(12) 1N’1111'1RNA'1'10NAL AI’I’LICA'I'EUN FUELISHEE) UNEHCR 1111111 PA'I‘EN'E‘ (:1:§()1’1€RA'I'10N 'I'REA'I'Y (NET)
`(1.9) W’Dfld Inteflectua} Pmperty
`Q
`Organizatinn
`Internati (131211. Burs an
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`11111111 11111111
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`
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`111111111111111111111111111111111111111L 11111111111111111111111111111111111
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`(413) 1nternat10na1 Pubiicatinn Date
`28 Slay 21115 (211.115.2815)
`
`W1P€31PGT
`
`(111) Internatimlal 13111111931101: Number
`
`11111 20151111776241 A1
`
`(51}
`
`InternatinnaE E‘atent Chasgification:
`CIZN 25/63 (2006.01):
`
`(21}
`
`International Agxlpiicatiun Number:
`
`PCP/US$21) 1 11/066920
`
`(22}
`
`(25}
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`(26}
`
`(30}
`
`(71)
`
`(’73)
`
`Entermztinmii Fiiing Date:
`21 November 2014 (21.1 1.2014)
`
`Fiiing Language:
`
`Pesbiiwaiim Language:
`
`Priority Data:
`61/907,860
`22 Novambezr 2013 (22.11.2013)
`
`Engliah
`
`English
`
`US
`
`[US/US]; One (Zireenway
`[ENA'E‘REX INC.
`Applicants:
`13132.91, Suite 930, “01181011, 1}; 77019 (US). 11111-1 EQARD
`OF REGENTS, THE UNIVERSITY OF TEXAS SYS-
`TEM: {US/US]; 201 Vv’ust 7th Street, Austin, TX 78701
`(US).
`
`'I‘UBVARU, Frank; 3028 Pasen (111310, Box
`Inventors:
`1555, Ranch] Santa Fe, CA 921157 (11S). FUEYG—NiAR—
`(12111111111), Juan; 5519 Edith Street, Houston, TX '
`.'
`
`
`(US).
`(iaNfiEU—RiANZANU, Cantfleiarisa; 5519 1:7 1.
`Sinai, 1101151011, TN 77081 (US), CG‘NRAD, Charks;
`
`15911 Harwick Drive, Sprig, TX 77379 (US), YUNG,
`“REL, Aii’md; 414 Bymn 81mm, Hausum, TX 770115
`(US). JEANG, Huang; 4050 N. Braeswond Boulevard,
`HHUSEL‘IH, TX 77025 (US).
`
`(74}
`
`(:1 :41; Mmgzm, anis &.
`Agents: CLOUGH, David, W’.
`Hocking; LLP, Attention: Docketing Department, 1111
`PennysyWania Avenue, N.W’.,
`“12151111133011, DC 20004
`(US),
`
`(8 11
`
`Designated States (zmiess arherwise indicated, for every
`kind L1,?"I'zati0/mi pr'meczion available): AE, AG, AL, AMI,
`AU, AT, AU, AZ, BA, BB, 116, 1311, EN, BR, BW, «w,
`112, CA, C11, CL, CN, CO, C,R CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, 131:1, 138,11, (18,00,613, GH, (3M, GT,
`HN, HR, 11L, 11), 1L, 1N,11{, 18,.11’, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG,
`MK, MN, MW, MX, MY, MZ, NA, NG, N1, NO, NZ. OM,
`PA, 1‘13, PG, 1‘11, PL, PT, 12A, RO, RS, RU, KW, SA, SC,
`SD, SE, SG, SK, SL, SM, 81, SV, SY, 111, 1.1, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(.154)
`
`Designated States (unless ofize'rwis'e indicated. fin” every
`kind 0f regimmi prmeainn available): AREPO (8W, G171,
`GM, K11, LR, LS, 1V1W, MZ, NA, R‘W’, SD, SL, S1 S2,
`1/,1113, 2M, Z‘W), Bums 12m {A M, AZ, BY, KG, KZ, RU,
`1,}, TM), 13111111):an (AL, AT, 1811., BG, CH, CY, CZ, 11-171,
`13K, 1313, LS, F1, FR, GB, GR, HR, RU, 111, S, 11, LT, LU,
`
`LV, MC, MK, MT, NI... NO, PL, PT, RC),
`‘3. SE]. S1, SK,
`SM, TR), OA1’1 (BF, 13.1, CF, CG, C1, CM, GA, (1N, (3Q,
`61W, KM, ML. MR, NE, SN, TD, TG).
`Hecflaratinns under Ruiés 41.17:
`
`as {a applicanti? entitlement to apply/for and be granted at
`patem (Rafe 4.1 71/55))
`
`(3,?" izwenrorsiz 2;; (Rule 4.1 7(ivjj
`i’uflflistned:
`
`with inferzmz‘innai search report (Arr. 217(3))
`
`before {he expiratz'afi 53f the 551726 limizfi)?‘ amending {he
`claims and to be repubiz‘shed in Jhe even! 1:5,?” receipt (3,?”
`amendments (Rule 482(k))
`
`
`
`141113111113 1
`
`24 by; 13151911011
`
`
`
`r5 .au.
`1115511102“: Dim-13041.”
`caning sewenm
`\g,
`
`(57} Abstract: Certain embodiments include the enhancement Of afiéctiveness 101' an adenoviral cancer therapy.
`
`
`
`we21115111171124A1111111111111111111111111111111111111111111111111111111111111
`
`

