polynucleotides, isolated adenovirus, virus-like particle, vector, composition, cell and in vitro me

专利摘要:
The present invention relates to new strains of adenovirus with high immunogenicity and without pre-existing immunity in the human population in general. The lack of pre-existing immunity is due to new hypervariable regions in the hexon protein of the adenoviral capsid. The new adenovirus strains also have an improved reproduction capacity. The present invention provides nucleotide sequences and amino acid sequences of these new adenovirus strains, as well as recombinant viruses, virus-like particles (VLPs) and vectors based on those strains. In addition, pharmaceutical compositions and medical uses are provided for therapy or prophylaxis of a disease and methods for producing an adenovirus or virus-like particles using the new sequences, recombinant viruses, virus-like particles and vectors.
公开号:BR112020000145A2
申请号:R112020000145-7
申请日:2018-07-05
公开日:2020-07-14
发明作者:Alfredo Nicosia；Stefano Colloca；Armin Lahm
申请人:Nouscom Ag；
IPC主号:

专利说明:

[001] [001] The present invention relates to new strains of adenovirus with high immunogenicity and without pre-existing immunity in the human population in general. The lack of pre-existing immunity is due to new hypervariable regions in the hexon protein of the adenoviral capsid. The new adenovirus strains also have an improved reproduction capacity. The present invention provides nucleotide sequences and amino acid sequences of these new adenovirus strains, as well as recombinant viruses, virus-like particles (VLPs, Virus Like Particles) and vectors based on those strains. In addition, pharmaceutical compositions and medical uses are provided for therapy or prophylaxis of a disease and methods for producing an adenovirus or virus-like particles using the new sequences, recombinant viruses, virus-like particles and vectors. BACKGROUND OF THE INVENTION
[002] [002] Adenoviruses (Ads) comprise a large family of double-stranded DNA viruses found in amphibians, birds and mammals that have an uninvolved icosahedral capsid structure (Straus, Adenovirus infections in humans; The Adenoviruses, 451-498, 1984 ; Hierholzer et al., J. Infect. Dis., 158: 804-813,1988; Schnurr and Dondero, Intervirology., 36: 79-83,1993; Jong et al., J. Clin. Microbiol., 37: 3940-3945: 1999). In contrast to retroviruses, adenoviruses can transduce numerous cell types from various mammalian species, including both dividing and non-dividing cells, without integrating into the host cell's genome.
[003] [003] In general, adenoviral DNA is typically very stable and remains episomal (for example, extrachromosomal), unless transformation or tumorigenesis occurs. In addition, adenoviral vectors can be propagated in high yield in well-defined production systems that are easily accessible for the pharmaceutical scale production of clinical grade compositions. These characteristics and their well-characterized molecular genetics make recombinant adenoviral vectors good candidates for use as vaccine carriers. The production of recombinant adenoviral vectors can rely on the use of a packaging cell line that is able to complement the functions of adenoviral gene products that may have been deleted or modified to be non-functional.
[004] [004] Currently, two well-characterized human adenovirus serotypes from subgroup C (ie hAd2 and hAd5) are widely used as sources of the viral backbone for most of the adenoviral vectors used in gene therapy. Human adenoviral vectors with replication defects have also been tested as carriers of vaccines for the delivery of a variety of immunogens derived from a variety of infectious agents. Experimental studies carried out on animals (for example, rodents, canines and non-human primates) indicate that recombinant human adenoviral vectors defective in replication carrying transgenes that encode immunogens, as well as other antigens, elicit humoral and cell-mediated immune responses against the transgenic product. In general, the researchers reported success using human adenoviral vectors as vaccine carriers in non-human experimental systems, using immunization protocols that use high doses of recombinant adenoviral vectors that are predicted to elicit immune responses; or using immunization protocols that employ the sequential administration of adenoviral vectors that are derived from different serotypes, but that carry the same transgenic product as booster immunizations (Mastrangeli, et al., Human Gene Therapy, 7: 79-87 (1996 )).
[005] [005] Vectors derived from adenoviruses of species C (for example, Ad5, Ad6 and ChAd63) are the most immunogenic (Colloca et al., Sci.
[006] [006] Thus, there is a need for adenoviral vectors with high immunogenicity and a low, or absent, pre-existing immunity in humans. Preferably, these adenoviral vectors have a high productivity in terms of replication. BRIEF DESCRIPTION OF THE INVENTION
[007] [007] In a first aspect, the invention provides an isolated polynucleotide that encodes an adenovirus hexon protein comprising: THE)
[008] [008] In a second aspect, the invention provides an isolated polynucleotide that encodes an adenovirus, preferably an incompetent replication adenovirus comprising the polynucleotide the first aspect.
[009] [009] In a third aspect, the invention provides at least one isolated adenoviral capsid polypeptide encoded by a polynucleotide isolated from the first aspect.
[010] [010] In a fourth aspect, the invention provides an adenovirus encoded by a polynucleotide isolated from the first aspect or an isolated adenovirus, preferably an incompetent replication adenovirus, comprising a polynucleotide isolated according to the first aspect and / or at least one polypeptide adenoviral capsid isolated according to the third aspect.
[011] [011] In a fifth aspect, the invention provides a virus-like particle (VLP) encoded by a polynucleotide isolated from the first aspect.
[012] [012] In a sixth aspect, the invention provides a vector comprising a polynucleotide isolated from the first aspect.
[013] [013] In a seventh aspect, the invention provides a composition comprising (i) an adjuvant, (ii) a polynucleotide isolated from the first or second aspect, at least one adenoviral capsid polypeptide isolated from the third aspect, a fourth aspect adenovirus, a virus-like particle of the fifth aspect, or a vector of the sixth aspect, and optionally (iii) a pharmaceutically acceptable excipient.
[014] [014] In an eighth aspect, the invention provides a cell comprising (i) an adjuvant, (ii) a polynucleotide isolated from the first or second aspect, at least one adenoviral capsid polypeptide isolated from the third aspect, a fourth aspect adenovirus, a virus-like particle of the fifth aspect, or a vector of the sixth aspect.
[015] [015] In a ninth aspect, the invention provides a polynucleotide isolated from the first or second aspect, at least an adenoviral capsid polypeptide isolated from the third aspect, an adenovirus from the fourth aspect, a virus-like particle from the fifth aspect, or a vector the sixth aspect and / or the composition of the seventh aspect for use in the treatment or prevention of a disease.
[016] [016] In a tenth aspect, the invention relates to an in vitro method for producing an adenovirus or an adenovirus-like particle, comprising the steps of; (i) expressing a polynucleotide isolated from the first or second aspect in a cell, so that an adenovirus or adenovirus-like particle is assembled in the cell, (ii) isolating the adenovirus or adenovirus-like particle from the cell, or of the medium surrounding the cell. DETAILED DESCRIPTION OF THE INVENTION
[017] [017] Before the present invention is described below in detail, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It is also necessary to understand that the terminology used in the present is used only for the purpose of describing examples of specific achievements, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used in the present have the same meaning as is commonly understood by a person skilled in the art.
[018] [018] Preferably, the terms used here are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger HGW, Nagel B. and Kölbl H., Eds., Helvetica Chimica Acta, CH-4010 Basel , Switzerland (1995), and as described in “Pharmaceutical Substances: Syntheses, Patents, Applications” by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999; the “Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals”, edited by Susan Budavari et al., CRC Press, 1996; and United States Pharmacopeia-25 / National Formulary-20, published by United States Pharmcopeial Convention, Inc., Rockville Md., 2001.
[019] [019] Throughout the specification and claims that follow, and unless the context dictates otherwise, the word "understand" and grammatical variations such as "comprising" and "understood", will be understood to imply the inclusion of a characteristic, whole or step or group of characteristics, integers or steps declared, but not excluding any other characteristic, whole or step or group of integers or steps. In the following passages, different aspects of the invention are defined in more detail. Each aspect thus defined may be combined with any other aspect or aspects, unless stated otherwise. In particular, any characteristic indicated as preferred or advantageous can be combined with any other characteristic or characteristics indicated as preferred or advantageous.
[020] [020] Several documents are cited throughout this specification. Each of the documents cited here (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.), whether mentioned above or below, is hereby incorporated in full by reference. Nothing here should be construed as an admission that the invention is not entitled to anticipate such disclosure by virtue of a previous invention. DESCRIPTION OF THE FIGURES
[021] [021] Figure 1: Schematic view of the BAC GAd-GAG A / L / S carrier vector.
[022] [022] Figure 2: Schematic view of plasmid GAdNou19 GAG (DE1E3) BAC with deletions of E1 and E3.
[023] [023] Figure 3: Schematic view of plasmid GAdNou20 GAG (DE1E3) BAC with deletions of E1 and E3.
[024] [024] Figure 4: Productivity of GADNOU19 and GADNOU20 in Hek293 cells compared to the reference Ad5 vector carrying the same expression cassette.
[025] [025] Figure 5: Immunogenicity of the GADNOU19 and GADNOU20 vectors that encode the GAG antigen. The immunological potency of the GADNOU19 GAG (DE1DE3) and GADNOU20 GAG (DE1DE3) vectors was determined by ELISpot for IFN-γ. T cell responses are measured three weeks after immunization with 3x107 and 3x106 vp of each vector. The number of T cells that produce IFN-γ per million splenocytes in response to stimulation with the immunodominant gag peptide encoding a CD8 + epitope is shown. NUCLEOTIDE AND AMINO ACID SEQUENCES
[026] [026] Table 1 below provides an overview of the sequences referred to here (GADNOU + number: isolated adenoviral strain; *:
[027] [027] Tables 2a and 2b below provide the genomic limits / coordinates of CDSs, RNAs and ITRs in GADNOU genomes. They apply to any reference to the genomic elements listed here in the tables and are incorporated according to preference in the respective realization examples.
[028] [028] E3_Orf2 * indicates a putative Open Reading Phase (ORF) with a GTG as the initial codon. rc indicates reverse complement. The products generated by splicing are indicated by multiple pairs of coordinates. GADNOU
[029] [029] E3_Orf2 * indicates a putative Open Reading Phase (ORF) with a GTG as the initial codon. rc indicates reverse complement. The products generated by splicing are indicated by multiple pairs of coordinates. GADNOU
[030] [030] The invention relates to several aspects, as set out above in the summary of the invention. These aspects comprise alternative modalities and preferred modalities, which are described below.
[031] [031] In a first aspect, the invention provides an isolated polynucleotide that encodes an adenovirus hexon protein, as defined in the brief description of the invention above.
[032] [032] In a preferred embodiment, variant HVRs have at least 90% and more preferably at least 95% sequence identity with the respective SEQ ID NO. As an alternative to the definition made according to the percentage level of sequence identity, the variant HVRs can be defined according to the number of amino acid mutations within the respective SEQ ID NO. The number of mutations is as follows: instead of at least 85% sequence identity, up to 4 mutations in any HVR1, up to 2 mutations in any HVR2, up to 1 mutation in any HVR3, up to 1 mutation in any HVR4, up to 2 mutations in any HVR5, up to 1 mutations in any HVR6, and up to 3 mutations in any HVR7; instead of at least 90% sequence identity, up to 2 mutations in any HVR1, up to 1 mutation in any HVR2, up to 1 mutation and preferably no mutation in any HVR3, up to 1 mutation in any HVR4, up to 1 mutation in any HVR5, up to 1 mutation and preferably no mutation in any HVR6, and up to 2 mutations in any HVR7; instead of at least 95% sequence identity, up to 1 mutation in any HVR1, up to 1 mutation and preferably no mutation in any HVR2, up to 1 mutation and preferably no mutation in any HVR3, up to 1 mutation and preferably no mutation in any HVR4 , up to 1 mutation and preferably without mutation in any HVR5, up to 1 mutation and preferably without mutation in any HVR6, and up to 1 mutation in any HVR7.
