1111111111111111 IIIIII IIIII 1111111111 11111 1111111111 111111111111111 IIIII IIIIII IIII 11111111
`US 20210106632Al
`
`c19) United States
`c12) Patent Application Publication
`KIM et al.
`
`(54) NOVEL RECOMBINANT PLASMA
`MEMBRANE-BASED VESICLE, FOR
`TREATING CANCER
`
`(71) Applicant: TANDEM CO., LTD., Seoul, (KR)
`
`(72)
`
`Inventors: Gi-Beom KIM, Seoul (KR); Yoo Soo
`YANG, Seoul (KR); In-San KIM,
`Seoul (KR); Gihoon NAM, Seoul (KR)
`
`(73) Assignee: TANDEM CO., LTD., Seoul, (KR)
`
`(21) Appl. No.:
`
`16/967,074
`
`(22) PCT Filed:
`
`Sep. 28, 2018
`
`(86) PCT No.:
`
`PCT /KR2018/011504
`
`§ 371 (c)(l),
`(2) Date:
`
`Aug. 3, 2020
`
`(30)
`
`Foreign Application Priority Data
`
`Sep. 28, 2017
`
`(KR) ........................ 10-2017-0126202
`
`c10) Pub. No.: US 2021/0106632 Al
`Apr. 15, 2021
`(43) Pub. Date:
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`A61K 351766
`A61P35/00
`C07K 16128
`A61K 45106
`(52) U.S. Cl.
`CPC ............ A61K 351766 (2013.01); A61P 35/00
`(2018.01); A61K 45106 (2013.01); C07K
`1612827 (2013.01); C07K 1612863 (2013.01)
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`(57)
`
`ABSTRACT
`
`The present invention relates to a recombinant plasma
`membrane-based vesicle, and more specifically to a recom(cid:173)
`binant plasma membrane-based vesicle comprising a
`VSV-G mutated protein in which histidine, the 162nd amino
`acid, has been substituted with arginine, and a pharmaceu(cid:173)
`tical composition for treating cancer comprising the recom(cid:173)
`binant plasma membrane-based vesicle.
`Specification includes a Sequence Listing.
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.001
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 1 of 20
`
`US 2021/0106632 Al
`
`FIG. I
`
`FIG. 2a
`
`Human P~
`globtn
`fntron
`
`mVSVG
`
`H1.un-an p:~
`gfobin Poly A
`
`pCMV
`
`Amp+
`
`[Construct]
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.002
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 2 of 20
`
`US 2021/0106632 Al
`
`FIG. 2b
`
`STT
`STT
`SEQff)NO: ti
`
`[Sequence]
`
`FIG.3
`
`300g, 10min
`
`t SPNT
`t SPNT
`
`2000g, 1 Omin
`
`10,000g. 30min
`f
`SPNT
`'V (J 2 11M filter
`
`2729g.
`Concentration with Amicon ultra centrifugal filter
`
`150f000g, 3 hr
`
`t
`t Peffet
`
`Exosomes
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.003
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 3 of 20
`
`US 2021/0106632 Al
`
`FIG.4
`
`mVSVG
`
`Alix
`
`C063
`
`TSG 101
`
`[Western blot]
`
`FIG. Sa
`
`[TEM]
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.004
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 4 of 20
`
`US 2021/0106632 Al
`
`FIG. Sb
`
`mmtm rnVSVG • Exo
`
`·w
`
`1000
`
`100
`01.-wneter {tm11
`c::::::a Con
`
`20
`
`ii§'. 15 I l~.
`
`s
`
`10
`
`100
`tHarMter (nmt
`[DLS]
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.005
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 5 of 20
`
`US 2021/0106632 Al
`
`FIG. 6a
`
`***
`
`*
`
`1.4
`
`1
`
`-
`
`(!) 1.2
`>
`(I)
`>
`~ 0.8
`I.&.
`:E 0.6
`Q)
`>
`i 0.4
`"i
`0:: 0.2
`
`0
`
`1.4
`
`1
`
`-
`
`c, 1.2
`>
`en
`>
`O 0.8
`Li:
`:; 0.6
`(l)
`>
`~ 0.4
`ii>
`0:: 0.2
`
`0
`
`*
`
`*
`r--7
`
`*
`**
`r--7
`
`2
`
`C)
`~ 1.5
`>
`.....
`0
`u:
`:;
`
`(I)
`>
`:;:;
`~ 0.5
`(l)
`0:::
`
`0
`
`1.8
`
`(/')
`
`(!) 1.5
`>
`> 1.2
`.....
`0
`~ 0.9
`
`(U
`-~ 0.6
`1u
`iii
`0::: 0.3
`
`0
`
`***
`
`***
`*
`r--7
`
`2.5
`
`(!)
