US 20160367695A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0367695A1 Wilson et al. (43) Pub. Date: Dec. 22, 2016
(54) POLYPEPTIDE CONSTRUCTS AND USES (30) Foreign Application Priority Data THEREOF Oct. 28, 2011 (AU) ...... 2011 9045O2 (71) Applicant: Teva Pharmaceuticals Australia Pty Ltd, Macquarie Park (AU) Publication Classification (72) Inventors: David S. Wilson, Freemont, CA (US); Sarah L. Pogue, Freemont, CA (US); (51) Int. Cl. Glen E. Mikesell, Pacifica, CA (US); A6II 47/48 (2006.01) Tetsuya Taura, Palo Alto, CA (US); C07K 6/28 (2006.01) Wouter Korver, Mountain View, CA (52) U.S. Cl. (US); Anthony G. Doyle, Drummoyne CPC ..... A61K 47/48269 (2013.01); C07K 16/2896 (AU); Adam Clarke, Five Dock (AU); (2013.01); C07K 231 7/565 (2013.01); C07K Matthew Pollard, Dural (AU): 2317/55 (2013.01); C07K 2317/92 (2013.01) Stephen Tran, Strathfield South (AU); Jack Tzu Chiao Lin, Redwood City, (57) ABSTRACT CA (US) (21) Appl. No.: 15/194,926 The present invention provides a polypeptide construct (22) Filed: Jun. 28, 2016 comprising a peptide or polypeptide signaling ligand linked to an antibody or antigen binding portion thereof which Related U.S. Application Data binds to a cell Surface-associated antigen, wherein the ligand (63) Continuation of application No. 14/262,841, filed on comprises at least one amino acid Substitution or deletion Apr. 28, 2014, which is a continuation of application which reduces its potency on cells lacking expression of said No. PCT/AU2012/001323, filed on Oct. 29, 2012. antigen. Patent Application Publication Dec. 22, 2016 Sheet 1 of 62 US 2016/0367695 A1
FIGURE 1:
EGENO EABOVES
Antibody heavy chain IgG
Antibody light chair IgG
Attenuated (Polypeptide Ligand
Peptide linker (0-50 a mino acids if length) Patent Application Publication Dec. 22, 2016 Sheet 2 of 62 US 2016/0367695 A1
FIGURE 2:
LEGEND EMBODEMENT
Antibody to cell surface protein attached to Attenuated Ligand (AL)
Receptor for ligand
Viv Cell Surface Antigen Patent Application Publication Dec. 22, 2016 Sheet 3 of 62 US 2016/0367695 A1
FIGURE 3:
Human CD38 (SEQ ID NO: 131) : 1 MANCEFSPVSGDKPCCRLSRRAOLCLGVSILVLILVVVLAVVVPRWRQOWSGPGTTKRFP 60 61. ETVLARCVKYTEIHPEMRHVDCOSWWDAFKGAFISKHPCNITEEDYOPLMKLGTOTV PCN 120 121. KILLWSRIKDLAHQFTQVORDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDC 180 181 SNNPVSV FWKTVSRREAEAACDVVHVMLNGSRSKIF DKNSTFGSVEVHNL QPEKVOTLEA 240 241 WVIHGGREDSRDLCQDPTIKELESTISKRNT OFSCKNTYRPDKET, OCVKNPEDSSCTSET 3 OO
underlined: extra Callular domain italic: trans IIleinbrane do Ilain
Patent Application Publication Dec. 22, 2016 Sheet 5 of 62 US 2016/0367695 A1
FIGURE Sa:
Heavy chain (SEQ ID NO:180) : 1 EVOLLESGGGLVOPGGSLRLSCAVSGETFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYY 60 61 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTV 120 12 LO 180 181 SSGLYSLSSVVTVPsssTGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGP 240 24, 3OO 301 I 360 361. TKNOWSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWO 420 421. DLPOTHSLGSRRTLMLLACMRRISLFSCLK 480 48. DRHDFGFPOEEFGNOFOKAETIPvt. HEMIQQIFNLFSTKDSSAAWDETILDKFYTETYQQ SA 541 LNDLEACVIQGWGVTETPLMKEDSILAVRKYFORITLYLKEKKYSPCAWEVVRDEIMRSF 600 601 SLSTNLQESLRSKE 64 Light chain (SEQ ID NO:134): 60 2O 180 181 LSKADY 24 double underlined: antibody heavy and light chains single underlined: cytokine single underlined & bold: attenuating mutation G005-HC-LO-IFNo.(A145D) igg4
Patent Application Publication Dec. 22, 2016 Sheet 8 of 62 US 2016/0367695A1
FIGURE 5d: Heavy chain (SEQ ID NO:324) : l QVCLKOSGPGIVQPSQSLSLTCTVSGESLTSYGVHWVRQPPGKGLEWLGWIWSGGSTDYN 60 61 SA 120 1.21 180 81 240 241 300 3O1 ------m 360 361 LTKNQVSLTCLVKG ESNGQPENNYKTTPPVL SRW 420 421 QQGNVFSCSVMHEAL TQKSLSLSPGKSGGGGSGGGGSGGGGSPWEPGEDSKDVAAP 480 481 HRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCF 540 541 QSGFNEETCLVKIITGLLEFEVYLEYLONRFESSEEQARAVOMSTKVLIQFLOKKAKNLD 600 601 AITTPDETINASLLTKLQAQNQWLQDMTTELILRSFKEFLQSSLEALRQM 650 Light chain (SEQ ID NO:312) :
1 SIVMTOTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPD 60 120 180 215 double underlined: antibody heavy and light chains Single underlined: cytokine single underlined si bold: attenuating mutation italic: i.inker sequence HB95-HC-L16-IL-6(RI79E) iggi
Patent Application Publication Dec. 22, 2016 Sheet 10 of 62 US 2016/0367695A1
FIGURE 6:
Non-Targeted IFNo. Activity
S Construct EC50(pM) g g - FNC. 19 s -- Rituximab-HC-L6-IFNOlgG1 5.9 : -e- isotype-HC-L6-FNC. igG1 8.0 E o -
0.0001 0.01 100 10000 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 11 of 62 US 2016/0367695A1
FIGURE 7:
Targeted IFNo. Activity (Daudi)
1500000 E Construct IC50(pM) 2. 1000000 -- Rituximab-HC-L6-IFNo. IgG1 0.40 -- IFNO. 13 500000 E
O 0.001 0.01 0.1 10 100 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 12 of 62 US 2016/0367695A1
FIGURE 8:
Targeted IFNo. Activity (Daudi) 1 25 OO O O 1000000 Construct IC50(pM) -- Rituximab-HC-L6-IFNo. IgG1 0.18 -er isotype-HC-L6-FNo. lgG1 2.2 2575O5 OOOOOO OOO OOO O 0.001 0.01 0.1 1 10 100 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 13 of 62 US 2016/0367695A1
FIGURE 9:
Non-Targeted IFNa Activity
6000 Construct EC50(pM) S -- FNC. 5.7 -- Rituximab-HC-L6-IFNa IgG1 22 2 4000 -- Rituximab-HC-L6-IFNg (R144A) gG1 2200 -- Rituximab-HC-L6-IFNo. (A145G) IgG1 2800 2000 -- Rituximab-HC-L6-IFNo. (R33A+YNS) IgG1 6100 -o- isotype-HC-L6-IFNo. IgG1 18 O 0.01 100 0000 OOOOOO IFNo. equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 14 of 62 US 2016/0367695A1
FIGURE 10:
Non-Targeted IFNo. Activity Construct EC50 5000 Rituximab-HC-L6-FNo, IgG1 5.6 is 4000 Rituximab-HC-6-FNg (R33A) gG1 ND o Rituximab-HC-6-FNo. (R144A + YNS) IgG1 1700 3000
is 2000 E 1000
O 10-2 100 102 104 106 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 15 of 62 US 2016/0367695A1
FIGURE 11:
Targeted IFNo. Activity (Daudi) 2000000 Construct IC50(pM) S -0- IFNo. 12
1500000 Rituximab-HC-L6-IFNo, IgG1 0.33 8 Rituximab-HC-L6-IFNa (R144A)lgG1 1.4 1000000 - Rituximab-HC-L6-IFNo. (A145G) IgG1 1.1 2 500000 Rituximab-HC-L6-IFNo. (R33AEYNS) IgG1 0.74 isotype-HC-L6-IFNo, IgG1 2.
O 0.0001 0.01 1 100 10000 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 16 of 62 US 2016/0367695A1
FIGURE 12:
Targeted ifno. Activity (Daudi) Construct EC50
1,5006 O Rituximab-HC-L6-IFNo. IgG1 0.2 Rituximab-HC-L6-FNo. (R33A) gG1 340 1000000 o Rituximab-HC-L6-IFNo. (R144A+YNS) IgG1 3.3
500000
0.0001 0.01 1 100 10000 1000000 IFNo equivalents (pM)
Patent Application Publication Dec. 22, 2016 Sheet 18 of 62 US 2016/0367695A1
FIGURE 14:
40000 -O- X355/02 IgG1 -- X355/04 IgG1 30000 -- X355/07 IgG1 s -v- X910/12 gC1 20000 -- X913/15 g61 10000 -er negative control gG1 rate anti FC O s S. s. N S S Patent Application Publication Dec. 22, 2016 Sheet 19 of 62 US 2016/0367695A1
FIGURE 15:
Non-Targeted IFNo. Activity
Construct EC50 (pM) -- FNO. 0.726 -e- GOO5-HCLO-IFNo. IgG4 195
O 0.0001 0.01 1 1OO 10000 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 20 of 62 US 2016/0367695A1
FIGURE 16:
Targeted IFNo. Activity (ARP1) 5OOOOO S. Construct IC50 o 400000 -- FNC. 14.7O 300000 -e- G005-HC-LO-IFNo.g64 4,079 8 200000 . 100000 {
O 0.01 1 100 10000 IFNo. equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 21 of 62 US 2016/0367695A1
FIGURE 17:
Non-Targeted FNot Activity
r Construct EC50
o 4000 -- FN 3O 30- -- G005-HC-L6-IFNa (R144A) igG4 11,000 8. - A - GD05-HC-LO-IFNa (R 144A) igG4 30,000 200 mir G005-Hc-L6-IFNa (R144A) igG1 2600 E 1000- G005-HC-LO-IFNoy (R144A) igG1 6800
0.000 OO1 10 IFNo. equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 22 of 62 US 2016/0367695A1
FIGURE 18:
Non-Targeted IFNot Activity
6000 Construct ...... EC50(pM) g4000 -O- FNC. O.O87 - - G005-HC-L0-IFNo. (A145G)lgG1 510 £2000 * - G005-HC-L6-IFNo. (A145G)lgG1 730 -- G005-HC-L6-IFNcy (A145G)lgG4 2200
0.0001 0.01 1 100 OOOO 1000000 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 23 of 62 US 2016/0367695A1
FIGURE 19:
Non-Targeted IFNo. Activity
25 - Construct ...... EC50(pM) 3 -- IFNO. 0.21 -0- G005-LC-L6-IFNo. (A145G) IgG1 12000 2 -6- G005-LC-LO-IFNo. (A145G) gG1 24000
-
0.0001 0.01 1 100 10000 1000000 IFNo, equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 24 of 62 US 2016/0367695A1
FIGURE 20:
Targeted IFNo. Activity (Daudi) 1500000 Construct IC50(pM) as n -o- FNo. 0.77 g 1OOOOOO - a G005-HC-L6-IFNo. (R144A) gG4 1.3 -- G005-HC-LO-IFNo. (R144A) gG4 2.7 2 500000 - GOO5-HC-L6-IFNo. (R.144A) g(G1 1.9 E re- G005-HC-LO-IFNo. (R144A) gG1 7.3
O 0.0001 0.01 100 10000 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 25 of 62 US 2016/0367695A1
FIGURE 21:
Targeted IFNo. Activity (Daudi) 2OOOOOO C50(pM) S. a 1500000 - - FNC. 0.48 g - - G005-HC-LO-IFNo. (A145G) gG1 O.74 1000000 -- G005-HC-L6-LFNo. (A145G) gCS1 1.0
S -- G005-HC-L6-IFNo. (A145G) igG4 0.59 E 500000 - 0. O.OOO 0.01 100 OOOO IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 26 of 62 US 2016/0367695A1
FIGURE 22:
Targeted IFNo. Activity (Daudi)
1500000 S n Construct ...... IC50(pM) 1000000 -- FNC. 11 : -- G005-LC-L6-IFNo. (A145G) lgG1 8.5 2 500000 -- G005-LC-LO-IFNo. (A145G) gG1 21 E m O 0.0001 0.01 f 100 1OOOO FNo. equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 27 of 62 US 2016/0367695A1
FIGURE 23:
Targeted IFNo. Activity (ARP-1)
600000 Construct ...... IC500p) 2. -- FNo. 6.O n * - G005-HC-L6-IFNo. (R144A) igG1 21 400000 rar G005-HC-LO-IFNo. (R144A) gG1 110 -- G005-HC-L6-IFNo. (R144A) g(G4 28 200000 -- G005-HC-LO-IFNo. (R144A) g(G4 85 E s -
0.01 1 100 0000 IFNot equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 28 of 62 US 2016/0367695A1
FIGURE 24:
Targeted IFNo. Activity (ARP-1)
500000 s 400000 Construct ...... IC50(pM) -o- FN 9.5 300000 -- G005-HC-L6-IFNo. (A145G) IgG1 4.0 3 de G005-HC-LO-IFNo. (A145G) gG1 8.0 2000 -- G005-HC-L6-IFNo. (A145G) gG4 4.4
-E 100000
0.01 1 100 10000 IFNot equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 29 of 62 US 2016/0367695A1
FIGURE 25:
Non-Targeted IFNot Activity
3.O O O Construct ...... EC50(pM) -- G005-HC-LO-IFNo. (R144A) lgG4 25,800 2O OO * - G005-HC-LO-IFNo. (R144S) gG4 108,000 -- G005-HC-LO-IFNo. (R144E) lgG4 >10 -- G005-HC-LO-IFNo. (R144G) IgG4 9,970 10OO
100 102 104 106 108 FNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 30 of 62 US 2016/0367695A1
FIGURE 26:
Non-Targeted F.No. Activity
3000 3. o Construct ...... EC50(pM) 2000 -i- G005-HC-LO-IFNo. (R144H) igG4 1,690 s is G005-HC-LO-IFNo. (R144K) ligG4 <100 i -- G005-HC-LO-IFNo. (R144N) igG4 431 1000 -- G005-HC-LO-IFNo. (R144Q) IgG4 3,500 5 to G005-HC-LO-IFNo. (R144T) igG4 30,800 O 100 102 104 106 108 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 31 of 62 US 2016/0367695A1
FIGURE 27:
Non-Targeted IFNo. Activity
Construct EC50(pM)
ce. G005-HC-LO-IFNo. (R144Y) ligG4 92,100 as -- G005-HC-LO-IFNo. (R1441) IgG4 159,000 2 ow G005-HC-LO-IFNo. (R144L)lgG4 26,700 3 -- G005-HC-LO-IFNo. (R144V) lgG4 86,900
E s
100 102 104 106 108 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 32 of 62 US 2016/0367695A1
FIGURE 28:
Non-Targeted IFNo. Activity
3500 Construct ... . EC50(pM) 2 2500 -- G005-HC-LO-IFNo. (A145G) gG4 2,040 3 - - G005-HC-LO-IFNo. (A145D) igG4 52,600 -&- G005-HC-LO-IFNa (A145E) lgG4 <100 i 1500 -- G005-HC-LO-IFNo. (A145H) igG4 24,900 -O FNoy 0.260 500
10-4 10-2 100 102 104 106 108 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 33 of 62 US 2016/0367695A1
FIGURE 29:
Non-Targeted IFNo. Activity
3500 3. ge- 250 W. ConstructVuisul------...... EC50(pM) 3. - - GOO5-HC-LO-IFNa (A1451) lgG4 28.9 -- GOO5-HC-LO-IFNa (A145K) gG4 662,000 i 1500 -- GOO5-HC-LO-IFNo. (A145L) IgG4 239 -- GOO5-HC-LO-IFNa (A145N) IgG4 309 500 -o- GOO5-HC-LO-IFNo. (A145Q) gG4 709
100 102 104 106 108 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 34 of 62 US 2016/0367695A1
FIGURE 30:
Non-Targeted IFNo. Activity
3500 S n 2500 Construct ...... EC50(pM) -- G005-HC-LO-IFNa (A145R) IgG4 >3x10 g -A G005-HC-LO-IFNa (A145T)lgG4 <2 8 1500 -- G005-HC-LO-IFNa (A145Y)lgG4 19,200 E 500
100 102 104 106 108 IFNo. equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 35 of 62 US 2016/0367695A1
FIGURE 31:
Targeted IFNo. Activity (ARP-1)
500000 34ooooo ( G005-HC-LO-IFNa (R144A) gG4 186 3300000 G005-HC-LO-IFNa (R144S) IgG4 290 3 G005-HC-LO-IFNa (R144T) g(G4 333 2000 F. G005-HC-LO-IFNa (R144Y) gG4 306 E 100000 G005-HC-LO-IFNa (R1441) IgG4 257 es G005-HC-LO-IFNo. (R144L) IgG4 191
0.01 1 100 10000 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 36 of 62 US 2016/0367695A1
FIGURE 32:
Targeted IFNa Activity (ARP-1) too Construct . . . . IC50(pM) Foo -- GOO5-HC-LO-IFNo. (A145G) IgG4 23.8 o -- G005-HC-LO-FNa (A145H) IgG4 113 Ézooloo -- G005-HC-LO-IFNo. (A145D) gG4 222 or G005-HC-LO-IFNo. (A145K) IgG4 174 2 -- G005-HC-LO-IFNa (A145Y) igG4 914 E 1000 -o- FNO. 10.8 - O 0.01 100 0000 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 37 of 62 US 2016/0367695A1
FIGURE 33:
Targeted FNa Activity (ARP-1) 500000 ars Construct IC50(pM) 400000 FN 8.91 X910/12-HC-LO-IFNO. (R144A) gG4 191 : 3OOOOO X355/02-HC-LO-IFNO. (R144A) IgG4 147 g - X913/15-HC-LO-FNo. (R144A) gG4 84.1 g 200000 X355/07-HC-LO-FNo. (R144A) IgG4 61.2 100000 G005-HC-LO-IFNo.w (R144A) igG4 220
0.01 1 100 10000 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 38 of 62 US 2016/0367695A1
FIGURE 34:
Targeted IFNo. Activity (ARP-1)
1000000 Construct ...... IC50(pM) 800000 -O- FN 43.0 vs. G005-HC-LO-IFNQ (A145D) IgG4 85.7 600000 -- X910/12-HC-LO-IFNo. (A145D) gG4 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 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 39 of 62 US 2016/0367695A1
FIGURE 35:
Non-Targeted IFNo. Activity
S Construct ...... EC50(pM) i -- FNo. 0.499 o - X910/12-HC-LO-IFNa (R144A) gG4 41,000 -- X913/15-HC-LO-IFNo. (R144A) gG4 29,600 -- X355/02-HC-LO-IFNo. (R144A) IgG4 70,500 -- X355/07-HC-LO-IFNa (R144A) gG4 70,300 -- G005-HC-LO-IFNo. (R144A) igG4 73,200 -
10-5 100 105 1010 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 40 of 62 US 2016/0367695A1
FIGURE 36:
Non-Targeted IFNo. Activity
as Construct...... EC50(pM) iO -o- FNO. 0.304 o -- X910/12-HC-LO-IFNa (A145D) igG4 22,500 2 -- X913/15-HC-LO-IFNo. (A145D) IgG4 31,800 -- X355/02-HC-LO-IFNo. (A145D) igG4 53,900 -- X355/07-HC-LO-IFNa (A145D) igG4 61,800 -o- G005-HC-LO-IFNo. (A145D) IgG4 47,100 -
10-5 100 105 1010 IFNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 41 of 62 US 2016/0367695A1
FIGURE 37:
Targeted IFNo. Activity (ARP-1)
Construct ...... IC50(pM) g -0- FNo. 16.1 -- R5D1-HC-LO-IFNo. (A145D)lgG4 51.3 i -- R5E8-HC-LO-IFNo. (A145D)lgG4 89.2 -- R10A2-HC-LO-IFNo. (A145D) igG4 32.7 5 -o- X355/02-HC-L0-IFNa (A145D)lgG4 104
0.01 1 100 10000 1000000 IFNot equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 42 of 62 US 2016/0367695A1
FIGURE 38:
Non-Targeted IFNo. Activity
Construct EC50 (pM) o -- FNo. 0.271 2 -- R5D1-HC-LO-IFNo. (A145D) gG4 49,500 -- R5E8-HC-LO-FNo. (A145D) IgG4 38,400 2 -- R1OA2-HC-LO-FNo. (A145D) gG4 29,600 -o- X355/02-HC-LO-FNo. (A145D) IgG4 81,600 -
10-5 100 105 1010 IFNo. equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 43 of 62 US 2016/0367695A1
FIGURE 39:
CD138 targeted: ARP-1
900000
800000
7OOOOO re- isotype-HC-LO-IFNo. (A145D) igG4 600000 -H nBTO62-HC-LO-IFNo. (A145D) igG4 500000
400000 0.01 1 100 10000 IFNo. equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 44 of 62 US 2016/0367695A1
FIGURE 40a:
MHC Class I targeted attenuated-IFNo.: ARP-1 cells 1000000 g5 Construct . . . . IC50(pM) a 2 g -- FN 16 : S -v- HB95-HC-LO-Ifno (A145D) IgG4 139 "A isotype-HC-LO-IFNo.(A145D) gG4 N/A Y
0. 1 100 OOOO IFNo equivalents (pM)
FIGURE 40b:
Targeted IFNa Activitya (ARP1) Construct C5.
800000 O Paliwizumab-HC-L6-IFNa (A145D) Fab 2410 2D 2-HC-6-IFNa (A145D) Fab 7.O. SCOOOO A H895-HC-LG-FMo (A145D) Fab (874 FN 42.8 400000
200000
10-5 100 05 10 0 FNo equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 45 of 62 US 2016/0367695A1
FIGURE 41:
Anti-MHC class I-IFN: Efficacy of targeted FN in virus infected cells
Éio Construct EC50 (pM) 2 75 v FNO 0.18 o E 50 HB95-HC-LO-IFNo.(A145D) gG4 1.15 O A isotype-HC-LO-IFNo.(A145D) igG4 19,461 8 25 S O 0.0001 0.01 ? OO 10000 OOOOOO IFNot equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 46 of 62 US 2016/0367695A1
FIGURE 42:
Non-Targeted IFNB Activity
4000 S Construct EC50 (pM) a 3000 -o- IFNB1 0.799 -- G005-HC-LO-IFNB IgG4 57.6 5 2000 o'- G005-HC-LO-IFNB (R35A) gG4 16,100
o E 1000 -
0.0001 0.01 1 100 10000 OOOOOO IFNEquivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 47 of 62 US 2016/0367695A1
FIGURE 43:
Targeted IFNB Activity (ARP-1)
500000 s Construct IC50(pM) 400000 -- IFN? 1 10.5 -- G005-HC-LO-IFNB (R35A) lgG4 46.9 300000 -- G005-HC-LO-IFNB IgG4 32.7 9 200000
-E 100000 O 0.01 OO 0000 fn Equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 48 of 62 US 2016/0367695A1
FIGURE 44:
Non-Targeted IL-4 Activity
2.0 Construct ...... EC50(pM) 1.5 -0- L-4 1.26 g -- J110-HC-L6-IL-4lgG1 6.88 1.O -- isotype-HCL6-IL-4(R880)lgG1 19,200 sos -- J110-HC-L6-IL-4(R88O) gG1 44,800 5
10-4 10-2 100 102 104 106 108 IL-4Equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 49 of 62 US 2016/0367695A1
FIGURE 45:
Targeted IL-4 Activity 50 g Construct IC50(pM) or L-4 114 f -- J110-HC-L6-L-4 IgG1 31.8 -- J110-HC-L6-IL-4(R880) igG1 46.1 -- isotype-HC-L6-L-4(R880) IgG1 >1000 SS
0.1 1 10 100 1000 10000 100000 IL-4Equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 50 of 62 US 2016/0367695A1
-6 Activation Assay 15 Construct IC50 (pM)
1.2 E was isotype-HC-1 16-116 gCS1 O.9 : is isotype-HC-L16-IL6 (R179E) gG182600 g 0.9 -- HE395-HC-1 16-i6 gG1 12.4 So - HB95-HC-L16-IL6 (R179E) g(Sf 86.7 i i-- 6 1.04 a 0.6
0.3
104 O2 109 102 104 Os Oe IL-6 Equivalents (pM) Patent Application Publication Dec. 22, 2016 Sheet 51 of 62 US 2016/0367695A1
FIGURE 47:
H929 subcutaneous MM model 2500 2000 wer Vehicle S2 -i. G005 (IgG1) 1500 -- FNO. 10 O O -v- isotype-HC-L6-IFNo. (A145G) IgG1 G005-HC-L6-IFNo. (A145G) gG1
Treatment No detectable tumors in Days post treatment start 10/10 mice on day 22 Patent Application Publication Dec. 22, 2016 Sheet 52 of 62 US 2016/0367695A1
FIGURE 48:
MM1S Systemic MM Model
100----- G005-HC-L6-IFNo. (A145G) lgG1
5 O
Treatment Days post tumor inoculation Patent Application Publication Dec. 22, 2016 Sheet 53 of 62 US 2016/0367695A1
FIGURE 49:
Daudi Subcutaneous NHL model 3000 & E. were Vehicle C 2000 or on GOO5 (ICG1(lgG1) g -0- FNo.
s -- G005-HC-L6-IFNo. (A145G) gG1 5 1000 -v- isotype-HC-L6-IFNo. (A145G)lgG1
s O 100 Treatment Days post cell inoculation Patent Application Publication Dec. 22, 2016 Sheet 54 of 62 US 2016/0367695A1
FIGURE SO:
H929 MM model: Dose titration
Vehicle 10mg/kg 1 mg/kg 0.1 mg/kg isotype-HC-L6-IFNo. (A145G)lgG1 0.01 mg/kg 10mg/kg 1mg/kg 0.1 mg/kg G005-HC-6-IFNo. (A145G) igG1 0.01 mg/kg O 20 40 60 i Days post treatment start Patent Application Publication Dec. 22, 2016 Sheet 55 of 62 US 2016/0367695A1
FIGURE 51:
H929 MM model: IgG1 vs IgG4 format, 3.5 mg/kg dosing
to Vehicle
-e- isotype-HC-L6-IFNo. (A145G) igG4 -- isotype-HC-L6-FNo. (A145G) igG1 -- G005-HC-L6-IFNo. (A145G) lgG1 -- G005-HC-L6-FNo. (A145G) IgG4
Days post treatment start Patent Application Publication Dec. 22, 2016 Sheet 56 of 62 US 2016/0367695A1
FIGURE 52:
H929 MM model: XO2-FNO. Variant s E 2000-...... 8 2 1500 area. Vehicle gs -- isotype-HC-LO-IFNo. (A145D) lgG4
g 1000 -H X355/02-HC-LO-IFNo. (A145D) lgG4 s 500 vs s 9 O 80 Treatment Phase Days post treatment start Patent Application Publication Dec. 22, 2016 Sheet 57 of 62 US 2016/0367695A1
FIGURE 53:
Targeted IFNo.2b vs Standard of Care 6. E E. Dexamethasone, 10mg/kg, daily, 28 days s 4mg/kg,Melphalan, daily, 5 days O --- Bortezomibk (Velcade), 1 mg/kg, 2X/week, 4 weeks Vs s G005-HC-LO-IFNo. (A145D) IgG4, e O 20 40 60 s 3mg/kg, 2XWeek, 5 weeks Days post treatment start Patent Application Publication Dec. 22, 2016 Sheet 58 of 62 US 2016/0367695A1
FIGURE 54:
Dosing regimen Study: 10 mg/kg dose
-E- 6doses isotype-HC-6-IFNo. (A145G) IgG4
-- 1 dose re 2 doses -- 4 doses G005-HC-L6-IFNo. (A145G) IgG4 -0- 6 doSeS O 2040 60 80 Days post treatment start Patent Application Publication Dec. 22, 2016 Sheet 59 of 62 US 2016/0367695A1
FIGURESS:
Dosing regimen Study: Large Tumor Start N E 2000.- ...... 5 5: 1500 -- Vehicle O s 1000 G005-HC-L6-IFNa (A145G) IgG4 Start at 730 mm 500 a O ; : " : " ": Days Post Grouping Patent Application Publication Dec. 22, 2016 Sheet 60 of 62 US 2016/0367695A1
FIGURE S6:
Colony Forming Assay: 3 normal human BM MNC
120 100 80 -O- G005-HC-LO-IFNo. (A145D) lgG4 60 rt 2D12-HC-LO-FNo. (A145D) gG4 40 wriko FNC. 20
0.0001 0.01 1 100 10000 IFN concentration (nM) —- 10,000X attenuation Patent Application Publication Dec. 22, 2016 Sheet 61 of 62 US 2016/0367695A1
FIGURE 57:
IP-10
3000
w8 FNo. -- isotype-HC-L6-1FNo. (A145G) igG1
0.001 0.01 0.1 1 10 100
1OOOX attenuation
MCP-1
4000 -- FNO 3000 -e- isotype-HC-L6-IFNo. (A145G) igG1 2000
1000
0.001 O.O1 O. 1 nM FNO ------5000X attenuation Patent Application Publication Dec. 22, 2016 Sheet 62 of 62 US 2016/0367695A1
FIGUREST:
MCP-3
-- IFNo.
-o- isotype-HC-L6-IFNo. (A145G)lgG1
0.001 0.01 0.1 1 10 100 nM FNO. ------2500X attenuation
IL-10. 15
ow. IFNO.
