
                 GGCTACACCAGATACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCGCCGACACCTCC 
               
               
                   
               
               
                 AAGAACACCGCCTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTAC 
               
               
                   
               
               
                 TGCTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCCAGGGCACCCTG 
               
               
                   
               
               
                 GTCACTGTGTCCTCTGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTCCAGC 
               
               
                   
               
               
                 AAGTCTACCTCTGGCGGCACCGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAG 
               
               
                   
               
               
                 CCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCC 
               
               
                   
               
               
                 GTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCTCTAGC 
               
               
                   
               
               
                 CTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGAC 
               
               
                   
               
               
                 AAGAAGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCATGCCCTGCCCCT 
               
               
                   
               
               
                 GAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCTCCAAAGCCTAAGGACACCCTGATG 
               
               
                   
               
               
                 ATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGATCCTGAA 
               
               
                   
               
               
                 GTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGG 
               
               
                   
               
               
                 GAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAC 
               
               
                   
               
               
                 TGGCTGAACGGCAAAGAGTATAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCTATC 
               
               
                   
               
               
                 GAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGGGAACCTCAGGTGTACACCCTGCCT 
               
               
                   
               
               
                 CCCAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTC 
               
               
                   
               
               
                 TACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAG 
               
               
                   
               
               
                 ACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTG 
               
               
                   
               
               
                 GACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTG 
               
               
                   
               
               
                 CACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCTGGCAAGTGATGA AAGCTT GGC 
               
               
                   
               
               
                 GCGCC 
               
               
                   
               
               
                 &gt;m5; Heavy chain: Y57A (Changed TAC for GCC) 
               
               
                 [SEQ ID No. 17] 
                   
               
               
                 TTAATT AAGCTT GCCACCATGGACTGGACCTGGCGGATCCTGTTTCTGGTGGCCGCTGCT 
                   
               
               
                   
               
               
                 ACCGGCACACACGCCGAGGTGCAGCTGGTGGAGTCTGGCGGAGGACTGGTGCAGCCTGGC 
               
               
                   
               
               
                 GGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACACCTACATCCAC 
               
               
                   
               
               
                 TGGGTCCGGCAGGCTCCAGGCAAGGGACTGGAATGGGTGGCCCGGATCTACCCTACCAAC 
               
               
                   
               
               
                 GGC  ACCAGATACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCGCCGACACCTCC 
               
               
                   
               
               
                 AAGAACACCGCCTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTAC 
               
               
                   
               
               
                 TGCTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCCAGGGCACCCTG 
               
               
                   
               
               
                 GTCACTGTGTCCTCTGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTCCAGC 
               
               
                   
               
               
                 AAGTCTACCTCTGGCGGCACCGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAG 
               
               
                   
               
               
                 CCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCC 
               
               
                   
               
               
                 GTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCTCTAGC 
               
               
                   
               
               
                 CTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGAC 
               
               
                   
               
               
                 AAGAAGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCATGCCCTGCCCCT 
               
               
                   
               
               
                 GAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCTCCAAAGCCTAAGGACACCCTGATG 
               
               
                   
               
               
                 ATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGATCCTGAA 
               
               
                   
               
               
                 GTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGG 
               
               
                   
               
               
                 GAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAC 
               
               
                   
               
               
                 TGGCTGAACGGCAAAGAGTATAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCTATC 
               
               
                   
               
               
                 GAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGGGAACCTCAGGTGTACACCCTGCCT 
               
               
                   
               
               
                 CCCAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTC 
               
               
                   
               
               
                 TACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAG 
               
               
                   
               
               
                 ACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTG 
               
               
                   
               
               
                 GACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTG 
               
               
                   
               
               
                 CACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCTGGCAAGTGATGA AAGCTT GGC 
               
               
                   
               
               
                 GCGCC 
               
               
                   
               
               
                 &gt;m6; Heavy chain: R59E (changed AGA for GAG) 
               
               
                 [SEQ ID No. 18] 
                   
               
               
                 TTAATT AAGCTT GCCACCATGGACTGGACCTGGCGGATCCTGTTTCTGGTGGCCGCTGCT 
                   
               
               
                   
               
               
                 ACCGGCACACACGCCGAGGTGCAGCTGGTGGAGTCTGGCGGAGGACTGGTGCAGCCTGGC 
               
               
                   
               
               
                 GGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACACCTACATCCAC 
               
               
                   
               
               
                 TGGGTCCGGCAGGCTCCAGGCAAGGGACTGGAATGGGTGGCCCGGATCTACCCTACCAAC 
               
               
                   
               
               
                 GGCTACACC  TACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCGCCGACACCTCC 
               
               
                   
               
               
                 AAGAACACCGCCTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTAC 
               
               
                   
               
               
                 TGCTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCCAGGGCACCCTG 
               
               
                   
               
               
                 GTCACTGTGTCCTCTGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTCCAGC 
               
               
                   
               
               
                 AAGTCTACCTCTGGCGGCACCGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAG 
               
               
                   
               
               
                 CCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCC 
               
               
                   
               
               
                 GTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCTCTAGC 
               
               
                   
               
               
                 CTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGAC 
               
               
                   
               
               
                 AAGAAGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCATGCCCTGCCCCT 
               
               
                   
               
               
                 GAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCTCCAAAGCCTAAGGACACCCTGATG 
               
               
                   
               
               
                 ATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGATCCTGAA 
               
               
                   
               
               
                 GTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGG 
               
               
                   
               
               
                 GAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAC 
               
               
                   
               
               
                 TGGCTGAACGGCAAAGAGTATAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCTATC 
               
               
                   
               
               
                 GAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGGGAACCTCAGGTGTACACCCTGCCT 
               
               
                   
               
               
                 CCCAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTC 
               
               
                   
               
               
                 TACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAG 
               
               
                   
               
               
                 ACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTG 
               
               
                   
               
               
                 GACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTG 
               
               
                   
               
               
                 CACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCTGGCAAGTGATGA AAGCTT GGC 
               
               
                   
               
               
                 GCGCC 
               
            
           
         
       
     
     Amino acid sequences constituting the antibody mutant chains are provided below. The signal peptide is indicated using lower case letters and is not included in the residue numbering. Mutated positions are italicized and in bold font. 
     
