USOO961 1322B2
(12) United States Patent (10) Patent No.: US 9,611,322 B2 Wilson, Jr. et al. (45) Date of Patent: Apr. 4, 2017
(54) FUSIONS OF ANTIBODIES TO CD38 AND 6,800,735 B2 10/2004 Whitty ATTENUATED INTERFERON ALPHA 7,091,321 B2 8, 2006 Gillies 7,670,595 B2 3/2010 Gillies (71) Applicant: Teva Pharmaceuticals Australia Pty 7,790.415 B2 9/2010 Gillies Ltd, Macquarie Park, NSW (AU) 8,980,267 B2 3/2015 Grewal et al. 9,139,634 B2 9/2015 Morrison (72) Inventors: David S. Wilson, Jr., Freemont, CA 2002fO164788 A1 11/2002 Ellis et al. (US); Sarah L. Pogue, Freemont, CA 2002.0193569 A1 12/2002 Hanna (US); Glen E. Mikesell, Pacifica, CA 2006/0269.516 A1 11/2006 Presta et al. (US); Tetsuya Taura, Palo Alto, CA 2010/0172868 A1 7/2010 Morrison et al. (US); Wouter Korver, Mountain View, 2010.018.9689 A1 7/2010 Chang et al. CA (US); Anthony G. Doyle, 2015,0203560 A1 7/2015 Grewel Drummoyne (AU); Adam Clarke, Five FOREIGN PATENT DOCUMENTS Dock (AU); Matthew Pollard, Dural (AU); Stephen Tran, Strathfield South WO 97241.37 7/1997 (AU); Jack Tzu Chiao Lin, Redwood WO 004O265 T 2000 WO O197844 12/2001 City, CA (US) WO 2005103O83 11/2005 (73) Assignee: Teva Pharmaceuticals Australia Pty WO 200609.9875 9, 2006 Ltd, Macquarie Park, NSW (AU) WO 2007042309 4/2007 WO 2008124086 10, 2008 (*) Notice: Subject to any disclaimer, the term of this WO 2009017823 2, 2009 patent is extended or adjusted under 35 WO 2011 154453 12/2011 WO 2012092612 T 2012 U.S.C. 154(b) by 0 days. WO 2013059.885 5, 2013 (21) Appl. No.: 14/262,841 WO 20131077.91 T 2013 WO 20140285O2 2, 2014 (22) Filed: Apr. 28, 2014 (65) Prior Publication Data OTHER PUBLICATIONS US 2014/0248238 A1 Sep. 4, 2014 Bork, 2000, Genome Research 10:398-400.* Related U.S. Application Data Bork et al., 1996, Trends in Genetics 12:425-427.* Wells, 1990, Biochemistry 29:8509-8517.* (63) Continuation of application No. Ngo et al., 1994. The Protein Folding Problem and Tertiary Struc PCT/AU2012/001323, filed on Oct. 29, 2012. ture Prediction, pp. 492-495.* Tomoyuki, et al., “Engineering the variable region of therapeutic (30) Foreign Application Priority Data IgG antibodies', MABS, Landes Bioscience, US, vol. 3, No. 3, May 1, 2011, p. 243-252. Oct. 28, 2011 (AU) ...... 2011 9045O2 Kodama, et al., “Mutated SEA-D227A-congated antibodies greatly enhance antitumor activity against MUC1-expressing bile duct (51) Int. Cl. carcinoma, Cancer Immunology, Immnotherapy, vol. 50, No. 10, C07K 6/28 (2006.01) Dec. 2001, pp. 539-548. C07K I6/46 (2006.01) Piehler, et al., “New structural and functional aspects of the Type 1 C07K I4/56 (2006.01) interferon-receptor interaction revealed by comprehensive muta C07K 4/54 (2006.01) tional analysis of the binding interface', vol. 275, No. 51, Dec. 2000, pp. 40425-40433. C07K I4/565 (2006.01) International Search Report and Written Opinion from related C07K I4/57 (2006.01) application PCT/AU2012/001323 dated Mar. 13, 2013. C07K 6/10 (2006.01) International Search Report and Written Opinion from related (52) U.S. Cl. application PCT/US2013/0386.59 dated Feb. 12, 2014. CPC ...... C07K 16/28 (2013.01); C07K 14/5406 (2013.01); C07K 14/54.12 (2013.01); C07K * cited by examiner 14/56 (2013.01); C07K 14/565 (2013.01); C07K 14/57 (2013.01); C07K 16/1027 Primary Examiner — Christine J Saoud (2013.01); C07K 16/2887 (2013.01); C07K Assistant Examiner — Jegatheesan Seharasey on I6/2896 (2013.01); C07K 16/46 (2013.01); (74) Attorney, Agent, or Firm — Stradley Ronon Stevens C07K 2317/24 (2013.01); C07K 23.17/33 & Young, LLP (2013.01); C07K 2317/92 (2013.01); C07K 2319/00 (2013.01) (57) ABSTRACT (58) Field of Classification Search The present invention provides a polypeptide construct None comprising a peptide or polypeptide signaling ligand linked See application file for complete search history. to an antibody or antigen binding portion thereof which binds to a cell Surface-associated antigen, wherein the ligand (56) References Cited comprises at least one amino acid Substitution or deletion which reduces its potency on cells lacking expression of said U.S. PATENT DOCUMENTS antigen. 5,055.289 A 10, 1991 Frincke 5,650,150 A 7, 1997 Gillies 18 Claims, 62 Drawing Sheets U.S. Patent Apr. 4, 2017 Sheet 1 of 62 US 9,611,322 B2
FIGURE 1
EGENO EWBOOMENS
Antibody heavy chain gG)
Antibody light chain gG)
Attenuated Polypeptide ligand
sist Peptide linker (0-50 a mino acids in length U.S. Patent Apr. 4, 2017 Sheet 2 of 62 US 9,611,322 B2
FIGURE 2
LEGEND EMBODEMENT
Antibody to cell surface protein attached to Attenuated Ligand (AL)
Receptor for ligand
Signaling VV Cell Surface Antigen U.S. Patent Apr. 4, 2017 Sheet 3 of 62 US 9,611,322 B2
FIGURE 3
Human CD38 (SEQ ID NO : 131) : 1 MANCEFSPVSGDKPCCRLSRRAOLCLGVSILVLILVVVLAVVVPRWRQOWSGPGTTKRFP 60 61 ETVLARCVKYTEIHPEMRHVDCOSVWDAF KGAFISKHPCNITEEDYQPIMKT, GTQTV PCN 120 121 KILLWSRIKDIAHOFTOVORDMFTLEDTLIGYLADDLTWCGEFNTSKINYOSCPDWRKDC 180 181 SNNP VSV FWKTVSRRFAEAACDVVHVMINGSRSKIF DKNSTFGSVEVHNLOPEKVOTLEA 240 241 WVIHGGREDSRDLCODPTIKELESIISKRNIOFSCKNIYRPDKELQCVKNPEDSSCTSEI 300
underlined: extra Cellular dOIlain italic: transmembrane domain
U.S. Patent Apr. 4, 2017 Sheet 7 of 62 US 9,611,322 B2
FIGURE Sc
Heavy chain (SEQ ID NO:214) : l EVOLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYY 60 61 ADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYECAKDKILWFGEPVFDYWGOGTLVTV 120 l21. SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYE PEPWTVSWNSGALTSGWHTFPAVLQ 18O 8. 240 24l FLFPPKPKDTLMISRTPEvTCvvvDvsoEDPEvoFNWYVDGVEVHNAKTKPREEQFNs 3OO 301 GOPREPOVYT 360 361 W. 420 421 EGNVFSCSVMHEALHNHYTOKSLSLSLGKMSYNLLGFLORSSNFOSOKLLWQLNGRLEYC A 80 481. LKDAMNFDIPEEIKOLOOFOKEDAALTIYEMLONIFAIFRODSSSTGWNETIVENLLANV 540 541 YHOINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEIL 600 60. RNFYFINRLTGYLRN 615 Light chain (SEQ ID NO: 134) : l. EIVITOSPATLSLSPGERATISCRASOSVSSYLAWYOOKPGQAPRILLIYDASNRATGIPA 60 6. 2O 121. SDEOLKSGTASVWCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDSTYSLSSTLT 18O 181 LSKADYEKHKWYACEWTOGLSSPWTKSFNRGEC 24 double underlined: antibody heavy and light Chains single underlined: cytokine single underlined & bold: attenuating mutation G005-HC-LO-IFNB(R35A) gCG4
U.S. Patent Apr. 4, 2017 Sheet 9 of 62 US 9,611,322 B2
FIGURE 5e
Heavy chain (SEQ ID NO:306) : 1 DVQLQESGPGLVKPSQSLSLTCTVTGHSITSDYAWNWIRQFPGDKLEWMGYISYSGYTTY 60 61. 2O 121. 18O 181 240 241. 3OO 30 360 361 Q 420 421. oGNVFscsvmHEALHNHYTOKSLSLsPGKSGGGGSHKCDITLQEIIKTLNstEQKTLCT 480 481. ELTVTDIFAASKNTTEKETFCRAATVLROFYSHHEKDTRCLGATAOOFHRHKOLIRFLKR 540 541 LDQNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS 584 Light chain (SEQ ID NO:300) :
60 S. SLOPEDFGSYYCOHFWSSPWTF KRTVAAPSVFIFPP 120 121 SDEQLKSGTASVVCLLNNEY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT 180 FNRGEC 214
doubleYv underlined:mm antibody heavy and light chains single underlined: cytokirie single underlined bold: attenuating mutation italic: linker sequence Ji IO-HC-L6-IL-4(R88O) IgGl U.S. Patent Apr. 4, 2017 Sheet 10 of 62 US 9,611,322 B2
FIGURE 6
Non-Targeted IFNo. Activity
5. Construct EC50(pM) - g -8- FNO 19 -- Rituximab-HC-L6-IFNo. IgG1 5.9 -e- isotype-HC-L6-IFNo. IgG1 8.0 E s -
0.0001 0.01 100 10000 IFNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 11 of 62 US 9,611,322 B2
FIGURE 7
Targeted IFNo. Activity (Daudi)
1500000 5 Construct ...... IC50(pM) 2 1000000 -- Rituximab-HC-L6-IFNo. IgG1 0.40 - A - FNC. 13 500000 E - O 0.001 0.01 0.1 1 10 100 IFNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 12 of 62 US 9,611,322 B2
FIGURE 8
Targeted IFNo. Activity (Daudi) 1 25 O O O O 1000000 Construct IC50(pM) -- Rituximab-HC-L6-IFNo. IgG1 0.18 "" isotype-HC-L6-LFNo. IgG1 2.2 2575O5 OOO OOO OOO OOO O 0.001 0.01 0.1 1 10 100 IFNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 13 of 62 US 9,611,322 B2
FIGURE 9
Non-Targeted IFNo. Activity
6000 Construct ...... EC50(pM) -b- FN 5.7 400 - Rituximab-HC-L6-IFNo. IgG1 22 -- Rituximab-HC-L6-IFNo. (R144A) gG1 2200 -- Rituximab-HC-L6-IFNo. (A145G) IgG1 2800 c 2000 -- Rituximab-HC-L6-IFNo. (R33AEYNS) IgG1 6100 -o- isotype-HC-L6-IFNo IgG1 18
0.01 ? 100 10000 1000000 IFNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 14 of 62 US 9,611,322 B2
FIGURE 10
Non-Targeted FNa Activity COnstruct EC50 5000 O Rituximab-HC-L6-IFNo, IgG1 5.6 is 4000 Rituximab-HC-L6-IFNo. (R33A) IgG ND i. o Rituximab-HC-L6-IFNo. (R144A+YNS) IgG1 1700 g 3000
2000 E 1000
O 10-2 100 102 104 106 IFNC equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 15 of 62 US 9,611,322 B2
FIGURE 11
Targeted IFNo. Activity (Daudi) 2000000 Construct IC50(pM) S -- FN 12
1500000 - Rituximab-HC-6-IFNa IgG1 0.33 g Rituximab-HC-L6-IFNa (R144A) gG1 1.4 5 1000000 Rituximab-HC-L6-IFNo. (A145G) gG1 1.1 2 Rituximab-HC-L6-FNo. (R33A+YNS) IgG1 0.74 E 500000 isotype-HC-L6-FNo. IgG1 2.1
0.0001 0.01 1 100 10000 IFNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 16 of 62 US 9,611,322 B2
FIGURE 12
Targeted ifno Activity (Daudi) Construct EC50
1,5006 O Rituximab-HC-L6-IFNo. IgG1 0.21 Rituximab-HC-L6-FNo. (R33A) IgG1 340 1000000 to Rituximab-HC-L6-FNo. (R144A+YNS) IgG1 3.3 O d O 500000 E -
O 0.0001 0.01 1 100 10000 1000000 IFNo equivalents (pM)
U.S. Patent Apr. 4, 2017 Sheet 18 of 62 US 9,611,322 B2
FIGURE 14
40000 -o- X355/02 lgG1 30000 -H X355/04 IgG1 -- X355/07 IgG1 s -v- X910/12 IgG1 20000 -- X913/15 IgG1 10000 -E negative controllgG1 oral anti FC O s SS s N NS S U.S. Patent Apr. 4, 2017 Sheet 19 of 62 US 9,611,322 B2
FIGURE 15
Non-Targeted IFNo. Activity
Construct EC50 (pM) -- FNO O.726 2000 -e- GOO5-HCLO-IFNolgG4 195
O 0.0001 0.01 1 100 10000 IFNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 20 of 62 US 9,611,322 B2
FIGURE 16
Targeted IFNo. Activity (ARP1) 500000 S. Construct IC50 o 400000 -0- IFNo. 14.70 300000 -e- GOO5-HC-L0-FNo. lgG4 4,079 8 » g 200000 100000 {d
O 0.01 1 100 10000 IFNot equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 21 of 62 US 9,611,322 B2
FIGURE 17
Non-Targeted IFNot Activity
Mr. 5000 Construct EC50
o 400 -- FN 3O 3. -- G005-HC-L6-IFNo. (R144A) igG4 11,000 8. -- G005-HC-LO-IFNa (R 144A) igG4 30,000 2000 mar G005-Hc-L6-IFNc (R144A) igG 1 2600 E 1000 G005-HC-LO-IFNo. (R144A) igG1 6800
| O. 1 1 FNo. equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 22 of 62 US 9,611,322 B2
FIGURE 18
Non-Targeted IFNo. Activity
6000 92S Construct ...... EC50(pM) g4000 -o- FNC. O.087 -- G005-HC-L0-IFNo. (A145G)lgG1 510 £2000 re. G005-HC-L6-IFNo. (A145G)lgG1 730 -- G005-HC-L6-IFNo. (A145G) gG4 2200 -
0.0001 0.01 1 100 OOOO 1000000 IFNot equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 23 of 62 US 9,611,322 B2
FIGURE 19
Non-Targeted IFNo. Activity
25 g Construct EC50(pM) 2 -o- FNo. 0.21 -0- G005-LC-L6-IFNo. (A145G) IgG1 12000 2 -6- G005-LC-LO-IFNa (A145G) gG1 24000
-
0.0001 0.01 1 100 10000 1000000 IFNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 24 of 62 US 9,611,322 B2
FIGURE 20
Targeted IFNo. Activity (Daudi) 15OOOOO Construct IC50(pM) S n -o- FNC. 0.77 1000000 • G005-HC-L6-IFNo. (R144A) gG4 13 g -- G005-HC-LO-IFNo. (R144A) igG4 2.7 2 500000 - G005-HC-L6-IFNo. (R144A) gG1 1.9 E -- G005-HC-LO-IFNa (R144A) igG1 7.3
O 0.0001 0.01 1 100 10000 IFNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 25 of 62 US 9,611,322 B2
FIGURE 21
Targeted IFNo. Activity (Daudi) 2OOOOOO Construct IC50(pM) 15OOOOO asu FNO 0.48 - G005-HC-LO-IFNo. (A145G) IgG1 0.74 1OOOOOO -- G005-HC-L6-IFNa (A145G) IgG1 1.0
-v- G005-HC-L6-IFNa (A145G) igG4 0.59 5OOOOO
OOOO 0.01 100 OOOO IFNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 26 of 62 US 9,611,322 B2
FIGURE 22
Targeted IFNo. Activity (Daudi)
15OOOOO S n Construct IC50(pM) 1000000 «Six FNO 11 -- G005-LC-L6-IFNo. (A145G) igG1 8.5 2 500000 -- G005-LC-LO-IFNo. (A145G) igG1 21 E - O 0.0001 0.01 1 100 10000 FNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 27 of 62 US 9,611,322 B2
FIGURE 23
Targeted IFNo. Activity (ARP-1)
600000 Construct IC50(pM) 3. -- FNo. 6.0 n -- G005-HC-L6-IFNo. (R144A) igG1 21 400000 & G005-HC-LO-IFNo. (R144A) lgG1 110 3. -- G005-HC-L6-IFNo. (R144A) igG4 28 200000 -- G005-HC-LO-IFNo. (R144A) igG4 85 E es -
0.01 1 100 10000 IFNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 28 of 62 US 9,611,322 B2
FIGURE 24
Targeted IFNo. Activity (ARP-1)
500000 S Construct ...... IC50 (pM) 4000 -O FNo. 9.5 : 300000 - - G005-HC-L6-IFNo. (A145G) gG1 4.0 3 ri- G005-HC-LO-IFNa (A145G) gG1 8.0 : 200000 -- G005-HC-L6-IFNo. (A145G) gG4 4.4 E 100000 l O 0.01 100 10000 IFNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 29 of 62 US 9,611,322 B2
FIGURE 25
Non-Targeted IFNot Activity
Construct...... EC50(pM) -- G005-HC-L0-IFNo. (R144A) lgG4 25,800 -i- G005-HC-L0-IFNo. (R144S) ligG4 108,000 -- G005-HC-L0-IFNo. (R144E) igG4 >105 -- G005-HC-LO-IFNo. (R144G) lgG4 9,970
100 102 104 106 108 IFNo. equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 30 of 62 US 9,611,322 B2
FIGURE 26
Non-Targeted FNa Activity
3000 S a; Construct . . . . EC50(pM) 2000 -e G005-HC-LO-IFNo. (R144H) igG4 1,690 s "r G005-HC-LO-IFNo. (R144K) lgG4 <100 -- G005-HC-LO-IFNo. (R144N) ligG4 431 1000 -- G005-HC-LO-IFNo. (R144Q) IgG4 3,500 5 -o- G005-HC-LO-IFNo. (R144T) lgG4 30,800 O 100 102 104 106 108 IFNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 31 of 62 US 9,611,322 B2
FIGURE 27
Non-Targeted IFNo. Activity
Construct ...... EC50(pM)
- G005-HC-LO-IFNo. (R144Y) ligG4 92,100 o -- G005-HC-L0-IFNo. (R1441) IgG4 159,000 3 -- G005-HC-LO-IFNo. (R144L)lgG4 26,700 3 -- G005-HC-LO-IFNo. (R144V) g(G4 86,900
S -
100 102 104 106 108 IFNo. equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 32 of 62 US 9,611,322 B2
FIGURE 28
Non-Targeted IFNo. Activity
3500 S Construct ...... EC50(pM) - E 2500 - G005-HC-LO-IFNo. (A145G) gG4 2,040 3 -5 G005-HC-LO-IFNo. (A145D) igG4 52,600 -& G005-HC-LO-IFNo. (A145E) lgG4 < 100 8 500 -- G005-HC-LO-IFNo. (A145H) IgG4 24,900 s -- FN 0.260 500
10-4 10-2 100 102 104 106 108 IFNo. equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 33 of 62 US 9,611,322 B2
FIGURE 29
Non-Targeted IFNo. Activity
3500
2500 ConstructConstr ...... EC50(pM) 8 - - G005-HC-LO-IFNo. (A1451) igG4 28.9 -- G005-HC-LO-IFNa (A145K) g(G4 662,000 1500 -- G005-HC-LO-IFNo. (A145L) gG4 239 -- GOO5-HC-LO-IFNo. (A145N) lgG4 309 500 -o- GOO5-HC-LO-IFNa (A145Q) IgG4 709
100 102 104 106 108 IFNot equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 34 of 62 US 9,611,322 B2
FIGURE 30
Non-Targeted IFNo. Activity
3500 S o 2500 Construct EC50(pM) - - G005-HC-LO-IFNo.(A145R) gG4 >3x10 -A G005-HC-LO-IFNa (A145T) IgG4 <2 3 1500 -- G005-HC-LO-IFNa (A145Y) gG4 19,200
500
100 102 104 106 08 IFNo. equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 35 of 62 US 9,611,322 B2
FIGURE 31
Targeted IFNot Activity (ARP-1)
500000 3400000es Construct IC50(pM) Z -- G005-HC-LO-IFNo. (R144A) lgG4 186 300000 -- G005-HC-LO-IFNo. (R144S) gG4 290 g -- G005-HC-LO-IFNo. (R144T) lgG4 333 2000 -- G005-HC-LO-IFNo. (R144Y) ligG4 306 E 100000 -- G005-HC-LO-IFNo. (R1441) IgG4 257 -o- G005-HC-LO-IFNo. (R144L) gG4 191
0.01 1 100 10000 IFNo. equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 36 of 62 US 9,611,322 B2
FIGURE 32
Targeted IFNo. Activity (ARP-1)
S 400000 Construct IC50(pM) a 300000 -- G005-HC-LO-IFNa (A145G) IgG4 23.8 g -- G005-HC-LO-IFNa (A145H) IgG4 113 200000 -- G005-HC-LO-IFNa (A145D) gG4 222 i -- G005-HC-LO-IFNa (A145K) gC4 174 -- G005-HC-LO-IFNa (A145Y) gG4 914 100000 -0- FNC. 10.8 - O 0.01 1 100 OOOO IFNa equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 37 of 62 US 9,611,322 B2
FIGURE 33
Targeted IFNo. Activity (ARP-1) 500000 as Construct IC50(pM)
Z -- X910/12-HC-LO-IFNo. (R144A) igG4 191 : 300000 -- X355/02-HC-LO-IFNo. (R144A) igG4 147 g -- X913/15-HC-LO-IFNo. (R144A) igG4 84.1 2000 ~ X355/07-HC-LO-IFNo. (R144A) gG4 61.2 100000 -- G005-HC-LO-IFNo. (R144A) IgG4 220 -
O.O1 1 100 10000 IFNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 38 of 62 US 9,611,322 B2
FIGURE 34
Targeted IFNo. Activity (ARP-1)
1000000
800000 -o- FNo. 43.0 -- G005-HC-LO-IFNo. (A145D) gG4 85.7 600000O -- X910/12-HC-LO-IFNo. (A145D) IgG4 336 400000 -- X913/15-HC-LO-IFNo. (A145D) IgG4 68.1 -- X355/02-HC-LO-IFNo. (A145D) IgG4 46.0 200000 -o- X355/07-HC-LO-IFNo. (A145D) IgG4 619
O 0.0001 0.01 1 100 10000 1000000 IFNa equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 39 of 62 US 9,611,322 B2
FIGURE 35
Non-Targeted IFNo. Activity
5. Construct EC50(pM) n -0. IFNO. 0.499 o • X910/12-HC-LO-IFNo. (R144A) gG4 41,000 -- X913/15-HC-LO-IFNo. (R144A) gG4 29,600 -- X355/02-HC-LO-IFNo. (R144A) gG4 70,500 -- X355/07-HC-LO-IFNo. (R144A) gG4 70,300 -o- G005-HC-L0-IFNo. (R144A) igG4 73,200 -
10-5 100 105 1010 IFNo equivalents (pM)
U.S. Patent Apr. 4, 2017 Sheet 41 of 62 US 9,611,322 B2
FIGURE 37
Targeted IFNo. Activity (ARP-1)
Construct ...... IC50(pM) -O FNo. 16.1 -- R5D1-HC-LO-IFNo. (A145D)lgG4 51.3 -- R5E8-HC-LO-IFNo. (A145D)lgG4 89.2 -- R10A2-HC-LO-IFNo. (A145D)lgG4 32.7 -o- X355/02-HC-LO-IFNo. (A145D) gG4 104
0.01 1 100 1OOOO 1000000 IFNo equivalents (pM)
U.S. Patent Apr. 4, 2017 Sheet 43 of 62 US 9,611,322 B2
FIGURE 39
CD138 targeted: ARP-1
90OOOO
800000
700000 ride isotype-HC-LO-IFNo. (A145D) igG4 600000 -H nBTO62-HC-LO-IFNo. (A145D) igG4 5OOOOO
400000 0.01 1 100 10000 IFNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 44 of 62 US 9,611,322 B2
FIGURE 40 (a) MHC Class targeted attenuated-IFNo.: ARP-1 cells 1000000
800000
400000 -- HB95-HC-LO-Ifnc.(A145D) gG4 139 -er isotype-HC-LO-IFNo.(A145D) lgG4 N/A 200000
0.01 1 100 10000 FNo equivalents (pM)
(b)
Targeted FNa Activity (ARP1) Costuct C5.
8. to Pativizumab-HC-6-FNa (A145D) Fab 2410 2D12-HC-L6-FNc. (A1450) Fab 17,000 SO A HE95-HC-L6-LFNa (A145D) Fab 8. FNo. 42.8 4.
2
(s f s 1916 FNa equivalents (pl.) U.S. Patent Apr. 4, 2017 Sheet 45 of 62 US 9,611,322 B2
FIGURE 41
Anti-MHC class I-IFN: Efficacy of targeted FN in virus infected cells
Éio Construct EC50 (pM) 2 75 v FNO 0.8 o 50 HB95-HC-LO-IFNo.(A145D) gG4 1.15 A isotype-HC-L0-FNo.(A145D) igG4 19,461 g 25 SS O O.OOO1 O,O 1 OO 10000 OOOOOO IFNo equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 46 of 62 US 9,611,322 B2
FIGURE 42
Non-Targeted IFNB Activity 4000 S Construct . EC50(pM) a 3000 -- IFNB1 0.799 3 : -- G005-HC-LO-IFNB IgG4 57.6 2000 / -- G005-HC-LO-IFNB (R35A) igG4 16,100 i / 5E 1000 ---...----J
0.0001 0.01 1 100 0000 1000000 IFN Equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 47 of 62 US 9,611,322 B2
FIGURE 43
Targeted IFNB Activity (ARP-1) ax 500000 Construct ...... IC50(pM)
-- G005-HC-LO-IFNB (R35A) gG4 46.9 300000 -- G005-HC-LO-IFNB IgG4 32.7 3 g 200000 E 100000
O.O. 100 0000 Ifn Equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 48 of 62 US 9,611,322 B2
FIGURE 44
Non-Targeted IL-4 Activity
2.0 Construct. EC50(pM) 1.5 -One L-4 1.26 31.0 -- J110-HC-L6-L-4lgG1 6.88 -- isotype-HC-L6-IL-4(R88O)lgG1 19,200 0.5 -- J110-HCL6-IL-4(R88O) gG1 44,800 a.
10-4 10-2 100 102 104 106 108 IL-4Equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 49 of 62 US 9,611,322 B2
FIGURE 45
Targeted IL-4 Activity 50 Construct IC50(pM) c 8. L-4 114 f -- J110-HC-6-IL-4 IgG1 31.8 -- J110-HC-L6-IL-4(R880) IgG1 46.1 -- isotype-HC-L6-L-4 (R880) gG1 >1000 SS
0.1 1 10 100 1000 10000 100000 IL-4 Equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 50 of 62 US 9,611,322 B2
FIGURE 46
IL-6 Activation Assay 15 Construct IC50 (pM) 12 -a isotype-HC-l. 16-L6 igg 1 10.9 -- isotype-HC-L16-IL6 (R179E) ligG182600 0.9 -- HE395-HC-L16-EL6 gCG1 12.4 s -- HB95-HC-L16-IL6 (R179E) igGf 86.7 -- 6 1.O.A. 0.6
10-4 O2 100 102 04 Os 108 Il-6 Equivalents (pM) U.S. Patent Apr. 4, 2017 Sheet 51 of 62 US 9,611,322 B2
FIGURE 47
H929 Subcutaneous MM model 2500 aE 2000 well. Vehicle E -i. G005 (IgG1) c 1500 -- FNC. 1000 -- isotype-HC-L6-IFNo. (A145G) lgG1 500 -- G005-HC-L6-FNo. (A145G) gG1 s 0 No detectable tumors in Days post treatment start 10/10 mice on day 22 U.S. Patent Apr. 4, 2017 Sheet 52 of 62 US 9,611,322 B2
FIGURE 48
MM1S Systemic MM Model
100----- G005-HC-L6-IFNo. (A145G) lgG1
5 O
an Days post tumor inoculation U.S. Patent Apr. 4, 2017 Sheet 53 of 62 US 9,611,322 B2
FIGURE 49
Daudi Subcutaneous NHL model
(30005 weso Vehicle d 2000 - on GOO5 (IgG1)(CG1 g -- FNO.
5 -- G005-HC-L6-IFNo. (A145G) IgG1 5 1000 -- isotype-HC-L6-IFNo. (A145G) lgG1
s 5 9 O SSSSSS 100 Treatment Days post cell inoculation U.S. Patent Apr. 4, 2017 Sheet 54 of 62 US 9,611,322 B2
FIGURE SO
H929 MM model: Dose titration
O Vehicle 10mg/kg 1 mg/kg 0.1 mg/kg isotype-HC-L6-IFNo. (A145G) gG1 0.01mg/kg 10mg/kg 1mg/kg 0.1mg/kg G005-HC-L6-IFNa (A145G)lgG1 0.01 mg/kg U.S. Patent Apr. 4, 2017 Sheet 55 of 62 US 9,611,322 B2
FIGURE S1
H929 MM model: IgG1 vs IgG4 format, 3.5 mg/kg dosing
0. Vehicle
-e- isotype-HC-6-IFNo. (A145G) gG4 -- isotype-HC-L6-IFNa (A145G) igG1 G005-HC-L6-IFNo. (A145G) gG1 G005-HC-L6-FNo. (A145G) gG4
Days post treatment start U.S. Patent Apr. 4, 2017 Sheet 56 of 62 US 9,611,322 B2
FIGURE 52
H929 MM model: X02-FNO. Variant
E 2000- ...... E. 1500 sea. Vehicle g -- isotype-HC-LO-IFNo. (A145D) igG4
g 1000 -H X355/02-HC-LO-IFNo. (A145D) lgG4 o 500 vs s d s 80 Treatment Phase Days post treatment start U.S. Patent Apr. 4, 2017 Sheet 57 Of 62 US 9,611,322 B2
FIGURE S3
Targeted IFNo.2b vs Standard of Care 6. E 5 oda DexamethaSone, : 10mg/kg, daily, 28 days E Melphalan, s ass 4mg/kg, daily, 5 days O re Bortezomib8 (Velcade), 1mg/kg, 2X/week, 4 weeks s s G005-HC-LO-IFNo. (A145D) igG4, s O 20 40 60 3mg/kg, 2X/week, 5 weeks Days post treatment start U.S. Patent Apr. 4, 2017 Sheet 58 of 62 US 9,611,322 B2
FIGURE 54
Dosing regimen Study: 10 mg/kg dose
6 N e - Vehicle g -- 6doses isotype-HC-L6-IFNo. (A145G)lgG4 -- 1 dose - - 2 doses 4 G005-HCL6-IFNo. (A145G)lgG4 -8- 6 doses S O 20 40 80 Days post treatment start U.S. Patent Apr. 4, 2017 Sheet 59 of 62 US 9,611,322 B2
FIGURESS
Dosing regimen Study: Large Tumor Start 6N E 2000-...... 5 5: 1500 or Vehicle O
1000 G005-HC-L6-IFNa (A145G) IgG4 Start at 730 mm 500 s X& 0 ; : " ' " ": Days Post Grouping U.S. Patent Apr. 4, 2017 Sheet 60 of 62 US 9,611,322 B2
FIGURE 56
Colony Forming Assay: 3 normal human BM MNC
120 100 80 -O- GOO5-HC-LO-IFNo. (A145D) lgG4 60 res 2D 12-HC-LO-FMo (A145D) gG4 40 wer FNO. 20
O.OOO1 0.01 1 1OO 10000 IFN concentration (nM) —- 10,000X attenuation U.S. Patent Apr. 4, 2017 Sheet 61 of 62 US 9,611,322 B2
FIGURE S7a
IP-10
w8- FNO. -- isotype-HC-L6-FNo. (A145G)lgG1
0.001 0.01 0.1 1 10 100
1000X attenuation
MCP-1
4000 -- FNo. 3000 -e- isotype-HC-L6-IFNo. (A145G) igG1 2000
1000
0.001 0.01 0.1 1 nM FNo. ------5000X attenuation U.S. Patent Apr. 4, 2017 Sheet 62 of 62 US 9,611,322 B2
FIGURE S7b.
MCP-3 800
-- FNC.
-o- isotype-HC-L6-1F No. (A145G) lgG1 5. 400 O
200
O 0.001 O.O1 0.1 10 100 nM FNO 2500X attenuation
IL-10, 15
wo. IFNC.
