Patent Application
20230002740
A Sequence Listing is provided herewith as a text file, “PC040318A_SeqListing_ST25.txt” created on Nov. 3, 2020 and having a size of 152 KB. The contents of the text file are incorporated by reference herein in their entirety.
The present disclosure relates to oncolytic viruses in general and to recombinant oncolytic vaccinia virus with enhanced anti-tumor properties in particular.
Oncolytic viruses (OVs) are viruses that replicate selectively or more efficiently in cancer cells than in non-cancer cells. This group of viruses includes viruses found in nature (e.g., wild-type or native virus), as well as viruses that are engineered from a native virus by gene disruptions or gene additions so as to improve its anti-tumor properties, such as tumor selectivity or preferential replication in tumor cells, host tropism, surface attachment, lysis, and spread. Live replicating OVs have been tested in clinical trials in a variety of human cancers. OVs can induce anti-tumor immune responses, as well as direct lysis of tumor cells (i.e., oncolysis). Common OVs include attenuated strains of Herpes Simplex Virus (HSV), Adenovirus (Ad), Measles Virus (MV), Coxsackie virus (CV), Vesicular Stomatitis Virus (VSV), and Vaccinia Virus (VV).
Vaccinia virus, the prototypical member of the Orthopoxvirus genus, replicates in the cytoplasm of a host cell. During replication, three morphologically and antigenically distinct forms of the virus are produced: the intracellular mature virions (IMV), the intracellular enveloped virions (IEV), and extracellular virions. A subset of IMV, the first infectious progeny produced, are trafficked to the trans-Golgi network (TGN), where they are enveloped with two additional membranes to produce IEV. IEV are transported through the cytoplasm to the cell periphery, where the outermost membrane fuses with the plasma membrane to release a double membraned form, termed EV. EV that remain on the cell surface are called cell-associated enveloped virion (CEV), while EV that are no longer attached to the cell surface are called extracellular enveloped virion (EEV). IMV is the most abundant infectious form and is thought to be responsible for spread between hosts; the CEV is believed to play a role in cell-to-cell spread; and the EEV is thought to be important for long range dissemination within the host organism.
VV contains a double-stranded DNA genome of approximately 200 kbp that is predicted to encode more than 200 open reading frames. Seven proteins encoded by the virus, namely, A33, A34, A36, A56, B5, F12, and F13, are unique to enveloped forms (IEV/EEV/CEV) of the virus. Vaccinia virus open reading frames are designated by a capital letter indicating a HindIII restriction endonuclease fragment, a number indicating the position in the HindIII fragment, and a letter (L or R) indicating the direction of transcription, e.g., A34R. The corresponding protein is designated by a capital letter and number, e.g., A34. Specific to the extracellular virion membrane, glycoproteins A33, A34, and B5 are exposed on the surface of EV and have roles during extracellular virion formation and subsequent infection.
The present disclosure provides a replication-competent, recombinant oncolytic vaccinia virus (hereinafter referred to as “oncolytic vaccinia virus,” or “OVV”) that comprises a nucleotide sequence encoding a variant viral polypeptide, wherein the variant viral polypeptide provides for enhanced virus spreading or enhanced EEV production. The term “enhanced” as used herein means the same as, and is used interchangeably with, “increased” in the present disclosure. In some aspects, the OVV comprises a nucleotide sequence encoding a variant viral polypeptide selected from a variant A33 polypeptide, a nucleotide sequence encoding a variant A34 polypeptide, a nucleotide sequence encoding variant B5 polypeptide, or any combination of the nucleotide sequences above, where the variant A33, variant A34, and variant B5 polypeptides comprise 1, 2, 3, 4, 5, 6, or more amino acid mutations (such as substitutions, deletions, or insertions) that provide for enhanced virus spreading or enhanced EEV production, compared with a corresponding polypeptide without the respective mutations. In some embodiments, the OVV is constructed based on strain Copenhagen. In other embodiments, the OVV is constructed based on strain Western Reserve.
The present disclosure further provides compositions comprising the OVV. The present disclosure also provides methods of inducing oncolysis in an individual having a tumor, the methods comprising administering to the individual an effective amount of the OVV of the present disclosure or a composition of the present disclosure. The disclosure also provides an OVV or composition of the present disclosure for use in methods of inducing oncolysis in an individual having a tumor, and the use of an OVV or composition of the present disclosure in the manufacture of a medicament for use in such methods.
Before describing the present invention, several terms used in the context of the present disclosure will be defined. In addition to these terms, others are defined elsewhere in the specification, as necessary. Unless otherwise expressly defined herein, terms of art used in this specification will have their art-recognized meanings.
The term “oncolytic” vaccinia virus refers to a vaccinia virus that preferentially infects and kills cancer cells, compared to normal (non-cancerous) cells.
The term “heterologous” refers to a nucleotide or polypeptide sequence that is not found in the native nucleic acid or protein, respectively. For example, in the context of a recombinant vaccinia virus of the present disclosure, a nucleic acid comprising a nucleotide sequence encoding a “heterologous” immunomodulatory polypeptide is a nucleic acid that is not found naturally in vaccinia virus, i.e., the encoded immunomodulatory polypeptide is not encoded by naturally-occurring vaccinia virus.
The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.
A “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent (e.g., a replication-competent, recombinant oncolytic vaccinia virus of the present disclosure), or combined amounts of two agents (e.g., a replication-competent, recombinant oncolytic vaccinia virus of the present disclosure and a second therapeutic agent), that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.
The term “variant” polypeptide refers to a polypeptide the amino acid sequence of which exhibits at least 90%, but less than 100%, identity with the amino acid sequence of a reference polypeptide; provided said variant has a biological activity as defined herein. The variant maybe arrived at by modification of the amino acid sequence of the reference polypeptide by such modifications as insertion, substitution, or deletion of one or more amino acids. Accordingly, the term “variant” polypeptide encompasses fragments of a reference polypeptide that comprises a sufficient number of contiguous amino acid residues to confer a desired biological property.
The term “substitution” refers to the replacement of one amino acid in a polypeptide with a different amino acid. In the context of the present disclosure, a substitution in a variant is indicated as: original amino acid-position-substituted amino acid. Accordingly, the notation “K151E” means, that the variant comprises a substitution of Lysine (K) with Glutamic acid (E) in the variant amino acid position corresponding to the amino acid in position 151 in the parent polypeptide.
Before the present invention is further described, it is to be understood that this invention is not limited to any particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a vaccinia virus” includes a plurality of such vaccinia viruses and reference to “the A33 polypeptide” includes reference to one or more A33 polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
Oncolytic Vaccinia Virus
The present disclosure provides a replication-competent, recombinant oncolytic vaccinia virus (also referred to as “oncolytic vaccinia virus,” or “OVV”) that exhibits desirable oncolytic properties. It can derive from any vaccinia virus strain, preferably Elstree, Wyeth, Copenhagen and Western Reserve strains. Unless otherwise indicated, the gene nomenclature used herein is that of Copenhagen vaccinia strain. The OVV may be derived from a parent vaccinia virus by altering one or more viral gene(s) that changes the production, activity, function, or any other properties of the gene product (such as deletion, insertion, substitution of one or more nucleotides within the viral gene or its regulatory elements) and/or inserting one or more transgenes that encode exogenous polypeptides (i.e., polypeptides that are not naturally expressed by the virus).
In some aspects, the present disclosure provides an OVV that comprises a nucleotide sequence encoding a variant viral polypeptide, wherein the variant viral polypeptide provides for one or more enhanced oncolytic properties of the virus, such as enhanced virus spreading or enhanced EEV production. Effects of a variant viral polypeptide on virus spreading or EEV production can be determined using the methods described in the Examples provided in the specification, as well as any other suitable methods known in the art.
In some embodiments, the OVV comprises a nucleotide sequence encoding a variant A33 polypeptide, wherein the variant A33 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A33 polypeptide (e.g., compared to a control vaccinia virus that comprises a nucleotide sequence encoding a wild-type A33 polypeptide, such as an A33 polypeptide having an amino acid sequence depicted in
In some cases, an OVV of the present disclosure comprises a) a nucleotide sequence encoding a variant A33 polypeptide, wherein the variant A33 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A33 polypeptide; b) a nucleotide sequence encoding an A34 polypeptide that does not include any amino acid substitutions that provide for enhanced viral spreading and/or enhanced production of EEV; and c) a nucleotide sequence encoding a B5 polypeptide that does not include any amino acid substitutions that provide for enhanced viral spreading and/or enhanced production of EEV. In some cases, an OVV of the present disclosure comprises: a) a nucleotide sequence encoding a variant A33 polypeptide, wherein the variant A33 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A33 polypeptide; b) a nucleotide sequence encoding a wild-type A34 polypeptide (e.g., an A34 polypeptide having a naturally-occurring amino acid sequence); and c) a nucleotide sequence encoding a wild-type B5 polypeptide (e.g., a B5 polypeptide having a naturally-occurring amino acid sequence).
In some cases, an OVV of the present disclosure comprises: a) a nucleotide sequence encoding a variant A34 polypeptide, wherein the variant A34 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A34 polypeptide; b) a nucleotide sequence encoding an A33 polypeptide that does not include any amino acid substitutions that provide for enhanced viral spreading and/or enhanced production of EEV (e.g., a wild-type A33 polypeptide); and c) a nucleotide sequence encoding a B5 polypeptide that does not include any amino acid substitutions that provide for enhanced viral spreading and/or enhanced production of EEV (e.g., a wild-type B5 polypeptide). In some cases, an OVV of the present disclosure comprises: a) a nucleotide sequence encoding a variant A34 polypeptide, wherein the variant A34 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A34 polypeptide; b) a nucleotide sequence encoding a wild-type A33 polypeptide (e.g., an A33 polypeptide having a naturally-occurring amino acid sequence); and c) a nucleotide sequence encoding a wild-type B5 polypeptide (e.g., a B5 polypeptide having a naturally-occurring amino acid sequence).
In some cases, an OVV of the present disclosure comprises: a) a nucleotide sequence encoding a variant B5 polypeptide, wherein the variant B5 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant B5 polypeptide; b) a nucleotide sequence encoding an A33 polypeptide that does not include any amino acid substitutions that provide for enhanced viral spreading and/or enhanced production of EEV; and c) a nucleotide sequence encoding an A34 polypeptide that does not include any amino acid substitutions that provide for enhanced viral spreading and/or enhanced production of EEV. In some cases, an OVV of the present disclosure comprises: a) a nucleotide sequence encoding a variant B5 polypeptide, wherein the variant B5 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant B5 polypeptide; b) a nucleotide sequence encoding a wild-type A33 polypeptide (e.g., an A33 polypeptide having a naturally-occurring amino acid sequence); and c) a nucleotide sequence encoding a wild-type A34 polypeptide (e.g., an A34 polypeptide having a naturally-occurring amino acid sequence).
In some cases, an OVV of the present disclosure comprises: a) a nucleotide sequence encoding a variant A33 polypeptide, wherein the variant A33 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A33 polypeptide (e.g., compared to a control vaccinia virus that comprises a nucleotide sequence encoding a wild-type A33 polypeptide, such as an A33 polypeptide having an amino acid sequence depicted in
In some cases, an OVV of the present disclosure comprises: a) a nucleotide sequence encoding a variant A33 polypeptide, wherein the variant A33 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A33 polypeptide (e.g., compared to a control vaccinia virus that comprises a nucleotide sequence encoding a wild-type A33 polypeptide, such as an A33 polypeptide having an amino acid sequence depicted in
In some cases, an OVV of the present disclosure comprises: a) a nucleotide sequence encoding a variant A34 polypeptide, wherein the variant A34 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A34 polypeptide (e.g., compared to a control vaccinia virus that comprises a nucleotide sequence encoding a wild-type A34 polypeptide, such as an A34 polypeptide having an amino acid sequence depicted in
In some cases, an OVV of the present disclosure comprises: a) a nucleotide sequence encoding a variant A33 polypeptide, wherein the variant A33 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A33 polypeptide (e.g., compared to a control vaccinia virus that comprises a nucleotide sequence encoding a wild-type A33 polypeptide, such as an A33 polypeptide having an amino acid sequence depicted in
In some cases, an OVV of the present disclosure exhibits increased viral spreading and/or increased EEV production, compared to a control vaccinia virus that does not comprise a nucleotide sequence encoding a variant A33 polypeptide and/or a variant A34 polypeptide and/or a variant B5 polypeptide, as described herein. For example, in some cases, an OVV of the present disclosure comprising a nucleotide sequence encoding a variant A33 polypeptide and/or a nucleotide sequence encoding a variant A34 polypeptide and/or a nucleotide sequence encoding a variant B5 polypeptide exhibits at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or more than 25-fold, greater spreading, compared to a control vaccinia virus that does not comprise the nucleotide sequence(s) encoding the variant A33 polypeptide and/or the variant A34 polypeptide and/or the variant B5 polypeptide. Whether a given variant A33 polypeptide, a given variant A34 polypeptide, a given B5 polypeptide, or a combination of two or three of a given A33 polypeptide, a given variant A34 polypeptide, and a given variant B5 polypeptide, provides for increased viral spreading, compared to a control, can be determined using any known method, including a method as described in the Examples herein, such as the two-stage infectivity assay with U-2 OS cells described in Example 2. The two-stage infectivity assay comprises the steps of (1) infecting the U-2 cells with different 3-fold dilutions of multiplicities of infection (MOI) of vaccinia virus, (2) collecting the supernatant 22 hours post-infection, (3) infecting a new plate of U-2 OS cells with the supernatant, and (4) measuring luciferase expressed from the virus 15 hours post-infection, wherein an increased levels of luciferase is indicative of increased virus spreading.
