US 200701.91272A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0191272 A1 Stemmer et al. (43) Pub. Date: Aug. 16, 2007 (54) PROTEINACEOUS PHARMACEUTICALS Publication Classification AND USES THEREOF (76) Inventors: Willem P.C. Stemmer, Los Gatos, CA (51) Int. Cl. (US); Volker Schellenberger, Palo A6II 38/16 (2006.01) Alto, CA (US); Martin Bader, C40B 40/08 (2006.01) Mountain View, CA (US); Michael C40B 40/10 (2006.01) Scholle, Mountain View, CA (US) C07K I4/47 (2006.01) (52) U.S. Cl. ................. 514/12: 435/7.1: 435/6; 530/324 Correspondence Address: WILSON SONSN GOODRCH & ROSAT 650 PAGE MILL ROAD (57) ABSTRACT PALO ALTO, CA 94304-1050 (US) (21) Appl. No.: 11/528,927 The present invention provides cysteine-containing scaf folds and/or proteins, expression vectors, host cell and (22) Filed: Sep. 27, 2006 display systems harboring and/or expressing such cysteine containing products. The present invention also provides Related U.S. Application Data methods of designing libraries of Such products, methods of (60) Provisional application No. 60/721,270, filed on Sep. screening Such libraries to yield entities exhibiting binding 27, 2005. Provisional application No. 60/721,188, specificities towards a target molecule. Further provided by filed on Sep. 27, 2005. Provisional application No. the invention are pharmaceutical compositions comprising 60/743,622, filed on Mar. 21, 2006. the cysteine-containing products of the present invention. Patent Application Publication Aug. 16, 2007 Sheet 1 of 46 US 2007/0191272 A1 Takara togra: Patent Application Publication Aug. 16, 2007 Sheet 2 of 46 US 2007/0191272 A1 FIG. 7 was 8Aas Furse actics in proteirss is awarage proteir Awsages lossei Patent Application Publication Aug. 16, 2007 Sheet 3 of 46 US 2007/0191272 A1 F.G. 14 P P LECHNOQSSQPPTTKTCPGETNCYKKVWRDHRGTIERGCGCPTVKPG IKLNCCTTDKCN 3 15 25 T5 36 35 45 45 55 55 6 N FIG. 18 LicYNCPNPTADCKTAVNCSSDFDACLITKAGLovnkcwKFEHCNFNDvTTRLRENELT. = . 1. 15 2b. 25 3. 4. s B top YYCCKKDLCN FNEQLEN 6 I 7 Patent Application Publication Aug. 16, 2007 Sheet 5 of 46 US 2007/0191272 A1 FIG. 24 L.was essIS FIG. 26 aessA Ex e--- A. sists is S. assistasiaataasaia8 was w ws seas a Patent Application Publication Aug. 16, 2007 Sheet 6 of 46 US 2007/0191272 A1 FG. 30 f |AH T.HH Patent Application Publication Aug. 16, 2007 Sheet 7 of 46 US 2007/0191272 A1 FIG. 36 Cysteine i. 2. & S 6 Bgk vgRDWFKETAgrRAKSL TSQKYRAN-SA Shk RSIDTIFKSRS----T SMKYRISFg : stecrisp 182frRENKFTSNTMVQQSSEQD-NYMKTN-rais-FioNKii & s 22 PST PKRNNDFSKSLAKKSKQT-EWIKKK-g SEN I pseudecin PNYNNDFSS SLAKKSKQT-EWIKKK-stra -FSHN HTX PixONDVYNgPDLKKQVGGGH-PIMK-D-S-STS-KSLTEK Eara sess & is a 's so is is a a 4 a 4: a is 8 s Patent Application Publication Aug. 16, 2007 Sheet 8 of 46 US 2007/0191272 A1 FIG. 41 n AS YKs CRyaw- seekyll'sww. T Eastess-------------------------------------------------------------r SS5's SSA ESS as 1-5 2-436 till s Yellvil tWe a WBALLIEUlstAAAAA Patent Application Publication Aug. 16, 2007 Sheet 9 of 46 US 2007/0191272 A1 rakhtawirraat A FAA 'reaweerses rwpackaw Patent Application Publication Aug. 16, 2007 Sheet 10 of 46 US 2007/0191272 A1 's JLC : SufusAAF Aki| Patent Application Publication Aug. 16, 2007 Sheet 11 of 46 US 2007/0191272 A1 Patent Application Publication Aug. 16, 2007 Sheet 12 of 46 US 2007/0191272 A1 FIG. 67 s d AR al Patent Application