Page 108 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 6
5.6. Conclusions 5.6. Conclusions 6.4. Conclusion and Outlook
WeThaevme detehvoedloopfeidnsvtarliloinugsanWtoyvereohlsmainvetrhdeoesdvidesultoeopveaidnatavibnaoridnoytue-sprnrnoaotlvepeienlpmctoiedntehjuotgadagsteitnosabaasnostelivbdeonodtny--espxtrepopot-esweindisceaorneajugates based o
SPOisCQcleaanrdlytevitarbazleinienlvigaartioiouns(SwfPoaOryCmsQ.oFnaironsfdtuotnefcttarilaol,zniwnaleizlafiogtiauotniodontfh(afaontrtaimbnoednxyop)fousrnecbdtyitofynurarotlshizienareticoionmtohbfeiananantitboibonoddy)yor by further c
CDR is labeled with high efficiency and homogeneity based on the standard SPOCQ conditions.
with sortase-based protweiitnh csonrjtuagsea-tbioansed (fopr rotediinfunctcionjaulgizatiion). (foSrpecifdicifaullnyc, tionalization).
Unfortunately, the method has limited applicability for the generation of functional antibody monofunctionalization was acmhioenvoefdunbcatsieodnaolnizatitownow-satasgaechaipepvreodacbhasetdarotingawtwitoh-isntatrgoedaupcptiroonach starting with i
conjugates due to the disruption of antigen binding. As an alternative, we explored the insertion
of a TCO moiety based on chemofoaseTlCeOctmiveoiSePtyObCaQsewditohnacBhCeNm-oPsEeGle3-cTtCivOelSinPkOeCrQ, awfteitrhwahBiCchNI-EPDEGDA3-TCO linker, after
of specific tyrosine-containing peptide loops in the antibody CH2 fragment. Indeed, SPOCQ was
can be employed for attachmecannt obfeaeMmepTlozy-beedafroinr gatptraocthemine,notliogfoanuMcleTozt-ibdea, roinr gsmpraolltmeino,leocluigloen. ucleotide, or sma
detected on both Tras-Y1 and Tras-Y2 antibodies, but the instability of both the Tras-Y1 and Tras-
To introduce these moieties Tfoastinetrroadnudcme othreseffmicoieinetiley,s iftawstaesr faonudndmothreatetfhfiecielenntglyt,hitofwtahsefound that the le
Y2 antibodies and the low yield during the conjugation clearly requires further optimization.
peptide spacer between the apnetipbtoidey sapnadcethr ebeGt4wY-eteang tphlaeyasnaticbroudcyiaal nrodlethine tGh4eY-etfafgicpielanycsy aofcrtuhceial role in the effici
reacFtiriostn,,thineploacratitciounlarofwthitehtryergoeasaricndteiotion,soeixnritdpcaatrinotibnceuolaflrtehwreeitdthe.rGmergüianraedlwtyaorldosxeiintd.eatlri.eorsneidpouofertehbdeyrtmesTrimydrui,newasliAth1yr2o1s,ine residue by
longSe1r22spancderTs1(2G34SalalnsdhoGw4SinGg4lSoh)nigsghiegrnDisAfpiRcaascnefotrlrsyb(sGop4teSheadSn6ind-gtaGug4psSGathn4Sed)eYsfibgfibncRief-itncacgnysto,lyaf stshpweerledl aincsgtgiouenpn.etrhaelleyfbfiectietenrcy of the reacti stability than conjugates formed in the CH2 region.19 The trade-off for higher stability might be
Thislatcwko-fsotexpidacotinojnugbaytmionTymr,Teahtshistohdtewwsot-asbtelsepu,blcesosensqjeuxegpnaotilsoyendfumtrateghtsehrmodeigxhwtetansdoestudbetsoeaqctuchesnsgtilbeylnefeufroratrthitoehnreeolaxftregneded to the ge trifuencztyimoneal(1a3n0tibkoDdai)e.sIfwtihthishtbrieigfchuonemcftfeiocsineanlcaiysn.stIuinbeo,thduiseinscagwsmeit,hoanhfoiigrmhsteerpfircfiocmtieuinschyisr.oiInontmtrhotidsyurcoaesiden,abasaesfei(rd4st3opnkrDoate)in is introduce
might be a viable alternative.24 Second, optimization of the installed tyrosine-bearing peptide