In some cases, an OVV of the present disclosure comprising a nucleotide sequence encoding a variant A33 polypeptide, a nucleotide sequence encoding a variant A34 polypeptide, a nucleotide sequence encoding a variant B5 polypeptide, or nucleotide sequences encoding any combination of variant A33, A34, and B5 polypeptide exhibits at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or more than 25-fold, greater EEV production, compared to a control vaccinia virus that comprise the nucleotide sequence(s) encoding the corresponding wild-type A33, A34, and B5 polypeptide. Whether a given variant A33 polypeptide, a given variant A34 polypeptide, a given variant B5 polypeptide, or a combination of two or three of a given A33 polypeptide, a given variant A34 polypeptide, and a given B5 polypeptide, provides for increased EEV production, compared to a control, can be determined using any known method, including a method as described in the Examples herein, such as the plaque assay described in Example 3. The plaque assay comprises the steps of (1) infecting one or more cell lines the viruses, (2) washing the cells after 1 hour of virus adsorption, (3) collecting the supernatant (potentially EEVs). at 24 hours post-infection, and (4) determining the number of infectious viruses produced in the supernatant via plaque assay.
In some cases, an OVV of the present disclosure exhibits increased viral spreading and/or increased EEV production, compared to a control vaccinia virus that does not comprise a nucleotide sequence encoding a variant A33 polypeptide and/or a variant A34 polypeptide as described herein. For example, in some cases, an OVV of the present disclosure comprising a nucleotide sequence encoding a variant A33 polypeptide and/or a nucleotide sequence encoding a variant A34 polypeptide exhibits at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or more than 25-fold, greater spreading, compared to a control vaccinia virus that does not comprise the nucleotide sequence(s) encoding the variant A33 polypeptide and/or the variant A34 polypeptide (e.g., compared to a control vaccinia virus that comprises a nucleotide sequence encoding a wild-type A33 polypeptide, such as an A33 polypeptide having an amino acid sequence depicted in
In some cases, an OVV of the present disclosure comprising a nucleotide sequence encoding a variant A33 polypeptide and/or a nucleotide sequence encoding a variant A34 polypeptide exhibits at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or more than 25-fold, greater EEV production, compared to a control vaccinia virus that does not comprise the nucleotide sequence(s) encoding the variant A33 polypeptide and/or the variant A34 polypeptide (e.g., compared to a control vaccinia virus that comprises a nucleotide sequence encoding a wild-type A33 polypeptide, such as an A33 polypeptide having an amino acid sequence depicted in
In some cases, an OVV of the present disclosure exhibits increased viral spreading and/or increased EEV production, compared to a control vaccinia virus that does not comprise a nucleotide sequence encoding a variant B5 polypeptide as described herein (e.g., compared to a control vaccinia virus that comprises a nucleotide sequence encoding a wild-type B5 polypeptide, such as a B5 polypeptide having an amino acid sequence depicted in
In some cases, an OVV of the present disclosure comprising a nucleotide sequence encoding a variant B5 polypeptide exhibits at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or more than 25-fold, greater EEV production, compared to a control vaccinia virus that does not include a nucleotide sequence encoding a variant B5 polypeptide (e.g., compared to a control vaccinia virus that comprises a nucleotide sequence encoding a wild-type B5 polypeptide, such as a B5 polypeptide having an amino acid sequence depicted in
In some cases, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or more than 75%, of the infectious virions produced using an OVV of the present disclosure are EEV. In some cases, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or more than 75%, of the total physical viral particles produced using an OVV of the present disclosure are EEV.
Variant A33 Polypeptides
Examples of wild-type amino acid sequences of A33 polypeptides of common strains of vaccinia virus are depicted in
In some cases, a variant A33 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A33 amino acid sequence depicted in
In some cases, a variant A33 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A33 amino acid sequence depicted in
In some cases, a variant A33 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A33 amino acid sequence depicted in
In some cases, a variant A33 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A33 amino acid sequence depicted in
In some cases, a variant A33 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A33 amino acid sequence depicted in
In some cases, a variant A33 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A33 amino acid sequence depicted in
In some cases, a variant A33 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A33 amino acid sequence depicted in
Variant A34 Polypeptides
Examples of wild-type amino acid sequences of A34 polypeptides of common strains of vaccinia virus are depicted in
In some cases, a variant A34 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A34 amino acid sequence depicted in
In some cases, a variant A34 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A34 amino acid sequence depicted in
In some cases, a variant A34 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A34 amino acid sequence depicted in
In some cases, a variant A34 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A34 amino acid sequence depicted in
In some cases, a variant A34 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A34 amino acid sequence depicted in
In some cases, a variant A34 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A34 amino acid sequence depicted in
In some cases, a variant A34 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A34 amino acid sequence depicted in
In some cases, a variant A34 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A34 amino acid sequence depicted in
In any of the above-described embodiments, a variant A34 polypeptide can have, at position 151 based on the numbering shown in
Combinations of Variant A33 Polypeptide and Variant A34 Polypeptide
As noted above, in some cases, an OVV of the present disclosure comprises: a) a nucleotide sequence encoding a variant A33 polypeptide, where the variant A33 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A33 polypeptide (e.g., compared to a control vaccinia virus that comprises a nucleotide sequence encoding a wild-type A33 polypeptide, such as an A33 polypeptide having an amino acid sequence depicted in
An OVV of the present disclosure that comprises nucleotide sequences encoding both a variant A33 polypeptide and a variant A34 polypeptide can comprise nucleotide sequences encoding any of the above-described variant A33 polypeptides and any of the above-described A34 polypeptides in any combination. Non-limiting examples of combinations are set out in Table 2.
Thus, e.g., in some cases, an OVV of the present disclosure comprises: a) a nucleotide sequence encoding a variant A33 polypeptide, where the variant A33 polypeptide comprises 1, 2, or 3 amino acid substitutions at M63, A88, and E129, as described above; and b) a nucleotide sequence encoding a variant A34 polypeptide, where the variant A34 polypeptide comprises 1, 2, 3, 4, or 5 amino acid substitutions at M66, F94, R84, R91, and T127, as described above.
The following are exemplary combinations and are not meant to be limiting.
A33(A88x) and A34(F94x)
As an example, an OVV of the present disclosure comprises a) a nucleotide sequence encoding a variant A33 polypeptide, where the variant A33 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A33 amino acid sequence depicted in
A33(E129x) AND A34(F94x) As an example, an OVV of the present disclosure comprises a) a nucleotide sequence encoding a variant A33 polypeptide, where the variant A33 polypeptide comprises an amino acid at position 129 other than Glu (e.g., the variant A33 polypeptide comprises Ala, Gly, lie, Leu, Pro, Val, Phe, Trp, Tyr, Asp, Arg, His, Lys, Ser, Thr, Cys, Met, Asn, or Gln at position 129; and b) a nucleotide sequence encoding a variant A34 polypeptide, where the variant A34 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A34 amino acid sequence depicted in
A33(A88x; and E129x) and A34(F94x)
As an example, an OVV of the present disclosure comprises: a) a nucleotide sequence encoding a variant A33 polypeptide, where the variant A33 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A33 amino acid sequence depicted in
Variant A56 Polypeptides
In any of the embodiments set out above, in some cases, an OVV of the present disclosure comprises a nucleotide sequence encoding a variant A56 polypeptide, where the A56 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A56 amino acid sequence depicted in
As an example, an OVV of the present disclosure comprises: a) a nucleotide sequence encoding a variant A34 polypeptide, where the variant A34 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A34 amino acid sequence depicted in
Variant B5 Polypeptides
In some embodiments, an OVV provided by the resent disclosure comprises a nucleotide sequence encoding a variant B5 polypeptide. Examples of wild-type amino acid sequences of vaccinia virus B5 polypeptides are depicted in
In some cases, a variant B5 polypeptide comprises a single amino acid substitution at one of N39, L90, N94, S197, S199, K229, V233, 1236, V238, T240, N241, E243, V247, D248, G250, D263, E268, E270, E272, S273, E275, and A276, where the amino acid numbering is based on the numbering shown in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In some cases, a variant B5 polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to a B5 amino acid sequence depicted in
In any one of the above-described embodiments, the replication-competent, recombinant oncolytic vaccinia virus can further include a nucleotide sequence encoding a variant A34 polypeptide, where the variant A34 polypeptide has a K151E substitution. For example, the variant A34 polypeptide can comprise an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity to an A34 amino acid sequence depicted in
Variant A34 Polypeptides and Variant B5 Polypeptides
In some cases, an OVV of the present disclosure comprises nucleotide sequences encoding both a variant A34 polypeptide and a variant B5 polypeptide; such an OVV can comprise nucleotide sequences encoding any of the above-described variant A34 polypeptides and any of the above-described B5 polypeptides in any combination. Non-limiting examples of combinations are set out in Table 2.
As one non-limiting example, an OVV of the present disclosure comprises: a) a nucleotide sequence encoding a variant A34 polypeptide, where the variant A34 polypeptide comprises an F94H substitution; and b) a nucleotide sequence encoding a variant B5 polypeptide, where the variant B5 polypeptide comprises an N39G substitution and an S273I substitution. As another non-limiting example, an OVV of the present disclosure comprises: a) a nucleotide sequence encoding a variant A34 polypeptide, where the variant A34 polypeptide comprises an F94H substitution and a K151E substitution; and b) a nucleotide sequence encoding a variant B5 polypeptide, where the variant B5 polypeptide comprises an N39G substitution and an S273I substitution.
Construction of the Oncolytic Vaccinia Viruses
An OVV of the present disclosure can be constructed from any of a variety of strains of vaccinia virus. Examples of vaccinia virus strain suitable for use include, but not limited to, the strains Lister, New York City Board of Health (NYBH), Wyeth, Copenhagen, Western Reserve (WR), Modified Vaccinia Ankara (MVA), EM63, Ikeda, Dalian, LIVP, Tian Tan, IHD-J, Tashkent, Bern, Paris, Dairen and derivatives the like. In some cases, an OVV of the present disclosure is a Copenhagen strain vaccinia virus. In some cases, an OVV of the present disclosure is a WR strain vaccinia virus.
The nucleotide sequences of the genomes of vaccinia viruses of various strains are known in the art. See, e.g., Goebel et al. (1990) Virology 179:247; Goebel et al. (1990) Virology 179:517. The nucleotide sequence of the Copenhagen strain vaccinia virus is known; see, e.g., GenBank Accession No. M35027. The nucleotide sequence of the WR strain vaccinia virus is known; see, e.g., GenBank Accession No. AY243312; and GenBank Accession No. NC_006998. The WR strain of vaccinia virus is available from the American Type Culture Collection (ATCC); ATCC VR-1354.