`

`W0 Elli 55/977624
`
`PC'I‘IUSZ-ilttifir’ifilmfi
`
`PATENT APPLICATEON
`
`FOR
`
`AiDENOVlRUS EXPRESSING ithlUNE CELL STENEULATORY RECEPTOR AGONIST S
`
`1.
`
`Field of Invention
`
`BACKGRGHND.
`
`{0‘01}
`
`The present invention relates generally to the Fields of oncology and cancer
`
`therapy. More particularly, it concerns replicative oncclytic viruses genetically modified to
`
`express an immune cell stimulatory receptor agonist such as 0X40 ligand (OX-fills).
`
`ll.
`
`flescri gtlon of Related Art
`
`{002]
`
`Cancer remains one of the leading causes of morbidity and mortality in humans
`
`worldwide. Although surgery, chemotherapy and radiation have been utilized with some
`
`success to cure cancer, novel strategies are needed. Viruses that replicate in tumor cells
`
`better than in normal cells have shown promise as oncolytie agents. The tertsibility' ol’ gene
`
`transfer and tumor lysis using adenoviruses has been well established.
`
`{003]
`
`There remains a need for additional anti-cancer therapeutics.
`
`SUI‘HI‘VEARY
`
`{004]
`
`The present invention relates to novel replicationucompetent oncolytic viruses
`
`expressing one or more immune cell stimulatory receptor agonists, pharmaceutical
`
`compositions comprising the replication—competent oneolytic adenovirus and their use in
`
`treating a variety of cancers.
`
`In preferred embodiments, the replication—competent oncolytie
`
`virus is an atlenovirus. The replieationncoinpetent oncolytie virus will present the immune
`
`cell stimulatory receptor agonist from the first replication cycle, triggering a persistent
`
`effector anti—tumor immune response by activating lymphocytes that recognize tumor
`
`antigens and reversing the immune suppressive environment surrounding the tumor. in
`
`certain aspects, administration of the replicationneompetent oncolytic virus such as
`
`adenovirus to a su'bj ect with cancer provides an enhanced and even synergistic anti—tumor
`
`

`

`‘WO 2015/077624
`
`PCT/U829} «titltififililtl
`
`immunity compared to the unmodified virus tie, not expressing an immune cell stimulatory
`
`receptor agonist) and the immune cell stimulatory receptor agonist when administered
`
`separately.
`
`in related aspects, the antiutumor effects of the replication—competent oncolytic
`
`virus persist even after clearance of the virus and even extend to one or more non—infected,
`
`tumors ,
`
`[titlSl
`
`In certain aspects, the replication—competent oncolytic vims expresses an immune
`
`cell stimulatory receptor agonist from a heterologous nucleic acid incorporated into a non-
`
`essential region of the viral genome.’ the heterologous nucleic acid comprising a nucleic acid
`
`sequence encoding the immune cell stimulatory receptor agonist‘ in some embodiments, the
`
`replication«competent oncolytic virus is an adenovirus and expression of the immune cell
`
`stimulatory receptor agonist is under the control of an endogenous adenovirus promoter such
`
`as the E3 promoter or a late adenoviral promoter such as the major late promoter.
`
`in other
`
`embodiments, the replication—competent oncolytic virus is an adenovirus and the nucleic acid
`
`encoding the immune cell stimulatory receptor agonist is under the control. of (Le. operatively
`
`linked to) a non—adenoviral transcriptional and/or translational control sequence such as an
`
`enhancer, promoter arid/or leader sequence from cytomegalovirus (CMV) (e.g. a CMV
`
`promoter), rous sarcoma virus (RSV) (eg. an RSV promoter) or simian virus 40 (SV49) (eg.
`
`an SV4G promoter). A "heterologous" region of the construct is an identifiable segment of
`
`nucleic acid within a larger nucleic acid molecule that is not found in association with the
`
`larger molecule in nature.
`
`[006}
`
`in several embodiments, the replication—competent oncolytic virus expresses an
`
`agonist of an immune cell stimulatory receptor selected from the group consisting of: CD28,
`
`0X40 (Cl) l34), glucocorticoid~induced TNqueceptor (G lTR), CDl37 (4—er8), and herpes
`
`virus entry mediator A (l‘l‘VEM). 0X40, Gl’l‘R, CDl 37 and HVEM are members ofthe
`
`tumor necrosis factor receptor (TNFR) tamily that are inducibly expressed upon T cell
`
`activation and accordingly induce costimulation on activated effector T cells and memory T
`
`cells. Stimulation through C928 must be induced by professional antigen presenting cells
`
`(APCs) such as dendritic cells and macrophages; costimulation through TNFR family
`
`members such as 0X40 and CD l 3’7 can be induced by expression of their respective ligands
`
`

