US00899.9335B2 (12) United States Patent (10) Patent No.: US 8,999,335 B2 Cojocaru et al. (45) Date of Patent: Apr. 7, 2015 (54) COMPOSITIONS AND METHODS FOR (56) References Cited TREATMENT OF DRUG RESISTANT MULTIPLE MYELOMA U.S. PATENT DOCUMENTS (75) Inventors: Gad S. Cojocaru, Ramat HaSharon (IL); 6,979,557 B2 12/2005 Isogai et al. Haiming Chen, West Hills, CA (US); 7,411,051 B2 8, 2008 Rosen et al. 2003/0219741 A1 11/2003 Isogai et al. James Berenson, Beverly Hills, CA 2004/0241803 A1 12/2004 Rosen et al. (US) 2006, OO68405 A1 3/2006 Diber et al. (73) Assignee: Compugen Ltd., Tel Aviv (IL) 2007, OO15163 A1 1/2007 Isogai et al. 2007/0O37741 A1 2/2007 Baldwin et al. (*) Notice: Subject to any disclaimer, the term of this 2007/0224663 A1 9, 2007 Rosen et al. patent is extended or adjusted under 35 2008/0057519 A1* 3/2008 McWhirter .................. 435/723 2010, O144538 A1 6, 2010 Belouchi et al. U.S.C. 154(b) by 0 days. 2010/0286.048 A1 11/2010 Rosen et al. (21) Appl. 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A6 IK39/00 (2006.01) ER for AU 2009325878 mailed Jul. 3, 2013. A 6LX39/395 (2006.01) Genbank accession No. AAH6O775. 1; from 2002. A6 IK3I/713 (2006.01) Genbank accession No. AAH 19600.1; from 2002. C07K 6/30 (2006.01) NCBI accession No. NP 689893.1; from 2007. CI2N IS/II3 (2010.01) Genbank Accession No. EAXO4974. 1; from 2001. GOIN33/574 (2006.01) Genbank Accession No. EAXO4975. 1; from 2001. Office action for corresponding Chinese Application No. A6 IK 45/06 (2006.01) 200980 155730.6. mailed on Mar. 15 2013. CI2O I/68 (2006.01) Office action for corresponding Chinese Application No. GOIN33/68 (2006.01) 200980 155730.6. mailed on Oct. 31 2013. (52) U.S. Cl. Office Action for IL 213300 mailed Mar. 23, 2014. CPC ........... A61K.39/3955 (2013.01); A61 K3I/713 (2013.01); A61 K39/39558 (2013.01); A61 K * cited by examiner 2039/505 (2013.01); C07K 16/3061 (2013.01); C07K 2317/73 (2013.01); C07K 2317/732 Primary Examiner — Daniel C Gamett (2013.01); C07K 2317/77 (2013.01); C07K (74) Attorney, Agent, or Firm — Graeser Associates 2319/30 (2013.01); C12N 15/1138 (2013.01); International Inc.; Dvorah Graeser CI2N 2310/11 (2013.01); C12N 2310/12 (2013.01); C12N 23 10/14 (2013.01); G0IN (57) ABSTRACT 33/57426 (2013.01); A61K 45/06 (2013.01): This invention relates to a novel target for production of CI2O I/6886 (2013.01); G0IN33/6893 immune and non-immune based therapeutics and for disease (2013.01) diagnosis. More particularly, the invention provides thera (58) Field of Classification Search peutic antibodies against TMEM154 antigens, which are dif CPC ........... A61 K31/713; A61K 2039/505; A61 K ferentially expressed in cancer, and diagnostic and therapeu 39/00; G01N 33/6893; C07K 16/3061; C07K tic usages, wherein the cancer is relates to multiple myeloma, 2317/73; C07K 2317/732; C07K 2317/77; including multiple myeloma precursor diseases. This inven C07K 2319/30; C07K 16/28: C07K 16/30; tion further relates to extracellular domains of TMEM154 C07K 2317/55; C12N 15/1138; C12N 2310/11; proteins and variants, and therapeutic usages thereof. C12N 2310/12: C12N 2310/14 See application file for complete search history. 11 Claims, 20 Drawing Sheets U.S. Patent Apr. 7, 2015 Sheet 5 of 20 US 8,999,335 B2 ATGCAGGCTCCCCGCGCAGCCCAGCTCGCCCTGGGACGCGCCGTCCCGCGGCCGG GGAAT AGAGGAATTAGAAAACCAGGAGATACAAC GTGGAATCGAAAGACCAAATAAA GTGACACCAAGCACATT GCGCAGTGACCACAAAGAAACATTAAATGCAAAATAAAT CTACCAACTTTGCTCCGGAGAAAATCAGTTAGAGTTATACTGAGGTGAACCCATTG ATTATGGTCCTCTACTTACCGTGGATTCCTGCAACAACAAAAAGAAAAAGAA CAAACAAGAACCCAGCCAAGGATCTCAGAGGCTTACAGACAATGAACTGGGAAG GAAAACGTGAAAGTCCCTATTTTGAGGAAGATACACCCCTGTATGGAAATTGAAAGGA AGAGCTTGAAAAGGATGAACAGCAGAAAGAAAGCCGACTTGAATGTACCTACCTT GAAGGAAGAGAAGGAATCAAATCACAACCCAAGGACAGTGAACCGACACAAAGACGAT GACGACAAGAA FG. 3 MOAPRAALVFALVIALVPVGRGNYEELENSGDTTVESERPNKVTIPSTFA AVTIKETLNANINSTNFAPDENOLEFILMVLIPLLVLLLLSWFLATY YKRKRTKOEPSSOGSQSALOTYELGSENVKVPFEEDTPSVMEEMEELD KWMNSMNRNADFECLPLKEEKESNHNPSDSESDYKDDDDK FG. 4 U.S. Patent Apr. 7, 2015 Sheet 6 of 20 US 8,999,335 B2 FG 5A FG FIG. 6A FIG. 6B U.S. Patent Apr. 7, 2015 Sheet 7 of 20 US 8,999,335 B2 FIG 7A FIG. 7B FIG. 8A FIG. 8B FG. 8C U.S. Patent Apr. 7, 2015 Sheet 8 of 20 US 8,999,335 B2 FIG. 9A - 1 FG. 9A - 2 FIG. 9A-3 FIG 9A" 4 U.S. Patent Apr. 7, 2015 Sheet 9 of 20 US 8,999,335 B2 FG. 9B-1 FIG. 9B-2 FG 9 B-3 U.S. Patent Apr. 7, 2015 Sheet 10 of 20 US 8,999,335 B2 FIG. 9C - 1 FIG. 9C - 2 FG. 9C-3 U.S. Patent Apr. 7, 2015 Sheet 12 of 20 US 8,999,335 B2 U.S. Patent Apr. 7, 2015 Sheet 13 of 20 US 8,999,335 B2 75 1. 