(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`6 February 2014 (06.02.2014)
`
`WIPOI PCT
`
`\9
`
`(10) International Publication Number
`
`WO 2014/022138 A2
`
`(51)
`
`International Patent Classification:
`A61K 48/00 (2006.01)
`A61K 39/395 (2006.01)
`
`(21)
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`International Application Number:
`
`PCT/USZOl3/051535
`
`(22)
`
`International Filing Date:
`
`(25)
`
`(26)
`
`(30)
`
`(72)
`(71)
`
`(74)
`
`(81)
`
`Filing Language:
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`Publication Language:
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`22 July 2013 (22.07.2013)
`
`English
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`English
`
`Priority Data:
`61/741,804
`
`30 July 2012 (30.07.2012)
`
`US
`
`Inventor; and
`Applicant : YEUNG, Alex Wah Hin [US/US]; 2 Nar-
`bonne, Newport Beach, California 92660 (US).
`
`Agent: YANG, James C.; STETINA BRUNDA GARRED
`& BRUCKER, 75 Enterprise, Suite 250, Aliso Viejo, Cali-
`fornia 92656 (US).
`
`Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, G11, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ,
`OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC,
`SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`KM, ML, MR, NE, SN, TD, TG).
`Published:
`
`without international search report and to be republished
`upon receipt ofthat report (Rule 48.2(g))
`
`(54) Title: LIVE AND IN—VIVO TUMOR SPECIFIC CANCER VACCINE SYSTEM DEVELOPED BY CO—ADMINISTRATION OF EITIIER
`AT LEAST TWO OR ALL THREE OF THE FOLLOWING COMPONENTS SUCH AS TUMOR CELLS, AN ONCOLYTIC VIRUS VECTOR
`WITH TRANSGEN'IC EXPRESSION OF GM—CSF AND AN IM1VIUNE CHECKPOINT MODULATOR
`
`A
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`Wild Type Ad 5
`
`Packaging signal
`rml ElA
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`EIB
`.
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`53
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`ITR
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`CGOO70
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`\ /
`Endogenous promoters
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`EZF—l
`Midi-Iii III! Illlll. ,
`
`
`
`1‘my
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`12.5, 6.7
`
`hGMCSF FADP, RIDu, RIDIS, 14.7
`
`Fig. 1
`
`(57) Abstract: The invention discloses a novel tumor—specific complete vaccine system generated in—vivo. This vaccine system is de—
`Veloped by the use of separated tumor cells inactivated by irradiation and the in-vivo interaction with an oncolytic viral vector with
`transgenic expression of GM—CSF, completed with immune checkpoint modulators ("lCM") such as co—stimulatory signals confirma—
`tion with an anti-CTLA4 antibody. Specifically there will be no pre-incubation or interaction of the either two or all three compon -
`ents before administration to the patient. One of such oncolytic virual vector examples is CG0070 (GM—CSF expressing condition—
`ally replication competent adenovirus). Mixing of the tumor-Viral-ICM components will take place just prior to administration to
`preserve the effects of the oncolytic process and subsequent immunotherapeutic responses to be live and in vivo from the very first
`beginning. This invention is a complete live and in-vivo cancer vaccine system ("CLIVS").
`
`
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`LIVE AND IN-VIVO TUMOR SPECIFIC CANCER VACCINE SYSTEM
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`DEVELOPED BY CO-ADMINISTRATION OF EITHER AT LEAST TWO OR
`
`ALL THREE OF THE FOLLOWING COMPONENTS SUCH AS TUMOR
`
`CELLS, AN ONCOLYTIC VIRUS VECTOR WITH TRANSGENIC
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`EXPRESSION OF GM-CSF AND AN IMMUNE CHECKPOINT
`
`MODULATOR
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`Applicant: Yeung, Alex Wah Hin, 2 Narbonne, Newport Beach, CA 92660, USA
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`10
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`Inventor: Yeung, Alex Wah IIin (US)
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`15
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`20
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`FIELD OF THE INVENTION
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`The present
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`invention relates to novel cancer vaccine combinations.
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`In
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`particular, the present invention relates to tumor-specific immunotherapeutic vaccine
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`system comprising irradiated tumor cells, an oncolytic viral expressing GM—CSF, and
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`an immune checkpoint regulatory molecule, kits comprising the tumor—specific
`
`immunotherapeutic vaccine system and methods of use therefor.
