Patent Application
20220162266
The present invention is in the field of viruses with improved properties, and more particularly of VSV viruses with improved properties, in particular for use in oncolytic cancer therapy, gene therapy, immunotherapy and selective delivery of a cargo to a chosen cell type, and relates to a mutant polypeptide comprising the amino acid sequence of the ectodomain of glycoprotein G of a Vesicular stomatitis virus (VSV) strain, wherein said ectodomain comprises the amino acid sequence as set forth in SEQ ID NO: 2 or a sequence having at least 50% of identity with the amino acid sequence as set forth in SEQ ID NO: 2, with at least one substitution of an amino acid residue selected from the group consisting of:
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. Oncogene 24, 7710-7719 (2005); Fernandez et al. J. Virol. 76, 895-904 (2002); Hastie et al. Virus Res. 176, 16-32 (2013)).
VSV genome consists of 11 kb single-stranded negative-sense RNA (
Glycoprotein G is the only protein responsible for VSV tropism. This tropism is wide due to the ubiquitous nature of LDL-R family members, allowing VSV to enter many cell types. This is why for example VSV glycoprotein G is used to pseudotype many retrovirals vectors. Nevertheless, despite this broad tropism, several cell types only have a low expression level or do not carry the VSV receptors (such as resting T cells, B cells, and CD341 cells) (Amirache. Blood 123, 1422-4 (2014))
Hence, there is a need for modified VSV viruses with an altered tropism. Such modified VSV viruses would infect cell types not carrying the natural VSV receptors.
In order to overcome this problem, the inventors sought to identify sites allowing the insertion of the sequence of a targeting moiety (such as a nanobody), specifically recognizing a given receptor, in the amino acid sequence of VSV glycoprotein G. In particular, this insertion should not interfere with the membrane fusion properties of glycoprotein G.
They analyzed several insertion sites and demonstrated that the amino-terminal site tolerated the insertion of a nanobody. The resulting first chimera (GNano described precisely in
In the context of the invention, the inventors then built a recombinant VSV, in which the gene encoding the wild-type glycoprotein G was replaced by that encoding GNano (first chimera). The amplification of this initial recombinant VSV virus was however very difficult; and only very low infectious titers ˜1.8·106 pfu/ml were obtained after amplification in BSR cells after 24 hours. Such a low titer is not compatible with industrial production of a targeted VSV virus intended for therapy.
The inventors then performed several passages of this recombinant virus on BSR cells to select mutations improving its amplification. After 10 passages, the sequencing of the genomes of the viral population showed the progressive invasion of this population by a variant containing two mutations in the nucleotide sequence of the glycoprotein, resulting in the change of the histidine residue in position 22 into asparagine (H22N substitution) and of the serine 422 residue in position into isoleucine (S422I substitution). The titer of the recombinant virus obtained after this optimization was around ˜108 pfu/ml.
Surprisingly, the inventors thus found that two mutations (H22N and S422I), in combination, allowed the recombinant virus (GNano H22N S422I, or improved chimera, see
Moreover, the inventors also found that substitution S422I was able to rescue G fusion properties, which were completely abolished by substitution H407A. In this context, they demonstrated that S422I facilitates G folding and stabilizes G prefusion form by showing that it improves LDL-R CR domains recognition (the structural transition toward the postfusion state disrupts the CR domain binding site).
In the context of GNano H22N, they also demonstrated that substitutions S422F, S422M, S422L or S422V have a similar phenotype as S422I.
The inventors also verified that, in the presence of H22N substitution and/or S422I, S422F, S422M, S422L or S422V substitution, a recombinant VSV expressing a fusion polypeptide consisting of VSV Indiana G signal peptide, a dipeptide linker, an anti-GFP nanobody, a GGGGSGGGGS (SEQ ID NO:81) linker and VSV Indiana G ectodomain with one of substitutions S422I, S422F, S422M, S422L and S422V, was able to form syncytia when exposed to low pH, between 5.5 and 6.3, similarly to wild-type VSV Indiana. This also suggests that the presence of a hydrophobic residue in position 422 is key in the regulation of the pH-dependent structural transition, and facilitates G folding and stabilizes the prefusion form of a fusion polypeptide of G with a nanobody inserted N-terminal of the ectodomain (see Example 3). This is also confirmed by the crystal structure of VSV G ectodomain, solved by the inventors. Indeed, the inventors surprisingly showed that the C-terminal part (from residue 407 to residue 429, more particularly the region located from residue 421 to residue 429, even more particularly the region from residue 421 to residue 425) largely interacts with the fusion domain and therefore contributes to the stabilization of the pre-fusion complex. The crystal structure further revealed that compensatory substitution of S422 residue with a hydrophobic residue (such as I, F, M, L or V) stabilizes the beta-hairpin structure (and thus the pre-fusion state) through hydrophobic interactions (see Example 4).
In a first aspect, the present invention thus relates to a mutant polypeptide comprising, or consisting essentially of, or consisting of, the amino acid sequence of the ectodomain of glycoprotein G of a vesicular stomatitis virus (VSV) strain, wherein said ectodomain comprises, or consists essentially of, or consists of, the amino acid sequence as set forth in SEQ ID NO: 2 or a sequence having at least 50% of identity with the amino acid sequence as set forth in SEQ ID NO: 2, with at least one substitution of an amino acid residue selected from the group consisting of:
The present invention also relates to fusion polypeptide comprising, or consisting essentially of, or consisting of, the mutant polypeptide according to the invention, and an additional peptide, polypeptide or protein, wherein said additional peptide, polypeptide or protein is inserted:
The present invention also relates to a nucleic acid molecule encoding the mutant polypeptide or the fusion polypeptide according to the invention.
The present invention also relates to a vector comprising at least one nucleic acid according to the invention or expressing the mutant polypeptide or the fusion polypeptide according to the invention.
The present invention also relates to a host cell containing or expressing the mutant polypeptide or the fusion polypeptide according to the invention, or containing the nucleic acid molecule according to the invention, or containing the vector (in particular a VSV vector) according to the invention.
The present invention also relates to a composition comprising, or consisting essentially of, or consisting of, the mutant polypeptide or the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, or any combination thereof.
The present invention also relates to therapeutic uses of the mutant polypeptide or the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention, or any combination thereof.
The present invention also relates to the mutant polypeptide according to the invention or the fusion polypeptide according to the invention for use for targeting a lipid membrane in a subject to a specific target, for instance a cell, in particular a cell to be killed, such as a cancer cell, wherein said mutant polypeptide or fusion polypeptide is anchored in said lipid membrane.
The present invention also relates to an in vitro use of the mutant polypeptide according to the invention or the fusion polypeptide according to the invention for targeting a lipid membrane to a specific target, for instance a cell, in particular a cell to be killed, such as a cancer cell, wherein said mutant polypeptide or fusion polypeptide is anchored in said lipid membrane.
Bottom: Flow cytometry analysis of surface expression of mutant glycoproteins G-H407A and G.H407A/S422I. The surface G protein expression was detected with monoclonal antibody 8G5F11 (KeraFAST) and a goat anti-mouse IgG secondary antibody conjugated to Alexa Fluor 488. A total of 10,000 cells were counted by flow cytometry. In each frame, the black curve corresponds to the fluorescence of untransfected cells, the blue curve corresponds to the fluorescence of cells transfected by pCAGGS plasmids encoding WT G, and the red curve corresponds to the fluorescence of cells transfected by pCAGGS plasmids encoding the indicated mutant. In each frame, the surface expression of mutant G is expressed as a percentage of that of WT G.
(A) Crystalline structure of the pre-fusion trimer of G1-440. The chain is traced up to residue 432. Residues 410 to 432 are in dark grey.
(B) Structure of segment 410-432. The two close-up detail the organization of segment 410-432 and its interactions with the fusion domain.
(C) Close-up view of residue S422 in G pre-fusion state.
