US 201700.95531A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0095531 A1 SCHREBER et al. (43) Pub. Date: Apr. 6, 2017 (54) COMPOSITIONS AND METHODS FOR on Dec. 4, 2015, provisional application No. 62/235, ADONING TYPE AND TYPE II 727, filed on Oct. 1, 2015. EXTRACELLULAR DOMAINSAS HETEROLOGOUS CHMERIC PROTEINS Publication Classification (71) Applicant: Heat Biologics, Inc., Durham, NC (US) (51) Int. Cl. A638/17 (2006.01) (72) Inventors: Taylor SCHREIBER, Durham, NC (52) U.S. Cl. (US); George FROMM, Durham, NC CPC ........ A6IK 38/1774 (2013.01); A61K 38/177 (US); Suresh DE SILVA, Durham, NC (2013.01) (US); Neal SCHILLING, Durham, NC (US) (57) ABSTRACT (21) Appl. No.: 15/281,196 The present invention relates to, interalia, compositions and methods, including chimeric proteins that find use in the (22) Filed: Sep. 30, 2016 treatment of disease. Such as immunotherapies for cancer and autoimmunity. In part, the invention provides, in various Related U.S. Application Data embodiments, fusions of extracellular domains of trans (60) Provisional application No. 62/372.574, filed on Aug. membrane proteins that can have stimulatory or inhibitory 9, 2016, provisional application No. 62/263.313, filed effects. Patent Application Publication Apr. 6, 2017 Sheet 1 of 41 US 2017/0095531A1 FGURE 1 ise 83 garag ise Eisenaries sister ce FIGURE 2 Patent Application Publication Apr. 6, 2017 Sheet 2 of 41 US 2017/0095531A1 FGURE 3 is Metab:re Festigierre cks esses - FIGURE 4 3888 88 Patent Application Publication Apr. 6, 2017 Sheet 3 of 41 US 2017/0095531A1 FGURE 5 2233 5 OOOO 1 5 O mPD1-FC nPO-FC-OX40 Patent Application Publication Apr. 6, 2017 Sheet 4 of 41 US 2017/0095531A1 FGURE 6 A. B. nP-Fc-OX40 l 3.50 300 & Media Control 2.50 & PD1-FC rt 2.00 8 nPD1-FC-OX40. l PD-Fc. Patent Application Publication Apr. 6, 2017. Sheet 5 of 41 US 2017/0095531A1 FGURE 7 8 mPD -FC-OX4OL PD-Fc-OX40 Patent Application Publication Apr. 6, 2017 Sheet 6 of 41 US 2017/0095531A1 FIGURE 8 10.0 8.0 6.O. an EP only 40 xxP) -FC-OX4OL 2.0 OO o 4 6 10 17 24 26 Days post-electroporation FIGURE 9 5 O 25 O asp only us-Pol-Fooxact O 6 1 17 20 Days post-electroporation Patent Application Publication Apr. 6, 2017 Sheet 7 of 41 US 2017/0095531A1 FIGURE 10 A. Mouse; PD1-Fc-ox404. B. 88: S.ww. 888s taggs: g . r 2000 OOO SO 253 Fc-bition rPD1-fc-OX40.