Patent Application
20240066081
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 25, 2020, is named 07039-1936WO1_SL.txt and is 1,096,580 bytes in size.
This document relates to canine distemper virus (CDV) hemagglutinin (H) and fusion (F) polypeptides. For example, this document relates to CDV H polypeptides, CDV F polypeptides, recombinant viruses (e.g., vesicular stomatitis viruses (VSVs)) containing CDV H polypeptides and/or CDV F polypeptides, nucleic acid molecules encoding a CDV H polypeptide and/or CDV F polypeptide, methods for making recombinant viruses (e.g., VSVs) containing CDV H polypeptides and/or CDV F polypeptides, and methods for using recombinant viruses (e.g., VSVs) containing CDV H polypeptides and/or CDV F polypeptides to treat cancer or infectious diseases.
Viruses such as VSVs, measles viruses (MeVs), and adenoviruses can be used as oncolytic viruses to treat cancer. Vesicular stomatitis virus (VSV) is a member of the Rhabdoviridae family. The VSV genome is a single molecule of negative-sense RNA that encodes five major polypeptides: a nucleocapsid (N) polypeptide, a phosphoprotein (P) polypeptide, a matrix (M) polypeptide, a glycoprotein (G) polypeptide, and a viral polymerase (L) polypeptide.
This document provides methods and materials related to CDV H and/or CDV F polypeptides. For example, this document provides CDV H polypeptides, CDV F polypeptides, recombinant viruses (e.g., vesicular stomatitis viruses (VSVs)) containing CDV H polypeptides and/or CDV F polypeptides, nucleic acid molecules encoding a CDV H polypeptide and/or CDV F polypeptide, methods for making recombinant viruses (e.g., VSVs) containing CDV H polypeptides and/or CDV F polypeptides, and methods for using recombinant viruses (e.g., VSVs) containing CDV H polypeptides and/or CDV F polypeptides to treat cancer or infectious diseases.
As described herein, CDV F polypeptides can be designed to have increased fusogenic activity when expressed by cells in combination with a CDV H polypeptide as compared to the level of fusogenic activity of a wild-type CDV F polypeptide expressed by comparable cells in combination with that CDV H polypeptide. For example, CDV F polypeptides designed to have a truncated signal peptide sequence can exhibit increased fusogenic activity when expressed by cells in combination with a CDV H polypeptide (e.g., a wild-type or de-targeted CDV H polypeptide) as compared to the level of fusogenic activity of a wild-type CDV F polypeptide containing a full length signal peptide sequence expressed by comparable cells in combination with that CDV H polypeptide. Such CDV F polypeptides can be incorporated into a virus to create a recombinant virus having the ability to increase fusogenic activity observed in cells infected by that virus.
As also described herein, CDV H polypeptides can be designed to be de-targeted such that they do not have the ability, when used in combination with an F polypeptide (e.g., a CDV F polypeptide), to enter cells via, or fuse cells via, a Nectin 4 polypeptide, a SLAMF1 polypeptide, or a virus receptor present on wild-type Vero cells. Such CDV H polypeptides can provide a platform for designing H polypeptides having the ability to be re-targeted to one or more targets of interest. For example, an H polypeptide provided herein can be further engineered to contain a binding sequence (e.g., a single chain antibody (scFv) sequence) having binding specificity for a target of interest such that a recombinant virus containing that re-targeted H polypeptide, and an F polypeptide, can infect cells expressing that target.
In addition, viruses such as VSVs can be designed to have a nucleic acid molecule that encodes a VSV N polypeptide, a VSV P polypeptide, a VSV M polypeptide, a CDV F polypeptide (e.g., a wild-type CDV F polypeptide or an engineered CDV F polypeptide described herein), a CDV H polypeptide (e.g., a wild-type CDV H polypeptide or an engineered CDV H polypeptide described herein), and a VSV L polypeptide. Such a nucleic acid molecule can lack a functional VSV G polypeptide and/or lack the nucleic acid sequence that encodes a full-length VSV G polypeptide. For example, a VSV provided herein can be designed to have a nucleic acid molecule that encodes a VSV N polypeptide, a VSV P polypeptide, a VSV M polypeptide, a CDV F polypeptide (e.g., a wild-type CDV F polypeptide or an engineered CDV F polypeptide described herein), a CDV H polypeptide (e.g., a wild-type CDV H polypeptide or an engineered CDV H polypeptide described herein), and a VSV L polypeptide and lacks the ability to encode a functional VSV G polypeptide. In some cases, a VSV provided herein can be designed to have a nucleic acid molecule that encodes a VSV N polypeptide, a VSV P polypeptide, a VSV M polypeptide, a CDV F polypeptide (e.g., a wild-type CDV F polypeptide or an engineered CDV F polypeptide described herein), a CDV H polypeptide (e.g., a wild-type CDV H polypeptide or an engineered CDV H polypeptide described herein), and a VSV L polypeptide with the nucleic acid sequence encoding the CDV F polypeptide and the CDV H polypeptide being located in the position where the nucleic acid sequence encoding a full-length VSV G polypeptide is normally located in a wild-type VSV. In some cases, a VSV provided herein can be designed to have a nucleic acid molecule where the nucleic acid sequence encoding a VSV G polypeptide is replaced with nucleic acid that encodes a CDV F polypeptide (e.g., a wild-type CDV F polypeptide or an engineered CDV F polypeptide described herein) and a CDV H polypeptide (e.g., a wild-type CDV H polypeptide or an engineered CDV H polypeptide described herein).
As described herein, VSV/CDV hybrids can be designed to have CDV selectivity and a rapid replication as observed with wild-type or parental VSV. In some cases, a VSV/CDV hybrid provided herein can be designed to have a preselected tropism. For example, CDV F and/or H polypeptides having knocked out specificity for Nectin-4 and/or SLAMF1 can be used. In such cases, a scFv or polypeptide ligand can be attached to, for example, the C-terminus of the CDV H polypeptide. In such cases, the scFv or polypeptide ligand can determine the tropism of the VSV/CDV hybrid. Examples of scFvs that can be used to direct VSV/CDV hybrids to cellular receptors (e.g., tumor associated cellular receptors) include, without limitation, anti-EGFR, anti-αFR, anti-CD46, anti-CD38, anti-HER2/neu, anti-EpCAM, anti-CEA, anti-CD20, anti-CD133, anti-CD117 (c-kit), and anti-CD138 and anti-PSMA scFvs. Examples of polypeptide ligands that can be used to direct VSV/CDV hybrids include, without limitation, urokinase plasminogen activator uPA polypeptides, cytokines such as IL-13 or IL-6, single chain T cell receptors (scTCRs), echistatin polypeptides, stem cell factor (SCF), EGF and integrin binding polypeptides.
In some cases, a VSV/CDV hybrid provided herein can have a nucleic acid molecule that includes a sequence encoding an interferon (IFN) polypeptide (e.g., a human IFN-β polypeptide), a sodium iodide symporter (NIS) polypeptide (e.g., a human NIS polypeptide), a fluorescent polypeptide (e.g., a GFP polypeptide), any appropriate therapeutic transgene (e.g., HSV-TK or cytosine deaminase), polypeptide that antagonizes host immunity (e.g., influenza NS1, HSVγ34.5, or SOCS1), or tumor antigen (e.g., cancer vaccine components). The nucleic acid encoding the IFN polypeptide can be positioned between the nucleic acid encoding the VSV M polypeptide and the nucleic acid encoding the VSV L polypeptide. Such a position can allow the viruses to express an amount of the IFN polypeptide that is effective to activate anti-viral innate immune responses in non-cancerous tissues, and thus alleviate potential viral toxicity, without impeding efficient viral replication in cancer cells. The nucleic acid encoding the NIS polypeptide can be positioned between the nucleic acid encoding the VSV M polypeptide and the VSV L polypeptide. Such a position of can allow the viruses to express an amount of the NIS polypeptide that (a) is effective to allow selective accumulation of iodide in infected cells, thereby allowing both imaging of viral distribution using radioisotopes and radiotherapy targeted to infected cancer cells, and (b) is not so high as to be toxic to infected cells. Positioning the nucleic acid encoding an IFN polypeptide between the nucleic acid encoding the VSV M polypeptide and the nucleic acid encoding the VSV L polypeptide and positioning the nucleic acid encoding a NIS polypeptide between the nucleic acid encoding the VSV M polypeptide and the VSV L polypeptide within the genome of a VSV can result in VSVs that are viable, that have the ability to replicate and spread, that express appropriate levels of functional IFN polypeptides, and that expression appropriate levels of functional NIS polypeptides to take up radio-iodine for both imaging and radio-virotherapy.
In general, one aspect of this document features a CDV F polypeptide having signal peptide sequence that is less than 75 amino acid residues in length. The signal peptide sequence can comprise no more than 75 amino acid residues of SEQ ID NO:6. The CDV F polypeptide can comprise SEQ ID NO:4 with the proviso that the CDV F polypeptide lacks at least amino acid residues 1 to 60 or lacks at least amino acid residues 1 to 105 of SEQ ID NO:4. A recombinant virus comprising the CDV F polypeptide and a CDV H polypeptide can exhibit increased fusogenic activity as compared to a comparable control recombinant virus comprising a full length, wild-type CDV F polypeptide and the CDV H polypeptide.
In another embodiment, this document features a nucleic acid molecule encoding a CDV F polypeptide. The CDV F polypeptide can have a signal peptide sequence that is less than 75 amino acid residues in length. The signal peptide sequence can comprise no more than 75 amino acid residues of SEQ ID NO:6. The CDV F polypeptide can comprise SEQ ID NO:4 with the proviso that the CDV F polypeptide lacks at least amino acid residues 1 to 60 or lacks at least amino acid residues 1 to 105 of SEQ ID NO:4. A recombinant virus comprising the CDV F polypeptide and a CDV H polypeptide can exhibit increased fusogenic activity as compared to a comparable control recombinant virus comprising a full length, wild-type CDV F polypeptide and the CDV H polypeptide.
