Patent Application: US-64259306-A

Abstract:
the present invention relates to synthetic dna sequence which encode one or more collections of homologous proteinspeptides , and methods for generating and applying libraries of these dna sequences . in particular , the invention relates to the preparation of a library of human - derived antibody genes by the use of synthetic consensus sequences which cover the structural repertoire of antibodies encoded in the human genome . furthermore , the invention relates to the use of a single consensus antibody gene as a universal framework for highly diverse antibody libraries .

Description:
useful libraries of ( poly ) peptides . in a first embodiment , the invention provides for a method of setting up nucleic acid sequences suitable for the creation of said libraries . in a first step , a collection of at least three homologous proteins is identified and then analyzed . therefore , a database of the protein sequences is established where the protein sequences are aligned to each other . the database is used to define subgroups of protein sequences which show a high degree of similarity in both the sequence and , if information is available , in the structural arrangement . for each of the subgroups a ( poly ) peptide sequence comprising at least one consensus sequence is deduced which represents the members of this subgroup ; the complete collection of ( poly ) peptide sequences represent therefore the complete structural repertoire of the collection of homologous proteins . these artificial ( poly ) peptide sequences are then analyzed , if possible , according to their structural properties to identify unfavorable interactions between amino acids within said ( poly ) peptide sequences or between said or other ( poly ) peptide sequences , for example , in multimeric proteins . such interactions are then removed by changing the consensus sequence accordingly . the ( poly ) peptide sequences are then analyzed to identify sub - elements such as domains , loops , helices or cdrs . the amino acid sequence is backtranslated into a corresponding coding nucleic acid sequence which is adapted to the codon usage of the host planned for expressing said nucleic acid sequences . a set of cleavage sites is set up in a way that each of the sub - sequences encoding the sub - elements identified as described above , is flanked by two sites which do not occur a second time within the nucleic acid sequence . this can be achieved by either identifying a cleavage site already flanking a sub - sequence of by changing one or more nucleotides to create the cleavage site , and by removing that site from the remaining part of the gene . the cleavage sites should be common to all corresponding sub - elements or sub - sequences , thus creating a fully modular arrangement of the sub - sequences in the nucleic acid sequence and of the sub - elements in the corresponding ( poly ) peptide . in a further embodiment , the invention provides for a method which sets up two or more sets of ( poly ) peptides , where for each set the method as described above is performed , and where the cleavage sites are not only unique within each set but also between any two sets . this method can be applied for the creation of ( poly ) peptide libraries comprising for example two a - helical domains from two different proteins , where said library is screened for novel hetero - association domains . in yet a further embodiment , at least two of the sets as described above , are derived from the same collection of proteins or at least a part of it . this describes libraries comprising for example , but not limited to , two domains from antibodies such as vh and vl , or two extracellular loops of transmembrane receptors . in another embodiment , the nucleic acid sequences set up as described above , are synthesized . this can be achieved by any one of several methods well known to the practitioner skilled in the art , for example , by total gene synthesis or by pcr - based approaches . in one embodiment , the nucleic acid sequences are cloned into a vector . the vector could be a sequencing vector , an expression vector or a display ( e . g . phage display ) vector , which are well known to those skilled in the art . any vector could comprise one nucleic acid sequence , or two or more nucleic sequences , either in different or the same operon . in the last case , they could either be cloned separately or as contiguous sequences . in one embodiment , the removal of unfavorable interactions as described above , leads to enhanced expression of the modified ( poly ) peptides . in a preferred embodiment , one or more sub - sequences of the nucleic acid sequences are replaced by different sequences . this can be achieved by excising the sub - sequences using the conditions suitable for cleaving the cleavage sites adjacent to or at the end of the sub - sequence , for example , by using a restriction enzyme at the corresponding restriction site under the conditions well known to those skilled in the art , and replacing the sub - sequence by a different sequence compatible with the cleaved nucleic acid sequence . in a further preferred embodiment , the different sequences replacing the initial sub - sequence ( s ) are genomic or rearranged genomic sequences , for example in grafting cdrs from non - human antibodies onto consensus antibody sequences for rapid humanization of non - human antibodies . in the most preferred embodiment , the different sequences are random sequences , thus replacing the sub - sequence by a collection of sequences to introduce variability and to create a library . the random sequences can be assembled in various ways , for example by using a mixture of mononucleotides or preferably a mixture of trinucleotides ( virnekäs et al ., 1994 ) during automated oligonucleotide synthesis , by error - prone pcr or by other methods well known to the practitioner in the art . the random sequences may be completely randomized or biased towards or against certain codons according to the amino acid distribution at certain positions in known protein sequences . additionally , the collection of random sub - sequences may comprise different numbers of codons , giving rise to a collection of sub - elements having different lengths . in another embodiment , the invention provides for the expression of the nucleic acid sequences from a suitable vector and under suitable conditions well known to those skilled in the art . in a further preferred embodiment , the ( poly ) peptides expressed from said nucleic acid sequences are screened and , optionally , optimized . screening may be performed by using one of the methods well known to the practitioner in the art , such as phage - display , selectively infective phage , polysome technology to screen for binding , assay systems for enzymatic activity or protein stability . ( poly ) peptides having the desired property can be identified by sequencing of the corresponding nucleic acid sequence or by amino acid sequencing or mass spectrometry . in the case of subsequent optimization , the nucleic acid sequences encoding the initially selected ( poly ) peptides can optionally be used without sequencing . optimization is performed by repealing the replacement of sub - sequences by different sequences , preferably by random sequences , and the screening step one or more times . the desired property the ( poly ) peptides are screened for is preferably , but not exclusively , selected from the group of optimized affinity or specificity for a target molecule , optimized enzymatic activity , optimized expression yields , optimized stability and optimized solubility . in one embodiment , the cleavage sites flanking the sub - sequences are sites recognized and cleaved by restriction enzymes , with recognition and cleavage sequences being either identical or different , the restricted sites either having blunt or sticky ends . the length of the sub - elements is preferably , but not exclusively ranging between 1 amino acid , such as one residue in the active site of an enzyme or a structure - determining residue , and 150 amino acids , as for whole protein domains . most preferably , the length ranges between 3 and 25 amino acids , such as most commonly found in cdr loops of antibodies . in one embodiment , the ( poly ) peptides have an amino acid pattern characteristic of a particular species . this can for example be achieved by deducing the consensus sequences from a collection of homologous proteins of just one species , most preferably from a collection of human proteins . since the ( poly ) peptides comprising consensus sequences are artificial , they have to be compared to the protein sequence ( s ) having the closest similarity to ensure the presence of said characteristic amino acid pattern . in one embodiment , the invention provides for the creation of libraries of ( poly ) peptides comprising at least part of members or derivatives of the immunoglobulin superfamily , preferably of member or derivatives of the immnoglobulins . most preferably , the invention provides for the creation of libraries of human antibodies , wherein said ( poly ) peptides are or are derived from heavy or light chain variable regions wherein said structural sub - elements are framework regions ( fr ) 1 , 2 , 3 , or 4 or complementary determining regions ( cdr ) 1 , 2 , or 3 . in a first step , a database of published antibody sequences of human origin is established where the antibody sequences are aligned to each other . the database is used to define subgroups of antibody sequences which show a high degree of similarity in both the sequence and the canonical fold of cdr loops ( as determined by analysis of antibody structures ). for each of the subgroups a consensus sequence is deduced which represents the members of this subgroup ; the complete collection of consensus sequences represent therefore the complete structural repertoire of human antibodies . these artificial genes are then constructed e . g . by total gene synthesis or by the use of synthetic genetic subunits . these genetic subunits correspond to structural sub - elements on the ( poly ) peptide level . on the dna level , these genetic subunits are defined by cleavage sites at the start and the end of each of the sub - elements , which are unique in the vector system . all genes which are members of the collection of consensus sequences are constructed such that they contain a similar pattern of corresponding genetic sub - sequences . most preferably , said ( poly ) peptides are or are derived from the hucal consensus genes : vκ1 , vκ2 , vκ3 , vκ4 , vλ1 , vλ2 , vλ3 , vh1a , vh1b , vh2 , vh3 , vh4 , vh5 , vh6 , cκ , cλ , ch1 or any combination of said hucal consensus genes . this collection of dna molecules can then be used to create libraries of antibodies or antibody fragments , preferably fv , disulphide - linked fv , single - chain fv ( scfv ), or fab fragments , which may be used as sources of specificities against new target antigens . moreover , the affinity of the antibodies can be optimized using pre - built library cassettes and a general procedure . the invention provides a method for identifying one or more genes encoding one or more antibody fragments which binds to a target , comprising the steps of expressing the antibody fragments , and then screening them to isolate one or more antibody fragments which bind to a given target molecule . preferably , an scfv fragment library comprising the combination of hucal vh3 and hucal vλ2 consensus genes and at least a random sub - sequence encoding the heavy chain cdr3 sub - element is screened for binding antibodies . if necessary , the modular design of the genes can then be used to excise from the genes encoding the antibody fragments one or more genetic sub - sequences encoding structural sub - elements , and replacing them by one or more second sub - sequences encoding structural sub - elements . the expression and screening steps can then be repeated until an antibody having the desired affinity is generated . particularly preferred is a method in which one or more of the genetic subunits ( e . g . the cdrs ) are replaced by a random collection of sequences ( the library ) using the said cleavage sites . since these cleavage sites are ( i ) unique in the vector system and ( ii ) common to all consensus genes , the same ( pre - built ) library can be inserted into all artificial antibody genes . the resulting library is then screened against any chosen antigen . binding antibodies are selected , collected and used as starting material for the next library . here , one or more of the remaining genetic subunits are randomized as described above . a further embodiment of the present invention relates to fusion proteins by providing for a dna sequence which encodes both the ( poly ) peptide , 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 to the one 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 for example 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 embodiment , the additional moiety may confer on its ( poly ) peptide partner a means of detection and / or purification . for example , the fusion protein could comprise the modified antibody 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 ; plückthun , 1994 ). by engineering one or more fused additional domains , antibody fragments or any other ( poly ) peptide can be assembled into larger molecules which also fall under the scope of the present invention . 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 . in a further embodiment , the random collection of sub - sequences ( the library ) is inserted into a singular nucleic acid sequence encoding one ( poly ) peptide , thus creating a ( poly ) peptide library based on one universal framework . preferably a random collection of cdr sub - sequences is inserted into a universal antibody framework , for example into the hucal h3λ2 single - chain fv fragment described above . in further embodiments , the invention provides for nucleic acid sequence ( s ), vector ( s ) containing the nucleic acid sequence ( s ), host cell ( s ) containing the vector ( s ), and ( poly ) peptides , obtainable according to the methods described above . in a further preferred embodiment , the invention provides for modular vector systems being compatible with the modular nucleic acid sequences encoding the ( poly ) peptides . the modules of the vectors are flanked by restriction sites unique within the vector system and essentially unique with respect to the restriction sites incorporated into the nucleic acid sequences encoding the ( poly ) peptides , except for example the restriction sites necessary for cloning the nucleic acid sequences into the vector . the list of vector modules comprises origins of single - stranded replication , origins of double - stranded replication for high - and low copy number plasmids , promotor / operator , repressor or terminator elements , resistance genes , potential recombination sites , gene iii for display on filamentous phages , signal sequences , purification and detection tags , and sequences of additional moieties the vectors are preferably , but not exclusively , expression vectors or vectors suitable for expression and screening of libraries . in another embodiment , the invention provides for a kit , comprising one or more of the list of nucleic acid sequence ( s ), recombinant vector ( s ), ( poly ) peptide ( s ), and vector ( s ) according to the methods described above , and suitable host cell ( s ) for producing the ( poly ) peptide ( s ). in a preferred embodiment , the invention provides for the creation of libraries of human antibodies . in a first step , a database of published antibody sequences of human origin is established : the database is used to define subgroups of antibody sequences which show a high degree of similarity in both the sequence and the canonical fold ( as determined by analysis of antibody structures ). for each of the subgroups a consensus sequence is deduced which represents the members of this subgroup ; the complete collection of consensus sequences represent therefore the complete structural repertoire of human antibodies . these artificial genes are then constructed by the use of synthetic genetic subunits . these genetic subunits correspond to structural sub - elements on the protein level . on the dna level , these genetic subunits are defined by cleavage sites at the start and the end of each of the subelements , which are unique in the vector system . all genes which are members of the collection of consensus sequences are constructed such that they contain a similar pattern of said genetic subunits . this collection of dna molecules can then be used to create libraries of antibodies which may be used as sources of specificities against new target antigens . moreover , the affinity of the antibodies can be optimised using pre - built library cassettes and a general procedure . the invention provides a method for identifying one or more genes encoding one or more antibody fragments which binds to a target , comprising the steps of expressing the antibody fragments , and then screening them to isolate one or more antibody fragments which bind to a given target molecule . if necessary , the modular design of the genes can then be used to excise from the genes encoding the antibody fragments one or more genetic sub - sequences encoding structural sub - elements , and replacing them by one or more second sub - sequences encoding structural sub - elements . the expression and screening steps can then be repeated until an antibody having the desired affinity is generated . particularly preferred is a method in which one or more of the genetic subunits ( e . g . the cdr &# 39 ; s ) are replaced by a random collection of sequences ( the library ) using the said cleavage sites . since these cleavage sites are ( i ) unique in the vector system and ( ii ) common to all consensus genes , the same ( pre - built ) library can be inserted into all artificial antibody genes . the resulting library is then screened against any chosen antigen . binding antibodies are eluted , collected and used as starting material for the next library . here , one or more of the remaining genetic subunits are randomised as described above . the term protein comprises monomeric polypeptide chains as well as homo - or heteromultimeric complexes of two or more polypeptide chains connected either by covalent interactions ( such as disulphide bonds ) or by non - covalent interactions ( such as hydrophobic or electrostatic interactions ). the amino acid sequences of three or more proteins are aligned to each other ( allowing for introduction of gaps ) in a way which maximizes the correspondence between identical or similar amino acid residues at all positions . these aligned sequences are termed homologous if the percentage of the sum of identical and / or similar residues exceeds a defined threshold . this threshold is commonly regarded by those skilled in the art as being exceeded when at least 15 % of the amino acids in the aligned genes are identical , and at least 30 % are similar . examples for families of homologous proteins are : immunoglobulin superfamily , scavenger receptor superfamily , fibronectin superfamilies ( e . g . type ii and iii ), complement control protein superfamily , cytokine receptor superfamily , cystine knot proteins , tyrosine kinases , and numerous other examples well known to one of ordinary skill in the art . using a matrix of al least three aligned amino acid sequences , and allowing for gaps in the alignment , it is possible to determine the most frequent amino acid residue at each position . the consensus sequence is that sequence which comprises the amino acids which are most frequently represented at each position . in the event that two or more amino acids are equally represented at a single position , the consensus sequence includes both or all of those amino acids . the consensus sequence is per se in most cases artificial and has to be analyzed in order to change amino acid residues which , for example , would prevent the resulting molecule to adapt a functional tertiary structure or which would block the interaction with other ( poly ) peptide chains in multimeric complexes . this can be done either by ( i ) building a three - dimensional model of the consensus sequence using known related structures as a template , and identifying amino acid residues within the model which may interact unfavorably with each other , or ( ii ) analyzing the matrix of aligned amino acid sequences in order to detect combinations of amino acid residues within the sequences which frequently occur together in one sequence and are therefore likely to interact with each other . these probable interaction - pairs are then tabulated and the consensus is compared with these “ interaction maps ”. missing or wrong interactions in the consensus are repaired accordingly by introducing appropriate changes in amino acids which minimize unfavorable interactions . structural sub - elements are stretches of amino acid residues within a protein /( poly ) peptide which correspond to a defined structural or functional part of the molecule . these can be loops ( e . g . cdr loops of an antibody ) or any other secondary