Modified adenoviruses
AssigneeGritstone Bio, Inc.
InventorsKarin Jooss, Ciaran Daniel Scallan, Leonid Gitlin
Life of this patent
Estimating the term…
Abstract
Disclosed herein are compositions that include modified adenoviruses. Also disclosed are nucleotides, cells, and methods associated with the compositions including their use as vaccines. Also disclosed herein are viral vectors using TET promoter system and methods of producing viruses having the same.
Claims145
An adenovirus vector comprising:
- an adenoviral backbone comprising one or more genes or regulatory sequences of an adenovirus genome, and
- wherein the adenoviral backbone comprises a partially deleted E4 gene with reference to the adenovirus genome, wherein the partially deleted E4 gene comprises a deleted or partially-deleted E4orf2 region and a deleted or partially-deleted E4orf3 region, and optionally a deleted or partially-deleted E4orf4 region, and
- optionally, wherein the adenovirus vector further comprises a cassette, the cassette comprising:
- (1) at least one payload nucleic acid sequence, optionally wherein the at least one payload nucleic acid sequence encodes a polypeptide, optionally wherein the polypeptide comprises an antigen, optionally wherein the antigen comprises:
- a MHC class I epitope,
- a MHC class II epitope,
- an epitope capable of stimulating a B cell response, or
- a combination thereof, and
- optionally wherein the at least one payload nucleic acid sequence further comprises a 5′ linker sequence and/or a 3′ linker sequence, and optionally wherein;
- (2) at least one promoter sequence operably linked to the at least one payload nucleic acid sequence,
- (3) optionally, at least one universal MHC class II antigen-encoding nucleic acid sequence;
- (4) optionally, at least one GPGPG-encoding linker sequence (SEQ ID NO:56);
- and
- (5) optionally, at least one polyadenylation sequence.
A chimpanzee adenovirus vector comprising a modified ChAdV68 sequence, wherein the modified ChAdV68 sequence comprises:
- (a) a partially deleted E4 gene of the E4 gene sequence shown in SEQ ID NO:1 and that lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO:1;
- and
- (b) one or more genes or regulatory sequences of the ChAdV68 sequence shown in SEQ ID NO:1, optionally wherein the one or more genes or regulatory sequences comprise at least one of the chimpanzee adenovirus inverted terminal repeat (ITR), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the sequence shown in SEQ ID NO:1;
- and
- optionally, wherein the chimpanzee adenovirus vector further comprises a cassette, wherein the cassette comprises at least one payload nucleic acid sequence, and wherein the cassette comprises at least one promoter sequence operably linked to the at least one payload nucleic acid sequence.
A chimpanzee adenovirus vector comprising a modified ChAdV68 sequence, wherein the modified ChAdV68 sequence comprises:
- (a) a partially deleted E4 gene of the E4 gene sequence shown in SEQ ID NO:1 and that lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO:1;
- (b) nucleotides 2 to 34,916 of the sequence shown in SEQ ID NO:1, wherein the partially deleted E4 gene is 3′ of the nucleotides 2 to 34,916, and optionally the nucleotides 2 to 34,916 additionally lack nucleotides 577 to 3403 of the sequence shown in SEQ ID NO:1 corresponding to an E1 deletion and/or lack nucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO:1 corresponding to an E3 deletion;
- and
- (c) nucleotides 35,643 to 36,518 of the sequence shown in SEQ ID NO:1, and wherein the partially deleted E4 gene is 5′ of the nucleotides 35,643 to 36,518, and optionally, wherein the chimpanzee adenovirus vector further comprises a cassette, wherein the cassette comprises at least one payload nucleic acid sequence, and wherein the cassette comprises at least one promoter sequence operably linked to the at least one payload nucleic acid sequence.