`

`‘WO 2015/077624
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`PCT/U829} «titltifiililil
`
`on ncnheinatcpoietic cells in the periphery.
`
`ln a preferred einbcdiinent, the replicaticn—
`
`competent cncolytic virus is an adenovirus.
`
`[007]
`
`CD28 is the most prominent costimulation receptor and is constitutively
`
`expressed on T cells and plays a critical role in stimulating naive T cells for proliferation,
`
`effecter function and differentiation,
`
`in one embodiment, the replicationmcnnipetent
`
`oncclytie virus {egn adenovims) expresses an agcnist cf a CD28 agnnist such as human
`
`C1180 (B7. l), GenBank Accession Nos. Niv’lflQOflQl (niRNA) and NP___095182 (protein) or
`
`CD86 (ll-7,2), GenBank Accession No. NMfll 75862 (mRNA) and accession new, P422081 in
`
`the Swiss—Fret database.
`
`[008]
`
`Gl'l‘R is expressed ccnstitutively at high levels on regulatory '1‘ cells and activated
`
`CD4+ and CD8+ T cells, Engagement of Gl'l‘R by its receptor GITR ligand (Gl'l‘RL) has
`
`1neen shown tc dampen the suppressive effects of regulatery T cells and cc—activate effector T
`
`cells.
`
`In one emhcdiment, the replicaticn~ccmpetent cncclylic virus (cg adencvirus)
`
`expresses an agonist chlTR such as human GITRL, NCBI database Entrez Gene ll): 8995.
`
`[009]
`
`4--lBB (CD37) is expressed on the surface of activated CD4+ and CD8+ T cells,
`
`on natural killer cells, monocytes and resting dendritic cells. Engagement of 4—188 with its
`
`ligand, 4—1813 ligand (AlmlBBL) plays a role in T cell survival and the establishment of long—
`
`term. immunclcgical memory and selectively promotes type i cytokines such as lL—Z, lFN—v
`
`and lNF-Ct
`
`in ene embediment, the replicatien—conipetent cncelytic virus (cg. adencvirns)
`
`expresses an agcnist of 4438 such as human 4—l BBL, the full amino acid sequence of
`
`which can be found under accession no. 1341273 in the Swiss-Fret database.
`
`{Gill}
`
`lilVlfiM is expressed in peripheral blood T cells, B cells and nmncctyes.
`
`Engagement cl l-IVEM with its receptcr LlGH’l‘ costimulates "ll and B—cell activation,
`
`upregnlates apeptotic genes and induces cytckine production, particularly, of lFN—Y and
`
`TNFOL, In one embodiment, the replication—competent oncolytic virus (cg. adenovirus)
`expresses an agcnist ct‘l—l’VEM such as human lymphotcxin—like (LIGHT), the full amino
`
`acid sequence of which can be found under accession. nc. (343557 in the Swiss~Pret database.
`
`