`> 2
`en
`>
`0 1.5
`Li:
`~
`(l)
`>
`;:;
`('Cl
`~ 0.5
`
`i
`
`0
`
`2
`
`(!)
`~ 1.5
`>
`....
`0
`Li:
`~
`(l)
`>
`:.::;
`~ 0.5
`(I)
`0::
`
`0
`
`[4T1-Luc]
`
`mVSVG-Exo
`
`[EL4-0va]
`
`mVSVG,Exo
`[CT26.CL25]
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.006
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 6 of 20
`
`US 2021/0106632 Al
`
`FIG. 6b
`
`4T1-Luc
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.007
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 7 of 20
`
`US 2021/0106632 Al
`
`FIG. 6c
`
`LDLR
`
`1S0kDa
`100kDa
`
`FIG. 7a
`
`4T1-Luc
`
`EL4.ova
`
`CT26.CL25
`
`7 39.6
`10.
`10
`
`1
`100 0.069
`
`3.47
`
`::!!
`0
`
`i IL
`C .. f
`
`0
`
`00 .
`"° :c
`
`Q.
`
`.. f
`
`0
`
`<(
`Q
`1a5
`IL
`::!:
`4
`10
`0
`C 1l
`2
`10
`
`10 ~;;o~n-;;2~-~7 ~~~;s
`
`Sf.I
`
`Deeored
`
`,
`
`10
`4
`10
`3
`
`1o!hs.2
`i 1\1_ - - -~ - - - - - -1
`IL
`:I:
`0
`5j
`10
`~ 10
`J2.7
`lO~J0-14
`10 '1:·W.-,C·•--.......-.-~•·H-· .. Y>Of ","""'•.-....... •
`4
`1a°
`1n2
`10
`1/
`Oeeo red
`
`2
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.008
`
`

`

`Patent Application Publication
`
`Apr. 15, 2021 Sheet 8 of 20
`
`US 2021/0106632 Al
`
`FIG. 7b
`
`4T1~Luc
`
`EL4.ova
`
`CT26.CL25
`
`*
`
`!40
`
`4T1-Luc
`
`FIG. 7c
`
`EL4.ova
`
`CT26.CL25
`
`140
`
`"''
`
`,0
`
`1
`pH 6.8 pH 7.4 pH 6.0 pH 14 pH 6.8 pH ?A i
`•
`l
`mVSYG,Exo
`Con.Em
`ButfP.t o:-tly
`
`1
`
`Q
`
`pH S,:8 pH 7.4 j µH "9.8 pH 7.4- • pH 6,8 pH 7.4
`mVSVG,£:X◊ ; Con.Exe
`EkUl~r only
`
`7 4 I 6.S
`6.S
`mVSVG EY.o I
`
`!'C,Exo
`
`74 I 6.8
`74 I
`l:luf1er only I
`
`,
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.009
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 9 of 20
`
`US 2021/0106632 Al
`
`FIG. Sa
`
`BMDM
`
`BMDC
`
`4T1-Luc
`
`***
`I ***
`***
`***ii***
`r-,r-,
`
`***
`***
`**
`I
`***ii***
`r········1r7
`
`18% •
`
`16%
`
`BMDC
`
`I ***
`***
`*** :! ***
`
`BMDM
`
`FIG. Sb
`
`EL4-0va
`
`***
`:
`***
`*'k*
`***:I***
`11...........-i
`*
`
`*
`
`l .
`·1~
`Ill
`
`mVSVG he
`
`;
`
`(e4 rxo
`
`tt? !
`
`30%
`
`-:;, 2$¾
`
`I::
`
`0
`O>
`J1!
`o.
`
`l0%
`
`•
`•
`:
`l
`·,¾,
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.010
`
`

`

`Patent Application Publication
`
`Apr. 15, 2021 Sheet 10 of 20
`
`US 2021/0106632 Al
`
`FIG. Sc
`
`BMOM
`
`BMOC
`
`CT26.Cl25
`
`***
`I ***
`***
`***Ii***
`r-,r-,
`
`18"
`
`16%
`
`14'<.
`
`lu"
`
`.! il:
`
`i
`f
`
`"'
`
`4"
`
`2"
`
`°"
`
`2
`
`0
`
`***
`! ***
`! ***
`*** r 7
`***!
`J I
`
`~ I
`
`14%
`
`ll.%
`
`ll0%
`
`'I)
`
`·;;
`0 >-0
`
`0
`0)
`(0
`
`.t::. a.
`
`8%
`
`&%
`
`4%
`
`2%
`
`0%
`
`FIG.9
`
`"' (:0
`C u
`0
`u..