In another embodiment, this document features a recombinant virus comprising a CDV F polypeptide. The CDV F polypeptide can have a signal peptide sequence that is less than 75 amino acid residues in length. The signal peptide sequence can comprise no more than 75 amino acid residues of SEQ ID NO:6. The CDV F polypeptide can comprise SEQ ID NO:4 with the proviso that the CDV F polypeptide lacks at least amino acid residues 1 to 60 or lacks at least amino acid residues 1 to 105 of SEQ ID NO:4. A recombinant virus comprising the CDV F polypeptide and a CDV H polypeptide can exhibit increased fusogenic activity as compared to a comparable control recombinant virus comprising a full length, wild-type CDV F polypeptide and the CDV H polypeptide.
In another embodiment, this document features a recombinant virus comprising a nucleic acid molecule. The nucleic acid molecule can encode a CDV F polypeptide. The CDV F polypeptide can have a signal peptide sequence that is less than 75 amino acid residues in length. The signal peptide sequence can comprise no more than 75 amino acid residues of SEQ ID NO:6. The CDV F polypeptide can comprise SEQ ID NO:4 with the proviso that the CDV F polypeptide lacks at least amino acid residues 1 to 60 or lacks at least amino acid residues 1 to 105 of SEQ ID NO:4. A recombinant virus comprising the CDV F polypeptide and a CDV H polypeptide can exhibit increased fusogenic activity as compared to a comparable control recombinant virus comprising a full length, wild-type CDV F polypeptide and the CDV H polypeptide.
In another embodiment, this document features a CDV H polypeptide comprising 454A, 464A, 479A, 494A, 510A, 520A, 525A, 526A, 527S, 528A, 529A, 537A, 539A, 547A, 548A, or a combination thereof according to the amino acid numbering of SEQ ID NO:2. The CDV H polypeptide can comprise a combination of two, three, four, five, or six of 454A, 464A, 479A, 494A, 510A, 520A, 525A, 526A, 527S, 528A, 529A, 537A, 539A, 547A, and 548A. The CDV H polypeptide can comprise a combination of seven, eight, nine, ten, or eleven of 454A, 464A, 479A, 494A, 510A, 520A, 525A, 526A, 527S, 528A, 529A, 537A, 539A, 547A, and 548A. The CDV H polypeptide can comprise a combination of 12, 13, or 14 of 454A, 464A, 479A, 494A, 510A, 520A, 525A, 526A, 527S, 528A, 529A, 537A, 539A, 547A, and 548A. The CDV H polypeptide can comprise 454A, 464A, 479A, 494A, 510A, 520A, 525A, 526A, 527S, 528A, 529A, 537A, 539A, 547A, and 548A. The CDV H polypeptide can comprise M437 according to the amino acid numbering of SEQ ID NO:5.
In another embodiment, this document features a CDV H polypeptide comprising the sequence set forth in
In another embodiment, this document features a nucleic acid molecule encoding a CDV H polypeptide. The CDV H polypeptide can comprise the sequence set forth in
In another embodiment, this document features a recombinant virus comprising a CDV H polypeptide. The CDV H polypeptide comprising the sequence set forth in
In another embodiment, this document features a recombinant virus comprising a nucleic acid molecule encoding a CDV H polypeptide. The CDV H polypeptide can comprise the sequence set forth in
The virus can comprise a nucleic acid molecule encoding a CDV F polypeptide. The CDV F polypeptide can have a signal peptide sequence that is less than 75 amino acid residues in length. The signal peptide sequence can comprise no more than 75 amino acid residues of SEQ ID NO:6. The CDV F polypeptide can comprise SEQ ID NO:4 with the proviso that the CDV F polypeptide lacks at least amino acid residues 1 to 60 or lacks at least amino acid residues 1 to 105 of SEQ ID NO:4. A recombinant virus comprising the CDV F polypeptide and a CDV H polypeptide can exhibit increased fusogenic activity as compared to a comparable control recombinant virus comprising a full length, wild-type CDV F polypeptide and the CDV H polypeptide.
In another embodiment, this document features a recombinant virus described herein that is a hybrid virus of (a) CDV and (b) VSV, MeV, or Adenovirus.
In another embodiment, this document features a replication-competent vesicular stomatitis virus comprising an RNA molecule, wherein the RNA molecule comprises a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a CDV F polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a CDV H polypeptide, and a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, wherein the RNA molecule lacks a nucleic acid sequence that is a template for a positive sense transcript encoding a functional VSV G polypeptide. The CDV F polypeptide can have a signal peptide sequence that is less than 75 amino acid residues in length. The signal peptide sequence can comprise no more than 75 amino acid residues of SEQ ID NO:6. The CDV F polypeptide can comprise SEQ ID NO:4 with the proviso that the CDV F polypeptide lacks at least amino acid residues 1 to 60 or lacks at least amino acid residues 1 to 105 of SEQ ID NO:4. A recombinant virus comprising the CDV F polypeptide and a CDV H polypeptide can exhibit increased fusogenic activity as compared to a comparable control recombinant virus comprising a full length, wild-type CDV F polypeptide and the CDV H polypeptide. The CDV H polypeptide can be a CDV H polypeptide described in one of the preceding paragraphs. The CDV H polypeptide can comprises an amino acid sequence of a single chain antibody. The single chain antibody can be a single chain antibody directed to CD19, CD20, CD38, CD46, EGFR, αFR, HER2/neu, or PSMA. The RNA molecule can comprise a nucleic acid sequence that is a template for a positive sense transcript encoding a NIS polypeptide.
In another embodiment, this document features a composition comprising a virus of any of the preceding paragraphs.
In another embodiment, this document features a nucleic acid molecule comprising a nucleic acid strand comprising a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a CDV F polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a CDV H polypeptide, and a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, wherein the nucleic acid strand lacks a nucleic acid sequence that is a template for a positive sense transcript encoding a functional VSV G polypeptide. The CDV F polypeptide can have a signal peptide sequence that is less than 75 amino acid residues in length. The signal peptide sequence can comprise no more than 75 amino acid residues of SEQ ID NO:6. The CDV F polypeptide can comprise SEQ ID NO:4 with the proviso that the CDV F polypeptide lacks at least amino acid residues 1 to 60 or lacks at least amino acid residues 1 to 105 of SEQ ID NO:4. A recombinant virus comprising the CDV F polypeptide and a CDV H polypeptide can exhibit increased fusogenic activity as compared to a comparable control recombinant virus comprising a full length, wild-type CDV F polypeptide and the CDV H polypeptide. The CDV H polypeptide can be a CDV H polypeptide described in one of the preceding paragraphs. The CDV H polypeptide can comprises an amino acid sequence of a single chain antibody. The single chain antibody can be a single chain antibody directed to CD19, CD20, CD38, CD46, EGFR, αFR, HER2/neu, or PSMA. The RNA molecule can comprise a nucleic acid sequence that is a template for a positive sense transcript encoding a NIS polypeptide.
In another embodiment, this document features a composition comprising a nucleic acid molecule of any of the preceding paragraphs.
In another embodiment, this document features a method for treating cancer. The method comprises administering a composition described herein (e.g., a composition containing a virus described herein) to a mammal comprising cancer cells, wherein the number of cancer cells within the mammal is reduced following the administration. The mammal can be a human. The cancer can be myeloma, melanoma, glioma, lymphoma, mesothelioma, lung cancer, brain cancer, stomach cancer, colon cancer, rectum cancer, kidney cancer, prostate cancer, ovary cancer, breast cancer, pancreas cancer, liver cancer, or head and neck cancer.
In another embodiment, this document features a method for inducing tumor regression in a mammal. The method comprises administering a composition described herein (e.g., a composition containing a virus described herein) to a mammal comprising a tumor, wherein the size of the tumor is reduced following the administration. The mammal can be a human. The cancer can be myeloma, melanoma, glioma, lymphoma, mesothelioma, lung cancer, brain cancer, stomach cancer, colon cancer, rectum cancer, kidney cancer, prostate cancer, ovary cancer, breast cancer, pancreas cancer, liver cancer, or head and neck cancer.
In another embodiment, this document features a method for rescuing replication-competent vesicular stomatitis viruses from cells. The vesicular stomatitis viruses comprise an RNA molecule comprising a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a CDV F polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a CDV H polypeptide, and a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV L polypeptide, wherein the RNA molecule lacks a nucleic acid sequence that is a template for a positive sense transcript encoding a functional VSV G polypeptide. The method comprises (a) inserting nucleic acid encoding the RNA molecule into the cells under conditions wherein replication-competent vesicular stomatitis viruses are produced, and (b) harvesting the replication-competent vesicular stomatitis viruses.
Unless otherwise defined, 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 pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides CDV F polypeptides. As described herein, a CDV F polypeptide can be designed such that virus particles containing the CDV F polypeptide together with a CDV H polypeptide exhibit enhanced fusogenic activity. For example, a CDV F polypeptide can be designed to contain a signal peptide sequence that is no longer than 75 amino acids in length. Typically, wild-type CDV F polypeptides contain a signal peptide sequence that is about 135 amino acids in length. An example of a 135 amino acid signal peptide sequence of a wild-type CDV F polypeptide is set forth in SEQ ID NO:6 (MHKEIPEKSRTRTHTQQDLPQQKSTEYTEIKTSRARHGITPAQRSTH YGPRTLDRLVCYIMNRAMSCKQASYRSDNIPAHGDHEGVVHHTPESVSQGARSQ LKRRTSNAINSGFQYIWLVLWCIGIASLFLCSKA). As described herein, truncating the signal peptide sequence of CDV F polypeptides such that it is no longer than 75 amino acids in length can result in CDV F polypeptides that, when part of viruses together with CDV H polypeptides, allow for increased fusogenic activity of the viruses as compared to the level of fusogenic activity exhibited by comparable control viruses containing a CDV F polypeptide having a full-length wild-type signal peptide sequence (e.g., SEQ ID NO:6).
