CN116940687A - Viral delivery of sialidases for the treatment of cancer - Google Patents

Viral delivery of sialidases for the treatment of cancer Download PDF

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CN116940687A
CN116940687A CN202280015793.7A CN202280015793A CN116940687A CN 116940687 A CN116940687 A CN 116940687A CN 202280015793 A CN202280015793 A CN 202280015793A CN 116940687 A CN116940687 A CN 116940687A
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海因茨•卢布利
娜塔莉亚•罗德里格斯•曼图阿诺
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Universitaet Basel
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Abstract

The present application relates to adenovirus-associated viruses comprising influenza-derived neuraminidase transgenes, for use alone or in combination with immune checkpoint inhibitors in the treatment of patients diagnosed with solid cancer.

Description

Viral delivery of sialidases for the treatment of cancer
The present application relates to an adeno-associated virus carrying a sialidase gene for use in the treatment of cancer.
The present application claims the benefit of european patent application EP21157979.2 filed at 18 of 2 nd of 2021 and european patent application EP21182465.1 filed at 29 of 6 th of 2021, both of which are incorporated herein by reference in their entirety.
Background
Therapeutic desialylation of the tumor microenvironment can lead to a dramatic decrease in immune-mediated growth in cancer. Sialidases (sialidases), also known as neuraminidases (neuraminidases), are enzymes found in a large number of organisms. Viral sialidases are the most studied sialidase structure.
Intratumoral injection of viruses carrying, for example, transgenes that produce immunostimulatory cytokines is an approved treatment option for patients with advanced melanoma and is currently being investigated for different cancer types including head and neck tumors, triple negative breast cancer and cutaneous lymphomas.
Based on the state of the art described above, it is an object of the present invention to provide means and methods for treating cancer or supplementing solid cancer treatment by administering sialidases. This object is achieved by the subject matter of the independent claims of the present description, further advantageous embodiments being described in the dependent claims, examples, figures and the general description of the present description.
Disclosure of Invention
The present invention relates to an adeno-associated virus (AAV) vector comprising a sialidase (Sia) transgene, which transgene specifically encodes an influenza-derived neuraminidase protein. A further aspect of the invention relates to the use of the neuraminidase-carrying adeno-associated virus (AAV-Sia) as a medicament for the treatment of cancer or for enhancing the effect of immunotherapy administered against cancer, in addition to the routes of administration and methods particularly relevant to the invention.
In another embodiment, the invention relates to a pharmaceutical composition comprising an AAV-Sia of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient, optionally further comprising a checkpoint inhibitor.
Terminology and definitions
For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall govern.
As used herein, the terms "comprising," "having," "containing," and "including," and other similar forms and grammatical equivalents thereof, are intended to have an equivalent meaning and are open ended, as one or more items following any of these terms are not intended to be an exhaustive list of such one or more items, or are intended to be limited to only the one or more items listed. For example, an article that "comprises" components A, B and C may consist of components A, B and C (i.e., comprise only) or may contain not only components A, B and C, but also one or more other components. Thus, it is intended and understood that "comprising" and its similar forms and grammatical equivalents include the disclosure of embodiments that "consist essentially of" or "consist of".
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure and subject to any clearly excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
References herein to "about" a value or parameter include (and describe) variations with respect to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".
As used herein, including in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally Sambrook et al, molecular cloning laboratory guidelines (Molecular Cloning: A Laboratory Manual), 4 th edition (2012), cold spring harbor laboratory press and Ausubel et al, fine programming molecular biology laboratory guidelines (Short Protocols in Molecular Biology), 5 th edition (2002), john wili father company (John Wiley & Sons, inc.).
The term gene refers to a polynucleotide comprising at least one Open Reading Frame (ORF) that, when transcribed and translated, is capable of encoding a specific polypeptide or protein. The polynucleotide sequence may be used to identify a larger fragment or full-length coding sequence of the gene to which it relates. Methods for isolating larger fragment sequences are known to those skilled in the art.
The term gene expression or expression, or alternatively the term gene product, may refer to either or both of the process of producing a nucleic acid (RNA) or producing a peptide or polypeptide and its products, as well as transcription and translation, respectively, or any intermediate process that modulates the processing of genetic information to produce a polypeptide product. The term gene expression may also be applied to transcription and processing of RNA gene products, such as regulatory RNAs or structural (e.g. ribosomal) RNAs. If the expressed polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells. Expression can be measured at the transcriptional and translational levels, in other words, at the mRNA and/or protein product levels.
Sequences that are similar or homologous (e.g., at least about 70% sequence identity) to sequences disclosed herein are also part of the invention. In some embodiments, the sequence identity at the amino acid level may be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. At the nucleic acid level, sequence identity may be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Alternatively, substantial identity exists when a nucleic acid segment will hybridize to a complementary sequence of a strand under selective hybridization conditions (e.g., very high stringency hybridization conditions). The nucleic acid may be present in whole cells, in cell lysates, or in partially purified or substantially pure form.
In the context of the present specification, the terms sequence identity and percent sequence identity refer to a single quantitative parameter that represents the result of a sequence comparison determined by comparing two aligned sequences, position by position. Methods for aligning sequences are well known in the art. The comparison of the sequences may be performed as follows: by the local homology algorithm of Smith and Waterman, application math progression (adv. Appl. Math.)) (2:482 (1981); by the global alignment algorithm of Needleman and Wunsch, journal of molecular biology (j.mol. Biol.) 48:443 (1970); search similarity methods by Pearson and Lipman, proc. Nat. Acad. Sci.) "85:2444 (1988); or by computerized implementations of these algorithms, including but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analysis is publicly available, for example, through the national center for Biotechnology information (http:// BLAST. Ncbi. Nrm. Nih. Gov /).
An example of an amino acid sequence comparison is the BLASTP algorithm, which uses the following default settings: the desired threshold: 10; word length: 3, a step of; maximum match within query range: 0; matrix: BLOSUM62; gap penalty: there is 11, extension 1; composition adjustment: the conditional component score matrix adjusts. One such example for comparing nucleic acid sequences is the BLASTN algorithm, which uses the following default settings: the desired threshold: 10; word length: 28; maximum match within query range: 0; match/mismatch score: 1.-2; gap penalty: linearity. Unless otherwise indicated, sequence identity values provided herein refer to values obtained using the BLAST program set (Altschul et al, J. Mol. Biol. 215:403-410 (1990)), which uses the default parameters described above for protein and nucleic acid comparisons, respectively.
References to identical sequences without specifying a percentage value means a sequence that is 100% identical (i.e., the same sequence).
The acronym AAV in the context of this specification relates to adeno-associated virus, a small non-pathogenic virus that infects humans and other primate species. The term AAV is synonymous with AAV virions and AAV viral particles, and relates to viral particles composed of at least one AAV capsid protein and AAV nucleic acid encapsulated with a capsid. AAV formulations for gene delivery purposes typically rely on co-infection with a helper virus, such as an adenovirus, for replication, although replication competent AAV may also be present. AAV refers to all subtypes or serotypes, and replicative and recombinant forms, unless otherwise specified. The term AAV encompasses wild-type serotypes such as serotypes AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and synthetic variants with particular tissue targeting preferences.
In the context of the present specification, the term transgene refers to a gene or genetic material that has been transferred from one organism to another, herein referred to as insertion of a neuraminidase gene within an AAV vector. According to an embodiment of the invention, the term encompasses ORFs encoding neuraminidases derived from the influenza RNA genome. Those skilled in the art are able to construct DNA genomic adenovirus vectors based on this information to produce DNA sequences encoding influenza neuraminidase proteins. In this context, the term may also refer to the transfer of expression of a genetic sequence into tissue of a patient infected with an AAV vector.
The term sialidase is used interchangeably with the term neuraminidase in this specification. In the context of the present invention, the term sialidase or neuraminidase refers to a family of glycoside hydrolases that catalyze cleavage of a non-reducing sialic acid residue attached to an oligosaccharide chain, which is a derivative of a neuraminic acid group.
In the context of the present invention, the term sialidase of an influenza-derived virus refers to one of the known variants of neuraminidase proteins naturally occurring in influenza viruses of the orthomyxoviridae family.
In the context of the present specification, the term checkpoint inhibitor, immune checkpoint inhibitor or checkpoint inhibitory antibody is intended to cover agents, in particular antibodies (or antibody-like molecules), which are known in the art as part of the immune checkpoint mechanism, capable of disrupting the signaling cascade leading to T cell inhibition upon T cell activation. Non-limiting examples of checkpoint inhibitors or checkpoint inhibitory antibodies include antibodies that inhibit the interaction of CTLA-4 (Uniprot P16410), PD-1 (Uniprot Q15116), PD-L1 (Uniprot Q9NZQ 7) or B7H3 (CD 276; uniprot Q5ZPR 3) with their natural ligands.
In the context of the present specification, the term antibody refers to an intact antibody, which includes, but is not limited to, immunoglobulin G-type (IgG), a-type (IgA), D-type (IgD), E-type (IgE), or M-type (IgM), any antigen binding fragment or single chain thereof, and related or derived constructs. Whole antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (V H ) And a heavy chain constant region (C) H ) The composition is formed. The heavy chain constant region of IgG consists of three domains, C H 1、C H 2 and C H 3. Each light chain is composed of a light chain variable region (abbreviated herein as V L ) And a light chain constant region (C L ) The composition is formed. The light chain constant region consists of one domain C L The composition is formed. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. The term encompasses antibody-like molecules such as so-called nanobodies or single domain antibodies, antibody fragments comprising or consisting of a single monomer variable antibody domain, or single chain variable fragment (ScFv) multimers.
As used herein, the term pharmaceutical composition refers to a compound of the invention or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the invention is provided in a form suitable for topical, parenteral or injectable administration.
As used herein, the term pharmaceutically acceptable carrier includes any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal), isotonic, absorption delaying, salt, preservative, drug stabilizer, binder, excipient, disintegrant, lubricant, dye, and the like, and combinations thereof known to those of skill in the art (see, e.g., remington: science and practice of pharmacy (Remington: the Science and Practice of Pharmacy), ISBN 0857110624).
As used herein, in one embodiment, the term treating (treating) any disease or disorder (e.g., cancer) or treatment of any disease or disorder (e.g., cancer) refers to ameliorating a disease or disorder (e.g., slowing or inhibiting or reducing the progression, spread, or growth rate of a malignant tumor disease). In another embodiment, "treatment" or "treatment" refers to reducing or improving at least one physical parameter, including those that may not be discernable by the patient. In another embodiment, "treatment" or "treatment" refers to modulating a disease or disorder on the body (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art unless specifically described below.
Detailed Description
The first aspect of the invention relates to a recombinant AAV vector comprising a transgene encoding an influenza-derived sialidase polypeptide. Throughout this specification, the term "AAV-Sia" is used synonymously with the term "recombinant AAV vector comprising a transgene encoding an influenza-derived sialidase polypeptide". In this sense, recombinant (recombiant) refers to an AAV vector comprising an alteration of an influenza-derived sialidase transgene.
Sialidase polypeptide according to the invention
Certain embodiments of the invention relate to AAV comprising a sialidase transgene encoding an influenza-derived neuraminidase polypeptide. Known variants of influenza neuraminidase polypeptides (listed in the UniProtKB 2021_01 knowledge database under ID: uniProtKB) are considered as viable alternatives to type a neuraminidase 1 (N1) incorporated in the AAV tested in the examples, provided that they have similar biological sialidase activity.
In some embodiments, AAV encodes a sialidase for which broad immunogenicity exists in humans due to local infection and immune procedures. In alternative embodiments, the sialidases are derived from influenza strains that more commonly infect different mammalian hosts (see representative examples in table 1). It is expected that this will lead to existing immunity derived from vaccination against AAV-Sia transduced tumor cells or natural infection. Suitable neuraminidase transgenes may be selected taking into account additional features such as immunogenicity, nucleic acid length (single stranded AAV vectors are suitable for transgenes of less than 4.4Kb, while double stranded capacity is half) and suitability for expression in human cells.
In some embodiments of AAV-Sia according to the invention, the sialidase transgene encodes an influenza a polypeptide having biological sialidase function equivalent to the polypeptide designated SEQ ID NO 001 in the context of recombinant AAV-Sia.
Transgenic sialidase activity can be measured in assays to determine the level of desialylation of tumor cells following transduction with AAV-Sia, as in assays measuring the presence of fluorescent lectins following exposure of tumor cell lines to AAV-Sia, as shown in fig. 2 of the examples. Induction of sialidase transgene expression in tumor cells transduced with AAV-Sia vectors cleaves sialic acid residues from the tumor cell surface, thereby increasing binding of peanut lectin (PNA) lectin as a detection agent. Desialylation can be defined as being at 2×10 compared to control vector lacking sialidase transgene or untransduced cells (fig. 2, dashed line) 5 PNA fluorescence intensity on AAV-Sia-transduced tumor cell lines increased by about 99% (B16D 5), 30% (EMT 6), 72% (MC 38) and 43% hela at viral dose of viral particles/target cells. Sialic acid removal can be assessed by SNA or MAA staining. Measurement of biological Activity according to the present invention is defined in the section entitled "biological Activity of AAV-Sia according to the present invention" below.
In certain embodiments of the AAV-SIa, the sialidase transgene encodes N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, or N11 neuraminidase (see representative examples in table 1).
In some embodiments of the AAV-SIa, the sialidase transgene is N2 influenza neuraminidase. In other embodiments, the sialidase transgene is N3 influenza neuraminidase. In other embodiments, the sialidase transgene is N4 influenza neuraminidase. In other embodiments, the sialidase transgene is N5 influenza neuraminidase. In other embodiments, the sialidase transgene is N6 influenza neuraminidase. In other embodiments, the sialidase transgene is N7 influenza neuraminidase. In other embodiments, the sialidase transgene is N8 influenza neuraminidase. In other embodiments, the sialidase transgene is N9 influenza neuraminidase. In other embodiments, the sialidase transgene is N10 influenza neuraminidase. In other embodiments, the sialidase transgene is N11 influenza neuraminidase. In specific embodiments, the AAV-Sia sialidase transgene encodes an influenza a N1 neuraminidase polypeptide.
In an alternative embodiment of AAV-SIa, the sialidase transgene encodes a type B Victoria (Victoria) lineage neuraminidase. In other embodiments, the sialidase transgene encodes a B-lineage neuraminidase (Yamagata).