`:E
`<U
`
`.~ -_!E 0.5
`
`0
`0:::
`
`0
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.011
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 11 of 20 US 2021/0106632 Al
`
`FIG. lOa
`
`+control
`......,coo .. exo
`,~--mvsva .. exo
`
`-
`
`1200
`
`1000
`~
`E
`.§. 800
`
`200
`
`18
`14
`11
`8
`5
`0
`Days after tumor inoculation
`
`FIG. lOb
`
`1.2
`
`1
`
`.c:
`0)
`'<ii
`~ 0.6
`
`.3: - 0.8
`... 0
`E 0.4
`:::::;
`l-
`
`0.2
`
`0
`
`+O
`(i«;-
`e:;.:s.
`
`0,;;.
`+O
`v
`~«;-
`cP
`14
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.012
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 12 of 20
`
`US 2021/0106632 Al
`
`FIG. lOc
`
`25
`
`20
`
`5
`
`..... control
`..... con-Exo
`,1:i--mVSVG-Exo
`
`o_.__ ____________ _
`
`5
`
`14
`11
`16
`8
`Days after tumor inoculation
`
`FIG. lla
`
`-11-Control
`-11-Con-Exo
`~mVSVG-Exo
`~wtVSVG-Exo
`
`1400
`
`1200
`~ s 1000
`§_
`~ 800
`'<ii
`0 600
`s
`i= 400
`
`200
`
`0
`
`9
`15
`18
`12
`6
`Days after tumor inoculation
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.013
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 13 of 20
`
`US 2021/0106632 Al
`
`FIG. llb
`
`*** ***
`* I
`*** r7
`
`!
`
`**I
`r7
`
`1,2
`
`0.2
`
`0
`
`FIG. llc
`
`30
`
`25
`
`5
`
`...... control
`~Con~Exo
`-~-wtVSVG-Exo
`,--m-,mVSVG-Exo
`
`o~ - - - - - - - - - - - -
`15
`6
`18
`12
`9
`Days after tumor inoculation
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.014
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 14 of 20 US 2021/0106632 Al
`
`FIG.12
`
`**
`***
`
`(!)
`
`> (/) > -1.2
`
`0 cr:
`:E
`Cl> > 0.6
`:.:: co
`4) c::
`
`FIG. 13a
`
`*
`
`10
`9
`8
`~ ~7
`+ ~ 6
`q 5
`~ 4
`N ::c 3
`2
`1
`0
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.015
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 15 of 20 US 2021/0106632 Al
`
`FIG. 13b
`
`**
`
`FIG.13c
`
`***
`
`1,6
`0 1,4
`"<'t' 8 1.2
`0
`1
`i 0.8
`g! 0.6
`:;:
`.!! 0.4
`Q.)
`.o:: 0.2
`0
`
`2
`1.8
`28 1.6
`0
`0 1.4 o 1.2
`
`1
`~ 0.8
`
`~ 0.6 & 0.4
`
`0.2
`0
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.016
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 16 of 20 US 2021/0106632 Al
`
`FIG. 13d
`
`Control
`
`wtVSVG-Exo
`
`rnVSVG-Exo
`
`FIG.13e
`
`140
`
`***
`
`e 120
`E
`!! 100
`<ii
`(.) to 80
`S 60
`0
`0 40
`z
`20
`0 .._. _____ _
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.017
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 17 of 20 US 2021/0106632 Al
`
`FIG. l4a
`
`***
`***
`
`140
`
`120
`
`_100
`
`E e> 80
`Q. -::;»- 60
`
`z
`LL
`
`40
`
`20
`
`Macrophage Dendritic cells
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.018
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 18 of 20 US 2021/0106632 Al
`
`FIG.14b
`
`***
`***
`***
`
`~
`
`1000
`
`0 soo
`
`800 -E
`a.. -1""' 400
`z u..
`
`200
`
`PBS
`
`Ova
`
`FIG.15a
`
`2400
`
`..... control
`
`2100
`j 1800
`S. 1500
`i 0 1200
`0 e soo
`?J. 600
`300
`0 ~~~L - - - - - - - -
`9
`12
`0
`6
`15
`18
`Days after tumor inoculation
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.019
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 19 of 20
`
`US 2021/0106632 Al
`
`FIG. 15b
`
`-ii-Control
`
`".ffi.--Con•Exo
`
`, .... mVSVG-Exo
`
`1800
`
`1500
`;;
`s
`.§_ 1200
`
`(I)
`N
`'iii 900
`...
`0
`S 600
`?:.
`
`300
`
`0
`
`17
`14
`11
`8
`5
`0
`Days after tumor inoculation
`
`FIG.15c
`
`-a-Anti-COS
`·-fil"Anti-CO8 + mVSVG-Exo
`
`··ili··r!gG + rnVSVG-Exo
`
`15
`12
`9
`6
`18
`Days after tumor inoculation
`
`1800
`
`1500
`M
`E
`.§. 1200
`
`<l)
`N
`·;; 900
`
`... 0
`E &oo
`::,
`1-
`
`300
`
`1.8
`'.§ 1.5
`:E 1,2
`O'>
`-~ 0.$
`
`g 0.6
`s I- 0.3
`
`0
`
`i
`1 ...
