Abstract:
The present invention relates to the modification of immunoglobulin superfamily (IgSF) domains, IgSF fragments and fusion proteins thereof, especially to the modification of antibody derivatives, so as to improve their solubility, and hence the yield, and ease of handling. The inventors have found that this can be achieved by making the region which comprised the interface with domains adjoined to said IgSF domain in a larger fragment or a full IgSF protein, and which becomes exposed in the IgSF domain, more hydrophilic by modification. The present invention describes DNA sequences encoding modified IgSF domains or fragments and fusion proteins thereof, vectors and hosts containing these DNA sequences, IgSF domains or fragments or fusion proteins obtainable by expressing said DNA sequences in suitable expression systems, and a method for modifying IgSF domains, so as to improve their solubility, expressibility and ease of handling.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a Continuation of International Application PCT/EP97/03792, filed Jul. 16, 1997, which claims priority from European patent application EP 96111441.0, filed Jul. 16, 1996. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to the modification of immunoglobuling superfamily (IgSF) domains and derivatives thereof so as to increase their solubility, and hence the yield, and ease of handling. 
     Small antibody fragments show exciting promise for use as therapeutic agents, diagnostic reagents, and for biochemical research. Thus, they are needed in large amounts, and the expression of antibody fragments, e.g. Fv, single-chain-Fv (scFv), or Fab in the periplasm of  E. coli  (Skerra &amp; Plückthun 1988; Better et al., 1988) is now used routinely in many laboratories. Expression yields vary widely, however, especially in the case of scFvs. While some fragments yield up to several mg of functional, soluble protein per litre and OD of culture broth in shake flask 25 culture (Carter et al., 1992, Plückthun et al. 1996), other fragments may almost exclusively lead to insoluble material, often found in so-called inclusion bodies. Functional protein may be obtained from the latter in modest yields by a laborious and time-consuming refolding process. The factors influencing antibody expression levels are still only poorly understood. 
     Folding efficiency and stability of the antibody fragments, protease lability and toxicity of the expressed proteins to the host cells often severely limit actual production levels, and several attempts have been tried to increase expression yields. For example, Knappik &amp; Plückthun (1995) have identified key residues in the antibody framework which influence expression yields dramatically. Similarly, Ullrich et al. (1995) found that point mutations in the CDRs can increase the yields in periplasmic antibody fragment expression. Nevertheless, these strategies are only applicable to a few antibodies. 
     The observations by Knappik &amp; Plückthun (1995) indicate that optimizing those parts of the antibody fragment which are not directly involved in antigen recognition can significantly improve folding properties and production yields of recombinant Fv and scFv constructs. The causes for the improved expression behavior lie in the decreased aggregation behavior of these molecules. For other molecules, fragment stability and protease resistance may also be affected. The understanding of how specific sequence modifications change these properties is still very limited and currently under active investigation. 
     Difficulties in expressing and manipulating protein domains may arise because amino acids which are normally buried within the protein structure become exposed when only a portion of the whole molecule is expressed. Aggregation may occur through interaction of newly solvent-exposed hydrophobic residues originally forming the contact regions between adjacent domains. Leistler and Perham (1994) could show that a certain domain of glutathione reductase may be expressed separately from its neighboring domains, but the protein showed non-specific association in vitro forming multimeric protein species. The introduction of hydrophilic residues instead of exposed hydrophobic amino acids could decrease this aggregation tendency and thus stabilize this isolated domain. Both wild type and modified domains were exclusively found in inclusion bodies and had to be refolded. Although in vitro experiments contributed a lot to define various intermolecular interactions, which drive folding processes, they are only of limited value in predicting the folding behaviour of different polypeptide chains in vivo (Gething &amp; Sambrook, 1992). Thus, Leistler and Perham do not teach or suggest how to increase expression yields of soluble protein domains. 
     In the case of antibodies, two chains comprising several domains dimerize, each domain consisting of a b-barrel whose two b-sheets are held together by a disulphide bond, forming the so-called immunoglobulin fold. Two domains, one variable domain (VL) and one constant domain (CL) are adjacent along the longitudinal axis in the light chain (VL-CL), and four domains, one variable domain (VH) and three constant domain (CH1 to CH3) are adjacent along the longitudinal axis in the heavy chain (VH-CH1-CH2-CH3). In the dimer formed by chains a and b, two such domains associate laterally: VLa with VHa, CLa with CH1a, VLb with VHb, CLb with CH1b CH2a with CH2b and CH3a with CH3b. In WO 92/01787 (Johnson et al., 1992), it is taught that isolated single domains, e.g. VH, can be modified in the former VL/VH interface region by exchanging hydrophobic residues by hydrophilic ones without changing the specificity of the parent domain. The rationale for WO 92/01787 was the assumption that exposed hydrophobic residues might lead to non-specific binding, interaction with surfaces and decreased stability. Data for increase in binding specificity was given, but increase in expression level was not shown. Furthermore, WO 92/01787 would not be applicable to any antibody fragment containing the complete antigen binding site, as it must contain VL and VH. In the case of T cell receptors, two chains (a and b) dimerize, each consisting of a variable (V) and a constant (C) domain with the immunoglobulin fold, and one transmembrane domain. In each chain, the variable and constant domains are adjacent along the longitudinal axis in the chains (Va-Ca; Vb-Cb) and associate laterally with the corresponding domains of the second chain (Va-Vb; Ca-Cb). 
     Various other molecules of the immunoglobulin superfamily, such as CD2, CD4, CD16, CD22, comprise only one chain, wherein two or more domains (variable and/or constant) with the immunoglobulin fold are adjacent along the longitudinal axis in the chains. 
     SUMMARY OF THE INVENTION 
     The present inventors have found that expression problems are largely associated with a part of the molecule that has hitherto not been regarded relevant for expression studies and which comprises the interface between adjacent domains within an immunoglobulin chain. This surprising finding forms the basis of the present invention, which provides a general solution to the problems associated with production of domains or fragments of the immunoglobulin superfamily (IgSF), especially antibody fragments, which exhibit poor solubility or reduced levels of expression. 
     In addition to lateral interactions between domains of different chains described above, there are well documented contacts between adjacent domains within individual chains along the longitudinal axis. For example, in the case of an antibody (Lesk &amp; Chothia, 1988), the “bottom” of VL makes contact with the “top” of CL, and, in a similar manner there are contacts between VH and CH1. The contacts at these inter-domain interfaces are probably essential for the compact arrangement of the Fab fragment, and, as is typical for such contacts, are at least partially hydrophobic in nature (Lesk &amp; Chothia, 1988). 
     The basis of the present invention is the surprising finding that the solubility (and hence the yield) of antibody fragments comprising at least one domain can be dramatically increased by decreasing the hydrophobicity of former interfaces at the “end” of said domain, where it would normally adjoin a second domain within a chain in a larger antibody fragment or full antibody. This is surprising and could not have been predicted from the prior art (WO 92/01787), because the size of the longitudinal interface, for example, in a scFv fragment, is much smaller than that between VH and VL, and therefore, the amino acids which make up the interfaces between VH and CH1 or between VL and CL in a Fab fragment represent a much smaller proportion of the total surface area of the scFv molecule, and would accordingly be expected to play less of a role in determining the physical properties of the molecule. 
     The present invention has the additional advantage that because the alterations effected in the molecules that lead to said decreased hydrophobicity of former interfaces are located at the most distant part of the domain from the CDRs, applying the invention is unlikely to have a deleterious effect on the binding properties of the molecule. This is not the case in WO 92/01787, where at least one modification is close to the CDRs and may therefore be expected to have an impact on antigen binding. Furthermore, WO 92/01787 cannot be applied to VL/VH heterodimers, as explained above. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 provides a space filling representation of the Fv fragment of the antibody 4-4-20. 
     FIG. 2 presents the variable/constant domain interface residues for VL (2a) and VH (2b). For 30 non-redundant Fab fragments taken from the Brookhaven Databank, the solvent accessible surface of the amino acid side chains was calculated in the context of an Fv and of an Fab fragment. The plot shows the relative reduction in accessible surface upon contact with the constant domains (overlay plot for all 30 Fv fragments). In the sequence alignment, residues contributing to the v/c interface are highlighted. The symbols indicate the relative reduction of solvent accessible surface upon removing the constant domains (symbols: no symbol &lt;1%; 1&lt;20%; n&lt;40%; s&lt;60%; t&lt;80%, and u 3  80%). Circles indicate those positions which are further analyzed (see Table 1). 
     FIG. 3 presents Western blots showing the insoluble (i) and soluble (s) fractions of cell extracts, prepared as described in Material and Methods, expressing the scFv fragments of the antibody 4-4-20. The amino acids substituted in the various mutants are given in Table 2. 
     FIG. 4 presents a Scatchard plot of the fluorescence titration of fluorescein (20 nM) with antibody (4 to 800 nM), measured at 510 nm. The value r was obtained from (F-Fo)/(FY-Fo), where F is the measured fluorescein fluorescence at a given antibody concentration, Fo is the fluorescence in the absence of antibody and FY when antibody is present in large excess. Note that r gives the saturation of fluorescein by antibody. (a) Titration of wt scFv, (b) titration of Flu4 (V84D). 
     FIG. 5 presents an overlay plot of the urea denaturation curves ((X) wt scFv, (o) Flu4). 
     FIG. 6 presents the thermal denaturation time courses at 40 and 44° C. for wt and Flu4 scFv fragment ((a) wt scFv at 40° C., (b) Flu4 at 40° C., (c) Flu4 at 44° C., (d) wt scFv at 44° C.). 
    