5 O 1.l 57O2 O5O5 2 5 as1. NE Y-E En YE NE nE NE Y.E nE NE H d O O O O O O Sa 5 to5 c.s is3 is 9is &is 3is TM21-ANTIBODY 103 100 FG. 12B ANNEXIN V U.S. Patent CNNNNNNNN í,y^^) IZUZ |u/370GIZWI |u/370OIIZWI FG, 13C U.S. Patent Apr. 7, 2015 Sheet 19 of 20 US 8,999,335 B2 U.S. Patent Apr. 7, 2015 Sheet 20 of 20 US 8,999,335 B2 s irer sS hs 5 O H. H. - races cr (r. O. O. He 48 a CN YO C 5 N. N. CN? electice a s CD ti - O C - s E. C-5 - 2 O CP s re C C C C C co c S. S2 CO d s CN C (TOLNOOO %) ALITIS WIN TO US 8,999,335 B2 1. 2 COMPOSITIONS AND METHODS FOR “magic bullets' that would deliver toxic agents. Such as drugs, TREATMENT OF DRUG RESISTANT toxins, enzymes and radioisotopes, specifically to the dis MULTIPLE MYELOMA eased site and leaving the non-target normal tissues unaf fected. Indeed, antibodies, and in particular antibody frag FIELD OF THE INVENTION ments, can function as carriers of cytotoxic Substances Such as radioisotopes, drugs and toxins. Immunotherapy with Such The present invention relates to compositions and methods immunoconjugates is more effective than with the naked for treatment of drug resistant cancer, and in particular, but antibody. not exclusively, to TMEM154 related polypeptides and poly With the advent of antibody engineering, small molecular nucleotides as Suitable targets and/or candidates for develop 10 weight antibody fragments exhibiting improved tumor pen ment of therapeutics and diagnostics, particularly for cancer etration have been generated. Such antibody fragments are therapy and treatment of immune related disorders. often conjugated to specific cytotoxic molecules and are designed to selectively deliver them to cancer cells. Still, solid BACKGROUND OF THE INVENTION tumors remain a formidable challenge for therapy, even with 15 immunoconjugated antibody fragments. Tumor antigens are ideally positioned as biomarkers and The new wave of optimization strategies involves the use of drug targets, and they play a critical role in the development of biological modifiers to modulate the impediments posed by novel strategies for active and passive immunotherapy agents, solid tumors. Thus, in combination to antibodies or their to be used as stand-alone therapies or in conjunction with conjugated antibody fragments, various agents are being used conventional therapies for cancer. Tumor antigens can be to improve the tumor blood flow, enhance vascular perme classified as either tumor-specific antigens (TSAs) where the ability, lower tumor interstitial fluid pressure by modulating antigens are expressed only in tumor cells and not in normal stromal cells and extracellular matrix components, upregu tissues, or tumor-associated antigens (TAAS) where the anti late expression of target antigens and improve penetration gens are overexpressed in tumor cells but nonetheless also and retention of the therapeutic agent. present at low levels in normal tissues. 25 Immunotherapy with antibodies represents an exciting TAAS and TSAS are validated as targets for passive (anti opportunity for combining with Standard modalities, such as body) therapy as well as active immunotherapy using strate chemotherapy, as well as combinations with diverse biologi gies to break immune tolerance and stimulate the immune cal agents to obtain a synergistic activity. Indeed, unconju system. The antigenic epitopes that are targeted by these gated mAbs are more effective when used in combination therapeutic approaches are present at the cell Surface, over 30 with other therapeutic agents, including other antibodies. expressed in tumor cells compared to non-tumor cells, and are Passive tumor immunotherapy uses the exquisite specific targeted by antibodies that block functional activity, inhibit ity and lytic capability of the immune system to target tumor cell proliferation, or induce cell death. specific antigens and treat malignant disease with a minimum There are a growing number of tumor-associated antigens of damage to normal tissue.