1111111111111111 IIIIII IIIII 1111111111 11111 1111111111 111111111111111 111111111111111 11111111
`US 20200216502Al
`
`c19) United States
`c12) Patent Application Publication
`Albertini et al.
`
`c10) Pub. No.: US 2020/0216502 Al
`Jul. 9, 2020
`(43) Pub. Date:
`
`(54) MUTATED GLYCOPROTEIN OF VESICULAR
`STOMATITIS VIRUS
`
`(30)
`
`Foreign Application Priority Data
`
`Sep. 22, 2017
`
`(EP) .................................. 17306255.5
`
`(71) Applicants:Centre National de la Recherche
`Scientifique (CNRS), Paris (FR);
`UNIVERSITE PARIS-SUD, Orsay
`(FR)
`
`(72)
`
`Inventors: Aurelie Albertini, Gometz Le Chatel
`(FR); Yves Gaudin, Paris (FR); Helene
`Raux, Antony (FR); Laura Belot,
`Maurepas (FR); Jovan Nikolic, Paris
`(FR)
`
`(21) Appl. No.:
`
`16/649,271
`
`(22) PCT Filed:
`
`Sep. 24, 2018
`
`(86) PCT No.:
`
`PCT/EP2018/075824
`
`§ 371 (c)(l),
`(2) Date:
`
`Mar. 20, 2020
`
`Publication Classification
`
`(51)
`
`(2006.01)
`(2006.01)
`
`Int. Cl.
`C07K 141005
`C12N 7100
`(52) U.S. Cl.
`CPC .............. C07K 141005 (2013.01); C12N 7100
`(2013.01); Cl2N 2760/20262 (2013.01); Cl2N
`2760/20232 (2013.01); Cl2N 2760/20245
`(2013.01); Cl2N 2760/20222 (2013.01)
`
`ABSTRACT
`(57)
`The invention relates to an isolated non-naturally occurring
`protein comprising the amino acid sequence as set forth in
`SEQ ID NO: 1, and wherein the amino acid in position 8, 4 7,
`209 and/or 354 is substituted by any amino acid different
`from the amino acid indicated at that position in said
`sequence SEQ ID NO: 1.
`Specification includes a Sequence Listing.
`
`D.
`
`A.
`
`B.
`
`C.
`
`Page 1 of 27
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`KELONIA EXHIBIT 1019
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`Patent Application Publication
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`Jul. 9, 2020 Sheet 1 of 10
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`US 2020/0216502 Al
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`D.
`
`A.
`
`B.
`
`C.
`
`Fig. 1
`
`7
`
`C.
`
`1 2 3 4 5 6
`
`75. _,,,, - ..
`
`25.
`
`◄ A.
`
`◄ B.
`.,.. D.
`
`Fig. 2
`
`I
`• I
`
`Fig. 3
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`Page 2 of 27
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`Patent Application Publication
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`Jul. 9, 2020 Sheet 2 of 10
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`US 2020/0216502 Al
`
`Figs. 4
`
`0.20 0 10 20 30 40 50 60
`
`0 10 20 30 40 50 60
`
`0 10 20 30 40 50 60 70 80
`
`0.10
`
`0.00
`
`0.00
`
`~~~-,\.~H4~ -0.04
`
`-0.10
`
`.,,,,,. _____ """""""'~ -0.12 .Jl-,,m-=----..,,,-,~
`
`-0.08
`
`0.0
`
`0.00
`
`-0.10
`
`-0.20
`
`0.0
`
`-2.0
`
`-4.0
`
`1.00
`
`0.50
`
`.,
`"' .,
`....
`
`-1.0
`
`-2.0
`
`-3.0
`
`o.oo~-------
`o.o 0.5 1.0 1.5 2.0
`
`1.0 1.5 2.0
`Fig. 5
`
`.5
`
`-5.o 0.0 0.5 1.0 1.5 2.0
`
`.5
`
`Page 3 of 27
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`Patent Application Publication
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`Jul. 9, 2020 Sheet 3 of 10
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`US 2020/0216502 Al
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`Figs. 6
`
`Fig. 7
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`Page 4 of 27
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`Patent Application Publication
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`Jul. 9, 2020 Sheet 4 of 10
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`US 2020/0216502 Al
`
`PHO
`
`. CTer
`
`Fig. 8
`
`Fig. 9
`
`SEQ ID NO: 323
`SEQ ID NO: 324
`
`Page 5 of 27
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`

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`Patent Application Publication
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`Jul. 9, 2020 Sheet 5 of 10
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`US 2020/0216502 Al
`
`A
`
`B
`
`Figs. 10
`
`83,5-¼
`
`C
`
`96.0%
`
`G
`
`B
`
`F
`
`87.0%
`
`I
`
`96.'1%
`
`I
`
`D
`
`H
`
`42.6%
`
`l.2%
`
`A
`
`E
`
`00,'1%
`
`,,/
`
`.,
`
`93,.4%
`
`,/
`
`7,7%
`
`J
`
`L
`
`M
`
`K
`
`N
`
`Figs. 11
`
`Page 6 of 27
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`Patent Application Publication
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`Jul. 9, 2020 Sheet 6 of 10
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`US 2020/0216502 Al
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`Fig. 12
`
`Figs. 13
`
`Page 7 of 27
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`Patent Application Publication
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`Jul. 9, 2020 Sheet 7 of 10
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`US 2020/0216502 Al
`
`A
`
`B
`
`C
`
`◄ 1.