In other embodiments of AAV-SIa, the AAV transgene encodes an influenza a neuraminidase polypeptide comprising or consisting of a polypeptide designated SEQ ID NO 002. In other embodiments, the AAV transgene encodes a neuraminidase polypeptide comprising or consisting of a polypeptide designated SEQ ID NO 003. In other embodiments, the AAV transgene encodes a neuraminidase polypeptide comprising or consisting of the polypeptide designated SEQ ID NO 004. In other embodiments, the AAV transgene encodes a neuraminidase polypeptide comprising or consisting of a polypeptide designated SEQ ID NO 005. In other embodiments, the AAV transgene encodes a neuraminidase polypeptide comprising or consisting of the polypeptide designated SEQ ID NO 006. In other embodiments, the AAV transgene encodes a neuraminidase polypeptide comprising or consisting of a polypeptide designated SEQ ID NO 007. In other embodiments, the AAV transgene encodes a neuraminidase polypeptide comprising or consisting of a polypeptide designated SEQ ID NO 008. In other embodiments, the AAV transgene encodes a neuraminidase polypeptide comprising or consisting of the polypeptide designated SEQ ID NO 009. In other embodiments, the AAV transgene encodes a neuraminidase polypeptide comprising or consisting of a polypeptide designated SEQ ID NO 010. These polypeptide sequences encode 9 common type a derived polypeptides (SEQ ID NOs 001 to 009) and type B derived neuraminidase polypeptides (SEQ ID NO 010). In particular embodiments that have proven effective against tumor growth in the examples, the AAV vector encodes influenza a neuraminidase 1 having the sequence SEQ ID NO 001.
In alternative embodiments, the AAV vector sialidase transgene encodes a polypeptide comprising or consisting of a polypeptide sequence that has ≡85% ≡90% ≡91% >,. Gtoreq.92% >,. Gtoreq.93% >, 94% >, more particularly ≡95% >,. Gtoreq.96% >,. Gtoreq.97% >,. Gtoreq.98% >, 99% identity to SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009 or SEQ ID NO 010, provided that the sialidase has at least 90% biological activity of the particular sialidase polypeptide sequence on which it is based.
In a specific alternative embodiment, the AAV vector sialidase transgene encodes or consists of a variant polypeptide comprising or consisting of a polypeptide sequence having ≡85%, specifically ≡90%, more specifically ≡95% identity to SEQ ID NO 001, and having at least 90% of the biological activity of the AAV vector expressed sialidase polypeptide sequence SEQ ID NO 001.
Nucleic acid sequences of sialidase AAV transgenes according to the invention
In a specific embodiment, the AAV-Sia sialidase transgene comprises a nucleic acid sequence encoding an N1 neuraminidase having the polypeptide sequence SEQ ID NO 001. In a further specific embodiment, the AAV-Sia sialidase transgene consists of a nucleic acid sequence encoding an N1 neuraminidase having the polypeptide sequence SEQ ID NO 001.
In other embodiments, the AAV-Sia sialidase transgene comprises or consists of a nucleic acid sequence encoding an influenza neuraminidase having the polypeptide sequence SEQ ID NO 002. In other embodiments, the AAV-Sia sialidase transgene comprises or consists of a nucleic acid sequence encoding an influenza neuraminidase having the polypeptide sequence SEQ ID NO 003. In other embodiments, the AAV-Sia sialidase transgene comprises or consists of a nucleic acid sequence encoding an influenza neuraminidase having the polypeptide sequence SEQ ID NO 004. In other embodiments, the AAV-Sia sialidase transgene comprises or consists of a nucleic acid sequence encoding an influenza neuraminidase having the polypeptide sequence SEQ ID NO 005. In other embodiments, the AAV-Sia sialidase transgene comprises or consists of a nucleic acid sequence encoding an influenza neuraminidase having the polypeptide sequence SEQ ID NO 006. In other embodiments, the AAV-Sia sialidase transgene comprises or consists of a nucleic acid sequence encoding an influenza neuraminidase having the polypeptide sequence SEQ ID NO 007. In other embodiments, the AAV-Sia sialidase transgene comprises or consists of a nucleic acid sequence encoding an influenza neuraminidase having the polypeptide sequence SEQ ID NO 008. In other embodiments, the AAV-Sia sialidase transgene comprises or consists of a nucleic acid sequence encoding an influenza neuraminidase having the polypeptide sequence SEQ ID NO 009. In other embodiments, the AAV-Sia sialidase transgene comprises or consists of a nucleic acid sequence encoding an influenza neuraminidase having the polypeptide sequence SEQ ID NO 010.
In a specific embodiment, the AAV-Sia carries a transgene comprising the sequence designated SEQ ID NO 011. In an alternative embodiment, the AAV-Sia sialidase transgene has a sequence designated SEQ ID NO 012. In a further embodiment, the AAV-Sia sialidase transgene has a sequence designated SEQ ID NO 013. In a further embodiment, the AAV-Sia sialidase transgene has a sequence designated SEQ ID NO 014. In a further embodiment, the AAV-Sia sialidase transgene has a sequence designated SEQ ID NO 015. In a further embodiment, the AAV-Sia sialidase transgene has a sequence designated SEQ ID NO 016. In a further embodiment, the AAV-Sia sialidase transgene has a sequence designated SEQ ID NO 017. In a further embodiment, the AAV-Sia sialidase transgene has a sequence designated SEQ ID NO 018. In a further embodiment, the AAV-Sia sialidase transgene has a sequence designated SEQ ID NO 019. In a further embodiment, the AAV-Sia sialidase transgene has a sequence designated SEQ ID NO 020.
In a specific embodiment, the AAV-Sia sialidase transgene consists of the nucleic acid sequence SEQ ID NO 011.
Alternative embodiments provide AAV2 vectors comprising sialidase transgenes encoding polypeptides comprising or consisting of variant sialidase polypeptide sequences having greater than or equal to 85%, specifically greater than or equal to 90%, more specifically greater than or equal to 95% sequence identity to an amino acid sequence selected from SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, or SEQ ID NO 010. According to these embodiments, the sialidase has at least 90% of the biological activity of SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, or SEQ ID NO 010. In a specific embodiment, the variant has at least 90% of the biological function of the polypeptide SEQ ID NO 001. In a specific embodiment, the AAV2 vector comprises or consists of a sialidase transgene encoding a polypeptide comprising or consisting of a polypeptide sequence having greater than or equal to 85%, specifically greater than or equal to 90%, more specifically greater than or equal to 95% sequence identity to the amino acid sequence designated SEQ ID NO 001, and having at least 90% of the biological functions of the sialidase of sequence SEQ ID NO 001.
Biological Activity of sialidases according to the invention
The biological activity of AAV-Sia according to the invention can be measured in an in vitro or in vivo system designed to determine the desialylation of AAV-Sia transduced tumor cells. Equivalent biological activity may be defined by at least a 1.2-fold increase in PNA binding induction compared to a vector lacking the sialidase transgene, and/or a decrease in MAA II and SNA binding to the surface of transduced tumor cells. Alternatively, a decrease in tumor development, growth rate, or spread in an animal model as shown in figures 2 and 3 of the examples may also be a measure of the biological activity of AAV-Sia.
Without further explanation, to assess whether the variant sialidases have equivalent biological activity, i.e. 90% of the biological activity of the sialidases described herein, the biological activity is determined in the following assay. A positive control recombinant AAV2 vector is generated comprising a transgene consisting of a nucleic acid encoding a sialidase polypeptide sequence provided herein, e.g., SEQ ID NO 011, to which the variant is to be compared, encoding the influenza neuraminidase 1 protein of SEQ ID NO 001 under the control of a CMV promoter as shown in fig. 1. An experimental recombinant AAV2 vector is generated by replacing the nucleic acid encoding SEQ ID NO 001 with a nucleic acid encoding a variant sialidase polypeptide to be tested. HEK293 cells were co-transfected with AAV plasmids carrying each gene of interest (GOI) and replication-helper plasmid DNA encoding the REP and CAP genes of wild-type AAV 2. Two days after transfection, cell pellet was harvested and virus was released by 3 freeze/thaw cycles. The virus was purified by CsCl-gradient ultracentrifugation, followed by desalting. Viral titers (GC/ml-genome copy number/ml) were then determined by real-time PCR measurement of the gene of interest. SDS-gel silver staining was used to confirm the purity of 90% AAV protein prior to use of the viral stock in the experiments. At 37℃and 5% CO 2 MC38, B16D5, sea Law (Hela) or EMT6 cells (preferably the latter) were grown in 10% glucose in Dulbecco's modified Eagle medium) in 0.1mL DMEM (Sigma-Aldrich) supplemented with 1% glutamine, 1% pyruvate, 1% nonessential amino acids, streptomycin penicillin (5000U/mL) and 10% fetal bovine serum (Sigma-Aldrich) 4 Individual cells/wells were plated in 96-well plates. Each virus stock in PBS 5% glycerol was diluted in cell culture medium and used at 2X 10 5 The multiplicity of infection (MOI) was applied to three wells of the plate. After 72 hours, the cell culture medium was removed and adherent cells were collected with trypsin and washed. Will beThe cell pellet from each well was resuspended in 10. Mu.g/ml PNA (peanut lectin, vector Biolabs) for 30 minutes. After washing 2 times in PBS, the cell pellet was incubated with streptavidin-PE (1:500, BD Biosciences) for 30 minutes. Cells were washed twice in PBS and fixed with IC fixation buffer (sameifeier). Flow cytometry was performed to assess the Mean Fluorescence Intensity (MFI) of PE on cells from each well. The average PNA-PE of duplicate wells comprising cells transduced with the test recombinant AAV2 vector is at least 90% of the average MFI of a duplicate sample of a positive control recombinant AAV2 vector encoding the neuraminidase polypeptide sequence defined herein, in particular the N1 polypeptide of sequence SEQ ID NO 001, confirming that the sample has 90% of the biological activity of the positive control AAV-Sia.
Sialidase viral vectors according to the invention
Another aspect of the invention relates to AAV vectors. In certain embodiments, the AAV is recombinant AAV1. In particular embodiments, the AAV is a recombinant AAV2 vector. AAV2 is the AAV vector most commonly used for transgene delivery with broad cell tropism. However, different engineered or naturally occurring serotypes with specific tissue tropism can be selected to match the desired tumor cell targets. For example, AAV6 may be selected to target tumors derived from airway epithelial cells, or AAV8 vectors may be selected to target liver cancer.
In certain embodiments, AAV-Sia is for use in treating liver cancer, and AAV-Sia is an AAV8 vector that can induce expression of influenza neuraminidase polypeptides.
In certain embodiments, AAV-Sia is for use in treating lung cancer, and AAV-Sia is an AAV6 vector that can induce expression of an influenza neuraminidase polypeptide.
In certain embodiments, the vector is a recombinant AAV2 vector that has been demonstrated to efficiently induce functional transgene expression in a range of cancer cell lines in vitro, and has anti-tumor effects in a mouse model of cancer derived from different tissues (fig. 2 and 3).
In certain embodiments, the AAV2 vector carries a transgene encoding an influenza a neuraminidase N1 polypeptide. In other embodiments, the AAV-Sia is an AAV2 vector carrying an N2 transgene. In other embodiments, the AAV-Sia is an AAV2 vector carrying an N3 transgene. In other embodiments, the AAV-Sia is an AAV2 vector carrying an N4 transgene. In other embodiments, the AAV-Sia is an AAV2 vector carrying an N5 transgene. In other embodiments, the AAV-Sia is an AAV2 vector carrying an N6 transgene. In other embodiments, the AAV-Sia is an AAV2 vector carrying an N7 transgene. In other embodiments, the AAV-Sia is an AAV2 vector carrying an N8 transgene. In other embodiments, the AAV-Sia is an AAV2 vector carrying an N9 transgene. In other embodiments, the AAV-Sia is an AAV2 vector carrying an N10 transgene. In other embodiments, the AAV-Sia is an AAV2 vector carrying an N11 transgene. In an alternative embodiment, the AAV-Sia is an AAV2 vector carrying a transgene encoding a type B victoria lineage neuraminidase. In yet another embodiment, the AAV-Sia is an AAV2 vector encoding a type B gable lineage neuraminidase.
In other embodiments, the AAV-Sia is AAV2 carrying a transgene encoding a polypeptide sequence comprising SEQ ID NO 001. In other embodiments, the AAV-Sia is AAV2 carrying a transgene consisting of a nucleic acid sequence encoding a protein designated SEQ ID NO 001. In an alternative embodiment, an AAV-Sia according to the invention is an AAV2 vector comprising or consisting of a nucleic acid sequence encoding a protein selected from those designated SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, or SEQ ID NO 010. In a specific embodiment, the AAV-Sia is an AAV2 vector encoding an influenza a neuraminidase having the sequence SEQ ID NO 001.
According to an alternative embodiment of the present invention, there is provided an AAV2 vector comprising a sialidase transgene encoding a variant sialidase polypeptide comprising or consisting of a polypeptide sequence having more than or equal to 85%, specifically more than or equal to 90%, more specifically more than or equal to 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, or SEQ ID NO 010. In a specific embodiment, the variant sialidase polypeptide according to this aspect of the invention has at least 90% of the biological activity of an N1 influenza neuraminidase of the sequence SEQ ID NO 001.
In other embodiments, AAV-Sia is AAV2 having a transgene comprising the nucleic acid SEQ ID NO 011. In an alternative embodiment, the AAV-Sia is AAV2 comprising a sialidase transgene consisting of the sequence SEQ ID NO 012. In an alternative embodiment, the AAV-Sia is AAV2 comprising a sialidase transgene consisting of the sequence SEQ ID NO 013. In an alternative embodiment, the AAV-Sia is AAV2 comprising a sialidase transgene consisting of the sequence SEQ ID NO 014. In an alternative embodiment, the AAV-Sia is AAV2 comprising a sialidase transgene consisting of sequence SEQ ID NO 015. In an alternative embodiment, the AAV-Sia is AAV2 comprising a sialidase transgene consisting of sequence SEQ ID NO 016. In an alternative embodiment, the AAV-Sia is AAV2 comprising a sialidase transgene consisting of the sequence SEQ ID NO 017. In an alternative embodiment, the AAV-Sia is AAV2 comprising a sialidase transgene consisting of sequence SEQ ID NO 018. In an alternative embodiment, the AAV-Sia is AAV2 comprising a sialidase transgene consisting of the sequence SEQ ID NO 019. In an alternative embodiment, the AAV-Sia is AAV2 comprising a sialidase transgene consisting of the sequence SEQ ID NO 020.
In alternative embodiments, AAV-Sia is AAV2 having a transgene with a sequence identity of ≡85%, specifically ≡90%, more specifically ≡95% compared to SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014, SEQ ID NO 015, SEQ ID NO 016, SEQ ID NO 017, SEQ ID NO 018, SEQ ID NO 019 or SEQ ID NO 020, provided that the sialidase activity of the polypeptide encoded by the transgene has at least 90% biological activity of SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009 or SEQ ID NO 010.
In a specific embodiment, AAV-Sia is AAV2 carrying a transgene comprising the nucleic acid sequence SEQ ID NO 011. In a more specific embodiment, AAV-Sia is AAV2 comprising a neuraminidase transgene consisting of the sequence SEQ ID NO 011.
AAV can coat the positive or negative strand of a sialidase sequence with a capsid into its single-stranded DNA genome to generate a virion, and thus the invention also encompasses complementary nucleic acid sequences of those specified according to this aspect of AAV-Sia.
In certain embodiments, the sialidase transgene is located 3' of and is under the control of a promoter sequence operable in mammalian cells. In certain embodiments, the promoter sequence is operable in human cells, particularly in malignant cancer cells. In certain embodiments, the promoter is a ubiquitous promoter. In certain embodiments, the promoter is a cell-specific promoter. In certain embodiments, the promoter is a CMV immediate early promoter. In certain embodiments, the AAV-Sia comprises a nucleic acid sequence encoding an influenza a or B neuraminidase under the control of a CMV immediate early promoter having the nucleic acid sequence SEQ ID NO 021.
