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(12) Patent Application Publication (10) Pub. No.: US 2014/0294765 A1 Cojocaru Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2014/0294765 A1 Cojocaru Et Al US 20140294765A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0294765 A1 Cojocaru et al. (43) Pub. Date: Oct. 2, 2014 (54) LSRANTIBODIES, AND USES THEREOF FOR Publication Classification TREATMENT OF CANCER (51) Int. Cl. (71) Applicant: Compugen Ltd., Tel Aviv-Yafo (IL) C07K 6/28 (2006.01) (72) Inventors: Gad S. Cojocaru, Ramat HaSharon (IL); GOIN33/574 (2006.01) Liat Dassa, Yehud (IL); Galit Rotman, A63/675 (2006.01) Herzliyya (IL); Ofer Levi, Moshav A6II 45/06 (2006.01) Mesisraelat Zion (IL); Andrew Pow, San A 6LX39/395 (2006.01) Francisco, CA (US); Shirley Sameach-Greenwald, Kfar Saba (IL); (52) U.S. Cl. Zurit Levine, Herzliyya (IL) CPC ................. C07K 16/28 (2013.01); A61K 45/06 (2013.01); A61 K39/3955 (2013.01); A61 K (73) Assignee: COMPUGEN LTD., Tel Aviv-Yafo (IL) 31/675 (2013.01); G0IN33/57492 (2013.01) USPC ..... 424/85.2: 424/139.1; 424/85.7; 424/85.6; (21) Appl. No.: 14/361,571 424/85.5; 424/133.1; 424/136.1; 435/7.23 (22) PCT Fled: Jun. 19, 2013 (86) PCT NO.: PCT/IL2013/050527 (57) ABSTRACT S371 (c)(1), (2), (4) Date: May 29, 2014 This invention relates to antibodies and antigen binding frag Related U.S. Application Data ments and conjugates containing same, and/or alternative (60) Provisional application No. 61/662,470, filed on Jun. scaffolds, specific for LSR molecules, which are suitable 21, 2012. drugs for immunotherapy and treatment of specific cancer. Patent Application Publication Oct. 2, 2014 Sheet 1 of 30 US 2014/0294765 A1 FG. A F.G. B 2 2 -72kDa -72ka B: AN B: A.N SR FAG (Abnova) F.G. 1C FG D of 2ka B: ANSR B: A.N SR (Abcam) (Sigma) Patent Application Publication Oct. 2, 2014 Sheet 2 of 30 US 2014/0294765 A1 s C were Patent Application Publication Oct. 2, 2014 Sheet 3 of 30 US 2014/0294765 A1 F.G. 3 1. HEK293T prESpuro3 2. HEK293T prESpuro3 human LSR skip4 Flag FG. 4 8 8 §§ ? 1. HEK293T prESpuro3? 2. HEK293T plRESpuro3 cyno WTLSR-Flag 3. HEK293 pirESpuro3 cyno skip4 SR Flag Patent Application Publication Oct. 2, 2014 Sheet 4 of 30 US 2014/0294765 A1 70. F.G. 5 C-O-K 2, CHO-K pcDNA3.t mouse WTSR Flag 3. HEK293 pcDNA3. 4. HEK293 peoNA3.1 mouse WTLSR Flag F.G. 6 2 3 4 5 6 7 8 9 2 1. HEK293T prESpuro3 human W. SR Flag + scrambled siRNA 2. HEK293T pirESpuro3 human WTSR Flag-LSR siRNA Patent Application Publication Oct. 2, 2014 Sheet 5 of 30 US 2014/0294765 A1 FG. 8A AN AG FG. 8B ANT SR (Abcam) FG. 8C ANTI LSR (Abnova) FG. 8D ANTSR (Sigma) Patent Application Publication Oct. 2, 2014 Sheet 6 of 30 US 2014/0294765 A1 06'0|+ Patent Application Publication Oct. 2, 2014 Sheet 7 of 30 US 2014/0294765 A1 Patent Application Publication Oct. 2, 2014 Sheet 8 of 30 US 2014/0294765 A1 Patent Application Publication Oct. 2, 2014 Sheet 9 of 30 US 2014/0294765 A1 Patent Application Publication Oct. 2, 2014 Sheet 10 of 30 US 2014/0294765 A1 Patent Application Publication Oct. 2, 2014 Sheet 11 of 30 US 2014/0294765 A1 NAO) Patent Application Publication Oct. 2, 2014 Sheet 12 of 30 US 2014/0294765 A1 EXPRESSION OF SR IN CHO RECOMBINANT CELS g CHO-K cDNA31 