`
`
`
`
`
`I 1111111111111111 1111111111 111111111111111 IIIII IIIII IIIII IIIIII IIII IIII IIII
`
`USO 10555981B2
`
`(IO) Patent No.: US 10,555,981 B2
`
`c12) United States Patent
`
`Feb.11,2020
`(45)Date of Patent:
`
`Silvestre et al.
`
`(54)ONCOLYTIC VIRUS FOR EXPRESSION OF
`
`IMMUNE CHECKPOINT MODULATORS
`
`C12N 7/00; C12N 9/1077; C12N 9/80;
`
`
`C12N 2710/24121; C12N 2710/24132;
`
`C12Y 204/02009; C12Y 305/01023
`
`
`
`
`(FR)
`
`(71)Applicant: Transgene SA, Illkirch Graffenstaden
`USPC ......................... 424/199.1, 93.6; 530/288.23
`
`
`
`
`
`See application file for complete search history.
`
`Inventors: Nathalie Silvestre, Ergersheim (FR);
`
`
`
`
`(72)
`
`
`Michel Geist, Brumath (FR); Karola
`
`
`Rittner, Strasbourg (FR);
`(56)
`
`Jean-Baptiste Marchand, Obemai
`
`
`(FR); Christine Thioudellet, Strasbourg
`(FR)
`
`
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`
`(73)
`
`
`
`
`
`7/1993 Winter
`5,225,539 A
`
`
`10/1995 Baum et al.
`
`5,457,035 A
`Assignee: Transgene S.A., Illkirch Graffenstaden
`
`6/1996 Queen et al.
`
`5,530,101 A
`
`
`6/ 1998 Hercend et al.
`
`5,773,578 A
`
`1/2001 Queen et al.
`
`6,180,370 Bl
`Notice: Subject to any disclaimer, the term ofthis
`
`( *)
`1/2006 Korman et al.
`6,984,720 Bl
`
`
`
`patent is extended or adjusted under 35
`
`9/2006 Hanson et al.
`
`7,109,003 B2
`
`U.S.C. 154(b) by O days.
`
`11/2007 Croft et al.
`7,291,331 Bl
`
`11/2009 Weinberg
`
`7,622,444 B2
`
`12/2010 Honjo et al.
`
`7,858,746 B2
`9/2011 Korman et al.
`
`8,017,114 B2
`3/2012 Hanson et al.
`
`8,143,379 B2
`
`7/2013 Hanson et al.
`8,491,895 B2
`2013/0177557 Al
`
`7/2013 Noelle et al.
`
`2015/0250837 Al *
`
`9/2015 Nolin . C07K 16/2818
`424/281.1
`
`
`(21)
`
`Appl. No.: 15/325,562
`
`
`(22)
`
`PCT Filed: Jul. 16, 2015
`
`PCT No.: PCT /EP2015/066263
`(86)
`
`§ 371 (c)(l),
`(2)Date: Jan. 11, 2017
`
`FOREIGN PATENT DOCUMENTS
`
`
`(87)
`
`PCT Pub. No.: WO2016/008976
`
`
`
`PCT Pub. Date: Jan. 21, 2016
`
`(65)
`
`
`
`Prior Publication Data
`
`
`
`US 2017/0157188 Al Jun. 8, 2017
`
`
`
`EA
`
`EP
`
`WO
`
`WO
`
`WO
`
`WO
`
`WO
`
`WO
`
`WO
`
`OTHER PUBLICATIONS
`
`
`
`
`
`Dias et al. (2012) Gene Therapy, vol. 19, 988-998. *
`
`
`
`
`013615 12/2007
`
`
`1 907 000 Bl 4/2008
`6/1997 WO 97/20574
`
`
`6/2003 WO 03/045197
`WO 03/082919
`10/2003
`WO 03/106498
`12/2003
`WO 2004/004771
`1/2004
`WO 2004/056875
`7/2004
`WO 2006/121168
`11/2006
`WO 2007/123737
`11/2007
`WO
`WO 2008/156712
`12/2008
`
`Jul. 16, 2014 (EP) ..................................... 14306153
`WO
`(Continued)
`
`
`
`
`
`(30) Foreign Application Priority Data
`
`(51) Int. Cl.
`
`A61K 351768 (2015.01)
`
`(2006.01)
`(2006.01)
`
`C07K 141535 (2006.01)
`(2006.01)
`
`C12N 9/10 (2006.01)
`(2006.01)
`(2006.01)
`
`C07K 16128
`
`A61K 9/00
`
`C12N 7100
`
`C12N 9/80
`
`Primary Examiner - Anne Marie S Wehbe
`
`
`A61K 39/00
`
`(52) U.S. Cl.
`
`
`
`
`(74)Attorney, Agent, or Firm - Buchanan Ingersoll &
`CPC .......... A61K 351768 (2013.01); A61K 9/0019
`
`
`Rooney PC
`
`
`(2013.01); C07K 141535 (2013.01); C07K
`
`
`
`1612896 (2013.01); C12N 7100 (2013.01);
`(57)
`
`C12N 9/1077 (2013.01); C12N 9/80
`
`
`
`(2013.01); C12Y 204/02009 (2013.01); C12Y
`The present invention provides an oncolytic virus compris­
`
`
`
`
`
`
`305/01023 (2013.01); A61K 2039/505
`
`
`
`
`ing nucleotide sequence( s) encoding one or more immune
`
`
`
`(2013.01); A61K 2039/5256 (2013.01); C07K
`
`
`
`checkpoint modulator(s). It also concerns a pharmaceutical
`
`
`
`16/2818 (2013.01); C07K 2317/51 (2013.01);
`
`
`
`
`composition comprising effective amount of said oncolytic
`
`
`C07K 2317/515 (2013.01); C07K 2317/55
`
`
`
`
`virus and, eventually, a pharmaceutically acceptable vehicle
`
`
`
`(2013.01); C07K 2317/622 (2013.01); Cl2N
`
`
`
`
`and its use for treating proliferative diseases such as cancers.
`
`
`2710/24121 (2013.01); Cl2N 2710/24132
`(2013.01)
`(58)
`Field of Classification Search
`
`
`CPC ................ A61K 35/769; A61K 9/0019; A61K
`
`
`
`
`
`2039/5256; C07K 16/2896; C07K 14/535;
`
`
`(Continued)
`
`ABSTRACT
`
`
`
`15 Claims, 7 Drawing Sheets
`
`
`
`
`
`Specification includes a Sequence Listing.
`
`TRANSGENE/BIOINVENT
`EXHIBIT 1002
`
`Page 1 of 29
`
`