    
     Table 1 describes the sequence variability of residues contributing to the v/c interface. Residue statistics are based on the variable domain sequences in the Kabat database (March 1996). Sequences which were &lt;90% complete were excluded from the analysis. Number of sequences analyzed: human VL kappa: 404 of 881, murine VL kappa: 1061 of 2239, human VL lambda: 223 of 409, murine VL lambda: 71 of 206, human VH: 663 of 1756, murine VH: 1294 of 3849. Position refers to the sequence position according to Kabat et al. 1991, %exp. (Fab) to the relative side chain accessibility in an Fab fragment as calculated by the program NACCESS (NACCESS v2.0 by Simon Hubbard, %exp. (ind.) to the relative side chain accessibility in the isolated VL or VH domain, %buried to the relative difference in side chain accessibility between Fv and Fab fragment. Consensus refers to the sequence consensus, and Distribution to the distribution of residue types. 
     Table 2 describes mutations introduced in the scFv fragment of the antibody 4-4-20. Each line represents a different protein carrying the mutations indicated. The residues are numbered according to Kabat et al. (1991). 
     Table 3 describes KD values of the different scFv mutants determined in fluorescence titration. The KD values are given in nM, the error was calculated from the Scatchard analysis (FIG.  4 ). # determined by Miklasz et al. (1995). 
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention relates to a modified immunoglobulin superfamily (IgSF) domain or fragment which differs from a parent IgSF domain or fragment in that the region which comprised or would comprise the interface with a second domain adjoined to said parent IgSF domain or fragment within the protein chain of a larger IgSF fragment or a full IgSF protein, and which is exposed in said parent IgSF domain or fragment in the absence of said second domain, is made more hydrophilic by modification. 
     In the context of the present invention, the term immunoglobulin superfamily (IgSF) domain refers to those parts of members of the immunoglobulin superfamily which are characterized by the immunoglobulin fold, said superfamily comprising the immunoglobulins or antibodies, and various other proteins such as T-cell receptors or integrins. The term IgSF fragment refers to any portion of a member of the immunoglobulin superfamily, said portion comprising at least one IgSF domain. The term adjoining domain refers to a domain which is contiguous with a first domain. The term interface refers to a region of said first domain where interaction with the adjoining domain takes place. The terms hydrophobic and hydrophilic refer to a physical property of amino acids, which can be estimated quantitatively: tabulated values of hydrophobicity for the twenty naturally-occurring amino acids are available (Nozaki &amp; Tanford, 1971; Casari &amp; Sippl, 1992; Rose &amp; Wolfenden, 1993). 
     The residues to be modified can be identified in a number of ways. For example, in one way, the solvent accessibilities (Lee &amp; Richards, 1971) of hydrophobic interface residues in said parent IgSF fragment compared to said larger IgSF fragment or full IgSF protein are calculated, with high accessibilities indicating highly exposed residues. In a second way, the number of van der Waals contacts of hydrophobic interface residues in said larger IgSF fragment or full IgSF protein is calculated. A large number for a residue of said parent domain indicates that it will be highly solvent-exposed in the absence of an adjoining domain. There are other ways of calculating or determining residues to be modified according to the present invention, and one of ordinary skill in the art will be able to identify and practice these ways. 
     By analyzing computer models of said parent IgSF fragment, interactions of said highly exposed residues within the fragment can be identified. Such interactions could stabilize the parent IgSF fragment. Residues, which interact closely with other hydrophobic residues and which can be identified by anyone of ordinary skill in the art, should not preferentially be mutated. 
     The modification referred to above may be effected in a number of ways which are well known to one skilled in the art. In a preferred embodiment, the modification is a substitution of one or more amino acids at the exposed interface, identified as described above, with amino acids which are more hydrophilic Alternatively, one or more amino acids can be inserted in said interface, or one or more amino acids can be deleted from said interface, so as to increase its overall hydrophilicity. Furthermore, any combination of substitution, insertion and deletion can be effected to reduce the hydrophobicity of said interface. Also comprised by the present invention is the possibility that the substitution or insertion comprises amino acids with a relatively high hydrophobicity value, or that the deletion comprises amino acids with relatively low hydrophobicity value, as long as the overall hydrophilicityvalue is increased in said interface region. Modifications such as substitution, insertion and deletion can be effected using standard methods which are well known to practitioners skilled in the art. By way of example, the skilled artisan can use either site-directed or PCR-based mutagenesis (Ho et al., 1989; Kunkel et al., 1991; Trower, 1994; Viville, 1994), or total gene synthesis (Prodromou &amp; Pearl, 1992) to effect the necessary modifications). In a further embodiment, the mutations may be obtained by random mutagenesis and screening of random mutants, using a suitable expression and screening system (see, for example, Stemmer, 1994; Crameri et al., 1996). 
     In a preferred embodiment, the amino acid(s) which replace(s) the more hydrophobic amino acids include Asn, Asp, Arg, Gln, Glu, Gly, His, Lys, Ser, and Thr. These are among the more hydrophilic of the 20 naturally-occurring amino acids, and have proven to be particularly effective in the application of the present invention. Said amino acids, alone or in combination, or in combination with other amino acids, can also be used to form the above mentioned insertion which makes the interface region more hydrophilic. 
     The parent IgSF domain or fragment referred to above can be one of several different types. In a preferred embodiment, said parent domain or fragment is derived from an antibody. In one embodiment, said parent antibody fragment comprises an Fv fragment. In this context, the term Fv fragment refers to a complex comprising the VL (variable light) and VH (variable heavy) portions of the antibody molecule. In a further embodiment, the parent antibody fragment may be a single-chain Fv fragment (scFv; Bird et al., 1988; Huston et al., 1988), in which the VL and VH chains are joined, in either a VL-VH, or VH-VL orientation, by a peptide linker. In yet a further embodiment, the parent antibody fragment may be an Fv fragment stabilized by an inter-domain disulphide bond. This is a structure which can be made by engineering into each chain a single cysteine residue, wherein said cysteine residues from two chains become linked through oxidation to form a disulphide (Glockshuber et al., 1990; Brinkmann et al., 1993). 
     In a most preferred embodiment, the interface region of the variable domains mentioned above comprises residues 9, 10, 12, 15, 39, 40, 41, 80, 81, 83, 103, 105, 106, 106A, 107, 108 for VL, and residues 9, 10, 11, 13, 14, 41, 42, 43, 84, 87, 69, 105, 108, 110, 112, 113 for VH according to the Kabat numbering system (Kabat et al., 1991). Said numbering system was established for the sequences of whole antibodies, but can be adapted correspondingly to describe the sequences of isolated antibody domains or antibody fragments, even in the case of scFv fragments, where VL and VH are connected via a peptide linker, and where the protein sequence from N- to C-terminus has to be numbered differently. This means that the Kabat numbering system is used in the present invention as a sequence description relative to the existing data on antibody sequences, not as an absolute description of actual positions within the antibody fragment sequences of interest. 
     In a further embodiment, said parent antibody fragment comprises a Fab fragment. In this context, the term Fab refers to a complex comprising the VL-CL (variable and constant light) and VH-CH1 (variable and first constant heavy) portions of the antibody molecule, and the term interface region refers to a region in the first constant domain of the heavy chain (CH1) which is, or would be adjoined to, the CH2 domain in a larger antibody fragment or full antibody. 
     In a still further embodiment, said parent IgSF fragment is a fusion protein of any of said domains or fragments and another protein domain, derived from an antibody or any other protein or peptide. The advent of bacterial expression of antibody fragments has opened the way to the construction of proteins comprising fusions between antibody fragments and other molecules. A further embodiment of the present invention relates to such fusion proteins by providing for a DNA sequence which encodes both the modified IgSF domain or fragment, as described above, as well as an additional moiety. Particularly preferred are moieties which have a useful therapeutic function. For example, the additional moiety may be a toxin molecule which is able to kill cells (Vitetta et al., 1993). There are numerous examples of such toxins, well known those skilled in the art, such as the bacterial toxins  Pseudomonas  exotoxin A, and diphtheria toxin, as well as the plant toxins ricin, abrin, modeccin, saporin, and gelonin. By fusing such a toxin to an antibody fragment, the toxin can be targeted to, for example, diseased cells, and thereby have a beneficial therapeutic effect. Alternatively, the additional moiety may be a cytokine, such as IL-2 (Rosenberg &amp; Lotze, 1986), which has a particular effect (in this case a T-cell proliferative effect) on a family of cells. In a further preferred embodiment, the additional moiety is at least part of a surface protein which may direct the fusion protein to the surface of an organism, for example, a cell or a phage, and thereby displays the IgSF partner. Preferably, the additional moiety is at least part of a coat protein of filamentous bacteriophages, most preferably of the geneIII protein. In a further embodiment, the additional moiety may confer on its IgSF partner a means of detection and/or purification. For example, the fusion protein could comprise the modified IgSF domain or fragment and an enzyme commonly used for detection purposes, such as alkaline phosphatase (Blake et al., 1984). There are numerous other moieties which can be used as detection or purification tags, which are well known to the practitioner skilled in the art. Particularly preferred are peptides comprising at least five histidine residues (Hochuli et al., 1988), which are able to bind to metal ions, and can therefore be used for the purification of the protein to which they are fused (Lindner et al., 1992). Also provided for by the invention are additional moieties such as the commonly used c-myc and FLAG tags (Hopp et al., 1988; Knappik &amp; Pluckthun, 1994). 
     By engineering one or more fused additional domains, IgSF domains or fragments can be assembled into larger molecules which also fall under the scope of the present invention. To the extent that the physical properties of the IgSF domain or fragment determine the characteristics of the assembly, the present invention provides a means of increasing the solubility of such larger molecules. For example, mini-antibodies (Pack, 1994) are dimers comprising two antibody fragments, each fused to a self-associating dimerization domain. Dimerization domains which are particularly preferred include those derived from a leucine zipper (Pack &amp; Plückthun, 1992) or helix-turn-helix motif (Pack et al., 1993). 
     All of the above embodiments of the present invention can be effected using standard techniques of molecular biology known to anyone skilled in the art. 
     The compositions described above may have utility in any one of a number of settings. Particularly preferred are diagnostic and therapeutic compositions. 
     The present invention also provides methods for making the compositions and compounds comprised therein described above. Particularly preferred is a method comprising the following steps: 
     i) analyzing the interface region of an IgSF domain for hydrophobic residues which are solvent-exposed using either a solvent-accessibility approach (Lee &amp; Richards, 1971), analysis of van der Waals interactions in the interface region, or similar methods which are well known to one skilled in the art, 
     ii) identifying one or more of the hydrophobic residues to be substituted by more hydrophilic residues, or one or more positions where hydrophilic residues or amino acid stretches enhancing the overall hydrophilicity of the interface region can be inserted into said interface region, or one or more positions where hydrophobic residues or amino acid stretches enhancing the overall hydrophobicity of the interface region can be deleted from said interface region, or any combination of said substitutions, said insertions, and said deletions to give one or more mutants of said parent IgSF domain, 
     iii) preparing DNA encoding mutants of said IgSF domain, characterized by the changes identified in ii), by using e.g. conventional mutagenesis or gene synthesis methods, said DNA being prepared either separately or as a mixture, 
     iv) introducing said DNA or DNA mixture in a vector system suitable for expression of said mutants, 
     v) introducing said vector system into suitable host cells and expressing said mutant or mixture of mutants, 
     vi) identifying and characterizing mutants which are obtained in higher yield in soluble form, and 
     vii) if necessary, repeating steps iii) to vi) to increase the hydrophilicity of said identified mutant or mutants further. 
     The host referred to above may be any of a number commonly used in the production of heterologous proteins, including but not limited to bacteria, such as  E. coli  (Ge et al, 1995), or  Bacillus subtilis  (Wu et al., 1993), fungi, such as yeasts (Horwitz et al., 1988; Ridder et al., 1995) or filamentous fungus (Nyyssönen et al., 1993), plant cells (Hiatt, 1990, Hiatt &amp; Ma, 1993; Whitelam et al., 1994), insect cells (Potter et al., 1993; Ward et al., 1995), or mammalian cells (Trill et al., 1995). 
     The invention also relates to a method for the production of an IgSF domain or fragment of the invention comprising culturing a host cell of the invention and isolating said domain or fragment. 
     The invention is now demonstrated by the following examples, which are presented for illustration only and are not intended to limit the scope of the invention. 
     EXAMPLES 
     i) Abbreviations 
     Abbreviations are defined as follows: CDR: complementarity determining region; dsFv: disulfide-linked Fv fragment; IMAC: immobilized metal ion affinity chromatography; IPTG: isopropyl-b-D-thiogalactopyranoside; i/s: ratio insoluble/soluble; H(X): heavy chain residue number X; L(X): light chain residue number X; NTA: nitrilo-triacetic acid; OD550: optical density at 550 nm; PDB: protein database; scFv: single-chain Fv fragment; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; v/c: variable/constant; wt: wild type. 
     ii) Material and Methods 
     (a) Calculation of Solvent Accessibility 
     Solvent accessible surface areas for 30 non-redundant Fab fragments and the Fv fragments derived from these by deleting the constant domain coordinates from the PDB file were calculated using the latest version, as of March 1996, of the program NACCESS (http://www.biochem.ucl.ac.uk/˜roman/naccess/naccess) based on the algorhithm described by Lee &amp; Richards (1971). 
     (b) scFv Gene Synthesis 
     The single-chain Fv fragment (scFv) in the orientation VL-linker-VH of the antibody 4-4-20 (Bedzyk et al., 1990) was obtained by gene synthesis (Prodromou and Pearl, 1992). The VL domain carries a three-amino acid long FLAG tag (Knappik and Plückthun, 1994). We have used two different linkers with a length of 15 (Gly4Ser)3 and 30 amino acids (Gly4Ser)6, respectively. The gene so obtained was cloned into a derivative of the vector pIG6 (Ge et al., 1995). The mutant antibody fragments were constructed by site-directed mutagenesis (Kunkel et al., 1987) using single-stranded DNA and up to three oligonucleotides per reaction. 
     (c) Expression 