The vaccinia virus used to construct an OVV of the present disclosure can include attenuated and/or tumor-selective vaccinia viruses. As used herein, “attenuated” means low toxicity (for example, low virus replication, low cytolytic activity, low cytotoxic activity) to normal cells (for example, non-tumor cells). As used herein, “tumor selective” means toxicity to tumor cells (for example, oncolytic) higher than that to normal cells (for example, non-tumor cell). Vaccinia viruses genetically modified to be deficient in the function of a specific protein or to suppress the expression of a specific gene or protein (Guse et al. (2011) Expert Opinion on Biological Therapy 11:595) may be used in an oncolytic virus of the present disclosure. For example, in order to increase tumor selectivity of vaccinia virus, vaccinia virus deficient in the function of vaccinia growth factor (VGF) (McCart et al. (2001) Cancer Research 61:8751); vaccinia virus having a modified vaccinia virus TK gene, a modified hemagglutinin (HA) gene, and a modified F3 gene or an interrupted F3 locus (WO 2005/047458), vaccinia virus deficient in the function of VGF and O1L (WO 2015/076422); vaccinia virus in which a target sequence of a microRNA whose expression is decreased in cancer cells is inserted into the 3′ noncoding region of the B5R gene (WO 2011/125469); vaccinia virus deficient in the function of HA and F14.5L (Zhang et al. (2007) Cancer Research 67:10038); vaccinia virus deficient in the function of ribonucleotide reductase (Gammon et al. (2010) PLoS Pathogens 6:e1000984); vaccinia virus deficient in the function of serine protease inhibitor (e.g., SPI-1, SPI-2) (Guo et al. (2005) Cancer Research 65:9991); vaccinia virus deficient in the function of SPI-1 and SPI-2 (Yang et al. (2007) Gene Therapy 14:638); vaccinia virus deficient in the function of ribonucleotide reductase genes F4L or 14L (Child et al. (1990) Virology 174:625; Potts et al. (2017) EMBO Mol. Med. 9:638); vaccinia virus deficient in the function of B18R (B19R in Copenhagen strain) (Symons et al. (1995) Cell 81:551; Kirn et al. (2007) PLoS Medicine 4:e353); vaccinia virus deficient in the function of A48R (Hughes et al. (1991) J. Biol. Chem. 266:20103); vaccinia virus deficient in the function of B8R (Verardi et al. (2001) J. Virol. 75:11); vaccinia virus deficient in the function of B15R (B16R in Copenhagen strain) (Spriggs et al. (1992) Cell 71:145); vaccinia virus deficient in the function of A41R (Ng et al. (2001) Journal of General Virology 82:2095); vaccinia virus deficient in the function of A52R (Bowie et al. (2000) Proc. Natl. Acad. Sci. USA 97:10162); vaccinia virus deficient in the function of F1L (Gerlic et al. (2013) Proc. Natl. Acad. Sci. USA 110:7808); vaccinia virus deficient in the function of E3L (Chang et al. (1992) Proc. Natl. Acad. Sci. USA 89:4825); vaccinia virus deficient in the function of A44R-A46R (Bowie et al. (2000) Proc. Natl. Acad. Sci. USA 97:10162); vaccinia virus deficient in the function of K1L (Bravo Cruz et al. (2017) Journal of Virology 91:e00524); vaccinia virus deficient in the function of A48R, B18R, C11R, and TK (Mejías-Pérez et al. (2017) Molecular 25 Therapy: Oncolytics 8:27); or vaccinia virus having mutations in the E3L and K3L regions (WO 2005/007824) may be used. Moreover, vaccinia virus deficient in the function of O1 L may be used (Schweneker et al. (2012) J. Virol. 86:2323). Moreover, vaccinia virus deficient in the extracellular region of B5R (Bell et al. (2004) Virology 325:425) or vaccinia virus deficient in the A34R region (Thirunavukarasu et al. (2013) Molecular Therapy 21:1024) may be used. Moreover, vaccinia virus deficient in interleukin-1b (IL-1b) receptor (WO 2005/030971) may be used. Such insertion of a foreign gene or deletion or mutation of a gene can be made, for example, by a known homologous recombination or site-directed mutagenesis. Moreover, vaccinia virus having a combination of two or more of such genetic modifications may be used in an OVV of the present disclosure.
As used herein, “being deficient” means that the gene region, or a gene product, specified by this term has reduced or no function and includes a deficiency resulting from one or more of: i) mutation (e.g., substitution, inversion, etc.) and/or truncation and/or deletion of the gene region; ii) mutation and/or truncation and/or deletion of a promoter region controlling expression of the gene region; and iii) mutation and/or truncation and/or deletion of a polyadenylation sequence such that translation of a polypeptide encoded by the gene region is reduced or eliminated. An OVV of the present disclosure that comprises a genetic alteration such that the replication-competent, recombinant oncolytic vaccinia virus is “deficient” in a given vaccinia virus gene exhibits reduced production and/or activity of a gene product (e.g., mRNA gene product; polypeptide gene product) of the gene; for example, the amount and/or activity of the gene product is less than 75%, less than 60%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the amount and/or activity of the same gene product produced by wild-type vaccinia virus, or by a control vaccinia virus that does not comprise the genetic alteration. For example, “being deficient” may be a result of the deletion in a region consisting of the specified gene region or the deletion in a neighboring gene region comprising the specified gene region. As an example, a mutation and/or truncation and/or deletion of a promoter region that reduces transcription of a gene region can result in deficiency. A gene region can also be rendered deficient through incorporation of a transcriptional termination element such that translation of a polypeptide encoded by the gene region is reduced or eliminated. A gene region can also be rendered deficient through use of a gene-editing enzyme or a gene-editing complex to reduce or eliminate transcription of the gene region. A gene region can also be rendered deficient through use of competitive reverse promoter/polymerase occupancy to reduce or eliminate transcription of the gene region. A gene region can also be rendered deficient by insertion of a nucleic acid into the gene region, thereby knocking out the gene region.
In some embodiments, an OVV provided by the present disclosure is vaccinia virus thymidine kinase (TK) deficient. In some cases, an OVV of the present disclosure comprises a deletion of all or a portion of the vaccinia virus TK coding region, such that the replication-competent, recombinant oncolytic vaccinia virus is TK deficient. For example, in some cases, an OVV of the present disclosure comprises a deletion in the J2R gene (i.e., gene that encodes viral thymidine kinase). See, e.g., Mejía-Perez et al. (2018) Mol. Ther. Oncolytics 8:27. In some cases, an OVV of the present disclosure comprises an insertion into the J2R region, thereby resulting in reduced vaccinia virus TK expression or activity.
An OVV of the present disclosure will in some instances comprise an A34R gene encoded an A34 polypeptide having a K151E substitution (i.e., comprising a modification that provides for a K151E substitution in the encoded polypeptide). See, e.g., Blasco et al. (1993) J. Virol. 67(6):3319-3325; and Thirunavukarasu et al. (2013) Mol. Ther. 21:1024. The A34R gene encodes vaccinia virus gp22-24.
In some cases, an OVV of the present disclosure comprises a heterologous nucleic acid comprising a nucleotide sequence encoding an immunomodulatory polypeptide (a heterologous immunomodulatory polypeptide). Examples of immunomodulatory polypeptide include cytokines (such as IL-2, and IL-12), granulocyte-macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-α).
An OVV of the present disclosure exhibits oncolytic activity. Examples of methods for evaluating whether a given virus exhibits oncolytic activity include a method for evaluating decrease of the survival rate of cancer cells by the addition of the virus. Examples of cancer cells to be used for the evaluation include the malignant melanoma cell RPMI-7951 (for example, ATCC HTB-66), the lung adenocarcinoma HCC4006 (for example, ATCC CRL-2871), the lung carcinoma A549 (for example, ATCC CCL-185), the lung carcinoma HOP-62 (for example, DCTD Tumor Repository), the lung carcinoma EKVX (for example, DCTD Tumor Repository), the small cell lung cancer cell DMS 53 (for example, ATCC CRL-2062), the lung squamous cell carcinoma NCI-H226 (for example, ATCC CRL-5826), the kidney cancer cell Caki-1 (for example, ATCC HTB-46), the bladder cancer cell 647-V (for example, DSMZ ACC 414), the head and neck cancer cell Detroit 562 (for example, ATCC CCL-138), the breast cancer cell JIMT-1 (for example, DSMZ ACC 589), the breast cancer cell MDA-MB-231 (for example, ATCC HTB-26), the breast cancer cell MCF7 (for example, ATCC HTB-22), the breast cancer HS-578T (for example, ATCC HTB-126), the breast ductal carcinoma T-47D (for example, ATCC HTB-133), the esophageal cancer cell OE33 (for example, ECACC 96070808), the glioblastoma U-87MG (for example, ECACC 89081402), the neuroblastoma GOTO (for example, JCRB JCRB0612), the myeloma RPMI 8226 (for example, ATCC CCL-155), the ovarian cancer cell SK-OV-3 (for example, ATCC HTB-77), the ovarian cancer cell OVMANA (for example, JCRB JCRB1045), the cervical cancer HeLa (for example, ATCC CCL-2), the colon cancer cell RKO (for example, ATCC CRL-2577), the colon cancer cell HT-29 (for example, ATCC HTB-38), the colon cancer Colo 205 (for example, ATCC CCL-222), the colon cancer SW620 (for example, ATCC CCL-227), the colorectal carcinoma HCT 116 (for example, ATCC CCL-247), the pancreatic cancer cell BxPC-3 (for example, ATCC CRL-1687), the bone osteosarcoma U-2 OS (for example, ATCC HTB-96), the prostate cancer cell LNCaP clone FGC (for example, ATCC CRL-1740), the hepatocellular carcinoma JHH-4 (for example, JCRB JCRB0435), the mesothelioma NCI-H28 (for example, ATCC CRL-5820), the cervical cancer cell SiHa (for example, ATCC HTB-35), and the gastric cancer cell Kato III (for example, RIKEN BRC RCB2088). Also suitable for use are cancer cells obtained from a patient. For example, colorectal cancer cells, breast cancer cells, or non-small lung cancer cells obtained from a patient can be used.
As noted above, an OVV of the present disclosure exhibits enhanced virus spreading and/or enhanced EEV production, compared to a control vaccinia virus that does not include a) a nucleotide sequence encoding a variant A33 polypeptide, as described herein (e.g., compared to a control vaccinia virus that comprises a nucleotide sequence encoding a wild-type A33 polypeptide, such as an A33 polypeptide having an amino acid sequence depicted in
Compositions
The present disclosure provides compositions comprising an OVV of the present disclosure. In some cases, the composition is a pharmaceutical composition. A pharmaceutical composition comprising an OVV of the present disclosure can further comprises a pharmaceutically acceptable carrier(s). As used herein, the terms “pharmacologically acceptable carrier” and “pharmacologically acceptable excipient” are used interchangeably and refer to any substance that is suitable for use in the formulation of the OVV for administration to a human. Such a carrier generally is mixed with an OVV of the present disclosure, and can be a solid, semi-solid, or liquid agent. The composition can be a solution or a suspension in the desired carrier or diluent. Any of a variety of pharmaceutically acceptable carriers can be used including, without limitation, aqueous media such as, e.g., distilled, deionized water, saline; solvents; dispersion media; coatings; antibacterial and antifungal agents; isotonic and absorption delaying agents; or any other inactive ingredient. Selection of a pharmacologically acceptable carrier can depend on the mode of administration. Except insofar as any pharmacologically acceptable carrier is incompatible with an OVV of the present disclosure, its use in pharmaceutically acceptable compositions is contemplated. Non-limiting examples of specific uses of such pharmaceutical carriers can be found in “Pharmaceutical Dosage Forms and Drug Delivery Systems” (Howard C. Ansel et al., eds., Lippincott Williams & Wilkins Publishers, 7th ed. 1999); “Remington: The Science and Practice of Pharmacy” (Alfonso R. Gennaro ed., Lippincott, Williams & Wilkins, 20th 2000); “Goodman & Gilman's The Pharmacological Basis of Therapeutics” Joel G. Hardman et al., eds., McGraw-Hill Professional, 10th ed. 2001); and “Handbook of Pharmaceutical Excipients” (Raymond C. Rowe et al., APhA Publications, 4th edition 2003).
A subject pharmaceutical composition can optionally include, without limitation, other pharmaceutically acceptable components, including, without limitation, buffers, preservatives, tonicity adjusters, salts, antioxidants, physiological substances, pharmacological substances, bulking agents, emulsifying agents, wetting agents, and the like. Various buffers and means for adjusting pH can be used to prepare a pharmaceutical composition disclosed in the present specification, provided that the resulting preparation is pharmaceutically acceptable. Such buffers include, without limitation, acetate buffers, citrate buffers, phosphate buffers, neutral buffered saline, phosphate buffered saline and borate buffers. It is understood that acids or bases can be used to adjust the pH of a composition as needed. Pharmaceutically acceptable antioxidants include, without limitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene. Useful preservatives include, without limitation, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate and a stabilized oxy chloro composition, for example, PURITE™. Tonicity adjustors suitable for inclusion in a subject pharmaceutical composition include, without limitation, salts such as, e.g., sodium chloride, potassium chloride, mannitol or glycerin and other pharmaceutically acceptable tonicity adjustor. It is understood that these and other substances known in the art of pharmacology can be included in a subject pharmaceutical composition.
Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.
A pharmaceutical composition of the present disclosure can comprise an OVV of the present disclosure in an amount of from about 102 plaque-forming units (pfu) per ml (pfu/ml) to about 104 pfu/ml, from about 104 pfu/ml to about 105 pfu/ml, from about 105 pfu/ml to about 106 pfu/ml, from about 106 pfu/ml to about 107 pfu/ml, from about 107 pfu/ml to about 108 pfu/ml, from about 108 pfu/ml to about 109 pfu/ml, from about 109 pfu/ml to about 1010 pfu/ml, from about 1010 pfu/ml to about 1011 pfu/ml, or from about 1011 pfu/ml to about 1012 pfu/ml.
Methods of Inducing Oncolysis
The present disclosure provides methods of inducing oncolysis in an individual having a tumor, the methods comprising administering to the individual an effective amount of an OVV of the present disclosure or a composition of the present disclosure. Administration of an effective amount of an OVV of the present disclosure, or a composition of the present disclosure, is also referred to herein as “virotherapy.” The present disclosure further provides an OVV or composition of the present disclosure for use in methods of inducing oncolysis in an individual having a tumor. The disclosure further provides the use of an OVV or composition of the present disclosure in the manufacture of a medicament for use in such methods.
In some cases, an “effective amount” of an OVV of the present disclosure is an amount that, when administered in 1, 2, or more doses to an individual in need thereof, reduces the number of cancer cells in the individual. For example, in some cases, an “effective amount” of an OVV of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, compared to the number of cancer cells in the individual before administration of the replication-competent, recombinant oncolytic vaccinia virus, or in the absence of administration with the replication-competent, recombinant oncolytic vaccinia virus. In some cases, an “effective amount” of an OVV of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual to undetectable levels. In some cases, an “effective amount” of an OVV of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, reduces the tumor mass in the individual.