or functional structure within the protein /( poly ) peptide ( domains . α - helices , β - sheets , framework regions of antibodies , etc .). a structural sub - element can be identified using known structures of similar or homologous ( poly ) peptides , or by using the above mentioned matrices of aligned amino acid sequences . here the variability at each position is the basis for determining stretches of amino acid residues which belong to a structural sub - element ( e . g . hypervariable regions of an antibody ). a sub - sequence is defined as a genetic module which is flanked by unique cleavage sites and encodes at least one structural sub - element . it is not necessarily identical to a structural sub - element . a short dna sequence which is used as a specific target for a reagent which cleaves dna in a sequence - specific manner ( e . g . restriction endonucleases ). cleavage sites are compatible with each other , if they can be efficiently ligated without modification and , preferably , also without adding an adapter molecule . a cleavage site is defined as unique if it occurs only once in a vector containing at least one of the genes of interest , or if a vector containing at least one of the genes of interest could be treated in a way that only one of the cleavage sites could be used by the cleaving agent . sequences deduced from the same part of one group of homologous proteins are called corresponding ( poly ) peptide sequences . a cleavage site in at least two corresponding sequences , which occurs at the same functional position ( i . e . which flanks a defined sub - sequence ), which can be hydrolyzed by the same cleavage tool and which yields identical compatible ends is termed a common cleavage site . a method which uses the unique cleavage sites and the corresponding cleavage reagents to cleave the target dna at the specified positions in order to isolate , remove or replace the genetic sub - sequence flanked by these unique cleavage sites . a method by which an existing sub - sequence is removed using the flanking cleavage sites of this sub - sequence , and a new sub - sequence or a collection of sub - sequences , which contain ends compatible with the cleavage sites thus created , is inserted . the term expression refers to in vivo or in vitro processes , by which the information of a gene is transcribed into mrna and then translated into a protein /( poly ) peptide . thus , the term expression refers to a process which occurs inside cells , by which the information of a gene is transcribed into mrna and then into a protein . the term expression also includes all events of post - translational modification and transport , which are necessary for the ( poly ) peptide to be functional . any method which allows isolation of one or more proteins /( poly ) peptides having a desired property from other proteins /( poly ) peptides within a library . a ( poly ) peptide sequence is assumed to exhibit an amino acid pattern characteristic for a species if it is deduced from a collection of homologous proteins from just this species . the igsf is a family of proteins comprising domains being characterized by the immunoglobulin fold . the igsf comprises for example t - cell receptors and the immunoglobulins ( antibodies ). a framework of an antibody variable domain is defined by kabat et al . ( 1991 ) as the part of the variable domain which serves as a scaffold for the antigen binding loops of this variable domain . the cdrs ( complementarity determining regions ) of an antibody consist of the antigen binding loops , as defined by kabat et al . ( 1991 ). each of the two variable domains of an antibody fv fragment contain three cdrs . acronym for human combinatorial antibody library . antibody library based on modular consensus genes according to the invention ( see example 1 ). any portion of an antibody which has a particular function , e . g . binding of antigen . usually , antibody fragments are smaller than whole antibodies . examples are fv , disulphide - linked fv , single - chain fv ( scfv ), or fab fragments . additionally , antibody fragments are often engineered to include new functions or properties . one single framework which can be used to create the full variability of functions , specificities or properties which is originally sustained by a large collection of different frameworks , is called universal framework . the process which leads to a tight and specific association between an antibody and a corresponding molecule or ligand is called binding . a molecule or ligand or any part of a molecukle or ligand which is recognized by an antibody is called the target . a method by which an existing sub - sequence is removed using the flanking cleavage sites of this sub - sequence , and a new sub - sequence or collection o : sub - sequences , which contains ends compatible with tow cleavage sites thus create , is inserted . any process which is used to combine synthetic or natural genetic sequences in a specific manner in order to get longer genetic sequences which contain at least parts of the used synthetic or natural genetic sequences . the corresponding amino acid sequences of two or more genes are aligned to each other in a way which maximizes the correspondence between identical or similar amino acid residues at all positions . these aligned sequences are termed homologous if the percentage of the gum of identical and / or similar residues exceeds a defined threshold . this threshold is commonly regarded by those skilled in the art as being exceeded when at least 15 per cent of the amino acids in the aligned genes are identical , and at least 30 per cent are similar . fig1 : flow chart outlining the process of construction of a synthetic human antibody library based on consensus sequences . fig2 : alignment of consensus sequences designed for each subgroup ( amino acid residues are shown with their standard one - letter abbreviation ). ( a ) kappa sequences , ( b ) lambda sequences and ( c ), heavy chain sequences . the positions are numbered according to kabat ( 1991 ). in order to maximize homology in the alignment , gaps (-) have been introduced in the sequence at certain positions . fig3 : gene sequences of the synthetic v kappa consensus genes . the corresponding amino acid sequences ( see fig2 ) as well as the unique cleavage sites are also shown . fig4 : gene sequences of the synthetic v lambda consensus genes . the corresponding amino acid sequences ( see fig2 ) as well as the unique cleavage sites are also shown . fig5 : gene sequences of the synthetic v heavy chain consensus genes . the corresponding amino acid sequences ( see fig2 ) as well as the unique cleavage sites are also shown . fig6 : oligonucleotides used for construction of the consensus genes . the oligos are named according to the corresponding consensus gene , e . g . the gene vk1 was constructed using the six oligonucleotides o1k1 to o1k6 . the oligonucleotides used for synthesizing the genes encoding the constant domains cκ ( oclk1 to 8 ) and ch1 ( och1 to 8 ) are also shown . fig7 a / b : sequences of the synthetic genes encoding the constant domains cκ ( a ) and ch1 ( b ). the corresponding amino acid sequences as well as unique cleavage sites introduced in these genes are also shown . fig7 c : functional map and sequence of module m24 comprising the synthetic cλ gene segment ( hucl lambda ). fig8 : sequence and restriction map of the synthetic gene encoding the consensus single - chain fragment vh3 - vκ2 . the signal sequence ( amino acids 1 to 21 ) was derived from the e . coli phoa gene ( skerra & amp ; plückthun , 1988 ). between the phoa signal sequence and the vh3 domain , a short sequence stretch encoding 4 amino acid residues ( amino acid 22 to 25 ) has been inserted in order to allow detection of the single - chain fragment in western blot or elisa using the monoclonal antibody m1 ( knappik & amp ; plückthun , 1994 ). the last 6 basepairs of the sequence were introduced for cloning purposes ( ecori site ). fig9 : plasmid map of the vector pig10 . 3 used for phage display of the h3κ2 scfv fragment . the vector is derived from pig10 and contains the gene for the lac operon repressor , lac , the artificial operon encoding the h3κ2 - gene3ss fusion under control of the lac promoter , the ipp terminator of transcription , the single - strand replication origin of the e . coli phage f1 ( f1_ori ), a gene encoding β - lactamase ( bla ) and the colei derived origin of replication . fig1 : sequencing results of independent clones from the initial library , translated into the corresponding amino acid sequences . ( a ) amino acid sequence of the vh3 consensus heavy chain cdr3 ( position 93 to 102 , kabat numbering ). ( b ) amino acid sequences of 12 clones of the 10 - mer library . ( c ) amino acid sequences of 11 clones of the 15 - mer library , single base deletion . fig1 : expression test of individual library members . ( a ) expression of 9 independent clones of the 1o - mer library . ( b ) expression of 9 independent clones of the 15 - mer library . the lane designated with m contains the size marker . both the gp3 - scfv fusion and the scfv monomer are indicated . fig1 : enrichment of specific phage antibodies during the panning against fitc - bsa . the initial as well as the subsequent fluorescein - specific sub - libraries were panned against the blocking buffer and the ratio of the phage eluted from the fitc - bsa coated well vs . that from the powder milk coated well from each panning round is presented as the “ specificity factor ”. fig1 : phage elisa of 24 independent clones after the third round of panning tested for binding on fitc - bsa . fig1 : competition elisa of selected fitc - bsa binding clones . the elisa signals ( od 405 nm ) of scfv binding without inhibition are taken as 1 00 % o . fig1 : sequencing results of the heavy chain cdr3s of independent clones after 3 rounds of panning against fitc - bsa , translated into the corresponding amino acid sequences ( position 93 to 102 . kabat numbering ). fig1 : coomassie - blue stained sds - page of the purified anti - fluorescein scfv fragments : m : molecular weight marker . a : total soluble cell extract after induction , b : fraction of the flow - through , c , d and e : purified scfv fragments 1ha - 3e4 , 1ha - 3e5 and 1ha - 3e 10 , respectively . fig1 : enrichment of specific phage antibodies during the panning against β - estradiol - bsa , testosterone - bsa , bsa , esl - 1 , interleukin - 2 , lymphotoxin - β , and ley - bsa after three rounds of panning . fig1 : elisa of selected esl - 1 and β - estradiol binding clones fig1 : selectivity and cross - reactivity of hucal