     Growth curves were obtained as follows: 20 ml of 2xYT medium containing 100 g/ml ampicillin and 25 g/ml streptomycin were inoculated with 250 1 of an overnight culture of  E. coli  JM83 harboring the plasmid encoding the respective antibody fragment and incubated at 24.5° C. until an OD550 of 0.5 was reached. IPTG (Biomol Feinchemikalien GmbH) was added to a final concentration of 1 mM and incubation was continued for 3 hours. The OD was measured every hour, as was the b-lactamase activity in the culture supernantant to quantify the degree of cell leakiness. Three hours after induction an aliquot of the culture was removed and the cells were lysed exactly as described by Knappik and Plückthun (1995). The b-lactamase activity was measured in the supernatant, in the insoluble and in the soluble fraction. The fractions were assayed for antibody fragments by reducing SDS-PAGE, with the samples normalized to OD and b-lactamase activity to account for possible plasmid loss as well as for cell leakiness. The gels were blotted and immunostained using the FLAG antibody Ml (Prickett et al., 1989) as the first antibody, an Fc-specific anti-mouse antiserum conjugated to horseradish peroxidase (Pierce) as the second antibody, using a chemoluminescent detection assay described elsewhere (Ge et al., 1995). 
     (d) Purification 
     Mutant scFv fragments were purified by a two-column procedure. After French press lysis of the cells, the raw  E. coli  extract was first purified by IMAC (Ni-NTA superflow, Qiagen) (20 mM HEPES, 500 mM NaCl, pH 6.9; step gradient of imidazole 10, 50 and 200 mM) (Lindner et al., 1992) and, after dialyzing the IMAC eluate against 20 mM MES pH 6.0, finally purified by cation exchange chromatography (S-Sepharose fast flow column, Pharmacia) (20 mM MES, pH 6.0; salt gradient 0-500 mM NaCl). Purity was controlled by Coomassie stained SDS-PAGE. The functionality of the scFv was tested by competition ELISA. 
     Because of its very poor solubility in the periplasmic system, the wt 4-4-20 was expressed as cytoplasmic inclusion bodies in the T7-based system (Studier &amp; Moffatt, 1986; Ge et al., 1995). The refolding procedure was carried out as described elsewhere (Ge et al., 1995). For purification, the refolding solution (21) was loaded over 10 h without prior dialysis onto a fluorescein affinity column, followed by a washing step with 20 mM HEPES, 150 mM NaCl, pH 7.5. Two column volumes of 1 mM fluorescein (sodium salt, Sigma Chemicals Co.) pH 7.5 were used to elute all functional scFv fragment. Extensive dialysis (7 days with 12 buffer changes) was necessary to remove all fluorescein. All purified scFv fragments were tested in gel filtration (Superose-12 column, Pharmacia SMART-System, 20 mM HEPES, 150 mM NaCl, pH 7.5). 
     (e) KD Determination by Fluorescence Titration 
     The concentrations of the proteins were determined photometrically using an extinction coefficient calculated according to Gill and von Hippel (1989). Fluorescence titration experiments were carried out by taking advantage of the intensive fluorescence of fluorescein. Two ml of 20 mM HEPES, 150 mM NaCl, pH 7.5 containing 10 or 20 nM fluorescein were placed in a cuvette with integrated stirrer. The excitation wavelength was 485 nm, emission spectra were recorded from 490 to 530 nm. Purified scFv (in 20 mM HEPES, 150 mM NaCl, pH 7.5) was added in 5 to 100 1 aliquots, and after a 3 min equilibration time a spectrum was recorded. All spectra were recorded at 20° C. The maximum of emission at 510 nM was used for determining the degree of complexation of scFv to fluorescein, seen as quenching as a function of the concentration of the antibody fragment. The KD value was determined by Scatchard analysis. 
     (f) Equilibrium denaturation measurement 
     Equilibrium denaturation curves were obtained by denaturation of 0.2 M protein in HEPES buffered saline (HBS) buffer (20 mM HEPES, 150 mM NaCl, 1 mM EDTA, pH 7.5) and increasing amounts of urea (1.0-7.5 M; 20 mM HEPES, 150 mM NaCl, pH 7.4; 0.25 M steps) in a total volume of 1.7 ml. After incubating the samples at 10° C. for 12 hours and an additional 3 hours at 20° C. prior to measurements, the fluorescence spectra were recorded at 20° C. from 320-360 nm with an excitation wavelength of 280 nm. The emission wavelength of the fluorescence peak shifted from 341 to 347 nm during denaturation and was used for determining the fraction of unfolded molecules. Curves were fitted according to Pace (1990). 
     (g) Thermal Denaturation 
     For measuring the thermal denaturation rates, purified scFv was dissolved in 2 ml HBS buffer to a final concentration of 0.5 M. The aggregation was followed for 2.5 h at 40° C. and at 44° C. by light scattering at 400 nm. 
     iii) Results 
     (a) Comparison of Known Antibody Sequences 
     Compared to other domain/domain interfaces in proteins, the interface between immunoglobulin variable and constant domains is not very tightly packed. A comparison of 30 non-redundant Fab structures in the PDB database showed that between the light chain variable and constant domain an area of 410 ±90 Å2 per domain is buried, while the heavy chain variable and constant domains interact over an area of 710 ±180 Å2. Some, but not all of the interface residues are hydrophobic, predominantly aliphatic. Generally, sequence conservation of the residues contributing to the v/c domain interface is not particularly high. Still, the v/c domain interface shows up as a marked hydrophobic patch on the surface of an Fv fragment (FIG.  1 ). 
     Solvent accessible surface areas for 30 non-redundant Fab fragments and their corresponding Fv fragments (derived from the Fab fragment by deleting the constant domain coordinates from the PDB file) were calculated using the program NACCESS (Lee &amp; Richards, 1971). Residues participating in the v/c domain interface were identified by comparing the solvent-accessible surface area of each amino acid side chain in the context of an Fv fragment to its accessible surface in the context of an Fab fragment. FIG. 2 shows a plot of the relative change in side chain accessibility upon deletion of the constant domains as a function of sequence position. Residues which show a significant reduction of side chain accessibility are also highlighted in the sequence alignment. To assess sequence variability in the positions identified in FIG. 2, the variable domain sequences collected in the Kabat database (status March 1996) were analyzed (Table 1). Of the 15 interface residues identified in the VL domain of the antibody 4-4-20 (FIG.  1  and Table 1), L9(leu), L12(pro), L15(leu), L40(pro), L83(leu), and L106(ile) are hydrophobic and therefore candidates for replacement. Of the 16 interface residues in the VH domain, Hll(leu), H14(pro), H41(pro), H84(val), H87(met) and H89(ile) were identified as possible candidates for substitution by hydrophilic residues in the scFv fragment of the antibody 4-4-20 (FIG.  1  and Table 1). 
     Not all of these hydrophobic residues are equally good candidates for replacements, however. While residues which are hydrophobic in one particular sequence but hydrophilic in many other sequences may appear most attractive, the conserved hydrophobic residues listed in Table 1 have also been investigated, since the evolutionary pressure which kept these conserved residues acted on the Fab fragment within the whole antibody, but not the isolated Fv portion. In this study, we did not replace the proline residues since proL40 and pro H41 form the hairpin turns at the bottom of the framework II region, while the conserved VL cis-proline L8 and proline residues H9 and H14 determine the shape of framework I of the immunoglobulin variable domains. 
     Excluding prolines, this leaves residues L9 (leu in 4-4-20, ser in most kappa chains), L15 (leu, usually hydrophobic), L83 (leu, usually val or phe) and L106 (Ile, as in 86% of all kappa chains) in the VL domain and Hll (leu as in 60% of all heavy chains), H84 (val, in other VH domains frequently ala or ser), H87 (met, usually ser) and H89 (ile, most frequently val) in VH as possible candidates for replacement in the 4-4-20 scFv fragment. 
     (b) Mutations in the 4-4-20 scFv 
     For the 4-4-20 scFv fragment some of the crucial residues identified in the sequence analysis described above are already hydrophilic, but nevertheless 9 residues are of hydrophobic nature (including pro12 in the light chain) (Table 1). We chose three residues for closer analysis by mutations. 
     Leu15 in VL is a hydrophobic amino acid in 98% of all kappa chains (Table 1). Leu11 is conserved in VH (Table 1) and is involved in v/c interdomain contacts (Lesk &amp; Chothia, 1988). In contrast, valine occurs very infrequently at position H84; mainly found at this position are threonine or serine and alanine (Table 1). As can be seen in FIG. 1, val84 is contributing to a large hydrophobic patch at the newly exposed surface of VH. All three positions were mutated into acidic residues, and L11 was also changed to asparagine (Table 2). 
     The scFv fragment was tested and expressed with two different linkers, the 15-mer linker (Gly4Ser)3 (Huston et al., 1995) and the same motif extended to 30 amino acids (Gly4Ser)6. All mutations were tested in both constructs. The in vivo results of the different mutations on solubility were identical, and therefore only the results of the 30-mer linker are described in more detail. The periplasmic expression experiments were carried out at 24.5° C., and all constructs were tested for soluble and insoluble protein by immunoblotting. The ratio of insoluble to soluble (i/s) protein was determined for every mutant. In FIG. 3 A-D, insoluble (lane 1) and soluble (lane 2) fractions of the wt scFv are shown. Nearly no soluble material occurs in periplasmic expression, which is consistent with previous reports of Bedzyk et al. (1990) and Denzin et al. (1991), who described earlier that the periplasmic expression of the wt scFv leads mainly to periplasmic inclusion bodies. 
     The single point mutation L15E in VL (Flul) shows no effect on the ratio i/s when compared with the wt (FIG. 3A, lane 3, 4). Mutating leu at position 11 in the heavy chain to asparagine (Flu2) also shows nearly no effect compared to the wt, whereas the subtitution with aspartic acid (Flu3) changes the i/s ratio to more soluble protein, but still this effect is not very dramatic. In contrast, the point mutation at position 84 (Flu4, FIG. 3B, lane 3, 4 and FIG. 3D, lane 3, 4) had a dramatic influence on the solubility of the scFv fragment of the antibody 4-4-20. The ratio i/s is changed to about 1:1, resulting in a 25-fold increase of soluble protein compared to the wt. 
     The combination of V84D with LIN or L11D (Flu5, Flu6) also changes the ratio i/s compared to the wt, but this ratio compared to V84D alone is not improved further (FIG.  3 B). Interestingly, the combination of Flu5 with the light chain mutation at position 15 (Flu9) leads to less soluble material (FIG. 3C lane 7,8) than Flu5 itself (FIG. 3B, lane 5, 6). The negative influence of the L15E mutations can also be seen in Flu8 (FIG. 3C, lane 5, 6) compared with Flu3 (FIG. 3A, lane 7, 8). In FIG. 3D the comparison of the wt (lane 1, 2 and 5, 6) and Flu4 (lane 3, 4 and 7, 8) is shown in both the 15-mer and the 30-mer construct. 
     The negative effect of L15E can be rationalized by looking at a model of the 4-4-20 scFv fragment. L15 is forming a hydrophobic pocket together with residues A80, L83, and L106. Apparently, L15 stabilizes the scFv fragment by hydrophobic interactions with its closest neighbours. Thus the exchange L15E for making the scFv fragment more hydrophilic and more soluble is made at the expense of the fragment stability. The analysis of hydrophobic interactions within a fragment should thereby by used to choose the solvent-exposed residues to be mutated in the case of any other antibody fragment. Combinations of various serine mutations in VH led to further improvements in the i/s ratio. The mutants FH15 (V84S, M87S, I89S) and FH20 (L11S, v84s, M87S, I89S) both showed more than 70% of soluble protein in immunoblots (data not shown). The negative effect of L15E 
     (c) Functional Expression and Purification 
     The oligomerization of scFv fragments as a function of linker length has been investigated previously. A continuous decrease in the amount of dimer and multimer formation as a function of linker length has been reported (Desplancq et al., 1994; Whitlow et al., 1994). While the (Gly4Ser)3 linker has been shown to lead to monomeric scFvs in many cases in the VH-VL direction, this is often not the case in the VL-VH direction. This is caused by an asymmetry in the VL/VH arrangement, leading to a longer distance between the end of VH and the N-terminus of VL than between C-terminus of VL and N-terminus of VH (Huston et al., 1995). Consequently, a linker of identical length may lead to different properties of the resulting molecules. 
     Since we have chosen to use the minimal pertubation FLAG (Knappik &amp; Plückthun, 1994) at the N-terminus of VL in our constructs and thus the VL-linker-VH orientation, we have investigated the use of longer linkers. In the periplasmic expression in  E. coli  no difference between the 15-mer and the 30-mer linker in the corresponding mutants is visible (FIG.  3 D), but when we attempted to purify the two Flu4 scFvs with long and short linker, a big discrepancy between the two constructs was found. The purification of the Flu4 mutant (V84D) with the 15-mer linker leads to very small amounts of partially purified protein (about 0.015 mg per liter and OD; estimated from SDS-PAGE after IMAC purification), whereas the 30-mer linker construct gives about 0.3 mg per liter and OD of highly pure functional protein. All mutants with 30-mer linker were tested in gel filtration and found to be monomeric (data not shown). 
     For further in vitro characterization five mutants were purified with the 30-mer linker, V84D (Flu4), V84D/L11D (Flu6), L11D (Flu3), and the serine mutants FH15 and FH20 (see iii(b)). A two-step chromatography, first using IMAC and then cation-exchange chromatography, led to homogeneous protein. The i/s ratio of the antibody fragments (FIG. 3) was also reflected in the purification yield of functional protein. The highly soluble mutant Flu4 (V84D) (FIG. 3B lane 3, 4) yielded about 0.3 mg purified and functional protein per liter and OD, Flu6 (L11D/V84D) (FIG. 3B lane 7, 8) yielded about 0.25 mg per liter and OD and Flu3 (less soluble material on the blot in FIG. 3A lane 7, 8) yielded 0.05 mg per liter and OD. The serine mutants FH15 and FH20 yielded 0.3 mg and 0.4 mg per liter and OD, respectively. The wt scFv of the antibody 4-4-20 did not give any soluble protein at all in periplasmic expression with either linker, and it was therefore expressed as cytoplasmic inclusion bodies, followed by refolding in vitro and fluorescein affinity chromatography. The refolded wt scFv was shown by gel filtration to be monomeric with the 30-mer linker (data not shown). 
     (d) Biophysical Properties of the Mutant scFvs 
     Since we changed amino acids which are conserved, it cannot be excluded that changes at these positions may be transmitted through the structure and have an effect on the binding constant, even though they are very far from the binding site (Chatellier et al., 1996). To eliminate this possibility, we determined the binding constant of the mutants Flu3, Flu4, Flu6 and the wt scFv. Fluorescence titration was used to determine KD in solution by using the quenching of the intrinsic fluorescence of fluorescein when it binds to the antibody. The fluorescence quenching at 510 nm was measured as a function of added scFv. The KD values (Table 3 and FIG. 4) obtained for all three mutant scFvs and the wt scFv are very similar and correspond very well to the recently corrected KD of the monoclonal antibody 4-4-20 (Miklasz et al., 1995). 
     To determine whether the mutations had an influence on the thermodynamic stability of the protein we determined the equilibrium unfolding curves by urea denaturation. V84D mutant and the wt scFv were used for this analysis, and in FIG. 5 an overlay plot is shown. The midpoint of both curves is at 4.1 M urea. Both curves were fitted by an algorithm for a two-state model described by Pace (1990), but the apparent small difference between the V84D mutant and the wt scFv is not of statistical significance. Aggregation of folding intermediates could be another explanation for the different in vivo results between the mutant scFvs and the wt scFv (FIG.  3 ). In the periplasm of  E. coli , the protein concentrations are assumed to be rather high (van Wielink &amp; Duine, 1990) and the aggregation effects could thus be pronounced. In order to estimate the aggregation behavior in vitro, we have measured the thermal aggregation rates at different temperatures. In FIG. 6 it is clearly seen that the wt scFv is significantly aggregating already at 44° C., whereas the mutant V84D tends to aggregate more slowly. The wt scFv is thus clearly more aggregation prone than the mutant scFv. This is very similar to the observations made with different mutations on the antibody McPC603 (Knappik and Plückthun, 1995), where no correlation was found between equilibrium denaturation curves and expression behavior, but a good correlation was found with the thermal aggregation rates. 
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             60 
           