-0- isotype-HC-L6-IFNo. (A145G) lgG1
1300X attenuation US 9,611,322 B2 1. 2 FUSIONS OF ANTIBODIES TO CD38 AND tolerated due to their “off target' biological activity. The ATTENUATED INTERFERON ALPHA off-target activity and associated poor tolerability also mean that some of these peptide ligand-based drugs cannot be RELATED APPLICATIONS administered at Sufficiently high dosages to produce optimal therapeutic effects on the target cells which mediate the This application is a continuation of International Patent therapeutic effect. Application No. PCT/AU2012/001323, filed on Oct. 29, Similarly, it has been known since the mid-1980s that 2012, which claims priority to Australian Patent Application interferons, in particular IFNC, are able to increase apoptosis No. 2011 904502, filed on Oct. 28, 2011, the contents of each and decrease proliferation of certain cancer cells. These application are incorporated herein by reference in their 10 biological activities are mediated by type I interferon recep entirety and for all purposes. tors on the surface of the cancer cells which, when stimu lated, initiate various signal transduction pathways leading REFERENCE TO ASEQUENCE LISTING to reduced proliferation and/or the induction of terminal differentiation or apoptosis. IFNC. has been approved by the This application includes a Sequence Listing Submitted 15 FDA for the treatment of several cancers including mela electronically as a text file named Pctau2012001323-se noma, renal cell carcinoma, B cell lymphoma, multiple q1.txt, created on Apr. 24, 2014, with a size of 1 megabyte. myeloma, chronic myelogenous leukemia (CML) and hairy The Sequence Listing is incorporated by reference herein. cell leukemia. A "direct’ effect of IFNC. on the tumour cells is mediated by the IFNC. binding directly to the type IIFN FIELD OF THE INVENTION receptor on those cells and stimulating apoptosis, terminal differentiation or reduced proliferation. One “indirect’ effect The present invention relates to polypeptide constructs of IFNC. on non-cancer cells is to stimulate the immune comprising mutated, attenuated polypeptide ligands system, which may produce an additional anti-cancer effect attached to antibodies, wherein the antibodies direct the by causing the immune system to reject the tumour. mutated ligands to cells that express on their Surfaces the 25 Unfortunately, the type I interferon receptor is also pres antigens to which said antibodies bind, as well as receptors ent on most non-cancerous cells. Activation of this receptor for said ligands. The invention further relates to methods of on such cells by IFNC. causes the expression of numerous treatment involving the use of these polypeptide constructs. pro-inflammatory cytokines and chemokines, leading to toxicity. Such toxicity prevents the dosing of IFNC. to a BACKGROUND OF THE INVENTION 30 subject at levels that exert the maximum anti-proliferative and pro-apoptotic activity on the cancer cells. Numerous peptide and polypeptide ligands have been Ozzello et al. (Breast Cancer Research and Treatment described to function by interacting with a receptor on a cell 25:265-76, 1993) described covalently attaching human Surface, and thereby stimulating, inhibiting, or otherwise IFNC. to a tumour-targeting antibody, thereby localizing the modulating a biological response, usually involving signal 35 direct inhibitory activity of IFNC. to the tumour as a way of transduction pathways inside the cell that bears the said reducing tumour growth rates, and demonstrated that Such receptor. Examples of Such ligands include peptide and conjugates have anti-tumour activity in a Xenograft model of polypeptide hormones, cytokines, chemokines, growth fac a human cancer. The mechanism of the observed anti-cancer tors, apoptosis-inducing factors and the like. Natural ligands activity was attributed to a direct effect of IFNC. on the can be either soluble or can be attached to the surface of 40 cancer cells, since the human IFNC. used in the experiments another cell. did not interact appreciably with the murine type I IFN Due to the biological activity of Such ligands, some have receptor, which could have lead to an indirect anti-cancer potential use as therapeutics. Several peptide or polypeptide effect. Because of this lack of binding of the human IFNC. to ligands have been approved by regulatory agencies as thera the murine cells, however, the authors could not evaluate the peutic products, including, for example, human growth 45 toxicity of the antibody-IFNC. conjugate relative to free hormone, insulin, interferon (IFN)-C2b. IFNC2a, IFNB, INFO. These authors used a chemical method to attach the erythropoietin, G-CSF and GM-CSF. Many of these and IFNC to the antibody. other ligands have demonstrated potential in therapeutic Alkan et al., (Journal of Interferon Research, volume 4, applications, but have also exhibited toxicity when admin number 3, p. 355-63, 1984) demonstrated that attaching istered to human patients. One reason for toxicity is that 50 human IFNC. to an antibody that binds to the Epstein-Barr most of these ligands trigger receptors on a variety of cells, virus (EBV) membrane antigen (MA) increased its antipro including cells other than those that mediate the therapeutic liferative activities towards cells that express the EBV-MA effect. For example, when IFNC2b is used to treat multiple antigen. This increased potency was dependent on both myeloma its utility resides, at least in part, in its binding to antigen expression by the target cells and the binding type I interferon receptors on the myeloma cells, which in 55 specificity of the antibody. The cell line tested was the turn triggers reduced proliferation and hence limits disease cancer cell line QIMR-WIL, a myeloblastic leukemia. The progression. Unfortunately, however, this IFN also binds to authors suggested that the attachment of IFNC. to an anti numerous other, normal cells within the body, triggering a body could be used as a treatment for cancer since it would variety of other cellular responses, some of which are reduce tumour growth. Alkan et al did not address the harmful (e.g. flu-like symptoms, neutropenia, depression). A 60 potential toxicity of these antibody-IFNC. conjugates arising consequence of Such “off target activity of ligands is that from their interactions with normal, antigen-negative cells. many ligands are not suitable as drug candidates. In this It is also known that the linkage between an antibody and context, “off target activity” refers to activity on the ligands IFNC may be accomplished by making a fusion protein natural receptor, but on the surface of cells other than those construct. For example, IDEC (WO01/97844) disclose a that mediate therapeutically beneficial effects. 65 direct fusion of human IFNC. to the C terminus of the heavy Even though some ligands. Such as IFNC2b, are approved chain of an IgG targeting the tumour antigen CD20. Other for the treatment of medical conditions, they are poorly groups have disclosed the use of various linkers between the US 9,611,322 B2 3 4 C-terminus of an IgG heavy chain and the IFNC. For receptor, is linked to an antibody that targets the mutated example, U.S. Pat. No. 7,456,257 discloses that the C-ter ligand to target cells which display the antibody's corre minus of an antibody heavy chain constant region may be sponding antigen, the ligand’s activity on target antigen connected to IFNC. via an intervening serine-glycine rich positive cells is maintained while the ligands activity on (S/G) linker of the sequence (GGGGS), where n may be 1, 5 non-target antigen-negative cells is substantially reduced. 2 or 3, and that there are no significant differences in the The net result is a ligand signaling molecule that has a much IFNC. activity of the fusion protein construct regardless of greater potency in activation of its receptors on antigen linker length. positive target cells compared to antigen-negative non-target Morrison et al. (US2011/0104112 A1; and Xuan C, Stew cells, which provides a means for reducing toxicity arising ard K.K., Timmerman J. M. Morrison SL. Targeted delivery 10 from off-target ligand activity. of interferon-C. via fusion to anti-CD20 results in potent Accordingly, a first aspect of the present invention pro antitumor activity against B-cell lymphoma. Blood 2010; vides a polypeptide construct comprising a peptide or poly 115:2864-71) also disclose IFNC. linked to the C-terminus of the heavy chain of a cancer-targeting IgG antibody, with an peptide signaling ligand linked to an antibody or antigen intervening S/G linker, and observed that the fusion of the 15 binding portion thereof which binds to a cell surface IgG and linker to the IFNC. reduced the activity of IFNC. on associated antigen, wherein the ligand comprises at least one cells that did not express the corresponding antigen on the amino acid substitution or deletion which reduces its cell surface. The decreased IFN activity of these fusion potency on cells lacking expression of said antigen. protein constructs was modest when compared to human In a second aspect, the present invention provides a non-fusion protein IFNC. (free IFNC.) acting on human cells, method of treating a tumour in a Subject, comprising admin but appeared to be more significant for murine IFNC. on istering to the Subject the polypeptide construct of the murine cells. The decrease in the activity of human IFNC. present invention. that results from fusing it to the C-terminus of an antibody, In a third aspect, the present invention provides use of the as observed by Morrison et al, and in U.S. Pat. No. 7,456, polypeptide construct of the present invention in the treat 257 is modest and is generally considered to be a disadvan 25 ment of cancer. tage since it reduces potency of the ligand. This disadvan In a fourth aspect, the present invention provides a tage was pointed out, for example, by Rossi et al (Blood Vol. composition comprising the polypeptide construct of the 114, No. 18, pp. 3864-71), who used an alternative strategy present invention and a pharmaceutically acceptable carrier of attaching the IFNC. to a tumor targeting antibody in Such or diluent. a way that no loss in IFNC. activity was observed. 30 In a fifth aspect, the present invention provides method of In general the prior art teaches to use a potent IFN and to reducing the potency of a peptide or polypeptide signaling target this IFN to cancer cells. While this approach results in ligand on an antigen negative cell which bears the ligand an increase in activity of the IFN against cancer cells, it does receptor whilst maintaining the potency of the ligand on an not address the issue of activity of the IFN on normal antigen positive cell which bears the ligand receptor to a “off-target cells. In prior art examples referred to above, the 35 greater extent when compared to the antigen negative cell, human IFNC. portion of the antibody-IFNC. fusion protein the method comprising modifying the ligand Such that the maintained a high proportion of native IFNC activity when ligand comprises at least one amino acid Substitution or exposed to human cells that do not express the correspond deletion which reduces its potency on the antigen negative ing antigen on their cell Surfaces. This activity may lead to cell and linking the modified ligand to an antibody or toxicity arising from the activation of non-cancerous, nor 40 antigen-binding portion thereof, wherein the antibody or mal (“off target') cells by the IFNC. portion of the fusion antigen binding portion thereof is specific for a cell Surface protein. Accordingly, there exists a need to decrease the associated antigen on the antigen positive cell but not on the “off-target activity of ligand-based drugs, while retaining antigen negative cell. the “on-target, therapeutic effect of such ligands. The Unlike the linking of a non-attenuated “native' or “wild maintenance of target-specific ligand activity and at the 45 type' human ligand to an antibody or antigen-binding por same time a reduction in non-target toxicity of ligand-based tion thereof, which typically results in from 1 to 15-fold therapeutic agents would create a greater therapeutic con higher potency of the ligand on antigen-positive compared centration window for therapeutically useful ligands. It to antigen-negative cells, the present invention demonstrates would for example be desirable to use human IFNC. in a that the attachment of mutated, attenuated forms of the form such that its activity can be directed to the cancer cells 50 ligand to the same antibody is able to generate higher while minimizing its effects on normal human cells. Ideally potency on antigen-positive cells compared to antigen nega the type I interferon receptor on the cancer cells would be tive cells. maximally stimulated, while the same receptor on non In one embodiment the signaling ligand is IFNC. or IFNB cancerous cells would experience minimal stimulation. and the polypeptide construct shows at least 10, at least 100, There is a need to target human IFNC. to the cancer cells in 55 at least 1,000, at least 10,000 or at least 100,000-fold greater Such a way that it has dramatically more activity on the selectivity towards antigen positive cells over antigen nega cancer cells, which display the antigen, than on the normal tive cells compared to free, wild-type ligand using the cells, which do not display the antigen. The same logic “off-target' assay and the “on target (ARP) or “on target applies to other potentially therapeutic ligands, e.g. other (Daudi) assays described herein. cytokines, peptide and polypeptide hormones, chemokines, 60 The present invention also provides an antibody-attenu growth factors, apoptosis-inducing factors and the like. ated ligand fusion proteins, wherein the attenuated ligand is IFNC. or IFNB and the wherein fusion protein construct, SUMMARY OF THE INVENTION when injected into a mouse with an established human tumor, can eliminate the tumor. The present inventors have found that when a peptide or 65 The present invention also provides an antibody-attenu polypeptide signaling ligand, having one or more mutations ated ligand fusion proteins, wherein the attenuated ligand is which substantially decrease the affinity of the ligand for its IFNC. or IFNB and wherein the fusion protein construct, US 9,611,322 B2 5 6 when injected into a mouse with an established human lymphoma. In particular embodiments the Subject is also tumor with a volume of over 500 cubic millimeters, can treated with a retinoid, such as all-trans retinoic acid. In eliminate the tumor. certain embodiments in which the cell surface associated The present invention also provides an antibody-attenu antigen is CD38, the tumour or cancer may be selected from ated ligand fusion proteins, wherein the attenuated ligand is multiple myeloma, non-Hodgkin’s lymphoma, chronic IFNC. or IFNB and wherein the fusion protein construct, myelogenous leukemia, chronic lymphocytic leukemia or when injected as a single one-time treatment into a mouse acute myelogenous leukemia. with an established human tumor, can eliminate the tumor. In embodiments in which the ligand is linked to an An antibody-attenuated ligand fusion proteins, wherein antibody, the antibody may be an IgG4. In particular the attenuated ligand is IFNC. or IFNB and wherein the 10 embodiments the IgG4 comprises an S228P amino acid fusion protein construct can eliminate both established substitution. myeloma tumors and established lymphoma tumors in a In certain embodiments in which the signaling ligand of OUS the polypeptide construct is an IFNC, the antibody or In each of these cases it is preferred that cell surface antigen binding portion thereof may bind to a cell Surface associated antigen is CD 38. 15 associated antigen on virally infected cells. In these embodi In one embodiment, the amino acid sequence of the ments the cell Surface associated antigen may be selected signaling ligand comprising at least one amino acid Substi from a virally encoded protein, phosphatidylserine or a tution or deletion has greater than 90% or greater than 95%, phosphatidylserine-binding protein. In embodiments in or greater than 96%, or greater than 97%, or greater than which the cell Surface associated antigen is phosphatidyl 98% or greater than 99% sequence identity with the wild serine or a phosphatidylserine-binding protein the construct type ligand amino acid sequence. may be used to treat Hepatitis C. In one embodiment, the construct is a fusion protein. In certain embodiments in which the signaling ligand of In certain embodiments the signaling ligand is linked to the polypeptide construct is IFNC. or IFNB, the cell surface the C-terminus of the heavy chain of the antibody or antigen associated antigen is selected from CD20, CD38, CD138 or binding portion thereof. In certain embodiments the signal 25 CS1. In certain embodiments in which the ligand is IFNC. or ing ligand is linked to the C-terminus of the light chain of the IFNB, the tumour or cancer may be selected from multiple antibody or antigen binding portion thereof. In either of myeloma, melanoma, renal cell carcinoma, chronic myel these embodiments, the ligand may be linked directly to the ogenous leukemia or hairy cell leukemia. C-terminus of the heavy or light chain of the antibody or In a particular embodiment the construct is G005-HC-L0 antigen binding portion thereof (ie without an intervening 30 IFNC. (A145D) IgG4. additional linker). In certain embodiments in which the signaling ligand of In one embodiment the cell surface associated antigen is the polypeptide construct is an IFNB, the cell surface asso Selected from class I MHC or PD-1. ciated antigen may be a T cell, a myeloid cell or an antigen In certain embodiments, the cell Surface-associated anti presenting cell cell Surface associated protein. gen is a myeloma associated antigen which is selected from 35 In certain embodiments in which the signaling ligand of the group consisting of CD38, HM1.24, CD56, CS1, the polypeptide construct is an IFNB, the cell surface asso CD138, CD74, IL-6R, Blys (BAFF), BCMA, HLA-SR, ciated antigen may be selected from the group consisting of Kininogen, beta2 microglobulin, FGFR3, ICAM-1, CD3, CD4, CD8, CD24, CD38, CD44, CD69, CD71, CD83, matriptase, CD52, EGFR, GM2, alpha-4-integrin, IFG-1R CD86, CD96, HLA-DR, PD-1, ICOS, CD33, CD115, and KIR, and the ligand is an IFNC. 40 CD11c, CD14, CD52 and PD-1. In these embodiments, the In one embodiment, the signaling ligand is selected from construct may be used to treat a disease characterized by any one of IFNC2b. IFNB, IL-4 or IL-6. excess inflammation, such as an autoimmune disease. In certain embodiments in which the signaling ligand is an In certain embodiments in which the signaling ligand of IFNC, the amino acid substitution or deletion may be at any the polypeptide construct is an IFNB, the at least one amino one or more of amino acid positions R33, R144 or A145. In 45 acid substitution or deletion is selected from the group certain embodiments the signaling ligand is an IFNC. and the consisting of R35A, R35T, E42K, M62I, G78S, A141Y. Substitution is selected from the group consisting of R144A A142T, E149K, R152H. In these embodiments, the IFNB (SEQID NO:30), R144S (SEQID NO:40), R144T (SEQID may also possess a C17S or C17A substitution. NO:41), R144Y (SEQ ID NO:43), R144I (SEQID NO:35), In certain embodiments the signaling ligand of the poly R144L (SEQ ID NO:37), A145D (SEQID NO:44), A145H 50 peptide construct is an IFNY. In these embodiments, the cell (SEQID NO:47), A145Y (SEQID NO:58), A145K (SEQID Surface associated antigen may be a tumor-associated anti NO:49), R33A+YNS (SEQ ID NO:65), R33A (SEQ ID gen. In other embodiments, the cell Surface associated NO:16) and R144A+YNS (SEQ ID NO:68). antigen may be selected from the group consisting of CD14. In certain embodiments in which the signaling ligand is an FSP1, FAP, PDGFR alpha and PDGFR beta. In these IFNC. and the cell surface associated antigen is CD38, the 55 embodiments, the construct may be used to treat a disease antibody is selected from any one of G003, G005, G024, characterized by excess fibrosis. MOR03077, MORO3079, MORO3080, MORO3100, In certain embodiments in which the signaling ligand of 38SB13, 38SB18, 38SB19, 38SB30, 38SB31, 38SB39, the polypeptide construct is an IFNY, the at least one amino OKT10, X355/02, X910/12, X355/07, X913/15, R5D1, acid substitution or deletion is selected from the group R5E8, R10A2, or an antigen binding portion thereof, or an 60 consisting of a deletion of residues A23 and D24, an S2OI antibody with greater than 95%, greater than 96%, greater substitution, an A23V substitution, a D21K substitution and than 97%, greater than 98% or at least 99% amino acid a D24A substitution. sequence identity with any one of R5D1, R5E8 or R10A2. In certain embodiments in which the signaling ligand of In certain embodiments in which the cell Surface associ the polypeptide construct is an IL-4, the cell Surface asso ated antigen is CD38, the signaling ligand of the polypeptide 65 ciated antigen is selected from the group consisting of CD3. construct is an IFNC, the treatment is for a cancer in a CD4, CD24, CD38, CD44, CD69, CD71, CD96, PD-1, Subject selected from multiple myeloma, a leukemia or a ICOS, CD52 and PD-1. US 9,611,322 B2 7 8 In certain embodiments in which the signaling ligand of FIG. 4 shows the amino acid sequences of certain exem the polypeptide construct is an IL-6, the cell Surface asso plary signaling ligands of the present invention: (a) human ciated antigen is selected from the group consisting of CD3. IFNo.2b, IFNB1, IFNB1b and IFNY; (b) IL-4 and IL-6. CD4, CD24, CD38, CD44, CD69, CD71, CD96, PD-1, FIG. 5 shows the amino acid sequences of certain anti ICOS, CD52 and PD-1. 5 body-attenuated ligand fusion proteins of the present inven In certain embodiments in which the signaling ligand of tion: (a) G005-HC-L0-IFNC. (A145D) IgG4; (b) nBT062 the polypeptide construct is an HGF, the cell surface asso HC-L0-IFNC. (A145D) IgG4; (c) G005-HC-L0-IFNB ciated antigen is selected from the group consisting of (R35A) IgG4; (d) HB95-HC-L16-IL-6 (R179E) IgG1; and ASGR1, ASGR2, FSP1, RTI 140/Ti-alpha, HTI56 and a (e) J110-HC-L6-IL-4 (R88O) IgG1. The nomenclature for 10 the fusion proteins is described in the examples. VEGF receptor. FIG. 6 shows the non-antibody-antigen-targeted inter In certain embodiments in which the signaling ligand of feron activity of IFNC2b, and of the antibody-IFN fusion the polypeptide construct is a TGFB, the cell surface asso protein constructs Rituximab-IFNC2b (Rituximab-HC-L6 ciated antigen is selected from the group consisting of CD3. IFNC. IgG1) and Palivizumab-IFNC2b (Isotype-HC-L6 CD4, CD8, CD24, CD38, CD44, CD69, CD71, CD83, 15 IFNC. IgG1) in the interferon activity assay described in the CD86, CD96, HLA-DR, PD-1, ICOS, CD33, CD115, examples below as the “off-target assay. Throughout the CD11c, CD14, CD52 and PD-1. figures “IFNC equivalents' refers to the molar concentration In certain embodiments in which the signaling ligand of of interferon molecules, either free or attached to an anti the polypeptide construct is an erythropoietin, the cell Sur body. “IFN' refers to free (non-fusion protein) wild-type face associated antigen is selected from the group consisting interferon. of CD241 the product of the RCHE gene, CD117 (c-kit), FIG. 7 shows the antibody-antigen-targeted interferon CD71 (transferrin receptor). CD36 (thrombospondin recep activity of the Rituximab-IFNC2b fusion protein construct tor), CD34, CD45RO, CD45RA, CD115, CD168, CD235, (Rituximab-HC-L6-IFNC. IgG1) compared with IFNC2b in CD236, CD237, CD238, CD239 and CD240. the antiproliferative assay described in the examples below In certain embodiments in which the signaling ligand of 25 as the “on target (Daudi) assay.” the polypeptide construct is an interleukin-10 and the cell FIG. 8 shows the antibody-antigen-targeted interferon Surface associated antigen is selected from the group con activity of the Rituximab-IFNC. fusion protein construct sisting of CD11c, CD33 or CD115, CD14, FSP1, FAP, or (Rituximab-HC-L6-IFNC. IgG1) compared with the non PDGFR (alpha or beta). targeted activity of Palivizumab-IFNC. fusion protein con In a sixth aspect there is provided anti-CD38 antibodies 30 struct (Isotype-HC-L6-IFNC. IgG1) in the “on-target (Daudi) with variable regions designated X910/12, X913/15, X355/ assay” described in the examples below. 02, X355/07, R5D1, R5E8, or R10A2, with sequences set FIG. 9 shows the non-antibody-antigen-targeted inter out as follows: feron activity of IFNC.2b, of the antibody-IFN fusion protein constructs Rituximab-IFNC2b (Rituximab-HC-L6-IFNC. 35 IgG1) and Palivizumab-IFNC2b (Isotype-HC-L6-IFNo. Name V. Sequence VfV sequence IgG1), and of certain variants of Rituximab-IFNC2b con X910,12 SEQ ID NO: 395 SEQ ID NO: 394 structs that have been mutated to reduce their interferon X913,15 SEQ ID NO:397 SEQ ID NO: 396 activity. The assay is described in the examples as the X355/01 SEQ ID NO: 421 SEQ ID NO: 420 “off-target assay”. X355/02 SEQ ID NO:391 SEQ ID NO: 390 40 FIG. 10 shows the non-antibody-antigen-targeted inter X3SSO4 SEQ ID NO: 423 SEQ ID NO: 422 X355/07 SEQ ID NO:393 SEQ ID NO: 392 feron activity of the antibody-IFN fusion protein constructs RSD1 SEQ ID NO:399 SEQ ID NO:398 Rituximab-IFNC2b (Rituximab-HC-L6-IFNC. IgG1) and of RSE8 SEQ ID NO: 401 SEQ ID NO: 400 two variants of Rituximab-IFNC2b that were mutated to R1OA2 SEQ ID NO: 403 SEQ ID NO: 402 reduce interferon activity. The assay is described in the 45 examples as the "off-target assay'. From these sequences the person skilled in the field can FIG. 11 shows the antibody-antigen-targeted interferon readily identify the CDR sequences using known methods. activity of the antibody-IFN fusion protein construct Ritux As will be recognized by the skilled worker these CDR imab-IFNC2b (Rituximab-HC-L6-IFNC. IgG1) and of vari sequences can be used in differing framework sequences to ants of Rituximab-IFNC2b constructs that have been those specified in the SEQ ID NO’s specified above. 50 mutated to reduce their interferon activity compared to the non-targeted activity of the Palivizumab-IFNC2b (Isotype BRIEF DESCRIPTION OF THE DRAWINGS HC-L6-IFNC. IgG1) fusion protein constructs and compared to IFNC2b. The assay is described in the examples as the “on FIG. 1 shows a schematic of the certain embodiments of target (Daudi) assay.” the present invention that comprise an antibody consisting of 55 FIG. 12 shows the antibody-antigen-targeted interferon 2 heavy chains and 2 light chain, in which one or two activity of the antibody-IFN fusion protein constructs Ritux attenuated signaling ligands is or are attached to each heavy imab-IFNo.2b (Rituximab-HC-L6-IFNC. IgG1) and of two chain or each light chain, or both. variants that were mutated to reduce interferon activity. The FIG. 2 shows a schematic illustrating one possible assay is described in the examples as the “on target (Daudi) approach for how the antibody-attenuated ligand fusion 60 assay.” proteins of the present invention cause signaling by activat FIG. 13 shows the sequences of certain novel human ing receptors on cells that display the antigen corresponding CD38 antibodies disclosed herein. to the said antibody on their cell surfaces. The fusion protein FIG. 14 shows the results of detection of binding of novel activates the receptor on the same cell that the antibody is human anti-CD38 antibodies to a CD38' cell line RPMI bound to, via its specific antigen. 65 8226 by flow cytometry. The X axis is the antibody concen FIG. 3 shows the amino acid sequences of the human tration in micrograms/ml and the y axis represents the mean CD38 (SEQ ID NO:131). fluorescence intensity. US 9,611,322 B2 10 FIG. 15 shows the non-antibody-antigen targeted IFN between the antibody heavy chain C-terminus and the N-ter activity of IFNC2b compared with an anti-CD38-IFNo. minus of the mutated IFN. The antibody variable regions of fusion protein construct (G005-HC-L0-IFNC. IgG4), based these fusion protein constructs are derived from antibody on the anti-CD38 antibody G005. The assay is described in G005. The assay is described in the examples as the “on the examples as the "off-target assay.” target (ARP) assay.” FIG. 16 shows the antiproliferative activity of IFNC2b vs FIG. 25 shows the non-antibody-antigen targeted IFN an anti-CD38-IFNC. fusion protein construct (G005-HC-L0 activity of various anti-CD38-IFNC. fusion protein con IFNC. IgG4) on the multiple myeloma cell line ARP-1 structs with different point mutations in the IFN portion. The (CD38). The assay is described in the examples as the “on antibody variable regions of these fusion protein constructs target (ARP) assay.” 10 FIG. 17 shows the non-antibody-antigen targeted IFN are derived from antibody G005. The assay is described in activity of IFNC2b vs various anti-CD38-IFNC. fusion pro the examples as the "off-target assay.” tein constructs bearing point mutations in the IFN portion. FIG. 26 shows the non-antibody-antigen targeted IFN The antibody variable regions of these fusion protein con activity of various anti-CD38-IFNC. fusion protein con structs were derived from antibody G005. The assay is 15 structs with different point mutations in the IFN portion. The described in the examples as the “off-target assay.” antibody variable regions of these fusion protein constructs FIG. 18 shows the non-antibody-antigen targeted IFN are derived from antibody G005. The assay is described in activity of IFNC2b vs various anti-CD38-IFNC. fusion pro the examples as the "off-target assay.” tein constructs bearing point mutations in the IFN portion. FIG. 27 shows the non-antibody-antigen targeted IFN The antibody variable regions of these fusion proteins were activity of various anti-CD38-IFNC. fusion protein con derived from antibody G005. The assay is described in the structs with different point mutations in the IFN portion. The examples as the "off-target assay.” antibody variable regions of these fusion protein constructs FIG. 19 shows the non-antibody-antigen targeted IFN are derived from antibody G005. The assay is described in activity of IFNC2b vs two anti-CD38-IFNC. fusion protein the examples as the "off-target assay.” constructs bearing point mutations in the IFN portion. The 25 FIG. 28 shows the non-antibody-antigen targeted IFN antibody variable regions of these fusion protein constructs activity of various anti-CD38-IFNC. fusion protein con are derived from antibody G005. The assay is described in structs with different point mutations in the IFN portion. The the examples as the "off-target assay.” antibody variable regions of these fusion protein constructs FIG. 20 shows the antiproliferative activity of IFNC2b vs are derived from antibody G005. The assay is described in anti-CD38-IFNC. fusion protein constructs with mutations in 30 the examples as the "off-target assay.” the IFN portion on the lymphoma cell line Daudi. The FIG. 29 shows the non-antibody-antigen targeted IFN antibody variable regions of these fusion protein constructs activity of IFNo.2b vs various anti-CD38-IFNC. fusion pro are derived from antibody G005. The assay is described in tein constructs with different point mutations in the IFN the examples as the "on target (Daudi) assay.” portion. The antibody variable regions of these fusion pro FIG. 21 shows the anti-proliferative activity of IFNC2b 35 tein constructs are derived from antibody G005. The assay and various anti-CD38-IFNC. fusion protein with the A145G is described in the examples as the “off-target assay.” mutation in the IFN portion. Fusion protein constructs have FIG. 30 shows the non-antibody-antigen targeted IFN either the 6 amino acid L6 linker or no linker (LO) and are activity of various anti-CD38-IFNC. fusion protein con of the IgG1 or IgG4 isotype. The antibody variable regions structs with different point mutations in the IFN portion. The of these fusion protein constructs are derived from antibody 40 antibody variable regions of these fusion protein constructs G005. The assay is described in the examples as the “on are derived from antibody G005. The assay is described in target (Daudi) assay'. the examples as the "off-target assay.” FIG. 22 shows the anti-proliferative activity of IFNC2b FIG. 31 shows the antiproliferative activity on the mul and two anti-CD38-IFNC. fusion protein with the A145G tiple myeloma cell line ARP-1 of anti-CD38-IFNC. fusion mutation in the IFN portion. Both fusion protein constructs 45 protein constructs with the various mutations in the IFN had the IFN portion linked to the C-terminus of the light portion. The antibody variable regions of these fusion pro chain, with either a six amino acid linker (L6) or no linker tein constructs are derived from antibody G005. The assay (LO). The antibody variable regions of these fusion protein is described in the examples as the “on target (ARP) assay.” constructs are derived from antibody G005. The assay is FIG. 32 shows the antiproliferative activity on the mul described in the examples as the “on target (Daudi) assay.” 