In the context of the present invention, the inventors surprisingly found that, while a recombinant VSV virus comprising a nanobody inserted between the signal peptide and the ectodomain of VSV Indiana glycoprotein G retained its fusion properties and infectivity, it was very difficult to amplify, not permitting an industrial use thereof. They further surprisingly found that insertion of at least one substitution in position 22 or position 422 of the ectodomain of VSV Indiana glycoprotein G, while retaining fusion properties and infectivity, permitted to dramatically increase the amplification of the recombinant virus, thus permitting an industrial use thereof (see Example 1). They also obtained results showing that the presence of a hydrophobic residue in position 422 is key in the regulation of the pH-dependent structural transition, and facilitates G folding and stabilizes the prefusion form of a fusion polypeptide of G with a nanobody inserted N-terminal of the ectodomain (see Examples 2, 3 and 4).
Mutant Polypeptide with Improved Production
The present invention thus firstly relates to a mutant polypeptide comprising, or consisting essentially of, or consisting of, the amino acid sequence of the ectodomain of glycoprotein G of a Vesicular stomatitis virus (VSV) strain, wherein said ectodomain comprises, or consists essentially of, or consists of, the amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence having at least 50% of identity with the amino acid sequence as set forth in SEQ ID NO: 2, with at least one substitution of an amino acid residue selected from the group consisting of:
In a preferred embodiment, the mutant polypeptide of the invention is an isolated and/or non-naturally occurring polypeptide.
Reference Sequences and Positions for Substitution
The mutant polypeptide is based on the amino acid sequence of the ectodomain of glycoprotein G of a Vesicular stomatitis virus (VSV) strain, which corresponds to the amino acid sequence of glycoprotein G without the signal peptide. In the present description, the reference VSV strain is the Indiana strain. The amino acid sequence of glycoprotein G of VSV Indiana strain is as set forth in SEQ ID NO:1. The amino acid sequence of the ectodomain of glycoprotein G of VSV Indiana strain is as set forth in SEQ ID NO:2, and corresponds to amino acids 17-511 of SEQ ID NO:1, amino acids 1-16 of SEQ ID NO:1 corresponding to the signal peptide. In a preferred embodiment, the mutant polypeptide according to the invention is derived from the ectodomain of glycoprotein G of VSV Indiana strain and thus comprises, or consists essentially of, or consists of, the amino acid sequence as set forth in SEQ ID NO: 2, with at least one substitution of an amino acid residue selected from the group consisting of any amino acid residue located from position 421 to position 429 and any amino acid residue located from position 17 to position 25 of SEQ ID NO:2, preferably any amino acid residue located from position 421 to position 425 and any amino acid residue located from position 17 to position 25 of SEQ ID NO:2, more preferably any amino acid residue located in positions 422 and 22 of SEQ ID NO:2. However, the mutant polypeptide according to the invention may also be derived from the ectodomain of glycoprotein G of a VSV strain other than Indiana or may comprise further mutations, provided that it comprises, or consists essentially of, or consists of, an amino acid sequence having at least 50% of identity, preferably at least 55% of identity, at least 60% of identity, at least 65% of identity, more preferably at least 70% of identity, at least 75% of identity, at least 78% of identity, at least 79% of identity, at least 80% of identity, at least 81% of identity, at least 82% of identity, at least 83% of identity, at least 84% of identity, at least 85% of identity, at least 86% of identity, at least 87% of identity, at least 88% of identity, at least 89% of identity, at least 90% of identity, at least 91% of identity, at least 92% of identity, at least 93% of identity, at least 94% of identity, at least 95% of identity, at least 96% of identity, at least 97% of identity, at least 98% of identity, or even at least 99% of identity with the amino acid sequence as set forth in SEQ ID NO: 2, with at least one substitution of an amino acid residue selected from the group consisting of any amino acid residue located from a position equivalent to position 421 to a position equivalent to position 429 of SEQ ID NO: 2 and any amino acid residue located from a position equivalent to position 17 to a position equivalent to position 25 of SEQ ID NO: 2, after optimal global alignment with SEQ ID NO:2, preferably any amino acid residue located from a position equivalent to position 421 to a position equivalent to position 425 of SEQ ID NO: 2 and any amino acid residue located from a position equivalent to position 17 to a position equivalent to position 25 of SEQ ID NO: 2, after optimal global alignment with SEQ ID NO:2, more preferably any amino acid residue located in positions equivalent to positions 422 and 22 of SEQ ID NO: 2, after optimal global alignment with SEQ ID NO:2. In this case, an optimal global alignment of SEQ ID NO:2 and of the initial amino acid sequence (for instance amino acid sequence of the ectodomain of glycoprotein G of a VSV strain other than Indiana, provided that this sequence has at least 50% of identity or any other preferred minimal percentage of identity defined above with SEQ ID NO:2 after optimal global alignment) is made, and the initial amino acid sequence has at least one substitution of an amino acid residue selected from the group consisting of any amino acid residue located from a position equivalent to position 421 to a position equivalent to position 429 of SEQ ID NO: 2 and any amino acid residue located from a position equivalent to position 17 to a position equivalent to position 25 of SEQ ID NO: 2, after optimal global alignment with SEQ ID NO:2, preferably any amino acid residue located from a position equivalent to position 421 to a position equivalent to position 425 of SEQ ID NO: 2 and any amino acid residue located from a position equivalent to position 17 to a position equivalent to position 25 of SEQ ID NO: 2, after optimal global alignment with SEQ ID NO:2, more preferably any amino acid residue located in positions equivalent to positions 422 and 22 of SEQ ID NO: 2, after optimal global alignment with SEQ ID NO:2.
The percent identities referred to in the context of the disclosure of the present invention are determined on the basis of an optimal global alignment of sequences to be compared, i.e., on an optimal alignment of the sequences taken in their entirety over their entire length using any algorithm well-known to a person skilled in the art, such as the algorithm of Needleman and Wunsch (1970). This sequence comparison may be performed using any software well-known to a person skilled in the art, for example the Emboss Needle software. The Emboss Needle software is for example available at https://www.ebi.ac.uk/Tools/psa/emboss_needle/. This software reads two input sequences and writes their optimal global sequence alignment to file. It uses the Needleman-Wunsch alignment algorithm to find the optimum alignment (including gaps) of two sequences along their entire length. The algorithm uses a dynamic programming method to ensure the alignment is optimum, by exploring all possible alignments and choosing the best. A scoring matrix is read that contains values for every possible residue or nucleotide match. Needle finds the alignment with the maximum possible score where the score of an alignment is equal to the sum of the matches taken from the scoring matrix, minus penalties arising from opening and extending gaps in the aligned sequences. The substitution matrix and gap opening and extension penalties are user-specified. In the context of the invention, in order to obtain an optimal global alignment, the Emboss Needle software may be used with default parameters, i.e. using the “Gap open” parameter equal to 10.0, the “Gap extend” parameter equal to 0.5, the “End gap penalty” parameter to “false”, the “End gap open” parameter to 10.0, and a “Blosum 62” matrix. When entering two amino acid sequences, the Emboss Needle software returns an optimal global alignment, as well as several values characterizing the alignment:
Once an optimal global alignment has been obtained between SEQ ID NO:2 and another initial sequence, the percentage of identity and position equivalent to specific position(s) of SEQ ID NO:2 are defined. In particular, a position equivalent to position N of SEQ ID NO:2 is the position of the other initial sequence that is aligned with position N of SEQ ID NO:2.