`
`150 • •
`
`100 ...
`
`75 .. ,
`
`·--------◄ 2.
`
`Fig.14
`
`100
`
`50
`
`0
`
`A
`
`B
`
`Fig.15
`
`Page 8 of 27
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`Patent Application Publication
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`Jul. 9, 2020 Sheet 8 of 10
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`US 2020/0216502 Al
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`Fig.16
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`Page 9 of 27
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`Patent Application Publication
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`Jul. 9, 2020 Sheet 9 of 10
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`US 2020/0216502 Al
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`55
`
`25
`
`G
`
`55
`
`M
`
`25
`
`Fig. 17
`
`G
`
`M
`
`HEK
`
`BSR
`
`1.0
`
`OS
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`1.0
`
`0.8
`
`0.6
`
`OA
`
`0.2
`
`0.0
`
`CHO
`
`1.0
`
`OS
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`1.0
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`Fig. 18
`
`S2
`
`Page 10 of 27
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`Patent Application Publication
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`Jul. 9, 2020 Sheet 10 of 10 US 2020/0216502 Al
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`Figs. 19
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`Page 11 of 27
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`US 2020/0216502 Al
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`Jul. 9, 2020
`
`1
`
`MUTATED GLYCOPROTEIN OF VESICULAR
`STOMATITIS VIRUS
`
`[0001] The invention relates to a mutated viral protein, in
`particular a muted protein originating from an oncolytic
`virus.
`[0002] Vesicular stomatitis virus (VSV) is an enveloped,
`negative-strand RNA virus that belongs to the Vesiculovirus
`genus of the Rhabdovirus family. It is an arbovirus which
`can infect insects, cattle, horses and pigs. In mammals, its
`ability to infect and kill tumor cells while sparing normal
`cells makes it a promising oncolytic virus for the treatment
`of cancer (Barber, 2005; Fernandez et al., 2002; Hastie et al.,
`2013).
`[0003] VSV genome encodes five structural proteins
`among which a single transmembrane glycoprotein (G). The
`glycoprotein is a classic type I membrane glycoprotein with
`an amino-terminal signal peptide, an ectodomain of about
`450 amino acids, a single alpha helical transmembrane
`segment and a small intraviral carboxy-terminal domain.
`The signal peptide is cleaved in the lumen of the endoplas(cid:173)
`mic reticulum and the native glycoprotein consists in the
`ectodomain, the transmembrane domain and the intraviral
`domain.
`[0004] G plays a critical role during the initial steps of
`virus infection (Albertini et al., 2012b ). First, it is respon(cid:173)
`sible for virus attachment to specific receptors. After bind(cid:173)
`ing, virions enter the cell by a clathrin-mediated endocytic
`pathway. In the acidic environment of the endocytic vesicle,
`G triggers the fusion between the viral and endosomal
`membranes, which releases the genome in the cytosol for the
`subsequent steps of infection. Fusion is catalyzed by a
`low-pH-induced large structural transition from a pre-to(cid:173)
`ward a post-fusion conformation which are both trimeric
`(Roche et al., 2006; Roche et al., 2007).
`[0005] The polypeptide chain of G ectodomain folds into
`three distinct domains which are the fusion domain (FD), the
`pleckstrin homology domain (PHD), and the trimerization
`domain (TrD). During the structural transition, the FD, the
`PHD and the TrD retain their tertiary structure. Neverthe(cid:173)
`less, they undergo large rearrangements in their relative
`orientation due to secondary changes in hinge segments (Sl
`to S5) which refold during the low-pH induced conforma(cid:173)
`tional change (Roche et al., 2006; Roche et al., 2007).