A CDV F polypeptide provided herein can contain a signal peptide sequence that is from 7 amino acids to 75 amino acids in length. For example, a CDV F polypeptide provided herein can contain a signal peptide sequence that is from 7 to 75 (e.g., from 7 to 70, from 7 to 65, from 7 to 60, from 7 to 55, from 7 to 50, from 7 to 45, from 7 to 40, from 7 to 35, from 7 to 30, from 7 to 25, from 10 to 75, from 15 to 75, from 20 to 75, from 25 to 75, from 35 to 75, from 45 to 75, from 50 to 75, from 55 to 75, from 65 to 75, from 20 to 60, from 25 to 50, from 30 to 60, or from 30 to 40) amino acids in length. A CDV F polypeptide provided herein can be produced by truncating a wild-type signal peptide sequence from its N-terminus, from its C-terminus, or from both its N-terminus and C-terminus or by deleting amino acids from in between the N-terminal and C-terminal regions of a wild-type signal peptide sequence. In some cases, a measles virus signal peptide sequence can be used for a signal peptide of a CDV F polypeptide described herein. Examples of signal peptide sequences of CDV F polypeptides provided herein include, without limitation, those set forth in Table 1.
In some cases, a CDV F polypeptide provided herein can be designed to lack the entire signal peptide sequence. For example, a CDV F polypeptide provided herein can have one of the amino acid sequences set forth in
A CDV F polypeptide provided herein can have any appropriate amino acid sequence provided that the CDV F polypeptide does not contain a signal peptide sequence longer than 75 amino acid residues in length. Examples of amino acid sequences of CDV F polypeptides that can be used as described herein include, without limitation, those amino acid sequences set forth in
This document also provides CDV H polypeptides. As described herein, a CDV H polypeptide can be designed such that viruses containing the CDV H polypeptide together with a CDV F polypeptide exhibit reduced or eliminated tropism for SLAMF1 polypeptides and/or Nectin-4 polypeptides as compared to viruses containing wild-type CDV H polypeptides. For example, a CDV H polypeptide can be designed to contain a mutation at one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, or 15) of amino acid positions 454, 460, 479, 494, 510, 520, 525, 526, 527, 528, 529, 537, 539, 547, and 548. Typically, viruses containing wild-type CDV H polypeptides (together with CDV F polypeptides) exhibit tropism for SLAMF1 polypeptides and Nectin-4 polypeptides such that the viruses infect SLAMF1-positive cells and Nectin-4-positive cells. As described herein, mutating one or more of amino acid positions P/S454, V/L/F460, L/F/W479, I494, I/L/V510, Y520, Y/N525, D/G526, I/V527, S/T528, R529, Y/D537, Y539, Y/F547, and Y/M548 of a CDV H polypeptide to a different amino acid (e.g., alanine) can reduce or eliminate the ability of viruses containing that CDV H polypeptide (together with a CDV F polypeptide) to infect SLAMF1-positive cells and/or Nectin-4-positive cells. Examples of CDV H polypeptides provided herein having reduced or eliminated tropism for SLAMF1 polypeptides and/or Nectin-4 polypeptides include, without limitation, those CDV H polypeptides set forth in
This document also provides recombinant viruses (e.g., VSVs) containing a CDV H polypeptide provided herein and/or a CDV F polypeptide provided herein as well as methods for making recombinant viruses (e.g., VSVs) containing a CDV H polypeptide provided herein and/or a CDV F polypeptide provided herein. For example, a recombinant virus (e.g., VSV) can be designed to include (a) a CDV H polypeptide provided herein and a wild-type CDV F polypeptide, (b) a wild-type CDV H polypeptide and a CDV F polypeptide provided herein, or (c) a CDV H polypeptide provided herein and a CDV F polypeptide provided herein. In some cases, a recombinant virus (e.g., VSV) can be designed to include a CDV H polypeptide having CDV H 5804 and a CDV F polypeptide having CDV F 22458/16.
This document also provides nucleic acid molecules encoding a CDV H polypeptide provided herein and/or nucleic acid molecules encoding a CDV F polypeptide provided herein. For example, a nucleic acid molecule (e.g., a vector) can be designed to encode a CDV H polypeptide provided herein and/or a CDV F polypeptide provided herein.
This document provides methods and materials related to VSVs. For example, this document provides replication-competent VSVs, nucleic acid molecules encoding replication-competent VSVs, methods for making replication-competent VSVs, and methods for using replication-competent VSVs to treat cancer or infectious diseases.
As described herein, a VSV can be designed to have a nucleic acid molecule that encodes a VSV N polypeptide, a VSV P polypeptide, a VSV M polypeptide, a CDV F polypeptide (e.g., a CDV F polypeptide provided herein), a CDV H polypeptide (e.g., a CDV H polypeptide provided herein), and a VSV L polypeptide, and does not encode a functional VSV G polypeptide. It will be appreciated that the sequences described herein with respect to a VSV are incorporated into a plasmid coding for the positive sense cDNA of the viral genome allowing generation of the negative sense genome of VSVs. Thus, it will be appreciated that a nucleic acid sequence that encodes a VSV polypeptide, for example, can refer to an RNA sequence that is the template for the positive sense transcript that encodes (e.g., via direct translation) that polypeptide.
The nucleic acid encoding the CDV F polypeptide and the CDV H polypeptide can be positioned at any location within the VSV genome. In some cases, the nucleic acid encoding the CDV F polypeptide and the CDV H polypeptide can be positioned downstream of the nucleic acid encoding the VSV M polypeptide. For example, nucleic acid encoding a CDV F polypeptide and nucleic acid encoding a CDV H polypeptide can be positioned between nucleic acid encoding a VSV M polypeptide and nucleic acid encoding a VSV L polypeptide.
Any appropriate nucleic acid encoding a CDV F polypeptide can be inserted into the genome of a VSV. For example, nucleic acid encoding a wild-type CDV F polypeptide or a CDV F polypeptide provided herein can be inserted into the genome of a VSV.
Any appropriate nucleic acid encoding a CDV H polypeptide can be inserted into the genome of a VSV. For example, nucleic acid encoding a wild-type H polypeptide or a H polypeptide provided herein can be inserted into the genome of a VSV. In some cases, nucleic acid encoding a CDV H polypeptide that lacks specificity for SLAMF1 and/or Nectin-4 can be inserted into the genome of a VSV. For example, nucleic acid encoding a CDV H polypeptide having one or more mutations set forth in Table 2 can be inserted into the genome of a VSV. In some cases, a VSV/CDV hybrid provided herein can be designed to have a preselected tropism. For example, CDV F and/or H polypeptides having knocked out specificity for SLAMF1 and/or Nectin-4 can be used such that a scFv or polypeptide ligand can be attached to, for example, the C-terminus of the CDV H polypeptide. In such cases, scFv or polypeptide ligand can determine the tropism of a VSV/CDV hybrid. Examples of scFvs that can be used to direct VSV/CDV hybrids to cellular receptors (e.g., tumor associated cellular receptors) include, without limitation, anti-EGFR, anti-CD46, anti-αFR, anti-PSMA, anti-HER-2, anti-CD19, anti-CD20, or anti-CD38 scFvs. Examples of polypeptide ligands that can be used to direct VSV/CDV hybrids include, without limitation, urokinase plasminogen activator uPA polypeptides, cytokines such as IL-13, single chain T cell receptors (scTCRs), echistatin polypeptides, and integrin binding polypeptides.
In some cases, the nucleic acid molecule of VSV provided herein can encode an IFN polypeptide, a fluorescent polypeptide (e.g., a GFP polypeptide), a NIS polypeptide, a therapeutic polypeptide, an innate immunity antagonizing polypeptide, a tumor antigen, or a combination thereof. Nucleic acid encoding an IFN polypeptide can be positioned downstream of nucleic acid encoding a VSV M polypeptide. For example, nucleic acid encoding an IFN polypeptide can be positioned between nucleic acid encoding a VSV M polypeptide and nucleic acid encoding a CDV F polypeptide or nucleic acid encoding a CDV H polypeptide. Such a position can allow the viruses to express an amount of IFN polypeptide that is effective to activate anti-viral innate immune responses in non-cancerous tissues, and thus alleviate potential viral toxicity, without impeding efficient viral replication in cancer cells.
Any appropriate nucleic acid encoding an IFN polypeptide can be inserted into the genome of a VSV. For example, nucleic acid encoding an IFN beta polypeptide can be inserted into the genome of a VSV. Examples of nucleic acid encoding IFN beta polypeptides that can be inserted into the genome of a VSV include, without limitation, nucleic acid encoding a human IFN beta polypeptide of the nucleic acid sequence set forth in GenBank® Accession No. NM_002176.2 (GI No. 50593016), nucleic acid encoding a mouse IFN beta polypeptide of the nucleic acid sequence set forth in GenBank® Accession Nos. NM_010510.1 (GI No. 6754303), BC119395.1 (GI No. 111601321), or BC119397.1 (GI No. 111601034), and nucleic acid encoding a rat IFN beta polypeptide of the nucleic acid sequence set forth in GenBank® Accession No. NM_019127.1 (GI No. 9506800).
Nucleic acid encoding a NIS polypeptide can be positioned downstream of nucleic acid encoding a CDV F polypeptide or nucleic acid encoding a CDV H polypeptide. For example, nucleic acid encoding a NIS polypeptide can be positioned between nucleic acid encoding a CDV F or H polypeptide and nucleic acid encoding a VSV L polypeptide. Such a position of can allow the viruses to express an amount of NIS polypeptide that (a) is effective to allow selective accumulation of iodide in infected cells, thereby allowing both imaging of viral distribution using radioisotopes and radiotherapy targeted to infected cancer cells, and (b) is not so high as to be toxic to infected cells.