`

`US 10,555,981 B2
`Page 2
`
`(56)
`
`References Cited
`
`FOREIGN PATENT DOCUMENTS
`
`WO WO 2009/0 14708
`WO WO 2009/065546
`WO WO 2009/065547
`WO WO 2009/114335
`WO WO 2012/110360
`WO WO 2013/043569
`WO WO 2014/022138
`WO WO 2014/047350
`
`1/2009
`5/2009
`5/2009
`9/2009
`8/2012
`3/2013
`2/2014
`3/2014
`
`OTHER PUBLICATIONS
`
`Dias et al. (2010) Clin. Canc. Res., vol. 16(9), 25402549.*
`Bauzon et al. (Feb. 1,2014), Frontiers in Immunology, vol. 5, 110.*
`Gammon et al. (2010) PbS Pathogens, vol. 6(7), 120.*
`Foloppe et al. (2008) Gene Therapy, vol. 15, 13611371.*
`Agata et al., Expression of the PD-i antigen on the surface of
`stimulated mouse T and B lymphocytes, 8(5) International Immu-
`nology 765-772 (1996).
`Andtbacka et al., OPTiM; A randomized phase III trial oftalimogene
`laherparepvec (T- VEC) versus subcutaneous (SC) granulocyte-
`macrophage colony-stimulating factor (GM-CSF)for the treatment
`of(tx) of unresected stage IIIB/C andlVmelanoma, 31 J. Clin Oncol
`1-2 (2013).
`Bauzon et al., Armed therapeutic viruses--a disruptive therapy on
`the horizon of cancer immunotherapy, 5(74) Frontiers in Immunol-
`ogy 1-10 (Feb. 24, 2014).
`Bedke et al., Targeted therapy in renal cell carcinoma: moving from
`molecular agents to specific immunotherapy, 32 World J. Urol
`3 1-38 (2014).
`Bennett et al., Program Death-i Engagement Upon TCR Activation
`Has Distinct Effects on Costimulation and Cytokine-Driven Prolf
`eration: Attenuation of ICOS, IL-4, and IL-2i, But Not CD28, IL-7,
`and IL-i5 Responses, 170 J. Immunol 711-718 (2003).
`Blank et al., Interaction of PD-Li on tumor cells with PD-i on
`tumor-specific T cells as a mechanism of immune evasion: impli-
`cations for tumor immunotherapy, 54 Cancer Immunol Immunother
`307-3 14 (2005).
`Blank, CU., The perspective of immunotherapy: new molecules and
`new mechanisms of action in immune modulation, 26(2) Co-
`Oncology 204-2 14 (Mar. 2014).
`Boviatsis et al., Antitumor activity and reporter gene transfer into
`rat brain neoplasms inoculated with herpes simplex virus vectors
`defective in thymidine kinase or ribonucleotide reductase, 1(5)
`Gene Therapy 323-33 1 (Sep. 1994).
`Breitbach et al., Targeted and Armed Oncolytic Poxviruses for
`Cancer: the Lead Example of JX-594, 13 Current Pharmaceutical
`Biotechnology 1768-1772 (2012).
`Broyles, S., Vaccinia Virus Encodes a functional of dUTPase, 195
`Virology 863-865 (1993).
`Brunet et al., A new member of the immunoglobulin superfamily—
`CTLA-4, 328 Nature 267-270 (Jul. 16, 1987).
`Cohen et al., ONYX-OlS Onyx Pharmaceuticals, 2(12) Current
`Opinion in Investigational Drugs 1770-1775 (2001).
`Carter et al., PD-i .PD-L inhibitory pathway affects both CD4* and
`CD8*T cells and is overcome by IL-2, 32 Eur. J. Immunol 634-643
`(2002).
`Chambers et al., Comparison of genetically engineered herpes
`simplex viruses for the treatment of brain tumors in a scid mouse
`model of human malignant glioma, 92 Proc. Nat!. Acad. Sci
`1411-1415 (Feb. 1995).
`Champiat et al., Incorporating Immune-Checkpoint Inhibitors into
`Systemic Therapy of NSCLC, 9(2) Journal of Thoracic Oncology
`144-153 (Feb. 2014).
`Darivach et al., Human Ig superfamily CTLA-4 gene: chromosomal
`localization and identity of protein sequence between murine and
`human CTLA-4 cytoplasmic domains, 18 Eur. J. Immunol. 1901-
`1905 (1988).
`
`Chernajovsky et al., Fighting cancer with oncolytic viruses, 332
`BMJ 170-172 (Jan. 21, 2006).
`Dias et al., Targeted cancer immunotherapy with oncolytic adenovirus
`coding for afully human monoclonal antibody specfic for CTLA-4,
`Gene Therapy 1-11 (2011).
`Dias et al., Targeted Chemotherapy for Head and Neck Cancer with