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`BACKGROUND OF THE INVENTION
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`An oncolytic virus with transgenic expression of GM-CSF, such as CG0070,
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`has been preliminary successfully when given intravesically in bladder cancer [V0046
`
`trial]. Long term complete responses lasting more than a few years have been reported
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`after only a six weekly course of therapy, raising possibilities that this agent is indeed
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`be able to mount a tumor specific immunotherapy against this cancer. To further
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`expand upon this theory, it is proposed here to use a unique way of interaction by a
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`live vaccine system for other cancers that are not easily accessible as those inside the
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`bladder. The first component is the patient’s own tumor cells (though allogeneic
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`tumor cells may also be used), taken from biopsy or from a surgical specimen. The
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`second component is an oncolytic virus with GM-CSF expression such as CG0070.
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`The third component is the immune checkpoint modulators, an example given is the
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`co-stimulatory signals confinnation molecule anti-CTLA4 antibody. Instead of pre-
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`incubation or interaction such as in all past methodologies of cancer vaccine to
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`generate antigens or virus attached, infected or modified tumor cells in—vitro, the
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`present novel tumor—virus—ICM vaccine or CLIVS will be developed, live and in—vivo,
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`by mixing the three components only just prior to administration to patients. This will
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`allow the full oncolytic and immunogenic effects to be within the real time reaction of
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`the patient’s own immune system. It is believed that such novel method of delivery
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`will enhance the chance of a tumor specific tumor immunotherapy that has never been
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`realized before.
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`In addition, other
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`immune checkpoint modulators, anti-suppressor cell
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`molecules or stimulation of sustaining co-confirmatory signals modulators can also be
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`co-administered to reinforce strong and long lasting tumor specific immune reactions.
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`Alternatively, immune checkpoint modulators confirmation may be omitted in certain
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`type of cancers.
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`SUMMARY OF THE INVENTION
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`In one aspect of the invention, a tumor-specific immunotherapeutic vaccine
`
`system comprising either at least two or all three components: separated tumor cells
`
`isolated and inactivated by irradiation, an oncolytic viral and a cancer specific vector
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`comprising a heterologous nucleic acid encoding GM—CSF and an immune checkpoint
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`modulator
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`(“ICM”), wherein the three components are admixed just prior
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`to
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`administration to patient without any pre—incubation are provided.
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`In another aspect of the invention, the immune checkpoint modulator (ICM) is
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`omitted from the vaccine system.
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`In certain aspects, the immune checkpoint molecule (ICM) is an anti—CTLA4
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`antibody.
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`In certain preferred embodiments, the anti—CTLA4 antibody is selected
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`from ipilimumab, tremilimuab and a single chain anti-CTLA-4 antibody.
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`In other aspects, the ICM is the 0X40 binding agent or agonist, or an OX40L
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`molecule that can maintain T cell proliferation beyond the first few days.
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`In another embodiment, the ICM are antibodies or modulators against PDl,
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`TIM3, B7—H3, B7—H4, LAG—3 and KIR or its ligands.
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`In another embodiment, the ICM component can be the combination of two or
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`more of ICMs as described in previous embodiments.
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`In some embodiments,
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`the separate tumor cells are collected and prepared
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`from an autologous, allogeneic or a combination of autologous and allogeneic cells.
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`'lhc autologous and allogeneic cells in certain embodiments may be prepared from
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`cell cultures. In other embodiments,
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`the tumor cells may have been modified to
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`secrete agents that will enhance immune modulation. One of these examples is the
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`GVAX autologous or allogeneic cancer cell therapy, the cells of which secrete GM-
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`CSF.
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`In another aspect, the oncolytic viral component is from an adenovirus, an
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`example is CG0070, an adenoviral vector with an E2F promoter and transgene
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`expression of GM-CSF.
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`In another aspect,
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`the oncolytic viral component is any one of the other
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`oncolytic viruses that will be able to express GM-CSF after transduction. Examples,
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`but not exclusively limited to this list, are Herpes Simplex Virus, Vaccinia virus,
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`Mumps virus, Reovirus and Newcastle Disease Virus.
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`In one aspect of the invention,
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`the CLIVS is given subcutaneously to the
`
`patient.
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`In another aspect of the invention, the CLIVS can be given by other routes to
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`the patient, such as epidermal, intramuscular, into lymphatic chains and other sites or
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`organs known to those familiar with the art and that may enhance immune response.