50 tiple myeloma cell line ARP-1 of IFNC2b vs anti-CD38 FIG. 23 shows the antiproliferative activity on the mul IFNC. fusion protein constructs with the various mutations in tiple myeloma cell line ARP-1 of IFNC2b vs anti-CD38 the IFN portion. The antibody variable regions of these IFNC. fusion protein constructs with the R144A mutation in fusion protein constructs are derived from antibody G005. the IFN portion. The experiment compares the potency of The assay is described in the examples as the "on target the fusion protein constructs as a function of isotype (IgG1 55 (ARP) assay.”. vs. IgG4) and the presence or absence of the L6 linker FIG. 33 shows the antiproliferative activity on the mul between the antibody heavy chain C-terminus and the N-ter tiple myeloma cell line ARP-1 of IFNC2b vs anti-CD38 minus of the mutated IFN. The antibody variable regions of IFNC. fusion protein constructs with the R144A mutation in these fusion protein constructs are derived from antibody the IFN portion. The experiment compares different anti G005. The assay is described in the examples as the “on 60 body variable regions in the context of the same mutated target (ARP) assay.” IFN fusion protein. The assay is described in the examples FIG. 24 shows the antiproliferative activity on the mul as the “on target (ARP) assay.” tiple myeloma cell line ARP-1 of IFNC2b vs anti-CD38 FIG. 34 shows the antiproliferative activity on the mul IFNC. fusion protein constructs with the A145G mutation in tiple myeloma cell line ARP-1 of IFNC2b vs anti-CD38 the IFN portion. The experiment compares the potency of 65 IFNC. fusion protein constructs with the A145D mutation in the fusion protein constructs as a function of isotype (IgG1 the IFN portion. The experiment compares different anti vs. IgG4) and the presence or absence of the L6 linker body variable regions in the context of the same mutated US 9,611,322 B2 11 12 IFN fusion protein construct. The assay is described in the assay is described in the examples as the “on target (ARP) examples as the “on target (ARP) assay.” assay.” “Ifn equivalents’ refers to the molar concentration of FIG. 35 shows the non-antibody-antigen targeted IFN interferon molecules, either free or attached to an antibody. activity of IFNC2b and various anti-CD38-IFNC. fusion FIG. 44 shows the non-antibody-antigen-targeted IL-4 protein constructs with the R144A mutation in the IFN activity “off-target (HB-IL4) assay of IL-4 and three portion. The experiment compares different antibody vari antibody-IL-4 fusion protein constructs: J1 10-HC-L6-IL-4 able regions in the context of the same mutated IFN fusion IgG1, an anti-PD1 antibody fused to wild type IL-4; J110 protein construct. The assay is described in the examples as HC-L6-IL-4 (R88O), which is identical to the previously the “off-target assay.” mentioned fusion protein construct except for the attenuat FIG. 36 shows the non-antibody-antigen targeted IFN 10 ing R88CR mutation in the IL-4 portion; and Isotype-HC-L6 activity of IFNC2b and various anti-CD38-IFNC. fusion protein constructs with the A145D mutation in the IFN IL-4 (R88O), based on the 2D12 antibody, which does not portion. The experiment compares different antibody vari bind to any of the cells used in the assays of the present able regions in the context of the same mutated IFN fusion invention, and is fused to the attenuated IL-4. “IL-4 equiva protein construct. The assay is described in the examples as 15 lents’ refers to the molar concentration of IL-4 molecules, the “off-target assay.” either free or attached to an antibody. FIG. 37 shows the antiproliferative activity on the mul FIG. 45 shows the “on target (Th1 diversion) assay” tiple myeloma cell line ARP-1 of IFNC2b vs anti-CD38 comparing the activity of IL-4 and three antibody-IL-4 IFNC. fusion protein constructs with the A145D mutation in fusion protein constructs: J110-HC-L6-IL-4 IgG1, an anti the IFN portion. The experiment compares different anti PD1 antibody fused to wild type IL-4; J110-HC-L6-IL-4 body variable regions in the context of the same mutated (R88O), which is identical to the previously mentioned IFN fusion protein construct. The assay is described in the fusion protein construct except for the attenuating R88C examples as the “on target (ARP) assay.” mutation in the IL-4 portion; and Isotype-HC-L6-IL-4 FIG. 38 shows the non-antibody-antigen targeted IFN (R88O), based on the 2D12 antibody, which does not bind activity of IFNC2b and various anti-CD38-IFNC. fusion 25 to any of the cells used in the assays of the present invention, protein constructs with the A145D mutation in the IFN and is fused to the attenuated IL-4. “IL-4 equivalents’ refers portion. The experiment compares different antibody vari to the molar concentration of IL-4 molecules, either free or able regions in the context of the same mutated IFN fusion attached to an antibody. protein construct. The assay is described in the examples as FIG. 46 shows the “IL-6 bioassay” comparing IL-6 with the “off-target assay.” 30 various antibody-IL-6 fusion protein constructs that either FIG. 39 shows the antiproliferative activity on the mul do bind to the target cells (based on the HB95 antibody, tiple myeloma cell line ARP-1 of two antibody-IFNo. fusion which binds to class I MHC on the target cells) or do not protein constructs with the A145D mutation in the IFN bind the target cells (based on the isotype control antibody portion. The nBT062 antibody binds CD138 whereas the 2D12), fused to either wild type IL-6 or IL-6 with the “isotype' antibody does not (it is derived from the antibody 35 attenuating R179E mutation. “IL-6 equivalents’ refers to the 2D12). The assay is described in the examples as the “on molar concentration of IL-6 molecules, either free or target (ARP) assay.” attached to an antibody. FIG. 40 (a) shows the antiproliferative activity on the FIG. 47 shows the effects of various compounds on the multiple myeloma cell line ARP-1 of IFNC2b and two growth of subcutaneous H929 myeloma tumors in SCID antibody-IFNC. fusion protein constructs with the A145D 40 mice. The bar labeled “treatment” shows the duration of mutation in the IFN portion. The HB95 antibody binds treatment with the compounds. The “isotype' antibody was human class I MHC (which is expressed on the ARP-1 cells) based on antibody 2D12. G005 is an anti-CD38 antibody. whereas the “isotype' antibody does not (it is derived from FIG. 48 shows the effects of various compounds on the antibody 2D12). Palivizumab, like 2D12, does not bind survival (Kaplan-Meier graph) of NOD-SCID mice systemi to the ARP-1 cells. (b) shows the same assay, comparing 45 cally inoculated with the human myeloma cell line MM1S. antibody-attenuated IFNC. fusion protein constructs in which The bar labeled “treatment’ shows the duration of treatment the antibody portion is a Fab fragment rather than a full size with the compounds. G005 is an anti-CD38 antibody. antibody. For both (a) and (b) panels, the assay is described FIG. 49 shows the effects of various compounds on the in the examples as the “on target (ARP) assay.” growth of subcutaneous Daudi lymphoma tumors in NOD FIG. 41 shows measurements of the antiviral activity of 50 SCID mice. The bar labeled “treatment’ shows the duration IFNC. and two antibody-IFNC. fusion protein constructs with of treatment with the compounds. The “isotype' antibody the A145D mutation in the IFN portion. This cytopathic was based on antibody 2D12. G005 is an anti-CD38 anti effect inhibition assay utilized the cell line A549 and the body. EMC virus. The HB95 antibody binds human class I MHC FIG. 50 shows the effects of an anti-CD38-attenuated (which is expressed on the A549 cells) whereas the “iso 55 IFNC. fusion protein construct (G005-HC-L6-IFNo. type' antibody derived from the antibody 2D12 does not. (A145G) IgG1) and an isotype control-attenuated IFNC. FIG. 42 shows the non-antibody-antigen targeted IFN fusion protein construct (Isotype-HC-L6-IFNC. (A145G) activity of IFNB, an anti-CD38-IFNB fusion protein con IgG1) on the growth of subcutaneous H929 myeloma tumors struct and an identical fusion protein construct but with the in SCID mice, at various doses. The bar labeled “treatment’ attenuating R35A mutation in the IFN portion. The assay is 60 shows the duration of treatment with the compounds. The described in the examples as the “off-target assay.” “isotype' antibody was based on antibody 2D12. FIG. 43 shows the antiproliferative activity on the mul FIG. 51 shows the effects of anti-CD38-attenuated IFNC. tiple myeloma cell line ARP-1 of IFNB, an anti-CD38-IFNB fusion protein constructs VS isotype control antibody-attenu fusion protein construct and an identical fusion protein ated IFNC. fusion protein constructs, on the growth of construct but with the attenuating R35A mutation in the IFN 65 subcutaneous H929 myeloma tumors in SCID mice. IgG1 is portion. The antibody variable regions of these fusion pro compared to IgG4 in the context of these fusion protein tein constructs are derived from the antibody G005. The constructs. The bar labeled “treatment shows the duration US 9,611,322 B2 13 14 of treatment with the compounds. The “isotype antibody be understood as meaning the amount of signaling activity was based on antibody 2D12. (or downstream effect thereof) at the high, plateau portion of FIG. 52 shows the effects of an anti-CD38-attenuated a dose-response curve, where further increases in the agent IFNC. fusion protein construct (X355/02-HC-L0-IFNo. does not further increase the amount of response). (A145D) IgG4) vs an isotype control antibody-attenuated “Specificity' as used herein is not necessarily an absolute IFNC. fusion protein constructs on the growth of subcuta designation but often a relative term signifying the degree of neous H929 myeloma tumors in SCID mice. The bar labeled selectivity of an antibody-ligand fusion protein construct for “treatment phase' shows the duration of treatment with the an antigen-positive cell compared to an antigen-negative compounds. The “isotype' antibody was based on antibody cell. Thus for example, a construct may be said to have 2D12. 10 “100-fold specificity for antigen-positive cells compared to FIG. 53 shows the effects of various compounds on the antigen-negative cells' and this would indicate that the growth of subcutaneous H929 myeloma tumors in SCID construct has 100-fold higher potency on cells that express mice. G005 is an anti-CD38 antibody. the antigen compared to cells that do not. In some cases, this FIG. 54 shows the effects of an anti-CD38-attenuated degree of specificity of a construct comparing antigen IFNC. fusion protein construct (G005-HC-L6-IFNo. 15 (A145G) IgG4) and an isotype control-attenuated IFNC. positive vs. antigen-negative cells may not be based on the fusion protein construct (Isotype-HC-L6-IFNC. (A145G) absolute ratio of potency of the construct on antigen-positive IgG4) on the growth of subcutaneous H929 myeloma tumors vs. antigen-negative cells, but of the potency of the construct in SCID mice, with several rounds of administration each at on each type of cell relative to the potency of the free, non a dose of 10 mg/kg. The “isotype' antibody was based on attenuated ligand on the same same type of cell. This “ratio antibody 2D12. of ratio” approach for quantifying the degree of specificity FIG. 55 shows the effects of an anti-CD38-attenuated of an antibody-ligand construct takes into consideration any IFNC. fusion protein construct (G005-HC-L6-IFNo. inherent differences in the potency of a ligand on different (A145G) IgG4) on the growth of subcutaneous H929 cell types and is examplefied by the calculations of Antigen myeloma tumors in SCID mice. Dosing (indicated by 25 Specificity Index (ASI) in Table 25. Assays for determining arrows) was initiated when the median tumor volume potency of antibody-ligand fusion constructs are exempli reached 730 mm. fied in the examples and include cell based assays for FIG. 56 shows the inhibition of colony formation from proliferation, apoptosis, phosphorylation of receptors and normal human bone marrow mononuclear cells (BMMNC) intracellular proteins, migration, differentiation (for by IFNa2b, an anti-CD38-attenuated IFNC. fusion protein 30 example, differentiation of naive CD4+ T cells into Th1. construct (G005-HC-L0-IFNC. (A145D) IgG4) and an iso Th17, Th2 VS. Treg cells), increases or decreases in gene type control antibody-attenuated IFNC. fusion protein con expression or gene product secretion into the media, etc. struct 2D12-HC-L0-IFNC. (A145D) IgG4. The antibody Accordingly, in a first aspect the present invention pro attenuated IFNC. fusion protein constructs show about vides a polypeptide construct comprising a peptide or poly 10,000-fold reduced potency in this assay. 35 peptide signaling ligand linked to an antibody or antigen FIG. 57 shows the effects of IFNC2b vs an antibody binding portion thereof which binds to a cell surface attenuated IFNC. fusion protein construct (Isotype-HC-L6 associated antigen wherein the ligand comprises at least one IFNC. (A145G) IgG1; the isotype variable regions are based amino acid substitution or deletion which reduces its on antibody 2D12) on cytokine production by human potency on cells lacking expression of said antigen. peripheral blood mononuclear cells (PBMCs). (a) IP-10 and 40 In one embodiment the present invention provides a MCP-1; (b) MCP-3 and IL-1C. polypeptide construct comprising IFN linked to an antibody or antigen binding portion thereof which binds to a tumour DETAILED DESCRIPTION OF THE associated antigen wherein the IFN comprises at least one INVENTION amino acid substitution or deletion which reduces its 45 potency on cells lacking expression of said antigen. Such a The constructs of the present invention are antibody polypeptide will be capable of exerting with high potency attenuated ligand constructs, which show an elevated anti the IFN's anti-proliferative activity on the antigen-positive gen-specificity index with respect to activating signaling tumor cells while exerting a much lower potency on the pathways due to the action of the attenuated ligand on a cell antigen-negative, non-tumour cells within the body. Surface receptor. These constructs are based on the Surpris 50 In a second aspect the present invention provides a ing discovery that, in the context of an antibody-ligand method of treating a tumour in a Subject comprising admin construct, the ligand portion can be mutated in Such a way istering to the Subject the polypeptide construct of the that the ligand activity on antigen-negative cells is dramati present invention. cally attenuated, while the ligand activity on antigen-posi The term “antibody-ligand construct” as used herein tive cells is only modestly, if at all, attenuated. Such con 55 refers to an antibody or antigen-binding fragment thereof structs display one, two, three, four or five orders of covalently attached to a signaling ligand that has been magnitude greater potency on antigen-positive cells com attenuated by one or more substitutions or deletions that pared to antigen negative cells than does the free ligand. In reduce the ligand’s potency on cells that do not express the one embodiment, the antibody-attenuated ligand construct antigen corresponding to the antibody. retains at least 1%, at least 10%, at least 20%, at least 30%, 60 The term “antibody', as used herein, broadly refers to any at least 40% or at least 50% of the potency on antigen immunoglobulin (Ig) molecule comprised of four polypep positive cells as the non-attenuated free (i.e. not attached to tide chains, two heavy (H) chains and two light (L) chains, an antibody) ligand. In addition, in one embodiment the or any functional fragment, mutant, variant, or derivation antibody-attenuated ligand construct retains at least 30%, at thereof, which retains the essential epitope binding features least 50%, at least 75% or at least 90% of the maximal 65 of an Ig molecule. Such mutant, variant, or derivative activity of the non-attenuated free (i.e. not attached to an antibody formats are known in the art, non-limiting embodi antibody) ligand; in this context, “maximal activity” should ments of which are discussed below. US 9,611,322 B2 15 16 In a full-length antibody, each heavy chain is comprised primate) grafted onto or inserted into FRS from a human of a heavy chain variable region (abbreviated herein as antibody (this type of antibody is also referred to a “CDR HCVR or VH) and a heavy chain constant region. The heavy grafted antibody'). Humanized antibodies also include pro chain constant region is comprised of three domains, CH1, teins in which one or more residues of the human protein are CH2 and CH3. Each light chain is comprised of a light chain modified by one or more amino acid Substitutions and/or one variable region (abbreviated herein as LCVR or VL) and a or more FR residues of the human protein are replaced by light chain constant region. The light chain constant region corresponding non-human residues. Humanized antibodies is comprised of one domain, CL. The VH and VL regions may also comprise residues which are found in neither the can be further subdivided into regions of hypervariability, human antibody or in the non-human antibody. Any addi termed complementarity determining regions (CDR), inter 10 tional regions of the protein (e.g., Fc region) are generally spersed with regions that are more conserved, termed frame human. Humanization can be performed using a method work regions (FR). Each VH and VL is composed of three known in the art, e.g., U.S. Pat. No. 5.225,539, U.S. Pat. No. CDRs and four FRs, arranged from amino-terminus to 6,054,297, U.S. Pat. No. 7,566,771 or U.S. Pat. No. 5,585, carboxy-terminus in the following order: FR1, CDR1, FR2, 089. The term “humanized antibody' also encompasses a CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can 15 Super-humanized antibody, e.g., as described in U.S. Pat. be of any type (e.g., IgG, IgE, IgM, Ig), IgA and IgY), class No. 7,732,578. (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or Subclass. The antibody described herein may be human. The term The term “antigen binding domain or “antigen binding “human antibody' as used herein refers to proteins having portion' of an antibody, as used herein, refers to one or more variable and, optionally, constant antibody regions found in fragments of an antibody or protein that retain the ability to humans, e.g. in the human germline or Somatic cells or from specifically bind to an antigen (e.g., CD38). It has been libraries produced using such regions. The “human' anti shown that the antigen-binding function of an antibody can bodies can include amino acid residues not encoded by be performed by fragments of a full-length antibody. Such human sequences, e.g. mutations introduced by random or antibody embodiments may also be bispecific, dual specific, site directed mutations in vitro (in particular mutations or multi-specific formats, specifically binding to two or 25 which involve conservative Substitutions or mutations in a more different antigens. Examples of binding fragments Small number of residues of the protein, e.g. in 1, 2, 3, 4 or encompassed within the term “antigen-binding portion of 5 of the residues of the protein). These “human antibodies' an antibody include (i) a Fab fragment, a monovalent do not necessarily need to be generated as a result of an fragment consisting of the VL, VH, CL and CH1 domains: immune response of a human, rather, they can be generated (ii) a F(ab')2 fragment, a bivalent fragment comprising two 30 using recombinant means (e.g., screening a phage display Fab fragments in addition to a portion of the hinge region, library) and/or by a transgenic animal (e.g., a mouse) linked by a disulfide bridge at the hinge region; (iii) an Fd comprising nucleic acid encoding human antibody constant fragment consisting of the VH and CH1 domains; (iv) an Fv and/or variable regions and/or using guided selection (e.g., fragment consisting of the VL and VH domains of a single as described in or U.S. Pat. No. 5,565,332). This term also arm of an antibody, (v) a domain antibody (dAb) (Ward et 35 encompasses affinity matured forms of Such antibodies. For al. 1989 Nature 341 544-6, Winter et al., PCT publication the purposes of the present disclosure, a human protein will WO 90/05144 A1 herein incorporated by reference), which also be considered to include a protein comprising FRS from comprises a single variable domain; and (vi) an isolated a human antibody or FRS comprising sequences from a complementarity determining region (CDR). Furthermore, consensus sequence of human FRS and in which one or more although the two domains of the Fv fragment, VL and VH, 40 of the CDRS are random or semi-random, e.g., as described are coded for by separate genes, they can be joined, using in U.S. Pat. No. 6,300,064 and/or U.S. Pat. No. 6,248,516. recombinant methods, by a synthetic linker that enables The antibody portions of polypeptides of the present them to be made as a single protein chain in which the VL invention may be full length antibodies of any class, pref and VH regions pair to form monovalent molecules (known erably IgG1, IgG2 or IgG4. The constant domains of Such as single chain Fv (scFV); see e.g., Bird et al. 1988 Science 45 antibodies are preferably human. The variable regions of 242 423-6; Huston et al. 1988 Proc Natl Acad Sci USA 85 Such antibodies may be of non-human origin or, preferably, 5879-83). Such single chain antibodies are also intended to be of human origin or be humanized. Antibody fragments be encompassed within the term “antigen-binding portion' may also be used in place of the full length antibodies. of an antibody. Other forms of single chain antibodies, such The term “antibody' also includes engineered antibodies. as diabodies, are also encompassed. Diabodies are bivalent, 50 As will be appreciated there are many variations of engi bispecific antibodies in which VH and VL domains are neered antibodies (e.g. mouse monoclonal, chimeric, expressed on a single polypeptide chain, but using a linker humanized and human monoclonal antibodies, single chain that is too short to allow for pairing between the two variable antibody fragments (ScPv's), minibodies, aptamers, domains on the same chain, thereby forcing the domains to as well as bispecific antibodies and diabodies as described pair with complementary domains of another chain and 55 above). creating two antigen binding sites (see e.g., Holliger, P., et Single variable region domains (termed dAbs) are, for al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, example, disclosed in (Ward et al., Nature 341: 544-546, R. J., et al., 1994, Structure 2:1121-1123). Such antibody 1989; Hamers-Casterman et al., Nature 363: 446-448, 1993; binding portions are known in the art (Kontermann and Davies & Riechmann, FEBS Lett. 339: 285-290, 1994). Dubel eds. Antibody Engineering 2001 Springer-Verlag. 60 Minibodies are small versions of whole antibodies, which New York. 790 pp., ISBN 3-540-41354-5). In an embodi encode in a single chain the essential elements of a whole ment the antibody binding portion is a Fab fragment. antibody. Suitably, the minibody is comprised of the VHand The antibody described herein may be may be a human VL domains of a native antibody fused to the hinge region ized antibody. The term “humanized antibody' shall be and CH3 domain of the immunoglobulin molecule as, for understood to refer to a protein comprising a human-like 65 example, disclosed in U.S. Pat. No. 5,837,821. variable region, which includes CDRs from an antibody In an alternate embodiment, the engineered antibody may from a non-human species (e.g., mouse or rat or non-human comprise non-immunoglobulin derived, protein frame US 9,611,322 B2 17 18 works. For example, reference may be made to (Ku & such as by reductive alkylation by reaction with an aldehyde Schutz, Proc. Natl. Acad. Sci. USA 92: 6552-6556, 1995) followed by reduction with NaBH; amidination with which discloses a four-helix bundle protein cytochrome methylacetimidate; acylation with acetic anhydride; carbam b562 having two loops randomized to create CDRs, which oylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene Sulphonic acid have been selected for antigen binding. (TNBS); acylation of amino groups with succinic anhydride There is a plethora of non-antibody recognition protein or and tetrahydrophthalic anhydride; and pyridoxylation of protein domain scaffolds that may be utilised as the antigen lysine with pyridoxal-5-phosphate followed by reduction binding domains in the constructs of this invention. These with NaBH. include scaffolds based on cytotoxic T lymphocyte-associ The guanidine group of arginine residues may be modi ated antigen 4 (CTLA-4) (Evibody; U.S. Pat. No. 7,166, 10 fied by the formation of heterocyclic condensation products 697); human transferrin (Trans-body); a three-helix bundle with reagents such as 2.3-butanedione, phenylglyoxal and from the Z-domain of Protein A (Affibody); a monomeric or glyoxal. trimeric human C-type lectin domain (Tetranectin); the tenth The carboxyl group may be modified by carbodiimide human fibronectin type III domain (AdNectin); the Kunitz activation via O-acylisourea formation followed by subse type domain of human or bovine trypsin inhibitor; insect 15 quent derivatisation, for example, to a corresponding amide. Defensin A (IICA29), APPI (Kuntiz domains); lipocalins, Sulphydryl groups may be modified by methods such as FABP. Bilin-binding protein, Apoloproptein D (Anticalins): carboxymethylation with iodoacetic acid or iodoacetamide; human C-crystallin or ubiquitin molecule (Affilin); trypsin performic acid oxidation to cysteic acid; formation of a inhibitor II (Microbody); O.2p8 or Ankyrin repeat (repeat mixed disulphides with other thiol compounds; reaction motif proteins), Charybdotoxin (Scorpion toxins), Min-23, with maleimide, maleic anhydride or other substituted Cellulose binding domain (Knottins); Neocarzinostatin, maleimide; formation of mercurial derivatives using 4-chlo CBM4-2 and Tendamistat. romercuribenzoate, 4-chloromercuriphenylsulphonic acid, Further, in addition to scaffolds provided for by antibody phenylmercury chloride, 2-chloromercuri-4-nitrophenol and derived domains or non-antibody folds as described above, other mercurials; carbamoylation with cyanate at alkaline there are naturally occurring ligand binding proteins or 25 pH. protein domains that may be utilised as the ligand binding Tryptophan residues may be modified by, for example, domains in this invention. For example, protein domains that oxidation with N-bromosuccinimide or alkylation of the possess ligand binding properties include extracellular indole ring with 2-hydroxy-5-nitrobenzyl bromide or sul domains of receptors, PDZ modules of signalling proteins, phenyl halides. Tyrosine residues on the other hand, may be Such as Ras-binding protein AF-6, adhesion molecules, and 30 altered by nitration with tetranitromethane to form a 3-ni enzymes. trotyrosine derivative. The present invention further encompasses chemical ana Modification of the imidazole ring of a histidine residue logues of amino acids in the Subject antibodies. The use of may be accomplished by alkylation with iodoacetic acid chemical analogues of amino acids is useful inter alia to 35 derivatives or N-carbethoxylation with diethylpyrocarbon stabilize the molecules such as if required to be administered ate. to a Subject. The analogues of the amino acids contemplated Examples of incorporating unnatural amino acids and herein include, but are not limited to, modifications of side derivatives during peptide synthesis include, but are not chains, incorporation of unnatural amino acids and/or their limited to, use of norleucine, 4-aminobutyric acid, 4-amino derivatives during peptide, polypeptide or protein synthesis 3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, and the use of crosslinkers and other methods which impose 40 t-butylglycine, norvaline, phenylglycine, ornithine, Sar conformational constraints on the proteinaceous molecule or cosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thie their analogues. nyl alanine and/or D-isomers of amino acids. A list of Examples of side chain modifications contemplated by the unnatural amino acid, contemplated herein is shown in Table present invention include modifications of amino groups 1. TABLE 1.
Non-conventional amino acid Code Non-conventional amino acid Code C-aminobutyric acid Abu L-N-methylalanine Nimala C-amino-O-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane Cpro L-N-methylasparagine NmaSn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisoleucine Nmile D-alanine Dal L-N-methyleucine Nimleu D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine Dgln L-N-methylnorvaline Nminva D-glutamic acid Dglu L-N-methylornithine Nmorn D-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline Nmpro D-leucine Deu L-N-methylserine Nmser D-lysine Dlys L-N-methylthreonine Nimthr D-methionine Dmet L-N-methyltryptophan Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr US 9,61 1,322 B2 19 20 TABLE 1-continued
Non-conventional amino acid Code Non-conventional amino acid Code D-phenylalanine Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle D-tryptophan L-norvaline Nwa D-tyrosine C.-methyl-aminoisobutyrate Maib D-valine C-methyl-y-aminobutyrate Mgabu D-O-methylalanine C.-methylcyclohexylalanine Mchexa D-O-methylarginine C.-methylcycopentylalanine Mcpen D-O-methylasparagine C.-methyl-C-napthylalanine Manap D-O-methylaspartate C.-methylpenicillamine Mpen D-O-methylcysteine N-(4-aminobutyl)glycine Nglu D-O-methylglutamine N-(2-aminoethyl)glycine Naeg D-O-methylhistidine N-(3-aminopropyl)glycine Norn D-O-methylisoleucine N-amino-C.-methylbutyrate Nmaabu D-O-methyleucine C-napthylalanine D-O-methyllysine N-benzylglycine D-O-methylmethionine N-(2-carbamylethyl)glycine D-O-methylornithine N-(carbamylmethyl)glycine D-O-methylphenylalanine N-(2-carboxyethyl)glycine D-O-methylproline N-(carboxymethyl)glycine D-O-methylserine N-cyclobutylglycine D-O-methylthreonine N-cycloheptylglycine Nchep D-O-methyltryptophan N-cyclohexylglycine Nchex D-O-methyltyrosine N-cyclodecylglycine Nicdec D-O-methylvaline N-cylcododecylglycine Nicdod D-N-methylalanine N-cyclooctylglycine Nicoct D-N-methylarginine N-cyclopropylglycine Nicpro D-N-methylasparagine N-cycloundecylglycine Ncund D-N-methylaspartate N-(2,2-diphenylethyl)glycine Nbhm D-N-methylcysteine N-(3,3-diphenylpropyl)glycine Nbhe D-N-methylglutamine N-(3-guanidinopropyl)glycine D-N-methylglutamate N-(1-hydroxyethyl)glycine Nthr D-N-methylhistidine N-(hydroxyethyl)glycine Nser D-N-methylisoleucine N-(imidazolylethyl)glycine Nhis D-N-methyleucine N-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine N-methyl-y-aminobutyrate Nmgabu N-methylcyclohexylalanine D-N-methylmethionine Dnmmet D-N-methylornithine N-methylcyclopentylalanine Nmcpen N-methylglycine D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate D-N-methylproline Dnimpro N-(1-methylpropyl)glycine D-N-methylserine Dnmser N-(2-methylpropyl)glycine D-N-methylthreonine Dnmthr D-N-methyltryptophan N-(1-methylethyl)glycine Nval D-N-methyltyrosine N-methyla-napthylalanine Nmanap D-N-methylvaline N-methylpenicillamine Nmpen Y-aminobutyric acid N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine N-(thiomethyl)glycine Ncys L-ethylglycine penicillamine Pen L-homophenylalanine L-C.-methylalanine Mala L-C.-methylarginine L-C.-methylasparagine Masin L-C.-methylaspartate L-C.-methyl-t-butylglycine Mtbug L-C.-methylcysteine L-methylethylglycine Metg L-C.-methylglutamine L-C.-methylglutamate Mglu L-C-methylhistidine L-C.-methylhomophenylalanine Mhphe L-C.-methylisoleucine N-(2-methylthioethyl)glycine Nmet L-C.-methyleucine L-C-methyllysine Mlys L-C.-methylmethionine L-C.-methylmorleucine Minle L-C.-methylnorvaline L-C-methylornithine Morn L-C.-methylphenylalanine L-C-methylproline Mpro L-C.-methylserine L-C-methylthreonine Mthr L-C.-methyltryptophan L-C-methyltyrosine Mtyr L-C-methylvaline L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine -carboxy-1-(2,2-diphenyl ethylamino)cyclopropane
