TW201346034A - Protecting modified viruses from neutralizing antibodies using the reovirus sigma 1 protein - Google Patents

Abstract

Provided herein is a modified, non-reovirus virus comprising a reovirus sigma 1 protein. Methods of making a modified, non-reovirus virus comprising a reovirus sigma 1 protein and methods of using same are also provided.

Description

Protection of modified viruses from neutralizing antibodies using the Rio virus Sigma-1 protein

The name Leo virus is derived from the head of the respiratory enteric orphan virus, reflecting the initial isolate from the human respiratory and intestinal tract but not associated with serious disease. The Leo virus has a viral cell attachment protein called the sigma 1 protein, which is encoded by the S1 gene and is responsible for binding the Leo virus to specific receptors on the cell surface.

Provided herein is a modified non-League virus comprising a Leovirus σ1 protein, wherein the Leovirus σ1 protein replaces a naturally-attached protein of the Feria virus, and wherein the modified virus does not comprise a naturally-attached protein of the Feria virus Any part of it.

Further provided is a method for preparing a non-Leovirus protected against a neutralizing antibody, comprising replacing a naturally-attached protein of a non-Leovirus with a Leovirus σ1 protein, wherein the full-length sequence of the naturally-attached protein of the Non-Ley virus is Leo virus σ1 protein replacement.

Also provided is a method of treating a cellular proliferative disorder in a mammal by administering to the individual having the proliferative disorder an effective amount of the modified non-Le lives virus. Modified non-Leovirus promotes increase Cells of a septic disorder (such as cells of a tumor) are substantially dissolved.

【Detailed description】

Provided herein is a modified non-League virus comprising a Leovirus σ1 protein, wherein the Leovirus σ1 protein replaces a naturally-attached protein of the Feria virus, and wherein the modified virus does not comprise a naturally-attached protein of the Feria virus Any part of it.

In the modified Feria virus disclosed herein, the Leovirus σ1 protein is attached to a vector cell that protects the virus from neutralizing antibodies during delivery in vivo. In the modified Feria virus disclosed herein, the Leovirus σ1 protein is attached to a vector cell that protects the virus from neutralizing antibodies during delivery to the tumor in vivo, such as during systemic delivery. The Feria virus can be, but is not limited to, an adenovirus, a poxvirus, a paramyxovirus, a herpes simplex virus, or a parapoxvirus. The modified non-Le lives virus can be an oncolytic virus.

Further provided is a method for preparing a non-Leovirus protected against a neutralizing antibody, comprising replacing a naturally-attached protein of a non-Leovirus with a Leovirus σ1 protein, wherein the full-length sequence of the naturally-attached protein of the Non-Ley virus is Leo virus σ1 protein replacement. In preparation In the method of porcine virus, the natural attachment of the virus with the ribovirus σ1 protein allows the virus to attach to the vector cells that protect the virus from neutralizing antibodies during delivery in vivo.

As used herein, the term sigma-1 protein refers to a polypeptide encoded by the S1 genomic segment of the Leo virus. The term Leovirus refers to any type or strain of the Leo virus. Thus, the sigma-1 protein can be from any strain or type of Leovirus. The term Leovirus refers to any virus classified as a genus of the genus Corona, whether it is a naturally occurring, modified or recombinant virus. The human Leu virus includes three serotypes: type 1 (strain Lang or T1L), type 2 (strain Jones, T2J), and type 3 (strain Deearing or strain Abney, T3D). The three serotypes can be readily identified based on neutralization and hemagglutinin inhibition assays. The Leo virus according to the disclosure may be a type 3 mammal, the Rio virus. Type 3 mammals are including, but not limited to, Dearing and Abney strains (T3D or T3A, respectively). See, for example, ATCC Registry Numbers VR-232 and VR-824. The Leo virus can be a naturally occurring or modified virus. A bacterium is naturally occurring when it can be isolated from sources in nature and has not been intentionally modified by humans in the laboratory. For example, the Leo virus can be from a field source, ie from a human that has been infected with the Leo virus. Thus, the sigma-1 protein is optionally derived from a naturally occurring modified or recombinant Rio virus. Depending on the case, the sigma-1 protein is derived from serotype 1, 2 or 3 Leovirus. Depending on the case, the sigma-1 protein is derived from the serotype 3 Leo virus. Depending on the case, the sigma-1 protein is from the Dearing strain or the Abney strain Leo virus. Depending on the case, the sigma-1 protein is derived from the Dearing strain Leo virus.