In alternative embodiments, AAV-Sia comprises a nucleic acid sequence encoding influenza a or B neuraminidase under the control of a human Ef1a promoter, since Ef1a has similar expression properties as the CMV promoter tested in the examples and is typically incorporated into commercially available replication defective AAV constructs.
The next aspect of the invention relates to an AAV vector comprising a transgene encoding an influenza a or B derived neuraminidase for use in medicine. In particular embodiments, AAV-Sia is used as a medicament for treating a patient diagnosed with cancer. In a more specific embodiment, AAV2 comprises a transgene encoding an N1 influenza neuraminidase polypeptide according to the above aspects.
In general, cancers are classified by the type of tissue from which they originate. Epithelial cell derived cancers, also known as malignant epithelial tumors, account for up to 90% of cancer cases. However, the immune response required against tumors derived from other types of solid tissue (also known as sarcomas) or tumors of mixed origin is similar. In one embodiment of this aspect of the invention, AAV-Sia as described above is provided to treat a form of solid cancer, e.g., a malignant epithelial tumor such as colon cancer, or a sarcoma such as melanoma. Particular embodiments relate to the use of AAV-Sia for treating a solid cancer selected from colon cancer, lung cancer, breast cancer or melanoma.
In certain embodiments, AAV-Sia is provided for use in intratumoral injection of a virus carrying a recombinant transgene is a cancer type of approved therapeutic regimen, including, for example, head and neck tumors, triple negative breast cancer, or cutaneous lymphomas.
In a specific embodiment, AAV-Sia for use in treating cancer according to the invention is used to treat a patient diagnosed with colon cancer, as simulated by the MC38 system.
In a specific embodiment, AAV-Sia for use in treating cancer according to the invention is used to treat a patient diagnosed with lung cancer, and effective desialylation is shown in example 7.
In a specific embodiment, AAV-Sia for use in treating cancer according to the invention is used to treat a patient diagnosed with breast cancer, as simulated by the transduced EMT6 cells in fig. 2. In a specific embodiment, AAV-Sia for use in treating cancer according to the invention is used to treat a patient diagnosed with triple negative breast cancer.
In a specific embodiment, AAV-Sia for use in treating cancer according to the invention is used to treat a patient diagnosed with melanoma, as simulated by the B16D5 cells and in vivo tumor models in fig. 2 and 3.
In a specific embodiment, AAV-Sia for use in treating cancer according to the invention is used to treat a patient diagnosed with cutaneous lymphoma.
In some embodiments, in particular embodiments, AAV-Sia for use in treating cancer according to the invention is used to treat a patient diagnosed with head and neck cancer.
In some embodiments, AAV-Sia is for use in treating a patient diagnosed with an epithelial cell derived tumor. In some embodiments, the cancer is pancreatic cancer. In an alternative embodiment, the cancer is prostate cancer.
In some embodiments, AAV-Sia is for use in a patient diagnosed with a cancer characterized by metastasis to a second location other than the primary tumor.
Particular embodiments relate to the use of AAV-Sia according to the invention for direct administration to a tumor, e.g. by infusion, by insertion of a micropump, or in a surgical procedure. In specific embodiments, the AAV-Sia is administered to a cancer patient by intratumoral injection. Thus, AAV-Sia according to this aspect of the invention can be reasonably expected to inhibit the growth of any type of solid tissue-derived cancer or malignancy, rather than, for example, diffuse blood cell-derived cancer.
The broad benefit of immunomodulatory effects of sialidases delivered by means of AAV vectors can be observed in efficacy when administered alone or in combination with the checkpoint inhibition forms shown in the examples (fig. 3).
Other embodiments of the invention relate to the use of AAV-Sia for treating cancer in a patient who has not been administered a checkpoint inhibitor within a medically relevant window of AAV-Sia use. In other words, AAV-Sia is administered to a patient without combination with an immunotherapeutic agent, such as a checkpoint inhibitor antibody, e.g., anti-PD-1 or anti-PD-L1.
In certain embodiments for use in treating an AAV-Sia for a patient diagnosed with cancer, the AAV-Sia is for use in a patient who has not yet been administered Chimeric Antigen Receptor (CAR) T cell therapy. In other words, AAV-Sia is administered to a patient without being combined with adoptive transfer or recombinant T cells specific for tumor antigens. In some embodiments, the AAV-Sia is for use in a patient who has not received CAR T cell therapy three months prior to AAV-Sia administration. In some embodiments, the AAV-Sia is for use in a patient that is not currently receiving CAR T cell therapy at the time of AAV-Sia administration. In some embodiments, the AAV-Sia is for use in a patient who is scheduled to receive CAR T cell therapy within three months of AAV-Sia administration.
In certain embodiments of AAV-Sia for use according to the invention as described in any aspect or embodiment described herein, the AAV-Sia is administered directly to the patient, without administration to a transgenic cell, such as a CAR-T cell, used to treat the patient. In particular embodiments, AAV-Sia can be administered to a patient after or concomitant with cell therapy. In certain more specific embodiments, AAV-Sia is administered to a patient who has not received cell therapy. The term cell therapy relates in particular to the administration of cells to a patient which are intended to target a tumor.
Combination therapy
An alternative embodiment relates to the treatment of cancer patients using AAV-Sia according to the preceding aspect of the invention in combination with an immunotherapy, in particular selected from anti-PD-1, anti-PD-L1 or anti-CTLA-4 checkpoint inhibitor antibodies.
In some embodiments, the AAV-Sia and the checkpoint inhibitor are delivered together as a combination drug administered directly into a solid tumor.
Other embodiments provide parenteral administration of AAV-Sia for intratumoral delivery and checkpoint immunotherapeutic agents. This encompasses the use of AAV-Sia to treat patients who have recently received, are currently receiving, or are scheduled to receive checkpoint inhibitors within a medically relevant window of AAV-Sia administration, e.g., within 2 months of AAV-Sia administration according to the invention.
Another aspect of the invention relates to a checkpoint inhibitor for use in the treatment of cancer, wherein the checkpoint inhibitor is provided for use with administration of an AAV-Sia according to any of the aspects of the invention provided above. In some embodiments, the patient is administered both agents simultaneously. In alternative embodiments, both are administered within a medically relevant window, e.g., within alternating weeks.
In particular embodiments, the immune checkpoint inhibitor is an inhibitor of the interaction of programmed cell death protein 1 (PD-1) with its receptor PD-L1. In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of the clinically available antibody drugs nivolumab (Bristol-Myers Squibb), CAS number 946414-94-4), palbociclizumab (Merck Inc.; CAS number 1374853-91-4), pittuzumab (CAS number 1036730-42-3), atilizumab (Roche control group (Roche), CAS number 1380723-44-3), and Avalumumab (Merck group (Merck KGaA), CAS number 1537032-82-8). In certain embodiments, the immune checkpoint inhibitor is ipilimumab (Yervoy; CAS number 477202-00-9).
In a specific embodiment, the checkpoint inhibitor is a non-agonist ligand for PD-1. In a more specific embodiment, the checkpoint inhibitor is a non-agonist antibody specific for PD-1.
Pharmaceutical treatment and dosage form
Similarly, methods of treating cancer in a patient in need thereof are within the scope of the invention, the methods comprising administering AAV-Sia to the patient, optionally also administering a checkpoint inhibitor according to the above description.
The term intratumoral administration according to the present specification refers to the provision of AAV-sialidases by direct administration into a solid tumor, or into a location near a tumor, or into a lymph node associated with a tumor. This may be achieved by a single injection or by intermittent or continuous infusion. In alternative embodiments, intratumoral administration is performed with surgical intervention, such as direct administration of AAV-Sia in solution to a biopsy or tumor resection site.
In certain embodiments, the checkpoint inhibitor is an antibody, antibody fragment, antibody-like molecule, or protein a domain-derived polypeptide. In some embodiments, the checkpoint inhibitor is an immunoglobulin consisting of two heavy chains and two light chains. In some embodiments, the checkpoint inhibitor is a single domain antibody that consists of an isolated variable domain from a heavy chain or a light chain. In some embodiments, the checkpoint inhibitor is a heavy chain antibody consisting of heavy chains only, such as antibodies found in camelids.
In certain embodiments, the checkpoint inhibitor is an antibody fragment. In certain embodiments, the checkpoint inhibitor is a Fab fragment, i.e., an antigen-binding fragment of an antibody, or a single chain variable fragment, i.e., a fusion protein of the heavy chain variable region and the light chain variable region of an antibody linked by a peptide linker.
Similarly, a dosage form for treating cancer or preventing cancer recurrence is provided comprising a non-agonist ligand of a checkpoint inhibitor molecule according to any of the above aspects or embodiments of the present invention.
Formulations for parenteral administration of checkpoint inhibitors according to the present invention may be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection formulations. Optionally, pharmaceutically acceptable carriers and/or excipients may be present.
Topical administration of an AAV-Sia or immune checkpoint inhibitor of the invention is also within the scope of the intratumoral use of the invention in connection with direct application to a skin cancer or skin lymphoma form. Those skilled in the art are aware of a wide range of possible formulations for providing topical formulations, as exemplified by the following: benson and Watkinson (editors), "topical and transdermal drug delivery: principles and practices (Topical and Transdermal Drug Delivery: principles and Practice) (1 st edition, wiley publishing company (Wiley) 2011, ISBN-13:978-0470450291); guy and Handcraft: transdermal drug delivery system: revisions and extensions (Transdermal Drug Delivery Systems: revised and Expanded) (version 2, CRC Press 2002, ISBN-13:978-0824708610); osborne and Amann (editors): local drug delivery formulation (Topical Drug Delivery Formulations) (1 st edition, CRC Press, 1989; ISBN-13:978-0824781835).
Pharmaceutical composition and administration
Another aspect of the invention relates to a pharmaceutical composition comprising an AAV-Sia according to any aspect of the invention related thereto. In particular embodiments, the composition is formulated for topical application to a tumor, or the immediate surroundings of a tumor, or a tumor draining lymph node. In particular embodiments, the compositions comprising AAV-Sia are formulated for intratumoral injection. In other embodiments, the composition comprises an AAV-Sia of the invention together with an immune checkpoint inhibitor, and a pharmaceutically acceptable carrier. In further embodiments, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.
In certain embodiments of the invention, the inventive AAV-Sia or immune checkpoint inhibitor of the invention is typically formulated into a pharmaceutical dosage form to provide an easily controlled drug dosage and to give the patient an elegant and easy-to-use product.
In embodiments of the invention involving topical use of an AAV-Sia or immune checkpoint inhibitor of the invention, the pharmaceutical composition comprising the active ingredient together with one or more solubilizing agents, stabilizers, tonicity enhancing agents, buffers and preservatives known to those skilled in the art is formulated in a manner suitable for topical administration, such as aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations for delivery, e.g., by aerosol or the like.
Pharmaceutical compositions comprising immune checkpoint inhibitors may be formulated for parenteral administration, for example by intravenous (i.v.) injection or infusion, intraperitoneal (i.p.), intradermal, subcutaneous or intramuscular administration.
The dosing regimen of the immune checkpoint inhibitor of the invention will vary depending on known factors such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, physical condition, health condition and weight of the recipient; the nature and extent of the symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the desired effect. In certain embodiments, the compounds of the present invention may be administered in a single daily dose, or the total daily dose may be administered in divided doses of two, three or four times a day.
In certain embodiments, for a subject of about 50 to 70kg, the pharmaceutical composition or combination of the invention may be a unit dose of about 1 to 1000mg of immune checkpoint inhibitor. Dosages for the number of AAV-Sia virus particles can be extrapolated from the animal model used in the examples or by similar therapeutic AAV vectors known in the art. The therapeutically effective dose of the virus, pharmaceutical composition or combination thereof will depend on the weight, age and individual condition, disorder or disease or severity thereof being treated. A physician or clinician of ordinary skill can readily determine the effective amount of each active ingredient necessary to prevent, treat or inhibit the progression of the condition or disease.
The pharmaceutical compositions of the present invention may be subjected to conventional pharmaceutical procedures such as sterilization, and/or may contain conventional inert diluents, lubricants or buffers, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifying agents and buffers and the like. They may be produced by standard processes, for example by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see, for example, l.lachman et al, theory and practice of industrial pharmacy (The Theory and Practice of Industrial Pharmacy), 4 th edition, 2013 (ISBN 8123922892).
Manufacturing method and treatment method according to the invention
As an additional aspect, the invention further encompasses the use of AAV-Sia and optionally an additional immune checkpoint inhibitor ligand according to aspects of the invention identified herein for use in a method of manufacturing a medicament for treating a solid tumor or preventing recurrence of a solid tumor.
Similarly, the invention encompasses methods of treating patients who have been diagnosed with a disease associated with a tissue-derived solid tumor. The method entails administering to a patient an effective amount of an AAV-Sia as identified herein, in particular by intratumoral administration, and optionally concurrently with, shortly after, or shortly before receiving an immune checkpoint inhibitor antibody as detailed herein, by parenteral administration within a medically relevant window.
Where alternatives to individual separable features, such as strains of viruses, sialidase sequences, checkpoint inhibitors, or medical indications are listed herein as "embodiments," for example, it is to be understood that such alternatives can be freely combined to form discrete embodiments of the invention disclosed herein. Thus, any alternative embodiment of a viral vector may be combined with any alternative embodiment of a checkpoint inhibitor, and these combinations may be combined with any of the medical indications mentioned herein.
The invention also relates to the following:
A. an adeno-associated virus (AAV-Sia) comprising a transgene encoding a sialidase derived from influenza virus.
B. The AAV-Sia of item a, wherein the sialidase is a neuraminidase selected from the group consisting of:
a.A influenza virus-derived neuraminidase, in particular N1, N2, N3, N4, N5, N6, N7, N8, N9, N10 or N11 neuraminidase; or alternatively
b.B type Victoria or mountain line influenza-derived neuraminidase;
in particular, wherein the sialidase is N1 neuraminidase.
C. The AAV-Sia of item a or B, wherein the sialidase comprises or consists of a sialidase polypeptide sequence selected from SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010;
Or the sialidase comprises or consists of a polypeptide sequence having greater than or equal to 85%, specifically greater than or equal to 90%, more specifically greater than or equal to 95% identity to the sialidase polypeptide sequence, wherein the polypeptide sequence has at least 90% of the biological activity of the sialidase polypeptide sequence;
in particular, wherein the sialidase comprises or consists of the polypeptide sequence SEQ ID NO 001.
D. The AAV-Sia according to any one of items a-C, wherein the transgene encoding a sialidase comprises or consists of a nucleic acid sequence encoding a sialidase polypeptide sequence selected from the group consisting of SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010;
or comprises or consists of a polypeptide sequence having an identity of more than or equal to 85%, specifically more than or equal to 90%, more specifically more than or equal to 95% to the sialidase polypeptide sequence, wherein the polypeptide sequence has at least 90% of the biological activity of the sialidase polypeptide sequence;
in particular, wherein the transgene comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014, SEQ ID NO 015, SEQ ID NO 016, SEQ ID NO 017, SEQ ID NO 018, SEQ ID NO 019, SEQ ID NO 020;
More specifically, wherein the transgene comprises or consists of the nucleic acid sequence SEQ ID NO 011.