A chimpanzee adenovirus vector comprising:
- a. a modified ChAdV68 sequence, wherein the modified ChAdV68 sequence comprises:
- (i) a partially deleted E4 gene of the E4 gene sequence shown in SEQ ID NO:1 and that lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO:1;
- (ii) nucleotides 2 to 34,916 of the sequence shown in SEQ ID NO:1, wherein the partially deleted E4 gene is 3′ of the nucleotides 2 to 34,916, and optionally the nucleotides 2 to 34,916 additionally lack nucleotides 577 to 3403 of the sequence shown in SEQ ID NO:1 corresponding to an E1 deletion and/or lack nucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO:1 corresponding to an E3 deletion;
- and
- (iii) and nucleotides 35,643 to 36,518 of the sequence shown in SEQ ID NO:1, and wherein the partially deleted E4 gene is 5′ of the nucleotides 35,643 to 36,518, and;
- b. a CMV-derived promoter sequence;
- c. an SV40 polyadenylation signal nucleotide sequence;
- and
- d. a cassette, the cassette comprising at least one at least one payload nucleic acid sequence encoding:
- at least one MHC class I epitope, optionally wherein the at least one MHC class I epitope comprises at least 2 distinct MHC class I epitopes linearly linked to each other and each optionally comprising:
- (A) at least one alteration that makes the encoded peptide sequence distinct from the corresponding peptide sequence encoded by a wild-type nucleic acid sequence, wherein the distinct MHC I epitope is 7-15 amino acids in length,
- (B) an N-terminal linker comprising a native N-terminal amino acid sequence of the distinct MHC I epitope that is at least 3 amino acids in length,
- (C) an C-terminal linker comprising a native C-terminal acid sequence of the distinct MHC I epitope that is at least 3 amino acids in length, or
- (D) combinations thereof,
- at least one MHC class II epitope, optionally wherein the at least one MHC class II epitope comprises at least 2 distinct MHC class II epitopes,
- at least one an epitope capable of stimulating a B cell response, or
- combinations thereof, and
- wherein the cassette is inserted within a deleted region of ChAdV68 and the CMV-derived promoter sequence is operably linked to the cassette.
The vector of any of the above claims, wherein the adenoviral backbone or modified ChAdV68 sequence further comprises a functional deletion in at least one gene selected from the group consisting of an adenovirus E1A, E1B, E2A, E2B, E3, L1, L2, L3, L4, and L5 gene with reference to the adenovirus genome or with reference to the sequence shown in SEQ ID NO:1, optionally wherein the adenoviral backbone or modified ChAdV68 sequence is fully deleted or functionally deleted in:
- (1) E1A and E1B;
- or (2) E1A, E1B, and E3 with reference to the adenovirus genome or with reference to the sequence shown in SEQ ID NO:1, optionally wherein the E1 gene is functionally deleted through an E1 deletion of at least nucleotides 577 to 3403 with reference to the sequence shown in SEQ ID NO:1 and optionally wherein the E3 gene is functionally deleted through an E3 deletion of at least nucleotides 27,125 to 31,825 with reference to the sequence shown in SEQ ID NO:1.
The vector of any of the above claims, wherein the cassette is present and is inserted in the vector at the E1 region, E3 region, and/or any deleted AdV region that allows incorporation of the cassette.
The vector of any of the above claims, wherein at least one of the at least one payload nucleic acid sequences encodes an antigen, wherein the antigen comprises:
- a MHC class I epitope,
- a MHC class II epitope,
- an epitope capable of stimulating a B cell response, or
- a combination thereof.
The vector of any of the above claims, wherein at least one of the at least one payload nucleic acid sequences encodes a polypeptide sequence capable of undergoing antigen processing into an epitope, optionally wherein the epitope is known or suspected to be presented by MHC class I on a surface of a cell, optionally wherein the surface of the cell is a tumor cell surface or an infected cell surface.
The vector of any of the above claims, wherein at least one of the at least one payload nucleic acid sequences encodes a polypeptide sequence or portion thereof that is presented by MHC class I and/or MHC class II on a surface of a cell, optionally wherein the surface of the cell is a tumor cell surface or an infected cell surface.
The vector of any of the above claims, wherein at least one of the at least one payload nucleic acid sequences encodes a polypeptide sequence or portion thereof comprising an epitope capable of stimulating a B cell response, optionally wherein the polypeptide sequence or portion thereof comprises a full-length protein, a protein domain, a protein subunit, or an antigenic fragment predicted or known to be capable of being bound by an antibody.
The vector of any of the above claims, wherein at least one of the at least one payload nucleic acid sequences encodes an infectious disease organism peptide selected from the group consisting of:
- a pathogen-derived peptide, a virus-derived peptide, a bacteria-derived peptide, a fungus-derived peptide, and a parasite-derived peptide.
The vector of any of the above claims, wherein at least one of the at least one payload nucleic acid sequences encodes an epitope with at least one alteration that makes the encoded epitope sequence distinct from the corresponding peptide sequence encoded by a wild-type nucleic acid sequence.