Crosslinkers can be used, for example, to stabilize 3D " as maleimido or dithio moiety (SH) or carbodiimide conformations, using homo-bifunctional crosslinkers such (COOH). as the bifunctional imido esters having (CH2)n spacer Using methods well known in the art to increase binding, groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccin by for example, affinity maturation, or to decrease immu imide esters and hetero-bifunctional reagents which usually 65 nogenicity by removing predicted MHC class II-binding contain an amino-reactive moiety Such as N-hydroxysuccin motifs. The therapeutic utility of the antibodies described imide and another group specific-reactive moiety Such herein can be further enhanced by modulating their func US 9,611,322 B2 21 22 tional characteristics, such as antibody-dependent cell-me receptors, including FcRn binding and serum half-life, are diated cytotoxicity (ADCC), complement-dependent cyto described in U.S Pat. Application Nos. 20090142340: toxicity (CDC), serum half-life, biodistribution and binding 2009 OO68175 and 20090092599. to Fc receptors or the combination of any of these. This The glycans linked to antibody molecules are known to modulation can be achieved by protein-engineering, glyco 5 influence interactions of antibody with Fc receptors and engineering or chemical methods. Depending on the thera glycan receptors and thereby influence antibody activity, peutic application required, it could be advantageous to including serum half-life (Kaneko, Nimmerjahn et al. 2006; either increase or decrease any of these activities. Jones, Papac et al. 2007; and Kanda, Yamada et al. 2007). An example of glyco-engineering used the Potelligent(R) Hence, certain glycoforms that modulate desired antibody method as described in Shinkawa T. et al., 2003 (J Biol 10 activities can confer therapeutic advantage. Methods for Chem 278: 3466-73). generating engineered glycoforms are known in the art and Numerous methods for affinity maturation of antibodies include but are not limited to those described in U.S. Pat. are known in the art. Many of these are based on the general Nos. 6,602,684; 7,326,681; 7,388,081 and WO 2008/ strategy of generating panels or libraries of variant proteins OO6554. by mutagenesis followed by selection and/or screening for 15 Extension of half-life by addition of polyethylene glycol improved affinity. Mutagenesis is often performed at the (PEG) has been widely used to extend the serum half-life of DNA level, for example by error prone PCR (Thie, Voedisch proteins, as reviewed, for example, by Fishburn 2008. et al. 2009), by gene shuffling (Kolkman and Stemmer As will be recognised it is possible to make conservative 2001), by use of mutagenic chemicals or irradiation, by use amino acid substitutions within the sequences of the current of mutator strains with error prone replication machinery invention. By “conservative substitution is meant amino (Greener 1996) or by somatic hypermutation approaches acids having similar properties. As used in this specification that harness natural affinity maturation machinery (Peled, the following groups of amino acids are to be seen as Kuang et al. 2008). Mutagenesis can also be performed at conservative substitutions: H. R and K; D, E, N and Q. V. I the RNA level, for example by use of QB replicase (Kop and L; C and M: S, T, P, A and G.; and F, Y and W. sidas, Roberts et al. 2006). Library-based methods allowing 25 The term 'cell Surface-associated antigen', as used screening for improved variant proteins can be based on herein, broadly refers to any antigen expressed on Surfaces various display technologies such as phage, yeast, ribosome, of cells, including infectious or foreign cells or viruses. bacterial or mammalian cells, and are well known in the art In certain aspects of the present invention, the polypeptide (Benhar 2007). Affinity maturation can be achieved by more constructs or compositions of the present invention may be directed/predictive methods for example by site-directed 30 used to treat patients with cancer. Cancers contemplated mutagenesis or gene synthesis guided by findings from 3D herein include: a group of diseases and disorders that are protein modeling (see for example Queen, Schneider et al. characterized by uncontrolled cellular growth (e.g. forma 1989 or U.S. Pat. No. 6,180,370 or U.S. Pat. No. 5,225,539). tion of tumor) without any differentiation of those cells into Methods of increasing ADCC have been described by specialized and different cells. Such diseases and disorders Ferrara, Brunker et al. 2006; Li, Sethuraman et al. 2006; 35 include ABL1 protooncogene, AIDS related cancers, acous Stavenhagen, Gorlatov et al. 2007; Shields, Namenuk et al. tic neuroma, acute lymphocytic leukaemia, acute myeloid 2001; Shinkawa, Nakamura et al. 2003; and WO 2008/ leukaemia, adenocystic carcinoma, adrenocortical cancer, OO6554. agnogenic myeloid metaplasia, alopecia, alveolar soft-part Methods of increasing CDC have been described by sarcoma, anal cancer, angiosarcoma, aplastic anaemia, astro Idusogie, Wong et al. 2001; Dall’Acqua, Cook et al. 2006; 40 cytoma, ataxia-telangiectasia, basal cell carcinoma (skin), Michaelsen, Aase et al. 1990; Brekke, Bremnes et al. 1993; bladder cancer, bone cancers, bowel cancer, brain stem Tan, Shopes et al. 1990; and Norderhaug, Brekke et al. 1991. glioma, brain and CNS tumors, breast cancer, CNS tumors, References describing methods of increasing ADCC and carcinoid tumors, cervical cancer, childhood brain tumors, CDC include Natsume. In et al. 2008. The disclosure of each childhood cancer, childhood leukaemia, childhood soft tis of these references is included herein by cross reference. 45 Sue Sarcoma, chondrosarcoma, choriocarcinoma, chronic A number of methods for modulating antibody serum lymphocytic leukaemia, chronic myeloid leukaemia, col half-life and biodistribution are based on modifying the orectal cancers, cutaneous T-Cell lymphoma, dermatofibro interaction between antibody and the neonatal Fc receptor sarcoma-protuberans, desmoplastic-Small-round-cell-tumor, (FcRn), a receptor with a key role in protecting IgG from ductal carcinoma, endocrine cancers, endometrial cancer, catabolism, and maintaining high serum antibody concen 50 ependymoma, esophageal cancer, Ewings sarcoma, extra tration. Dall’Acqua et al describe substitutions in the Fc hepatic bile duct cancer, eye cancer, eye: melanoma, retino region of IgG1 that enhance binding affinity to FcRn, blastoma, fallopian tube cancer, fanconi anemia, fibrosar thereby increasing serum half-life (Dall’Acqua, Woods et al. coma, gall bladder cancer, gastric cancer, gastrointestinal 2002) and further demonstrate enhanced bioavailability and cancers, gastrointestinal-carcinoid-tumor, genitourinary modulation of ADCC activity with triple substitution of 55 cancers, germ cell tumors, gestational-trophoblastic-disease, M252Y/S254T/T256E (Dall’Acqua, Kiener et al. 2006). See glioma, gynaecological cancers, hematological malignan also U.S. Pat. Nos. 6,277,375; 6,821,505; and 7,083,784. cies, hairy cell leukaemia, head and neck cancer, hepatocel Hinton et all have described constant domain amino acid lular cancer, hereditary breast cancer, histiocytosis, Hodg substitutions at positions 250 and 428 that confer increased kin's disease, human papillomavirus, hydatidiform mole, in vivo half-life (Hinton, Johlfs et al. 2004). (Hinton, Xiong 60 hypercalcemia, hypopharynx cancer, intraocular melanoma, et al. 2006). See also U.S. Pat. No. 7,217,797. Petkova et al islet cell cancer, Kaposi's sarcoma, kidney cancer, Langer have described constant domain amino acid substitutions at han's-cell-histiocytosis, laryngeal cancer, leiomyosarcoma, positions 307, 380 and 434 that confer increased in vivo leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, half-life (Petkova, Akilesh et al. 2006). See also Shields et liver cancer, lung cancer, lymphedema, lymphoma, Hodg all 2001 and WO 2000/42072. Other examples of constant 65 kin's lymphoma, non-Hodgkin’s lymphoma, male breast domain amino acid Substitutions which modulate binding to cancer, malignant-rhabdoid-tumor-of-kidney, medulloblas Fc receptors and Subsequent function mediated by these toma, melanoma, merkel cell cancer, mesothelioma, meta US 9,611,322 B2 23 24 static cancer, mouth cancer, multiple endocrine neoplasia, itic organs such as pancreas, brain, spleen and liver mycosis fungoides, myelodysplastic syndromes, multiple (Koguma, T., Biochim. Biophys. Acta 1223:160, 1994.) myeloma, myeloproliferative disorders, nasal cancer, CD38 is a multifunctional ectoenzyme that is involved in nasopharyngeal cancer, nephroblastoma, neuroblastoma, transmembrane signaling and cell adhesion. It is also known neurofibromatosis, nijmegen breakage syndrome, non-mela as cyclic ADP ribose hydrolase because it can transform noma skin cancer, non-Small-cell-lung-cancer—(NSCLC), NAD" and NADP" into cADPR, ADPR and NAADP, ocular cancers, oesophageal cancer, oral cavity cancer, oro depending on extracellular pH. These products induce Ca"- pharynx cancer, osteosarcoma, ostomy ovarian cancer, pan mobilization inside the cell which can lead to tyrosine creas cancer, paranasal cancer, parathyroid cancer, parotid phosphorylation and activation of the cell. CD38 is also a gland cancer, penile cancer, peripheral-neuroectodermal 10 receptor that can interact with a ligand, CD31. Activation of receptor via CD31 leads to intracellular events including tumors, pituitary cancer, polycythemia Vera, prostate cancer, Ca" mobilization, cell activation, proliferation, differentia rare-cancers-and-associated-disorders, renal cell carcinoma, tion and migration (reviewed in Deaglio, S., Trends in Mol. retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson Med. 14:210, 2008.) syndrome, salivary gland cancer, sarcoma, Schwannoma, 15 CD38 is expressed at high levels on multiple myeloma Sezary syndrome, skin cancer, Small cell lung cancer cells, in most cases of T- and B-lineage acute lymphoblastic (SCLC), Small intestine cancer, soft tissue sarcoma, spinal leukemias, some acute myelocytic leukemias, follicular cen cord tumors, squamous-cell-carcinoma-(skin), stomach can ter cell lymphomas and T lymphoblastic lymphomas. (Mala cer, synovial sarcoma, testicular cancer, thymus cancer, vasi, F. J. Clin Lab Res. 22:73, 1992). More recently, CD38 thyroid cancer, transitional-cell-cancer-(bladder), transi expression has become a reliable prognostic marker in tional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic can B-lineage chronic lymphoblastic leukemia (B-CLL) (Ibra cer, urethral cancer, urinary system cancer, uroplakins, uter him, S., Blood. 98:181, 2001 and Durig, J., Leuk. Res. ine sarcoma, uterus cancer, vaginal cancer, Vulva cancer, 25:927, 2002). Independent groups have demonstrated that Waldenstrom's-macroglobulinemia and Wilms tumor. In an B-CLL patients presenting with a CD38 clone are charac embodiment the tumor is selected from a group of multiple 25 terized by an unfavorable clinical course with a more myeloma or non-hodgkin’s lymphoma. advance stage of disease, poor responsiveness to chemo As contemplated for the treatment of cancer, the antibody therapy and shorter survival time (Morabito. F., Haemato portions of the constructs of the present invention may bind logica. 87:217, 2002). The consistent and enhanced expres to tumour-associated antigens, i.e., cell Surface antigens that sion of CD38 on lymphoid tumors makes this an attractive are selectively expressed by cancer cells or over-expressed 30 target for therapeutic antibody technologies. in cancer cells relative to most normal cells. There are many Preferred antigens for the development of antibody-at tumour-associated antigens (TAAS) known in the art. Non tenuated ligand fusion protein constructs which target cancer limiting examples of TAAS include enzyme tyrosinase; are antigens which show selective or greater expression on melanoma antigen GM2; alphafetoprotein (AFP); carcino the cancer cells than on most other, non-transformed cells embryonic antigen (CEA); Mucin 1 (MUC1); Human epi 35 within the body. Non-protein examples of Such antigens dermal growth factor receptor (Her2/Neu); T-cell leukemia/ include, sphingolipids, ganglioside GD2 (Saleh et al., 1993, lymphoma 1 (TCL1) oncoprotein. Exemplary TAAS J. Immunol. 151, 3390-3398), ganglioside GD3 (Shitara et associated with a number of different cancers are telomerase al., 1993, Cancer Immunol. Immunother. 36:373-380), gan (hTERT); prostate-specific membrane antigen (PSMA); uro glioside GM2 (Livingston et al., 1994, J. Clin. Oncol. kinase plasminogen activator and its receptor (uPA/uPAR); 40 12:1036-1044), ganglioside GM3 (Hoon et al., 1993, Cancer vascular endothelial growth factor and its receptor (VEGF/ Res. 53:5244-5250) and Lewis, lewis' and lewis' carbo VEGFR); extracellular matrix metalloproteinase inducer hydrate antigens that can be displayed on proteins or gly (EMMPRIN/CD147); epidermal growth factor (EGFR): colipids. Examples of protein antigens are HER-2/neu, platelet-derived growth factor and its receptor (PDGF/ human papillomavirus-E6 or -E7, MUC-1; KS 1/4 pan PDGFR) and c-kit (CD117). 45 carcinoma antigen (Perez and Walker, 1990, J. Immunol. A list of other TAAs is provided in US 2010/0297076, the 142:3662-3667: Bumal, 1988, Hybridoma 7(4):407-415): disclosure of which is included herein by reference. Of ovarian carcinoma antigen CA125 (Yu et al., 1991, Cancer particular interest are cell Surface antigens associated with Res. 51(2):468-475); prostatic acid phosphate (Tailor et al., multiple myeloma cells, including but not limited to CD38, 1990, Nucl. Acids Res. 18(16):4928); prostate specific anti CD138, CS1, and HM1.24. In one embodiment an antigen 50 gen (Henttu and Vihko, 1989, Biochem. Biophys. Res. for antibody-attenuated ligand constructs, for example, an Comm. 160(2):903-910; Israeli et al., 1993, Cancer Res. antibody-attenuated interferon construct, is CD38. 53:227-230); melanoma-associated antigen p97 (Estin et al., CD38 is a 46 kDa type II transmembrane glycoprotein. It 1989, J. Natl. Cancer Instit. 81 (6):445-446); melanoma has a short N-terminal cytoplasmic tail of 20 amino acids, a antigen gp75 (Vijayasardahl et al., 1990, J. Exp. Med. single transmembrane helix and a long extracellular domain 55 171 (4): 1375-1380); prostate specific membrane antigen; of 256 amino acids (Bergsagel, P. Blood: 85:436, 1995 and carcinoembryonic antigen (CEA) (Foon et al., 1994, Proc. Liu, Q., Structure, 13:1331, 2005). It is expressed on the Am. Soc. Clin. Oncol. 13:294), MUC16 (antibodies include surface of many immune cells including CD4 and CD8 MJ-170, MJ-171, MJ-172 and MJ-173 U.S. Pat. No. 7,202, positive T cells, B cells, NK cells, monocytes, plasma cells 346, 3A5 U.S. Pat. No. 7,723.485). NMB (U.S. Pat. No. and on a significant proportion of normal bone marrow 60 8,039,593), malignant human lymphocyte antigen-APO-1 precursor cells (Malavasi, F. Hum. Immunol. 9:9, 1984). In (Bernhard et al., 1989, Science 245:301-304); high molecu lymphocytes, however, the expression appears to be depen lar weight melanoma antigen (HMW-MAA) (Natali et al., dent on the differentiation and activation state of the cell. 1987, Cancer 59:55-63; Mittelman et al., 1990, J. Clin. Resting T and B cells are negative while immature and Invest. 86:2136-2144); Burkitt's lymphoma antigen-38.13; activated lymphocytes are predominantly positive for CD38 65 CD19 (Ghetie et al., 1994, Blood 83:1329-1336); human expression (Funaro, A. J. Immunol. 145:2390, 1990). Addi B-lymphoma antigen-CD20 (Ref et al., 1994, Blood tional studies indicate mRNA expression in non-hemopoe 83:435-445); GICA 19-9 (Herlyn et al., 1982, J. Clin. US 9,611,322 B2 25 26 Immunol. 2:135), CTA-1 and LEA; CD33 (Sgouros et al., BR96 mAb (Trail et al., 1993, Science 261:212-215), mAbs 1993, J. Nucl. Med. 34:422-430); oncofetal antigens such as against the CD40 antigen, such as S2C6 mAb (Francisco et alphafetoprotein for liver cancer or bladder tumor oncofetal al., 2000, Cancer Res. 60:3225-3231) or other anti-CD40 antigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188); antibodies, such as those disclosed in U.S. Patent Publication differentiation antigens such as human lung carcinoma anti Nos. 2003-0211100 and 2002-0142358; mAbs against the gen L6 or L20 (Hellstrom et al., 1986, Cancer Res. 46:3917 CD30 antigen, such as AC 10 (Bowen et al., 1993, J. Immu 3923); antigens of fibrosarcoma; human leukemia T cell nol. 151:5896-5906: Wahl et al., 2002 Cancer Res.62(13): antigen-Gp37 (Bhattacharya-Chatterjee et al., 1988, J. 3736-42) or MDX-0060 (U.S. Patent Publication No. 2004 Immunol. 141:1398-1403); tumor-specific transplantation 0006215) and mAbs against the CD70 antigen, such as 1F6 type of cell-surface antigen (TSTA) such as virally-induced 10 tumor antigens including T-antigen, DNA tumor virus and mAb and 2F2 mAb (see, e.g., U.S. Patent Publication No. envelope antigens of RNA tumor viruses; neoglycoproteins, 2006-0083736) or antibodies 2H5, 10B4, 8B5, 18E7, 69A7 breast cancer antigens such as EGFR (Epidermal growth (U.S. Pat. No. 8,124,738). Other antibodies have been factor receptor), polymorphic epithelial mucin (PEM) (Hilk reviewed elsewhere (Franke et al., 2000, Cancer Biother. ens et al., 1992, Trends in Bio. Chem. Sci. 17:359); poly 15 Radiopharm. 15:45976; Murray, 2000, Semin. Oncol. 27:64 morphic epithelial mucin antigen; human milk fat globule 70; Breitling, F., and Dubel, S. Recombinant Antibodies, antigen; colorectal tumor-associated antigens such as TAG John Wiley, and Sons, New York, 1998). 72 (Yokata et al., 1992, Cancer Res. 52:3402-3408), CO In certain embodiments, useful antibodies can bind to a 17-1A (Ragnhammar et al., 1993, Int. J. Cancer 53:751 receptor or a complex of receptors expressed on a target cell. 758); differentiation antigens (Feizi, 1985, Nature 314:53 The receptor or receptor complex can comprise an immu 57) such as I(Ma) found in gastric adenocarcinomas, noglobulin gene Superfamily member, a major histocompat SSEA-1 found in myeloid cells, VEP8, VEP9, Myl, VIM ibility protein, a cytokine receptor, a TNF receptor super D5, M18 and M39 found in breast epithelial cancers, Ds family member, a chemokine receptor, an integrin, a lectin, found in colorectal cancer, TRA-1-85 (blood group H), C14 a complement control protein, a growth factor receptor, a found in colonic adenocarcinoma, F3 found in lung adeno 25 hormone receptor or a neuro-transmitter receptor. Non carcinoma, AH6 found in gastric cancer, Y hapten found in limiting examples of appropriate immunoglobulin Super embryonal carcinoma cells, TL5 (blood group A), E1 series family members are CD2, CD3, CD4, CD8, CD19, CD22, (blood group B) antigens found in pancreatic cancer, FC 10.2 CD79, CD90, CD152/CTLA-4, PD-1, B7-H4, B7-H3, and found in embryonal carcinoma cells, gastric adenocarci ICOS. Non-limiting examples of suitable TNF receptor noma antigen, CO-514 (blood group Le') found in adeno 30 superfamily members are TACI, BCMA, CD27, CD40, carcinoma, NS-10 found in adenocarcinomas, CO-43 (blood CD95/Fas, CD134/OX40, CD137/4-1BB, TNFR1, TNFR2, group Le’), G49 found in A431 cells, 19.9 found in colon RANK, osteoprotegerin, APO 3, Apo2/TRAIL R1, TRAIL cancer; gastric cancer mucins; R found in melanoma, MH2 R2, TRAIL R3, and TRAIL R4. Non-limiting examples of (blood group ALe”/Le’) found in colonic adenocarcinoma, suitable integrins are CD11a, CD11b, CD11c, CD18, CD29, 4.2. D1.1, OFA-1, G, OFA-2 and M1:22:25:8 found in 35 CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, embryonal carcinoma cells and SSEA-3 and SSEA-4. CD103 and CD104. Non-limiting examples of suitable HMW-MAA (SEQ ID NO:433), also known as melanoma lectins are S type, C type, and I type lectin. Examples of chondroitin Sulfate proteoglycan, is a membrane-bound pro antibodies to CEA are shown in Table 2. tein of 2322 residues which is overexpressed on over 90% of the Surgically removed benign nevi and melanoma lesions 40 (Camploi, et. al. Crit. Rev Immunol. 24:267, 2004). TABLE 2 Accordingly it may be a potential target cell Surface asso CEA Antibodies ciated antigen. Other example cancer antigens for targeting with fusion Ab Clones Patent Assignee Comments protein constructs of the present invention include (exem 45 COL-1 U.S. Pat. No. 6,417,337 The Dow Chemical Humanized plary cancers are shown in parentheses): CD5 (T-cell leu Company kemia/lymphoma), CA15-3 (carcinomas), CA19-9 (carcino 806.077 U.S. Pat. No. 6,903.203 AstraZeneca UK Ltd. Humanized mas), L6 (carcinomas), CA 242 (colorectal), placental T84.66 U.S. Pat. No. 7,776,330 City of Hope Humanized alkaline phosphatase (carcinomas), prostatic acid phos phatase (prostate), MAGE-1 (carcinomas), MAGE-2 (car 50 Antibodies that bind the CD22 antigen expressed on cinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), human B cells include, for example, HD6, RFB4, UV22-2, transferrin receptor (carcinomas), p97 (melanoma), MUC1 To 15, 4KB128 and a humanized anti-CD22 antibody (hLL2) (breast cancer), MART 1 (melanoma), CD20 (non Hodgkin’s (see, e.g., Li et al. (1989) Cell. Immunol. 111: 85-99; Mason lymphoma), CD52 (leukemia), CD33 (leukemia), human et al. (1987) Blood 69: 836-40; Behr et al. (1999) Clin. chorionic gonadotropin (carcinoma), CD38 (multiple 55 Cancer Res. 5: 3304s-3314s; Bonardi et al. (1993) Cancer myeloma), CD21 (B-cell lymphoma), CD22 (lymphoma), Res. 53: 3015-3021). CD25 (B-cell Lymphoma), CD37 (B-cell lymphoma), CD45 Antibodies to CD33 include, for example, HuM195 (see, (acute myeloblastic leukemia), HLA-DR (B-cell lym e.g., Kossman et al. (1999) Clin. Cancer Res. 5: 2748–2755: phoma), IL-2 receptor (T-cell leukemia and lymphomas), U.S. Pat. No. 5,693,761) and CMA-676 (see, e.g., Sieverset CD40 (lymphoma), various mucins (carcinomas), P21 (car 60 al., (1999) Blood 93: 3678-3684). cinomas), MPG (melanoma), Ep-CAM (Epithelial Tumors), Illustrative anti-MUC-1 antibodies include, but are not Folate-receptor alpha (Ovarian), A33 (Colorectal), G250 limited to Mc5 (see, e.g., Peterson et al. (1997) Cancer Res. (renal), Ferritin (Hodgkin lymphoma), de2-7 EGFR (glio 57: 1103-1108; Ozzello et al. (1993) Breast Cancer Res. blastoma, breast, and lung), Fibroblast activation protein Treat. 25: 265-276), and hCTMO1 (see, e.g., Van H of et al. (epithelial) and tenascin metalloproteinases (glioblastoma). 65 (1996) Cancer Res. 56: 5179-5185). Some specific, useful antibodies include, but are not limited Illustrative anti-TAG-72 antibodies include, but are not to, BR64 (Trail et al., 1997, Cancer Research 57:100 105), limited to CC49 (see, e.g., Pavlinkova et al. (1999) Clin. US 9,611,322 B2 27 28 Cancer Res. 5: 2613-2619), B72.3 (see, e.g., Divgi et al. copies per cell. Methods for determining copy number of a (1994) Nucl. Med. Biol. 21:9-15), and those disclosed in cell Surface antigen are well known and readily available to U.S. Pat. No. 5,976,531. a person of skill in the art, for example the method provided Illustrative anti-HM1.24 antibodies include, but are not by Jilana (Am J Clin Pathol 118:560-566, 2002) limited to a mouse monoclonal anti-HM1.24 and a human It may be advantageous for the cell Surface-associated ized anti-HM1.24 IgG1 kappa antibody (see, e.g., Ono et al. antigen to be expressed in a configuration on the cell Surface (1999) Mol. Immuno. 36:387-395). such that the polypeptide construct is able to contact both the In certain embodiments the targeting moiety comprises an cell Surface antigen and the ligand receptor on the target cell. anti-HER2 antibody. The erBB 2 gene, more commonly Accordingly, in particular embodiments the cell Surface known as (Her-2/neu), is an oncogene encoding a trans 10 membrane receptor. Several antibodies have been developed associated antigen has an extracellular domain having a against Her-2/neu, including trastuzumab (e.g., HERCEP molecular weight of less than 240 kD. TINTM: Fornier et al. (1999) Oncology (Huntingt) 13: 647 It may be advantageous for the antibody or antigen 58), TAB-250 (Rosenblum et al. (1999) Clin. Cancer Res. 5: binding portion thereof to bind to the cell surface associated 865-874), BACH-250 (Id.), TA1 (Maier et al. (1991) Cancer 15 antigen with Sufficient affinity to facilitate ligand binding to Res. 51: 5361-5369), and the mabs described in U.S. Pat. the ligand receptor on the cell Surface. Accordingly, in Nos. 5,772.997: 5,770,195 (mAb 4D5; ATCC CRL 10463); particular embodiments of the present invention the poly and U.S. Pat. No. 5,677,171. peptide constructs exhibit an antigen binding affinity, as A number of antibodies have been developed that spe measured by EC50, of from 50 nM, from 25 nM, from 10 cifically bind HER2 and some are in clinical use. These nM or from 5 nM to 0.1 p.M. include, for example, trastuzumab (e.g., HERCEPTINTM, As described in U.S. Pat. Nos. 6,512,097 and 5,977,322, Fornier et al. (1999) Oncology (Huntingt) 13: 647-658), other anti-EGFR family member antibodies can readily be TAB-250 (Rosenblum et al. (1999) Clin. Cancer Res. 5: produced by shuffling light and/or heavy chains followed by 865-874), BACH-250 (Id.), TA1 (see, e.g., Maier et al. one or more rounds of affinity selection. Thus in certain (1991) Cancer Res. 51: 5361-5369), and the antibodies 25 embodiments, this invention contemplates the use of one, described in U.S. Pat. Nos. 5,772,997; 5,770, 195, and 5,677, two, or three CDRs in the VL and/or VH region that are 171. CDRs described in the above-identified antibodies and/or Other fully human anti-HER2/neu antibodies are well the above identified publications. known to those of skill in the art. Such antibodies include, In various embodiments the targeting antibody or antigen but are not limited to the C6 antibodies such as C6.5, DPL5, 30 binding portion thereof comprises an antibody or antigen G98A, C6MH3-B1, B1D2, C6VLB, C6VLD, C6VLE, binding portion thereof that specifically or preferentially C6VLF, C6MH3-D7, C6MH3-D6, C6MH3-D5, C6MH3 binds CD20. Anti-CD20 antibodies are well known to those D3, C6MH3-D2, C6MH3-D1, C6MH3-C4, C6MH3-C3, of skill and include, but are not limited to Rituximab, C6MH3-B9, C6MH3-B5, C6MH3-B48, C6MH3-B47, Ibritumomab, and Tositumomab, AME-133v (Applied C6MH3-B46, C6MH3-B43, C6MH3-B41, C6MH3-B39, 35 Molecular Evolution). Ocrelizumab (Roche), Ofatumumab C6MH3-B34, C6MH3-B33, C6MH3-B31, C6MH3-B27, (Genmab), TRU-015 (Trubion) and IMMU-106 (Immuno C6MH3-B25, C6MH3-B21, C6MH3-B20, C6MH3-B2, medics). C6MH3-B16, C6MH3-B15, C6MH3-B11, C6MH3-B1, WO 2010/105290 discloses an antibody designated C6MH3-A3, C6MH3-A2, and C6ML3-9. These and other “SC104 together with a range of humanised variants which anti-HER2/neu antibodies are described in U.S. Pat. Nos. 40 bind an antigen expressed on a range of tumour cells. 6,512,097 and 5,977,322, in PCT Publication WO 97/00271, In an embodiment, the antibody attachment and attenu in Schier et al. (1996) J Mol Biol 255: 28-43, Schier et al. ating mutation in the ligand increases the antigen-specificity (1996) J Mol Biol 263: 551-567, and the like. index (ASI) by greater than 10-fold, preferably greater than More generally, antibodies directed to various members 50-fold, preferably greater than 100-fold, preferably greater of the epidermal growth factor receptor family are well 45 than 1000-fold, or preferably greater than 10,000 fold. The Suited for use as targeting antibodies or antigen binding antigen-specificity index (ASI) is defined herein as the fold portions thereof in the constructs of the present invention. increased potency in signaling activity of the polypeptide Such antibodies include, but are not limited to anti-EGF-R construct of the invention relative to the free non-mutated antibodies as described in U.S. Pat. Nos. 5,844,093 and polypeptide ligand on target antigen-positive cells multi 5,558,864, and in European Patent No. 706,799A. Other 50 plied by the fold decreased potency in signaling activity illustrative anti-EGFR family antibodies include, but are not relative to the free non-mutated polypeptide ligand on target limited to antibodies such as C6.5, C6ML3-9, C6MH3-B1, antigen-negative cells. The term “potency’ in this context C6-B1D2, F5, HER3A5, HER3.F4, HER3.H1, HER3.H3, may be quantitatively represented by the EC50 value, which HER3. E12, HER3.B12, EGFR.E.12, EGFR.C10, is the mathematical midpoint of a dose-response curve, in EGFR.B11, EGFR.E.8, HER4.B4, HER4.G4, HER4.F4, 55 which the dose refers to the concentration of ligand or HER4A8, HER4.B6, HER4.D4, HER4.D7, HER4D11, antibody-ligand construct in an assay, and response refers to HER4.D12, HER4.E3, HER4.E7, HER4 F8 and HER4.C7 the quantitative response of the cells to the signaling activity and the like (see, e.g., U.S. Patent publications US 2006/ of the ligand at a particular dose. Thus, for example, when 0099205 A1 and US 2004/0071696 A1 which are incorpo a first compound is shown to possess an EC50 (expressed for rated herein by reference). 60 example in Molar units) that is 10-fold lower than a second It may be advantageous for the cell Surface-associated compound's EC50 on the same cells, typically when mea antigen to be expressed at Sufficient levels on the target cell Sured by the same method, the first compound is said to have that a sufficiently therapeutic amount of polypeptide con a 10-fold higher potency. Conversely, when a first com struct is presented to ligand receptors on the target cell pound is shown to possess an EC50 that is 10-fold higher Surface. Accordingly, in particular embodiments, the cell 65 than a second compound's EC50 on the same cells, typically Surface associated antigen is expressed at a density of when measured by the same method, the first compound is greater than 12,600 copies per cell or greater than 15,000 said to have a 10-fold lower potency. US 9,611,322 B2 29 30 While the large majority of antibodies tested showed believe that the relatively low copy number of this target efficient targeting of attenuated IFNC, the present inventors may in some cases, depending on other factors such as identified examples of two antigens where targeting attenu epitope position, the receptor density of the IFN receptors, ated IFNC. to a target-expressing cell line did not exhibit an etc., limit the potency of the antibody-attenuated IFN fusion protein constructs. It is, however, possible that other anti ASI that was appreciably greater than for the free, non bodies that target other epitopes on this antigen may support mutated ligand. The first example is demonstrated by the the targeted IFN activity, or that other cells with low copy antigen CSPG4 (also known as HMW-MAA, high molecu numbers of CD33 may nevertheless respond to such fusion lar weight melanoma-associated antigen). We tested two protein constructs due to higher intrinsic IFN sensitivity, for different anti-HMW-MAA-antibody-IFNC. fusion protein example. Despite this possibility it is preferred that the constructs in on-target proliferation assays using A375 or 10 antibody or antigen binding portion thereof of the polypep CHL-1 cell lines. We did not see inhibitory activity with tide construct of the present invention binds an antigen either cell line or antibody at the doses tested (EC50s-21 wherein the antigen is present on the cell at a density of nM). The extracellular domain of this antigen is exception greater than 12,600 copies per cell, preferably greater than ally large (extracellular domain MW approx. 240 kD-450 15,000 copies per cell. kD depending on glycosylation). It is possible that certain 15 Another example of an antibody-attenuated fusion protein antibody-IFN fusion protein constructs that bind to very construct in which the antibody did not provide sufficient large antigens may be sterically restricted from simultane targeting to the cancer cells was an anti-GM2 ganglioside ously interacting with the IFN receptors on the same cells. antibody attached to an attenuated IFNC. In this case, the It is, however, possible that other antibodies that target other antibody was to a carbohydrate epitope and, as typical of epitopes of this antigen may support the targeted IFN such antibodies, had a low affinity (EC50 for binding target activity. Despite this possibility it is preferred that the cells was ~50 nM by flow cytometry). Therefore, preferred antibody or antigen binding portion thereof of the polypep embodiments of the present invention show high affinity tide construct of the present invention binds an antigen binding to their antigens, with EC50s preferably below 50 wherein the extracellular domain thereof has a molecular 25 nM, more preferably below 25 nM, still more preferably weight of less than 240 kD. below 10 nM and ideally below 5 nM. In addition, preferred A second example of an antibody-attenuated IFNC. fusion embodiments comprise antibodies that bind to protein and protein construct that did not show potent activity was based peptide epitopes rather than carbohydrate epitopes. on an antibody which binds to the myeloid antigen CD33. Multiple myeloma is of particular interest for certain CD33 is expressed at a relatively low level on KG-1 cells, 30 embodiments of the present invention, namely fusion pro reported as 12,600 copies per cell (Sutherland, MAbs. 1(5): tein constructs comprising antibodies to multiple myeloma 481-490, 2009). Treatment of KG-1 cells with an anti-CD33 antigens and attenuated IFN peptides. Table 3 lists examples antibody-attenuated IFNC. fusion protein construct failed to of multiple myeloma antigens and antibodies, with a refer inhibit proliferation at all doses tested (IC50>76 nM). We ence to antibody sequences. TABLE 3 Examples of Ab in preclinical or clinical Clinical trial Target development Sequence citation reference CD40 Dacetuzumab SGN-40 USPTO Granted NCTOO664898 & U.S. Pat. No. 7,666.422 NCTOO525447 CD40 Lucatumumab HCD-122 USPTOH2OO70098.718 NCTOO231166 CHIR12.12 HM124 XmAb5592 humanized + Fc USPTO#20100104557 1999, Ozaki, Blood, 93: 3922 CDS6 HN901-DM1 1994, Roguska et al., NCTOO3462SS & BB-10901 PNAS 91: 969-973 NCTOO991S62 CS1 Elotuzumab Huluco.3 USPTO Granted NCTOO742S60 U.S. Pat. No. 7,709,610 &NCT00726869 CD138 BTO62 USPTO H2O090175863 2008, Tassone, Blood, 104:3688 Milatuzumab Immu-110 Granted NCT00421525, Stein U.S. Pat. No. 7,312,318 et. Al. 2007 and 2009 Tocilizumab MRA Granted 2007, Yoshio U.S. Pat. No. 5,795,965 Hoshino, Canc Res, 67; 871 Trail-R1 Mapatumumab, anti-DR4 Granted NCTOO315757 U.S. Pat. No. 7,252,994 Trail-R2 (DR5, Lexatumumab, ETR2-STO1, Granted 2006, Menoret, APO-2) anti-DR5 U.S. Pat. No. 6,872,568 Blood, 132; 1356 Baff Belimumab LY2127399 Granted U.S. Pat. No. 7,317,089 ICOSL AMG-SS7 USPTO application Number 2008O166352 BCMA SG1 USPTO application 2007, Ryan, Mol Number 2012008266 Cancer Ther, 6: 3009 HLA-DR 1DO9C3 USPTO Granted 2007, Carlo-Stella, U.S. Pat. No. 7,521,047 Canc. Res., Kininogen C11C1 USPTO Granted 2006, Sainz, Canc U.S. Pat. No. 4,908,431 Immunol Immunother US 9,611,322 B2 31 32 TABLE 3-continued Examples of Ab in preclinical or clinical Clinical trial Target development Sequence citation reference 32microglobulin ATCC Cat iHB-149 2007, Yang, Blood, 110: 3028; 2009, Clin Can Res, 15: 951 FGFR3 Pro-OO1 USPTO Grante 2006, Trudel, Blood, U.S. Pat. No. 8,187,601 2: 4908 ICAM-1 cUV3 USPTO Grante 2004, Smallshaw, J U.S. Pat. No. 7,943,744 Immunother: 2006, Coleman Matriptase M24-DOX USPTO Grante 2010, Bertino, AACR U.S. Pat. No. 7,355,015 abstract no. 2596 CD2O Rituxan and others U.S. patent NCTOO2S82O6 & application No.: NCT00505895 US 2010.O189729 A1 CD52 Campath-1H USPTO Grante NCTOO62S144 U.S. Pat. No. 6,569.430 EGFR Erbitux (Emma-1) USPTO Grante NCTOO3681.21 U.S. Pat. No. 6,217,866 GM2 BIW-8962 USPTO Grante Biowa, no ref U.S. Pat. No. 6,872,392 C4-integrin natalizumab USPTO Grante NCTOO67S428 U.S. Pat. No. 5,840,299 IGF-1R CD-751, 871 figitumumab USPTO Grante Lacy, J. Clin. Oncol, U.S. Pat. No. 7,700,742 26:3196 (TBD-need to connect Ab 4.9.2 to CD751,871) KIR IPH2101 USPTO Granted NCT00552396; U.S. Pat. No. 8,119,775 2009, ASCO abs. 09 AB-3032;
