I 1111111111111111 1111111111 1111111111 1111111111 11111 11111 lll111111111111111 US010188716B2 c12) United States Patent (IO) Patent No.: US 10,188,716 B2 Bann et al. (45) Date of Patent: Jan.29,2019 (54) PROTECTIVE ANTIGEN COMPLEXES Williams et al (Protein Science. 2009. 18: 2277-2286).* WITH INCREASED STABILITY AND USES Chadegani, Fatemeh, et al., F Nuclear Magnetic Resonance and THEREOF Crystallographic Studies of 5-Fluorotryptophan-Labeled Anthrax Protective Antigen and Effects of the Receptor on Stability, Copyright (71) Applicants:Wichita State University, Wichita, KS 2014 American Chemical Society, Open Access on Jan. 3, 2015, (US); Case Western Reserve Biochemistry 2014, 53, 690-701 (12 pages). University, Cleveland, OH (US) Wimalasena, D. Shyamili et al, Evidence That Histidine Protonation of Receptor-Bound Anthrax Protective Antigen is a Trigger for Pore (72) Inventors: James G. Bann, Wichita, KS (US); Formation, Biochemistry, 2010, 49 (33), pp. 6973-6983, DOI: Masaru Miyagi, Cleveland, OH (US) 10.1021/bil00647z, Publication Date (Web): Jul. 22, 2010, Copyright © 2010 American Chemical Society (11 pages). (73) Assignees: Wichita State University, Wichita, KS Rajapaksha, Maheshinie et al., pH effects on binding between the (US); Case Western Reserve anthrax protective antigen and the host cellular receptor CMG2, University, Cleveland, OH (US) Protein Science: A Publication ofthe Protein Society.2012;21( 10): 1467- 1480. doi: 10.1002/pro.2136 (14 pages). ( *) Notice: Subject to any disclaimer, the term ofthis Williams, Alexander S et al., Domain 4 of the anthrax protective patent is extended or adjusted under 35 antigen maintains structure and binding to the host receptor CMG2 U.S.C. 154(b) by O days. at low pH, Protein Science 2009, 18: 2277-2286. doi:10.1002/pro. 238, Aug. 31, 2009 (10 pages). (21) Appl. No.: 15/886,112 Hu, Lei et al., Comparison of the Structural Stability and Dynamic Properties of Recombinant Anthrax Protective Antigen and Its (22) Filed: Feb. 1, 2018 2-Fluorohistidine-LabeledAnalogue, Journal of Pharmaceutical Sci­ ences, vol. 101, No. 11, Nov. 2012, doi: 10.1002/jps.23294. Epub (65) Prior Publication Data Aug. 21, 2012 (11 pages). US 2018/0169208 Al Jun. 21, 2018 Mullangi, Vennela et al, Long-Range Stabilization of Anthrax Protective Antigen upon Binding to CMG2, Biochemistry, 2014, 53 Related U.S. Application Data (38), pp. 6084-6091, DOI: 10.1021/bi500718g, Publication Date (Web): Sep. 3, 2014 Copyright© 2014 American Chemical Society (62) Division of application No. 14/987,505, filed on Jan. (8 pages). 4, 2016. Zhang, Jeney et al., Stabilization of vaccines and antibiotics in silk (60) Provisional application No. 62/099,356, filed on Jan. and eliminating the cold chain, Proc Natl Acad Sci U SA. Jul. 24, 2, 2015. 