Publication Aug. 16, 2007 Sheet 13 of 46 US 2007/0191272 A1 ALAEGQS CGVYTERCAQGLRCL PRODEEKPLHALLHGRGVCLNEKS 13 2O 25 3O 3S 4O 45 SO S5. Active site:- V BS Patent Application Publication Aug. 16, 2007 Sheet 14 of 46 US 2007/0191272 A1 FIG. 81 FIG. 83 FIG. 84 AscNGvcs PFEMPP cors ACRCI Pvg Lv I GYCRNP sc 3. lb. 5 2b 25 35 FG. 85 Patent Application Publication Aug. 16, 2007 Sheet 15 of 46 US 2007/0191272 A1 aws. Yearsaw FG. 94 A w a - - - - - - - - - - - - - - - a a is a error Vs Arka ce-Yee is escalarate criss E away pms WEochy s y s ss s dis Patent Application Publication Aug. 16, 2007 Sheet 16 of 46 US 2007/0191272 A1 FIG. 95 saww.saw-sursres Eks. TrAccor cpcrks ppg.cgcroy Rekcts AckscLc Rs procRico or cyps 2 3. 4. s al s FIG. 99 - P LRoc LP copogkorcFGP s cccbecace vocale Aircea is NYL ps Pcces. Geep cosgesR 25. - - - - - 3. a. s s 37 75 s s Patent Application Publication Aug. 16, 2007 Sheet 20 of 46 US 2007/0191272 A1 36 kektCanast tunnthdlicts yistclvksg ggCdnrtCan aptntinda CeayftgnnC 421 itksgggovtnttcaaitle aacvknSSgstcfwdtaSSSckdktcvnapatnthdlcq 481 pfintctwins tsagcvektcensivlaicid kdtsSraciw kgkcykkocylassattha 54. FIG. 113 .2a. leaglessells Elsiegels FIG. 114 elesslyas ' aasassesses O ars s y ps s sease . s FIG. 116 MGRSNSLRLL FLFCLLLALS TAASRRLKROCGCSNFCNCO COPVFFAOS 50 LPACSCQQAPICQPQCPRAE INSDCSATCv RACIPSCSKSTGNTFACSTT 100 CESTCDKTCA SAAQQAMSHIQVSPPNPQPLVPIAAAPTVD DSCQNVCONV 150 CQGACVSQNSPPAVCQQTCR QSCQFGCATN EQLPTTSSTS TNAPTIKITL 200 NINDAYFDSN CAPKCTOSCHSOCISOGNPAASCSNSCNTE CSDKCSTRPV 250 QAovoaiaPQ QVQITIPQTC QSRCENRCLSTCTASQPSVCAPSCSTACQL 300 SCDSGAATAPQSTNPTPVE WRALCTPTCMPQCLPSCTATTTTTTTOTPY 350 Patent Application Publication Aug. 16, 2007 Sheet 21 of 46 US 2007/0191272 A1 FIG. 117 N-cap Library I Library II F.G. 118 F.G. 119 1H Second cross-over First cross-over Patent Application Publication Aug. 16, 2007 Sheet 22 of 46 US 2007/0191272 A1 FIG. 120 10 2O 3O 40 50 library VESCEQYTSCGECLGSGDPH-CGWCVLENRCTRRSDCQRAEEPNRWASSISQCVKL 1ss GPGCRHFLTCSMCLRAPRFMGCGWC-GGVCSRQHEC-DGGWWQDS-CPPP. 10 20 30 40 FIG. 121 FIG. 122 | || is a s ass series Patent Application Publication Aug. 16, 2007 Sheet 23 of 46 US 2007/0191272 A1 FIG. 124 Cysteine-Rich Repeat Proteins Natural repeat Protein Repeat domain Repeat domain Non-Repeat domain Repeat contain N-termis linker . N-tettina Cap module -optional, depending Repeat on bonding pattem often 1-2 fewer Cys Modeo unit (optional)E, since it does not link to previous module. Spiral AA backbone library 1 Library 2 library 3 Binding Binding Binding Site 1 Site 2 Site 3 Repeat Protein Affinity Maturation: Different Walking Processes Radotte Select, Select, AdditionModule "S" "S" Repeat Protein --- . Select, Select, Module "S" i Randgnize next Replacement Randotzed Selected Randomized Unselected Selected Randonized Unselected Modules Modules Modules Modules Modules Modules Modules FIG 125 Granulin Repeat Proteins Repeat unit: - - - 3C6C5CC8CC6CCSCC5C6C2 CCXCCX or N- N-1 V-1 CX5CCXC on 1-3 2-5 4-7 6-9 8-11 1 0-12 XCCXCC, where Highest disulfide density in database X = 5,6,7,8 Repeat unit F 4. Cys Nat Struct Biol. 1996 Sep;3(9):747-52. The hairpin stack fold, a novel protein architecture for a new family of protein growth factors granuins have shot antibaralled beta-sheets that may impose sourie degree of sequence conservation Hrabal FR, Chen Z, Jarnes S. Bennett