CD38 is of particular interest as an antibody target for through 1,000 amino acids in length, which bind to particu fusion protein constructs of the present invention. Antibod lar cell surface molecules (“receptors') on certain cells and ies to CD38 include for example, AT13/5 (see, e.g., Ellis et thereby transmit a signal or signals within those cells. al. (1995) J. Immunol. 155: 925-937), HB7, and the like. 35 Exemplary signaling ligands and polypeptide signaling Table 4 discloses several known CD38 antibodies that may ligands contemplated by the present invention include, but be used in this context: are not limited to cytokines, chemokines, growth factors, hormones, neurotransmitters, and apoptosis inducing fac TABLE 4 tOrS. 40 Non-limiting examples of suitable cytokines include the Company Clone names Sequence citation Ref interleukin’s IL-1, IL-2 IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, Genmab, GOO3. GOO5, GO24 WO 2006/099875 De Weers, M., IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, Janssen (Daratumumab) A1 J Immunol. 186: Biotech Inc 1840, 2011 IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL 25, MorphoSys MORO3077, U.S. 2009.O123950 IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, Il-35 AG MORO3079, A1 45 and their subfamilies; the interferon (IFN) subfamily includ MORO3O80, MORO3100 ing Interferon type I (IFN-O. (IFNA1, IFNA2, IFNA4, (MOR202) IFNA5, IFNA6. IFNA7, IFNA8, IFNA10, IFNA13, Sanofi- 38SB13, 38SB18, U.S. 2009/0304710 IFNA14, IFNA16, IFNA17, IFNA21), IFN-B (IFN-?31 Aventis 38SB19, 38SB30, A1 (IFNB1) and IFN-B3 (IFNB3)), IFN-co (IFNW1), IFNWP2, US. LLC. 38SB31, 38SB39 50 (SAR650984) IFNWP4, IFNWP5, IFNWP9, IFNWP15, IFNWP18, and Tenovus UK Chimeric OKT10 U.S. 2010/0285004 A1 Stevenson, F., IFNWP19 and IFNK), Interferon type II (IFN-Y) and Inter Parental hybridoma Blood. 77: feron type III (IFN-epsilon, -kappa, -omega, -delta, -tau, and ATCC accession: 1071, 1991 CRL-8O22 -gamma) and interferon-like molecules (limitin, IL-28A, Immunogen HB7-Ricin Hybridoma: ATCC Goldmacher, 55 IL-28B, and IL-29; the IL-1 family including IL-1C, IL-1B. HB-136 V., Blood, 84: the IL-1 Receptor antagonist (IL-1RA) and IL1F5, IL1F6, 3017, 1994 IL1 F7, IL1F8, IL1F9 and IL1F10 and the IL-17 family including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (IL-25), The term “Signaling ligand’ as used herein broadly and IL-17F. In an embodiment the peptide or polypeptide includes any ligand involved in the activation of cell sig 60 signaling ligand is selected from the group consisting of an naling pathways, including any molecule capable of acti IFN, IL-4 and IL-6. In an embodiment the peptide or vating or inhibiting cell surface receptors. The term should polypeptide signaling ligand is selected from the group also be understood as including reference to molecules that consisting of IFNC, IFNC2b. IFNB1, IFNB1b and IFNY. can pass through the lipid bilayer of the cell membrane to Preferably the sequence of IFNC. is selected from SEQ ID activate cell signaling pathways within the cell. The term 65 NOS 1 to 3, 80 to 90, 434 and 435. "polypeptide signaling ligand’ as used herein refers to Exemplary chemokines include, for example, RANTES, peptide and polypeptide sequences of length 6 amino acids MCAF, MIP1-alpha, IP-10, monocyte chemoattractant pro US 9,611,322 B2 33 34 tein-1 (MCP-1 or CCL2), interleukin-8 (IL-8), CXCL13, which conjugates free amino groups and thiols from Cys XCL1 (lymphotactin-C), XCL2 (lymphotactin-B) and frac residues, benzidine (BDB) which links to Tyr residues, talkine (CXCL1). periodate which attaches to carbohydrate groups and isoth Non-limiting examples of growth factors include, for iocyanate. The use of commercial chemical conjugation kits example, Adrenomedullin (AM), Angiopoietin (Ang), Auto is contemplated. crine motility factor, Bone morphogenetic proteins (BMPs), In some embodiments, labels are attached via spacer arms Brain-derived neurotrophic factor (BDNF), Epidermal of various lengths to reduce potential steric hindrance. For growth factor (EGF), Erythropoietin (EPO), Fibroblast example, a chemical linker may be used between the ligand growth factor (FGF), Glial cell line-derived neurotrophic and the antibody. Exemplary linker sequences will be readily factor (GDNF), Granulocyte colony-stimulating factor 10 (G-CSF), Granulocyte macrophage colony-stimulating fac ascertained by those of skill in the art, and are likely to tor (GM-CSF), Growth differentiation factor-9 (GDF9), include linkers such as C6, C7 and C12 amino modifiers and Hepatocyte growth factor (HGF), Hepatoma-derived growth linkers comprising thiol groups. factor (HDGF). Insulin-like growth factor (IGF), Migration The antibody-ligand fusion protein constructs of the pres stimulating factor, Myostatin (GDF-8), Nerve growth factor 15 ent invention have mutations or deletions in the ligand that (NGF) and other neurotrophins, Platelet-derived growth render the ligands less active in stimulating their receptors factor (PDGF). Thrombopoietin (TPO), Transforming on cells that lack cell Surface expression of the antigen to growth factor alpha (TGF-C.), Transforming growth factor which the antibody binds. beta (TGF-B), Tumor necrosis factor-alpha (TNF-C.), Vas In one aspect of the present invention, the ligand is an cular endothelial growth factor (VEGF), placental growth interferon, examples of which are type I interferons (IFN-C. factor (PIGF), IL-1-Cofactor for IL-3 and IL-6, IL-2-T-cell (alpha), IFN-?3 (beta), IFN-K (kappa), IFN-8 (delta), IFN-e growth factor, IL-3, IL-4, IL-5, IL-6 and IL-7. (epsilon), IFN-T (tau), IFN-() (omega), and IFN- (Zeta, also Exemplary apoptosis inducing factors include FasL and known as limitin), type II interferons (IFN-Y) or type III TRAIL. interferons (IFN-1, IFN-2 and IFN-3) (Pestka, Immu Exemplary hormones include peptide hormones such as 25 nological Reviews 202(1):8-32, 2004). TRH and vasopressin, protein hormones such as insulin and Type I interferons all signal through the Type I interferon growth hormone, glycoprotein hormones such as Luteiniz receptor, which is made of of IFNAR1 and IFNAR2. Sig ing hormone, follicle-stimulating hormone and thyroid naling occurs when a type I IFN binds IFNAR1 and stimulating hormone, Lipid and phospholipid-derived hor IFNAR2, thus bringing them together into a complex with mones such as steroid hormones e.g. testosterone and 30 cortisol, Sterol hormones such as calcitriol, eicosanoids Such the IFN. This initiates a cascade of intracellular events (the as prostaglandins. “signaling') which leads, among other things, to changes in Non-limiting examples of Suitable neurotransmitters the expression of numerous interferon regulated genes. include monoamines and other biogenic amines: dopamine Details of the intracellular signaling events triggered by (DA), norepinephrine (noradrenaline; NE, NA), epinephrine 35 activation of the type I interferon receptor is described, for (adrenaline), histamine, serotonin (SE, 5-HT), Somatostatin, example, by Platanias, (Nature Reviews 5:375-86. 2005). substance P, opioid peptides and acetylcholine (ACh), Type I interferons include various interferon-alphas. Known The linkage between the antibody and the ligand could be human interferon-alphas are IFNC.1b, C2C, C23, C4b, C5, made via a simple peptide bond by creating a fusion protein C6, C7, C8, C10, C.1a/13, C. 14, C16, C17, Ovö, O21, C2c, and between the ligand and the heavy or light chain, or both, of 40 C.44a. Some embodiments comprise IFN C2b, the sequence the antibody. The ligand could be attached at either the N of which, SEQID NO:3, is shown in FIG. 4. IFNs have been or C-terminus of either the heavy or the light chain of the approved in several forms for several indications, as out antibody, with or without an intervening linker peptide lined in Table 5 (which also shows lists of approved IFNB sequence. In an embodiment the ligand is linked to the and Ys): antibody or antigen binding portion thereof via a peptide 45 bond. In one embodiment, the ligand is linked to the TABLE 5 C-terminus of the heavy chain of a human, humanized or chimeric IgG1, IgG2 or IgG4, either directly or with an Generic Name Trade name Approved for treatment intervening linker of 1 to 20 amino acids in length. interferon alpha 2a Roferon A Hep C, CML, Hairy cell The mutated polypeptide ligands may be attached to the 50 Leukemia, NHL, Kaposi's antibody or antibody fragment by means of chemical con S8CO8. interferon alpha 2b Intron A/Reliferon Hep C, Hep B, Hairy cell, jugation, non-covalent protein-protein interactions, or by Uniferon melanoma, leukemia, genetic fusion. Methods for conjugating the ligands NHL, Kaposi's sarcoma described herein with antibodies may be readily accom Human leukocyte Multiferon Melanoma, viral plished by one of ordinary skill in the art. As will be readily 55 (CO and malignant (HuIFN-C-Le) disease ascertained, commonly used chemical coupling methods interferon beta 1a, liquid Rebif Multiple Sclerosis may be utilized to link ligands to antibodies via for example, interferon beta1a, AvoneX Multiple Sclerosis free amino, carboxylic acid, or Sulfhydryl groups. Ligands yophylized interferon beta1a, Cinnovex Multiple Sclerosis can also be linked to antibodies via Carbonyls ( CHO); biogeneric (Iran) these aldehyde groups can be created by oxidizing carbo 60 interferon beta1b Betaseron/Betaferon Multiple Sclerosis hydrate groups in glycoproteins. interferon beta1b, Ziferon Multiple Sclerosis Some commonly used cross-linking reagents include glu biosimilar (Iran) PEGylated interferon Pegasys Hepatitis B and C taraldehyde which links protein or peptide molecules to the alpha 2a N-terminal or aliphatic amine groups of peptides or poly PEGylated interferon Reiferon Retard Hep C, Hep B, Hairy cell, peptides, carbodiimide (EDC) which attaches proteins or 65 alpha 2a (Egypt) melanoma, leukemia, peptides to the C-terminus or side chain carboxyl groups of NHL, Kaposi's sarcoma proteins or peptides, succinimide esters (e.g. MBS, SMCC) US 9,611,322 B2 35 36 TABLE 5-continued TABLE 7 Relative affinity of interferon mutants to their receptors Generic Name Trade name Approved for treatment
PEGylated interferon PegIntron Hepatitis and melanoma Generic Name Trade name Approved for treatment alpha 2b PEGylated interferon Pegetron Hepatitis C 10 alpha 2b plus ribavirin (Canada) Interferon alfacon-1 Infergen Hepatitis C Interferon alpha n3 Alferon N Genital warts 15 Interferon gamma Actimmune Chronic granulo matous disease
Non-limiting examples of mutations in IFNC2b that can be used to reduce its potency are described in Tables 6 and 7, based on the sequence of human IFNC2b (SEQ ID NO:3):
TABLE 6 25 Relative biological activities of interferon mutants relative anti- relative anti viral activity proliferative activity 30
35
40 N65A, L80A, Y85A, Y89A, R120A ND Y85A, Y89A, R120A O.91 D114A, R120A O.83 L117A, R120A 1.4 L117A, R120A, K121A O.14 O.91 45 R120A, K121A 1.7 R120E, K121E 1.3 A(161-165) O.S3
These mutants have known reductions in binding to the 50 type 1 interferon receptor IFNAR1 or IFNAR2, and/or have known reductions in IFNC potency based on cell-based assayS. The data in these tables was disclosed in the following references: 55 Piehler, Jacob, Roisman, Laila C., Schreiber, Gideon (2000). New structural and functional aspects of the Type I interferon-receptor interaction revealed by comprehen sive mutational analysis of the binding interface. J. Biol. Chen. 275: 40425-40433. 60 Jaitin, Diego A, Roisman, Laila C, Jaks, Eva, Gavutis, Martynas, Piehler, Jacob, Van der Heyden, Jose, Uze, Gilles, Schreiber, Gideon (2006). Inquiring into the dif ferential action of interferons (IFNs): an IFN-O2 mutant with enhanced affinity to IFNAR1 is functionally similar 65 to IFN-B. Mol. Cell. Biol. 26: 1888-1897. Slutzki, Michal, Jaitin, Diego A. Yehezkel, Tuval Ben, Schreiber, Gideon (2006). Variations in the unstructured US 9,611,322 B2 37 38 C-terminal tail of interferons contribute to differential tic approaches, such as ABVD (Adriamycin (doxorubicin), receptor binding and biological activity.J. Mol. Biol. 360: bleomycin, vinblastine, and dacarbazine), or Stanford V 1019-1030. (doxorubicin, bleomycin, vinblastine, Vincristine, mechlo Kalie, Eyal, Jaitin, Diego A., Abramovich, Renne, Schreiber, rethamine, etoposide, prednisone), or BEACOPP (doxoru Gideon (2007). An interferon C.2 mutant optimized by 5 bicin, bleomycin, Vincristine, cyclophosphamide, procarba phage display for IFNAR1 binding confers specifically Zine, etoposide, prednisone). enhanced antitubor activities. J. Biol. Chem. 282: 11602 In the case of non-Hodgkin’s lymphoma or other lympho 11611. mas, it is contemplated that the constructs of the present Pan, Manjing, Kalie, Eyal, Scaglione, Brian J. Raveche, invention may be administered in combination current thera Elizabeth S., Schreiber, Gideon, Langer, Jerome A. 10 peutic approaches. Examples of drugs approved for non (2008). Mutation of to IFNAR-1 receptor binding site of Hodgkin lymphoma include Abitrexate (Methotrexate), human IFN-C2 generates Type IIFN competitive antago Adriamycin PFS (Doxorubicin Hydrochloride), Adriamycin nists. Biochemistry 47: 12018-12027. RDF (Doxorubicin Hydrochloride), Ambochlorin (Chloram Kalie, Eyal, Jaitin, Diego A., Podoplelova, Yulia, Piehler, bucil), Amboclorin (Chlorambucil), Arranon (Nelarabine), Jacob, Schreiber, Gideon (2008). The Stability of the 15 Bendamustine Hydrochloride, Bexxar (Tositumomab and ternary interferon-receptor complex rather than the affin Iodine I 131 Tositumomab), Blenoxane (Bleomycin), Bleo ity to the individual subunits dictates differential biologi mycin, Bortezomib, Chlorambucil, Clafen (Cyclophosph cal activities. J. Biol. Chem. 283: 32925-32936. amide), Cyclophosphamide, Cytoxan (Cyclophosphamide), The abbreviation “YNS” is sometimes used herein to Denileukin Diftitox, DepoCyt (Liposomal Cytarabine), represent IFNC, variants including the following mutation: 20 Doxorubicin Hydrochloride, DTIC-Dome (Dacarbazine), H57Y, E58N and Q61S. Folex (Methotrexate), Folex PFS (Methotrexate), Folotyn The present invention also contemplates combinations of (Pralatrexate), Ibritumomab Tiuxetan, Istodax (Ro the abovementioned mutations or deletions in IFNC. midepsin), Leukeran (Chlorambucil), Linfolizin (Chloram The invention also contemplates the combination of the bucil), Liposomal Cytarabine, Matulane (Procarbazine constructs of the present invention with other drugs and/or 25 Hydrochloride), Methotrexate, Methotrexate LPF (Metho in addition to other treatment regimens or modalities such as trexate), Mexate (Methotrexate), Mexate-AQ (Methotrex radiation therapy or surgery. When the contructs of the ate), Mozobil (Plerixafor), Nelarabine, Neosar (Cyclophos present invention are used in combination with known phamide), Ontak (Denileukin Diftitox), Plerixafor, therapeutic agents the combination may be administered Pralatrexate, Rituxan (Rituximab), Rituximab, Romidepsin, either in sequence (either continuously or broken up by 30 Tositumomab and Iodine I 131 Tositumomab, Treanda (Ben periods of no treatment) or concurrently or as an admixture. damustine Hydrochloride), Velban (Vinblastine Sulfate), In the case of cancer, there are numerous known anticancer Velcade (Bortezomib), and Velsar (Vinblastine Sulfate), agents that may be used in this context. Treatment in Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vin combination is also contemplated to encompass the treat cristine Sulfate, Vorinostat, Zevalin (Ibritumomab Tiux ment with either the construct of the invention followed by 35 etan), Zolinza (Vorinostat). Examples of drug combinations a known treatment, or treatment with a known agent fol used in treating non-Hodgkin lymphoma include CHOP lowed by treatment with the construct of the invention, for (C=Cyclophosphamide, H=Doxorubicin Hydrochloride example, as maintenance therapy. For example, in the treat (Hydroxydaunomycin), O=Vincristine Sulfate (Oncovin), ment of cancer it is contemplated that the constructs of the P=Prednisone); COPP (C=Cyclophosphamide, present invention may be administered in combination with 40 O=Vincristine Sulfate (Oncovin), P=Procarbazine Hydro an alkylating agent (Such as mechlorethamine, cyclophos chloride, P-Prednisone); CVP (C=Cyclophosphamide, phamide, chlorambucil, ifosfamidecysplatin, or platinum V=Vincristine Sulfate, P=Prednisone); EPOCH containing alkylating-like agents such as cysplatin, carbo (E=Etoposide, P-Prednisone, O=Vincristine Sulfate (On platin and oxaliplatin), an antimetabolite (such as a purine or covin), C=Cyclophosphamide, H=Doxorubicin Hydrochlo pyrimidine analogue or an antifolate agent, Such as azathio- 45 ride (Hydroxydaunomycin)); ICE (I-Ifosfamide, prine and mercaptopurine), an anthracycline (such as C=Carboplatin, E=Etoposide) and R-CHOP (R-Rituximab, Daunorubicin, Doxorubicin, Epirubicin Idarubicin, Valrubi C=Cyclophosphamide, H-Doxorubicin Hydrochloride (Hy cin, Mitoxantrone, or anthracycline analog), a plant alkaloid droxydaunomycin), O=Vincristine Sulfate (Oncovin), (such as a Vinca alkaloid or a taxane, Such as Vincristine, P=Prednisone. Vinblastine, Vinorelbine, Vindesine, paclitaxel or 50 Combination of retinoids with interferon-based fusion Dosetaxel), a topoisomerase inhibitor (Such as a type I or protein constructs is also contemplated. Retinoids are a type II topoisomerase inhibitor), a Podophyllotoxin (such as family of molecules that play a major role in many biologi etoposide or teniposide), or a tyrosine kinase inhibitor (Such cal functions including growth, vision, reproduction, epithe as imatinib mesylate, Nilotinib, or Dasatinib). lial cell differentiation and immune function (Meyskens, F. In the case of the treatment of multiple myeloma, it is 55 et al. Crit. Rev. Oncol Hematol 3:75, 1987, Herold, M. et al. contemplated that the constructs of the present invention Acta Dermatovener 74:29 1975). Early preclinical studies may be administered in combination with current therapies, with the retinol all-trans retinoic acid or ATRA, either alone Such as steroids such as dexamethasone, proteasome inhibi or in combination with other agents, demonstrated activity tors (such as bortezomib or carfilzomib), immunomodula against acute promyelocytic leukemia (APL), myelodys tory drugs (such as thalidomide, lenalidomide or pomalido- 60 plastic syndrome, chronic myelogenous leukemia (CML), mide), or induction chemotherapy followed by autologous mycosis fungoides and multiple myeloma (reviewed in haematopoietic stem cell transplantation, with or without Smith, M. J. Clin. Oncol. 10:839, 1992). These studies led other chemotherapeutic agents such as Melphalan hydro to the approval of ATRA for the treatment of APL. Currently chloride or the chemotherapeutic agents listed above. there are over 100 clinical trials evaluating the activity of In the case of the treatment of Hodgkin’s lymphoma, it is 65 ATRA in combination with other therapies for the treatment contemplated that the constructs of the present invention of hematological malignancies, kidney cancers, lung can may be administered in combination with current therapeu cers, squamous cell carcinomas and more. Of particular US 9,611,322 B2 39 40 interest and pertaining directly to this invention are the Therefore, in one aspect of the present invention, an studies demonstrating enhanced efficacy of interferon-C. attenuated IFN-B is attached to an anti-CD3 antibody that treatment when combined with ATRA. This is described for targets all T cells, which includes CD4+, CD8+, Treg, Th1. mantle cell lymphoma (Col. J. et al. Cancer Res. 72:1825, Th2 and Th17 cells. This comprehensive approach ensures 2012), renal cell carcinoma (Aass, N. et al. J. Clin. Oncol. 5 full coverage of all T cells, as all of these cell types have 23:4172, 2005; Motzer, R. J. Clin. Oncol. 18:2972, 2000), reported roles in MS pathogenesis and are affected by IFN-B CML, melanoma, myeloma and renal cell carcinoma (Kast, treatment (Dhib-Jalbut, 2010 Neurology 74:S17: Prinz, R. Cancer Biology and Therapy, 7:1515, 2008) and breast 2010 Trends Mol Med 16:379; Graber, 2010 Clin Neurol cancer (Recchia, F. et al. J. Interferon Cytokine Res. 15:605, Neurosurg 112:58 and Loma, 2011 Curr Neuropharmacol 1995). We would therefor predict enhanced activity of our 10 targeted attenuated IFNs when combined with therapeutic 9:409). Examples of CD3 antibodies that may be incorpo dosing of ATRA in the clinic. In addition, Mehta (Mol rated into the fusion protein constructs of the present inven Cancer Ther 3(3):345-52, 2004) demonstrated that in vitro tion are listed in Table 8. treatment of leukemia cells with retinoic acid induced expression of CD38 antigen. Thus, the enhanced efficacy of 15 TABLE 8 interferon plus the induced expression of the target CD38 would indicate a combination therapy of ATRA with our CD3 Antibodies anti-CD38 antibody-attenuated IFNC. in the treatment of Ab Clones Patent Assignee Comments IFN-sensitive cancers that express CD38 or may be induced by ATRA to express CD38. Example of such cancers are TFNSO U.S. Pat. No. 7,994,289 BTG Humanized CD3A122 International multiple myeloma, non-Hodgekin’s lymphoma, CML and M291 U.S. Pat. No. 7,381,803 PDL Humanized AML. BioPharma In addition, while the above constructs are based on 28F1, 27H5, U.S. Pat. No. 7,728,114 Novimmune Human IFNC2b, the mutations or deletions could also be made in 23F10, 15C3 S.A. the context of any of the other IFNC's or IFNB. In another embodiment of the present invention, the type I IFN is an 25 IFNB. IFN-?3 is approved for the treatment of multiple Alternatively, an attenuated IFN-B-anti-CD4 fusion pro sclerosis (MS). IFN-B could be attenuated by mutation or tein construct presents a more restrictive approach, but deletion and then attached to an antibody that targets cells would target autoreactive and regulatory T cells, including involved in the pathogenesis of this disease. IFN-?3 is an Th1 and Th17 cells and CD4"CD25 Treg cells. In addition, effective drug in MS, but its use is associated with adverse 30 events, including injection site inflammation, flu-like symp subsets of dendritic cells (DCs) also express CD4 and direct toms, leukocytopenia, liver dysfunction and depression, therapeutic effects of IFN-B on DCs have been disclosed leading to discontinuation in a Subset of patients. By direct (Shinohara, 2008 Immunity 29:68; Dann, 2012 Nat Neuro ing IFN-B activity directly to pathogenic cells, these adverse sci 15: 98). Examples of CD4 antibodies that may be incor events may be avoided. porated into the fusion protein constructs of the present Pathogenesis of MS is thought to be initiated and pro 35 invention are listed in Table 9. gressed by a number of events, including innate activation of dendritic and microglial cells through toll-like receptors, an TABLE 9 imbalance between pro-inflammatory and anti-inflamma tory/regulatory cytokines, differentiation of CD4+ T cells CD4 Antibodies into Th1 and Th17 phenotypes, activation of Th1 cells by 40 antigen presenting cells (APCs), reduction in the number of Ab Clones Patent Assignee Comments regulatory T (Treg) cells and migration of activated immune CE9.1 U.S. Pat. No. 7,452,534 Biogen Idec Non-human cells across the blood-brain barrier (BBB). The primary primate drivers of the clinical episodes of the disease are thought to variable 45 regions be autoreactive, myelin-specific Th1 cells (reviewed in TRX1 U.S. Pat. No. 7,541,443 Tolerrx Humanized Gandhi, 2010 J Neuroimmunol 221:7: Boppana, 2011 Mt 1E11, 1G2, 6G5, U.S. Pat. No. 8,231,877 GenPharm Human Sinai J Med 78:207; Loma, 2011 Curr Neuropharmacol 9:409). In an embodiment of the invention, an attenuated version of IFN-B may be attached to an antibody targeting a cell 50 A role for CD8" T cells in MS has been reported (Friese, surface marker specific for T cells, for the treatment of 2005 Brain 128:1747: Friese, 2009 Ann Neurol 66:132), as multiple Sclerosis or other autoimmune indications where well as a direct effect of IFN-B on CD8+ T cells in MS IFN-B may be effective. Direct effects of IFN-B on T cells patients (Zafranskaya, 2006 Immunol 121:29). Therefore, include inhibition of proliferation (Rep., 1996 J Neuroim directing an attenuated IFN-?3 directly to CD8" T cells with munol 67:111), downregulation of the co-stimulatory mol 55 an anti-CD8 antibody may result in clinical benefits for MS ecule CD40L (Teleshova, 2000 Scand J. Immunol. 51:312), patients. Examples of CD8 antibodies are shown in Table 10. decrease of metaloproteinase activity leading to reduced migration across the BBB (Stuve, 1996 Ann Neurol 40:853; TABLE 10 Uhm, 1999 Ann Neurol 46:319), induction of apoptosis by upregulating intracellular CTLA-4 and cell Surface Fas 60 CD8 Antibodies molecules (Hallal-Longo, 2007 J Interferon Cytokine Res 27:865), downregulation of anti-apoptotic proteins (Sharief, Ab Clones Patent Assignee Comments 2001 J Neuroimmunol. 120:199: Sharief, 2002 J Neuroim 37B1, 8G6 U.S. Pat. No. 7,247.474 Ortho-McNeil Hybridomas Pharmaceutical, deposited at munol. 129:224), and restoration of Treg function (De Inc. ATCC Andres, 2007 J Neuroimmunol 182:204; Korporal, 2008 65 (HB-12441, Arch Neurol 65:1434; Sarasella, 2008 FASEB J 22:3500; HB-12657) Chen, 2012 J Neuroimmunol 242:39). US 9,611,322 B2 41 42 TABLE 10-continued In another embodiment of the invention, an attenuated version of IFN-B can be fused to an antibody targeting cell CD8 Antibodies surface markers of myeloid cells, known to contribute to MS pathogenesis by driving T cell activation and differentiation. Ab Clones Patent Assignee Comments For example, the pan-myeloid markers CD33, CD115, or the OKT8 U.S. Pat. No. 4,361,550 Ortho Hybridoma dendritic cell marker CD11c may be targeted. A broad Pharmaceutical deposited at targeting approach may be preferred, for example, using Corporation ATCC antibodies against CD33 or CD115, since the exact contri (CRL-8014) Several US2009.O3O4659 Baylor Research bution of each of the myeloid cell subsets to MS disease examples Institute pathogenesis and response to IFN-B has been disputed 10 (Prinz, 2008 Immunity 28:675; Shinohara, 2008 Immunity 29:68; Dann, 2012 Nat Neurosci 15: 98). Antibodies to CD33 Markers of activated T cells, including, but not limited to that could be used in the present invention include My9-6 CD25, CD38, CD44, CD69, CD71, CD83, CD86, CD96, (U.S. Pat. No. 7,557, 189), any of 14 antibodies described in HLA-DR, ICOS and PD-1, also represent attractive targets US patent application US2012/0082670, or the antibody for this approach, since activated T cells are thought to be 15 known as huM195 (U.S. Pat. No. 5,693,761). Antibodies to the main drivers of autoreactivity resulting in demyelination CD115 that could be used include Abl and Ab16 (U.S. Pat. in MS (Gandhi, 2010 J Neuroimmunol 221:7: Boppana, No. 8,206,715) or CXIIG6 (US2011/0178278). An example 2011 Mt Sinai J Med 78:207; Loma, 2011 Curr Neurophar of a CD11c antibody that could be used according to the macol 9:409). Antibodies targeting any of these antigens present invention is mab 107 (U.S. Pat. No. 7,998,738, could be attached to an attenuated IFNB. Examples antibod ATCC deposit number PTA-11614). The attenuated IFN-?3 ies that could be used in the present invention include the could alternatively be directed to the CD14 antigen, present following: CD71 antibodies include BA120g (U.S. Pat. No. primarily on macrophages. Examples of CD14 antibodies 7,736,647) and various antibodies mentioned in Wang et al are shown in Table 13. (Di Yi Jun Yi Da Xue Xue Bao (Academic journal of the first medical college of PLA) 22(5):409–411, 2002). Examples of 25 TABLE 13 antibodies to CD83 include 20B08, 6G05, 20D04, 11G05, CD14 Antibodies 14C12, 96G08 and 95F04 (U.S. Pat. No. 7,700,740). An example of an antibody to CD86 includes 1G10H6D10 Ab Clones Patent Assignee Comments (U.S. Pat. No. 6,071,519). HLA-DR antibodies include 4C1 U.S. Pat. No. 6,245,897 Seikagaku Mouse Ab HD3, HD4, HD6, HD7, HD8 and HD10 (U.S. Pat. No. 30 Corporation 7,262,278), DN1921 and DN1924 (US2005/0208048). One F1024-1-3 U.S. Pat. No. 7,264,967 Mochida Humanized, Pharmaceutical inhibits attractive target along these lines could be PD-1, which is Co. CD14, TLR expressed on recently activated T cells. Ideally, a non binding antagonizing antibody could be used, such as the J1 10 F1024, F1031- U.S. Pat. No. 8,252,905, Mochida Part of antibody discussed in further detail below. 35 13-2 US2008,0286290 Pharmaceutical fusion Examples of antibodies to ICOS include JMabs (U.S. Pat. Co. proteins No. 6,803,039) and JMab 136 (US2011/0243929). with Further of these examples of antibodies to these targets protease are shown in the Tables 11 and 12 40 In yet another embodiment, targeting CD52-expressing TABLE 11 cells would deliver IFN-B to all lymphocytes and, in addi tion, to monocytes and peripheral dendritic cells (Buggins, CD25 Antibodies 2002 Blood 100:1715; Ratzinger, 2003 Blood 101: 1422), Ab Clones Patent Assignee Comments which are the key APCs responsible for proliferation and 45 differentiation of autoreactive T cells in MS. This approach Anti-tac Abs U.S. Pat. No. 5,530,101 PDL, Inc daclizumab would direct the activity of IFN-B to the key cell types RFTS U.S. Pat. No. 6,521,230 Novartis Chimeric, AG inhibits known to be directly affected by IFN-B and would facilitate MLR its therapeutic activity in MS. Examples of CD52 antibodies AB1, AB7, AB11, U.S. Pat. No. 8,182,812 Genmab Human that could be used according to the present invention AB12 (or U.S. Pat. No. 7,438,907) A/S antibodies, 50 prevent include, but are not limited to DIVHv5/DIVKv2 (U.S. Pat. CD25-IL-2 No. 7,910,104), any of the CD52 antibodies disclosed in interaction (US2012/0100152) or CAMPATH. and inhibit Any of the above mentioned, antibody-targeted attenuated MLR IFNB fusion protein constructs may have therapeutic activity 55 in the context of other inflammatory and autoimmune dis eases beyond multiple Sclerosis, due to their common under TABLE 12 lying immunological etiologies. Autoimmune diseases contemplated herein include inter CD44 Antibodies alia alopecia areata, ankylosing spondylitis, antiphospho 60 lipid syndrome, autoimmune Addison's disease multiple Ab Clones Patent Assignee Comments Sclerosis, autoimmune disease of the adrenal gland, autoim H90 US2007/0237761 Chimeric mune hemolytic anemia, autoimmune hepatitis, autoimmune 1A9, 2D1, 14G9, US2O10,0092484 Human oophoritis and orchitis, Behcet’s disease, bullous pemphig SACK-1 U.S. Pat. No. 7,816,500 Sackstein Binds CD44 oid, cardiomyopathy, celiac sprue-dermatitis, chronic glycoforms 65 fatigue syndrome (CFIDS), chronic inflammatory demyeli nating, chronic inflammatory polyneuropathy, Churg Strauss syndrome, cicatricial pemphigoid, crest syndrome, US 9,611,322 B2 43 44 cold agglutinin disease, Crohn's disease, irritable bowel TABLE 14-continued syndrome, inflammatory bowel disease, dermatitis herpeti formis, discoid lupus, essential mixed cryoglobulinemia, IFNB activity-attenuating mutations fibromyalgia, glomerulonephritis, Grave's disease, Guillain FNbeta Fold Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibro mutations attenuation Reference sis, idiopathic thrombocytopenia purpura (ITP), IgA neph E42K >10 2 ropathy, insulin dependent diabetes (Type I), lichen planus, DS4N 1.4 2 lupus, Meniere's disease, mixed connective tissue disease, M62I 8.7 2 multiple Sclerosis, myasthenia gravis, myocarditis, pemphi G78S 6.2 2 gus Vulgaris, pernicious anemia, polyarteritis nodosa, poly 10 K123 2.5 1 C141Y >25 2 chondritis, polyglancular syndromes, polymyalgia rheu A142T >10 2 matica, polymyositis and dermatomyositis, pochitis, R147A C17S** 1.7 3 primary agammaglobulinemia, primary biliary cirrhosis, E149K >5 2 psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheu R1S2H 4.7 2 matic fever, rheumatoid arrthritis, Sarcoidosis, Scleroderma, 15 *based on anti-proliferation activity Sjogren's syndrome, stiff-man syndrome, systemic lupus **the C17S mutation was made in order to remove the unpaired cysteine in the native erythematosus, Takayasu arteritis, temporal arteritis/giant sequence of DFNB1 cell arteritis, ulcerative colitis, uveitis, vasculitis and Vit References: iligo. Of particular interest is Behcet’s disease and chronic (1) Runkel, L. Pfeffer, L., Lewerenz, M., Mogensen, K. uveitic macular edema and other types of uveitis, since IFNC. has been shown to render therapeutic benefit (Deuter, Dev (1998). Differences in Activity between C. and B Type I Ophthalmol. 51:90-7. 2012) Interferons Explored by Mutational Analysis. J. Biol. Examples of inflammatory disease conditions contem Chem. 273: 8003-8008 plated by the present disclosure include but are not limited (2) Stewart, A. G., Adair, J. R., Catlin, G., Hynes, C., Hall, to those disease and disorders which result in a response of 25 J. Davies, J., Dawson, K. & Porter, A. G. (1987). Chemi redness, Swelling, pain, and a feeling of heat in certain areas cal mutagenesis of human interferon-beta: construction, that is meant to protect tissues affected by injury or disease. expression in E. coli, and biological activity of Sodium Inflammatory diseases which can be treated using the meth bisulfite-induced mutations. DNA 6: 119-128. ods of the present disclosure, include, without being limited (3) In-house results to, acne, angina, arthritis, aspiration pneumonia, disease, 30 In still another embodiment of the present invention, the empyema, gastroenteritis, inflammation, intestinal flu, NEC, IFN is IFN-J., (WO 2007/029041 A2), which may be used necrotizing enterocolitis, pelvic inflammatory disease, phar for any of the applications described more thoroughly for yngitis, PID, pleurisy, raw throat, redness, rubor, Sore throat, IFNo. or IFNB. stomach flu and urinary tract infections, chronic inflamma Type I IFNs can have anti-cancer activity based on a tory demyelinating polyneuropathy, chronic inflammatory 35 direct stimulation of the type I IFN receptor on cancer cells. demyelinating polyradiculoneuropathy, chronic inflamma This has been shown for numerous types of cancer including tory demyelinating polyneuropathy, chronic inflammatory multiple myeloma, melanoma, B cell lymphoma, non-small demyelinating polyradiculoneuropathy. cell lung cancer, renal cell carcinoma, hairy cell leukemia, The sequence of human interferon-?31 is shown below: chronic myelogenous leukemia, ovarian cancer, fibrosar (SEQ ID NO: 191) coma, cervical cancer, bladder cancer, astrocytoma, pancre