Any appropriate nucleic acid encoding a NIS polypeptide can be inserted into the genome of a VSV. For example, nucleic acid encoding a human NIS polypeptide can be inserted into the genome of a VSV. Examples of nucleic acid encoding NIS polypeptides that can be inserted into the genome of a VSV include, without limitation, nucleic acid encoding a human NIS polypeptide of the nucleic acid sequence set forth in GenBank® Accession Nos. NM_000453.2 (GI No. 164663746), BC105049.1 (GI No. 85397913), or BC105047.1 (GI No. 85397519), nucleic acid encoding a mouse NIS polypeptide of the nucleic acid sequence set forth in GenBank® Accession Nos. NM_053248.2 (GI No. 162138896), AF380353.1 (GI No. 14290144), or AF235001.1 (GI No. 12642413), nucleic acid encoding a chimpanzee NIS polypeptide of the nucleic acid sequence set forth in GenBank® Accession No. XM_524154 (GI No. 114676080), nucleic acid encoding a dog NIS polypeptide of the nucleic acid sequence set forth in GenBank® Accession No. XM_541946 (GI No. 73986161), nucleic acid encoding a cow NIS polypeptide of the nucleic acid sequence set forth in GenBank® Accession No. XM_581578 (GI No. 297466916), nucleic acid encoding a pig NIS polypeptide of the nucleic acid sequence set forth in GenBank® Accession No. NM_214410 (GI No. 47523871), and nucleic acid encoding a rat NIS polypeptide of the nucleic acid sequence set forth in GenBank® Accession No. NM_052983 (GI No. 158138504).
The nucleic acid sequences of a VSV provided herein that encode a VSV N polypeptide, a VSV P polypeptide, a VSV M polypeptide, and a VSV L polypeptide can be from a VSV Indiana strain as set forth in GenBank® Accession Nos. NC_001560 (GI No. 9627229) or can be from a VSV New Jersey strain.
In one aspect, this document provides VSVs containing a nucleic acid molecule (e.g., an RNA molecule) having (e.g., in a 3′ to 5′ direction) a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV N polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV P polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV M polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a CDV F polypeptide, a nucleic acid sequence that is a template for a positive sense transcript encoding a CDV H polypeptide, and a nucleic acid sequence that is a template for a positive sense transcript encoding a VSV L polypeptide while lacking a nucleic acid sequence that is a template for a positive sense transcript encoding a functional VSV G polypeptide. Such VSVs can infect cells (e.g., cancer cells) and be replication-competent.
Any appropriate method can be used to insert nucleic acid (e.g., nucleic acid encoding a CDV F polypeptide, nucleic acid encoding a CDV H polypeptide, nucleic acid encoding an IFN polypeptide, and/or nucleic acid encoding a NIS polypeptide) into the genome of a VSV. For example, the methods described elsewhere (Schnell et. al., PNAS, 93:11359-11365 (1996), Obuchi et al., J. Virol., 77(16):8843-56 (2003)); Goel et al., Blood, 110(7):2342-50 (2007)); and Kelly et al., J. Virol., 84(3):1550-62 (2010)) can be used to insert nucleic acid into the genome of a VSV. Any appropriate method can be used to identify VSVs containing a nucleic acid molecule described herein. Such methods include, without limitation, PCR and nucleic acid hybridization techniques such as Northern and Southern analysis. In some cases, immunohistochemistry and biochemical techniques can be used to determine if a VSV contains a particular nucleic acid molecule by detecting the expression of a polypeptide encoded by that particular nucleic acid molecule.
In another aspect, this document provides nucleic acid molecules that encode a VSV N polypeptide, a VSV P polypeptide, a VSV M polypeptide, a CDV F polypeptide, a CDV H polypeptide, and a VSV L polypeptide, while lacking the ability to encode a functional VSV G polypeptide. For example, a nucleic acid molecule provided herein can be a single nucleic acid molecule that includes a nucleic acid sequence that encodes a VSV N polypeptide, a nucleic acid sequence that encodes a VSV P polypeptide, a nucleic acid sequence that encodes a VSV M polypeptide, a nucleic acid sequence that encodes a CDV F polypeptide, a nucleic acid sequence that encodes a CDV H polypeptide, and a nucleic acid sequence that encodes a VSV L polypeptide, while lacking a nucleic acid sequence that encodes a functional VSV G polypeptide.
In another aspect, this document provides nucleic acid molecules that encode a VSV N polypeptide, a VSV P polypeptide, a VSV M polypeptide, an IFN polypeptide, a CDV F polypeptide, a CDV H polypeptide, a NIS polypeptide, and a VSV L polypeptide, while lacking the ability to encode a functional VSV G polypeptide. For example, a nucleic acid molecule provided herein can be a single nucleic acid molecule that includes a nucleic acid sequence that encodes a VSV N polypeptide, a nucleic acid sequence that encodes a VSV P polypeptide, a nucleic acid sequence that encodes a VSV M polypeptide, a nucleic acid sequence that encodes an IFN polypeptide, a nucleic acid sequence that encodes a CDV F polypeptide, a nucleic acid sequence that encodes a CDV H polypeptide, a nucleic acid sequence that encodes a NIS polypeptide, and a nucleic acid sequence that encodes a VSV L polypeptide, while lacking the ability to encode a functional VSV G polypeptide.
The term “nucleic acid” as used herein encompasses both RNA (e.g., viral RNA) and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. A nucleic acid can be double-stranded or single-stranded. A single-stranded nucleic acid can be the sense strand or the antisense strand. In addition, a nucleic acid can be circular or linear.
This document also provides method for treating cancer (e.g., to reduce tumor size, inhibit tumor growth, or reduce the number of viable tumor cells), methods for inducing host immunity against cancer, and methods for treating an infectious disease such as HIV or measles. For example, a recombinant virus (e.g., a VSV) provided herein can be administered to a mammal having cancer to reduce tumor size, to inhibit cancer cell or tumor growth, to reduce the number of viable cancer cells within the mammal, and/or to induce host immunogeneic responses against a tumor. A recombinant virus (e.g., a VSV) provided herein can be propagated in host cells in order to increase the available number of copies of that virus, typically by at least 2-fold (e.g., by 5- to 10-fold, by 50- to 100-fold, by 500- to 1,000-fold, or even by as much as 5,000- to 10,000-fold). In some cases, a recombinant virus (e.g., a VSV) provided herein can be expanded until a desired concentration is obtained in standard cell culture media (e.g., DMEM or RPMI-1640 supplemented with 5-10% fetal bovine serum at 37° C. in 5% CO2). A viral titer typically is assayed by inoculating cells (e.g., Vero cells) in culture.
Recombinant viruses (e.g., VSVs) provided herein can be administered to a cancer patient by, for example, direct injection into a group of cancer cells (e.g., a tumor) or intravenous delivery to cancer cells. A recombinant virus (e.g., a VSV) provided herein can be used to treat different types of cancer including, without limitation, myeloma (e.g., multiple myeloma), melanoma, glioma, lymphoma, mesothelioma, and cancers of the lung, brain, stomach, colon, rectum, kidney, prostate, ovary, breast, pancreas, liver, and head and neck.
Recombinant viruses (e.g., VSVs) provided herein can be administered to a patient in a biologically compatible solution or a pharmaceutically acceptable delivery vehicle, by administration either directly into a group of cancer cells (e.g., intratumorally) or systemically (e.g., intravenously). Suitable pharmaceutical formulations depend in part upon the use and the route of entry, e.g., transdermal or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the virus is desired to be delivered to) or from exerting its effect For example, pharmacological compositions injected into the blood stream should be soluble.
While dosages administered will vary from patient to patient (e.g., depending upon the size of a tumor), an effective dose can be determined by setting as a lower limit the concentration of virus proven to be safe and escalating to higher doses of up to 1012 pfu, while monitoring for a reduction in cancer cell growth along with the presence of any deleterious side effects. A therapeutically effective dose typically provides at least a 10% reduction in the number of cancer cells or in tumor size. Escalating dose studies can be used to obtain a desired effect for a given viral treatment (see, e.g., Nies and Spielberg, “Principles of Therapeutics,” In Goodman & Gilman's The Pharmacological Basis of Therapeutics, eds. Hardman, et al., McGraw-Hill, N Y, 1996, pp 43-62).
Recombinant viruses (e.g., VSVs) provided herein can be delivered in a dose ranging from, for example, about 103 pfu to about 1012 pfu (e.g., about 105 pfu to about 1012 pfu, about 106 pfu to about 1011 pfu, or about 106 pfu to about 1010 pfu). A therapeutically effective dose can be provided in repeated doses. Repeat dosing is appropriate in cases in which observations of clinical symptoms or tumor size or monitoring assays indicate either that a group of cancer cells or tumor has stopped shrinking or that the degree of viral activity is declining while the tumor is still present. Repeat doses can be administered by the same route as initially used or by another route. A therapeutically effective dose can be delivered in several discrete doses (e.g., days or weeks apart) and in one embodiment, one to about twelve doses are provided. Alternatively, a therapeutically effective dose of recombinant viruses (e.g., VSVs) provided herein can be delivered by a sustained release formulation. In some cases, a recombinant virus (e.g., a VSV) provided herein can be delivered in combination with pharmacological agents that facilitate viral replication and spread within cancer cells or agents that protect non-cancer cells from viral toxicity. Examples of such agents are described elsewhere (Alvarez-Breckenridge et al., Chem. Rev., 109(7):3125-40 (2009)).