The vector of any of the above claims, wherein at least one of the at least one payload nucleic acid sequences encodes a MHC class I epitope or MHC class II epitope with at least one alteration that makes the encoded peptide sequence distinct from the corresponding peptide sequence encoded by a wild-type nucleic acid sequence, optionally wherein the encoded polypeptide sequence or portion thereof has increased binding affinity to, increased binding stability to, and/or an increased likelihood of presentation on its corresponding MHC allele relative to the translated, corresponding wild-type nucleic acid sequence.
The vector of any of the above claims, wherein at least one of the at least one payload nucleic acid sequences encodes a full-length protein, a protein domain, or a protein subunit.
The vector of any of the above claims, wherein at least one of the at least one payload nucleic acid sequences encodes an antibody, a cytokine, a chimeric antigen receptor (CAR), a T-cell receptor, and a genome-editing system nuclease.
The vector of any of the above claims, wherein at least one of the at least one payload nucleic acid sequences comprises a non-coding nucleic acid sequence.
The vector of any of the above claims, wherein the expression of each of the at least one payload nucleic acid sequences is driven by the at least one promoter.
The vector of any of the above claims, wherein the at least one payload nucleic acid sequence comprises at least 2-400 MHC class I and/or class II antigen-encoding nucleic acid sequences, wherein, when administered to the subject and translated, at least one of the MHC class I or class II antigens are presented on antigen presenting cells resulting in an immune response targeting at least one of the antigens on a cell surface, and optionally wherein the expression of each of the at least 2-400 MHC class I or class II antigen-encoding nucleic acid sequences is driven by the at least one promoter.
The vector of any of the above claims, wherein each MHC class I epitope is independently between 8 and 35 amino acids in length, optionally 7-15, 9-17, 9-25, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 amino acids in length.
The vector of any of the above claims, wherein the at least one promoter sequence is a regulatable promoter, optionally wherein the regulatable promoter is a tetracycline (TET) repressor protein (TETr) controlled promoter, optionally wherein the regulatable promoter comprises multiple TET operator (TETo) sequences 5′ or 3′ of a RNA polymerase binding sequence of the promoter.
The vector of any of the above claims, wherein the at least one promoter sequence is constitutive.
The vector of any of the above claims, wherein the cassette further comprises at least one poly-adenylation (polyA) sequence operably linked to at least one of the at least one payload nucleic acid sequences, optionally wherein the polyA sequence is located 3′ of the at least one payload nucleic acid sequence.
The vector of any of the above claims, wherein the cassette further comprises at least one of:
- an intron sequence, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence, an internal ribosome entry sequence (IRES) sequence, a nucleotide sequence encoding a 2A self-cleaving peptide sequence, a nucleotide sequence encoding a Furin cleavage site, a nucleotide sequence encoding a TEV cleavage site, or a sequence in the 5′ or 3′ non-coding region known to enhance the nuclear export, stability, or translation efficiency of mRNA that is operably linked to at least one of the at least one payload nucleic acid sequences.
The vector of any of the above claims, wherein the cassette comprises a reporter gene, including but not limited to, green fluorescent protein (GFP), a GFP variant, secreted alkaline phosphatase, luciferase, or a luciferase variant.
The vector of any of the above claims, wherein the vector further comprises one or more payload nucleic acid sequences encoding at least one immune modulator, optionally wherein the at least one immune modulator inhibits an immune checkpoint molecule.
The vector of any of the above claims, wherein at least one of the at least one payload nucleic acid sequences are selected by performing the steps of:
- (a) obtaining at least one of exome, transcriptome, or whole genome nucleotide sequencing data from a tumor cell, an infected cell, or an infectious disease organism, wherein the nucleotide sequencing data is used to obtain data representing peptide sequences of each of a set of antigens;
- (b) inputting the peptide sequence of each antigen into a presentation model to generate a set of numerical likelihoods that each of the antigens is presented by one or more of the MHC alleles on a cell surface, optionally a tumor cell surface or an infected cell surface, the set of numerical likelihoods having been identified at least based on received mass spectrometry data;
- and
- (c) selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a set of selected antigens which are used to generate the at least one payload nucleic acid sequence.
The vector of any of the above claims, wherein each of the at least one payload nucleic acid sequences are selected by performing the steps of:
- (a) obtaining at least one of exome, transcriptome, or whole genome nucleotide sequencing data from a tumor cell, an infected cell, or an infectious disease organism, wherein the nucleotide sequencing data is used to obtain data representing peptide sequences of each of a set of antigens;
- (b) inputting the peptide sequence of each antigen into a presentation model to generate a set of numerical likelihoods that each of the antigens is presented by one or more of the MHC alleles on a cell surface, optionally a tumor cell surface or an infected cell surface, the set of numerical likelihoods having been identified at least based on received mass spectrometry data;
- and
- (c) selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a set of selected antigens which are used to generate each of the at least one payload nucleic acid sequences.