`a Chimeric Oncolytic Adenovirus Coding for Bfunctional Suicide
`Protein FCUi, 16(9) Clin Cancer Res 2540-2549 (May 1, 2010).
`Dong et al., B 7-Hi pathway and its role in the evasion of tumor
`immunity, 81 J. Mo! Med 281-287 (2003).
`Dong et al., Tumor-associated B 7-Hi promotes T-cell apoptosis: A
`potential mechanism of immune evasion, 8(8) Nature Medicine
`793-800 (Aug. 2002).
`Du et al., Tumor-specfic oncolytic adenoviruses expressing granu-
`locyte macrophage colony-stimulating factor or anti-CTLA4 anti-
`body for the treatment of cancers, 21 Cancer Gene Therapy 340-348
`(2014).
`Engeland et al., CTLA-4 and PD-Li CheckpointBlockade Enhances
`Oncolytic Measles Virus Therapy, 22(11) Molecular Therapy 1949-
`1959 (Nov. 2014).
`Engeland et al., Measles Ti/us Mediated Immune Checkpoint Block-
`ade Enhances Canc; liiiiiiiiiioiiotherapy, 22( Supplement 1) Molecu-
`lar Therapy (May 2014) (abstract only).
`Foloppe et al., Targeted delivery of a suicide gene to human
`colorectal tumors by a conditionally replicating vaccine virus, 15
`Gene Therapy 1361-1371 (2008).
`Freeman et al., Engagement of the PD-i Immunoinhibitory Recep-
`tor by a Novel B 7 Family Member Leads to Negative Regulation of
`Lymphocyte Activation, 192(7) J. Exp. Med. 1027-1034 (Oct. 2,
`2000).
`Freeman et al., Phase 1/11 Trial ofIntravenous ND V-HUJ Oncolytic
`Virus in Recurrent Glioblastoma Multforme, 13(1) Molecular Therapy
`21-228 (Jan. 2006).
`Gammon et al., Vaccinia Virus-Encoded Ribonucleotide Reductase
`Subunits Are D[ferentially Required for Replication and Pathogen-
`esis, 6(7) PLoS Pathogens 1-20 (Jul. 2010).
`Geevarghese et al., Phase 1/11 Study of Oncolytic Herpes Simplex
`Virus NVi 020 in Patients with Extensively Pretreated Refractory
`Colorectal Cancer Metastatic to theLiver, 21 Human Gene Therapy
`1119-1128 (Sep. 2010).
`Guse et al., Oncolytic vaccinia virus for the treatment of cancer,
`11(3) Expert Opin. Biol. Ther. 595-608 (2011).
`Hermiston, T., A demand for next-generation oncolytic adenovirus,
`8(4) Current Opinion in Molecular Therapeutics 322-330 (Aug.
`2006).
`Kaufmann et al., Chemovirotherapy ofMalignant Melanoma with a
`Targeted and Armed Oncoytic Measles Virus,133 Journal of Inves-
`tigative Dermatology 1034-1042 (2013).
`Khuri et al., A controlled trial of intratumoral ONYX-0i5, a
`selectively-replicating adenovirus, in combination with cisplatin
`and 5-fluorouracil in patients with recurrent head and neck cancer,
`6(8) Nature Medicine 879-885 (Aug. 2000).
`Kim et al., Replication-selective virotherapy for cancer: Biological
`principles, risk management and future directions, 7(7) Nature
`Medicine 78 1-787 (Jul. 2001).
`Kim et al., Targeted and armed oncolytic poxviruses: a novel
`multi-mechanistic therapeutic class for cancer, 9 Nature 64-7 1 (Jan.
`2009).
`Leach et al., Enhancement of Antitumor Immunity by CTLA-4
`Blockade, 271 Science 1734-1736 (Mar. 22, 1996).
`Lorence et al., Phase i Clinical Experience Using Intravenous
`Administration ofPV70i, an Oncolytic Newcastle Disease Virus, 7
`Current Cancer Drug Targets 157-167 (2007).
`Martuza et al., Experimental Therapy of Human Glioma by Means
`of a Genetically Engineered Virus Mutant, 252 Science 854-856
`(Oct. 3, 1990).
`McDonald et al., A measles virus vaccine strain derivative as a
`novel oncolytic agent against breast cancer, 99 Breast Cancer
`Research and Treatment 177-184 (2006).
`Mineta et a!, Treatment of Malignant Gliomas Using Ganciclovir-
`hypersensitive, Ribonucleotide Reductase-dejicient Herpes Simplex
`Viral Mutant, 54 Cancer Research 3963-3966 (Aug. 1, 1994).
`
`Page 2 of 29
`
`