Recombinant viruses (e.g., VSVs) provided herein can be administered using a device for providing sustained release. A formulation for sustained release of recombinant viruses (e.g., VSVs) provided herein can include, for example, a polymeric excipient (e.g., a swellable or non-swellable gel, or collagen). A therapeutically effective dose of recombinant viruses (e.g., VSVs) provided herein can be provided within a polymeric excipient, wherein the excipient/virus composition is implanted at a site of cancer cells (e.g., in proximity to or within a tumor). The action of body fluids gradually dissolves the excipient and continuously releases the effective dose of virus over a period of time. Alternatively, a sustained release device can contain a series of alternating active and spacer layers. Each active layer of such a device typically contains a dose of virus embedded in excipient, while each spacer layer contains only excipient or low concentrations of virus (i.e., lower than the effective dose). As each successive layer of the device dissolves, pulsed doses of virus are delivered. The size/formulation of the spacer layers determines the time interval between doses and is optimized according to the therapeutic regimen being used.
In some cases, recombinant viruses (e.g., VSVs) provided herein can be directly administered. For example, a virus can be injected directly into a tumor (e.g., a breast cancer tumor) that is palpable through the skin. Ultrasound guidance also can be used in such a method. Alternatively, direct administration of a virus can be achieved via a catheter line or other medical access device, and can be used in conjunction with an imaging system to localize a group of cancer cells. By this method, an implantable dosing device typically is placed in proximity to a group of cancer cells using a guidewire inserted into the medical access device. An effective dose of a recombinant virus (e.g., a VSV) provided herein can be directly administered to a group of cancer cells that is visible in an exposed surgical field.
In some cases, recombinant viruses (e.g., VSVs) provided herein can be delivered systemically. For example, systemic delivery can be achieved intravenously via injection or via an intravenous delivery device designed for administration of multiple doses of a medicament. Such devices include, but are not limited to, winged infusion needles, peripheral intravenous catheters, midline catheters, peripherally inserted central catheters, and surgically placed catheters or ports.
The course of therapy with a recombinant virus (e.g., a VSV) provided herein can be monitored by evaluating changes in clinical symptoms or by direct monitoring of the number of cancer cells or size of a tumor. For a solid tumor, the effectiveness of virus treatment can be assessed by measuring the size or weight of the tumor before and after treatment. Tumor size can be measured either directly (e.g., using calipers), or by using imaging techniques (e.g., X-ray, magnetic resonance imaging, or computerized tomography) or from the assessment of non-imaging optical data (e.g., spectral data). For a group of cancer cells (e.g., leukemia cells), the effectiveness of viral treatment can be determined by measuring the absolute number of leukemia cells in the circulation of a patient before and after treatment. The effectiveness of viral treatment also can be assessed by monitoring the levels of a cancer specific antigen. Cancer specific antigens include, for example, carcinoembryonic antigen (CEA), prostate specific antigen (PSA), prostatic acid phosphatase (PAP), CA 125, alpha-fetoprotein (AFP), carbohydrate antigen 15-3, and carbohydrate antigen 19-4.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Vero African green monkey kidney cells (Vero; American type culture collection [ATCC], Cat. #CCL-81) and their derivatives (either expressing Nectin-4 (Noyce et al., Virology, 436(1):210-20 (2013)), SLAMF1 (Tatsuo et al., Nature, 406(6798):893-7 (2000)), dog SLAMF1 (von Messling et al., J. Virol., 77(23):12579-91 (2003)), or a membrane-anchored single-chain antibody specific for a hexahistidine peptide (SEQ ID NO: 21) (Nakamura et al., Nat. Biotechnol., 23(2):209-14 (2005)) were kept in Dulbecco's modified Eagle's medium (DMEM) (GE Healthcare Life Sciences, Cat. #SH30022.01) supplemented with 5% (vol./vol.) heat-inactivated fetal bovine serum (FBS, Gibco) and 0.5 mg/mL of Geneticin (G418; Coming) for Vero/NECTIN-4 and Vero/SLAMF1 or 1 mg/mL Zeocin (ThermoFisher) for Vero/dogSLAMF1. Human kidney epithelial cells (HEK293T) cells, obtained from Dr. Cosset (Universitd de Lyon), baby hamster kidney cells (BHK, ATCC, Cat. #CCL-10), human glioblastoma U-87 MG cells ((ATCC, Cat. #HTB-14), and SKOV3ip.1 human ovarian tumor cells were maintained in DMEM plus 10% FBS. Chinese hamster ovary cells (CHO) cells, CHO-CD46, and CHO-EGFR (Nakamura et al., Nat. Biotechnol., 22(3):331-6 (2004)), CHO-SLAMF1 (Tatsuo et al., Nature, 406(6798):893-7 (2000)), CHO-dogSLAMF1 (Seki et al., J. Virol., 77(18):9943-50 (2003)), CHO-NECTIN4 (Liu et al., J. Virol., 88(4):2195-204 (2014)), CHO-CD38 (Peng et al. Blood, 101:2557-62 (2003), CHO-HER2/neu (Hasegawa et al., J. Virol., 81(23): 13149-57 (2007), Burkitt's B cell lymphoma Ramos (ATCC, Cat. #CRL-1596), and Raji cells (ATCC, Cat. #CCL-86) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Corning Inc., Cat. #10-040-CV, Coming, NY, United States) as described elsewhere.
Plasmids and Construction of Full-Genome rMeV
To generate CDV 22458/16 expression plasmids, total RNA was extracted from CDV 22458/16 isolate-infected Vero/dog SLAMF1 cells (passage 1) using RNeasy Mini Kit (Qiagen, Hilden, Germany). Both CDV-H and CDV-F genes were reverse transcribed with SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Cat.#11752050) and amplified by PCR with the following primers:
PCR products were sequenced directly by Sanger (Genewiz, Plainfield NJ, USA) and cloned into pJET1.2 vector (Thermo Fisher). CDV H open reading frame (
Expression plasmids for CDV H/F Onderstepoort vaccine and 5804 isolate (von Messling et al., J. Virol., 75(14):6418-27 (2001)), as well as the MeV Nse are described elsewhere (Cathomen et al., J. Virol., 72(2):1224-34 (1998)). Retargeted versions of the H protein were generated by inserting the homologous PacI/SfiI-digested PCR product into the pTNH6 vectors (Nakamura et al., Nat. Biotechnol., 23(2):209-14 (2005); and Nakamura et al., Nat. Biotechnol., 22(3):331-6 (2004)). Site-directed mutagenesis (QuickChange Site-Directed Mutagenesis Kit, Agilent Technologies, Santa Clara CA, USA) was used to ablate tropism in H as well as to remove a SpeI site in CDV-F and to introduce truncations in the cytoplasmic tails.
Envelope-exchange rMeVs were produced by shuttling the PacI/SpeI and NarI/PacI region of the corresponding expression plasmids. Rescue of rMeVs was carried out employing the START system (Nakamura et al., Nat. Biotechnol., 23(2):209-14 (2005)).
Cells were transfected using Fugene HD (PROMEGA, Fitchburg WI, USA) or TransIT-LT1 transfection reagent (Mirus Bio LLC, Madison WI, USA). For quantitative fusion assay, the dual-split reporter system (Kondo et al., J. Biol. Chem., 285(19):14681-8 (2010); and Ishikawa et al., Protein Eng. Des. Sel., 25(12):813-20 (2012)) was used as described elsewhere (Muñoz-Alia et al., Viruses, 11(8), pii: E688, doi: 0.3390/v11080688 (2019)), using BHK cells as effector cells. For semiquantitative assessment of fusion, Vero cells and derivates were transfected with 1 μg each of H and F expression plasmids and stained 1 day later with Hema-Quik (Thermo Fisher Scientific, Cat. #123-745). Images were obtained with a microscope (Eclipse Ti-S; Nikon) at 4× magnification. Alternatively, a GFP expression plasmid was included for additional visualization of syncytia formation. For assessment of the level of H polypeptide, transfected cells were analyzed by flow cytometry or CELISA using a 6×His tag monoclonal antibody (Miltenyi Biotec, Cat. #130-120-787 or Thermo Fisher Scientific, Cat. #MA1-135), as described elsewhere (Muñoz-Alia et al., Viruses, 11(8), pii: E688, doi: 10.3390/v11080688 (2019); and Saw et al., Methods, 90:68-75 (2015)). For total protein expression by flow cytometry, cells were treated with eBioscience Intracellular Fixation & Permeabilization buffer (Thermofisher, Cat. #88-8823-88).
Virus preparations were heated in the presence of dithiothreitol, fractionated into 4-12% Bis-Tris polyacrylamide gel, and transferred to polyvinylidene fluoride membranes. Blots were analyzed with anti-MeV-Hcyt (Cathomen et al., J. Virol., 72(2):1224-34 (1998)), anti-MeV-N(Toth et al., J. Virol., 83(2):961-8 (2009)), or anti-His tag (Genscript, Piscataway NJ, USA, Cat. #A01857-40) antibodies and probed with a conjugated secondary rabbit antibody (ThermoFisher, Cat. #31642). The blots were incubated with SuperSignal Wester Pico chemiluminescent substrate (ThermoFisher) and analyzed with a ChemiDoc Imaging Sytem (Bio-Rad).
A fluorescence focus reduction neutralization assay was used as described elsewhere (Munoz-Alia et al., J. Virol., 91(11): e00209-17 (2017)). The polyclonal Anti-Canine Distemper Virus, Lederle Avirulent (antiserum, Ferret), was obtained through BEI resources (NR-4025). The human sera used was pooled from 60 to 80 donors who were specifically blood type AB (Valley Biomedical Products & Services, Inc, Cat. #HS1017, Lot #C80553).
