Patent Application: US-81120709-A

Abstract:
the invention relates to methods of making fc - heterodimeric proteins or polypeptides . the invention also relates to the fc - heterodimeric proteins or polypeptides themselves , including the individual polypeptide components that comprise the heterodimer . nucleic acids encoding such polypeptides , expression vectors , and host cells . moreover , the invention relates to pharmaceutical compositions comprising one of more fc - heterodimeric proteins or polypeptides .

Description:
a total of 48 antibody crystal structures which had co - ordinates corresponding to the fc region were identified from the protein data bank ( pdb ) ( bernstein , koetzle et al . 1977 ) using a structure based search algorithm ( ye and godzik 2004 ). examination of the identified fc crystal structures revealed that the structure determined at highest resolution corresponds to the fc fragment of rituximab bound to a minimized version of the b - domain from protein a called z34c ( pdb code : 1 l6x ). the biological fc homodimer structure for 1 l6x was generated using the deposited fc monomer co - ordinates and crystal symmetry . two methods were used to identify the residues involved in the ch3 - ch3 domain interaction : ( i ) contact as determined by distance limit criterion and ( ii ) solvent accessible surface area analysis . according to the contact based method , interface residues are defined as residues whose side chain heavy atoms are positioned closer than a specified limit from the heavy atoms of any residues in the second chain . though 4 . 5 å distance limit is preferred , one could also use longer distance limit ( for example , 5 . 5 å ) in order to identify the interface residues ( bahar and jernigan 1997 ). the second method involves calculating solvent accessible surface area ( asa ) of the ch3 domain residues in the presence and absence of the second chain ( lee and richards 1971 ). the residues that show difference (& gt ; 1 å 2 ) in asa between the two calculations are identified as interface residues . both the methods identified similar set of interface residues . further , they were consistent with the published work ( miller 1990 ). table 1 lists twenty four interface residues identified based on the contact criterion method , using the distance limit of 4 . 5 å . these residues were further examined for structural conservation . for this purpose , 48 fc crystal structures identified from the pdb were superimposed and analyzed by calculating root mean square deviation for the side chain heavy atoms . the residue designations are based on the eu numbering scheme of kabat , which also corresponds to the numbering in the protein data bank ( pdb ). fig3 shows the ch3 domain interface along with the structurally conserved , buried (% asa ≦ 10 ), and exposed (% asa & gt ; 10 ) positions (% asa refers to ratio of observed asa to the standard asa of amino acids ; ( lee and richards 1971 )). conservation of interface residues among human and mouse igg subclasses as well as among other ig classes was also examined through sequence comparisons ( fig4 ). a positions involving interaction between oppositely charged residues are indicated in bold . due to the 2 - fold symmetry present in the ch3 — ch3 domain interaction , each pair - wise interaction is represented twice in the structure ( for example , asp a 356 - - - lys b 439 ′ & amp ; lys a 439 - - - asp b 356 ′; fig5 ) b arg355 and lys360 positions ( shown in italics ) could also be used for enhancing electrostatic steering effects though they are not involved in interaction with oppositely charged residues . at neutral ph (= 7 . 0 ), asp and glu residues are negatively charged and lys , arg and h is are positively charged . these charged residues can be used to promote heterodimer formation and at the same time hinder homodimers . attractive interaction takes place between opposite charges and repulsive interaction occurs between like charges . the method presented here makes use of the attractive and repulsive interactions for promoting heterodimer and hindering homodimer , respectively , by carrying out site directed mutagenesis of charged interface residues . examination of the identified ch3 domain interface residues ( table 1 ) reveals four unique charge residue pairs involved in the domain - domain interaction ( asp356 - - - lys439 ′, glu357 - - - second lys370 ′, lys392 - - - asp399 ′, asp399 - - - lys409 ′; residue numbering in the chain is indicated by prime ′). these charge pairs are not necessarily involved in charge - charge interaction in the crystal structure used here ( 1l6x ), since crystal structure is an end product in the protein folding reaction pathway and it represents structure in the crystalline state . it is assumed here that in order to have electrostatic steering effects it is sufficient if the residues are close in space as defined by the distance limit criterion ( 4 . 