`

`US 10,555,981 B2
`Page 3
`
`(56)
`
`References Cited
`
`OTHER PUBLICATIONS
`
`Okazaki et al., New regulatory co-receptors: Inducible co-
`stimulator and PD-i, 17 Autoimmunity 779-782 (2002).
`Phuangsab et al., Newcastle disease virus therapy of human tumor
`xenografis: antitumor effects of local or systemic administration,
`172 Cancer Letters 27-36 (2001).
`Presta et al., Humanization of an Anti-Vascular Endothelial Growth
`Factor Monoclonal Antibody for the Therapy of Solid Tumors and
`Other Disorders, 57 Cancer Research 4593-4599 (Oct. 15, 1997).
`Pyles et al., Evidence that the Herpes Simplex Virus Type i Uracil
`DNA Glycosylase Is Required for Efficient
`ral Replication and
`Latency in the Murine Nervous System, 68(8) Journal of Virology
`4963-4972 (Aug. 1994).
`Qureshi et al., Trans-endocytosis of CD8O and CD86: a molecular
`basis for the cell extrinsic function of CTLA-4, 332(6029) Science
`600-603 (2011).
`Rudlin et al., Phase I Clinical Study of Seneca Valley Virus
`(SVV-OOi), a Replication-Competent Picornavirus, in Advance Solid
`Tumors with Neuroendocrine Features, 17(4) Clin Cancer Res
`888-895 (Feb. 15, 2011).
`Senzer et al., Phase II Clinical Trial of a Granulocyte-Macrophase
`Colony-Stimulating Factor-Encoding, Second-Generation Oncolytic
`Herpesvirus in Patents with Unresectable Metastatic Melanoma,
`27(34) Journal of Clinical Oncology 5763-577 1 (Dec. 1, 2009).
`Shi et al., Tumor-specific oncolytic adenoviruses expressing granu-
`locyte macrophage colony-stimulatingfactor or anti-CTLA4 anitbody
`for the treatment of cancers, 21 Cancer Gene Therapy 340-348
`(2014).
`Stojdl et al., Exploiting tumor-specfic defects in the interferon
`pathway with a previously unknown oncolytic virus, 6(7) Nature
`Medicine 821-825 (Jul. 2000).
`Stojdl et al., VSV strains with defects in their ability to shutdown
`innate immunity are potent systemic anti-cancer agents, 4 Cancer
`Cell 263-275 (2003).
`Thorne, S.H. et al., Immunotherapeutic potential of oncolytic vac-
`cinia virus, 4 Frontiers in Oncology 1-5 (Jun. 2014).
`Thorne et al., Rational strain selection and engineering creates a
`broad-spectrum, systemically effective oncolytic poxvirus, JX-963,
`117(11) The Journal of Clinical Investigation 3350-3358 (Novem-
`ber 2007).
`Topalian et al., Targeting the PD-i/B 7-Hi(PD-Li) pathway to
`activate anti-tumor immunity, 24(2) Curr. Opin. Immunol. 207-2 12
`(Apr. 2012).
`Wang et al., VISTA, a novel mouse Ig superfamily ligand that
`negatively regulates Tcell responses, 208(3)J. Exp. Med. 577-592
`(Mar. 14, 2011).
`Wong et al., Oncolytic Viruses for Cancer Therapy: Overcoming the
`Obstacles, 2 Viruses 78-106 (2010).
`
`Xia, et al., Phase III randomized clinical trial on intratumoral
`injection of EiB gene-deleted adenovirus (Hi Oi) combined with
`cispliatin-based chemotherapy in treating squamous cell cancer of
`head and neck or esophagus, 23(12) Al Zheng 1666-1670 (Dec.
`2004) (abstract only).
`Zhang et al., Eradication of Solid Human Breast Tumors in Nude
`Mice with an Intravenously Inject Light-Emitting Oncolytic Vac-
`cinia Virus, 67(20) Cancer Research 10038-10046 (Oct. 15, 2007).
`International Search Report dated Sep. 9,2015, and Written Opinion
`in corresponding PCT Application No. PCT/EP2O1S/066353.
`Kleinpeter et al., Vectorization in an oncolytic vaccinia virus of an
`antibody, a Fab and a scFv against programmed cell death-i
`(PD-i) allows their intraturomal delivery and an improved tumor-
`growth inhibition, 5(10) Oncoimmunology e1220467-e1220467-14
`(2016).
`Potts et al., Oncolytic viruses in the Treatment of Bladder Cancer,
`2012 Advanced in Urology 1-11 (2012).
`Anti-tumoral effect of a vaccinia virus VV-TK-RRJ43, encoding
`murine monoclonal anti-PDi antibody J43, Annex 1 (May 11,
`2018).
`Comparative analysis of the replication capacity of three vaccinia
`viruses in primary versus cancerous cells: the Vaccinia virus
`VVTGi 7i3 7 deleted for I4L gene compared to VVTGi5466 and
`VVTGi 7i0i with intact I4L gene, in human hepatocellular carci-
`noma cells and human primary hepatocytes, Annex 2 (May 11,
`2018).
`Russian Inquiry with English Translation dated Aug. 22, 2018.
`Cory et al., Regulation of ribonucleotide reductase activity in
`mammalian cells, 53-54(1-2) Mo!. Cell. Biochem. 257-266 (1983)
`(Abstract).
`Duxbury et al., RNA interference targeting the M2 subunit of
`ribonucleotide reductase enhances pancreatic adenocarcinoma
`chemosensitivity to gemcitabine, 23 Oncogene 1539-1549 (2004).
`H. L. Elford et al., Effect ofMethotrexate and 5-Fluorodeoxyuridine
`on Robinucleotide Reductase Activity in Mammalian Cells, 37
`Cancer Research 4389-4394 (Dec. 1977).
`Guse et al., Oncolytic vaccinia virus for the treatment of cancer,
`Expert Opinion on Biological Therapy 1-14 (Feb. 2011).
`D. Kim et al., Targeted and armed oncolytic poxviruses: a novel
`multi-mechanistic therapeutic class for cancer, 9 Nature 64-7 1 (Jan.
`2009).
`P. K!einpeter et al., Vectorization in an oncolytic vaccinia virus of
`an antibody, a Fab and a scFv against programmed cell death-i
`(PD-i) allows their intratumoral delivery and an improved tumor-
`growth inhibition, 5(10) Oncoimmunology 1-14 (2016).
`P. K!einpeter et al., By Binding CD8O and CD86, the Vaccinia Virus
`M2 Protein Blocks Their Interactions with both CD28 and CTLA4
`and Potentiates CD8O Binding to PD-Li, 93(11) Journal of Viro!-
`ogy 1-18 (Jun. 2019).
`
`* cited by examiner
`
`Page 3 of 29
`
`