`[0006] Recently, it has been shown that low-density lipo(cid:173)
`protein receptor (LDL-R) and other members of this recep(cid:173)
`tor family serve as VSV receptors (Finkelshtein et al., 2013).
`[0007] The LDL-R is a type I transmembrane protein
`which regulates cholesterol homeostasis in mammalian cells
`(Brown and Goldstein, 1986). LDL-R removes cholesterol
`carrying lipoproteins from plasma circulation. Ligands
`bound extracellularly by LDL-R at neutral pH are internal(cid:173)
`ized and then released in the acidic environment of the
`endosomes leading to their subsequent lysosomal degrada(cid:173)
`tion. The receptor then recycles back to the cell surface.
`ligand-binding
`LDL-R ectodomain is composed of a
`domain, an epidermal growth factor (EGF) precursor homol(cid:173)
`ogy domain and a C-terminal domain enriched in 0-linked
`oligosaccharides. The ligand binding domain is made of 7
`cysteine-rich repeats (CR! to CR7, FIG. 1). Each repeat is
`made of approximately 40 amino acids and contains 6
`cysteine residues, engaged in 3 disulfide bridges, and an
`acidic residues cluster that coordinates a Ca2
`+ ion. The
`
`intracellular release of the cargo is driven by a low-pH(cid:173)
`induced conformational change of LDL-R from an open to
`a closed conformation.
`[0008] The LDL-R gene family consists of trans-mem(cid:173)
`brane receptors that reside on the cell-surface, are involved
`in endocytic uptake of lipoproteins, and require Ca2
`+ for
`ligand binding. All these receptors have in common several
`CR repeats (up to several tens), EGF precursor-like repeats,
`a membrane-spamiing region and an intracellular domain
`containing at least one internalization signal sequence. They
`are found ubiquitously in all animals including insects.
`[0009] VSV-G has been widely used for pseudotyping
`other viruses and VSV-G-pseudotyped lentiviruses (VSV(cid:173)
`G-LVs) exhibit the same broad tropism as VSV.
`[0010] On the other hand, VSV-G-LVs do not allow effi(cid:173)
`cient gene transfer into unstimulated T cells, B cells, and
`hematopoietic stem cells, as they have a very low expression
`level of LDL-R (Amirache et al., 2014).
`[0011] The broad tropism ofVSV and VSV-G LVs, due to
`the ubiquitous distribution of the LDL-R receptor family
`members, is a limitation of their therapeutic use. This is
`particularly the case in oncotherapy when one wants to
`target specifically tumor cells.
`[0012] One aim of the invention is to obviate this draw(cid:173)
`back.
`[0013] One aim of the invention is to provide a new
`mutated VSV-G protein deficient in one of its properties in
`order to specifically target this protein.
`[0014] Another aim of the invention is to provide a new
`VSV expressing such a protein and its use in oncotherapy.
`[0015] The invention relates to an isolated non-naturally
`occurring protein comprising, consisting essentially of or
`consisting of the amino acid sequence as set forth in SEQ ID
`NO: 1, which corresponds to (which is) the amino acid
`sequence of the ectodomain of VSV Indiana strain,
`[0016] wherein at least one of the amino acids at positions
`8, 47, 209 and 354, said numbering being made from the
`position of the first amino acid in the sequence SEQ ID
`NO: 1, is substituted by an amino acid different from the
`amino acid indicated at that position in said sequence SEQ
`ID NO: 1,
`[0017] or any homologous protein derived from said pro(cid:173)
`tein as set forth in SEQ ID NO: 1 by substitution, addition or
`deletion of at least one amino acid, provided that the derived
`protein retains at least 70% of identity with the amino acid
`sequence as set forth in SEQ ID NO: 1, and said derived
`protein retaining the ability to induce membrane fusion and
`retaining the ability to interact with LDL membrane recep(cid:173)
`tor,
`[0018] wherein at least one amino acid, of said homolo(cid:173)
`gous protein located at a position equivalent to the positions
`8, 47, 209 and 354 of said sequence SEQ ID NO: 1, is
`substituted by an amino acid distinct from the amino acid
`indicated at that position in the sequence SEQ ID NO: 1,
`[0019]
`said
`isolated non-naturally occurring protein
`retaining the ability to induce membrane fusion and being
`unable to interact with LDL membrane receptor.
`[0020]
`In a preferred embodiment, the amino acid at
`position 8 in SEQ ID NO: 1 of the isolated non-naturally
`occurring protein of invention (said numbering being made
`from the position of the first amino acid in the sequence SEQ
`ID NO: 1 ), or at the position equivalent in the homologous
`protein derived from said protein as set forth in SEQ ID
`NO: 1 of the invention, cannot be a Y residue.