The vector of any of the above claims, wherein the cassette comprises junctional epitope sequences formed by adjacent sequences in the cassette.
The vector of any of the above claims, wherein the cassette does not encode a non-therapeutic MHC class I or class II epitope, wherein the non-therapeutic epitope is predicted to be displayed on an MHC allele of a subject.
The composition of any one of the above claims, wherein each of the MHC class I and/or class II epitopes is predicted or validated to be capable of presentation by at least one HLA allele present in at least 5% of a human population.
The composition of any one of the above claims, wherein each of the MHC class I and/or class II epitopes is predicted or validated to be capable of presentation by at least one HLA allele, wherein each antigen/HLA pair has an antigen/HLA prevalence of at least 0.01% in a human population.
The composition of any one of the above claims, wherein each of the MHC class I and/or class II epitopes is predicted or validated to be capable of presentation by at least one HLA allele, wherein each antigen/HLA pair has an antigen/HLA prevalence of at least 0.1% in a human population.
The composition of any one of the above claims, wherein the at least one payload nucleic acid sequence encoding the polypeptide is codon optimized relative to a native nucleic acid sequence directly extracted from a subject tissue or sample.
A pharmaceutical composition comprising the vector of any of the above claims and a pharmaceutically acceptable carrier.
An isolated nucleotide sequence comprising the cassette of any of the above vector claims and a gene of the sequence of SEQ ID NO:1, optionally wherein the gene is selected from the group consisting of the chimpanzee adenovirus ITR, E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the sequence shown in SEQ ID NO:1, and optionally wherein the nucleotide sequence is cDNA.
A kit comprising the vector of any of the above vector claims and instructions for use.
A method of manufacturing the vector of any of the above vector claims, the method comprising:
- obtaining a plasmid sequence comprising the adenovirus vector or chimpanzee adenovirus vector;
- transfecting the plasmid sequence into one or more host cells;
- and
- isolating the vector from the one or more host cells.
A method for stimulating an immune response in a subject, the method comprising administering to the subject an adenovirus vector comprising:
- an adenoviral backbone comprising one or more genes or regulatory sequences of an adenovirus genome, and
- wherein the adenoviral backbone comprises a partially deleted E4 gene with reference to the adenovirus genome, wherein the partially deleted E4 gene comprises a deleted or partially-deleted E4orf2 region and a deleted or partially-deleted E4orf3 region, and optionally a deleted or partially-deleted E4orf4 region, and
- wherein the adenovirus vector further comprises a cassette, the cassette comprising:
- (1) at least one payload nucleic acid sequence, optionally wherein the at least one payload nucleic acid sequence encodes a polypeptide, optionally wherein the polypeptide comprises an antigen, optionally wherein the antigen comprises:
- a MHC class I epitope,
- a MHC class II epitope,
- an epitope capable of stimulating a B cell response, or
- a combination thereof, and
- optionally wherein the at least one payload nucleic acid sequence further comprises a 5′ linker sequence and/or a 3′ linker sequence, and optionally wherein;
- (2) at least one promoter sequence operably linked to the at least one payload nucleic acid sequence,
- (3) optionally, at least one universal MHC class II antigen-encoding nucleic acid sequence;
- (4) optionally, at least one GPGPG-encoding linker sequence (SEQ ID NO:56);
- and
- (5) optionally, at least one polyadenylation sequence.
A method for treating a subject with a disease, optionally wherein the disease is cancer or an infection, the method comprising administering to the subject an adenovirus vector comprising:
- an adenoviral backbone comprising one or more genes or regulatory sequences of an adenovirus genome, and
- wherein the adenoviral backbone comprises a partially deleted E4 gene with reference to the adenovirus genome, wherein the partially deleted E4 gene comprises a deleted or partially-deleted E4orf2 region and a deleted or partially-deleted E4orf3 region, and optionally a deleted or partially-deleted E4orf4 region, and
- wherein the adenovirus vector further comprises a cassette, the cassette comprising:
- (1) at least one payload nucleic acid sequence, optionally wherein the at least one payload nucleic acid sequence encodes a polypeptide, optionally wherein the polypeptide comprises an antigen, optionally wherein the antigen comprises:
- a MHC class I epitope,
- a MHC class II epitope,
- an epitope capable of stimulating a B cell response, or
- a combination thereof, and
- optionally wherein the at least one payload nucleic acid sequence further comprises a 5′ linker sequence and/or a 3′ linker sequence, and optionally wherein;
- (2) at least one promoter sequence operably linked to the at least one payload nucleic acid sequence,
- (3) optionally, at least one universal MHC class II antigen-encoding nucleic acid sequence;
- (4) optionally, at least one GPGPG-encoding linker sequence (SEQ ID NO:56);
- and
- (5) optionally, at least one polyadenylation sequence.
