Patent Application: US-24095103-A

Abstract:
the present invention provides a method for producing a polynucleotide sequence encoding an antibody variable domain , the variable domain comprising complementarity - determining regions located within a selected framework , the method comprising the steps of providing at least one nucleic acid molecule encoding one or more cdrs and associated framework regions , amplifying at least one cdr - encoding portion of the nucleic acid molecule of step using one or more pairs of oligonucleotides as amplification primers and assembling a polynucleotide sequence encoding an antibody variable domain by combining the amplified cdr - encoding nucleotide sequences produced in step with nucleotide sequences encoding said master framework , wherein the oligonucleotide primers of step comprise nucleotide sequences which differ from the corresponding nucleotide sequences encoding said master framework . the invention further provides an antibody library , such as a phage display library , and methods of making the same .

Description:
the present invention represents a development of the technology presented in wo98 / 32845 , söderlind et al . ( 1999 ) immunotechnology 4 , 279 - 285 , and jirholt et al . ( 1998 ) gene 215 , 471 - 476 , all of which are incorporated herein in their entirety , particularly for the purpose of describing generally the methods and conditions used for amplifying cdrs from a cdna library containing antibody - encoding sequences , and methods , materials and conditions for reassembling the cdrs thus amplified into the master framework by overlap extension pcr . the method may further comprise the step of expressing the resulting antibody encoded by the assembled nucleotide sequence and screening for desired properties . again , this is described in detail in the above - mentioned references . the resulting expressed antibody can be screened for desired characteristics . for example it may be desirable to alter its ability to specifically bind to an antigen or to improve its binding properties in comparison to the parent antibody . once more , this is described in detail in the above - mentioned references . preferably the oligonucleotides used for amplification primers have at least two nucleic acid residues different from a corresponding portion of the nucleic acid sequence encoding the master framework . more preferably there are at least 3 , 4 , 5 , 6 , 7 , 8 , 10 or 12 different nucleic acid residues . in an alternative definition , the amplification primers preferably have no more than about 95 % sequence identity with a corresponding portion of the nucleic acid sequence encoding the master framework , more preferably no more than about 90 %, 85 %, 80 %, 70 % or 60 % sequence identity . in conventional cdr implantation , the amplification primers may include a small number of nucleotides encoding one or more amino acid residues of the adjoining end of the cdr ( e . g . three nucleotides , encoding one cdr residue ). this applies also to the present invention , and in such cases , the nucleotides of the cdr may be discounted when determining the number of nucleotide differences between the primer and the master framework . bearing in mind the teaching herein , and given in the cited references on the basic cdr - implantation technique , the skilled person will be able to design primers for amplifying the cdrs and , if necessary or desired , for modifying the amplification products to make their framework regions more similar to the selected master framework . where a particular germline gene is to be targeted , highly specific primers may be desired , for example based closely on the sequence encoding the parts of the framework regions of that gene which flank the cdr or cdrs to be amplified . the sequences of different germline genes are available from the vbase sequence directory ( url : http :// www . mrc - cpe . cam . ac . uk / imt - doc / public / intro . html ) or from the dnaplot directory ( url : http :// www . genetik . uni - koeln . de : 80 / dnaplot / vsearch human . html ). similarly , the primers can be designed to amplify cdrs from a particular germline gene family , by designing primers based on the consensus sequence of genes of that family . for example , a consensus sequence can be defined as the sequence of bases found at & gt ; 90 % of loci of a particular germline family . such sequences may include degenerate sites , indicating that different individual sequences have different nucleotides at that site . there may nevertheless be some common feature of the nucleotide residues which appear at such a degenerate site ; such sites are designated r ( purine ; bases g and a ), y ( pyrimidine ; c , t ), m ( amino ; a , c ), k ( keto ; t , g ), s ( strong ; c , g ), w ( weak , a , t ), b ( not a ), d ( not c ), h ( not g ) or v ( not t ). a site where no common feature is evident is designated n ( any ). primers based on consensus sequences including such designations may be degenerate , i . e . a population of primers is made to include all possible combinations consistent with the consensus sequence , or where appropriate artificial bases which mimic particular sets of bases may be included within a homogeneous population of primers . information ascribing germline genes to germline gene families ( such as the variable heavy germline gene families v h 1 , v h 2 , v h 3 , v h 4 , v h 5 , v h 6 and v h 7 ) is available from the vbase directory referred to above . similarly , it is possible also to design the primers to amplify cdrs from a plurality of germline gene families , using a consensus sequence of germline genes from said plurality of families . however , it will generally be preferred to target a particular germline gene or family . with this in mind , the skilled person will be able to design appropriate primers depending on the specificity required preferably