HP, Nif. and prevent full randomization as it may be necessary to use amino acids that are capable of beta-aheet formation. Natural granuins have some length variation in the loops which may of ?tay not be irthportant for folding. One solution is to define a short corsensus replaat motifie (CCs) arwd another solution is to define a long repeat consensus motif that retains the naturallength variation (ie coscC8ccescC5cc5). Patent Application Publication Aug. 16, 2007 Sheet 24 of 46 US 2007/0191272 A1 FIG, 126. Re peat Proteins Approach 1 Rarizatio: -DegreeSelectingth dependent on (get it ofstructural positions variation) App roach 2 Sirrultaneous Stepwise New Synthetic lib alonization Cycle 1 diverse Sequences Cycle 3 Randomization requires carefit balancing of the length AA arc degree AAposition of randomization. The actual numbers depend on how sensitive the structure is to perturtation. synthetic mutagenesis is preferred because it is well-controlled. However, Cycle 4 its difficus to synthetically re-mutagenize an area that was synthetically mutagenized before, the reason is that one needs to re-mutagenize a whole pool of cores, each having a different sequence, which requires sequencing all of the dones and dividing the pool into families of related cores. Resynthesis of a family allows retention of a family-specific sequence motif, but any detailbeyond the nati is destroyed. Thus, approaches that re-mutagenizes sequences blindly, without requiring sequence information, are much preferred. Cwcle 5 NA shufflic. error-prone PCR and Tutator strains art such big approachers. y the best corportise is to synthetically mutageize each past of a sequerce only once, Multiple cycles of Litagenesis are generatyroxuired to accumulate sufficiant binding activity, but each cycle targets a different part of the protein aequence. it is possible to use recombination to permutate previously selected sequences. Other approaches: D However, binding proteirs derived fram random sequence tend to bind to different epitopes, belong to different sequence families and differ in their register, F.Cistricts avaney of approaches. one can randomize only one face of a protein instead of the whole surface. ots can randomize alterating blocks of 2,368,739 or 1 artino acids, eiths successively or simultaneously. Antifreeze Protein-derived Repeat Protein FIG. 127 Natural repeat proteins are imperfect repeats but comparison of the repeats can be used to define a consensus sequence that can be repeated as rany tungna sequence: times as desired. he diagrams below show how two different solutions can - - be derived from the same original sequence. Both likely work but one may have - - - advantages. g QCTGGADCTSCTGACTGCGNCPNAV.......... at Unit S - Patent Application Publication Aug. 16, 2007 Sheet 25 of 46 US 2007/0191272 A1 | FIG. 128 Simple Designs for Spiral Repeat Protein Scaffolds Disulfide Bond. Examples of +3: Types Öxcxcke - Antifreeze Protein -3 XCXCXCX - Granulin On Oen More different disulfidelinkages result in less efficient folding. The simplest linkages Are preferred, such as the +3 patten shown. -3-4-4 to Affinity Maturation of Repeat Proteins Randomization: -Sequence t length get structural variation) -degree dependent on is of positions -Examples below do not use recombination Approach 3 Approach 4 Approach 5 Approach 6 Cycle 1 E.