(g/ml) Patent Application Publication Apr. 6, 2017 Sheet 8 of 41 US 2017/0095531A1 FIGURE 10 (continued) Activatedcate Primaryrimary Mouseviouse Spenocytes AT: (PO-11, F. 1.4 PC. .98% .9% E. 93.5% s s 2. s: s kis S. 10 8-X. g E FSocie: s g 8 s 1 * * to 8 is? * 33 6 Po-lice:s PO-3. Cells 6 ofEE population ing -> mP-3-APCas ais +3344; 3 cessceis +500g-FR) FP; k +38.30sis.F.C. cells i-FP--SGorg FP) *-i-4 ce's 45ts FP * +338.50 ccs --Stig FP} Patent Application Publication Apr. 6, 2017 Sheet 9 of 41 US 2017/0095531A1 FIGURE 11 A. MC38 Tumor rowth 2. an intreated crys? x tox40 (n:5) s P- (-5) GK4 fee)- n:5) x rinfl-Fc-CX40L naS} - - - - - - - - - - - - - - - - - - - - - - - - - - - --. OP-2 s ::first Session. 2. rete: 9:7: s :cox43. 35: : :Sc-X30s: ; Secondary Tumor intreated 2 neS 0. (OXAG 2 ho3) SS QP-2 (rs1 cQX40ACP.L. 2 (nies -a, / x mPD-Fc-QX40.2 in-4) :28:Fiasraelection : 50 mPDitc-OX40L 2. s 1. 5 25 3. 35 4. as 5 55 O 65 Days Postumor inocutation Patent Application Publication Apr. 6, 2017. Sheet 10 of 41 US 2017/0095531 A1 FIGURE 11 (continued) Periphera Blood B. C. Flow Cytometry D. Serum Cytokines Ox: an sNFy SO É4 s Os S so s s NF 90s C 203 O 5 & S. 2 25, 38, 35.30 45 5 33 SO 85 Days Post Tumor inoculation Patent Application Publication Apr. 6, 2017. Sheet 11 of 41 US 2017/0095531 A1 FIGURE 11 (continued) E, 80 or Size G, Memory Phenotyping on Day 13 Splenocytes Bay i3 Kics 2ks cks arra 8 sternary Prea; or s Shr-e Eter Mentory ce: 60 5 a 40 5 : 3. 8. s 2 is. S x s & & 8.8 s is pK. s exes & 3. F. .3 trate CD4 f CD8 Ratio ina Muttipley Tissues to 8treated ld Day 33 Peripheral Blood Speers 8 mPD1-fc-OX40t S. s say 2. day 3 3. E, 8 mPD1-Fe-GTR. i. w CB2a....O. a. ...OX43 3 *: C-3 d 2 & 53 S 3 2 8 Patent Application Publication Apr. 6, 2017 Sheet 12 of 41 FIGURE 11 (continued) C2S: Co. Cacer Day A. 3. x sox C2 tutor 1. Sess 8jection cytosines (S^0 ces {3 g) A natysis J. id: cytokine-P -o-> Cytokine-PE MoMo Patent Application Publication Apr. 6, 2017. Sheet 13 of 41 US 2017/0095531 A1 FIGURE t (continued) w X axe w xx 3:2 8 8 3 : 8 3 S & 3.8 s S as 'cs stics inciatio 3. i s S is . & S 3 & S & 3 as stor sociats Patent Application Publication US 2017/0095531A1 Patent Application Publication Apr. 6, 2017. Sheet 15 of 41 US 2017/0095531 A1 FIGURE3 A. B, Overlay from Thermo prep i&isig: ...ESA 3. cÉing: 3. 838ss ce E.35.& 8::c:::::: 8:12 ote 8ackgroufti Background fastic: 88c.gif a. s.3 k3 Patent Application Publication Apr. 6, 2017. Sheet 16 of 41 US 2017/0095531 A1 FIGURE 13 (continued) C f & CX43-is 3888-is 8,8. OX40-His 8.8 w8 .3 C 94 SL-279252 m 3.3 was 3888-FS3:3: 8 S232S2 . 