       
         
           
               
               
            
               
                 Anti-HER2 Light chain wild-type 
                   
               
               
                 [SEQ ID No. 19] 
                   
               
               
                 .........|.........|.........|.........|.........|.........| 
                   
               
               
                   
               
               
                 mvlqtqvfislllwisgaygDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP 
               
               
                   
               
               
                 GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ 
               
               
                   
               
               
                 GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ 
               
               
                   
               
               
                 ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 Anti-HER2 Heavy chain wild-type 
               
               
                 [SEQ ID No. 20] 
                   
               
               
                 .........|.........|.........|.........|.........|.........| 
                   
               
               
                   
               
               
                 mdwtwrilflvaaatgthaEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP 
               
               
                   
               
               
                 GKGLEWVARTYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGG 
               
               
                   
               
               
                 DGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW 
               
               
                   
               
               
                 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK 
               
               
                   
               
               
                 SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY 
               
               
                   
               
               
                 VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK 
               
               
                   
               
               
                 AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
               
               
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 Mutant sequences 
               
               
                 &gt;m1; Light chain: F53N 
               
               
                 [SEQ ID No. 21] 
                   
               
               
                 mvlqtqvfislllwisgaygDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP 
                   
               
               
                   
               
               
                 GKAPKLLIYSAS  LYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ 
               
               
                   
               
               
                 GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ 
               
               
                   
               
               
                 ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 &gt;m2; Light chain: Y92A 
               
               
                 [SEQ ID No. 22] 
                   
               
               
                 mvlqtqvfislllwisgaygDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP 
                   
               
               
                   
               
               
                 GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQH  TTPPTFGQ 
               
               
                   
               
               
                 GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ 
               
               
                   
               
               
                 ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 &gt;m3; Light chain: F53N, Y92A 
               
               
                 [SEQ ID No. 23] 
                   
               
               
                 mvlqtqvfislllwisgaygDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP 
                   
               
               
                   
               
               
                 GKAPKLLIYSAS  LYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQH  TTPPTFGQ 
               
               
                   
               
               
                 GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ 
               
               
                   
               
               
                 ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 &gt;m4; Light chain: N30D 
               
               
                 [SEQ ID No. 24] 
                   
               
               
                 mvlqtqvfislllwisgaygDIQMTQSPSSLSASVGDRVTITCRASQDV  TAVAWYQQKP 
                   
               
               
                   
               
               
                 GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ 
               
               
                   
               
               
                 GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ 
               
               
                   
               
               
                 ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
               
                 &gt;m5; Heavy chain: Y57A 
               
               
                 [SEQ ID No. 25] 
                   
               
               
                 mdwtwrilflvaaatgthaEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP 
                   
               
               
                   
               
               
                 GKGLEWVARTYPTNG  TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGG 
               
               
                   
               
               
                 DGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW 
               
               
                   
               
               
                 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK 
               
               
                   
               
               
                 SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY 
               
               
                   
               
               
                 VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK 
               
               
                   
               
               
                 AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
               
               
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
               
                   
               
               
                 &gt;m6; Heavy chain: R59E 
               
               
                 [SEQ ID No. 26] 
                   
               
               
                 mdwtwrilflvaaatgthaEVQLVESGGGLVQPGGSLRLSCAASGENIKDTYTHWVRQAP 
                   
               
               
                   
               
               
                 GKGLEWVARIYPTNGYT  YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGG 
               
               
                   
               
               
                 DGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW 
               
               
                   
               
               
                 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK 
               
               
                   
               
               
                 SCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY 
               
               
                   
               
               
                 VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK 
               
               
                   
               
               
                 AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
               
               
                 DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 
               
            
           
         
       
     
     REFERENCES 
     
         
         TS1. Cho, H. S., Mason, K., Ramyar, K. X., Stanley, A. M., Gabelli, S. B., Denney, D. W., and Leahy, D. J. (2003) Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab, Nature 421, 756-760. 
         TS2. Li, Z. and Scheraga, H. A. (1987) Monte Carlo-minimization approach to the multiple-minima problem in protein folding, Proc. Nat. Acad. Sci. U.S.A. 84, 6611-6615. 
         TS3. Naim, M., Bhat, S., Rankin, K. N., Dennis, S., Chowdhury, S. F., Siddiqi, I., Drabik, P., Sulea, T., Bayly, C. I., Jakalian, A., and Purisima, E. O. (2007) Solvated Interaction Energy (SIE) for Scoring Protein-Ligand Binding Affinities. 1. Exploring the Parameter Space, J. Chem. Inf. Model. 47, 122-133. 
         TS4. Selzer, T., Albeck, S., and Schreiber, G. (2000) Rational design of faster associating and tighter binding protein complexes, Nat. Struct. Mol. Biol. 7, 537-541.