[033] [033] As known in the prior art, for example, from Bradley et al. (J Virol., 2012 jan; 86 (2): 1267-72), neutralizing adenovirus antibodies target the hypervariable regions of the hexon and, by replacing the HVR regions of an adenovirus with seroprevalence, that adenovirus can bypass the system immune system in the immune host. Thus, although the above HVRs can be used with the respective hexon proteins defined below, they are of independent use for those hexon proteins and also for the penton and fiber proteins below, namely replacing the hexon HVRs in a different adenovirus with other hexon, penton and / or fiber.
[034] [034] In a preferred embodiment, the hexon protein according to A) comprises an amino acid sequence according to SEQ ID NO: 46 or a variant thereof with at least 85% sequence identity with SEQ ID NO: 46,
[035] [035] In a preferred embodiment, hexon variants have at least 90%, and preferably at least 95%, 96%, 97%, 98% or 99% sequence identity with the respective SEQ ID NO. Alternatively to the definition by a percentage level of sequence identity, the hexon variants can be defined by having a number of amino acid mutations within the respective SEQ ID NO. The number of mutations is as follows: instead of at least 85% sequence identity, up to 143 mutations in any hexon; instead of at least 90% sequence identity, up to 95 mutations in any hexon; instead of at least 95% sequence identity, up to 47 mutations in any hexon; instead of at least 96% sequence identity, up to 38 mutations in any hexon; instead of at least 97% sequence identity, up to 28 mutations in any hexon; instead of at least 98% sequence identity, up to 19 mutations in any hexon; instead of at least 99% sequence identity, up to 9 mutations in any hexon. It should be understood that the hexon variants do not have less sequence identity or more mutations in their HVRs than those defined for the respective HVRs above.
[036] [036] In one embodiment, the polynucleotide isolated from the first aspect further encodes an adenoviral penton protein comprising an amino acid sequence according to SEQ ID NO: 51 or 52, or a variant thereof with at least 85% identity with SEQ ID NO: 51 or 52. In a preferred embodiment, penton variants have at least 90%, and preferably at least 95%, 96%, 97%, 98% or 99% sequence identity with the respective SEQ ID NO. As an alternative to defining by a percentage level of sequence identity, the penton variants can be defined by having a number of amino acid mutations within the respective SEQ ID NO. The number of mutations is as follows: instead of at least 85% sequence identity, up to 97 mutations in any penton; instead of at least 90% sequence identity, up to 65 mutations in any penton; instead of at least 95% sequence identity, up to 32 mutations in any penton; instead of at least 96% sequence identity, up to 26 mutations in any penton; instead of at least 97% sequence identity, up to 19 mutations in any penton; instead of at least 98% sequence identity, up to 13 mutations in any penton; instead of at least 99% sequence identity, up to 6 mutations in any penton.
[037] [037] Preferably, the penton variants of SEQ ID NOs: 51 and 52 do not have D and, preferably, a G in position 289 and do not have D and, preferably, an N in position 341. More preferably, the variant of SEQ ID NO: 52 also does not have A and more preferably has a T in position 442.
[038] [038] In another example, the polynucleotide isolated from the first aspect (i.e., close to hexon and possibly to the penton protein) further encodes an adenoviral fiber protein comprising an amino acid sequence according to SEQ ID NO: 53 or 54, or a variant thereof with at least 85% sequence identity to SEQ ID NO: 53 or 54. In a preferred embodiment, fiber protein variants are at least 90%, and preferably at least 95%, 96%, 97%, 98% or 99% sequence identity with the respective SEQ ID NO. Alternatively to the definition by a percentage level of sequence identity, fiber variants can be defined as having a number of amino acid mutations within the respective SEQ ID NO. The number of mutations is as follows: instead of at least 85% sequence identity, up to 89 mutations in any fiber; instead of at least 90% sequence identity, up to 59 mutations in any fiber; instead of at least 95% sequence identity, up to 29 mutations in any fiber; instead of at least 96% sequence identity, up to 23 mutations in any fiber; instead of at least 97% sequence identity, up to 17 mutations in any fiber; instead of at least 98% sequence identity, up to 11 mutations in any fiber; instead of at least 99% sequence identity, up to 5 mutations in any fiber.
[039] [039] Preferably, the fiber proteins of SEQ ID NO: 53 do not have A and preferably have a P at position 181, do not have V and preferably have an I at position 474 and / or do not have the insertion of an S and, preferably, they do not have the amino acid insertion between positions 4 and 5.
[040] [040] In another embodiment, the polynucleotide isolated from the first aspect (i.e., close to hexon and possibly to the penton protein and / or fiber) further encodes a VA RNA II non-coding RNA comprising an amino acid sequence according to SEQ ID NO: 57, or a variant thereof with at least 85% sequence identity to SEQ ID NO: 57. Alternatively or additionally, it can encode a non-coding VA RNA I RNA comprising a nucleotide sequence according to SEQ ID NO: 55 or 56, or a variant thereof with at least 85% sequence identity to SEQ ID NO: 55 or 56, respectively. In a preferred embodiment example, VA RNA variants have at least 90%, and preferably at least 95%, 96%, 97%, 98% or 99% sequence identity with the respective SEQ ID NO. Alternatively to the definition by a percentage level of sequence identity, VA RNA variants can be defined by having a number of nucleotide mutations within the respective SEQ ID NO. The number of mutations is as follows: instead of at least 85% sequence identity, up to 25 mutations in VA RNA I and up to 26 mutations in VA RNA II; instead of at least 90% sequence identity, up to 16 mutations in VA RNA I and up to 17 mutations in VA RNA II; instead of at least 95% sequence identity, up to 8 mutations in any VA RNA; instead of at least 96% sequence identity, up to 6 mutations in any VA RNA; instead of at least 97% sequence identity, up to 5 mutations in any VA RNA; instead of at least 98% sequence identity, up to 3 mutations in any VA RNA; instead of at least 99% sequence identity, up to 1 mutation in any VA RNA.
[041] [041] Preferably, the VA RNA II variant of SEQ ID NO: 57 (a) does not have C in position 79 and / or does not have A in position 80 and, preferably, a T in position 79 and / or G in position 80 and (b) does not have A in position 81, and preferably a G in position 81. The VA RNA I variant of SEQ ID NO: 55 preferably does not have G in position 80 and preferably has an A in position 80.
[042] [042] A VA RNA according to the invention leads to improved production of adenovirus or adenovirus-like particles, as shown in Example 5.
[043] [043] It is preferable that the polynucleotide of the first aspect further comprises other adenoviral genes and nucleotide segments, which are adjacent to the hexon, penton and / or fiber gene in the adenovirus genome, using SEQ ID NOs: 1-10 as a reference. It is particularly preferred that the polynucleotide also comprises sequences necessary for packaging the polynucleotide into an adenoviral particle.
[044] [044] It is generally preferred that the polynucleotide isolated from the first aspect comprises at least one of the following: (a) a 5 'adenoviral end, preferably a 5' adenoviral inverted terminal repeat; (b) an adenoviral Ela region, or a fragment of said region selected from regions 13S, 12S and 9S; (c) an adenoviral Elb region, or a fragment of said region selected from the group consisting of the small T, large T and IX regions; (d) an adenoviral VA RNA region; or a fragment thereof selected from the group consisting of the VA RNA I and VA RNA II regions; (e) an E2b adenoviral region, or a fragment of said region selected from the group consisting of the small pTP, Polymerase and IVa2 regions;
[045] [045] These elements may be from the same adenovirus as the HVRs and / or hexon of the polynucleotide of the first aspect according to Table 1 (ie, from the same GADNOU) or from a different adenovirus, in particular from a different species, for example example, a human adenovirus, to form a chimeric adenovirus.
[046] [046] In some examples of carrying out the polynucleotide mentioned above, it may be desirable that the polynucleotide does not comprise one or more genomic regions, as described above (as in (a) to (m), such as, for example, the E3 region and / or E4) and / or it may be desirable for the polynucleotide to comprise an adenoviral gene that contains a deletion and / or mutation that renders said gene non-functional. In these preferred embodiments, suitable adenoviral regions are modified to not include the region (s) / gene (s) mentioned above or to make the selected regions / genes non-functional. One possibility of making genes non-functional is to introduce one or more artificial stop codons (for example, TAA) into the open reading phase of these genes. Methods for rendering the virus defective in replication are well known in the art (see, for example, Brody et al, 1994 Ann NY Acad Sci., 716: 90-101). A deletion can provide space for inserting transgenes, preferably into an expression cassette, such as a minigene cassette, as described in the present invention. In addition, deletions can be used to generate adenoviral vectors that are unable to replicate without the use of a packaging cell line or helper virus, as is well known in the art. Thus, a final recombinant adenovirus comprising a polynucleotide as described above that comprises one or more specified deletions of genes / regions or loss of function mutations can provide a safer recombinant adenovirus, for example, for gene therapy or vaccination.
[047] [047] Although the polynucleotide may not comprise at least one genomic region / gene as described in the present invention (such as, for example, E1, E3 and / or E4 region), specifically E1A, E1B, E2A, E2B, E3 ORF1, E3 ORF2 , E3 ORF3, E3 ORF4, E3 ORF5, E3 ORF6, E3 ORF7, E3 ORF8, E3 ORF9, E4 ORF6 / 7, E4 ORF6, E4 ORF5, E4 ORF4, E4 ORF3, E4 ORF2 and / or E4 ORF1, preferably E1A , E1B, E2A, E2B, E3 and / or E4, and / or can comprise an adenoviral gene that comprises a deletion and / or mutation that makes the at least one gene non-functional, it is desirable to keep the Ela and / or Elb region intact . An intact E1 region can be located at its native location in the adenoviral genome or placed at the site of a deletion in the native adenoviral genome (for example, in the E3 region).
[048] [048] In a preferred embodiment, the polynucleotide isolated from the first aspect further encodes one or more, preferably all, of the following adenoviral proteins: protein VI, protein VIII, protein IX, protein IIIa and protein IIIa and protein IVa2.
[049] [049] A skilled adenovirus technician is aware of how to determine the open reading frames that encode the adenoviral proteins specified above. He also knows the structure of adenoviral genomes and can map, without undue burden, the individual adenoviral regions and ORFs described in the present invention to any adenoviral genome.
[050] [050] In another embodiment, the polynucleotide isolated from the first aspect further encodes one or more heterologous proteins or fragments thereof. The one or more heterologous proteins or fragments thereof are preferably non-adenoviral proteins or fragments thereof. In an example of a preferred embodiment, the one or more non-adenoviral proteins or fragments thereof are one or more antigenic proteins or fragments thereof. Preferably, the one or more heterologous proteins or fragments thereof are part of one or more expression cassettes. Sequences encoding the heterologous protein and preferably an expression cassette comprising such (such) sequence (s) encoding the heterologous protein can be inserted, for example, into the deleted regions of an adenoviral genome defined in the present invention. An exemplary heterologous protein is the polypeptide according to SEQ ID NO: 74 or a variant thereof with at least 85% sequence identity to SEQ ID NO: 74.
[051] [051] In a second aspect, the invention provides an isolated polynucleotide that encodes an adenovirus, which comprises a polynucleotide of the first aspect, preferably comprising an adenoviral genome according to any of SEQ ID NOs: 1-10, or a variant with at least 85% sequence identity with SEQ ID NOs 1 to 10, respectively.