2012; 109(30): 11981-11986. Published online Jul. 9, 2012. doi: 10.1073/pnas.1206210109 PMCID: PMC3409735 (6 pages). (51) Int. Cl. Office Action in corresponding U.S. Appl. No. 14/987,505, dated A61K 39/002 (2006.01) Nov. 8, 2017. A61K 39107 (2006.01) A61K 39/40 (2006.01) * cited by examiner A61K 45106 (2006.01) A61K 39/00 (2006.01) Primary Examiner - Jennifer E Graser (52) U.S. Cl. (74) Attorney, Agent, or Firm - Hovey Williams LLP CPC A61K 39107 (2013.01); A61K 39/40 (2013.01); A61K 45106 (2013.01); A61K (57) ABSTRACT 2039/6031 (2013.01); A61K 2039/622 Immunogenic compositions against Bacillus anthracis com­ (2013.01) prising a stabilized protective antigen complex are dis­ (58) Field of Classification Search closed. The stabilized complex comprises protective antigen None protein and capillary morphogenesis protein-2, with the See application file for complete search history. capillary morphogenesis protein-2 being bound to the pro­ tective antigen protein along a binding interface. The stabi­ (56) References Cited lized protective antigen complex has increased thermal and structural stability, along with resistance to premature pro­ U.S. PATENT DOCUMENTS teolytic degradation. Methods of using the same to induce an immunogenic response in a subject against B. anthracis 7,731,979 B2 6/2010 Bann infection are also disclosed. OTHER PUBLICATIONS 19 Claims, 11 Drawing Sheets (3 of 11 Drawing Sheet(s) Filed in Color) Rajapaksha et al (Protein Science. 2012. 21:1467-1480).* Mullangi et al (Biochemistry. 2014. 53: 6084-6091 ). * Specification includes a Sequence Listing. U.S. Patent Jan.29,2019 Sheet 1 of 11 US 10,188,716 B2 A so B 8000 -+.t- 5Hr* PA-280nm 0000 ,.....:;.-5FTro PA-'.!05nr, 4(/ ~WTPA __..., WT PA-2f,Cnm ~ ----·SFTrpPA ........_WTPA-29!inm 'S... .woo I 1 .,"' zooo ·e 30 s<.> "'<!I Z' <> 0 "' ~ ;fl t -toOO Iu.. % -4000 !ii; -imoo-----..... ___..._ ______, lW 340 SllO 350 400 420 440 1$1) 190 200 210 220 ~I) 2>W 2~0 200 W;,vel2no1h {llm) W«1el~lh tnmJ C 1~0 D eo ,. "' ~~SHrpPll.,,, 1$ 00 "' ... j ;!! ~ S; ,. ¥.., 150 <:l .,,<( "'g ~ ....... W-fPA ? • ,. l 50 n ~ E -!:>- :5FTfpf>A ;!! " ,!~" ~- w l3!'.I 1 ~t 140 ti> o' ;t~· ......... ➔ \ ~ ~ 3 <( ,u .. rn s,::: ia· 20. A. "'.. 3 :g .. ,: "- ! PA,, ~ } ;: ,., e· ti 40 z ~- ~ .. X ·;.. i • !::i 70 no m •;e 1$ ~: ~ z ~ j< . • s t $ "QI ... w • • "1 <O" ' 'lltM(M) ' ' :;> 1 "' t 1$ '<$ t2!l "' 30 wf 00. ? i U.S. Patent Jan.29,2019 Sheet 2 of 11 US 10,188,716 B2 A -42 -43 -44 -45 -46 -47 -48 -49 -50 ppm B ~ Unfolded ~ 7.2M 5.SM 4.0M 3.2 M 2.1 M 1.9M 1.3M 0.6M No urea -42 -48 ·49 -so ppm C PA03 3.2 M urea PA'° 3.2 M urea PA20 No urea ·42 .43 .44 -45 ··46 .47 -48 .49 -50 ppm Fig. 2 U.S. Patent Jan.29,2019 Sheet 3 of 11 US 10,188,716 B2 I I I -42 -43 -44 -45 -46 -47 -48 -49 -50 ppm / Trp206Tyr I I I I I I j I I I I I I I I I I I j I I j I