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.020
`
`

`

`Patent Application Publication Apr. 15, 2021 Sheet 20 of 20 US 2021/0106632 Al
`
`FIG.15d
`
`... control
`-§-Con-Exo
`"~ "mVSVG-Exo
`
`1200
`
`-1000
`M
`E
`_§_ 800
`
`(1)
`
`N 600
`·;
`!..
`0
`E 400
`::;
`I-
`
`18
`15
`12
`9
`6
`0
`Days after tumor inoculation
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.021
`
`

`

`US 2021/0106632 Al
`
`Apr. 15, 2021
`
`NOVEL RECOMBINANT PLASMA
`MEMBRANE-BASED VESICLE, FOR
`TREATING CANCER
`
`TECHNICAL FIELD
`
`[0001] The present invention relates to a novel recombi(cid:173)
`nant plasma membrane-based vesicle, and more specifically,
`to a novel recombinant plasma membrane-based vesicle for
`treating cancer.
`
`BACKGROUND ART
`
`[0002] Cancer refers to a group of diseases associated with
`abnormal cell growth with a potential for invasion and
`metastasis to other parts of the body. As of 2015, it is known
`that there are more than 90 million cancer patients world(cid:173)
`wide, with about 14 million new cancer patients occurring
`every year. Cancer accounts for 15.7% of causes of human
`deaths. The most frequently occurring cancers are lung
`cancer, prostate cancer, colon cancer, and stomach cancer in
`men, and breast cancer, colon cancer, lung cancer, and
`uterine cervical cancer in women.
`[0003] For
`therapeutic
`various
`cancer
`treatment,
`approaches are attempted, including chemotherapy using
`various anticancer agents, radiation therapy by irradiation,
`antibody therapy targeting particular in vivo molecules
`associated with cancer, etc. However, anticancer agents used
`in chemotherapy or irradiation have serious side effects
`because these treatments also affect normal cells and often
`result in treatment failure or recurrence because cancer cells
`can acquire resistance to anticancer agents.
`[0004] Recently, the anti-cancer immunotherapy using the
`immune system of our body has been showing a surprising
`effect in clinical treatment. However, due to the complexity
`of cancer, the anti-cancer immunotherapy has a limitation in
`that the therapy is only effective in less than about 30% of
`cancer patients. This is because cancer cells are recognized
`as "self' by our body's immune cells, and therefore, it is
`important for cancer cells to be recognized as "non-self' by
`immune cells, so that the phagocytosis of cancer cells can be
`induced thereby inducing an amplified immune response.
`[0005] Exosomes are cell-derived vesicles present in all
`biological liquids, including blood, urine, and culture media
`of cell cultures, also called extracellular vesicles or microve(cid:173)
`sicles. Exosomes are known to have a size between 30 nm
`and 100 nm, and are secreted from cells when the multive(cid:173)
`sicular body fuses with a cell membrane, secreted directly
`through a cell membrane, or are budding directly from a cell
`membrane. Exosomes are known to play an important role
`in various processes such as coagulation, intercellular sig(cid:173)
`naling, and metabolic waste management. Exosomes have
`important advantages as drug carriers compared to lipo(cid:173)
`somes or polymer-type nanoparticles in that exosomes have
`compositions similar to those of the cells themselves of the
`human body and in that exosomes are non-immunogenic
`(Ha et al., Acta Pharm. Sin. B. 6(4): 287-296, 2016). In this
`regard, various attempts have been made using exosomes so
`that anticancer drugs (e.g., doxorubicin) are delivered to a
`tumor tissue (Tian et al., Biomaterials. 35:2383-2390,
`2014); paclitaxel and doxorubicin pass through the blood
`brain barrier and are delivered to the brain (Yang et al.,
`Pharm. Res. 32:2003-2014, 2015); for treatment of Parkin(cid:173)
`son's disease, catalase passes through the blood brain barrier
`and is delivered to the brain (Haney et al., J. Control
`
`Release. 207: 18-30, 2015); or for treatment of cancer,
`siRNAs specific to certain genes are delivered (Shtam et al.,
`Cell Commun. Signal. 11: 88, 2013), etc.
`[0006] An extracellular vesicle refers to a structure in the
`form of a particle, in which various biomolecules (e.g.,
`proteins, nucleic acid molecules (e.g., RNA), lipids of
`various functions, etc.) released or secreted from cells into
`an extracellular environment are enclosed in a cell mem(cid:173)
`brane of a lipid bilayer identical to the cell membrane of the
`cell from which the various biomolecules are derived. The
`extracellular vesicle refers to a plasma membrane-based
`vesicle having an average diameter of 100 nm to 1 µm,
`which is larger than that of an exosome normally having a
`size of 30 nm to 100 nm.