-0- isotype-HC-L6-IFNo. (A145G) gG1
100
1300X attenuation US 2016/0367695 A1 Dec. 22, 2016
POLYPEPTIDE CONSTRUCTS AND USES natural receptor, but on the surface of cells other than those THEREOF that mediate therapeutically beneficial effects. 0006 Even though some ligands, such as IFNC2b, are CROSS REFERENCE TO RELATED approved for the treatment of medical conditions, they are APPLICATIONS poorly tolerated due to their “off target' biological activity. 0001. This application is a continuation of U.S. patent The off-target activity and associated poor tolerability also application Ser. No. 14/262,841, filed on Apr. 28, 2014, mean that some of these peptide ligand-based drugs cannot which is a continuation of International Patent Application be administered at Sufficiently high dosages to produce No. PCT/AU2012/001323, filed on Oct. 29, 2012, which optimal therapeutic effects on the target cells which mediate claims priority to Australian Patent Application No. the therapeutic effect. 2011 904502, filed on Oct. 28, 2011, the contents of each 0007 Similarly, it has been known since the mid-1980's application are incorporated herein by reference in their that interferons, in particular IFNC, are able to increase entirety and for all purposes. apoptosis and decrease proliferation of certain cancer cells. These biological activities are mediated by type I interferon receptors on the surface of the cancer cells which, when REFERENCE TO ASEQUENCE LISTING stimulated, initiate various signal transduction pathways 0002 This application includes a Sequence Listing sub leading to reduced proliferation and/or the induction of mitted electronically as a text file named Pctau201200 1323 terminal differentiation or apoptosis. IFNC. has been seql.txt, created on Apr. 24, 2014, with a size of 1 megabyte. approved by the FDA for the treatment of several cancers The Sequence Listing is incorporated by reference herein. including melanoma, renal cell carcinoma, B cell lym phoma, multiple myeloma, chronic myelogenous leukemia FIELD OF THE INVENTION (CML) and hairy cell leukemia. A “direct” effect of IFNC. on the tumour cells is mediated by the IFNC. binding directly to 0003. The present invention relates to polypeptide con the type I IFN receptor on those cells and stimulating structs comprising mutated, attenuated polypeptide ligands apoptosis, terminal differentiation or reduced proliferation. attached to antibodies, wherein the antibodies direct the One “indirect’ effect of IFNC. on non-cancer cells is to mutated ligands to cells that express on their Surfaces the stimulate the immune system, which may produce an addi antigens to which said antibodies bind, as well as receptors tional anti-cancer effect by causing the immune system to for said ligands. The invention further relates to methods of reject the tumour. treatment involving the use of these polypeptide constructs. 0008 Unfortunately, the type I interferon receptor is also present on most non-cancerous cells. Activation of this BACKGROUND OF THE INVENTION receptor on such cells by IFNC. causes the expression of 0004 Numerous peptide and polypeptide ligands have numerous pro-inflammatory cytokines and chemokines, been described to function by interacting with a receptor on leading to toxicity. Such toxicity prevents the dosing of a cell Surface, and thereby stimulating, inhibiting, or other IFNC. to a subject at levels that exert the maximum anti wise modulating a biological response, usually involving proliferative and pro-apoptotic activity on the cancer cells. signal transduction pathways inside the cell that bears the 0009 Ozzello et al. (Breast Cancer Research and Treat said receptor. Examples of Such ligands include peptide and ment 25:265-76, 1993) described covalently attaching polypeptide hormones, cytokines, chemokines, growth fac human IFNC to a tumour-targeting antibody, thereby local tors, apoptosis-inducing factors and the like. Natural ligands izing the direct inhibitory activity of IFNC. to the tumour as can be either soluble or can be attached to the surface of a way of reducing tumour growth rates, and demonstrated another cell. that such conjugates have anti-tumour activity in a Xenograft 0005. Due to the biological activity of such ligands, some model of a human cancer. The mechanism of the observed have potential use as therapeutics. Several peptide or poly anti-cancer activity was attributed to a direct effect of IFNC. peptide ligands have been approved by regulatory agencies on the cancer cells, since the human IFNC. used in the as therapeutic products, including, for example, human experiments did not interact appreciably with the murine growth hormone, insulin, interferon (IFN)-C2b. IFNC2C. type I IFN receptor, which could have lead to an indirect IFNB, erythropoietin, G-CSF and GM-CSF. Many of these anti-cancer effect. Because of this lack of binding of the and other ligands have demonstrated potential in therapeutic human IFNC. to the murine cells, however, the authors could applications, but have also exhibited toxicity when admin not evaluate the toxicity of the antibody-IFNC. conjugate istered to human patients. One reason for toxicity is that relative to free INFO. These authors used a chemical method most of these ligands trigger receptors on a variety of cells, to attach the IFNC. to the antibody. including cells other than those that mediate the therapeutic 0010 Alkan et al., (Journal of Interferon Research, Vol effect. For example, when IFNC2b is used to treat multiple ume 4, number 3, p. 355-63, 1984) demonstrated that myeloma its utility resides, at least in part, in its binding to attaching human IFNC. to an antibody that binds to the type I interferon receptors on the myeloma cells, which in Epstein-Barr virus (EBV) membrane antigen (MA) turn triggers reduced proliferation and hence limits disease increased its antiproliferative activities towards cells that progression. Unfortunately, however, this IFN also binds to express the EBV-MA antigen. This increased potency was numerous other, normal cells within the body, triggering a dependent on both antigen expression by the target cells and variety of other cellular responses, some of which are the binding specificity of the antibody. The cell line tested harmful (e.g. flu-like symptoms, neutropenia, depression). A was the cancer cell line QIMR-WIL, a myeloblastic leuke consequence of Such “off target activity of ligands is that mia. The authors suggested that the attachment of IFNC. to many ligands are not suitable as drug candidates. In this an antibody could be used as a treatment for cancer since it context, “off target activity” refers to activity on the ligands would reduce tumour growth. Alkan et at did not address the US 2016/0367695 A1 Dec. 22, 2016 potential toxicity of these antibody-IFNC. conjugates arising cells in Such a way that it has dramatically more activity on from their interactions with normal, antigen-negative cells. the cancer cells, which display the antigen, than on the 0011. It is also known that the linkage between an anti normal cells, which do not display the antigen. The same body and IFNC may be accomplished by making a fusion logic applies to other potentially therapeutic ligands, e.g. protein construct. For example, IDEC (WO01/97844) dis other cytokines, peptide and polypeptide hormones, close a direct fusion of human IFNC to the C terminus of the chemokines, growth factors, apoptosis-inducing factors and heavy chain of an IgG targeting the tumour antigen CD20. the like. Other groups have disclosed the use of various linkers between the C-terminus of an IgG heavy chain and the SUMMARY OF THE INVENTION IFNC. For example, U.S. Pat. No. 7,456,257 discloses that 0014. The present inventors have found that when a the C-terminus of an antibody heavy chain constant region peptide or polypeptide signaling ligand, having one or more may be connected to IFNC. via an intervening serine-glycine mutations which substantially decrease the affinity of the rich (S/G) linker of the sequence (GGGGS), where n may ligand for its receptor, is linked to an antibody that targets be 1, 2 or 3, and that there are no significant differences in the mutated ligand to target cells which display the anti the IFNC. activity of the fusion protein construct regardless body's corresponding antigen, the ligands activity on target of linker length. antigen-positive cells is maintained while the ligand’s activ 0012 Morrison et al. (US2011/0104112 A1; and Xuan C, ity on non-target antigen-negative cells is Substantially Steward K K, Timmerman J. M. Morrison S L. Targeted reduced. The net result is a ligand signaling molecule that delivery of interferon-C. via fusion to anti-CD20 results in has a much greater potency in activation of its receptors on potent antitumor activity against B-cell lymphoma. Blood antigen-positive target cells compared to antigen-negative 2010: 115:2864-71) also disclose IFNC. linked to the C-ter non-target cells, which provides a means for reducing tox minus of the heavy chain of a cancer-targeting IgG antibody, icity arising from off-target ligand activity. with an intervening S/G linker, and observed that the fusion 0015. Accordingly, a first aspect of the present invention of the IgG and linker to the IFNC. reduced the activity of provides a polypeptide construct comprising a peptide or IFNC. on cells that did not express the corresponding antigen polypeptide signaling ligand linked to an antibody or antigen on the cell surface. The decreased IFN activity of these binding portion thereof which binds to a cell surface fusion protein constructs was modest when compared to associated antigen, wherein the ligand comprises at least one human non-fusion protein IFNC. (free IFNC) acting on amino acid substitution or deletion which reduces its human cells, but appeared to be more significant for murine potency on cells lacking expression of said antigen. IFNC. on murine cells. The decrease in the activity of human IFNC. that results from fusing it to the C-terminus of an 0016. In a second aspect, the present invention provides antibody, as observed by Morrison et al, and in U.S. Pat. No. a method of treating a tumour in a Subject, comprising 7,456,257 is modest and is generally considered to be a administering to the Subject the polypeptide construct of the disadvantage since it reduces potency of the ligand. This present invention. disadvantage was pointed out, for example, by Rossi et at 0017. In a third aspect, the present invention provides use (Blood vol. 114, No. 18, pp. 3864-71), who used an alter of the polypeptide construct of the present invention in the native strategy of attaching the IFNC to a tumor targeting treatment of cancer. antibody in such a way that no loss in IFNC activity was 0018. In a fourth aspect, the present invention provides a observed. composition comprising the polypeptide construct of the 0013. In general the prior art teaches to use a potent IFN present invention and a pharmaceutically acceptable carrier and to target this IFN to cancer cells. While this approach or diluent. results in an increase in activity of the IFN against cancer 0019. In a fifth aspect, the present invention provides cells, it does not address the issue of activity of the IFN on method of reducing the potency of a peptide or polypeptide normal “off-target cells. In prior art examples referred to signaling ligand on an antigen negative cell which bears the above, the human IFNC. portion of the antibody-IFNC. fusion ligand receptor whilst maintaining the potency of the ligand protein maintained a high proportion of native IFNC. activity on an antigen positive cell which bears the ligand receptor when exposed to human cells that do not express the to a greater extent when compared to the antigen negative corresponding antigen on their cell Surfaces. This activity cell, the method comprising modifying the ligand Such that may lead to toxicity arising from the activation of non the ligand comprises at least one amino acid Substitution or cancerous, normal (“off target') cells by the IFNC. portion of deletion which reduces its potency on the antigen negative the fusion protein. Accordingly, there exists a need to cell and linking the modified ligand to an antibody or decrease the “off-target activity of ligand-based drugs, antigen-binding portion thereof, wherein the antibody or while retaining the “on-target, therapeutic effect of such antigen binding portion thereof is specific for a cell Surface ligands. The maintenance of target-specific ligand activity associated antigen on the antigen positive cell but not on the and at the same time a reduction in non-target toxicity of antigen negative cell. ligand-based therapeutic agents would create a greater thera 0020. Unlike the linking of a non-attenuated “native' or peutic concentration window for therapeutically useful “wild-type' human ligand to an antibody or antigen-binding ligands. It would for example be desirable to use human portion thereof, which typically results in from 1 to 15-fold IFNC. in a form such that its activity can be directed to the higher potency of the ligand on antigen-positive compared cancer cells while minimizing its effects on normal human to antigen-negative cells, the present invention demonstrates cells. Ideally the type I interferon receptor on the cancer that the attachment of mutated, attenuated forms of the cells would be maximally stimulated, while the same recep ligand to the same antibody is able to generate higher tor on non-cancerous cells would experience minimal stimu potency on antigen-positive cells compared to antigen nega lation. There is a need to target human IFNC. to the cancer tive cells. US 2016/0367695 A1 Dec. 22, 2016
0021. In one embodiment the signaling ligand is IFNC. or consisting of R144A (SEQ ID NO:30), R144S (SEQ ID IFNB and the polypeptide construct shows at least 10, at NO:40), R144T (SEQID NO:41), R144Y (SEQ ID NO:43), least 100, at least 1,000, at least 10,000 or at least 100,000 R144I (SEQ ID NO:35), R144L (SEQ ID NO:37), A145D fold greater selectivity towards antigen positive cells over (SEQID NO:44), A145H (SEQID NO:47), A145Y (SEQID antigen negative cells compared to free, wild-type ligand NO:58), A145K (SEQ ID NO:49), R33A+YNS (SEQ ID using the “off-target' assay and the “on target (ARP) or “on NO:65), R33A (SEQID NO:16) and R144A+YNS (SEQ ID target (Daudi) assays described herein. NO:68). 0022. The present invention also provides an antibody 0034. In certain embodiments in which the signaling attenuated ligand fusion proteins, wherein the attenuated ligand is an IFNC. and the cell Surface associated antigen is ligand is IFNC. or IFNB and the wherein fusion protein CD38, the antibody is selected from any one of G003, G005, construct, when injected into a mouse with an established G024, MOR03077, MOR03079, MOR03080, MORO3100, human tumor, can eliminate the tumor. 385B13, 385B18, 385B19, 385B30, 38SB31, 38SB39, 0023 The present invention also provides an antibody OKT10, X355/02, X910/12, X355/07, X913/15, R5D1, attenuated ligand fusion proteins, wherein the attenuated R5E8, R10A2, or an antigen binding portion thereof, or an ligand is IFNC. or IFNB and wherein the fusion protein antibody with greater than 95%, greater than 96%, greater construct, when injected into a mouse with an established than 97%, greater than 98% or at least 99% amino acid human tumor with a volume of over 500 cubic millimeters, sequence identity with any one of R5D1, R5E8 or R10A2. can eliminate the tumor. 0035. In certain embodiments in which the cell surface 0024. The present invention also provides an antibody associated antigen is CD38, the signaling ligand of the attenuated ligand fusion proteins, wherein the attenuated polypeptide construct is an IFNC, the treatment is for a ligand is IFNC. or IFNB and wherein the fusion protein cancer in a Subject selected from multiple myeloma, a construct, when injected as a single one-time treatment into leukemia or a lymphoma. In particular embodiments the a mouse with an established human tumor, can eliminate the Subject is also treated with a retinoid, such as all-trans tumor. retinoic acid. In certain embodiments in which the cell 0025. An antibody-attenuated ligand fusion proteins, Surface associated antigen is CD38, the tumour or cancer wherein the attenuated ligand is IFNC. or IFNB and wherein may be selected from multiple myeloma, non-Hodgkin’s the fusion protein construct can eliminate both established lymphoma, chronic myelogenous leukemia, chronic lym myeloma tumors and established lymphoma tumors in a phocytic leukemia or acute myelogenous leukemia. OUS 0036. In embodiments in which the ligand is linked to an 0026. In each of these cases it is preferred that cell antibody, the antibody may be an IgG4. In particular Surface-associated antigen is CD 38. embodiments the IgG4 comprises an S228P amino acid 0027. In one embodiment, the amino acid sequence of the substitution. signaling ligand comprising at least one amino acid Substi 0037. In certain embodiments in which the signaling tution or deletion has greater than 90% or greater than 95%, ligand of the polypeptide construct is an IFNC, the antibody or greater than 96%, or greater than 97%, or greater than or antigen binding portion thereof may bind to a cell Surface 98% or greater than 99% sequence identity with the wild associated antigen on virally infected cells. In these embodi type ligand amino acid sequence. ments the cell Surface associated antigen may be selected 0028. In one embodiment, the construct is a fusion pro from a virally encoded protein, phosphatidylserine or a tein. phosphatidylserine-binding protein. In embodiments in 0029. In certain embodiments the signaling ligand is which the cell Surface associated antigen is phosphatidyl linked to the C-terminus of the heavy chain of the antibody serine or a phosphatidylserine-binding protein the construct or antigen binding portion thereof. In certain embodiments may be used to treat Hepatitis C. the signaling ligand is linked to the C-terminus of the light 0038. In certain embodiments in which the signaling chain of the antibody or antigen binding portion thereof. In ligand of the polypeptide construct is IFNC. or IFNB, the cell either of these embodiments, the ligand may be linked surface associated antigen is selected from CD20, CD38, directly to the C-terminus of the heavy or light chain of the CD138 or CS1. In certain embodiments in which the ligand antibody or antigen binding portion thereof (ie without an is IFNC. or IFNB, the tumour or cancer may be selected from intervening additional linker). multiple myeloma, melanoma, renal cell carcinoma, chronic 0030. In one embodiment the cell surface associated myelogenous leukemia or hairy cell leukemia. antigen is selected from class I MHC or PD-1. 0039. In a particular embodiment the construct is G005 0031. In certain embodiments, the cell surface-associated HC-L0-IFNC. (A145D) IgG4. antigen is a myeloma associated antigen which is selected 0040. In certain embodiments in which the signaling from the group consisting of CD38, HM1.24, CD56, CS1, ligand of the polypeptide constrct is an IFNB, the cell CD138, CD74, IL-6R, Blys (BAFF), BCMA, HLA-SR, Surface associated antigen may be a T cell, a myeloid cell or Kininogen, beta2 microglobulin, FGFR3, ICAM-1, an antigen presenting cell Surface associated protein. matriptase, CD52, EGFR, GM2, alpha-4-integrin, IFG-1R 0041. In certain embodiments in which the signaling and KIR, and the ligand is an IFNC. ligand of the polypeptide construct is an IFNB, the cell 0032. In one embodiment, the signaling ligand is selected Surface associated antigen may be selected from the group from any one of IFNC2b. IFNB, IL-4 or IL-6. consisting of CD3, CD4, CD8, CD24, CD38, CD44, CD69, 0033. In certain embodiments in which the signaling CD71, CD83, CD86, CD96, HLA-DR, PD-1, ICOS, CD33, ligand is an IFNC, the amino acid substitution or deletion CD115, CD11c, CD14, CD52 and PD-1. In these embodi may be at any one or more of amino acid positions R33. ments, the construct may be used to treat a disease charac R144 or A145. In certain embodiments the signaling ligand terized by excess inflammation, such as an autoimmune is an IFNC. and the substitution is selected from the group disease. US 2016/0367695 A1 Dec. 22, 2016
0042. In certain embodiments in which the signaling -continued ligand of the polypeptide construct is an IFNB, the at least one amino acid substitution or deletion is selected from the Name V. Sequence VfV sequence group consisting of R35A, R35T, E42K, M62I, G78S, X355/02 SEQ ID NO:391 SEQ ID NO: 390 A141Y, A142T, E149K, R152H. In these embodiments, the X355,04 SEQ ID NO: 423 SEQ ID NO: 422 IFNB may also possess a C17S or C17A substitution. X355/07 SEQ ID NO:393 SEQ ID NO: 392 0043. In certain embodiments the signaling ligand of the RSD1 SEQ ID NO:399 SEQ ID NO:398 polypeptide construct is an IFNY. In these embodiments, the RSE8 SEQ ID NO: 401 SEQ ID NO: 400 cell Surface associated antigen may be a tumor-associated R1OA2 SEQ ID NO: 403 SEQ ID NO: 402 antigen. In other embodiments, the cell Surface associated antigen may be selected from the group consisting of CD14. 0.052 From these sequences the person skilled in the field FSP1, FAP, PDGFR alpha and PDGFR beta. In these can readily identify the CDR sequences using known meth embodiments, the construct may be used to treat a disease ods. As will be recognized by the skilled worker these CDR characterized by excess fibrosis. sequences can be used in differing framework sequences to 0044. In certain embodiments in which the signaling those specified in the SEQ ID NO’s specified above. ligand of the polypeptide construct is an IFNY, the at least one amino acid substitution or deletion is selected from the BRIEF DESCRIPTION OF THE DRAWINGS group consisting of a deletion of residues A23 and D24, an S20I substitution, an A23V substitution, a D21K substitution 0053 FIG. 1 shows a schematic of the certain embodi and a D24A substitution. ments of the present invention that comprise an antibody 0045. In certain embodiments in which the signaling consisting of 2 heavy chains and 2 light chain, in which one ligand of the polypeptide construct is an IL-4, the cell or two attenuated signaling ligands is or are attached to each Surface associated antigen is selected from the group con heavy chain or each light chain, or both. sisting of CD3, CD4, CD24, CD38, CD44, CD69, CD71, 0054 FIG. 2 shows a schematic illustrating one possible CD96, PD-1, ICOS, CD52 and PD-1. approach for how the antibody-attenuated ligand fusion 0046. In certain embodiments in which the signaling proteins of the present invention cause signaling by activat ligand of the polypeptide construct is an IL-6, the cell ing receptors on cells that display the antigen corresponding Surface associated antigen is selected from the group con to the said antibody on their cell surfaces. The fusion protein sisting of CD3, CD4, CD24, CD38, CD44, CD69, CD71, activates the receptor on the same cell that the antibody is CD96, PD-1, ICOS, CD52 and PD-1. bound to, via its specific antigen. 0047. In certain embodiments in which the signaling 0055 FIG. 3 shows the amino acid sequences of the ligand of the polypeptide construct is an HGF, the cell human CD38 (SEQ ID NO:131). Surface associated antigen is selected from the group con 0056 FIGS. 4a-4b show the amino acid sequences of sisting of ASGR1, ASGR2, FSP1, RTI 140/Ti-alpha, HTI56 certain exemplary signaling ligands of the present invention: and a VEGF receptor. (4a) human IFNC2b. IFNB1, IFNB1b and IFNY; and (4b) 0048. In certain embodiments in which the signaling IL-4 and IL-6. ligand of the polypeptide construct is a TGFB, the cell 0057 FIGS. 5a-5e show the amino acid sequences of Surface associated antigen is selected from the group con certain antibody-attenuated ligand fusion proteins of the sisting of CD3, CD4, CD8, CD24, CD38, CD44, CD69, present invention: (5a) G005-HC-L0-IFNC. (A145D) IgG4; CD71, CD83, CD86, CD96, HLA-DR, PD-1, ICOS, CD33, (5b) nBT062-HC-L0-IFNo. (A145D) IgG4; (5c) G005-HC CD115, CD11c, CD14, CD52 and PD-1. L0-IFNB (R35A) IgG4; (5d) HB95-HC-L16-IL-6 (R179E) 0049. In certain embodiments in which the signaling IgG1; and (5e) J110-HC-L6-IL-4 (R88O) IgG1. The nomen ligand of the polypeptide construct is an erythropoietin, the clature for the fusion proteins is described in the examples. cell Surface associated antigen is selected from the group 0.058 FIG. 6 shows the non-antibody-antigen-targeted consisting of CD241 the product of the RCHE gene, CD117 interferon activity of IFNC.2b, and of the antibody-IFN (c-kit), CD71 (transferrin receptor), CD36 (thrombospondin fusion protein constructs Rituximab-IFNC2b (Rituximab receptor), CD34, CD45RO, CD45RA, CD115, CD168, HC-L6-IFNC. IgG1) and Palivizumab-IFNC2b (Isotype-HC CD235, CD236, CD237, CD238, CD239 and CD240. L6-IFNC. IgG1) in the interferon activity assay described in 0050. In certain embodiments in which the signaling the examples below as the “off-target assay. Throughout the ligand of the polypeptide construct is an interleukin-10 and figures “IFNC equivalents' refers to the molar concentration the cell Surface associated antigen is selected from the group of interferon molecules, either free or attached to an anti consisting of CD11c, CD33 or CD115, CD14, FSP1, FAP or body. “IFN' refers to free (non-fusion protein) wild-type PDGFR (alpha or beta). interferon. 0051. In a sixth aspect there is provided anti-CD38 0059 FIG. 7 shows the antibody-antigen-targeted inter antibodies with variable regions designated X910/12, X913/ feron activity of the Rituximab-IFNC2b fusion protein con 15, X355/02, X355/07, R5D1, R5E8, or R10A2, with struct (Rituximab-HC-L6-IFNC. IgG1) compared with sequences set out as follows: IFNC2b in the antiproliferative assay described in the examples below as the “on target (Daudi) assay.” 0060 FIG. 8 shows the antibody-antigen-targeted inter Name V. Sequence VV Sequence feron activity of the Rituximab-IFNC. fusion protein con X910,12 SEQ ID NO: 395 SEQ ID NO: 394 struct (Rituximab-HC-L6-IFNC. IgG1) compared with the X913,15 SEQ ID NO:397 SEQ ID NO: 396 non-targeted activity of Palivizumab-IFNC. fusion protein X355/01 SEQ ID NO: 421 SEQ ID NO: 420 construct (Isotype-HC-L6-IFNC. IgG1) in the “on-target (Daudi) assay” described in the examples below. US 2016/0367695 A1 Dec. 22, 2016
0061 FIG. 9 shows the non-antibody-antigen-targeted (0072 FIG. 20 shows the antiproliferative activity of interferon activity of IFNC.2b, of the antibody-IFN fusion IFNC2b vs anti-CD38-IFNC. fusion protein constructs with protein constructs Rituximab-IFNC2b (Rituximab-HC-L6 mutations in the IFN portion on the lymphoma cell line IFNC. IgG1) and Palivizumab-IFNC2b (Isotype-HC-L6 Daudi. The antibody variable regions of these fusion protein IFNC. IgG1), and of certain variants of Rituximab-IFNC2b constructs are derived from antibody G005. The assay is constructs that have been mutated to reduce their interferon described in the examples as the “on target (Daudi) assay.” activity. The assay is described in the examples as the (0073 FIG. 21 shows the anti-proliferative activity of “off-target assay”. IFNC2b and various anti-CD38-IFNC. fusion protein with 0062 FIG. 10 shows the non-antibody-antigen-targeted the A145G mutation in the IFN portion. Fusion protein interferon activity of the antibody-IFN fusion protein con constructs have either the 6 amino acid L6 linker or no linker structs Rituximab-IFNC2b (Rituximab-HC-L6-IFNC. IgG1) (LO) and are of the IgG1 or IgG4 isotype. The antibody and of two variants of Rituximab-IFNC2b that were mutated variable regions of these fusion protein constructs are to reduce interferon activity. The assay is described in the derived from antibody G005. The assay is described in the examples as the "off-target assay'. examples as the "on target (Daudi) assay'. 0063 FIG. 11 shows the antibody-antigen-targeted inter (0074 FIG. 22 shows the anti-proliferative activity of feron activity of the antibody-IFN fusion protein construct IFNC2b and two anti-CD38-IFNC. fusion protein with the Rituximab-IFNC2b (Rituximab-HC-L6-IFNC. IgG1) and of A145G mutation in the IFN portion. Both fusion protein variants of Rituximab-IFNC2b constructs that have been constructs had the IFN portion linked to the C-terminus of mutated to reduce their interferon activity compared to the the light chain, with either a six amino acid linker (L6) or no non-targeted activity of the Palivizumab-IFNC2b (Isotype linker (LO). The antibody variable regions of these fusion HC-L6-IFNC. IgG1) fusion protein constructs and compared protein constructs are derived from antibody G005. The to IFNC2b. The assay is described in the examples as the “on assay is described in the examples as the “on target (Daudi) target (Daudi) assay.” assay.” 0064 FIG. 12 shows the antibody-antigen-targeted inter (0075 FIG. 23 shows the antiproliferative activity on the feron activity of the antibody-IFN fusion protein constructs multiple myeloma cell line ARP-1 of IFNC2b vs anti-CD38 Rituximab-IFNC2b (Rituximab-HC-L6-IFNC. IgG1) and of IFNC. fusion protein constructs with the R144A mutation in two variants that were mutated to reduce interferon activity. 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 (Daudi) assay.” vs. IgG4) and the presence or absence of the L6 linker 0065 FIG. 13 shows the sequences of certain novel between the antibody heavy chain C-terminus and the N-ter human CD38 antibodies disclosed herein. minus of the mutated IFN. The antibody variable regions of 0066 FIG. 14 shows the results of detection of binding of these fusion protein constructs are derived from antibody novel human anti-CD38 antibodies to a CD38' cell line G005. The assay is described in the examples as the “on RPMI-8226 by flow cytometry. The X axis is the antibody target (ARP) assay.” concentration in micrograms/ml and they axis represents the mean fluorescence intensity. 0076 FIG. 24 shows the antiproliferative activity on the 0067 FIG. 15 shows the non-antibody-antigen targeted multiple myeloma cell line ARP-1 of IFNC2b vs anti-CD38 IFN activity of IFNC2b compared with an anti-CD38-IFNo. IFNC. fusion protein constructs with the A145G mutation in fusion protein construct (G005-HC-L0-IFNC. IgG4), based the IFN portion. The experiment compares the potency of on the anti-CD38 antibody G005. The assay is described in the fusion protein constructs as a function of isotype (IgG1 the examples as the "off-target assay.” vs. IgG4) and the presence or absence of the L6 linker 0068 FIG. 16 shows the antiproliferative activity of between the antibody heavy chain C-terminus and the N-ter IFNC2b vs an anti-CD38-IFNC. fusion protein construct minus of the mutated IFN. The antibody variable regions of (G005-HC-LO-IFNC. IgG4) on the multiple myeloma cell these fusion protein constructs are derived from antibody line ARP-1 (CD38"). The assay is described in the examples G005. The assay is described in the examples as the “on as the “on target (ARP) assay.” target (ARP) assay.” 0069 FIG. 17 shows the non-antibody-antigen targeted 0077 FIG. 25 shows the non-antibody-antigen targeted IFN activity of IFNC2b vs various anti-CD38-IFNC. fusion IFN activity of various anti-CD38-IFNC. fusion protein protein constructs bearing point mutations in the IFN por constructs with different point mutations in the IFN portion. tion. The antibody variable regions of these fusion protein The antibody variable regions of these fusion protein con constructs were derived from antibody G005. The assay is structs are derived from antibody G005. The assay is described in the examples as the “off-target assay.” described in the examples as the “off-target assay.” 0070 FIG. 18 shows the non-antibody-antigen targeted 0078 FIG. 26 shows the non-antibody-antigen targeted IFN activity of IFNC2b vs various anti-CD38-IFNC. fusion IFN activity of various anti-CD38-IFNC. fusion protein protein constructs bearing point mutations in the IFN por constructs with different point mutations in the IFN portion. tion. The antibody variable regions of these fusion proteins The antibody variable regions of these fusion protein con were derived from antibody G005. The assay is described in structs are derived from antibody G005. The assay is the examples as the "off-target assay.” described in the examples as the “off-target assay.” 0071 FIG. 19 shows the non-antibody-antigen targeted 007.9 FIG. 27 shows the non-antibody-antigen targeted IFN activity of IFNC2b vs two anti-CD38-IFNC. fusion IFN activity of various anti-CD38-IFNC. fusion protein protein constructs bearing point mutations in the IFN por constructs with different point mutations in the IFN portion. tion. The antibody variable regions of these fusion protein The antibody variable regions of these fusion protein con constructs are derived from antibody G005. The assay is structs are derived from antibody G005. The assay is described in the examples as the “off-target assay.” described in the examples as the “off-target assay.” US 2016/0367695 A1 Dec. 22, 2016