Examples of VSV strains other than Indiana also include 7 serotypes such as Maraba, Cocal, Morreton, Alagoa, New Jersey., and Carajas strains. The non-mutated (wild-type) amino acid sequences of the full-length glycoprotein G and of the ectodomain of glycoprotein G of these strains and Indiana strain are presented in Table 1 below:
MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWK
MLRLFLFCFLALGAHSKFTIVFPHHQKGNWK
MNFLLLTFIVLPLCSHAKFSIVFPQSQKGNWK
MLVLYLLLSLLALGAQCKFTIVFPHNQKGNW
MTPAFILCMLLAGSSWAKFTIVFPQSQKGDW
MLSYLIFALVVSPILGKIEIVFPQHTTGDWKRV
MKMKMVIAGLILCIGILPAIGKITISFPQSLKGD
An optimal global alignment of the non-mutated the amino acid sequences of the ectodomain of the glycoprotein G of VSV strain Indiana versus each of Maraba, Cocal, Morreton, Alagoa, New Jersey, and Carajas strains is presented in
The alignments of
A multiple amino acid sequences alignment of the ectodomains of the glycoprotein G of VSV strains Indiana, Maraba, Cocal, Morreton, Alagoa, New Jersey, and Carajas is presented in
In particular, positions 17-25 of SEQ ID NO:2 contain conserved amino acids in positions 17-18, 21 and 23-25. A consensus sequence of positions 17-25 of amino sequences of the ectodomain of glycoprotein G of VSV strains Indiana, Maraba, Cocal, Morreton, Alagoa, New Jersey, and Carajas may be defined as VPX1X2YX3YCP (SEQ ID NO:15), wherein:
Similarly, positions 421-429 of SEQ ID NO:2 contain conserved amino acids in positions 421 and 426-429. A consensus sequence of positions 421-429 of amino sequences of the ectodomain of glycoprotein G of VSV strains Indiana, Maraba, Cocal, Morreton, Alagoa, New Jersey, and Carajas may be defined as EX4X5X6X7GDTG (SEQ ID NO:16), wherein:
Therefore, in a preferred embodiment, the mutant polypeptide according to the invention comprises, or consists essentially of, or consists of:
wherein:
is(are) substituted.
Moreover, amino acids conserved in all amino sequences of the ectodomain of glycoprotein G of VSV strains Indiana, Maraba, Cocal, Morreton, Alagoa, New Jersey, and Carajas correspond to positions 1, 4, 6-7, 12, 14, 17-18, 21, 23-25, 27, 29, 31, 33, 46-4755-56, 58-60, 64, 66-69, 71-75, 77, 79, 81-82, 87, 91-92, 96, 102, 107-111, 114-116, 119, 121, 127, 130, 132, 134, 137-139, 141, 143, 145, 151, 153, 158, 160, 162-165, 167, 177, 199, 204, 206-208, 210, 212, 219, 221, 224, 228, 231, 236-237, 241, 253, 259, 262, 268, 271-272, 274-277, 279-281, 283-285, 287-290, 299, 301, 304, 307-311, 313-314, 316-318, 320-321, 323, 325-326, 330, 332, 338, 345, 349, 354, 356-357, 360, 362, 368-372, 374, 376, 379, 381, 383-384, 388, 390, 393, 395, 407-408, 413, 421, 426-429, 431-434, 436, 439-443, 445, 447, 456, 467, 487, and 489-490 of SEQ ID NO:2. In the following, these positions are referred to as “conserved positions”.
Therefore, in some embodiments of the mutated polypeptide according to the invention, the amino acid sequence having at least 50% of identity with the amino acid sequence as set forth in SEQ ID NO: 2, after optimal global alignment with SEQ ID NO:2, has the same amino acids as SEQ ID NO: 2 in the above defined conserved positions.
An exception may be made for conserved positions 47 and 354, when it is desired to alter the ability of the ectodomain to bind to the low-density lipoprotein receptor (LDL-R) (see below section entitled “Further optional mutations”). In this case, the amino acid sequence having at least 50% of identity (or any other minimal % of identity disclosed above) with the amino acid sequence as set forth in SEQ ID NO: 2, after optimal global alignment with SEQ ID NO:2, may have the same amino acids as SEQ ID NO: 2 in the above defined conserved positions, except for possible additional substitution(s) in position 47 and/or 354 of SEQ ID NO:2.
Type of Amino Acid for Substitution
While the substitute amino acid(s) in the mutant polypeptide according to the invention may be selected from any amino acid different from the original amino acid, depending on the location(s) of the substitution(s), some particular types of amino acids are preferred.
In particular, when the mutant polypeptide according to the invention comprises a substitution in any amino acid residue located from position 421 to position 429 of SEQ ID NO:2 or equivalent position after optimal global alignment with SEQ ID NO:2, the original amino acid is preferably substituted by a hydrophobic amino acid, preferably selected for the group consisting of glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), phenylalanine (F) and tryptophan (W), more preferably isoleucine (I), leucine (L), methionine (M), valine (V) and phenylalanine (F), even more preferably isoleucine (I). Indeed, the present inventors have shown that the presence of a hydrophobic amino acid in any amino acid residue located from position 421 to position 429 of SEQ ID NO:2 or equivalent position after optimal global alignment with SEQ ID NO:2 results in the stabilization of the glycoprotein G prefusion conformation.
When the mutant polypeptide according to the invention comprises a substitution in any amino acid residue located from position 17 to position 25 of SEQ ID NO:2 or equivalent position after optimal global alignment with SEQ ID NO:2, the original amino acid is preferably substituted by a polar amino acid, preferably selected from the group consisting of asparagine (N), glutamine (Q), cysteine (C), tyrosine (Y), threonine (T), serine (S), more preferably asparagine (N). Indeed, it is believed that the presence of a polar amino acid in any amino acid residue located from position 17 to position 25 of SEQ ID NO:2 or equivalent position after optimal global alignment with SEQ ID NO:2 results in the stabilization of the glycoprotein G prefusion conformation.
When the mutant polypeptide according to the invention comprises a substitution in any amino acid residue located from position 17 to position 25 of SEQ ID NO:2 or equivalent position after optimal global alignment with SEQ ID NO:2, and also a substitution in any amino acid residue located from position 421 to position 429 of SEQ ID NO:2 or equivalent position after optimal global alignment with SEQ ID NO:2:
Preferred Mutant Polypeptides
Some preferred mutant polypeptides according to the invention are based on the amino acid sequence of the ectodomain of glycoprotein G of VSV Indiana strain, and may be selected from:
Most preferred mutant polypeptides based on the amino acid sequence of the ectodomain of glycoprotein G of VSV Indiana strain are those comprising at least a substitution in position 422 (SEQ ID NO: 18 to 27 or a mutant polypeptide comprising one of these amino acid sequences and further comprising a signal peptide in N-terminal, preferably the signal peptide corresponding to positions 1-16 of SEQ ID NO:1).
The amino acid sequences SEQ ID NO:17 to 27 of preferred mutant polypeptide derived from VSV Indiana glycoprotein G ectodomain are presented in Table 3 below.
Other preferred mutant polypeptides according to the invention are based on the amino acid sequence of the ectodomain of glycoprotein G of VSV Maraba strain, and may be selected from:
Most preferred mutant polypeptides based on the amino acid sequence of the ectodomain of glycoprotein G of VSV Maraba strain are those comprising at least a substitution in position 422 (SEQ ID NO: 29 to 38 or a mutant polypeptide comprising one of these amino acid sequences and further comprising a signal peptide in N-terminal, preferably the signal peptide corresponding to positions 1-16 of SEQ ID NO:3).
The amino acid sequences SEQ ID NO:28 to 38 of preferred mutant polypeptide derived from VSV Maraba glycoprotein G ectodomain are presented in Table 4 below.
Other preferred mutant polypeptides according to the invention are based on the amino acid sequence of the ectodomain of glycoprotein G of VSV Cocal strain, and may be selected from:
Most preferred mutant polypeptides based on the amino acid sequence of the ectodomain of glycoprotein G of VSV Cocal strain are those comprising at least a substitution in position 422 (SEQ ID NO: 40 to 49 or a mutant polypeptide comprising one of these amino acid sequences and further comprising a signal peptide in N-terminal, preferably the signal peptide corresponding to positions 1-17 of SEQ ID NO:5).
The amino acid sequences SEQ ID NO:39 to 49 of preferred mutant polypeptide derived from VSV Cocal glycoprotein G ectodomain are presented in Table 5 below.