[052] [052] In an example of a preferred embodiment, it encodes an incompetent replicating adenovirus, preferably comprising an adenoviral genome according to any of SEQ ID NOs: 1-10 that does not have one or more of the E1A, E1B genomic regions / genes , E2A, E2B, E3 and E4.
[053] [053] Most preferably, it encodes a recombinant adenovirus, preferably comprising an adenovirus genome according to any of SEQ ID NOs: 1-10, or a variant sequence with at least 85% sequence identity with SEQ ID NOs: 1-10, respectively, preferably in which one or more heterologous protein or fragments thereof are inserted (carrier adenovirus). Preferably, one or more heterologous proteins or fragments thereof are inserted replacing one or more of the E1A, E1B, E2A, E2B, E3 ORF1, E3 ORF2, E3 ORF3, E3 ORF4, E3 ORF5, E3 ORF6, E3 genomic regions / genes ORF7, E3 ORF8, E3 ORF9, E4 ORF6 / 7, E4 ORF6, E4 ORF5, E4 ORF4, E4 ORF3, E4 ORF2 and E4 ORF1, more preferably E1, E3 and / or E4. The heterologous proteins or fragments thereof are preferably inserted as part of an expression cassette.
[054] [054] In an exemplary embodiment, the invention provides an isolated polynucleotide that encodes an adenovirus, which comprises a polynucleotide according to SEQ ID NO: 72 or 73, or a variant thereof with at least 85% identity sequence with SEQ ID NO: 72 or 73, respectively.
[055] [055] In a preferred embodiment example, the adenoviral genome variants have, instead of at least 85%, at least 90%, and preferably at least 95%, 96%, 97%, 98%, 99%, 99.5 or 99 0.9% sequence identity with the respective SEQ ID NO: 1, at least 90%, and preferably at least 95%, 96%, 97%, 98%, 99%, 99.5 or 99.9% sequence identity with SEQ ID NO 2, at least 90%, and preferably at least 95%, 96%, 97%, 98%, 99%, 99.5 or 99.9% identity sequence with SEQ ID NO 3, at least 90%, and preferably at least 95%, 96%, 97%, 98%, 99%, 99.5 or 99.9% sequence identity with the respective SEQ ID NO 4, at least 90%, and preferably at least 95%, 96%, 97%, 98%, 99%, 99.5 or 99.9% sequence identity with the respective SEQ ID NO 5, at least 90% , and preferably at least 95%, 96%, 97%, 98%, 99%, 99.5 or 99.9% of sequence identity with the respective SEQ ID NO 6, at least 90%, and preferably at least 95 %, 96 %, 97%, 98%, 99%, 99.5 or 99.9% sequence identity with the respective SEQ ID NO 7, at least 90%, and preferably at least 95%, 96%, 97%, 98 %, 99%, 99.5 or 99.9% sequence identity with the respective SEQ ID NO 8, at least 90%, and preferably at least 95%, 96%, 97%, 98%, 99%, 99 , 5 or 99.9% sequence identity with the respective SEQ ID NO 9, or at least 90%, and preferably at least 95%, 96%, 97%, 98%, 99%, 99.5 or 99, 9% sequence identity with the respective SEQ ID NO 10, (in each case, taking into account the deletions defined above).
[056] [056] In one embodiment, the polynucleotide isolated from the second aspect encodes a recombinant adenovirus, where at least one adenoviral genomic region of the recombinant adenovirus is derived from an adenovirus that does not comprise the hexon or hexon protein HVRs as defined above ( chimeric adenovirus). Preferably, the chimeric adenovirus is mainly chimeric or preferably only for the HVR hexon or hexon protein and optionally also for the penton protein and / or fiber as defined in the present invention. In other words, the polynucleotide encodes the hexon HVRs or hexon protein as defined above and, optionally, also for the penton and / or fiber as defined above, but one or more, preferably all other genomic regions are derived from a different adenovirus, particularly a different adenovirus according to SEQ ID NOs 1-10. The different adenovirus is preferably an adenovirus found naturally in a different host, more preferably a human adenovirus. This polynucleotide preferably also encodes one or more non-adenoviral heterologous proteins or fragments thereof, as defined above.
[057] [057] In general, it is preferred that the adenovirus is incompetent to replicate. For this purpose, it is preferable that the adenovirus does not have one or more of the E1A, E1B, E2A, E2B, E3 and E4 genomic regions or includes an exclusion and / or mutation in it that processes the genomic region or an expression product encoded by it be non-functional.
[058] [058] In a particularly preferred embodiment, the polynucleotide isolated from the first or the second aspect, in all its variants described in the present invention, may have a functionally impaired IVa2 gene, preferably a deletion or null mutation therein. This gene is involved in the packaging of viral DNA and its compromise leads to the production of virus-like particles. In this embodiment example, the polynucleotide isolated from the first or second aspect preferably encodes one or more non-adenoviral B cell epitopes and / or T cell epitopes.
[059] [059] In a third aspect, the invention provides at least one isolated adenoviral capsid polypeptide encoded by a polynucleotide of the first or second aspect. The at least one isolated adenoviral capsid polypeptide comprises at least one hexon with the HVRs defined above, preferably the hexon protein defined above, and optionally also the penton and / or the fiber protein defined above.
[060] [060] At least one isolated adenoviral capsid polypeptide can be obtained by expression in a cell. The expressed polypeptide can be optionally purified using standard techniques. For example, cells can be mechanically lysed or subjected to osmotic shock before being subjected to precipitation and chromatography steps, the nature and sequence of which will depend on the recombinant material to be recovered.
[061] [061] In a fourth aspect, the invention provides an adenovirus (also called an adenovirus vector or adenoviral vector in the present invention) comprising the polynucleotide isolated from the first or second aspect and / or the adenoviral capsid polypeptides from the third aspect.
[062] [062] In an exemplary embodiment, the invention provides an adenovirus comprising a polynucleotide according to SEQ ID NO: 72 or 73, or a variant thereof having at least 85% sequence identity with SEQ ID NO: 72 or 73, respectively.
[063] [063] The adenovirus may or may not comprise a polynucleotide of the first or second aspect. If this polynucleotide is not present in the adenovirus, it is preferable that it be supplied in trans (that is, by a genetic element other than the adenovirus genome incorporated in the adenovirus). It is generally provided by an auxiliary construct (for example, a plasmid or virus) or by the genome of an auxiliary construct in a packaging host cell (complementary cell as described in the present invention). It is further preferred that polynucleotides provided in trans are not included in the genome incorporated into the adenovirus, including homologues or other variant sequences of those polynucleotides. For example, if the polynucleotide provided in trans comprises a hexon, penton and / or fiber gene, the genome incorporated in the adenovirus does not comprise any polynucleotide that encodes a hexon, penton and / or fiber protein, respectively. More preferably, the polynucleotide provided in trans encodes at least one adenoviral capsid polypeptide as defined in the third aspect, that is, a hexon with the HVRs defined in the first or second aspect, preferably the hexon protein defined in the first or second aspect, and optionally also the penton protein and / or fiber (s) defined in the first or second aspect.
[064] [064] In the construction of adenovirus vectors for delivering a gene to a host, for example, a human cell or another mammalian cell, a series of adenovirus nucleic acid sequences can be used. For example, all or a portion of the early adenovirus-delayed E3 gene can be deleted from the adenovirus sequence that is part of the recombinant virus. The simian E3 function is believed to be irrelevant to the function and production of the recombinant virus particle. In some realization examples, adenovirus vectors can also be constructed with a deletion of at least the ORF6 region of the E4 gene and, more desirable, due to the redundancy in the function of that region, of the entire E4 region. Yet another vector of the present invention contains a deletion in the early delayed E2a gene. Deletions can also be made in any of the late genes from L1 to L5 of the simian adenovirus genome. Likewise, deletions in the intermediate genes IX and IVa2 can be useful for some purposes. Other deletions can be made in other structural or non-structural genes in adenoviruses. The deletions discussed above can be used individually, that is, an adenovirus sequence for use in the present invention can contain deletions only in a single region. Alternatively, deletions of whole genes or parts of them effective to destroy their biological activity can be used in any combination. For example, the adenovirus sequence may have deletions from the E1 and E4 regions, or from the E1, E2a and E3 regions, or from the E1 and E3 regions, or from the E1, E2a and E4 regions, with or without E3 deletion, and so on. onwards. Such deletions can be used in combination with other adenoviral gene mutations, such as temperature-sensitive mutations, to achieve a desired result.
[065] [065] An adenoviral vector without essential adenoviral sequences (for example, a selected region of Ela, Elb, E2a, E2b, E4 ORF6, L1 or L4) can be grown in the presence of missing adenoviral gene products, necessary for infectivity and spread of an adenoviral particle. These helper functions can be provided by culturing the adenoviral vector in the presence of one or more helper constructs (for example, a plasmid or virus) or a packaging host cell (complementary cell, as described in the present invention). See, for example, the techniques described for the preparation of a “minimal” human adenovirus vector in WO96 / 13597).
[066] [066] Useful constructs contain selected adenovirus gene sequences that complement the respective genes that are deleted and / or that are not expressed by the vector and the cell in which the vector is transfected. In one embodiment, the helper construct is defective in replication and contains essential and optionally additional adenovirus genes.
[067] [067] Auxiliary constructs can also be formed in polycation conjugates as described in Wu et al., J. Biol. Chem. 264: 16985-16987 (1989); K. J. Fisher and J. M. Wilson, Biochem. J., 299: 49 (April 1, 1994). An auxiliary construct can optionally contain a reporter gene. Several reporter genes are known in the art. The presence of a reporter gene in the auxiliary construct that is different from the transgene in the adenovirus vector allows both the adenovirus and the auxiliary construct to be monitored independently. This second reporter can be used to facilitate the separation between the resulting recombinant adenovirus and the auxiliary construct after purification.
[068] [068] To generate recombinant adenoviruses (Ad) with deletions in any of the genes described in the context of the preferred embodiments in the present invention, the function of the deleted gene region, if essential for virus replication and infectivity, is preferably provided to the recombinant virus by a cell or auxiliary construct, that is, a complementation or packaging cell. In many circumstances, a construct / cell that expresses human E1 can be used to transcomplement the vector used to generate the recombinant adenoviruses. This is particularly advantageous because, due to the diversity between the polynucleotide sequences of the invention and the human adenovirus E1 sequences found in the currently available packaging cells / constructs, the use of the human E1 containing packaging cells / constructs will prevent the generation of adenovirus replication authorities during the replication and production process. However, in certain circumstances, it will be desirable to use a construct / cell that expresses the products of the E1 gene for the production of a recombinant adenovirus with an E1 deletion.
[069] [069] If desired, the sequences provided in the present invention can be used to generate an auxiliary construct / cell or cell line that expresses at least the adenovirus E1 gene from an adenovirus according to any of the SEQ IDs NOS: 1-10 under the transcriptional control of a promoter for expression in a selected stem cell line, such as a HeLa cell. Inducible or constitutive promoters can be employed for this purpose. Examples of promoters are provided, for example, in the examples described in the present invention. Such E1-expressing cells are useful in the generation of E1-deleted vectors from recombinant adenoviruses. Additionally, or alternatively, the invention provides constructs / cells that express one or more adenoviral gene products, for example, Ela, Elb, E2a and / or E4 ORF6, preferably Ad5 E4 ORF6, which can be constructed using essentially the same procedures for use in the generation of recombinant adenoviral vectors. Such constructs / cells can be used to transcomplement adenovirus vectors excluded into essential genes that encode these products or to provide auxiliary functions necessary for packaging an auxiliary-dependent virus (for example, adeno-associated virus).