`
`Page 12 of 27
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`

`

`US 2020/0216502 Al
`
`Jul. 9, 2020
`
`2
`
`[0021]
`In a preferred embodiment, the amino acid at
`position 209 in SEQ ID NO: 1 of the isolated non-naturally
`occurring protein of invention (said numbering being made
`from the position of the first amino acid in the sequence SEQ
`ID NO: 1 ), or at the position equivalent in the homologous
`protein derived from said protein as set forth in SEQ ID
`NO: 1 of the invention, cannot be a H residue.
`[0022] Thus, in other words, the invention relates to an
`isolated non-naturally occurring protein comprising the
`amino acid sequence as set forth in SEQ ID NO: 1, which
`corresponds to (which is) the amino acid sequence of the
`ectodomain of VSV Indiana strain,
`[0023] wherein at least one of the amino acids at positions
`8, 47, 209 and 354, said numbering being made from the
`position of the first amino acid in the sequence SEQ ID NO:
`1, is substituted by an amino acid different from the amino
`acid indicated at that position in said sequence SEQ ID NO:
`1,
`[0024] wherein the substitution at position 8 is by any
`amino acid different from the amino acid indicated at that
`position in said sequence SEQ ID NO: 1, except Y, and
`[0025] wherein the substitution at position 209 is by any
`amino acid different from the amino acid indicated at that
`position in said sequence SEQ ID NO: 1, except H,
`[0026] or any homologous protein derived from said pro(cid:173)
`tein as set forth in SEQ ID NO: 1 by substitution, addition
`or deletion of at least one amino acid, provided that the
`derived protein retains at least 70% of identity with the
`amino acid sequence as set forth in SEQ ID NO: 1, and said
`derived protein retaining the ability to induce membrane
`fusion and retaining the ability to interact with LDL mem(cid:173)
`brane receptor,
`[0027] wherein at least one amino acid, of said homolo(cid:173)
`gous protein located at a position equivalent to the positions
`8, 47, 209 and 354 of said sequence SEQ ID NO: 1, is
`substituted by an amino acid distinct from the amino acid
`indicated at that position in the sequence SEQ ID N° 1,
`[0028] wherein the substitution of the amino acid located
`at a position equivalent to the position 8 is by any amino acid
`distinct from the amino acid indicated at that position in the
`sequence SEQ ID N° 1, except Y, and
`[0029] wherein the substitution of the amino acid located
`at a position equivalent to the position 209 is by any amino
`acid distinct from the amino acid indicated at that position
`in the sequence SEQ ID N° 1, except H,
`[0030]
`said
`isolated non-naturally occurring protein
`retaining the ability to induce membrane fusion and being
`unable to interact with LDL membrane receptor.
`[0031] Advantageously, the invention relates to an isolated
`non-naturally occurring protein comprising, consisting
`essentially of or consisting of the amino acid sequence as set
`
`forth in SEQ ID NO: 1, which corresponds to (which is) the
`amino acid sequence of the ectodomain of VSV Indiana
`strain,
`[0032] wherein the amino acid at position 8, or at position
`47, or at position 209, or at position 354, or at both positions
`8 and 4 7, or at both positions 8 and 209, or at both positions
`8 and 354, or at both positions 47 and 209, or at both
`positions 47 and 354, or at both positions 209 and 354, or at
`the positions 8 and 47 and 209, or at the positions 8 and 47
`and 354, or at the positions 8 and 209 and 354, or at the
`positions 47 and 209 and 354, or at the position 8 and 47 and
`209 and 354, said numbering being made from the position
`of the first amino acid in the sequence SEQ ID NO: 1, are
`substituted by any amino acid different from the amino acid
`found in SEQ ID NO: 1,
`[0033] or any homologous protein derived from said pro(cid:173)
`tein as set forth in SEQ ID NO: 1 by substitution, addition or
`deletion of at least one amino acid, provided that the derived
`protein retains at least 70% of identity with the amino acid
`sequence as set forth in SEQ ID NO: 1, and said derived
`protein retaining the ability to induce membrane fusion and
`retaining the ability to interact with LDL membrane recep(cid:173)
`tor,
`[0034] wherein the amino acid, of said homologous pro(cid:173)
`tein, located at a position equivalent to position 8, or to
`position 47, or to position 209, or to position 354, or to both
`positions 8 and 47, or to both positions 8 and 209, or to both
`positions 8 and 354, or to both positions 47 and 209, or to
`both positions 47 and 354, or to both positions 209 and 354,
`orto the positions 8 and 47 and 209, or to the positions 8 and
`47 and 354, or at the positions 8 and 209 and 354, or to the
`positions 47 and 209 and 354, or to the position 8 and 47 and
`209 and 354, are substituted by any amino acid different
`from the amino acid found in SEQ ID NO: 1,
`[0035]
`in particular the amino acid at position 8 is substi(cid:173)
`tuted by any amino acid except H, and preferably except Y,
`[0036]
`the amino acid at position 47 is substituted by any
`amino acid except K,
`[0037]
`the amino acid at position 209 is substituted by any
`amino acid except Y and preferably except H,
`[0038]
`the amino acid at position 354 is substituted by any
`amino acid except R,
`[0039]
`the numbering being made from the position of the
`first amino acid in the sequence SEQ ID NO: 1,
`[0040]
`said
`isolated non-naturally occurring protein
`retaining the ability to induce membrane fusion and being
`unable to interact with LDL membrane receptor.