5 å ). it must also be noted here that due to the 2 - fold symmetry present in the ch3 - ch3 domain interaction , each unique interaction will be represented twice in the structure ( for example , asp399 - - - lys409 ′ & amp ; lys409 - - - asp399 ′; fig5 ). the four pairs were ranked according to the extent of solvent accessibility ( asa analysis ) ( lee and richards 1971 ). in lys409 - - - asp399 ′ case , both the residues were structurally conserved as well as buried . in other three pairs case , at least one of the partner is solvent exposed (% asa & gt ; 10 ). therefore , for the example herein , the lys409 - - - asp399 ′ pair was chosen for site directed mutagenesis . the strategy is schematically shown in fig6 . in the wild type , k409 - - - d399 ′ interaction favors both heterodimer and homodimer formation . a single mutation switching the charge polarity ( k409e ; positive to negative charge ) in the first chain leads to unfavorable interactions for the formation of the first chain homodimer . the unfavorable interactions arise due to the repulsive interactions occurring between the same charges ( negative - - - negative ; d399 - - - k409e & amp ; k409e - - - d399 ). a similar mutation switching the charge polarity ( d399 ′ k ; negative to positive charge ) in the second chain leads to unfavorable interactions ( k409 ′ - - - d399 ′ k & amp ; d399 ′ k - - - k409 ′) for the second chain homodimer formation . but , at the same time , these two mutations ( k409e & amp ; d399 ′ k ) lead to favorable interactions ( k409e - - - d399 ′ k & amp ; d399 - - - k409 ′) for the heterodimer formation . the electrostatic steering effects on heterodimer formation and homodimer discouragement can be further enhanced by mutation of additional charge residues which may or may not be paired with an oppositely charged residue in the second chain , such as arg355 and lys360 , as shown in fig6 d . the mutations shown in fig6 are for the purpose of illustration only . table 2 lists many possible mutations involving charge change , and the mutations can be combined to enhance the electrostatic effects . also be used . for example lys409 - - - asp399 ′ interaction pair mutations b histidine ( his ) could also be added to this list of positively charged a these single residue mutations could be combined with the table 2a each positively charged residue ( lys and arg ) can be mutated to two negatively charged residues ( asp or glu ) and vice versa , and as a result the method described here provides numerous combinations . it must be stated here that different combinations will have diverse effect on the quaternary ( homodimer / heterodimer ) structure formation depending on surrounding residues at the mutation site and role of water molecules . the amino acid histidine ( h is ) is positively charged at neutral ph and therefore mutation to h is also contemplated . however , mutating negatively charged residues ( asp or glu ) to h is will lead to increase in side chain volume which may cause steric issues . further , histidine proton donor - and acceptor - form depends on the localized environment . these issues should be taken into consideration during the design strategy . because the interface residues are highly conserved in human and mouse igg subclasses , electrostatic steering effects can be applied to human or mouse igg1 , igg2 , igg3 , or igg4 . this strategy can also be extended to modifying uncharged residues to charged residues at the ch3 domain interface . a similar strategy involving charge residue mutations can also be used to enhance homodimers and hinder heterodimer formation when two different heavy chains are co - expressed ( fig7 ). in order to assess the stability of the charge residue mutants , egad software was used to estimate the ch3 - ch3 domain binding free energy . by optimizing parameters used in the calculation , pokala and handel could predict the effects of nearly 400 mutations on protein - protein complex formation within 1 . 0 kcal / mol error ( pokala and handel 2005 ). egad was used to roughly compare the binding free energy of various mutations made at the ch3 domain interface . table 3 lists computed binding free energy ( δδg ) for the interface charge residue mutants . the binding free energy of a mutant is defined as δδg mut = μ ( δg mut − δg wt ). where , μ (= 0 . 1 , in general ) is the scaling factor used to normalize the predicted changes in binding affinity to have a slope of 1 when comparing with the experimental energies ( pokala and handel 2005 ). the free energy of dissociation ( δg ) is defined as the energy difference between the complex ( δg bound ) and free states ( δg free ). the comparison shows that charged residue mutations affect the stability to a much lesser extent compared to the knobs - into - holes mutations . for comparison , melting temperatures reported for the wild type and knobs - into - holes mutants are given . the melting temperatures were measured by carter and coworkers using only the ch3 domain construct ( atwell , ridgway et al . 