CDV envelope glycoproteins share a 36% (H polypeptide) and 66% (F polypeptide) amino acid homology with those of MeV. The open reading frame for the H and F polypeptides were obtained from a first-passage wild-type CDV isolate SPA.Madrid/22458/16 (CDV 22458/16) from post-mortem tissues from moribund dog (SPA.Madrid/22458/16). Maximum-likelihood phylogenetic analysis of fill-length hemagglutinin genes showed that CDV H polypeptide of 22458/16 grouped within the artic clade (
Using a reporter gene to more accurately identify syncytia formation, the above fusogenic phenotype was confirmed for the heterotypic wild-type combination CDV H5804 polypeptide and CDV F22458/16 polypeptide (
Since co-transfection of wild-type CDV H/F complexes, but not the Ondertepoort vaccine-derived H/F, resulted in syncytia formation in a specific receptor-dependent manner, the following was performed to determine whether receptor usage could be expanded to alternative receptors. As a target receptor, CD38 was chosen and for this purpose a CD38-specific scFv was displayed at the carboxy-terminal domain of the attachment protein (
The CDV H polypeptides described in the preceding paragraph could still use human Nectin-4 as a receptor. To disrupt a Nectin-4 interaction, nucleic acid encoding a CDV H polypeptide containing a Y539A mutation was produced.
To assess whether differences in the binding affinity of the ligand displayed to the ectodomain of the CDV H polypeptide influenced fusogenicity, Her2/neu-specific scFv binders as well as affibodies molecules (Hasegawa et al. J. Virol., 81(23): 13149-57 (2007); Wikman et al. Protein Eng. Des. Sel., 17(5):455-62 (2004); and Orlova et al. Cancer Res., 66(8):4339-48 (2006)) were displayed.
Different CD46-specific scFv binders were displayed on CDV H polypeptides in an attempt to obtain scFv-CDV H polypeptides supporting a similar fusion level to those of a MeV H Nse strain Cell surface expression levels were compared (
The neutralization sensitivity of Stealth 2.0 was studied using pooled serum from 20-30 American donors. As control, CDV antisera was used.
The following was performed to confirm the virus tropism endowed by the new envelope since virus-entry could occurred in the absence of evident fusion. Virus-derived GFP autofluorescence was observed when CHO cells expressed the receptors CD46, Nectin-4, and either canine or human SLAMF1 (
The following was performed to assess the use of Stealth 2.0 as an oncolytic agent. SCID mice bearing U266.B1 tumors were treated with a single intravenous dose of either MeV or Stealth 2.0 (
The following was performed to investigate the suitability of CDV H/F complexes to govern the tropism of VSVs, which are a member of the genus Lysasavirus, family Rhadoviridae. A VSV engineered to express interferon-beta (IFN-β) and the sodium iodide symporter (NIS), VSV-hIFNβ-NIS, (Naik et al. Mol. Cancer Ther., 17(1):316-326 (2018)) was obtained and modified by replacing the VSV-G polypeptide with CDV H and F polypeptides using the techniques described elsewhere (Ayala-Breton et al. Hum. Gene Ther., 23(5):484-91 (2012)). CDV F22458/16 polypeptide and either the parental CDV H5804 polypeptide (VSV-CDVFH-GFP) or the CDV H polypeptide retargeted against EGFR (VSV-CDVFHaal-αEGFR-GFP) or CD38 (VSV-CDVFHaal-αCD38-GFP) receptors were used (
To confirm that the new envelope complex governed virus tropism, a panel of CHO cells expressing specific receptors were infected. As shown in
The use of this system as an oncolytic vector also was assessed in vivo (
The following was performed to confirm that CD38 and EGFR targeting in the context of MeV can be achieved using CDV F and H polypeptides (
The in vivo oncolytic activity of CD38 and EGFR-targeted MeV were also investigated. SKOV3ip.1 tumors were implanted either subcutaneously or intraperitoneally into athymic nude mice, followed by virus treatment using the same route (
This example repeats some of the information and results from Example 1 in addition to providing additional results.
The MeV coat was replaced with an alternate viral coat that would enable the virus to evade neutralization by anti-measles antibodies. To do this, wild-type CDV was selected. While a strain of CDV approved for vaccine use exists (the Onderstepoort strain), this strain can use a currently unidentified receptor in addition to SLAMF1 and Nectin-4 (
The following was performed to identify the most fusogenic CDV H/F glycoprotein pair. Different H/F combinations from the 5804P and SPA.Madrid/16 (hereafter named 5804 and SPA, respectively) isolates were transiently expressed in Vero cells expressing either SLAMF1 or NECTIN4 and assessed qualitatively the degree of cellular fusion (syncitia formation) induced by the viral proteins. Fusion activity for the CDV-H/F pairs was not observed when the 135 aa-signal peptide was maintained in CDV-F (
Based on this set of experiments, the hyperfusogenic CDV-H 5804/F SPA pair was selected for further investigation and modification.
The Strength of the CDV H/F Interaction Inversely Correlates with the Cell-to-Cell Fusion Efficiency
The enhanced cell membrane fusion observed for the CDV-H 5804/F SPA pair might be related to a lower binding avidity at the H/F interface. This was based on the observation that H/F dissociation is essential for the fusion process (Plemper et al., J. Virol., 76(10):5051-61 (2002); and Bradel-Tretheway et al., J. Virol., 93(13) (2019)). In order to test the hypothesis, the relative strengths of association different combinations of CDV-H and F proteins were evaluated by coimmunoprecipitation (co-IP) assays. To facilitate detection, CDV-F SPA was fused to a FLAG-tag, which had no effect on the bioactivity of the protein (
The CDV-H proteins described above could still use NECTIN4 as a receptor (
Given that the CDV-H protein selected as described above could be efficiently retargeted to CD38 by fusing a CD38-specific scFv, this protein was next retargeted to CD46 via display of a CD46-specific scFv. It was postulated that the C-terminal display on CDV-H of an scFv that recognized CD46 with sufficiently high binding affinity would result in CD46-mediated cell-to-cell fusion activity similar to that induced by the MeV H/F complex. To test this, an anti-CD46 scFv with high affinity for CD46 was identified by assessing binding to purified CD46 of several different scFv variants isolated from a phage antibody display library (
The following was performed to determine whether fusion of a scFv to the CDV-H protein can support CD46-dependent fusion and if so, how CD46 binding affinity would affect cell fusion. The primary approach was to perform quantitative fusion assays for the detargeted CDV H [5804 (Y539)] and retargeted CDV-H [5804 (Y539)-scFv]/F SPA pairs and to compare them to the unmodified MeV/F complex on CHO cells and CHO cells expressing either NECTIN4 or CD46. All the proteins were expressed at comparable levels (
It was conclude from this set of experiments that there exists a binding affinity threshold for CD46-mediated cell-cell fusion through the retargeted CDV H/F complexes and above this threshold there is a positive correlation between binding affinity and intercellular fusion.
The CD46-Targeted CDV Envelope Glycoproteins are Efficiently Incorporated into MeV Virions and Mediate Virus Entry in Accordance with their Binding Affinity
The following was performed to investigate whether higher receptor affinity translated into higher virus infectivity. To begin to address this question, a panel of isogenic MeVs were generated where the MeV coat was replaced with CDV-F SPA together with CDV-H 5804 (Y539A) displaying low (K1), intermediate (N1E) and high (A09) affinity scFv specific for CD46 (
Based on its superior CD46-dependent virus entry, Stealth-A09 (this virus was referred to as Stealth 2.0 in Example 1) was selected for further characterization. Stealth-A09 replicated in Vero-αHIS cells but not in the parental Vero cell line, indicating efficient virus replication through the HIS-pseudo receptor and a lack of interaction with CD46 from African green monkey (
In keeping with a recent report showing that CDV-H does not bind human SLAMF1 (Fukuhara et al., Viruses, 11(8) (2019)), entry of MeV-Stealth-A09 into CHO-hSLAMF1 was not observed (
Collectively, these results indicate that the MeV H/F glycoproteins can be exchanged with the CD46-retargeted CDV-H/F glycoproteins and that cell entry is dependent on receptor affinity.
The following was performed to determine the antitumor potential of Stealth viruses and the role of CD46 binding affinity in vivo. For this, athymic mice bearing peritoneally disseminated SKOV3ip.1 tumors expressing the firefly luciferase gene (SKOV3ip.Fluc) were treated with a single intraperitoneal dose of saline or 106 TCID50 of Stealth-N1E and Stealth-A09 (n=5). Tumor burden was then monitored using in vivo bioluminescence imaging (
The following was performed to assess the utility of MeV-Stealth-A09 over MeV as an oncolytic agent. For this, we initially left untreated (PBS-treated group) or treated severe combined immunodeficiency (SCID) mice bearing subcutaneous human myeloma xenografts (derived from U266.B1 cells) with a suboptimal intravenous dose of MeV-Stealth or MeV. The tumors in the PBS-treated group continued to grow exponentially, and all mice had to be sacrificed because of the tumor burden by day 12 (
Next, the therapeutic effect of MeV-Stealth-A09 in prolonging survival in the presence of measles-immune serum was evaluated. Before embarking on this in vivo study, the neutralization sensitivity of recombinant viruses was first evaluated in vitro. The results, presented in
These results demonstrate that CD46 targeting drives oncolyis in both a xenograft myeloma model and an orthotropic model of ovarian cancer, and that exchange of the MeV coat by the homologous CDV-H/F fusion apparatus shields MeV from MeV-immune human serum.