Sigma-1 polypeptides include, but are not limited to, SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 and are available under GenBank Accession Nos. M10262.1, JQ599138, and ACV52070.1. As discussed in more detail below, a polypeptide can include one or more modifications. The sigma-1 polypeptide is modified to have increased infectivity, as appropriate. See, for example, SEQ ID NO: 3, which is available under GenBank Accession No. JQ599138 (Shmulevitz et al, J. Virol. 86(13): 7403-13 (2012)).

It will be appreciated that nucleic acids encoding their peptide, polypeptide or protein sequences, variants and fragments thereof are also disclosed. This will include all degenerate sequences associated with a particular polypeptide sequence, i.e., all nucleic acids having sequences encoding a particular polypeptide sequence, as well as all nucleic acids encoding the disclosed variants and derivatives of the polypeptide sequences, including degenerate nucleic acids. Thus, although specific nucleic acid sequences may not be described herein, it is to be understood that each and every sequence is actually disclosed and described herein via the disclosed polypeptide sequences. Furthermore, nucleic acids encoding sigma-1 proteins include, but are not limited to, SEQ ID NO: 4 and SEQ ID NO: 5 and SEQ ID NO: 6, and are available under GenBank accession numbers GQ468272.1, M10262.1, and JQ599138. .

With respect to all peptides, polypeptides and proteins, including fragments thereof, it will be appreciated that other modifications may be present in the amino acid sequence of the agent provided as a polypeptide that do not alter the nature or function of the peptide, polypeptide or protein. Such modifications include, for example, conservative amino acid substitutions and are discussed in more detail below. Thus, the provided agent comprising the polypeptide or nucleic acid can be further modified and varied as long as the desired function is maintained. It will be appreciated that a manner of defining any of the known modifications and derivatives or possible occurrences of the nucleic acid sequences and proteins disclosed herein is accomplished by defining modifications and derivatives in accordance with the identity with particular known sequences. It is expressly disclosed that the polypeptides provided herein have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity polypeptides. Those skilled in the art will readily understand how to determine the identity of two polypeptides. For example, consistency can be calculated after aligning the two sequences to bring the consistency to its highest level.

Another way to calculate consistency can be performed by the disclosed algorithm. The optimal alignment for the sequences used for comparison can be partially consistent by Smith and Waterman, Adv. Appl. Math 2:482 (1981). Sexual algorithm, by Needleman and Wunsch, J. Mol Biol. 48: 443 (1970) Consistency Alignment Algorithm, by Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988) Sex search method, computerized execution program by such algorithm (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software suite (575 Science Dr., Madison, WI)), or by inspection .

For example, by Zuker, Science 244: 48-52 (1989); Jaeger et al, Proc. Natl. Acad. Sci. USA 86: 7706-7710 (1989); Jaeger et al, Methods Enzymol. 183: 281-306 The algorithm disclosed in (1989) obtains the same type of identity of nucleic acids, which are incorporated herein by reference for at least the reference to the related materials. It should be understood that any method is generally available and in some cases the results of such methods may vary, but the skilled artisan will appreciate that if consistency is obtained using at least one of these methods, the sequence will be referred to as The consistency is described and disclosed herein.