For example, in some cases, an “effective amount” of an OVV of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, reduces the tumor mass in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, compared to the tumor mass in the individual before administration of the replication-competent, recombinant oncolytic vaccinia virus, or in the absence of administration with the replication-competent, recombinant oncolytic vaccinia virus.
In some cases, an “effective amount” of an OVV of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, increases survival time of the individual. For example, in some cases, an “effective amount” of an OVV of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, increases survival time of the individual by at least 1 month, at least 2 months, at least 3 months, from 3 months to 6 months, from 6 months to 1 year, from 1 year to 2 years, from 2 years to 5 years, from 5 years to 10 years, or more than 10 years, compared to the expected survival time of the individual in the absence of administration with the replication-competent, recombinant oncolytic vaccinia virus.
In some cases, an “effective amount” of an OVV of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, induces a durable anti-tumor immune response, e.g., an anti-tumor immune response that provides for reduction in tumor cell number and/or tumor mass and/or tumor growth for at least 1 month, at least 2 months, at least 6 months, or at least 1 year.
A suitable dosage can be determined by an attending physician or other qualified medical personnel, based on various clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, tumor burden, and other relevant factors.
An OVV of the present disclosure can be administered in an amount of from about 102 plaque-forming units (pfu) to about 104 pfu, from about 104 pfu to about 105 pfu, from about 105 pfu to about 106 pfu, from about 106 pfu to about 107 pfu, from about 107 pfu to about 108 pfu, from about 108 pfu to about 109 pfu, from about 109 pfu to about 1010 pfu, from about 1010 pfu to about 1011 pfu, or from about 1011 pfu to about 1012 pfu per dose.
In some cases, an OVV of the present disclosure is administered in a total amount of from about 1×109 pfu to 5×1012 pfu. In some cases, an OVV of the present disclosure is administered in a total amount of from about 1×109 pfu to about 5×109 pfu, from about 5×109 pfu to about 1010 pfu, from about 1010 pfu to about 5×1010 pfu, from about 5×1010 pfu to about 1011 pfu, from about 1011 pfu to about 5×1011 pfu, from about 5×1011 pfu to about 1012 pfu, or from about 1012 pfu to about 5×1012 pfu. In some cases, an OVV of the present disclosure is administered in a total amount of about 2×1010 pfu.
In some cases, an OVV of the present disclosure is administered in an amount of from about 1×108 pfu/kg patient weight to about 1×1010 pfu/kg patient weight. In some cases, an OVV of the present disclosure is administered in an amount of from about 1×108 pfu/kg patient weight to about 5×108 pfu/kg patient weight, from about 5×108 pfu/kg patient weight to about 109 pfu/kg patient weight, from about 109 pfu/kg patient weight to about 5×109 pfu/kg patient weight, or from about 5×109 pfu/kg patient weight to about 1010 pfu/kg patient weight. In some cases, an OVV of the present disclosure is administered in an amount of 1×108 pfu/kg patient weight. In some cases, an OVV of the present disclosure is administered in an amount of 2×108 pfu/kg patient weight. In some cases, an OVV of the present disclosure is administered in an amount of 3×108 pfu/kg patient weight. In some cases, an OVV of the present disclosure is administered in an amount of 4×108 pfu/kg patient weight. In some cases, an OVV of the present disclosure is administered in an amount of 5×108 pfu/kg patient weight. In some cases, an OVV of the present disclosure is administered in an amount of 109 pfu/kg patient weight. In some cases, an OVV of the present disclosure is administered in an amount of 5×109 pfu/kg patient weight.
In some cases, multiple doses of an OVV of the present disclosure are administered. The frequency of administration of an OVV of the present disclosure can vary depending on any of a variety of factors, e.g., severity of the symptoms, etc. For example, in some embodiments, an OVV of the present disclosure is administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).
The duration of administration of an OVV of the present disclosure, e.g., the period of time over which a multimeric polypeptide of the present disclosure, an OVV of the present disclosure is administered, can vary, depending on any of a variety of factors, e.g., patient response, etc. For example, an OVV of the present disclosure can be administered over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more.
An OVV of the present disclosure is administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.
Conventional and pharmaceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intratracheal, intrathecal, intracranial, subcutaneous, intradermal, topical application, intravenous, intraarterial, intraperitoneal, intrabladder, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the replication-competent, recombinant oncolytic vaccinia virus and/or the desired effect. An OVV of the present disclosure can be administered in a single dose or in multiple doses.
In some cases, an OVV of the present disclosure is administered intravenously. In some cases, an OVV of the present disclosure is administered intramuscularly. In some cases, an OVV of the present disclosure is administered locally. In some cases, an OVV of the present disclosure is administered intratumorally. In some cases, an OVV of the present disclosure is administered peritumorally. In some cases, an OVV of the present disclosure is administered intracranially. In some cases, an OVV of the present disclosure is administered subcutaneously. In some cases, an OVV of the present disclosure is administered intra-arterially. In some cases, an OVV of the present disclosure is administered intraperitoneally. In some cases, an OVV of the present disclosure is administered via an intrabladder route of administration. In some cases, an OVV of the present disclosure is administered intrathecally.
Combinations
In some cases, an OVV of the present disclosure is administered in combination with another cancer therapy, such as surgery (e.g., surgical removal of cancerous tissue), radiation therapy, bone marrow transplantation, chemotherapeutic treatment, antibody treatment, biological response modifier treatment, immunotherapy treatment, and certain combinations of the foregoing. In some cases, a method of the present disclosure comprises: a) administering to an individual in need thereof an OVV of the present disclosure, or a composition comprising same; and b) administering to the individual a second cancer therapy. In some cases, the second cancer therapy is selected from chemotherapy, biological therapy, radiotherapy, immunotherapy, hormone therapy, anti-vascular therapy, cryotherapy, toxin therapy, oncolytic virus therapy (e.g., an oncolytic virus other than an OVV of the present disclosure), a cell therapy, and surgery.
Radiation therapy includes, but is not limited to, x-rays or gamma rays that are delivered from either an externally applied source such as a beam, or by implantation of small radioactive sources.
Suitable antibodies for use in cancer treatment include, but are not limited to, e.g., trastuzumab (Herceptin), bevacizumab (Avastin™), cetuximab (Erbitux™) panitumumab (Vectibix™), Ipilimumab (Yervoy™), rituximab (Rituxan), alemtuzumab (Lemtrada™), Ofatumumab (Arzerra™), Oregovomab (OvaRex™), Lambrolizumab (MK-3475), pertuzumab (Perjeta™), ranibizumab (Lucentis™) etc., and conjugated antibodies, e.g., gemtuzumab ozogamicin (Mylortarg™), Brentuximab vedotin (Adcetris™), 90Y-labelled ibritumomab tiuxetan (Zevalin™)1311-labelled tositumoma (Bexxar™), etc. Suitable antibodies for use in cancer treatment include, but are not limited to, e.g., Ipilimumab targeting CTLA-4 (as used in the treatment of Melanoma, Prostate Cancer, RCC); Tremelimumab targeting CTLA-4 (as used in the treatment of CRC, Gastric, Melanoma, NSCLC); Nivolumab targeting PD-1 (as used in the treatment of Melanoma, NSCLC, RCC); MK-3475 targeting PD-1 (as used in the treatment of Melanoma); Pidilizumab targeting PD-1 (as used in the treatment of Hematologic Malignancies); BMS-936559 targeting PD-L1 (as used in the treatment of Melanoma, NSCLC, Ovarian, RCC); MED14736 targeting PD-L1; MPDL33280A targeting PD-L1 (as used in the treatment of Melanoma); Rituximab targeting CD20 (as used in the treatment of Non-Hodgkin's lymphoma); Ibritumomab tiuxetan and tositumomab (as used in the treatment of Lymphoma); Brentuximab vedotin targeting CD30 (as used in the treatment of Hodgkin's lymphoma); Gemtuzumab ozogamicin targeting CD33 (as used in the treatment of Acute myelogenous leukaemia); Alemtuzumab targeting CD52 (as used in the treatment of Chronic lymphocytic leukaemia); IGN101 and adecatumumab targeting EpCAM (as used in the treatment of Epithelial tumors (breast, colon and lung)); Labetuzumab targeting CEA (as used in the treatment of Breast, colon and lung tumors); huA33 targeting gpA33 (as used in the treatment of Colorectal carcinoma); Pemtumomab and oregovomab targeting Mucins (as used in the treatment of Breast, colon, lung and ovarian tumors); CC49 (minretumomab) targeting TAG-72 (as used in the treatment of Breast, colon and lung tumors); cG250 targeting CAIX (as used in the treatment of Renal cell carcinoma); J591 targeting PSMA (as used in the treatment of Prostate carcinoma); MOv18 and MORAb-003 (farletuzumab) targeting Folate-binding protein (as used in the treatment of Ovarian tumors); 3F8, ch14.18 and KW-2871 targeting Gangliosides (such as GD2, GD3 and GM2) (as used in the treatment of Neuroectodermal tumors and some epithelial tumors); hu3S193 and IgN311 targeting Le y (as used in the treatment of Breast, colon, lung and prostate tumors); Bevacizumab targeting VEGF (as used in the treatment of Tumor vasculature); IM-2C6 and CDP791 targeting VEGFR (as used in the treatment of Epithelium-derived solid tumors); Etaracizumab targeting Integrin_V_3 (as used in the treatment of Tumor vasculature); Volociximab targeting Integrin_5_1 (as used in the treatment of Tumor vasculature); Cetuximab, panitumumab, nimotuzumab and 806 targeting EGFR (as used in the treatment of Glioma, lung, breast, colon, and head and neck tumors); Trastuzumab and pertuzumab targeting ERBB2 (as used in the treatment of Breast, colon, lung, ovarian and prostate tumors); MM-121 targeting ERBB3 (as used in the treatment of Breast, colon, lung, ovarian and prostate, tumors); AMG 102, METMAB and SCH 900105 targeting MET (as used in the treatment of Breast, ovary and lung tumors); AVE1642, IMC-A12, MK-0646, R1507 and CP 751871 targeting IGF1R (as used in the treatment of Glioma, lung, breast, head and neck, prostate and thyroid cancer); KB004 and IIIA4 targeting EPHA3 (as used in the treatment of Lung, kidney and colon tumors, melanoma, glioma and hematological malignancies); Mapatumumab (HGS-ETR1) targeting TRAILR1 (as used in the treatment of Colon, lung and pancreas tumors and hematological malignancies); HGS-ETR2 and CS-1008 targeting TRAILR2; Denosumab targeting RANKL (as used in the treatment of Prostate cancer and bone metastases); Sibrotuzumab and F19 targeting FAP (as used in the treatment of Colon, breast, lung, pancreas, and head and neck tumors); 81C6 targeting Tenascin (as used in the treatment of Glioma, breast and prostate tumors); Blinatumomab (Blincyto; Amgen) targeting CD3 (as used in the treatment of ALL); pembrolizumab targeting PD-1 as used in cancer immunotherapy; 9E10 antibody targeting c-Myc; and the like.