k k k k 1. MSYNLLGFLQ RSSNFOCOKL LWOLNGRLEY CLKDRMNFDI PEEIKOLOOF SO
k k k 51 OKEDAALTIY EMLONIFAIF RODSSSTGWN ETIVENLLAN WYHOINHLKT 1 OO
101 WLEEKLEKED FTRGKLMSSL HLKRYYGRIL HYLKAKEYSH CAWTIWRWEI 150
k 151 LRNFYFINRL TGYLRN 166
Using the numbering scheme above (residues 1-166), atic cancer, etc (Borden, Cancer Research 42:4948-53, 1982; known mutations (at positions indicated by asterisks) in Chawla-Sarkar, Clinical Cancer Research 7: 1821-31, 2001; human IFNB that reduce its activity include those listed in 55 Morgensen, Int J. Cancer 28:575-82, 1981; Otsuka, British Table 14. Journal of Haematology 103:518-529, 1998: Lindner, J of Interferon and Cytokine Research 17:681-693, 1997: Cara TABLE 1.4 glia, Cell Death and Differentiation 6:773-80, 1999; Ma, World J. Gastroenterol 11 (10): 1521-8, 2005). One of skill in IFNB activity-attenuating mutations 60 the art will recognize that the present invention has many IFNbeta Fold aspects resulting from combining antibodies to tumor asso mutations attenuation Reference ciated antigens with mutated type I interferons, and that the resulting fusion protein constructs may be used to reduce the wild type 1 R27A 3.3 1 proliferation of various interferon-sensitive cancers that R35A - C17S 28O 3 65 express the corresponding tumor associated antigens. It will R35T 10 1 also be appreciated that type I interferons can be combined with other agents to further improve their effectiveness. US 9,611,322 B2 45 46 Type I interferons can also display anti-viral properties. PS on their cell surfaces, so an antibody (or alternatively a IFNC.2b, for example, has been FDA-approved for the direct or indirect PS binding protein) to PS, attached to an treatment of chronic hepatitis C infections, and may have attenuated IFN, could also have use in the treatment of utility in treating other viral infections as well. Pegylated certain cancers. IFN-Cl is currently part of the standard of care regimen for It should be understood that antibody-targeted attenuated hepatitis C, according to American and European guidelines, IFNW could also be used in much the same way as IFNC. for but results in side effects in over 80% of patients, often the targeting virally infected cells (S. V. Kotenko, G. Gal leading to discontinuation of treatment (Aman, 2012; Cal lagher, V. V. Baurin et al., “IFN-us mediate antiviral pro Varuso, 2011). In one aspect of the present invention, a type tection through a distinct class II cytokine receptor com IIFN with an attenuating mutation is attached to an antibody 10 plex,” Nature Immunology, vol. 4, no. 1, pp. 69-77, 2003). that binds to virally infected cells. The antigen to be recog In one embodiment Type II IFNs, namely INFY, may also nized by the above referenced antibody could be a viral be attenuated and attached to antibodies that direct them to protein that is transiently expressed on the host cell Surface, specific cell types. IFNY has anti-proliferative properties or it could be an endogenous host cell-produced antigen that towards cancer cells (Kalvakolanu, Histol. Histopathol is exposed on the cell Surface to a greater extent after viral 15 15:523-37, 2000; Xu, Cancer Research 58:2832-7, 1998: infection than before infection. Exemplary viral proteins that Chawla-Sarkar, Apoptosis 8:237-49, 2003: Schiller, J Inter could serve as targets for the antibody portion include but feron Research 6:615-25, 1986). Sharifi has described how are not limited to, Hepatitis C viral envelope glycoproteins, to make a fusion protein in which an IFNY has been fused to E1 and E2: Hepatitis B surface antigen (HBSAg); Herpes the C-terminus of a tumor-targeting antibody (Sharifi, virus viral envelope glycoproteins B, C, D, E, G, H, I, J, K, Hybridoma and Hybridomics 21(6):421-32, 2002). In this L, M and UL32, and envelope protein UL49A. Human reference, Sharifi disclosed how to produce antibody-IFNY immune deficiency virus (HIV) Envelope proteins glyco fusion proteins in mammalian cells and showed that both the protein (gp) 120 and gp41, Adenoviruses knob domain of antibody and the IFN were functional. Alternatively, a the fiber protein; Varicella-zoster virus envelope glycopro single-chain dimer version of IFNY, as described by Lander teins (gB, gC, gE. g. g. gK, gL); Epstein-barr virus viral 25 (J Mol. Biol. 2000 May 26; 299(1): 169-79) may be used in glycoprotein gp350 and viral protein BMRF-2: Human the fusion protein. In addition to IFNY's anti-proliferative cytomegalovirus UL 16: Parvovirus B19 viral capsid pro effect on the targeted tumor cells, it may also have another teins VP1-3: Human astrovirus structural proteins, e.g. effect specifically on breast cancer cells: IFNY has been VP26, VP29 and VP32; Noroviruses structural protein VP1 shown to restore antiestrogen sensitivity to breast cancer and capsid protein VP2, Poliovirus viral capsid proteins 30 cells (Mol Cancer Ther. 2010 May: 9(5): 1274-1285) and so VP0, VP1, VP2, VP3 and VP4; Rhinovirus viral capsid an attenuated-IFNY attached to a breast cancer antigen proteins VP1,VP2, VP3 and VP4; and dengue virus virus antibody may be therapeutically useful in combination with particle proteins capsid (C), pre-membrane/membrane antiestrogen therapy. By attenuating IFNY via mutation, a (prM/M) and envelope (E). more cancer-selective form of IFNY may be produced. Two In one embodiment, IFN-O. activity may be targeted with 35 attenuating mutations in IFNY have been described by Was an antibody that binds, directly or indirectly via an inter chutza (Eur J. Biochem. 256:303-9, 1998), namely des mediate protein Such as annexin V or beta2-glycoprotein 1, (A23, D24), in which residues A23 and D24 are deleted, and to phosphatidylserine (PS), a phospholipid component of the des-(N25, G26), in which residues N25 and G26 are deleted. inner leaflet of cellular membranes. Cells undergoing apop The des-(A23, D24) mutant has an ~18-fold reduced affinity tosis, however, or cells infected with viruses, expose PS on 40 for the IFNY receptor compared to wild type IFNY, and had the outer membrane, where it becomes accessible to anti a ~100-fold reduced antiviral activity compared to wild type bodies. PS is exposed on the surface of cancer cells (Reidl, IFNY. The des-(N25, G26) variant had a ~140-fold reduced L. et al., J Immunol. 14:3623, 1991), the vascular endothe affinity for the IFNY receptor compared to wild type IFNY, lium in tumors (Ran, S. et al., Cancer Res.62:6132. 2002: and had a ~ 10-fold reduced antiviral activity compared to He, J. et al., Clin Cancer Res. 15:6871, 2009), and virus 45 wild type IFNY. Examples of fusion proteins comprising infected cells (Soares, M. et al., Nat. Med. 14:1357, 2008). antibodies to tumor cell Surface targets and attenuated An antibody indirectly (via beta2 glycoprotein 1) targeting mutants of IFNY include the following: Rituximab may be PS, bavituximab, has been described. It mediates antibody used as fusion protein with one of these attenuated IFNY dependent cytotoxicity and is effective in a number of in using a 7 amino acid linker described by Sharifi to produce Vivo cancer models, including human breast and lymphoma 50 the fusion protein construct “Rituximab-HC-L7-IFN) (A Xenografts and a ratglioblastoma model, as well as in viral A23.D24) IgG1,” composed of SEQ ID NOS:378 (heavy disease models (Ran, S. et al., Clin Cancer Res; 11:1551, chain) and 276 (light chain)). Such a fusion protein construct 2005; He, J. et al., Clin Cancer Res. 15:6871, 2009: Soares, would be expected to have potent anti-proliferative activity M. et al., Nat. Med. 14:1357, 2008). Currently, it is being against CD20 malignancies such as B cell lymphomas. developed as a therapeutic antibody for lung cancer treat 55 Other attenuated mutants of IFNY that may be appropriate ment (DeRose, P. et al., Immunotherapy. 3:933, 2011; Ger for fusing to a cell-targeting antibody were described by ber, D. et al., Clin Cancer Res. 17:6888, 2011). Alternative Lundell (J. Biol. Chem. 269(23):16159-62, 1994), namely antibodies may be based on the variable regions from the S20I (-50x reduced affinity), D21 K (-100x reduced affin anti-PS antibody 9D2 (Cancer Res Nov. 1, 2002 62: 6132). ity), A23Q (-2.500-fold reduced binding), A23V (-2,000 Yet another alternative for targeting PS would be to replace 60 fold reduced binding) and D24A (~4-fold reduced binding). the antibody Fab portions with a natural PS-binding protein These attenuated IFNY may be used as fusions in combina such annexin V or beta2-glycoprotein 1. An anti-PS anti tion with anti-CD38 antibodies, to generate the fusion pro body (or alternatively a director indirect PS binding protein) tein construct “X355/02-HC-L7-IFNY (S20I) IgG1” (com fused with an attenuated version of IFN-C. would target posed of SEQ ID NOS:380 (heavy chain) and 226 (light IFN-Cl activity to PS expressing virus-infected cells without 65 chain)) or “R10A2-HC-L7-IFNY (D21K) IgG1 (composed displaying the systemic safety issues related to IFN-C. of SEQ ID NOS:382 (heavy chain) and 270 (light chain)). Certain tumor cells, such as lung cancer cells, also express Other attenuating mutations in IFNY that may be exploited US 9,611,322 B2 47 48 for the current invention were described by Fish (Drug Des TABLE 1.5 Deliv. 1988 February; 203):191-206.) Targeted attenuated IFNY may also be used to treat various FAP Antibodies indications characterized by pathological fibrosis, including Ab Clones Patent Assignee Comments kidney fibrosis, liver fibrosis and idiopathic pulmonary MFP5, BIBH1 US2009,0304718 Boehringer Humanized fibrosis (IPF). IPF is a chronic, progressive form of lung Ingelheim USA disease, characterized by fibrosis of unknown cause, occur Corporation Many US2O12.0128591 Bacac et al. Humanized ring primarily in older adults. Despite the medical need, F19 US2OO3,O143229 Boehringer there has been little progress in the development of effective 10 Ingelheim therapeutic strategies (O'Connell, 2011 Adv Ther 28:986). International Pulmonary fibrosis can also be induced by exposure to GmbH drugs, particles, microorganisms or irradiation. The follow ing relates to both IPF and lung fibrosis induced by known agents and potentially for treatment of fibrosis in other types 15 TABLE 16 of organs, including liver and kidney. PDGFR-C and - Antibodies Fibroblasts play a key role in fibrotic diseases of the lung and their activation leads to collagen disposition, resulting in Ab Clones Patent Assignee Comments excessive Scarring and destruction of the lung architecture. 2.175.3, U.S. Pat. No. 7,754,859 AstraZeneca AB Human abs against Yet there is little information on the origin of these patho 2.499.1, PDGFRC 2.998.2 genic fibroblasts, though several precursor cell types have IMC-3G3 US2012 OO27767 Imclone LLC Human abs against been proposed, including bone marrow progenitors, mono PDGFRC cytes, circulating fibrocytes, and endogeneous cells. Such as 2C5 US2012 OO221267 Imclone LLC Human abs against resident mesenchymal and epithelial cells (Stevens, 2008 25 PDGFRB Proc Am Thorac Soc 5:783; King, 2011 Lancet 378:1949). CD14" monocytes from peripheral blood are able to In a preclinical model of liver fibrosis, IFN-Y was deliv differentiate into fibrocytes, the precursors of fibroblasts, ered to hepatic stellate cells, the equivalent of fibroblasts and and this process is inhibited by interferon-Y (IFN-Y). A direct responsible for secreting collagen in liver fibrosis, through effect of IFN-Y on monocytes was demonstrated in in vitro 30 liposomes targeting PDGFR-B, thereby enhancing the anti differentiation studies, Supporting the strategy of targeting fibrotic effects of IFN-Y (Li, 2012 J Control Release 159: an attenuated form of IFN-Y to CD14" monocytes for the 261). These data support the concept and the potential treatment of fibrotic disease (Shao, 2008 J Leukoc Biol therapeutic benefit gained by delivering IFN-y activity 83:1323). directly to fibroblasts in fibrotic diseases, including IPF and 35 liver fibrosis, and validate PDGFR-B as a target for this Experimental evidence exists that IFN-Y is capable of approach. inhibiting proliferation and activation of fibroblasts (Rogli The present invention also contemplates the attenuation ani, 2008 Ther Adv. Respir Dis 2:75) and this fact has and antibody-based targeting of type III IFNs, including exploited Successfully in preclinical models to reduce scar IFNA1 (IL29), IFN 2 (IL28A), and IFN.3 (IL28B) (S. V. ing and fibrosis. Clinical trials in IPF patients studying the 40 Kotenko, G. Gallagher, V. V. Baurin et al., “IFN-us mediate benefit of subcutaneously administered IFN-Y failed to reach antiviral protection through a distinct class II cytokine primary endpoints for survival benefits (O'Connell, 2011 receptor complex,” Nature Immunology, Vol. 4, no. 1, pp. Adv. Ther 28:986: King, 2011). Current approaches focus on 69-77, 2003. P. Sheppard, W. Kindsvogel, W. Xu, et al., direct delivery of recombinant IFN-y through inhalation of “IL-28, IL-29 and their class II cytokine receptor IL-28R.' an aerosol form (Diaz, 2012 JAerosol Med Pulm Drug Deliv 45 Nature Immunology, vol. 4, no. 1, pp. 63-68, 2003). These 25:79), such that the lungs may achieve sufficient IFN-y IFNs act through receptors composed of the IFNXR1 chain activity to produce benefit at an overall safe systemic dose. (also known as IL28RC) and the IL10R2 chain (shared with IL 10, IL22, and IL26 receptor complexes A. Lasfar, W. Delivering IFN-Y activity directly to fibroblasts could be Abushahba, M. Balan, and K. A. Cohen-Solal, “Interferon a powerful method to increase clinical response to this agent 50 lambda: a new Sword in cancer immunotherapy,” Clinical and at the same time reduce its side effects. Fusing attenu and Developmental Immunology, vol. 2011, Article ID ated IFN-Y to antibodies targeting fibroblast specific markers 349575, 11 pages, 2011). IFNWRs are expressed on most could facilitate this approach. There are several fibroblast cell types and mediate similar signalling pathways as the cell surface molecules that are enriched in fibroblasts. These type I IFNs. The antiviral activity of w IFNs has been include, for example, fibroblast specific protein (FSP1; 55 demonstrated against several viruses including HBV and Strutz, 1995 J Cell Biol 130:393), fibroblast activation HCV (E. M. Coccia, M. Severa, E. Giacomini et al., “Viral protein (FAP; Park, 1999 J Biol Chem 274:36505; Acharya, infection and toll-like receptor agonists induce a differential 2006 Hum Pathol 37:352), and platelet derived growth expression of type I and W interferons in humans plasma factor receptors (PDGFR-C. and -3; Trojanowska, 2008 cytoid and monocyte-derived dendritic cells.' European Rheumatology (Oxford) 47S5:2). Expression of these mol 60 Journal of Immunology, vol. 34, no. 3, pp. 796-805, 2004; ecules is elevated in lung biopsies obtained from IPF M. D. Robek, B. S. Boyd, and F. V. Chisari, “Lambda patients and they have been directly implicated as drug interferon inhibits hepatitis B and C virus replication.” targets in IPF or its pathogenesis (Lawson, 2005 Am J Respir Journal of Virology, vol. 79, no. 6, pp. 3851-3854, 2005; N. Crit. Care Med 171:899: Acharya, 2006 Hum Pathol 37:352: Ank, H. West, C. Bartholdy, K. Eriksson, A. R. Thomsen, Abdollahi, 2005 J Exp Med201:925). Examples of antibod 65 and S. R. Paludan, “Lambda interferon (IFN-w), a type III ies to FAP and the PDGF receptors are shown in Tables 15 IFN, is induced by viruses and IFNs and displays potent and 16. antiviral activity against select virus infections in vivo.” US 9,611,322 B2 49 50 Journal of Virology, vol. 80, no. 9, pp. 4501-4509, 2006: S. Hepatogastroenterology 54:2092). Clinical use of IL-10 is E. Doyle, H. Schreckhise, K. Khuu-Duong et al., “Interleu hampered by its short half-life and a PEGylated version has kin-29 uses a type 1 interferon-like program to promote shown promising pharmacokinetic improvements and effi antiviral responses in human hepatocytes.' Journal of Hepa cacy in a preclinical model of fibrosis (Mattos, 2012 J tology, vol. 44, no. 4, pp. 896-906, 2006: T. Marcello, A. Control Release 162:84). Targeting IL-10 activity through Grakoui, G. Barba-Spaeth et al., “Interferons C. and inhibit fusion with an antibody directing it to fibroblasts could hepatitis C virus replication with distinct signal transduction result in therapeutic benefits in fibrotic diseases, including and gene regulation kinetics.” Gastroenterology, Vol. 131, lung and liver fibrosis. Antibodies against fibroblast specific no. 6, pp. 1887-1898, 2006). Clinical studies with IFN) for proteins such as fibroblast activation protein and platelet 10 derived growth factor receptors, as described above in the the treatment of hepatitis C have shown promise (E. L. description of IFN-y-targeting, could deliver attenuated Ramos, “Preclinical and clinical development of pegylated IL-10 directly to fibroblasts. interferon-lambda 1 in chronic hepatitis C. Journal of Recombinant erythropoietin (EPO) is a widely used and Interferon and Cytokine Research, vol. 30, no. 8, pp. 591 effective hormone for the treatment of anemia, often in 595, 2010). One aspect of the present invention is to target 15 cancer patients. It acts by signaling through the EPO recep a mutated, attenuated for of an IFNW towards virally infected tor (EPOR), which is not only expressed by cells of the cells, using for example the targeting antibodies describe hematopoietic system, but also on non-hematopoietic cells, above for the targeting of an attenuated form of IFNC. including cells from various tumor types. Many studies have Mutated, attenuated forms of an IFNW could also be used to examined the role of EPO and EPO-R stimulation in cancer target cancer cells, as described in more detail for IFNC. models in vitro and in vivo, and a number of studies have above. demonstrated a stimulatory effect on tumor growth, either Non-IFN ligands are also contemplated in the present directly on cancer cells, or through increased angiogenesis in invention and may also be attenuated by mutation and then the tumors (reviewed in Jelkmann, 2008 Crit. Rev. Oncol targeted to specific cell types by antibodies or fragments Hematol 67:39). In several clinical trials, treatment with thereof. The anti-inflammatory cytokine interleukin-10 (IL 25 EPO has been associated with increased tumor growth and 10) plays a central role during innate and adaptive immune decreased Survival, leading to the recommendation and responses. IL-10 forms a homodimer and binds to the IL-10 blackbox warning to limit and monitor the exposure of EPO receptor complex expressed on APCs, leading to reduced in cancer patients as much as clinically feasible (Farrell, expression of MHC class II and reduced production of 2004 The Oncologist 9:18; Jelkmann, 2008 Crit. Rev. Oncol 30 Hematol 67:39: Elliott, 2012). pro-inflammatory cytokines and chemokines, thereby inhib Erythropoiesis is a multi-step process, in which pluripo iting T cell development and differentiation. However, IL-10 tent stem cells undergo tightly controlled differentiation and has also been implicated in inducing the proliferation of proliferation steps. An intermediate cell type in this process, several immune cells, including B cells (Hofmann, 2012 is the colony-forming-unit-erythroid (CFU-E) cell, which Clin Immunol 143: 116). 35 expresses high levels of EPOR, depends on EPO for survival Reduced expression of IL-10 is associated with a number and appears to be the main cell type in the differentiation of autoimmune disorders in humans and rodents, including process with this dependency (Elliott, 2008 Exp Hematol psoriasis, inflammatory bowel disease and rheumatoid 36:1573). arthritis. Mice deficient in IL-10 develop chronic entero Targeting EPO activity to CFU-E cells using specific colitis, which can be prevented by the administration of 40 markers would substantially reduce the effect of EPO on IL-10, but the clinical translation of these findings resulted cancer and other non-hematopoietic cells, while maintaining in a number of failed trials in patients. One explanation of the ability to drive erythrocyte formation and increase these failures is that the local IL-10 concentrations may be hemoglobin levels. Genome-wide analysis of CFU-E cells too low, even at maximum tolerable systemic administration revealed several potential candidate cellular markers, (Herfarth, 2002 Gut 50:146). Another explanation may be 45 including Rh-associated glycoproteins, e.g. CD241 and the immunostimulatory effect of IL-10 on B cells and the members of the Rh blood group system, e.g. the product of resulting production of the pro-inflammatory IFN-y, as was the RCHE gene (Terszowski, 2005 Blood 105:1937). demonstrated in IL-10-treated Crohn's disease patients Additional example surface markers expressed on CFU (Tilg, 2002 Gut 50:191). Es, and several other intermediates of erythropoiesis, Fusing attenuated IL-10 to an antibody specific for APCs, 50 include CD117 (c-kit), CD71 (transferrin receptor) and e.g. targeting dendritic cells through CD11c, or more CD36 (thrombospondin receptor) (Elliott, 2012 Biologics broadly expressed myeloid markers, like CD33 or CD115, 6:163), but these markers are overexpressed in certain would decrease systemically active biologic activity and at cancer cells as well, as they are all involved in general the same time increase the targeted local active concentra growth and proliferation, and therefore represent less attrac tions of IL-10. In addition, the demonstrated pro-inflamma 55 tive targets for targeting EPO activity in cancer patients, but tory effect through B cells would be decreased or eliminated. this approach may benefit patients with tumors not express The production of antibody-IL 10 fusion proteins have been ing these targets. CD117 antibodies include SR-1 (U.S. Pat. described previously (Schwager Arthritis Res Ther. 11(5): No. 7,915,391) and antibodies DSM ACC 2007, 2008 and R142, 2009). 2009 (U.S. Pat. No. 5,545,533). Other antigens for targeting Evidence exists for an anti-fibrotic role of IL-10 in 60 of an attenuated EPO include CD34, CD45RO, CD45RA, various models. A hallmark of fibrosis is the overproduction CD115, CD168, CD235, CD236, CD237, CD238, CD239 and deposition of collagen produced by fibroblasts, resulting and CD240. in scarring tissue formation. IL-10 directly inhibits extra Fusing EPO activity to an antibody would also greatly cellular matrix synthesis by human fibroblasts (Reitamo, increase the extent of the therapeutic activity. The half-life 1994 J Clin Invest 94:2489) and is anti-fibrotic in a rat 65 of recombinant EPO is about 5 hours in humans and this hepatic fibrosis model through downregulation of TGF-B would likely be increased to weeks when attenuated EPO is (Shi, 2006 World J. Gastroenterol 12:2357; Zhang, 2007 fused to an antibody. This approach could benefit patients US 9,611,322 B2 51 52 treated for anemia, who are dosed typically multiple times Diversion towards Th2 may provide a therapeutic benefit per week, often through intravenous injections. Importantly, in certain types of diseases. Delivery of IL-4 to CD4 T cells it has been shown that the therapeutic response to EPO is could accomplish this, or IL-4 activity could be targeted to primarily controlled by the length of time EPO concentra macrophages to protect from immunopathology (Ghoreschi, tions are maintained, and not by the concentration levels 2007 Clin Dermatol 25:574; Hunig, 2010 Med Microbiol Immunol 199:239). (Elliott, 2008 Exp Hematol 36:1573). Attenuating mutations in IL-4 that may be exploited in the Another example is transforming growth factor B (TGF design of antibody-attenuated IL-4 fusion protein constructs B) which is a critical factor in the regulation of T cell of the present invention include those listed in Table 17. mediated immune responses and the induction of immune 10 tolerance. TGF-B knockout mice die from multifocal inflam TABLE 17 mation and autoimmune disorders, suggesting an immuno ECso T cell suppressive effect (Shull, 1992 Nature 359:693). However, IL4 Variant Korx 103 Sl proliferation (nM) TGF-B also has been shown to induce fibrotic disease through a prominent role in extracellular matrix regulation 15 IL4 2.1 O.12 ISR 8.7 and by promoting fibroblast migration, proliferation and T6D 15 activation (Rosenbloom, 2010 Ann Intern Med 152: 159; E9C 270 3.1 Wynn, 2011 J Exp Med 208:1339; King, 2011 Lancet R81E 6.1 K84D 9.3 378:1949). R88O 140 2.5 In the presence of TGF-E, CD4"CD25 naive T cells can R88A 760 8.1 be converted into Treg cells, which can Suppress antigen N89R 6.1 specific T cell expansion in vivo and prevent allergic patho W91D 8.5 genesis in a murine asthma model (Chen, 2003 J Exp Med ND. No specific binding found. 198:1875). Inflammatory responses also contribute to the 25 transition of acute liver disease and perpetuation into The IL-4 mutants in this table, and their binding proper chronic fibrosis and cirrhosis and TGF-B may help dampen ties and biological activity, were described by Wang Y. Shen these responses through its effect on Treg differentiation B and Sebald W. Proc. Natl. Acad. Sci. USA 1997 Mar. 4; (Dooley, 2012 Cell Tissue Res 347:245). Similarly, TGF-B 94(5): 1657-62. 30 In yet another example, Interleukin-6 (IL-6) may also be directed to naive T cells in inflammatory bowel disease attenuated and targeted to specific cell types. A mechanism could lead to control and suppression of inflammation by which tumors can evade anti-tumor immunity is by (Feagins, 2010 Inflamm Bowel Dis 16:1963). recruiting Treg cells to the tumor microenvironment, result Targeting TGF-B specifically to CD4 T cells may lever ing in tolerance at tumor sites. IL-6 is a cytokine involved age the anti-inflammatory potential of TGF-B, while mini 35 in regulating the balance between Treg and Th17 cells and mizing its pro-fibrotic properties, and could provide a novel induces the development of Th17 cells, while it inhibits Treg strategy to combat autoimmune disorders. Alternatively, differentiation (Kimura, 2010 Eur J. Immunol 40:1830). TGF-B could be targeted soley to activated T cells using a T IL-6, by skewing the terminal differentiation of naive cell activation marker, as described above for the discussion CD4 T cells towards the Th17 lineage, or reprogramming of IFNB targeting. One attractive target along these lines 40 of Th17 cells, has the potential to reverse tumor-associated could be, for example, PD-1, which is expressed on recently immune Suppression by Treg cells in the context of cancer, activated CD4 T cells. Ideally, a non-antagonizing antibody thereby enabling the immune system to control the tumors. could be used, such as the J110 antibody discussed in further This strategy has proven Successful in a murine model of detail below. 45 pancreatic cancer in which mice injected with tumor cells Another example is Interleukin-4 (IL-4) which is a expressing IL-6 demonstrated a significant delay in tumor cytokine that induces the differentiation of naive CD4+ T growth and enhanced Survival, accompanied by an increase cells into Th2 cells. Upon activation, Th2 cells produce more in Th17 cells in the tumor microenvironment, compared to IL-4, and as a result, IL-4 is considered a main driver of mice bearing tumors not expressing IL-6 (Gnerlich, 2010 J Th2-mediated immune responses. The concept of a Th1/Th2 50 Immunol 185:4063). imbalance (favoring Th1) contributing to autoimmune and Adoptive transfer of T cells is an effective treatment for other inflammatory diseases was first postulated in the 1980s solid (Rosenberg, 2011 Clin Cancer Res 17:4550) and (reviewed in Kidd, 2003 Altern Med Rev 8:223), and indeed, hematologic (Kochenderfer, 2012 Blood 119:2709) malig a role of Th1/Th17 cells as drivers of disease in psoriasis nancies. Analysis of five different clinical trials in which (Ghoreschi, 2007 Clin Dermatol 25:574), certain types of 55 adoptive T cell transfer was employed using a variety of inflammatory bowel disease, in particular Crohn's disease preconditioning regimens revealed that the depth and dura tion of Treg depletion correlates with clinical response rate, (Sanchez-Munoz, 2008 World J. Gastroenterol 14:4280), or highlighting the important role of residual Tregs controlling severe versus mild forms of asthma (Hansbro, 2011 Br J the anti-tumor response (Yao, 2012 Blood 119:56.88). In Pharmacol 163:81), has been documented. 60 mice, a direct link between Surviving Tregs and efficacy of In preclinical models of infectious diseases, deviation of adoptive transfer therapy strongly supports these clinical the immune response away from Th1 to Th2 and activation observations (Baba, 2012 Blood 120:2417). of macrophages by IL-4 protected from immunopathology The importance of Tregs in controlling anti-tumor activity (Hunig, 2010 Med Microbiol Immunol 199:239), and IL-4 is further exemplified by a significant increase in the therapy of psoriasis patients resulted in an induction of Th2 65 humoral response to peptide vaccination in glioblastoma differentiation and an improvement in clinical scores (Gho patients after depletion of Tregs with the anti-IL-2 receptor reschi, 2003 Nat Med 9:40). antibody daclizumab (Sampson, 2012 PloS ONE 7:e31046). US 9,611,322 B2 53 54 Taken together, the published data strongly support a role 1997 Am J Respir Crit. Care Med 156:1937; Mizuno, 2005 for Tregs in inhibiting the immune response against tumors. FASEB J 19:580), in an obstructive nephropathy model in By directing IL-6 activity to CD4" cells in order to stimulate mice (Yang, 2003 Am J Physiol Renal Physiol 284:F349) Th17 differentiation and decrease Treg formation, enhanced and in liver fibrosis models in rats (Matsuda, 1997 Hepa anti-tumor responses are expected. These may be achieved 5 tology 26:81); HGF also prevents fibrosis in cardiomyo with or without accompanying vaccination Strategies. Fus pathic hamsters (Nakamura, 2005 Am J Physiol Heart Circ ing attenuated IL-6 to an antibody against a T cell antigen Physiol 288:H2131). (e.g. targeting CD4) or an activated T cell antigen (Such as Limitations of HGF as a therapeutic include its short PD-1) would provide a comprehensive delivery directly to half-life, which requires Supra-physiological systemic con the target cells. 10 centrations to reach locally effective levels, and the role of Attenuated mutants of IL-6 include those listed in Table its receptor, MET, in cancer. MET can activate oncogenic 18. pathways in epithelial cells. Both of these limitations may be overcome by generation of an antibody-HGF fusion protein TABLE 1.8 construct and targeting it to regenerating or fibrotic tissue. 