Protein modifications include amino acid sequence modifications. Modifications in the amino acid sequence may be naturally occurring in a dual gene variant (eg, due to genetic polymorphism), may be due to environmental influences (eg, exposure to ultraviolet light), or may be intervened by humans (eg, by breeding DNA) Sequence mutations are induced, such as induced point mutants, deletion mutants, insertion mutants, and substitution mutants. Such modifications may result in changes in the amino acid sequence, provide silent mutations, modify restriction sites, or provide other specific mutations. Amino acid sequence modifications typically fall into one or more of the following three categories: substitution modifications, insertion modifications or deletion modifications. Inserts include intra-sequence insertions of amine and/or terminal fusions as well as single or multiple amino acid residues. Insertions will typically be insertions that are less than the insertion of the amine or carboxy terminal fusion, for example from about one to four residues. The deletion is characterized by the removal of one or more amino acid residues from the protein sequence. Usually, no more than about 2 to 6 residues are deleted at any position within the protein molecule. base. Amino acid substitutions are typically single residue substitutions, but can occur at many different positions at once; insertions will typically be from about 1 to 10 amino acid residues; and deletions will range from about 1 to 30 residues. Inside. Deletions or insertions are preferably carried out in an adjacent pairwise manner, i.e., 2 residues are deleted or 2 residues are inserted. Substitutions, deletions, insertions, or any combination thereof can be combined to obtain the final construct. The mutation must not leave the sequence outside of the reading frame and preferably will not create a complementary region that will produce a secondary mRNA structure. A substitution modification is a modification in which at least one residue has been removed and a different residue is inserted into its position. Such substitutions are generally made according to Table 1 below and are referred to as conservative substitutions.

Modifications are made in the nucleic acid of the virus using a number of methods known in the art. For example, site-directed mutagenesis can be used to modify nucleic acid sequences. One of the most common site-directed mutagenesis induction methods is induced by oligonucleotide directed mutagenesis. In the induction of oligonucleotide-directed mutagenesis, an oligonucleotide encoding a change in the desired sequence is affixed to a strand of the relevant DNA and used as a primer for initiation of DNA synthesis. In this way, oligonucleotides containing sequence changes are incorporated into the newly synthesized strand. See, for example, Kunkel, 1985, Proc. Natl. Acad. Sci. USA, 82: 488; Kunkel et al, 1987, Meth. Enzymol., 154: 367; Lewis and Thompson, 1990, Nucl. Acids Res., 18: 3439 Bohnsack, 1996, Meth. Mol. Biol., 57:1; Deng and Nickoloff, 1992, Anal. Biochem., 200:81; and Shimada, 1996, Meth. Mol. Biol., 57: 157. Other methods are routinely used in the art to introduce modifications into the sequence. For example, a modified nucleic acid can be produced using PCR or chemical synthesis, or a polypeptide having a desired amino acid sequence change can be chemically synthesized. See, for example, Bang and Kent, 2005, Proc. Natl. Acad. Sci. USA, 102: 5014-9 and references therein. Selection of cell types (eg, human cells) to which the virus does not normally grow and/or chemical mutation induction (see, eg, Rudd and Lemay, 2005, J. Gen. Virology, 86: 1489-97) can also be used in viruses. A modification is produced in the nucleic acid.

The non-Ley virus used in the provided method includes, but is not limited to, a virus belonging to the following families: myoviridae, siphoviridae, podoviridae, capsid virus Family (tectiviridae), the family of the genus Corticoviridae, the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus Phycodnaviridae, baculoviridae, herpesviridae, adenoviridae, papovaviridae, polyoxyribidididae (polydnaviridae), inoviridae, microviridae, geminiviridae, circoviridae, parvoviridae, hepatnaviridae , retroviridae, cystoviridae, birnaviridae, paramyxoviridae, bullet Virology family (rhabdoviridae), fibroviridae, filoviridae, Orthomyxoviridae, bunyaviridae, arenaviridae, leviviridae, picaraviridae, sequiviridae, Cowpea mosaic virus family (comoviridae), potato Y virus family (potyviridae), caliciviridae (caliciviridae), astroviridae, nodaviridae, tetraviridae, tomato cluster Dwarf virus family (tombusviridae), coronaviridae, flaviviridae, togaviridae, and birnaviridae. These and other viral immunoprotective viruses and reconstituted or recombinant non-Leoviruses are also covered by the methods provided. Several non-Leoviruses are discussed below, and one of ordinary skill in the art can use other non-Leo viruses to implement the methods of the present invention in light of the disclosure herein and the knowledge available in the art.