`[0041] Advantageously, the invention relates to an isolated
`non-naturally occurring protein comprising or consisting
`essentially of or consisting of the amino acid sequence as set
`forth in SEQ ID NO: 1,
`
`KVKG@SNLISMDITFFSEDGELSS@EG~ETGGKl'jgK!i'!JQYIQKHWI:¥
`
`Page 13 of 27
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`

`

`US 2020/0216502 Al
`
`Jul. 9, 2020
`
`3
`
`-continued
`Rl!:JPSGvJ!11mEMJ'[lKDLFAAARFPEIQPEGSSQ;ISlff,QTSvi;J\1~~S~E!l:)
`
`~ RAGLPI ~LS[IL~I~TRCTJI181VDIAAl!JI LSRMV
`
`AQVFEJ!IBHIQIJBASQLPDqmSLF~~L~Sl!1JKl2JSIASFFFil]GLI
`
`IGLFLVLl8iVGIHLCIKLKHTKKRQIY~I~RLGK
`
`[0042] or any protein derived from said protein as set forth
`in SEQ ID NO: 1 by substitution, addition or deletion of at
`least one amino acid, provided that said protein derived
`protein derived from said protein as set forth in SEQ ID NO:
`1 retains the boxed amino acids as shown above,
`[0043] wherein the amino acid at position 8, or at position
`4 7, or at position 209, or at position 354, or at both positions
`8 and 47, or at both positions 8 and 209, or at both positions
`8 and 354, or at both positions 47 and 209, or at both
`positions 47 and 354, or at both positions 209 and 354, or at
`the positions 8 and 47 and 209, or at the positions 8 and 47
`and 354, or at the positions 8 and 209 and 354, or at the
`positions 47 and 209 and 354, or at the position 8 and 47 and
`209 and 354, or the corresponding positions in the protein
`derived from said protein as set forth in SEQ ID NO: 1, are
`substituted by any amino acid different from the amino acid
`found in SEQ ID NO: 1,
`in particular the amino acid at position 8 is substi(cid:173)
`[0044]
`tuted by any amino acid except H, and preferably except Y,
`the amino acid at position 47 is substituted by any
`[0045]
`amino acid except K,
`the amino acid at position 209 is substituted by any
`[0046]
`amino acid except Y and preferably except H,
`the amino acid at position 354 is substituted by any
`[0047]
`amino acid except R,
`the numbering being made from the position of the
`[0048]
`first amino acid in the sequence SEQ ID NO: 1,
`said
`isolated non-naturally occurring protein
`[0049]
`retaining the ability to induce membrane fusion and is
`unable to interact with LDL membrane receptor.
`[0050] Advantageously, the invention relates to an isolated
`non-naturally occurring protein comprising, consisting
`essentially of or consisting of the amino acid sequence as set
`forth in SEQ ID NO: 1, which corresponds to (which is) the
`amino acid sequence of the ectodomain of VSV Indiana
`strain,
`[0051] wherein the amino acid at position 47 or at position
`354, or at both positions 47 and 354, said numbering being
`
`made from the pos1t10n of the first amino acid in the
`sequence SEQ ID NO: 1, are substituted by any amino acid,
`in particular by any amino acid except K or R,
`[0052] or any homologous protein derived from said pro(cid:173)
`tein as set forth in SEQ ID NO: 1 by substitution, addition or
`deletion of at least one amino acid, provided that the derived
`protein retains at least 70% of identity with the amino acid
`sequence as set forth in SEQ ID NO: 1, and said derived
`protein retaining the ability to induce membrane fusion and
`retaining the ability to interact with LDL membrane recep(cid:173)
`tor,
`[0053] wherein the amino acid, of said homologous pro(cid:173)
`tein, located at a position equivalent to position 47 or to
`position 354, or to both positions 47 and 354, are substituted
`by any amino acid, in particular any amino acid except K or
`R,
`the numbering being made from the position of the
`[0054]
`first amino acid in the sequence SEQ ID NO: 1,
`said
`isolated non-naturally occurring protein
`[0055]
`retaining the ability to induce membrane fusion and being
`unable to interact with LDL membrane receptor.