`[0007] A cell-derived nanovesicle, which is a nano-sized
`vesicle surrounded by a plasma membrane which is a
`nano-sized cell membrane component formed by artificial
`methods ( e.g., an extrusion process where cells are passed
`through microfluidic channels, a multi-stage filtration pro(cid:173)
`cess, etc.), refers to a plasma membrane-based vesicle
`distinguished from an exosome or extracellular vesicle
`secreted by a cell and formed naturally.
`[0008] Meanwhile, vesicular stomatitis virus glycoprotein
`(VSV-G) is the only virus glycoprotein present in the virion
`membrane of vesicular stomatitis virus and acts as a protein
`for adhesion and fusion of the virus into a target cell. The
`VSV-G protein is a transmembrane protein including two
`N-linked glycans, which can initiate a membrane fusion
`event in a low-pH-dependent manner when no other virus
`protein is present. Since VSV-G proteins can form a com(cid:173)
`plex with a nucleic acid molecule such as DNA, VSV-G
`proteins have been used as a carrier for direct gene transfer,
`or have been used effectively in gene therapy by producing
`more stable and high-titer pseudotyped murine leukemia
`virus (MLV)-based retrovirus and lentivirus-based vectors.
`Recently, however, it has been suggested that VSV-G pro(cid:173)
`teins be available for use to deliver various proteins, in
`addition to genes, to heterologous cells (Mangeot et al., Mal.
`Ther. 19(9): 1656-1666, 2011 ).
`[0009] As a result of the research on the structures and
`functions of the VSV-G proteins, it was previously con(cid:173)
`firmed that histidine, which is the 60'h
`, the 162nd
`, and the
`407 th amino acid residues based on the mature protein from
`which the signal sequence was removed, forms a cluster and
`acts as a pH sensor (Roche et al., Science, 313: 187-191,
`2006; Roche et al., Science, 315: 843-848, 2007), and
`recently, it was reported that when the 162nd amino acid
`residue, histidine, among the above histidine residues is
`mutated into arginine (H162R), it induces a membrane
`fusion at pH 6.8, which is a physiological pH surrounding
`cancer cells, and promotes the death of cancer cells upon
`administration of neural stem cells expressing the VSV-G
`mutant (H162R) (Zhu et al., Mal. Ther. 21 (8): 1492-1497,
`2013).
`[0010] However, the method disclosed in the related art is
`a technology difficult to apply to the actual clinical envi(cid:173)
`ronment in that the supply of neural stem cells is difficult,
`and that the cancer cell killing effect is not very significant.
`
`DISCLOSURE OF THE INVENTION
`
`Technical Problem
`
`[0011] The objects of the present invention are to solve
`various problems including the above problems, and an
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.022
`
`

`

`US 2021/0106632 Al
`
`Apr. 15, 2021
`
`2
`
`object of the present invention is to provide a recombinant
`plasma membrane-based vesicle for safe cancer treatment
`which does not operate under conditions outside the cancer
`microenvironment while efficiently killing cancer cells on
`their own without an anticancer agent. In particular, the
`object of the present invention is to provide a recombinant
`plasma membrane-based vesicle which enables "xenogeni(cid:173)
`zation" so that immune cells can recognize cancer cells as an
`"enemy". However, these objects are illustrative, and the
`scope of the present invention is not limited thereby.
`
`Technical Solution
`
`[0012] According to an aspect of the invention, there is
`provided a recombinant plasma membrane-based vesicle,
`wherein a VSV-G mutant protein in which the l 6rd amino
`acid, histidine, is substituted with arginine is introduced into
`the membrane.
`[0013] According to another aspect of the invention, there
`is provided a pharmaceutical composition for treating cancer
`containing the recombinant plasma membrane-based vesicle
`as an active ingredient.
`[0014] Moreover, according to still another aspect of the
`invention, there is provided a pharmaceutical composition
`for treating cancer containing a recombinant plasma mem(cid:173)
`brane-based vesicle, in which a virus-derived fusogenic
`membrane protein is introduced into the membrane, as an
`active ingredient.
`[0015] Moreover, according to still another aspect of the
`invention, there is provided a recombinant plasma mem(cid:173)
`brane-based vesicle, wherein a VSV-G mutated protein in
`which the 162nd amino acid, histidine, is substituted with
`arginine is introduced into the membrane; or the use of a
`recombinant plasma membrane-based vesicle, in which a
`virus-derived fusogenic membrane protein is introduced into
`the membrane, for the preparation of a cancer therapeutic
`agent.