0080 FIG. 28 shows the non-antibody-antigen targeted body variable regions in the context of the same mutated IFN activity of various anti-CD38-IFNC. fusion protein IFN fusion protein construct. The assay is described in the constructs with different point mutations in the IFN portion. examples as the “on target (ARP) assay.” The antibody variable regions of these fusion protein con 0090 FIG. 38 shows the non-antibody-antigen targeted structs are derived from antibody G005. The assay is IFN activity of IFNC2b and various anti-CD38-IFNC. fusion described in the examples as the “off-target assay.” protein constructs with the A145D mutation in the IFN 0081 FIG. 29 shows the non-antibody-antigen targeted portion. The experiment compares different antibody vari IFN activity of IFNC2b vs various anti-CD38-IFNC. fusion able regions in the context of the same mutated IFN fusion protein constructs with different point mutations in the IFN protein construct. The assay is described in the examples as portion. The antibody variable regions of these fusion pro the “off-target assay.” tein constructs are derived from antibody G005. The assay (0091 FIG. 39 shows the antiproliferative activity on the is described in the examples as the “off-target assay.” multiple myeloma cell line ARP-1 of two antibody-IFNC. 0082 FIG. 30 shows the non-antibody-antigen targeted fusion protein constructs with the A145D mutation in the IFN activity of various anti-CD38-IFNC. fusion protein IFN portion. The nBT062 antibody binds CD138 whereas constructs with different point mutations in the IFN portion. the “isotype' antibody does not (it is derived from the The antibody variable regions of these fusion protein con antibody 2D12). The assay is described in the examples as structs are derived from antibody G005. The assay is the “on target (ARP) assay.” described in the examples as the “off-target assay.” 0092 FIGS. 40a-40b show the antiproliferative activity I0083 FIG. 31 shows the antiproliferative activity on the on the multiple myeloma cell line ARP-1 of IFNC2b and two multiple myeloma cell line ARP-1 of anti-CD38-IFNC. antibody-IFNC. fusion protein constructs with the A145D fusion protein constructs with the various mutations in the mutation in the IFN portion. The HB95 antibody binds IFN portion. The antibody variable regions of these fusion human class I MHC (which is expressed on the ARP-1 cells) protein constructs are derived from antibody G005. The whereas the “isotype' antibody does not (it is derived from assay is described in the examples as the “on target (ARP) the antibody 2D12). Palivizumab, like 2D12, does not bind assay.” to the ARP-1 cells. FIG. 40b shows the same assay, com 0084 FIG. 32 shows the antiproliferative activity on the paring antibody-attenuated IFNC. fusion protein constructs multiple myeloma cell line ARP-1 of IFNC2b vs anti-CD38 in which the antibody portion is a Fab fragment rather than IFNC. fusion protein constructs with the various mutations in a full size antibody. For both FIGS. 40a and 40b, the assay the IFN portion. The antibody variable regions of these is described in the examples as the "on target (ARP) assay.” fusion protein constructs are derived from antibody G005. The assay is described in the examples as the "on target 0093 FIG. 41 shows measurements of the antiviral activ (ARP) assay.”. ity of IFNC. and two antibody-IFNC. fusion protein con I0085 FIG. 33 shows the antiproliferative activity on the structs with the A145D mutation in the IFN portion. This multiple myeloma cell line ARP-1 of IFNC2b vs anti-CD38 cytopathic effect inhibition assay utilized the cell line A549 IFNC. fusion protein constructs with the R144A mutation in and the EMC virus. The HB95 antibody binds human class the IFN portion. The experiment compares different anti I MHC (which is expressed on the A549 cells) whereas the body variable regions in the context of the same mutated “isotype' antibody derived from the antibody 2D12 does IFN fusion protein. The assay is described in the examples not. as the “on target (ARP) assay.” 0094 FIG. 42 shows the non-antibody-antigen targeted I0086 FIG. 34 shows the antiproliferative activity on the IFN activity of IFNB, an anti-CD38-IFNB fusion protein multiple myeloma cell line ARP-1 of IFNC2b vs anti-CD38 construct and an identical fusion protein construct but with IFNC. fusion protein constructs with the A145D mutation in the attenuating R35A mutation in the IFN portion. The assay the IFN portion. The experiment compares different anti is described in the examples as the “off-target assay.” body variable regions in the context of the same mutated (0095 FIG. 43 shows the antiproliferative activity on the IFN fusion protein construct. The assay is described in the multiple myeloma cell line ARP-1 of IFNB, an anti-CD38 examples as the “on target (ARP) assay.” IFNB fusion protein construct and an identical fusion protein 0087 FIG. 35 shows the non-antibody-antigen targeted construct but with the attenuating R35A mutation in the IFN IFN activity of IFNC2b and various anti-CD38-IFNC. fusion portion. The antibody variable regions of these fusion pro protein constructs with the R144A mutation in the IFN tein constructs are derived from the antibody G005. The portion. The experiment compares different antibody vari assay is described in the examples as the “on target (ARP) able regions in the context of the same mutated IFN fusion assay.” “Ifn equivalents’ refers to the molar concentration of protein construct. The assay is described in the examples as interferon molecules, either free or attached to an antibody. the “off-target assay.” 0096 FIG. 44 shows the non-antibody-antigen-targeted 0088 FIG. 36 shows the non-antibody-antigen targeted IL-4 activity “off-target (HB-IL4) assay of IL-4 and three IFN activity of IFNC2b and various anti-CD38-IFNC. fusion antibody-IL-4 fusion protein constructs: J1 10-HC-L6-IL-4 protein constructs with the A145D mutation in the IFN IgG1, an anti-PD1 antibody fused to wild type IL-4; J110 portion. The experiment compares different antibody vari HC-L6-IL-4 (R88O), which is identical to the previously able regions in the context of the same mutated IFN fusion mentioned fusion protein construct except for the attenuat protein construct. The assay is described in the examples as ing R88CR mutation in the IL-4 portion; and Isotype-HC-L6 the “off-target assay.” IL-4 (R88O), based on the 2D12 antibody, which does not I0089 FIG. 37 shows the antiproliferative activity on the bind to any of the cells used in the assays of the present multiple myeloma cell line ARP-1 of IFNC2b vs anti-CD38 invention, and is fused to the attenuated IL-4. “IL-4 equiva IFNC. fusion protein constructs with the A145D mutation in lents’ refers to the molar concentration of IL-4 molecules, the IFN portion. The experiment compares different anti either free or attached to an antibody. US 2016/0367695 A1 Dec. 22, 2016
0097 FIG. 45 shows the “on target (Th1 diversion) 01.06 FIG. 54 shows the effects of an anti-CD38-attenu assay” comparing the activity of IL-4 and three antibody ated IFNC. fusion protein construct (G005-HC-L6-IFNo. IL-4 fusion protein constructs: J1 10-HC-L6-IL-4 IgG1, an (A145G) IgG4) and an isotype control-attenuated IFNC. anti-PD1 antibody fused to wild type IL-4; J110-HC-L6 fusion protein construct (Isotype-HC-L6-IFNC. (A145G) IL-4 (R88O), which is identical to the previously mentioned IgG4) on the growth of subcutaneous H929 myeloma tumors fusion protein construct except for the attenuating R88C in SCID mice, with several rounds of administration each at mutation in the IL-4 portion; and Isotype-HC-L6-IL-4 a dose of 10 mg/kg. The “isotype' antibody was based on (R88O), based on the 2D12 antibody, which does not bind antibody 2D12. to any of the cells used in the assays of the present invention, 01.07 FIG.55 shows the effects of an anti-CD38-attenu and is fused to the attenuated IL-4. “IL-4 equivalents’ refers ated IFNC. fusion protein construct (G005-HC-L6-IFNo. to the molar concentration of IL-4 molecules, either free or (A145G) IgG4) on the growth of subcutaneous H929 attached to an antibody. myeloma tumors in SCID mice. Dosing (indicated by 0098 FIG. 46 shows the “IL-6 bioassay” comparing IL-6 arrows) was initiated when the median tumor volume with various antibody-IL-6 fusion protein constructs that reached 730 mm. either do bind to the target cells (based on the HB95 (0.108 FIG. 56 shows the inhibition of colony formation antibody, which binds to class I MHC on the target cells) or from normal human bone marrow mononuclear cells (BM do not bind the target cells (based on the isotype control MNC) by IFNo.2b, an anti-CD38-attenuated IFNC. fusion antibody 2D12), fused to either wild type IL-6 or IL-6 with protein construct (G005-HC-L0-IFNC. (A145D) IgG4) and the attenuating R179E mutation. “IL-6 equivalents’ refers to an isotype control antibody-attenuated IFNC. fusion protein the molar concentration of IL-6 molecules, either free or construct2D12-HC-L0-IFNC. (A145D) IgG4. The antibody attached to an antibody. attenuated IFNC. fusion protein constructs show about 0099 FIG. 47 shows the effects of various compounds on 10,000-fold reduced potency in this assay. the growth of subcutaneous H929 myeloma tumors in SCID 0109 FIGS. 57a and 57b show the effects of IFNo.2b vs mice. The bar labeled “treatment' shows the duration of an antibody-attenuated IFNC. fusion protein construct (Iso treatment with the compounds. The “isotype' antibody was type-HC-L6-IFNC. (A145G) IgG1; the isotype variable based on antibody 2D12. G005 is an anti-CD38 antibody. regions are based on antibody 2D12) on cytokine production 0100 FIG. 48 shows the effects of various compounds on by human peripheral blood mononuclear cells (PBMCs). survival (Kaplan-Meier graph) of NOD-SCID mice systemi (57a) IP-10 and MCP-1: (57b) MCP-3 and IL-1C. cally inoculated with the human myeloma cell line MM1S. The bar labeled “treatment’ shows the duration of treatment DETAILED DESCRIPTION OF THE with the compounds. G005 is an anti-CD38 antibody. INVENTION 0101 FIG. 49 shows the effects of various compounds on 0110. The constructs of the present invention are anti the growth of Subcutaneous Daudi lymphoma tumors in body-attenuated ligand constructs, which show an elevated NOD-SCID mice. The bar labeled “treatment shows the antigen-specificity index with respect to activating signaling duration of treatment with the compounds. The “isotype' pathways due to the action of the attenuated ligand on a cell antibody was based on antibody 2D12. G005 is an anti Surface receptor. These constructs are based on the Surpris CD38 antibody. ing discovery that, in the context of an antibody-ligand 01.02 FIG. 50 shows the effects of an anti-CD38-attenu construct, the ligand portion can be mutated in Such a way ated IFNC. fusion protein construct (G005-HC-L6-IFNo. that the ligand activity on antigen-negative cells is dramati (A145G) IgG1) and an isotype control-attenuated IFNC. cally attenuated, while the ligand activity on antigen-posi fusion protein construct (Isotype-HC-L6-IFNC. (A145G) tive cells is only modestly, if at all, attenuated. Such con IgG1) on the growth of subcutaneous H929 myeloma tumors structs display one, two, three, four or five orders of in SCID mice, at various doses. The bar labeled “treatment’ magnitude greater potency on antigen-positive cells com shows the duration of treatment with the compounds. The pared to antigen negative cells than does the free ligand. In “isotype' antibody was based on antibody 2D12. one embodiment, the antibody-attenuated ligand construct (0103 FIG. 51 shows the effects of anti-CD38-attenuated retains at least 1%, at least 10%, at least 20%, at least 30%, IFNC. fusion protein constructs Vs isotype control antibody at least 40% or at least 50% of the potency on antigen attenuated IFNC. fusion protein constructs, on the growth of positive cells as the non-attenuated free (i.e. not attached to subcutaneous H929 myeloma tumors in SCID mice. IgG1 is an antibody) ligand. In addition, in one embodiment the compared to IgG4 in the context of these fusion protein antibody-attenuated ligand construct retains at least 30%, at constructs. The bar labeled “treatment shows the duration least 50%, at least 75% or at least 90% of the maximal of treatment with the compounds. The “isotype antibody activity of the non-attenuated free (i.e. not attached to an was based on antibody 2D12. antibody) ligand; in this context, “maximal activity” should 0104 FIG. 52 shows the effects of an anti-CD38-attenu be understood as meaning the amount of signaling activity ated IFNC. fusion protein construct (X355/02-HC-L0-IFNo. (or downstream effect thereof) at the high, plateau portion of (A145D) IgG4) vs an isotype control antibody-attenuated a dose-response curve, where further increases in the agent IFNC. fusion protein constructs on the growth of subcuta does not further increase the amount of response). neous H929 myeloma tumors in SCID mice. The bar labeled 0111. “Specificity' as used herein is not necessarily an “treatment phase' shows the duration of treatment with the absolute designation but often a relative term signifying the compounds. The “isotype' antibody was based on antibody degree of selectivity of an antibody-ligand fusion protein 2D12. construct for an antigen-positive cell compared to an anti 0105 FIG. 53 shows the effects of various compounds on gen-negative cell. Thus for example, a construct may be said the growth of subcutaneous H929 myeloma tumors in SCID to have “100-fold specificity for antigen-positive cells com mice. G005 is an anti-CD38 antibody. pared to antigen-negative cells' and this would indicate that US 2016/0367695 A1 Dec. 22, 2016
the construct has 100-fold higher potency on cells that and VL regions can be further subdivided into regions of express the antigen compared to cells that do not. In some hyperVariability, termed complementarity determining cases, this degree of specificity of a construct comparing regions (CDR), interspersed with regions that are more antigen-positive vs. antigen-negative cells may not be based conserved, termed framework regions (FR). Each VH and on the absolute ratio of potency of the construct on antigen VL is composed of three CDRs and four FRS, arranged from positive vs. antigen-negative cells, but of the potency of the amino-terminus to carboxy-terminus in the following order: construct on each type of cell relative to the potency of the FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobu free, non attenuated ligand on the same type of cell. This lin molecules can be of any type (e.g., IgG, IgE, IgM, Ig|D. "ratio of ratio” approach for quantifying the degree of IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and specificity of an antibody-ligand construct takes into con IgA2) or subclass. sideration any inherent differences in the potency of a ligand 0118. The term “antigen binding domain or “antigen on different cell types and is exemplified by the calculations binding portion of an antibody, as used herein, refers to one of Antigen Specificity Index (ASI) in Table 25. Assays for or more fragments of an antibody or protein that retain the determining potency of antibody-ligand fusion constructs ability to specifically bind to an antigen (e.g., CD38). It has are exemplified in the examples and include cell based been shown that the antigen-binding function of an antibody assays for proliferation, apoptosis, phosphorylation of can be performed by fragments of a full-length antibody. receptors and intracellular proteins, migration, differentia Such antibody embodiments may also be bispecific, dual tion (for example, differentiation of naive CD4+ T cells into specific, or multi-specific formats, specifically binding to Th1. Th17, Th2 vs. Treg cells), increases or decreases in two or more different antigens. Examples of binding frag gene expression or gene product secretion into the media, ments encompassed within the term “antigen-binding por etc. tion of an antibody include (i) a Fab fragment, a monova 0112 Accordingly, in a first aspect the present invention lent fragment consisting of the VL, VH, CL and CH1 provides a polypeptide construct comprising a peptide or domains; (ii) a F(ab')2 fragment, a bivalent fragment com polypeptide signaling ligand linked to an antibody or antigen prising two Fab fragments in addition to a portion of the binding portion thereof which binds to a cell surface hinge region, linked by a disulfide bridge at the hinge region; associated antigen wherein the ligand comprises at least one (iii) an Fd fragment consisting of the VHand CH1 domains: amino acid substitution or deletion which reduces its (iv) an Fv fragment consisting of the VL and VH domains potency on cells lacking expression of said antigen. of a single arm of an antibody, (v) a domain antibody (dAb) 0113. In one embodiment the present invention provides (Ward et al. 1989 Nature 341 544-6, Winter et al., PCT a polypeptide construct comprising IFN linked to an anti publication WO 90/05144 Al herein incorporated by refer body or antigen binding portion thereof which binds to a ence), which comprises a single variable domain; and (vi) an tumour associated antigen wherein the IFN comprises at isolated complementarity determining region (CDR). Fur least one amino acid substitution or deletion which reduces thermore, although the two domains of the Fv fragment, VL its potency on cells lacking expression of said antigen. Such and VH, are coded for by separate genes, they can be joined, a polypeptide will be capable of exerting with high potency using recombinant methods, by a synthetic linker that the IFN's anti-proliferative activity on the antigen-positive enables them to be made as a single protein chain in which tumor cells while exerting a much lower potency on the the VL and VH regions pair to form monovalent molecules antigen-negative, non-tumour cells within the body. (known as single chain Fv (sclv); see e.g., Bird et al. 1988 0114. In a second aspect the present invention provides a Science 242 423-6; Huston et al. 1988 Proc Natl Acad Sci method of treating a tumour in a subject comprising admin USA 85 5879-83). Such single chain antibodies are also istering to the Subject the polypeptide construct of the intended to be encompassed within the term “antigen present invention. binding portion of an antibody. Other forms of single chain 0115 The term “antibody-ligand construct as used antibodies, such as diabodies, are also encompassed. Dia herein refers to an antibody or antigen-binding fragment bodies are bivalent, bispecific antibodies in which VH and thereof covalently attached to a signaling ligand that has VL domains are expressed on a single polypeptide chain, but been attenuated by one or more substitutions or deletions using a linker that is too short to allow for pairing between that reduce the ligand’s potency on cells that do not express the two domains on the same chain, thereby forcing the the antigen corresponding to the antibody. domains to pair with complementary domains of another 0116. The term “antibody', as used herein, broadly refers chain and creating two antigen binding sites (see e.g., to any immunoglobulin (Ig) molecule comprised of four Holliger, P., et al., 1993, Proc. Natl. Acad. Sci. USA polypeptide chains, two heavy (H) chains and two light (L) 90:6444-6448; Poljak, R. J., et al., 1994, Structure 2:1121 chains, or any functional fragment, mutant, variant, or 1123). Such antibody binding portions are known in the art derivation thereof, which retains the essential epitope bind (Kontermann and Dubel eds. Antibody Engineering 2001 ing features of an Ig molecule. Such mutant, variant, or Springer-Verlag. New York. 790 pp., ISBN 3-540-41354-5). derivative antibody formats are known in the art, non In an embodiment the antibody binding portion is a Fab limiting embodiments of which are discussed below. fragment. 0117. In a full-length antibody, each heavy chain is 0119 The antibody described herein may be may be a comprised of a heavy chain variable region (abbreviated humanized antibody. The term “humanized antibody' shall herein as HCVR or VH) and a heavy chain constant region. be understood to refer to a protein comprising a human-like The heavy chain constant region is comprised of three variable region, which includes CDRs from an antibody domains, CH1, CH2 and CH3. Each light chain is comprised from a non-human species (e.g., mouse or rat or non-human of a light chain variable region (abbreviated herein as LCVR primate) grafted onto or inserted into FRS from a human or VL) and a light chain constant region. The light chain antibody (this type of antibody is also referred to a “CDR constant region is comprised of one domain, CL. The VH grafted antibody'). Humanized antibodies also include pro US 2016/0367695 A1 Dec. 22, 2016
teins in which one or more residues of the human protein are frameworks. For example, reference may be made to (Ku & modified by one or more amino acid Substitutions and/or one Schutz, Proc. Natl. Acad. Sci. USA 92: 6552-6556, 1995) or more FR residues of the human protein are replaced by which discloses a four-helix bundle protein cytochrome corresponding non-human residues. Humanized antibodies b562 having two loops randomized to create CDRs, which may also comprise residues which are found in neither the have been selected for antigen binding. human antibody or in the non-human antibody. Any addi 0.126 There is a plethora of non-antibody recognition tional regions of the protein (e.g., Fc region) are generally protein or protein domain scaffolds that may be utilised as human. Humanization can be performed using a method the antigen binding domains in the constructs of this inven known in the art, e.g., U.S. Pat. No. 5.225,539, U.S. Pat. No. tion. These include scaffolds based on cytotoxic T lympho 6,054,297, U.S. Pat. No. 7,566,771 or U.S. Pat. No. 5,585, cyte-associated antigen 4 (CTLA-4) (Evibody; U.S. Pat. No. 089. The term “humanized antibody' also encompasses a 7,166.697); human transferrin (Trans-body); a three-helix Super-humanized antibody, e.g., as described in U.S. Pat. bundle from the Z-domain of Protein A (Affibody); a mono No. 7,732,578. meric or trimeric human C-type lectin domain (Tetranectin); 0120. The antibody described herein may be human. The the tenth human fibronectin type III domain (AdNectin); the term “human antibody” as used herein refers to proteins Kunitz-type domain of human or bovine trypsin inhibitor; having variable and, optionally, constant antibody regions insect Defensin A (IICA29), APPI (Kuntiz domains); lipoca found in humans, e.g. in the human germline or Somatic cells lins, FABP. Bilin-binding protein, Apoloproptein D (Anti or from libraries produced using Such regions. The “human” calins); human C-crystallin or ubiquitin molecule (Affilin); antibodies can include amino acid residues not encoded by trypsin inhibitor II (Microbody); O.2p8 or Ankyrin repeat human sequences, e.g. mutations introduced by random or (repeat-motif proteins), Charybdotoxin (Scorpion toxins), site directed mutations in vitro (in particular mutations Min-23, Cellulose binding domain (Knottins); Neocarzinos which involve conservative Substitutions or mutations in a tatin, CBM4-2 and Tendamistat. Small number of residues of the protein, e.g. in 1, 2, 3, 4 or I0127. Further, in addition to scaffolds provided for by 5 of the residues of the protein). These “human antibodies' antibody-derived domains or non-antibody folds as do not necessarily need to be generated as a result of an described above, there are naturally occurring ligand bind immune response of a human, rather, they can be generated ing proteins or protein domains that may be utilised as the using recombinant means (e.g., screening a phage display ligand binding domains in this invention. For example, library) and/or by a transgenic animal (e.g., a mouse) protein domains that possess ligand binding properties comprising nucleic acid encoding human antibody constant include extracellular domains of receptors, PDZ modules of and/or variable regions and/or using guided selection (e.g., signalling proteins, such as Ras-binding protein AF-6, adhe as described in or U.S. Pat. No. 5,565,332). This term also sion molecules, and enzymes. encompasses affinity matured forms of Such antibodies. For I0128. The present invention further encompasses chemi the purposes of the present disclosure, a human protein will cal analogues of amino acids in the Subject antibodies. The also be considered to include a protein comprising FRS from use of chemical analogues of amino acids is useful interalia a human antibody or FRS comprising sequences from a to stabilize the molecules such as if required to be admin consensus sequence of human FRS and in which one or more istered to a subject. The analogues of the amino acids of the CDRS are random or semi-random, e.g., as described contemplated herein include, but are not limited to, modi in U.S. Pat. No. 6,300,064 and/or U.S. Pat. No. 6,248,516. fications of side chains, incorporation of unnatural amino 0121 The antibody portions of polypeptides of the pres acids and/or their derivatives during peptide, polypeptide or ent invention may be full length antibodies of any class, protein synthesis and the use of crosslinkers and other preferably IgG1, IgG2 or IgG4. The constant domains of methods which impose conformational constraints on the such antibodies are preferably human. The variable regions proteinaceous molecule or their analogues. of Such antibodies may be of non-human origin or, prefer I0129. Examples of side chain modifications contem ably, be of human origin or be humanized. Antibody frag plated by the present invention include modifications of ments may also be used in place of the full length antibodies. amino groups such as by reductive alkylation by reaction 0122) The term “antibody' also includes engineered anti with an aldehyde followed by reduction with NaBH; amidi bodies. As will be appreciated there are many variations of nation with methylacetimidate; acylation with acetic anhy engineered antibodies (e.g. mouse monoclonal, chimeric, dride; carbamoylation of amino groups with cyanate; trini humanized and human monoclonal antibodies, single chain trobenzylation of amino groups with 2, 4, 6-trinitrobenzene variable antibody fragments (ScPv's), minibodies, aptamers, sulphonic acid (TNBS); acylation of amino groups with as well as bispecific antibodies and diabodies as described Succinic anhydride and tetrahydrophthalic anhydride; and above). pyridoxylation of lysine with pyridoxal-5-phosphate fol 0123. Single variable region domains (termed dAbs) are, lowed by reduction with NaBH4. for example, disclosed in (Ward et al., Nature 341: 544-546, 0.130. The guanidine group of arginine residues may be 1989; Hamers-Casterman et al., Nature 363: 446-448, 1993; modified by the formation of heterocyclic condensation Davies & Riechmann, FEBS Lett. 339: 285-290, 1994). products with reagents such as 2,3-butanedione, phenylgly 012.4 Minibodies are small versions of whole antibodies, oxal and glyoxal. which encode in a single chain the essential elements of a I0131 The carboxyl group may be modified by carbodi whole antibody. Suitably, the minibody is comprised of the imide activation via O-acylisourea formation followed by VH and VL domains of a native antibody fused to the hinge Subsequent derivatisation, for example, to a corresponding region and CH3 domain of the immunoglobulin molecule as, amide. for example, disclosed in U.S. Pat. No. 5,837,821. I0132) Sulphydryl groups may be modified by methods 0.125. In an alternate embodiment, the engineered anti Such as carboxymethylation with iodoacetic acid or iodoac body may comprise non-immunoglobulin derived, protein etamide; performic acid oxidation to cysteic acid; formation US 2016/0367695 A1 Dec. 22, 2016
of a mixed disulphides with other thiol compounds; reaction I0134) Modification of the imidazole ring of a histidine with maleimide, maleic anhydride or other substituted residue may be accomplished by alkylation with iodoacetic maleimide; formation of mercurial derivatives using 4-chlo acid derivatives or N-carbethoxylation with diethylpyrocar romercuribenzoate, 4-chloromercuriphenylsulphonic acid, bonate. phenylmercury chloride, 2-chloromercuri-4-nitrophenol and 0.135 Examples of incorporating unnatural amino acids other mercurials; carbamoylation with cyanate at alkaline and derivatives during peptide synthesis include, but are not pH. limited to, use of norleucine, 4-aminobutyric acid, 4-amino 0.133 Tryptophan residues may be modified by, for 3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, example, oxidation with N-bromosuccinimide or alkylation t-butylglycine, norvaline, phenylglycine, ornithine, Sar of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or cosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thie Sulphenyl halides. Tyrosine residues on the other hand, may nyl alanine and/or D-isomers of amino acids. A list of be altered by nitration with tetranitromethane to form a unnatural amino acid, contemplated herein is shown in Table 3-nitrotyrosine derivative. 1. TABLE 1.