Other preferred mutant polypeptides according to the invention are based on the amino acid sequence of the ectodomain of glycoprotein G of VSV Morreton strain, and may be selected from:
Most preferred mutant polypeptides based on the amino acid sequence of the ectodomain of glycoprotein G of VSV Morreton strain are those comprising at least a substitution in position 422 (SEQ ID NO: 51 to 60 or a mutant polypeptide comprising one of these amino acid sequences and further comprising a signal peptide in N-terminal, preferably the signal peptide corresponding to positions 1-17 of SEQ ID NO:7).
The amino acid sequences SEQ ID NO:50 to 60 of preferred mutant polypeptide derived from VSV Morreton glycoprotein G ectodomain are presented in Table 6 below.
Other preferred mutant polypeptides according to the invention are based on the amino acid sequence of the ectodomain of glycoprotein G of VSV Alagoa strain, and may be selected from:
Most preferred mutant polypeptides based on the amino acid sequence of the ectodomain of glycoprotein G of VSV Alagoa strain are those comprising at least a substitution in position 422 (SEQ ID NO: 62 to 71 or a mutant polypeptide comprising one of these amino acid sequences and further comprising a signal peptide in N-terminal, preferably the signal peptide corresponding to positions 1-17 of SEQ ID NO:9).
The amino acid sequences SEQ ID NO:61 to 71 of preferred mutant polypeptide derived from VSV Alagoa glycoprotein G ectodomain are presented in Table 7 below.
Other preferred mutant polypeptides according to the invention are based on the amino acid sequence of the ectodomain of glycoprotein G of VSV New Jersey strain, and may be selected from:
The amino acid sequences SEQ ID NO:72 to 75 of preferred mutant polypeptide derived from VSV New Jersey glycoprotein G ectodomain are presented in Table 8 below.
Other preferred mutant polypeptides according to the invention are based on the amino acid sequence of the ectodomain of glycoprotein G of VSV Carajas strain, and may be selected from:
The amino acid sequences SEQ ID NO:76 to 79 of preferred mutant polypeptide derived from VSV Carajas glycoprotein G ectodomain are presented in Table 9 below.
Thus, the mutant polypeptide of the invention advantageously comprises, or consists essentially of, or consists of, an amino acid sequence selected from the group consisting of SEQ ID NO: 17-79, preferably selected from the group consisting of SEQ ID NO: 17-72 and SEQ ID NO: 74-79.
Further Optional Mutations
The mutant polypeptide according to the invention is derived from the amino acid sequence of the ectodomain of glycoprotein G of a VSV strain, and thus comprises, or consists essentially of, or consists of, an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence having at least 50% of identity (or any other minimal % of identity disclosed above) with the amino acid sequence as set forth in SEQ ID NO: 2, with at least one substitution of an amino acid residue as defined above.
The mutant polypeptide according to the invention may further contain one or more (such as 1, 2, or more) additional substitutions at additional positions of SEQ ID NO:2, or equivalent positions after optimal global alignment with SEQ ID NO:2.
When it is desired not to alter the function of the ectodomain, the possible one or more additional substitutions will preferably be conservative, meaning that the substitute amino acid has a structure that is similar to that of the original amino acid and is therefore unlikely to change the biological activity of the polypeptide. Examples of such conservative substitutions are presented in Table 10 below:
In addition, as explained above, the alignment presented in
However, in some cases, it may be desired to alter the function of the ectodomain, for instance in order to alter the ability of the ectodomain to bind to the low-density lipoprotein receptor (LDL-R), as described in the application filed under number PCT/EP2018/075824 and in Nikolic et al. Nat Commun. 2018 Mar. 12; 9(1):1029. This permits to alter the tropism of a VSV expressing an ectodomain unable to interact with LDL membrane receptor.
Therefore, in an embodiment of the present invention, the mutant polypeptide according to the invention further comprises at least one substitution of an amino acid residue selected from the group consisting of amino acid residues located at positions 8, 47, 209 and 354 of SEQ ID NO:2 or amino acid residues located at positions equivalent to positions 8, 47, 209 and 354 of SEQ ID NO:2 after optimal global alignment with SEQ ID NO:2.
For substitution in position 8 or equivalent position after optimal global alignment with SEQ ID NO:2, the original amino acid is preferably substituted by any amino acid except histidine (H), glutamine (Q) or tyrosine (Y), more preferably the original amino acid is substituted by alanine (A).
For substitution in position 47 of SEQ ID NO:2 or equivalent position after optimal global alignment with SEQ ID NO:2, the original amino acid is preferably substituted by any amino acid except lysine (K) or arginine (R), more preferably the original amino acid is substituted by alanine (A) or glutamine (Q).
For substitution in position 209 or equivalent position after optimal global alignment with SEQ ID NO:2, the original amino acid is preferably substituted by any amino acid except tyrosine (Y) or histidine (H), more preferably the original amino acid is substituted by alanine (A).
For substitution in position 354 or equivalent position after optimal global alignment with SEQ ID NO:2, the original amino acid is preferably substituted by any amino acid except lysine (K) or arginine (R), more preferably the original amino acid is substituted by alanine (A) or glutamine (Q).
Positions 47 and 354 of SEQ ID NO:2 or equivalent positions after optimal global alignment with SEQ ID NO:2 are preferred for obtaining an ectodomain unable to interact with LDL membrane receptor. When it is desired that the mutant polypeptide according to the invention is unable to interact with LDL membrane receptor; the mutant polypeptide further thus preferably comprises a substitution at position 47 of SEQ ID NO:2 or equivalent position after optimal global alignment with SEQ ID NO:2, a substitution at position 354 of SEQ ID NO:2 or equivalent position after optimal global alignment with SEQ ID NO:2, or two substitutions at positions 47 and 354 of SEQ ID NO:2 or equivalent positions after optimal global alignment with SEQ ID NO:2. More preferably, the substitutions at position(s) 47 and/or 354 are those defined as preferred above.
Fusion Polypeptide
The present invention also relates to a fusion polypeptide comprising, or consisting essentially of, or consisting of, the mutant polypeptide of the invention, and an additional peptide, polypeptide or protein, wherein said additional peptide, polypeptide or protein is inserted:
preferably said additional peptide, polypeptide or protein is inserted in N-terminal of the mutant polypeptide or between the amino acids at positions 351 and 352 of SEQ ID NO:2 or equivalent positions after optimal global alignment with SEQ ID NO:2.
The additional peptide, polypeptide or protein may have various functions, but may notably be used in order to target a recombinant VSV virus to specific target cells. In this case, any additional peptide, polypeptide or protein that is able to specifically bind to an antigen expressed at the extracellular surface of the target cells may be used. In particular, the additional peptide, polypeptide or protein may comprise at least part of a ligand of a cellular receptor.
When the target cell expresses at its surface one of the components of a (receptor-ligand) couple (for instance: EGF-EGFR, TNFa-TNFaR, PD1-PDL1 or PDL2, etc. . . . ) (which will happen often, since molecules expressed at the cell surface generally have a cognate receptor or ligand), then at least part (provided that it is still able to bind its cognate receptor or ligand) of the second component of the (receptor-ligand) couple (i.e. the natural receptor or ligand of the targeted cell surface antigen) may be used.
Alternatively, an antibody, a functional antibody fragment.
By “antibody” or “immunoglobulin” is meant a molecule comprising at least one binding domain for a given antigen and a constant domain comprising an Fc fragment capable of binding to Fc receptors (FcR). In most mammals, like humans and mice, an antibody consists of four polypeptide chains: two heavy chains and two light chains bound together by a variable number of disulfide bridges providing flexibility to the molecule. Each light chain consists of a constant domain (CL) and a variable domain (VL); the heavy chains consisting of a variable domain (VH) and three or four constant domains (CH1 to CH3 or CH1 to CH4) according to the isotype of the antibody. In a few rare mammals, such as camels and llamas, the antibodies consist of only two heavy chains, each heavy chain comprising a variable domain (VH) and a constant region.
The variable domains are involved in antigen recognition, while the constant domains are involved in the biological, pharmacokinetic and effector properties of the antibody.