antibodies : in the diagonal specific binding of hucal antibodies can be seen , off - diagonal signals show non - specific cross - reactivity . fig2 : sequencing results of the heavy chain cdr3s of independent clones after 3 rounds of panning against β - estradiol - bsa , translated into the corresponding amino acid sequences ( position 93 to 102 , kabat numbering ). one clone is derived from the 10 mer library . fig2 : sequencing results of the heavy chain cdr3s of independent clones after 3 rounds of panning against testosterone - bsa , translated into the corresponding amino acid sequences ( position 93 to 102 , kabat numbering ). fig2 : sequencing results of the heavy chain cdr3s of independent clones after 3 rounds of panning against lymphotoxin - β , translated into the corresponding amino acid sequences ( position 93 to 102 , kabat numbering ). one clone comprises a 14 mer cdr , presumably introduced by incomplete coupling of the trinucleotide mixture during oligonucleotide synthesis . fig2 : sequencing results of the heavy chain cdr3s of independent clones after 3 rounds of panning against esl - 1 , translated into the corresponding amino acid sequences ( position 93 to 102 , kabat numbering ). two clones are derived from the 10 mer library . one clone comprises a 16 mer cdr , presumably introduced by chain elongation during oligonucleotide synthesis using trinucleotides . fig2 : sequencing results of the heavy chain cdr3s of independent clones after 3 rounds of panning against bsa , translated into the corresponding amino acid sequences ( position 93 to 102 , kabat numbering ). fig2 : schematic representation of the modular pcal vector system . fig2 a : list of restriction sites already used in or suitable for the modular hucal genes and pcal vector system . fig2 : list of the modular vector elements for the pcal vector series : shown are only those restriction sites which are part of the modular system . fig2 : functional map and sequence of the multi - cloning site module ( mcs ) fig2 : functional map and sequence of the pmcs cloning vector series . fig2 : functional map and sequence of the pcal module m1 ( see fig2 ). fig3 : functional map and sequence of the pcal module m7 - iii ( see fig2 ). fig3 : functional map and sequence of the pcal module m9 - ii ( see fig2 ). fig3 : functional map and sequence of the pcal module m11 - ii ( see fig2 ). fig3 : functional map and sequence of the pcal module m14 - ext2 ( see fig2 ). fig3 : functional map and sequence of the pcal module m17 ( see fig2 ). fig3 : functional map and sequence of the modular vector pcal4 . fig3 a : functional maps and sequences of additional pcal modules ( m2 , m3 , m7i , m7ii , m8 , m10ii , m 11ii , m12 , m13 , m19 , m20 , m21 , m41 ) and of low - copy number plasmid vectors ( pcalo1 to pcalo3 ). fig3 b : list of oligonucleotides and primers used for synthesis of pcal vector modules . fig3 : functional map and sequence of the β - lactamase cassette for replacement of cdrs for cdr library cloning . fig3 : oligo and primer design for vκ cdr3 libraries fig3 : oligo and primer design for vλ cdr3 libraries fig3 : functional map of the pbs13 expression vector series . fig4 : expression of all 49 hucal scfvs obtained by combining each of the 7 vh genes with each of the 7 vl genes ( pbs13 , 30 ° c . ): values are given for the percentage of soluble vs . insoluble material , the total and the soluble amount compared to the combination h3κ2 , which was set to 100 %. in addition , the corresponding values for the mcpc603 scfv are given . table 1 : summary of human immunoglobulin germline sequences used for computing the germline membership of rearranged sequences . ( a ) kappa sequences , ( b ) lambda sequences and ( c ), heavy chain sequences . ( 1 ) the germline name used in the various calculations , ( 2 ) the references number for the corresponding sequence ( see appendix for sequence related citations ), ( 3 ) the family where each sequence belongs to and ( 4 ), the various names found in literature for germline genes with identical amino acid sequences . table 2 : rearranged human sequences used for the calculation of consensus sequences . ( a ) kappa sequences , ( b ) lambda sequences and ( c ), heavy chain sequences the table summarized the name of the sequence ( 1 ), the length of the sequence in amino acids ( 2 ), the germline family ( 3 ) as well as the computed germline counterpart ( 4 ). the number of amino acid exchanges between the rearranged sequence and the germline sequence is tabulated in ( 5 ), and the percentage of different amino acids is given in ( 6 ). column ( 7 ) gives the references number for the corresponding sequence ( see appendix for sequence related citations ). table 3 : assignment of rearranged v sequences to their germline counterparts . ( a ) kappa sequences , ( b ) lambda sequences and ( c ), heavy chain sequences . the germline genes are tabulated according to their family ( 1 ), and the number of rearranged genes found for every germline gene is given in ( 2 ). table 4 : computation of the consensus sequence of the rearranged v kappa sequences . ( a ), v kappa subgroup 1 , ( b ), v kappa subgroup 2 , ( c ), v kappa subgroup 3 and ( d ), v kappa subgroup 4 . the number of each amino acid found at each position is tabulated together with the statistical analysis of the data . ( 1 ) amino acids are given with their standard one - letter abbreviations ( and b means d or n , z means e or q and x means any amino acid ). the statistical analysis summarizes the number of sequences found at each position ( 2 ), the number of occurrences of the most common amino acid ( 3 ), the amino acid residue which is most common at this position ( 4 ), the relative frequency of the occurrence of the most common amino acid ( 5 ) and the - number of different amino acids found at each position ( 6 ). table 5 : computation of the consensus sequence of the rearranged v lambda sequences . ( a ), v lambda subgroup 1 , ( b ), v lambda subgroup 2 , and ( c ), v lambda subgroup 3 . the number of each amino acid found at each position is tabulated together with the statistical analysis of the data . abbreviations are the same as in table 4 . table 6 : computation of the consensus sequence of the rearranged v heavy chain sequences . ( a ), v heavy chain subgroup 1a , ( b ), v heavy chain subgroup 1b , ( c ), v heavy chain subgroup 2 , ( d ), v heavy chain subgroup 3 , ( e ). v heavy chain subgroup 4 , ( f ), v heavy chain subgroup 5 , and ( g ), v heavy chain subgroup 6 . the number of each amino acid found at each position is tabulated together with the statistical analysis of the data . abbreviations are the same as in table 4 . the following example describes the design of a fully synthetic human combinatorial antibody library ( hucal ), based on consensus sequences of the human immunoglobulin repertoire , and the synthesis of the consensus genes . the general procedure is outlined in fig1 . in a first step , sequences of variable domains of human immunoglobulins have been collected and divided into three sub bases : v heavy chain ( vh ), v kappa ( vκ ) and v lambda ( vλ ). for each sequence , the gene sequence was then translated into the corresponding amino acid sequence . subsequently , all amino acid sequences were aligned according to kabat et al . ( 1991 ). in the case of vλ sequences , the numbering system of chuchana et al . ( 1990 ) was used . each of the three main databases was then divided into two further sub bases : the first sub base contained all sequences derived from rearranged v genes , where more than 70 positions of the sequence were known . the second sub base contained all germline gene segments ( without the d - and j - minigenes ; pseudogenes with internal stop codons were also removed ). in all cases , where germline sequences with identical amino acid sequence but different names were found , only one sequence was used ( see table 1 ). the final databases of rearranged sequences contained 386 , 149 and 674 entries for vκ , vλ and vh , respectively . the final databases of germline sequences contained 48 , 20 and 141 entries for vκ , vλ and vh , respectively . p 1 . 1 . 2 assignment of sequences to subgroups the sequences in the three germline databases where then grouped according to sequence homology ( see also tomlinson et al ., 1992 , williams & amp ; winter , 1993 , and cox et al ., 1994 ). in the case of vκ , 7 families could be established . vλ was divided into 8 families and vh into 6 families . the vh germline genes of the vh7 family ( van dijk et al ., 1993 ) were grouped into the vh1 family , since the genes of the two families are highly homologous . each family contained different numbers of germline genes , varying from 1 ( for example vh6 ) to 47 ( vh3 ). for each of the 1209 amino acid sequences in the databases of rearranged genes , the nearest germline counterpart , i . e . the germline sequence with the smallest number of amino acid differences was then calculated . after the germline counterpart was found , the number of somatic mutations which occurred in the rearranged gene and which led to amino acid exchanges could be tabulated . in 140 cases , the germline counterpart could not be calculated exactly , because more than one germline gene was found with an identical number of amino acid exchanges . these rearranged sequences were removed from the database . in a few cases , the number of amino acid exchanges was found to be unusually large (& gt ; 20 for vl and & gt ; 25 for vh ), indicating either heavily mutated rearranged genes or derivation from germline genes not present in the database . since it was not possible to distinguish between these two possibilities , these sequences were also removed from the database . finally , 12 rearranged sequences were removed from the database because they were found to have very unusual cdr lengths and composition or unusual amino acids at canonical positions ( see below ). in summary , 1023 rearranged sequences out of 1209 ( 85 %) could be clearly assigned to their germline counterparts ( see table 2 ). after this calculation , every rearranged gene could be arranged in one of the families established for the germline genes . now the usage of each germline gene , i . e . the number of rearranged genes which originate from each germline gene , could be calculated ( see table 2 ). it was found that the usage was strongly biased towards a subset of germline genes , whereas most of the germline genes were not present as rearranged genes in the database and therefore apparently not used in the immune system ( table 3 ). this observation had already been reported in the case of vκ ( cox , et al ., 1994 ). all germline gene families , where no or only very few rearranged counterparts could be assigned , were removed from the database , leaving 4 vκ , 3 v ), and 6 vh families . 