Non-conventional Non-conventional amino acid Code amino acid Code C-aminobutyric acid Abu L-N-methylalanine Nimala C-amino-C.-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane Cpro L-N-methylasparagine NmaSn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Alb 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 Dglin 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 Dphe L-N-methylvaline Nmval Dpro L-N-methylethylglycine Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle Dtrp L-norvaline Nwa Dtyr C.-methyl-aminoisobutyrate Maib Dval C.-methyl-y-aminobutyrate Mgabu D-C.-methylalanine Dmala C.-methylcyclohexylalanine Mchexa D-O-methylarginine Dmarg C.-methylcylcopentylalanine Mcpen D-O-methylasparagine DmaSn C.-methyl-C-napthylalanine Manap D-C.-methylaspartate Dmasp C.-methylpenicillamine Mpen D-C.-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D-O-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg D-C.-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn D-C.-methylisoleucine Dmile N-amino-C.-methylbutyrate Nmaabu D-C.-methyleucine Dmleu C.-napthylalanine Anap D-C.-methyllysine Dmlys N-benzylglycine Nphe D-C.-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln D-C.-methylornithine Dmorn N-(carbamylmethyl)glycine Nasin D-O-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu D-C.-methylproline Dmpro N-(carboxymethyl)glycine Nasp D-C.-methylserine Dmser N-cyclobutylglycine Nicbut D-C.-methylthreonine Dmthr N-cycloheptylglycine Nchep D-C.-methyltryptophan Dmitrp N-cyclohexylglycine Nchex D-C.-methyltyrosine Dmity N-cyclodecylglycine Nicdec D-C.-methylvaline Dmval N-cylcododecylglycine Nicdod D-N-methylalanine Dnmala N-cyclooctylglycine Nicoct D-N-methylarginine Dnmarg N-cyclopropylglycine Nicpro D-N-methylasparagine DnmaSn N-cycloundecylglycine Ncund D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe D-N-methylglutamine Dnmglin N-(3-guanidinopropyl)glycine Narg D-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine Nthr D-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine Nser D-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine Nhis D-N-methyleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp US 2016/0367695 A1 Dec. 22, 2016 11
TABLE 1-continued
Non-conventional Non-conventional amino acid Code amino acid Code D-N-methyllysine Dnmlys N-methyl-y-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnimpro N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Neu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen Y-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine Pen L-homophenylalanine Hphe L-C.-methylalanine Mala L-C.-methylarginine Marg L-C.-methylasparagine Masin L-C.-methylaspartate Masp L-C.-methyl-t-butylglycine Mtbug L-C.-methylcysteine Mcys L-methylethylglycine Metg L-C.-methylglutamine Mglin L-C.-methylglutamate Mglu L-C-methylhistidine Mhis L-C.-methylhomophenylalanine Mhphe L-C.-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-C.-methyleucine Meu L-C.-methyllysine Mlys L-C.-methylmethionine Mmet L-C.-methylmorleucine Minle L-C.-methylnorvaline Mnva L-C.-methylornithine Morn L-C.-methylphenylalanine Mphe L-C.-methylproline Mpro L-C.-methylserine MSer L-C.-methylthreonine Mthr L-C.-methyltryptophan Mtrp L-C.-methyltyrosine Mtyr L-C-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl)carbamylmethyl)glycine Nnbhm N-(N-(3,3-diphenylpropyl)carbamylmethyl)glycine Nnbhe -carboxy-1-(2,2-diphenyl- Nmbc ethylamino)cyclopropane
0.136 Crosslinkers can be used, for example, to stabilize approaches that harness natural affinity maturation machin 3D conformations, using homo-bifunctional crosslinkers ery (Peled, Kuang et al. 2008). Mutagenesis can also be Such as the bifunctional imido esters having (CH2)n spacer performed at the RNA level, for example by use of Qf3 groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccin replicase (Kopsidas, Roberts et al. 2006). Library-based imide esters and hetero-bifunctional reagents which usually methods allowing screening for improved variant proteins contain an amino-reactive moiety Such as N-hydroxysuccin can be based on various display technologies such as phage, imide and another group specific-reactive moiety Such as yeast, ribosome, bacterial or mammalian cells, and are well maleimido or dithio moiety (SH) or carbodiimide (COOH). known in the art (Benhar 2007). Affinity maturation can be 0.137 Using methods well known in the art to increase achieved by more directed/predictive methods for example binding, by for example, affinity maturation, or to decrease by site-directed mutagenesis or gene synthesis guided by immunogenicity by removing predicted MHC class II-bind findings from 3D protein modeling (see for example Queen, ing motifs. The therapeutic utility of the antibodies Schneider et al. 1989 or U.S. Pat. No. 6,180,370 or U.S. Pat. described herein can be further enhanced by modulating No. 5,225,539). their functional characteristics, such as antibody-dependent 0140 Methods of increasing ADCC have been described cell-mediated cytotoxicity (ADCC), complement-dependent by Ferrara, Brunker et al. 2006; Li, Sethuraman et al. 2006; cytotoxicity (CDC), serum half-life, biodistribution and Stavenhagen, Gorlatov et al. 2007; Shields, Namenuk et al. binding to Fc receptors or the combination of any of these. 2001; Shinkawa, Nakamura et al. 2003; and WO 2008/ This modulation can be achieved by protein-engineering, OO6554. glyco-engineering or chemical methods. Depending on the 0141 Methods of increasing CDC have been described therapeutic application required, it could be advantageous to by Idusogie, Wong et al. 2001; Dall’Acqua, Cook et al. either increase or decrease any of these activities. 2006; Michaelsen, Aase et al. 1990; Brekke, Bremnes et al. 0138 An example of glyco-engineering used the Potel 1993; Tan, Shopes et al. 1990; and Norderhaug, Brekke et al. ligent(R) method as described in Shinkawa T. et al., 2003 (J 1991. Biol Chem 278: 3466-73). 0.142 References describing methods of increasing 0.139. Numerous methods for affinity maturation of anti ADCC and CDC include Natsume. In et al. 2008. The bodies are known in the art. Many of these are based on the disclosure of each of these references is included herein by general strategy of generating panels or libraries of variant cross reference. proteins by mutagenesis followed by selection and/or 0.143 A number of methods for modulating antibody screening for improved affinity. Mutagenesis is often per serum half-life and biodistribution are based on modifying formed at the DNA level, for example by error prone PCR the interaction between antibody and the neonatal Fc recep (Thie, Voedisch et al. 2009), by gene shuffling (Kolkman and tor (FcRn), a receptor with a key role in protecting IgG from Stemmer 2001), by use of mutagenic chemicals or irradia catabolism, and maintaining high serum antibody concen tion, by use of mutator strains with error prone replication tration. Dall’Acqua et al describe substitutions in the Fc machinery (Greener 1996) or by somatic hypermutation region of IgG1 that enhance binding affinity to FcRn, US 2016/0367695 A1 Dec. 22, 2016
thereby increasing serum half-life (Dall’Acqua, Woods et al. mor, ductal carcinoma, endocrine cancers, endometrial can 2002) and further demonstrate enhanced bioavailability and cer, ependymoma, esophageal cancer, Ewing's sarcoma, modulation of ADCC activity with triple substitution of extra-hepatic bile duct cancer, eye cancer, eye: melanoma, M252Y/S254T/T256E (Dall’Acqua, Kiener et al. 2006). See retinoblastoma, fallopian tube cancer, fanconi anemia, fibro also U.S. Pat. Nos. 6,277,375; 6,821,505; and U.S. Pat. No. sarcoma, gallbladder cancer, gastric cancer, gastrointestinal 7,083,784. Hinton et all have described constant domain cancers, gastrointestinal-carcinoid-tumor, genitourinary amino acid substitutions at positions 250 and 428 that confer cancers, germ cell tumors, gestational-trophoblastic-disease, increased in vivo half-life (Hinton, Johlfs et al. 2004). glioma, gynaecological cancers, hematological malignan (Hinton, Xiong et al. 2006). See also U.S. Pat. No. 7,217, cies, hairy cell leukaemia, head and neck cancer, hepatocel 797. Petkova et al have described constant domain amino lular cancer, hereditary breast cancer, histiocytosis, Hodg acid substitutions at positions 307, 380 and 434 that confer increased in vivo half-life (Petkova, Akilesh et al. 2006). See kin's disease, human papillomavirus, hydatidiform mole, also Shields etal 2001 and WO 2000/42072. Other examples hypercalcemia, hypopharynx cancer, intraocular melanoma, of constant domain amino acid Substitutions which modulate islet cell cancer, Kaposi's sarcoma, kidney cancer, Langer binding to Fc receptors and Subsequent function mediated by han's-cell-histiocytosis, laryngeal cancer, leiomyosarcoma, these receptors, including FcRn binding and serum half-life, leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, are described in U.S. Pat. Application Nos 20090142340: liver cancer, lung cancer, lymphedema, lymphoma, Hodg 2009 OO68175 and 20090092599. kin's lymphoma, non-Hodgkin’s lymphoma, male breast 0144. The glycans linked to antibody molecules are cancer, malignant-rhabdoid-tumor-of-kidney, medulloblas known to influence interactions of antibody with Fc recep toma, melanoma, merkel cell cancer, mesothelioma, meta tors and glycan receptors and thereby influence antibody static cancer, mouth cancer, multiple endocrine neoplasia, activity, including serum half-life (Kaneko, Nimmerjahn et mycosis fungoides, myelodysplastic syndromes, multiple al. 2006; Jones, Papac et al. 2007; and Kanda, Yamada et al. myeloma, myeloproliferative disorders, nasal cancer, 2007). Hence, certain glycoforms that modulate desired nasopharyngeal cancer, nephroblastoma, neuroblastoma, antibody activities can confer therapeutic advantage. Meth neurofibromatosis, nijmegen breakage syndrome, non-mela ods for generating engineered glycoforms are known in the noma skin cancer, non-Small-cell-lung-cancer-(NSCLC), art and include but are not limited to those described in U.S. ocular cancers, oesophageal cancer, oral cavity cancer, oro Pat. Nos. 6,602,684; 7,326,681; 7,388,081 and WO 2008/ pharynx cancer, osteosarcoma, ostomy ovarian cancer, pan OO6554. creas cancer, paranasal cancer, parathyroid cancer, parotid 0145 Extension of half-life by addition of polyethylene gland cancer, penile cancer, peripheral-neuroectodermal glycol (PEG) has been widely used to extend the serum tumors, pituitary cancer, polycythemia Vera, prostate cancer, half-life of proteins, as reviewed, for example, by Fishburn rare-cancers-and-associated-disorders, renal cell carcinoma, 2008. retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson 0146. As will be recognised it is possible to make con syndrome, salivary gland cancer, sarcoma, Schwannoma, servative amino acid Substitutions within the sequences of Sezary syndrome, skin cancer, Small cell lung cancer the current invention. By “conservative substitution' is (SCLC), Small intestine cancer, Soft tissue sarcoma, spinal meant amino acids having similar properties. As used in this cord tumors, squamous-cell-carcinoma-(skin), stomach can specification the following groups of amino acids are to be cer, synovial sarcoma, testicular cancer, thymus cancer, seen as conservative substitutions: H, Rand K; D, E, N and thyroid cancer, transitional-cell-cancer-(bladder), transi Q: V. I and L; C and M. S. T. P. A and G.; and F, Y and W. tional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic can 0147 The term “cell surface-associated antigen”, as used cer, urethral cancer, urinary system cancer, uroplakins, uter herein, broadly refers to any antigen expressed on Surfaces ine sarcoma, uterus cancer, vaginal cancer, Vulva cancer, of cells, including infectious or foreign cells or viruses. Waldenstrom's-macroglobulinemia and Wilms’ tumor. In an 0148. In certain aspects of the present invention, the embodiment the tumor is selected from a group of multiple polypeptide constructs or compositions of the present inven myeloma or non-hodgkin’s lymphoma. tion may be used to treat patients with cancer. Cancers 0149. As contemplated for the treatment of cancer, the contemplated herein include: a group of diseases and dis antibody portions of the constructs of the present invention orders that are characterized by uncontrolled cellular growth may bind to tumour-associated antigens, i.e., cell Surface (e.g. formation of tumor) without any differentiation of those antigens that are selectively expressed by cancer cells or cells into specialized and different cells. Such diseases and over-expressed in cancer cells relative to most normal cells. disorders include ABL1 protooncogene, AIDS related can There are many tumour-associated antigens (TAAS) known cers, acoustic neuroma, acute lymphocytic leukaemia, acute in the art. Non-limiting examples of TAAS include enzyme myeloid leukaemia, adenocystic carcinoma, adrenocortical tyrosinase; melanoma antigen GM2, alphafetoprotein cancer, agnogenic myeloid metaplasia, alopecia, alveolar (AFP); carcinoembryonic antigen (CEA); Mucin 1 (MUC1); Soft-part sarcoma, anal cancer, angiosarcoma, aplastic anae Human epidermal growth factor receptor (Her2/Neu); T-cell mia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma leukemia/lymphoma 1 (TCL1) oncoprotein. Exemplary (skin), bladder cancer, bone cancers, bowel cancer, brain TAAS associated with a number of different cancers are stem glioma, brain and CNS tumors, breast cancer, CNS telomerase (hTERT); prostate-specific membrane antigen tumors, carcinoid tumors, cervical cancer, childhood brain (PSMA); urokinase plasminogen activator and its receptor tumors, childhood cancer, childhood leukaemia, childhood (uPA/uPAR); vascular endothelial growth factor and its Soft tissue sarcoma, chondrosarcoma, choriocarcinoma, receptor (VEGF/VEGFR); extracellular matrix metallopro chronic lymphocytic leukaemia, chronic myeloid leukaemia, teinase inducer (EMMPRIN/CD147); epidermal growth fac colorectal cancers, cutaneous T-Cell lymphoma, dermatofi tor (EGFR); platelet-derived growth factor and its receptor brosarcoma-protuberans, desmoplastic-Small-round-cell-tu (PDGF/PDGFR) and c-kit (CD117). US 2016/0367695 A1 Dec. 22, 2016
0150. A list of other TAAs is provided in US 2010/ (Hoon et al., 1993, Cancer Res. 53:5244-5250) and Lewis, 0297076, the disclosure of which is included herein by lewis' and lewis' carbohydrate antigens that can be dis reference. Of particular interest are cell Surface antigens played on proteins or glycolipids. Examples of protein associated with multiple myeloma cells, including but not antigens are HER-2/neu, human papillomavirus-E6 or -E7. limited to CD38, CD138, CS1, and HM1.24. In one embodi MUC-1; KS 1/4 pan-carcinoma antigen (Perez and Walker, ment an antigen for antibody-attenuated ligand constructs, 1990, J. Immunol. 142:3662-3667: Bumal, 1988, for example, an antibody-attenuated interferon construct, is Hybridoma 7(4):407-415); ovarian carcinoma antigen CD38. CA125 (Yu et al., 1991, Cancer Res. 51(2):468-475); pro 0151. CD38 is a 46 kDa type II transmembrane glyco static acid phosphate (Tailor et al., 1990, Nucl. Acids Res. protein. It has a short N-terminal cytoplasmic tail of 20 18(16):4928); prostate specific antigen (Henttu and Vihko, amino acids, a single transmembrane helix and a long 1989, Biochem. Biophys. Res. Comm. 160(2):903-910; extracellular domain of 256 amino acids (Bergsagel, P. Israeli et al., 1993, Cancer Res. 53:227-230); melanoma Blood: 85:436, 1995 and Liu, Q., Structure, 13:1331, 2005). associated antigen p97 (Estin et al., 1989, J. Natl. Cancer It is expressed on the Surface of many immune cells includ Instit. 81 (6):445-446); melanoma antigen gp75 (Vijayasar ing CD4 and CD8 positive T cells, B cells, NK cells, dahl et al., 1990, J. Exp. Med. 171 (4): 1375-1380); prostate monocytes, plasma cells and on a significant proportion of specific membrane antigen; carcinoembryonic antigen normal bone marrow precursor cells (Malavasi, F. Hum. (CEA) (Foon et al., 1994, Proc. Am. Soc. Clin. Oncol. Immunol. 9:9, 1984). In lymphocytes, however, the expres 13:294), MUC16 (antibodies include MJ-170, MJ-171, sion appears to be dependent on the differentiation and MJ-172 and MJ-173 U.S. Pat. No. 7,202,346, 3A5 U.S. activation state of the cell. Resting T and B cells are negative Pat. No. 7,723.485). NMB (U.S. Pat. No. 8,039,593), while immature and activated lymphocytes are predomi malignant human lymphocyte antigen-APO-1 (Bernhard et nantly positive for CD38 expression (Funaro, A. J. Immu al., 1989, Science 245:301-304); high molecular weight nol. 145:2390, 1990). Additional studies indicate mRNA melanoma antigen (BMW-MAA) (Natali et al., 1987, Can expression in non-hemopoeitic organs such as pancreas, cer 59:55-63; Mittelman et al., 1990, J. Clin. Invest. brain, spleen and liver (Koguma, T., Biochim. Biophys. Acta 86:2136-2144); Burkitt's lymphoma antigen-38.13: CD19 1223:160, 1994.) (Ghetie et al., 1994, Blood 83:1329-1336); human B-lym 0152 CD38 is a multifunctional ectoenzyme that is phoma antigen-CD20 (Reffet al., 1994, Blood 83:435-445): involved in transmembrane signaling and cell adhesion. It is GICA 19-9 (Herlyn et al., 1982, J. Clin. Immunol. 2:135), also known as cyclic ADP ribose hydrolase because it can CTA-1 and LEA: CD33 (Sgouros et al., 1993, J. Nucl. Med. transform NAD" and NADP into cADPR, ADPR and 34:422-430); oncofetal antigens such as alpha-fetoprotein NAADP, depending on extracellular pH. These products for liver cancer or bladder tumor oncofetal antigen (Hell induce Ca"-mobilization inside the cell which can lead to strom et al., 1985, Cancer. Res. 45:2210-2188); differentia tyrosine phosphorylation and activation of the cell. CD38 is tion antigens such as human lung carcinoma antigen L6 or also a receptor that can interact with a ligand, CD31. L20 (Hellstrom et al., 1986, Cancer Res. 46:3917-3923): Activation of receptor via CD31 leads to intracellular events antigens of fibrosarcoma; human leukemia T cell antigen including Ca" mobilization, cell activation, proliferation, Gp37 (Bhattacharya-Chatterjee et al., 1988, J. Immunol. differentiation and migration (reviewed in Deaglio, S., 141:1398-1403); tumor-specific transplantation type of cell Trends in Mol. Med. 14:210, 2008.) Surface antigen (TSTA) Such as virally-induced tumor anti 0153 CD38 is expressed at high levels on multiple gens including T-antigen, DNA tumor virus and envelope myeloma cells, in most cases of T- and B-lineage acute antigens of RNA tumor viruses; neoglycoproteins, breast lymphoblastic leukemias, some acute myelocytic leukemias, cancer antigens such as EGFR (Epidermal growth factor follicular center cell lymphomas and T lymphoblastic lym receptor), polymorphic epithelial mucin (PEM) (Hilkens et phomas. (Malavasi, F. J. Clin Lab Res. 22:73, 1992). More al., 1992, Trends in Bio. Chem. Sci. 17:359); polymorphic recently, CD38 expression has become a reliable prognostic epithelial mucin antigen; human milk fat globule antigen; marker in B-lineage chronic lymphoblastic leukemia colorectal tumor-associated antigens such as TAG-72 (B-CLL) (Ibrahim, S., Blood. 98:181, 2001 and Durig, J., (Yokata et al., 1992, Cancer Res. 52:3402-3408), CO 17-1A Leuk. Res. 25:927, 2002). Independent groups have dem (Ragnhammar et al., 1993, Int. J. Cancer 53:751-758); onstrated that B-CLL patients presenting with a CD38" differentiation antigens (Feizi, 1985, Nature 314:53-57) clone are characterized by an unfavorable clinical course Such as I(Ma) found in gastric adenocarcinomas, SSEA-1 with a more advance stage of disease, poor responsiveness found in myeloid cells, VEP8, VEP9, Myl, VIM-D5, M18 to chemotherapy and shorter survival time (Morabito. F., and M39 found in breast epithelial cancers, Ds found in Haematologica. 87:217, 2002). The consistent and enhanced colorectal cancer, TRA-1-85 (blood group H). C14 found in expression of CD38 on lymphoid tumors makes this an colonic adenocarcinoma, F3 found in lung adenocarcinoma, attractive target for therapeutic antibody technologies. AH6 found in gastric cancer, Y hapten found in embryonal 0154 Preferred antigens for the development of anti carcinoma cells, TL5 (blood group A), E1 series (blood body-attenuated ligand fusion protein constructs which tar group B) antigens found in pancreatic cancer, FC 10.2 found get cancer are antigens which show selective or greater in embryonal carcinoma cells, gastric adenocarcinoma anti expression on the cancer cells than on most other, non gen, CO-514 (blood group Le') found in adenocarcinoma, transformed cells within the body. Non-protein examples of NS-10 found in adenocarcinomas, CO-43 (blood group Le'), Such antigens include, sphingolipids, ganglioside GD2 G49 found in A431 cells, 19.9 found in colon cancer; gastric (Saleh et al., 1993, J. Immunol. 151, 3390-3398), ganglio cancer mucins; R found in melanoma, MH2 (blood group side GD3 (Shitara et al., 1993, Cancer Immunol. Immu ALe”/LeY) found in colonic adenocarcinoma, 4.2. D1.1, nother. 36:373-380), ganglioside GM2 (Livingston et al., OFA-1, G, OFA-2 and M1:22:25:8 found in embryonal 1994, J. Clin. Oncol. 12:1036-1044), ganglioside GM3 carcinoma cells and SSEA-3 and SSEA-4. HMW-MAA US 2016/0367695 A1 Dec. 22, 2016
(SEQ ID NO:433), also known as melanoma chondroitin CD49e, CD49f, CD103 and CD104. Non-limiting examples Sulfate proteoglycan, is a membrane-bound protein of 2322 of suitable lectins are S type, C type, and I type lectin. residues which is overexpressed on over 90% of the surgi Examples of antibodies to CEA are shown in Table 2. cally removed benign nevi and melanoma lesions (Camploi, et. al. Crit Rev Immunol. 24:267, 2004). Accordingly it may TABLE 2 be a potential target cell Surface associated antigen. 0155. Other example cancer antigens for targeting with CEA Antibodies fusion protein constructs of the present invention include Ab (exemplary cancers are shown in parentheses): CD5 (T-cell Clones Patent Assignee Comments leukemia/lymphoma), CA15-3 (carcinomas), CA19-9 (car COL-1 U.S. Pat. No. 6,417,337 The Dow Chemical Humanized cinomas), L6 (carcinomas), CA 242 (colorectal), placental Company alkaline phosphatase (carcinomas), prostatic acid phos 806.077 U.S. Pat. No. 6,903,203 AstraZeneca UK Ltd. Humanized phatase (prostate), MAGE-1 (carcinomas), MAGE-2 (car T84.66 U.S. Pat. No. 7,776,330 City of Hope Humanized cinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), transferrin receptor (carcinomas), p97 (melanoma), MUC1 0157 Antibodies that bind the CD22 antigen expressed (breast cancer), MARTI (melanoma), CD20 (non Hodgkin’s on human B cells include, for example, HD6, RFB4, UV22 lymphoma), CD52 (leukemia), CD33 (leukemia), human 2. To 15, 4KB128 and a humanized anti-CD22 antibody chorionic gonadotropin (carcinoma), CD38 (multiple (hLL2) (see, e.g., Li et al. (1989) Cell. Immunol. 111: 85-99; myeloma), CD21 (B-cell lymphoma), CD22 (lymphoma), Mason et al. (1987) Blood 69: 836-40; Behr et al. (1999) CD25 (B-cell Lymphoma), CD37 (B-cell lymphoma), CD45 Clin. Cancer Res. 5: 3304s-3314s: Bonardi et al. (1993) (acute myeloblastic leukemia), HLA-DR (B-cell lym Cancer Res. 53: 3015-3021). phoma), IL-2 receptor (T-cell leukemia and lymphomas), 0158 Antibodies to CD33 include, for example, CD40 (lymphoma), various mucins (carcinomas), P21 (car HuM195 (see, e.g., Kossman et al. (1999) Clin. Cancer Res. cinomas), MPG (melanoma), Ep-CAM (Epithelial Tumors), 5: 2748–2755; U.S. Pat. No. 5,693,761) and CMA-676 (see, Folate-receptor alpha (Ovarian), A33 (Colorectal), G250 e.g., Sievers et al., (1999) Blood 93: 3678-3684). (renal), Ferritin (Hodgkin lymphoma), de2-7 EGFR (glio 0159 Illustrative anti-MUC-1 antibodies include, but are blastoma, breast, and lung), Fibroblast activation protein not limited to Mc5 (see, e.g., Peterson et al. (1997) Cancer (epithelial) and tenascin metalloproteinases (glioblastoma). Res. 57: 1103-1108; Ozzello et al. (1993) Breast Cancer Res. Some specific, useful antibodies include, but are not limited Treat. 25: 265-276), and hCTMO1 (see, e.g., Van Hof et al. to, BR64 (Trail et al., 1997, Cancer Research 57:100 105), (1996) Cancer Res. 56: 5179-5185). BR96 mAb (Trail et al., 1993, Science 261:212-215), mAbs (0160 Illustrative anti-TAG-72 antibodies include, but are against the CD40 antigen, such as S2C6 mAb (Francisco et not limited to CC49 (see, e.g., Pavlinkova et al. (1999) Clin. al., 2000, Cancer Res. 60:3225-3231) or other anti-CD40 Cancer Res. 5: 2613-2619), B72.3 (see, e.g., Divgi et al. antibodies, such as those disclosed in U.S. Patent Publication (1994) Nucl. Med. Biol. 21:9-15), and those disclosed in Nos. 2003-0211100 and 2002-0142358; mAbs against the U.S. Pat. No. 5,976,531. CD30 antigen, such as AC 10 (Bowen et al., 1993, J. Immu 0.161 Illustrative anti-HM1.24 antibodies include, but are nol. 151:5896-5906: Wahl et al., 2002 Cancer Res.62(13): not limited to a mouse monoclonal anti-HM1.24 and a 3736-42) or MDX-0060 (U.S. Patent Publication No. 2004 humanized anti-HM1.24 IgGlkappa antibody (see, e.g., Ono 0006215) and mAbs against the CD70 antigen, such as 1F6 et al. (1999) Mol. Immuno. 36:387-395). mAb and 2F2 mAb (see, e.g., U.S. Patent Publication No. 0162. In certain embodiments the targeting moiety com 2006-0083736) or antibodies 2H5, 10B4, 8B5, 18E7, 69A7 prises an anti-HER2 antibody. The erBB 2 gene, more (U.S. Pat. No. 8,124,738). Other antibodies have been commonly known as (Her-2/neu), is an oncogene encoding reviewed elsewhere (Franke et al., 2000, Cancer Biother. a transmembrane receptor. Several antibodies have been Radiopharm. 15:45976; Murray, 2000, Semin. Oncol. 27:64 developed against Her-2/neu, including trastuzumab (e.g., 70; Breitling, F., and Dubel, S. Recombinant Antibodies, HERCEPTINTM: Fornier et al. (1999) Oncology (Huntingt) John Wiley, and Sons, New York, 1998). 13: 647-58), TAB-250 (Rosenblum et al. (1999) Clin. Can 0156. In certain embodiments, useful antibodies can bind cer Res. 5: 865-874), BACH-250 (Id.), TA1 (Maier et al. to a receptor or a complex of receptors expressed on a target (1991) Cancer Res. 51:5361-5369), and the mAbs described cell. The receptor or receptor complex can comprise an in U.S. Pat. Nos. 5,772,997; 5,770,195 (mAb 4D5; ATCC immunoglobulin gene Superfamily member, a major histo CRL 10463); and U.S. Pat. No. 5,677,171. compatibility protein, a cytokine receptor, a TNF receptor 0163 A number of antibodies have been developed that Superfamily member, a chemokine receptor, an integrin, a specifically bind HER2 and some are in clinical use. These lectin, a complement control protein, a growth factor recep include, for example, trastuzumab (e.g., HERCEPTINTM., tor, a hormone receptor or a neuro-transmitter receptor. Fornier et al. (1999) Oncology (Huntingt) 13: 647-658), Non-limiting examples of appropriate immunoglobulin TAB-250 (Rosenblum et al. (1999) Clin. Cancer Res. 5: superfamily members are CD2, CD3, CD4, CD8, CD19, 865-874), BACH-250 (Id.), TA1 (see, e.g., Maier et al. CD22, CD79, CD90, CD152/CTLA-4, PD-1, B7-H4, (1991) Cancer Res. 51: 5361-5369), and the antibodies B7-H3, and ICOS. Non-limiting examples of suitable TNF described in U.S. Pat. Nos. 5,772,997; 5,770, 195, and 5,677, receptor superfamily members are TACI, BCMA, CD27. 171. CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, TNFR1, 0164. Other fully human anti-HER2/neu antibodies are TNFR2, RANK, osteoprotegerin, APO 3, Apo2/TRAIL R1, well known to those of skill in the art. Such antibodies TRAIL R2, TRAIL R3, and TRAIL R4. Non-limiting include, but are not limited to the C6 antibodies such as examples of suitable integrins are CD11a, CD11b, CD11c, C6.5, DPL5, G98A, C6MH3-B1, B1D2, C6VLB, C6VLD, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, C6VLE, C6VLF, C6MH3-D7, C6MH3-D6, C6MH3-D5, US 2016/0367695 A1 Dec. 22, 2016
C6MH3-D3, C6MH3-D2, C6MH3-D1, C6MH3-C4, antigen binding portion thereof that specifically or prefer C6MH3-C3, C6MH3-B9, C6MH3-B5, C6MH3-B48, entially binds CD20. Anti-CD20 antibodies are well known C6MH3-B47, C6MH3-B46, C6MH3-B43, C6MH3-B41, to those of skill and include, but are not limited to Ritux C6MH3-B39, C6MH3-B34, C6MH3-B33, C6MH3-B31, imab, Ibritumomab, and Tositumomab, AME-133v (Applied C6MH3-B27, C6MH3-B25, C6MH3-B21, C6MH3-B20, Molecular Evolution). Ocrelizumab (Roche), Ofatumumab C6MH3-B2, C6MH3-B16, C6MH3-B15, C6MH3-B11, (Genmab), TRU-015 (Trubion) and IMMU-106 (Immuno C6MH3-B1, C6MH3-A3, C6MH3-A2, and C6ML3-9. medics). These and other anti-HER2/neu antibodies are described in U.S. Pat. Nos. 6,512,097 and 5,977,322, in PCT Publication (0171 WO 2010/105290 discloses an antibody designated WO 97/00271, in Schier et al. (1996) J Mol Biol 255: 28-43, “SC104 together with a range of humanised variants which Schier et al. (1996) J Mol Biol 263: 551-567, and the like. bind an antigen expressed on a range of tumour cells. 0.165 More generally, antibodies directed to various 0172. In an embodiment, the antibody attachment and members of the epidermal growth factor receptor family are attenuating mutation in the ligand increases the antigen well Suited for use as targeting antibodies or antigen binding specificity index (ASI) by greater than 10-fold, preferably portions thereof in the constructs of the present invention. greater than 50-fold, preferably greater than 100-fold, pref Such antibodies include, but are not limited to anti-EGF-R erably greater than 1000-fold, or preferably greater than antibodies as described in U.S. Pat. Nos. 5,844,093 and 10,000 fold. The antigen-specificity index (ASI) is defined 5,558,864, and in European Patent No. 706,799A. Other herein as the fold increased potency in signaling activity of illustrative anti-EGFR family antibodies include, but are not the polypeptide construct of the invention relative to the free limited to antibodies such as C6.5, C6ML3-9, C6MH3-B1, non-mutated polypeptide ligand on target antigen-positive C6-B1D2, F5, HER3A5, HER3.F4, HER3.H1, HER3.H3, cells multiplied by the fold decreased potency in signaling HER3. E12, HER3.B12, EGFR.E.12, EGFR.C10, EGFR. activity relative to the free non-mutated polypeptide ligand B11, EGFR.E.8, HER4.B4, HER4.G4, HER4.F4, HER4A8, on target antigen-negative cells. The term “potency in this HER4.B6, HER4.D4, HER4D7, HER4.D11, HER4D12, context may be quantitatively represented by the EC50 HER4.E3, HER4.E7, HER4.F8 and HER4C7 and the like value, which is the mathematical midpoint of a dose-re (see, e.g., U.S. Patent publications US 2006/0099205 A1 and sponse curve, in which the dose refers to the concentration US 2004/0071696 A1 which are incorporated herein by of ligand or antibody-ligand construct in an assay, and reference). response refers to the quantitative response of the cells to the (0166 It may be advantageous for the cell surface-asso signaling activity of the ligand at a particular dose. Thus, for ciated antigen to be expressed at Sufficient levels on the example, when a first compound is shown to possess an target cell that a Sufficently therapeutic amount of polypep EC50 (expressed for example in Molar units) that is 10-fold tide construct is presented to ligand receptors on the target lower than a second compound's EC50 on the same cells, cell Surface. Accordingly, in particular embodiments, the typically when measured by the same method, the first cell Surface associated antigen is expressed at a density of compound is said to have a 10-fold higher potency. Con greater than 12,600 copies per cell or greater than 15,000 versely, when a first compound is shown to possess an EC50 copies per cell. Methods for determining copy number of a that is 10-fold higher than a second compound's EC50 on cell Surface antigen are well known and readily available to the same cells, typically when measured by the same a person of skill in the art, for example the method provided method, the first compound is said to have a 10-fold lower by Jilana (Am J Clin Pathol 118:560-566, 2002) potency. 0167. It may be advantageous for the cell surface-asso 0173 While the large majority of antibodies tested ciated antigen to be expressed in a configuration on the cell showed efficient targeting of attenuated IFNC, the present Surface such that the polypeptide construct is abe to contact inventors identified examples of two antigens where target both the cell Surface antigen and the ligand receptor on the ing attenuated IFNC. to a target-expressing cell line did not target cell. Accordingly, in particular embodiments the cell exhibit an ASI that was appreciably greater than for the free, Surface associated antigen has an extracellular domain hav non-mutated ligand. The first example is demonstrated by ing a molecular weight of less than 240 kD. the antigen CSPG4 (also known as BMW-MAA, high 0.168. It may be advantageous for the antibody or antigen molecular weight melanoma-associated antigen). We tested binding portion thereof to bind to the cell surface associated two different anti-HMW-MAA-antibody-IFNC. fusion pro antigen with Sufficient affinity to facilitate ligand binding to tein constructs in on-target proliferation assays using A375 the ligand receptor on the cell Surface. Accordingly, in or CHL-1 cell lines. We did not see inhibitory activity with particular embodiments of the the present invention the either cell line or antibody at the doses tested (EC50s-21 polypeptide constructs exhibit an antigen binding affinity, as nM). The extracelluar domain of this antigen is exception measured by EC50, of from 50 nM, from 25 nM, from 10 ally large (extracellular domain MW approx. 240 kD-450 nM or from 5 nM to 0.1 p.M. kD depending on glycosylation). It is possible that certain (0169. As described in U.S. Pat. Nos. 6.512,097 and antibody-IFN fusion protein constructs that bind to very 5,977.322, other anti-EGFR family member antibodies can large antigens may be sterically restricted from simultane readily be produced by shuffling light and/or heavy chains ously interacting with the IFN receptors on the same cells. followed by one or more rounds of affinity selection. Thus It is, however, possible that other antibodies that target other in certain embodiments, this invention contemplates the use epitopes of this antigen may support the targeted IFN of one, two, or three CDRs in the VL and/or VH region that activity. Despite this possibility it is preferred that the are CDRs described in the above-identified antibodies and/ antibody or antigen binding portion thereof of the polypep or the above identified publications. tide construct of the present invention binds an antigen 0170 In various embodiments the targeting antibody or wherein the extracellular domain thereof has a molecular antigen binding portion thereof comprises an antibody or weight of less than 240 kD. US 2016/0367695 A1 Dec. 22, 2016 16