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`In other aspects of the invention, kits comprising: separated tumor cells
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`isolated and inactivated by irradiation, an oncolytic viral and a cancer specific vector
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`comprising a heterologous nucleic acid encoding GM—C SF and an immune checkpoint
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`modulator (“ICM”), and a packaging insert containing directions for use are provided.
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`In other aspects of the invention,
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`tumor cell preparation kits comprising:
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`materials and methods to conduct
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`tumor dissociation and preparation, enzymatic
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`and/or virus vector transduction agents, cryopreservation vials etc. and a packaging
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`insert containing directions for use.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`Figure 1 is a schematic diagram of CG0070 and wild type (wt) adenovirus
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`type 5. CG0070 is based on adenovirus serotype 5 and the endogenous Ela promoter
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`and E3 19kD coding region have been replaced by the human E2F-1 promoter and a
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`cDNA coding region of human GM-CSRF, respectively.
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`Figure 2.2.1.1 illustrates two bar charts. Selective Ela gene transcription and
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`GM—CSF production in normal Wi38 fibroblasts and Wi38-VA13 cells. A. Selective
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`Ela gene transcription. Wi38 (normal Rb pathway) and Wi38—VA13 (defective Rb
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`pathway) cells were mock infected or infected with CG0070 at 100 or 1000 viral
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`particles (Vp) per cell (ppc) for 1 hour on ice followed by incubation at 37°C to
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`synchronize viral uptake. Quantitative RT—PCR for Ela 111RNA was performed 24
`
`hours post infection. Ela RNA levels were normalized to hexon DNA copy number
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`determined at 4 hours post—infection.
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`>“p<0.01 t—test, Ela RNA in Wi38—VA13 vs.
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`Ela RNA in Wi38 infected with the same vector. B. Selective GM—DXF production.
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`The supernatants from CG0070-infected Wi38 and Wi38-VA13 cells (100 ppc) were
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`analyzed for human GM—CSF 15 24 hours by ELISA.
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`>“p0<0.01, t-test, GM-DSF
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`level in Wi38-VA13 cells vs. Wi38 cells.
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`Figure 2.2.2.1 illustrates a bar chart. Productivity of CG0070 and wild type
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`adenovirus in Rb pathway—defective human bladder TCC cells and normal cells.
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`Monolayers of 293, human bladder TCC cell lines (RT4, SW780, UC14 and 253J B—
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`V cells) and human normal cells (fibroblasts MRC5 and aortic endothelial hAEC)
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`were infected with either CG0070 or wild type adenovirus at a MOI of 2 plaque
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`forming units (pfu)/cell. Cells were harvested 72 hours after infection and Virus titers
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`were determined by plaque assay on 293 cells. The average of duplicate titers from
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`two independent experiments was determined and normalized on 293 cells.
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`Figure 2 illustrates
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`a graph.
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`Anti-tumor efficacy of CG0070 in the
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`subcutaneous 253J BOV bladder TCC xenograft model. NCR.nude mice bearing
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`subcutaneous tumors received intratumoral injections of saline or CG0070 five times
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`as indicated by the arrows (SD 1, 3, 5, 8 and 10). The group average tumor volumes
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`i standard deviation (n=10 per group) are shown for mice that received saline or 3 X
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`108 Vp, 3 X 109 or 3 X 1010 vp of CG0070 per injection. No significant difference in
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`anti—tumor efficacy was observed among three treatment groups during the course of
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`the study. All of the CG0070-treated groups were all statistically different from the
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`saline treated group (p<0.001, t-test) on SD 60. Between SD 43 and SD 47, 4/10
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`animals in the PBS group were euthanized due to tumor volume resulting in a drop in
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`the average tumor volume in this group.
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`Figures 2.3.2A—C are reproductions of test mice images. Anti-tumor efficacy
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`of CG0070 in SW780—Luc orthotopic bladder tumor model. Following establishment
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`of orthotopic bladder tumors, mice were treated intravesically with 50 uL of either
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`PBS or CG0070 at the dose indicated in the figure. Mice were imaged every week
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`following intraperitoneal injection of Luciferin. The images shown were taken on SD
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`1 (A-C) and SD 32 (C) or SD 42 (B) for all animals except as indicated in the figure.
