Clonal strains of attenuated vaccinia viruses and methods of use thereof
Inventors
Szalay, Aladar A. • Chen, Nanhai G. • Yu, Yong A. • Zhang, Qian
Assignees
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Publication Number
US-11149254-B2
Publication Date
2021-10-19
Expiration Date
Abstract
Clonal strains of vaccinia viruses are provided. Also provided are methods of identifying and isolating attenuated and oncolytic clonal strains from virus preparations. Modified recombinant forms of the clonal strains also are provided. The clonal strains and virus preparations can be used for diagnostic and therapeutic methods, in particular for therapy and diagnosis or monitoring treatment of proliferative disorders, including neoplastic diseases, such as, but are not limited to, solid tumors and blood cancers.
Core Innovation
The invention relates to isolated clonal LIVP strain isolates derived from LIVP vaccinia virus preparations that are substantially homogeneous in genome. The clonal LIVP strains are defined by specific nucleotide regions and/or high sequence identity requirements, and their genomes are not modified to contain non-viral heterologous nucleic acid containing an open reading frame encoding a non-viral heterologous protein.
The clonal LIVP strains differ from a reference LIVP sequence set forth in SEQ ID NO:10 while retaining similarity to selected nucleotide regions of SEQ ID NOs:1, 2, 4, 5, 6, 7, and 8. Compared to GLV-1h68, the clonal strains exhibit greater anti-tumorigenicity and/or reduced toxicity, as evaluated by assays of anti-tumorigenicity and toxicity and subsequent selection of isolates meeting the comparative performance.
The disclosure further addresses recombinant LIVP virus strains derived from clonal LIVP strains. These descendants include deletion of nucleic acid, replacement of nucleic acid with heterologous nucleic acid, or insertion of heterologous nucleic acid, where the recombinant strain retains the ability to replicate while being modified in genomic sequence compared to the clonal LIVP strain.
Claims Coverage
The claim set covers isolated clonal LIVP strains, a cell culture or isolated cell comprising a clonal LIVP strain, and recombinant LIVP virus strains derived from clonal LIVP strains. Across the independent claims, the inventive subject matter centers on sequence-defined, non-heterologous-ORF clonal LIVP strains with greater anti-tumorigenicity and/or reduced toxicity versus GLV-1h68, with recombinant modifications that retain replication ability.
Sequence-defined clonal LIVP strain with no non-viral heterologous protein-encoding ORF
An isolated clonal LIVP strain whose genome is not modified to contain non-viral heterologous nucleic acid containing an open reading frame encoding a non-viral heterologous protein.
Clonal genome comprises specified SEQ ID nucleotide regions or high-identity sequences
The genome comprises the sequence of nucleotides selected from among nucleotides 2,256-181,114 of SEQ ID NO:1, nucleotides 11,243-182,721 of SEQ ID NO:2, nucleotides 6,264-181,390 of SEQ ID NO:4, nucleotides 7,044-181,820 of SEQ ID NO:5, nucleotides 6,674-181,409 of SEQ ID NO:6, nucleotides 6,716-181,367 of SEQ ID NO:7, or nucleotides 6,899-181,870 of SEQ ID NO:8; and sequences having at least 99% sequence identity to those regions.
Improved anti-tumorigenicity and/or reduced toxicity versus GLV-1h68
The clonal LIVP strain has greater anti-tumorigenicity or reduced toxicity or both compared to GLV-1h68, wherein GLV-1h68 has the sequence of nucleotides set forth in SEQ ID NO:9.
Clonal genome is at least 95% identical to SEQ ID NO:10 but does not comprise SEQ ID NO:10
The genome comprises a sequence of nucleotides that has at least 95% sequence identity with the sequence of nucleotides set forth in SEQ ID NO:10 but does not comprise the sequence of nucleotides set forth in SEQ ID NO:10.
Assay-and-select production tied to toxicity and anti-tumorigenicity versus GLV-1h68
The isolated clonal strain is produced by or obtainable by preparing a clonal isolate from an LIVP virus sample, assaying the clonal isolate for toxicity, assaying the clonal isolate for anti-tumorigenicity, and selecting the clonal isolate that exhibits greater anti-tumorigenicity and/or reduced toxicity compared to GLV-1h68.
Clonal LIVP genome comprises SEQ ID NOS: 1, 2, 4, 5, 6, 7 and 8
A pharmaceutical composition comprising an isolated clonal LIVP strain wherein the genome comprises the sequence of nucleotides set forth in any of SEQ ID NOS:1, 2, 4, 5, 6, 7 and 8.
Cell culture or isolated cell comprising sequence-constrained clonal LIVP and improved performance
A cell culture or isolated cell comprising a clonal LIVP strain wherein the genome does not contain non-viral heterologous nucleic acid containing an open reading frame encoding a non-viral heterologous protein; the clonal strain has greater anti-tumorigenicity or reduced toxicity or both compared to GLV-1h68; and the genome comprises a sequence having at least 95% sequence identity with the sequence of nucleotides set forth in SEQ ID NO:10 but does not comprise the sequence of nucleotides set forth in SEQ ID NO:10.
Recombinant LIVP derived from a clonal LIVP strain while retaining replication ability
A recombinant LIVP virus strain that is a derivative of a clonal LIVP strain, comprising deletion of nucleic acid or replacement of nucleic acid with heterologous nucleic acid or insertion of heterologous nucleic acid in the genome of the clonal LIVP strain, whereby the recombinant strain retains the ability to replicate, but is modified in its genomic sequence compared to the genome of the clonal LIVP strain.
Recombinant strain retains improved anti-tumorigenicity and/or reduced toxicity versus GLV-1h68
The recombinant LIVP virus strain has greater anti-tumorigenicity or reduced toxicity or both compared to GLV-1h68, wherein GLV-1h68 has the sequence of nucleotides set forth in SEQ ID NO:9.
Across the independent claims, the coverage centers on isolated clonal LIVP strains with sequence-defined genomes that lack non-viral heterologous protein-encoding ORFs and show greater anti-tumorigenicity and/or reduced toxicity compared to GLV-1h68. The claim set further covers cell culture or isolated cell embodiments and recombinant derivatives obtained by deletion, replacement, or insertion of heterologous nucleic acid while retaining replication ability and preserving the comparative performance constraints.
Stated Advantages
Greater anti-tumorigenicity compared to GLV-1h68.
Reduced toxicity compared to GLV-1h68.
Documented Applications
Treating proliferative disorders, including cancers and metastases.
Diagnostic/monitoring uses for proliferative disorders, including cancers and metastases, including detection/monitoring by imaging components and marker genes referenced in the document.
Use in combination therapies and kit-based therapeutic/diagnostic combinations for treating and detecting proliferative disorders.
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