0.174. A second example of an antibody-attenuated IFNC. 0.175. Another example of an antibody-attenuated fusion fusion protein construct that did not show potent activity protein construct in which the antibody did not provide was based on an antibody which binds to the myeloid antigen CD33. CD33 is expressed at a relatively low level on Sufficient targeting to the cancer cells was an anti-GM2 KG-1 cells, reported as 12,600 copies per cell (Sutherland, ganglioside antibody attached to an attenuated IFNC. In this MAbs. 1(5): 481-490, 2009). Treatment of KG-1 cells with case, the antibody was to a carbohydrate epitope and, as an anti-CD33 antibody-attenuated IFNC. fusion protein con typical of such antibodies, had a low affinity (EC50 for struct failed to inhibit proliferation at all doses tested binding target cells was ~50 nM by flow cytometry). There (IC50>76 nM). We believe that the relatively low copy number of this target may in Some cases, depending on other fore, preferred embodiments of the present invention show factors such as epitope position, the receptor density of the high affinity binding to their antigens, with EC50s preferably IFN receptors, etc, limit the potency of the antibody-attenu below 50 nM, more preferably below 25 nM, still more ated IFN fusion protein constructs. It is, however, possible preferably below 10 nM and ideally below 5 nM. In addi that other antibodies that target other epitopes on this antigen tion, preferred embodiments comprise antibodies that bind may support the targeted IFN activity, or that other cells with to protein and peptide epitopes rather than carbohydrate low copy numbers of CD33 may nevertheless respond to epitopes. such fusion protein constructs due to higher intrinsic IFN sensitivity, for example. Despite this possibility it is pre 0176 Multiple myeloma is of particular interest for cer ferred that the antibody or antigen binding portion thereof of tain embodiments of the present invention, namely fusion the polypeptide construct of the present invention binds an protein constructs comprising antibodies to multiple antigen wherein the antigen is present on the cell at a density myeloma antigens and attenuated IFN peptides. Table 3 lists of greater than 12,600 copies per cell, preferably greater examples of multiple myeloma antigens and antibodies, with than 15,000 copies per cell. a reference 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 & Patent #7,666.422 NCT00525.447 CD40 Luca tumumab HCD-122 USPTOH2O07OO98718 NCTOO231166 CHIR12.12 HM124 XmAb5592 humanized + Fc USPTOH2O100104.557 1999, Ozaki, Blood, 93: 3922 CDS6 HN901-DM1 994, Roguska et al., NCTOO3462SS & BB-10901 PNAS 91: 969-973 NCTOO991S62 CS1 Elotuzumab HuLuc63 USPTO Granted NCTOO742560 Patent #7,709,610 &NCTOO726869 CD138 BTO62 USPTO #2009.0175863 2008, Tassone, Blood, 104:3688 CD74 Milatuzumab Immu-110 US. Granted Patentii NCT00421525, Stein 7,312,318 et. Al. 2007 and 2009 IL-6R Tocilizumab MRA US Granted Patent 2007, Yoshio #5,795,965 Hoshino, Canc Res, 67; 871 Trail-R1 Mapatumumab, anti-DR4 US Granted Patent # NCT00315757 7,252,994 Trail-R2 (DR5, Lexatumumab, ETR2-STO1, US Granted Patent # 2006, Menoret, APO-2) anti-DR5 6,872,568 Blood, 132; 1356 Baff Belimumab LY2127399 US Granted Patent # 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 1D09C3 USPTO Granted 2007, Carlo-Stella, Pantent # 7,521,047 Canc. Res., Kininogen C11C1 USPTO Granted 2006, Sainz, Canc Patent # 4,908,431 Immunol Immunother 32microglobulin ATCC Cat iHB-149 2007, Yang, Blood, 110: 3028; 2009, Clin Can Res, 15: 951 FGFR3 Pro-OO1 USPTO Granted 2006, Trudel, Blood, Patent # 8,187,601 2: 4908 ICAM-1 cUV3 USPTO Granted 2004, Smallshaw, J Patent # 7,943,744 Immunother; 2006, Coleman Matriptase M24-DOX USPTO Granted 2010, Bertino, AACR Patent #7,355,015 abstract no. 2596 CD2O Rituxan and others U.S. Patent NCTOO2S82O6 & Application Number: NCT00505895 US 2010.O189729 A1 US 2016/0367695 A1 Dec. 22, 2016 17
TABLE 3-continued Examples of Ab in preclinical or clinical Clinical trial Target development Sequence citation reference CD52 Campath-1H USPTO Granted NCTOO62S144 Patent #6,569.430 EGFR Erbitux (Emma-1) USPTO Granted NCTOO3681.21 Patent #6,217,866 GM2 BIW-8962 USPTO Granted Biowa, no ref Patent # 6,872,392 C4-integrin natalizumab USPTO Granted NCTOO67S428 Patent # 5,840,299 IGF-1R CD-751,871 figitumumab USPTO Granted Lacy, J. Clin. Oncol, Patent # 7,700,742 26: 31.96 (TBD - need to connect Ab 4.9.2 to CD751,871) KIR IPH2101 USPTO Granted NCT00552396; Patent # 8,119,775 2009, ASCO abs. 09 AB-3032;
0177. CD38 is of particular interest as an antibody target 0179 Non-limiting examples of suitable cytokines for fusion protein constructs of the present invention. Anti include the interleukin’s IL-1, IL-2 IL-3, IL-4, IL-5, IL-6, bodies to CD38 include for example, AT13/5 (see, e.g., Ellis IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, et al. (1995) J. Immunol. 155: 925-937), HB7, and the like. IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, Table 4 discloses several known CD38 antibodies that may IL-24, IL 25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, be used in this context: IL-32, IL-33, 11-35 and their subfamiles; the interferon TABLE 4 Company Clone names Sequence citation Ref Genmab, GOO3. GOO5, GO24 WO 2006/099875 A1 De Weers, M., J Janssen Biotech (Daratumumab) Immunol. Inc 186: 1840, 2011 MorphoSys AG MORO.3077, US 2009.O123950 A1 MORO3079, MORO3O80, MORO3100 (MOR202) Sanofi-Aventis 38SB13, 38SB18, US 2009,030471.0 A1 US. LLC. 38SB19, 38SB30, 38SB31, 38SB39 (SAR650984) Tenovus UK Chimeric OKT10 US 2010.028SOO4 A1 Stevenson, F., Parental hybridoma Blood. 77: 1071, ATCC accession: CRL- 1991 8022 Immunogen HB7-Ricin Hybridoma: ATCC HB- Goldmacher, V., 136 Blood, 84: 3017, 1994
0.178 The term “Signaling ligand’ as used herein broadly (IFN) subfamily including Interferon type I (IFN-C. (IFNA1, includes any ligand involved in the activation of cell sig IFNA2, IFNA4, IFNA5, IFNA6. IFNA7, IFNA8, IFNA10, naling pathways, including any molecule capable of acti IFNA13, IFNA14, IFNA16, IFNA17, IFNA21), IFN-B vating or inhibiting cell surface receptors. The term should (IFN-B1 (IFNB1) and IFN-B3 (IFNB3)), IFN-co ((IFNW1), also be understood as including reference to molecules that IFNWP2, IFNWP4, IFNWP5, IFNWP9, IFNWP15, can pass through the lipid bilayer of the cell membrane to IFNWP18, and IFNWP19 and IFNK), Interferon type II activate cell signaling pathways within the cell. The term (IFN-Y) and Interferon type III (IFN-epsilon, -kappa, "polypeptide signaling ligand’ as used herein refers to -omega, -delta, -tau, and -gamma) and interferon-like mol peptide and polypeptide sequences of length 6 amino acids ecules (limitin, IL-28A, IL-28B, and IL-29; the IL-1 family through 1,000 amino acids in length, which bind to particu including IL-1C., IL-1B, the IL-1 Receptor antagonist (IL lar cell surface molecules (“receptors') on certain cells and 1RA) and IL1F5, IL1F6, IL1F7, IL1F8, IL1F9 and IL1F10 thereby transmit a signal or signals within those cells. and the IL-17 family including IL-17A, IL-17B, IL-17C, Exemplary signaling ligands and polypeptide signaling IL-17D, IL-17E (IL-25), and IL-17F. In an embodiment the ligands contemplated by the present invention include, but peptide or polypeptide signaling ligand is selected from the are not limited to cytokines, chemokines, growth factors, group consisting of an IFN, IL-4 and IL-6. In an embodi hormones, neurotransmitters, and apoptosis inducing fac ment the peptide or polypeptide signaling ligand is selected tOrS. from the group consisting of IFNC, IFNC2b. IFNB1, US 2016/0367695 A1 Dec. 22, 2016
IFNB1b and IFNY. Preferably the sequence of IFNC is genetic fusion. Methods for conjugating the ligands selected from SEQ ID NOs 1 to 3, 80 to 90, 434 and 435. described herein with antibodies may be readily accom 0180 Exemplary chemokines include, for example, plished by one of ordinary skill in the art. As will be readily RANTES, MCAF, MIP1-alpha, IP-10, monocyte chemoat ascertained, commonly used chemical coupling methods tractant protein-1 (MCP-1 or CCL2), interleukin-8 (IL-8), may be utilized to link ligands to antibodies via for example, CXCL13, XCL1 (lymphotactin-a), XCL2 (lymphotactin-3) free amino, carboxylic acid, or Sulfhydryl groups. Ligands and fractalkine (CXCL1). can also be linked to antibodies via Carbonyls ( CHO); 0181. Non-limiting examples of growth factors include, these aldehyde groups can be created by oxidizing carbo for example, Adrenomedullin (AM), Angiopoietin (Ang), hydrate groups in glycoproteins. Autocrine motility factor, Bone morphogenetic proteins 0187. Some commonly used cross-linking reagents (BMPs), Brain-derived neurotrophic factor (BDNF), Epi include glutaraldehyde which links protein or peptide mol dermal growth factor (EGF), Erythropoietin (EPO), Fibro ecules to the N-terminal or aliphatic amine groups of pep blast growth factor (FGF), Glial cell line-derived neuro tides or polypeptides, carbodiimide (EDC) which attaches trophic factor (GDNF), Granulocyte colony-stimulating proteins or peptides to the C-terminus or side chain carboxyl factor (G-CSF), Granulocyte macrophage colony-stimulat groups of proteins or peptides, Succinimide esters (e.g. ing factor (GM-CSF), Growth differentiation factor-9 MBS, SMCC) which conjugates free amino groups and (GDF9), Hepatocyte growth factor (HGF), Hepatoma-de thiols from Cys residues, benzidine (BDB) which links to rived growth factor (HDGF). Insulin-like growth factor Tyr residues, periodate which attaches to carbohydrate (IGF), Migration-stimulating factor, Myostatin (GDF-8), groups and isothiocyanate. The use of commercial chemical Nerve growth factor (NGF) and other neurotrophins, Plate conjugation kits is contemplated. let-derived growth factor (PDGF). Thrombopoietin (TPO), 0188 In some embodiments, labels are attached via Transforming growth factor alpha (TGF-C.), Transforming spacer arms of various lengths to reduce potential steric growth factor beta (TGF-B), Tumor necrosis factor-alpha hindrance. For example, a chemical linker may be used (TNF-C.), Vascular endothelial growth factor (VEGF), pla between the ligand and the antibody. Exemplary linker cental growth factor (P1GF), IL-1-Cofactor for IL-3 and sequences will be readily ascertained by those of skill in the IL-6, IL-2-T-cell growth factor, IL-3, IL-4, IL-5, IL-6 and art, and are likely to include linkers such as C6, C7 and C12 IL-7. amino modifiers and linkers comprising thiol groups. 0182 Exemplary apoptosis inducing factors include FasL 0189 The antibody-ligand fusion protein constructs of and TRAIL. the present invention have mutations or deletions in the 0183 Exemplary hormones include peptide hormones ligand that render the ligands less active in stimulating their Such as TRH and vasopressin, protein hormones Such as receptors on cells that lack cell Surface expression of the insulin and growth hormone, glycoprotein hormones such as antigen to which the antibody binds. Luteinizing hormone, follicle-stimulating hormone and thy 0190. In one aspect of the present invention, the ligand is roid-stimulating hormone, Lipid and phospholipid-derived an interferon, examples of which are type I interferons hormones such as steroid hormones e.g. testosterone and (IFN-C. (alpha), IFN-? (beta), IFN-K (kappa), IFN-ö (delta), cortisol, Sterol hormones such as calcitriol, eicosanoids Such IFN-e (epsilon), IFN-T (tau), IFN-() (omega), and IFN as prostaglandins. (Zeta, also known as limitin), type II interferons (IFN-Y) or 0184. Non-limiting examples of suitable neurotransmit type III interferons (IFN-1, IFN-2 and IFN-3) (Pestka, ters includemonoamines and other biogenic amines: dop Immunological Reviews 202(1):8-32, 2004). amine (DA), norepinephrine (noradrenaline; NE, NA), epi 0191 Type I interferons all signal through the Type I nephrine (adrenaline), histamine, serotonin (SE, 5-HT), interferon receptor, which is made of IFNAR1 and IFNAR2. Somatostatin, Substance P, opioid peptides and acetylcholine Signaling occurs when a type I IFN binds IFNAR1 and (ACh), IFNAR2, thus bringing them together into a complex with 0185. The linkage between the antibody and the ligand the IFN. This initiates a cascade of intracellular events (the could be made via a simple peptide bond by creating a fusion “signaling') which leads, among other things, to changes in protein between the ligand and the heavy or light chain, or the expression of numerous interferon regulated genes. both, of the antibody. The ligand could be attached at either Details of the intracellular signaling events triggered by the N- or C-terminus of either the heavy or the light chain activation of the type I interferon receptor is described, for of the antibody, with or without an intervening linker peptide example, by Platanias, (Nature Reviews 5:375-86. 2005). sequence. In an embodiment the ligand is linked to the Type I interferons include various interferon-alphas. Known antibody or antigen binding portion thereof via a peptide human interferon-alphas are bond. In one embodiment, the ligand is linked to the (0192 IFNC.1b, C2C, C2fB, C4b, C.5, C6, C7, C8, C.10, C-terminus of the heavy chain of a human, humanized or C.1a/13, C. 14, C.16, C17, Ovö C.21, C.2c and C4a. Some chimeric IgG1, IgG2 or IgG4, either directly or with an embodiments comprise IFNC.2b, the sequence of which, intervening linker of 1 to 20 amino acids in length. SEQ ID NO:3, is shown in FIG. 4a. IFNs have been 0186 The mutated polypeptide ligands may be attached approved in several forms for several indications, as out to the antibody or antibody fragment by means of chemical lined in Table 5 (which also shows lists of approved IFNB conjugation, non-covalent protein-protein interactions, or by and Ys): TABLE 5 Generic Name Trade name Approved for treatment Interferon alpha 2a ROFERON (RA (Hoffman-La Hep C, CML, Hairy cell Roche Inc., Nutley, NJ) Leukemia, NHL, Kaposi's S8CO8. US 2016/0367695 A1 Dec. 22, 2016 19
TABLE 5-continued Generic Name Trade name Approved for treatment interferon alpha 2b Intron A Reliferon/Uniferon Hep C, Hep B, Hairy cell, melanoma, leukemia, NHL, Kaposi's sarcoma Human leukocyte Interferon MULTIFERON (R) (Viranative AB, Melanoma, viral and (HuIFN-D-Le) Umea Sweden) malignant disease interferon beta 1a, liquid REBIF (R) (Ares Trading Multiple Sclerosis S.A., Aubonne Switzerland) interferon beta1a, AVONEX (R) (Biogen, Inc., Multiple Sclerosis yophylized Cambridge, MA) interferon beta1a, Cinnovex Multiple Sclerosis biogeneric (Iran) interferon beta1b BETASERON (R)/Betaferon (Bayer Multiple Sclerosis Pharma Aktiengesellshaft, Berlin Germany) interferon beta1b, Ziferon Multiple Sclerosis biosimilar (Iran) PEGylated interferon alpha PEGASYS (R) (Hoffman-La Roche Hepatitis B and C 2a nc., Nutley, NJ) PEGylated interferon alpha Reiferon Retard Hep C, Hep B, Hairy cell, 2a (Egypt) melanoma, leukemia, NHL, Kaposi's sarcoma PEGylated interferon alpha PEGINTRON (R) (Merck Sharpe & Hepatitis and melanoma 2b Dome Corp., Kenilworth, NJ) PEGylated interferon alpha Pegetron Hepatitis C 2b plus ribavirin (Canada) interferon alfacon-1 NFERGEN (R) (Amgen, Inc., Hepatitis C Thousand Oaks, CA) interferon alpha n3 ALFERON N (R) (Hemispherx Genital warts Biopharma, Inc., Philadelphia, PA) interferon gamma ACTIMMUNE (R) (Genentech, Chronic granulomatous Inc., San Francisco, CA) disease
0193 Non-limiting examples of mutations in IFNC2b TABLE 6-continued that can be used to reduce its potency are described in Tables 6 and 7, based on the sequence of human IFNC2b (SEQ ID Relative biological activities of interferon mutants NO:3): relative anti relative anti-viral proliferative TABLE 6 activity activity - Relative biological activities of intereon mans SA 9. 89.7 relative anti- SA I al relative anti-viral proliferative activity activity H57Y, E58N, Q61S, L30A O.34 O.13 H57Y, E58N, Q61S, R33A O.O73 O.OO82 FNC2b wild type 1 1 H57Y, E58N, Q61S, M148A O.45 O.94 L15A O.O79 O.29 H57Y, E58N, Q61S, L153A 1.06 2.3 R22A O.9 N65A, L80A, Y85A, Y89A O.O12 O.OOO9 R23A 0.4 O49 N65A, L80A, Y85A, Y89A, D114A O.019 O.OOOS S2SA O.76 0.7 N65A, L80A, Y85A, Y89A, L117A O.OOO3 TABLE 7-continued (0198 Slutzki, Michal, Jaitin, Diego A. Yehezkel, Tuval Ben, Schreiber, Gideon (2006). Variations in the unstruc Relative affinity of interferon mutants to their receptors tured C-terminal tail of interferons contribute to differen tial receptor binding and biological activity. J. Mol. Biol. 360: 1019-1030. 0199 Kalie, Eyal, Jaitin, Diego A., Abramovich, Renne, Schreiber, Gideon (2007). An interferon C.2 mutant opti mized by phage display for IFNAR1 binding confers specifically enhanced antitubor activities. J. Biol. Chem. 282: 116O2-11611. 0200 Pan, Manjing, Kalie, Eyal, Scaglione, Brian J., Raveche, Elizabeth S., Schreiber, Gideon, Langer, Jerome A. (2008). Mutation of to IFNAR-1 receptor binding site of human IFN-C2 generates Type I IFN competitive antagonists. Biochemistry 47: 12018-12027. 0201 Kalie, Eyal, Jaitin, Diego A., Podoplelova, Yulia, Piehler, Jacob, Schreiber, Gideon (2008). The Stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities. J. Biol. Chem. 283: 32925-32936. 0202 The abbreviation “YNS” is sometimes used herein to represent IFNC. variants including the following mutation: H57Y, E58N and Q61S. 0203 The present invention also contemplates combina tions of the abovementioned mutations or deletions in IFNC. 0204 The invention also contemplates the combination of the constructs of the present invention with other drugs and/or in addition to other treatment regimens or modalities such as radiation therapy or surgery. When the contructs of the present invention are used in combination with known therapeutic agents the combination may be administered either in sequence (either continuously or broken up by periods of no treatment) or concurrently or as an admixture. In the case of cancer, there are numerous known anticancer agents that may be used in this context. Treatment in combination is also contemplated to encompass the treat ment with either the construct of the invention followed by a known treatment, or treatment with a known agent fol N65A, L80A, Y85A, Y89A, R120A ND lowed by treatment with the construct of the invention, for Y85A, Y89A, R120A O.91 example, as maintenance therapy. For example, in the treat D114A, R120A O.83 L117A, R120A 1.4 ment of cancer it is contemplated that the constructs of the L117A, R120A, K121A O.14 O.91 present invention may be administered in combination with R120A, K121A 1.7 an alkylating agent (Such as mechlorethamine, cyclophos R120E, K121E 1.3 phamide, chlorambucil, ifosfamidecysplatin, or platinum A(161-165) O.S3 containing alkylating-like agents such as cysplatin, carbo platin and oxaliplatin), an antimetabolite (such as a purine or 0194 These mutants have known reductions in binding pyrimidine analogue or an antifolate agent, Such as azathio to the type 1 interferon receptor IFNAR1 or IFNAR2, and/or prine and mercaptopurine), an anthracycline (such as have known reductions in IFNC. potency based on cell-based Daunorubicin, Doxorubicin, Epirubicin Idarubicin, Valrubi assayS. cin, Mitoxantrone, or anthracycline analog), a plant alkaloid (such as a vinca alkaloid or a taxane, Such as Vincristine, 0195 The data in these tables was disclosed in the Vinblastine, Vinorelbine, Vindesine, paclitaxel or following references: Dosetaxel), a topoisomerase inhibitor (Such as a type I or 0196. Piehler, Jacob, Roisman, Laila C., Schreiber, type II topoisomerase inhibitor), a Podophyllotoxin (such as Gideon (2000). New structural and functional aspects of etoposide or teniposide), or a tyrosine kinase inhibitor (Such the Type I interferon-receptor interaction revealed by as imatinib mesylate, Nilotinib, or Dasatinib). comprehensive mutational analysis of the binding inter 0205. In the case of the treatment of multiple myeloma, face. J. Biol. Chen. 275: 40425-40433. it is contemplated that the constructs of the present invention 0.197 Jaitin, Diego A, Roisman, Laila C, Jaks, Eva, may be administered in combination with current therapies, Gavutis, Martynas, Piehler, Jacob, Van der Heyden, Jose, Such as steroids such as dexamethasone, proteasome inhibi Uze, Gilles, Schreiber, Gideon (2006). Inquiring into the tors (such as bortezomib or carfilzomib), immunomodula differential action of interferons (IFNs): an IFN-C2 tory drugs (such as thalidomide, lenalidomide or pomalido mutant with enhanced affinity to IFNAR1 is functionally mide), or induction chemotherapy followed by autologous similar to IFN-B. Mol. Cell. Biol. 26: 1888-1897. haematopoietic stem cell transplantation, with or without US 2016/0367695 A1 Dec. 22, 2016 other chemotherapeutic agents such as Melphalan hydro (APL), myelodysplastic syndrome, chronic myelogenous chloride or the chemotherapeutic agents listed above. leukemia (CML), mycosis fungoides and multiple myeloma 0206. In the case of the treatment of Hodgkin’s lym (reviewed in Smith, M. J. Clin. Oncol. 10:839, 1992). These phoma, it is contemplated that the constructs of the present studies led to the approval of ATRA for the treatment of invention may be administered in combination with current APL. Currently there are over 100 clinical trials evaluating therapeutic approaches, such as ABVD (Adriamycin (doxo the activity of ATRA in combination with other therapies for rubicin), bleomycin, vinblastine, and dacarbazine), or Stan the treatment of hematological malignancies, kidney can ford V (doxorubicin, bleomycin, vinblastine, Vincristine, cers, lung cancers, squamous cell carcinomas and more. Of mechlorethamine, etoposide, prednisone), or BEACOPP particular interest and pertaining directly to this invention (doxorubicin, bleomycin, Vincristine, cyclophosphamide, are the studies demonstrating enhanced efficacy of inter procarbazine, etoposide, prednisone). feron-C. treatment when combined with ATRA. This is 0207. In the case of non-Hodgkin’s lymphoma or other described for mantle cell lymphoma (Col. J. et al. Cancer lymphomas, it is contemplated that the constructs of the Res. 72:1825, 2012), renal cell carcinoma (Aass, N. et al. J. present invention may be administered in combination cur Clin. Oncol. 23:4172, 2005: Motzer, R. J. Clin. Oncol. rent therapeutic approaches. Examples of drugs approved 18:2972, 2000), CML, melanoma, myeloma and renal cell for non-Hodgkin lymphoma include Abitrexate (Methotrex carcinoma (Kast, R. Cancer Biology and Therapy, 7:1515, ate), Adriamycin PFS (Doxorubicin Hydrochloride), Adri 2008) and breast cancer (Recchia, F. et al. J. Interferon amycin RDF (Doxorubicin Hydrochloride), Ambochlorin Cytokine Res. 15:605, 1995). We would therefor predict (Chlorambucil), Amboclorin (Chlorambucil), Arranon enhanced activity of our targeted attenuated IFNs when (Nelarabine), Bendamustine Hydrochloride, Bexxar (Tosi combined with therapeutic dosing of ATRA in the clinic. In tumomab and Iodine I 131 Tositumomab), Blenoxane (Bleo addition, Mehta (Mol Cancer Ther 3(3):345-52, 2004) dem mycin), Bleomycin, Bortezomib, Chlorambucil, Clafen (Cy onstrated that in vitro treatment of leukemia cells with clophosphamide), Cyclophosphamide, Cytoxan retinoic acid induced expression of CD38 antigen. Thus, the (Cyclophosphamide), Denileukin Diftitox, DepoCyt (Lipo enhanced efficacy of interferon plus the induced expression somal Cytarabine), Doxorubicin Hydrochloride, DTIC of the target CD38 would indicate a combination therapy of Dome (Dacarbazine), Folex (Methotrexate), Folex PFS ATRA with our anti-CD38 antibody-attenuated IFNC. in the (Methotrexate), Folotyn (Pralatrexate), Ibritumomab Tiux treatment of IFN-sensitive cancers that express CD38 or etan, Istodax (Romidepsin), Leukeran (Chlorambucil), Lin may be induced by ATRA to express CD38. Example of such folizin (Chlorambucil), Liposomal Cytarabine, Matulane cancers are multiple myeloma, non-Hodgekin’s lymphoma, (Procarbazine Hydrochloride), Methotrexate, Methotrexate CML and AML. LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ 0209. In addition, while the above constructs are based (Methotrexate), Mozobil (Plerixafor), Nelarabine, Neosar on IFNC2b, the mutations or deletions could also be made (Cyclophosphamide), Ontak (Denileukin Diftitox), Plerixa in the context of any of the other IFNC.s or IFNB. In another for, Pralatrexate, Rituxan (Rituximab), Rituximab, embodiment of the present invention, the type I IFN is an Romidepsin, Tositumomab and Iodine I 131 Tositumomab, IFNB. IFN-B is approved for the treatment of multiple Treanda (Bendamustine Hydrochloride), Velban (Vinblas sclerosis (MS). IFN-B could be attenuated by mutation or tine Sulfate), Velcade (Bortezomib), and Velsar (Vinblastine deletion and then attached to an antibody that targets cells Sulfate), Vinblastine Sulfate, Vincasar PFS (Vincristine Sul involved in the pathogenesis of this disease. IFN-?3 is an fate), Vincristine Sulfate, Vorinostat, Zevalin (Ibritumomab effective drug in MS, but its use is associated with adverse Tiuxetan), Zolinza (Vorinostat). Examples of drug combi events, including injection site inflammation, flu-like symp nations used in treating non-Hodgkin lymphoma include toms, leukocytopenia, liver dysfunction and depression, CHOP (C=Cyclophosphamide, H=Doxorubicin Hydrochlo leading to discontinuation in a Subset of patients. By direct ride (Hydroxydaunomycin), O=Vincristine Sulfate (On ing IFN-B activity directly to pathogenic cells, these adverse covin), P=Prednisone); COPP (C=Cyclophosphamide, events may be avoided. O=Vincristine Sulfate (Oncovin), P=Procarbazine Hydro 0210 Pathogenesis of MS is thought to be initiated and chloride, P=Prednisone); CVP (C=Cyclophosphamide, progressed by a number of events, including innate activa V=Vincristine Sulfate, P=Prednisone); EPOCH tion of dendritic and microglial cells through toll-like recep (E=Etoposide, P=Prednisone, O=Vincristine Sulfate (On tors, an imbalance between pro-inflammatory and anti covin), C=Cyclophosphamide, H=Doxorubicin Hydrochlo inflammatory/regulatory cytokines, differentiation of CD4+ ride (Hydroxydaunomycin)); ICE (I-Ifosfamide, T cells into Th1 and Th17 phenotypes, activation of Th1 C=Carboplatin, E=Etoposide) and R-CHOP (R-Rituximab, cells by antigen presenting cells (APCs), reduction in the C=Cyclophosphamide, H-Doxorubicin Hydrochloride (Hy number of regulatory T (Treg) cells and migration of acti droxydaunomycin), O=Vincristine Sulfate (Oncovin), vated immune cells across the blood-brain barrier (BBB). P=Prednisone. The primary drivers of the clinical episodes of the disease 0208 Combination of retinoids with interferon-based are thought to be autoreactive, myelin-specific Th1 cells fusion protein constructs is also contemplated. Retinoids are (reviewed in Gandhi, 2010 J Neuroimmunol 221:7: Bop a family of molecules that play a major role in many pana, 2011 Mt Sinai J Med 78:207; Loma, 2011 Curr biological functions including growth, vision, reproduction, Neuropharmacol 9:409). epithelial cell differentiation and immune function (Mey 0211. In an embodiment of the invention, an attenuated skens, F. et al. Crit Rev. Oncol Hematol 3:75, 1987, Herold, version of IFN-?3 may be attached to an antibody targeting M. et al. Acta Dermatovener 74:29 1975). Early preclinical a cell surface marker specific for T cells, for the treatment of studies with the retinol all-trans retinoic acid or ATRA, multiple Sclerosis or other autoimmune indications where either alone or in combination with other agents, demon IFN-B may be effective. Direct effects of IFN-B on T cells strated activity against acute promyelocytic leukemia include inhibition of proliferation (Rep., 1996 J Neuroim US 2016/0367695 A1 Dec. 22, 2016 22 munol 67:111), downregulation of the co-stimulatory mol with an anti-CD8 antibody may result in clinical benefits for ecule CD40L (Teleshova, 2000 Scand J Immunol. 51:312), MS patients. Examples of CD8 antibodies are shown in decrease of metaloproteinase activity leading to reduced Table 10. migration across the BBB (Stuve, 1996 Ann Neurol 40:853; Uhm, 1999 Ann Neurol 46:319), induction of apoptosis by TABLE 10 upregulating intracellular CTLA-4 and cell Surface Fas molecules (Hallal-Longo, 2007 J Interferon Cytokine Res CD8 Antibodies 27:865), downregulation of anti-apoptotic proteins (Sharief, 2001 J Neuroimmunol. 120:199: Sharief, 2002 J Neuroim Ab Clones Patent Assignee Comments munol. 129:224), and restoration of Treg function (De 37B1, 8G6 U.S. Pat. No. Ortho-McNeil Hybridomas Andres, 2007 J Neuroimmunol 182:204; Korporal, 2008 7,247,474 Pharmaceutical, Inc. deposited at ATCC Arch Neurol 65:1434; Sarasella, 2008 FASEB J 22:3500; (HB-12441, HB Chen, 2012 J Neuroimmunol 242:39). 12657) 0212. Therefore, in one aspect of the present invention, OKT8 U.S. Pat. No. Ortho Hybridoma an attenuated IFN-B is attached to an anti-CD3 antibody that 4,361,550 Pharmaceutical deposited at ATCC targets all T cells, which includes CD4+, CD8+, Treg, Th1. Corporation (CRL-8014) Th2 and Th17 cells. This comprehensive approach ensures Several US2009, Baylor Research full coverage of all T cells, as all of these cell types have examples O3O4659 Institute reported roles in MS pathogenesis and are affected by IFN-B treatment (Dhib-Jalbut, 2010 Neurology 74:S17: Prinz, 2010 Trends Mol Med 16:379; Graber, 2010 Clin Neurol 0215 Markers of activated T cells, including, but not Neurosurg 112:58 and Loma, 2011 Curr Neuropharmacol limited to CD25, CD38, CD44, CD69, CD71, CD83, CD86, 9:409). Examples of CD3 antibodies that may be incorpo CD96, HLA-DR, ICOS and PD-1, also represent attractive rated into the fusion protein constructs of the present inven targets for this approach, since activated T cells are thought tion are listed in Table 8. to be the main drivers of autoreactivity resulting in demy elination in MS (Gandhi, 2010 J Neuroimmunol 221:7; TABLE 8 Boppana, 2011 Mt Sinai J Med 78:207; Loma, 2011 Curr CD3 Antibodies Neuropharmacol 9:409). Antibodies targeting any of these antigens could be attached to an attenuated IFNB. Examples Ab Clones Patent Assignee Comments antibodies that could be used in the present invention TFNSO U.S. Pat. No. 7,994,289 BTG International Humanized include the following: CD71 antibodies include BA120g CD3A122 (U.S. Pat. No. 7,736,647) and various antibodies mentioned M291 U.S. Pat. No. 7,381,803 PDL BioPharma Humanized in Wang et at (Di Yi Jun Yi Da Xue Xue Bao (Academic 28F1, 27H5, U.S. Pat. No. 7,728,114 Novimmune S.A. Human 23F10, 15C3 journal of the first medical college of PLA) 22(5):409–411, 2002). Examples of antibodies to CD83 include 20B08, 6G05, 20D04, 11G05, 14C12, 96G08 and 95F04 (U.S. Pat. 0213 Alternatively, an attenuated IFN-B-anti-CD4 No. 7,700,740). An example of an antibody to CD86 fusion protein construct presents a more restrictive approach, but would target autoreactive and regulatory T includes 1G10H6D10 (U.S. Pat. No. 6,071,519). HLA-DR cells, including Th1 and Th17 cells and CD4"CD25 Treg antibodies include HD3, HD4, HD6, HD7, HD8 and HD 10 cells. In addition, subsets of dendritic cells (DCs) also (U.S. Pat. No. 7,262,278), DN1921 and DN1924 (US2005/ express CD4 and direct therapeutic effects of IFN-B on DCs 0208048). One attractive target along these lines could be have been disclosed (Shinohara, 2008 Immunity 29:68; PD-1, which is expressed on recently activated T cells. Dann, 2012 Nat Neurosci 15:98). Examples of CD4 anti Ideally, a non-antagonizing antibody could be used, such as bodies that may be incorporated into the fusion protein the J110 antibody discussed in further detail below. constructs of the present invention are listed in Table 9. 