`

`U.S. Patent
`
`Feb. 11, 2020
`
`Sheet 1 of 7
`
`US 10,555,981 B2
`
`Figure 1
`
`r
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`
`Page 4 of 29
`
`

`

`U.S. Patent
`
`Feb. 11, 2020
`
`Sheet 2 of 7
`
`US 10,555,981 B2
`
`Figure 2
`
`Page 5 of 29
`
`

`

`U.S. Patent
`
`Feb. 11, 2020
`
`Sheet 3 of 7
`
`US 10,555,981 B2
`
`Figure 3
`
`100
`
`90
`
`80
`
`70
`
`>.
`4-'
`.a 60
`
`50
`
`j5 40
`U
`'0
`
`0"' E
`
`0
`
`-
`_
`-
`1
`WRTG18616 WRTG18618 WRTG18621 WRTG18O11
`
`-
`
`MOIO.0001
`
`• MOI 0.001
`
`Page 6 of 29
`
`

`

`U.S. Patent
`
`Feb. 11, 2020
`
`Sheet 4 of 7
`
`US 10,555,981 B2
`
`Figure 4
`
`Heavy chain J43 sequence:
`
`MGLGLQWVFFVALLKGVHCEVRLLESGGGLVKPEGSLKLSCVASGFTFSD
`
`YFMSWVRQAPGKGLEWVAHIYTKSYNYATYYSGSVKGRFTISRDDSRSMV
`
`YLOM NN LRTEDTATYYCTRDGSGYPSLDFWGQGTQVTVSSATTTAPSVYP
`
`LAPACDSTTSTTDTVTLGCLVKGYF PE PVTVSWNSGALTSGVHTFPSVLH
`
`SG LYSLSSSVTVPSSTWPKQPITCNVAH PASSTKVDKKI E PRTDTDTCPN
`
`PPDPCPTCPTPDLLGGPSVFIFPPKPKDVLMISLTPKITCVVVDVSEEEP
`
`DVQFNWYVN NVED KTAQTETRQRQYNSTYRVVSVLPI KHQDWMSG KVFKC
`
`KVN NNALPSPIEKTISKPRGQVRVPQIYTFPPPI EQTVKKDVSVTCLVTG
`
`FLPQDIHVEWESNGQPQPEQNYKNTQPVLDSDGSYFLYSKLNVPKSRWDQ
`
`G DS FTCS VIH [A LH NH H MTKTIS RS LG N
`
`Light chain J43 sequence:
`
`MAWTPG I FMVLSYLTGSFSYELTQPPSASVNVG ETVKITCSG DQLPKYFA
`
`DWFHQRSDQTILQVIYDDNKRPSGI PERISGSSSGTTATLTI RDVRAEDE
`
`GDYYCFSGYVDSDSKLYVFGSGTQLTVLGGPKSSPKVTVFPPSPEELRTN
`
`KATLVCLVN DFYPGSATVTWKANGATI N DGVKTTKPSKQGQNYMTSSYLS
`
`LTADQWKSH NRVSCQVTHEGETVEKSLSPAECL
`
`Page 7 of 29
`
`