CHO-KimSR F.G. 14B Patent Application Publication Oct. 2, 2014 Sheet 13 of 30 US 2014/0294765 A1 a\ six-w wr. sea s well Patent Application Publication Oct. 2, 2014 Sheet 14 of 30 US 2014/0294765 A1 FG 16A 38 1. HepG2C3A scrambled siRNA 2. HepG2C3A+LSR siRNA F.G. 6B 38 29-hscramed Si RNA 2. - 29-SR S RNA Patent Application Publication Oct. 2, 2014 Sheet 15 of 30 US 2014/0294765 A1 KEY - Scrambled siRNA+ Culture medium - - - SR SIRNA+Culture medium - Scrambled siRNA+808 Sup --- SR siRNA+808 Sup 100 101 102 103 10? FL-H FG. 17A KEY. ... Scrambled siRNA+Culture medium - - - SR S RNA+ Culture medium - Scrambled siRNA+8C8 Sup ---.SR siRNA+808 Sup F.G. 17B Patent Application Publication Oct. 2, 2014 Sheet 17 of 30 US 2014/0294765 A1 SEQID NO. 219,8C3 Light chain. DNA sequence (381 bp) Leadersequence-FR1-CDR-FR2-CDR2-FR3-CDR3-FR4 AGATGTCCCGCCAGTTCCTGGTCCCTGIGCTCGTTTCAAGGACCAGATGIGATATCCAGATGACACAGACACATCCTCCCTGTCTGCCTCTCT GGGAGACAGAGTCACCATCAGTGCAGGGCAAGTAGGACAAGCAAATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGA TCTACACACATCAAGAAACAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACTATAGCAACCTGGAACAAGA AGATATTGCCACTTACTTTTGCCAACAGGAAGAAGCACCGGGACGTTCGGGGAGGCACCAAGCTGGAAATCAAA SEQID NO. 220,8C3 Light chain. Amino acids sequence (27 AA) leader sequence-FR1-CDR-FR2-CDR2-FR3-CDR3-FR4 MMSSAGFIGLLCFQGFRCDIOMTOTSSLSASLGDRYTSCRASGDISNYLNWYOOKPDGTVKLYYTSRLHSGVPSRFSGSGSGTDYSTSNLEQEDIAT YFCQQDSKHPWTFGGGTKLEK SEQID NO. 230, 8C3 Light CDR DNA secuence AGGGCAAGCAGGACAAGCAATAAAAC SEQID NO. 231, 8C3 Light CDR2 DNA sequence TACACATCAAGAAACTA SEQDNC; 232, 8C3 Light CDR3DNA sequence CAACAGGAAGAAGCATCCGTGGACG SEQD NO. 233,8C3 Light chain CDR1 amino acid sequence RASQDSNYLN SEQID NO. 234,8C3 Light chain CDR2 aminoacidsecuence YTSRS SEQDNC; 235,8C8 Light chain CDR3 aminoacid sequence QQDSKHPWT FG. 8B Patent Application Publication Oct. 2, 2014 Sheet 18 of 30 US 2014/0294765 A1 F.G. 19A FG 9B Patent Application Publication Oct. 2, 2014 Sheet 19 of 30 US 2014/0294765 A1 ( SNOO ( Ed +---+---+ ZZ'$!)TOHINOOEA||ISOd– (/9(†7)YJS?~ SNOO Patent Application Publication Oct. 2, 2014 Sheet 20 of 30 US 2014/0294765 A1 H—SIB)($*$)/,'$1GB£7||— NOO Patent Application Publication Oct. 2, 2014 Sheet 21 of 30 US 2014/0294765 A1 C c\) on oo N to Lo sit or c\, f : - - - - - - - - - - (eaS5/ N3Old) - W Patent Application Publication Oct. 2, 2014 Sheet 22 of 30 US 2014/0294765 A1 EXP, OO4 OO2 2h 8h 82 8: 82 8h 82h 8, 8.2h 8h 82, 8, 82 8 82h 8 8h LPS+FNg FG. 22A EXP2 O25 0.2 0.5 0.1 0.05 EXP3 CO2 0.15 F.G. 22C Patent Application Publication Oct. 2, 2014 Sheet 24 of 30 US 2014/0294765 A1 EXP, OOO2 O.0015 OOO OOOO5 O 2h 8, 8.2h 8h 182h 8 82 8h 82 8 82h 8 82h 8, 182 8h 8th PS+FNg FG. 24A EXP2 0.0035 0.003 COO25 OOO2 0.005 0.00 COOO5 O 2h 8h 182h 8, 182h 8, 182h 8, 182h 8 82h 8h 82h 8, 1828, 18h LPS+IFNg F.G. 24B EXP3 O.O2 OOO OOO OOOO3 FG. 24C Patent Application Publication Oct. 2, 2014 Sheet 25 of 30 US 2014/0294765 A1 60 ZZ Cyno LSR Hek O GFP M Hek FG. 25 Patent Application Publication Oct. 2, 2014 Sheet 26 of 30 US 2014/0294765 A1 (2) ueeNO99 808 Patent Application Publication Oct. 2, 2014 Sheet 27 of 30 US 2014/0294765 A1 00\0\]['00 OuÁSTIO'#0–IIS---- Patent Application Publication Oct. 2, 2014 Sheet 29 of 30 US 