The non-Leovirus is a virus selected from the group consisting of adenovirus, acne virus, paramyxovirus, herpes simplex virus or parapoxvirus, as the case may be. Depending on the situation, the non-Ley virus contains only one modification, ie the naturally-attached protein of the non-Leovirus is replaced by the Leovirus σ-1 protein. As used herein, the term natural refers to the source of the molecule. As used herein, the term naturally-attached protein refers to a protein that specifically binds to one or more molecules, such as a receptor, on a cell. Attaching proteins helps the virus enter the cell. The attached protein is a capsid protein, as appropriate. Some non-Leoviruses exhibit more than one attached protein. As provided herein, the sigma-1 protein can replace one, two, three, four, and similar or all naturally-attached proteins represented by the Feria virus. Depending on the situation, the sigma-1 protein replaces all naturally attached proteins of the Feria virus.

Thus, promoters for controlling the expression of sigma-1 proteins can be obtained from a variety of sources. The Leovirus σ-1 protein can be expressed from a naturally attached protein promoter or from a sigma-1 protein promoter. Depending on the situation In addition, the promoter is obtained from a genome such as a virus (such as polyoma virus, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, and cytomegalovirus), or from a heterologous mammalian promoter ( For example, the beta actin promoter is obtained. In the Feria virus, the sigma-1 protein can be expressed from its natural promoter (i.e., the sigma-1 promoter) or from a promoter of a naturally attached protein. The entire naturally attached protein is replaced by a sigma-1 protein, as appropriate. Optionally, the entire naturally attached protein is replaced by the sigma-1 protein and its promoter. The promoter of the naturally attached protein remains intact, as appropriate.

For example, the non-League virus can be an adenovirus that attaches to a cellular target via an adenoviral fiber protein. Thus, depending on the situation, the sigma-1 protein replaces the entire adenoviral fiber protein. Depending on the situation, the non-Leo virus is a acne virus. Naturally attached proteins of the acne virus include, but are not limited to, A27L and H3L proteins. As described herein, the Leovirus sigma-1 protein can replace one or all of these attachment proteins of the acne virus. At least one at least one naturally attached protein is replaced by a sigma-1 protein. As the case may be, as discussed above, the Leovirus σ-1 protein is expressed from its natural promoter. The non-Leovirus is a paramyxovirus, such as Newcastle disease virus, as appropriate. Naturally attached proteins of paramyxovirus include, but are not limited to, hemagglutinin-neuraminase (HN), hemagglutinin glycoprotein (H), glycoprotein (G), and fusion (F) proteins. As described herein, the Leovirus σ-1 protein can replace one or all of such attachment proteins of the paramyxovirus.

The Non-Ley virus contains one or more other modifications, as appropriate. For example, an adenovirus can be a mutant adenovirus carrying a 24 base pair deletion in the E1A region (Fueyo, J., et al., Oncogene 19(1): 2-12 (2000)), such as the δ24 virus. . Adenovirus is mutated in the VAI or VAII region, as appropriate. Optionally, the non-Le lives virus is a delta 24 virus or an adenovirus mutated in the VAI or VAII region, wherein the naturally attached protein (i.e., the adenoviral fiber protein) is replaced with the Leovirus σ-1 protein. Optionally, the acne virus contains one or more other modifications selected from the group consisting of: K3L and / Or modifications in the E3L region, modifications in the thymidine kinase (TK) gene, modifications in the vaccinia growth factor (VGF) gene, and combinations thereof. Optionally, the non-League virus is a herpes simplex virus comprising one or more other modifications including, but not limited to, mutations in the gamma 134.5 gene. Depending on the situation, the non-Leovirus is a parapoxvirus. As the case may be, the parapoxvirus is a parapoxvirus orf virus that is mutated in the OV20.0L gene.

The non-Leovirus providing the sigma-1 protein comprising the naturally-attached protein of the replacement of the non-Ley virus can be prepared using methods known to those skilled in the art. For example, non-Li can be prepared as described in many standard laboratory manuals, such as Sambrook et al., Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012). Austrian virus. The nucleic acid sequence of the sigma-1 protein may comprise the mature polypeptide coding sequence alone, or the mature polypeptide coding sequence in frame with other coding sequences, such as those encoding the leader or secretion sequence. Nucleic acid sequences may also contain non-coding 5' and 3' sequences, such as non-translated sequences of transcription, splicing and polyadenylation signals, ribosome binding sites, and sequences that stabilize mRNA.