[070] [070] In general, when delivering an adenovirus vector by transfection, the vector is delivered in an amount of about 0.1 μg to about 100 μg of DNA, and preferably about 10 to about 50 μg of DNA up to about 1 x 104 cells to about 1 x 103 cells and preferably about 105 cells. However, the relative amounts of the vector's DNA to the host cells can be adjusted, taking into account factors such as the selected vector, the delivery method and the selected host cells. The introduction of the vector into a host cell can be achieved by any means known in the art or as described in the present invention, including transfection, infection and, for example, using CaPO4 transfection or electroporation.
[071] [071] For the construction and assembly of the desired recombinant adenovirus, the adenovirus vector can, in one example, be transfected in vitro in the presence of an auxiliary construct in the packaging cell line, allowing for homologous recombination between the helper sequences and the adenovirus vector sequences, allowing the adenovirus-transgene sequences in the vector to be replicated and packaged in virion capsids, resulting in recombinant viral vector particles, as is well known in the art. A recombinant adenovirus of the invention is useful, for example, in transferring a selected transgene to a selected host cell.
[072] [072] In an example of a preferred embodiment, adenovirus of the fourth aspect has a seroprevalence in less than 5% of human subjects and, preferably, no seroprevalence in human subjects, more preferably no seroprevalence in human subjects who have not previously been in contact with a non-human large ape adenovirus, more preferably with one or more, particularly all adenoviruses according to SEQ ID NOs: 1-10. In this context, it is preferable that human beings belong to an ethnic group selected among Europeans, indigenous peoples of Africa, Asians, indigenous peoples of America and Oceania. Methods for identifying the ethnic origin of a human subject are comprised in the state of the art (see, for example, WO2003 / 102236).
[073] [073] In another example of a preferred embodiment of a recombinant adenovirus, the adenovirus DNA is able to enter a mammalian target cell, that is, it is infectious. An infectious recombinant adenovirus of the invention can be used as a vaccine and for gene therapy as also described in the present invention. Thus, in another embodiment, it is preferred that the recombinant adenovirus comprises a molecule for delivery to a target cell.
[074] [074] The molecule for delivery to a target cell is preferably a heterologous polynucleotide, but it can also be a polypeptide or a small chemical compound, preferably having a therapeutic or diagnostic activity. In a particularly preferred embodiment, the molecule for delivery to a target cell is a heterologous polynucleotide comprising a 5 'adenovirus inverted terminal repeat sequence (ITR) and a 3' ITR. It will be evident to the specialist that the molecular size of the molecule must be chosen so that the capsid can form around and package the molecule, when the recombinant adenovirus is produced, for example, in a packaging cell. Thus, preferably the heterologous gene is a minigene that can have, for example, up to 7000 and at most up to 8000 base pairs.
[075] [075] In a fifth aspect, the invention provides a virus-like particle (VLP) encoded by a polynucleotide of the first or second aspect. Consequently, the VLP comprises at least one adenoviral capsid polypeptide isolated according to the third aspect.
[076] [076] According to the definition of VLP below, the VLP of the fifth aspect comprises substantially non-viral genomic DNA. VLPs, including adenovirus VLPs, have been used for vaccination, gene therapy or for direct administration of drugs, for example, anticancer drugs (Chroboczek et al., ACTA ABP BIOCHIMICA POLONICA, Vol. 61, No. 3/2014 ). Consequently, the VLP of the fifth aspect may comprise one or more epitopes of non-adenoviral B cells and / or non-adenoviral T cells, one or more non-adenoviral genes for gene therapy and / or one or more pharmaceutical agents, for example, anticancer agents. In one embodiment, the VLP preferably incorporates one or more non-adenoviral B cell epitopes and / or incorporates one or more non-adenoviral T cell epitopes.
[077] [077] In a sixth aspect, the invention provides a vector comprising a polynucleotide of the first or second aspect. In an example of a preferred embodiment, the vector is a plasmidial vector, for example, an expression vector. A plasmid vector can advantageously be used to generate a recombinant adenovirus as described in the present invention. As the sequence information for the new hexon, penton and fiber proteins and the VA RNAs of the invention are provided, said recombinant adenovirus is obtained, for example, by constructing a recombinant adenovirus that is encoded by the polynucleotide of the first or second aspect and any other adenoviral genomic region. Methods for constructing recombinant adenoviruses are well known in the art. Techniques useful for the preparation of recombinant adenoviruses are, for example, reviewed in Graham & Prevec, 1991 in: Methods in Molecular Biology: Gene Transfer and Expression Protocols, (Ed. Murray, EJ.), P. 109; and Hitt et al., 1997 “Human Adenovirus Vectors for Gene Transfer into Mammalian Cells” Advances in Pharmacology 40: 137-206. Additional methods are described in WO 2006/086284.
[078] [078] In order to express a polynucleotide of the first or second aspect, it is possible to subclone said polynucleotide in an expression vector that contains a strong promoter for direct transcription, preferably with an expression cassette. Suitable bacterial promoters are well known in the art, for example, E. coli, Bacillus sp. and Salmonella, and kits for such expression systems are commercially available. Likewise, eukaryotic expression systems for mammalian cells, yeast and insect cells are well known in the art and are also commercially available. See below for more details on the expression cassettes.
[079] [079] The particular expression vector useful for carrying genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells can be used. Standard bacterial expression vectors include plasmids such as plasmids based on pBR322, pSKF,
[080] [080] In a seventh aspect, the invention provides a composition comprising (i) an adjuvant, (ii) a polynucleotide isolated from the first or second aspect, at least one adenoviral capsid polypeptide isolated from the third aspect, the fourth aspect adenovirus, a virus-like particle of the fifth aspect, or a vector of the sixth aspect, and optionally (iii) a pharmaceutically acceptable excipient. Preferably, the adjuvant is an agonist of a receptor selected from the group consisting of type I cytokine receptors, type II cytokine receptors, TNF receptors, vitamin D receptor acting as a transcription factor and Toll-like receptors 1 ( TLR1), TLR-2, TLR 3, TLR4, TLR5, TLR-6, TLR7 and TLR9.
[081] [081] A composition comprising an adjuvant can be used as a vaccine, for example, for humans. For example, an activation of specific receptors can stimulate an immune response. Such receptors are known to those skilled in the art and comprise, for example, cytokine receptors, in particular type I cytokine receptors, type II cytokine receptors, TNF receptors; and vitamin D receptor acting as a transcription factor; and Toll-like receptors (Toll-like) 1 (TLR1), TLR-2, TLR3, TLR4, TLR5, TLR-6, TLR7 and TLR9. Agonists to these receptors have adjuvant activity, that is, they are immunostimulators. In an example of a preferred embodiment, the composition adjuvant may be one or more Toll-like receptor agonists. In a more preferred embodiment example, the adjuvant is a Toll-like 4 receptor agonist. In a specific embodiment example, the adjuvant is a Toll-like 9 receptor agonist. For adjuvant examples, see below. In addition, preferred pharmaceutically acceptable excipients are mentioned below.
[082] [082] In an eighth aspect, the invention provides a cell comprising a polynucleotide of the first or second aspect, and at least one adenoviral capsid polypeptide isolated from the third aspect, a fourth aspect adenovirus, a virus-like particle of the fifth aspect, or a vector of the sixth aspect.
[083] [083] Preferably, the cell is a host cell that expresses at least one adenoviral gene, or preferably all adenoviral genes, which are excluded or rendered non-functional, as explained above, to render the adenovirus incompetent to replicate. By the expression of this at least one gene, the host cell preferably allows replication of the incompetent replication adenovirus in another way. In one embodiment, the host cell expresses at least one adenoviral gene selected from the group consisting of E1A, E1B, E2A, E2B, E3 and E4. In particular, this at least one adenoviral gene is deleted or rendered non-functional in the adenoviral genome. This complement cell can be used for the propagation and rescue of incompetent replication adenoviruses, as they do not have, for example, one of the gene products mentioned above.
[084] [084] A cell can be selected from a bacterial cell, such as an E. coli cell, a yeast cell, such as Saccharomyces cerevisiae or Pichia pastoris, a plant cell, an insect cell, such as SF9 cells or Hi5 or a mammalian cell. Preferred examples of mammalian cells are Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK 293), HELA cells, human hepatoma cells (eg, Huh7.5), human Hep G2 hepatoma cells, Hep3B human hepatoma cells and the like.
[085] [085] If the cell comprises a polynucleotide according to the first or the second aspect, that polynucleotide may be present in the cell (i) in a freely dispersed form or (ii) integrated into the cell genome or mitochondrial DNA.
[086] [086] In another preferred embodiment, the cell is a host cell, preferably a 293 cell or a PER.C6 ™ cell, which expresses at least one adenoviral gene selected from the group consisting of E1a, E1b, E2a , E2b, E4, L1, L2, L3, L4 and L5.
[087] [087] Standard transfection methods can be used to produce bacterial, mammalian, yeast or insect cell lines. Any of the well-known procedures for introducing foreign polynucleotide sequences into host cells can be used. For example, commercially available liposome transfection kits, such as Lipofectamine® (Invitrogen), commercially available lipid-based transfection kits, such as Fugene (Roche Diagnostics), polyethylene glycol transfection, phosphate precipitation calcium, gene weapon (biolistics), electroporation, or viral infection and any of the other known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell can be used. It is only necessary that the specific genetic engineering procedure used is capable of successfully introducing at least one gene into the host cell capable of expressing the receptor.
[088] [088] Further examples of the cell are described in relation to the fourth aspect of the invention above.
[089] [089] In a ninth aspect, the invention provides a polynucleotide of the first or second aspect, at least an adenoviral capsid polypeptide isolated from the third aspect, the adenovirus of the fourth aspect, a virus-like particle of the fifth aspect, or a vector of the sixth aspect and / or the composition of the seventh aspect for use in the treatment or prevention of a disease. In one embodiment, treatment or prevention is by vaccination. In another example, the treatment is by gene therapy.
[090] [090] Adenoviruses are known to be useful in gene therapy and as vaccines. Pre-clinical and clinical studies have demonstrated the feasibility of vector design, robust expression of antigens and protective immunity using this system. Thus, an example of a preferred use is vaccination, for example, of humans. Detailed instructions on how adenoviruses are used and prepared for vaccination are provided as ample literature understood in the prior art and known to the person skilled in the art. Viral vectors, for example, based on a large non-human ape adenovirus represent an alternative to the use of human-derived Ad vectors for the development of genetic vaccines (Farina SF, J Virol. 2001 Dec; 75 (23): 11603- 13 .; Fattori E, Gene Ther.
[091] [091] This is due to the new adenovirus capsid protein sequences, including hexon, penton and fiber proteins, but in particular new hexon HVR sequences that represent the adenovirus epitopes most exposed to the surface. Consequently, it is expected that no or very few neutralizing antibodies specific to capsid proteins and in particular hexon HVRs according to the invention will be present in human blood sera. Thus, an advantage of the new sequences is that they can be used to improve adenoviruses of the prior art, which were designed, for example, for medical purposes. For example, the sequences can be used to, for example, replace one or more of the major structural capsid proteins or, in particular, only the hexon HVRs of a different adenovirus, for example, an adenovirus of the prior art, to obtain adenovirus recombinants enhanced with reduced seroprevalence in humans (chimeric adenoviruses). Like the new sequences and therefore the adenoviruses that have been redesigned as described, they will not find a significant inhibitory immune response in humans when administered, so that transduction efficiency and infectivity will be improved. Thus, it is hoped that such improved adenoviruses will be more effective vaccines, since entry into host cells and expression of antigens will not be impaired by any significant neutralizing antibody titer.