Baby hamster kidney cells (BHK, Cat. #CCL-10, ATCC, Manassas, VA, USA), Human kidney epithelial cells (HEK293T) obtained from Dr. Franųcois-Loïc Cosset (Universitd de Lyon), and the human ovarian cancer cell line SKOV3ip.1-Fluc (Mader et al., Clin. Cancer Res., 15(23):7246-55 (2009)) were maintained in Dulbecco's modified Eagle's medium (DMEM; Cat. #SH30022.01, GE Healthcare Life, Pittsburgh, PA, USA) supplemented with 5% fetal bovine serum (FBS; Cat. #10437-028; Thermo Fisher Scientific, Waltham, MA, USA). Vero African green monkey kidney cells (Vero, ATCC, Cat. #CCL-81) and their derivatives (expressing human NECTIN-4 (Noyce et al., Virology, 436(1):210-20 (2013)), human SLAMF1 (Ono et al., J. Virol., 75(9):4399-401 (2001)) or a membrane-anchored single-chain variable fragment (scFv) specific for a hexahistidine peptide (6×HIS-tag (SEQ ID NO: 21)) (Nakamura et al., Nat. Biotechnol., 23(2):209-14 (2005)) were cultured in DMEM (Cat. #SH30022.01, GE Healthcare Life Sciences) as described elsewhere (Munoz-Alia et al., Viruses, 11(8) (2019)). Vero cells constitutively expressing the canine SLAMF1 molecule (Vero-dogSLAMF1) were generated by transduction and puromycin selection of a second-generation lentiviral vector (kindly provided by Dr. Lukkana Suksanpaisan [Imanis Life Science, Rochester, MN, USA]) encoding, under the control of the spleen focus-forming virus promoter, a codon-optimized SLAMF1 molecule from Canis lupus familiaris (GenBank NP_001003084.1) with an N-terminal FLAG-tag sequence (DYKDDDD (SEQ ID NO: 28)). Cells were maintained in DMEM supplemented with 5% FBS. The Chinese hamster ovary (CHO) cell line, CHO-CD46 cells, CHO-hSLAMF1 cells, CHO-dogSLAMF1 cells, CHO-NECTIN4 cells, CHO-αHIS cells, CHO-CD38 cells and the human myeloma cell line U266.B1 (kindly provided to us by Dr. David Dingli [Mayo Clinic, Rochester, MN]) were grown in RPMI 1640 medium supplemented with 10% FBS. Cells were incubated at 37° C. in 5% CO2 with saturating humidity.
To generate canine distemper virus (CDV) SPA.Madrid/22458/16 expression plasmids, total RNA was extracted from CDV SPA.Madrid/22458/16 isolate-infected Vero/dog SLAMF1 cells (passage 1) using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Both the CDV-hemagglutinin (H) and CDV-fusion (F) genes were reverse transcribed with SuperScript III Reverse Transcriptase (Cat. #11752050, Thermo Fisher Scientific) and amplified by PCR with the following primers: CDVH7050(+): 5′-AGAAAACTTAGGGCTCAGGTAGTCC;-3′ (SEQ ID NO: 22) CDVH8949(−): 5′-TCGTCTGTAAGGGATTTCTCACC-3′ (SEQ ID NO: 23); CDVF4857(+): 5′-AGGACATAGCAAGCCAACAGG-3′ (SEQ ID NO: 24) and CDVH7050(−): 5′-GGACTACCTGAGCCCTAAGTTTCT-3′ (SEQ ID NO: 25). PCR products were sequenced directly by Sanger sequencing (Genewiz, Plainfield NJ, USA) and cloned into the pJET1.2 vector (Thermo Fisher Scientific). Next, the CDV-H open reading frame was PCR amplified with a forward primer (5′-CCG GTA GTT AAT TAA AAC TTA GGG TGC AAG ATC ATC GAT AAT GCT CTC CTA CCA AGA TAA GGT G-3′ (SEQ ID NO: 26)) and a reverse primer (5′-CTA TTT CAC ACT AGT GGG TAT GCC TGA TGT CTG GGT GAC ATC ATG TGA TTG GTT CAC TAG CAG CCT CAA GGT TTT GAA CGG TTA CAG GAG-3′ (SEQ ID NO: 27)) and cloned into a PacI and SpeI-restricted (New England Biolabs, Ipswich, MA, USA) pCG vector (Cathomen et al., J. Virol., 72(2):1224-34 (1998)) using an InFusion HD kit (Takara, Shinagawa, Tokyo, Japan). The primers contained the PacI and SpeI restriction sites (underlined) as well as the coding sequence for the untranslated region of MeV-H (italics). Similarly, the CDV-F open reading frame (amino acid residues 136-662) was cloned into the HpaI/SpeI-restricted pCG-CDV-F plasmid (von Messling et al., J. Virol., 75(14):6418-27 (2001)). The resulting plasmid pCG-CDV-F SPA.Madrid/22458/16 contained coding sequences for the MeV-F untranslated region and signal peptide.
Expression plasmids for the CDV-H/F Onderstepoort vaccine and 5804P isolate (von Messling et al., J. Virol., 75(14):6418-27 (2001)), as well as MeV Nse strain, were described elsewhere (Cathomen et al., J. Virol., 72(2):1224-34 (1998)). The signal peptide for CDV-F 5804 was replaced with heterologous MeV-F as described above for CDV-F SPA.Madrid/22458/16. The open reading frames of the Nipah-G and Nipah-F glycoprotein genes were amplified from purchased RNA templates (Cat. #NR-37391, BEI Resources), and the Nipah-F gene (GenBank AF212302.2) was inserted into the pCG vector using the NarI and PacI sites. Retargeted versions of the H/G proteins were generated by inserting the homologous PacI/SfiI-digested PCR product into pCGHX α-CD38 (Peng et al., Blood, 101(7):2557-62 (2003)). Insertion of the coding sequence for an scFv recognizing CD46 was performed by exchanging the anti-CD38 scFv via the SfiI and NotI restriction sites. Site-directed mutagenesis (QuickChange Site-Directed Mutagenesis Kit, Agilent Technologies, Santa Clara CA, USA) was used to ablate the tropism of H and remove the SpeI site in CDV-F.
The viruses used in this example were derived from the molecular cDNA clone of the Moraten/Schwart vaccine strain pB(+)MVvac2(ATU)P, with an additional transcriptional unit downstream of the phosphoprotein gene (Cathomen et al., J. Virol., 72(2):1224-34 (1998); and Munoz-Alia et al., Viruses, 11(8) (2019)). To avoid plasmid instability upon propagation in bacteria and increase virus rescue efficiency, the plasmid backbone was replaced with the pSMART LCkan vector (Cat. #40821-1; Lucigen, Middleton, WI, USA), with an optimized T7 promoter followed by a self-cleaving hammerhead ribozyme (Hrbz) (Beaty et al., mSphere, 2(2) (2017); and Munoz-Alia et al., Viruses, 11(8) (2019)). eGFP or firefly luciferase were cloned into the infectious clone by using the unique MluI/AatII restriction sites. Rescue of rMeVs was carried out employing the START system (Nakamura et al., Nat. Biotechnol., 23(2):209-14 (2005)).
A plasmid encoding the CD46-Fc fusion protein was produced by fusing the CD46 ectodomain (residues 35-328) with the Fc domain of IgG1 (pfc1-hg1e3; InvivoGen, San Diego, CA, USA). The scFvs K1, K2, and A09 were designed with the VL and VH sequences separated by a GSSGGSSSG flexible linker (SEQ ID NO: 29), codon-optimized, synthesized and cloned into pUC57-Kan (GenScript). A fourth scFv (NIE) was designed with the VH and VL sequences separated by an SSGGGGS linker (SEQ ID NO: 30), codon-optimized, synthesized by Creative Biolabs (Shirley, NY) and cloned into pCDNA3.1+(Invitrogen). For the IgG constructs, scFvs were cloned into the unique AgeI and KpnI sites of pHL-FcHIS (Cat. #99846, Addgene, Cambridge, MA, USA), harboring the coding sequence for a secretion signal and a C-terminal human Fc region followed by a 6×HIS-tag (SEQ ID NO: 21). The recombinant proteins were expressed by transfecting Expi293F suspension cells (Thermo Fisher) in serum-free Expi293 expression medium (Thermo Fisher) in shaker flasks following the manufacturer's instructions. The culture supematants containing the recombinant proteins were collected and passed through a Protein G chromatography cartridge (Cat.#89926, ThermoFisher). Bound recombinant proteins were eluted with 0.1 M glycine (pH 2.0), followed by immediate neutralization with 1 M Tris (pH 8.0), and the isolated proteins were concentrated with an Amicon Ultra centrifugal concentrator (Millipore Sigma, Burlington, MA, USA). CD46 and NECTIN4 were released from the Fc region by incubation with HRV 3C Protease (Thermo Fisher) at a 1:200 ratio. A final purification step was performed using a Superdex 75 10/300 gel filtration column (GE Healthcare) equilibrated in phosphate-buffered saline (PBS). Protein concentrations were calculated from the protein extinction coefficient as determined from the amino acid composition.
Cells were transfected using Fugene HD (PROMEGA, Fitchburg WI, USA) or TransIT-LT1 transfection reagent (Mirus Bio LLC, Madison WI, USA). For a quantitative fusion assay, a dual-split reporter system (Kondo et al., J. Biol. Chem., 285(19):14681-8 (2010); and Ishikawa et al., Protein Eng. Des. Sel., 25(12):813-20 (2012)) was used as described elsewhere (Munoz-Alia et al., Viruses, 11(8) (2019)), using BHK cells as the effector cells. For semiquantitative assessment of fusion, Vero cells and derivative cell lines were transfected with a total of 0.1 μg of DNA (1:1 ratio of H and F expression plasmids), including a GFP expression plasmid for added visualization of syncitia formation. Images were obtained with a microscope (Eclipse Ti-S; Nikon) at 40×or 100× magnification.
For assessment of the level of the H polypeptide, transfected cells were analyzed by flow cytometry or cellular enzyme-linked immunosorbent assay (CELISA) using an anti-6×HIS-tag monoclonal antibody (Cat. #130-120-787, Miltenyi Biotec or Cat. #MA1-135, Thermo Fisher Scientific), as described elsewhere (Munoz-Alia et al., Viruses, 11(8) (2019); and Saw et al., Methods, 90:68-75 (2015)). For analysis of total protein expression by flow cytometry (FACSCanbt, BD Biosciences, San Jose, CA, USA), cells were treated with the eBioscience Intracellular Fixation & Permeabilization buffer (Cat. #88-8823-88, Thermo Fisher Scientific).