15 This strategy would produce a therapeutic with a much ECso in XG-1 growth Binding stimulation assay longer half-life directed primarily at the relevant cells types. IL6 Variant (% of wild type) (pg/ml) Clinical trials have investigated the therapeutic potential IL6 100 600 and regenerative activity of HGF, or HGF mimetics, in F74E 1 Low activity hepatic failure, chronic leg ulcers, limb ischemic, peripheral F78E 5 Low activity arterial disease, cardiovascular disease after myocardial R168M 2 Low activity R179E None detected Low activity infarction and neurological diseases (de Andrade 2009 Curr R179W None detected Low activity Opin Rheumatol 21:649; Nakamura, 2011 J Gastroenterol Hepatol 26:188: Madonna, 2012 Thromb Haemost 107: 25 656). These IL-6 mutants and their properties were described by Liver fibrosis, typically the result of chronic liver damage Kalai Met. al. Blood. February 15: 89(4): 1319-1333 caused by infections or alcohol abuse, is, like fibrosis in Another example is hepatocyte growth factor (HGF) other organs, characterized by excessive accumulation of discovered as a mitogen for hepatocytes (reviewed in Naka extracellular matrix, including collagen produced by (myo) mura, 2010 Proc Jpn. Acad Ser B Phys Biol Sci 86:588). 30 Hepatocyte growth factor is a pleiotropic cytokine that fibroblasts. Damaged hepatocytes release inflammatory regulates cell growth and motility, playing a central role in cytokines and the resulting inflammatory milieu stimulates angiogenesis and tissue generation and repair in many the transformation of hepatic stellate cells (HSC) into fibro Organs. blasts, producing collagen. The accumulation of extracellu HGF acts through its receptor, MET, which is expressed 35 lar matrix proteins results in Scar tissue, which leads to liver on epithelial and endothelial cells. Binding of HGF to MET cirrhosis (Bataller, 2005 J Clin Invest 115:209). Evidence results in a number of intracellular phosphorylation and exists for a direct effect of HGF on hepatocytes and HSC in signaling events, leading to a variety of biological responses vitro (Kwiecinski, 2012 PloS One 6:e24568; Namada, 2012 including migration, proliferation and morphogenesis. J Cell Physiol DOI 10.1002/jcp.24143). Targeting HGF Essential for embryogenesis, HGF’s primary function in the 40 specifically to hepatocytes or HSC may result in a thera adult is tissue repair (Nakamura, 2010 Proc Jpn Acad Ser B peutic benefit in liver fibrosis patients, while eliminating the Phys Biol Sci 86:588). unwanted systemic effects of HGF. HGF has been shown to alter the fate of epithelial cells Possible membrane proteins for hepatocytes include, for and reduce epithelial-mesenchymal transition (EMT) example, ASGR1, a subunit of the asialoglycoprotein, used through its intereference with TGF-B signaling, antagoniz 45 as a target for liver specific drug delivery (Stockert, 1995 ing the process of fibroblastogenesis (Shukla, 2009 Am J Physiol Rev 75:591), or alternatively the other subunit of Respir Cell Mol Biol 40:643). After organ injury, TGF-f this receptor, ASGR2. Fibroblast-specific protein (FSP1) drives conversion of HGF-producing fibroblasts into colla expression is increased after liver injury and may be used to gen-producing myofibroblasts, while HGF in turn inhibits target fibroblasts or inflammatory macrophages in fibrotic TGF-B production by myofibroblasts (Mizuno, 2004 Am J 50 liver tissue (Osterreicher, 2011 Proc Natl Acad Sci USA Physiol Renal Physiol 286:F134). Exogeneous HGF, or 108:308). mimetics activating the MET receptor, act by restoring this In lung fibrosis patients, the loss of pulmonary architec imbalance imposed by tissue injury, and are therefore con ture is characterized by a loss of alveolar epithelial cells, the sidered promising drug candidates for treating damaged persistent proliferation of activated fibroblasts and the exten tissues and fibrotic diseases (Nakamura, 2010 Proc Jpn. Acad 55 sive alteration of the extracellular matrix (Panganiban, 2011 Ser B Phys Biol Sci 86:588). Acta Pharmacol Sin 32:12). Initially studied in models for liver damage and hepatitis To treat lung fibrosis, HGF activity may be delivered to (Roos, 1992 Endocrinology 131:2540; Ishiki, 1992 Hepa alveolar epithelial cells by attenuating it (by mutation) and tology 16:1227), HGF subsequently demonstrated therapeu attaching it to an antibody against a specific cell Surface tic benefits in many additional damaged organs, including 60 protein on these cells, such as RTI40/Tic. or HTI56 (McEl pulmonary, gastrointestinal, renal and cardiovascular mod roy, 2004 Eur Respir J 24:664). els of injury and fibrosis (Nakamura, 2011 J Gastroenterol Endothelial cell-specific markers, including VEGF recep Hepatol 26:188). tors (Stuttfeld, 2009 IUBMB Life 61:915) may be used for In in vivo model systems of fibrosis, HGF prevents the targeting blood vessels for endothelial cell layer enhance progression of fibrotic changes and reduces collagen accu 65 ment for a number of pathologic indications, including mulation when administered prophylactically or therapeuti hindlimb ischemia. Examples of VEGF receptor antibodies cally in murine lungs exposed to bleomycin (Yaekashiwa, are shown in Table 19. US 9,611,322 B2 55 56 TABLE 19 Methods of Screening mutated ligands to determine potency includes assaying for the presence of a complex VEGFR Antibodies between the ligand and the target. One form of assay Ab Clones Patent Assignee Comments involves competitive binding assays. In Such competitive binding assays, the target is typically labeled. Free target is AC88 U.S. Pat. No. 8,128,932 Shanghai Human Aosaiersi anti-VEGFR2 separated from any putative complex and the amount of free Biotech Co., mAb (i.e. uncomplexed) label is a measure of the binding of the Ltd agent being tested to target molecule. One may also measure Antibody 1, US2012,OOS8126 Imclone LLC Anti-VEGFR3 Antibody 2 Abs 10 the amount of bound, rather than free, target. It is also 6A6 US2O11 OO651.76 Korea Human, possible to label the compound rather than the target and to Research anti-VEGFR measure the amount of compound binding to target in the Institute of BioScience presence and in the absence of the drug being tested. and One example of a cell free assay is a binding assay. Whilst BioTechnology 15 not directly addressing function, the ability of a modulator to bind to a target molecule in a specific fashion is strong evidence of a related biological effect. For example, binding Many other examples of signaling ligands are also known of a molecule to a target may, in and of itself, be inhibitory, in the art and may, as described in the non-limiting exem due to steric, allosteric or charge-charge interactions. The plary embodiments above, be attenuated and attached to an target may be either free in solution, fixed to a Support, antibody (or fragment thereof) that binds to an antigen on expressed in or on the surface of a cell. Either the target or specific target cells, thereby allowing the ligand to generate the compound may be labeled, thereby permitting determi its biological signal on those target cells to a much greater nation of binding. Usually, the target will be the labeled degree than it generates its signal on antigen-negative cells. species, decreasing the chance that the labeling will interfere Examples of ligands that have a direct negative effect on 25 with or enhance binding. Competitive binding formats can tumor proliferation include TNFC., TRAIL, Fas Ligand, be performed in which one of the agents is labeled, and one IFNB IFNY or IFN), which can be targeted to various tumor may measure the amount of free label versus bound label to cell surface antigens as discussed above for INFO. determine the effect on binding. In many of the aspects of the present invention, specific Depending on the assay, culture may be required. The cell mutations in various ligands are explicitly mentioned. There 30 is examined using any of a number of different physiologic are, however, methods well known in the art for identifying assays. Alternatively, molecular analysis may be performed, other mutations in signalling ligands numerous methods for for example, protein expression, mRNA expression (includ mutagenesis of proteins are known in the art. Such methods ing differential display of whole cell or polyA RNA) and include random mutagenesis for example, exposing the others. Non-limiting examples of in vitro biological assays 35 that can be used to screen protein variants are shown in the protein to UV radiation or mutagenic chemicals and select Examples below and also include apoptosis assays, migra ing mutants with desired characteristics. Random mutagen tion assays, invasion assays, caspase-activation assays, esis may also be done by using doped nucleotides in cytokine production assays and the like. oligonucleotides synthesis, or conducting a PCR reaction in The present invention also provides compositions com conditions that enhance misincorporation of nucleotide, 40 prising the polypeptides of the present invention. These thereby generating mutants. Another technique is site-di compositions can further comprise at least one of any rected mutagenesis which introduces specific changes to the Suitable auxiliary, such as, but not limited to, diluent, binder, DNA. One example of site directed mutagenesis is using stabiliser, buffers, salts, lipophilic solvents, preservative, mutagenic oligonucleotides in a primer extension reaction adjuvant or the like. Pharmaceutically acceptable auxiliaries with DNA polymerase. This method allows for point muta 45 are preferred. Non-limiting examples of, and methods of tion, or deletion or insertion of small stretches of DNA to be preparing such sterile solutions are well known in the art, introduced at specific sites. The site-directed approach may Such as, but not limited to, Gennaro, Ed., Remington's be done systematically in Such technique as alanine Scanning Pharmaceutical Sciences, 18th Edition, Mack Publishing mutagenesis whereby residues are systematically mutated to Co. (Easton, Pa.) 1990. Pharmaceutically acceptable carriers alanine and its effect on the peptide's activity is determined. 50 can be routinely selected that are suitable for the mode of Each of the amino acid residues of the peptide is analyzed administration, solubility and/or stability of the antibody in this manner to determine the important regions of the composition as well known in the art or as described herein. peptide. Pharmaceutical excipients and additives useful in the present composition include but are not limited to proteins, Another example is combinatorial mutagenesis which 55 peptides, amino acids, lipids, and carbohydrates (e.g., Sug allows the screening of a large number of mutants for a ars, including monosaccharides, di-, tri-, tetra-, and oli particular characteristic. In this technique, a few selected gosaccharides; derivatised Sugars such as alditols, aldonic positions or a short stretch of DNA may be exhaustively acids, esterified Sugars and the like; and polysaccharides or modified to obtain a comprehensive library of mutant pro Sugar polymers), which can be present singly or in combi teins. One approach of this technique is to excise a portion 60 nation, comprising alone or in combination 1-99.99% by of DNA and replaced with a library of sequences containing weight or Volume. Exemplary protein excipients include all possible combinations at the desired mutation sites. The serum albumin, such as human serum albumin (HSA), segment may be at an enzyme active site, or sequences that recombinant human albumin (rIA), gelatin, casein, and the have structural significance or immunogenic property. A like. Representative amino acids which can also function in segment however may also be inserted randomly into the 65 a buffering capacity include alanine, glycine, arginine, gene in order to assess the structural or functional signifi betaine, histidine, glutamic acid, aspartic acid, cysteine, cance of particular part of protein. lysine, leucine, isoleucine, Valine, methionine, phenylala US 9,611,322 B2 57 58 nine, aspartame, and the like. One preferred amino acid is EXAMPLES OF THE INVENTION histidine. A second preferred amino acid is arginine. Carbohydrate excipients suitable for use in the invention Production of Antibody-IFNC. Fusion Protein Constructs include, for example, monosaccharides, such as fructose, Expression Vectors: maltose, galactose, glucose, D-mannose, Sorbose, and the The DNA encoding the rituximab (Anderson et al., U.S. like; disaccharides, such as lactose, Sucrose, trehalose, cel Pat. No. 5,843,439, Dec. 1, 1998) and palivizumab (John lobiose, and the like; polysaccharides, such as raffinose, son, U.S. Pat. No. 5,824.307, Oct. 20, 1998) variable regions melezitose, maltodextrins, dextrans, starches, and the like; were generated from 18 (heavy chain) and 16 (light chain) and alditols, such as mannitol. Xylitol, maltitol, lactitol, DNA oligonucleotides, which were designed according to 10 the published amino acid sequences, by PCR-based gene xylitol sorbitol (glucitol), myoinositol and the like. Preferred assembly. The DNA encoding the variable regions of the carbohydrate excipients for use in the present invention are G005 anti-CD38 and nBTO62 anti-CD138 monoclonal anti mannitol, trehalose, and raffinose. bodies were drawn from the publications by De Weers et al. Antibody compositions can also include a buffer or a pH (U.S. Pat. No. 7,829,673) and by Daelken et al. (WO adjusting agent; typically, the buffer is a salt prepared from 15 2009/080832), respectively, and subjected to be synthesized an organic acid or base. Representative buffers include by Integrated DNA Technology, Inc. (Coralville, Iowa) after organic acid salts, such as salts of citric acid, ascorbic acid, the sequence modification to eliminate rare codons and gluconic acid, carbonic acid, tartaric acid, Succinic acid, unpreferred restriction sites. acetic acid, or phthalic acid; Tris, tromethamine hydrochlo The DNA sequences encoding the variable regions of ride, or phosphate buffers. Preferred buffers for use in the anti-human HLA (HB95), anti-human PD-1 (J110) and present compositions are organic acid salts, such as citrate. anti-yellow fever virus (2D12) monoclonal antibodies were Additionally, the compositions of the invention can determined after cloning from hybridoma W6/32 (ATCC include polymeric excipients/additives. Such as polyvi HB-95, Barnstable et al. (1978), Cell 14:9-20), J110 (Inter nylpyrrolidones, ficols (a polymeric Sugar), dextrates (e.g., national Patent Organism Depositary FERM-8392, Iwai et cyclodextrins, such as 2-hydroxypropyl-3-cyclodextrin), 25 al. (2002), Immunol. Lett, 83:215-220) and 2D12 (ATCC polyethylene glycols, flavoring agents, antimicrobial agents, CRL-1689, Schlesinger et al. (1983), Virol. 125:8-17), Sweeteners, antioxidants, antistatic agents, Surfactants (e.g., respectively, using the SMART RACE cDNA Amplification polysorbates such as “TWEENR 20” and “TWEENR 80), kit (Clontech, Mountain View, Calif.) and Mouse Ig-Primer lipids (e.g., phospholipids, fatty acids), Steroids (e.g., cho Sets (Novagen/EMD Chemicals, San Diego, Calif.). The lesterol), and chelating agents (e.g., EDTA). 30 sequence determination and Sub-cloning of the newly iso These and additional known pharmaceutical excipients lated anti-CD38 antibodies is described in the following and/or additives suitable for use in the antibody composi Sections. tions according to the invention are known in the art, e.g., as The DNA encoding human interferon-C.2b (IFNC2b: listed in “Remington: The Science & Practice of Pharmacy’. amino acid sequence of SEQ ID NO:3) was isolated from 19th ed., Williams & Williams, (1995), and in the “Physi 35 genomic DNA of a HEK cell line by PCR. The sequences of cian's Desk Reference', 52 nd ed., Medical Economics, human interferon-B1 (IFNB1, SEQID NO:91), human inter Montvale, N.J. (1998), the disclosures of which are entirely leukin-4 (IL-4, SEQ ID NO:119) and human interleukin-6 incorporated herein by reference. Preferred carrier or excipi (IL-6, SEQ ID NO:123) were designed from the protein ent materials are carbohydrates (e.g., Saccharides and aldi sequences such as NP 002167, NP 000580 and tols) and buffers (e.g., citrate) or polymeric agents. 40 NP 000591, respectively, and synthesized by Integrated Throughout this specification the word “comprise', or DNA Technology, Inc. (Coralville, Iowa) or GenScript USA variations such as “comprises” or “comprising, will be Inc. (Piscataway, N.J.) using methods commonly known to understood to imply the inclusion of a stated element, those of skill in the art. Alterations of the cytokine integer or step, or group of elements, integers or steps, but sequences, for example the addition of linkers or point not the exclusion of any other element, integer or step, or 45 mutations, were introduced to the cytokine genes using group of elements, integers or steps. overlap extension PCR techniques well known in the art. All publications mentioned in this specification are herein The cytokine-endoding gene fragments were then cloned incorporated by reference. Any discussion of documents, into the pTT5 expression vector (Durocher, Nucleic Acids acts, materials, devices, articles or the like which has been Research volume 30, number 2, pages E1-9, 2002) contain included in the present specification is solely for the purpose 50 ing either a human IgG1 heavy chain complete or partial of providing a context for the present invention. It is not to constant region (such as Swissprot accession number be taken as an admission that any or all of these matters form PO1857), a human IgG4 heavy chain constant region (such part of the prior art base or were common general knowledge as Swissprot accession number PO1861 incorporating sub in the field relevant to the present invention as it existed in stitution S228P), human Ig kappa constant region (Swissprot Australia or elsewhere before the priority date of each claim 55 accession number PO1834) or human Ig lambda constant of this application. region (Swissprot accession number POCG05) either as a It must be noted that, as used in the Subject specification, naked Igor as a cytokine gene fusion form using overlap the singular forms “a”, “an and “the include plural aspects extension PCR techniques and restriction sites according to unless the context clearly dictates otherwise. Thus, for cloning methods well known by those skilled in the art. example, reference to 'a' includes a single as well as two or 60 Production of IgG and IgG Interferon Fusion Protein Con more; reference to “an includes a single as well as two or Structs: more; reference to “the includes a single as well as two or DNA plasmids encoding the IgGs and IgG-cytokine more and so forth. fusion protein constructs were prepared using Plasmid Plus Having generally described the invention, the same will Maxi kit (Qiagen, Valencia, Calif.) and then transfected into be more readily understood by reference to the following 65 HEK293-6E cells (CNRC, Montreal, Canada) grown in F17 examples, which are provided by way of illustration and are synthetic medium supplemented with 0.1% Pluronic F-68, 4 not intended as limiting. mM L-glutamine (Invitrogen, Carlsbad, Calif.) using a com US 9,611,322 B2 59 60 mercially available transfection reagent and OptiMEM aliquoted into each well of a 96 well round bottom tissue medium (Invitrogen, Carlsbad, Calif.). After allowing for culture plate (hereafter, “experimental plate') (TPP, expression for 6 days in an incubator supplied with 5% CO cath92067). On a separate, sterile 96 well plate (hereafter, and gentle shaking, the culture media was isolated and "dilution plate'; Costar, Corning, N.Y. catfi3879), test Subjected to IgG affinity purification using Protein G-aga 5 articles were serially diluted in duplicate in RPMI 1640+ rose beads (GE Healthcare, Piscataway, N.J.). Purified IgG 10% FBS. Then, 50 pul?well was transferred from the dilution and IgG-cytokine fusion protein constructs were then con plate to the experimental plate. The experimental plate was centrated and buffer-exchanged to phosphate buffered saline then incubated for four days at 37° C. with 5% CO. (PBS) pH 7.4 using Amicon Ultra centrifugal filter devices A mixture of the manufacturer-Supplied assay buffer and (Millipore, Billerica, Mass.), followed by protein concen 10 assay substrate (hereafter, “CellTilterGlo reagent, mixed tration determination using a NanoDrop 2000 spectropho according to the manufacturers instructions) was added to tometer (Thermo Scientific, Waltham, Mass.). the experimental plate at 100 ul/well. The plate was shaken Although different antibody-cytokine fusion protein con for two minutes. Then, 100 ul/well was transferred from the structs were expressed in the HEK system with differing experimental plate to a 96 well flat bottom white opaque yields, several of them, in particular several of those based 15 plate (hereafter, “assay plate’: BD Biosciences, Franklin on IFNC, were produce at at least 100 mg/L of media, Lakes, N.J. cati35 3296). The content of the assay plate was showed high solubility and did not aggregate as determined then allowed to stabilize in the dark for 15 minutes at room by size exclusion chromatography. temperature. The plate was read on a Victor 3V Multilabel The amino acid sequences of the antibodies and antibody Counter (Perkin Elmer, Waltham, Mass., modeli1420-041) ligand construct fusion protein constructs are described on the luminometry channel and the luminescence was below. For antibody-cytokine fusion protein constructs in measured. Results are presented as "relative luminescence which the cytokine was fused to the C-terminus of the heavy units (RLU)”. or light chain, the following naming convention was used: Data was analyzed using Prism 5 (Graphpad, San Diego, name of mab-linkage to heavy chain (“HC) or light chain Calif.) using non-linear regression and three parameter ("LC)-Linker name-ligand name (mutation) iso 25 curve fit to determine the midpoint of the curve (EC50). For type. each test article, potency relative to free IFNC.2b (or some Thus for example the construct “Rituximab-HC-L6-IFNC. other form of IFN with a known potency relative to IFNC2b) (A145G) IgG1 is the antibody rituximab, with IFNC2b was calculated as a ratio of EC50s. (with the A145G point mutation), linked to the C-terminus One of ordinary skill in the art will appreciate that there of the IgG1 heavy chain, with an intervening linker L6. 30 are many other commonly used assays for measuring cell viability that could also be used. The linkers used in the experiments were as follows: “On target (ARP) assay” (also sometimes referred to L0: no linker (direct fusion of the C-terminus of an antibody herein as a “targeted assay’): The multiple myeloma cell line chain with the N-terminus of the cytokine) ARP-1 was a gift from Bart Barlogie MD, PhD, Director of 35 the Myeloma Institute at the University of Arkansas Medical (SEQ ID NO: 132) Center (Little Rock, Ak.). It is described in Hardin J. et al., L6: SGGGGS (Interleukin-6 prevents dexamethasone-induced myeloma (SEQ ID NO: 133) cell death. Blood: 84:3063, 1994). ARP-1 cells (CD38") L16 : SGGGGSGGGGSGGGGS were used to test CD38 targeting antibody-IFN fusion pro 40 tein constructs. Culture and assay conditions were the same Method for Measuring Antigen-Targeted Activity of Anti as for Daudi-based assay outlined above, with the following body-IFNC. Fusion Protein Constructs exceptions: ARP-1 was cultured to a density of 4.0x10 to "On target (Daudi) assay': This assay was used to quan 6.0x10 cells/ml. ARP-1 concentration was adjusted to 1.0x tify the anti-proliferative activity of IFNs and antibody-IFN 10 cells/ml prior to assay. fusion protein constructs on cells that display that antigen 45 Method for Measuring Non-Antigen-Targeted Activity of corresponding to the antibody to which the IFN is fused, and Antibody-IFNC. Fusion Protein Constructs may be used as part of the assay for calculating the antigen “Off-target assay” (also sometimes referred to herein as sensitivity index (ASI) defined herein. Daudi cells express the “not-targeted assay): The iLite assay from PBL Inter both CD20 and CD38 as cell surface associated antigens. feron Source (Piscataway, N.J., Cati51100), was performed The viability of cells was measured using the reagent 50 largely as described by the manufacturer with the addition of CellTiter-Glo (R), Cat #G7570, from Promega (Madison, a human IgG blocking step. The iLite cell line is described Wis.). This is a luminescence-based assay that determines by the manufacturer as “a stable transfected cell line derived the viability of cells in culture based on quantitation of ATP. from a commercially available pro-monocytic human cell The signal strength is proportional to the number of viable line characterized by the expression of MHC Class II cells in a microtiter plate well. The details of the assay are 55 antigens, in particular the human lymphocyte antigen (HLA as follows: DR), on the cell surface.” The cell line contains a stably Daudi cells (obtained from ATCC, Manassas, Va.) were transfected luciferase gene, the expression of which is driven cultured in a T75 flask (TPP, Trasadingen, Switzerland, by an interferon-response element (IRE), which allows for cath90076) to a preferred density of between 0.5x10 and interferon activity to be quantified based on luminescence 0.8x10 viable cells/ml in RPMI 1640 (Mediatech, Inc., 60 output. The manufacturer-supplied iLite plate (hereafter Manassas, Va., cat #10-040-CV) with 10% Fetal Bovine “assay plate”) and diluent were removed from the -80° C. Serum (FBS; Hyclone, Logan, Utah cathi SH30070.03). freezer and allowed to equilibrate to room temperature. Cells were harvested by centrifuging at 400 g for five Then, 50 ul of the diluent was added per well to the assay minutes, decanting the Supernatant, and resuspending the plate. The vial of manufacturer-supplied reporter cells was cell pellet in RPMI 1640+10% FBS. Cells were then counted 65 removed from the -80° C. freezer and thawed in a 37° C. and the density was adjusted to 3.0x10 cells/ml in RPMI water bath. Then, 25ul aliquiots of cells were dispensed into 1640+10% FBS. Then, 50 ul of the cell suspension was each well of the assay plate. Next, 12.5ul of 8 mg/ml human US 9,611,322 B2 61 62 IgG that was diluted into RPMI 1640+10% FBS (Sigma reported by other (for example in U.S. Pat. No. 7,456,257). Chemicals, St. Louis, Mo.; cathi I4506) was added per well. Neither Palivizumab nor Rituximab alone (without the The contents were mixed and incubated at 37° C. for 15 fusion to an interferon) showed any activity in this assay minutes. On a separate “dilution plate,' test articles were (data not shown). serially diluted in duplicate in RPMI 1640+10% FBS. Then, To determine whether the antibody-IFNC2b fusion pro 12.5ul of the test articles were transferred from the dilution tein constructs had enhanced activity relative to free IFNC2b plate to the assay plate. The assay plate was then incubated on cells that do display the corresponding antigen on their at 37° C. with 5% CO, for 17 hours. The manufacturer cell surface, their effect on Daudi cells, which display the supplied assay buffer and substrate were removed from the CD20 antigen of Rituximab, but which do not display the -80°C. freezer and allowed to equilibrate to room tempera 10 RSV F protein antigen corresponding to Palivizumab, was ture for two hours. The manufacturer-supplied assay buffer examined. The assay used in this case, described above as was added to the manufacturer-Supplied Substrate vial and "Method for measuring antigen-targeted activity of anti mixed well according to the manufacturers instructions to body-IFNC. fusion protein constructs” or simply the “on create the “luminescence solution.” Then, 100 ul of the target (Daudi) assay, measured the effect of the test sub luminescence Solution was added to each well of the assay 15 stances on the viability of Daudi cells. With these cells, the plate. The plate was shaken for 2 minutes. The plate was Rituximab-IFNC2b fusion protein construct (Rituximab then incubated at room temperature for 5 minutes in the dark HC-L6-IFNC. IgG1) was 3.25-fold (1.3/0.4—3.25) more and finally read on a Victor 3V Multilabel Counter on a potent than free IFNC2b (FIG. 7). In other words, the luminometry channel and the luminescence measured and attachment of Rituximab to IFNC2b resulted in slightly presented as RLU. The data was analyzed with Graphpad reduced (3.1-fold) activity towards antigen-negative cells Prism 5 as described for the"on-target (Daudi) assay.” (FIG. 6) but slightly increased (3.25-fold) activity towards above. To test anti-CD38 antibody-IFN fusion protein con antigen-positive cells (FIG. 7). Overall, the antibody attach structs in the iLite assay, the manufacturer-Supplied diluent ment therefore increased the antigen-specificity index (ASI). was Supplemented with 2 mg/ml human IgG and 0.5 mg/ml defined as the fold increased potency relative to free IFNC2b anti-CD38 antibody (same antibody clone being tested as an 25 on antigen-positive cells multiplied by the fold decreased antibody-IFN fusion protein construct, to block any binding potency relative to free IFNC.2b on antigen-negative cells, of the anti-CD38 antibody-IFN fusion protein constructs to by 10-fold (3.1x3.25) in this experiment. A repeat of the the CD38 expressed on the iLite cells). experiments measured an ASI of 14, as shown in Table 20, Results: Antigen-Specificity of Antibody-IFNC. Fusion Pro row 2. The EC50 (mathematical midpoint of the dose tein Constructs 30 response curve) was used as a measure of potency in the FIG. 6 shows the interferon activity of free IFNo.2b (SEQ calculations presented here. In other words, when compound ID NO:3: “IFNo” in figure) as well as IFNo.2b fused to the A showed an EC50 that is 10-fold lower than compound B, C-terminus of the heavy chain of two different antibodies it was said to have a 10-fold higher potency. (rituximab and palivizumab, an isotype control antibody), as The results presented in FIG. 8 are consistent with anti acting on a the iLite cell line. This cell line does not display 35 body-based targeting relying on antibody-antigen reactivity: the antigen for either of these antibodies, so this assay the Rituximab-IFNC. fusion protein construct (Rituximab reveals the potency of various IFNC2b-containing proteins HC-L6-IFNC-IgG1) was 12-fold (2.2/0.18–12) more potent in the absence of antibody-antigen-based targeting. The in reducing viability of the CD20 Daudi cells than the details of this assay are described above under the heading Palivizumab-IFNC. fusion protein construct (Isotype-HC "Method for measuring non-antigen-targeted activity of 40 L6-IFNC-IgG1), the antigen for which is not present on the antibody-IFNC. fusion protein constructs” and is hereafter Daudi cells. abbreviated as the “off-target assay.” “Rituximab-HC-L6 The modest reduction in IFNC activity that occurred as a IFNC. IgG1 refers to the CD20-targeting chimeric antibody result of linking it to an antibody may not be sufficient to Rituximab, in which the light chain (SEQ ID NO:276) is prevent the toxicity of the IFNC. component of the construct unaltered but the IgG1 class heavy chain (SEQID NO:277) 45 in human Subjects. Various mutations were therefore intro has, attached to its C terminus, a 6 amino acid linker duced into IFNC2b in order to reduce its activity and sequence (“L6:” SGGGGS, SEQ ID NO:132), followed by toxicity. For example, five different mutant versions of the sequence for IFNC2b (SEQ ID NO:3); this heavy IFNC.2b were generated and, in each case, linked to the chain-linker-IFNC. sequence is shown as SEQ ID NO:280. C-terminus of the heavy chain of Rituximab via the six “Isotype-HC-L6-IFNC. IgG1 refers to the RSV-targeting 50 amino acid linker L6, which has the sequence SGGGGS humanized antibody Palivizumab, in which the light chain (SEQ ID NO:132). These constructs were compared to the (SEQ ID NO:290) is unaltered but the IgG1 class heavy Rituximab-wild type IFN fusion protein construct, Ritux chain (SEQ ID NO:291) has, attached to its C terminus, a 6 imab-HC-L6-IFNC. IgG1 (as also used in the experiments amino acid linker sequence (“L6: SGGGGS, SEQ ID shown in FIGS. 6-8). The five mutant versions were R144A, NO:132), followed by the sequence for IFNC2b (SEQ ID 55 A145G, R33A+YNS, R33A and R144A+YNS. The NO:3); this heavy chain-linker-IFNC2b sequence is shown sequences of these variants are described below. The degree as SEQ ID NO:294. In this assay, free IFNC2b showed an of expected reduced affinity for the type I interferon recep ECso for activating gene expression through an interferon tors based on previous characterization by others of IFN response element (IRE) of 1.9 pM. By attaching IFNC2b to mutants, and the amount of expected attenuation in inter Rituximab, there was a 3.1-fold (5.9/1.9–3.1) decrease in its 60 feron activity, are shown in Tables 6 and 7, above. potency. A similar, modest decrease in potency was observed FIGS. 9, 10 and Table 20 show the degree of reduced when IFNC.2b was linked to Palivizumab. Again, the cell interferon activity for each of these Rituximab-attenuated line used in this study did not have the antigen correspond IFNC2b fusion protein constructs relative to free, wild type ing to either of these antibodies on its cell Surface, demon IFNC.2b, on antigen-negative (i.e. CD20-negative) cells. The strating that attachment of an IgG to the N-terminus of 65 R144A mutant of the Rituximab-IFNC2b fusion protein IFNC2b caused a modest (3-4x) decrease in its non-antigen construct (composed of SEQID NOS:282 (heavy chain) and targeted IFN activity. This is consistent with what has been 276 (light chain)) showed 386-fold reduced interferon activ US 9,611,322 B2 63 64 ity (2200/5.7–386). The A145G and R33A+YNS versions activity on antigen-positive cells (FIGS. 11 and 12) relative (composed of the heavy chains of SEQ ID NOS:284 and to their reduced potency on antigen-negative cells (FIGS. 9 286, respectively, each of which are combined with the light and 10). With the exception of the R33A version of the chain of SEQID NO:276) showed 491-fold (2800/5.7=491) fusion protein construct, discussed below, the attenuating and 1,071-fold (6100/5.7=1,071) reduced activity, respec 5 mutations caused a 384-1,160-fold decrease in interferon tively. FIG. 10 shows the degree of reduced interferon activity relative to free wild type IFNC2b on antigen activity for the R144A+YNS fusion protein construct (com negative cells, but showed 0.23-1.2-fold of the potency of posed of SEQ ID NOS:288 (heavy chain) and 276 (light wild type IFNC.2b on antigen-positive cells. The R33A mutated fusion protein construct, which had undetectable chain)) to be 303-fold (1700/5.6–303) relative to the Rituxu IFN activity in the absence of antibody-antigen targeting, mab fusion protein construct lacking the IFN mutations 10 still showed significant activity in the presence of antibody (Rituximab-HC-L6-IFNC. IgG1); since Rituximab-HC-L6 targeting; the potency of the R33A version of the fusion IFNC. IgG1 is 3.8-fold less potent on antigen negative cells protein construct was 1,620-fold lower than the same fusion than free, wild type IFNC2b (data from FIG.9; 22/5.7–3.8), protein construct lacking this attenuating mutation in the this means that the R144A+YNS version of the fusion on-target assay (340/0.21=1,620-fold attenuation). This is in protein construct was 1,150-fold less potent than free, wild 15 stark contrast to the at least 100,000-fold attenuation caused type IFNC (303x3.8=1,150). The R33A version of the fusion by the same mutation in the absence of antibody-based protein construct (composed of SEQ ID NOS:436 (heavy targeting (FIG. 10). These results are summarized in Table chain) and 276 (light chain)) was attenuated to Such a high 2O. degree that it showed no detectable activity in the non To determine whether this dramatic difference in the targeted assay. ability of the mutations in the IFNC. component of the fusion TABLE 20 Targeted Potency Relative Non-Targeted Potency Antigen-Specificity to free IFNC2b (EC50 Relative to free IFNC2b Index (ASI: Fusion protein IFNC2b EC50 Fusion (EC50 IFNo.2b/EC50 calculated as construct Test protein construct) Fusion protein construct) Column Af Article Column A Column B Column B) Ritux-IFNC2b 3.6 O.26 14 Ritux-IFNC2b O.86 O.OO26 330 (R144A) Ritux-IFNC2b 1.2 O.OO20 600 (A145G) Ritux-IFNC2b 1.6 O.OOO93 1,700 (R33A + YNS) Ritux-IFNC2b 0.0022* No detectable activity in ND (R33A) non-targeted assay Ritux-IFNC2b 0.23% O.OOO86* 270 (R144A + YNS) *Free DFNo.2b was not tested on the same day as the test articles in these rows. Therefore, these measurements are based on a comparison of the test article with Rituximab-HC-L6-IFNo. IgG1, which was assayed on the same day and same plate, multiplied by a correction factor based on the relative activity of IFNo.2b vs Rituximab HC-L6-LFNo. IgG1 (i.e. data shown in the second row from the top) measured on a different day,