[092] [092] It is preferred that the vaccine comprises an adjuvant. Preferred immunological adjuvants are mentioned in the present invention and can be used in that vaccine.
[093] [093] If the use is a vaccination, a recombinant adenovirus of the invention can be administered in an immunologically and / or prophylactically effective dose, which is preferably 1 x 10 8 to 1 x 10 11 viral particles (ie 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 2.5 x1010 or 5 x 1010 particles).
[094] [094] In addition, for a vaccination that requires a booster dose, it is preferable to apply a heterologous “initial dose-boost” vaccination methodology: In vaccination, the polynucleotide of the first or second aspect, at least least one adenoviral capsid polypeptide isolated from the third aspect, the fourth aspect adenovirus, a virus-like particle from the fifth aspect, or a sixth aspect vector and / or the seventh aspect composition can be used for the initial immunization (priming) or for boosting immunization, in particular for a heterologous prime-boost vaccination. In an example of a preferred embodiment of the initial dose-boost vaccination strategy, two different vaccines can be used, for example, adenovirus, in which it is particularly advantageous that the polynucleotide of the first or second aspect, at least one adenoviral capsid polypeptide isolated from the third aspect, adenovirus of the fourth aspect, a virus-like particle of the fifth aspect, or a vector of the sixth aspect and / or the composition of the seventh aspect is used as a booster vaccine due to lack or neutralizing antibodies, for example, in humans humans.
[095] [095] A recombinant adenovirus prepared using a polynucleotide or recombinant adenoviral protein or a fragment according to the invention can be used to transduce a host cell with a polynucleotide, for example, DNA. Thus, an adenovirus preferably with poor replication, although infectious (i.e., capable of entering a host cell) can be prepared to express any custom protein or polypeptide in a host cell. Thus, in an example of a preferred embodiment, the therapy mentioned in the use according to the invention is gene therapy. Gene therapy can be an in vivo, ex vivo or in vitro gene therapy. Preferably, it is a somatic gene therapy. If an isolated polynucleotide, an isolated protein, a vector, a recombinant adenovirus and / or a pharmaceutical composition according to the invention is used for gene therapy and is administered to a subject to be treated, it is preferable that it is administered in a sufficiently high dose. large so that the treatment results in one or more cells of the patient being transfected, that is, transduced. If a recombinant adenovirus and / or a pharmaceutical composition according to the invention is administered by any of the preferred means of administration disclosed in the present invention, it is preferred that an effective dose, which is preferably 1 x 10 8 to 5 x 10 11 viral particles (i.e., 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 2.5 x 1010, 5 x 1010, 1 x 1011 or, more preferably, 5 x 1011 particles) is administered. In preferred embodiment examples, the heterologous polynucleotide that is comprised in the recombinant adenovirus of the invention is capable of expressing a protein or polypeptide in a host cell of the subject, wherein the protein or polypeptide comprises a signal peptide that affects the secretion of the protein or polypeptide of said host cell. For example, a patient in need of a particular protein can be treated using an adenovirus of the present invention that comprises a cDNA that encodes a secretible form of that protein.
[096] [096] In an example of further use of the present invention, a polynucleotide of the first or second aspect, at least one polypeptide of the adenoviral capsid isolated from the third aspect, the adenovirus of the fourth aspect, a particle similar to the virus of the fifth aspect, or a vector of the sixth aspect and / or the composition of the seventh aspect (hereinafter referred to as the pharmaceutical product according to the invention) is formulated to further comprise one or more diluents; conveyors; excipients, including fillers, binders, lubricants, glidants, disintegrants and adsorbents; and / or pharmaceutically acceptable preservatives.
[097] [097] The pharmaceutical product according to the invention can be administered by several well-known routes of administration, including oral, rectal, intragastric and parenteral administration, for example, intravenous, intramuscular, intranasal, intradermal, subcutaneous and similar routes. Parenteral, intramuscular and intravenous administration is preferred. Preferably, the pharmaceutical product according to the invention is formulated as a syrup, solution for infusion or injection, tablet, capsule, lozenge, liposome, suppository, plaster, band-aid, delayed capsule, powder, or a slow release formulation. Preferably, the diluent is water, buffer, buffered saline or a saline solution and the carrier is preferably selected from the group consisting of cocoa butter and vitebesol.
[098] [098] Particular preferred dosage forms for administering the pharmaceutical product according to the invention during use of the present invention are forms suitable for injectable use and include sterile aqueous solutions or dispersions and sterile powder for the extemporaneous preparation of sterile injectable solutions or dispersions . Typically, such a solution or dispersion will include a solvent or dispersion medium, containing, for example, aqueous solutions buffered with water, for example, biocompatible buffers, ethanol, polyol, such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures, surfactants or oils vegetables.
[099] [099] Infusion or injection solutions can be performed by any number of techniques recognized in the prior art, including, among others, the addition of preservatives, such as antibacterial or antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid or thimerosal. In addition, isotonic agents, such as sugars or salts, in particular sodium chloride, can be incorporated into infusion or injection solutions.
[0100] [0100] The preferred diluents of the present invention are water, acceptable physiological buffers, acceptable physiological saline solutions or saline solutions. Preferred vehicles are cocoa butter and vitebesol. Excipients that can be used with the various pharmaceutical forms of the medicament according to the invention can be chosen from the following non-limiting list: a) binders such as lactose, mannitol, crystalline sorbitol, dibasic phosphates, calcium phosphates, sugars, microcrystalline cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, polyvinylpyrrolidone and the like; b) lubricants such as magnesium stearate, talc, calcium stearate, zinc stearate, stearic acid, hydrogenated vegetable oil, leucine, glycerides and sodium stearyl fumarates; c) disintegrants such as starches, croscarmellose, sodium methylcellulose, agar, bentonite, alginic acid, carboxymethylcellulose, polyvinylpyrrolidone and the like.
[0101] [0101] Other suitable excipients can be found in the Handbook of Pharmaceutical Excipients, published by the American Pharmaceutical Association.
[0102] [0102] Certain amounts of the pharmaceutical product according to the invention are preferred for therapy or prophylaxis of a disease.
[0103] [0103] In a tenth aspect, the present invention relates to an in vitro method for producing an adenovirus or an adenovirus-like particle, comprising the steps of: (i) expressing an isolated polynucleotide from the first or second aspect in one cell, so that an adenovirus or an adenovirus-like particle is assembled in the cell,
[0104] [0104] The method optionally comprises an additional step before step (i) of introducing the polynucleotide isolated from the first or second aspect or a vector of the sixth aspect into the cell, for example, as described above.
[0105] [0105] It is generally preferred that the isolated polynucleotide encodes a fourth aspect adenovirus or a fifth aspect virus-like particle. Adenovirus is preferentially incompetent to replicate. The cell is preferably a seventh aspect cell. If the isolated polynucleotide encodes a replication-incompetent adenovirus, it is preferable that the cell is a helper cell or comprises an helper construct (eg, an helper plasmid or helper virus, for example, which is transduced with a helper construct, preferably infected with a helper virus, before or during step (i)), as described in the present invention, so that the helper cell or helper construct, respectively, expresses the genomic genes / regions that make the adenovirus incompetent to replicate.
[0106] [0106] "So that an adenovirus or adenovirus-like particle is assembled in the cell" means that in step (i), all the genes necessary for the assembly of the adenovirus or adenovirus-like particle, as described in the present invention, are expressed in the cell.
[0107] [0107] In one preferred embodiment, the isolated polynucleotide encodes a VA RNA II non-coding RNA and / or a VA RNA I non-coding RNA, as defined above. A VA RNA according to the invention leads to improved production of adenovirus or adenovirus-like particles obtained from the method as shown in Example 1. DEFINITIONS AND OTHER EXAMPLES OF CARRYING OUT THE INVENTION
[0108] [0108] Below are some definitions of terms frequently used in this specification. These terms will, in each case of their use, in the rest of the specification, the meaning and preferred meanings defined respectively.
[0109] [0109] As used in the present invention, the term "isolated" refers to a molecule that is substantially free of other molecules with which it is naturally associated. In particular, isolated means that the molecule is not in an animal body or in an animal body sample.
[0110] [0110] The term "polynucleotide" is intended to refer to a nucleic acid, that is, a biological molecule composed of a plurality of nucleotides. It includes DNA, RNA and synthetic analogues, for example, PNA. DNA is preferred.
[0111] [0111] The term “Open Reading Phase” (ORF, acronym for English Open Reading Frame) refers to a sequence of nucleotides that can be translated into amino acids. An ORF typically contains a start codon, a subsequent region usually with a length that is a multiple of 3 nucleotides, but does not contain a stop codon (TAG,
[0112] [0112] As used in the present invention, the terms "protein", "peptide", "polypeptide", "peptides" and "polypeptides" are used interchangeably. These terms refer to naturally occurring peptides, for example, naturally occurring proteins and synthesized peptides that can include naturally occurring or non-naturally occurring amino acids. Peptides can also be chemically modified by modifying a side chain or a natural or unnatural amino-terminal or free carboxy-terminal amino acid.
[0113] [0113] An adenovirus (Ad) is an icosahedral non-enveloped virus that has been identified in several hosts of birds and mammals.
[0114] [0114] The adenoviral virion has icosahedral symmetry and, depending on the serotype, diameter from 60 to 90 nm. The icosahedral capsid comprises three main proteins, hexon (II), penton base (III) and a knobbed fiber protein (IV) (W.C. Russel, J. Gen. Virol., 81: 2573-2604 (2000)).
[0115] [0115] One aspect of the pre-existing immunity seen in humans is humoral immunity, which can result in the production and persistence of specific antibodies to adenoviral proteins. The humoral response caused by the adenovirus is mainly directed against the hypervariable regions of the hexon structural protein. Adenoviruses isolated from large non-human apes are closely related to adenoviruses isolated from humans, as demonstrated by their efficient propagation in cells of human origin.
[0116] [0116] The capsid can be modified as described in the present invention by incorporating non-adenoviral polypeptides, such as epitopes for T and / or B cells.
[0117] [0117] The term "hexon protein" refers to the hexon (II) protein comprised in an adenovirus. A hexon protein or a variant thereof according to the invention has the same function as a hexon protein or a fragment thereof in an infectious adenovirus virus.
[0118] [0118] The term “hypervariable region” (HVR) refers to domains with high sequence variation between strains, located on the surface exposed to the hexon protein solvent, exposed outside the viral capsid. They are the main determinants of neutralizing antibodies. HVRs can be identified, for example, by sequence alignment with other hexon proteins.
[0119] [0119] By "adenoviral penton protein", we mean the base penton protein (III) comprised in an adenovirus. An adenoviral penton protein is characterized by being located in the corners of the capsid's icosahedral symmetry. A penton protein or a variant thereof according to the invention has the same function as a penton protein in an infectious adenovirus virus. Thus, an adenovirus comprising said penton protein or its variant preferably as a capsid protein is capable of entering a host cell, which can be tested as described above. In addition, a functional penton protein has an affinity for the adenovirus fiber protein. The qualified technician is well aware of how to test protein-protein affinities. To determine whether a first protein is able to bind to a second protein, it can use, for example, a yeast double hybrid system genetic assay or a biochemical assay, such as a pull-down assay, an enzyme-linked immunosorbent assay. (ELISA), a fluorescence activated cell separation assay (FACS) or a plasmon resonance assay. When using pull-down or plasmon resonance assays, it is useful to fuse at least one of the proteins to an affinity marker, such as HIS, GST, or other, as is well known in the state of the art in biochemistry.