`[0016] According to still another aspect of the invention,
`there is provided a method for treating cancer in a subject
`with cancer, which includes administering the recombinant
`plasma membrane-based vesicle or one or more pharmaceu(cid:173)
`tical compositions among those described above to the
`subject with cancer.
`
`Advantageous Effects
`
`[0017] According to an embodiment of the present inven(cid:173)
`tion constituted as described above, the present invention
`can effectively treat cancer without resorting to a complex
`mechanism such as gene transfer.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0018] FIG. 1 is a schematic diagram schematically illus(cid:173)
`trating a mechanism of action of a recombinant exosome,
`which includes a mutant VSV-G Hl62R (hereinafter abbre(cid:173)
`viated as "m VSV-G") that induces anticancer immune
`effects in a cancer cell microenvironment (pH 6.8), accord(cid:173)
`ing to an embodiment of the present invention.
`[0019] FIG. 2a is a plasmid map illustrating a schematic
`structure of the plasmid DNA for producing a recombinant
`exosome according to an embodiment of the present inven(cid:173)
`tion, and FIG. 2b shows amino acid sequences illustrating
`the wild-type VSV-G protein and the mutation at position
`162 of a mutated VSV-G protein for producing a recombi-
`
`nant exosome according to an embodiment of the present
`invention, and the nucleic acid sequences of the polynucle(cid:173)
`otides encoding the same.
`[0020] FIG. 3 is a process chart illustrating a process for
`producing a recombinant exosome including m VSV-G
`according to an embodiment of the present invention.
`[0021] FIG. 4 shows the results of Western blot analysis of
`a recombinant exosome according to an embodiment of the
`present invention and the cell extracts transfected with the
`m VSV-G gene construct so as to produce the recombinant
`exosome.
`[0022] FIG. Sa is an image photographed with a transmis(cid:173)
`sion electron microscope of a recombinant exosome includ(cid:173)
`ing an m VSV-G prepared according to an embodiment of the
`present invention, and FIG. Sb shows a series of histograms
`illustrating analysis results of particle size of the recombi(cid:173)
`nant exosome by dynamic light scattering analysis.
`[0023] FIG. 6a shows a series of graphs illustrating the
`results of confirming the degree of membrane fusion with
`cancer cells according to pH change when three types of
`cancer cells (4Tl-Luc, EL4-Ova, and CT26.CL25) was
`treated with the mVSVG-Exo according to an embodiment
`of the present invention (*: P<0.05; **: P<0.01; ***: P<0.
`001); FIG. 6b shows a series of fluorescence microscopic
`images of the 4Tl-Luc cells after staining the 4Tl-Luc cells
`using an anti-VSV-G antibody and an anti-Cadherin anti(cid:173)
`body (green), when 4Tl-Luc cells were treated with the
`mVSVG-Exo according to an embodiment of the present
`invention, as to whether there was a membrane fusion with
`cancer cells according to pH change; and FIG. 6c shows an
`image illustrating the analysis results by Western blot with
`regard to the expression oflow density lipoprotein receptors
`(LDLRs), which are known as VSV-G receptors, on various
`cell surfaces, including those of three types of cancer cells
`(i.e., 4Tl-Luc, EL4-Ova, and CT26.CL25).
`[0024] FIG. 7a shows a series of histograms illustrating
`the results of flow cytometry, to observe whether the
`mVSVG-Exo according to an embodiment of the present
`invention, after the fusion with three types of cancer cells
`(4Tl-Luc, EL4-Ova, and CT26.CL25) on their surfaces,
`promoted fusions between the cancer cells according to pH
`change; FIG. 7b shows a series of graphs illustrating the
`results of quantitative measurements of the results of FIG.
`7a; and FIG. 7c shows a series of graphs illustrating the
`results of cell viability analysis to confirm whether the
`mVSVG-Exo according to an embodiment of the present
`invention directly induces death of cancer cells ( 4 Tl-Luc,
`EL4-Ova, and CT26.CL25) (*: P<0.05; **: P<0.01).
`[0025] FIG. Sa shows a series of graphs illustrating the
`quantification of the analysis results of fluorescence micro(cid:173)
`scopic images, in which the effects of the recombinant
`exosome including the mVSV-G according to an embodi(cid:173)
`ment of the present invention and the control exosome
`(Con-Exo) on the phagocytic action by macrophages and
`dendritic cells exerted on 4Tl-Luc breast cancer cells were
`analyzed; FIG. Sb shows a series of graphs illustrating the
`quantification of the analysis results of fluorescence micro(cid:173)
`scopic images, in which the effects of the recombinant
`exosome including the mVSV-G according to an embodi(cid:173)
`ment of the present invention and the control exosome
`(Con-Exo) on the phagocytic action by macrophages and
`dendritic cells exerted on EL4-Ova lymphoma cells were
`analyzed; and FIG. Sc shows a series of graphs illustrating
`the quantification of the analysis results of fluorescence
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.023
`
`

`

`US 2021/0106632 Al
`
`Apr. 15, 2021
`
`3
`
`microscopic images, in which the effects of the recombinant
`exosome including the mVSV-G according to an embodi(cid:173)
`ment of the present invention and the control exosome
`(Con-Exo) on the phagocytic action by macrophages and
`dendritic cells exerted on CT46.CL25 colon cancer cells
`were analyzed(*: P<0.05; **: P<0.01; ***: P<0.001).