In some cases, a method of the present disclosure comprises administering: a) an effective amount of an OVV of the present disclosure; and b) an anti-PD-1 antibody. In some cases, a method of the present disclosure comprises administering: a) an effective amount of an OVV of the present disclosure; and b) an anti-PD-L1 antibody. Examples of anti-PD-1 and anti-PD-L1 antibodies that may useful in the combination therapy of the present disclosure include, but are not limited to, pembrolizumab (Keytruda®; MK-3475), Nivolumab (Opdivo®; BMS-926558; MDX1106), Pidilizumab (CT-011), AMP-224, AMP-514 (MEDI-0680), PDR001, and PF-06801591 (also known as sasanlimab or RN888), BMS-936559 (MDX1105), durvalumab (MED14736; IMFINZI®), Atezolizumab (MPDL33280A; TECENTRIQ®), MSB0010718C, BCD-100 (BIOCAD Biopharmaceutical Company), tislelizumab (BGB-A317, BeiGene Ltd./Celgene Corporation), genolimzumab (CBT-501, CBT Pharmaceuticals), CBT-502 (CBT Pharmaceuticals), GLS-010 (Harbin Gloria Pharmaceuticals Co., Ltd.), sintilimab (IB1308, Innovent Biologics, Inc.), WBP3155 (CStone Pharmaceuticals Co., Ltd.), AMP-224 (GlaxoSmithKline plc), BI 754091 (Boehringer Ingelheim GmbH), BMS-936559 (Bristol-Myers Squibb Company), CA-170 (Aurigene Discovery Technologies), FAZ053 (Novartis AG), spartalizumab (PDR001, Novartis AG), LY3300054 (Eli Lilly & Company), MED10680 (AstraZeneca PLC), PDR001 (Novartis AG), cemiplimab (LIBTAYO®, REGN2810, Regeneron Pharmaceuticals, Inc.), camrelizumab (SHR-1210, Incyte Corporation), TSR-042 (Tesaro, Inc.), AGEN2034 (Agenus Inc.), CX-072 (CytomX Therapeutics, Inc.), JNJ-63723283 (Johnson & Johnson), MGD013 (MacroGenics, Inc.), AN-2005 (Adlai Nortye), ANA011 (AnaptysBio, Inc.), ANB011 (AnaptysBio, Inc.), AUNP-12 (Pierre Fabre Medicament S.A.), BBI-801 (Sumitomo Dainippon Pharma Co., Ltd.), BION-004 (Aduro Biotech), CA-327 (Aurigene Discovery Technologies), CK-301 (Fortress Biotech, Inc.), ENUM 244C8 (Enumeral Biomedical Holdings, Inc.), FPT155 (Five Prime Therapeutics, Inc.), FS118 (F-star Alpha Ltd.), hAb21 (Stainwei Biotech, Inc.), J43 (Transgene S.A.), JTX-4014 (Jounce Therapeutics, Inc.), KD033 (Kadmon Holdings, Inc.), KY-1003 (Kymab Ltd.), MCLA-134 (Merus B.V.), MCLA-145 (Merus B.V.), PRS-332 (Pieris AG), SHR-1316 (Atridia Pty Ltd.), STI-A1010 (Sorrento Therapeutics, Inc.), STI-A1014 (Sorrento Therapeutics, Inc.), STI-A1110 (Les Laboratoires Servier), and XmAb20717 (Xencor, Inc.). Additional anti-PD-1 and anti-PD-L1 antibodies ca nbe found in, e.g., Sunshine and Taube (2015) Curr. Opin. Pharmacol. 23:32; and Heery et al. (2017) The Lancet Oncology 18:587; Iwai et al. (2017) J. Biomed. Sci. 24:26; Hu-Lieskovan et al. (2017) Annals of Oncology 28: issue Suppl. 5, mdx376.048; WO2016/092419 and U.S. Patent Publication No. 2016/0159905.
In some cases, a suitable antibody is a bispecific antibody, e.g., a bispecific monoclonal antibody. Catumaxomab, blinatumomab, solitomab, pasotuxizumab, and flotetuzumab are non-limiting examples of bispecific antibodies suitable for use in cancer therapy. See, e.g., Chames and Baty (2009) MAbs 1:539; and Sedykh et al. (2018) Drug Des. Devel. Ther. 12:195.
Biological response modifiers suitable for use in connection with the methods of the present disclosure include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine/threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that bind specifically to a tumor antigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6) interferon-α; (7) interferon-γ; (8) colony-stimulating factors; (9) inhibitors of angiogenesis; and (10) antagonists of tumor necrosis factor.
Chemotherapeutic agents are non-peptidic (i.e., non-proteinaceous) compounds that reduce proliferation of cancer cells, and encompass cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones.
Agents that act to reduce cellular proliferation are known in the art and widely used. Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.
Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatine, and gemcitabine.
Suitable natural products and their derivatives, (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins), include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.; antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinone glycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclic immunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, etc.; and the like.
Other anti-proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.
Microtubule affecting agents that have antiproliferative activity are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones including but not limited to, eopthilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.
Hormone modulators and steroids (including synthetic analogs) that are suitable for use include, but are not limited to, adrenocorticosteroids, e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocortical suppressants, e.g. aminoglutethimide; 17α-ethinylestradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex®. Estrogens stimulate proliferation and differentiation, therefore compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids may inhibit T cell proliferation.
Other chemotherapeutic agents include metal complexes, e.g. cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines, e.g. N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Other anti-proliferative agents of interest include immunosuppressants, e.g. mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline); etc.
“Taxanes” include paclitaxel, as well as any active taxane derivative or pro-drug. “Paclitaxel” (which should be understood herein to include analogues, formulations, and derivatives such as, for example, docetaxel, TAXOL™ TAXOTERE™ (a formulation of docetaxel), 10-desacetyl analogs of paclitaxel and 3′N-desbenzoyl-3′N-t-butoxycarbonyl analogs of paclitaxel) may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtained from a variety of commercial sources, including for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T-1912 from Taxus yannanensis).
Paclitaxel should be understood to refer to not only the common chemically available form of paclitaxel, but analogs and derivatives (e.g., Taxotere™ docetaxel, as noted above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).
Cell therapy includes chimeric antigen receptor (CAR) T cell therapy (CAR-T therapy); natural killer (NK) cell therapy; dendritic cell (DC) therapy (e.g., DC-based vaccine); T cell receptor (TCR) engineered T cell-based therapy; and the like.
Immune checkpoint inhibitors are known in the art and include antibodies specific for immune checkpoint polypeptide. For example, an immune checkpoint inhibitors include, e.g., an antibody specific for an immune checkpoint polypeptide selected from CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2.
Antibodies, e.g., monoclonal antibodies, that are specific for immune checkpoints and that function as immune checkpoint inhibitors, are known in the art. See, e.g., Wurz et al. (2016) Ther. Adv. Med. Oncol. 8:4; and Naidoo et al. (2015) Ann. Oncol. 26:2375.
Suitable anti-immune checkpoint antibodies include, but are not limited to, nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck), pidilizumab (Curetech), AMP-224 (GlaxoSmithKline/Amplimmune), MPDL3280A (Roche), MDX-1105 (Medarex, Inc./Bristol Myer Squibb), MEDI-4736 (Medimmune/AstraZeneca), arelumab (Merck Serono), ipilimumab (YERVOY, (Bristol-Myers Squibb), tremelimumab (Pfizer), pidilizumab (CureTech, Ltd.), IMP321 (Immutep S.A.), MGA271 (Macrogenics), BMS-986016 (Bristol-Meyers Squibb), lirilumab (Bristol-Myers Squibb), urelumab (Bristol-Meyers Squibb), PF-05082566 (Pfizer), IPH2101 (Innate Pharma/Bristol-Myers Squibb), MEDI-6469 (Medlmmune/AZ), CP-870,893 (Genentech), Mogamulizumab (Kyowa Hakko Kirin), Varlilumab (CellDex Therapeutics), Galiximab (Biogen Idec), AMP-514 (Amplimmune/AZ), AUNP 12 (Aurigene and Pierre Fabre), Indoximod (NewLink Genetics), NLG-919 (NewLink Genetics), INCB024360 (Incyte); KN035; and combinations thereof.
Cancers
Cancer cells that may be treated by methods and compositions of the present disclosure include cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, spinal cord, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; pancreatic cancer; rectal cancer; and hairy cell leukemia.
Tumors that can be treated using a method of the present disclosure include, e.g., a brain cancer tumor, a head and neck cancer tumor, an esophageal cancer tumor, a skin cancer tumor, a lung cancer tumor, a thymic cancer tumor, a stomach cancer tumor, a colon cancer tumor, a liver cancer tumor, an ovarian cancer tumor, a uterine cancer tumor, a bladder cancer tumor, a testicular cancer tumor, a rectal cancer tumor, a breast cancer tumor, or a pancreatic cancer tumor.
In some cases, the tumor is a colorectal adenocarcinoma. In some cases, the tumor is non-small cell lung carcinoma. In some cases, the tumor is a triple-negative breast cancer. In some cases, the tumor is a solid tumor. In some cases, the tumor is a liquid tumor. In some cases, the tumor is recurrent. In some cases, the tumor is a primary tumor. In some cases, the tumor is metastatic.
A variety of subjects are suitable for treatment with a subject method of treating cancer. Suitable subjects include any individual, e.g., a human or non-human animal who has cancer, who has been diagnosed with cancer, who is at risk for developing cancer, who has had cancer and is at risk for recurrence of the cancer, who has been treated with an agent other than a an oncolytic vaccinia virus of the present disclosure for the cancer and failed to respond to such treatment, or who has been treated with an agent other than an oncolytic vaccinia virus of the present disclosure for the cancer but relapsed after initial response to such treatment.
Vaccinia Virus Immunogenic Compositions
The present disclosure provides a composition comprising an OVV comprising one or more of: a) a nucleotide sequence encoding a variant A33 polypeptide, wherein the variant A33 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A33 polypeptide (e.g., compared to a control vaccinia virus that comprises a nucleotide sequence encoding a wild-type A33 polypeptide, such as an A33 polypeptide having an amino acid sequence depicted in
To induce or enhance an immune response in an individual to a cancer antigen, an OVV of the present disclosure is administered to an individual in need thereof. Subjects suitable for treatment include those described above. In some cases, the replication-competent, recombinant oncolytic vaccinia virus is administered to an individual in need thereof in a low dose, e.g., from about 102 plaque-forming units (pfu) to about 104 pfu, from about 104 pfu to about 105 pfu, or from about 105 pfu to about 106 pfu per dose. In some cases, the replication-competent, recombinant oncolytic vaccinia virus is administered to an individual in need thereof in a dose of from about 106 pfu to about 1012 pfu, e.g., in a dose of from about 106 pfu to about 107 pfu, from about 107 pfu to about 108 pfu, from about 108 pfu to about 109 pfu, from about 109 pfu to about 1010 pfu, from about 1010 pfu to about 1011 pfu, or from about 1011 pfu to about 1012 pfu.
An OVV of the present disclosure can be administered to an individual in need thereof in a pharmaceutical composition, e.g., the pharmaceutical composition can comprise: a) an OVV of the present disclosure; and b) a pharmaceutically acceptable excipient. Thus, the present disclosure provides a pharmaceutical composition comprising: a) an OVV of the present disclosure; and b) a pharmaceutically acceptable excipient. Suitable pharmaceutically acceptable excipients are as described above. In some cases, the pharmaceutical composition comprises an adjuvant. Suitable adjuvants include, but are not limited to, alum, aluminum phosphate, aluminum hydroxide, MF59 (4.3% w/v squalene, 0.5% w/v Tween 80™ 0.5% w/v Span 85), CpG-containing nucleic acid (where the cytosine is unmethylated), monophosphoryl lipid A (MPL), 3-Q-desacyl-4′-monophosphoryl lipid A (3DMPL), and the like.
An OVV of the present disclosure can be administered to an individual in need thereof via any suitable route of administration, e.g., a route of administration as described above. For example, a recombinant vaccinia virus of the present disclosure can be administered to an individual in need thereof via an intramuscular, an intravenous, a subcutaneous route of administration.
Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-72 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:
Aspect 1. An OVV comprising one or more of:
a) a nucleotide sequence encoding a variant A33 polypeptide, wherein the variant A33 polypeptide provides for enhanced viral spreading and/or enhanced production of extracellular enveloped virion (EEV), compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A33 polypeptide;
b) a nucleotide sequence encoding a variant A34 polypeptide, wherein the variant A34 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A34 polypeptide; and
c) a nucleotide sequence encoding a variant B5 polypeptide, wherein the variant B5 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant B5 polypeptide.
Aspect 2. The replication-competent, recombinant oncolytic vaccinia virus of aspect 1, comprising:
a) a nucleotide sequence encoding a variant A33 polypeptide, wherein the variant A33 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A33 polypeptide; and
b) a nucleotide sequence encoding a variant A34 polypeptide, wherein the variant A34 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A34 polypeptide.
Aspect 3. The replication-competent, recombinant oncolytic vaccinia virus of aspect 1, comprising:
a) a nucleotide sequence encoding a variant A33 polypeptide, wherein the variant A33 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A33 polypeptide;
b) a nucleotide sequence encoding a variant A34 polypeptide, wherein the variant A34 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant A34 polypeptide; and
c) a nucleotide sequence encoding a variant B5 polypeptide, wherein the variant B5 polypeptide provides for enhanced viral spreading and/or enhanced production of EEV, compared to a control vaccinia virus that does not comprise the nucleotide sequence encoding the variant B5 polypeptide.
Aspect 4. The replication-competent, recombinant oncolytic vaccinia virus of any one of aspects 1-3, wherein the variant A33 polypeptide comprises a substitution of 1, 2, or 3 of M63, A88, and E129.
Aspect 5. The replication-competent, recombinant oncolytic vaccinia virus of aspect 4, wherein the substitution is one or more of an M63R substitution, an A88D substitution, and an E129M substitution.
Aspect 6. The replication-competent, recombinant oncolytic vaccinia virus of aspect 5, wherein the variant A33 polypeptide comprises an A88D substitution and an E129M substitution.
Aspect 7. The replication-competent, recombinant oncolytic vaccinia virus of any one of aspects 4-6, wherein the vaccinia virus comprises a nucleotide sequence encoding a variant A34 polypeptide, wherein the variant A34 polypeptide comprises a K151E substitution.
Aspect 8. The replication-competent, recombinant oncolytic vaccinia virus of any one of aspects 1-3, wherein the variant A34 polypeptide comprises a substitution of 1, 2, 3, 4, or 5 of M66, F94, R84, R91, and T127.
Aspect 9. The replication-competent, recombinant oncolytic vaccinia virus of aspect 8, wherein the substitution is one or more of an M66T substitution, an F94H substitution, an R84G substitution, an R91A substitution, an R91S substitution, and a T127E substitution.