Three micrograms (1 μg of H and 2 μg of F) of total DNA were transfected into HEK293T cells (4e5 cells). After 24 hours, the cells were washed twice with PBS and treated with the cross-linker 3-3′-diothiobis (sulfosuccinimidyl propionate) (DTSSP; Cat. #21578, Thermo Fisher Scientific) at 1 mM, followed by quenching with 20 mM Tris/HCl (pH 7.4) and lysis with 0.4 mL of M-PER mammalian protein extraction reagent (Thermo Fisher Scientific) containing a 1× Halt protease and phosphatase inhibitor cocktail (Cat. #1861281, Thermo Fisher Scientific). Soluble fractions were collected after centrifugation at 10,000×g for 10 minutes at 4° C., and one-thirtieth of the volume was set aside as the cell lysate input. The rest was incubated with 0.5 μg of anti-FLAG monoclonal antibody M2 (Sigma-Aldrich) and EZview red protein G affmity gel (Sigma-Aldrich, St. Louis, MO, USA). The precipitated material was washed (20 mM Tris-HCl, pH 7.4, 140 mM sodium chloride) and denatured by boiling in laemmli buffer containing β-mercaptoethanol.
Samples were fractioned by gel electrophoresis on a 4 to 12% NuPAGE Bis-tris gel (Thermo Fisher) and transferred to polyvinylidene difluoride (PVDF) membranes using an iBLOT 2 dry blotting system (Cat. #IB21001, Thermo Fisher Scientific). The protein material was detected though incubation with the antibodies anti-MeV-H606 (Hudacek et al., Cancer Gene Therapy, 20(2):109-16 (2013)), anti-MeV-F431 (von Messling et al., J. Virol., 78(15):7894-903 (2004)), anti-Fcyt (von Messling et al., J. Virol., 75(14):6418-27 (2001)), anti-MeV-N(Toth et al., J. Virol., 83(2):961-8 (2009)), anti-HIS (Cat. #A01857-40, GenScript, Piscataway NJ, USA), anti-β-actin (Cat. #A3854, Sigma-Aldrich), and anti-CD46 (Cat. #sc-7056, Santa Cruz, Dallas TX, USA). Immunoblots were visualized using a rabbit horseradish peroxidase (HRP)-conjugated secondary antibody and KwikQuant Imager (Kindle Bioscience LLC, Greenwich CT, USA). Representative results of two independent repeats are shown. Band quantification was carried out using the KwikQuant Image Analyzer 1.4. (Cat. #D1016, Kindle Biosciences, LLC)
An enzyme-linked immunosorbent assay (ELISA) was used to measure the binding of scFvs to CD46. Nunc-Immuno MicroWell 96-well solid plates were coated overnight at 4° C. with 1 μg of purified CD46 or N4 in 0.05 M carbonate-bicarbonate buffer, pH 9.6. (Cat. #E107, Bethyl Laboratories, Montgomery TX, USA). Purified scFv-Fc fusion proteins were then diluted in PBS and added at a concentration of 12.5 μg/mL. Bound antibodies were detected with a secondary anti-human IgG (Fc-specific) HRP-conjugated antibody (1:70,000; Cat. #A0170, Sigma-Aldrich). In parallel, 125 ng of scFv-Fc fusion protein was first bound to wells, and protein levels were monitored by measuring the optical density (OD490 nm) after incubation with the secondary antibody alone.
The interaction between the scFv A09, 2B10, K1, K2, and CD46 was measured using series S CM5 sensor chips on a Biacore T-100 system (GE Healthcare, Waukesha, WI, USA). For A09, N1E, K1, and K2, 50 μg/mL of an anti-FC antibody (MAB1302, EMD Millipore, Burlington, MA, USA) diluted into 10 mM NaAcetate, pH 4.5, were immobilized to the active and the reference channel of the CM5 chip using amine coupling kit reagents (EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide), NHS (N-hydroxysuccinimide) and ethanolamine). The immobilization of the antibody resulted in ˜12000 response units. The interactions between CD46 and the anti-FC antibody captured scFv were measured at 25° C. with a data rate of 10 Hz using HBS EP buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20). Each binding cycle began with the loading of 15 μg/mL of the scFv onto the active channel for 300 s at a flow rate of 10 μL/min. After 100 s of buffer wash and a 120 s stabilization period, CD46 (concentration range 50 nM-1000 nM for A09, 37.5 nM-100 nM for K2 and 50-1000 nM for N1E and K1) was flown over the active and the reference channel for 100 s at a flow rate of 40 μL/min. The association phase was followed by a 200 s dissociation period followed by a 60 s injection of 10 mM Glycine pH 2.0 at a flow rate of 30 μL/min, to regenerate the surface immobilized anti-FC antibody. All sensograms were fitted with a 1:1 binding model using the Biacore T100 evaluation software v2.04.
For virus infections, cells were infected at the indicated MOIs for 90 min at 37° C. in Opti-MEM I reduced-serum medium. After the absorption phase, we removed the inoculum, washed, and added viral growth media (DMEM+5% FBS). When using eGFP-expressing viruses, fluorescence microscopy photographs were taken 48 hours post-infection. For infections with Fluc-expressing viruses, luciferase expression was measured using an Infinite M200 Pro multimode microplate reader (Tecan Trading AG) after adding 0.5 mM of D-Luciferin to the infected cells.
For virus growth kinetic analysis, Vero cells and derivative cell lines seeded in 6-well plates 16-18 hours prior to infection were infected at a multiplicity of infection (MOI) of 0.03 for 90 min in Opti-MEM (Cat. #31985070, Thermo Fisher Scientific). The inoculum was then removed, and the cell monolayers were washed three times with Dulbecco's phosphate-buffered saline (DPBS; Cat. #MT-21-031-CVRF, Mediatech, Inc., Manassas, VA, USA), and the medium was replaced with 1 mL of DMEM supplemented with 5% FBS. At the indicated time points, cell supernatants were collected, and cells were scraped into 1 mL of Opti-MEM, followed by 3 freeze/thaw cycles. Cell debris was removed by centrifugation (2,000×g for 5 min), and virus titers were determined in Vero-αHIS cells.
Four to six-week-old male and female HuCD46Ge-IFNARKO mice (Mrkic et al., J. Virol., 74(3):1364-72 (2000)), deficient for type I IFN receptor and transgenically expressing human CD46, were inoculated intraperitoneally (i.p.) with 1×105 TCID50 particles of MeV or Stealth-A09. On day 28, serum samples were collected and stored at −20° C. until assessed for neutralizing antibodies.
A fluorescence-based plaque reduction microneutralization (PRMN) assay was carried out as described elsewhere (Munoz-Alia et al., J. Virol., 91(11) (2017)). Briefly, Vero-αHIS cells were seeded in a 96-well plate, and serial dilutions of serum samples were premixed for 1 hour at 37° C. with virus inoculum before they were added to the cells. The data were plotted as the log(dilution of serum) vs. the normalized response (variable slope) present with GraphPad software (Prism 8), and the neutralization dose 50% was calculated (ND50). Inclusion of the 3rd World Health Organization International serum standard (3IU/mL) enabled conversion of antibody titers to mIU/mL by calculation of the unitage constant (Haralambieva et al., Vaccine, 29(27):4485-91 (2011)). Pooled human serum from 60-80 donors that had blood type AB (Cat. #HS1017; Lot #C80553, Valley Biomedical Inc., Winchester, VA, USA) was used. The following reagents were obtained from the NIH Defense and Emerging Infections Research Resources Repository, NIAID, NIH: polyclonal anti-MeV antibody, Edmonston, (antiserum, Guinea Pig), NR-4024 and polyclonal anti-CDV Lederle Avirulent (antiserum, Ferret), NR-4025.
A lack of cross-neutralization between measles virus and Stealth was assessed (
To establish subcutaneous tumors, 6-week-old female severe combined immunodeficiency (SCID) mice were injected in the right flank with 1×107 U266.B1 tumor cells. When the tumor reached 0.5 cm in diameter, mice received a single intravenous dose of MeV (n=5) or Stealth (n=5) at 1×10 5 50% tissue culture infectious dose (TCID50). Control mice (n=5) were injected with an equal volume of PBS. Animals were euthanized when the tumors ulcerated or when the burden reached 20% of the body weight. Tumor diameter was measured every other day, and tumor volume was calculated with the formula length×length×width×0.5.
To establish an orthotopic model of ovarian cancer, 5×106 SKOV3ip.1 cells expressing firefly luciferase (SKOV3ip.1-Fluc) were injected into the peritoneal cavity of athymic nude mice. Ten days later, the animals received 600 mIU of measles-immune serum (Cat. #HS1017; Lot #C80553, Valley Biomedical Inc.) or an equal volume of saline, and three hours later, they were treated with a single intraperitoneal dose (1×106 TCID50) of MeV (n=5) or Stealth (n=5). The mice in the control group received a similar volume of Vero cell lysates (n=5). For the therapy experiment, 5×106 SKOV3ip.1-Fluc cells were implanted instead. The tumor burden was monitored weekly through in vivo bioluminescence imaging using an IVIS Spectrum instrument (Perking Elmer, Waltham, MA, USA). The mice were euthanized at the end of the study (80 days), when they developed ascites or had lost 20% of their body weight. Statistical comparisons among groups were performed with the log-rank (Mantel-Cox) test, and p<0.05 was considered statistically significant.
Statistical analyses were performed with GraphPad Prism 8.3.1 version for Mac OS X. Significant differences among groups were determined using one-way analysis of variance (ANOVA) with Holm-Sidak's multiple comparison test Survival data were analyzed using the Kaplan-Meier method, and the log-rank test was used to identify significant differences among groups.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application is a National Stage application under 35 U.S.C. § 371 of International Application No. PCT/US2020/055100, having an International Filing Date of Oct. 9, 2020, which claims the benefit of U.S. Provisional Application Ser. No. 62/913,111, filed Oct. 9, 2019. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/055100 | 10/9/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62913111 | Oct 2019 | US |