By “functional antibody fragment” is meant an antibody fragment retaining the antigen-binding domain and thus having the same antigen specificity as the original antibody. In most mammals, such functional antibody fragments comprise the fragments Fv, ScFv, Fab, F(ab′)2, Fab′, and scFv-Fc. In the case of camels and llamas, in which the antibodies consist of only two heavy chains, each heavy chain comprising a variable domain (referred to as VHH) and a constant region, such functional antibody corresponds to the VHH fragment, also known as nanobody.
In the context of the invention, the additional peptide, polypeptide or protein will preferably be a peptide or relatively short polypeptide (preferably less than 200 amino acids), in order not to alter the function of the mutated polypeptide according to the invention. As a result, when antibodies or functional antibody fragments are the selected type of additional peptide, polypeptide or protein, functional antibody fragments will preferably be used. While any suitable functional antibody fragment may be used in the context of the invention, a nanobody (or VHH fragment) will preferably be used, because of its small size, globular shape and robustness.
Depending on the targeted cell, the antigen targeted at the cell surface will vary. For instance, when a recombinant VSV virus expressing the mutant polypeptide according to the invention is used as on oncolytic virus, the mutated polypeptide according to the invention may be fused as described above with an antibody or functional antibody fragment (in particular a nanobody) targeting the G ectodomain to a cancer cell antigen. For instance, an anti-HER2 or anti-MUC18 or anti-EGFR or anti-CD20 or anti-CD52 antibody or functional antibody fragment (in particular a nanobody) may be used. In another context, an antibody or functional antibody fragment (in particular a nanobody) targeting an inhibitory immune checkpoint (such as PD1 or its ligands PD-L1 and PD-L2, or CTLA4) may be used, when it is desired to target immune cells expressing such inhibitory immune checkpoints. Also, an antibody or functional antibody fragment (in particular a nanobody) targeting an inhibitory immune checkpoint (such as PD1 or its ligands PD-L1 and PD-L2, or CTLA4) may be used, when it is desired to target immune cells expressing such inhibitory immune checkpoints. Likewise, an antibody or functional antibody fragment (in particular a nanobody) targeting the B-lymphocyte antigen CD19 may be used, when it is desired to target B cells. An exemplary nanobody directed to GFP has the amino acid sequence SEQ ID NO:80
When the additional peptide, polypeptide or protein (preferably a functional antibody fragment such as a nanobody) is inserted in N-terminal of the mutant polypeptide according to the invention, it may be fused directly to the N-terminal extremity of the mutant polypeptide, or separated from the N-terminal extremity of the mutant polypeptide by a linker peptide, preferably it is separated from the N-terminal extremity of the mutant polypeptide by a first linker peptide.
Typically, linkers are 1 to 30 amino acids long peptides composed of amino acid residues such as glycine (G), serine (S), threonine (T), asparagine (N), glutamine (Q), alanine (A) proline (P), and/or phenylalanine (F). Preferred linkers in the context of this invention comprise 2 to 15 amino acids, with a preference for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids.
The first linker peptide between the additional peptide, polypeptide or protein (preferably a functional antibody fragment such as a nanobody) and the mutated polypeptide according to the invention is 2 to 15 amino acids long, preferably 4 to 12 or 6 to 10 amino acids long, and notably 8 amino acids long. Alternatively or in combination, its amino acid sequence is preferably constituted of amino acids selected from glycine (G), serine (S), threonine (T), asparagine (N), glutamine (Q), alanine (A), proline (P), and phenylalanine (F), more preferably selected from glycine (G), serine (S), threonine (T), and alanine (A), or from glycine (G), serine (S), threonine (T), or from glycine (G), serine (S), and alanine (A), even more preferably from glycine (G) and serine (S). In a preferred embodiment, the first linker peptide is constituted of 6 to 10 amino acids selected from glycine (G) and serine (S), and may notably be of amino acid sequence GGGGSGGGGS (SEQ ID NO:81).
When the additional peptide, polypeptide or protein (preferably a functional antibody fragment such as a nanobody) is inserted in N-terminal of the mutant polypeptide according to the invention, the fusion polypeptide may further comprise, in N-terminal of the additional peptide, polypeptide or protein (preferably a functional antibody fragment such as a nanobody), a signal peptide in order to address the fusion polypeptide to the virus envelope (when the fusion polypeptide is expressed by a virus) or to the cell membrane (when the fusion polypeptide is expressed by a host cell).
In this case, the fusion polypeptide according to the invention comprises, or consists essentially of, or consists of, from N- to C-terminal: a signal peptide, optionally a second linker peptide, an additional peptide, polypeptide or protein (preferably a functional antibody fragment such as a nanobody), optionally a first linker peptide, and the mutated polypeptide according to the invention. In this fusion polypeptide, the first linker peptide, when present, is as described above. The second linker peptide (between the signal peptide and the additional peptide, polypeptide or protein (preferably a functional antibody fragment such as a nanobody)), when present, is 2 to 15 amino acids long, preferably 2 to 6 amino acids long, 2 to 4 amino acids long, and notably 2 or 3 amino acids long. Alternatively or in combination, its amino acid sequence is preferably constituted of amino acids selected from glycine (G), serine (S), threonine (T), asparagine (N), glutamine (Q), alanine (A), proline (P), phenylalanine (F), more preferably selected from serine (S), threonine (T), asparagine (N), glutamine (Q), and phenylalanine (F), and in particular from serine (S), threonine (T), asparagine (N), and glutamine (Q), or from glutamine (Q) and serine (S), or glutamine (Q) and phenylalanine (F). In a preferred embodiment, the second linker peptide is constituted of 2 to 4 amino acids selected from serine (S), threonine (T), asparagine (N), glutamine (Q), and phenylalanine (F), and in particular from serine (S), threonine (T), asparagine (N), and glutamine (Q), or from glutamine (Q) and serine (S), or glutamine (Q) and phenylalanine (F), and may notably be of amino acid sequence QF or QS.
In a preferred embodiment, the fusion polypeptide according to the invention comprises a first linker peptide or a second linker peptide, and more preferably both a first linker peptide and a second linker peptide.
When the fusion polypeptide according to the invention comprises both a first linker peptide and a second linker peptide:
In a preferred embodiment, when the fusion polypeptide according to the invention comprises both a first linker peptide and a second linker peptide, the first linker peptide (between the additional peptide, polypeptide or protein (preferably a functional antibody fragment such as a nanobody) and the mutated polypeptide according to the invention) has the amino acid sequence GGGGSGGGGS (SEQ ID NO:81)., and the second linker peptide (between the signal peptide and the additional peptide, polypeptide or protein (preferably a functional antibody fragment such as a nanobody)) has the amino acid sequence QF or QS.
An exemplary preferred fusion polypeptide has the sequence SEQ ID NO:82
Nucleic Acid Molecule
The present invention also relates to a nucleic acid molecule encoding the mutant polypeptide or the fusion polypeptide according to the invention.
All the different nucleic sequences encoding a particular amino acid sequence, because of degeneration of the genetic code, are within the scope of the invention.
In particular, the sequence of a nucleic acid according to the invention may be optimized to promote the expression thereof in a host cell or any other production host. Indeed, there are in general several three-nucleotide combinations encoding the same amino acid (except for methionine and tryptophan), called synonymous codons. However, some of these combinations are in general used preferentially by a cell or a given organism (this is referred to as genetic code usage bias). This preference depends notably on the producing organism from which the cell is derived. Consequently, when a protein derived from one or more organisms is produced in a heterologous organism or a cell of such a heterologous organism, it may be useful to modify the nucleic sequence encoding the protein to use mainly the preferred codons of the heterologous organism. Data are available in the literature concerning the use of codons preferred by different species and a person skilled in the art knows how to optimize the expression of a given protein in a heterologous organism or a cell of a heterologous organism.
Preferred nucleic acid molecules include those based on the natural nucleotide sequence encoding the ectodomain of glycoprotein G of VSV Indiana strain (SEQ ID NO:83), mutated at one or more nucleotides in order to generate a substitution as defined above. Preferred nucleic acid molecules include those having a nucleotide sequence selected from SEQ ID NO:83 to SEQ ID NO:86, which are more precisely defined in Table 11 below.