1 . 2 . 2 analysis of cdr conformations the conformation of the antigen binding loops of antibody molecules , the cdrs , is strongly dependent on both the length of the cdrs and the amino acid residues located at the so - called canonical positions ( chothia & amp ; lesk , 1987 ). it has been found that only a few canonical structures exist , which determine the structural repertoire of the immunoglobulin variable domains ( chothia et al ., 1989 ). the canonical amino acid positions can be found in cdr as well as framework regions . the 13 used germline families defined above ( 7 vl and 6 vh ) were now analyzed for their canonical structures in order to define the structural repertoire encoded in these families . in 3 of the 4 vκ families ( vκ1 , 2 and 4 ), one different type of cdr1 conformation could be defined for every family . the family vκ3 showed two types of cdr1 conformation : one type which was identical to vκ1 and one type only found in vκ3 . all vκ cdr2s used the same type of canonical structure . the cdr3 conformation is not encoded in the germline gene segments . therefore , the 4 vκ families defined by sequence homology and usage corresponded also to 4 types of canonical structures found in vκ germline genes . the 3 vλ families defined above showed 3 types of cdr1 conformation , each family with one unique type . the vλ1 family contained 2 different cdr1 lengths ( 13 and 14 amino acids ), but identical canonical residues , and it is thought that both lengths adopt the same canonical conformation ( chothia & amp ; lesk , 1987 ). in the cdr2 of the used vλ germlines , only one canonical conformation exists , and the cdr3 conformation is not encoded in the germline gene segments . therefore , the 3 vλ families defined by sequence homology and usage corresponded also to 3 types of canonical structures . the structural repertoire of the human vh sequences was analyzed in detail by chothia et al ., 1992 . in total , 3 conformations of cdr1 ( h1 - 1 , h1 - 2 and h1 - 3 ) and 6 conformations of cdr2 ( h2 - 1 , h2 - 2 , h2 - 3 , h2 - 4 , h2 - 5 and h2 - x ) could be defined . since the cdr3 is encoded in the d - and j - minigene segments , no particular canonical residues are defined for this cdr . all the members of the vh1 family defined above contained the cdr1 conformation h1 - 1 , but differed in their cdr2 conformation : the h2 - 2 conformation was found in 6 germline genes , whereas the conformation h2 - 3 was found in 8 germline genes . since the two types of cdr2 conformations are defined by different types of amino acid at the framework position 72 , the vh1 family was divided into two subfamilies : vh1a with cdr2 conformation h2 - 2 and vh1b with the conformation h2 - 3 . the members of the vh2 family all had the conformations h1 - 3 and h2 - 1 in cdr1 and cdr2 , respectively . the cdr1 conformation of the vh3 members was found in all cases to be h1 - 1 , but 4 different types were found in cdr2 ( h2 - 1 , h2 - 3 , h2 - 4 and h2 - x ). in these cdr2 conformations , the canonical framework residue 71 is always defined by an arginine . therefore , it was not necessary to divide the vh3 family into subfamilies , since the 4 types of cdr2 conformations were defined solely by the cdr2 itself . the same was true for the vh4 family . here , all 3 types of cdr1 conformations were found , but since the cdr1 conformation was defined by the cdr itself ( the canonical framework residue 26 was found to be glycine in all cases ), no subdivisions were necessary . the cdr2 conformation of the vh4 members was found to be h2 - 1 in all cases . all members of the vh5 family were found to have the conformation h1 - 1 and h2 - 2 , respectively . the single germline gene of the vh6 family had the conformations h1 - 3 and h2 - 5 in cdr1 and cdr2 , respectively . in summary , all possible cdr conformations of the vκ and vλ genes were present in the 7 families defined by sequence comparison . from the 12 different cdr conformations found in the used vh germline genes , 7 could be covered by dividing the family vh1 into two subfamilies , thereby creating 7 vh families . the remaining 5 cdr conformations ( 3 in the vh3 and 2 in the vh4 family ) were defined by the cdrs themselves and could be created during the construction of cdr libraries . therefore , the structural repertoire of the used human v genes could be covered by 49 ( 7 × 7 ) different frameworks . the 14 databases of rearranged sequences ( 4 vκ , 3 vλ and 7 vh ) were used to compute the hucal consensus sequences of each subgroup ( 4 hucal - vκ , 3 hucal - vλ , 7 hucal - vh , see table 4 , 5 and 6 ). this was done by counting the number of amino acid residues used at each position ( position variability ) and subsequently identifying the amino acid residue most frequently used at each position . by using the rearranged sequences instead of the used germline sequences for the calculation of the consensus , the consensus was weighted according to the frequency of usage . additionally , frequently mutated and highly conserved positions could be identified . the consensus sequences were cross - checked with the consensus of the germline families to see whether the rearranged sequences were biased at certain positions towards amino acid residues which do not occur in the collected germline sequences , but this was found not to be the case . subsequently , the number of differences of each of the 1 4 consensus sequences to each of the germline sequences found in each specific family was calculated . the overall deviation from the most homologous germline sequence was found to be 2 . 4 amino acid residues ( s . d . = 2 . 7 ), ensuring that the “ artificial ” consensus sequences can still be considered as truly human sequences as far as immunegenicity is concerned . so far , only sequence information was used to design the consensus sequences . since it was possible that during the calculation certain artificial combinations of amino acid residues have been created , which are located far away in the sequence but have contacts to each other in the three dimensional structure , leading to destabilized or even misfolded frameworks , the 14 consensus sequences were analyzed according to their structural properties . it was rationalized that all rearranged sequences present in the database correspond to functional and therefore correctly folded antibody molecules . hence , the most homologous rearranged sequence was calculated for each consensus sequence . the positions where the consensus differed from the rearranged sequence were identified as potential “ artificial residues ” and inspected . the inspection itself was done in two directions . first , the local sequence stretch around each potentially “ artificial residue ” was compared with the corresponding stretch of all the rearranged sequences . if this stretch was found to be truly artificial , i . e . never occurred in any of the rearranged sequences , the critical residue was converted into the second most common amino acid found at this position and analyzed again . second , the potentially “ artificial residues ” were analyzed for their long range interactions . this was done by collecting all available structures of human antibody variable domains from the corresponding pdb files and calculating for every structure the number and type of interactions each amino acid residue established to each side - chain . these “ interaction maps ” were used to analyze the probable side - chain / side - chain interactions of the potentially “ artificial residues ”. as a result of this analysis , the following residues were exchanged ( given is the name of the gene , the position according to kabat &# 39 ; s numbering scheme , the amino acid found at this position as the most abundant one and the amino acid which was used instead ): the process described above provided the complete consensus sequences derived solely from the databases of rearranged sequences . it was rationalized that the cdr1 and cdr2 regions should be taken from the databases of used germline sequences , since the cdrs of rearranged and mutated sequences are biased towards their particular antigens . moreover , the germline cdr sequences are known to allow binding to a variety of antigens in the primary immune response , where only cdr3 is varied . therefore , the consensus cdrs obtained from the calculations described above were replaced by germline cdrs in the case of vh and vκ . in the case of vλ , a few amino acid exchanges were introduced in some of the chosen germline cdrs in order to avoid possible protease cleavage sites as well as possible structural constraints . hucal gene cdr1 cdr2 hucal - vh1a vh1 - 12 - 1 vh1 - 12 - 1 hucal - vh1b vh1 - 13 - 16 vh1 - 13 - 6 , - 7 , - 8 , - 9 hucal - vh2 vh2 - 31 - 10 , - 11 , - 12 , - 13 vh2 - 31 - 3 , - 4 hucal - vh3 vh3 - 13 - 8 , - 9 , - 10 vh3 - 13 - 8 , - 9 , - 10 hucal - vh4 vh4 - 11 - 7 to - 14 vh4 - 11 - 8 , - 9 , - 11 , - 12 , - 14 , - 16 vh4 - 31 - 17 , - 18 , - 19 , - 20 hucal - vh5 vh5 - 12 - 1 , - 2 vh5 - 12 - 1 , - 2 hucal - vh6 vh6 - 35 - 1 vh6 - 35 - 1 hucal - vκ1 vκ1 - 14 , - 15 vκ1 - 2 , - 3 , - 4 , - 5 , - 7 , - 8 , - 12 , - 13 , - 18 , - 19 hucal - vκ2 vκ2 - 6 vκ2 - 6 hucal - vκ3 vκ3 - 1 , - 4 vκ3 - 4 hucal - vκ4 vκ4 - 1 vκ4 - 1 hucal - vλ1 humlv117 , dpl5 dpl5 hucal - vλ2 dpl11 , dpl12 dpl12 hucal - vλ3 dpl23 humlv318 in the case of the cdr3s , any sequence could be chosen since these cdrs were planned to be the first to be replaced by oligonucleotide libraries . in order to study the expression and folding behavior of the consensus sequences in e . coli , it would be useful to have all sequences with the same cdr3 , since the influence of the cdr3s on the folding behavior would then be identical in all cases . the dummy sequences qqhyttpp and arwggdgfyamdy were selected for the vl chains ( kappa and lambda ) and for the vh chains , respectively . these sequences are known to be compatible with antibody folding in e . coli ( carter et al ., 1992 ). the final outcome of the process described above was a collection of 14 hucal amino acid sequences , which represent the frequently used structural antibody repertoire of the human immune system ( see fig2 ). these sequences were back - translated into dna sequences . in a first step , the back - translation was done using only codons which are known to be frequently used in e . coli . these gene sequences were then used for creating a database of all possible restriction endonuclease sites , which could be introduced without changing the corresponding amino acid sequences . using this database , cleavage sites were selected which were located at the flanking regions of all sub - elements of the genes ( cdrs and framework regions ) and which could be introduced in all hucal vh , vκ or vλ genes simultaneously at the same position . in a few cases it was not possible to find cleavage sites for all genes of a subgroup . when this happened , the amino acid sequence was changed , if this was possible according to the available sequence and structural information . this exchange was then analyzed again as described above . in total , the following 6 amino acid residues were exchanged during this design ( given is the name of the gene , the position according to kabat &# 39 ; s numbering scheme , the amino acid found at this position as the most abundant one and the amino acid which was used instead ): in one case ( 5 ′- end of vh framework 3 ) it was not possible to identify a single cleavage site for all 7 vh genes . two different type of cleavage sites were used instead : bsteii for hucal vh1a , vh1b , vh4 and vh5 , and nspv for hucal vh2 , vh3 , vh4 and vh6 . several restriction endonuclease sites were identified , which were not located at the flanking regions of the sub - elements but which could be introduced in every gene of a given group without changing the amino acid sequence . these cleavage sites were also introduced in order to make the system more flexible for further improvements . finally , all but one remaining restriction endonuclease sites were removed in every gene sequence . the single cleavage site , which was not removed was different in all genes of a subgroup and could be therefore used as a “ fingerprint ” site to ease the identification of the different genes by restriction digest . the designed genes , together with the corresponding amino acid sequences and the group - specific restriction endonuclease sites are shown in fig3 and 5 , respectively . the consensus genes were synthesized using the method described by prodromou & amp ; pearl , 1992 , using the oligonucleotides shown in fig6 . gene segments encoding the human constant domains cκ , cλ and ch1 were also synthesized , based on sequence information given by kabat et al ., 1991 ( see fig6 and fig7 ). since for both the cdr3 and the framework 4 gene segments identical sequences were chosen in all hucal vκ , vλ and vh genes , respectively , this part was constructed only once , together with the corresponding gene segments encoding the constant domains . the pcr products were cloned into pcr - script ks (+) ( stratagene , inc .) or pzero - 1 ( invitrogen , inc .) and verified by sequencing . a combination of two of the synthetic consensus genes was chosen after construction to test whether binding antibody fragments can be isolated from a library based on these two consensus frameworks . the two genes were cloned as a single - chain fv ( scfv ) fragment , and a vh - cdr3 library was inserted . in order to test the library for the presence of functional antibody molecules , a selection procedure was carried out using the small hapten fluorescein bound to bsa ( fitc - bsa ) as antigen . in order to test the design of the consensus genes , one randomly chosen combination of synthetic light and heavy gene ( hucal - vκ2 and hucal - vh3 ) was used for the construction of a single - chain antibody ( scfv ) fragment . briefly , the gene segments encoding the vh3 consensus gene and the ch1 gene segment including the cdr3 - framework 4 region , as well as the vκ2 consensus gene and the cκ gene segment including the cdr3 - framework 4 region were assembled yielding the gene for the vh3 - ch1 fd fragment and the gene encoding the vκ2 - cκ light chain , respectively . the ch1 gene segment was then replaced by an oligonucleotide cassette encoding a 20 - mer peptide linker with the sequence agggsggggsggggsggggs . the two oligonucleotides encoding this linker were 5 ′- tcagcgggtggcggttctggcggcggtgggagcggtggcggtggttc - tggcggtggtggttccgatatcggtccacgtacgg - 3 ′ and 5 ′- aattccgtacgtggaccgatatcggaaccaccaccgccagaaccaccgccaccgctcccaccgc cgccagaaccgccacccgc - 3 ′, respectively . finally , the hucal - vκ2 gene was inserted via ecorv and bsiwi into the plasmid encoding the hucal - vh3 - linker fusion , leading to the final gene hucal - vh3 - vκ2 , which encoded the two consensus sequences in the single - chain format vh - linker - vl . the complete coding sequence is shown in fig8 . phagemid pig10 . 3 ( fig9 ) was constructed in order to create a phage - display system ( winter et al ., 1994 ) for the h3κ2 scfv gene . briefly , the ecori / hindiii restriction fragment in the phagemid vector pig10 ( ge et al ., 1995 ) was replaced by the c - myc followed by an amber codon ( which encodes an glutamate in the amber - suppresser strain xl1 blue and a stop codon in the non - suppresser strain jm83 ) and a truncated version of the gene iii ( fusion junction at codon 249 , see lowman et al ., 1991 ) through pcr mutagenesis . heavy chain cdr3 libraries of two lengths ( 10 and 15 amino acids ) were constructed using trinucleotide codon containing oligonucleotides ( virnekäs et al ., 1994 ) as templates and the oligonucleotides complementing the flanking regions as primers . to concentrate only on the cdr3 structures that appear most often in functional antibodies , we kept the salt - bridge of r h94 and o h101 in the cdr3 loop . for the 15 - mer library , both phenylalanine and methionine were introduced at position 100 since these two residues were found to occur quite often in human cdr3s of this length ( not shown ). for the same reason , valine and tyrosine were introduced at position 102 . all other randomized positions contained codons for all amino acids except cystein , which was not used in the trinucleotide mixture . the cdr3 libraries of lengths 10 and 15 were generated from the pcr fragments using oligonucleotide templates o3hcdr103t ( 5 ′- gatacggccgtgtattattgcgcgcgt ( tri ) 6 gattattggggccaaggcaccctg - 3 ′) and o3hcdr153t ( 5 ′- gatacggccgt gtattattgcgcgcgt ( tri ) 10 ( ttt / atg ) gat ( gtt / tat ) tggg - gccaaggcaccctg - 3 ′), and primers o3hcdr35 ( 5 ′- gatacggccgtgtatta - ttgc - 3 ′) and o3hcdr33 ( 5 ′- cagggtgccttggcccc - 3 ′), where tri are trinucleotide mixtures representing all amino acids without cystein , ( ttt / atg ) and ( gtt / tat ) are trinucleotide mixtures encoding the amino acids phenylalanine / methionine and valine / lyrosine , respectively . the potential diversity of these libraries was 4 . 7 × 10 7 and 3 . 4 × 10 10 for 10 - mer and 15 - mer library , respectively . the library cassettes were first synthesized from pcr amplification of the oligo templates in the presence of both primers : 25 pmol of the oligo template o3hcdr103t or o3hcdr153t , 50 pmol each of the primers o3hcdr35 and o3hcdr33 , 20 nmol of dntp , 10 × buffer and 2 . 5 units of pfu dna polymerase ( stratagene ) in a total volume of 100 μl for 30 cycles ( 1 minute at 92 ° c ., 1 minute at 62 ° c . and 1 minute at 72 ° c .). a hot - start procedure was used . the resulting mixtures were phenol - extracted , ethanol - precipitated and digested overnight with eagi and styi . the vector pig10 . 3 - sch3κ2cat , where the eagi - styi fragment in the vector pig10 . 3 - sch3κ2 encoding the h - cdr3 was replaced by the chloramphenicol acetyltransferase gene ( cat ) flanked with these two sites , was similarly digested . the digested vector ( 35 μg ) was gel - purified and ligated with 100 μg of the library cassette overnight at 16 ° c . the ligation mixtures were isopropanol precipitated , air - dried and the pellets were redissolved in 100 μl of ddh2o . the ligation was mixed with 1 ml of freshly prepared electrocompetent xl1 blue on ice . 20 rounds of electroporation were performed and the transformants were diluted in soc medium , shaken at 37 ° c . for 30 minutes and plated out on large lb plates ( amp / tel / glucose ) at 37 ° c . for 6 - 9 hrs . the number of transformants ( library size ) was 3 . 2 × 10 7 and 2 . 3 × 10 7 for the 10 - mer and the 15 - mer library , respectively . the colonies were suspended in 2xyt medium ( amp / tet / glucose ) and stored as glycerol culture . in order to test the quality of the initial library , phagemids from 24 independent colonies ( 12 from the 10 - mer and 12 from the 15 - mer library , respectively ) were isolated and analyzed by restriction digestion and sequencing . the restriction analysis of the 24 phagemids indicated the presence of intact vector in all cases . sequence analysis of these clones ( see fig1 ) indicated that 22 out of 24 contained a functional sequence in their heavy chain cdr3 regions . 1 out of 12 clones of the 10 - mer library had a cdr3 of length 9 instead of 10 , and 2 out of 12 clones of the 15 - mer library had no open reading frame , thereby leading to a non - functional scfv ; one of these two clones contained two consecutive inserts , but out of frame ( data not shown ). all codons introduced were presented in an even distribution . expression levels of individual library members were also measured . briefly , 9 clones from each library were grown in 2xyt medium containing amp / tet / 0 . 5 % glucose at 37 ° c . overnight . next day , the cultures were diluted into fresh medium with amp / tet . at an od 600 nm of 0 . 4 , the cultures were induced with 1 mm of iptg and shaken at rt overnight . then the cell pellets were suspended in 1 ml of pbs buffer + 1 mm of edta . the suspensions were sonicated and the supernatants were separated on an sds - page under reducing conditions , blotted on nylon membrane and detected with anti - flag m1 antibody ( see fig1 ). from the nine clones of the 10 - mer library , all express the scfv fragments . moreover , the gene iii / scfv fusion proteins were present in all cases . among the nine clones from the 15 - mer library analyzed , 6 / 9 ( 67 %) led to the expression of both scfv and the gene iii / scfv fusion proteins . more importantly , all clones expressing the scfvs and gene iii / scfv fusions gave rise to about the same level of expression . phages displaying the antibody libraries were prepared using standard protocols . phages derived from the 10 - mer library were mixed with phages from the 15 - mer library in a ratio of 20 : 1 ( 1 × 10 10 cfu / well of the 10 - mer and 5 × 10 8 cfu / well of the 15 - mer phages , respectively ). subsequently , the phage solution was used for panning in elisa plates ( maxisorp , nunc ) coated with fitc - bsa ( sigma ) at concentration of 100 μg / ml in pbs at 4 ° c . overnight . the antigen - coated wells were blocked with 3 % powder milk in pbs and the phage solutions in 1 % powder milk were aoced to each well and the plate was shaken at rt for 1 hr . the wells were then washed with pbst and pbs ( 4 times each with shaking at rt for 5 minutes ). the bound phages were eluted with 0 . 