Also provided is a method of treating a cell proliferative disorder in a mammal comprising administering to a subject having a proliferative disorder an effective amount of a virus comprising riovirus σ1 under conditions which substantially cause lysis of the cell causing the proliferative disorder Modified oncolytic non-Leovirus.

As used herein, the term proliferative disorder refers to any cellular disorder in which cell proliferation is faster than normal tissue growth. Proliferative disorders include, but are not limited to, neoplasms, which are also known as tumors. Tumors can include, but are not limited to, pancreatic cancer, breast cancer, brain cancer (eg, glioblastoma), lung cancer, prostate cancer, colorectal cancer, thyroid cancer, kidney cancer, adrenal cancer, liver cancer, neurofibromatosis 1 and leukemia. A tumor can be a solid tumor (such as a sarcoma or cancer) or affect the cancerous growth of the hematopoietic system. Other proliferative disorders include, but are not limited to, neurofibromatosis.

The modified non-Le lives virus described herein can be included in a pharmaceutical composition along with a pharmaceutically acceptable carrier. Such pharmaceutical compositions may include, for example, one or more chemotherapeutic agents and/or one or more immunosuppressive agents. Representative routes of administration include, for example, direct injection, intravenous, intravascular, intrathecal, intramuscular, subcutaneous, intraperitoneal, topical, oral, rectal, transvaginal, nasal or by inhalation. Methods of treating a proliferative disorder as described herein can be followed by one or more of the following procedures, such as surgery, chemotherapy, radiation therapy, and immunosuppressive therapy.

A virus having a modified sequence as disclosed herein is administered in an amount sufficient to treat a proliferative disorder (e.g., "effective amount"). When one or more symptoms or clinical signs of a virus-affected condition with a modified sequence are administered to a proliferating cell as compared to a sign or symptom in the absence of treatment, including, for example, increased cell lysis (eg, "oncolytic") The proliferative disorder can be "treated" by reducing the number of proliferating cells, reducing the size or progression of the tumor, and alleviating the pain associated with the tumor. As used herein, the term "oncolytic" means that at least 10% of proliferating cells are solubilized (eg, at least 20%, 30%, 40%, 50%, or 75% of the cells are solubilized). The percentage of dissolution can be determined, for example, by measuring a decrease in the size of the tumor or the number of proliferating cells in the mammal, or by measuring the amount of dissolution of the cells in vitro (e.g., by biopsy of proliferating cells).

An effective amount of a virus having a modified sequence is determined on an individual basis and based, at least in part, on the particular virus used; the size, age, sex of the individual; and the size and other characteristics of the proliferating cells. For example, for treating humans, about 10 ³ to 10 ¹² plaque forming units (PFU) of virus are used, depending on the type, size and number of proliferating cells or tumors present. An effective amount can range from about 1.0 pfu/kg body weight to about 10 ¹⁵ pfu/kg body weight (e.g., from about 10 ² pfu/kg body weight to about 10 ¹³ pfu/kg body weight). The virus is administered in a single dose or in multiple doses (eg, two, three, four, six or more doses). Multiple doses are administered simultaneously or sequentially (e.g., over a period of days or weeks). Treatment with a virus having a modified sequence lasts for several days to several months or until disease is attenuated.

As used herein, a pharmaceutically acceptable carrier includes a solid, semi-solid or liquid material that acts as a vehicle, carrier or medium for the modified virus. Thus, for example, a composition comprising a virus having a modified sequence is in the form of a troche, a pill, a powder, an ingot, a sachet, a cachet, an elixir, a suspension, an emulsion, a solution, a syrup, an aerosol. (in solid form or in a liquid medium), ointments containing, for example, up to 10% by weight of active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

Some examples of suitable carriers include phosphate buffered saline or another physiologically acceptable buffer, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, Alginate, tragacanth, gelatin, calcium citrate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The pharmaceutical compositions may additionally include, but are not limited to, lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl hydroxybenzoate and propyl hydroxybenzoate; Sweeteners; and flavoring agents. The pharmaceutical compositions of the present invention can be formulated to provide rapid, sustained or delayed release of a virus having a modified sequence following administration by a procedure known in the art. Other formulations suitable for use in pharmaceutical compositions are also found in Remington: The Science and Practice of Pharmacy (2003, Gennaro and Gennaro ed., Lippincott Williams and Wilkens), in addition to the representative formulations described below.