`

`‘WO 2015/077624
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`PCT/U829} «titltifillilll
`
`[011]
`
`in a preferred embodiment, the replication—competent oncolytic virus comprises a
`
`heterologous nucleic acid encoding an 0X40 agonist. An 0X40 agonist interacts with the
`
`X040 receptor on eg. activated T cells during or shortly after priming by a tumor or
`
`adenoviral antigen and results in an enhanced and prolonged immune response to the tumor,
`
`Preferably, the (“Di—40 agonist is expressed on the surface of the host cell {e.g., tumor cell)
`
`tollowing infection of the cell with the replication competent oncolytic virus.
`
`in one
`
`preferred embodiment, the replicationucompetent oncolytic virus is an adenovirus comprising
`
`a heterologous nucleic acid encoding an 0X40 agonist.
`
`[GlZ]
`
`in a particularly pre “erred embodiment, the replicationucompetent oncolytic virus
`
`comprises a heterologous nucleic acid encoding 0X40 ligand (OX-4&4. or nglI-l) or an 0X40
`
`receptor—binding fragment of OXI—llll, or an OXI—llll, fusion protein such as those described in
`
`US Patent No. 7,959,925, the content o f which is incorporated herein by reference.
`
`in one
`
`particularly preferred embodiment, the replication~competent oncolytic virus is an adenovirus
`
`comprising a heterologous nucleic acid encoding OX4OL, OX40L, also known as gp34, like
`
`other TNF superfamily members, exists as a homotrimer on the surface o l‘" activated B cells,
`
`T cells, dendritic cells and endothelial cells. Binding ot‘OXi’lOL to 0X48 (CD134) sustains
`
`the initial CDZS-mediated T cell response and promotes both T~cell tliiferentiatitin and
`
`survival. in particular, engagement of OK40 by its natural ligand OX4GL or other 0X40
`
`agonists has been shown to provide key signals that can augment CD4 and CD8 Tmcell
`
`responses. 0X46 signaling also controls regulatory T cell differentiation and suppressive
`
`function,
`
`importantly, numerous studies have highlighted the ability of UX4€l~specific
`
`agonists to enhance antitumor immunity or ameliorate autoimmune disease, respectively. On
`
`the basis of these studies, the development of OX40- and OX4GL—specific reagents has been
`
`pursued for clinical use. Studies over the past decade have demonstrated that 0X49 agonists
`
`enhance antimtumor immunity in preclinical models using immunogenic tumors; however,
`
`treatment of poorly immunogenic tumors has been less successful. Combining strategies that
`
`prime tumor—specific T cells together with QK4O signaling could generate and maintain a
`
`therapeutic anti~tumor immune response. The amino acid sequence of human OX4OL is
`
`described at GenBank Accession Number NP_003317,1. Full cDNA encoding human
`
`OX4OL is at NCBl Reference Sequence: NM_003326.3, Additional OX4QL sequences are
`
`

`

`“/0 20152077624
`
`PCT/U820} #066920
`
`further disclosed in eg. SwissFrot Accession Number P235l 0. Human OX40L shares 46%
`
`amino acid sequence identity with its mouse counterpart.
`
`[013]
`
`Other 0X40 agonists that can be expressed by the replication~competent oncolytic
`
`adenovirus include antibodies against 0X40 such as those described in US F’atent Nos.
`
`6,312,700, 7,504,101 , 7,291,33 l, and 7,807,156, the entire contents of each of which are
`
`incorporated herein by reference. Specific nonwlimiting examples ofOXL‘lO antibody include
`
`ll2F32, lift/'8, llZYSS, ll2‘i’131, ilZZS, mAh 3i5, inAhlfil, inAh 262, 1137, ACTSS,
`
`mAh Lille and inAh 0X86. cher 0X40 agonists include those described in US. Patent
`
`Application Publication No. l_l82006028l072, the entire content of wl’iich is incorporated
`
`herein by reference.
`
`[til-4]
`
`DNA encoding an immune cell stimulatory receptor agonist can be inserted e.g. at
`
`any nonessential location in the oncolytic virus so long as the oncolytic virus remains
`
`replication competent. in one embodiment, the oncolytic virus is an adenovirus with a
`
`heterologous nucleic acid comprising a sequence encoding an immune cell stimulatory
`
`receptor agonist inserted downstream of the adenovirus iiher gene whereby expression of the
`
`encoded protein is driven by the adenoviras major late promoter.
`
`in a preferred embodiment,
`
`a heterologous nucleic acid comprising a sequence encoding an immune cell stimulatory
`
`receptor agonist is inserted in the E3 region of a replication—competent adenovirus backbone.
`
`The E3 region is nonessential for viral replication; however, the E3 proteins play a role in
`
`regulating host immune response.
`
`'l‘he replicationcompetent adenovirus can comprise a full
`
`or partial i313 deletion. For example, the replication~competent adenovirus can comprise
`
`deletions of one, two, three or more open reading frames (GRFs) in the E3 region and the
`
`heteroiogous nucleic acid inserted in its place.
`
`in one embodiment, the gpint and 6.7K genes
`
`are deleted and the heterologous nucleic acid is inserted into a gplel/ofli; deleted E3 region.
`
`in a related embodiment, the region between the :9ng restriction enzyme sites at 783 and
`
`85.8 map units of adenovirus type 5 genome may be deleted and the heterologous nucleic
`
`acid inserted into the deleted E3 region, as described in Bett et al., J. Virol, 67(10)::391L
`
`5922 (1993), the contents of which are incorporated herein by reference.
`
`in related aspects,
`
`the full E3 region is deleted from the replication—competent adenovirus backbone and the
`
`heterologous nucleic acid is inserted into a location containing the hill E3 deletion.
`
`in a
`
`U1
`
`