[0120] [0120] The term "fiber protein" refers to the knobbed fiber (IV) protein comprised in an adenovirus. A fiber protein or a variant thereof according to the invention has the same function as a fiber protein or a fragment thereof in an infectious adenovirus virus. Thus, an adenovirus comprising said fiber protein or fiber variant protein preferably as a capsid protein is capable of entering a host cell, which can be tested as described above. In addition, a functional fiber protein has an affinity for an adenovirus penton protein. In addition, a functional adenoviral fiber protein in its glycosylated form is able to trimerize. Thus, it is also preferred that the variant form is capable of being glycosylated and / or forming a trimer. Affinity, including trimerization, can be tested as described above, and glycosylation assays are also well known in the art.
[0121] [0121] "VA RNA" (RNA associated with viruses) is a type of non-coding RNA found in adenovirus. It plays a role in regulating translation. There are two copies of this RNA called VAI or VA RNA I and VAII or VA RNA II. The two VA RNA genes are distinct genes in the adenovirus genome. VA RNA I is the main species with VA RNAII expressed at a lower level. Neither of the transcripts is polyadenylated and both are transcribed by PolIII.
[0122] [0122] The term "identity" or "identical" in the context of polynucleotide, polypeptide or protein sequences refers to the number of residues in the two sequences that is identical when the sequences are aligned for maximum matching. Specifically, the sequence identity percentage of two sequences, whether nucleic acid or amino acids, is the number of exact matches between two aligned sequences, divided by the length of the shortest sequence and multiplied by 100. The alignment tools that can be used to align two strings are well known to those skilled in the art and can, for example, be obtained on the World Wide Web, for example, Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) for polypeptide alignments or MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/) or MAFFT (http://www.ebi.ac.uk/Tools/msa/mafft/) for alignments polynucleotides or WATER (http://www.ebi.ac.uk/Tools/psa/ emboss_water /) for polynucleotide and polypeptide alignments. Alignments between two sequences can be performed using standard parameter settings, for example, for MAFFT, preferably: Matrix: Blosum62, Gap Open 1.53, Gap Extend 0.123, for the alignment of polynucleotides with WATER, preferably: MATRIX: DNAFULL, Gap Open: 10.0, Gap Extend 0.5 and for the alignment of polypeptides with WATER, preferably: Matrix: BLOSUM62, Gap Open: 10.0, Gap Extend: 0.5. Those skilled in the art understand that it may be necessary to introduce gaps in any sequence to produce satisfactory alignment. The “best sequence alignment” is defined as the alignment that produces the largest number of identical residues aligned while having the minimum number of gaps.
[0123] [0123] The term "variant", with respect to a polypeptide, generally refers to a modified version of the polypeptide, for example, a mutation, so that one or more amino acids of the polypeptide can be deleted, inserted, modified and / or replaced. In general, the variant is functional, which means that an adenovirus comprising the functional variant is capable of infecting a host cell. More specific functions are defined in the present invention and take precedence over the general definition. A "mutation" or "amino acid mutation" can be a substitution, deletion and / or insertion of amino acids ("e" can be applied if there is more than one mutation). Preferably, the mutation is a substitution (i.e., a conservative or non-conservative amino acid substitution), more preferably a conservative amino acid substitution. In some embodiments, a substitution also includes the exchange of a naturally occurring amino acid with a non-naturally occurring amino acid. A conservative substitution comprises the replacement of one amino acid with another amino acid having a chemical property similar to the amino acid that is substituted. Preferably, the conservative substitution is a substitution selected from the group consisting of: (i) a substitution of a basic amino acid with a different basic amino acid; (ii) a replacement of an acidic amino acid with a different acidic amino acid; (iii) a replacement of an aromatic amino acid with a different aromatic amino acid; (iv) a replacement of a non-polar aliphatic amino acid with another non-polar aliphatic amino acid; and (v) a replacement of an uncharged polar amino acid with a different uncharged polar amino acid.
[0124] [0124] A basic amino acid is preferably selected from the group consisting of arginine, histidine and lysine. An acidic amino acid is preferably aspartate or glutamate. An aromatic amino acid is preferably selected from the group consisting of phenylalanine, tyrosine and tryptophan. A non-polar aliphatic amino acid is preferably selected from the group consisting of glycine, alanine, valine, leucine, methionine and isoleucine. An uncharged polar amino acid is preferably selected from the group consisting of serine, threonine, cysteine, proline, asparagine and glutamine. In contrast to a conservative amino acid substitution, a non-conservative amino acid substitution is the exchange of an amino acid for any amino acid that does not fall within the conservative substitutions of (i) to (v) described above.
[0125] [0125] The means for determining the sequence identity are described above.
[0126] [0126] The amino acids of a protein can also be modified, for example, they can be chemically modified. For example, the side chain or a free amino-terminal or carboxy-terminal portion of an amino acid of the protein or polypeptide can be modified, for example, by glycosylation, amidation, phosphorylation, ubiquitination, etc. Chemical modification can also occur in vivo, for example, in a host cell, as is well known in the art. For example, a suitable chemical modification motif, for example, a glycosylation sequence motif present in the protein's amino acid sequence, will cause the protein to be glycosylated. Unless a modification leads to a change in the identity of a modified amino acid (for example, a substitution or deletion), a modified polypeptide is within the scope of the polypeptide as mentioned with respect to some SEQ ID NO :, that is, it is not a variant polypeptide as defined in the present invention.
[0127] [0127] The term "variant", with respect to a polynucleotide, refers in general to a modified version of the polynucleotide, for example, a mutation, so that one or more nucleotides of the polynucleotide can be deleted, inserted, modified and / or replaced. In general, the variant is functional, which means that an adenovirus comprising the functional variant is capable of infecting a host cell. More specific functions are defined in the present invention and take precedence over the general definition. A "mutation" can be a substitution, deletion and / or insertion of nucleotides (and "e" can be applied if there is more than one mutation). Preferably, the mutation is a substitution, more preferably, it causes an amino acid substitution, and more preferably it causes a conservative amino acid substitution.
[0128] [0128] An "antigenic protein or its fragment" (where the fragment is also antigenic) is capable of eliciting an immune response in a mammal. Preferably, it is a tumor antigen or an antigen derived from a pathogen. The term "pathogen" refers to any organism that can cause disease in a subject. The pathogen includes, but is not limited to, bacteria, protozoa, fungi, nematodes, viroids, viruses and parasites, in which each pathogen is capable, alone or in conjunction with another pathogen, of causing disease in vertebrates, including but not limited to mammals, and including, but not limited to, humans. As used in the present invention, the term "pathogen" also encompasses organisms that may not be pathogenic in a non-immunocompromised host, but that are pathogenic in an immunocompromised host.
[0129] [0129] In general, the adenoviral genome is well characterized. There is a general conservation in the general organization of the adenoviral genome in relation to the specific open reading phases similarly positioned, for example, the location of the genes E1A, E1B, E2A, E2B, E3, E4, LI, L2, L3, L4 and L5 of each virus. Each end of the adenoviral genome comprises a sequence known as an inverted terminal repeat (ITR), necessary for viral replication. The virus also comprises a virus-encoded protease, needed to process some of the structural proteins needed to produce infectious virions. The structure of the adenoviral genome is described based on the order in which viral genes are expressed after transduction in host cells. More specifically, viral genes are referred to as early (E, early English) or late (L, late English) genes, according to the occurrence of transcription before or after the start of DNA replication. In the initial phase of transduction, the adenovirus E1A, E1B, E2A, E2B, E3 and E4 genes are expressed to prepare the host cell for viral replication. During the late stage of the infection, the expression of the late L1-L5 genes, which encode the structural components of the virus particles, are activated.
[0130] [0130] The term "vector", as used in the present invention, includes any vectors known to the person skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus (Ad) vectors (for example, example, Ad5, Ad11, Ad26 non-replicating vectors, Ad35, Ad49, ChAd3, ChAd4, ChAd5, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37 , ChAd38, ChAd44, ChAd63 and ChAd82 or competent replication vectors Ad4 and Ad7 known in the prior art, for example, in WO 2005/071093 A2), adenoassociated virus vectors (AAV) (for example, AAV type 5), vectors alphavirus (for example, Venezuelan equine encephalitis virus (VEE), Sindbis virus (SIN), Semliki forest virus (SFV) and VEE-SIN chimeras), herpes virus vectors, measles virus vectors, virus vectors smallpox (for example, vaccinia virus, vaccinia virus, vaccini virus modified Ankara (MVA), NYVAC (derived from the Copenhagen strains of the vaccinia virus) and avipox: canarypox (canarypox virus) (ALVAC) and fowlpox (Avian Bouba) (FPV) vectors and vesicular stomatitis virus, particles similar to viruses or bacterial spores. A vector also includes expression vectors, cloning vectors and vectors that are useful for generating recombinant adenoviruses in host cells.
[0131] [0131] As mentioned above, a "heterologous protein or fragment thereof" may be a non-adenoviral protein or a fragment thereof, in particular an antigenic protein or a fragment thereof.
[0132] [0132] The term "expression cassette" refers to a nucleic acid molecule that comprises at least one nucleic acid sequence that must be expressed, along with its transcription and translation control sequences. Changing the expression cassette will cause the vector in which it is embedded to direct the expression of a different sequence or combination of sequences. Because the restriction locations are preferably designed to be present at the 5 'and 3' ends, the cassette can be easily inserted, removed or replaced with another cassette. Preferably, an expression cassette includes cis regulatory elements for efficient expression of a given gene, such as promoter, initiation site and / or polyadenylation site. More specifically in relation to the present invention, an expression cassette contains all the additional elements necessary for the expression of the polynucleotide of the first or the second aspect in the host cells. Thus, a typical expression cassette contains a promoter operatively linked to the polynucleotide of the first or second aspect and the necessary signals for efficient polyadenylation of the transcript, ribosome binding and translation termination sites. Additional elements of the cassette may include, for example, facilitators / enhancers. An expression cassette must also contain a transcription termination region downstream of the structural gene to provide efficient termination. The termination region can be obtained from the same gene as the promoter sequence or it can be obtained from different genes.
[0133] [0133] As used in the present invention, the term
[0134] [0134] The term "replication competent" recombinant adenovirus (AdV) refers to an adenovirus that can replicate in a host cell in the absence of any recombinant helper proteins comprised in the cell. Preferably, a "replication-competent" or "replication-competent" adenovirus comprises the following essential intact or functional early genes: E1A, E1B, E2A, E2B, E3 and E4. Wild-type adenoviruses isolated from a particular animal will be competent for replication in that animal.
[0135] [0135] The term recombinant AdV “with replication defect” or “replication incompetent” refers to an adenovirus that has become unable to replicate because it was designed to comprise at least one functional deletion, that is, a deletion that impairs function of a gene without removing it completely, for example, introduction of artificial stop codons, deletion or mutation of active sites or domains of interaction, mutation or deletion of a regulatory sequence of a gene etc., or a complete deletion of a gene that encodes a genetic product essential for viral replication, such as one or more of the adenoviral genes selected from E1, E2, E3 and E4. The recombinant adenoviruses of the invention are preferably defective in replication.
[0136] [0136] The term "recombinant adenovirus" refers in particular to an adenovirus that is modified to comprise a heterologous polynucleotide and / or heterologous polypeptide sequence. “Heterologist” can mean from another adenovirus strain, in particular a strain from a different host (for example, a human host, therefore, from a human adenovirus such as Ad3 or Ad5) or from a non-adenoviral organism, such as an antigen derived from a pathogen as described in the present invention, or from human, as a human tumor antigen. Thus, the term comprises chimeric adenoviruses and transporters, respectively.