1 m triethylamine ( tea ) at rt for 10 minutes . the eluted phage solutions were immediately neutralized with ½ the volume of 1 m tris - cl , ph 7 . 6 . eluted phage solutions ( ca . 450 μl ) were used to infect 5 ml of xl1 blue cells at 37 ° c . for 30 min . the infected cultures were then plated out on large lb plates ( amp / tet / glucose ) and allowed to grow at 37 ° c . until the colonies were visible . the colonies were suspended in 2xyt medium and the glycerol cultures were made as above described . this panning round was repeated twice , and in the third round elution was carried out with addition of fluorescein in a concentration of 100 μg / ml in pbs . the enrichment of specific phage antibodies was monitored by panning the initial as well as the subsequent fluorescein - specific sub - libraries against the blocking buffer ( fig1 ). antibodies with specificity against fluorescein were isolated after 3 rounds of panning . one of the criteria for the successful biopanning is the isolation of individual phage clones that bind to the targeted antigen or hapten . we undertook the isolation of anti - fitc phage antibody clones and characterized them first in a phage elisa format . after the 3rd round of biopanning ( see above ), 24 phagemid containing clones were used to inoculate 100 μl of 2xyt medium ( amp / tet / glucose ) in an elisa plate ( nunc ), which was subsequently shaken at 37 ° c . for 5 hrs . 100 μl of 2xyt medium ( amp / tet / 1 mm iptg ) were added and shaking was continued for 30 minutes . a further 100 μl of 2xyt medium ( amp / tet ) containing the helper phage ( 1 × 10 9 cfu / well ) was added and shaking was done at rt for 3 hrs . after addition of kanamycin to select for successful helper phage infection , the shaking was continued overnight . the plates were then centrifuged and the supernatants were pipetted directly into elisa wells coated with 100 μl fitc - bsa ( 100μg / ml ) and blocked with milk powder . washing was performed similarly as during the panning procedure and the bound phages were detected with anti - m13 antibody - pod conjugate ( pharmacia ) using soluble pod substrate ( boehringer - mannheim ). of the 24 clones screened against fitc - bsa , 22 were active in the elisa ( f ;; g . 1 3 ). the initial libraries of similar titer gave rise to no detectable signal . specificity for fluorescein was measured in a competitive elisa . periplasmic fractions of five fitc specific scfvs were prepared as described above . western blotting indicated that all clones expressed about the same amount of scfv fragment ( data not shown ). elisa was performed as described above , but additionally , the periplasmic fractions were incubated 30 min at rt either with buffer ( no inhibition ), with 10 mg / ml bsa ( inhibition with bsa ) or with 10 mg / ml fluorescein ( inhibition with fluorescein ) before adding to the well . binding scfv fragment was detected using the anti - flag antibody m1 . the elisa signal could only be inhibited , when soluble fluorescein was added , indicating binding of the scfvs was specific for fluorescein ( fig1 ). the heavy chain cdr3 region of 20 clones were sequenced in order to estimate the sequence diversity of fluorescein binding antibodies in the library ( fig1 ). in total , 16 of 20 sequences ( 80 %) were different , showing that the constructed library contained a highly diverse repertoire of fluorescein binders . the cdr3s showed no particular sequence homology , but contained on average 4 arginine residues . this bias towards arginine in fluorescein binding antibodies had already been described by barbas et al ., 1992 . e . coli jm83 was transformed with phagemid dna of 3 selected clones and cultured in 0 . 5 l 2xyt medium . induction was carried out with 1 mm iptg at od 600 nm = 0 . 4 and growth was continued - with vigorous shaking at rt overnight . the cells were harvested and pellets were suspended in pbs buffer and sonicated . the supernatants were separated from the cell debris via centrifugation and purified via the biologic system ( bio - rad ) by with a poros ® mc 20 column ( imac , perseptive biosystems , inc .) coupled with an ion - exchange chromatography column . the ion - exchange column was one of the poros ® hs , cm or hq or pi 20 ( perseptive biosystems , inc .) depended on the theoretical pi of the scfv being purified . the ph of all the buffers was adjusted to one unit lower or higher than the pi of the scfv being purified throughout . the sample was loaded onto the first imac column , washed with 7 column volumes of 20 mm sodium phosphate , 1 m nacl and 10 mm imidazole . this washing was followed by 7 column volumes of 20 mm . sodium phosphate and 10 mm imidazole . then 3 column volumes of an imidazole gradient ( 10 to 250 mm ) were applied and the eluent was connected directly to the ion - exchanger . nine column volumes of isocratic washing with 250 mm imidazole was followed by 15 column volumes of 250 mm to 100 mm and 7 column volumes of an imidazole / nacl gradient ( 100 to 10 mm imidazole , 0 to 1 m nacl ). the flow rate was 5 ml / min . the purity of scfv fragments was checked by sds - page coomassie staining ( fig1 ). the concentration of the fragments was determined from the absorbance at 280 nm using the theoretically determined extinction coefficient ( gill & amp ; von hippel , 1989 ). the scfv fragments could be purified to homogeneity ( see fig1 ). the yield of purified fragments ranged from 5 to 10 mg / l / od . in order to test the library used in example 2 further , a new selection procedure was carried out using a variety of antigens comprising β - estradiol , testosterone , lewis - y epitope ( ley ), interleukin - 2 ( il - 2 ), lymphotoxin - β ( lt - β ), e - selectin ligand - 1 ( esl - 1 ), and bsa . the library and all procedures were identical to those described in example 2 . the elisa plates were coated with β - estradiol - bsa ( 100 pg / ml ), testosterone - bsa ( 100 μg / ml ), ley - bsa ( 20 μg / ml ) il - 2 ( 20 μg / ml ), esl - 1 ( 20 μg / ml ) and bsa ( 100 μg / ml ), lt - β ( denatured protein , 20 μg / ml ). in the first two rounds , bound phages were eluted with 0 . 1 m triethylamine ( tea ) at rt for 10 minutes . in the case of bsa , elution after three rounds of panning was carried out with addition of bsa in a concentration of 100 μg / ml in pbs . in the case of the other antigens , third round elution was done with 0 . 1 m triethylamine . in all cases except ley , enrichment of binding phages could be seen ( fig1 ). moreover , a repetition of the biopanning experiment using only the 15 - mer library resulted in the enrichment of ley - binding phages as well ( data not shown ). clones binding to β - estradiol , testosterone , ley , lt - β , esl - 1 and bsa were further analyzed and characterized as described in example 2 for fitc . elisa data for anti - β - estradiol and anti - esl - 1 antibodies are shown in fig1 . in one experiment . selectivity and cross - reactivity of binding scfv fragments were tested . for this purpose , an elisa plate was coated with fitc , testosterone , β - estradiol , bsa , and esl - 1 , with 5 wells for each antigen arranged in 5 rows , and 5 antibodies , one against each of the antigens , were screened against each of the antigens . fig1 shows the specific binding of the antibodies to the antigen it was selected for , and the low cross - reactivity with the other four antigens . the sequencing data of several clones against β - estradiol ( 34 clones ), testosterone ( 12 clones ), lt - β ( 23 clones ), esl - 1 ( 34 clones ), and bsa ( 10 clones ) are given in fig2 to 24 . to be able to take advantage of the modularity of the consensus gene repertoire , a vector system had to be constructed which could be used in phage display screening of hucal libraries and subsequent optimization procedures . therefore , all necessary vector elements such as origins of single - stranded or double - stranded replication , promotor / operator , repressor or terminator elements , resistance genes , potential recombination sites , gene iii for display on filamentous phages , signal sequences , or detection tags had to be made compatible with the restriction site pattern of the modular consensus genes . fig2 shows a schematic representation of the pcal vector system and the arrangement of vector modules and restriction sites therein . fig2 a shows a list of all restriction sites which are already incorporated into the consensus genes or the vector elements as part of the modular system or which are not yet present in the whole system . the latter could be used in a later stage for the introduction of or within new modules . a series of vector modules was constructed where the restriction sites flanking the gene sub - elements of the hucal genes were removed , the vector modules themselves being flanked by unique restriction sites . these modules were constructed either by gene synthesis or by mutagenesis of templates . mutagenesis was done by add - on pcr , by site - directed mutagenesis ( kunkel et at ., 1991 ) or multisite oligonucleotide - mediated mutagenesis ( sutherland et al ., 1995 ; perlak , 1990 ) using a pcr - based assembly method . fig2 contains a list of the modules constructed . instead of the terminator module m9 ( hindiii - ipppaci ), a larger cassette m9ii was prepared to introduce fsei as additional restriction site . m9ii can be cloned via hindiii / bsrgi . all vector modules were characterized by restriction analysis and sequencing . in the case of module m11 - ii , sequencing of the module revealed a two - base difference in positions 164 / 65 compared to the sequence database of the template . these two different bases ( ca → gc ) created an additional banii site . since the same two - base difference occurs in the 11 origin of other bacteriophages , it can be assumed that the two - base difference was present in the template and not created by mutagenesis during cloning . this banii site was removed by site - directed mutagenesis , leading to module m11 - iii . the bsssi site of module m14 could initially not be removed without impact on the function of the coie1 origin , therefore m14 - ex12 was used for cloning of the first pcal vector series . fig2 to 34 are showing the functional maps and sequences of the modules used for assembly of the modular vector pcal4 ( see below ). the functional maps and sequences of additional modules can be found in fig3 a . fig3 b contains