`

`‘WO 2015/077624
`
`PCT/U829} ditltifillilil
`
`particularly preferred embodiment the present invention provides a Delta—24 or Delta-24m
`
`RGD adenovirus comprising a heterologous nucleic acid inserted in place of a partially or
`
`completely deleted E3 region, wherein the heterologous nucleic acid comprises a sequence
`
`encoding an 0X49 agonistfi preferably 0X40L and expression of the 0X49 agonist is under
`
`the control of a non—adenoyiral promoter such as a CM‘V promoter.
`
`[(3l5}
`
`Certain embodiments are directed to methods o t‘ treating cancer comprising
`
`administering to a tumor a replication competent oncolytic Virus (e. g. adenovirus) expressing
`
`one or more immune cell stimulatory receptor agonists as described above or a
`
`pharmaceutical composition comprising the replication-—cornpetent oncolytic virus.
`
`in certain
`
`aspects, the methods comprise administering to a tumor a Delta-24 adenoyirus comprising a
`
`heterologous nucleic acid comprising a nucleic acid sequence encoding an immune cell
`
`stimulatory receptor agonist inserted into a non—essential region of the Delta-24 adenoyirus
`
`backbone,
`
`in a preferred embodiment part of the E3 region or all of the E5 region of the
`
`Delta—24 adenoviius genome is deleted and replaced with the heterlogous nucleic acid in a
`
`particularly preferred embodiment? the present invention provides a method for treating
`
`cancer (cg, glioina) in a human subject by administering to the subject a l)elta—24~RGD
`
`adenoviros comprising a heterologous nucleic acid comprising a nucleic acid sequence
`
`encoding immune cell stimulatory receptor agonist (e.g. 03:401..) into a non-essential region
`
`of the adenovirus backbone (eg. a deleted E3 region)” in some embodiments? the human
`
`subject exhibits a 'l‘hl interlueltine pattern.
`
`in other embodimentss the human subject
`
`exhibits a Th2 interleukine pattern. A subj ect is determined to exhibit a ’l‘hZ interleukine
`
`pattern if the subject has an lL~lZ/’lL—4 ratio ot’less than about 20, less than about 15, or less
`
`than about 10., Subjects exhibiting a 'l‘hl interleukine pattern will generally exhibit an lL~
`
`lLZ/‘lL—4 ratio of greater than 20 and in some cases greater than 50, greater than lGG and even
`
`greater than 300. The lL~lZ/lL—4 ratio can be determined in the subject by obtaining a
`
`sample from the subject (eg a blood or serum sampleli contacting the sample with
`
`antibodies against anl2 and Ila—LlL and determining the amount of lL--12 and le4 in the
`
`sample as a function of the amount of binding of the antibodies to their respective antigens
`
`tog by ELlSAL
`
`o
`
`

`

`‘WO 2015/077624
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`PCT/US$33 «iiiltififililii
`
`{016]
`
`in related embodimentst one or more Th1 stimulating agents is co~administered
`
`with the replication competent oncolytic virus expressing one or more immune cell
`
`stimulatory receptor agonists as described above to treat cancer (erg. gliohlastoma) in a
`
`subject. In some embodiments, the subject has an IL— lZ/lL~4 ratio of less than about 20 (ie.
`
`exhibits a Th2 interluekine pattern).
`
`in other embodiments, the subject has an lL-iZ/iL-lt
`
`ratio of greater than about '20 (i.e. exhibits a Th1 interleukine pattern). Th1 stimulating
`
`agents includeg without limitation; (i) Thl cytoltines such as lL—12p70, lL-Z and lFN—y, (ii)
`
`agents that increase production of Th1 cytolcines such as REVLIMID (lenalidomide) (iii)
`
`agents that suppress regulatory T cells (e.g. all<ylating agents such as temozolomide (4~
`
`methyl~5~oxo~ 293,4,658~pentazahicyclo [4.3.0] nona~2,799—triene— 9~earboxarnide),
`
`cyclophosphamide ((RS‘)—N,N-bis{2-chioroethyl)-1,3,2~oxazaphosphinan~2—amine 2—oxide),
`
`lomustine (CCNU; N—(2~chloroethyi)—N'—cyclohexyl~N—nitrosourea), bis--
`
`chloroethylnitrosourea (BCNUL meiphalan hydrochloride (4
`
`[bis{chioroethybaminolphenyialanine), busulfan (butanewl fludiyi dimethanesulfonate),
`
`mechiorethamine {nitrogen mustard), chiorambucil, itbsfamide, streptozocin, dacarbazine
`
`(DTIC), thiotepa, altretamine (hexamethyimelamine), cisplatin, carboplatin, and oxalaplatin)
`
`and (iv) agents that stimulate cell mediated immune response (cg: lpilimumahg
`
`Tremelimumabg MDX~1106, MIC-3475, AMP~224, Pidilizumah, and MDXui 105‘). Preferred
`
`'l‘hl stimulating agents to for comadministration with a replication competent oncolytic virus
`
`of the invention include lE‘N—y (preferably recombinant) and temozolomidec The replication
`
`competent oneolytic virus of the invention and a Thl. stimulating agent may be separatelya
`
`concurrently or consecutively administered to a subject with cancer to treat the cancer.
`
`in
`
`one embodiment the Thl stimulating agent is administered to the subj cot and thereafter the
`
`replicationmcornpetent oncolytic virus is administered.
`
`in other related embodiments, a
`
`composition or kit is provided comprising (i) a Thl stimulating agent and (ii) a replication”
`
`competent oncoiytic adenovirus expressing one or more immune cell stimulatory receptor
`
`agonists as herein described, each in an amount effective to treat cancer in a subject in
`
`combination with the other.
`
`in a preferred embodiment, the composition or kit comprises (i)
`
`a 'l‘hl stimulating agent selected from the group consisting of: recombinant HEN—r,
`
`temozoiomide, CCNU, BCNU, meiphalan hydrochloride and busuli‘an and (ii) a replication—
`
`