[0137] [0137] As used in the present invention, the term "virus-like particle" or "VLP" refers to an empty non-replicating viral envelope, derived in this case from an adenovirus. VLPs are generally composed of one or more viral proteins, such as, among others, proteins referred to as capsid proteins, coating, envelope, surface and / or envelope. They contain functional viral proteins responsible for cell penetration by the virus, which ensures efficient cell entry. VLPs can form spontaneously after recombinant protein expression in an appropriate expression system. Specific methods for producing VLPs are known in the art. Adenovirus VLPs, in particular, can be produced by impairing functionally, for example, deleting or introducing a null mutation in the Iva2 gene of an adenovirus, involved in the packaging of viral DNA (Ostapchuk et al. J Virol. 2011 jun; 85 (11 ): 5524-5531). The presence of VLPs can be detected using conventional techniques known in the art, such as electron microscopy, X-ray crystallography and the like. See, for example, Baker et al., Biophys. J. (1991) 60: 1445-1456; Hagensee et al., J. Virol. (1994) 68: 4503-4505. For example, cryoelectronic microscopy can be performed on vitrified aqueous samples of the VLP preparation in question and images recorded under appropriate exposure conditions.
[0138] [0138] "Substantially no viral genomic DNA" included in a VLP means that there is not enough viral genomic DNA in the VLP or enough viral DNA in the VLP to allow replication of the virus in a VLP-infected cell, and there is no DNA expression that would complement the DNA in the VLP, so that virus replication can occur.
[0139] [0139] In addition to the above, an "epitope", also known as an antigenic determinant, is the segment of a macromolecule recognized by the immune system, specifically by antibodies, B cells or T cells. In the context of the present invention it is preferred that the term "Epitope" refers to the segment of protein or polyprotein that is recognized by the immune system. Epitopes generally consist of chemically active surface clusters of molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by the fact that the bond to the former, but not to the latter, is lost in the presence of denaturing solvents.
[0140] [0140] A "non-adenoviral T cell epitope" is an epitope that can be displayed on the surface of an antigen presenting cell, where it is attached to an MHC molecule. In humans, professional antigen presenting cells are specialized to present MHC class II peptides, while most nucleated somatic cells have MHC class I peptides. T cell epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length, whereas MHC class II molecules have longer peptides, from 13 to 17 amino acids in length.
[0141] [0141] A "non-adenoviral B cell epitope" is an epitope that is recognized as a three-dimensional structure on the surface of native B-cell antigens.
[0142] [0142] B and T cell epitopes can be predicted with in silico tools, for example, the IEDB Analysis Resource online B or T cell prediction tools.
[0143] [0143] The term "has one or more non-adenoviral B cell epitopes" means that one or more epitopes are incorporated into the capsid so that they are recognized by B cells. The term "incorporates one or more B / T cell epitopes non-adenoviral ”means that the epitope is contained in the VLP without being incorporated into the capsid or is incorporated into the capsid. If it is incorporated into the capsid, it may or may not be presented to the outside, in order to be recognized by immune cells.
[0144] [0144] An "immunological adjuvant" or simply "adjuvant" is a substance that accelerates, prolongs and / or improves the quality and / or strength of an immune response to an antigen / immunogen, compared to the administration of the antigen alone, thus reducing the amount of antigen / immunogen needed in a given vaccine and / or the frequency of injection needed to generate an appropriate immune response to the antigen / immunogen of interest. Examples of adjuvants that can be used in the context of the composition according to the present invention are precipitates similar to aluminum hydroxide gel (alum); AlPO 4; alhydrogel; bacterial products from the outer membrane of Gram-negative bacteria, in particular monophosphoryl lipid A (MPLA), lipopolysaccharides (LPS), muramyl dipeptides and their derivatives; incomplete Freund's adjuvant; liposomes, in particular neutral liposomes, liposomes containing the composition and optionally cytokines; non-ionic block copolymers; Adjuvant ISCOMATRIX (Drane et al., 2007); Unmethylated DNA comprising CpG dinucleotides (CpG motif), in particular CpG
[0145] [0145] The term "vaccination" in the context of the present invention is an active immunization, which is an induction of a specific immune response by the administration (for example, subcutaneous, intradermal, intramuscular, oral, nasal) of an antigen (substance that the The immune system of the vaccinated individual recognizes it as foreign and is therefore immunogenic) in an appropriate immunogenic formulation. The antigen is therefore used as a trigger for the immune system to build a specific immune response to the antigen. A vaccination within the scope of the present invention can, in principle, be carried out both in the therapeutic and in the prophylactic sense. It includes vaccination against pathogens as described in the present invention to treat or prevent infectious diseases or vaccination to treat or prevent non-infectious diseases, such as cancer. In the case of non-infectious diseases, the antigen is preferably a cell membrane antigen,
[0146] [0146] "Initial immunization" or "priming", as used in the present invention, refers to the administration of a vaccine to induce / generate an immune response in a mammal and "boosting immunization" or "boosting" refers to administration of a vaccine to improve an immune response in a mammal. The term "heterologous initial immunization-booster" means that the vaccine to induce / generate the immune response (priming) in a mammal and the vaccine to improve the immune response (boosting) in a mammal are different. The heterologous initial-boost immunization strategy is useful if a subject, for example, the patient has developed antibodies against a first vector and reinforcement is needed. In this context, a first vaccine (prime) and a second vaccine (boost), for example, adenovirus, are sufficiently different if the antibody response induced during the initial immunization (priming) by the first vaccine does not prevent more than 70% or preferably more 80% of the second vaccine particle administered to increase entry into the cell nucleus of the animal that has undergone boosting immunization.
[0147] [0147] The term "gene therapy" can be broadly defined as the concept of targeted introduction of foreign genetic material into a cell, tissue or organ to correct defective genes, with the aim of improving a patient's clinical condition. As used in the present invention, the term "gene therapy" preferably refers to "somatic therapy" rather than "germ line therapy", which would induce inheritable changes passed from generation to generation, so that somatic therapy restricts the effect therapeutic to the treated individual. Gene therapy, preferably somatic therapy, can be further discriminated against by a quick and easy direct gene transfer to the organism (“in vivo”) or by a sophisticated, but more specific and controllable gene transfer to explanted tissues or cells (“Ex vivo” or “in vitro”), which are reimplanted after treatment.
[0148] [0148] The term "neutralizing antibody" refers to an antibody that binds to an adenovirus epitope and prevents it from producing a productive infection in a host cell or prevents the transduction of a target cell with an incompetent replication vector that expresses a transgene, for example, adenovirus DNA is able to enter a cell, in particular a host cell.
[0149] [0149] Various modifications and variations of the invention will be evident to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in relation to examples of specific preferred embodiments, it should be understood that the present invention, as claimed, should not be unduly limited to such specific examples. In fact, it is intended that various modifications in the modes described for carrying out the invention, which are obvious to those skilled in the relevant fields, are included and covered by the present invention.
[0150] [0150] The invention is described by means of the following examples that should be interpreted as merely illustrative and not limiting the scope of the invention.
[0151] [0151] The construction of the vectors pGADNOU19 and pGADNOU20 continued with the steps provided below. The vectors pGADNOU19 and pGADNOU20 were derived from wild-type Adenovirus strains isolated from stool samples obtained from large healthy non-human apes, using standard procedures. Wild type viruses were isolated by inoculating monolayers of HEK 293 and A549 cells with extracts of feces. Cell monolayers were observed daily for the appearance of a cytopathic effect. Samples with positive results by observation under a microscope were collected and then the cells were lysed by freeze-thaw (-70ºC / 37ºC). The clarified cell lysate was used for the propagation of the virus through the infection of fresh cells grown in monolayers. After two passages of virus amplification, the adenovirus was purified using standard procedures. The viral genome was extracted from viruses purified by digestion with SDS / proteinase K, followed by extraction with phenol-chloroform. The purified adenoviral DNA was cloned into a plasmid carrier vector to be modified by performing the following deletions in the viral genome: 1) deletion of the E1 region (from 461 bp to 3402 bp) of the viral genome 2) deletion of the E3 region (from 28472 bp to 31996 bp) of the viral genome.
[0152] [0152] The genomes of purified DNA from the GADNOU viruses were first sequenced and then the DNA sequence information used to construct a carrier vector to clone the entire GAd genome by homologous recombination. The transporter vector was designed to introduce exclusion from the E1 region (nucleotide coordinates: 461-3402).
[0153] [0153] The shuttle plasmid (shuttle) was designed to contain restriction enzyme sites (PmeI) that are present only at the end of both ITRs to allow the release of viral DNA from plasmid DNA.
[0154] [0154] GADNOU with genomic DNA was isolated by digestion with Proteinase K followed by extraction with phenol / chloroform. The vectors pGADNOU19 and pGADNOU20 were obtained by homologous recombination in the E. coli strain BJ5183. The cloning of the viral DNA was obtained by co-transforming the cells of the E.coli strain BJ5183 with purified WT viral DNA and the BAC GAd-GAG A / L / S shuttle. The homologous recombination between the pIX genes, the right ITR DNA sequences present at the ends of the BAC shuttle (digested with AscI) and the viral genomic DNA allowed its insertion in the BAC vector, while excluding the E1 region that was replaced by the cassette of expression, generating the vectors ΔE1 / GAG (BAC) GADNOU19 GAG BAC and GADNOU20 GAG BAC. The screening was performed by restriction analysis and PCR sequencing in the hexon region.
[0155] [0155] The construction strategy was based on two consecutive steps, as described below: a) Replacement of the E3 region by the Amp-LacZ-SacB selection cassette: The Amp-LacZ-SacB selection cassette was obtained from the BAC GAD -GAG A / G / S shuttle by PCR using the FW oligonucleotides (5'- GGATTACACCAAGATCTTTGCTGTC
[0156] [0156] The productivity of the two adenoviral vectors of large non-human apes, GADNOU19 and GADNOU 20, carrying the E1 deletion and expressing the GAG antigen was evaluated in Hek293 adherent cells. Productivity was assessed by infecting adherent T25 cells with purified viruses using multiplicity of infection (MOI) 100 and MOI 300 vp / cells, compared to the reference Ad5 vector carrying the same expression cassette. The infected cells were collected three days after infection, when the total cytopathic effect was evident; the virus was released from the infected cells by three cycles of freezing / thawing (-70ºC / 37ºC) and the lysates were then clarified by centrifugation. The clarified lysates were quantified by quantitative PCR with primers and a probe complementary to the CMV promoter region. The oligonucleotide sequences are as follows: CMVfw - 5'- CATCTACGTATTAGTCATCGCTATTACCA-3 '(SEQ ID NO: 69), CMVrv - 5'- GACTTGGAAATCCCCGTGAGT-3' (SEQ ID NO: 70), probe CMVFGGGGTGA- 3 '(SEQ ID NO: 71). The qPCRs were performed in an ABI Prism 7900 - Applied Biosystem sequence detector. The resulting specific productivity expressed in viral particles per cell (vp / cell) of GADNOU19 and GADNOU expressing GAG was significantly higher than the reference vector Ad5 carrying the same expression cassette (Figure 1).
