US 20150037359A1 (19) United States (12) Patent Application Publication oo) Pub. No.: US 2015/0037359 Al Schellenberger et al. (43) Pub. Date: Feb. 5,2015 (54) XTEN CONJUGATE COMPOSITIONS AND filed on Jun. 18, 2012, provisional application No. METHODS OF MAKING SAME 61/709,942, filed on Oct. 4, 2012. (71) Applicant: AMUNIX OPERATING, INC., Mountain View, CA (US) Publication Classification (72) Inventors: Volker Schellenberger, Palo Alto, CA (US); Vladimir Podust, Castro Valley, (51) Int. Cl. CA (US); Chia-Wei Wang, Milpitas, CA A61K47/48 (2006.01) (US); Bryant McLaughlin, Millbrae, A61K38/24 (2006.01) CA (US); Bee-Cheng Sim, Mountain (52) U.S. Cl. View, CA (US); Sheng Ding, Redwood CPC ........... A61K47/48246 (2013.01); A61K 38/24 City, CA (US); Chen Gu, Fremont, CA (2013.01) (US) USPC ........ 424/178.1; 435/68.1; 435/69.1; 506/10; (73) Assignee: AMUNIX OPERATING, INC., 514/1.1; 514/9.7; 514/11.7; 530/345; 530/350; Mountain View, CA (US) 530/391.7 (21) Appl. No.: 14/381,199 (57) ABSTRACT (22) PCT Filed: Feb. 27, 2013 The present invention relates to extended recombinant (86) PCT No.: PCT/US2013/028116 polypeptide (XTEN) compositions, conjugate compositions § 371 (c)(1), comprising XTEN and XTEN linked to cross-linkers useful (2) Date: Aug. 26, 2014 for conjugation to pharmacologically active payloads, meth­ ods of making highly purified XTEN, methods of making Related U.S. Application Data XTEN-Iinker and XTEN-payload conjugates, and methods (60) Provisional application No. 61/634,312, filed on Feb. of using the XTEN-cross-linker and XTEN-payload compo­ 27, 2012, provisional application No. 61/690,187, sitions. Patent Application Publication Feb. 5, 2015 Sheet I of 119 US 2015/0037359 Al FIG. 1A FIG. 1B :?¾¾¾¾?¾¾¾¾¾¾¾¾¾^¾¾¾¾^¾¾¾¾¾^¾¾¾¾^¾¾¾¾^¾¾¾¾¾^¾¾¾¾^¾¾¾¾¾¾¾¾¾^¾¾¾¾^¾¾¾¾¾^¾¾¾¾^¾¾¾¾^¾¾¾¾¾^¾¾¾¾ Cys*! FIG. 1E FIG. 1C f w w Z H2N1Ii:<c-:cc<c-:cccc<cccc<c-:cc<c-:cccc<c-:cc<c-:cccc-:cccc<c-:cc<c-:cccc<cccc<c-:cc<c-:cccc<c-:cc<c-:cccc-:cccc<c-:c'' X^^^^C-j-j-j'jC-j-j-j-j^-j^C-j-j-j'j^-j-j^CCCCCCCCCCC^CCCCCCCCCCCCCCCC^CCCCCCCCCCCCCCCC^CCCCCCCCCCC H2N*.cccccccccccccc<ccccccccccc<cccccccccccccccc<cccccc<cccc<cccc<c-:cccc<cccc<cccccc-:cccc<cccc<c-:cccc< CM CM CM CM X X X X Z Z Z Z H2NdZj^C-j-j-j^^-jj^-j'jC-j-j-j^^-jjC-j^C-j-jCCCC^CCCCCCCCCCCCCC^CCCCCCCCC^CCCCCCCCCCCCCC^CCCC-: FIG. 1D LI A\.\a!AVV.W .W .VVMAVAV.V.W^^^ i I I2l N X X X X :yw>>'£ ¥ h2n€ FIG. I PatentApplication Publication Feb. 5, 2015 Sheet 2 of 119 US 2015/0037359 Al Amine NHSester Amide NHS compound FIG. 2 PatentApplication Publication Feb. 5, 2015 Sheet 3 of 119 US 2015/0037359 Al Sulfhydryl-containing M aleim ide Thioether bond molecule compound FIG. 3 PatentApplication Publication Feb. 5, 2015 Sheet 4 of 119 US 2015/0037359 Al O O pH >7 5 + 1 * 2 ------------ > + IH x N V N H H Sulfhyd ryl-containing Iodoacetyl Thioether bond molecule compound FIG. 4 Patent Application Publication Feb. 5, 2015 Sheet 5 of 119 US 2015/0037359 Al Sulfhydryl-conta ining Pyridyl disulfide Disulfide bond Pyridine 2-thione molecule compound FIG. 5 Patent Application Publication Feb. 5, 2015 Sheet 6 of 119 US 2015/0037359 Al \ Cf- \ + A. OH I R1 Carbo'yiats containing molecule reactive ester \ / N B. !,R2 -NH2 I H o - Acylisourea x Amine Amide reactive ester FIG. 6 PatentApplication Publication Feb. 5, 2015 Sheet 7 of 119 US 2015/0037359 Al FIG. 7 Patent Application Publication Feb. 5, 2015 Sheet 8 of 119 US 2015/0037359 Al FIG. 8 Patent Application Publication Feb. 5, 2015 Sheet 9 of 119 US 2015/0037359 Al + FIG. 9 PatentApplication Publication Feb. 5, 2015 Sheet 10 of 119 US 2015/0037359 Al IR1 NH2 I r2 H v5nSS.