`

`U.S. Patent
`
`Feb. 11, 2020
`
`Sheet 5 of 7
`
`US 10,555,981 B2
`
`Figure 5
`
`.1
`
`1
`
`1
`
`C -
`
`_
`
`J
`
`H
`
`Page 8 of 29
`
`

`

`U.S. Patent
`
`Feb. 11, 2020
`
`Sheet 6 of 7
`
`US 10,555,981 B2
`
`Figure 6A
`
`Mean concentration of J43 in serum
`
`WRTG18618 Sc
`
`WRTG18618 IT
`
`ü J43 lOpg
`
`J43 1pg
`
`day after injection
`
`Page 9 of 29
`
`

`

`U.S. Patent
`
`Feb. 11, 2020
`
`Sheet 7 of 7
`
`US 10,555,981 B2
`
`Figure 6B
`
`-
`
`C)
`
`Mean concentration of J43 in
`tumor homogenate
`3800
`
`4000
`
`3000
`
`2000
`
`1000
`
`LIWRI86I8IT
`
`0 J43 lOpg
`
`J431pg
`
`700
`
`410
`
`500
`250 [1!L
`
`51
`
`day after injection
`
`Page 10 of 29
`
`

`

`US 10,555,981 B2
`
`1
`ONCOLYTIC VIRUS FOR EXPRESSION OF
`IMMUNE CHECKPOINT MODULATORS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a U.S. National Stage Application
`pursuant to 35 U.S.C. § 371 of International Patent Appli-
`cation PCT/EP2015/066263, filed on Jul. 16, 2015, and
`published as WO 2016/008976 on Jan. 21, 2016, which
`claims priority to European Patent Application 14306153.9,
`filed on Jul. 16, 2014, all of which are incorporated herein
`by reference in their entireties for all purposes.
`
`FIELD OF THE INVENTION
`
`The present invention generally relates to the field of
`oncolytic virotherapy and more specifically to compositions
`and methods to treat, prevent, or inhibit proliferative dis-
`eases, especially cancer. Embodiments include an oncolytic
`virus comprising nucleotide sequence(s) encoding one or
`more immune checkpoint modulator(s). Embodiments also
`include a pharmaceutical composition comprising such
`oncolytic virus and, eventually, a pharmaceutically accept-
`able vehicle and its use for treating proliferative diseases
`such as cancers.
`Cancer is caused by both external factors (e.g. tobacco,
`infectious organisms, alimentary habits, chemicals, and
`radiation) and internal factors (e.g. inherited mutations,
`hormones, immune conditions, and mutations that occur
`from metabolism). Each year, cancer is diagnosed in more
`than 12 million subjects worldwide. In industrialized coun-
`tries, approximately one person out five will die of cancer.
`Although a vast number of chemotherapeutics exist, they are
`often ineffective, especially against malignant and meta-
`static tumors that establish at a very early stage of the
`disease. Moreover, antitumor immunity is often ineffective
`due to the fact that tumor cells have evolved mechanisms to
`escape host defense. One of the major mechanisms of
`immune suppression is a process known as "T-cell exhaus-
`tion", which results from chronic exposure to antigens and
`is characterized by the upregulation of inhibitory receptors.
`These inhibitory receptors serve as immune checkpoints in
`order to prevent uncontrolled immune reactions. Various
`immune checkpoints acting at different levels of T cell
`immunity have been described in the literature, including
`programmed cell death protein 1 (PD-i) and its ligands
`PD-Li and PD-L2, CTLA-4 (cytotoxic T-lymphocyte asso-
`ciated protein-4), LAG3, B and T lymphocyte attenuator,
`T-cell immunoglobulin, mucin domain-containing protein 3
`(TIM-3), and V-domain immunoglobulin suppressor of T
`cell activation.
`Whatever the mechanism of action, these immune check-
`points can inhibit the development of an efficient anti-tumor
`immune response. There is increasing interest in the possible
`therapeutic benefits of blocking such immune checkpoints as
`a means of inhibiting immune system tolerance to tumors
`and thus rescue exhausted antitumor T cells (Leach et al.,
`1996, Science 271: 1734-6). A vast number of antagonistic
`antibodies have been developed during the last decade (e.g.
`anti Tim3, -PD-Li, -CTLA-4, -PDi, etc) and most impor-
`tantly, some have been associated with objective clinical
`responses in cancer patients. Antibodies targeting CTLA-4
`are already marketed (e.g. Ipilimumab, Yervoy, Bristol-
`Myers Squibb) for metastatic melanoma. BMS reported that