A method for stimulating an immune response in a subject, the method comprising administering to the subject an adenovirus vector comprising a modified ChAdV68 sequence, wherein the modified ChAdV68 sequence comprises:
- (a) a partially deleted E4 gene of the E4 gene sequence shown in SEQ ID NO:1 and that lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO:1;
- and
- (b) one or more genes or regulatory sequences of the ChAdV68 sequence shown in SEQ ID NO:1, optionally wherein the one or more genes or regulatory sequences comprise at least one of the chimpanzee adenovirus inverted terminal repeat (ITR), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the sequence shown in SEQ ID NO:1;
- and
- wherein the chimpanzee adenovirus vector further comprises a cassette, wherein the cassette comprises at least one payload nucleic acid sequence, and wherein the cassette comprises at least one promoter sequence operably linked to the at least one payload nucleic acid sequence.
A method for treating a subject with a disease, optionally wherein the disease is cancer or an infection, the method comprising administering to the subject an adenovirus vector comprising a modified ChAdV68 sequence, wherein the modified ChAdV68 sequence comprises:
- (a) a partially deleted E4 gene of the E4 gene sequence shown in SEQ ID NO:1 and that lacks at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO:1;
- and
- (b) one or more genes or regulatory sequences of the ChAdV68 sequence shown in SEQ ID NO:1, optionally wherein the one or more genes or regulatory sequences comprise at least one of the chimpanzee adenovirus inverted terminal repeat (ITR), E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of the sequence shown in SEQ ID NO:1;
- and
- wherein the chimpanzee adenovirus vector further comprises a cassette, wherein the cassette comprises at least one payload nucleic acid sequence, and wherein the cassette comprises at least one promoter sequence operably linked to the at least one payload nucleic acid sequence.
A method of producing a virus, wherein the virus is produced using any of the above vector claims.
A method of producing a virus, wherein the method comprises the steps of:
- a. providing a viral vector comprising a cassette, the cassette comprising:
- (i) at least one payload nucleic acid sequence, optionally wherein the at least one payload nucleic acid sequence encodes a polypeptide, optionally wherein the polypeptide comprises an antigen, optionally wherein the antigen comprises:
- a MHC class I epitope,
- a MHC class II epitope,
- an epitope capable of stimulating a B cell response, or
- a combination thereof, and
- optionally wherein the at least one payload nucleic acid sequence further comprises a 5′ linker sequence and/or a 3′ linker sequence, and optionally wherein;
- (ii) at least one promoter sequence operably linked to the at least one payload nucleic acid sequence, wherein the at least one promoter is a tetracycline (TET) repressor protein (TETr) controlled promoter,
- (iii) optionally, at least one MHC class II antigen-encoding nucleic acid sequence;
- (iv) optionally, at least one GPGPG-encoding linker sequence (SEQ ID NO:56);
- and
- (v) optionally, at least one polyadenylation sequence;
- b. providing a cell engineered to express the TETr protein;
- and
- c. contacting the viral vector with the cell under conditions sufficient for production of the virus.
A viral vector comprising a cassette, the cassette comprising:
- (i) at least one payload nucleic acid sequence, optionally wherein the at least one payload nucleic acid sequence encodes a polypeptide, optionally wherein the polypeptide comprises an antigen, optionally wherein the antigen comprises:
- a MHC class I epitope,
- a MHC class II epitope,
- an epitope capable of stimulating a B cell response, or
- a combination thereof, and
- optionally wherein the at least one payload nucleic acid sequence further comprises a 5′ linker sequence and/or a 3′ linker sequence, and optionally wherein;
- (ii) at least one promoter sequence operably linked to the at least one payload nucleic acid sequence, wherein the at least one promoter is a tetracycline (TET) repressor protein (TETr) controlled promoter,
- (iii) optionally, at least one MHC class II antigen-encoding nucleic acid sequence;
- (iv) optionally, at least one GPGPG-encoding linker sequence (SEQ ID NO:56);
- and
- (v) optionally, at least one polyadenylation sequence.
Dependencies are inferred from the claim text.