Aspect 10. The replication-competent, recombinant oncolytic vaccinia virus of aspect 9, wherein the variant A34 polypeptide comprises a K151E substitution.
Aspect 11. The replication-competent, recombinant oncolytic vaccinia virus of aspect 9, wherein the variant A34 polypeptide comprises an F94H substitution.
Aspect 12. The replication-competent, recombinant oncolytic vaccinia virus of aspect 10, wherein the variant A34 polypeptide comprises an F94H substitution and a K151E substitution.
Aspect 13. The replication-competent, recombinant oncolytic vaccinia virus of aspect 2,
wherein the variant A33 polypeptide comprises a substitution of one or more of M63, A88, and E129; and
wherein the variant A34 polypeptide comprises a substitution of one or more of M66, F94, R84, R91, and T127.
Aspect 14. The replication-competent, recombinant oncolytic vaccinia virus of aspect 13, wherein the variant A33 polypeptide comprises one or more of an M63R substitution, an A88D substitution, and an E129M substitution; and wherein the variant A34 polypeptide comprises one or more of an M66T substitution, an F94H substitution, an R84G substitution, an R91S substitution, an R91A substitution and a T127E substitution.
Aspect 15. The replication-competent, recombinant oncolytic vaccinia virus of aspect 14, wherein the variant A34 polypeptide comprises a K151E substitution.
Aspect 16. The replication-competent, recombinant oncolytic vaccinia virus of aspect 14, wherein the variant A33 polypeptide comprises an A88D substitution; and wherein the variant A34 polypeptide comprises an F94H substitution.
Aspect 17. The replication-competent, recombinant oncolytic vaccinia virus of aspect 14, wherein the variant A33 polypeptide comprises an E129M substitution and wherein the variant A34 polypeptide comprises an F94H substitution.
Aspect 18. The replication-competent, recombinant oncolytic vaccinia virus of aspect 14, wherein the variant A33 polypeptide comprises an A88D substitution and an E129M substitution, and wherein the variant A34 polypeptide comprises an F94H substitution.
Aspect 19. The replication-competent, recombinant oncolytic vaccinia virus of aspect 15, wherein the variant A33 polypeptide comprises an A88D substitution and an E129M substitution, and wherein the variant A34 polypeptide comprises a K151E substitution.
Aspect 20. The replication-competent, recombinant oncolytic vaccinia virus of aspect 15, wherein the variant A33 polypeptide comprises an A88D substitution, and wherein the variant A34 polypeptide comprises an F94H substitution and a K151E substitution.
Aspect 21. The replication-competent, recombinant oncolytic vaccinia virus of aspect 15, wherein the variant A33 polypeptide comprises an E129M substitution, and wherein the variant A34 polypeptide comprises an F94H substitution and a K151E substitution.
Aspect 22. The replication-competent, recombinant oncolytic vaccinia virus of aspect 15, wherein the variant A33 polypeptide comprises an A88D substitution and an E129M substitution, and wherein the variant A34 polypeptide comprises an F94H substitution and a K151E substitution.
Aspect 23. The replication-competent, recombinant oncolytic vaccinia virus of aspect 1 or aspect 3, wherein the variant B5 polypeptide comprises 1, 2, 3, 4, or more amino acid substitutions at positions of N39, L90, N94, S199, K229, V233, 1236, V238, T240, N241, E243, V247, D248, G250, D263, E268, E270, D272, S273, D275, and A276.
Aspect 24. The replication-competent, recombinant oncolytic vaccinia virus of aspect 23, wherein the variant B5 polypeptide comprises an S197F or an S197V substitution.
Aspect 25. The replication-competent, recombinant oncolytic vaccinia virus of aspect 23, wherein the variant B5 polypeptide comprises an S199M substitution.
Aspect 26. The replication-competent, recombinant oncolytic vaccinia virus of aspect 23, wherein the variant B5 polypeptide comprises an S273L substitution or an S273I substitution.
Aspect 27. The replication-competent, recombinant oncolytic vaccinia virus of aspect 23, wherein the variant B5 polypeptide comprises an N39G substitution.
Aspect 28. The replication-competent, recombinant oncolytic vaccinia virus of aspect 23, wherein the variant B5 polypeptide comprises an N94T substitution.
Aspect 29. The replication-competent, recombinant oncolytic vaccinia virus of aspect 23, wherein the variant B5 polypeptide comprises an L90R substitution and an S273V substitution.
Aspect 30. The replication-competent, recombinant oncolytic vaccinia virus of aspect 23, wherein the variant B5 polypeptide comprises an N39G substitution and an S273I substitution.
Aspect 31. The replication-competent, recombinant oncolytic vaccinia virus of aspect 23, wherein the variant B5 polypeptide comprises a K229C substitution and an S273L substitution.
Aspect 32. The replication-competent, recombinant oncolytic vaccinia virus of aspect 23, wherein the variant B5 polypeptide comprises an D263A substitution, an E270S substitution, an E272G substitution, and an E275F substitution.
Aspect 33. The replication-competent, recombinant oncolytic vaccinia virus of aspect 23, wherein the variant B5 polypeptide comprises an I236P substitution, a V238R substitution, a T240R substitution, and an E243G substitution.
Aspect 34. The replication-competent, recombinant oncolytic vaccinia virus of aspect 23, wherein the variant B5 polypeptide comprises a V233D substitution, an I236L substitution, a V238W substitution, a T240Y substitution, and an E243R substitution.
Aspect 35. The replication-competent, recombinant oncolytic vaccinia virus of aspect 23, wherein the variant B5 polypeptide comprises an D263V substitution, an E268T substitution, an E270G substitution, an E272P substitution, and an E275S substitution.
Aspect 36. The replication-competent, recombinant oncolytic vaccinia virus of aspect 23, wherein the variant B5 polypeptide comprises an N241T substitution, an E243V substitution, a V247S substitution, a G250R substitution, and an A276F substitution.
Aspect 37. The replication-competent, recombinant oncolytic vaccinia virus of aspect 23, wherein the variant B5 polypeptide comprises an N241G substitution, an E243S substitution, a V247W substitution, a D248Y substitution, a G250A substitution, and an A276F substitution.
Aspect 38. The replication-competent, recombinant oncolytic vaccinia virus of any one of aspects 1-37, comprising a nucleotide sequence encoding a variant A34 polypeptide, wherein the variant A34 polypeptide comprises a K151E substitution Aspect 39. The replication-competent, recombinant oncolytic vaccinia virus of any one of aspects 1-38, comprising a nucleotide sequence encoding a variant A56 polypeptide.
Aspect 40. The replication-competent, recombinant oncolytic vaccinia virus of aspect 39, wherein the variant A56 polypeptide comprises a substitution of amino acid 269.
Aspect 41. The replication-competent, recombinant oncolytic vaccinia virus of aspect 40, wherein the substitution of amino acid 269 is an I269F substitution. Aspect 42. The replication-competent, recombinant oncolytic vaccinia virus of any one of aspects 1-41, wherein the vaccinia virus comprises a heterologous nucleic acid comprising a nucleotide sequence encoding an immunomodulatory polypeptide.
Aspect 43. The replication-competent, recombinant oncolytic vaccinia virus of any one of aspects 1-41, wherein the vaccinia virus comprises a modification that results in lack of J2R expression and/or function.
Aspect 44. The replication-competent, recombinant oncolytic vaccinia virus of any one of aspects 1-41, wherein the vaccinia virus comprises both: i) a heterologous nucleic acid comprising a nucleotide sequence encoding an immunomodulatory polypeptide; and ii) a modification that results in lack of J2R expression and/or function.
Aspect 45. A composition comprising:
a) the vaccinia virus of any one of aspects 1-44; and
b) a pharmaceutically acceptable excipient.
Aspect 46. A method of inducing oncolysis in an individual having a tumor, the method comprising administering to the individual an effective amount of the vaccinia virus of any one of aspects 1-44, or the composition of aspect 45.
Aspect 47. The method of aspect 46, wherein said administering comprises administering a single dose of the virus or the composition.
Aspect 48. The method of aspect 47, wherein the single dose comprises at least 106 plaque forming units (pfu) of the vaccinia virus.
Aspect 49. The method of aspect 47, wherein the single dose comprises from 109 to 1012 pfu of the vaccinia virus.
Aspect 50. The method of aspect 46, wherein said administering comprises administering multiple doses of the vaccinia virus or the composition.
Aspect 51. The method of aspect 50, wherein the vaccinia virus or the composition is administered every other day.
Aspect 52. The method of aspect 50, wherein the vaccinia virus or the composition is administered once per week.
Aspect 53. The method of aspect 50, wherein the vaccinia virus or the composition is administered every other week.
Aspect 54. The method of any one of aspects 46-53, wherein the tumor is a brain cancer tumor, a head and neck cancer tumor, an esophageal cancer tumor, a skin cancer tumor, a lung cancer tumor, a thymic cancer tumor, a stomach cancer tumor, a colon cancer tumor, a liver cancer tumor, an ovarian cancer tumor, a uterine cancer tumor, a bladder cancer tumor, a testicular cancer tumor, a rectal cancer tumor, a breast cancer tumor, or a pancreatic cancer tumor.
Aspect 55. The method of any one of aspects 46-53, wherein the tumor is colorectal cancer.
Aspect 56. The method of any one of aspects 46-53, wherein the tumor is non-small cell lung cancer.
Aspect 57. The method of any one of aspects 46-53, wherein the tumor is breast cancer.
Aspect 58. The method of aspect 5576, wherein the tumor is a triple-negative breast cancer.
Aspect 59. The method of any one of aspects 46-58, wherein the tumor is a solid tumor.
Aspect 60. The method of any one of aspects 46-58, wherein the tumor is a liquid tumor.
Aspect 61. The method of any one of aspects 46-60, wherein the tumor is recurrent.
Aspect 62. The method of any one of aspects 46-60, wherein the tumor is a primary tumor.
Aspect 63. The method of any one of aspects 46-62, wherein the tumor is metastatic.
Aspect 64. The method of any one of aspects 46-63, further comprising administering to the individual a second cancer therapy.
Aspect 65. The method of aspect 64, wherein the second cancer therapy is selected from chemotherapy, biological therapy, radiotherapy, immunotherapy, hormone therapy, anti-vascular therapy, cryotherapy, toxin therapy, oncolytic virus therapy, a cell therapy, gene therapy, and surgery.
Aspect 66. The method of aspect 64, wherein the second cancer therapy is an immune checkpoint inhibitor.
Aspect 67. The method of aspect 66, wherein the immune checkpoint inhibitor is an antibody specific for an immune checkpoint inhibitor selected from CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2.
Aspect 68. The method of any one of aspects 46-67, wherein the individual is immunocompromised.
Aspect 69. The method of any one of aspects 46-68, wherein said administering of the vaccinia virus or the composition is intra-arterial, intraperitoneal, intrabladder, or intrathecal.
Aspect 70. The method of any one of aspects 46-68, wherein said administering of the vaccinia virus or the composition is intratumoral.
Aspect 71. The method of any one of aspects 46-68, wherein said administering of the vaccinia virus or the composition is peritumoral.
Aspect 72. The method of any one of aspects 46-68, wherein said administering of the vaccinia virus or the composition is intravenous.
Aspect 73 The vaccinia virus of any one of aspects 1-44, or the composition of aspect 45 for use in a method of inducing oncolysis in an individual having a tumor.
Aspect 74. Use of the vaccinia virus of any one of aspects 1-44, or the composition of aspect 45 in the manufacture of a medicament for use in a method of inducing oncolysis in an individual having a tumor.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
A. Generation of Vaccinia Virus Library Backbones
Backbone viruses were generated to facilitate cloning, reactivation, and construction of multiple libraries into loci of interest within the vaccinia virus genome. Backbone viruses contained the selectable markers herpes simplex virus-thymidine kinase (HSV-TK) and mCherry fusion protein (codon optimized for vaccinia virus expression) under the control of the synthetic early late promoter (pSEL). Donor DNA containing the pSEL-HSV-TK/mCherry cassette flanked by homing endonuclease sites I-Scel and I-Ceul, and unique pairs of Aarl sites in upstream and downstream regions of homology to 1) the A33R/A34R loci, 2) the A56R locus, or 3) the B5R locus were generated via overlapping polymerase chain reaction (PCR) with specific primers for each region (IDT) and Phusion-HF (Thermo Fisher). PCR products were purified using Zymo DNA clean-and-concentrator-5 kit (ZymoResearch) and blunt-cloned into the DNA shuttle vector supplied by the Zero Blunt™ TOPO™ PCR Cloning Kit (Thermo Fisher). DNA was sequenced by Elim Biopharmaceuticals (Hayward, Calif.). Viral genomic DNA from Copenhagen vaccinia virus containing a J2R deletion and insertion of firefly luciferase-2A-eGFP driven by the pSEL promoter, was extracted and cut at one of the three loci of interest. The selectable markers replaced each loci of interest: 1) A33R/A34R loci, 2) A56R locus, and 3) the B5R locus of the vaccinia virus genome using viral reactivation. Briefly, VERO-B4 cells (DSMZ) were infected with Shope fibroma virus (SFV; ATCC) for 2 hours at room temperature, washed and allowed to recover at 37° C. and 5% CO2 for 1 hour. During recovery, transfection mixture containing genomic DNA and donor DNA were mixed with Lipofectamine 2000 (Thermo Fisher) according to manufacturer instructions. The infected cells were transfected with the transfection mixture at 37° C. and 5% CO2 for 2 days. Functional virus was recovered by serial dilution and plaque isolation from BSC-40 cells (Earl et al. (1998) Curr. Protocol. Mol. Biol. 43:16.17.1.). Virus plaques were DNA extracted using QuickExtract (Epicentre/Lucigen) and Sanger sequenced before and after 3 rounds of plaque purification by Elim Biopharmaceuticals. Sequence verified viruses were amplified in HeLa S3 cells (DSMZ), and purified as described below. DNA was extracted (Cotter et al, (2015) Curr. Protocol Mol. Biol. 39:14A.3.1.) and summited for whole genome sequencing (WGS) (Seqmatic Hayward, Calif.). WGS analysis was performed in-house using Geneious desktop software (Biomatters Ltd.)