E. The AAV-Sia of any one of claims a to D, wherein the transgene is comprised within a viral expression element comprising the transgene operably linked to a promoter sequence that confers transgene expression in mammalian cells, particularly wherein the promoter sequence comprises or consists of a cytomegalovirus promoter, more particularly wherein the promoter sequence comprises or consists of the nucleic acid sequence SEQ ID NO 021.
F. The AAV-Sia according to any one of items a to E, wherein the recombinant adeno-associated virus is a replication-defective recombinant adeno-associated virus, in particular a replication-defective adeno-associated virus type 2.
G. The AAV-Sia according to any one of items a to F, for use in treating cancer or preventing cancer recurrence in a patient in need thereof.
H. The AAV-Sia according to item G, wherein the cancer is a solid cancer, in particular a solid cancer selected from colon cancer, lung cancer, breast cancer and melanoma.
I. AAV-Sia according to clause G or H, wherein the AAV-Sia is administered directly into a tumor, particularly wherein the AAV-Sia is administered by intratumoral injection.
J. AAV-Sia according to clauses G-I, wherein the patient is scheduled to receive, is currently receiving, or has recently been administered a checkpoint inhibitor.
K. The AAV-Sia of item J, wherein the checkpoint inhibitor is an antibody specific for a checkpoint molecule selected from PD-1 or PD-L1, more specifically an antibody targeting PD-1.
An AAV-Sia according to clause J or K, wherein the checkpoint inhibitor is administered parenterally.
A pharmaceutical composition for use in treating cancer or preventing cancer recurrence in a patient in need thereof, comprising:
a. AAV-Sia according to any one of items a to I, and optionally
b. Checkpoint inhibitors, particularly PD-1 or PD-L1 ligands, more particularly PD-1 ligands.
A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the AAV-Sia of any one of items a-L or the pharmaceutical composition of item M by intratumoral injection.
Further embodiments and advantages may be obtained by further illustrating the invention by the following examples and figures. These examples are intended to illustrate the invention and not to limit its scope.
Drawings
Table 1 lists representative sialidase protein sequences and representative genomic RNA sialidase sequences isolated from humans, birds and Ma Suzhu.
FIG. 1 shows the design of AAV2-Sia constructs. AAV2 viruses were produced by co-transfecting HEK293 cells with AAV plasmids containing neuraminidase sequences and helper plasmids. Neuraminidase (sialidase) is obtained from influenza a H1N1 virus, with a constitutive CMV promoter. In AAV plasmids, the REP and CAP genes of wild-type AAV are deleted, leaving 2 Inverted Terminal Repeat (ITR) copies.
FIG. 2 shows the in vitro desialylation of AAV-Sia tumor cells. Tumor cell lines A B D5, BMC 38, C EMT6 and D sea Lai 10 4 Individual cells/wells were seeded in 96-well plates (n-1). After addition of AAV-Sia at the indicated concentrations/cell (multiplicity of infection, MOI), 72 hours, the cells were isolated with trypsin and stained with PNA (peanut lectin) bound to the desialylated glycans or MAA II (Maackia amurensis) and SNA bound to sialic acid residues in the cell surface. PNA, MAAII or SNA (American elderberry (Sucumbus Nigra)) average fluorescence intensity (MFI) was obtained using flow cytometry.
Figure 3 shows that AAV2-Sia inhibits tumor growth and enhances checkpoint inhibitor treatment. MC38 or B16D5 cells were subcutaneously injected into the flank of C57Bl6 mice. Tumor growth was measured 3 times per week until about 1500mm was reached 3 Ethical endpoint tumor volume of (c). When the tumor reaches about 50mm 3 At the beginning of AAV2-Sia treatment or control AAV2-null (AAV 2-null) treatment, 4 doses (10) were administered every 2 to 3 days 10 GC), diluted in 50 μl PBS and injected intratumorally (i.t.). The a-PD1 antibody was also used as a combination therapy at four doses (10 mg/kg, i.p.). a-PD1 treatment starts with a second dose of AAV, once every 3 to 4 days. Tumor growth a and survival B in MC38 colon cancer mouse model: AAV2-Sia (n-6), AAV2-null (n-5), AAV2-Sia+a-PD1 (n-5), AAV 2-null+a-PD 1 (n-6), a-PD1 (n-5), and no treatment (n-5). C tumor growth and D survival in the acd-1 resistant B16D5 melanoma mouse model: AAV2-Sia (n-10), AAV2-null (n-11), AAV2-Sia+a-PD1 (n-4), AAV 2-null+a-PD 1 (n-4), and no treatment (n-9). Results are expressed as mean ± SEM. P-values were calculated using a two-way Anova (Bonferroni Test). For survival curves, p-values were calculated using the Gehan-Breslow-Wilcoxon test. * P < 0.05, P < 0.01 and P < 0.001.
Figure 4 shows the growth of AAV-Sia virus against untreated distant tumors. AAV-Sia or AAV2-null controls were intratumorally injected into subcutaneously transplanted MC38 tumors (treated) while contralateral tumors (distant) were untreated. Direct injection of AAV2-Sia into tumors at both ipsilateral (fig. 4A) and contralateral (fig. 4B) tumor sites was demonstrated to have the expected anti-tumor effect compared to AAV2-null (vector) control animals.
Figure 5 shows the effect of specific tumor cell-specific sialidases on in vitro AAV-Sia transduction of primary non-small cell lung cancer (NSCLC) tumor samples from 3 patients. The isolated primary tumor cells were treated with 2 different concentrations of AAV2-Sia virus (10 4 Or 10 5 Virus/cell) transduction for 5 days. PNA levels were measured using flow cytometry analysis, showing that cancer cells within the CD45 negative region (FIG. 5A) were effectively desialylated after treatment, while CD45+ cells (immune cells) were unaffected (fig. 5B).
Examples
Example 1: method of
Virus constructs
The influenza-derived neuraminidase 1 polypeptide is inserted into an AAV serotype 2 (AAV 2) virus, which comprises capsids and ITRs from AAV2, as shown in fig. 1. All recombinant AAV viruses and clones were performed by Vector BioLabs (production lot number: 190715#45, purchase order number: HL_2019_02). Briefly, HEK293 cells were co-transfected with AAV plasmids carrying a gene of interest (GOI) and replication helper plasmid DNA comprising AAV2 capsids and replication genes. The selected GOI is neuraminidase 1 (type a H1N1 influenza virus derived N1, SEQ ID NO 011, reference sequence No. NC 026434.1) under the control of CMV (promoter SEQ ID NO 21) (fig. 1). In the AAV plasmid, REP and CAP genes of wild-type AAV were deleted, leaving 2 ITR copies (about 145 bp/each). AAV 2-null control vectors were prepared in tandem (Vector BioLabs # 7026). 2 days after transfection, HEK293 cell pellet was harvested and virus was released by 3 freeze/thaw cycles. The virus was purified by CsCl-gradient ultracentrifugation, followed by desalting. Viral titers (GC/ml-genome copy number/ml) were determined by real-time PCR. The purity of AAV proteins was analyzed using SDS-gel silver staining, and only those AAV preparations with > 90% purity were used in the experiments.
Virus stock solution
The virus stock was stored at-80 ℃ avoiding repeated freeze-thaw cycles. Viral stocks were diluted in PBS 5% glycerol and in cell culture medium for in vitro experiments or PBS for in vivo treatment.
Cell culture
At 37℃and 5% CO 2 The tumor cell lines were cultured in DMEM (darbek modified eagle medium, sigma aldrich) supplemented with 1% glutamine, 1% pyruvate, 1% non-essential amino acids, streptomycin penicillin (5000U/ml) and 10% fetal bovine serum (sigma aldrich), 25mM glucose. B16D5, EMT6, MC38 and sea Law cell lines were grown at 10 4 Individual cells/well were seeded in 6-well plates (n-1). AAV-Sia was added at various concentrations/cell (multiplicity of infection, MOI), and after 72 hours the cells were isolated with trypsin and stained with PNA (peanut lectin, vector Biolabs).
Flow cytometry
Following AAV2-Sia infection, cell lines were isolated and stained with biotinylated PNA (10. Mu.g/ml), MAAII or SNA for 30 min, washed 2 times with PBS and incubated with streptavidin-PE for 30 min (1:500, BD biosciences). Cells were washed twice and fixed with IC fixation buffer (sameimer). Acquisition was performed using a Fortessa LSR II flow cytometer (BD bioscience). Analysis was performed using Flowjo and Prism software (Graphpad).
Animal experiment
The mouse experiments were approved by the local ethics committee (bazier city, switzerland). In vivo experiments were performed using 8 to 10 week old male C57Bl/6 mice. MC 38-or B16D 5-containing cell suspensions (5X 10) 5 ) 200 μl of PBS was injected subcutaneously into the flank of the mice. Tumor growth and overall health were monitored 3 times per week until the tumor reached about 1500mm 3 Maximum size (late stage tumor). When the tumor reaches about 50mm 3 At the beginning of AAV2-Sia treatment, 4 doses (10) were administered every 3 to 4 days 9 GC). Blank vector was used as control (AAV 2-null). All viruses were injected intratumorally (i.t.), diluted in 50 μl PBS. In the experiments shown, anti-PD 1 antibodies, clone RMP1-14 (BioXcell) were also used as combination therapy at four doses (10 mg/kg, i.p.). a-PD1 treatment was also initiated every 3 to 4 days with a second dose of AAV.
Primary tumor cells
The original samples were obtained from a university hospital chest surgery department authorized by the local ethics committee (EKNZ 2018-01990). To prepare single cell suspensions, tumors were collected, surgical samples were mechanically dissociated, followed by digestion with cell digests (accutase) (PAAs laboratory, germany), collagenase type IV (wogonington, USA), hyaluronidase (Sigma, USA) and DNase type IV (Sigma, USA) for 1 hour at 37 ℃ with constant stirring. Digested primary tumors were grown in 24 well plates at 5X 10 4 The individual cells/wells were incubated overnight and samples were incubated on the same day with AAV-Sia (10 4 Or 10 5 Individual viruses/cells) for 5 days. Adherent and non-adherent cells were collected on day 5 and stained with biotinylated PNA for analysis by flow cytometry as above.
Example 2: AAV2-NA design
Adeno-associated virus (AAV 2-sia) is engineered such that it can produce sialidases when transduced into mammalian cells. The influenza neuraminidase N1 gene (SEQ ID NO 011, encoding SEQ ID NO 001) is amplified/synthesized and inserted by ligation into a commercial replication defective AAV serotype 2 virus in an expression cassette under the control of a constitutive CMV promoter (fig. 1). Vectors lacking the N1 gene insert were used as controls (AAV 2-null).
Example 3: AAV2-NA removal of SA in vitro
In vitro analysis of tumor cell infection was performed. To determine whether AAV-Sia induced functional expression of sialidases in transduced cells, the level of desialylation was analyzed by flow cytometry and direct PNA staining to test the enzymatic activity, i.e. desialylation of in vitro transduced tumor cells. This demonstrates that upon increased exposure to AAV2-SIa, AAV2-SIa induced desialylation of tumor cells, as shown by increased lectin PNA (peanut lectin) binding to desialylated glycan residues (fig. 2). Sialylation was also performed using lectins MAA II (Maackia amurensis) and SNA (Sambucus Nigra) that bind directly to sialic acid residues, and transduced cells continued to decrease binding (fig. 2).
Example 4: AAV2-Sia inhibition of tumor growth in preclinical mouse models
The efficacy of these AAV2-Sia viruses was tested by intratumoral administration of the virus into subcutaneously transplanted BI6D5 melanoma and MC38 colon cancer derived tumors. AAV2-Sia virus was applied to a syngeneic tumor model of tumor cells subcutaneously implanted in C57Bl6 mice, where anti-tumor effects were observed by comparing AAV2-Sia to AAV 2-null treatment (FIG. 3).
Example 5: AAV2-Sia has additional benefits for checkpoint inhibitor treatment
In the MC38 and B15D5 models, combination therapies of AAV2-Sia and a-PD1 antibodies were tested by co-administering the a-PD1 antibody after a second dose of AAV 2-Sia. This combination showed additional benefit in the responsive MC38 tumor model, where reduced tumor growth rate and increased survival of AAV2-Sia versus AAV 2-sia+agd1 were observed compared to the control AAV 2-null group and AAV 2-null+a-PD 1 group (fig. 3). On day 35, AAV-Sia treated animals showed a significant 50% survival advantage compared to anti-PD-1 treatment alone. Unexpected synergistic survival advantages are conferred by treatment with a combination of both AAV-Sia and a non-agonist anti-PD-1 antibody according to the invention. After day 40, approximately 80% of animals receiving systemic injection of anti-PD 1 antibodies in combination with topical AAV-Sia administration were alive, while none of the animals survived in the groups receiving either treatment alone. Melanoma B16 tumors, a cancer model that is more resistant to immunotherapy, showed no significant decrease in tumor burden upon immune checkpoint blockade when paired with AAV 2-null. However, AAV2-Sia driven a small but significant decrease in tumor growth rate, which was enhanced by the addition of the aPD-1 treatment, indicating that AAV2-Sia induced susceptibility to immunotherapy-resistant cancers. Combination treatment also conferred synergistic survival advantage on day 34 after implantation of the treatment resistant tumor, with 50% of animals surviving to this time point compared to only 20% in the control group, and no survival in the anti-PD 1 treated group.
Example 6: AAV2-Sia limits distal tumor growth
Intratumoral administration of AAV-Sia was demonstrated above to deliver local effects at tumors receiving injections. The inventors then interrogated whether AAV-Sia, which has the potential to eliminate the growth of secondary, metastatic tumors at distant sites within the host, activates systemic adaptive immunity. AAV2-Sia virus was tested for efficacy in untreated distant tumors by intratumoral administration of the virus into subcutaneously transplanted MC38 tumors (treated), whereas contralateral tumors (distant) were untreated. In comparison to AAV 2-null (vector) control animals (fig. 4A), direct injection of AAV2-Sia into tumors was demonstrated to have the expected anti-tumor effect. Importantly, growth of contralateral tumors that did not directly receive virus was also inhibited, indicating that the systemic anti-tumor immune response generated by local AAV-Sia administration could combat tumor growth in the whole host to control metastatic cancer (fig. 4B).