The nucleic acid molecule according to the invention may further contain any modification aimed to improve cloning and/or expression of the encoded mutated polypeptide or fusion polypeptide according to the invention, as well as its folding and stability.
Vector
The present invention also relates to a vector comprising at least one nucleic acid according to the invention or expressing the mutant polypeptide or the fusion polypeptide according to the invention.
Such a vector comprises the elements necessary for the expression of said nucleic sequence, and notably a promoter, a transcription initiation codon, termination sequences, and suitable transcription regulatory sequences. These elements vary according to the host used for the expression and are easily selected by persons skilled in the art based on their general knowledge.
In the context of the invention, the term “vector” has to be understood broadly as including plasmid and viral vectors. Vectors which are appropriate in the context of the present invention, include, without limitation, bacteriophage, plasmid or cosmid vectors for expression in prokaryotic host cells such as bacteria (e.g. E. coli, BCG or Listeria); vectors for expression in yeast (e.g. Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris); baculovirus vectors for expression in insect cell systems (e.g. Sf9 cells); as well as plasmid and viral vectors for expression in higher eukaryotic cells or subjects. Typically, such vectors are commercially available (e.g. in Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.) or available from depositary institutions such as the American Type Culture Collection (ATCC, Rockville, Md.) or have been the subject of numerous publications describing their sequence, organization and methods of producing, allowing the skilled person to apply them.
A “plasmid vector” as used herein refers to a replicable DNA construct. Usually plasmid vectors contain selectable marker genes that allow host cells carrying the plasmid vector to be selected for or against in the presence of a corresponding selective drug. A variety of positive and negative selectable marker genes are known in the art. By way of illustration, an antibiotic resistance gene can be used as a positive selectable marker gene that allows a host cell to be selected in the presence of the corresponding antibiotic. Suitable plasmid vectors are commercially available.
The term “viral vector” as used herein refers to a nucleic acid vector that includes at least one element of a virus genome and may be packaged into a viral particle or to a viral particle. The terms “virus”, “virions”, “viral particles” and “viral vector particle” are used interchangeably to refer to viral particles that are formed when the nucleic acid vector is transduced into an appropriate cell or cell line according to suitable conditions allowing the generation of viral particles. In the context of the present invention, the term “viral vector” has to be understood broadly as including nucleic acid vector (e.g. DNA viral vector) as well as viral particles generated thereof. The term “infectious” refers to the ability of a viral vector to infect and enter into a host cell or subject.
The viral vector may preferably be a Vesicular stomatitis virus (VSV) vector comprising at least one nucleic acid according to the invention or expressing the mutant polypeptide or the fusion polypeptide according to the invention.
VSV is an enveloped, negative-strand RNA virus that belongs to the Vesiculovirus genus of the Rhabdovirus family. VSV negative-strand RNA genome encodes five structural proteins and comprises, from 3′ to 5′: a leader region, the genes for the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G), the polymerase protein (L), and the trailer region. A mutated VSV vector according to the invention may thus be obtained by replacing the natural wild-type sequence encoding the natural wild-type glycoprotein G by a nucleic acid sequence according to the invention, encoding a mutant polypeptide or a fusion polypeptide according to the invention. Standard techniques for generating mutated VSV vectors may be used for this purpose. In particular, reverse genetic for the recovery of recombinant viruses may be used.
With respect to other regions of the VSV genome (leader, genes encoding the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G), the polymerase protein (L), and trailer region), the mutated VSV vector may contain any suitable natural or artificial sequence. In particular, sequences of the complete genome of various Indiana strain isolates (Rodriguez et al. J Gen Virol. 2002 October; 83(Pt 10):2475-83) and of New Jersey strain isolates (Velazquez-Salinas et al. Genome Announc. 2017 Sep. 14; 5(37)) are known in the art. In addition, various VSV strains are available at the American Type Culture Collection (ATCC) under references VR-1238, VR-1239, VR-159, and VR-1415 to VR-1421.
In an embodiment, the vector and preferably the VSV vector according to the invention may preferably be oncolytic, meaning that it preferentially infects and kills cancer cells. All strains of VSV previously listed (Indiana, Maraba, Cocal, Morreton, Alagoa, New Jersey, and Carajas) are potentially oncolytic and may thus be used.
Alternatively or in combination, the vector and preferably the VSV vector according to the invention may also be used for gene therapy, immunotherapy and selective delivery of a cargo to a chosen cell type.
When used for gene therapy, the vector and preferably the VSV vector according to the invention further encodes a gene of therapeutic interest, in order to restore the activity of this gene in cells that are deficient for this activity.
When used for immunotherapy, the vector and preferably the VSV vector according to the invention further encodes at least one antigen of interest and/or molecules involved in regulation of immune responses, such as cytokines, adjuvants, immune checkpoint modulators, antibodies targeting cytokines . . . .
When intended for delivery of a cargo to a chosen cell type such as unstimulated T cells, B cells, and hematopoietic cells, the recombinant mutated glycoprotein can be inserted at the surface of VLP containing a therapeutic molecule a marker or any drug.
According to a preferred embodiment, the viral vector according to the invention (in particular the VSV vector) is in the form of infectious viral particles. Typically, such viral particles are produced by a process comprising the steps of (i) introducing the viral vector of the invention into a suitable producer cell, (ii) culturing said producer cell under suitable conditions allowing the production of said infectious viral particle, (iii) recovering the produced viral particles from the culture of said producer cell, and (iv) optionally purifying said recovered viral particle.
Host Cell
The present invention also relates to a host cell containing or expressing the mutant polypeptide or the fusion polypeptide according to the invention, or containing the nucleic acid molecule according to the invention, or containing the vector (in particular a VSV vector) according to the invention.
When the host cell contains the vector (in particular a VSV vector) according to the invention, the host cell is preferably an eukaryotic cell suitable for VSV replication, such as BSR cells (a clone of BHK-21: Baby Hamster Kidney cells; ATCC CCL-10), BHK-21 cells (ATCC CCL-10), or Vero cells (ATCC CCL-81).
Composition
The present invention also relates to a composition comprising, or consisting essentially of, or consisting of, the mutant polypeptide or the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, or any combination thereof.
Preferably, the composition is a pharmaceutical composition which comprises a therapeutically effective amount of the active agent(s) (the mutant polypeptide or the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, or any combination thereof), and one or more pharmaceutically acceptable vehicle(s).
As used herein, a “pharmaceutically acceptable vehicle” is intended to include any and all carriers, solvents, diluents, excipients, adjuvants, dispersion media, coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like, compatible with administration in a subject and in particular in a human.
As used herein a “therapeutically effective amount” is a dose sufficient for the intended use.
The composition preferably comprises a vector according to the invention, and more particularly a VSV vector according to the invention.
Therapeutic/In Vivo Uses
The present invention also relates to a vector according to the invention (in particular a VSV vector according to the invention, preferably an oncolytic VSV vector according to the invention) or a (pharmaceutical) composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention, preferably an oncolytic VSV vector according to the invention), for use as a drug.
The present invention also relates to a vector according to the invention (in particular a VSV vector according to the invention, preferably an oncolytic VSV vector according to the invention) or a (pharmaceutical) composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention, preferably an oncolytic VSV vector according to the invention), for use for treating cancer.
The present invention also relates to the use of a vector according to the invention (in particular a VSV vector according to the invention, preferably an oncolytic VSV vector according to the invention) or of a (pharmaceutical) composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention, preferably an oncolytic VSV vector according to the invention), for the manufacture of a drug for treating cancer.
The present invention also relates to the use of a vector according to the invention (in particular a VSV vector according to the invention, preferably an oncolytic VSV vector according to the invention) or of a (pharmaceutical) composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention, preferably an oncolytic VSV vector according to the invention), for treating cancer.
The present invention also relates to a method for treating cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector according to the invention (in particular a VSV vector according to the invention, preferably an oncolytic VSV vector according to the invention) or of a (pharmaceutical) composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention, preferably an oncolytic VSV vector according to the invention).