`[0026] FIG. 9 shows a series of graphs in which, to
`examine whether VSVG can act as a TLR4 agonist and
`activate the functions of dendritic cells, the recombinant
`exosome including the mVSV-G (mVSVG-Exo) according
`to an embodiment of the present invention and the control
`group exosome (Con-Exo) were treated to bone marrow(cid:173)
`derived dendritic cells, respectively, and the change in the
`relative expression rate of CD40 (left) and CDS6 (right) in
`the bone marrow-derived dendritic cells were recorded (*:
`P<0.05; **: P<0.01).
`[0027] FIG. lOa is a graph comparing the size of cancer
`cells over time in 4Tl-Luc breast cancer tumor model
`animals (Balb/c, 7-week-old female mice), in which the
`recombinant exosome (200 µg) including the m VSV-G
`according to an embodiment of the present invention was
`administered (a square represents a control group, a circle
`represents a control group exosome excluding them VSV-G,
`and a triangle represents a recombinant exosome including
`the m VSV-G according to an embodiment of the present
`invention); FIG. 10b is a graph illustrating the measurement
`results of the weight of cancer tissues extracted by sacrific(cid:173)
`ing experimental animals 16 days after cancer cell injection
`in FIG. lOa; and FIG. 10c is a graph illustrating the change
`in the weight of the animals used in the experiment illus(cid:173)
`trating(*: P<0.05; **: P<0.01; ***: P<0.001).
`[0028] FIG. lla is a graph illustrating the comparison of
`the size of cancer cells over time in EL4-Ova lymphoma
`tumor model animals (C57BL/6, 7-week-old female mice),
`in which the recombinant exosome (200 µg) including the
`mVSV-G according to an embodiment of the present inven(cid:173)
`tion was administered (a square represents a control group,
`a circle represents a control group exosome excluding the
`m VSV-G, and a triangle represents a recombinant exosome
`including the m VSV-G according to an embodiment of the
`present invention); FIG. llb is a graph illustrating the
`measurement results of the weight of cancer tissues
`extracted by sacrificing experimental animals 16 days after
`cancer cell injection in FIG. lla; and FIG. llc is a graph
`illustrating the change in the weight of the animals used in
`the experiment(*: P<0.05; **: P<0.01; ***: P<0.001).
`[0029] FIG. 12 is a graph illustrating the results of flow
`cytometry, in which a recombinant exosome including the
`m VSV-G (m VSVG-Exo) according to an embodiment of the
`present invention, a recombinant exosome including the
`wild-type VSV-G (wtVSVG-Exo ), a control group exosome
`(Con-Exo ), and PBS as the control group were administered
`into the cancer tissues of tumor model animals (EL4-Ova(cid:173)
`injected C57BL/6 mice), and the extracted cancer tissues
`were converted into single cells, stained with an anti-VSV-G
`antibody, and then subjected to flow cytometry analysis (*:
`P<0.05; **: P<0.01; ***: P<0.001 ).
`[0030] FIG. 13a is a graph illustrating the results of flow
`cytometry, in which a recombinant exosome including the
`m VSV-G (m VSVG-Exo) according to an embodiment of the
`present invention, a recombinant exosome including the
`wild-type VSV-G (wtVSVG-Exo ), a control group exosome
`(Con-Exo ), and PBS as the control group were administered
`into the cancer tissues of tumor model animals (EL4-Ova-
`
`injected C57BL/6 mice), and the extracted tumor-draining
`lymph nodes were converted into single cells, and subjected
`to flow cytometry analysis using an anti-CDllc antibody
`and an anti-H2kb-Ova antibody, which are dendritic cell
`markers; FIG. 13b is a graph illustrating the results of flow
`cytometry in which the extracted tumor-draining lymph
`nodes were converted into single cells, and then subjected to
`flow cytometry analysis using an anti-CDllc antibody and
`an anti-CD40 antibody; FIG. 13c is a graph illustrating the
`results of flow cytometry in which the extracted tumor(cid:173)
`draining lymph nodes were converted into single cells, and
`then subjected to flow cytometry analysis using an anti(cid:173)
`CDllc antibody and an anti-CDS6 antibody(*: P<0.05; **:
`P<0.01; ***: P<0.001); FIG. 13d shows a series of images
`illustrating the analysis results of the degree of infiltration of
`CDS T cells in tumor tissues performed using a fluorescence
`microscope after staining tumor tissue slices with an anti(cid:173)
`CDS antibody; and FIG. 13e is a graph illustrating the results
`of the quantification of the degree of infiltration of cancer
`tissue of CDS T cells into cancer tissue in FIG. 13d (***:
`P<0.001).