`

`‘WO 2015/077624
`
`PCT/U829} «titltififiilii
`
`competent oncolytic adenovirus (e. g. Delta~24 or Delta—Zit—RGD) expressing an (Di-40
`
`agenist (eg. GXIlDL).
`
`[017]
`
`in certain embodimentse a replication—eoinpetent oncolytic virus (eg. adenovirus)
`
`is provided that expresses a PD—Ll or PD—i antagonist.
`
`in some embodiments? the.
`
`replication—competent oncolytic virus express a PEP-Ll or PD—l antagonist in addition. to
`
`expressing an immune cell stimulatory receptor agonist. in other ernhodirnents,a the
`
`replieanon—competent oneolytic virus expresses a PlLLLl or PD—l antagonist but does not
`
`express an immune cell stimulatory receptor agonist. PEP—Ll has been identified as a
`
`negative regulator of antitunior T cells and is expressed in up to 50% of human cancer.
`
`Binding of PD-Ll on tumor cells to PD—i on activated effector T cells results in activation of
`
`PI?» kinase-signaling cascade which in turn blocks the production of cytotoxic mediators
`
`required for killing tumor cells. As used herein, a PILL} or l’Dui antagonist is a molecule
`
`that dismpts the interaction between PDle and Fill. in one aspect, the replication—
`
`competent oncolytic virus is an adenovirus that comprises heterologous nucleic acid
`
`encoding a PD—Ll or PD-l antagonist inserted into a nonuessential region of the adenovirus
`
`genome.
`
`in related aspects, the heterologous nucleic acid encodes an anti—PD—Ll antibody
`
`such as MPDL3280A5 or an anti-PD“ l antibody such as nivoliunab or lainhroliatunab.
`
`in
`
`other embodiments? the heterologous nucleic acid encodes a PD—Ll or fill antagonist such
`
`as those described in US Patent Application Puhlication Nos. 29t39/92l740l3 20l10195068
`
`and 2012025l537 and US Fatent No. 8,2l7jl493 the contents of each which are incorporated
`
`herein by reference.
`
`in certain embodiments: a method for treating cancer (eg. a glionia) in a
`
`human is provided comprising administering an effective amount ot‘a replication-competent
`
`oncolytic virus expressing a PD—Ll and/or l’D-i antagonist.
`
`in a preferred embodiment the
`
`replicationucompetent oncolytie virus is an adenovims expressing a PD-Ll and/or E’Dml
`
`antagonist.
`
`in one preferred embodiment, the adenovirus is Delta~24 or Delta-24v ROD
`
`adenovirus.
`
`3:018]
`
`in certain embodiments, the replication—competent oncolytic virus, in addition to
`
`expressing an immune cell stimulatory receptor agonists also expresses one or more tumor
`
`antigens on its surface.
`
`in certain. aspects, 1, 29 3, 4, or 5 antigens are expressed on the
`
`surface of the virus, for example? by inserting nucleic acid encoding each antigen into a
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`‘WO 2015/077624
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`PCT/U826} dititififiiltl
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`separate gene encoding an adenovims surface protein.
`
`in a preferred embodiment, the turner
`
`associated antigen(s) are EGFvall (epidermal growth factor receptor variant ill) and/or NY-
`
`ESO-i (New Yorlt oesophageai squarnos cell carcinoma 1). The tumor antigens can he
`
`expressed as part of the capsid or fiber, or produced as exogenous proteins linked to
`
`autophagy-related proteins such as LC3 to increase the presentation ot‘the exogenous protein
`
`during the adenovirai infection and replication. Targeting multiple antigens will help
`
`generate a consistent and effective immune response.
`
`[019]
`
`Tumor associated antigens (TAA) include, but are not limited to tumor associated
`
`antigens that have been identified as occurring in patients with brain cancers such as giiornas
`
`representative examples of which include: Alli/l2 (absent in melanoma 2), Bl‘vlll (B‘Mll
`
`polycomh ring finger oncogene), COXQ (cyclooxygenase--2), TRPul (tyrosine related
`
`protein 2) TRPnZ (tyrosine related protein 2), GPth) (glycoprotein 100), EGFR‘vlll
`
`(epidermal growth factor receptor variant ill), EZHZ (enhancer of zeste hornolog 2), LlCAlVl
`
`(human Ll cell adhesion molecule), Livin, Livinfi, BARF—3 (multidrug resistance protein 3),
`
`Nestin, OLIGZ (oligodendrocyte transcription factor), SOXZ (SRY-related HMS—box 2),
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`ARTl (antigen recognized by T cells 1), ART4 (antigen recognised by T cells 4'), SARTI
`
`(squamous cell carcinoma antigen recognized by T celis 1), SAR‘TZ, SARTSZ, B»cyclin, h—
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`eatenin, Glil (gliorna—associated oncogene horniog l), Cat/ml (caveolin—l), cathepsin B,
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`CD74 (cluster of Differentiation 74), E-cadherin (epithelial calcium-dependent adhesion),
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`EphAE/Eck (EFH receptor AZ/epithelial kinase), Fra-l/Fosl l (res—related antigen 1), GAGE—
`
`l (C: antigen 1), Ganglioside/GDZ, GnT—V, Bl,6~N (acetylglucosaniinyltranst‘erase-V),
`
`HerZ/neu (human epidermal growth factor receptor 2), are? (nuclear prolifermion—associated
`
`antigen of antibody Ki67), Ku70/80 (human Kn heterodimer proteins subunits), lL—l 3Ra2
`
`(interleukin—13 receptor subunit alpha—2), MAGE-A, (inclanoma—associated antigen 1),
`
`MAGEvAfi (melanoma—associated antigen 3), NY—ESO-l (New York oesophageal squarnos
`
`cell carcinoma 1), MART-l (melanoma antigen recognized by T cells), FROXl (prospero
`
`horneobox protein 1), PSCA (prostate stem. cell antigen), SOXlQ (SRY-related HMG~box
`
`l0), SOXl l, Survivin, UPAR (urokinase—type plasminogen activator receptor, and i-Vlil
`
`(Wilms” tumor protein 1). The replication—conipetent oncolytic Virus (eg. adenovirus) may
`
`express the full length tumor associated antigen or an innnnnogenic peptide thereof.
`
`