1997 ). for the knobs - into - holes mutants , decrease in enthalpy was also observed in the differential scanning calorimetry experiments . a not all possible charge - charge pairs were considered for the binding free energy calculation . wild type is listed for comparison . δg is defined as energy difference between the complex and free states . the binding free energy of a mutant ( δδg mut ) is defined as difference between the mutant ( δg mut ) and wild type ( δg wt ) free energies . fig2 depicts several embodiments comprising fc heterodimeric molecules , from bispecific antibodies to heterodimeric receptor complexes . the two heavy chains of heterodimeric fc molecules can be fused with proteins and / or domains that have different functionalities . for example , fusing fabs that bind to different antigens will lead to bispecifc antibodies ( bsabs ). fusing two different single - chain fv ( scfv ; variable light and heavy chains joined by a flexible peptide linker ) domains will lead to bispecific maxibodies . further , domains or proteins that interact for functional reasons can also be fused with heterodimeric fc for the purpose of developing functional assays or for therapeutic uses . for instance , in the hematopoietic receptor family gp130 is known to interact with other receptors such as leukemia inhibitory factor receptor ( lifr ). the extra cellular domain ( ecd ) of gp130 can be fused to the first heavy chain of fc and the ecd of lifr can be fused to the second fc heavy chain , which will lead to formation of gp130 - lifr complex that is likely to mimic the biological state . since fcrn binding site is located in the fc region , fc fusion molecules are likely to have extended serum half - life — a feature that distinguishes fc heterodimeric molecules from other heterodimeric molecules such as leucine zipper fusion proteins ( liu , caderas et al . 2001 ). it is not essential to have different functionalities attached to the two heavy chains of the fc heterodimer . a monobody can also be created ( fig2 ). in certain embodiments , e . g ., when producing bispecific antibodies , multiple different light chains may be co - expressed with the multiple different heavy chains . to increase the fidelity of each light chain binding to the proper heavy chain thereby maintaining specificity of the antibody “ arm ,” the ch1 domains of one or more of the heavy chains and the constant region of one or more of the light chains can be engineered to favor dimerization . preferably , this is accomplished using an electrostatic steering technique similar to that described above for the ch3 domains the interaction of the kappa light chain sequence corresponding to the protein data bank ( pdb ) deposition code 1n0x ( seq id no : 25 ) and the lambda light chain corresponding to ( pdb ) deposition code 7fab ( seq id no : 26 ) with the heavy chain sequence corresponding to the ch1 domain of igg1 ( seq id no : 27 ) was analyzed . the lambda light chain - heavy chain contacts within the interface are shown in table 4 . a contacting residues were identified using 4 . 5 å distance limit criterion . the light and heavy chain numbering scheme corresponds to that in the deposited co - ordinates file ( pdb code : 7fab ). the kappa light chain - heavy chain contacts within the interface are shown in table 5 . a contacting residues were identified using 4 . 5 å distance limit criterion . the light chain numbering scheme corresponds to that in the deposited co - ordinates file ( pdb code : 1n0x ). the heavy chain numbering scheme corresponds to that in the table 4 . in certain embodiments , lys 125 of the lambda chain is mutated to a negatively charged amino acid and a corresponding mutation is made in a heavy chain at asp148 , changing the residue to a positively charged amino acid . alternatively , or in addition , glu119 of the lambda chain is mutated to a positively charged amino acid a corresponding mutation is made in a heavy chain at lys213 , changing the residue to a negatively charged amino acid . the analysis of the light chain - heavy chain interaction revealed positions in which charge pairs could be introduced into the sequence to enhance binding of a specific light and heavy chain pair . these positions include thr112 of lambda and ala141 of the heavy chain , glu 156 of lambda and ser176 of the heavy chain , and ser171 of lambda and ser183 of the heavy chain and other positions shown in table 4 and 5 in bold face . this example demonstrates that ch3 domains can be engineered to favor heterodimerization while disfavoring homodimerization using electrostatic steering effects . a maxibody — dummy fc construct as shown in fig8 ( a ) was made having charge residue mutations at the ch3 domain interface . the formation of homodimer and heterodimer yield was assessed through sds polyacrylamide gel electrophoresis . because the maxibody has a higher molecular weight compared to dummy fc , the heterodimer ( maxibody - dummy fc ) and homodimers ( maxibody - maxibody & amp ; dummy fc - dummy fc ) have different mobility on the sds - page facilitating the identification of the various pairings ( fig8 ( b )). a rat anti - mouse nkg2d antibody , designated m315 , was generated through conventional hybridoma fusions and the dna sequences encoding the variable heavy chain ( vh ) and variable light chain ( vl ) were used to construct m315 scfv - fc using previously described method ( gilliland , norris , et al . 