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`Figure 2.3.3 illustrates two graphs. Serum GM-CSF expression. CG0070 and
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`Ar20-1004 (2 X 1010 vp/injection) were injected intratumorally on SD 1, 3, and 5 as
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`indicated by arrows. Mice were bled and tumors were removed on indicated SD, and
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`serum (panel A) and tumor extracts (panel B) were prepared and assayed for GM- CSF
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`using human— or murine—specific ELISAs. No GM-CSF was detected in mice injected
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`with saline (not shown). Each data point represents the average i standard deviation
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`of 5 mice.
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`Figure 2.3.4 illustrates a single graph. Anti-tumor efficacy in established
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`CMT—64 tumors. CMT-64 tumors were established subcutaneously in C57Bl/6 mice.
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`When the tumors reached an average of 120 mm3, saline or adenovirus (2 X 1010 vp)
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`was injected once daily or three consecutive days, as indicated by the arrows.
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`Treatments are indicated in the graph insets. Data represent
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`the average tumor
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`volume and standard error of the mean (n=9 per group). Asterisks indicate p<0.05
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`versus saline. Asterisks below a symbol indicates significance only for the treatment
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`group directly above the symbol whereas the asterisk above symbols indicates
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`significance for all groups below the symbol compared to saline injection.
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`Figure 2.3.5 illustrates a bar chart. Tumor—draining lymph node enrichment of
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`CDllc+ cells in the CMT-64 tumor model following treatment with Ar20-1004.
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`CMT—64 tumor-bearing C57Bl/6 mice were injected daily with HBSS (saline) or 1 X
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`1010 particles/injection of Ar20-1004 or Ar20-lO6l for 4 days. Four days after the
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`last
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`injection, mice were euthanized and the tumor—draining lymph nodes (right
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`inguinal lymph node) were collected from each mouse. Single ell suspensions were
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`prepared and stained with an anti-mouse CDllc monoclonal antibody. Each bar
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`represents the man percent positive cell staining i SD (n:lO per group). The actual
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`percent positive staining (background subtracted) is indicated above the bars. The
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`asterisk indicated p<0.001 compared to HBSS or Ar20— 1061, by one—way ANOVA.
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`DETAILED DESCRIPTION OF THE INEVTION
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`Unless defined otherwise, all technical and scientific terms used herein have
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`the same meaning as is commonly understood by one of skill in the art to which this
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`invention belongs. All patents and publications referred to herein are incorporated by
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`reference.
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`Cancer treatment vaccines, in various combinations and methodologies, have
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`been developed in the past few decades without much clinical success. The human
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`immune system of innate and adaptive immunity is an extremely complex system that
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`has never been successfully utilized to fight against cancer. One explanation is, since
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`cancers are usually developed within the later part of life, the development of an
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`immunological response to counteract cancer is not vital to the survival of the fittest
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`theory in the evolutionary process. In all likelihood, the different aspects of the human
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`immune system are not designed specifically for that purpose.
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`This invention relates to the preparation of a complete live and in-vivo tumor-
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`virus vaccine system, and its use in immunotherapy of tumor
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`treatment and
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`metastases. This complete live and in—vivo cancer vaccine system (CLIVS) is then
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`able to develop the specific cancer immunotherapeutic effects.
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`Even after extensive removal of the primary tumor it is still a problem to
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`prevent the formation of metastases either due to growing out of micro-metastases
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`already present at the time of surgery, or to the formation of new metastases by tumor
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`cells or tumor stem cells that have not been removed completely or being re-attached
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`after surgery. In essence, for later stages of cancer, surgery and/or radiotherapy can
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`only take care of the macroscopic lesions, while most patients will have their cancers
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`grow back and not amenable to further therapies.
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`More recently FDA has approved two immunotherapeutic agents against
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`prostate cancer and melanoma. The first agent, Provenge, utilizes GM—CSF fusion
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`molecule with a prostatic antigen to activate the mononuclear or antigen presenting
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`cells of late stage patients in-Vitro and is able to lengthen the overall survival of these
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`patients. The second agent is an anti—CTLA 4 monoclonal antibody that had shown a
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`profound enhancing effect on immune checkpoint modulators signals in T effector
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`cell generation for melanoma patients. An oncolytic virus CG0070 was also been
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`shown to have a long—term complete response effect in bladder cancer patients after
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`one series of six weekly intravesical treatments [V0046 clinical study report].