`In one embodiment, said isolated non-naturally
`[0056]
`occurring protein further comprises a substitution of the
`amino acid at position 8, or at position 209, or at both
`positions 8 and 209, by any amino acid, said numbering
`being made from the position of the first amino acid in the
`sequence SEQ ID NO: 1,
`[0057] preferably wherein the amino acid at position 8 is
`substituted by any amino acid except H or Y, and
`[0058] preferably wherein the amino acid at position 209
`is substituted by any amino acid except H or Y.
`[0059] Advantageously, the invention relates to an isolated
`non-naturally occurring protein as defined above compris(cid:173)
`ing, consisting essentially of or consisting of the amino acid
`sequence as set forth in SEQ ID NO: 1,
`
`IBJF'Il]VIDJNQJ@'SKNJ'lllSNY~sg:JS~DLIGTAIQVKtvrn,HKAIQA@')
`
`KVKG@SNLISMDITFFSEDGELSS@EG~ETGGKl'jgK!i'!JQYIQKHWI:¥
`
`Page 14 of 27
`
`

`

`US 2020/0216502 Al
`
`Jul. 9, 2020
`
`4
`
`-continued
`Rl!:JPSGvJ!11mEMJ'[lKDLFAAARFPEIQPEGSSQ;ISAJBSQTSvi;J\1~~S~E!l:)
`
`~ RAGLPI ~LS[IL~I~TRCTJI181VDIAAl!JI LSRMV
`
`AQVFEJ!IBHIQIJBASQLPDqmSLF~~L~Sl!1JKl2JSIASFFFil]GLI
`
`IGLFLVLl8iVGIHLCIKLKHTKKRQIY~I~RLGK
`
`[0060] or any protein derived from said protein as set forth
`in SEQ ID NO: 1 by substitution, addition or deletion of at
`least one amino acid, provided that said protein derived
`protein derived from said protein as set forth in SEQ ID NO:
`1 retains the boxed amino acids as shown above,
`[0061] wherein the amino acid at position 47 or at position
`354, or at both positions 47 and 354, or the corresponding
`positions in the protein derived from said protein as set forth
`in SEQ ID NO: 1, are substituted by any amino acid, in
`particular by any amino acid except K or R,
`[0062]
`the numbering being made from the position of the
`first amino acid in the sequence SEQ ID NO: 1,
`[0063]
`said
`isolated non-naturally occurring protein
`retaining the ability to induce membrane fusion and is
`unable to interact with LDL membrane receptor.
`[0064]
`In one embodiment, said isolated non-naturally
`occurring protein further comprises a substitution of the
`amino acid at position 8, or at position 209, or at both
`positions 8 and 209, by any amino acid distinct from the
`amino acid indicated at that position in the sequence SEQ ID
`N° 1 or at the position equivalent in said homologous
`protein, said numbering being made from the position of the
`first amino acid in the sequence SEQ ID NO: 1,
`[0065] preferably wherein the amino acid at position 8 is
`substituted by any amino acid except H or Y, and
`[0066] preferably wherein the amino acid at position 209
`is substituted by any amino acid except H or Y.
`[0067] The invention is based on the unexpected obser(cid:173)
`vation made by the inventors that a substitution of at least
`one amino acid residues at positions 8, 47, 209 or 354, or the
`combination of two or three or the four amino acids, affects
`the ability of VSV G protein to interact with its receptor
`(LDL membrane receptor) but retain its property to induce
`membrane fusion, in particular at low pH.
`[0068] The invention encompasses proteins containing the
`amino acid sequence SEQ ID NO: 1, which corresponds to
`the native form of the Indiana strain ofVSV, and which lacks
`the signal peptide. The invention also encompasses any G
`protein from VSV strains provided that said protein retains
`the amino acids that are represented with a box in SEQ ID
`NO: 1.
`[0069] The G proteins form VSV strains may differ by
`addition, substitution or insertion of at least one amino acid
`which are not the amino acid represented with a bow in SEQ
`ID NO: 1.