Example 7: AAV-Sia Security features
Immune cells have been demonstrated to mediate local and systemic protection against tumors in AAV-Sia treated animals, and researchers considered what effect the sialidase expression of virus-infected cells might have on immune cells. T cells are an important component of anti-tumor immune responses, particularly in patients receiving checkpoint inhibition. As with tumor cells, the membrane lipids of T cells are modified with glycans containing terminal sialic acid residues, and such residues are involved in almost every aspect of T cell fate and function, from cell maturation, differentiation, and migration to cell survival and cell death. Ideally, once an AAV-Sia of a primary tumor has a limited enzymatic effect on bystander cells, such as infiltrating immune cells, an AAV-Sia composition for use in treating cancer will induce specific desialylation of the cancer cells so as not to interfere with protective immune cell migration and effector function acquisition. To assess any bystander effect of intratumoral expressed sialidases AAV-Sia was used for in vitro infection of mixed cell cultures of cd45+ immune cells and CD 45-cancer cells obtained from tissue samples derived from digested tumor samples of 3 different lung cancer patients using lectin PNA. Primary tumor cells were treated with 2 different concentrations of AAV2-Sia virus (10 4 Or 10 5 Virus/cell) transduction for 5 days. Flow cytometry analysis of PNA levels after viral transduction demonstrated that cancer cells within the CD45 negative compartment (fig. 5A) underwent effective desialylation after treatment, whereas cd45+ cells (immune cells) were unaffected (fig. 5B). Thus, AAV-Sia exhibits high levels of sialidase specificity for tumor cells and has less immunity in mixed primary cell populations obtained from patient samplesCell action. In the case of cancer, this specific effect makes AAV-Sia a desirable alternative to other forms of sialidase administration due to the lack of bystander effects on the recruitment and effector functions of local protective immune cell responses.
Neuraminidase Database for storing data SEQ proteins SEQ ID DNA
Influenza A H1N1 Uniprot:C3W6G3 001 011
Influenza A H3N2 Reference sequence: NC_007368.1 002 012
Influenza A H7N3 Uniprot:P03476 003 013
Influenza A H8N4 Uniprot:P03477 004 014
Influenza A H6N5 Uniprot:P03478 005 015
H11N6 influenza Uniprot:Q6XV27 006 016
H7N7 influenza Uniprot:P88838 007 017
H3N8 influenza Uniprot:Q07599 008 018
H11N9 influenza Uniprot:P03472 009 019
Influenza B Reference sequence: NC_002209.1 010 020
Table 1 shows representative sialidase protein sequences and representative genomic RNA sialidase sequences isolated from humans, birds and Ma Suzhu.
Sequence listing
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Asn Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Gly Thr Asp
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Asn Asn Phe Ser Ile Lys Gln Asp Ile Val Gly Ile Asn Glu Trp Ser
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Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly
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Cys Val Thr Gly Asp Asp Lys Asn Ala Thr Ala Ser Phe Ile Tyr Asn
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Gly Arg Leu Val Asp Ser Ile Val Ser Trp Ser Lys Lys Ile Leu Arg
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Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val
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Met Thr Asp Gly Ser Ala Ser Gly Lys Ala Asp Thr Lys Ile Leu Phe
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Ile Glu Glu Gly Lys Ile Ile His Thr Ser Thr Leu Ser Gly Ser Ala
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Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg
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Cys Val Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Ile Val Asp
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Ile Asn Ile Lys Asp Tyr Ser Ile Val Ser Ser Tyr Val Cys Ser Gly
305 310 315 320
Leu Val Gly Asp Thr Pro Arg Lys Asn Asp Ser Ser Ser Ser Ser His
325 330 335
Cys Leu Asp Pro Asn Asn Glu Glu Gly Gly His Gly Val Lys Gly Trp
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Ala Phe Asp Asp Gly Asn Asp Val Trp Met Gly Arg Thr Ile Ser Glu
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Lys Leu Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Glu Gly Trp Ser
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Lys Pro Asn Ser Lys Leu Gln Ile Asn Arg Gln Val Ile Val Asp Arg
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Gly Asn Arg Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser
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Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Val Val Asn Thr Thr
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Thr Val Ile His Glu Lys Ile Gly Asn His Gln Thr Val Ile His Pro
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Thr Ile Thr Thr Pro Ala Val Pro Asn Cys Ser Asp Thr Ile Ile Thr
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Tyr Asn Asn Thr Val Ile Asn Asn Ile Thr Thr Thr Ile Ile Thr Glu
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Phe Phe Pro Phe His Lys Asp Asn Ala Ile Arg Leu Gly Glu Asn Lys
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Ala Val Thr Asp Gly Pro Ala Ala Asn Ser Ala Asp His Arg Val Tyr
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Trp Ile Arg Glu Gly Arg Ile Val Lys Tyr Glu Asn Val Pro Lys Thr
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Arg Ile Asn Asn Glu Thr Ile Leu Glu Thr Gly Tyr Val Cys Ser Lys
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Asp Ser Pro Ser Asn Ile Asn Gly Gly Pro Gly Val Lys Gly Phe Gly
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Gly Ile Ile Thr Asp Thr Leu Lys Ser Trp Lys Gly Asn Ile Met Arg
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Thr Gln Glu Ser Glu Cys Val Cys Gln Asp Glu Phe Cys Tyr Thr Leu
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Ile Thr Asp Gly Pro Ser Asn Ala Gln Ala Phe Tyr Lys Ile Leu Lys
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Ile Arg Lys Gly Lys Ile Val Ser Val Lys Asp Val Asn Ala Thr Gly
260 265 270
Phe His Phe Glu Glu Cys Ser Cys Tyr Pro Ser Gly Thr Asp Val Glu
275 280 285
Cys Val Cys Arg Asp Asn Trp Arg Gly Ser Asn Arg Pro Trp Ile Arg
290 295 300
Phe Asn Ser Asp Leu Asp Tyr Gln Ile Gly Tyr Val Cys Ser Gly Ile
305 310 315 320
Phe Gly Asp Asn Pro Arg Pro Val Asp Gly Ile Gly Ser Cys Asn Ser
325 330 335
Pro Val Asn Asn Gly Lys Gly Arg Tyr Gly Val Lys Gly Phe Ser Phe
340 345 350
Arg Tyr Gly Asp Gly Val Trp Ile Gly Arg Thr Lys Ser Leu Glu Ser
355 360 365
Arg Ser Gly Phe Glu Met Val Trp Asp Ala Asn Gly Trp Val Ser Thr
370 375 380
Asp Lys Asp Ser Asn Gly Val Gln Asp Ile Ile Asp Asn Asn Asn Trp
385 390 395 400
Ser Gly Tyr Ser Gly Ser Phe Ser Ile Arg Trp Glu Thr Thr Gly Arg
405 410 415
Asn Cys Thr Val Pro Cys Phe Trp Val Glu Met Ile Arg Gly Gln Pro
420 425 430
Lys Glu Lys Thr Ile Trp Thr Ser Gly Ser Ser Ile Ala Phe Cys Gly
435 440 445
Val Asn Ser Asp Thr Thr Gly Trp Ser Trp Pro Asp Gly Ala Leu Leu
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Pro Phe Asp Ile Asp Lys
465 470
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Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ala Ser Leu Gly
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Ile Ile Ser Val Thr Lys Asp Asn Lys Val His Ile Cys Asn Thr Thr
35 40 45
Glu Val Tyr Asn Glu Thr Val Arg Val Glu Thr Val Val Ile Pro Val
50 55 60
Asn Asn Thr Ile Tyr Leu Asn His Glu Pro Glu Phe Leu Asn Asn Thr
65 70 75 80
Glu Pro Leu Cys Asp Val Ser Gly Phe Ala Ile Val Ser Lys Asp Asn
85 90 95
Gly Ile Arg Ile Gly Ser Arg Gly His Ile Phe Val Ile Arg Glu Pro
100 105 110
Phe Val Ser Cys Gly Pro Ser Glu Cys Arg Thr Phe Phe Leu Thr Gln
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Gly Ala Leu Leu Asn Asp Lys His Ser Asn Asn Thr Val Lys Asp Arg
130 135 140
Ser Pro Tyr Arg Ala Leu Met Ser Val Pro Leu Gly Ser Ser Pro Asn
145 150 155 160
Ala Tyr Gln Ala Lys Phe Glu Ser Val Gly Trp Ser Ala Thr Ala Cys
165 170 175
His Asp Gly Lys Lys Trp Met Ala Ile Gly Val Ser Gly Ala Asp Asp
180 185 190
Asp Ala Tyr Ala Val Ile His Tyr Gly Gly Val Pro Thr Asp Val Ile
195 200 205
Arg Ser Trp Arg Lys Gln Ile Leu Arg Thr Gln Glu Ser Ser Cys Val
210 215 220
Cys Ile Lys Gly Glu Cys Tyr Trp Val Met Thr Asp Gly Pro Ala Asn
225 230 235 240
Asn Gln Ala Ser Tyr Lys Ile Phe Lys Ser Gln Lys Gly Met Val Val
245 250 255
Asp Glu Lys Glu Ile Ser Phe Gln Gly Gly His Ile Glu Glu Cys Ser
260 265 270
Cys Tyr Pro Asn Met Gly Lys Val Glu Cys Val Cys Arg Asp Asn Trp
275 280 285
Asn Gly Met Asn Arg Pro Ile Leu Ile Phe Asp Glu Lys Leu Glu Tyr
290 295 300
Glu Val Gly Tyr Leu Cys Ala Gly Ile Pro Thr Asp Thr Pro Arg Val
305 310 315 320
Gln Asp Ser Ser Phe Thr Gly Ser Cys Thr Asn Ala Val Gly Arg Ser
325 330 335
Gly Thr Asn Asn Tyr Gly Val Lys Gly Phe Gly Phe Arg Gln Gly Asn
340 345 350
Ser Val Trp Ala Gly Arg Thr Ile Ser Val Ser Ser Arg Ser Gly Phe
355 360 365
Glu Val Leu Leu Ile Glu Asp Gly Trp Ile Arg Pro Ser Lys Thr Ile
370 375 380
Ser Lys Lys Val Glu Val Leu Asn Asn Lys Asn Trp Ser Gly Tyr Ser
385 390 395 400
Gly Ala Phe Thr Ile Pro Thr Ala Met Thr Ser Lys Asn Cys Ile Val
405 410 415
Pro Cys Phe Trp Leu Glu Met Ile Arg Gly Lys Pro Glu Glu Arg Thr
420 425 430
Ser Ile Trp Thr Ser Ser Ser Ser Thr Val Phe Cys Gly Val Ser Ser
435 440 445
Glu Val Pro Gly Trp Ser Trp Asp Asp Gly Ala Ile Leu Pro Phe Asp
450 455 460
Ile Asp Lys Met
465
<210> 6
<211> 470
<212> PRT
<213> influenza A virus
<400> 6
Met Asn Pro Asn Gln Lys Ile Ile Cys Ile Ser Ala Thr Gly Met Thr
1 5 10 15
Leu Ser Val Val Ser Leu Leu Val Gly Ile Ala Asn Leu Gly Leu Asn
20 25 30
Ile Gly Leu His Tyr Lys Val Gly Asp Thr Pro Asn Val Asn Ile Pro
35 40 45
Asn Val Asn Gly Thr Asn Ser Thr Thr Thr Ile Ile Asn Asn Asn Thr
50 55 60
Gln Asn Asn Phe Thr Asn Ile Thr Asn Ile Ile Gln Ser Lys Gly Gly
65 70 75 80
Glu Arg Thr Phe Leu Asn Leu Thr Lys Pro Leu Cys Glu Val Asn Ser
85 90 95
Trp His Ile Leu Ser Lys Asp Asn Ala Ile Arg Ile Gly Glu Asp Ala
100 105 110
His Ile Leu Val Thr Arg Glu Pro Tyr Leu Ser Cys Asp Pro Gln Gly
115 120 125
Cys Arg Met Phe Ala Leu Ser Gln Gly Thr Thr Leu Arg Gly Arg His
130 135 140
Ala Asn Gly Thr Ile His Asp Arg Ser Pro Phe Arg Ala Leu Ile Ser
145 150 155 160
Trp Glu Met Gly Gln Ala Pro Ser Pro Tyr Asn Thr Arg Val Glu Cys
165 170 175
Ile Gly Trp Ser Ser Thr Ser Cys His Asp Gly Met Ser Arg Met Ser
180 185 190
Ile Cys Met Ser Gly Pro Asn Asn Asn Ala Ser Ala Val Val Trp Tyr
195 200 205
Gly Gly Arg Pro Ile Thr Glu Ile Pro Ser Trp Ala Gly Asn Ile Leu
210 215 220
Arg Thr Gln Glu Ser Glu Cys Val Cys His Lys Gly Val Cys Pro Val
225 230 235 240
Val Met Thr Asp Gly Pro Ala Asn Asn Arg Ala Ala Thr Lys Ile Ile
245 250 255
Tyr Phe Lys Glu Gly Lys Ile Gln Lys Ile Glu Glu Leu Ala Gly Asn
260 265 270
Ala Gln His Ile Glu Glu Cys Ser Cys Tyr Gly Ala Gly Gly Val Ile
275 280 285
Lys Cys Ile Cys Arg Asp Asn Trp Lys Gly Ala Asn Arg Pro Val Ile
290 295 300
Thr Ile Asp Pro Glu Met Met Thr His Thr Ser Lys Tyr Leu Cys Ser
305 310 315 320