           
             1 
             113 
             PRT 
             Murine 
           
            1
Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Val Val Ser Leu Gly
  1               5                  10                  15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
             20                  25                  30
Gly Lys Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
         35                  40                  45
Lys Val Leu Ile Tyr Ile Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
     50                  55                  60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp
 65                  70                  75                  80
Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn
                 85                  90                  95
Glu Asp Pro Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Met Arg
            100                 105                 110
Arg
 
           
             2 
             108 
             PRT 
             Murine 
           
            2
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
  1               5                  10                  15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Tyr Tyr Met
             20                  25                  30
Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr
         35                  40                  45
Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
     50                  55                  60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu
 65                  70                  75                  80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Pro Ile
                 85                  90                  95
Thr Phe Gly Val Gly Thr Lys Leu Asp Leu Lys Thr
            100                 105
 
           
             3 
             108 
             PRT 
             Murine 
           
            3
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
  1               5                  10                  15
Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn
             20                  25                  30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
         35                  40                  45
Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
     50                  55                  60
Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser
 65                  70                  75                  80
Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr
                 85                  90                  95
Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Arg
            100                 105
 
           
             4 
             113 
             PRT 
             Murine 
           
            4
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
  1               5                  10                  15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
             20                  25                  30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
         35                  40                  45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
     50                  55                  60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Tyr Leu Lys Ile
 65                  70                  75                  80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
                 85                  90                  95
Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
            100                 105                 110
Arg
 
           
             5 
             106 
             PRT 
             Homo sapiens 
           
            5
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
  1               5                  10                  15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Trp
             20                  25                  30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
         35                  40                  45
Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
     50                  55                  60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
 65                  70                  75                  80
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Phe
                 85                  90                  95
Gly Pro Gly Thr Lys Val Asp Ile Lys Arg
            100                 105
 
           
             6 
             108 
             PRT 
             Murine 
           
            6
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly
  1               5                  10                  15
Glu Thr Val Thr Ile Thr Cys Thr Ala Ser Gly Asn Ile His Asn Tyr
             20                  25                  30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
         35                  40                  45
Tyr Tyr Thr Thr Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
     50                  55                  60
Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro
 65                  70                  75                  80
Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Arg
                 85                  90                  95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
            100                 105
 
           
             7 
             109 
             PRT 
             Murine 
           
            7
Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
  1               5                  10                  15
Glu Lys Val Thr Met Ala Cys Arg Ala Ser Ser Ser Val Ser Ser Thr
             20                  25                  30
Tyr Leu His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Leu
         35                  40                  45
Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
     50                  55                  60
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Tyr Ile Ser Ser Val Glu
 65                  70                  75                  80
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro
                 85                  90                  95
Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
            100                 105
 
           
             8 
             114 
             PRT 
             Murine 
           
            8
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
  1               5                  10                  15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser
             20                  25                  30
Gly Lys Arg Lys Asn Phe Leu Thr Trp Tyr His Gln Lys Pro Gly Gln
         35                  40                  45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
     50                  55                  60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
 65                  70                  75                  80
Ile Thr Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr Tyr Cys Gln Asn
                 85                  90                  95
Asp Tyr Ser His Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
            100                 105                 110
      Lys Arg
 
           
             9 
             108 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence Humanized
      murine 
             
           
            9
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
  1               5                  10                  15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
             20                  25                  30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
         35                  40                  45
Tyr Ser Ala Ser Phe Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
     50                  55                  60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
 65                  70                  75                  80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
                 85                  90                  95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
            100                 105
 