*-"\ / ^SSSS'- \ / + N N H HI H Hydrazine Aldehyde Hydrazone FIG. 10 Patent Application Publication Feb. 5, 2015 Sheet 11 of 119 US 2015/0037359 Al O O 1¾ -NH2 + M3' + H+ I r* IR U V. H Acyl azide Amine Amide FIG. 11 PatentApplication Publication Feb. 5, 2015 Sheet 12 of 119 US 2015/0037359 Al O IR- Z4Proteir*1. S ’ O ,SH IProteto2 Z4Proteio1 I P ro te w 1 t Protein2 * NH2 FIG. 12 PatentApplication Publication Feb. 5, 2015 Sheet 13 of 119 US 2015/0037359 Al I: N-SShift 2:Thiol Mediated Cleavage -SO3-CH2-SH H2N \ Payload XTEN I 3: Peptide Attack 4: N-S Shift ( Payload FIG. 13 PatentApplication Publication Feb. 5, 2015 Sheet 14 of 119 US 2015/0037359 Al FIG. 14 PatentApplication Publication Feb. 5, 2015 Sheet 15 of 119 US 2015/0037359 Al FIG. 15 PatentApplication Publication Feb. 5, 2015 Sheet 16 of 119 US 2015/0037359 Al N -term inus GGGv iProtein1, x s GGGSLPETGXn I: Protein2 C-term inus S ortase V iProtein1.. Gggslpetgggn iProtein2. FIG. 16 Patent Application Publication Feb. 5,2015 Sheet 17 of 119 US 2015/0037359 Al UDLa./ ^¾¾¾¾¾¾¾¾¾¾^¾¾¾¾¾¾¾¾¾^¾¾¾¾¾¾¾¾¾^¾¾¾¾¾¾^^ ::^SS5S5^^SS5S5^^SS5S55^SS5S55^SS5S55^^S5S55^^S5S55^ Alkyne- @ 1 NMflIrimiHp HoN£1 N swnsnsnwnw noUC-J :;^:-^¾¾¾¾^¾¾¾¾¾¾¾¾¾^¾¾¾¾¾¾¾¾¾^¾¾¾¾¾¾¾¾¾^¾¾¾¾¾¾¾¾¾^¾¾¾^ B ^Payload AJ ^Payload AJ ^Payload A j ^Payload aJ ^Payload AJ ^Payload AJ ^Payload Aj ^Payload AJ UdlaJ ^ « DBCOHt ^Payload AJ ^Payload AJ ^Payload Aj ^Payload AJ ; ;^SgS^S^SSSSSS^S^SSSSSS^^^|sSSSS^^^SSSSssss^SSSSSSi^S^^SSSSSSgfe^^ Azidei A z k te ^ ^PayIoadAj ^Payload AJ ^Payload AJ rili\yi IC Alkyne ^PayloadAj ^Payload AJ ^Payload A J ^ Payload AJ MVIaIeimide- NMateiimte- ^Payload aJ ^Payload aJ ^Payload AJ ^Payload AJ _^555^^S555555^^S555555^H^^^^^?5$wS555555$^S555555^w5i r!555555^^^55555^^^55555^^^55555^^^55555^^^55555^^^5S55^ HoNHi- FIG. 17 Patent Application Publication Feb. 5, 2015 Sheet 18 of 119 US 2015/0037359 Al 2B f NMaIeimide SH DBCO NMaIeimide SH ’SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSWSSSSSSSSS Azide' Azide' NMaIeimide SH Akynei Alkyne' NMaIeimide SH NMaIeimide NMaleimide' m am NMaleimide SH H2N1 Azide NH2 >SSSSSSSSSSSSS????????????¥99999?^^^^^SSSSSSSSSSSSSSSSSSSSSSS???W????????« H2N- DBCXD- Azide NH2 ^55555555555555555555555555555555555555555555555555555555555555555555555555555; Azide' Azide NH2 "^5555555555555555555%¾¾¾¾¾¾¾¾¾¾??????????????????????????????¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾ NMaleimide Alkyne—] Akyne i H2N NMaleimide' Akyne Akynei ^¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾^^ H2N ^¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾^^ Akyne ___________ L-...... NMaleimide ^¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾?????¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾^ FIG. 18 PatentApplication Publication Feb. 5, 2015 Sheet 19 of 119 US 2015/0037359 Al A Reactive Groups Conjugation Links IAr-Br FIG. 19 PatentApplication Publication Feb. 5, 2015 Sheet 20 of 119 US 2015/0037359 Al Recombinant production JlL XTEN precursor 1 A XTEN precursor 2 CON /CO coJ [ m NON /N> XTEN precursor 3 XTEN precursor 4 XTEN precursor 5 FIG. 20 PatentApplication Publication Feb. 5, 2015 Sheet 21 of 119 US 2015/0037359 Al FIG. 21 Patent Application Publication Feb. 5, 2015 Sheet 22 of 119 US 2015/0037359 Al A B PrecursorXTEN I Add linker Add Payload 1H--N '21 - ''5 J Add linker Add Payload Add linker :|s :' Add Payload FIG. 22 Patent Application Publication Feb. 5,2015 Sheet 23 of 119 VS 2015/0037359 A Precursor XTEN I Add linker W AddPayioad 5 B precursorXTEN Patent Application Publication Feb. 5, 2015 Sheet 24 of 119 US 2015/0037359 Al f Payload A FIG. 24 PatentApplication Publication Feb. 5, 2015 Sheet 25 of 119 US 2015/0037359 Al Payload A Payload B v J V ^ B Payload B Payload B Payload B Payload A ^Payload B^ ^Payload B^ Vayload B^ Vayload B^ S \ Payload A Payload B Payload B J ^Payload B Payload B J ^Payload B V J FIG. 25 PatentApplication Publication Feb. 5, 2015 Sheet 26 of 119 US 2015/0037359 Al ^Payload fpayload A Payload BJ ^Payload B J Payload A Payload AJ ^Payload B Payload B C \ Payload A Payload A Payload A v J Payload B Payload B |PayloadB Payload A Payload A Payload A Payload B ^Payload B J Payload B B (! ) Payload A Payload A Payload B Payload B Payload B Payload B Payload B Payload B f Payload BI (PayloadB Payload B Payload b ! y v J v J v J v J v J FIG. 26 PatentApplication Publication Feb. 5, 2015 Sheet 27 of 119 US 2015/0037359 Al A H2N' Payload B Payload B H2N' Payload A Payload A Payload B Payload B ^¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾^^ i C/5 B H2N- .¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾¾^ Payload B Payload B 5¾??¾¾ H2N- | | r ' s i i r / \ / N f \ r \ Payload A Payload A Payload B Payload B ^ ____ J L _____ J < _____J < _____ J FIG. 27 Patent Application Publication Feb. 5,2015 Sheet 28 of 119 US 2015/0037359 Al FIG. 28 Patent Application Publication Feb. 5, 2015 Sheet 29 of 119 US 2015/0037359 Al X X X X H2NHi ^Payload A*j ^Payload ^PayIoadAJ Vayload A J ^Payload A^ ^Payload A J Precursor 2 lit. FIG. 29 PatentApplication Publication Feb. 5, 2015 Sheet 30 of 119 US 2015/0037359 Al A ^Payload ^Payload Payload A ; ; ' < i a r -b ^ , m o m 1 - Payload A Payload A Payload A Payload A Payload A Payload A \ M o m x : FIG. 30 PatentApplication Publication Feb. 5, 2015 Sheet 31 of 119 US 2015/0037359 Al ^Payload A^ ^ Payload A ^P ayIoad A^ ^Payload A j ^PayIoadAJ ^PayIoadAJ (Payfoad A ayload A^PayIoad A^PayIoad AJ ^ •■:: ::: :■: ::J :> .3:r.. * 0 t * - r ^Payload A^ ^Payload A J ^Payload A^ ^Payload A~j ^Payload A^ ^Payload A^ J jg s S K S K S isS s^ q o q rW:v: gM\ S » # 9 ^Payload A^ ^Payload AJ ^Payload AJ ^Payload A ^P ayIoad A ^PayIoad A^ * 1 1 = ^ FIG. 31 Patent Application Publication Feb. 5, 2015 Sheet 32 of 119 US 2015/0037359 Al ^Payload A J ^Payload A J ^Payload A J ^Payload A J ^Payload A J ^Payload A^ r • ” ” s':::: 5¾ ___ _ „ 555ff5555$$$S5555555ff5® UttMtoi ^ (g PBOl^Bdj Q Q FIG.