`[0070] Regarding the numbering of the amino acid, this
`numbering is in the invention conventionally based on the
`numbering of the amino acids of the native form of the G
`protein ofVSV G Indiana as set forth in SEQ ID NO: 1. The
`skilled person knows the sequence alignment algorithms and
`
`programs (ClustalW for instance) and could easily compare
`the sequences of different G proteins and recalculate the
`exact position for a determined G protein compared to the
`numbering obtain in SEQ ID NO: 1. For sake of clarity, the
`amino acid at positions 8, 47, 209 and 354 are indicated in
`bold in the above SEQ ID NO: 1.
`[0071] The invention encompasses proteins containing the
`amino acid sequence SEQ ID NO: 1. The invention also
`encompasses any homologous G protein from VSV strains
`provided that said protein retains at least 70% of identity
`with the amino acid sequence SEQ ID NO: 1.
`[0072] By "at least 70% of identity", it is meant in the
`invention 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%,
`78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%,
`88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%,
`98%, 99% and 100% of identity with the sequence SEQ ID
`NO: 1.
`[0073] Regarding the percentage of identity, it is defined
`by the percentage of amino acid residues of SEQ ID NO: 1
`which align with the same amino acid in the sequence of the
`homologous protein. The sequence alignment is performed
`using dedicated algorithms and programs (such as ClustalW,
`for instance).
`[0074] Therefore, the protein according to the invention
`may derive from the following amino acid sequences:
`[0075] SEQ ID NO: 2, the full length VSV G protein
`from Indiana strain, by substitution of the amino acids
`at position 63, or at position 370, or both by any amino
`acid except K or R,
`[0076] SEQ ID NO: 3, the ectodomain of the VSV G
`protein from Marraba strain, by substitution of the
`amino acids at position 47, or at position 354, or both
`by any amino acid except K or R,
`[0077] SEQ ID NO: 4, the full length VSV G protein
`from Marraba strain, by substitution of the amino acids
`at position 63, or at position 370, or both by any amino
`acid except K or R,
`[0078] SEQ ID NO: 5, the ectodomain of the VSV G
`protein from New Jersey strain, by substitution of the
`amino acids at position 47, or at position 358, or both
`by any amino acid except K or R,
`[0079] SEQ ID NO: 6, the full length VSV G protein
`from New Jersey strain, by substitution of the amino
`acids at position 63, or at position 374, or both by any
`amino acid except K or R,
`[0080] SEQ ID NO: 7, the ectodomain of the VSV G
`protein from Carajas strain, by substitution of the
`amino acids at position 47, or at position 358, or both
`by any amino acid except K or R,
`
`Page 15 of 27
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`US 2020/0216502 Al
`
`Jul. 9, 2020
`
`5
`
`[0081] SEQ ID NO: 8, the full length VSV G protein
`from Carajas strain, by substitution of the amino acids
`at position 63, or at position 374, or both by any amino
`acid except K or R,
`[0082] SEQ ID NO: 9, the ectodomain of the VSV G
`protein fromA!agoa strain, by substitution of the amino
`acids at position 47, or at position 354, or both by any
`amino acid except K or R,
`[0083] SEQ ID NO: 10, the full length VSV G protein
`from Alagoa strain, by substitution of the amino acids
`at position 64, or at position 371, or both by any amino
`acid except
`[0084] K or R,
`[0085] SEQ ID NO: 11, the ectodomain of the VSV G
`protein from Coca! strain, by substitution of the amino
`acids at position 47, or at position 354, or both by any
`amino acid except K or R, and
`[0086] SEQ ID NO: 12, the full length VSV G protein
`from Coca! strain, by substitution of the amino acids at
`position 64, or at position 371, or both by any amino
`acid except K or R.
`[0087] SEQ ID NO: 13, the ectodomain of the VSV G
`protein from Morreton strain, by substitution of the
`amino acids at position 47, or at position 354, or both
`by any amino acid except K or R, and
`[0088] SEQ ID NO: 14, the full length VSV G protein
`from Morreton strain, by substitution of the amino
`acids at position 64, or at position 371, or both by any
`amino acid except K or R.
`[0089] Via the crystallographic characterization of the G
`protein, the inventors showed that residues K47 and R354
`are highly critical for the interaction with the LDL derived
`receptor. When one or both residues are substituted by
`another amino acid residue have physical and chemical
`different properties, the resulting G protein loses its capacity
`to interact with cellular receptor. By contrast, the same
`resulting protein, in appropriate condition of pH retains its
`fusogenic property.