`from 1800 melanoma patients treated with ipilimumab 22%
`are still alive 3 years later. Antibody therapies with anti
`
`10
`
`(e.g.
`
`(e.g. MPDL328OA, Roche), anti PD-i
`PD-Li
`Nivolumab, BMS) are also ongoing.
`Another therapeutic approach that is emerging in the field
`of cancer is oncolytic viruses (Hermiston, 2006, Curr. Opin.
`Mol. Ther. 8: 322-30). Oncolytic viruses are capable of
`selective replication in dividing cells (e.g. cancer cell) while
`leaving non dividing cells (e.g. normal cells) unharmed. As
`the infected dividing cells are destroyed by lysis, they
`release new infectious virus particles to infect the surround-
`ing dividing cells. Cancer cells are ideal hosts for many
`viruses because they have the antiviral interferon pathway
`inactivated or have mutated tumour suppressor genes that
`enable viral replication to proceed unhindered (Chernajo-
`vsky et al., 2006, British Med. J. 332: 170-2). A number of
`viruses including adenovirus, reovirus, measles, herpes sim-
`15 plex, Newcastle disease virus and vacciia have now been
`clinically tested as oncolytic agents.
`Some viruses are naturally oncolytic (such as reovirus and
`the Seneca valley picornavirus) while others are engineered
`for tumor selectivity by modifying the viral genome. Such
`20 modifications include functional deletions in essential viral
`genes, the use of tumor- or tissue-specific promoters to
`control the viral gene expression and tropism modification to
`redirect virus to the cancer cell surface.
`The first oncolytic virus to be approved by a regulatory
`25 agency was a genetically modified adenovirus named Hi0i
`(Shanghai Sunway Biotech) that gained approval in 2005
`from China's State Food and Drug Administration (SFDA)
`for the treatment of head and neck cancer. Another oncolytic
`adenovirus, named ONYX-015 is in ongoing clinical trials
`30 for the treatment of various solid tumors (in phase III for the
`treatment of recurrent head and neck cancer) (Cohen et al.,
`2001, Curr. Opin. Investig. Drugs 2: 1770-5). As another
`example, oncolytic herpes simplex 1 (T-VEC) was geneti-
`cally engineered to attenuate the virus virulence, increase
`35 selectivity for cancer cells and enhance antitumor immune
`response (through GM-CSF expression). Clinical efficacy in
`unresectable melanoma has been demonstrated in Phase II
`and Phase III clinical trials (Senzer et al, 2009, J. Clin.
`Oncol. 27: 5763-71).
`40 Vaccinia viruses (VV) possess many of the key attributes
`necessary for use in oncolytic virotherapy such as natural
`tropism for tumors, strong lytic ability, short life cycle with
`rapid cell-to-cell spread, highly efficient gene expression
`and a large cloning capacity. In addition, they have been
`45 delivered to millions of individuals during the smallpox
`eradication campaign without major safety concerns. In this
`respect, a TK and VGF double deleted VV (Wyeth strain)
`expressing GM-CSF (named JX-963) showed significant
`cancer selectivity in tumor bearing mice (Thorne et al.,
`50 2007, JClin Invest. 117: 3350-8). Onthe same line, JX-594,
`a TK-deleted VV (Wyeth strain) armed with GM-CSF, has
`shown promising clinical data, and a randomized Phase III
`trial in hepatocellular carcinoma is expected to start soon.
`Combination therapies involving oncolytic virus and
`55 immune checkpoint inhibitors have been described in the
`literature. WO20i4/022138 describes the combination of
`irradiated tumor cells, an oncolytic adenovirus and an anti
`CTLA4 antibody for use for treating bladder or prostate
`cancer. WO20i4/047350 envisages a recombinant oncolytic
`60 virus with a gene encoding an anti-PD-i antibody inserted in
`the viral genome without providing any working example
`that would support utility of such an oncolytic virus.
`
`TECHNICAL PROBLEM
`
`65
`
`One may expect that cancer will continue to be a serious
`global health threat for many years due to the high number
`
`Page 11 of 29
`
`