Surprisingly, when the amount of interferon activity of protein constructs to Substantially reduce its activity on these highly attenuated rituxumab-mutant IFNC2b fusion antigen-negative cells as compared to antigen-positive cells protein constructs was measured on antigen-positive cells could be extended to other fusion protein constructs target (Daudi, CD20), there was generally very little attenuation 45 ing other antigens, antibodies targeting the multiple compared to the wild type IFNC2b version of the Ritux myeloma antigen CD 38 (SEQ ID NO:131) were fused to imab-IFNC2b fusion protein construct (FIGS. 11-12), and both wild type and attenuated forms of IFNC. and charac thus the mutated interferons still possessed the ability to terized. Some of these experiments were performed using activate the IFN receptor on “on-target cells whilst having the antibody G005 (De Weers et al. (U.S. Pat. No. 7,829, a greatly reduced ability to activate it on "off-target cells. 50 673)); the sequences of the heavy and light chains for this For example, the R33A+YNS version of the construct was human antibody are shown as SEQ ID NOS: 135 and 134, only 2.2-fold (0.74/0.33=2.2) less active than the Ritux respectively. imab-IFNC2b wild type construct on the antigen-positive In addition, several novel human and rat antibodies (Daudi) cells. This was in contrast to the 277-fold (6100/ against CD38 were produced, as described below. 22–277: FIG.9) reduced activity on antigen-negative cells. 55 Development of Novel CD38 Antibodies The mutations in the IFNC2b, in the context of the Ritux Formatting CD38 Constructs for Expression imab-IFNC2b fusion protein construct, caused a substan The extracellular domains (ECD) of human and cynomol tially greater attenuation of activity on antigen-negative cells gus monkey CD38 proteins were each formatted to include than on antigen-positive cells. As a result, the Rituximab a cleavable N-terminal leader sequence, an AvitagTM, a HC-L6-IFNC2b (R33A+YNS) IgG1 fusion protein con 60 poly-histidine tag and a thrombin cleavage site to yield struct exhibited a substantially greater antigen-specificity proteins SEQ ID NO:127 and 128 respectively. These were index (ASI, 1,700-fold) compared to Rituximab-HC-L6 back-translated into DNA sequences and synthesized de IFNC2b IgG1 (10- to 14-fold) or free IFNC2b (1-fold, by novo by assembly of synthetic oligonucleotides by methods definition), Suggesting that its off-target effects in vivo will known by those with skill in the art. Following gene be substantially reduced. 65 synthesis, the genes were subcloned into vector pTT5 Other Rituximab-IFNC2b constructs with mutations in (Durocher, Nucleic Acids Research volume 30, number 2, the IFNC2b portion also showed surprisingly little reduced pages E1-9, 2002) to yield constructs to produce soluble US 9,611,322 B2 65 66 secreted forms of these proteins via transient expression in biotinylation of CD38 ED proteins was confirmed using a HEK293E cells (Durocher, supra). combination of polyacrylamide gel electrophoresis and Construction of Vectors for Antibody Expression Western blotting. Western blots were probed using Strepta Heavy and light chain variable region sequences were vidin-HRP (BD Biosciences, San Diego, Calif.) and devel subcloned into variants of the vector pTT5 containing either 5 oped using TMB (Sigma-Aldrich, St. Louis, Mo.). For each a human IgG1 heavy chain constant region (such as Swis antigen, monomeric biotinylated CD38 ED was detected. sprot accession number PO1857), a human IgG4 heavy Generation of Anti-CD38 Antibodies by Phage Display chain constant region (such as Swissprot accession number FAbs that bind to both human and cynomolgus monkey P01861 incorporating substitution S228P), human kappa CD38 EDs were isolated from a naive phagemid library constant region (Swissprot accession number P01834) or 10 human lambda region (Swissprot accession number comprising approximately 2.5x10' individual human FAb P0CG05) to yield full length antibody chains. fragments. Methods of generating phage antibody fragment Transient Expression of Constructs in HEK293-6E Cells libraries are discussed in “Phage display: A Practical HEK293-6E cells were cultured in complete cell growth Approach” (Eds. Clackson and Lowman: Oxford University media (1 L of F17 medium (InvitrogenTM), 9 mL of Pluronic 15 Press, Oxford, UK) and “Antibody Phage Display Methods F68 (InvitrogenTM), 2 mM Glutamine containing 20% (w/v) and Protocols’ (Eds. O'Brien and Aitken; Humana Press Tryptone NI (Organotechnie R) with Geneticin (50 mg/mL, Inc, NJ 07512). Briefly, antibody heavy and light chain InvitrogenTM) at 50 ul/100 mL culture). The day before variable regions were amplified based on RNA from donor transfection, cells were harvested by centrifugation and samples. Antibody heavy and light chain variable regions resuspended in fresh media (without Geneticin). The next were then inserted into phagemid vectors to generate a day DNA was mixed with a commercial transfection reagent library of antibody fragments fused to a phage coat protein. and the DNA transfection mix added to the culture drop The antibody library used herein was a high diversity naive wise. The culture was incubated overnight at 37°C. with 5% phagemid library that expressed antibody fragments in the CO and 120 rpm without Geneticin. The next day, 12.5 mL Fab format. of Tryptone was added along with 250 ul of Geneticin per 25 Anti-CD38 FAbs were isolated from the phage display 500 mL culture. The culture was incubated at 37° C., 5% library over the course of two panning campaigns (i.e. CO and 120 rpm. After 7 days, the supernatant was har discrete phage display experiments with different reagents or vested by centrifugation and was ready for purification. panning conditions). The general protocol followed the Expression and Purification of Antibodies method outlined by Marks et al. (Marks, J. D. & Bradbury, Transient co-expression of heavy and light chains in 30 A., 2004, Methods Mol Biol, 248, 161-76). HEK293-6E cells (as described above) generated antibodies Each phage display campaign involved three rounds of that were subsequently purified by protein A chromatogra panning. For each round, ~2.5x10" phage particles were phy. Briefly, supernatants derived from these transfections blocked by mixing 1:1 with blocking buffer (4% skim milk were adjusted to pH 7.4 before being loaded onto a HiTrap in PBS, pH 7.4) and incubating for 1 hr at room temperature. Protein A column (5 mL, GE Healthcare). The column was 35 The blocked phage library was then pre-depleted for any washed with 50 mL of 1xPBS (pH 7.4). Elution was biotinylated protein tag motif binders used in panning performed using 0.1 M citric acid pH 2.5. The eluted through incubation for 45 mins with 50-200 pmols of an antibody was desalted using Zeba Desalting columns irrelevant antigen containing an identical biotinylated tag (Pierce) into 1xPBS (pH 7.4). The antibodies were analyzed motif. Tag- and streptavidin-binders were captured by add using SDS-PAGE. The concentration of the antibody was 40 ing an excess (75-300 uL) of streptavidin-coated Dynabeads determined using the BCA assay kit (Pierce). (Invitrogen), which were blocked as described for the Purification of Histidine-Tagged Proteins from Tissue Cul library. The beads (including tag- and streptavidin-binders ture Supernatants attached to them) were immobilized using a magnet and Immobilized metal ion affinity chromatography (IMAC) discarded. was used to purify human and cynomolgous monkey CD38 45 Library panning was conducted by mixing the blocked extracellular domain (ED) proteins from tissue culture and pre-depleted library with 50-200 pmols of biotinylated Supernatants. Briefly, protein Supernatants were diluted in recombinant CD38 ED in a 2 mL microcentrifuge tube and binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 30 rotating for 2 hrs at room temperature. Then, 100 uL of mM imidazole, pH 7.4) before being loaded onto a His streptavidin-coupled Dynabeads (Invitrogen, Carlsbad, TrapTM FF column (1 mL, GE Healthcare). The column was 50 Calif.) were added and the mixture was incubated a further washed with 5 mL of binding buffer (pH 7.4) and elution 15 minutes as described previously. Non-specifically bound was performed using 20 mM sodium phosphate, 0.5 M phage were removed using a series of washes. Each wash NaCl, 500 mM imidazole, pH 7.4. The eluted proteins were involved pulling the bead complexes out of the solution onto desalted and buffer exchanged using Amicon Ultra-15 cen the tube wall using a magnetic rack, aspirating the Super trifugal filter unit with Ultracel-10 membrane (Millipore) 55 natant and then re-suspending the beads in freshwash buffer. into 1xPBS (pH 7.4). The absorbance at 280 nm (As) of This was repeated multiple times with either PBS wash the protein was assessed using a Nanodrop spectrophotom buffer (1xPBS with 0.5% skim milk) or PBS-T wash buffer eter and readings corrected using the predicted extinction (1xPBS supplemented with 0.05% TWEEN-20 Sigma coefficients to determine protein concentrations. Aldrich, St. Louis, Mo. and 0.5% skim milk). Phage that Biotinylation of Antigens for Phage Display 60 remained bound after the washing process were eluted from The AvitagTM motifs of human and cynomolgus monkey the biotinylated-CD38 ED-bead complexes by incubation CD38 EDs were biotinylated according to manufacturers with either a twenty-fold excess of non-biotinylated CD38 directions (Avidity LLC, Aurora, Colo.). Excess unconju ED for 1 hr at room temperature or 0.5 mL of 100 mM gated biotin was removed from the biotinylated proteins by triethylamine (TEA) (Merck Chemicals, Darmstadt) for 20 desalting into 1xPBS using a 7 KD molecular weight cut off 65 mins at room temperature. TEA-eluted output phage were (MWCO) Zeba spin column (Thermo Scientific, Logan, neutralized by the addition of 0.25 mL of 1 M Tris-HCl pH Utah) according to manufacturers instructions. Successful 7.4 (Sigma-Aldrich, St. Louis, Mo.). US 9,611,322 B2 67 68 At the end of the first and second rounds of panning, the molgus monkey CD38 ED were isolated and sequenced. Of output phage were added to a 10 mL culture of exponentially approximately 1,000 FAbs screened for binding to human growing TG1 E. coli (2x yeast-tryptone (2YT) growth and cynomolgus monkey CD38 ED, six genetically unique media) and allowed to infect the cells during a 30 minute FAbs were identified. Table 21 summarises the FAb incubation at 37° C. without shaking, then with shaking at sequence data obtained. The variable regions of Some of 250 rpm for 30 additional minutes. The phagemids encoding these antibodies are shown in FIG. 13. the phage display output were then rescued as phage par ticles following a standard protocol (Marks, J. D. & Brad TABLE 21 bury, A., 2004, Methods Mol Biol, 248, 161-76). At the end of the third panning round, TG1 cells were infected with 10 Campaign output phage and were plated on 2YT agar (Supplemented Number FAb name V. Sequence VfV sequence with 2% glucose and 100 ug/mL carbenicillin) at a sufficient 1 X910/12 SEQ ID NO: 395 SEQ ID NO: 394 dilution to produce discrete E. coli colonies. These colonies 1 X913/15 SEQ ID NO:397 SEQ ID NO: 396 were used to inoculate 1 mL liquid cultures to allow expres 2 X355/01 SEQ ID NO: 421 SEQ ID NO: 420 15 2 X355/02 SEQ ID NO:391 SEQ ID NO: 390 sion of FAb fragments for use in Screening experiments. 2 X355/04 SEQ ID NO: 423 SEQ ID NO: 422 ELISA-Based Screening of FAbs for CD38 Binding 2 X355/07 SEQ ID NO:393 SEQ ID NO: 392 Each individual E. coli colony was used to express a FAb that could be screened for CD38 ED-binding activity. Colo nies were inoculated into 1 mL starter cultures (Supple All FAbs were converted into IgG1 format by cloning into mented with 100 ug/mL carbenicillin and 2% glucose) in the pTT5 vectors (described above), expressed in HEK293 96-well deep-well plates (Costar) and incubated overnight at 6E cells and the resulting IgGs purified by protein A affinity 37°C. with shaking at 350 rpm (Innova R44 shaker; 1 inch chromatography as described above. orbit). These starter cultures were diluted 1:100 into a 1 mL Assessing Binding of IgGs to Human CD38 Positive Cell expression culture (2YT supplemented with 100 g/mL Line RPMI-8226 carbenicillin) and grown to an optical density (600 nm) of 25 The ability of the phage derived antibodies to bind the 0.5-0.8. FAb expression was induced by adding isopropyl model human CD38 positive myeloma cell line RPMI-8226 beta-D-thiogalactopyranoside (IPTG) to a final concentra (obtained from the Health Protection Agency Culture Col tion of 1 mM. Cultures were incubated at 25°C. for 16 hrs. lections, Porton Down, Salisbury, SP4 0JG, UK) in flow FAb samples were prepared by harvesting cells by cen cytometry-based assays was tested. Briefly, viable RPMI trifugation (2,000 g, 10 mins) and performing a lysozyme 30 8226 cells (2x10, as judged by trypan blue exclusion) were extraction. The cell pellet was resuspended in 200 uL of lysis incubated with each antibody or with a human IgG isotype buffer (160 ug/mL lysozyme, 10 ug/mL RNase A, 5ug/mL control antibody preparation (Sigma-Aldrich, St. Louis, DNase and complete protease inhibitors (Roche, Nutley, Mo.) at various concentrations in 100 ul of FACS buffer N.J.)) and shaken at 400 rpm for 30 minutes at 21° C. (PBS plus 1% fetal calf serum, FCS) in 96 well plates for 20 Following addition of a further 100 ul of lysis buffer, the 35 minutes on ice in the dark. Cells were washed twice with reactions were incubated for a further 30 minutes as described previously. Clarified lysates were isolated follow FACS buffer before incubation for 20 minutes in 100 ul of ing centrifugation at 3,000 g for 10 minutes and stored at 4 FACS buffer containing goat anti-human IgG (Fc-specific, C. until required. conjugated to fluorescein isothiocyanate, FITC: Sigma-Al To Screen by enzyme-linked immunosorbent assay 40 drich, St. Louis, Mo.). After washing with FACS buffer, cells (ELISA) for human CD38 ED-binders derived from the were resuspended in FACS buffer and analysed for antibody phage display biopanning, human CD38 extracellular binding by flow cytometry on a FACS Canto (BD Biosci domain (ED) (produced in HEK 293-6E cells and bioti ences, San Diego, Calif.) using EV, side scatter and FL-1 nylated as described above) was captured on Streptavidin gating. Results are expressed as mean fluorescent intensity coated ELISA plates (Nunc) at 1 lug/mL. Plates were then 45 (MFI) plotted against protein concentration (FIG. 14). washed and individual FAb samples (prepared as described Generation of Anti-CD38 Antibodies by Genetic Immuni above) were added to individual wells on the ELISA plates. Zation FAbs were allowed to bind the captured CD38 ED for an Monoclonal antibodies against human CD38 ED were hour at room temperature and then washed three times with generated by genetic immunization with corresponding con PBS-T (1xPBS supplemented with 0.1% Tween R20). FAbs 50 ventional protein immunization of rats. For genetic immu that bound to CD38 ED were detected by incubation for 30 nization, the DNA sequence of human CD38 ED is provided minutes at room temperature with an anti-V5-HRP conju in SEQ ID NO:129. The corresponding conceptually trans gated antibody (Invitrogen, Carlsbad, Calif.) to detect the V5 lated protein sequence is given in SEQ ID NO: 130. The tag fused to the C-terminus of the FAb heavy chain. Plates DNA sequence of SEQ ID NO:129 was cloned into a were washed to remove unbound antibody and the assay 55 signal developed by incubation with 50 uL 3.3',5,5'-Tetram plasmid for genetic immunization using restriction enzyme ethylbenzidine (Sigma-Adrich, St. Louis, Mo.) and quench technology. Expression of the resulting plasmid allowed the ing with 50 uL 1 MHC1. Assay signals were read at A450 secretion of soluble CD38 ED tagged by a c-myc epitope at nm using a microplate reader (BMG Labtech). Results were the N- or C-terminus. The c-myc epitope was utilized to expressed as the raw A450 nm value, where any signal 60 confirm expression of CD38 ED. 2-fold greater than the average assay background was Rats were then immunized six times with the plasmid defined as positive'. using a Helios gene gun (Bio-Rad, Germany) according to In later assays FAb cross-reactivity with cynomolgus a published procedure (Kilpatrick et al., Hybridoma 17: monkey CD38 ED was assessed by coating biotinylated 569-576, 1998). One week after the last application of the cynomolgus monkey CD38 ED onto streptavidin coated 65 immunization plasmid, each rat was boosted by intradermal ELISA plates and proceeding as described above. Plasmids injection of untagged recombinant human CD38 ED. encoding FAbs cross-reactive with both human and cyno Untagged human CD38 ED for this purpose was produced US 9,611,322 B2 69 70 by removing the protein tags from SEQ ID NO:127 by TABLE 22-continued thrombin cleavage followed by purification over a size exclusion column. Binding to FACS binding to Rat Binding to Human Cynomolgus monkey RPMI-8226 cells Four days later, the rats were sacrificed and their lym antibody CD38 ED (ELISA) CD38 ED (ELISA) (MFI) phocytes fused with myeloma cells using polyethylene gly 5 R8A7 Y N S994 col (HybriMaxTM, Sigma-Aldrich, Germany), seeded at 100, R9B6 Y N 146 000 cells per well in 96-well microtiter plates and grown in R9C7 Y N 143 DMEM medium supplemented with 10% fetal bovine serum R9C10 Y N 645 and HAT additive for hybridoma selection (Kilpatricket al., R9ES Y N 179 10 R9GS Y N 2717 1998, supra). R1OA2 Y Y 4470 Screening Hybridoma Supernatants for Human and Cyno R1OA9 Y N 12807 molgus Monkey CD-38 Cross-Reactivity R1OB10 Y Y 858 Duplicate 100 uL samples of each hybridoma Supernatant were coated onto separate wells of a maxisorp ELISA plate FACS binding background MFI average was 153 (Nunc Plasticware. Thermo Scientific, Rochester, N.Y. 15 Molecular Characterisation of Rat Antibodies 14625, USA) through incubation at room temperature for an Six rat antibody hybridomas R5D1, R7F11, R5E8, hour. Plates were washed three times in 1xRBS-T and R10A2, R1OB10 and R7H11 were selected for molecular subsequently blocked by addition of 2% BSA/1xRBS. Fol characterization. RNA extraction from pelleted hybridoma lowing incubation for 1 hour at room temperature, plates cells of each clone was performed using TRI reagent were washed as described previously. To one well of each rat (Sigma-Aldrich, St. Louis, Mo.) according to manufactur antibody duplicate well was added 0.1 ug of biotinylated er's directions. The variable regions of each antibody were human CD38 in a final volume of 100 uL, 1xPBS. To the amplified using Rapid Amplification of cDNA Ends (RACE) second well of each rat antibody duplicate well was added reverse transcription polymerase chain reaction (RT-PCR) 0.1 g of biotinylated cynomolgus monkey CD38 ED in a methodology according to manufacturer's directions (Clon final volume of 100 uL 1xPBS. Plate wells were washed as 25 tech Mountain View, Calif. SMART RACE kit; Ambion described previously prior to detection of bound biotinylated Life Technologies Foster City, Calif. RLM-RACE kit). CD38 ED using a Streptavidin-HRP conjugate (BD Biosci Gene-specific reverse PCR primers to amplify the rat heavy ences, San Diego, Calif.). Plates were washed as above to chain variable domains by 5'-RACE were designed to anneal remove unbound Streptavidin-HRP conjugate and the assay to the available rat heavy chain constant region sequences. 30 Similarly, gene specific reverse PCR primers to amplify the signal developed by incubation with 50 uL 3.3',5,5'-Tetram rat light chains were designed to anneal to the rat kappa ethylbenzidine (Sigma-Aldrich) and quenching with 50 uL 1 chain constant region sequences, while further primers were M HC1. Assay signals were read at Also nm using a designed to anneal to the rat lambda chain constant region microplate reader (BMG Labtech, Cary, N.C.). Of the 15 Sequences. hybridoma Supernatants tested, all fifteen bound human 5'-RACE PCR was performed according to manufactur CD38 ED and seven bound cynomolgus monkey CD38 ED 35 er's directions (Life Technologies: Clontech) using Pful J1 (Table 22) as determined by ELISA. The cross-reactive tral I polymerase (Agilent). Following 5'-RACE PCR, prod antibodies are referred to as R5D1, R7F11, R5E8, R10A2, ucts were separated by agarose gel electrophoresis and R1OB10, R3A6 and R7H11. bands of approximately the predicted size based on the Flow Cytometry Binding of Rat Antibodies to Human CD38 location of the reverse primer in the constant region were Positive Cell Line RPMI-8226 40 excised from the gels. DNA was purified from agarose gel Viable RPMI-8226 cells (2x10, as judged by trypan blue slices using a Qiaquick spin gel extraction kit (Qiagen) exclusion) were incubated with 100 uL of rat hybridoma according to manufacturers instructions. Insert DNA was supernatant for 20 minutes on ice in the dark. Cells were cloned and propagated in E. coli using a StrataClone Blunt washed twice with FACS buffer (1xPBS plus 1% FCS) PCR Cloning Kit (Agilent, Santa Clara, Calif.) according to before incubation for 20 minutes in 100 ul of FACS buffer 45 manufacturers instructions. Single colonies from transfor containing anti-rat IgG-FITC conjugate (Sigma-Aldrich). mations were cultured and plasmid DNA prepared using a After washing cells in FACS buffer, they were resuspended GenEluteTM plasmid miniprep kit (Sigma-Aldrich, St. Louis, in FACS buffer and analysed for antibody-binding by flow Mo.). DNA inserts were sequenced and antibody variable cytometry on a FACS Canto (BD Biosciences, San Diego, regions identified in the conceptually translated protein 50 Sequences. Calif.) using EV, side scatter and FL-1 gating. Results were Vectors were constructed using the rat antibody variable expressed as mean fluorescent intensity (MFI). Of the 15 rat region sequences grafted onto human IgG1 constant antibodies exhibiting positive binding to human CD38 ED sequences for the heavy chain variable region and, human by ELISA, five showed weak or negligible binding to CD38 kappa or lambda backbones (keeping the same light chain expressed on the human myeloma cell line RPMI-8226 by isotype as in the rat antibodies). The resulting variable FACS (Table 22). 55 region sequences of each clone are listed in Table 23. Subsequent co-expression of the corresponding heavy- and TABLE 22 light chains in HEK293-6E cells, in the context of the pTT5 vectors, was followed by protein A purification of the Binding to FACS binding to resulting IgGs as described above. Rat Binding to Human Cynomolgus monkey RPMI-8226 cells 60 antibody CD38 ED (ELISA) CD38 ED (ELISA) (MFI) TABLE 23 R3A6 Y Y 279 RSD1 Y Y 122O7 Rat antibody Light chain isotype Heavy chain (VH) Light chain (VL) RSE8 Y Y 106.18 R7F4 Y N 310 RSD1 Kappa SEQ ID NO:399 SEQ ID NO:398 R7F11 Y Y 11897 65 RSE8 Kappa SEQ ID NO: 401 SEQ ID NO: 400 R7H11 Y Y 68O R1OA2 Kappa SEQ ID NO: 403 SEQ ID NO: 402 US 9,611,322 B2 71 72 TABLE 23-continued Anti-CD38-Attenuated IFN Fusion Protein Constructs To determine whether the surprising result obtained with Rat antibody Light chain isotype Heavy chain (VH) Light chain (VL) an anti-CD20 antibody fused to an attenuated IFNC. could be R1OB10 Lambda SEQ ID NO: 425 SEQ ID NO: 424 replicated with other antibodies, and in particular antibodies R7H11 Lambda SEQ ID NO: 427 SEQ ID NO: 426 5 targeting an antigen unrelated to CD20, fusion protein R7F11 kappa SEQ ID NO: 429 SEQ ID NO: 428 constructs comprising the fully human IgG1:kappa anti CD38 antibody G005 (composed of SEQ ID NOS:135 Affinity of Anti-CD38 Antibodies for Human and Cynomol gus Monkey CD38 (heavy chain) and 134 (light chain)) and IFNC. (SEQ ID The binding affinities of a selection of the antibodies 10 NO:3), with or without various attenuating mutations was produced against human and cynomolgus monkey CD38 made. FIG. 15 shows the results of the “off target assay” (as were measured. Briefly, using a Biacore T200, Protein A was described above) using the iLite kit. Because faint CD38 immobilized onto Flow Cell (FC) 1 (FC1) and FC2 (or signal was observed on the iLite cell line by flow cytometry alternatively FC3 and FC4) of a CM5 research grade sensor (not shown), the CD38 antigen was blocked by the addition chip using amine coupling, giving approximately 2000 RU. 15 of excess naked (e.g. without IFN or IFN variants fused to FC1 was used as a blank throughout the experiments. The it) anti-CD38 antibody for all iLite experiments using anti experiments were run in HBS-P buffer (0.01 M HEPES pH CD38-IFN fusion protein constructs; in each case, the con 7.4, 0.15 MNaCl, 0.005% v/v Surfactant P20). At a flow rate centration of blocking naked CD38 antibody used was 0.5 of 20 ul/min, 20 ul of 5 g/mL of antibody was passed over mg/ml. Also in each case, the same antibody clone being FC2. Human CD38 ED or separately, cynomolgus monkey assayed as an IFN or IFN-variant fusion protein construct CD38 ED was passed over the surface of FC1 and FC2 at was used to block any interaction with CD38. concentrations ranging from 25 nM to 200 nM. Regenera FIG. 15 shows the off target activity of free wild type tion of the surface was performed using 10 mM Glycine, pH IFNC2b (IFNC.(SEQID NO:3) vs. wild type IFNC2b fused 1.0. The FC1 sensorgram data was subtracted from FCS and the curves were fitted using a 1:1 Langmuir equation to 25 to the C-terminus of the CD38 antibody G005 (De Weers et generate the k, k, and K values. This data shows that al. (U.S. Pat. No. 7,829,673). The latter fusion protein cross-reactivity for human and cynomolgus monkey CD38 construct (G005-HC-L0-IFNC. IgG4) was of IgG4:kappa is maintained on conversion of the human phage-derived isotype and had no intervening linker between the C-termi Fabs into human IgGs and rat antibodies into chimeric nus of the heavy chain and the first residue of the IFNC. and rat-human IgGs (Table 24). 30 is described by SEQ ID NOS:150 (heavy chain) and 134 (light chain). As illustrated in FIG. 15, the anti CD38 TABLE 24 antibody-non-attenuated IFNC2b fusion protein construct was 27-fold less potent (19.5/0.726–27) than free IFNC.2fB in Antibody CD38 Ligand ka (1/Ms) x 10 kd (1/s) K (nM) the off-target assay (e.g. in the absence of CD38-targeting). X355/02 IgG1 Human 1.18 O.OOO892 7.6 35 FIG. 16 shows a comparison between the same two con X355/02 IgG1 Cynomolgus O.834 O.002282 27.4 structs in the “on target (ARP1) assay, in which the X355/07 IgG1 Human 1.15 O.OO132 11.4 X355/07 IgG1 Cynomolgus 11.3 0.01375 12.2 anti-CD38 antibody was allowed to bind to CD38, which R10A2 IgG1 Human 6.6 O.OOO4.79 0.7 was expressed at high levels on the ARP-1 cell line. The R10A2 IgG1 Cynomolgus 8.98 O.OO1928 2.2 G005-HC-L0-IFNC. IgG4 fusion protein construct was 3.6- R5D1 IgG1 Human 2.43 O.OOO239 10 40 fold (14.7/4.08=3.6) more potent than free IFNC.2b, presum R5D1 IgG1 Cynomolgus 11.1 O.OO1102 1.O R5E8 IgG1 Human 4.OS O.00118 2.9 ably due to the targeted delivery of the IFN to the CD38" R5E8 IgG1 Cynomolgus 4.52 O.OO1898 4.2 myeloma cells. Therefore, the G005-HC-L0-IFNC. IgG4 fusion protein construct has an antigen specificity index (ASI) of 97 (27x3.6–97; Table 25). TABLE 25 Antigen Specificity ECSO On ECSOIFNOf Index Target ECSO TA EC50 Off ECSOIFNOf Column Test Article (pM) ARP- (On Target; Target (pM) EC50 TA (Off 3. (TA) 1 ARP-1) iLite Target; iLite) Column 5 FNC. 10.8 1.OO O.260 1.00 1.OO GOOS-HC-LO 3.6* 0.037* * 97 FNC IgG4 GOOS-HC-LO 186 O.0581 25,800 1.01 x 10 5,750 FNC (R144A) gG4 GOOS-HC-LO 290 O.O372 1.08 x 10 2.41 x 10 15.400 FNC (R144S) gG4 GOOS-HC-LO ND ND >10 ND ND FNC (R144E) gG4 GOOS-HC-LO ND ND 9,970 2.61 x 10 ND FNC (R144G) gG4 US 9,611,322 B2 73 74 TABLE 25-continued Antigen Specificity ECSO On ECSOIFNOf Index Target ECSO TA EC50 Off ECSOIFNOf Column Test Article (pM) ARP- (On Target; Target (pM) EC50 TA (Off 3. (TA) 1 ARP-1) iLite Target; iLite) Column 5 GOOS-HC-LO- ND ND 1,690 1.54 x 10 ND FNC (R144H) gG4 GOOS-HC-LO- ND ND <100 ND ND FNC (R144K) gG4 GOOS-HC-LO- ND ND 431 O.OOO603 ND FNC (R144N) gG4 GOOS-HC-LO- ND ND 3,500 7.43 x 10 ND FNC (R144Q) gG4 GOOS-HC-LO- 333 O.O324 30,800 8.44 x 10 3,840 FNC (R144T) gG4 GOOS-HC-LO- 306 O.O3S3 92,100 2.82 x 10 12,500 FNC (R144Y) gG4 GOOS-HC-LO- 257 O.O420 1.59 x 10 1.64 x 10 25,600 FNC (R144I) gG4 GOOS-HC-LO- 191 O.OS6S 26,700 9.74 x 10 5,800 FNC (R144L) gG4 GOOS-HC-LO- ND ND 86,900 2.99 x 10 ND FNC (R144V) gG4 GOOS-HC-LO- 23.8 O.454 2,040 O.OOO127 3,570 FNC (A145G) gG4 GOOS-HC-LO- 222 O.0486 52,600 4.94 x 10 9,840 FNC (A145D) gG4 GOOS-HC-LO- ND ND <100 ND ND FNC (A145E) gG4 GOOS-HC-LO- 113 O.O956 24,900 1.04 x 10 9,190 FNC (A145H) gG4 GOOS-HC-LO- ND ND 28.9 O.OO900 ND FNC (A1451) gG4 GOOS-HC-LO- 174 O.O621 6.62 x 10 3.93 x 107 1.58 x 10 FNC (A145K) gG4 GOOS-HC-LO- ND ND 239 O.OO109 ND FNC (A145L) gG4 GOOS-HC-LO- ND ND 309 OOOO841 ND FNC (A145N) gG4 GOOS-HC-LO- ND ND 709 O.OOO367 ND FNC (A145Q) gG4 GOOS-HC-LO- ND ND >106 ND ND FNC (A145R) gG4 GOOS-HC-LO- ND ND <2 ND ND FNC (A145T) gG4 GOOS-HC-LO- 91.4 O.118 19,200 1.35 x 10 8,740 FNC (A145Y)
60 In order to determine whether the ASI could be increased, IgG isotypes (IgG1 and IgG4) and linker lengths (L0, no as was observed for the anti-CD20-IFNC. fusion protein linker; L6, 6 amino acid linker (SGGGGS, SEQ ID constructs, several variants were constructed by attenuating NO:132)) were made. The off-target assay and two types of the IFN portion of the anti-CD38-IFNC. fusion protein construct by mutation. Numerous different attenuating muta- 65 on-target assays (using Daudi and ARP-1), both described in tions were made in the context of the G005 or other CD38 detail above, were run and the results are shown in FIGS. monoclonal antibodies. In addition, constructs of different 17-38 and tabulated in Tables 25-33. The discussion below