Lys Val Leu Thr Asp Thr Ser Arg Pro Asn Asp Pro Thr Asn Gly Asn
325 330 335
Cys Asp Ala Pro Ile Thr Gly Gly Ser Pro Asp Pro Gly Val Lys Gly
340 345 350
Phe Ala Phe Leu Asp Gly Glu Asn Ser Trp Leu Gly Arg Thr Ile Ser
355 360 365
Lys Asp Ser Arg Ser Gly Tyr Glu Met Leu Lys Val Pro Asn Ala Glu
370 375 380
Thr Asp Ile Gln Ser Gly Pro Ile Ser Asn Gln Val Ile Val Asn Asn
385 390 395 400
Gln Asn Trp Ser Gly Tyr Ser Gly Ala Phe Ile Asp Tyr Trp Ala Asn
405 410 415
Lys Glu Cys Phe Asn Pro Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg
420 425 430
Pro Lys Glu Ser Ser Val Leu Trp Thr Ser Asn Ser Ile Val Ala Leu
435 440 445
Cys Gly Ser Lys Lys Arg Leu Gly Ser Trp Ser Trp His Asp Gly Ala
450 455 460
Glu Ile Ile Tyr Phe Glu
465 470
<210> 7
<211> 469
<212> PRT
<213> influenza A virus
<400> 7
Met Asn Pro Asn Gln Lys Leu Phe Ala Ser Ser Gly Ile Ala Ile Ala
1 5 10 15
Leu Gly Ile Ile Asn Leu Leu Ile Gly Ile Ser Asn Met Ser Leu Asn
20 25 30
Ile Ser Leu Tyr Ser Lys Gly Glu Asn His Lys Ser Asp Asn Leu Thr
35 40 45
Cys Thr Asn Ile Asn Gln Asn Asn Thr Thr Met Val Asn Thr Tyr Ile
50 55 60
Asn Asn Thr Thr Ile Ile Asp Lys Asn Thr Lys Met Glu Asn Pro Gly
65 70 75 80
Tyr Leu Leu Leu Asn Lys Ser Leu Cys Asn Val Glu Gly Trp Val Val
85 90 95
Ile Ala Lys Asp Asn Ala Ile Arg Phe Gly Glu Ser Glu Gln Ile Ile
100 105 110
Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Leu Ser Cys Lys Met
115 120 125
Tyr Ala Leu His Gln Gly Thr Thr Ile Arg Asn Lys His Ser Asn Gly
130 135 140
Thr Thr His Asp Arg Thr Ala Phe Arg Gly Leu Ile Ser Thr Pro Leu
145 150 155 160
Gly Asn Pro Pro Thr Val Ser Asn Ser Glu Phe Ile Cys Val Gly Trp
165 170 175
Ser Ser Thr Ser Cys His Asp Gly Val Ser Arg Met Thr Ile Cys Val
180 185 190
Gln Gly Asn Asn Glu Asn Ala Thr Ala Thr Val Tyr Tyr Asn Lys Arg
195 200 205
Leu Thr Thr Thr Ile Lys Thr Trp Ala Lys Asn Ile Leu Arg Thr Gln
210 215 220
Glu Ser Glu Cys Val Cys His Asn Ser Thr Cys Val Val Val Met Thr
225 230 235 240
Asp Gly Pro Ala Asn Asn Gln Ala Phe Thr Lys Val Ile Tyr Phe His
245 250 255
Lys Gly Thr Ile Ile Lys Glu Glu Pro Leu Lys Gly Ser Ala Lys His
260 265 270
Ile Glu Glu Cys Ser Cys Tyr Gly His Asn Gln Arg Val Thr Cys Val
275 280 285
Cys Arg Asp Asn Trp Gln Gly Ala Asn Arg Pro Val Ile Glu Ile Asp
290 295 300
Met Asn Asn Leu Glu His Thr Ser Arg Tyr Ile Cys Thr Gly Val Leu
305 310 315 320
Thr Asp Thr Ser Arg Pro Lys Asp Lys Ala Ile Gly Glu Cys Phe Asn
325 330 335
Pro Ile Thr Gly Ser Pro Gly Ala Pro Gly Ile Lys Gly Phe Gly Phe
340 345 350
Leu Asn Glu Asn Asn Thr Trp Leu Gly Arg Thr Ile Ser Pro Lys Leu
355 360 365
Arg Ser Gly Phe Glu Met Leu Lys Ile Pro Asn Ala Gly Thr Asp Pro
370 375 380
Asp Ser Lys Ile Lys Glu Arg Gln Glu Ile Val Gly Asn Asp Asn Trp
385 390 395 400
Ser Gly Tyr Ser Gly Ser Phe Ile Asp Tyr Trp Asn Asp Asn Ser Glu
405 410 415
Cys Tyr Asn Pro Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Glu
420 425 430
Glu Ala Lys Tyr Val Glu Trp Thr Ser Asn Ser Leu Ile Ala Leu Cys
435 440 445
Gly Ser Pro Ile Pro Val Gly Ser Gly Ser Phe Pro Asp Gly Ala Gln
450 455 460
Ile Lys Tyr Phe Ser
465
<210> 8
<211> 470
<212> PRT
<213> influenza A virus
<400> 8
Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Ser Leu Gly
1 5 10 15
Leu Val Val Phe Asn Val Leu Leu His Val Val Ser Ile Ile Val Thr
20 25 30
Val Leu Val Leu Gly Lys Gly Gly Asn Asn Gly Ile Cys Asn Glu Thr
35 40 45
Val Val Arg Glu Tyr Asn Glu Thr Val Arg Ile Glu Lys Val Thr Gln
50 55 60
Trp His Asn Thr Asn Val Val Glu Tyr Val Pro Tyr Trp Asn Gly Gly
65 70 75 80
Thr Tyr Met Asn Asn Thr Glu Ala Ile Cys Asp Ala Lys Gly Phe Ala
85 90 95
Pro Phe Ser Lys Asp Asn Gly Ile Arg Ile Gly Ser Arg Gly His Ile
100 105 110
Phe Val Ile Arg Glu Pro Phe Val Ser Cys Ser Pro Ile Glu Cys Arg
115 120 125
Thr Phe Phe Leu Thr Gln Gly Ser Leu Leu Asn Asp Lys His Ser Asn
130 135 140
Gly Thr Val Lys Asp Arg Ser Pro Phe Arg Thr Leu Met Ser Val Glu
145 150 155 160
Val Gly Gln Ser Pro Asn Val Tyr Gln Ala Arg Phe Glu Ala Val Ala
165 170 175
Trp Ser Ala Thr Ala Cys His Asp Gly Lys Lys Trp Met Thr Val Gly
180 185 190
Val Thr Gly Pro Asp Ser Lys Ala Val Ala Val Ile His Tyr Gly Gly
195 200 205
Val Pro Thr Asp Val Val Asn Ser Trp Ala Gly Asp Ile Leu Arg Thr
210 215 220
Gln Glu Ser Ser Cys Thr Cys Ile Gln Gly Asp Cys Tyr Trp Val Met
225 230 235 240
Thr Asp Gly Pro Ala Asn Arg Gln Ala Gln Tyr Arg Ile Tyr Lys Ala
245 250 255
Asn Gln Gly Arg Ile Ile Gly Gln Thr Asp Ile Ser Phe Asn Gly Gly
260 265 270
His Ile Glu Glu Cys Ser Cys Tyr Pro Asn Asp Gly Lys Val Glu Cys
275 280 285
Val Cys Arg Asp Gly Trp Thr Gly Thr Asn Arg Pro Val Leu Val Ile
290 295 300
Ser Pro Asp Leu Ser Tyr Arg Val Gly Tyr Leu Cys Ala Gly Ile Pro
305 310 315 320
Ser Asp Thr Pro Arg Gly Glu Asp Thr Gln Phe Thr Gly Ser Cys Thr
325 330 335
Ser Pro Met Gly Asn Gln Gly Tyr Gly Val Lys Gly Phe Gly Phe Arg
340 345 350
Gln Gly Thr Asp Val Trp Met Gly Arg Thr Ile Ser Arg Thr Ser Arg
355 360 365
Ser Gly Phe Glu Ile Leu Arg Ile Lys Asn Gly Trp Thr Gln Thr Ser
370 375 380
Lys Glu Gln Ile Arg Lys Gln Val Val Val Asp Asn Leu Asn Trp Ser
385 390 395 400
Gly Tyr Ser Gly Ser Phe Thr Leu Pro Val Glu Leu Ser Gly Lys Asp
405 410 415
Cys Leu Val Pro Cys Phe Trp Val Glu Met Ile Arg Gly Lys Pro Glu
420 425 430
Glu Lys Thr Ile Trp Thr Ser Ser Ser Ser Ile Val Met Cys Gly Val
435 440 445
Asp Tyr Glu Val Ala Asp Trp Ser Trp His Asp Gly Ala Ile Leu Pro
450 455 460
Phe Asp Ile Asp Lys Met
465 470
<210> 9
<211> 470
<212> PRT
<213> influenza A virus
<400> 9
Met Asn Pro Asn Gln Lys Ile Leu Cys Thr Ser Ala Thr Ala Leu Val
1 5 10 15
Ile Gly Thr Ile Ala Val Leu Ile Gly Ile Thr Asn Leu Gly Leu Asn
20 25 30
Ile Gly Leu His Leu Lys Pro Ser Cys Asn Cys Ser His Ser Gln Pro
35 40 45
Glu Ala Thr Asn Ala Ser Gln Thr Ile Ile Asn Asn Tyr Tyr Asn Asp
50 55 60
Thr Asn Ile Thr Gln Ile Ser Asn Thr Asn Ile Gln Val Glu Glu Arg
65 70 75 80
Ala Ile Arg Asp Phe Asn Asn Leu Thr Lys Gly Leu Cys Thr Ile Asn
85 90 95
Ser Trp His Ile Tyr Gly Lys Asp Asn Ala Val Arg Ile Gly Glu Asp
100 105 110
Ser Asp Val Leu Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Asp
115 120 125
Glu Cys Arg Phe Tyr Ala Leu Ser Gln Gly Thr Thr Ile Arg Gly Lys
130 135 140
His Ser Asn Gly Thr Ile His Asp Arg Ser Gln Tyr Arg Ala Leu Ile
145 150 155 160
Ser Trp Pro Leu Ser Ser Pro Pro Thr Val Tyr Asn Ser Arg Val Glu
165 170 175
Cys Ile Gly Trp Ser Ser Thr Ser Cys His Asp Gly Lys Thr Arg Met
180 185 190
Ser Ile Cys Ile Ser Gly Pro Asn Asn Asn Ala Ser Ala Val Ile Trp
195 200 205
Tyr Asn Arg Arg Pro Val Thr Glu Ile Asn Thr Trp Ala Arg Asn Ile
210 215 220
Leu Arg Thr Gln Glu Ser Glu Cys Val Cys His Asn Gly Val Cys Pro
225 230 235 240
Val Val Phe Thr Asp Gly Ser Ala Thr Gly Pro Ala Glu Thr Arg Ile
245 250 255
Tyr Tyr Phe Lys Glu Gly Lys Ile Leu Lys Trp Glu Pro Leu Ala Gly
260 265 270
Thr Ala Lys His Ile Glu Glu Cys Ser Cys Tyr Gly Glu Arg Ala Glu
275 280 285
Ile Thr Cys Thr Cys Arg Asp Asn Trp Gln Gly Ser Asn Arg Pro Val
290 295 300
Ile Arg Ile Asp Pro Val Ala Met Thr His Thr Ser Gln Tyr Ile Cys
305 310 315 320
Ser Pro Val Leu Thr Asp Asn Pro Arg Pro Asn Asp Pro Thr Val Gly
325 330 335
Lys Cys Asn Asp Pro Tyr Pro Gly Asn Asn Asn Asn Gly Val Lys Gly
340 345 350
Phe Ser Tyr Leu Asp Gly Val Asn Thr Trp Leu Gly Arg Thr Ile Ser
355 360 365
Ile Ala Ser Arg Ser Gly Tyr Glu Met Leu Lys Val Pro Asn Ala Leu
370 375 380
Thr Asp Asp Lys Ser Lys Pro Thr Gln Gly Gln Thr Ile Val Leu Asn
385 390 395 400
Thr Asp Trp Ser Gly Tyr Ser Gly Ser Phe Met Asp Tyr Trp Ala Glu
405 410 415
Gly Glu Cys Tyr Arg Ala Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg
420 425 430
Pro Lys Glu Asp Lys Val Trp Trp Thr Ser Asn Ser Ile Val Ser Met
435 440 445
Cys Ser Ser Thr Glu Phe Leu Gly Gln Trp Asp Trp Pro Asp Gly Ala
450 455 460
Lys Ile Glu Tyr Phe Leu
465 470
<210> 10
<211> 466
<212> PRT
<213> influenza B virus
<400> 10
Met Leu Pro Ser Thr Val Gln Thr Leu Thr Leu Leu Leu Thr Ser Gly
1 5 10 15
Gly Val Leu Leu Ser Leu Tyr Val Ser Ala Ser Leu Ser Tyr Leu Leu
20 25 30
Tyr Ser Asp Val Leu Leu Lys Phe Ser Ser Thr Lys Thr Thr Ala Pro
35 40 45
Thr Met Ser Leu Glu Cys Thr Asn Ala Ser Asn Ala Gln Thr Val Asn
50 55 60
His Ser Ala Thr Lys Glu Met Thr Phe Pro Pro Pro Glu Pro Glu Trp
65 70 75 80
Thr Tyr Pro Arg Leu Ser Cys Gln Gly Ser Thr Phe Gln Lys Ala Leu
85 90 95
Leu Ile Ser Pro His Arg Phe Gly Glu Ile Lys Gly Asn Ser Ala Pro
100 105 110
Leu Ile Ile Arg Glu Pro Phe Val Ala Cys Gly Pro Lys Glu Cys Arg
115 120 125
His Phe Ala Leu Thr His Tyr Ala Ala Gln Pro Gly Gly Tyr Tyr Asn
130 135 140
Gly Thr Arg Lys Asp Arg Asn Lys Leu Arg His Leu Val Ser Val Lys
145 150 155 160
Leu Gly Lys Ile Pro Thr Val Glu Asn Ser Ile Phe His Met Ala Ala
165 170 175
Trp Ser Gly Ser Ala Cys His Asp Gly Arg Glu Trp Thr Tyr Ile Gly
180 185 190
Val Asp Gly Pro Asp Asn Asp Ala Leu Val Lys Ile Lys Tyr Gly Glu
195 200 205
Ala Tyr Thr Asp Thr Tyr His Ser Tyr Ala His Asn Ile Leu Arg Thr
210 215 220
Gln Glu Ser Ala Cys Asn Cys Ile Gly Gly Asp Cys Tyr Leu Met Ile
225 230 235 240
Thr Asp Gly Ser Ala Ser Gly Ile Ser Lys Cys Arg Phe Leu Lys Ile
245 250 255
Arg Glu Gly Arg Ile Ile Lys Glu Ile Leu Pro Thr Gly Arg Val Glu
260 265 270
His Thr Glu Glu Cys Thr Cys Gly Phe Ala Ser Asn Lys Thr Ile Glu
275 280 285
Cys Ala Cys Arg Asp Asn Ser Tyr Thr Ala Lys Arg Pro Phe Val Lys
290 295 300
Leu Asn Val Glu Thr Asp Thr Ala Glu Ile Arg Leu Met Cys Thr Lys
305 310 315 320
Thr Tyr Leu Asp Thr Pro Arg Pro Asp Asp Gly Ser Ile Ala Gly Pro
325 330 335
Cys Glu Ser Asn Gly Asp Lys Trp Leu Gly Gly Ile Lys Gly Gly Phe
340 345 350
Val His Gln Arg Met Ala Ser Lys Ile Gly Arg Trp Tyr Ser Arg Thr
355 360 365
Met Ser Lys Thr Asn Arg Met Gly Met Glu Leu Tyr Val Lys Tyr Asp
370 375 380
Gly Asp Pro Trp Thr Asp Ser Asp Ala Leu Thr Leu Ser Gly Val Met
385 390 395 400
Val Ser Ile Glu Glu Pro Gly Trp Tyr Ser Phe Gly Phe Glu Ile Lys
405 410 415
Asp Lys Lys Cys Asp Val Pro Cys Ile Gly Ile Glu Met Val His Asp
420 425 430
Gly Gly Lys Asp Thr Trp His Ser Ala Ala Thr Ala Ile Tyr Cys Leu
435 440 445
Met Gly Ser Gly Gln Leu Leu Trp Asp Thr Val Thr Gly Val Asp Met
450 455 460
Ala Leu
465
<210> 11
<211> 1410
<212> DNA
<213> influenza A virus
<400> 11
atgaatccaa accaaaagat aataaccatt ggttcggtct gtatgacaat tggaatggct 60
aacttaatat tacaaattgg aaacataatc tcaatatgga ttagccactc aattcaactt 120
gggaatcaaa atcagattga aacatgcaat caaagcgtca ttacttatga aaacaacact 180
tgggtaaatc agacatatgt taacatcagc aacaccaact ttgctgctgg acagtcagtg 240
gtttccgtga aattagcggg caattcctct ctctgccctg ttagtggatg ggctatatac 300
agtaaagaca acagtgtaag aatcggttcc aagggggatg tgtttgtcat aagggaacca 360
ttcatatcat gctccccctt ggaatgcaga accttcttct tgactcaagg ggccttgcta 420
aatgacaaac attccaatgg aaccattaaa gacaggagcc catatcgaac cctaatgagc 480
tgtcctattg gtgaagttcc ctctccatac aactcaagat ttgagtcagt cgcttggtca 540
gcaagtgctt gtcatgatgg catcaattgg ctaacaattg gaatttctgg cccagacaat 600
ggggcagtgg ctgtgttaaa gtacaacggc ataataacag acactatcaa gagttggaga 660
aacaatatat tgagaacaca agagtctgaa tgtgcatgtg taaatggttc ttgctttact 720
gtaatgaccg atggaccaag taatggacag gcctcataca agatcttcag aatagaaaag 780
ggaaagatag tcaaatcagt cgaaatgaat gcccctaatt atcactatga ggaatgctcc 840
tgttatcctg attctagtga aatcacatgt gtgtgcaggg ataactggca tggctcgaat 900
cgaccgtggg tgtctttcaa ccagaatctg gaatatcaga taggatacat atgcagtggg 960