`

`US 10,555,981 B2
`
`3
`of causal factors that may act together or separately to
`initiate or promote the development of a cancer. Moreover,
`malignant and especially metastatic tumors are often resis-
`tant to conventional therapies explaining the significant
`morbidity of some cancers.
`Thus, there is an important need to develop more effective
`approaches, for improving prevention and treatment of such
`proliferative diseases, and especially metastatic cancers. The
`present invention provides a unique product combining
`oncolysis for killing dividing cells and immune checkpoint
`for breaking cancer-associated immune tolerance.
`This technical problem is solved by the provision of the
`embodiments as defined in the claims.
`Other and further aspects, features and advantages of the
`present invention will be apparent from the following
`description of the presently preferred embodiments of the
`invention. These embodiments are given for the purpose of
`disclosure.
`
`SUMMARY OF THE INVENTION
`
`The present invention concerns an oncolytic virus com-
`prising inserted in its genome one or more nucleic acid
`molecule(s) encoding one or more immune checkpoint
`modulator(s).
`The oncolytic virus is preferably selected from the group
`consisting of reovirus, New Castle Disease virus (NDV),
`vesicular stomatitis virus (VSV), measles virus, influenza
`virus, Sinbis virus, adenovirus, poxvirus and herpes virus
`(HSV) and the like. In one embodiment, the oncolytic virus
`is a vaccinia virus. In a preferred embodiment, the vaccinia
`virus is engineered to lack thymidine kinase activity (e.g. the
`genome of said VV has an inactivating mutation in J2R gene
`to produce a defective TK phenotype). Alternatively or in
`combination, the vaccinia virus is engineered to lack RR
`activity (e.g. the genome of said VV has an inactivating
`mutation in I4L and/or F4L gene to produce a defective RR
`phenotype).
`In one embodiment, the vaccinia virus further expresses at
`least one therapeutic gene, in particular a gene encoding a
`suicide gene product and/or an immunostimulatory protein.
`In one embodiment, the encoded one or more immune
`checkpoint modulator(s) is an antagonist molecule that
`antagonizes the activity of PD-i, PD-Li or CTLA4 with a
`specific preference for an anti PD-i antibody and/or an anti
`CTLA4 antibody.
`The present invention further provides a composition
`comprising said oncolytic virus, eventually with a pharma-
`ceutical acceptable vehicle. In one embodiment, the com-
`position is formulated for intravenous or intratumoral
`administration.
`The present invention also concerns the use of said
`oncolytic virus or composition thereof for treating a prolif-
`erative disease as well as a method of treatment relying on
`the administration of an effective amount of said oncolytic
`virus or composition thereof In one embodiment, the pro-
`liferative disease treated by the method of the invention is
`cancer and especially melanoma, renal cancer, prostate
`cancer, breast cancer, colorectal cancer, lung cancer and
`liver cancer. In one embodiment, the use or method com-
`prises an additional step in which a pharmaceutically accept-
`able amount of a prodrug is administered to said mammal.
`The administration of said prodrug takes place preferably at
`least 3 days after the administration of said oncolytic virus
`or virus composition.
`
`4
`DETAILED DESCRIPTION
`
`15
`
`20
`
`The present invention concerns an oncolytic virus com-
`prising inserted in its genome one or more nucleic acid
`5 molecule(s) encoding one or more immune checkpoint
`modulator(s).
`Definitions
`As used throughout the entire application, the terms "a"
`and "an" are used in the sense that they mean "at least one",
`10 "at least a first", "one or more" or "a plurality" of the
`referenced components or steps, unless the context clearly
`dictates otherwise. For example, the term "a cell" includes
`a plurality of cells, including mixtures thereof
`The term "one or more" refers to either one or a number
`above one (e.g. 2, 3, 4, 5, etc).
`The term "and/or" wherever used herein includes the
`meaning of "and", "or" and "all or any other combination of
`the elements connected by said term".
`The term "about" or "approximately" as used herein
`means within 20%, preferably within i 0%, and more pref-
`erably within 5% of a given value or range.
`As used herein, when used to define products, composi-
`tions and methods, the term "comprising" (and any form of
`25 comprising, such as "comprise" and "comprises"), "having"
`(and any form of having, such as "have" and "has"),
`"including" (and any form of including, such as "includes"
`and "include") or "containing" (and any form of containing,
`such as "contains" and "contain") are open-ended and do not
`30 exclude additional, unrecited elements or method steps.
`Thus, a polypeptide "comprises" an amino acid sequence
`when the amino acid sequence might be part of the final
`amino acid sequence of the polypeptide. Such a polypeptide
`can have up to several hundred additional amino acids
`35 residues. "Consisting essentially of' means excluding other
`components or steps of any essential significance. Thus, a
`composition consisting essentially of the recited compo-
`nents would not exclude trace contaminants and pharma-
`ceutically acceptable carriers.Apolypeptide "consists essen-
`40 tially of' an amino acid sequence when such an amino acid
`sequence is present with eventually only a few additional
`amino acid residues. "Consisting of' means excluding more
`than trace elements of other components or steps. For
`example, a polypeptide "consists of' an amino acid
`45 sequence when the polypeptide does not contain any amino
`acids but the recited amino acid sequence.
`The terms "polypeptide", "peptide" and "protein" refer to
`polymers of amino acid residues which comprise at least
`nine or more amino acids bonded via peptide bonds. The
`50 polymer can be linear, branched or cyclic and may comprise
`naturally occurring and/or amino acid analogs and it may be
`interrupted by non-amino acids. As a general indication, if
`the amino acid polymer is more than 50 amino acid residues,
`it is preferably referred to as a polypeptide or a protein
`55 whereas if it is 50 amino acids long or less, it is referred to
`as a "peptide".
`Within the context of the present invention, the terms
`"nucleic acid", "nucleic acid molecule", "polynucleotide"
`and "nucleotide sequence" are used interchangeably and
`60 define a polymer of any length of either polydeoxyribo-
`nucleotides (DNA) (e.g. cDNA, genomic DNA, plasmids,
`vectors, viral genomes, isolated DNA, probes, primers and
`any mixture thereof) or polyribonucleotides (RNA) (e.g.
`mRNA, antisense RNA, SiRNA) or mixed polyribo-
`65 polydeoxyribonucleotides. They encompass single or
`double-stranded, linear or circular, natural or synthetic,
`mo