B. Generation of Vaccinia Virus A33R, A34R, A56R, and B5R DNA Libraries
Libraries containing single amino acid substitutions were generated via PCR. Template DNA containing the Copenhagen vaccinia virus sequences for A33R and A34R (containing a K151E substitution), A56R, or B5R were synthesized (Genescript) for each locus with unique Aarl and Sfil cloning sites inserted upstream of the first gene's promoter and downstream of the last gene's terminator. NNK libraries spanning the length of the genes were designed for A33R, A34R, A56R, and B5R coding sequences. Degenerate NNK-containing primers (IDT) tiled across the coding regions of each protein were used to generate NNK variants for each locus by PCR.
Homology between forward and reverse primers permitted self-assembly of NNK variants for each codon using NEBuilder® HiFi DNA Assembly (NEB). Assembly reactions were transformed into Endura chemically competent E. coli (Lucigen) and grown on selective agar media (Luria Broth with 100 μg/mL carbenicillin or LB+Carb, Teknova). Colonies were counted and transformations were continued until ˜20× colony forming units (CFUs) per NNK was achieved for each codon in each locus. Colonies were recovered from the plates by flooding and scraping with liquid media LB+Carb (Teknova), aliquoted, and frozen in 30% glycerol. One aliquot for each codon was inoculated into a 96-well plate containing LB+50 μg/mL Carb, shaking overnight at 37° C. and 900 rpm; then miniprepped using the Nucleospin 96 Plasmid Transfection-grade Kit (Macherey-Nagel). Minipreps were diluted 1:100 in sterile water; then the donor DNA for each codon was amplified with specific primers (IDT) for A33R/A34R, A56R, and B5R respectively using Phusion-HF (Thermo Fisher). PCR amplicons for each randomized codon were quantified using the AccuClear Ultra High Sensitivity dsDNA quantification kit (Biotium) on a Spectramax M5 plate reader (Molecular Devices) and sequenced by Elim Biopharmaceuticals (Hayward, Calif.). Sequencing results were analyzed in-house using Geneious (Biomatters Ltd.). Verified NNK PCR products were normalized by concentration, pooled and used as donor DNA to recombine into vaccinia virus. Libraries containing randomized regions of interest within the B5R genes were synthesized by Genewiz using a degenerate synthesis process at the defined regions of randomization. Library diversity estimates and allele frequencies at the DNA library stage were estimated for all the libraries.
C. Generation of Vaccinia Virus A33, A34, A56, and B5 Viral Libraries
The libraries were inserted into the vaccinia virus genome using the same strategy described for vaccinia backbone generation or viral reactivation. Briefly, VERO-B4 cells (DSMZ) were infected with Shope fibroma virus (SFV, ATCC) and transfected with digested viral genomic DNA and donor DNA. Vaccinia virus viral particles were recovered 24 hours later. Diversity immediately after virus reactivation was estimated by isolating plaques, extracting DNA with Quick Extract (Epicentre/Lucigen), and sequencing PCR amplicons as described above. Viruses from each library were amplified in HeLa S3 cells. Virus seed stocks were analyzed for diversity by amplifying each locus and submitting for amplicon next-generation sequencing (NGS, SeqMatic). Analysis of the diversity present in each locus-specific library was facilitated by the free command-line program VirVarSeq (at https://omictools(dot)com(forward slash)virvarseq-tool)) and post-processing using Microsoft Excel. Amplified viral libraries were purified by sucrose gradient and ultracentrifugation (Cotter et al. (2015) Curr. Protocol Mol. Biol. 39:14A.3.1), as described below. Purified viral libraries were stored at −80° C. and titered in duplicate by plaque assay, adding serial dilutions of purified virus to BSC-40 cells (ATCC) as described below (Earl et al. (1998) Curr. Protocol. Mol. Biol. 43:16.17.1.).
D. Directed Evolution
Several in vitro and in vivo directed evolution programs were undertaken to identify variants capable of increased EEV production and virus spreading in combination with transduction of various tumor types. The programs are summarized in Table 1. The general directed evolution process is summarized in
For multiple rounds of the directed evolution process, sequencing of a representative fraction of the library was performed using a combination of amplicon next-generation sequencing (NGS), Sanger sequencing of individual plaques, and whole genome sequencing (WGS). The frequency, expressed as a percentage of the total population, of the most prevalent variants increased significantly over the course of the selections.
E. Plasmid Construction
Plasmids containing variant A33R and/or A34R sequences were generated using gene synthesis techniques. A sequence encoding each of the substitution variants described below was submitted to GenScript for gene synthesis and inserted into the pUC57-mini vector. The amino acid sequences encoded by the variant A33 and A34 sequences are annotated in in the Brief Description of
F. Viruses and Cells
Vaccinia virus Copenhagen IGV-007 was constructed by homologous recombination insertion of a luciferase-2A-GFP cassette under the control of the pSEL promoter into the thymidine kinase viral gene J2R of the Copenhagen strain of vaccinia virus. The expression of the luciferase reporter gene was confirmed by luminescence using the Bright-Glo™ Luciferase Assay System (Promega) and a Spectramax M5 (Molecular Devices). GFP expression was confirmed via fluorescent microscopy.
Vaccinia virus Copenhagen strains containing the K151E substitution in A34 (IGV-006), the A88D substitution in A33 (IGV-060), the E129M substitution in A33 (IGV-061), the F94H substitution in A34 (IGV-062), the A88D and E129M substitutions in A33 (IGV-063), the A88D substitution in A33 and the F94H substitution in A34 (IGV-064), the E129M substitution in A33 and the F94H substitution in A34 (IGV-065), and the A88D and E129M substitutions in A33 and the F94H substitution in A34 (IGV-066) were constructed by reactivation and homologous recombination of the synthesized genes into the A33R/A34R gene region of IGV-007. Vaccinia virus Copenhagen strains containing the A88D substitution in A33 and the K151E substitution in A34 (IGV-067), the E129M substitution in A33 and the K151E substitution in A34 (IGV-068), the F94H and K151E substitutions in A34 (IGV-073), the A88D and E129M substitutions in A33 and the K151E substitution in A34 (IGV-069), the A88D substitution in A33 and the F94H and K151E substitutions in A34 (IGV-070), the E129M substitution in A33 and the F94H and K151E substitutions in A34 (IGV-071), and the A88D and E129M substitutions in A33 and the F94H and K151E substitutions in A34 (IGV-072) were constructed by reactivation and homologous recombination of the synthesized genes into the A33R/A34R gene region of IGV-006. Successful insertion of the variant A33R gene and/or A34R gene into IGV-007 or IGV-006 was verified by Sanger sequencing of individual isolated plaques. Viruses were amplified and purified as described below.
Vaccinia virus Western Reserve strains containing the K151E substitution in A34 (IGV-117), the F94H and K151E substitutions in A34 (IGV-118), the A88D substitution in A33 and the F94H and K151E substitutions in A34 (IGV-119), and the A88D and E129M substitutions in A33 and the F94H and K151E substitutions in A34 (IGV-120) were constructed by reactivation and homologous recombination of the synthesized genes into the A33R/A34R gene region of IGV-013. Vaccinia virus Western Reserve (WR; ATCC) IGV-013 was constructed by homologous recombination insertion of a luciferase-2A-GFP cassette under the control of the pSEL promoter with a tetracycline operator into the thymidine kinase viral gene J2R of the WR strain of vaccinia virus. The expression of the luciferase reporter gene was confirmed by luminescence using the Bright-Glo™ Luciferase Assay System (Promega) and a Spectramax M5 (Molecular Devices). GFP expression was confirmed via fluorescent microscopy.
HeLa, U-2 OS, and BSC-40 cells were obtained from ATCC. A549, HCT 116, Colo205, and MDA-MB-231 cells were obtained from the NCI DCTD Repository of Tumors and Tumor Cell Lines. HeLa S3 and VERO-B4 cells were obtained from DSMZ. Cancer patient-derived primary cells (Breast, Colorectal, and Lung) were obtained from Conversant Bio. Human Umbilical Vein Endothelial Cells (HUVEC) were obtained from Lonza. RK-13 cells were obtained from Sigma-Aldrich.
G. Virus Amplification and Purification
HeLa S3 cells (DSMZ) were infected by adding virus to the monolayer and incubating for 1 hour in media with 2.5% FBS. Following infection, fresh media was added and the infected cells monolayer was incubated for 48 hours to allow for virus replication and amplification. Following incubation, the cells were harvested and collected by centrifugation. Cells were lysed by mechanical disruption with a Dounce homogenizer (Wheaton). Virus purification was accomplished with a 24% to 40% sucrose gradient and ultracentrifugation. Purified virus was aliquoted, stored at −80° C. and titered in duplicate by plaque assay, adding serial dilutions of purified virus to BSC-40 cells or U-2 OS cells (ATCC) as previously described (Earl et al. (1998) Curr. Protocol. Mol. Biol. 43:16.17.1.).
H. Virus Titering by Plaque Assay
Virus titer was determined by ten-fold serial dilutions, with a final dilution of 10-9 of the stock concentrated, purified virus. The virus dilutions were used to infect BSC-40 cells or U-2 OS cells to determine the number of plaque forming units per mL (PFU/mL). 1 mL of each serial dilution was applied in duplicate to wells containing a confluent monolayer of cells in a standard 6-well microplate (BD Falcon). Cells were infected for an hour, washed with fresh media, and overlaid with a solution of fresh media containing 1.5% carboxymethylcellulose (Teknova). Following 48 or 72 hours of incubation, the media was removed, and the cells were fixed and stained with a 20% ethanol solution containing 0.1% crystal violet (Sigma). The stock titer was then determined by counting the number of plaques in each well, averaging between duplicate titers, and adjusting for the dilution factor.
I. Infectious Virus in Supernatant of Human Tumor Cells
Virus replication in the tumor cell lines A549 (NCI), Colo205 (NCI), MDA MB 231 (NCI), HT-29 (NCI), SW-620 (NCI), and HeLa (DSMZ) was determined by infecting a monolayer of cells with virus at an MOI of 1 or 3 for 1 hour in triplicate. Following infection, the viral inoculum was washed 3 times and replaced with fresh media. Virus produced in the supernatant and cells were harvested separately into media at 24 hours post-infection. The virus in the supernatant was titered immediately in duplicate using the plaque assay protocol described above. The virus contained in the cell pellet was frozen and stored at −80° C.
J. Comet Assay
Monolayers of BSC-40 cells seeded in 6-well plates were infected with 1 mL of 10-fold serial dilutions of virus for 1 hour. The infected cells were washed 3 times, replenished with fresh media and incubated for 48 or 72 hours at a 40° angle at 37° C. Cell monolayers were stained with a 20% ethanol solution containing 0.1% crystal violet (Sigma) for 2 hours to visualize comets.
K. Spreading Assay
For a two-stage infectivity assay, U-2 OS (osteosarcoma), RK-13 (rabbit kidney), VERO-B4 (African green monkey kidney), A549 (lung adenocarcinoma) or MDA-MB-231 (breast cancer) cells were infected with a range of MOIs for 1 hour. The infected cells were washed 3 times and incubated for 20-24 hours, at which time the supernatant was collected. The supernatant was clarified from cell debris by low speed centrifugation and used to infect a new plate of the corresponding cells seeded in 96-well white wall plate (Greiner). Fifteen or twenty four hours post-infection on the second plate, luciferase activity was assessed on a Spectramax M5 spectrophotometer using the Bright-Glo Luciferase Assay System (Promega).
L. EEVs Quantification
Virus samples containing enrichment of intracellular mature virus (IMV) particles or enrichment of extracellular enveloped virus (EEV) particles were separated and purified using a CsCl density gradient ultracentrifugation method. Virus was overlaid on a 25% to 30% CsCl gradient and centrifuged for 18 hours at 175,000 rcf. Bands containing the enriched fractions were extracted at defined locations within the density gradient, and CsCl was removed. Samples were incubated with a conjugated anti-B5 antibody prior to quantification. Virus sized particles (VSP) and VSP containing the B5 antigen (an indicator of EEV) were quantified using a Micro flow cytometer (Apogee).