`[0090]
`In the invention, the protein is isolated, which
`means that the protein has been isolated from its natural
`context. The protein is non-naturally occurring, which
`means that the only way to obtain this protein is to carry out
`a substitution, in a laboratory, by using technological meth(cid:173)
`ods man-made, well known in the art.
`[0091] More advantageously, the invention relates to the
`isolated non-naturally occurring protein previously dis(cid:173)
`closed, wherein said protein comprises, or consists essen(cid:173)
`tially of or consists of one of the following amino acid
`sequence:
`[0092] SEQ ID NO: 15-20
`[0093] SEQ ID NO: 21-26
`[0094] SEQ ID NO: 27-32
`[0095] SEQ ID NO: 33-38
`[0096] SEQ ID NO: 39-44
`[0097] SEQ ID NO: 45-50, and
`[0098] SEQ ID NO: 51-56,
`[0099] wherein the amino acid at position 47 or at position
`354, or at both positions 47 and 354, or the corresponding
`positions in the protein derived from said protein as set forth
`in SEQ ID NO: 1, are any amino acid except Kor R. In other
`words, in the invention the amino acids Xaa corresponds to
`any amino acid except R or K.
`
`[0100] SEQ ID NO: 15 corresponds to the ectodomain of
`the VSV G protein from Indiana strain having a substitution
`at position 47, by any amino acid except Kor R.
`[0101] SEQ ID NO: 16 corresponds to the ectodomain of
`the VSV G protein from Indiana strain having a substitution
`at position 354, by any amino acid except K or R.
`[0102] SEQ ID NO: 17 corresponds to the ectodomain of
`the VSV G protein from Indiana strain having a substitution
`at positions 47 and 354, by any amino acid except K or R.
`[0103] SEQ ID NO: 18 corresponds to the full length VSV
`G protein from Indiana strain having a substitution at
`position 63, by any amino acid except K or R.
`[0104] SEQ ID NO: 19 corresponds to the full length VSV
`G protein from Indiana strain having a substitution at
`position 370, by any amino acid except Kor R.
`[0105] SEQ ID NO: 20 corresponds to the full length VSV
`G protein from Indiana strain having a substitution at
`positions 63 and 370, by any amino acid except Kor R.
`[0106] SEQ ID NO: 21 corresponds to the ectodomain of
`the VSV G protein from Marraba strain having a substitution
`at position 47, by any amino acid except Kor R.
`[0107] SEQ ID NO: 22 corresponds to the ectodomain of
`the VSV G protein from Marraba strain having a substitution
`at position 354, by any amino acid except K or R.
`[0108] SEQ ID NO: 23 corresponds to the ectodomain of
`the VSV G protein from Marraba strain having a substitution
`at positions 47 and 354, by any amino acid except K or R.
`[0109] SEQ ID NO: 24 corresponds to the full length VSV
`G protein from Marraba strain having a substitution at
`position 63, by any amino acid except K or R.
`[0110] SEQ ID NO: 25 corresponds to the full length VSV
`G protein from Marraba strain having a substitution at
`position 370, by any amino acid except Kor R.
`[0111] SEQ ID NO: 26 corresponds to the full length VSV
`G protein from Marraba strain having a substitution at
`positions 63 and 370, by any amino acid except Kor R.
`[0112] SEQ ID NO: 27 corresponds to the ectodomain of
`the VSV G protein from New Jersey strain having a substi(cid:173)
`tution at position 47, by any amino acid except Kor R.
`[0113] SEQ ID NO: 28 corresponds to the ectodomain of
`the VSV G protein from New Jersey strain having a substi(cid:173)
`tution at position 358, by any amino acid except K or R.
`[0114] SEQ ID NO: 29 corresponds to the ectodomain of
`the VSV G protein from New Jersey strain having a substi(cid:173)
`tution at positions 47 and 358, by any amino acid except K
`or R.
`[0115] SEQ ID NO: 30 corresponds to the full length VSV
`G protein from New Jersey strain having a substitution at
`position 63, by any amino acid except K or R.
`[0116] SEQ ID NO: 31 corresponds to the full length VSV
`G protein from New Jersey strain having a substitution at
`position 374, by any amino acid except Kor R.
`[0117] SEQ ID NO: 32 corresponds to the full length VSV
`G protein from New Jersey strain having a substitution at
`positions 63 and 374, by any amino acid except Kor R.
`[0118] SEQ ID NO: 33 corresponds to the ectodomain of
`the VSV G protein from Carajas strain having a substitution
`at position 47, by any amino acid except Kor R.
`[0119] SEQ ID NO: 34 corresponds to the ectodomain of
`the