           
             10 
             112 
             PRT 
             Murine 
           
            10
Asp Ile Val Leu Thr Gln Ser Pro Gly Ser Leu Ala Val Ser Leu Gly
  1               5                  10                  15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Asp Asp Asp
             20                  25                  30
Gly Asn Ser Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
         35                  40                  45
Lys Leu Leu Ile Tyr Arg Ser Ser Asn Leu Ile Ser Gly Ile Pro Asp
     50                  55                  60
Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn
 65                  70                  75                  80
Asp Pro Val Glu Ala Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
                 85                  90                  95
Gln Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg
            100                 105                 110
 
           
             11 
             111 
             PRT 
             Murine 
           
            11
Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu
  1               5                  10                  15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser
             20                  25                  30
Asn Tyr Ala Asn Trp Tyr Gln Glu Lys Pro Asp His Leu Phe Thr Gly
         35                  40                  45
Leu Ile Glu Glu Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe
     50                  55                  60
Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala
 65                  70                  75                  80
Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn
                 85                  90                  95
His Trp Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
            100                 105                 110
 
           
             12 
             114 
             PRT 
             Murine 
           
            12
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
  1               5                  10                  15
Glu Lys Val Thr Met Ser Cys Thr Ser Ser Gln Ser Leu Phe Asn Ser
             20                  25                  30
Gly Lys Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
         35                  40                  45
Pro Pro Lys Val Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
     50                  55                  60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
 65                  70                  75                  80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
                 85                  90                  95
Asp Tyr Ser Asn Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu
            100                 105                 110
Lys Arg
 
           
             13 
             113 
             PRT 
             Murine 
           
            13
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
  1               5                  10                  15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
             20                  25                  30
Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Ala Gly Gln Ser
         35                  40                  45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
     50                  55                  60
Asp Thr Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
 65                  70                  75                  80
Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln Thr
                 85                  90                  95
Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
            100                 105                 110
Arg
 
           
             14 
             109 
             PRT 
             Murine 
           
            14
Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr
  1               5                  10                  15
Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn
             20                  25                  30
Tyr Ala Asn Trp Tyr Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu
         35                  40                  45
Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe Ser
     50                  55                  60
Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln
 65                  70                  75                  80
Thr Glu Asp Glu Ala Arg Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu
                 85                  90                  95
Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
            100                 105
 
           
             15 
             113 
             PRT 
             Murine 
           
            15
Asp Val Leu Met Thr Gln Thr Pro Ile Ser Ile Pro Val Ser Leu Gly
  1               5                  10                  15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Gly
             20                  25                  30
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
         35                  40                  45
Pro Lys Leu Leu Ile Tyr Ser Ile Ser Ser Arg Phe Ser Gly Val Pro
     50                  55                  60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
 65                  70                  75                  80
Ser Arg Val Gln Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
                 85                  90                  95
Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
            100                 105                 110
Thr
 
           
             16 
             108 
             PRT 
             Murine 
           
            16
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
  1               5                  10                  15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Tyr Asn Tyr
             20                  25                  30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
         35                  40                  45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
     50                  55                  60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Asn Gln
 65                  70                  75                  80
Glu Asp Met Ala Thr Tyr Ile Cys Gln Gln Gly Asn Thr Leu Pro Phe
                 85                  90                  95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg
            100                 105
 
           
             17 
             111 
             PRT 
             Homo sapiens 
           
            17
Pro Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Leu Gly Gln
  1               5                  10                  15
Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
             20                  25                  30
Asn Tyr Val Ser Trp Tyr Gln Gln His Ala Gly Lys Ala Pro Lys Val
         35                  40                  45
Leu Ile Tyr Glu Val Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe
     50                  55                  60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu
 65                  70                  75                  80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Glu Gly Ser
                 85                  90                  95
Asp Asn Phe Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly
            100                 105                 110
 
           
             18 
             108 
             PRT 
             Anous minutus 
             
               Noddy Tern 
             
           
            18
Asp Ile Val Met Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly
  1               5                  10                  15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala
             20                  25                  30
Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
         35                  40                  45
Tyr Trp Ala Ser Thr Arg His Ile Gly Val Pro Asp Arg Phe Ala Gly
     50                  55                  60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala
 65                  70                  75                  80
Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Trp
                 85                  90                  95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
            100                 105
 
           
             19 
             113 
             PRT 
             Murine 
           
            19
Glu Leu Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly
  1               5                  10                  15
Asp Gln Ala Ser Ile Ser Cys Arg Pro Ser Gln Ser Leu Val His Ser
             20                  25                  30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
         35                  40                  45
Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
     50                  55                  60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile
 65                  70                  75                  80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Gly
                 85                  90                  95
Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
            100                 105                 110
Arg
 
           
             20 
             113 
             PRT 
             Murine 
           
            20
Asp Val Val Met Thr Gln Ile Pro Leu Ser Leu Pro Val Asn Leu Gly
  1               5                  10                  15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Ile His Ser
             20                  25                  30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
         35                  40                  45
Pro Lys Leu Leu Met Tyr Lys Val Ser Asn Arg Phe Tyr Gly Val Pro
     50                  55                  60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
 65                  70                  75                  80
Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln Ser
                 85                  90                  95
Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
            100                 105                 110
Arg
 
           
             21 
             108 
             PRT 
             Murine 
           
            21
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
  1               5                  10                  15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
             20                  25                  30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
         35                  40                  45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
     50                  55                  60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu His
 65                  70                  75                  80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Ser Thr Leu Pro Arg
                 85                  90                  95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
            100                 105
 
           
             22 
             111 
             PRT 
             Homo sapiens 
           
            22
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
  1               5                  10                  15
Arg Val Thr Ile Ser Cys Ser Gly Thr Ser Ser Asn Ile Gly Ser Ser
             20                  25                  30
Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Met Ala Pro Lys Leu Leu
         35                  40                  45
Ile Tyr Arg Asp Ala Met Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
     50                  55                  60
Gly Ser Lys Ser Gly Ala Ser Ala Ser Leu Ala Ile Gly Gly Leu Gln
 65                  70                  75                  80
Ser Glu Asp Glu Thr Asp Tyr Tyr Cys Ala Ala Trp Asp Val Ser Leu
                 85                  90                  95
Asn Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly
            100                 105                 110
 
           
             23 
             107 
             PRT 
             Murine 
           
            23
Glu Ile Val Leu Thr Gln Ser Pro Ala Ile Thr Ala Ala Ser Leu Gly
  1               5                  10                  15
Gln Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Lys
             20                  25                  30
Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Pro Trp Ile Tyr
         35                  40                  45
Glu Ile Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
     50                  55                  60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Asn Thr Met Glu Ala Glu
 65                  70                  75                  80
Asp Ala Ala Ile Tyr Tyr Cys Gln Gln Trp Thr Tyr Pro Leu Ile Thr
                 85                  90                  95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
            100                 105
 
           
             24 
             106 
             PRT 
             Murine 
           
            24
Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
  1               5                  10                  15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met
             20                  25                  30
Tyr Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
         35                  40                  45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
     50                  55                  60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Thr Glu
 65                  70                  75                  80
Asp Ala Ala Glu Tyr Tyr Cys Gln Gln Trp Gly Thr Asn Pro Thr Phe
                 85                  90                  95
Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
            100                 105
 
           
             25 
             113 
             PRT 
             Murine 
           
            25
Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
  1               5                  10                  15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Asn Gln Thr Ile Leu Leu Ser
             20                  25                  30
Asp Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
         35                  40                  45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
     50                  55                  60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
 65                  70                  75                  80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
                 85                  90                  95
Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
            100                 105                 110
Arg
 
           
             26 
             114 
             PRT 
             Murine 
           
            26
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly
  1               5                  10                  15
Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
             20                  25                  30
Gly Asn Gln Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
         35                  40                  45
Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Glu Val
     50                  55                  60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
 65                  70                  75                  80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
                 85                  90                  95
Asp His Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
            100                 105                 110
Lys Arg
 
           
             27 
             108 
             PRT 
             Murine 
           
            27
Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly
  1               5                  10                  15
Asn Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Asn Asn
             20                  25                  30
Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile
         35                  40                  45
Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
     50                  55                  60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr
 65                  70                  75                  80
Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Tyr
                 85                  90                  95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
            100                 105
 
           
             28 
             113 
             PRT 
             Murine 
           
            28
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
  1               5                  10                  15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
             20                  25                  30
Gln Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gln Lys Pro Gly Gln Ser
         35                  40                  45
Pro Lys Val Lys Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
     50                  55                  60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
 65                  70                  75                  80
Ser Arg Val Glu Ala Glu Asp Leu Gly Met Tyr Phe Cys Ser Gln Ser
                 85                  90                  95
Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
            100                 105                 110
Arg
 
           
             29 
             107 
             PRT 
             Murine 
           
            29
Asp Ile Gln Met Thr Gln Ile Pro Ser Ser Leu Ser Ala Ser Leu Gly
  1               5                  10                  15
Asp Arg Val Ser Ile Ser Cys Arg Ala Ser Gln Asp Ile Asn Asn Phe
             20                  25                  30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Ile Lys Leu Leu Ile
         35                  40                  45
Tyr Phe Thr Ser Arg Ser Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
     50                  55                  60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
 65                  70                  75                  80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Ala Leu Pro Arg
                 85                  90                  95
Thr Phe Gly Gly Thr Lys Leu Glu Ile Lys Arg
            100                 105
 
           
             30 
             105 
             PRT 
             Homo sapiens 
           
            30
Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln Thr Ala
  1               5                  10                  15
Arg Ile Thr Cys Ser Ala Asn Ala Leu Pro Asn Gln Tyr Ala Tyr Trp
             20                  25                  30
Tyr Gln Gln Lys Pro Gly Arg Ala Pro Val Met Val Ile Tyr Lys Asp
         35                  40                  45
Thr Gln Arg Pro Ser Gly Ile Pro Gln Arg Phe Ser Ser Ser Thr Ser
     50                  55                  60
Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu Asp Glu
 65                  70                  75                  80
Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Asn Ser Ala Ser Ile Phe Gly
                 85                  90                  95
Gly Gly Thr Lys Leu Thr Val Leu Gly
            100                 105
 
           
             31 
             119 
             PRT 
             Murine 
           
            31
Gln Val Lys Leu Gln Glu Ser Gly Pro Ala Val Ile Lys Pro Ser Gln
  1               5                  10                  15
Ser Leu Ser Leu Thr Cys Ile Val Ser Gly Phe Ser Ile Thr Arg Thr
             20                  25                  30
Asn Tyr Cys Trp His Trp Ile Arg Pro Gly Lys Gly Leu Glu Trp Met
         35                  40                  45
Gly Arg Ile Cys Tyr Glu Glu Ser Ile Tyr Tyr Ser Pro Ser Ile Lys
     50                  55                  60
Ser Arg Ser Thr Ile Ser Arg Asp Thr Ser Leu Asn Lys Phe Phe Ile
 65                  70                  75                  80
Gln Leu Ile Ser Val Thr Asn Glu Asp Thr Ala Met Tyr Tyr Cys Ser
                 85                  90                  95
Arg Glu Asn His Met Tyr Glu Thr Tyr Phe Asp Val Trp Gly Gln Gly
            100                 105                 110
Thr Thr Val Thr Val Ser Ser
        115
 