attttcggag acaatccacg ccctaatgat aagacaggca gttgtggtcc agtatcgtct 1020
aatggagcaa atggagtaaa agggttttca ttcaaatacg gcaatggtgt ttggataggg 1080
agaactaaaa gcattagttc aagaaacggt tttgagatga tttgggatcc gaacggatgg 1140
actgggacag acaataactt ctcaataaag caagatatcg taggaataaa tgagtggtca 1200
ggatatagcg ggagttttgt tcagcatcca gaactaacag ggctggattg tataagacct 1260
tgcttctggg ttgaactaat cagagggcga cccaaagaga acacaatctg gactagcggg 1320
agcagcatat ccttttgtgg tgtaaacagt gacactgtgg gttggtcttg gccagacggt 1380
gctgagttgc catttaccat tgacaagtaa 1410
<210> 12
<211> 1410
<212> DNA
<213> influenza A virus
<400> 12
atgaatccaa atcaaaagat aataacgatt ggctctgttt ctctcaccat ttccacaata 60
tgcttcttca tgcaaattgc catcctgata accactgtaa cattgcattt caagcaatat 120
gaattcaact cccccccaaa caaccaagtg atgctgtgtg aaccaacaat aatagaaaga 180
aacataacag agatagtgta tctgaccaac accaccatag agaaggaaat gtgccccaaa 240
ctagcagaat acagaaattg gtcaaagccg caatgtgaca ttacaggatt tgcacctttt 300
tctaaggaca attcgattag gctttccgct ggtggggaca tctgggtgac aagagaacct 360
tatgtgtcat gcgaccctga caagtgttac caatttgccc ttggacaggg aacaacacta 420
aacaacgtgc attcaaatga cacagtacat gataggaccc cttatcggac cctattgatg 480
aatgaattag gtgttccatt tcatctgggg accaagcaag tgtgcatagc atggtccagc 540
tcaagttgtc acgatggaaa agcatggctg catgtttgtg taacggggga tgataaaaat 600
gcaactgcta gcttcattta caatgggagg cttgtagata gtattgtttc atggtccaaa 660
aaaatcctca ggacccagga gtcagaatgc gtttgtatca atggaacttg tacagtagta 720
atgactgatg ggagtgcttc aggaaaagct gatactaaaa tactattcat tgaggagggg 780
aaaatcattc atactagcac attgtcagga agtgctcagc atgtcgagga gtgctcctgc 840
tatcctcgat atcctggtgt cagatgtgtc tgcagagaca actggaaagg ctccaatagg 900
cccatcgtag atataaacat aaaggattat agcattgttt ccagttatgt gtgctcaggg 960
cttgttggag acacacccag aaaaaacgac agctccagca gtagccattg cttggatcct 1020
aacaatgaag aaggtggtca tggagtgaaa ggctgggcct ttgatgatgg aaatgacgtg 1080
tggatgggaa gaacgatcag cgagaagtta cgctcaggat atgaaacctt caaagtcatt 1140
gaaggctggt ccaaacctaa ttccaaattg cagataaata ggcaagtcat agttgacaga 1200
ggtaataggt ccggttattc tggtattttc tctgttgaag gcaaaagctg catcaatcgg 1260
tgcttttatg tggagttgat aaggggaaga aaagaggaaa ctgaagtctt gtggacctca 1320
aacagtattg ttgtgttttg tggcacctca ggtacatatg gaacaggctc atggcctgat 1380
ggggcggaca tcaatctcat gcctatataa 1410
<210> 13
<211> 1410
<212> DNA
<213> influenza A virus
<400> 13
atgaatccaa atcagaagat aataacaatc ggggtagtga acactactct atcaacaata 60
gcccttctca ttggagtggg aaatctggtt ttcaacacag tcatacatga gaaaataggg 120
aatcaccaaa cagtgattca cccaacaata acgactcctg cagtaccaaa ctgcagtgac 180
actataataa catacaacaa cactgtgata aacaacataa caacaacaat aataactgaa 240
gcggaaaggc tttttaagcc tccactgccg ctgtgcccct tccgaggatt cttccctttt 300
cacaaggaca atgcaatacg attgggtgag aacaaggacg tcatagtcac aagagaacct 360
tatgttagct gcgataatga caattgttgg tccttcgctc tcgcacaagg agcactgtta 420
gggactaaac atagcaatgg aaccatcaaa gacagaacac cgtataggtc tctaatccga 480
ttcccaatag gaacagcccc agtactggga aattacaagg agatatgcat tgcttggtcg 540
agtagcagtt gctttgacgg gaaagagtgg atgcatgtat gcatgacagg gaacgataat 600
gatgcaagtg cccaaataat atatgcaggg agaatgacag actccatcaa atcatggaga 660
aaggacatac taaggaccca agagtccgaa tgtcaatgca ttggcggaac ttgtgttgtt 720
gctgttacag atggccctgc tgctaatagt gcagatcata gggtttattg gatacgggag 780
gggagaatag tgaagtatga aaatgtccct aaaacaaaga tacaacactt agaagagtgt 840
tcctgctatg tggacatcga tgtgtactgt atatgcaggg acaattggaa gggttccaac 900
agaccttgga tgagaatcaa caacgagacc atactggaaa caggatatgt gtgcagcaaa 960
tttcactcag acactcccag gccagctgac ccctcaacag tatcatgtga ttctccaagc 1020
aacattaatg gaggacccgg agttaaggga tttggcttca aagccggcaa tgatgtgtgg 1080
ttgggcagga cagtgtcaac tagtggtaga tcaggctttg aaatcatcaa agttacagat 1140
gggtggatca actctcccaa tcacgccaaa tcagttacac aaacactagt gtccaacaat 1200
gattggtcag gctattcagg tagtttcatt gtcaaaacca agggctgttt ccagccctgc 1260
ttttatgtcg aacttatacg aggaaggccc aacaagaatg atgatgtctc ttggacaagc 1320
aatagtatag ttactttctg tgggttagac aatgaacctg gatcgggaaa ttggccggat 1380
ggttccaaca ttgggtttat gcccaagtaa 1410
<210> 14
<211> 1413
<212> DNA
<213> influenza A virus
<400> 14
atgaatccaa atcagaaaat cataaccatc ggtagtgcga gtattgtatt aacaacaata 60
gggttgctcc tccagataac aagtttatgc tcaatatggt ttagccatta caaccaagtg 120
acccaaccgc atgaacaagc ctgttcgaac aacacaacga attactataa tgagaccttt 180
gtcaatgtca ccaatgtgca gaacaactat accacaataa ttgaaccctc agcccccaat 240
gtggttcact actctagtgg aagagacttg tgcccagtaa aggggtgggc acctctgagt 300
aaggacaatg gaattagaat tggatcccga ggcgaagtat ttgtcatacg ggagcctttc 360
atatcatgtt ccattagtga atgcaggact tttttcttaa ctcagggagc cctcctcaat 420
gacaagcact caaatggaac agtaaaagac aggagtccct ttcgtacatt aatgagttgt 480
cctatgggag ttgccccctc tcctagcaac agccgctttg aatctgtggc atggtctgcc 540
actgcatgca gtgatggacc cggttggtta acattaggga tcactggccc agatgctact 600
gctgtggcag tactgaaata caatggtata ataacagaca cgttaaaaag ctggaagggt 660
aatattatgc gaacacaaga gtctgaatgc gtatgccagg atgaattttg ttatacctta 720
ataacagacg gaccgtccaa cgcacaagct ttctataaga tactaaagat cagaaaaggg 780
aaaatagtaa gtgtgaagga tgtgaatgca acagggtttc attttgaaga atgttcctgt 840
tacccgagcg ggacagatgt tgaatgtgtc tgcagggaca attggcgggg gagcaatcga 900
ccgtggataa gattcaacag tgatcttgat taccaaattg gctatgtatg tagtgggata 960
tttggggaca atcccaggcc tgtggatggc ataggctcgt gtaacagccc agtcaacaat 1020
gggaaaggaa gatatggagt gaagggattc agctttagat atggggatgg tgtttggata 1080
gggaggacaa aaagcttgga atccagaagc ggttttgaga tggtctggga tgctaatggg 1140
tgggtatcga cagacaagga ttcaaatggt gtacaggata ttatagacaa caacaattgg 1200
tctggttaca gcggaagctt cagcattaga tgggagacaa caggcaggaa ttgcactgtc 1260
ccatgttttt gggttgagat gataagaggg cagcctaaag aaaaaaccat atggactagt 1320
ggtagtagca ttgcattctg tggtgttaat tctgatacca caggttggtc atggcccgat 1380
ggcgctctgt tgccctttga catagacaag taa 1413
<210> 15
<211> 1407
<212> DNA
<213> influenza A virus
<400> 15
atgaatccaa atcagaaaat aataacaatt ggctcagcat cgttgggatt agtcattttc 60
aacattcttc ttcatgtggc atcaataacc ttgggaataa tatcagtaac caaagacaac 120
aaagtacata tctgtaatac gactgaggtg tataatgaaa cggtgagagt ggaaacagtg 180
gtaatccctg tcaacaacac catatatttg aatcatgagc cagaattcct caacaacacg 240
gaacctctct gtgatgtatc gggatttgcc attgtttcca aggataatgg gatcagaata 300
ggttcaagag ggcacatatt tgttataaga gagccctttg tatcttgtgg cccttcagaa 360
tgtaggacct ttttcttaac gcagggtgcc ttgctgaatg acaagcattc aaataacaca 420
gtaaaagaca ggagtcccta tcgggcattg atgagtgtac cattgggatc ctctcctaat 480
gcctaccaag ccaaattcga gtccgtagga tggtctgcaa cagcctgcca tgatgggaaa 540
aaatggatgg ccataggggt gagtggtgca gacgatgacg cttatgctgt tattcattat 600
gggggagtgc cgacagatgt gataaggtca tggaggaagc aaatactaag aacacaagag 660
tcttcgtgtg tttgcataaa gggagagtgt tattgggtaa tgacggacgg tccagcgaat 720
aatcaagcaa gttacaaaat cttcaaatct cagaagggta tggtagttga tgaaaaagaa 780
atttcattcc aaggtgggca cattgaagaa tgttcctgtt atcccaacat ggggaaagtg 840
gagtgtgttt gcagggacaa ttggaacgga atgaatagac caattctgat attcgatgaa 900
aagcttgagt atgaagtcgg ttatctatgc gctggaattc caacagacac cccaagagtt 960
caagacagca gcttcactgg ctcatgcacc aatgcagttg ggaggagtgg gacaaacaat 1020
tatggagtaa agggattcgg ctttagacaa ggaaatagtg tatgggcggg aagaacaatt 1080
agtgtttctt cacggagtgg atttgaggtt ctattgatag aggacgggtg gattagaccg 1140
agtaaaacca ttagcaaaaa ggttgaggtt ttgaacaata agaattggtc gggatacagc 1200
ggggctttta ccatccccac tgcaatgaca agcaagaact gtatagttcc atgcttttgg 1260
ctggagatga ttagggggaa accagaggag aggactagca tttggacttc aagtagctcc 1320
accgtgtttt gtggtgtttc cagtgaggtc ccaggatggt cgtgggatga tggagcaatt 1380
cttccatttg acatcgataa gatgtag 1407
<210> 16
<211> 1413
<212> DNA
<213> influenza A virus
<400> 16
atgaatccaa atcagaagat aatatgcatt tcagccacag gaatgacact atcggtagta 60
agcctcctag taggaatcgc caatttaggt ctaaacatcg gactccatta taaagtaggt 120
gatacaccaa atgtgaatat tccaaacgta aatgggacca attcaacaac aacaataatc 180
aacaacaaca ctcagaataa tttcacaaat atcactaata ttatacagag caaaggtgga 240
gaaagaacat ttctaaacct aactaagccc ctatgtgaag taaactcatg gcacatccta 300
tcaaaggaca atgcaataag aataggagag gatgctcata tcctggtcac aagagagccc 360
tatctatctt gcgatccaca agggtgcagg atgtttgctc taagtcaagg cacaacactc 420
agagggcgac atgcaaatgg gactatacat gataggagtc cgttcagagc ccttataagc 480
tgggaaatgg gtcaagcacc cagtccatat aatactaggg ttgagtgcat agggtggtca 540
agcacatcat gccatgatgg catgtcaaga atgtcaatat gcatgtcagg accgaacaac 600
aatgcatcag cagtggtgtg gtatgggggt aggccaataa cagaaattcc atcatgggca 660
gggaatattc taagaaccca agaatcagag tgtgtgtgcc ataaaggagt ttgtccagta 720
gtcatgacag atggcccagc aaacaataga gcagcaacca agataatcta tttcaaggaa 780
gggaaaatac agaaaattga agaactagca ggaaatgctc aacacattga agaatgctcc 840
tgctatggag caggaggagt gatcaaatgc atctgcagag acaattggaa gggagcaaat 900
agaccagtaa tcactataga ccccgagatg atgacccata caagcaagta tttatgctca 960
aaggtcctaa ctgatacgag tcgtcccaat gaccccacta atggaaactg tgacgcgcca 1020
ataacaggag ggagcccaga tcctggagtc aaggggttcg cattcctaga tggagaaaat 1080
tcatggcttg ggaggacaat tagcaaagat tccagatcag gttatgaaat gttaaaggtc 1140
ccaaatgctg aaactgatat ccaatcgggc ccaatctcaa accaggtgat tgtcaacaac 1200
caaaactggt cgggatactc aggggcattc atcgactact gggcaaacaa agaatgcttc 1260
aatccctgct tttatgtgga actaatcaga gggaggccca aagagagtag tgtactgtgg 1320
acttcaaata gcatagtagc tctctgtgga tccaaaaagc gattaggatc atggtcctgg 1380
catgatggtg ctgaaatcat ctacttcgag tag 1413
<210> 17
<211> 1410
<212> DNA
<213> influenza A virus
<400> 17
atgaatccta atcagaaact ctttgcatca tccggaatag caatagcact aggaataata 60
aatctcctaa taggaatatc caatatgagt ttaaatatat ctctatactc aaaaggagaa 120
aaccacaaaa gtgataacct gacatgcaca aatatcaatc aaaacaatac caccatggta 180
aatacgtaca tcaataacac aacaataatt gacaaaaata caaagatgga gaaccctggt 240
tatctactgc tgaataaaag tctatgcaac gttgaggggt gggttgtaat agcaaaggat 300
aatgcaatta gatttgggga aagcgaacaa attatagtaa ctagagaacc ctatgtctca 360
tgtgatcctc taagttgtaa aatgtatgcc ctacatcaag gtacaacaat cagaaacaag 420
cattcaaatg gtaccaccca cgatagaaca gccttcagag gacttatctc taccccatta 480
ggcaaccccc caacagtgag caacagtgag ttcatatgtg ttggatggtc aagcacaagc 540
tgccatgatg gagtaagcag aatgacaatt tgcgtacaag gaaacaatga aaatgctact 600
gcaacggtgt attacaacaa gaggcttaca accactatta aaacatgggc taaaaacatt 660
ttaagaaccc aagaatctga gtgtgtctgt cataacagta cttgtgtagt ggtgatgact 720
gatgggcccg caaataacca ggcattcaca aaggtaatat actttcataa aggaacgata 780
ataaaagaag aaccactaaa aggttcagcc aaacacatag aagaatgttc ttgttatggt 840
cataatcaaa gagtgacttg tgtttgcaga gacaattggc agggtgcaaa tagacccgtt 900
atagaaattg acatgaataa tttagaacac acaagtagat atatatgcac aggagtatta 960
acagacacca gtagacccaa agataaagca ataggggaat gcttcaatcc tattactggg 1020
agtcctggtg caccaggaat aaaaggtttc gggttcctaa atgagaataa tacctggctg 1080
gggagaacaa tcagccccaa attgaggagt ggatttgaaa tgctgaagat acctaatgct 1140
gggactgacc cagattccaa aataaaagaa agacaagaaa tagttggtaa tgacaattgg 1200
tcaggctatt ccggaagttt cattgattat tggaatgaca acagtgaatg ctacaatcca 1260
tgcttctatg tagaattaat tagaggaagg cctgaagaag caaaatatgt tgaatggacc 1320
agtaacagcc taattgcact atgtgggagc ccaatcccag ttgggtctgg atctttccct 1380
gatggggcac aaatcaaata cttttcgtaa 1410
<210> 18
<211> 1413
<212> DNA
<213> influenza A virus
<400> 18
atgaatccaa atcagaaaat aataaccatt ggctccatat cattgggatt ggttgtattt 60
aatgttctac tgcatgtcgt gagcatcata gtaacagtat tagtcctggg aaaaggtgga 120
aacaatggaa tctgtaatga gacagtagtg agggaataca acgagacagt taggatcgag 180