at least one primer of the or each pair used to initially amplify the cdrs is at least 15 nucleotides in length , more preferably at least 18 , still more preferably at least 21 or 24 , optionally at least 30 , 36 or 42 . preferably , however , the primer is no more than 42 nucleotides in length , more preferably no more than 36 or 30 , more preferably no more than 27 . preferably the method will be used to implant cdrs at all three positions in the variable domain , since this leads to maximum variability , and ultimately more useful libraries . however , the method is not limited to this , and if desired ( for example to optimise a previously obtained antibody ), the method may be used to implant only one or two cdrs . in such cases , nucleic acid encoding the invariant cdr ( s ) will be included in the overlap extension pcr step , in addition to the newly amplified cdrs and the nucleic acid encoding the selected master framework . the present invention is not to be construed as limited to implanting cdrs from immunoglobulin genes of the same general type as the master framework ( e . g . implanting v h cdrs into v h master framework ), although this is a preferred embodiment of the invention . rather , the invention in its broader aspects includes the implantation of cdr - encoding nucleic acid from any type of immunoglobulin gene which has a variable region as defined above into a master framework which is independently of any such type of immunoglobulin superfamily gene . for example , vλ cdrs may be inserted into a v h master framework and vice versa . moreover , any members of the immunoglobulin superfamily having analogous structures to cdrs and frs may provide the cdrs and / or master frs of the invention , the above description being applicable mutatis mutandis . the term “ antibody ” is used herein in its broadest sense , to include also antibody fragments having a variable domain which includes cdrs flanked by framework regions . examples of antibody fragments having such variable domains are the fab fragment consisting of the v l , v h , c l and c h 1 domains ; the fd fragment consisting of the v h , and c h 1 domains ; the fv fragment consisting of the v l and v h domains of a single arm of an antibody ; the dab fragment which consists of a v h domain ; and the f ( ab ′) 2 fragment , a bivalent fragment including two fab fragments linked by a disulphide bridge at the hinge region . single chain fv fragments are also included . any desired master framework regions ( or “ framework regions ( frs ) of a selected type ”) may be utilised in the present invention . in particular , they may be selected to be highly compatible with the bacterial expression system , and phage system , to be employed , thus ensuring a high degree of functional protein display . favoured examples are framework regions from the dp - 47 and dpl - 3 germline genes ( of the v h 3 and vλ germline gene families , respectively ). it is now generally agreed that the cdr - loops , which build up the surfaces of antibody combining sites , can be grouped into a limited number of so - called canonical structures , depending on their conformation after folding . the pioneering work in this area was performed by cothia and lesk ( 1987 ) who classified cdr 1 and 2 in the heavy chain and cdr 1 - 3 in the light chain into a few basic structures . the concept of canonical structures is the result of extensive analyses of empirically determined and analysed antibody structures . the determinants for the canonical conformations are the lengths of the loops , key residues in the loops and key residues in the adjacent framework sequences ( chothia et al . 1992 ; tomlinson et al . 1995 ; al - lazikani et al . 1997 ). for example , the human vκ sequences can be grouped in 6 canonical structures for the cdrl1 loop , 1 canonical structure for the cdrl2 loop and 5 canonical structures for the cdrl3 loop . similarly , the human v h sequences can be grouped in 3 canonical structures for the cdrh1 loop and 4 canonical structures for the cdrh2 loop . the cdrh3 loop has not yet been classified in distinct canonical classes , most probably due to its inherited length variation which leads to unique properties regarding flexibility . however , recently it was demonstrated that this cdr also is built from structure elements forming a basic torso near , and to some extent including , the framework region , and an apical head region that sometimes includes an additional shoulder ( morea et al . 1998 ). it is conceivable that nature has developed different types of canonical structures to deal with the multitude of antigenic structures the immune systems may encounter . nature also presents these structures in the context of different framework structures . thus , a particular cdr - loop is found in combination with a certain framework ( vbase ). there also seems to be a bias to which canonical structures are used in order to create suitable surfaces , complementary to different types of antigens . in particular , loops with canonical structures building up a flat surface seem to yield surfaces that bind well to large protein antigens . these loops have a propensity to be rather short whereas longer loops are preferentially found in antibodies specific for smaller molecules e . g . haptens ( lara - ochoa et al . 1996 ). not all loops seem to be equally important in creating variability in the surfaces . of course h3 is of major importance in this respect but also h2 and l1 determine the surfaces to a great extent ( vargas - madrazo et al . 