`

`“7G 2&15/{377624
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`PCT/{ISleld/dfidgfli
`
`[(320]
`
`in one aspect, the replication—competent oncolytic virus, in addition to expressing
`
`an immune cell stimulatory receptor agonist, also expresses EGERVHI or an immunogenic
`
`peptide thereof on its surface. The sequence ot‘EGFRVlll is described in US. Patent No.
`
`6,4§§,498, the content of which is hereby incorporated by reference,
`
`lrnmunogenic
`
`EGFRVlll peptides include those described in U S l’atent Application Publication No.
`
`2009/0155282, the content of which is hereby incorporated by reference, particularly those at
`
`paragraph [0362} and Tables 4.14.3, Preferably, the oncolytic virus is an adenovirus and
`
`EGFRVlll or an intniunogenic peptide thereof is inserted into the gene encoding the fiber
`
`protein, preferably in the H1 loop, Nucleic acid encoding EGFRVHI or an irnrnunogenic
`
`peptide thereof may be inserted into genes encoding one or more surface proteins of any
`
`adenovirus. The term “immunogenie EGFvall peptide” as used herein means a peptide of
`
`suitable length eg. at least it) or iii. amino acids and up to ii 20, 25 or 30 amino acids or
`
`more which spans the mutated spiicejunction of the corresponding EGFRVHI protein,
`
`preferably human. EGFRViii.
`
`in a preferred embodiment, the nucleic acid inserted into an
`
`adenovirus surface protein encodes an 8—20 amino acid peptide consisting of, consisting
`
`essentially of, or comprising the sequence EK liGN‘i’VV.
`
`in a particularly preferred
`
`embodiment, the EGFRVHI iminunogenie peptide is LEEKKGNYVVT (SEQ ll) NO: 4) and
`
`is inserted into the gene encoding the fiber protein, preferably in the l-ll loop.
`
`in other
`
`embodiments, nucleic acid encoding the entire EGFRViii extracellular domain is inserted
`
`into a gene encoding a surface protein of the adenovirus.
`
`[0le
`
`in a related aspect, the replica‘tionvcompetent oncolytic virus, in addition to
`
`expressing an immune cell stimulatory receptor agonist, also expresses NY—ESG~l (GenBank
`
`ll87459.l) or an immunogcnie peptide thereot‘ (cg. SLLMerlTQCFlJPVF) on its surface,
`
`Preferably, the replica‘tionvcompetent oncolytic virus is an adenovirus and the nucleic acid
`
`encoding NY—ESOml or an immunogenic peptide thereot‘is inserted into a gene encoding a
`
`surface protein, whereby the adenovirus expresses a chimeric surface protein. comprising the
`
`NY~ESO«1 or an immunogenic peptide thereof.
`
`in one aspect, nucleic acid encoding NY—
`
`ESG—l or an immunogenic peptide thereol‘is inserted into the hyper—variable region 5 of the
`
`gene encoding the hexon of the adenovirus.
`
`lO
`
`