2014/0294765 A1 s (IWW) 69CO Patent Application Publication Oct. 2, 2014 Sheet 30 of 30 US 2014/0294765 A1 |u/ºffzIHonD.Inø TT3M/STT-30000GZZ TT3M/S?300000GDJ 08 OŽ 0I 0 NOI8-N % WOO % + 69CO HST-£67(1700-£6? |u/ºfizIHonD.Inø 08 9 0 09 09 07 08 WO O % -- 69CO WOO % -- 69CO US 2014/0294765 A1 Oct. 2, 2014 LSR ANTIBODIES, AND USES THEREOF FOR novel agents that are capable of modulating costimulatory TREATMENT OF CANCER signals, without compromising the immune systems ability to defend against pathogens, are highly advantageous for FIELD OF THE INVENTION treatment and prevention of such pathological conditions. 0001. This invention relates to LSR (lipolysis stimulated 0006 Costimulatory pathways play an important role in lipoprotein receptor)-specific antibodies, antibody frag tumor development. Interestingly, tumors have been shown to evade immune destruction by impeding T cell activation ments, conjugates and compositions comprising same, for through inhibition of co-stimmulatory factors in the treatment of cancer. B7-CD28 and TNF families, as well as by attracting regula tory T cells, which inhibit anti-tumor T cell responses (see BACKGROUND OF THE INVENTION Wang (2006) Immune Suppression by Tumor Specific CD4+ 0002 Naive T cells must receive two independent signals Regulatory T cells in Cancer. Semin Cancer. Biol. 16:73-79; from antigen-presenting cells (APC) in order to become pro Greenwald, et al. (2005) The B7 Family Revisited. Ann. Rev. ductively activated. The first, Signal 1, is antigen-specific and Immunol. 23:515-48: Watts (2005) TNF/TNFR Family occurs when T cell antigen receptors encounter the appropri Members in Co-stimulation of T Cell Responses Ann. Rev. ate antigen-MHC complex on the APC. The fate of the Immunol. 23:23-68; Sadum, et al. (2007) Immune Signatures immune response is determined by a second, antigen-inde of Murine and Human Cancers Reveal Unique Mechanisms pendent signal (Signal 2) which is delivered through a T cell of Tumor Escape and New Targets for Cancer Immuno costimulatory molecule that engages its APC-expressed therapy. Clin. Cane. Res. 13(13): 4016-4025). Such tumor ligand. This second signal could be either stimulatory (posi expressed co-stimulatory molecules have become attractive tive costimulation) or inhibitory (negative costimulation or cancer biomarkers and may serve as tumor-associated anti coinhibition). In the absence of a costimulatory signal, or in gens (TAAS). Furthermore, costimulatory pathways have the presence of a coinhibitory signal, T-cell activation is been identified as immunologic checkpoints that attenuate T impaired or aborted, which may lead to a state of antigen cell dependent immune responses, both at the level of initia specific unresponsiveness (known as T-cell anergy), or may tion and effector function within tumor metastases. As engi result in T-cell apoptotic death. neered cancer vaccines continue to improve, it is becoming 0003 Costimulatory molecule pairs usually consist of clear that such immunologic checkpoints are a major barrier ligands expressed on APCs and their cognate receptors to the vaccines’ ability to induce therapeutic anti-tumor expressed on T cells.
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