The first selection identified variants capable of enhanced spreading and EEV production following infection of different primary cancer cells (colon, breast and lung) and VEGF stimulated endothelial cells. Two variants were identified that demonstrated an increased frequency within the sequenced population (
The impact of the substitution variants on viral spreading and EEV production was assessed in vitro using BSC-40 (African green monkey kidney) and U-2 OS (human osteosarcoma) cell lines. Copenhagen vaccinia viruses containing the wild-type A33 and A34 sequences (IGV-007), a K151E substitution in A34 (IGV-006; a known mutation to increase spreading and EEV production), the A88D and E129M substitutions in A33 and a K151E substitution in A34 (IGV-051), or the F94H and K151E substitutions in A34 (IGV-052) were generated and manufactured as described in Example 1. The comet assay was performed as described in Example 1. The presence of longer comet tails (
The impact of the substitution variants on infectious virus generated in the supernatant (EEV production) in different human cancer cell lines was assessed in vitro using A549 cells (lung adenocarcinoma), MDA-MB-231 cells (breast adenocarcinoma), Colo205 cells (colorectal adenocarcinoma), and HeLa cells (cervical cancer). Copenhagen vaccinia viruses containing the wild-type A33 and A34 sequences (IGV-007), a K151E substitution in A34 (IGV-006; a known mutation to increase virus spreading and EEV production), the A88D and E129M substitutions in A33 and a K151E substitution in A34 (IGV-051), or the F94H and K151E substitutions in A34 (IGV-052) were generated and manufactured as described in Example 1. All cell lines listed above were infected with a MOI of 1 and washed thoroughly 3 times after 1 hour of virus adsorption. At 24 hours post-infection, the supernatant was collected, and the number of infectious viruses produced in the supernatant (potentially EEVs) was determined via plaque assay, as described in Example 1. In all four cell lines, the substitution variants containing the A88D and E129M substitutions in A33 and a K151E substitution in A34 (IGV-051) or the F94H and K151E substitutions in A34 (IGV-052) produced a higher number of infectious viruses in the supernatant (potentially EEVs) (
The impact of the substitution variants on vaccinia virus spreading in different human cancer cell lines was assessed in vitro using A549 cells (lung adenocarcinoma), and MDA-MB-231 cells (breast adenocarcinoma). Copenhagen vaccinia viruses containing the wild-type A33 and A34 sequences (IGV-007), a K151E substitution in A34 (IGV-006; a known mutation to increase virus spreading and EEV production), the A88D and E129M substitutions in A33 and a K151E substitution in A34 (IGV-051), or the F94H and K151E substitutions in A34 (IGV-052) were generated and manufactured as described in Example 1. A549 cells (lung adenocarcinoma), and MDA-MB-231 cells (breast adenocarcinoma) were infected with serial dilutions of MOIs of all the viruses tested, and the cells were washed 3 times after 1 hour. The supernatant was collected at 22 hours post-infection, clarified, and used to infect a new plate of cells. After 15 hours post-infection, the luciferase activity in cells on the second plate was assessed by quantifying luminescence using Bright-Glo Luciferase assay system (Promega). In both cell lines, the substitution variants containing the A88D and E129M substitutions in A33 and a K151E substitution in A34 (IGV-051) or the F94H and K151E substitutions in A34 (IGV-052) led to higher luciferase detection (
The A33 and A34 mutations identified in IGV-051 and IGV-52 viruses were used to engineer new viruses using IGV-006 and IGV-007 as viral backbones in all possible combinations, as described in Example 1. The evaluation of all single substitutions and combinations of the A88D, E129M, F94H, and K151E mutations enabled the identification of additional combinations and substitutions that conferred spreading advantages. The impact of the single or multiple substitutions on EEV production and viral spreading was assessed in vitro using BSC-40 (African green monkey kidney) and U-2 OS (human osteosarcoma) cells lines, as described in Example 1. Copenhagen vaccinia viruses containing the wild-type A33 and A34 sequences (IGV-007), a K151E substitution in A34 (IGV-006; a known mutation to increase virus spreading and EEV production), an A88D substitution in A33 (IGV-060), an E129M substitution in A33 (IGV-061), an F94H substitution in A34 (IGV-062), the A88D and E129M substitutions in A33 (IGV-063), an A88D substitution in A33 and an F94H substitution in A34 (IGV-064), an E129M substitution in A33 and an F94H substitution in A34 (IGV-065), the A88D and E129M substitutions in A33 and an F94H substitution in A34 (IGV-066), an A88D substitution in A33 and a K151E substitution in A34 (IGV-067), an E129M substitution in A33 and a K151E substitution in A34 (IGV-068), the A88D and E129M substitutions in A33 and a K151E substitution in A34 (IGV-069), an A88D substitution in A33 and the F94H and K151E substitutions in A34 (IGV-070), an E129M substitution in A33 and the F94H and K151E substitutions in A34 (IGV-071), the A88D and E129M substitutions in A33 and the F94H and K151E substitutions in A34 (IGV-072), or the F94H and K151E substitutions in A34 (IGV-073) were generated and manufactured as described in Example 1. The comet assay was performed as described in Example 1. In this assay, combinations of substitutions that included F94H consistently yielded longer and more widespread comet tails (
To further assess the substitution variants' impact on EEV total physical particle production and specific infectivity in cancer cells, the number of physical and infectious EEVs produced in HeLa S3 cells (cervical adenocarcinoma) at 24 hours post-infection was determined. Supernatant from infected HeLa S3 cells was collected and purified by cesium chloride (CsCl) gradients to obtain purified EEVs. The infectious EEVs were titered by plaque assay as described in Example 1. The physical number of virion sized particles (VSP) and the percentage of those particles stained with B5 antibody was quantified by Apogee. The viral variants containing the A88D and E129M substitutions in A33 and a K151E substitution in A34 (IGV-051), the F94H and K151E substitutions in A34 (IGV-052), and an A88D substitution in A33 and the F94H and K151E substitutions in A34 (IGV-070) produced a higher percentage of physical EEV particles, measured by antibody staining for the EEV membrane protein B5 when compared to Copenhagen vaccinia virus containing the wild-type A33 and A34 sequences (IGV-007) (
The impact of the viral variants on EEV production and viral spreading in a Western Reserve (WR) vaccinia virus strain was assessed in vitro using RK-13 (rabbit kidney) and VERO-B4 (African green monkey kidney) cell lines. WR vaccinia virus containing the wild-type A33 and A34 sequences (IGV-013), a K151E substitution in A34 (IGV-117; a known mutation to increase virus spreading and EEV production), the F94H and K151E substitutions in A34 (IGV-118), the A88D substitution in A33 and the F94H and K151E substitutions in A34 (IGV-119), and the A88D and E129M substitutions in A33 and the F94H and K151E substitutions in A34 (IGV-120) were generated and manufactured as described in Example 1. The spread of the variants was assessed using the spreading assay described in Example 1. When compared to WR vaccinia virus containing the wild-type A33 and A34 sequences (IGV-013), the variant vaccinia viruses generated a higher degree of relative light units (RLU), indicating enhanced viral spreading (
The next selection identified variants capable of enhanced virus spreading and EEV production following infection of HCT 116 colorectal human cancer cells. Four variants were identified that demonstrated an increased frequency within the sequenced population (
The impact of the substitution variants on EEV production and viral spreading was assessed in vitro using BSC-40 (African green monkey kidney) and U-2 OS (human osteosarcoma) cell lines. Copenhagen vaccinia viruses containing the wild-type A33 and A34 sequences (IGV-007), a K151E substitution in A34 (IGV-006; a known mutation to increase virus spreading and EEV production), R91S and K151E substitutions in A34 and I269F substitution in A56 (IGV-084), T127E and K151E substitutions in A34 (IGV-085), R84G and K151E substitutions in A34 (IGV-086), or R91A and K151E substitutions in A34 (IGV-087) were generated and manufactured as described in Example 1. The comet assay was performed as described in Example 1. The presence of longer comet tails (
The next selection identified variants capable of enhanced virus spreading and EEV production following infection of Colo 205 colorectal human cancer cells. Two variants were identified that demonstrated an increased frequency within the sequenced population (
The following selections identified variants capable of enhanced virus spreading and EEV production following infection of MDA-MB-231 breast cancer cells in the absence or presence of serum from donors vaccinated with vaccinia virus. Five total variants were identified, three in the absence of serum and two in the presence of serum, that demonstrated an increased frequency within the sequenced population (
The impact of the substitution variants on EEV production and viral spreading was assessed in vitro using BSC-40 (African green monkey kidney) and U-2 OS (human osteosarcoma) cell lines. Copenhagen vaccinia viruses containing the wild-type A33, A34, and B5 sequences (IGV-007), a K151E substitution in A34 (IGV-006; a known mutation to increase virus spreading and EEV production), the K229C and S273L substitutions in B5 (IGV-110), the N39G and S273I substitutions in B5 (IGV-112), the S199M substitution in B5 (IGV-116), the L90R and S273V substitutions in B5 (IGV-114), or the S273I substitution in B5 (IGV-111) were generated and manufactured as described in Example 1. The comet assay was performed as described in Example 1. The presence of longer comet tails (
The impact of the substitution variants on infectious virus particle production in the supernatant (EEV production) in different human cancer cells lines was assessed in vitro using MDA-MB-231 cells (breast adenocarcinoma), MCF7 cells (breast adenocarcinoma), T47D cells (breast carcinoma), and HCT 116 cells (colorectal carcinoma). Copenhagen vaccinia viruses containing the wild-type A33, A34, and B5 sequences (IGV-007), a K151E substitution in A34 (IGV-006; a known mutation to increase virus spreading and EEV production), and vaccinia virus variants containing a combination of a K151E substitution in A34 and the K229C and S273L substitutions in B5 (IGV-110), the S273I substitution in B5 (IGV-111), the N39G and S273I substitutions in B5 (IGV-112), the L90R and S273V substitutions in B5 (IGV-114), or the S199M substitution in B5 (IGV-116) were generated and manufactured as described in Example 1. All cell lines listed above were infected with a MOI of 3 and washed thoroughly 3 times after 1 hour of virus adsorption. At 24 hours post-infection, the supernatant was collected and the number of infectious virus (EEV) particles produced was determined by performing a plaque assay as described in Example 1. In all four cell lines, the substitution variants containing a combination of a K151E substitution in A34 and the K229C and S273L substitutions in B5 (IGV-110), the S273I substitution in B5 (IGV-111), the N39G and S273I substitutions in B5 (IGV-112), the L90R and S273V substitutions in B5 (IGV-114), or the S199M substitution in B5 (IGV-116) produced a higher number of infectious virus particles in the supernatant, suggesting an increase in EEV particle formation (
In vivo selections performed in mouse cancer models identified variants capable of enhanced spreading and EEV production following infection of mice containing human patient-derived cancer cells with vaccinia virus. Eight total variants were identified, two using a patient-derived xenograft (PDX) mouse bilateral model of colorectal cancer and six using a patient-derived xenograft (PDX) mouse bilateral model of colorectal cancer that also contained a humanized blood system, that demonstrated an increased frequency within the sequenced population (
The impact of the substitution variants on EEV production and viral spreading was assessed in vitro using BSC-40 (African green monkey kidney) and U-2 OS (human osteosarcoma) cell lines. Copenhagen vaccinia viruses containing the wild-type A33, A34, and B5 sequences (IGV-007), a K151E substitution in A34 (IGV-006; a known mutation to increase virus spreading and EEV production), and variants containing the combination of a K151E substitution in A34 with I236P, V238R, T240R, and E243G substitutions in B5 (IGV-101), V233D, I236L, V238W, T240Y, and E243R substitutions in B5 (IGV-102), D263V, E268T, E270G, E272P, and E275S substitutions in B5 (IGV-103), an N94T substitution in B5 (IGV-104), an M63R substitution in A33 and an M66T substitution in A34 (IGV-105), N241G, E243S, V247W, D248Y, G250A, and A276F substitutions in B5 (IGV-106), D263A, E270S, E272G, and E275F substitutions in B5 (IGV-107), or N241T, E243V, V247S, G250R, and A276F substitutions in B5 (IGV-108) were generated and manufactured as described in Example 1. The comet assay was performed as described in Example 1. The presence of longer comet tails (
A summary of all vaccinia virus substitution variants identified through directed evolution is provided in Table 2.
In embodiments that refer to a method of treatment as described herein, such embodiments are also further embodiments for use in that treatment, or alternatively for the manufacture of a medicament for use in that treatment.
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
This application claims the benefit of U.S. Provisional Application No. 62/947,200 filed Dec. 12, 2019, U.S. Provisional Application No. 62/947,202 filed Dec. 12, 2019, and U.S. Provisional Application No. 62/947,204 filed Dec. 12, 2019. The disclosure of each of the provisional applications is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2020/061707 | 12/9/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62947204 | Dec 2019 | US | |
| 62947202 | Dec 2019 | US | |
| 62947200 | Dec 2019 | US |