`

`“7G 2&15/{377624
`
`PCT/HSZQld/fifidllztl
`
`[022}
`
`insertion. of nucleic acids encoding> the tumor antigens into adenovirus genes
`
`should he done “in frame” such that the virus expresses the tumor antigen on its surface.
`
`{023]
`
`Certain aspects do not require the complete resection of the tumor, which is a
`
`limiting factor in recruitment ot‘patients in other approaches. Furthermore, certain aspects of
`
`the current methods and compositions have the potential to generate memory in the immune
`
`system and, preventing or reducing the probability of tumor recurrence,
`
`[024]
`
`The term "replication cornpeten " refers to any viral vector that is not deficient in
`
`any gene fitnction required for viral replication in specific cells or tissues, The vector must
`
`be capable ofrepllcating and being packaged, but might replicate only conditionally in
`
`specific cells or tissues. Replication competent adenoviral vectors oi” the present invention.
`
`are engineered as described herein to reduce or eliminate their ability to replicate in normal.
`
`cells while retaining their ability to replicate efficiently in specific tumor disease cell types.
`
`Typically, a replication competent adenovirus comprises enough of the El, E2, and E4
`
`regions that the adenovirus is capable of replicating and being packaged without the need for
`
`elements to be supplied in trans.
`
`[025]
`
`The term "therapeutic henelit" or “treatment” refers to anything that promotes or
`
`enhances the well—being of the suhject with respect to the medical treatment ot'his/her
`
`condition, which includes treatment ot‘pre~cancer, cancer, and hyperproliferative diseases. A
`
`list oi‘nonexhaustive examples of this includes extension of the suhj ect’s lite hy any period of
`
`time, decrease or delay in the neoplastic development of the disease, decrease in
`
`hyperprcliferation, reduction in tumor growth, delay of metastases, reduction in cancer cell
`
`or tumor cell proliferation rate, and a decrease in pain to the subject that can he attributed to
`
`the subject‘s condition.
`
`[026]
`
`A ”T regulatory cell” or “regulatory T cell" refers to a cell that can inhibit a T cell
`
`response. Regulatory T cells express the transcription factor FoxpB, which is not upregulated
`
`upon '1" cell activation and discriminates regulatory T cells from activated effector cells.
`
`Regulatory "i" cells are identified by the cell surface markers C1323, CD45RBS C'FLA-fih and
`
`Gl’l‘R. Regulatory T cell development is induced by myeloid suppressor cell activity. Several
`
`regulatory T cell subsets have been identified that have the ability to inhibit autoimmune and
`
`ii
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`

`

`‘WO 2015/077624
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`PCT/USZG‘E «titltififiilil
`
`chronic inflammatory responses and to maintain immune tolerance in tumor“ bearing hosts.
`
`These subsets include interleukin l0— (lL~l0—) secreting T regulatory type i (Til) cells,
`
`transforming growth factor—B— (TGF—B—) secreting T helper type 3 (Th3) cells, and "natural"
`
`Cl)4+/CDZS+ Tregs (Tm) (Fehervari and Sakaguclii. 3. Clin. invest. 2004, l
`
`l4: l209— lZl7;
`
`Chen et al, Science,
`
`l994l, 265: l237—l240; Groux et al. Nature. l997, 389: 737—742).
`
`[027]
`
`As used herein, an ”agonist," eg, an 0X40 agonist, is a molecule which enhances
`
`the biological activity of its target, e,g,, 0X40. in certain aspects 0X40 agonists, comprising,
`
`e. g,, anti—0X40 antibodies or 0X40 ligand compositions, substantially enhance the biological
`
`activity ot‘OXZlO. Desirably, the biological activity is enhanced lay l0%, 20%, 30%, 50%,
`
`70%, 80%, 90%, 95%, or even 100%. in certain. aspects, 0X40 agonists as disclosed herein
`
`include 0X40 binding molecules, eg. binding polypeptides, anti—0X40 antibodies, OX40L,
`
`or fragments or derivatives of these molecules.
`
`{028]
`
`Other embodiments of the invention are discussed tlnoughout this application.
`
`Any embodiment discussed with respect to one aspect of the invention applies to other
`
`aspects of the invention as well and vice versa. Each embodiment described herein is
`
`understood to be embodiments of the invention that are applicable to all aspects ofthe
`
`invention.
`
`it is contemplated that any embodiment discussed herein can be implemented
`
`with res