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`An object of the present invention is to prepare a complete tumor-viral-ICM
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`live (tumor and viral vector both) in-vivo (vaccine generated in patient) vaccine
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`system with immune checkpoint modulators,
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`such as co-stimulatory signals
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`confirmation by an anti—CTLA4 antibody, which can selectively induce a specific
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`immune response of the patient towards the tumor, and thereby enable a systemic
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`effect against residual primary tumor or in metastatic lesions.
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`Normally tumor—specific immune T lymphocytes in cancer patients, even
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`when they are present, only occur at low frequency among the lymphocytes. The
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`likely reason is that
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`the antigenicity and immunogenicity of tumor antigens is
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`generally weak, as well as the presence of overwhelming amount of suppressor
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`activities through cytokines and regulatory cells such as Treg etc.
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`It is now found that the activation of the immune system could be crucially
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`improved by combining different specific immunogenic components. The older
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`concepts of using nonspecific components to booster specific components were found
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`to have little success, as the ability for a human body to generate very specific
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`immunological
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`responses
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`against
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`its own cells
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`(most cancer cells
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`are not
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`immunogenic enough to be different from normal cells) is limited by nature. Such an
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`action derived from non-specific immunological component, even if generated, will
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`be short-lived.
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`The present
`
`invention is
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`to introduce a network of specific immune
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`components that have never been tried in combination before to overcome the natural
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`and complicated suppressing activities of the human body and to produce specific
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`immunotherapeutic effects against its own cancer cells.
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`The first specific component in the present invention is to use the autologous
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`tumor cells isolated from the rcscctcd tumor by mechanical and cnzymatical methods.
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`Since cancer cells, particular in metastatic sites, are heterogenous mixtures of
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`different clones of cells undergoing rapid replications and frequent mutations, it is
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`always best to have a specific component that may adapt to these changes while or
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`when they do occur. Autologous tumor cells can be prepared from the original
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`surgical specimen, biopsies or from removal of metastatic lesions later on. One of the
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`advantages of this vaccine is that this component can be changed according to the
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`patient’s response and the availability of tumor samples. Thus a tumor—viral live and
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`in—vivo vaccine system generated in the primary tumor phase may be different than
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`the one generated later on, using tumor cells from metastatic sites. The ultimate goal,
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`of course,
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`is to adapt the immunotherapeutic response according to the prevailing
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`tumor types, an advantage that cannot be found in recent development of pathway—
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`targeted therapy or monoclonal antibody—directed therapy.
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`The second specific component is the live and replicative competent cancer
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`specific oncolytic viral vector. One of such examples is the CG0070, which has a
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`promoter of the Ela early viral gene using the cancer specific E2F group of
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`transcriptional proteins. As recent studies showed, E2F protein is active in most
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`cancer and progenitor cells, and not
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`just only associated with RB pathway
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`deficiencies. This component is different from other tumor-viral vaccines in the past.
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`All of these past cancer vaccines are either non—specific, meaning they cause lysis of
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`normal cells or are not administered as a live viral vaccine system without irradiation.
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`The third specific component of the present
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`tumor-viral
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`live and in-vivo
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`vaccine system is that the oncolytic virus will be able to generate GM—CSF,
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`the
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`crucial cytokine that enable dendritic cell and other antigen presenting cells to mature
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`and be able to both sample and then to cross-present tumor agents to CD4 cells. Even
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`though antigen presenting cells are involved in non specific immune activities, the
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`availability of suitable and sufficient amounts of GM-CSF in situ will move the
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`direction of the immune response towards a more specific manner, namely in the Thl
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`and Thl7 pathway, if there are sufficient as well as immunogenic tumor antigens
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`present. Few past tumor—viral or tumor-infective agents were based on GM-CSF
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`transgenic expression and even if they did, the vaccines were not delivered with a
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`replicative competent form of the virus.
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`The fourth and one of the most differentiating components of this tumor-viral
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`live and in—vivo cancer vaccine system is that the generation of the vaccine will be
`
`developed in-vivo with immune checkpoint modulators, such as the use of co-
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`stimulatory signals confirmation by an anti-CTLA4 antibody. And that is why the
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`invention is named as a complete vaccine system rather than an already manufactured
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`or in-vitro defined vaccine. This sets the current vaccine apart from the closest
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`patented tumor—viral vaccine as shown in US patent 5273745, whereby the tumor—
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`viral vaccine has to be incubated beforehand in serum free media and then irradiated
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`again to confer non-replication of the viral vector. The viral part of the virally adhered
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`tumor cells will be viewed more or less as an adjuvant, such that
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`it can elicit
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`cytokines, mainly interferon (described in the patent) and be a non—specific
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`component
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`in this immunothcrapeutic approach.