0216 Examples of antibodies to ICOS include JMabs (U.S. Pat. No. 6,803,039) and JMab 136 (US2011/0243929). TABLE 9 0217. Further of these examples of antibodies to these CD4 Antibodies targets are shown in the Tables 11 and Ab Clones Patent Assignee Comments TABLE 11 CE9.1 U.S. Pat. No. 7,452,534 Biogen Idec Non-human primate CD25 Antibodies variable regions Ab Clones Patent Assignee Comments TRX1 U.S. Pat. No. 7,541,443 Tolerrx Humanized 1E11, 1G2, 6G5, U.S. Pat. No. 8,231,877 GenPharm Human Anti-tac Abs U.S. Pat. No. PDL, Inc daclizumab 5,530,101 RFTS U.S. Pat. No. Novartis AG Chimeric, inhibits 6,521,230 MLR 0214) A role for CD8" T cells in MS has been reported AB1, AB7, AB11, U.S. Pat. No. Genmab A/S Human antibodies, AB12 8,182,812 (or prevent CD25-IL-2 (Friese, 2005 Brain 128:1747: Friese, 2009 Ann Neurol U.S. Pat. No. interaction and 66:132), as well as a direct effect of IFN-Bon CD8+ T cells 7,438,907) inhibit MLR in MS patients (Zafranskaya, 2006 Immunol 121:29). There fore, directing an attenuated IFN-B directly to CD8" T cells US 2016/0367695 A1 Dec. 22, 2016 TABLE 12 antibodies that could be used according to the present invention include, but are not limited to DIVHv5/DIVKv2 CD44 Antibodies (U.S. Pat. No. 7,910,104), any of the CD52 antibodies disclosed in (US2012/0100152) or CAMPATH. Ab Clones Patent Assignee Comments 0220 Any of the above mentioned, antibody-targeted H90 US2007/0237761 Chimeric attenuated IFNB fusion protein constructs may have thera 1A9, 2D1, 14G9, US2010/0092484 Human peutic activity in the context of other inflammatory and autoimmune diseases beyond multiple Sclerosis, due to their SACK-1 U.S. Pat. No. 7,816,500 Sackstein Binds CD44 common underlying immunological etiologies. glycoforms 0221 Autoimmune diseases contemplated herein include inter alia alopecia areata, ankylosing spondylitis, antiphos pholipid syndrome, autoimmune Addison's disease multiple 0218. In another embodiment of the invention, an attenu Sclerosis, autoimmune disease of the adrenal gland, autoim ated version of IFN-B can be fused to an antibody targeting mune hemolytic anemia, autoimmune hepatitis, autoimmune cell surface markers of myeloid cells, known to contribute to oophoritis and orchitis, Behcet’s disease, bullous pemphig MS pathogenesis by driving T cell activation and differen oid, cardiomyopathy, celiac sprue-dermatitis, chronic tiation. For example, the pan-myeloid markers CD33, fatigue syndrome (CFIDS), chronic inflammatory demyeli CD115, or the dendritic cell marker CD11c may be targeted. nating, chronic inflammatory polyneuropathy, Churg A broad targeting approach may be preferred, for example, Strauss syndrome, cicatricial pemphigoid, crest syndrome, using antibodies against CD33 or CD115, since the exact cold agglutinin disease, Crohn's disease, irritable bowel contribution of each of the myeloid cell subsets to MS syndrome, inflammatory bowel disease, dermatitis herpeti disease pathogenesis and response to IFN-B has been dis formis, discoid lupus, essential mixed cryoglobulinemia, puted (Prinz, 2008 Immunity 28:675; Shinohara, 2008 fibromyalgia, glomerulonephritis, Grave's disease, Guillain Immunity 29:68; Dann, 2012 Nat Neurosci 15:98). Antibod Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibro ies to CD33 that could be used in the present invention sis, idiopathic thrombocytopenia purpura (ITP), IgA neph include My9-6 (U.S. Pat. No. 7,557, 189), any of 14 anti ropathy, insulin dependent diabetes (Type I), lichen planus, bodies described in US patent application US2012/0082670, lupus, Meniere's disease, mixed connective tissue disease, or the antibody known as huM195 (U.S. Pat. No. 5,693,761). multiple Sclerosis, myasthenia gravis, myocarditis, pemphi Antibodies to CD115 that could be used include Ab1 and guS Vulgaris, pernicious anemia, polyarteritis nodosa, poly Ab16 (U.S. Pat. No. 8,206,715) or CXIIG6 (US2011/ chondritis, polyglancular syndromes, polymyalgia rheu 0178278). An example of a CD11c antibody that could be matica, polymyositis and dermatomyositis, pochitis, used according to the present invention is mab 107 (U.S. Pat. primary agammaglobulinemia, primary biliary cirrhosis, No. 7,998,738, ATCC deposit number PTA-11614). The psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheu attenuated IFN-B could alternatively be directed to the CD14 matic fever, rheumatoid arrthritis, Sarcoidosis, Scleroderma, antigen, present primarily on macrophages. Examples of Sjogren's syndrome, stiff-man syndrome, systemic lupus CD14 antibodies are shown in Table 13. erythematosus, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis and Vit TABLE 13 iligo. Of particular interest is Behcet’s disease and chronic CD14 Antibodies uveitic macular edema and other types of uveitis, since IFNC. has been shown to render therapeutic benefit (Deuter, Dev Ab Clones Patent Assignee Comments Ophthalmol. 51:90-7. 2012) 4C1 U.S. Pat. No. 6,245,897 Seikagaku Mouse Ab 0222 Examples of inflammatory disease conditions con Corporation templated by the present disclosure include but are not F1024-1-3 U.S. Pat. No. 7,264,967 Mochida Humanized, limited to those disease and disorders which result in a Pharmaceutical inhibits response of redness, Swelling, pain, and a feeling of heat in Co. CD14iTLR certain areas that is meant to protect tissues affected by binding injury or disease. Inflammatory diseases which can be F1024, U.S. Pat. No. 8,252,905, Mochida Part of fusion F1031-13-2 US Pharmaceutical proteins with treated using the methods of the present disclosure, include, 2008 O28629O Co. protease without being limited to, acne, angina, arthritis, aspiration pneumonia, disease, empyema, gastroenteritis, inflamma tion, intestinal flu, NEC, necrotizing enterocolitis, pelvic 0219. In yet another embodiment, targeting CD52-ex inflammatory disease, pharyngitis, PID, pleurisy, raw throat, pressing cells would deliver IFN-B to all lymphocytes and, redness, rubor, Sore throat, stomach flu and urinary tract in addition, to monocytes and peripheral dendritic cells infections, chronic inflammatory demyelinating polyneu (Buggins, 2002 Blood 100:1715: Ratzinger, 2003 Blood ropathy, chronic inflammatory demyelinating polyradicu 101: 1422), which are the key APCs responsible for prolif loneuropathy, chronic inflammatory demyelinating polyneu eration and differentiation of autoreactive T cells in MS. ropathy, chronic inflammatory demyelinating This approach would direct the activity of IFN-B to the key polyradiculoneuropathy. cell types known to be directly affected by IFN-?3 and would 0223) The sequence of human interferon-?31 is shown facilitate its therapeutic activity in MS. Examples of CD52 below: (SEQ ID NO: 191) k k k k 1 MSYNLLGFLQ RSSNFOCOKL LWOLNGRLEY CLKDRMNFDI PEEIKOLOOF SO k k k 51 OKEDAALTIY EMLONIFAIF RODSSSTGWN ETIVENLLAN WYHOINHLKT 1 OO US 2016/0367695 A1 Dec. 22, 2016 24 - Continued k 101 WLEEKLEKED FTRGKLMSSL HLKRYYGRIL HYLKAKEYSH CAWTIWRWEI 150 k 151 LRNFYFINRL TGYLRN 166 0224. Using the numbering scheme above (residues associated antigens with mutated type I interferons, and that 1-166), known mutations (at positions indicated by aster the resulting fusion protein constructs may be used to reduce isks) in human IFNB that reduce its activity include those the proliferation of various interferon-sensitive cancers that listed in Table 14. express the corresponding tumor associated antigens. It will also be appreciated that type I interferons can be combined TABLE 1.4 with other agents to further improve their effectiveness. 0230 Type I interferons can also display anti-viral prop IFNB activity-attenuating mutations erties. IFNC.2b, for example, has been FDA-approved for the FNbeta Fold treatment of chronic hepatitis C infections, and may have mutations attenuation Reference utility in treating other viral infections as well. Pegylated IFN-Cl is currently part of the standard of care regimen for wild type 1 R27A 3.3 1 hepatitis C, according to American and European guidelines, R35A - C17S 28O 3 but results in side effects in over 80% of patients, often R35T 10 1 leading to discontinuation of treatment (Aman, 2012; Cal E42K >10 2 Varuso, 2011). In one aspect of the present invention, a type DS4N 1.4 2 M62I 8.7 2 IIFN with an attenuating mutation is attached to an antibody G78S 6.2 2 that binds to virally infected cells. The antigen to be recog K123 2.5 1 nized by the above referenced antibody could be a viral C141Y >25 2 protein that is transiently expressed on the host cell Surface, A142T >10 2 R147A C17S** 1.7 3 or it could be an endogenous host cell-produced antigen that E149K >5 2 is exposed on the cell Surface to a greater extent after viral R1S2H 4.7 2 infection than before infection. Exemplary viral proteins that *based on anti-proliferation activity could serve as targets for the antibody portion include but **the C17S mutation was made in order to remove the unpaired cysteine in the native are not limited to, Hepatitis C viral envelope glycoproteins, sequence of DFNB1 E1 and E2: Hepatitis B surface antigen (HBSAg); Herpes virus viral envelope glycoproteins B, C, D, E, G, H, I, J, K, REFERENCES L, M and UL32, and envelope protein UL49A. Human immune deficiency virus (HIV) Envelope proteins glyco 0225 (1) Runkel, L., Pfeffer, L., Lewerenz, M., protein (gp) 120 and gp41, Adenoviruses knob domain of Mogensen, K. (1998). Differences in Activity between C. the fiber protein; Varicella-Zoster virus envelope glycopro and B Type I Interferons Explored by Mutational Analy teins (gB, gC, gE. g. g. gK, gL); Epstein-barr virus viral Sis. J. Biol. Chem. 273: 8003-8008 glycoprotein gp350 and viral protein BMRF-2: Human 0226 (2) Stewart, A. G., Adair, J. R., Catlin, G., Hynes, cytomegalovirus UL 16: Parvovirus B19 viral capsid pro C., Hall, J., Dav ies, J., Dawson, K. & Porter, A. G. teins VP1-3: Human astrovirus structural proteins, e.g. (1987). Chemical mutagenesis of human interferon-beta: VP26, VP29 and VP32; Noroviruses structural protein VP1 construction, expression in E. coli, and biological activity and capsid protein VP2; Poliovirus viral capsid proteins of sodium bisulfite-induced mutations. DNA 6: 119-128. VP0, VP1, VP2, VP3 and VP4; Rhinovirus viral capsid 0227 (3) In-house results proteins VP1,VP2, VP3 and VP4; and dengue virus virus 0228. In still another embodiment of the present inven particle proteins capsid (C), pre-membrane/membrane tion, the IFN is IFN-J (WO 2007/029041 A2), which may be (prM/M) and envelope (E). used for any of the applications described more thoroughly 0231. In one embodiment, IFN-O. activity may be tar for IFNC, or IFN3. geted with an antibody that binds, directly or indirectly via 0229. Type IIFNs can have anti-cancer activity based on an intermediate protein Such as annexin V or beta2-glyco a direct stimulation of the type I IFN receptor on cancer protein 1, to phosphatidylserine (PS), a phospholipid com cells. This has been shown for numerous types of cancer ponent of the inner leaflet of cellular membranes. Cells including multiple myeloma, melanoma, B cell lymphoma, undergoing apoptosis, however, or cells infected with non-Small cell lung cancer, renal cell carcinoma, hairy cell viruses, expose PS on the outer membrane, where it leukemia, chronic myelogenous leukemia, ovarian cancer, becomes accessible to antibodies. PS is exposed on the fibrosarcoma, cervical cancer, bladder cancer, astrocytoma, surface of cancer cells (Reidl, L. et al., JImmunol. 14:3623, pancreatic cancer, etc (Borden, Cancer Research 42:4948 1991), the vascular endothelium in tumors (Ran, S. et al., 53, 1982: Chawla-Sarkar, Clinical Cancer Research 7: 1821 Cancer Res.62:6132. 2002: He, J. et al., Clin Cancer Res. 31, 2001; Morgensen, Int J. Cancer 28:575-82, 1981; 15:6871, 2009), and virus-infected cells (Soares, M. et al., Otsuka, British Journal of Haematology 103:518-529, 1998: Nat Med. 14:1357, 2008). An antibody indirectly (via beta2 Lindner, J of Interferon and Cytokine Research 17:681-693, glycoprotein 1) targeting PS, bavituximab, has been 1997: Caraglia, Cell Death and Differentiation 6:773-80, described. It mediates antibody-dependent cytotoxicity and 1999; Ma, World J. Gastroenterol 11(10): 1521-8, 2005). One is effective in a number of in vivo cancer models, including of skill in the art will recognize that the present invention has human breast and lymphoma xenografts and a rat glioblas many aspects resulting from combining antibodies to tumor toma model, as well as in viral disease models (Ran, S. et al., US 2016/0367695 A1 Dec. 22, 2016 Clin Cancer Res; 11:1551, 2005; He, J. et al., Clin Cancer attenuated IFNY using a 7 amino acid linker described by Res. 15:6871, 2009: Soares, M. et al., Nat Med. 14:1357, Sharifi to produce the fusion protein construct “Rituximab 2008). Currently, it is being developed as a therapeutic HC-L7-IFNY(ALA23.D24) IgG1,” composed of SEQ ID antibody for lung cancer treatment (DeRose, P. et al., NOS:378 (heavy chain) and 276 (light chain)). Such a fusion Immunotherapy. 3:933, 2011; Gerber, D. et al., Clin Cancer protein construct would be expected to have potent anti Res. 17:6888, 2011). Alternative antibodies may be based on proliferative activity against CD20 malignancies such as B the variable regions from the anti-PS antibody 9D2 (Cancer cell lymphomas. Other attenuated mutants of IFNY that may Res Nov. 1, 2002 62: 6132). Yet another alternative for be appropriate for fusing to a cell-targeting antibody were targeting PS would be to replace the antibody Fab portions described by Lundell (J Biol. Chem. 269(23):16159-62, with a natural PS-binding protein such annexin V or beta2 1994), namely S20I (-50x reduced affinity), D21 K (-100x glycoprotein 1. An anti-PS antibody (or alternatively a direct reduced affinity), A23Q (-2.500-fold reduced binding), or indirect PS binding protein) fused with an attenuated A23V (~2,000-fold reduced binding) and D24A (~4-fold version of IFN-C. would target IFN-C. activity to PS express reduced binding). These attenuated IFNY may be used as ing virus-infected cells without displaying the systemic fusions in combination with anti-CD38 antibodies, to gen safety issues related to IFN-C. Certain tumor cells, such as erate the fusion protein construct “X355/02-HC-L7-IFNY lung cancer cells, also express PS on their cell Surfaces, so (S20I) IgG1 (composed of SEQID NOS:380 (heavy chain) an antibody (or alternatively a direct or indirect PS binding and 226 (light chain)) or “R10A2-HC-L7-IFNY(D21K) protein) to PS, attached to an attenuated IFN, could also IgG1” (composed of SEQ ID NOS:382 (heavy chain) and have use in the treatment of certain cancers. 270 (light chain)). Other attenuating mutations in IFNY that 0232. It should be understood that antibody-targeted may be exploited for the current invention were described by attenuated IFN, could also be used in much the same way as Fish (Drug Des Deliv. 1988 February; 203):191-206.) IFNC. for the targeting virally infected cells (S. V. Kotenko, 0234 Targeted attenuated IFNY may also be used to treat G. Gallagher, V. V. Baurin et al., “IFN-us mediate antiviral various indications characterized by pathological fibrosis, protection through a distinct class II cytokine receptor including kidney fibrosis, liver fibrosis and idiopathic pull complex. Nature Immunology, vol. 4, no. 1, pp. 69-77. monary fibrosis (IPF). IPF is a chronic, progressive form of 2003). lung disease, characterized by fibrosis of unknown cause, 0233. In one embodiment Type II IFNs, namely INFy, occurring primarily in older adults. Despite the medical may also be attenuated and attached to antibodies that direct need, there has been little progress in the development of them to specific cell types. IFNY has anti-proliferative prop effective therapeutic strategies (O'Connell, 2011 Adv Ther erties towards cancer cells (Kalvakolanu, Histol. Histo 28:986). Pulmonary fibrosis can also be induced by expo pathol 15:523-37, 2000; Xu, Cancer Research 58:2832-7. Sure to drugs, particles, microorganisms or irradiation. The 1998; Chawla-Sarkar, Apoptosis 8:237-49, 2003: Schiller, J following relates to both IPF and lung fibrosis induced by Interferon Resarch 6:615-25, 1986). Sharifi has described known agents and potentially for treatment of fibrosis in how to make a fusion protein in which an IFNY has been other types of organs, including liver and kidney. fused to the C-terminus of a tumor-targeting antibody 0235 Fibroblasts play a key role in fibrotic diseases of (Sharifi, Hybridoma and Hybridomics 21(6):421-32, 2002). the lung and their activation leads to collagen disposition, In this reference, Sharifi disclosed how to produce antibody resulting in excessive Scarring and destruction of the lung IFNY fusion proteins in mammalian cells and showed that architecture. Yet there is little information on the origin of both the antibody and the IFN were functional. Alterna these pathogenic fibroblasts, though several precursor cell tively, a single-chain dimer version of IFNY, as described by types have been proposed, including bone marrow progeni Lander (J Mol Biol. 2000 May 26; 299(1): 169-79) may be tors, monocytes, circulating fibrocytes, and endogeneous used in the fusion protein. In addition to IFNY's anti cells, such as resident mesenchymal and epithelial cells proliferative effect on the targeted tumor cells, it may also (Stevens, 2008 Proc Am Thorac Soc 5:783; King, 2011 have another effect specifically on breast cancer cells: IFNY Lancet 378:1949). has been shown to restore antiestrogen sensitivity to breast 0236 CD14" monocytes from peripheral blood are able cancer cells (Mol Cancer Ther. 2010 May: 9(5): 1274-1285) to differentiate into fibrocytes, the precursors of fibroblasts, and so an attenuated-IFNY attached to a breast cancer and this process is inhibited by interferon-Y (IFN-Y). A direct antigen antibody may be therapeutically useful in combina effect of IFN-Y on monocytes was demonstrated in in vitro tion with antiestrogen therapy. By attenuating IFNY via differentiation studies, Supporting the strategy of targeting mutation, a more cancer-selective form of IFNY may be an attenuated form of IFN-Y to CD14" monocytes for the produced. Two attenuating mutations in IFNY have been treatment of fibrotic disease (Shao, 2008 J Leukoc Biol described by Waschutza (Eur J. Biochem. 256:303-9, 1998), 83: 1323). namely des-(A23, D24), in which residues A23 and D24 are 0237 Experimental evidence exists that IFN-Y is capable deleted, and des-(N25, G26), in which residues N25 and of inhibiting proliferation and activation of fibroblasts (Ro G26 are deleted. The des-(A23, D24) mutant has an ~18-fold gliani, 2008 Ther Adv Respir Dis 2:75) and this fact has reduced affinity for the IFNY receptor compared to wild type exploited Successfully in preclinical models to reduce scar IFNY, and had a ~100-fold reduced antiviral activity com ing and fibrosis. Clinical trials in IPF patients studying the pared to wild type IFNY. The des-(N25, G26) variant had a benefit of subcutaneously administered IFN-Y failed to reach ~140-fold reduced affinity for the IFNY receptor compared to primary endpoints for survival benefits (O'Connell, 2011 wild type IFNY, and had a ~10-fold reduced antiviral activity Adv. Ther 28:986: King, 2011). Current approaches focus on compared to wild type IFNY. Examples of fusion proteins direct delivery of recombinant IFN-y through inhalation of comprising antibodies to tumor cell Surface targets and an aerosol form (Diaz, 2012J Aerosol Med Pulm Drug Deliv attenuated mutants of IFNY include the following: Ritux 25:79), such that the lungs may achieve sufficient IFN-y imab may be used as fusion protein with one of these activity to produce benefit at an overall safe systemic dose. US 2016/0367695 A1 Dec. 22, 2016 26 0238. Delivering IFN-y activity directly to fibroblasts 2003). These IFNs act through receptors composed of the could be a powerful method to increase clinical response to IFNAR1 chain (also known as IL28RC.) and the IL10R2 this agent and at the same time reduce its side effects. Fusing chain (shared with IL 10, IL22, and IL26 receptor complexes attenuated IFN-Y to antibodies targeting fibroblast specific A. Lasfar, W. Abushahba, M. Balan, and K. A. Cohen-Solal, markers could facilitate this approach. There are several “Interferon lambda: a new sword in cancer immunotherapy.” fibroblast cell surface molecules that are enriched in fibro Clinical and Developmental Immunology, vol. 2011, Article blasts. These include, for example, fibroblast specific protein ID 349575, 11 pages, 2011). IFNARs are expressed on most (FSP1; Strutz, 1995 J Cell Biol 130:393), fibroblast activa cell types and mediate similar signalling pathways as the tion protein (FAP; Park, 1999 J Biol Chem 274:36505; type I IFNs. The antiviral activity of w IFNs has been Acharya, 2006 Hum Pathol 37:352), and platelet derived demonstrated against several viruses including HBV and growth factor receptors (PDGFR-C. and -3; Trojanowska, HCV (E. M. Coccia, M. Severa, E. Giacomini et al., “Viral 2008 Rheumatology (Oxford) 47S5:2). Expression of these infection and toll-like receptor agonists induce a differential molecules is elevated in lung biopsies obtained from IPF expression of type I and W interferons in humans plasma patients and they have been directly implicated as drug cytoid and monocyte-derived dendritic cells.' European targets in IPF or its pathogenesis (Lawson, 2005 Am J Respir Journal of Immunology, vol. 34, no. 3, pp. 796-805, 2004; Crit Care Med 171:899: Acharya, 2006 Hum Pathol 37:352: M. D. Robek, B. S. Boyd, and F. V. Chisari, “Lambda Abdollahi, 2005 J Exp Med201:925). Examples of antibod interferon inhibits hepatitis B and C virus replication.” ies to FAP and the PDGF receptors are shown in Tables 15 Journal of Virology, vol. 79, no. 6, pp. 3851-3854, 2005; N. and 16. Ank, H. West, C. Bartholdy, K. Eriksson, A. R. Thomsen, and S. R. Paludan, “Lambda interferon (IFN-w), a type III TABLE 1.5 IFN, is induced by viruses and IFNs and displays potent FAP Antibodies antiviral activity against select virus infections in vivo.” Journal of Virology, vol. 80, no. 9, pp. 4501-4509, 2006: S. Ab Clones Patent Assignee Comments E. Doyle, H. Schreckhise, K. Khuu-Duong et al., “Interleu MFP5, BIBH1 US2009/0304718 Boehringer Humanized kin-29 uses a type 1 interferon-like program to promote Ingelheim USA antiviral responses in human hepatocytes,” Journal of Hepa Corporation tology, vol. 44, no. 4, pp. 896-906, 2006: T. Marcello, A. Many US2012O128591 Bacac et al. Humanized Grakoui, G. Barba-Spaeth et al., “Interferons C. and inhibit F19 US2003/0143229 Boehringer Ingelheim hepatitis C virus replication with distinct signal transduction International GmbH and gene regulation kinetics.” Gastroenterology, Vol. 131, no. 6, pp. 1887-1898, 2006). Clinical studies with IFN) for the treatment of hepatitis C have shown promise (E. L. TABLE 16 PDGFR-C and - Antibodies Ab Clones Patent Assignee Comments 2.175.3, 2.499.1, U.S. Pat. No. 7,754,859 AstraZeneca AB Human abs against 2.998.2 PDGFRC IMC-3G3 US2O120027767 Imclone LLC Human abs against PDGFRC 2C5 US2O12.00221267 Imclone LLC Human abs against PDGFRB 0239. In a preclinical model of liver fibrosis, IFN-y was Ramos, “Preclinical and clinical development of pegylated delivered to hepatic stellate cells, the equivalent of fibro interferon-lambda 1 in chronic hepatitis C. Journal of blasts and responsible for secreting collagen in liver fibrosis, Interferon and Cytokine Research, Vol. 30, no. 8, pp. 591 through liposomes targeting PDGFR-B, thereby enhancing 595, 2010). One aspect of the present invention is to target the anti-fibrotic effects of IFN-Y (Li, 2012J Control Release a mutated, attenuated for of an IFNW towards virally infected 159:261). These data support the concept and the potential cells, using for example the targeting antibodies describe therapeutic benefit gained by delivering IFN-y activity above for the targeting of an attenuated form of IFNC. directly to fibroblasts in fibrotic diseases, including IPF and Mutated, attenuated forms of an IFNW could also be used to liver fibrosis, and validate PDGFR-B as a target for this target cancer cells, as described in more detail for IFNC. approach. above. 0240. The present invention also contemplates the attenu 0241. Non-IFN ligands are also contemplated in the ation and antibody-based targeting of type III IFNs, includ present invention and may also be attenuated by mutation ing IFN 1 (IL29), IFN 2 (IL28A), and IFN.3 (IL28B) (S. and then targeted to specific cell types by antibodies or V. Kotenko, G. Gallagher, V. V. Baurin et al., “IFN-us fragments thereof. The anti-inflammatory cytokine interleu mediate antiviral protection through a distinct class II kin-10 (IL-10) plays a central role during innate and adap cytokine receptor complex. Nature Immunology, Vol. 4, no. tive immune responses. IL-10 forms a homodimer and binds 1, pp. 69-77, 2003. P. Sheppard, W. Kindsvogel, W. Xu, et to the IL-10 receptor complex expressed on APCs, leading al., “IL-28, IL-29 and their class II cytokine receptor to reduced expression of MHC class II and reduced produc IL-28R.” Nature Immunology, vol. 4, no. 1, pp. 63-68, tion of pro-inflammatory cytokines and chemokines, thereby US 2016/0367695 A1 Dec. 22, 2016 27 inhibiting T cell development and differentiation. However, 2004 The Oncologist 9:18; Jelkmann, 2008 Crit Rev. Oncol IL-10 has also been implicated in inducing the proliferation Hematol 67:39: Elliott, 2012). of several immune cells, including B cells (Hofmann, 2012 0246 Erythropoiesis is a multi-step process, in which Clin Immunol 143: 116). pluripotent stem cells undergo tightly controlled differen 0242 Reduced expression of IL-10 is associated with a tiation and proliferation steps. An intermediate cell type in number of autoimmune disorders in humans and rodents, this process, is the colony-forming-unit-erythroid (CFU-E) including psoriasis, inflammatory bowel disease and rheu cell, which expresses high levels of EPOR, depends on EPO matoid arthritis. Mice deficient in IL-10 develop chronic for survival and appears to be the main cell type in the enterocolitis, which can be prevented by the administration differentiation process with this dependency (Elliott, 2008 of IL-10, but the clinical translation of these findings Exp Hematol 36:1573). resulted in a number of failed trials in patients. One expla 0247 Targeting EPO activity to CFU-E cells using spe nation of these failures is that the local IL-10 concentrations cific markers would substantially reduce the effect of EPO may be too low, even at maximum tolerable systemic on cancer and other non-hematopoietic cells, while main administration (Herfarth, 2002 Gut 50:146). Another expla taining the ability to drive erythrocyte formation and nation may be the immunostimulatory effect of IL-10 on B increase hemoglobin levels. Genome-wide analysis of cells and the resulting production of the pro-inflammatory CFU-E cells revealed several potential candidate cellular IFN-y, as was demonstrated in IL-10-treated Crohn's disease markers, including Rh-associated glycoproteins, e.g. CD241 patients (Tilg, 2002 Gut 50:191). and members of the Rh blood group system, e.g. the product 0243 Fusing attenuated IL-10 to an antibody specific for of the RCHE gene (Terszowski, 2005 Blood 105:1937). APCs, e.g. targeting dendritic cells through CD11c, or more 0248. Additional example surface markers expressed on broadly expressed myeloid markers, like CD33 or CD115, CFU-Es, and several other intermediates of erythropoiesis, would decrease systemically active biologic activity and at include CD117 (c-kit), CD71 (transferrin receptor) and the same time increase the targeted local active concentra CD36 (thrombospondin receptor) (Elliott, 2012 Biologics tions of IL-10. In addition, the demonstrated pro-inflamma 6:163), but these markers are overexpressed in certain tory effect through B cells would be decreased or eliminated. cancer cells as well, as they are all involved in general The production of antibody-IL 10 fusion proteins have been growth and proliferation, and therefore represent less attrac described previously (Schwager Arthritis Res Ther. 11(5): tive targets for targeting EPO activity in cancer patients, but R142, 2009). this approach may benefit patients with tumors not express 0244 Evidence exists for an anti-fibrotic role of IL-10 in ing these targets. CD117 antibodies include SR-1 (U.S. Pat. various models. A hallmark of fibrosis is the overproduction No. 7,915,391) and antibodies DSM ACC 2007, 2008 and and deposition of collagen produced by fibroblasts, resulting 2009 (U.S. Pat. No. 5,545,533). Other antigens for targeting in scarring tissue formation. IL-10 directly inhibits extra of an attenuated EPO include CD34, CD45RO, CD45RA, cellular matrix synthesis by human fibroblasts (Reitamo, CD115, CD168, CD235, CD236, CD237, CD238, CD239 1994 J Clin Invest 94:2489) and is anti-fibrotic in a rat and CD240. hepatic fibrosis model through downregulation of TGF-B 0249 Fusing EPO activity to an antibody would also (Shi, 2006 World J. Gastroenterol 12:2357; Zhang, 2007 greatly increase the extent of the therapeutic activity. The Hepatogastroenterology 54:2092). Clinical use of IL-10 is half-life of recombinant EPO is about 5 hours in humans and hampered by its short half-life and a PEGylated version has this would likely be increased to weeks when attenuated shown promising pharmacokinetic improvements and effi EPO is fused to an antibody. This approach could benefit cacy in a preclinical model of fibrosis (Mattos, 2012 J patients treated for anemia, who are dosed typically multiple Control Release 162:84). Targeting IL-10 activity through times per week, often through intravenous injections. Impor fusion with an antibody directing it to fibroblasts could tantly, it has been shown that the therapeutic response to result in therapeutic benefits in fibrotic diseases, including EPO is primarily controlled by the length of time EPO lung and liver fibrosis. Antibodies against fibroblast specific concentrations are maintained, and not by the concentration proteins such as fibroblast activation protein and platelet levels (Elliott, 2008 Exp Hematol 36:1573). derived growth factor receptors, as described above in the 0250) Another example is transforming growth factor B description of IFN-y-targeting, could deliver attenuated (TGF-3) which is a critical factor in the regulation of T IL-10 directly to fibroblasts. cell-mediated immune responses and the induction of 0245 Recombinant erythropoietin (EPO) is a widely immune tolerance. TGF-B knockout mice die from multifo used and effective hormone for the treatment of anemia, cal inflammation and autoimmune disorders, Suggesting an often in cancer patients. It acts by signaling through the EPO immunosuppressive effect (Shull, 1992 Nature 359:693). receptor (EPOR), which is not only expressed by cells of the However, TGF-B also has been shown to induce fibrotic hematopoietic system, but also on non-hematopoietic cells, disease through a prominent role in extracellular matrix including cells from various tumor types. Many studies have regulation and by promoting fibroblast migration, prolifera examined the role of EPO and EPO-R stimulation in cancer tion and activation (Rosenbloom, 2010 Ann Intern Med models in vitro and in vivo, and a number of studies have 152:159; Wynn, 2011 J Exp Med 208:1339; King, 2011 demonstrated a stimulatory effect on tumor growth, either Lancet 378:1949). directly on cancer cells, or through increased angiogenesis in (0251. In the presence of TGF-B, CD4"CD25 naive T the tumors (reviewed in Jelkmann, 2008 Crit Rev. Oncol cells can be converted into Treg cells, which can Suppress Hematol 67:39). In several clinical trials, treatment with antigen-specific T cell expansion in Vivo and prevent allergic EPO has been associated with increased tumor growth and pathogenesis in a murine asthma model (Chen, 2003 J Exp decreased Survival, leading to the recommendation and Med 198:1875). Inflammatory responses also contribute to blackbox warning to limit and monitor the exposure of EPO the transition of acute liver disease and perpetuation into in cancer patients as much as clinically feasible (Farrell, chronic fibrosis and cirrhosis and TGF-B may help dampen US 2016/0367695 A1 Dec. 22, 2016 28 these responses through its effect on Treg differentiation 0257 The IL-4 mutants in this table, and their binding (Dooley, 2012 Cell Tissue Res 347:245). Similarly, TGF-B properties and biological activity, were described by Wang directed to naive T cells in inflammatory bowel disease Y. Shen B and Sebald W. Proc. Natl. Acad. Sci. USA 1997 could lead to control and Suppression of inflammation March 4: 94(5): 1657-62. (Feagins, 2010 Inflamm Bowel Dis 16:1963). 