Since the VSV virus is an oncolytic virus, it may be used in the treatment of various types of cancers, including prostate cancers, breast cancers, pancreatic cancers and melanoma (Bishnoi. Viruses 10, (2018)).
The present invention also relates to a vector according to the invention (in particular a VSV vector according to the invention) or a (pharmaceutical) composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention), for use in gene therapy or immunotherapy (in particular as a vaccine).
The present invention also relates to the use of a vector according to the invention (in particular a VSV vector according to the invention) or of a (pharmaceutical) composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention), for the manufacture of a drug for gene therapy or immunotherapy (in particular as a vaccine).
The present invention also relates to the use of a vector according to the invention (in particular a VSV vector according to the invention) or of a (pharmaceutical) composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention), for gene therapy or immunotherapy (in particular as a vaccine).
The present invention also relates to a method of gene therapy or immunotherapy in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector according to the invention (in particular a VSV vector according to the invention) or of a (pharmaceutical) composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention).
Vectors according to the invention (and in particular VSV vectors according to the invention) used in gene therapy or immunotherapy are preferably as disclosed above in “Vector” section.
Gene therapy may be used each time it is necessary to correct deficient gene activity in deficient cells.
Immunotherapy may be used for the treatment of various types of diseases, including cancers, infectious diseases, and inflammatory diseases (in particular autoimmune diseases).
The present invention also relates to a vector according to the invention (in particular a VSV vector according to the invention) or a (pharmaceutical) composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention), for use for selective delivery in vivo of a cargo to a chosen cell type.
The present invention also relates to the use of a vector according to the invention (in particular a VSV vector according to the invention) or of a (pharmaceutical) composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention), for the manufacture of a drug for selective delivery in vivo of a cargo to a chosen cell type.
The present invention also relates to the use of a vector according to the invention (in particular a VSV vector according to the invention) or of a (pharmaceutical) composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention), for selective delivery in vivo of a cargo to a chosen cell type.
The present invention also relates to a method for selective delivery in vivo of a cargo to a chosen cell type in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector according to the invention (in particular a VSV vector according to the invention) or of a (pharmaceutical) composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention). Vectors according to the invention (and in particular VSV vectors according to the invention) used for selective delivery in vivo of a cargo to a chosen cell type are preferably as disclosed above in “Vector” section.
The present invention also relates to the mutant polypeptide according to the invention or the fusion polypeptide according to the invention or the vector according to the invention or the host cell according to the invention or of a composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention) or of a combination thereof, for use for targeting a lipid membrane in a subject to a specific target, for instance a cell, in particular a cell to be killed (such as a cancer cell) or unstimulated T cells, B cells, and hematopoietic cells, wherein said mutant polypeptide or fusion polypeptide is anchored in said lipid membrane.
In Vitro Uses
The present invention also relates to the in vitro use of the mutant polypeptide according to the invention or the fusion polypeptide according to the invention or the vector according to the invention (in particular a VSV vector according to the invention) or the host cell according to the invention, or of a composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention) or of a combination thereof, for selective delivery of a cargo to a chosen cell type, such as unstimulated T cells, B cells, and hematopoietic cells.
Vectors according to the invention (and in particular VSV vectors according to the invention) used for selective in vitro delivery of a cargo to a chosen cell type are preferably as disclosed above in “Vector” section.
The present invention also relates to an in vitro use of the mutant polypeptide according to the invention or the fusion polypeptide according to the invention or the vector according to the invention or the host cell according to the invention or of a composition according to the invention (in particular a composition comprising a vector according to the invention, and more particularly a composition comprising VSV vector according to the invention) or of a combination thereof, for targeting a lipid membrane to a specific target, for instance a cell, in particular a cell to be killed (such as a cancer cell) or unstimulated T cells, B cells, and hematopoietic cells, wherein said mutant polypeptide or fusion polypeptide is anchored in said lipid membrane.
The following examples merely intend to illustrate the present invention.
Materials and Methods
Construction of GNano (First Chimera)
GNano constructions were created starting from the cloned VSV G gene (Indiana strain) in the pCAGGS plasmid. pCAGGS plasmids containing the desired coding GNano sequence with the nanobody inserted at various position were generated using Gibson assembly method. The empty vector pCAGGS was linearized using EcoRI restriction enzyme. Then 3 PCR products with overlapping parts were generated. The product I is the fragment of G before the insertion site of the nanobody. The product II is the nanobody gene. The product III is the fragment of G after the insertion site. PCR products and linearized vector were combined and joint by incubation with Gibson Assembly® Master Mix (NEB).
Obtention and Amplification of Recombinant VSV Virus in BSR Cells
Recombinant VSV were obtained as described in Schnell et al. J. virol. 1996. A Plasmid pVSVFL(+)-GNano expressing the 11,161-nucleotide (nt) positive-strand full-length VSV RNA sequence with the mutant GNano in place of the wt VSV G was generated using two unique restriction sites present on pVSVFL(+), MluI In the 5′ noncoding sequence of the G gene and NheI present in a sequence introduced between the G gene and the L gene. BSR cells were infected with vaccinia virus at a moi of 10. After one hour plasmids pBS-N, pBS-P, pBS-L and pcDNA3.1-G, respectively, encoding N, P, L and G proteins plus the pVSVFL(+)-GNano plasmid were transfected into the cells using lipofectamine 2000 (Invitrogen) in the presence of 10 μg/ml AraC (Sigma). Plasmids and amounts were as follows: 5 μg of pVSVFL(+)_GNano, 2.5 μg of pBS-N, 2 μg of pBS-P, 1 μg of pCDNA3.1-G and 0.5 μg of pBS-L. After 48 h of incubation at 37° C. in 5% CO2, supernatants from transfected cells were collected and debris were pelleted from the cell lysates by centrifugation. Supernatants were filtered through 0.2 μm to remove vaccinia virus, and 2.5 ml of this filtrate was added to approximately 106 BSR cells in a 3.5-cm-diameter plate. After 24 h, the supernatant containing the recombinant VSV was clarified by centrifugation and stored for further use at −80°.
Titer in pfu/ml of the obtained recombinant VSV supernatants were determined by plaque assay in 6 well plates on BSR cells by counting the plaques in each wheel 24 h after infection using crystal violet coloration.
Then, the harvested recombinant virus was amplified by infecting 3 106 BSR cells at moi 0.1 on a 6 cm-diameter cell plate. This new supernatant was tittered by plaque assay and stored for further use at −80°.
To obtain higher titers, 3 106 BSR cells in a 6-cm-diameter plate were infected with 10 000 pfu in order to favor apparition of mutation susceptible to increase the growth rate of the recombinant virus. Ten subsequent passages of the viral supernatant on BSR cells were made. Titer was again estimated using plaque assay.
Titration by Plaque Assay
The day before, 106 BSR cells were seed in 6 wheel plate to ensure a ˜90% confluent monolayer the day after. Then cells were infected with 200 μL of tenfold serial dilutions for 1 h at room temperature. After the infection, cells are overlaid with immobilizing medium added directly to the inoculums containing 0.8% agarose in the wheel.
After addition of the overlay, plates are incubated at 37° C. in 5% CO2 for 24 h. To determine viral titer plaques are counted in wells containing from 5 to 100 plaques. The viral titer is determined as follow: pfu/ml=(average number of plaque)/(dilution×volume of dilution added to the plate).
Sequencing of the G Gene in the Viral Population
3 106 BSR cells on a 6 cm-diameter plate were infected at MOI 3 and total RNA extracts was done using TRIzol (Invitrogen). Then RT-PCR using the primer TTTTTTTTTTTTCAT (SEQ ID NO: 87) specific of all viral mRNAs and was performed on total RNA extracts. Then VSVG gene was amplified by PCR and the PCR product obtained was sent to sequencing to identify compensatory mutations.