           
             32 
             117 
             PRT 
             Murine 
           
            32
Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
  1               5                  10                  15
Ser Gln Ser Gln Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr
             20                  25                  30
Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu
         35                  40                  45
Glu Trp Met Gly Tyr Met Ser Tyr Ser Gly Ser Thr Arg Tyr Asn Pro
     50                  55                  60
Ser Leu Arg Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln
 65                  70                  75                  80
Phe Phe Leu Gln Leu Lys Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr
                 85                  90                  95
Phe Cys Ala Arg Gly Trp Pro Leu Ala Tyr Trp Gly Gln Gly Thr Gln
            100                 105                 110
Val Ser Val Ser Glu
        115
 
           
             33 
             115 
             PRT 
             Murine 
           
            33
Val Gln Leu Gln Gln Ser Asp Ala Glu Lys Val Lys Pro Gly Ala Ser
  1               5                  10                  15
Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His Ala
             20                  25                  30
Ile His Trp Ala Lys Gln Lys Pro Glu Gln Gly Leu Glu Trp Ile Gly
         35                  40                  45
Tyr Ile Ser Pro Gly Asn Asp Asp Ile Lys Tyr Asn Glu Lys Phe Lys
     50                  55                  60
Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met
 65                  70                  75                  80
Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Lys
                 85                  90                  95
Arg Ser Thr Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
            100                 105                 110
Val Ser Ser
        115
 
           
             34 
             119 
             PRT 
             Murine 
           
            34
Glu Val Gln Pro Val Glu Thr Gly Gly Gly Leu Val Gln Pro Lys Gly
  1               5                  10                  15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asn Thr Asn
             20                  25                  30
Ala Asn Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
         35                  40                  45
Ala Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val
     50                  55                  60
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Asn Met Leu Tyr
 65                  70                  75                  80
Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys
                 85                  90                  95
Val Arg Asp Gln Thr Gly Thr Ala Trp Phe Ala Tyr Trp Gly Gln Gly
            100                 105                 110
Thr Leu Val Thr Val Ser Ala
        115
 
           
             35 
             126 
             PRT 
             Homo sapiens 
           
            35
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Phe Gly Arg
  1               5                  10                  15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr
             20                  25                  30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
         35                  40                  45
Ser Gly Ile Ser Trp Asp Ser Ser Ser Ile Gly Tyr Ala Asp Ser Val
     50                  55                  60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
 65                  70                  75                  80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Leu Tyr Tyr Cys
                 85                  90                  95
Val Lys Gly Arg Asp Tyr Tyr Asp Ser Gly Gly Tyr Phe Thr Val Ala
            100                 105                 110
Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
        115                 120                 125
 
           
             36 
             116 
             PRT 
             Murine 
           
            36
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
  1               5                  10                  15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr
             20                  25                  30
Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
         35                  40                  45
Gly Met Ile Trp Gly Asp Gly Asn Thr Asp Tyr Asn Ser Ala Leu Lys
     50                  55                  60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
 65                  70                  75                  80
Lys Met Asn Ser Leu His Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala
                 85                  90                  95
Arg Glu Arg Asp Tyr Arg Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu
            100                 105                 110
Thr Val Ser Ser
        115
 
           
             37 
             119 
             PRT 
             Murine 
           
            37
Asp Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala
  1               5                  10                  15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Ser Leu Pro Gly His
             20                  25                  30
Asn Ile Asn Trp Ile Val Gln Arg Asn Gly Lys Ser Leu Glu Trp Ile
         35                  40                  45
Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Asn Phe Asn Pro Lys Phe
     50                  55                  60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Leu Tyr
 65                  70                  75                  80
Met His Leu Thr Ser Leu Gln Ser Glu Asp Ser Ala Val Tyr Tyr Cys
                 85                  90                  95
Ala Arg Arg Arg Asp Gly Asn Tyr Gly Phe Thr Tyr Trp Gly Gln Gly
            100                 105                 110
Thr Leu Val Thr Val Ser Ala
        115
 
           
             38 
             120 
             PRT 
             Murine 
           
            38
Glu Val Leu Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly
  1               5                  10                  15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe
             20                  25                  30
Gly Met Ser Trp Val Arg His Thr Pro Asp Lys Arg Leu Glu Trp Val
         35                  40                  45
Ala Thr Ile Ser Asn Gly Gly Gly Tyr Thr Tyr Tyr Gln Asp Ser Val
     50                  55                  60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe
 65                  70                  75                  80
Leu Glu Met Thr Ser Leu Lys Ser Glu Asp Ala Gly Leu Tyr Tyr Cys
                 85                  90                  95
Ala Arg Arg Glu Arg Tyr Asp Glu Lys Gly Phe Ala Tyr Trp Gly Arg
            100                 105                 110
Gly Thr Leu Val Thr Val Ser Ala
        115                 120
 
           
             39 
             120 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence  Humanized
      murine 
             
           
            39
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gln Gln
  1               5                  10                  15
Phe Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
             20                  25                  30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
         35                  40                  45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
     50                  55                  60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Leu Tyr
 65                  70                  75                  80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                 85                  90                  95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Val Trp Gly Gln
            100                 105                 110
Gly Thr Leu Val Thr Val Ser Ser
        115                 120
 
           
             40 
             115 
             PRT 
             Murine 
           
            40
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln
  1               5                  10                  15
Ser Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Tyr
             20                  25                  30
Gly Met Gly Val Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu
         35                  40                  45
Trp Leu Ala His Ile Phe Trp Asp Gly Asp Lys Arg Tyr Asn Pro Ser
     50                  55                  60
Leu Lys Ser Arg Leu Lys Ile Ser Lys Asp Thr Ser Asn Asn Gln Val
 65                  70                  75                  80
Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr
                 85                  90                  95
Cys Val Gln Glu Gly Tyr Ile Tyr Trp Gly Gln Gly Thr Ser Val Thr
            100                 105                 110
Val Ser Ser
        115
 
           
             41 
             122 
             PRT 
             Murine 
           
            41
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
  1               5                  10                  15
Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Leu Leu Ile Ser Asn
             20                  25                  30
Gly Val His Trp Val Arg Gln Pro Pro Gln Lys Gly Leu Glu Trp Leu
         35                  40                  45
Gly Val Ile Trp Ala Gly Gly Asn Thr Asn Tyr Asn Ser Ala Leu Met
     50                  55                  60
Ser Arg Val Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
 65                  70                  75                  80
Lys Met Lys Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala
                 85                  90                  95
Arg Asp Phe Tyr Asp Tyr Asp Val Phe Tyr Tyr Ala Met Asp Tyr Trp
            100                 105                 110
Gly Gln Gly Thr Ser Val Thr Val Ser Ser
        115                 120
 
           
             42 
             120 
             PRT 
             Murine 
           
            42
Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly
  1               5                  10                  15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Tyr
             20                  25                  30
Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val
         35                  40                  45
Ala Thr Ile Ser Asn Gly Gly Gly Tyr Thr Tyr Tyr Pro Asp Ser Val
     50                  55                  60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
 65                  70                  75                  80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Ser Ala Met Tyr Tyr Cys
                 85                  90                  95
Ala Arg Arg Glu Arg Tyr Asp Glu Asn Gly Phe Ala Tyr Trp Gly Gln
            100                 105                 110
Gly Thr Leu Val Thr Val Ser Ala
        115                 120
 
           
             43 
             118 
             PRT 
             Murine 
           
            43
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
  1               5                  10                  15
Val Arg Met Ser Cys Lys Ser Ser Gly Tyr Ile Phe Thr Asp Phe Tyr
             20                  25                  30
Met Asn Trp Val Arg Gln Ser His Gly Lys Ser Leu Asp Tyr Ile Gly
         35                  40                  45
Tyr Ile Ser Pro Tyr Ser Gly Val Thr Gly Tyr Asn Gln Lys Phe Lys
     50                  55                  60
Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met
 65                  70                  75                  80
Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala
                 85                  90                  95
Gly Ser Ser Gly Asn Lys Trp Ala Met Asp Tyr Trp Gly His Gly Ala
            100                 105                 110
Ser Val Thr Val Ser Ser
        115
 
           
             44 
             114 
             PRT 
             Murine 
           
            44
Glu Val Thr Leu Val Glu Ser Gly Gly Asp Ser Val Lys Pro Gly Gly
  1               5                  10                  15
Ser Leu Lys Lys Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Glu
             20                  25                  30
Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
         35                  40                  45
Ala Thr Thr Leu Ser Gly Gly Gly Phe Thr Phe Tyr Ser Ala Ser Val
     50                  55                  60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Asn Leu Tyr
 65                  70                  75                  80
Leu Gln Leu Asn Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Phe Cys
                 85                  90                  95
Ala Ser His Arg Phe Val His Trp Gly His Gly Thr Leu Val Thr Val
            100                 105                 110
Ser Ala
 
           
             45 
             118 
             PRT 
             Murine 
           
            45
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Ser
  1               5                  10                  15
Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Thr Tyr
             20                  25                  30
Ala Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile
         35                  40                  45
Gly Leu Ile Ser Pro Ser Ser Gly Tyr Thr Ser Tyr Asn Gly Glu Phe
     50                  55                  60
Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
 65                  70                  75                  80
Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
                 85                  90                  95
Ala Arg Val Met Gly Glu Gln Tyr Phe Asp Phe Trp Gly Ala Gly Thr
            100                 105                 110
Thr Val Thr Val Ser Ser
        115
 
           
             46 
             119 
             PRT 
             Murine 
           
            46
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
  1               5                  10                  15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr
             20                  25                  30
Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
         35                  40                  45
Gly Phe Ile Arg Asn Lys Ala Asp Gly Tyr Thr Thr Glu Tyr Ser Ala
     50                  55                  60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile
 65                  70                  75                  80
Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Ser Ala Thr Tyr
                 85                  90                  95
Tyr Cys Thr Arg Asp Pro Tyr Gly Pro Ala Ala Tyr Trp Gly Gln Gly
            100                 105                 110
Thr Leu Val Thr Val Ser Ala
        115
 
           
             47 
             117 
             PRT 
             Homo sapiens 
           
            47
Pro Leu Val Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
  1               5                  10                  15
Ala Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Asn Thr Ile
             20                  25                  30
Leu Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
         35                  40                  45
Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Gly Asn Pro Ser
     50                  55                  60
Leu Lys Ser Arg Val Thr Ile Ser Val Asn Thr Ser Lys Asn Gln Phe
 65                  70                  75                  80
Tyr Ser Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
                 85                  90                  95
Cys Ala Arg Val Pro Leu Val Val Asn Pro Trp Gly Gln Gly Thr Leu
            100                 105                 110
Val Thr Val Ser Ser
        115
 