0258. In yet another example, Interleukin-6 (IL-6) may also be attenuated and targeted to specific cell types. A 0252 Targeting TGF-3 specifically to CD4 T cells may mechanism by which tumors can evade anti-tumor immunity leverage the anti-inflammatory potential of TGF-B, while is by recruiting Treg cells to the tumor microenvironment, minimizing its pro-fibrotic properties, and could provide a resulting in tolerance at tumor sites. IL-6 is a cytokine novel strategy to combat autoimmune disorders. Alterna involved in regulating the balance between Treg and Th17 tively, TGF-B could be targeted soley to activated T cells cells and induces the development of Th17 cells, while it using a T cell activation marker, as described above for the inhibits Treg differentiation (Kimura, 2010 Eur J. Immunol discussion of IFNB targeting. One attractive target along 40: 1830). these lines could be, for example, PD-1, which is expressed 0259 IL-6, by skewing the terminal differentiation of on recently activated CD4 T cells. Ideally, a non-antagoniz naive CD4 T cells towards the Th17 lineage, or reprogram ing antibody could be used, such as the J110 antibody ming of Th17 cells, has the potential to reverse tumor discussed in further detail below. associated immune Suppression by Treg cells in the context 0253) Another example is Interleukin-4 (IL-4) which is a of cancer, thereby enabling the immune system to control the cytokine that induces the differentiation of naive CD4+ T tumors. cells into Th2 cells. Upon activation, Th2 cells produce more 0260 This strategy has proven successful in a murine IL-4, and as a result, IL-4 is considered a main driver of model of pancreatic cancer in which mice injected with Th2-mediated immune responses. The concept of a Th1/Th2 tumor cells expressing IL-6 demonstrated a significant delay imbalance (favoring Th1) contributing to autoimmune and in tumor growth and enhanced Survival, accompanied by an other inflammatory diseases was first postulated in the 1980s increase in Th17 cells in the tumor microenvironment, (reviewed in Kidd, 2003 Altern Med Rev 8:223), and indeed, compared to mice bearing tumors not expressing IL-6 (Gn a role of Th1/Th17 cells as drivers of disease in psoriasis erlich, 2010 J Immunol 185:4063). (Ghoreschi, 2007 Clin Dermatol 25:574), certain types of 0261 Adoptive transfer of T cells is an effective treat inflammatory bowel disease, in particular Crohn's disease ment for solid (Rosenberg, 2011 Clin Cancer Res 17:4550) (Sanchez-Munoz, 2008 World J. Gastroenterol 14:4280), or and hematologic (Kochenderfer, 2012 Blood 119:2709) severe versus mild forms of asthma (Hansbro, 2011 Br J malignancies. Analysis of five different clinical trials in Pharmacol 163:81), has been documented. which adoptive T cell transfer was employed using a variety of preconditioning regimens revealed that the depth and 0254. In preclinical models of infectious diseases, devia duration of Treg depletion correlates with clinical response tion of the immune response away from Th1 to Th2 and rate, highlighting the important role of residual Tregs con activation of macrophages by IL-4 protected from immu trolling the anti-tumor response (Yao, 2012 Blood 119: nopathology (Hunig, 2010 Med Microbiol Immunol 199: 5688). In mice, a direct link between surviving Tregs and 239), and IL-4 therapy of psoriasis patients resulted in an efficacy of adoptive transfer therapy strongly supports these induction of Th2 differentiation and an improvement in clinical observations (Baba, 2012 Blood 120:2417). clinical scores (Ghoreschi, 2003 Nat Med 9:40). 0262 The importance of Tregs in controlling anti-tumor 0255 Diversion towards Th2 may provide a therapeutic activity is further exemplified by a significant increase in the benefit in certain types of diseases. Delivery of IL-4 to CD4" humoral response to peptide vaccination in glioblastoma T cells could accomplish this, or IL-4 activity could be patients after depletion of Tregs with the anti-IL-2 receptor targeted to macrophages to protect from immunopathology antibody daclizumab (Sampson, 2012 PloS ONE 7:e31046). (Ghoreschi, 2007 Clin Dermatol 25:574; Hunig, 2010 Med 0263 Taken together, the published data strongly support Microbiol Immunol 199:239). a role for Tregs in inhibiting the immune response against tumors. By directing IL-6 activity to CD4 cells in order to 0256 Attenuating mutations in IL-4 that may be stimulate Th17 differentiation and decrease Treg formation, exploited in the design of antibody-attenuated IL-4 fusion enhanced anti-tumor responses are expected. These may be protein constructs of the present invention include those achieved with or without accompanying vaccination strate listed in Table 17. gies. Fusing attenuated IL-6 to an antibody against a T cell antigen (e.g. targeting CD4) or an activated T cell antigen TABLE 17 (such as PD-1) would provide a comprehensive delivery IL4 Variant Korx 103 Sl ECso T cell proliferation (nM) directly to the target cells. IL4 2.1 O.12 0264. Attenuated mutants of IL-6 include those listed in ISR 8.7 Table 18. T6D 15 E9C 270 3.1 TABLE 1.8 R81E 6.1 K84D 9.3 ECso in XG-1 growth R88O 140 2.5 stimulation assay R88A 760 8.1 IL6 Variant Binding (% of wild type) (pg/ml) N89R 6.1 W91D 8.5 IL6 100 600 F74E 1 Low activity ND. No specific binding found. F78E 5 Low activity US 2016/0367695 A1 Dec. 22, 2016 29 TABLE 18-continued 0271 Limitations of HGF as a therapeutic include its short half-life, which requires Supra-physiological systemic ECso in XG-1 growth concentrations to reach locally effective levels, and the role stimulation assay of its receptor, MET, in cancer. MET can activate oncogenic IL6 Variant Binding (% of wild type) (pg/ml) pathways in epithelial cells. Both of these limitations may be R168M 2 Low activity overcome by generation of an antibody-HGF fusion protein R179E None detected Low activity construct and targeting it to regenerating or fibrotic tissue. R179W None detected Low activity This strategy would produce a therapeutic with a much longer half-life directed primarily at the relevant cells types. 0272 Clinical trials have investigated the therapeutic 0265. These IL-6 mutants and their properties were potential and regenerative activity of HGF, or HGF mimet described by Kalai Met. al. Blood. 1997 Feb. 15: 89(4): 1319-1333 ics, in hepatic failure, chronic leg ulcers, limb ischemia, 0266. Another example is hepatocyte growth factor peripheral arterial disease, cardiovascular disease after myo (HGF) discovered as a mitogen for hepatocytes (reviewed in cardial infarction and neurological diseases (de Andrade Nakamura, 2010 Proc Jpn. Acad Ser B Phys Biol Sci86:588). 2009 Curr Opin Rheumatol 21:649; Nakamura, 2011 J Hepatocyte growth factor is a pleiotropic cytokine that Gastroenterol Hepatol 26:188: Madonna, 2012 Thromb regulates cell growth and motility, playing a central role in Haemost 107:656). angiogenesis and tissue generation and repair in many 0273 Liver fibrosis, typically the result of chronic liver Organs. damage caused by infections or alcohol abuse, is, like 0267 HGF acts through its receptor, MET, which is fibrosis in other organs, characterized by excessive accumu expressed on epithelial and endothelial cells. Binding of lation of extracellular matrix, including collagen produced HGF to MET results in a number of intracellular phospho by (myo) fibroblasts. Damaged hepatocytes release inflam rylation and signaling events, leading to a variety of bio matory cytokines and the resulting inflammatory milieu logical responses including migration, proliferation and stimulates the transformation of hepatic stellate cells (HSC) morphogenesis. Essential for embryogenesis, HGF’s pri into fibroblasts, producing collagen. The accumulation of mary function in the adult is tissue repair (Nakamura, 2010 extracellular matrix proteins results in Scar tissue, which Proc Jpn. Acad Ser B Phys Biol Sci 86:588). leads to liver cirrhosis (Bataller, 2005 J Clin Invest 115:209). 0268 HGF has been shown to alter the fate of epithelial Evidence exists for a direct effect of HGF on hepatocytes cells and reduce epithelial-mesenchymal transition (EMT) through its intereference with TGF-B signaling, antagoniz and HSC in vitro (Kwiecinski, 2012 PloS One 6:e24568: ing the process of fibroblastogenesis (Shukla, 2009 Am J Namada, 2012 J Cell Physiol DOI 10.1002/jcp.24143). Respir Cell Mol Biol 40:643). After organ injury, TGF-f Targeting HGF specifically to hepatocytes or HSC may drives conversion of HGF-producing fibroblasts into colla result in a therapeutic benefit in liver fibrosis patients, while gen-producing myofibroblasts, while HGF in turn inhibits eliminating the unwanted systemic effects of HGF. TGF-B production by myofibroblasts (Mizuno, 2004 Am J 0274 Possible membrane proteins for hepatocytes Physiol Renal Physiol 286:F134). Exogeneous HGF, or include, for example, ASGR1, a subunit of the asialoglyco mimetics activating the MET receptor, act by restoring this protein, used as a target for liver specific drug delivery imbalance imposed by tissue injury, and are therefore con (Stockert, 1995 Physiol Rev 75:591), or alternatively the sidered promising drug candidates for treating damaged other subunit of this receptor, ASGR2. Fibroblast-specific tissues and fibrotic diseases (Nakamura, 2010 Proc Jpn. Acad protein (FSP1) expression is increased after liver injury and Ser B Phys Biol Sci 86:588). may be used to target fibroblasts or inflammatory macro 0269. Initially studied in models for liver damage and phages in fibrotic liver tissue (Osterreicher, 2011 Proc Natl hepatitis (Roos, 1992 Endocrinology 131:2540; Ishiki, 1992 Acad Sci USA 108:308). Hepatology 16:1227), HGF subsequently demonstrated 0275. In lung fibrosis patients, the loss of pulmonary therapeutic benefits in many additional damaged organs, architecture is characterized by a loss of alveolar epithelial including pulmonary, gastrointestinal, renal and cardiovas cells, the persistent proliferation of activated fibroblasts and cular models of injury and fibrosis (Nakamura, 2011 J the extensive alteration of the extracellular matrix (Pangani Gastroenterol Hepatol 26:188). ban, 2011 Acta Pharmacol Sin 32:12). 0270. In in vivo model systems of fibrosis, HGF prevents 0276. To treat lung fibrosis, HGF activity may be deliv the progression of fibrotic changes and reduces collagen ered to alveolar epithelial cells by attenuating it (by muta accumulation when administered prophylactically or thera tion) and attaching it to an antibody against a specific cell peutically in murine lungs exposed to bleomycin (Yaeka surface protein on these cells, such as RTI40/Tia or HTI56 shiwa, 1997 Am J Respir Crit Care Med 156:1937: Mizuno, (McElroy, 2004 Eur Respir J 24:664). 2005 FASEB J 19:580), in an obstructive nephropathy 0277 Endothelial cell-specific markers, including VEGF model in mice (Yang, 2003 Am J Physiol Renal Physiol receptors (Stuttfeld, 2009 IUBMB Life 61:915) may be used 284:F349) and in liver fibrosis models in rats (Matsuda, for targeting blood vessels for endothelial cell layer 1997 Hepatology 26:81); HGF also prevents fibrosis in enhancement for a number of pathologic indications, includ cardiomyopathic hamsters (Nakamura, 2005 Am J Physiol ing hindlimb ischemia. Examples of VEGF receptor anti Heart Circ Physiol 288:H2131). bodies are shown in Table 19. TABLE 19 VEGFR Antibodies Ab Clones Patent Assignee Comments AC88 U.S. Pat. No. 8,128,932 Shanghai Aosaiersi Human anti-VEGFR2 Biotech Co., Ltd mAb Antibody 1, US2012,OOS8126 Imclone LLC Anti-VEGFR3 Abs Antibody 2 US 2016/0367695 A1 Dec. 22, 2016 30 TABLE 19-continued VEGFR Antibodies Ab Clones Patent Assignee Comments 6A6 US2O11 OO651.76 Korea Research Human, anti-VEGFR Institute of BioScience and BioTechnology 0278 Many other examples of signaling ligands are also separated from any putative complex and the amount of free known in the art and may, as described in the non-limiting (i.e. uncomplexed) label is a measure of the binding of the exemplary embodiments above, be attenuated and attached agent being tested to target molecule. One may also measure to an antibody (or fragment thereof) that binds to an antigen the amount of bound, rather than free, target. It is also on specific target cells, thereby allowing the ligand to possible to label the compound rather than the target and to generate its biological signal on those target cells to a much measure the amount of compound binding to target in the greater degree than it generates its signal on antigen-nega presence and in the absence of the drug being tested. tive cells. Examples of ligands that have a direct negative 0282 One example of a cell free assay is a binding assay. effect on tumor proliferation include TNFO, TRAIL, Fas Whilst not directly addressing function, the ability of a Ligand, IFNB, IFNY or IFNW, which can be targeted to modulator to bind to a target molecule in a specific fashion various tumor cell Surface antigens as discussed above for is strong evidence of a related biological effect. For example, INFO binding of a molecule to a target may, in and of itself, be 0279. In many of the aspects of the present invention, inhibitory, due to steric, allosteric or charge-charge interac specific mutations in various ligands are explicitly men tions. The target may be either free in solution, fixed to a tioned. There are, however, methods well known in the art support, expressed in or on the surface of a cell. Either the for identifying other mutations in signalling ligands numer target or the compound may be labeled, thereby permitting ous methods for mutagenesis of proteins are known in the determination of binding. Usually, the target will be the art. Such methods include random mutagenesis for example, labeled species, decreasing the chance that the labeling will exposing the protein to UV radiation or mutagenic chemi interfere with or enhance binding. Competitive binding cals and selecting mutants with desired characteristics. Ran formats can be performed in which one of the agents is dom mutagenesis may also be done by using doped nucleo labeled, and one may measure the amount of free label tides in oligonucleotides synthesis, or conducting a PCR versus bound label to determine the effect on binding. reaction in conditions that enhance misincorporation of 0283 Depending on the assay, culture may be required. nucleotide, thereby generating mutants. Another technique The cell is examined using any of a number of different is site-directed mutagenesis which introduces specific physiologic assays. Alternatively, molecular analysis may be changes to the DNA. One example of site directed muta performed, for example, protein expression, mRNA expres genesis is using mutagenic oligonucleotides in a primer sion (including differential display of whole cell or polyA extension reaction with DNA polymerase. This method RNA) and others. Non-limiting examples of in vitro bio allows for point mutation, or deletion or insertion of small logical assays that can be used to screen protein variants are stretches of DNA to be introduced at specific sites. The shown in the Examples below and also include apoptosis site-directed approach may be done systematically in Such assays, migration assays, invasion assays, caspase-activa technique as alanine Scanning mutagenesis whereby resi tion assays, cytokine production assays and the like. dues are systematically mutated to alanine and its effect on 0284. The present invention also provides compositions the peptide's activity is determined. Each of the amino acid comprising the polypeptides of the present invention. These residues of the peptide is analyzed in this manner to deter compositions can further comprise at least one of any mine the important regions of the peptide. Suitable auxiliary, such as, but not limited to, diluent, binder, 0280 Another example is combinatorial mutagenesis stabiliser, buffers, salts, lipophilic solvents, preservative, which allows the screening of a large number of mutants for adjuvant or the like. Pharmaceutically acceptable auxiliaries a particular characteristic. In this technique, a few selected are preferred. Non-limiting examples of, and methods of positions or a short stretch of DNA may be exhaustively preparing such sterile solutions are well known in the art, modified to obtain a comprehensive library of mutant pro Such as, but not limited to, Gennaro, Ed., Remington's teins. One approach of this technique is to excise a portion Pharmaceutical Sciences, 18th Edition, Mack Publishing of DNA and replaced with a library of sequences containing Co. (Easton, Pa.) 1990. Pharmaceutically acceptable carriers all possible combinations at the desired mutation sites. The can be routinely selected that are suitable for the mode of segment may be at an enzyme active site, or sequences that administration, solubility and/or stability of the antibody have structural significance or immunogenic property. A composition as well known in the art or as described herein. segment however may also be inserted randomly into the 0285 Pharmaceutical excipients and additives useful in gene in order to assess the structural or functional signifi the present composition include but are not limited to cance of particular part of protein. proteins, peptides, amino acids, lipids, and carbohydrates 0281 Methods of screening mutated ligands to determine (e.g., Sugars, including monosaccharides, di-, tri-, tetra-, and potency includes assaying for the presence of a complex oligosaccharides; derivatised Sugars such as alditols, aldonic between the ligand and the target. One form of assay acids, esterified Sugars and the like; and polysaccharides or involves competitive binding assays. In Such competitive Sugar polymers), which can be present singly or in combi binding assays, the target is typically labeled. Free target is nation, comprising alone or in combination 1-99.99% by US 2016/0367695 A1 Dec. 22, 2016 weight or Volume. Exemplary protein excipients include plural aspects unless the context clearly dictates otherwise. serum albumin, such as human serum albumin (HSA), Thus, for example, reference to “a” includes a single as well recombinant human albumin (rIA), gelatin, casein, and the as two or more; reference to “an includes a single as well like. Representative amino acids which can also function in as two or more; reference to “the includes a single as well a buffering capacity include alanine, glycine, arginine, as two or more and so forth. betaine, histidine, glutamic acid, aspartic acid, cysteine, 0293 Having generally described the invention, the same lysine, leucine, isoleucine, Valine, methionine, phenylala will be more readily understood by reference to the follow nine, aspartame, and the like. One preferred amino acid is ing examples, which are provided by way of illustration and histidine. A second preferred amino acid is arginine. are not intended as limiting. 0286 Carbohydrate excipients suitable for use in the invention include, for example, monosaccharides, such as Examples of the Invention fructose, maltose, galactose, glucose, D-mannose, Sorbose, and the like; disaccharides, such as lactose, Sucrose, treha Production of Antibody-IFNC. Fusion Protein lose, cellobiose, and the like; polysaccharides. Such as Constructs raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol. Xylitol, maltitol, Expression Vectors: lactitol, xylitol sorbitol (glucitol), myoinositol and the like. 0294 The DNA encoding the rituximab (Anderson et al., Preferred carbohydrate excipients for use in the present U.S. Pat. No. 5,843,439, Dec. 1, 1998) and palivizumab invention are mannitol, trehalose, and raffinose. (Johnson, U.S. Pat. No. 5,824.307, Oct. 20, 1998) variable 0287 Antibody compositions can also include a buffer or regions were generated from 18 (heavy chain) and 16 (light a pH adjusting agent; typically, the buffer is a salt prepared chain) DNA oligonucleotides, which were designed accord from an organic acid or base. Representative buffers include ing to the published amino acid sequences, by PCR-based organic acid salts, such as salts of citric acid, ascorbic acid, gene assembly. The DNA encoding the variable regions of gluconic acid, carbonic acid, tartaric acid, Succinic acid, the G005 anti-CD38 and nBTO62 anti-CD138 monoclonal acetic acid, or phthalic acid; Tris, tromethamine hydrochlo antibodies were drawn from the publications by De Weers et ride, or phosphate buffers. Preferred buffers for use in the al. (U.S. Pat. No. 7,829,673) and by Daelken et al. (WO present compositions are organic acid salts, such as citrate. 2009/080832), respectively, and subjected to be synthesized 0288 Additionally, the compositions of the invention can by Integrated DNA Technology, Inc. (Coralville, Iowa) after include polymeric excipients/additives, such as polyvi the sequence modification to eliminate rare codons and nylpyrrolidones, ficols (a polymeric Sugar), dextrates (e.g., unprefered restriction sites. cyclodextrins, such as 2-hydroxypropyl-3-cyclodextrin), 0295 The DNA sequences encoding the variable regions polyethylene glycols, flavoring agents, antimicrobial agents, of anti-human HLA (HB95), anti-human PD-1 (J110) and Sweeteners, antioxidants, antistatic agents, Surfactants (e.g., anti-yellow fever virus (2D12) monoclonal antibodies were polysorbates such as “TWEENR 20” and “TWEENR 80), determined after cloning from hybridoma W6/32 (ATCC lipids (e.g., phospholipids, fatty acids), Steroids (e.g., cho HB-95, Barnstable et al. (1978), Cell 14:9-20), J110 (Inter lesterol), and chelating agents (e.g., EDTA). national Patent Organism Depositary FERM-8392, Iwai et 0289. These and additional known pharmaceutical al. (2002), Immunol. Lett, 83:215-220) and 2D12 (ATCC excipients and/or additives suitable for use in the antibody CRL-1689, Schlesinger et al. (1983), Virol. 125:8-17), compositions according to the invention are known in the respectively, using the SMART RACE cDNA Amplification art, e.g., as listed in “Remington: The Science & Practice of kit (Clontech, Mountain View, Calif.) and Mouse Ig-Primer Pharmacy”, 19th ed., Williams & Williams, (1995), and in Sets (Novagen/EMD Chemicals, San Diego, Calif.). The the “Physician's Desk Reference', 52nd ed., Medical Eco sequence determination and Sub-cloning of the newly iso nomics, Montvale, N.J. (1998), the disclosures of which are lated anti-CD38 antibodies is described in the following entirely incorporated herein by reference. Preferred carrier sections. or excipient materials are carbohydrates (e.g., Saccharides 0296. The DNA encoding human interferon-C2b and alditols) and buffers (e.g., citrate) or polymeric agents. (IFNC2b; amino acid sequence of SEQ ID NO:3) was 0290 Throughout this specification the word “comprise’, isolated from genomic DNA of a HEK cell line by PCR. The or variations such as “comprises' or “comprising, will be sequences of human interferon-131 (IFNB1, SEQ ID understood to imply the inclusion of a stated element, NO:91), human interleukin-4 (IL-4, SEQ ID NO:119) and integer or step, or group of elements, integers or steps, but human interleukin-6 (IL-6, SEQID NO:123) were designed not the exclusion of any other element, integer or step, or from the protein sequences such as NP 002167, NP 000580 group of elements, integers or steps. and NP 000591, respectively, and synthesized by Integrated 0291 All publications mentioned in this specification are DNA Technology, Inc. (Coralville, Iowa) or GenScript USA herein incorporated by reference. Any discussion of docu Inc. (Piscataway, N.J.) using methods commonly known to ments, acts, materials, devices, articles or the like which has those of skill in the art. Alterations of the cytokine been included in the present specification is solely for the sequences, for example the addition of linkers or point purpose of providing a context for the present invention. It mutations, were introduced to the cytokine genes using is not to be taken as an admission that any or all of these overlap extension PCR techniques well known in the art. matters form part of the prior art base or were common 0297. The cytokine-endoding gene fragments were then general knowledge in the field relevant to the present cloned into the pTT5 expression vector (Durocher, Nucleic invention as it existed in Australia or elsewhere before the Acids Research volume 30, number 2, pages E1-9, 2002) priority date of each claim of this application. containing either a human IgG1 heavy chain complete or 0292. It must be noted that, as used in the subject partial constant region (such as Swissprot accession number specification, the singular forms “a”, “an and “the include PO1857), a human IgG4 heavy chain constant region (such US 2016/0367695 A1 Dec. 22, 2016 32 as Swissprot accession number PO1861 incorporating sub IFN fusion protein constructs on cells that display that stitution S228P), human Ig kappa constant region (Swissprot antigen corresponding to the antibody to which the IFN is accession number PO1834) or human Ig lambda constant fused, and may be used as part of the assay for calculating region (Swissprot accession number POCG05) either as a the antigen-sensitivity index (ASI) defined herein. Daudi naked Ig or as a cytokine gene fusion form using overlap cells express both CD20 and CD38 as cell surface associated extension PCR techniques and restriction sites according to antigens. The viability of cells was measured using the cloning methods well known by those skilled in the art. reagent CellTiter-Glo R., Cat #G7570, from Promega (Madi son, Wis.). This is a luminescence-based assay that deter Production of IgG and IgG Interferon Fusion Protein mines the viability of cells in culture based on quantitation Constructs: of ATP. The signal strength is proportional to the number of 0298 DNA plasmids encoding the IgGs and IgG-cy viable cells in a microtiter plate well. The details of the assay tokine fusion protein constructs were prepared using Plas are as follows: mid Plus Maxi kit (Qiagen, Valencia, Calif.) and then 0304 Daudi cells (obtained from ATCC, Manassas, Va.) transfected into HEK293-6E cells (CNRC, Montreal, were cultured in a T75 flask (TPP, Trasadingen, Switzerland, Canada) grown in F17 synthetic medium supplemented with cath90076) to a preferred density of between 0.5x10 and 0.1% Pluronic F-68, 4 mM L-glutamine (Invitrogen, Carls 0.8x10 viable cells/ml in RPMI 1640 (Mediatech, Inc., bad, Calif.) using a commercially available transfection Manassas, Va., cat #10-040-CV) with 10% Fetal Bovine reagent and OptiMEM medium (Invitrogen, Carlsbad, Serum (FBS; Hyclone, Logan, Utah cathi SH30070.03). Calif.). After allowing for expression for 6 days in an Cells were harvested by centrifuging at 400 g for five incubator supplied with 5% CO and gentle shaking, the minutes, decanting the Supernatant, and resuspending the culture media was isolated and subjected to IgG affinity cell pellet in RPMI 1640+10% FBS. Cells were then counted purification using Protein G-agarose beads (GE Healthcare, and the density was adjusted to 3.0x10 cells/ml in RPMI Piscataway, N.J.). Purified IgG and IgG-cytokine fusion 1640+10% FBS. Then, 50 ul of the cell suspension was protein constructs were then concentrated and buffer-ex aliquoted into each well of a 96 well round bottom tissue changed to phosphate buffered saline (PBS) pH 7.4 using culture plate (hereafter, “experimental plate') (TPP, Amicon Ultra centrifugal filter devices (Millipore, Billerica, cath92067). On a separate, sterile 96 well plate (hereafter, Mass.), followed by protein concentration determination "dilution plate'; Costar, Corning, N.Y. catfi3879), test using a NanoDrop 2000 spectrophotometer (Thermo Scien articles were serially diluted in duplicate in RPMI 1640+ tific, Waltham, Mass.). 