1995 ). using the cdr - implantation technology it has unexpectedly been found that some of the selected antibodies comprised cdrs with canonical structures that are not normally found in the used framework . these antibodies are functional since they bind their antigen with high affinity ( example 1 ). thus , using a single framework it is possible to create functional variability in antibody combining sites that is based on canonical loops that are atypical in a certain framework context a library based on such a concept would have advantages over more conventional libraries since it can harbour antibodies with a wide variety of topologies and at the same time be highly efficient in the selected host system ( e . g . e . coli ). furthermore , the binding characteristics of antibodies could be improved using shuffling of selected cdrs in order to recombine the most optimal cdrs into a single antibody molecule . as will be appreciated , cdr - implantation technology permits shuffling of 1 to 6 cdrs at the same time and has been used on the basis of the library presented herein in the examples to improve affinities of selected antibodies more than 30 times in a single step . the present invention may therefore lead to novel combinations of classes of canonical structure , for example by combining canonical structures of classes that are not normally found in genes of the same germline family . for example , by incorporating cdrh2 cdrs into the cdrl2 position of a vκ chain , variability from 4 classes of canonical structure can be accessed in this position , whereas in the natural vκ antibody , there is only one class of canonical structure used in the cdrl2 position . preferably , the amplification primers are designed to amplify cdrs of a greater number of classes of canonical structure than the number of classes of canonical structure found in the germline gene family to which the master framework belongs , or cdrs of different classes of canonical structure from those found in the germline gene family to which the framework belongs . predictions of the canonical structure adopted by a particular cdr may be determined using an online tool available at url : the master framework need not be a naturally occurring one , but may for example have been optimised , e . g . for the expression or phage system to be used , or to reduce antigenicity in vivo . the cdrs , having been amplified , may be subject to mutagenesis , e . g . using error - prone pcr , before being incorporated into the master framework ( e . g . as described in wo98 / 32845 ), though this is not generally preferred since naturally occurring cdrs are less likely than artificial ones to be antigenic . “ percent (%) nucleic acid sequence identity ” is defined as the percentage of nucleic acid residues in a candidate sequence that are identical with the nucleic acid residues in the sequence with which it is being compared , after aligning the sequences and introducing gaps , if necessary , to achieve the maximum percent sequence identity , and not considering any conservative substitutions as part of the sequence identity . the percent identity values used herein were generated by the blastn module of wu - blast - 2 ( which was obtained from altschul et al . ( 1996 ); url : http :// blast . wustl / edu / blast / readme . html ). wu - blast - 2 uses several search parameters , most of which are set to the default values . the adjustable parameters are set with the following values : overlap span = 1 , overlap fraction = 0 . 125 . a percent nucleic acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “ longer ” sequence in the aligned region , multiplied by 100 . the “ longer ” sequence is the one having the most actual residues in the aligned region ( gaps introduced by wu - blast - 2 to maximize the alignment score are ignored ). the following examples are provided for the better understanding of the invention , and make reference to the accompanying figures , in which : [ 0110 ] fig1 ( parts a to d ) shows the incorporation of a cdrh2 loop from germline gene dp - 29 into a framework of dp - 47 . [ 0111 ] fig2 ( parts a to e ) shows the incorporation of a cdrh2 loop from germline gene dp - 73 into a framework of dp - 47 . primers that are different from corresponding sequences in the dp - 47 framework are used to amplify cdrs from different germline genes . in example 1a , the master framework and the framework of the gene from which the cdr is amplified ( dp - 29 ) are sufficiently similar that the thus - amplified sequence can be incorporated into a dp - 47 framework without further modification . in example 1b , the frameworks are more dissimilar , and the thus - amplified sequence is further modified to make it more similar to the dp - 47 framework before it is incorporated therein . this is achieved by use of primers that successively bring the framework regions that flank the cdrs into conformity with the selected framework in a designed and planned iterative process . in this way , it is possible to pick up cdr - loops that have canonical structures that are atypical of the selected dp - 47 framework . when , as here , it is desired to incorporate a specific cdr into the master framework , it can be advantageous to determine the homology ( i e . percentage identity ) between the selected framework and the framework surrounding the atypical cdr to be incorporated into the selected framework . of course , if one is using primers of a known sequence to “ fish ” for cdrs in a library , it is more important to determine the homology between the primers and the framework sequence . the degree of homology determines the number of pcr amplification steps necessary to obtain the atypical cdr in the selected framework . this means that a lower degree of homology will result in several sequential pcr steps to convert the original fr flanking the atypical cdr into the sequence of the selected fr . [ 0116 ] fig1 shows the sequences and steps involved in the amplification of dp - 29 cdrh2 , and its incorporation into nucleic acid encoding framework of dp - 47 . part a shows nucleic acid sequences encoding portions of the framework regions flanking the dp - 47 and dp - 29 cdrh2 loops and the deduced amino acid sequences . nucleotide