           
             48 
             120 
             PRT 
             Anous Minutus 
             
               Noddy Tern 
             
           
            48
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
  1               5                  10                  15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
             20                  25                  30
Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
         35                  40                  45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Gly Glu Glu Phe
     50                  55                  60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Asn
 65                  70                  75                  80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Phe Phe Cys
                 85                  90                  95
Ala Arg Gly Glu Asp Asn Phe Gly Ser Leu Ser Asp Tyr Trp Gly Gln
            100                 105                 110
Gly Thr Thr Val Thr Val Ser Ser
        115                 120
 
           
             49 
             116 
             PRT 
             Murine 
           
            49
Arg Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Met Lys Pro Gly Ala
  1               5                  10                  15
Ser Val Gln Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Glu Tyr
             20                  25                  30
Trp Ile Glu Trp Val Lys Glu Arg Pro Gly His Gly Leu Glu Trp Ile
         35                  40                  45
Gly Glu Ile Leu Pro Gly Ser Gly Arg Thr Asn Tyr Arg Glu Lys Phe
     50                  55                  60
Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
 65                  70                  75                  80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
                 85                  90                  95
Thr Arg Gly Tyr Ser Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser Val
            100                 105                 110
Thr Val Ser Ala
        115
 
           
             50 
             119 
             PRT 
             Murine 
           
            50
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
  1               5                  10                  15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr
             20                  25                  30
Gly Val Asn Trp Val Lys Glu Ala Pro Gly Lys Glu Leu Lys Trp Met
         35                  40                  45
Gly Trp Ile Asn Ile Tyr Thr Gly Glu Pro Thr Tyr Val Asp Asp Phe
     50                  55                  60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
 65                  70                  75                  80
Leu Glu Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
                 85                  90                  95
Thr Arg Gly Asp Tyr Val Asn Trp Tyr Phe Asp Val Trp Gly Ala Gly
            100                 105                 110
Thr Thr Val Thr Val Ser Ser
        115
 
           
             51 
             124 
             PRT 
             Murine 
           
            51
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ala Gly Ser
  1               5                  10                  15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
             20                  25                  30
Gly Val Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
         35                  40                  45
Gly Tyr Ile Asn Pro Gly Lys Gly Tyr Leu Ser Tyr Asn Glu Lys Phe
     50                  55                  60
Lys Gly Lys Thr Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr
 65                  70                  75                  80
Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ala Ala Val Tyr Phe Cys
                 85                  90                  95
Ala Arg Ser Phe Tyr Gly Gly Ser Asp Leu Ala Val Tyr Tyr Phe Asp
            100                 105                 110
Ser Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
        115                 120
 
           
             52 
             126 
             PRT 
             Homo sapiens 
           
            52
Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg
  1               5                  10                  15
Ser Leu Arg Leu Ser Cys Ser Ser Ser Gly Phe Ile Phe Ser Ser Tyr
             20                  25                  30
Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
         35                  40                  45
Ala Ile Ile Trp Asp Asp Gly Ser Asp Gln His Tyr Ala Asp Ser Val
     50                  55                  60
Lys Gly Arg Phe Thr Ile Ser Arg Asn Asp Ser Lys Asn Ser Leu Phe
 65                  70                  75                  80
Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys
                 85                  90                  95
Ala Arg Asp Gly Gly His Gly Phe Cys Ser Ser Ala Ser Cys Phe Gly
            100                 105                 110
Pro Asp Tyr Trp Gly Gln Gly Thr Pro Val Thr Val Ser Ser
        115                 120                 125
 
           
             53 
             118 
             PRT 
             Murine 
           
            53
Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
  1               5                  10                  15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Lys Tyr
             20                  25                  30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
         35                  40                  45
Gly Glu Ile His Pro Asp Ser Gly Thr Ile Asn Tyr Thr Pro Ser Leu
     50                  55                  60
Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
 65                  70                  75                  80
Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
                 85                  90                  95
Ala Arg Leu His Tyr Tyr Gly Tyr Asn Ala Tyr Trp Gly Gln Gly Thr
            100                 105                 110
Leu Val Thr Val Ser Ala
        115
 
           
             54 
             115 
             PRT 
             Murine 
           
            54
Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala Ser
  1               5                  10                  15
Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr Trp
             20                  25                  30
Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly
         35                  40                  45
Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr His Glu Arg Phe Lys
     50                  55                  60
Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr Met
 65                  70                  75                  80
Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Leu
                 85                  90                  95
His Gly Asn Tyr Asp Phe Asp Gly Trp Gly Gln Gly Thr Thr Leu Thr
            100                 105                 110
Val Ser Ser
        115
 
           
             55 
             119 
             PRT 
             Murine 
           
            55
Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly
  1               5                  10                  15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Cys
             20                  25                  30
Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
         35                  40                  45
Ala Gly Ile Ser Ser Gly Gly Ser Tyr Thr Phe Tyr Pro Asp Thr Val
     50                  55                  60
Lys Gly Arg Phe Ile Ile Ser Arg Asn Asn Ala Arg Asn Thr Leu Ser
 65                  70                  75                  80
Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys
                 85                  90                  95
Thr Arg Tyr Ser Ser Asp Pro Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
            100                 105                 110
Thr Thr Leu Thr Val Ser Ser
        115
 
           
             56 
             122 
             PRT 
             Murine 
           
            56
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
  1               5                  10                  15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe
             20                  25                  30
Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile
         35                  40                  45
Ala Ala Ser Arg Asn Lys Gly Asn Lys Tyr Thr Thr Glu Tyr Ser Ala
     50                  55                  60
Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser Ile
 65                  70                  75                  80
Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr
                 85                  90                  95
Tyr Cys Ala Arg Asn Tyr Tyr Gly Ser Thr Trp Tyr Phe Asp Val Trp
            100                 105                 110
Gly Ala Gly Thr Thr Val Thr Val Ser Ser
        115                 120
 
           
             57 
             113 
             PRT 
             Murine 
           
            57
Asp Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
  1               5                  10                  15
Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Asp
             20                  25                  30
Tyr Trp Ser Trp Ile Arg Lys Phe Pro Gly Asn Arg Leu Glu Tyr Met
         35                  40                  45
Gly Tyr Val Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
     50                  55                  60
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu
 65                  70                  75                  80
Asp Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
                 85                  90                  95
Asn Trp Asp Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
            100                 105                 110
Ala
 
           
             58 
             118 
             PRT 
             Murine 
           
            58
Glu Val Gln Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg
  1               5                  10                  15
Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr
             20                  25                  30
Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
         35                  40                  45
Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp
     50                  55                  60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
 65                  70                  75                  80
Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr
                 85                  90                  95
Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr
            100                 105                 110
Ser Val Thr Val Ser Ser
        115
 
           
             59 
             121 
             PRT 
             Murine 
           
            59
Glu Val Gln Leu Gln Gln Ser Gly Val Glu Leu Val Arg Ala Gly Ser
  1               5                  10                  15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn
             20                  25                  30
Gly Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
         35                  40                  45
Gly Tyr Asn Asn Pro Gly Asn Gly Tyr Ile Ala Tyr Asn Glu Lys Phe
     50                  55                  60
Lys Gly Lys Thr Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
 65                  70                  75                  80
Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
                 85                  90                  95
Ala Arg Ser Glu Tyr Tyr Gly Gly Ser Tyr Lys Phe Asp Tyr Trp Gly
            100                 105                 110
Gln Gly Thr Thr Leu Thr Val Ser Ser
        115                 120
 
           
             60 
             121 
             PRT 
             Homo sapiens 
           
            60
Ala Val Gln Leu Val Gln Ala Gly Gly Gly Val Val Gln Pro Gly Arg
  1               5                  10                  15
Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Asn Tyr
             20                  25                  30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
         35                  40                  45
Ala Val Ile Trp Tyr Asn Gly Ser Arg Thr Tyr Tyr Gly Asp Ser Val
     50                  55                  60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Arg Thr Leu Tyr
 65                  70                  75                  80
Met Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys
                 85                  90                  95
Ala Arg Asp Pro Asp Ile Leu Thr Ala Phe Ser Phe Asp Tyr Trp Gly
            100                 105                 110
Gln Gly Val Leu Val Thr Val Ser Ser
        115                 120
 
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Sequence variability of residues contributing to the v/c interface 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Position 
                 L9 
                 L10 
                 L12 
                 L15 
                 L39 
                 L40 
                 L41 
                 L80 
               
               
                 % exp. (FAB) 
                 91 
                 59 
                 41 
                 48 
                 37 
                 64 
                 114 
                 57 
               
               
                 % exp. (ind.) 
                 91 
                 65 
                 47 
                 49 
                 37 
                 82 
                 121 
                 74 
               
               
                 % buried v/c 
                  0 
                 10 
                 12 
                  0 
                  1 
                 22 
                  3 
                 21 
               
             
          
           
               
                   
                 kappa 
                 lambda 
                 kappa 
                 kappa 
                 lambda 
                 kappa 
                 lambda 
                 kappa 
                 lambda 
                 kappa 
                 lambda 
                 kappa 
                 lambda 
                 kappa 
                 lambda 
               
             
          
           
               
                 Species 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
               
             
          
           
               
                 Seq. 4-4-20 
                 Leu 
                 Ser 
                 Pro 
                 Leu 
                 Lys 
                 Pro 
                 Gly 
                 Ala 
               
             
          
           
               
                 Consensus 
                 Ser 
                 Ala 
                 Ser 
                 Ala 
                 Thr 
                 Ser 
                 Ser 
                 Ser 
                 Ser 
                 Thr 
                 Pro 
                 Leu 
                 Pro 
                 Pro 
                 Lys 
                 Lys 
                 Lys 
                 Lys 
                 Pro 
                 Pro 
                 Pro 
                 Pro 
                 Gly 
                 Gly 
                 Gly 
                 Asp 
                 Pro 
                 Ala 
                 Ala 
                 Thr 
               
               
                   
               
               
                 Distribution: 
               
               
                 Asp 
                 8 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 10 
                 3 
                 96 
                 0 
                 0 
                 0 
                 0 
               
               
                 Glu 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 4 
                 0 
                 0 
                 0 
                 8 
                 0 
                 0 
               
               
                 Lys 
                 0 
                 6 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 83 
                 92 
                 30 
                 96 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Arg 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 14 
                 7 
                 11 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 1 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 His 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 20 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 3 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Thr 
                 0 
                 4 
                 0 
                 1 
                 49 
                 6 
                 0 
                 3 
                 0 
                 93 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 3 
                 17 
                 94 
               
               
                 Ser 
                 43 
                 24 
                 90 
                 4 
                 48 
                 60 
                 84 
                 50 
                 99 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 2 
                 19 
                 5 
                 0 
                 0 
                 1 
                 0 
                 0 
                 9 
                 6 
                 19 
                 0 
               
               
                 Asn 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Gln 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 4 
                 1 
                 0 
                 3 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 6 
                 0 
                 0 
               
               
                 Gly 
                 25 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 99 
                 80 
                 90 
                 3 
                 0 
                 0 
                 0 
                 0 
               
               
                 Ala 
                 11 
                 41 
                 7 
                 90 
                 0 
                 0 
                 4 
                 23 
                 0 
                 0 
                 0 
                 8 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 2 
                 0 
                 16 
                 64 
                 56 
                 4 
               