aaggtaactc aatggcacaa tactaatgta gtcgaatatg tgccgtattg gaatgggggc 240
acttacatga ataatacaga agcaatatgt gatgccaagg gctttgcacc tttttccaag 300
gacaatggaa taagaattgg ttccagggga catatttttg ttataaggga gcccttcgtc 360
tcttgttcac ctatagagtg cagaactttc ttcctcaccc aggggtccct gctcaatgac 420
aaacactcaa atggaacagt gaaggatagg agcccattca gaactctcat gagtgtcgaa 480
gtgggccaat cacccaatgt atatcaagcc agatttgaag ctgtggcgtg gtcagcaaca 540
gcctgtcacg atgggaagaa gtggatgaca gttggtgtaa cagggccaga ttccaaggca 600
gtagcagtaa ttcactatgg aggagtgcct actgatgtcg ttaattcctg ggcaggagat 660
atattgagga ctcaagagtc atcttgcact tgcatccaag gtgattgcta ttgggtaatg 720
actgacggcc ccgccaatag gcaggcacag tatagaatat acaaagcaaa tcaaggcaga 780
ataattggcc agacggatat cagttttaat ggaggacata ttgaagagtg ttcttgctat 840
cctaatgatg gcaaagtgga atgtgtatgt agagacggtt ggactggaac caacaggcct 900
gtactagtca tctcgcctga tctctcttac agagttgggt acttatgtgc agggattcct 960
agtgatactc caagagggga agatacccaa tttactggct cgtgcactag ccccatgggg 1020
aatcaggggt atggtgtaaa aggctttgga tttcggcagg gaactgatgt atggatgggg 1080
cggacaatta gtcgaacctc caggtcagga tttgagatat taaggataaa gaatggttgg 1140
acacagacaa gtaaagagca gattagaaaa caggtagttg ttgacaattt gaattggtcg 1200
ggatacagtg gatctttcac tctgccagta gaattgtctg ggaaagactg cctggttccc 1260
tgtttttggg tagaaatgat caggggcaag ccagaagaga aaacaatctg gacttccagc 1320
agttccattg tgatgtgtgg agttgattat gaagttgccg attggtcatg gcacgatgga 1380
gctattcttc cctttgacat cgataagatg taa 1413
<210> 19
<211> 1413
<212> DNA
<213> influenza A virus
<400> 19
atgaatccaa atcagaagat tctatgcact tctgccactg ctctcgtaat aggcacaatt 60
gcagtactca taggaataac gaacttagga ttgaacatag gactacatct gaaaccgagc 120
tgcaattgct cacactcaca acccgaagca accaatgcaa gccaaacaat aataaataac 180
tattataatg acacaaacat cacccagata agtaatacca acattcaggt agaggaaagg 240
gcaattagag atttcaataa cttgaccaaa gggctctgta ctataaattc atggcacata 300
tatgggaaag acaatgcggt gagaattggg gaggactcag atgttttagt cacaagagaa 360
ccctatgtct cctgtgaccc agatgagtgc aggttctatg ctctcagcca agggacaaca 420
atcagaggaa aacactcaaa tggaacaata cacgataggt ctcaatatcg tgccctgata 480
agctggccat tgtcatcacc gcccacagta tacaacagca gagtggaatg cattggatgg 540
tcaagtacta gttgtcatga tggcaaaacc aggatgtcaa tatgcatatc aggcccgaac 600
aataacgcat cagcagtgat ctggtacaat agaaggcctg tgacagaaat caacacatgg 660
gcccgaaaca tactaaggac acaagaatct gaatgcgtat gccacaacgg tgtctgcccg 720
gtagtgttca cagatgggtc tgccactgga cctgcagaaa caagaatata ctattttaaa 780
gaagggaaga tcttaaaatg ggaacctctg gctggaactg ctaagcatat cgaagaatgc 840
tcatgctacg gagagcgagc agagattact tgcacgtgta gggataattg gcaaggctca 900
aatagaccag taattcggat agatccagtg gcgatgacac atactagtca gtatatatgt 960
agccctgttc tcacagataa cccccgaccg aatgacccaa ctgtaggtaa gtgtaacgac 1020
ccttatccag gcaataacaa caatggggtc aaagggtttt catatctgga tggagttaat 1080
acttggctag ggaggacaat aagcatagct tcaagatccg gatatgagat gctaaaggtg 1140
ccaaatgcat tgacagacga taagtcaaag cccactcaag gtcagacaat cgtcttaaac 1200
actgactgga gtggttacag tgggtccttc atggactatt gggctgaggg ggaatgctac 1260
cgagcgtgtt tttacgtgga gttaatacgt gggagaccta aggaggataa agtgtggtgg 1320
accagtaata gtatagtatc gatgtgttcc agcacagaat tccttggaca atgggactgg 1380
cctgatgggg ctaaaataga gtacttcctc taa 1413
<210> 20
<211> 1557
<212> DNA
<213> influenza B virus
<400> 20
agcagaagca gagcatattc ttagaactga agtgaacagg ccaaaaatga acaatgctac 60
cttcaactgt acaaacatta accctattac tcacatcagg gggagtatta ttatcactat 120
atgtgtcagc ctcattgtca tacttattgt attcggatgt attgctaaaa ttttcatcaa 180
caaaaacaac tgcaccaaca atgtcattag agtgcacaaa cgcatcaaat gcccagactg 240
tgaaccattc tgcaacaaaa gagatgacat ttccaccccc agagccggag tggacatacc 300
ctcgtttatc ttgccagggc tcaacctttc agaaggcact cctaattagc cctcataggt 360
tcggagagat caaaggaaac tcagctccct tgataataag agaacctttt gttgcttgtg 420
gaccaaaaga atgcagacac tttgctctga cccattatgc agctcagccg gggggatact 480
acaatggaac aagaaaggac agaaacaagc tgaggcatct agtatcagtc aaattgggaa 540
aaatcccaac tgtggaaaac tccattttcc acatggcagc ttggagcgga tccgcatgcc 600
atgatggtag agaatggaca tatatcggag ttgatggtcc tgacaatgat gcattggtca 660
aaataaaata tggagaagca tatactgaca catatcattc ctatgcacac aacatcctaa 720
gaacacaaga aagtgcctgc aattgcatcg ggggagattg ttatcttatg ataacagacg 780
gctcagcttc aggaattagt aaatgcagat ttcttaaaat tagagagggt cgaataataa 840
aagaaatact tccaacagga agagtggagc acactgaaga gtgcacatgc gggttcgcca 900
gcaataaaac catagaatgt gcctgtagag acaacagtta cacagcaaaa agaccctttg 960
tcaaattaaa tgtggaaact gatacagctg aaataagatt gatgtgcaca aagacttatc 1020
tagacactcc cagaccggat gatggaagca tagcagggcc ttgcgaatct aatggagaca 1080
agtggcttgg aggcatcaaa ggaggattcg tccatcaaag aatggcatct aagattggaa 1140
gatggtactc ccgaacgatg tctaaaacta acagaatggg gatggaactg tatgtaaagt 1200
atgatggtga cccatggact gacagtgatg ctcttactct tagtggagta atggtttcca 1260
tagaagaacc tggttggtat tcttttggct tcgaaataaa ggacaagaaa tgtgatgtcc 1320
cttgtattgg gatagagatg gtacacgatg gtggaaaaga tacttggcat tcagctgcaa 1380
cagccattta ctgtttgatg ggctcaggac aattgctatg ggacactgtc acaggcgttg 1440
atatggcttt ataatagagg aatggttgga tctgttctaa accctttgtt cctattttat 1500
ttgaacagtt gttcttacta gatttaattg tttctgaaaa atgctcttgt tactact 1557
<210> 21
<211> 655
<212> DNA
<213> artificial sequence
<220>
<223> CMV promoter
<400> 21
cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc 60
aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 120
aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 180
tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 240
gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 300
cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 360
tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg 420
gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc 480
cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg 540
taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat 600
aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaatt 655

Claims (15)

1. An adeno-associated virus (AAV-Sia) comprising a transgene encoding a sialidase, wherein the sialidase is derived from influenza virus, for use in treating cancer or preventing cancer recurrence in a patient in need thereof.
2. The AAV-Sia for use according to claim 1, wherein the sialidase is a neuraminidase selected from:
a.A influenza virus-derived neuraminidase, in particular N1, N2, N3, N4, N5, N6, N7, N8, N9, N10 or N11 neuraminidase; or alternatively
b.B type Victoria or mountain line influenza-derived neuraminidase;
in particular, wherein the sialidase is N1 neuraminidase.
3. AAV-Sia for use according to claim 1 or 2, wherein the sialidase comprises or consists of a sialidase polypeptide sequence selected from SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009 or SEQ ID NO 010;
or the sialidase comprises or consists of a polypeptide sequence having more than or equal to 85%, specifically more than or equal to 90%, more specifically more than or equal to 95% identity to the sialidase polypeptide sequence selected from the group consisting of SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, wherein the polypeptide sequence has at least 90% of the biological activity of the sialidase polypeptide sequence;
in particular, wherein the sialidase comprises or consists of the polypeptide sequence SEQ ID NO 001; or the sialidase comprises or consists of a polypeptide sequence having an identity of more than or equal to 85%, in particular more than or equal to 90%, more in particular more than or equal to 95% compared to SEQ ID NO 001, and has at least 90% of the biological activity of the sialidase polypeptide sequence SEQ ID NO 001.
4. The AAV-Sia for use according to any one of claims 1 to 3, wherein the transgene encoding a sialidase comprises or consists of a nucleic acid sequence encoding a sialidase polypeptide sequence selected from SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, or SEQ ID NO 010;
or comprises or consists of a polypeptide sequence having more than or equal to 85%, specifically more than or equal to 90%, more specifically more than or equal to 95% identity to the sialidase polypeptide sequence selected from the group consisting of SEQ ID NO 001, SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, SEQ ID NO 005, SEQ ID NO 006, SEQ ID NO 007, SEQ ID NO 008, SEQ ID NO 009, SEQ ID NO 010, wherein the polypeptide sequence has at least 90% of the biological activity of the sialidase polypeptide sequence;
in particular, wherein the transgene comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO 011, SEQ ID NO 012, SEQ ID NO 013, SEQ ID NO 014, SEQ ID NO 015, SEQ ID NO 016, SEQ ID NO 017, SEQ ID NO 018, SEQ ID NO 019 or SEQ ID NO 020;
More specifically, wherein the transgene comprises or consists of the nucleic acid sequence SEQ ID NO 011.
5. The AAV-Sia for use according to claim 1 to 4, wherein said transgene is comprised within a viral expression element comprising said transgene operably linked to a promoter sequence conferring transgene expression in mammalian cells, in particular wherein said promoter sequence comprises or consists of a cytomegalovirus promoter,
more specifically, wherein the promoter sequence comprises or consists of the nucleic acid sequence SEQ ID NO 021.
6. The AAV-Sia for use according to any one of claims 1-5, wherein the adeno-associated virus is a replication-defective recombinant adeno-associated virus.
7. The AAV-Sia for use according to any one of claims 1-6, wherein the adeno-associated virus is an adeno-associated type 2 virus.
8. AAV-Sia for use according to any one of claims 1 to 7, wherein the cancer is a solid cancer, in particular a solid cancer selected from liver cancer, prostate cancer, pancreatic cancer, colon cancer, cervical cancer, lung cancer, breast cancer and melanoma.
9. The AAV-Sia for use of any one of claims 1-8, wherein the cancer is characterized by metastatic cancer.
10. The AAV-Sia for use according to any one of claims 1 to 9, wherein the AAV-Sia is administered directly into a tumor, in particular wherein the AAV-Sia is administered by intratumoral injection.
11. The AAV-Sia for use according to any one of claims 1 to 10, wherein the patient is scheduled to receive, is currently receiving, or has recently been administered a checkpoint inhibitor.
12. The AAV-Sia for use according to claim 11, wherein the checkpoint inhibitor is an antibody specific for a checkpoint molecule selected from PD-1 or PD-L1, more particularly an antibody specific for PD-1.
13. The AAV-Sia for use of claim 11 or 12, wherein the checkpoint inhibitor is administered parenterally.
14. A pharmaceutical composition for use in treating cancer or preventing cancer recurrence in a patient in need thereof, comprising an AAV-Sia according to any one of claims 1-13.
15. The pharmaceutical composition for use according to claim 14, formulated for intratumoral administration.
CN202280015793.7A 2021-02-18 2022-02-18 Viral delivery of sialidases for the treatment of cancer Pending CN116940687A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP21157979.2 2021-02-18
EP21182465.1 2021-06-29
EP21182465 2021-06-29
PCT/EP2022/054067 WO2022175446A1 (en) 2021-02-18 2022-02-18 Viral delivery of a sialidase to treat cancer

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