`[0031] FIG. 14a is a graph illustrating the results of
`analysis, in which a recombinant exosome including the
`m VSV-G (m VSVG-Exo) according to an embodiment of the
`present invention, a recombinant exosome including the
`wild-type VSV-G (wtVSVG-Exo), a control group exosome
`(Con-Exo ), and PBS as the control group were administered
`into the cancer tissues of tumor model animals (EL4-Ova(cid:173)
`injected C57BL/6 mice), and CD Ile-positive dendritic cells
`and F4/S0-positive macrophages were isolated from the
`extracted tumor tissue and co-cultured with OT-1 CDS T
`cells isolated from the spleens of OT-1 transgenic mice at a
`1:5 ratio, respectively, and the expression level of INF-y of
`the culture medium was analyzed by ELISA assay; and FIG.
`14b is a graph illustrating the results of analysis, in which the
`splenocytes, in which the spleen tissues extracted from the
`experimental animals were converted into single cells, were
`treated with PBS as the control group and ovalbumin (10
`µg/ml) (i.e., a cancer-specific antigen) for 24 hours, and then
`the expression level of INF-y of the culture medium was
`analyzed by ELISA assay (*: P<0.05; **: P<0.01; ***:
`P<0.001).
`[0032] FIGS. 15a and 15b show a graph (left) illustrating
`the measurement results, in which a recombinant exosome
`including the mVSV-G (mVSVG-Exo) according to an
`embodiment of the present invention, a control group exo(cid:173)
`some (Con-Exo ), and PBS as the control group were admin(cid:173)
`istered into tumor tissues of nude mice, in which cancer was
`induced by subcutaneously injecting EL4-Ova cancer cells
`thereto, and the volume of tumor tissue was measured over
`time, and a graph (right) illustrating the measurement
`results, in which mice were sacrificed on the 17th day after
`cancer cell injection and the weight of the extracted tumor
`tissues was measured. FIG. 15a shows the experimental
`results according to a low dose (two times of administration:
`100 µg each); FIG. 15b shows the results of the adminis(cid:173)
`tration according to a high dose (four times of administra(cid:173)
`tion: 100 µg each) using the same model; and FIG. 15c
`shows a graph (left) illustrating the measurement results, in
`which a recombinant exosome including the m VSV-G
`(m VSVG-Exo) according to an embodiment of the present
`invention was administered into the tumor tissues of
`C57BL/6 mice, to which an anti-CDS antibody was intrap(cid:173)
`eritoneally administered at 3 day intervals starting from one
`
`Kelonia v. Interius
`PGR2024-00008
`Interius Exhibit 2001.024
`
`

`

`US 2021/0106632 Al
`
`Apr. 15, 2021
`
`4
`
`day before the cancer cell injection and EL4-Ova cancer
`cells were injected subcutaneously so as to induce cancer,
`and the volume of tumor tissue over time was measured; and
`a graph (right) illustrating the measurement results of weight
`of the tumor tissues extracted from the mice sacrificed on the
`18th day after the cancer cell injection; and a recombinant
`IgG was used as the control antibody(***: P<0.001). FIG.
`15d shows a graph (left) illustrating the measurement
`results, in which a recombinant exosome including the
`m VSV-G (m VSVG-Exo) according to an embodiment of the
`present invention, a control group exosome (Con-Exo), and
`PBS as the control group were administered into tumor
`tissues ofBATF3 KO mice (which lack CD103 and CDS),
`in which cancer was induced by subcutaneous injection of
`EL4-Ova cancer cells, and then the volume of tumor tissue
`over time was measured; and a graph (right) illustrating the
`measurement results of weight of the tumor tissues extracted
`from the mice sacrificed on the 17th day after the cancer cell
`injection.
`
`MODE FOR CARRYING OUT THE INVENTION
`
`[0033] Definition of Terms
`[0034] As used herein, the term "fusogenic membrane
`protein" refers to a virus-derived membrane protein which
`plays a key role in inducing the adhesion and membrane
`fusion of a virus to a host cell for the penetration of the virus
`into the host cell. A representative example is an envelope
`glycoprotein derived from the vesicular stomatitis virus
`(VSV-G).
`[0035] As used herein, the term "an envelope glycoprotein
`derived from the