1996 ). the sequence of m315 scfv - fc ( seq id no : 1 ) and huigg1 fc ( seq id no : 2 ) were cloned into the ptt5 mammalian expression vector and the two constructs were used to co - transfect 293 - 6e cells to assess the formation fc / scfv - fc heterodimer relative to fc homodimer and scfv - fc homodimer . the charge residue pairs in the ch3 region identified through computational analysis were changed to amino acid of opposite charge polarity on either human igg1fc ( dummy ) or m315 scfv - fc ( mxb ) constructs . the mutations , which are listed in table 6 , were generated using the quikchange ® mutagenesis kit from stratagene and verified by dna sequencing . the mutations are denoted by wild type residue followed by the position using the kabat numbering system ( kabat et al ., sequences of proteins of immunological interest , national institutes of health , bethesda , md ., ed , 5 , [ 1991 ]), which is consistent with the crystal structure ( pdb code : 1l6x ) numbering scheme , and then the replacement residue in single letter code . the fc sequence used in these two constructs was derived from human igg1 non -( a ) allotype , which has a glu at position 356 and a met at position 358 . the ch3 sequences from the crystal structure are from a different igg1 allotype , which has an asp at position 356 and a leu at position 368 . dna was transfected into human embryonic kidney cell line 293 - 6e using lipofectamine ™ 2000 reagent ( invitrogen ). the cell culture supernatant was harvested 3 - 4 days after transfection and analyzed on sds - page gels under non - reduced condition . the gel was then transferred to nitrocellulose membrane and subject to western analysis using peroxidase - conjugated goat anti - human igg antibody ( jackson immunoresearch laboratories ) and results are shown in fig1 . co - transfection of expression vector for m315 scfv - fc ( mxb ) together with dummy fc resulted in the formation of scfv - fc / fc heterodimer as well as scfv - fc homodimer and fc homodimer . the ratio of scfv - fc / fc heterodimer to scfv - fc homodimer and fc homodimer is close to 1 : 1 : 1 when the wild type ch3 sequence is used . the introduction of one charge pair mutation k409d on dummy fc and d399 ′ k on m315 maxibody significantly increased the ratio of scfv - fc / fc heterodimer relative to scfv - fc homodimer as well as fc homodimer . similar enhancement of heterodimer formation was also observed for other mutant variants such as k409d / d399 ′ r , k409e / d399 ′ k and k409e / d399 ′ r ( fig9 ), further underscore the importance of charge polarity complementation for the formation of fc heterodimers . ( the wild type m315 scfv - fc construct used in this study has an extra tag at the carboxyl terminal of fc , so it migrates slower on the sds - page gel .) when additional mutations were introduced at charge residues that are located near k409 such as k360 and k392 , a further increase of heterodimer formation was observed ( fig1 ). for example , the combination k409d ; k392d on dummy fc with d399 ′ k on m315 maxibody showed increased ratio of heterodimer to homodimers , likely due to the disruption of fc homodimer . a 25 kd band correspond to the size of fc monomer was detected on all transfections using k409d ; k392d dummy fc ( data not shown ). adding another mutation such as d356 ′ k or d357 ′ k on top of d399 ′ k variant of m315 maxibody showed additional improvement . the combination of k409d ; k392d on dummy fc with d399 ′ k ; d356 ′ k on m315 maxibody resulted almost exclusive formation of heterodimer . other combinations such as k409d ; k392d / d399 ′ k ; d357 ′ k and k409d ; k370d / d399 ′ k ; d357 ′ k also offered significant improvement over the k409d / d399 ′ k variant . this example demonstrates that ch3 domains containing certain triple charge - pair mutations were unable to form homodimers when expressed alone but were capable of forming heterodimers when co - expressed . mutants were made and cells transfected as described in example 1 . when the constructs were co - transfected , a 1 : 1 ratio of plasmids were used . the results are shown in fig1 . heterodimer and homodimers were detected by western blot using goat - anti - human fc hrp conjugated antibody . interestingly , fc - containing molecules having triple mutations wherein positive - charged residues were changed to negative - charged residues ( k409d , k392d , k370d or k409d , k392d , k439d ) were unable to be detected when expressed alone . similarly , fc - containing molecules having triple mutations wherein negative - charged residues were changed to positive - charged residues ( d399k , e356k , e357k ) were unable to be detected when expressed alone . when co - expressed with an fc - containing molecule having mutations of opposite charge polarity , however , heterodimers only were detected . throughout this invention application , it is to be understood that use of a term in the singular may imply , where appropriate , use of respective term in the plural , and vice versa . atwell , s ., j . b . ridgway , et al . 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