               
                 Cys 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Pro 
                 0 
                 2 
                 0 
                 0 
                 0 
                 0 
                 11 
                 20 
                 0 
                 0 
                 45 
                 28 
                 84 
                 96 
                 0 
                 0 
                 0 
                 0 
                 94 
                 77 
                 92 
                 96 
                 0 
                 0 
                 0 
                 0 
                 74 
                 9 
                 2 
                 1 
               
               
                 Val 
                 1 
                 0 
                 2 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 41 
                 10 
                 0 
                 0 
                 0 
                 0 
                 3 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 2 
                 5 
                 0 
               
               
                 Ile 
                 0 
                 0 
                 0 
                 3 
                 0 
                 24 
                 0 
                 0 
                 0 
                 1 
                 2 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Leu 
                 11 
                 19 
                 0 
                 0 
                 0 
                 2 
                 0 
                 0 
                 0 
                 0 
                 11 
                 49 
                 14 
                 4 
                 0 
                 0 
                 24 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
               
               
                 Met 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 3 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Phe 
                 0 
                 1 
                 0 
                 0 
                 2 
                 7 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 3 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Tyr 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 3 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 0 
                 0 
               
               
                 Trp 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
               
             
          
           
               
                 Position 
                 L81 
                 L83 
                 L103 
                 L105 
                 L106 
                 L106A 
                 L107 
                 L108 
               
               
                 % exp. (FAB) 
                 70 
                 17 
                 42 
                 19 
                 14 
                 56 
                 56 
                 47 
               
               
                 % exp. (ind.) 
                 74 
                 38 
                 58 
                 57 
                 28 
                 65 
                 65 
                 103  
               
               
                 % buried v/c 
                  5 
                 46 
                 26 
                 66 
                 56 
                 15 
                 15 
                 55 
               
             
          
           
               
                   
                 kappa 
                 lambda 
                 kappa 
                 lambda 
                 kappa 
                 lambda 
                 kappa 
                 lambda 
                 kappa 
                 lambda 
                 lambda 
                 kappa 
                 lambda 
                 kappa 
                 lambda 
               
             
          
           
               
                 Species 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
               
             
          
           
               
                 Seq. 4-4-20 
                 Glu 
                 Leu 
                 Lys 
                 Glu 
                 Ile 
                   
                 Lys 
                 Arg 
               
             
          
           
               
                 Consensus 
                 Glu 
                 Glu 
                 Glu 
                 Glu 
                 Phe 
                 Leu 
                 Glu 
                 Glu 
                 Lys 
                 Lys 
                 Lys 
                 Lys 
                 Glu 
                 Glu 
                 Thr 
                 Thr 
                 Ile 
                 Ile 
                 Val 
                 Val 
                 Leu 
                 Leu 
                 Lys 
                 Lys 
                 Gly 
                 Gly 
                 Arg 
                 Arg 
                 Gln 
                 Gln 
               
               
                   
               
               
                 Distribution: 
               
               
                 Asp 
                 10 
                 7 
                 4 
                 0 
                 0 
                 0 
                 0 
                 15 
                 0 
                 0 
                 0 
                 0 
                 12 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Glu 
                 86 
                 93 
                 69 
                 82 
                 0 
                 0 
                 99 
                 68 
                 0 
                 0 
                 2 
                 0 
                 84 
                 99 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Lys 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 88 
                 98 
                 83 
                 100 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 94 
                 97 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Arg 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 8 
                 1 
                 6 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 1 
                 8 
                 3 
                 97 
                 100 
                 0 
                 0 
               
               
                 His 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 6 
               
               
                 Thr 
                 0 
                 0 
                 0 
                 0 
                 0 
                 3 
                 0 
                 0 
                 2 
                 0 
                 2 
                 0 
                 0 
                 0 
                 97 
                 100 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Ser 
                 0 
                 0 
                 0 
                 0 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 5 
                 5 
                 0 
                 0 
                 0 
                 0 
               
               
                 Asn 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 4 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Gln 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 95 
                 54 
               
               
                 Gly 
                 1 
                 0 
                 17 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 83 
                 92 
                 1 
                 0 
                 2 
                 0 
               
               
                 Ala 
                 1 
                 0 
                 0 
                 0 
                 0 
                 31 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
               
               
                 Cys 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Pro 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 6 
               
               
                 Val 
                 0 
                 0 
                 1 
                 0 
                 20 
                 12 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 3 
                 1 
                 100 
                 100 
                 2 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Ile 
                 0 
                 0 
                 0 
                 0 
                 6 
                 11 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 88 
                 73 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Leu 
                 0 
                 0 
                 0 
                 0 
                 1 
                 32 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 25 
                 0 
                 0 
                 97 
                 97 
                 0 
                 0 
                 3 
                 0 
                 1 
                 0 
                 0 
                 0 
               
               
                 Met 
                 0 
                 0 
                 6 
                 0 
                 0 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 3 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Phe 
                 0 
                 0 
                 0 
                 0 
                 72 
                 7 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Tyr 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Trp 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
               
             
          
           
               
                 Position 
                 H9 
                 H10 
                 H11 
                 H13 
                 H14 
                 H41 
                 H42 
                 H43 
                 H84 
                 H87 
                 H89 
                 H105 
                 H108 
                 H110 
                 H112 
                 H113 
               
               
                 % exp. (FAB) 
                 29 
                 65 
                 31 
                 69 
                 36 
                 72 
                 111 
                 65 
                 76 
                 28 
                 34 
                 72 
                 52 
                 26 
                 25 
                 72 
               
               
                 % exp. (ind.) 
                 33 
                 72 
                 71 
                 73 
                 36 
                 72 
                 115 
                 78 
                 77 
                 31 
                 37 
                 82 
                 64 
                 55 
                 68 
                 78 
               
               
                 % buried v/c 
                  7 
                  8 
                 58 
                  5 
                  0 
                  0 
                  3 
                  2 
                  2 
                  8 
                  1 
                  1 
                 18 
                 52 
                 62 
                  7 
               
             
          
           
               
                 Species 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
                 hu 
                 mu 
               
             
          
           
               
                 Seq. 4-4-20 
                 Gly 
                 Gly 
                 Leu 
                 Gln 
                 Pro 
                 Pro 
                 Glu 
                 Lys 
                 Val 
                 Met 
                 Ile 
                 Gln 
                 Ser 
                 Thr 
                 Ser 
                 Ser 
               
             
          
           
               
                 Consensus 
                 Gly 
                 Pro 
                 Gly 
                 Glu 
                 Leu 
                 Leu 
                 Lys 
                 Lys 
                 Pro 
                 Pro 
                 Ala 
                 Arg 
                 Pro 
                 Pro 
                 Gly 
                 Gly 
                 Ala 
                 Ser 
                 Thr 
                 Ser 
                 Val 
                 Val 
                 Gln 
                 Gln 
                 Leu 
                 Thr 
                 Thr 
                 Thr 
                 Ser 
                 Ser 
                 Ser 
                 Ser 
               
               
                   
               
               
                 Distribution: 
               
               
                 Asp 
                 0 
                 0 
                 5 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Glu 
                 2 
                 0 
                 27 
                 54 
                 0 
                 0 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 19 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Lys 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 59 
                 54 
                 0 
                 0 
                 0 
                 3 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 10 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Arg 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 3 
                 18 
                 0 
                 0 
                 1 
                 36 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 5 
                 0 
                 3 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 His 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 11 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Thr 
                 1 
                 2 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 13 
                 1 
                 1 
                 0 
                 0 
                 4 
                 12 
                 96 
                 46 
                 4 
                 15 
                 0 
                 4 
                 23 
                 51 
                 89 
                 99 
                 0 
                 0 
                 0 
                 0 
               
               
                 Ser 
                 1 
                 1 
                 0 
                 2 
                 0 
                 0 
                 0 
                 0 
                 2 
                 3 
                 10 
                 18 
                 2 
                 3 
                 8 
                 0 
                 18 
                 70 
                 2 
                 51 
                 0 
                 0 
                 0 
                 0 
                 0 
                 23 
                 2 
                 0 
                 98 
                 100 
                 97 
                 77 
               
               
                 Asn 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 3 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Gln 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 34 
                 22 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 80 
                 83 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Gly 
                 42 
                 29 
                 61 
                 38 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 90 
                 76 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
               
               
                 Ala 
                 33 
                 31 
                 3 
                 1 
                 0 
                 0 
                 0 
                 5 
                 1 
                 8 
                 59 
                 7 
                 1 
                 0 
                 1 
                 2 
                 55 
                 12 
                 0 
                 1 
                 0 
                 0 
                 0 
                 11 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 22 
               
               
                 Cys 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Pro 
                 21 
                 36 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 96 
                 85 
                 18 
                 14 
                 94 
                 83 
                 1 
                 0 
                 14 
                 1 
                 0 
                 0 
                 0 
                 0 
                 2 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 2 
                 0 
               
               
                 Val 
                 0 
                 1 
                 0 
                 1 
                 38 
                 1 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 4 
                 0 
                 0 
                 0 
                 76 
                 50 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
               
               
                 Ile 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
                 5 
                 13 
                 0 
                 0 
                 0 
                 0 
                 7 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Leu 
                 0 
                 0 
                 0 
                 0 
                 60 
                 95 
                 0 
                 0 
                 0 
                 3 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 6 
                 7 
                 0 
                 0 
                 63 
                 25 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Met 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 7 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 7 
                 13 
                 0 
                 0 
                 9 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Phe 
                 0 
                 0 
                 0 
                 0 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 6 
                 0 
                 0 
                 0 
                 0 
                 2 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Tyr 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Trp 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Mutations introduced in the scFv fragment of the 
               
               
                 antibody 4-4-20 
               
             
          
           
               
                   
                 L15E (V L ) 
                 L11N (V H ) 
                 L11D (V H ) 
                 V84D (V H ) 
               
               
                   
                   
               
             
          
           
               
                 Flu 1 
                  
                   
                   
                   
               
               
                 Flu 2 
                   
                  
               
               
                 Flu 3 
                   
                   
                  
               
               
                 Flu 4 
                   
                   
                   
                  
               
               
                 Flu 5 
                   
                  
                   
                  
               
               
                 Flu 6 
                   
                   
                  
                  
               
               
                 Flu 7 
                  
                  
               
               
                 Flu 8 
                  
                   
                  
               
               
                 Flu 9 
                  
                  
                   
                  
               
               
                 Flu 4 short 
                   
                   
                   
                  
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 K D  values of the different scFv mutants determined in 
               
               
                 fluorescence titration 
               
             
          
           
               
                   
                 Flu wt 
                 Flu 3 
                 Flu 4 
                 Flu 6 
                 Flu wt # 
               
               
                   
                   
               
             
          
           
               
                 K D  (nM) 
                 80 ± 7 
                 60 ± 12 
                 70 ± 10 
                 75 ± 13 
                 90