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`In this present
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`tumor-viral-lCM
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`system, the viral vector is replicate competent, though limited and specific only to
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`cancer cells or RB defective pathway cells, and this replicative process will elicit the
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`live and in—vivo system that has never been described. The most distinctive features
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`is, as the tumor lysis is happening in Vivo, the vaccine is formed from the interaction
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`of these components (tumor and live Virus), thereby enabling a stable, sufficient
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`supply of immediate cancer cell death proteins such as tumor associated or tumor
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`specific antigens that are vital to the stimulation of a specific tumor response. In
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`recent studies, it has been shown that not only are these cancer antigens important, but
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`the proteins released from cancer cell death,
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`together with the right cytokine or
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`chemokine environment will make this an ideal situation for the antigen presenting
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`cells, mainly dendritic cell, now primed by the GM-CSF mentioned above,
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`to
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`stimulate a successful and sustaining specific tumor
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`response. The real
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`time
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`happening of these events and the immune checkpoint modulators with co—stimulatory
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`signals confirmation by an anti-CTLA antibody will increase the chance of success in
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`this new invention.
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`This complete live and in-Vivo cancer vaccine system or CLIVS is also
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`obviously different from the intra—tumoral injection of oncolytic viruses with GM—
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`CSF expression. The delivery of oncolytic viruses by the intra-tumoral route has the
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`advantage that no tumor cell preparation in the in-vitro setting is necessary, but has
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`obvious disadvantages such as excessive and uncontrollable leakage of virus vectors
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`such that the dose of the Viral vector has to be increased to compensate for such a loss,
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`while there will still be no guarantee such a dose can reach the ideal multiplicity of
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`infectious ratio related to tumor cells. Most likely there will also be a lack of exposure
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`and unpredictable receptor or adherence interaction between tumor cells and the Virus
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`vector, disruptive tumor blood supply, difficulty in the access of most visceral tumors
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`and the potential spreading of live tumor cells into other parts of the body during
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`injections. Established tumors are also expected to have increased suppressor cell
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`activities and hostile environment for such therapy. It will also be difficult to calculate
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`the effective dose or to add other effective and supplemental immunotherapeutic
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`agents such as anti—CTLA4 antibodies to intra—tumoral
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`injections because of the
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`inherent messy and unpredictable nature of the procedure. Furthermore, intratumoral
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`injection of replication competent oncolytic Viral therapy will not be applicable to the
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`adjuvant setting when immunotherapy will have the best predictable effectiveness by
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`preventing recurrence.
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`As can be shown in recent studies, 1L6 and TGFB are primarily responsible for
`
`the auto-immune process development in a number of experimental animal models.
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`The Thl7 pathway and the activation and proliferation of CD4 cells associated in this
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`process is driven by the presence of sufficient and immunogenic antigens, together
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`with the availability of mature antigen presenting cells.
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`In our previous study
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`[V0046], one of the important cytokines associated with tumor response is 1L6
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`(results not shown). In this novel CLIVS approach, the cancer specific oncolytic virus
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`vector expressing GM—CSF (e. g. CG0070) will be responsible, hypothetically, for the
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`purely immunogenic effect enabling the helper T cells pathway shift from a mainly
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`Th1 to a mainly Thl7, with the presence of apoptotic tumor cells, its associated or
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`specific antigens and mature antigen presenting cells
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`from GM—CSF on-site
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`expression. It will be the first time that any oncolytic viral vector is utilized only for
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`its immunogenic effect, since the oncolytic effect is not meaningful because the tumor
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`cells have already been irradiated and will not be able to proliferate. The use of the
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`CG0070 immunogenic effect is also novel because it is hypothesized that this effect
`
`will be the T helper cell pathway type shift, by Th1 to Thl7, and not the usual
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`expected viral
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`immunological effects such as causing a cytokine inflammatory
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`reaction for innate cell killing, meaning more or less as an adj uvant and tumor cell
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`antigens production. As a matter of fact and exactly the opposite of the usual theory of
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`how to use oncolytic viral vectors, it will be beneficial for the patient to develop
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`neutralizing antibodies against the viral vector being used for the above T helper
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`pathway shift, as the immune system will then be focused onto the necessity of
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`eliciting the Thl7 cytokines and the necessary T effector cells and/or antibodies
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`generation for the auto-immune cancer therapy.