8. i. TT's a 10,383 - Fic-Riitic hi-fc-X4Qingfin Patent Application Publication Apr. 6, 2017. Sheet 17 of 41 US 2017/0095531A1 FGRE 14 A. Binding to PE-3 B. Sinding to PD-2 C. Binding to OX40 38 : we'PE.Fcept. Crit 250.sy 8. :asio-fc Citi 2sory; x wS-2922 S.C. rhi S-2733S. 230 & 8 s g i. s c -Sswarara S 50 SSO -250 2SO 58 SS -250 25 5. SO time seconds Eime issconds: Eime seconds , Binding to FcR1A E. Binding to FcRn ka K K S. : S9. Sarpe: orate;iffs): (strate; fs; birding:y: k as a SEE 8.32 25 g 400 g 3 : 3.33E-S 837 E. SS s:g 300 g33. Cat: {500 & g soo. S.g33. Cat S.O.M3 P3: Ec & S-2792.52 SfR::M $3:33.25on 8 s 290 S 3. 23 as w3. Y s u 20 433 *x. -28 a8 is -3 Time seconds ite seconds Patent Application Publication Apr. 6, 2017. Sheet 18 of 41 US 2017/0095531 A1 FIGURE 14 (continued) G. Binding to PD-1 hi. Binding to PD-2 i. Binding to OX40 SOO as solic Cit}(338.8 8. as PI3-fc.cnt: 325 na: 58 ex40.-Fi: Cat 825 fsw / & co-3-cr-CX43. 2S: n, :... cop-cR-X40: 253 M3 : co-fcRii-OX42S,3 33 2 8 asyaraas s 250 S3 73 25 253 388 f -2S 350 s 75 Tirse (seconds: ine secords Eime seconds) As a s KS . Binding58 to FcR1A K. S.Binding s to FcRn2 -- san fe: tort-rate::fits off-rate::1fs: th:riting; it : an igg: inti{SOCniv.; S. w.... $ 400 3. Wi-FR-S.ision 3 : : e in g SO to iggs: cryi (5083 ; : ce:Fisk-F8-x4. ''::: & s 200 {s0} n S. as : sigs: 30.4....yw -23 20 --- c -2. 2 4. s Tina seconds fine seconds} Patent Application Publication Apr. 6, 2017. Sheet 19 of 41 US 2017/0095531A1 W O art trar 38 4. w Xenograft. Histology 838-y:ce:388 s Serum taif-life (EISA \ age 2: P.S.to hrs FC Capture 13.8 hrs (K45 Capture sons: F. SSA &3-8:a: Fi . E. C 8. S. 3 is time hours N. intraturoraihai-life Flow Cytometry Haifa e. pt-33 33 -N : - \, 3 20 - Vs - i a. & A. SS-PO-1wPE-3. .e38,Csis R. 38 R. 338 28S fine hours Patent Application Publication Apr. 6, 2017. Sheet 20 of 41 US 2017/0095531 A1 FIGURE 5 Celi Line Development to Test in Vitro Fusion Protein Binding Jurkat Parentat 0.83% CHO-K1 Parental 0.77% CHO-K1 Parentai 0.86% x JurkatfhoX40 96.8% Ho-KafhpD-, 97.4% : CHO-Ki?hCD47 72.4% 18p 3 O or "r OX40-APCw- D--APCsa). CD4-AC web Patent Application Publication Apr. 6, 2017 Sheet 21 of 41 US 2017/0095531A1 FIGURE 15 (continued) hPD-Fc-OX40L isotype contral x g A. as 250 rig i as C-K. Parents 62.5 g. g & CHO-Ki?h-l. 3. a ac a s s s S.g a cy as ea Shes t KO-APC re E aircast Pass -x-girkat{OX40 & g OXA-APCM Patent Application Publication Apr. 6, 2017. Sheet 22 of 41 US 2017/0095531 A1 FIGURE 15 (continued) Cia-C-OX4, , & Esotsie Cott: an - 3 & -- Cis-X:3:? s o 3 •r 3. BX3-8aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa-h gfi. 3. aspairst $3 ea: 8. -x skat{X4 r recreer 8 884. A w. giffiti. Patent Application Publication Apr. 6, 2017. Sheet 23 of 41 US 2017/0095531 A1 FIGURE 15 (continued) Human S-2792.52 Binding in vitro to PB-1 Expressing Celis ..