10% FBS. Then, 50 ul/well was transferred from the dilution 0299. Although different antibody-cytokine fusion pro plate to the experimental plate. The experimental plate was tein constructs were expressed in the HEK system with then incubated for four days at 37° C. with 5% CO. differing yields, several of them, in particular several of 0305. A mixture of the manufacturer-supplied assay buf those based on IFNC, were produce at at least 100 mg/L of fer and assay substrate (hereafter, “CellTilterGlo reagent'. media, showed high solubility and did not aggregate as mixed according to the manufacturers instructions) was determined by size exclusion chromatography. added to the experimental plate at 100 The plate was shaken 0300. The amino acid sequences of the antibodies and for two minutes. Then, 100 ul/well was transferred from the antibody-ligand construct fusion protein constructs are experimental plate to a 96 well flat bottom white opaque described below. For antibody-cytokine fusion protein con plate (hereafter, “assay plate’: BD Biosciences, Franklin structs in which the cytokine was fused to the C-terminus of Lakes, N.J. cati35 3296). The content of the assay plate was the heavy or light chain, the following naming convention then allowed to stabilize in the dark for 15 minutes at room was used: temperature. The plate was read on a Victor 3V Multilabel name of mab-linkage to heavy chain (“HC) or light chain Counter (Perkin Elmer, Waltham, Mass., modeli1420-041) ("LC)-Linker name-ligand name (mutation) iso on the luminometry channel and the luminescence was type. measured. Results are presented as "relative luminescence 0301 Thus for example the construct “Rituximab-HC units (RLU)”. L6-IFNC. (A145G) IgG1 is the antibody rituximab, with 0306 Data was analyzed using Prism 5 (Graphpad, San IFNC.2b (with the A145G point mutation), linked to the Diego, Calif.) using non-linear regression and three param C-terminus of the IgG1 heavy chain, with an intervening eter curve fit to determine the midpoint of the curve (EC50). linker L6. For each test article, potency relative to free IFNC.2b (or 0302) The linkers used in the experiments were as fol some other form of IFN with a known potency relative to lows: IFNC2b) was calculated as a ratio of EC50s. L0: no linker (direct fusion of the C-terminus of an antibody 0307 One of ordinary skill in the art will appreciate that chain with the N-terminus of the cytokine) there are many other commonly used assays for measuring cell viability that could also be used. (SEQ ID NO: 132) 0308 "On target (ARP) assay” (also sometimes referred L6 : SGGGGS to herein as a “targeted assay’): The multiple myeloma cell line ARP-1 was a gift from Bart Barlogie MD, PhD, Director (SEQ ID NO: 133) of the Myeloma Institute at the University of Arkansas L16 : SGGGGSGGGGSGGGGS Medical Center (Little Rock, Ak.). It is described in Hardin J. et al., (Interleukin-6 prevents dexamethasone-induced Method for Measuring Antigen-Targeted Activity of myeloma cell death. Blood: 84:3063, 1994). ARP-1 cells Antibody-IFNC. Fusion Protein Constructs (CD38") were used to test CD38 targeting antibody-IFN 0303 “On target (Daudi) assay’: This assay was used to fusion protein constructs. Culture and assay conditions were quantify the anti-proliferative activity of IFNs and antibody the same as for Daudi-based assay outlined above, with the US 2016/0367695 A1 Dec. 22, 2016 following exceptions: ARP-1 was cultured to a density of to the C-terminus of the heavy chain of two different 4.0x10 to 6.0x10 cells/ml. ARP-1 concentration was antibodies (rituximab and palivizumab, an isotype control adjusted to 1.0x10 cells/ml prior to assay. antibody), as acting on a the iLite cell line. This cell line Method for Measuring Non-Antigen-Targeted Activity of does not display the antigen for either of these antibodies, so Antibody-IFNC. Fusion Protein Constructs this assay reveals the potency of various IFNC2b-containing 0309 “Off-target assay” (also sometimes referred to proteins in the absence of antibody-antigen-based targeting. herein as the “not-targeted assay): The iLite assay from The details of this assay are described above under the PBL Interferon Source (Piscataway, N.J., Cath51100), was heading "Method for measuring non-antigen-targeted activ performed largely as described by the manufacturer with the ity of antibody-IFNC. fusion protein constructs” and is addition of a human IgG blocking step. The iLite cell line is hereafter abbreviated as the “off-target assay.” “Rituximab described by the manufacturer as “a stable transfected cell HC-L6-IFNC. IgG1 refers to the CD20-targeting chimeric line derived from a commercially available pro-monocytic antibody Rituximab, in which the light chain (SEQ ID human cell line characterized by the expression of MHC NO:276) is unaltered but the IgG1 class heavy chain (SEQ Class II antigens, in particular the human lymphocyte anti ID NO:277) has, attached to its C terminus, a 6 amino acid gen (HLA-DR), on the cell surface.” The cell line contains linker sequence (“L6:” SGGGGS, SEQ ID NO:132), fol a stably transfected luciferase gene, the expression of which lowed by the sequence for IFNC2b (SEQ ID NO:3); this is driven by an interferon-response element (IRE), which heavy chain-linker-IFNC. sequence is shown as SEQ ID allows for interferon activity to be quantified based on NO:280. “Isotype-HC-L6-IFNC. IgG1” refers to the RSV luminescence output. The manufacturer-Supplied iLite plate targeting humanized antibody Palivizumab, in which the (hereafter “assay plate') and diluent were removed from the light chain (SEQID NO:290) is unaltered but the IgG1 class -80°C. freezer and allowed to equilibrate to room tempera heavy chain (SEQ ID NO:291) has, attached to its C ture. Then, 50 ul of the diluent was added per well to the terminus, a 6 amino acid linker sequence (“L6: SGGGGS, assay plate. The vial of manufacturer-Supplied reporter cells SEQ ID NO:132), followed by the sequence for IFNC2b was removed from the -80° C. freezer and thawed in a 37° (SEQ ID NO:3); this heavy chain-linker-IFNC2b sequence C. water bath. Then, 25ul aliquiots of cells were dispensed is shown as SEQ ID NO:294. In this assay, free IFNC2b into each well of the assay plate. Next, 12.5 ul of 8 mg/ml showed an ECs for activating gene expression through an human IgG that was diluted into RPMI 1640+10% FBS interferon response element (IRE) of 1.9 pM. By attaching (Sigma Chemicals, St. Louis, Mo.; catil 14506) was added IFNC2b to Rituximab, there was a 3.1-fold (5.9/1.9–3.1) per well. The contents were mixed and incubated at 37° C. decrease in its potency. A similar, modest decrease in for 15 minutes. On a separate “dilution plate,” test articles potency was observed when IFNC2b was linked to Palivi were serially diluted in duplicate in RPMI 1640+10% FBS. Zumab. Again, the cell line used in this study did not have Then, 12.5 ul of the test articles were transferred from the the antigen corresponding to either of these antibodies on its dilution plate to the assay plate. The assay plate was then cell Surface, demonstrating that attachment of an IgG to the incubated at 37° C. with 5% CO, for 17 hours. The manu N-terminus of IFNC2b caused a modest (3-4x) decrease in facturer-supplied assay buffer and substrate were removed its non-antigen-targeted IFN activity. This is consistent with from the -80° C. freezer and allowed to equilibrate to room what has been reported by other (for example in U.S. Pat. temperature for two hours. The manufacturer-supplied assay No. 7,456,257). Neither Palivizumab nor Rituximab alone buffer was added to the manufacturer-supplied substrate vial (without the fusion to an interferon) showed any activity in and mixed well according to the manufacturers instructions this assay (data not shown). to create the “luminescence solution.” Then, 100 ul of the 0311. To determine whether the antibody-IFNC2b fusion luminescence Solution was added to each well of the assay protein constructs had enhanced activity relative to free plate. The plate was shaken for 2 minutes. The plate was IFNC.2b on cells that do display the corresponding antigen then incubated at room temperature for 5 minutes in the dark on their cell surface, their effect on Daudi cells, which and finally read on a Victor 3V Multilabel Counter on a display the CD20 antigen of Rituximab, but which do not luminometry channel and the luminescence measured and display the RSV F protein antigen corresponding to Palivi presented as RLU. The data was analyzed with Graphpad Zumab, was examined. The assay used in this case, described Prism 5 as described for the"on-target (Daudi) assay.” above as "Method for measuring antigen-targeted activity of antibody-IFNC. fusion protein constructs” or simply the above. To test anti-CD38 antibody-IFN fusion protein con “on-target (Daudi) assay, measured the effect of the test structs in the iLite assay, the manufacturer-Supplied diluent substances on the viability of Daudi cells. With these cells, was supplenmented with 2 mg/ml human IgG and 0.5 mg/ml the Rituximab-IFNC2b fusion protein construct (Rituximab anti-CD38 antibody (same antibody clone being tested as an HC-L6-IFNC. IgG1) was 3.25-fold (1.3/0.4—3.25) more antibody-IFN fusion protein construct, to block any binding potent than free IFNC2b (FIG. 7). In other words, the of the anti-CD38 antibody-IFN fusion protein constructs to attachment of Rituximab to IFNC2b resulted in slightly the CD38 expressed on the iLite cells). reduced (3.1-fold) activity towards antigen-negative cells (FIG. 6) but slightly increased (3.25-fold) activity towards Results: Antigen-Specificity of Antibody-IFNC. Fusion antigen-positive cells (FIG. 7). Overall, the antibody attach Protein Constructs ment therefore increased the antigen-specificity index (ASI). 0310 FIG. 6 shows the interferon activity of free IFNC2b defined as the fold increased potency relative to free IFNC2b (SEQ ID NO:3: “IFNC” in figure) as well as IFNC2b fused on antigen-positive cells multiplied by the fold decreased US 2016/0367695 A1 Dec. 22, 2016 34 potency relative to free IFNC.2b on antigen-negative cells, TABLE 20 by 10-fold (3.1x3.25) in this experiment. A repeat of the Targeted Non-Targeted experiments measured an ASI of 14, as shown in Table 20, Potency Relative Potency Relative row 2. The EC50 (mathematical midpoint of the dose to free IFNC2b to free IFNC2b Antigen response curve) was used as a measure of potency in the (EC50 IFNo.2b? (EC50 IFNC2b/ Specificity EC50 Fusion EC50 Fusion Index (ASI: calculations presented here. In other words, when compound Fusion protein protein protein calculated A showed an EC50 that is 10-fold lower than compound B, construct Test construct) construct) as Column Af it was said to have a 10-fold higher potency. Article Column A Column B Column B) 0312 The results presented in FIG. 8 are consistent with Ritux-IFNC2b 3.6 O.26 14 antibody-based targeting relying on antibody-antigen reac Ritux-IFNC2b O.86 O.OO26 330 (R144A) tivity: the Rituximab-IFNC. fusion protein construct (Ritux Ritux-IFNC2b 1.2 O.OO2O 600 imab-HC-L6-IFNC.-IgG1) was 12-fold (2.2/0.18–12) more (A145G) potent in reducing viability of the CD20 Daudi cells than Ritux-IFNC2b 1.6 O.OOO93 1,700 (R33A + YNS) the Palivizumab-IFNC. fusion protein construct (Isotype Ritux-IFNC2b 0.0022* No ND HC-L6-IFNC-IgG1), the antigen for which is not present on (R33A) detectable the Daudi cells. activity in non-targeted 0313. The modest reduction in IFNC activity that assay occurred as a result of linking it to an antibody may not be Ritux-IFNC2b 0.23% O.OOO86* 270 sufficient to prevent the toxicity of the IFNC. component of (R144A + YNS) *Free DFNo.2b was not tested on the same day as the test articles in these rows. Therefore, the construct in human Subjects. Various mutations were these measurements are based on a comparison of the test article with Rituximab-HC therefore introduced into IFNC2b in order to reduce its L6-DFNC. IgG1, which was assayed on the same day and same plate, multiplied by a correction factor based on the relative activity of DFNo.2b vs Rituximab-HC-L6-IFNo. activity and toxicity. For example, five different mutant IgG1 (i.e., data shown in the second row from the top) measured on a different day, versions of IFNC2b were generated and, in each case, linked to the C-terminus of the heavy chain of Rituximab via the six 0315 Surprisingly, when the amount of interferon activ amino acid linker L6, which has the sequence SGGGGS ity of these highly attenuated rituxumab-mutant IFNC2b (SEQ ID NO:132). These constructs were compared to the fusion protein constructs was measured on antigen-positive the Rituximab-wild type IFN fusion protein construct, cells (Daudi, CD20), there was generally very little attenu Rituximab-HC-L6-IFNC. IgG1 (as also used in the experi ation compared to the wild type IFNC2b version of the ments shown in FIGS. 6-8). The five mutant versions were Rituximab-IFNC2b fusion protein construct (FIGS. 11-12), R144A, A145G, R33A+YNS, R33A and R144A+YNS. The and thus the mutated interferons still possessed the ability to sequences of these variants are described below. The degree activate the IFN receptor on “on-target cells whilst having of expected reduced affinity for the type I interferon recep a greatly reduced ability to activate it on "off-target cells. tors based on previous characterization by others of IFN For example, the R33A+YNS version of the construct was mutants, and the amount of expected attenuation in inter only 2.2-fold (0.74/0.33=2.2) less active than the Ritux feron activity, are shown in Tables 6 and 7, above. imab-IFNC2b wild type construct on the antigen-positive 0314 FIGS. 9, 10 and Table 20 show the degree of (Daudi) cells. This was in contrast to the 277-fold (6100/ reduced interferon activity for each of these Rituximab 22–277: FIG.9) reduced activity on antigen-negative cells. attenuated IFNC2b fusion protein constructs relative to free, The mutations in the IFNC2b, in the context of the Ritux wild type IFNC.2b, on antigen-negative (i.e. CD20-negative) imab-IFNC2b fusion protein construct, caused a substan cells. The R144A mutant of the Rituximab-IFNC2b fusion tially greater attenuation of activity on antigen-negative cells protein construct (composed of SEQ ID NOS:282 (heavy than on antigen-positive cells. As a result, the Rituximab chain) and 276 (light chain)) showed 386-fold reduced HC-L6-IFNC2b (R33A+YNS) IgG1 fusion protein con interferon activity (2200/5.7–386). The A145G and R33A+ struct exhibited a Substantially greater antigen-specificity YNS versions (composed of the heavy chains of SEQ ID index (ASI, 1,700-fold) compared to Rituximab-HC-L6 NOS:284 and 286, respectively, each of which are combined IFNC2b IgG1 (10- to 14-fold) or free IFNC2b (1-fold, by with the light chain of SEQ ID NO:276) showed 491-fold definition), Suggesting that its off-target effects in vivo will (2800/5.7=491) and 1,071-fold (6100/5.7=1,071) reduced be substantially reduced. activity, respectively. FIG. 10 shows the degree of reduced 0316. Other Rituximab-IFNC2b constructs with muta interferon activity for the R144A+YNS fusion protein con tions in the IFNC2b portion also showed surprisingly little struct (composed of SEQ ID NOS:288 (heavy chain) and reduced activity on antigen-positive cells (FIGS. 11 and 12) 276 (light chain)) to be 303-fold (1700/5.6–303) relative to relative to their reduced potency on antigen-negative cells the Rituxumab fusion protein construct lacking the IFN (FIGS. 9 and 10). With the exception of the R33A version of mutations (Rituximab-HC-L6-IFNC. IgG1); since Ritux the fusion protein construct, discussed below, the attenuating imab-HC-L6-IFNC. IgG1 is 3.8-fold less potent on antigen mutations caused a 384-1,160-fold decrease in interferon negative cells than free, wild type IFNC2b (data from FIG. activity relative to free wild type IFNC2b on antigen 9; 22/5.7–3.8), this means that the R144A+YNS version of negative cells, but showed 0.23-1.2-fold of the potency of the fusion protein construct was 1,150-fold less potent than wild type IFNC.2b on antigen-positive cells. The R33A free, wild type IFNC. (303x3.8=1,150). The R33A version of mutated fusion protein construct, which had undetectable the fusion protein construct (composed of SEQID NOS:436 IFN activity in the absence of antibody-antigen targeting, (heavy chain) and 276 (light chain)) was attenuated to Such still showed significant activity in the presence of antibody a high degree that it showed no detectable activity in the targeting; the potency of the R33A version of the fusion non-targeted assay. protein construct was 1,620-fold lower than the same fusion US 2016/0367695 A1 Dec. 22, 2016 protein construct lacking this attenuating mutation in the Geneticin per 500 mL culture. The culture was incubated at on-target assay (340/0.21=1,620-fold attenuation). This is in 37° C., 5% CO and 120 rpm. After 7 days, the supernatant stark contrast to the at least 100,000-fold attenuation caused was harvested by centrifugation and was ready for purifi by the same mutation in the absence of antibody-based cation. targeting (FIG. 10). These results are summarized in Table 2O. Expression and Purification of Antibodies 0317. To determine whether this dramatic difference in the ability of the mutations in the IFNC. component of the 0322 Transient co-expression of heavy and light chains fusion protein constructs to Substantially reduce its activity in HEK293-6E cells (as described above) generated anti on antigen-negative cells as compared to antigen-positive bodies that were subsequently purified by protein A chro cells could be extended to other fusion protein constructs matography. Briefly, Supernatants derived from these trans targeting other antigens, antibodies targeting the multiple fections were adjusted to pH 7.4 before being loaded onto a myeloma antigen CD 38 (SEQ ID NO:131) were fused to HiTrap Protein A column (5 mL, GE Healthcare). The both wild type and attenuated forms of IFNC. and charac column was washed with 50 mL of 1xPBS (pH 7.4). Elution terized. Some of these experiments were performed using was performed using 0.1 M citric acid pH 2.5. The eluted the antibody G005 (De Weers et al. (U.S. Pat. No. 7,829, antibody was desalted using Zeba Desalting columns 673)); the sequences of the heavy and light chains for this (Pierce) into 1xPBS (pH 7.4). The antibodies were analyzed human antibody are shown as SEQ ID NOS:135 and 134, using SDS-PAGE. The concentration of the antibody was respectively. determined using the BCA assay kit (Pierce). Purification of Histidine-Tagged Proteins from Tissue Cul 0318. In addition, several novel human and rat antibodies ture Supernatants against CD38 were produced, as described below. 0323 Immobilized metal ion affinity chromatography (IMAC) was used to purify human and cynomolgous mon Development of Novel CD38 Antibodies key CD38 extracellular domain (ED) proteins from tissue Formatting CD38 Constructs for Expression culture Supernatants. Briefly, protein Supernatants were diluted in binding buffer (20 mM sodium phosphate, 0.5 M 0319. The extracellular domains (ECD) of human and NaCl, 30 mM imidazole, pH 7.4) before being loaded onto cynomolgus monkey CD38 proteins were each formatted to a HisTrapTMFF column (1 mL, GE Healthcare). The column include a cleavable N-terminal leader sequence, an Avi was washed with 5 mL of binding buffer (pH 7.4) and tagTM, a poly-histidine tag and a thrombin cleavage site to elution was performed using 20 mM sodium phosphate, 0.5 yield proteins SEQID NO:127 and 128 respectively. These M. NaCl, 500 mM imidazole, pH 7.4. The eluted proteins were back-translated into DNA sequences and synthesized were desalted and buffer exchanged using Amicon Ultra-15 de novo by assembly of synthetic oligonucleotides by meth centrifugal filter unit with Ultracel-10 membrane (Millipore) ods known by those with skill in the art. Following gene into 1xPBS (pH 7.4). The absorbance at 280 nm (As) of synthesis, the genes were subcloned into vector pTT5 the protein was assessed using a Nanodrop spectrophotom (Durocher, Nucleic Acids Research volume 30, number 2, eter and readings corrected using the predicted extinction pages E1-9, 2002) to yield constructs to produce soluble coefficients to determine protein concentrations. secreted forms of these proteins via transient expression in HEK293E cells (Durocher, supra). Biotinylation of Antigens for Phage Display 0324. The AvitagTM motifs of human and cynomolgus Construction of Vectors for Antibody Expression monkey CD38 EDs were biotinylated according to manu 0320 Heavy and light chain variable region sequences facturer's directions (Avidity LLC, Aurora, Colo.). Excess were subcloned into variants of the vector pTT5 containing unconjugated biotin was removed from the biotinylated either a human IgG1 heavy chain constant region (Such as proteins by desalting into 1xPBS using a 7 KD molecular Swissprot accession number PO1857), a human IgG4 heavy weight cut off (MWCO) Zeba spin column (Thermo Scien chain constant region (such as Swissprot accession number tific, Logan, Utah) according to manufacturers instructions. P01861 incorporating substitution S228P), human kappa Successful biotinylation of CD38 ED proteins was con constant region (Swissprot accession number P01834) or firmed using a combination of polyacrylamide gel electro human lambda region (Swissprot accession number phoresis and Western blotting. Western blots were probed P0CG05) to yield full length antibody chains. using Streptavidin-HRP (BD Biosciences, San Diego, Calif.) and developed using TMB (Sigma-Aldrich, St. Louis, Transient Expression of Constructs in HEK293-6E Cells Mo.). For each antigen, monomeric biotinylated CD38 ED 0321) HEK293-6E cells were cultured in complete cell was detected. growth media (1 L of F17 medium (InvitrogenTM), 9 mL of Pluronic F68 (InvitrogenTM), 2 mM Glutamine containing Generation of Anti-CD38 Antibodies by Phage Display 20% (w/v) Tryptone NI (Organotechnie R) with Geneticin 0325 FAbs that bind to both human and cynomolgus (50 mg/mL, InvitrogenTM) at 50 ul/100 mL culture). The day monkey CD38 EDs were isolated from a naive phagemid before transfection, cells were harvested by centrifugation library comprising approximately 2.5x10' individual and resuspended in fresh media (without Geneticin). The human FAb fragments. Methods of generating phage anti next day DNA was mixed with a commercial transfection body fragment libraries are discussed in “Phage display: A reagent and the DNA transfection mix added to the culture Practical Approach” (Eds. Clackson and Lowman: Oxford drop-wise. The culture was incubated overnight at 37° C. University Press, Oxford, UK) and “Antibody Phage Dis with 5% CO and 120 rpm without Geneticin. The next day, play Methods and Protocols” (Eds. O'Brien and Aitken; 12.5 mL of Tryptone was added along with 250 ul of Humana Press Inc, NJ 07512). Briefly, antibody heavy and US 2016/0367695 A1 Dec. 22, 2016 36 light chain variable regions were amplified based on RNA colonies. These colonies were used to inoculate 1 mL liquid from donor samples. Antibody heavy and light chain vari cultures to allow expression of FAb fragments for use in able regions were then inserted into phagemid vectors to screening experiments. generate a library of antibody fragments fused to a phage coat protein. The antibody library used herein was a high diversity naive phagemid library that expressed antibody ELISA-Based Screening of FAbs for CD38 Binding fragments in the Fab format. 0330. Each individual E. coli colony was used to express 0326 Anti-CD38 FAbs were isolated from the phage a FAb that could be screened for CD38 ED-binding activity. display library over the course of two panning campaigns Colonies were inoculated into 1 mL starter cultures (Supple (i.e. discrete phage display experiments with different mented with 100 g/mL carbenicillin and 2% glucose) in reagents or panning conditions). The general protocol fol 96-well deep-well plates (Costar) and incubated overnight at lowed the method outlined by Marks et al. (Marks, J. D. & 37° C. with shaking at 350 rpm (Innova R44 shaker; 1 inch Bradbury, A., 2004, Methods Mol Biol. 248, 161-76). orbit). These starter cultures were diluted 1:100 into a 1 mL 0327 Each phage display campaign involved three expression culture (2YT supplemented with 100 ug/mL rounds of panning. For each round, ~2.5x10" phage par carbenicillin) and grown to an optical density (600 nm) of ticles were blocked by mixing 1:1 with blocking buffer (4% 0.5-0.8. FAb expression was induced by adding isopropyl skim milk in PBS, pH 7.4) and incubating for 1 hr at room beta-D-thiogalactopyranoside (IPTG) to a final concentra temperature. The blocked phage library was then pre-de tion of 1 mM. Cultures were incubated at 25° C. for 16 hrs. pleted for any biotinylated protein tag motif binders used in 0331 FAb samples were prepared by harvesting cells by panning through incubation for 45 mins with 50-200 pmols centrifugation (2,000 g, 10 mins) and performing a of an irrelevant antigen containing an identical biotinylated lysozyme extraction. The cell pellet was resuspended in 200 tag motif. Tag- and Streptavidin-binders were captured by uL of lysis buffer (160 ug/mL lysozyme, 10 g/mL RNase A, adding an excess (75-300 uL) of streptavidin-coated Dyna 5 lug/mL DNase and complete protease inhibitors (Roche, beads (Invitrogen), which were blocked as described for the Nutley, N.J.)) and shaken at 400 rpm for 30 minutes at 21° library. The beads (including tag- and streptavidin-binders C. Following addition of a further 100 ul of lysis buffer, the attached to them) were immobilized using a magnet and reactions were incubated for a further 30 minutes as discarded. described previously. Clarified lysates were isolated follow 0328 Library panning was conducted by mixing the ing centrifugation at 3,000 g for 10 minutes and stored at 4 blocked and pre-depleted library with 50-200 pmols of C. until required. biotinylated recombinant CD38 ED in a 2 mL microcentri 0332 To screen by enzyme-linked immunosorbent assay fuge tube and rotating for 2 hrs at room temperature. Then, (ELISA) for human CD38 ED-binders derived from the 100 uL of streptavidin-coupled Dynabeads (Invitrogen, phage display biopanning, human CD38 extracellular Carlsbad, Calif.) were added and the mixture was incubated domain (ED) (produced in HEK 293-6E cells and bioti a further 15 minutes as described previously. Non-specifi nylated as described above) was captured on Streptavidin cally bound phage were removed using a series of washes. coated ELISA plates (Nunc) at 1 lug/mL. Plates were then Each wash involved pulling the bead complexes out of the washed and individual FAb samples (prepared as described Solution onto the tube wall using a magnetic rack, aspirating above) were added to individual wells on the ELISA plates. the Supernatant and then re-suspending the beads in fresh FAbs were allowed to bind the captured CD38 ED for an wash buffer. This was repeated multiple times with either hour at room temperature and then washed three times with PBS wash buffer (1XPBS with 0.5% skim milk) or PBS-T PBS-T (1xPBS supplemented with 0.1% Tween R20). FAbs wash buffer (1xPBS supplemented with 0.05% TWEEN-20 that bound to CD38 ED were detected by incubation for 30 Sigma-Aldrich, St. Louis, Mo. and 0.5% skim milk). minutes at room temperature with an anti-V5-HRP conju Phage that remained bound after the washing process were gated antibody (Invitrogen, Carlsbad, Calif.) to detect the V5 eluted from the biotinylated-CD38 ED-bead complexes by tag fused to the C-terminus of the FAb heavy chain. Plates incubation with either a twenty-fold excess of non-bioti were washed to remove unbound antibody and the assay nylated CD38 ED for 1 hr at room temperature or 0.5 mL of signal developed by incubation with 50 uL 3.3',5,5'-Tetram 100 mM triethylamine (TEA) (Merck Chemicals, Darm ethylbenzidine (Sigma-Adrich, St. Louis, Mo.) and quench stadt) for 20 mins at room temperature. TEA-eluted output ing with 50 uL 1 MHC1. Assay signals were read at A450 phage were neutralized by the addition of 0.25 mL of 1 M nm using a microplate reader (BMG Labtech). Results were Tris-HCl pH 7.4 (Sigma-Aldrich, St. Louis, Mo.). expressed as the raw A450 nm value, where any signal 0329. At the end of the first and second rounds of 2-fold greater than the average assay background was panning, the output phage were added to a 10 mL culture of defined as positive'. exponentially growing TG1 E. coli (2x yeast-tryptone (2YT) 0333. In later assays FAb cross-reactivity with cynomol growth media) and allowed to infect the cells during a 30 gus monkey CD38 ED was assessed by coating biotinylated minute incubation at 37° C. without shaking, then with cynomolgus monkey CD38 ED onto streptavidin coated shaking at 250 rpm for 30 additional minutes. The ELISA plates and proceeding as described above. Plasmids phagemids encoding the phage display output were then encoding FAbs cross-reactive with both human and cyno rescued as phage particles following a standard protocol molgus monkey CD38 ED were isolated and sequenced. Of (Marks, J. D. & Bradbury, A., 2004, Methods Mol Biol, 248, approximately 1,000 FAbs screened for binding to human 161-76). At the end of the third panning round, TG1 cells and cynomolgus monkey CD38 ED, six genetically unique were infected with output phage and were plated on 2YT FAbs were identified. Table 21 summarises the FAb agar (Supplemented with 2% glucose and 100 ug/mL car sequence data obtained. The variable regions of Some of benicillin) at a sufficient dilution to produce discrete E. coli these antibodies are shown in FIG. 13. US 2016/0367695 A1 Dec. 22, 2016 37 TABLE 21 glycol (HybriMaxTM, Sigma-Aldrich, Germany), seeded at 100,000 cells per well in 96-well microtiter plates and grown Campaign Number FAb name V sequence V.K.V. sequence in DMEM medium supplemented with 10% fetal bovine 1 X910/12 SEQ ID NO: 395 SEQ ID NO: 394 serum and HAT additive for hybridoma selection (Kilpatrick 1 X913/15 SEQ ID NO:397 SEQ ID NO: 396 et al., 1998, supra). 2 X355/01 SEQ ID NO: 421 SEQ ID NO: 420 2 X355/02 SEQ ID NO:391 SEQ ID NO: 390 Screening Hybridoma Supernatants for Human and 2 X355/04 SEQ ID NO: 423 SEQ ID NO: 422 2 X355/07 SEQ ID NO:393 SEQ ID NO:392 Cynomolgus Monkey CD-38 Cross-Reactivity 0339 Duplicate 100 uL samples of each hybridoma 0334 All FAbs were converted into IgG1 format by Supernatant were coated onto separate wells of a maxisorp cloning into the pTT5 vectors (described above), expressed ELISA plate (Nunc Plasticware. Thermo Scientific, Roch in HEK293-6E cells and the resulting IgGs purified by ester, N.Y. 14625, USA) through incubation at room tem protein A affinity chromatography as described above. perature for an hour. Plates were washed three times in 1xPBS-T and subsequently blocked by addition of 2% Assessing Binding of IgGs to Human CD38 Positive Cell BSA/1xpBS. Following incubation for 1 hour at room Line RPMI-8226 temperature, plates were washed as described previously. To one well of each rat antibody duplicate well was added 0.1 0335. The ability of the phage derived antibodies to bind ug of biotinylated human CD38 in a final volume of 100 LL the model human CD38 positive myeloma cell line RPMI 1xPBS. To the second well of each rat antibody duplicate 8226 (obtained from the Health Protection Agency Culture well was added 0.1 ug of biotinylated cynomolgus monkey Collections, Porton Down, Salisbury, SP4 OJG, UK) in flow CD38 ED in a final volume of 100 uL 1XPBS. Plate wells cytometry-based assays was tested. Briefly, viable RPMI were washed as described previously prior to detection of 8226 cells (2x10, as judged by trypan blue exclusion) were bound biotinylated CD38 ED using a Streptavidin-HRP incubated with each antibody or with a human IgG isotype conjugate (BD Biosciences, San Diego, Calif.). Plates were control antibody preparation (Sigma-Aldrich, St. Louis, washed as above to remove unbound Streptavidin-HRP Mo.) at various concentrations in 100 ul of FACS buffer conjugate and the assay signal developed by incubation with (PBS plus 1% fetal calf serum, FCS) in 96 well plates for 20 50 uL 3.3',5,5'-Tetramethylbenzidine (Sigma-Aldrich) and minutes on ice in the dark. Cells were washed twice with quenching with 50 uL 1 M HC1. Assay signals were read at FACS buffer before incubation for 20 minutes in 100 ul of As nm using a microplate reader (BMG Labtech, Cary, FACS buffer containing goat anti-human IgG (Fc-specific, N.C.). Of the 15 hybridoma supernatants tested, all fifteen conjugated to fluorescein isothiocyanate, FITC: Sigma-Al bound human CD38 ED and seven bound cynomolgus drich, St. Louis, Mo.). After washing with FACS buffer, cells monkey CD38 ED (Table 22) as determined by ELISA. The were resuspended in FACS buffer and analysed for antibody cross-reactive antibodies are referred to as R5D1, R7F11, binding by flow cytometry on a FACS Canto (BD Biosci R5E8, R10A2, R1OB10, R3A6 and R7H11. ences, San Diego, Calif.) using EV, side scatter and FL-1 gating. Results are expressed as mean fluorescent intensity Flow Cytometry Binding of Rat Antibodies to Human CD38 (MFI) plotted against protein concentration (FIG. 14). Positive Cell Line RPMI-8226 Generation of Anti-CD38 Antibodies by Genetic (0340 Viable RPMI-8226 cells (2x10, as judged by Immunization trypan blue exclusion) were incubated with 100 uL of rat hybridoma Supernatant for 20 minutes on ice in the dark. 0336 Monoclonal antibodies against human CD38 ED Cells were washed twice with FACS buffer (1xPBS plus 1% were generated by genetic immunization with corresponding FCS) before incubation for 20 minutes in 100 ul of FACS conventional protein immunization of rats. For genetic buffer containing anti-rat IgG-FITC conjugate (Sigma-Al immunization, the DNA sequence of human CD38 ED is drich). After washing cells in FACS buffer, they were provided in SEQID NO: 129. The corresponding conceptu resuspended in FACS buffer and analysed for antibody ally translated protein sequence is given in SEQID NO: 130. binding by flow cytometry on a FACS Canto (BD Biosci The DNA sequence of SEQ ID NO:129 was cloned into a ences, San Diego, Calif.) using EV, side scatter and FL-1 plasmid for genetic immunization using restriction enzyme gating. Results were expressed as mean fluorescent intensity technology. Expression of the resulting plasmid allowed the (MFI). Of the 15 rat antibodies exhibiting positive binding secretion of soluble CD38 ED tagged by a c-myc epitope at to human CD38 ED by ELISA, five showed weak or the N- or C-terminus. The c-myc epitope was utilized to negligible binding to CD38 expressed on the human confirm expression of CD38 ED. myeloma cell line RPMI-8226 by FACS (Table 22). 0337. Rats were then immunized six times with the plasmid using a Helios gene gun (Bio-Rad, Germany) according to a published procedure (Kilpatrick et al., TABLE 22 Hybridoma 17: 569-576, 1998). One week after the last Binding to application of the immunization plasmid, each rat was Cynomolgus FACS binding to boosted by intradermal injection of untagged recombinant Rat Binding to Human monkey RPMI-8226 cells human CD38 ED. Untagged human CD38 ED for this antibody CD38 ED (ELISA) CD38 ED (ELISA) (MFI) purpose was produced by removing the protein tags from R3A6 Y Y 279 RSD1 Y Y 122O7 SEQ ID NO:127 by thrombin cleavage followed by purifi RSE8 Y Y 10618 cation over a size exclusion column. R7F4 Y N 310 0338 Four days later, the rats were sacrificed and their R7F11 Y Y 11897 lymphocytes fused with myeloma cells using polyethylene