sortase ligation followed by sIoEDrtDasAe, alingadtiaonsefcoollnodwepdrobteyinIEvDiDa AS,PaOnCdQafoslelocowned pbryotIeEiDnDvAia. ISnPOCQ followed b
can lead to better conjugation yields. The Y1 and Y2-tags were based on the S6 tag,20 which was
addition, we have shown thadtdtihtieonv,awleenchyaovef tsheowrensutlhtaintgthtreifuvnacletinocnyalofantthiebordeiseusltcinagn tbreifunctional antibo
optimized for PPTases. As seen on the AT1413 antibodies, the tyrosine simply needs to be
carefully tailored towards eithcearedfualllydtiavialolernedt ftuonwcatirodnsaeliiztahteior nduoanl bdoivtahlelingthftuanncdtiohnealvizyacthioaninosn, both light and h
exposed for oxidation by mTyr. Therefore, the loop could be shortened to minimize protein
or dual monovalent functionaolirzadtuioanl mononoorvtawleonstrfucntcutrioanllyaldiziastinocntohneaovrytwchoasintrsu.cOtuthraelrlyfodrimstaintcst heavy chains. Ot
structure perturbation. Inserting the exact same cysteine loop as present in AT1413 at the
or protein-to-antibody ratios hoarvperobtecino-mtoe-annotiboedeynrinatvieostihgavtedbehceorem,ebuntoatrbeeoebnviinovuessltyigaalstoedathere, but are obvi
positions identified by Grünewald et. al. might also lead to more stable and higher yielding hand. hand.
conjugates. Finally, it might even be possible to modify a single exposed amino acid residue to a
tyrosine, to this end single-point mutation of the amino acids where S6-tag insertion led to high
While functional tests with theWbhi-ilaenfduntrcitfiuoncatliotensatlsawntitibhothdeiebsip-raensdentrtiefudnhcetrioeninalaarentoibnogdoiensgp(breustented herein are o
DARs to a tyrosine residue might be sufficient, or alternatively careful analysis of the antibody
not ready for presenting), weneont vriesaiodny tfhoer pgreensernatiinogn),owf etriefunnvicstiionathl eangteibnoedraietsioin oafmtroifduunlcatrional antibodies in
crystal structures may reveal optimal naturally exposed sites for mutation to tyrosine and
fashion will allow for rapid prfoadsuhciotinonwiollfablilsopwecfoifricrAapDiCdsporrodimucmtiuonoocfytboiskpineecsif,icasAwDeClsl oasr imamnyunocytokines, as w oxidation by mushroom tyrosinase. To perform all this however, rather than expressing
other interesting multifunctionthaleranintitbeoredsyticnognmstruultcitfsu.nTcthiiosnwalillanetnibaboldeytchoenrsatrpuidcts.crTeheinsinwgilloefnable the rapid s antibodies in high quantities, a large quantity of small-scale expressions to optimize conditions
varying bispecifics based on e.vga.raynintigbenisptaercgifeicts,blianskedr otynpe.sga. nandtliegnegntthasr,gaentds,dlinffkeerretnytppeasyalonaddlsengths, and differe might be preferable. A possible option to facilitate this issue is using an automated Protein
to improve cell targeting and tsoubimsepqruoevnetcterlel atatmrgentitn. g and subsequent treatment. Expression and Purification Platform (PEPP).25
5.76. S.5u.pSpuoprptoinrgtiinngfoinrfmoramtioa5nt.i7o.nSupporting information
TheTShuepSpuoprptionrgtiIngfoInrmfoartmioantiiosTnahviesaSailuvaapbipleaobfrrlteinefgroeIfnecfohframcrhgaeatriaogtentaihsteatevhnaeidleaonbfdletohffirsethmeisoanfmucahsncarurigspectr.aipttt.he end of this manuscript.
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