[0157] [0157] Justification for improving productivity: The adenoviral genomes of the invention belong to the adenovirus group C, which are known to have a high immunological potency. At the same time, group C viruses are characterized by relatively low productivity. The inventors found that the adenoviral genomes of the invention contain a particular genomic characteristic that is different from many other group C adenoviruses. The characteristic is represented by a pair of non-coding RNAs present in the genome (so-called virus-associated RNAs I and II ( VA RNAs I and II)), each about 170 nucleotides in length and separated by about 60 nucleotides. In general, VA RNAs I and II are present, but there are cases (group A viruses and some group B viruses) in which only VA RNA I is present. It is known that these RNAs are related to the interference of the virus in the cellular defense mechanism.
[0158] [0158] When analyzing the known adenovirus sequences, the inventors found that the VA RNAs I and II of the genomes of the invention do not resemble the VA RNAs I and II sequences of other group C adenoviruses (for example, Ad5 and Ad2 humans, as well as many adenoviruses isolated from chimpanzees belonging to group C), but instead resemble the VA RNAs I and II of groups B and E. The mean sequence identities of the VA RNA I and II sequences within and between the groups were calculated and are shown in Table 3 below.
[0159] [0159] Average percentage of VA RNA I sequence identity in groups C *, B, E, D, A, C and VA RNA II in groups C *, B, E, D, C and the average percentage of identity of sequence between group C * in relation to the other groups. The C * group represents GADNOU viruses according to the invention.
[0160] [0160] Therefore, it is believed that these RNAs lead to greater replication of the viruses. According to the inventors' best knowledge, VA RNAs have not yet been correlated with improved adenovirus productivity.
[0161] [0161] The immunogenicity of two GADNOU (GADNOU19 GAG (DE1E3), SEQ ID NO: 72 and GADNOU20 GAG (DE1E3), SEQ ID NO: 73) vectors encoding HIV-1 gag (SEQ ID NO: 74) was evaluated in BALB / c mice. Six animals per group were immunized intramuscularly with increasing doses of each GADNOU vector. The ELISpot assay was performed on splenocytes collected after three weeks, using a 9-mer peptide encoding the H-2 Kd CD8 + epitope of the main HIV gag (AMQMLKETI) as antigen. The data show strong immunogenicity induced by both vectors at the highest tested dose of 3x10 7 vp (viral particles). Also at the lowest dose of 3x106 vp, the two vectors were still able to induce a specific T cell response to the HIV-1 gag in 50% of the vaccinated mice (Figure 5).

权利要求:
Claims (15)
[1]
1. Isolated POLINUCLEOTIDE which encodes a hexon adenovirus protein, characterized by comprising: A) (i) an HVR1 comprising an amino acid sequence according to SEQ ID NO: 11, or a variant thereof with at least 85% identity of sequence and without A at position 27, (ii) an HVR2 comprising an amino acid sequence according to SEQ ID NO: 12, or a variant thereof with at least 85% sequence identity and without L at position 1, (iii) an HVR3 comprising an amino acid sequence according to SEQ ID NO: 13, or a variant thereof with at least 85% sequence identity and without V in position 7, (iv) an HVR4 comprising a sequence of amino acids according to SEQ ID NO: 14, or a variant thereof with at least 85% sequence identity, (v) an HVR5 comprising an amino acid sequence according to SEQ ID NO: 15, or a variant of same with at least 85% sequence identity, (vi) an HVR6 comprising u an amino acid sequence according to SEQ ID NO: 16, or a variant thereof with at least 85% sequence identity, and
(vii) an HVR7 comprising an amino acid sequence according to SEQ ID NO: 17, or a variant thereof with at least 85% sequence identity and without I in position 1; or
B)
(i) an HVR1 comprising an amino acid sequence according to SEQ ID NO: 18, or a variant thereof with at least 85% sequence identity and without V in position 8, without D in position
12, without E in position 13, and / or without L in position 14,
(ii) an HVR2 comprising an amino acid sequence according to SEQ ID NO: 19, or a variant thereof with at least 85% sequence identity and without D at position 10,
(iii) an HVR3 comprising an amino acid sequence according to SEQ ID NO: 20, or a variant thereof with at least 85% sequence identity and without T in position 6,
(iv) an HVR4 comprising an amino acid sequence according to SEQ ID NO: 21, or a variant thereof with at least 85% sequence identity and without L at position 9,
(v) an HVR5 comprising an amino acid sequence according to SEQ ID NO: 22, or a variant thereof with at least 85% sequence identity and without T in position 3, (vi) an HVR6 comprising a sequence of amino acids according to SEQ ID NO: 23, or a variant thereof with at least 85% sequence identity and without I at position 9, and
(vii) an HVR7 comprising an amino acid sequence according to SEQ ID NO: 24, or a variant thereof with at least 85% sequence identity and without I at position 8; or
Ç)
(i) an HVR1 comprising an amino acid sequence according to SEQ ID NO: 25, or a variant thereof with at least 85% sequence identity,
(ii) an HVR2 comprising an amino acid sequence according to SEQ ID NO: 26, or a variant thereof with at least 85% sequence identity,
(iii) an HVR3 comprising an amino acid sequence according to SEQ ID NO: 27, or a variant thereof with at least 85% sequence identity and without V in position 7,
(iv) an HVR4 comprising an amino acid sequence according to SEQ ID NO: 28, or a variant thereof with at least 85% sequence identity and without E in position 10,
(v) an HVR5 comprising an amino acid sequence according to SEQ ID NO: 29, or a variant thereof with at least 85% sequence identity and without T in position 3,
(vi) an HVR6 comprising an amino acid sequence according to SEQ ID NO: 30, or a variant thereof with at least 85% sequence identity and without I in position 9, and
(vii) an HVR7 comprising an amino acid sequence according to SEQ ID NO: 31, or a variant thereof with at least 85% sequence identity and without I in position 8 and / or without T in position
11, or
D)
(i) an HVR1 comprising an amino acid sequence according to SEQ ID NO: 32, or a variant thereof with at least 85% sequence identity,
(ii) an HVR2 comprising an amino acid sequence according to SEQ ID NO: 33, or a variant thereof with at least 85% sequence identity,
(iii) an HVR3 comprising an amino acid sequence according to SEQ ID NO: 34, or a variant thereof with at least 85% sequence identity and without T at position 6,
(iv) an HVR4 comprising an amino acid sequence according to SEQ ID NO: 35, or a variant thereof with at least 85% sequence identity and without Q in position 6 and / or without E in position 10, ( v) an HVR5 comprising an amino acid sequence according to SEQ ID NO: 36, or a variant thereof with at least 85% sequence identity and without T in position 3,
(vi) an HVR6 comprising an amino acid sequence according to SEQ ID NO: 37, or a variant thereof with at least 85% sequence identity and without K in position 1, and
(vii) an HVR7 comprising an amino acid sequence according to SEQ ID NO: 38, or a variant thereof with at least 85% sequence identity and without I at position 8; or
AND)
(i) an HVR1 comprising an amino acid sequence according to SEQ ID NO: 39, or a variant thereof with at least 85% sequence identity and without A at position 27,
(ii) an HVR2 comprising an amino acid sequence according to SEQ ID NO: 40, or a variant thereof with at least 85% sequence identity,
(iii) an HVR3 comprising an amino acid sequence according to SEQ ID NO: 41, or a variant thereof with at least 85% sequence identity,
(iv) an HVR4 comprising an amino acid sequence according to SEQ ID NO: 42, or a variant thereof with at least 85% sequence identity,
(v) an HVR5 comprising an amino acid sequence according to SEQ ID NO: 43, or a variant thereof with at least 85% sequence identity, (vi) an HVR6 comprising an amino acid sequence according to SEQ ID NO: 44, or a variant thereof with at least 85% sequence identity, and (vii) an HVR7 comprising an amino acid sequence according to SEQ ID NO: 45, or a variant thereof with at least 85 % sequence identity and without I in position 1.
[2]
2. Isolated POLYNUCLEOTIDE, according to claim 1, characterized by the hexon protein comprising; A) an amino acid sequence according to SEQ ID NO: 46, or a variant thereof with at least 85% sequence identity, B) an amino acid sequence according to SEQ ID NO: 47, or a variant of the same with at least 85% sequence identity, C) an amino acid sequence according to SEQ ID NO: 48, or a variant thereof with at least 85% sequence identity, D) an amino acid sequence of according to SEQ ID NO: 49, or a variant thereof with at least 85% sequence identity, and / or E) an amino acid sequence according to SEQ ID NO: 50, or a variant thereof with at least minus 85% sequence identity.
[3]
3. Isolated POLYNUCLEOTIDE according to any one of claims 1 to 2, characterized in that it further encodes an adenoviral penton protein comprising an amino acid sequence according to SEQ ID NO: 51 or 52, or a variant thereof with at least 85% sequence identity.
[4]
4. Isolated polynucleotide according to any one of claims 1 to 3, characterized in that it further encodes an adenoviral fiber protein comprising an amino acid sequence according to SEQ ID NO: 53 or 54, or a variant thereof with at least 85% sequence identity.
[5]
5. Isolated POLINUCLEOTIDE, according to any one of claims 1 to 4, characterized in that it further encodes a non-coding VA RNA II comprising a nucleotide sequence according to SEQ ID NO: 57 or a variant thereof with at least 85 % sequence identity and / or a non-coding VA RNA I comprising a nucleotide sequence according to SEQ ID NO: 55 or 56, or a variant thereof with at least 85% sequence identity.
[6]
6. Isolated polynucleotide encoding an adenovirus, preferably an incompetent replication adenovirus, characterized in that it comprises the polynucleotide of any one of claims 1 to 5.
[7]
7. Isolated POLINUCLEOTIDE, according to claim 6, characterized in that the adenovirus is a chimeric adenovirus and / or an adenovirus that carries a non-adenoviral gene, protein or fragment.
[8]
8. POLYPEPTIDE of the isolated adenoviral capsid, characterized in that it is encoded by a polynucleotide isolated from any of claims 1 to 4.
[9]
9. ISOLATED ADENOVIRUS, preferably an incompetent replicating adenovirus, characterized in that it comprises an isolated polynucleotide according to any one of claims 1 to 7 and / or at least one adenoviral capsid polypeptide isolated according to claim 8.
[10]
10. VIRUS-LIKE PARTICLE, characterized by being encoded by an isolated polynucleotide according to any one of claims 1 to 7.
[11]
11. VECTOR, characterized in that it comprises an isolated polynucleotide according to any one of claims 1 to 7.
[12]
12. COMPOSITION, characterized in that it comprises: (i) an adjuvant, (ii) an isolated polynucleotide according to any one of claims 1 to 7, at least one adenoviral capsid polypeptide isolated according to claim 8, an adenovirus of according to claim 9, a virus-like particle according to claim 10, or a vector according to claim 11, and optionally (iii) a pharmaceutically acceptable excipient.
[13]
13. CELL, characterized by understanding; a polynucleotide according to any one of claims 1 to 7, at least one isolated adenoviral capsid polypeptide according to claim 8, an adenovirus according to claim 9, a virus-like particle according to claim 10, or a vector according to claim 11.
[14]
POLINUCLEOTIDE, according to any one of claims 1 to 7, isolated adenoviral capsid polypeptide according to claim 8, adenovirus according to claim 9, virus-like particle according to claim 10, or vector of according to claim 11 and / or the composition according to claim 12, characterized by use in the treatment or prevention of a disease.
[15]
15. IN VITRO METHOD FOR THE PRODUCTION OF A
ADENOVIRUS OR A PARTICLE LIKE ADENOVIRUS,
characterized by understanding the steps of:
(i) expressing a polynucleotide isolated from any one of claims 1 to 7 in a cell, so that an adenovirus or adenovirus-like particle is assembled in the cell,
(ii) isolating the adenovirus or adenovirus-like particle from the cell or the medium surrounding the cell.

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