Results
A recombinant VSV virus, in which the gene encoding the wild-type glycoprotein G was replaced by that encoding GNano (first chimera, consisting of the signal peptide of VSV Indiana G protein, a two amino acids QF linker, an anti-GFP nanobody of sequence SEQ ID NO: 80, a 10 amino acids linker GGGGSGGGGS (SEQ ID NO:81) and wild-type VSV Indiana G protein ectodomain (SEQ ID NO:2), see top of
Several passages of this recombinant virus on BSR cells were then performed in order to select mutations improving its amplification. After 10 passages, the sequencing of the genomes of the viral population showed the progressive invasion of this population by a variant containing two mutations in the nucleotide sequence of the glycoprotein resulting in the change of:
The titer of the recombinant virus obtained after this optimization was around ˜108 pfu/ml.
The resulting improved chimera (Mutated GNano, see bottom of
The titers obtained in BSR cells at 24 hours after infection are presented in Table 12 below:
It is worth noting that a third mutation subsequently appeared in about 50% of cases in the sequence encoding the dipeptide linker consisting of glutamine and phenylalanine (QF) and located between the signal peptide of VSV glycoprotein G and the inserted nanobody. This mutation led to the replacement of phenylalanine to serine (QF->QS; data not shown).
Importantly, this third mutation had no effect on the kinetics and on the viral titer (data not shown). Indeed, the titer of the recombinant viruses having the 3 mutations was still ˜108 pfu/ml. Therefore, this third mutation did not affect the improved amplification of the recombinant virus in BSR cells.
The insertion of mutations H22N and S422I in the ectodomain of VSV Indiana glycoprotein G surprisingly resulted in a dramatic increase in the ability to amplify a recombinant VSV virus comprising a GFP targeted glycoprotein G (by insertion of an anti-GFP nanobody).
In an attempt to identify the histidines which play the role of pH sensitive molecular switch, we replaced the histidines of G ectodomain by an alanine.
Particularly, in VSV Indiana G, in the prefusion form of the protomer, there is a cluster of four histidines (H60, H162, H407) (
Materials and Methods
Plasmids and Cloning.
Point mutations were created starting from the cloned VSV G gene (Indiana Mudd-Summer strain) in the pCAGGS plasmid. Briefly, forward and reverse primers containing the desired mutation were combined separately with one of the primers flanking the G gene to generate two PCR products. These two G gene fragments overlap in the region containing the mutation and were assembled into the pCAGGS linearized vector (by EcoRI) using Gibson assembly reaction kit (New England Biolabs).
Cells and Antibodies.
BSR, clones of BHK-21 (baby hamster kidney; ATCC CCL-10), and HEK-293T (human embryonic kidney expressing simian virus 40 T antigen [SV40T]; ATCC CRL-3216) cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 8% fetal calf serum (FCS). Mouse monoclonal antibody directed against G ectodomain was supplied by KeraFAST (8G5F11);
Transfection.
For fusion assays and indirect immunofluorescence, BSR cells, grown in six-well plate at 70% confluence, were transfected by the phosphate calcium method (5 μg of the appropriate plasmid per well). For pseudotyped virus production, HEK-293 cells were grown in 10-cm dishes; they were transfected by 6 μg of the appropriate plasmid with polyethylenimine (PEI; Sigma-Aldrich).
Cell Surface Expression.
In order to quantify the expression of G protein on the cell surface, HEK-293 cells plated on six-well dishes at 70% confluence were transfected as described above. At 24 h after transfection, cells were collected by scraping into 1 mM EDTA-PBS, followed by centrifugation at 600 g for 5 min. Cells were incubated with a 1:2,000 dilution of mouse monoclonal anti-G ectodomain antibody (8G5F11; KeraFAST) in PBS on ice for 1 h. Cells were washed twice in PBS, fixed at 4° C. in paraformaldehyde, incubated with a 1:100 dilution of goat anti-mouse Alexa Fluor 488 (Invitrogen) on ice for 1 h, and rinsed in PBS. After resuspension in 500 ml of 0.5 mM EDTA-PBS, the fluorescence of 10,000 cells from each population was determined by flow cytometry using a BD Accuri C6 fluorescence-activated cell sorter (FACS). The mean fluorescence intensity (MFI) of the transfected cells expressing G was quantified by flow cytometry. The relative cell surface expression of transfected cells was determined as follows: (MFI for the mutant)/(MFI for the WT). For each mutant, the percentage given in the bottom of
Results
Mutation H407A completely abolishes G fusion properties. This mutation is lethal for the virus. Therefore, the recombinant viruses (VSV G H407A) cannot be generated spontaneously. We have employed a complementation strategy to support its growth.
Briefly, the recovery of the recombinant VSV was supported by expression of functional WT VSV G protein in trans from a transfected plasmid. To generate virus particles containing only the fusion-defective VSV G expressed from the viral genome, viruses present in the supernatant were amplified in cells that lack the trans-complementing VSV G plasmid. Sequence analysis of independent clones revealed two distinct compensatory mutations: one of them resulted in the replacement of serine 422 by an isoleucine (leading to the double mutant VSV G H407A S422I).
The comparison of the characteristics of VSV G H407A with those of VSV G H407A/54221 revealed that, in the context of mutation H407A, S422I improves the efficiency of VSV G transport efficiency and G recognition by a soluble form of LDL-R CR2 domain (
Materials and Methods
Cell-Cell Fusion Assay.
BSR cells plated on glass coverslips at 70% confluence were cotransfected with pCAGGS plasmids encoding wild-type (WT) G or mutant G, and P-GFP plasmid encoding the phosphoprotein of Rabies virus fused to GFP (cytoplasmic marker). Twenty-four hours after transfection, cells were incubated with fusion buffer (DMEM+10 mM MES) at various pH values (from 5.0 to 6.5) for 10 min at 37°. Cells were then washed once and incubated with DMEM+10 mM HEPES-NaOH buffered at pH 7.4, 1% BSA at 37° C. for 1 h. Cells were fixed with 4% paraformaldehyde in 1×PBS for 15 min. Cells nuclei were stained with DAPI, and syncytium formation was analyzed with Zeiss Axio vert 200 fluorescence microscope with a 20× lens.
Results
Fusion properties of VSV G Indiana WT and VSV GNano after optimization (ie. Mutation of residue H22 to N and residue S422 to I, F, M, L, or V) were analysed in cell-cell fusion assay. For this, BSR cells were transfected with pCAGGS plasmids expressing VSV G (either WT or mutant) and P-GFP (a protein exclusively cytoplasmic allowing an easy observation of syncytia). At 24 h post-transfection, the cells were exposed for 10 min to DMEM adjusted to the indicated pH, which was then replaced by DMEM at pH 7.4. The cells were then kept at 37° C. for 1 h before fixation.
Cells expressing wild-type G protein formed massive syncytia when exposed to low pH, between 5.5 and 6.3 (
The results are consistent with the fact that the presence of a hydrophobic residue in position 422 is key in the regulation of the pH-dependent structural transition. This strongly suggests that the mutation S422I (or S422F, S422M, S422L or S422V, or equivalent substitutions in equivalent positions of G ectodomain of other VSV strains) facilitates G folding and stabilizes the prefusion form of GNano.
Results
The structure of WT Indiana VSV G ectodomain (G1-440 comprising residues 1 to 440) was determined at 2.1 Å resolution. The ectodomain was crystallized in its trimeric pre-fusion conformation and its structure was determined at 2.1 Å resolution (
This X-ray structure of VSV G ectodomain reveals that the C-terminal part (from residue 407 to residue 425) largely interacts with the fusion domain and therefore contributes to the stabilization of the pre-fusion complex. Particularly relevant for the present invention, is the fact that, in this pre-fusion structure, residue S422 is pointing toward F424 and L430. Therefore, the compensatory mutation S422I stabilizes the beta-hairpin structure (and thus the pre-fusion state) through hydrophobic interactions between 1422 and residues F424 and L430. This also explained why the replacement of S422 by other hydrophobic residues (L, M, V or F) has the same effect (i.e. stabilizes the pre-fusion conformation).
| Number | Date | Country | Kind |
|---|---|---|---|
| 19305317.0 | Mar 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/057144 | 3/16/2020 | WO | 00 |