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`As a summary,
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`the novelty of the present live and in-vivo tumor specific
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`cancer vaccine system is based on the following facts.
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`Firstly,
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`the tumor and viral components are completely separated until the
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`moment of administration to the patient. No pre-incubation or mixture of these
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`components for any measurable amount of time before treatment. All past cancer
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`vaccines consisting of tumor cells and virus vectors have all
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`involved in-vitro
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`manipulation.
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`The viral component is cancer specific and replicative competent.
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`The transgenic expression of GM-CSF is strategically happening at the tumor
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`lytic site.
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`The generation of the vaccine is live and in—vivo within the patient’s body,
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`capturing all
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`the necessary cellular, cytokine and chemokine and antigenic
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`components during cancer cell death to be sampled and cross—presented by the GM-
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`CSF primed and matured antigen presenting or dendritic cells and the immune
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`checkpoint modulators signal confirmation by the anti-CTLA4 antibody.
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`The easy and dependable access of producing tumor-viral-ICM interactions in
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`the proposed complete live and in-vivo cancer vaccine system to most cancer patients
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`is also immensely different from the setting of intra-tumoral injection of virus vectors.
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`The administration of the CLIVS approach will also be novel since this will be
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`the first time that a cancer vaccine may be given, with parts of or with all of its
`
`components, in multiple doses at the same or different injection sites, by more than
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`once per week. The normal administration schedule of the CLIVS system will likely
`
`be an intradermal or subcutaneous injection of all or parts of its vaccine components
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`once per week as a cycle and then the same cycle being repeated every two to three
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`weeks for four (4) to six (6) cycles as one course. To further enhance and sustain the
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`cancer specific immune response, the CLIVS system may also be administered twice
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`or more per week as a cycle, and/or with injections of the vaccine at the same or
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`different physical sites during each cycle. This “tandem” or two doses per week cycle
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`will then be repeated every two to three weeks for a total of four (4) to six (6) cycles
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`as a complete course of treatment.
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`Finally, yet another aspect of novelty of the system, the why and how to
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`implement the system, is based on the theory that the cancer specific oncolytic virus
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`used is purely to act as an immunogenic agent (not for its oncolytic effect like most
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`being developed are). That immunogenic agent effect is also novel in that it is aimed
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`at a T helper cell type shift, and not as an adjuvant or tumor antigens production like
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`in the past.
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`METHODS:
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`TUMOR CELLS:
`
`Preparation of Tumor Cells:
`
`For the usual surgical specimen, a piece of the tumor is removed for
`
`pathological classification and the main tumor cell mass is then placed into a tube
`
`with HBSS containing gentamycin and stored at 8°C. Within about 8—12 hours, the
`
`fresh tumor specimens are carried to the laboratory, where they are further
`
`dissociated. The tumor specimens are cut into smaller pieces, usually in 1 cm cubes
`
`with a scalpel. They are then incubated in an enzyme solution at 37°C. The usual
`
`enzymatic solution most effective is
`
`a mixture of collagenase, DNase, and
`
`hyaluronidase. After incubation the resulting suspension is filtered through a nylon
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`mesh with a pore of 40mm. These steps are repeated until all the main fraction of the
`
`tumor specimen has been dissolved. The resulting cell suspension is then washed
`
`three times in HBSS and then ready for cryopreservation.
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`Cryopreservation and Thawing of Tumor Cells:
`
`Tumor cells isolated in this manner are then frozen in 10% human serum
`
`albumin and 10% DMSO and stored in aliquots of 107 cells in liquid nitrogen. Cell
`
`freezing can be performed in a freezing computer Kryo 10 series 11 (Messer-
`
`Griesheim). On the day of the planned vaccination, the cells are carefully thawed in
`
`warm medium with the addition of 10% human serum albumin and then washed three
`
`times in this medium.
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`Inactivation of the Tumor Cells:
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`The tumor cells proliferative capacity is inactivated with 200Gy using a
`
`telecobalt source prior to administration.
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`VIRAL VECTOR:
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`Preparation of an oncolytic and cancer specific viral vector with transgenic
`
`expression of GM-CSF:
`
`While there are many viral vectors that are cancer specific and conditional
`
`replicative competent, they are usually