a list of oligonucleotides and primers used for the synthesis of the modules . to be able to assemble the individual vector modules , a cloning vector pmcs containing a specific multi - cloning site ( mcs ) was constructed . first , an mcs cassette ( fig2 ) was made by gene synthesis . this cassette contains all those restriction sites in the order necessary for the sequential introduction of all vector modules and can be cloned via the 5 ′- hindiii site and a four base overhang at the 3 ′- end compatible with an aatii site . the vector pmcs ( fig2 ) was constructed by digesting puc19 with aatii and hindiii , isolating the 2174 base pair fragment containing the bla gene and the coie1 origin , and ligating the mcs cassette . this was cloned step by step by restriction digest of pmcs and subsequent ligation of the modules m1 ( via aatii / xbai ), m7iii ( via ecori / hindiii ), and m9ii ( via hindiii / bsrgi ), and m11 - ii ( via bsrgi / nhei ). finally , the bla gene was replaced by the cat gene module m17 ( via aatii / bgiii ), and the wild type coie1 origin by module m14 - ext2 ( via bgiii / nhei ). fig3 is showing the functional map and the sequence of pcal4 . a series of low - copy number plasmid vectors was constructed in a similar way using the p15a module m12 instead of the coie1 module m14 - ext2 . fig3 a is showing the functional maps and sequences of the vectors pcalo1 to pcalo3 . all 49 combinations of the 7 hucal - vh and 7 hucal - vl consensus genes were assembled as described for the hucal vh3 - vk2 scfv in example 2 and inserted into the vector pbs12 , a modified version of the plisc series of antibody expression vectors ( skerra et a /., 1991 ). for replacement of cdrs , a universal β - lactamase cloning cassette was constructed having a multi - cloning site at the 5 ′- end as well as at the 3 ′- end . the 5 ′- multi - cloning site comprises all restriction sites adjacent to the 5 ′- end of the hucal vh and vl cdrs , the 3 ′- multi - cloning site comprises all restriction sites adjacent to the 3 ′ end of the hucal vh and vl cdrs . both 5 ′- and 38 ′- multi - cloning site were prepared as cassettes via add - on pcr using synthetic oligonucleotides as 5 ′- and 3 ′- primers using wild type β - lactamase gene as template . fig3 shows the functional map and the sequence of the cassette bla - mcs . the vl - cdr3 libraries comprising 7 random positions were generated from the pcr fragments using oligonucleotide templates vκ1 & amp ; vκ3 , vκ2 and vκ4 and primers o_k3l — 5 and o_k3l — 3 ( fig3 ) for the vκ genes , and vλ and primers o_l3l — 5 ( 5 ′- gcagaaggcgaacgtcc - 3 ′) and o_l3la — 3 ( fig3 ) for the vλ genes . construction of the cassettes was performed as described in example 2 . 3 . each of the 49 single - chains was subcloned into pcal4 via xbai / ecori and the vl - cdr3 replaced by the β - lactamase cloning cassette via bbsi / msci , which was then replaced by the corresponding vl - cdr3 library cassette synthesized as described above . this cdr replacement is described in detail in example 2 . 3 where the cat gene was used . the vh - cdr3 libraries were designed and synthesized as described in example 2 . 3 . 5 . 6 cloning of hucal scfv genes with vl - and vh - cdr3 libraries each of the 49 single - chain vl - cdr3 libraries was digested with bsshii / styi to replace vh - cdr3 . the “ dummy ” cassette digested with bsshii / styi was inserted , and was then replaced by a corresponding vh - cdr3 library cassette synthesized as described above . expression and toxicity studies were performed using the scfv format vh - linker - vl . all 49 combinations of the 7 hucal - vh and 7 hucal - vl consensus genes assembled as described in example 5 were inserted into the vector pbs13 , a modified version of the plisc series of antibody expression vectors ( skerra et al ., 1991 ). a map of this vector is shown in fig3 . e . coli jm83 was transformed 49 times with each of the vectors and stored as glycerol stock . between 4 and 6 clones were tested simultaneously , always including the clone h3κ2 , which was used as internal control throughout . as additional control , the mcpc603 scfv fragment ( knappik & amp ; plückthun , 1995 ) in pbs13 was expressed under identical conditions . two days before the expression test was performed , the clones were cultivated on lb plates containing 30 μg / ml chloramphenicol and 60 mm glucose . using this plates an 3 ml culture ( lb medium containing 90 μg chloramphenicol and 60 mm glucose ) was inoculated overnight at 37 ° c . next day the overnight culture was used to inoculate 30 ml lb medium containing chloramphenicol ( 30 μg / ml ). the starting od 600 nm was adjusted to 0 . 2 and a growth temperature of 30 ° c . was used . the physiology of the cells was monitored by measuring every 30 minutes for 8 to 9 hours the optical density at 600 nm . after the culture reached an od 600 nm of 0 . 5 , antibody expression was induced by adding iptg to a final concentration of 1 mm . a 5 ml aliquot of the culture was removed after 2 h of induction in order to analyze the antibody expression . the cells were lysed and the soluble and insoluble fractions of the crude extract were separated as described in knappik & amp ; plückthun , 1995 . the fractions were assayed by reducing sds - page with the samples normalized to identical optical densities . after blotting and immunostaining using the α - flag antibody m1 as the first antibody ( see ge et al ., 1994 ) and an fc - specific anti - mouse antiserum conjugated to alkaline phosphatase as the second antibody , the lanes were scanned and the intensities of the bands of the expected size ( appr . 30 kda ) were quantified densitometrically and tabulated relative to the control antibody ( see fig4 ). a l - cdr3 library cassette was prepared from the oligonucleotide template cdr3l ( 5 ′- tggaagctgaagacgtgggcgtgtattattgccagcag ( tr5 )( tri ) 4 ccg ( tri )- tttggccagggtacgaaagtt - 3 ′) and primer 5 ′- aactcgtaccctggcc - 3 ′ for synthesis of the complementary strand , where ( tri ) was a trinucleotide mixture representing all amino acids except cys , ( tr5 ) comprised a trinucleotide mixture representing the 5 codons for ala , arg , his , ser , and tyr . a h - cdr2 library cassette was prepared from the oligonucleotide template cdrsh ( 5 ′- agggtctcgagtgggtgagc ( tri ) att ( tri ) 2 - 3 ( 6 ) 2 ( tri ) acc ( tri ) tatgcggatagcgtgaaaggccgttttaccatttcacgtgataattcgaaaaacacca - 3 ′), and primer 5 ′- tggtgtttttcgaattattatca - 3 ′ for synthesis of the complementary strand , where ( tri ) was a trinucleotide mixture representing all amino acids except cys , ( 6 ) comprised the incorporation of ( a / g ) ( a / c / g ) t , resulting in the formation of 6 codons for ala , asn , asp , gly , ser , and thr , and the length distribution being obtained by performing one substoichiometric coupling of the ( tri ) mixture during synthesis , omitting the capping step normally used in dna synthesis . dna synthesis was performed on a 40 nmole scale , oligos were dissolved in te buffer , purified via gel filtration using spin columns ( s - 200 ), and the dna concentration determined by od measurement at 260 nm ( od 1 . 0 = 40 μg / ml ). 10 nmole of the oligonucleotide templates and 12 nmole of the corresponding primers were mixed and annealed at 80 ° c . for 1 min , and slowly cooled down to 37 ° c . within 20 to 30 min . the fill - in reaction was performed for 2 h at 37 ° c . using klenow polymerase ( 2 . 0 μl ) and 250 nmole of each dntp . the excess of dntps was removed by gel filtration using nick - spin columns ( pharmacia ), and the double - stranded dna digested with bbsi / msci ( l - cdr3 ), or xhoi / sfui ( h - cdr2 ) over night at 37 ° c . the cassettes were purified via nick - spin columns ( pharmacia ), the concentration determined by od measurement , and the cassettes aliquoted ( 15 pmole ) for being stored at − 80 ° c . dna was prepared from the collection of fitc binding clones obtained in example 2 ( approx . 10 4 to clones ). the collection of scfv fragments was isolated via xbai / ecori digest . the vector pcal4 ( 100 fmole , 10 μg ) described in example 4 . 3 was similarly digested with xbai / ecori , gel - purified and ligated with 300 fmole of the scfv fragment collection over night at 16 ° c . the ligation mixture was isopropanol precipitated , air - dried , and the pellets were redissolved in 100 μl of dd h 2 o . the ligation mixture was mixed with 1 ml of freshly prepared electrocompetent scs 101 cells ( for optimization of l - cdr3 ), or xl1 blue cells ( for optimization of h - cdr2 ) on ice . one round of electroporation was performed and the transformants were eluted in soc medium , shaken at 37 ° c . for 30 minutes , and an aliquot plated out on lb plates ( amp / tet / glucose ) at 37 ° c . for 6 - 9 hrs . the number of transformants was 5 × 10 4 . vector dna ( 100 μg ) was isolated and digested ( sequence and restriction map of sch3κ2 see fig8 ) with bbsi / msci for optimization of l - cdr3 , or xhoi / nspv for optimization of h - cdr2 . 10 μg of purified vector fragments ( 5 pmole ) were ligated with 15 pmole of the l - cdr3 or h - cdr2 library cassettes over night at 16 ° c . the ligation mixtures were isopropanol precipitated , air - dried , and the pellets were redissolved in 100 μl of dd h 2 o . the ligation mixtures were mixed with 1 ml of freshly prepared electrocompetent xl1 blue cells on ice . electroporation was performed and the transformants were eluted in soc medium and shaken at 37 ° c . for 30 minutes . an aliquot was plated out on lb plates ( amp / tet / glucose ) at 37 ° c . for 6 - 9 hrs . the number of transformants ( library size ) was greater than 10 8 for both libraries . the libraries were stored as glycerol cultures . this was performed as described for the initial h3κ2 h - cdr3 library in example 2 . 1 . optimized scfvs binding to fitc could be characterized and analyzed as described in example 2 . 2 and 2 . 3 , and further rounds of optimization could be made if necessary . barbas iii , c . f ., bain , j . d ., hoekstra , d . m . & amp ; lerner , r . a ., pnas 8 , 4457 - 4461 ( 1992 ). better , m ., chang , p ., robinson , r . & amp ; horwitz , a . h ., science 240 , 1041 - 1043 ( 1988 ). blake , m . s ., johnston , k . h ., russel - jones , g . j . & amp ; gotschlich , e . c ., anal . biochem 136 , 175 - 179 ( 1984 ). carter , p ., kelly , r . f ., rodrigues , m . l ., snedecor , b ., covrrubias , m ., velligan , m . d ., wong , w . l . t ., rowland , a . m ., kotts , c . e ., carver , m . e ., yang , m ., bourell , j . h ., shepard , h . m . & amp ; henner , d ., bio / technology 10 , 163 - 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