I I I I ' I ' -42 -43 -44 -45 -46 -47 -48 -49 -50 ppm Fig. 3 U.S. Patent Jan.29,2019 Sheet 4 of 11 US 10,188,716 B2 A FTrp226 Fftp 477 •·• C Fig. 4 U.S. Patent Jan.29,2019 Sheet 5 of 11 US 10,188,716 B2 k--+TCEP ' i I , • I ' I , • ' ' j , ' ' ' I I ' ,44 ,45 ,46 ,47 ,43 Fig. 5 U.S. Patent Jan.29,2019 Sheet 6 of 11 US 10,188,716 B2 A ..,-unfolded pH 5 pH 5.5 pH6 pH 7 ✓ FTrp346 -42 -43 -44 -45 -46 -47 I -48 -j9 -so ppm B I pH8 i l I J I i I I I i i I I I I i I I I I i I I J I j J I » » j i I I I i I I I t i I I 1 I j J I I -42 -43 -44 -45 -46 -47 -48 -49 -50 ppm Fig. 6 U.S. Patent Jan.29,2019 Sheet 7 of 11 US 10,188,716 B2 Unfolded A I I j I I I I I I ~ I I I I I I • I I ' I I ' ' I I ' ' ' I I I I ' I I I I I I 11 ' ' I I I ' ' I -42 -43 -44 -4s -46 -47 -48 -4r -so ppm 1 B I FTrp346 +CMG2 i ( I t I I ' i I I I I I I I I I I j i I • I j ' I I j ' J I j j • I I I ' I I I I i I I I I I • -42 -43 -44 -45 -46 -47 -48 -49 -50 ppm Fig. 7 U.S. Patent Jan.29,2019 Sheet 8 of 11 US 10,188,716 B2 H86 H263 H336 (CMG2) Fig. 8 U.S. Patent Jan.29,2019 Sheet 9 of 11 US 10,188,716 B2 A) His211 B) His253 o.orn O.OiO 6 0.005 -.:.:: (WOO 0.000 0 2 3 4 5 0 2 3 4 5 Gdn-HC! (M) C) His336 D) His616 ,:: • 0.010 0.010 • 0000 O.®O ..,.,.,_,_:_,--,,--.,--,--,---,--.,..--..,---,--...,--..,...--, 0 2 3 4 5 0 2 3 4 5 Gdn-HCI {M} Gdn-HCl (M} Fig. 9 U.S. Patent Jan.29,2019 Sheet 10 of 11 US 10,188,716 B2 120 100 80 60 20 --------------------- 0 1 2 2.5 3 4 Gdn HCl (M) Fig.10 e• 00 • ~ ~ ~ ~ o.oo, (A) His86 .. (B) His263 (C) His304 /! C ~ • ... --.,.-•-,;:, = ,,...,-p-----c,·-·•0 0.03 / .d./~: ,,// 0.04 . ,,// 0.010 >" ..... 0.02 '7~ / l / /_,/ ~ :!::,. - '.E ~ .;,,; 'b ,/ ~ ? 0.02 ~ OJJ05 N 0.01 '"\,Ci .,,// N ~// ,;,/ .---,-··•-··'·····/''''.·· 0 o~ ___ o_ ... __ ......... ··" ~_,.. .... \,Ci 0.00 0.00 0.000-l I- 1----,---....--~--,-----,..---, 4 6 8 4 6 8 10 4 6 8 10 pH* pH* pH * rJJ ('D=­ 0.020 (E) His336 0.06 (F) His597 ('D..... _... 9 .... (D) His310 ,,..----~ ~,,_.,.,,. .... 0.02 , 0.015 , 0 0 ,,~;,,,"" . .... 0.04 , .... / I / ..... .... .-:--' 0.010 //. 9- 0 ~ /­ .... l ~ 0.01 ~/ E, f 5 !/0 .;,,; ;:,,:; 0.02 .:,,: 0.005 : . /,/ 9/ .. 0 • 0 ....l __..,...- • -?"'" 0.000 ~----.---•-•-s............... d s.--.---• 0.00 ~. r.,;_ g 0,001 t--_,....---,---.----.-------, I I 4 6 8 10 4 6 8 10 4 6 8' 10 "'""' pH* pf-I* pH * '"= 00"'""' 00 Fig. 11 ~ 0--,"'""' =N US 10,188,716 B2 1 2 PROTECTIVE ANTIGEN COMPLEXES metalloprotease that can kill macrophages or, at lower WITH INCREASED STABILITY AND USES concentrations, prevent macrophages from secreting cytok­ THEREOF ines, resulting in a compromised host immunity. PA is a channel forming polypeptide that allows entry of EF and LF CROSS-REFERENCE TO RELATED 5 across membranes into the cell, a step that is critical for the APPLICATIONS pathogenesis of anthrax.