matches are denoted by the symbol i . as will be seen , there are some mismatches : 8 of 36 nucleotides and 7 of 27 nucleotides in the two flanking portions shown , respectively . part b shows amplification primers (“# 1 primers ”) identical to the nucleic acid encoding portions of the framework regions flanking the dp - 29 cdrh2 loops , aligned with the double - stranded dp - 29 coding sequence . part c shows the amplification product (“# 1 product ”) of the first pcr step ( which was shown in part b ). conditions for amplification are as for cdr amplification in wo98 / 32845 . the # 1 product is identical to the coding sequence of dp - 29 . aligned with this are primers (“# 2 primers ”) for a second pcr step . these are identical to the nucleic acid encoding corresponding portions of the framework regions flanking the dp - 47 cdrh2 loops . consequently the same mismatches are apparent as in part a . part d shows the product (“# 2 product ”) of the second pcr step . this has the framework regions of dp - 47 ( the master framework ) and the cdrh2 loop of dp - 29 . thus , there is sufficient sequence identity - between the framework regions of dp - 47 and dp - 29 flanking the cdrh2 loop for the loop to be switched from one framework to the other in a single pcr step . the dp - 29 germline gene encodes a cdrh2 of canonical class 4 ( vbase ), whereas the cdrh2 of dp - 47 is of canonical class 3 ( vbase ). the second pcr step could be performed as an overlap extension pcr step , since the primer used is identical to the master framework sequence into which the cdr is intended to be incorporated , for example using the conditions ( and other primers ) set out in wo98 / 32845 . an iterative process of sequential pcr amplifications is used to insert a cdr into a dp - 47 master framework from a germline gene ( dp - 73 ) which has significantly different sequences encoding the portions of the framework regions flanking the cdr . in this example the homology between the dp - 47 v h framework , adjacent to cdrh2 , and the dp - 73 framework is too low to allow for direct amplification ( e . g . in an overlap extension pcr step ) using primers wholly identical to dp - 47 . thus , several individual pcr steps are used , each step using a unique primer pair . the primers are successively modified to become more homologous to the dp - 47 primer . in this process it is important to carefully choose the proper distribution of the base modifications . fig2 shows this process . the underlined sequence is where the greatest differences occur between dp - 47 and dp - 73 . bold letters denote residues in the primers which are identical to those in dp - 47 , the master framework . parts a and b are analogous to the same parts of fig1 . again , there are mismatches between the sequences encoding the portions of framework which flank the dp - 47 and dp - 73 cdrh2 loops , 13 mismatches out of 42 nucleotides and 9 mismatches out of 27 in the two flanking sequences , respectively . in part c , instead of using primers identical to dp - 47 , primers which are chimaeras of dp - 47 and dp - 73 are used , to introduce changes into the framework regions of the amplified dp - 73 fragment , to bring them partly into conformity with those of dp - 47 . so , rather than there being no mismatches between the primers and the dp - 47 sequences ( as in example 1a ), there are still some mismatches , though fewer than before , i . e . 2 in each flanking sequence . part d shows the amplification product (“# 2 product ”) of the second pcr step , aligned with primers (“# 3 primers ”) identical to corresponding portions of the dp - 47 framework as with example 1a , such primers could be used in overlap extension pcr . the third amplification step ( analogously to the second in example 1a ) leads to a fragment incorporating cdrh2 of dp - 73 in a framework of dp - 47 . in particular , in the second pcr step ( which uses the # 2 primers , shown in part c ), the following base substitutions are made in the upper pcr primer relative to the # 1 primer , used in the first pcr step ( shown in part b ): at position 35 ( counted from the 5 ′ end of the primer ) g is changed to c ; at position 37 a is changed to g and at position 38 t is changed to c . this results in a higher degree of homology than if a primer homologous to dp - 47 was to be used directly in the second pcr step . the intermediate pcr product from the second pcr step therefore still contains bases that are homologous to the dp73 sequence ( g36 and c39 ). these bases will then not prime in the third pcr step , since the upper primer used in this step is 100 % homologous to the dp47 sequence . however , bases 34 , 37 and 38 of the amplification product (“# 2 product ”) of the second amplification step are now homologous to the dp - 47 sequence and this homology will give an annealing of 6 of 8 bases at the 3 ′ end ( underlined ) of the upper primer in the third pcr step ( shown in part d ). this is to be compared to 3 out of 8 bases at the 3 ′ end between dp47 and dp73 . such an increase in homology greatly facilitates the successful production of a dna sequence comprising the dp - 73 cdrh2 in the dp - 47 framework . using the principles of this method any cdr can be transferred to any given and selected framework resulting in composite antibody molecules that possess combinations of natural cdr - loops and hence possibly also canonical structures , that can not be found in nature . thus , combination of atypical but natural cdr - loops gives a basis for generation of an enormous variability in the antibody combining site and the created variants may be captured in large libraries using e . g . phage ( marks et al . 1991 ), ribosome ( hanes and pluckthun , 1997 ) or covalent ( wo98 / 37186 ) display technologies . adkins j c , spencer c m ( 1998 ) drugs 56 ( 4 ): 619 - 26 ; 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