For the preparation of solid compositions such as troches, the virus having the modified sequence is mixed with a pharmaceutical carrier to form a solid composition. The lozenge or pill is optionally coated or otherwise compounded to provide a dosage form that provides the advantage of prolonged action. For example, tablets or pills contain internals A dose component and an external dose component, the latter being in the form of a capsule on top of the former. The two components are separated, for example, by an enteric layer that is resistant to disintegration in the stomach and allows the internal components to pass intact into the duodenum or delayed release. A variety of materials are used in such enteric layers or coatings, including mixtures of various polymeric acids and polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

Liquid formulations for oral administration or for injection comprising a virus having a modified sequence typically include an aqueous solution, a suitable flavored syrup, an aqueous or oily suspension, and an edible oil such as corn oil, cottonseed oil, Flavored sesame oil, coconut oil or peanut oil) as well as tinctures and similar pharmaceutical agents.

Compositions for inhalation or insufflation include solutions and suspensions, and powders, in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof. These liquid or solid compositions optionally contain suitable pharmaceutically acceptable excipients as described herein. Such compositions achieve local or systemic effects, for example, by oral or nasal respiratory routes. The composition in a pharmaceutically acceptable solvent is atomized by using an inert gas. The nebulized solution is for example inhaled directly from a spray device, from a self-attached mask or from an intermittent positive pressure ventilator. The solution, suspension or powder composition is administered orally or nasally, e.g., from a device that delivers the formulation in an appropriate manner.

Another formulation for use in the methods taught herein employs a transdermal delivery device ("patch"). These transdermal patches are used for continuous or discontinuous infusion of a Leo virus having a modified sequence. The construction and use of a transdermal patch for delivering a pharmaceutical agent is carried out according to methods known in the art. See, for example, U.S. Patent No. 5,023,252. These patches are constructed for continuous, pulsatile or on-demand delivery of a Leo virus with a modified sequence.

A virus having a modified sequence is optionally subjected to chemical or biochemical pretreatment prior to administration (e.g., prior to incorporation into a pharmaceutical composition) (e.g., by using, for example, chymotrypsin) Or trypsin protease treatment). Pretreatment with a protease removes the coat or capsid of the virus and can be used to increase the infectivity of the virus. Alternatively or additionally, a virus having a modified sequence is coated in a liposome or micelle to reduce or prevent an immune response in a mammal that has developed immunity to the virus. These viruses are called "immunoprotective viruses." See, for example, U.S. Patent Nos. 6,565,831 and 7,014,847.

A virus having a modified sequence or a pharmaceutical composition comprising the virus can be packaged into a kit. The kit is expected to include one or more chemotherapeutic agents and/or anti-antiviral antibodies, as appropriate. The pharmaceutical compositions are formulated, for example, in unit dosage form. The term "unit dosage form" refers to a physical individual unit suitable as a single dosage for use in human subjects and other mammals, each unit containing a predetermined amount of a virus having a modified sequence calculated to produce the desired therapeutic effect, and a pharmaceutically acceptable Accept the suitable carrier.

Details of one or more aspects of the modified non-Ley virus or related methods are set forth in the accompanying drawings and the following description. Other features, objects, and advantages of the virus and methods will be apparent from the drawings and detailed description and claims. Therefore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety.

Although oncolytic viruses represent a novel cytotoxic and immunogenic approach to the treatment of cancer, how to circumvent the antiviral immune response and its transmission to tumors beyond their selectivity to normal tissues has not been studied in humans. In the present study, patients were treated with a single cycle of intravenous ribavirin followed by planned surgery to remove colorectal cancer metastases from the liver. Tracking The viral genome in the loop showed that after the infusion, the Leo virus was detected in both plasma and blood mononuclear cells and platelet cell compartments. Although there are baseline levels of neutralizing antibodies in all patients, the function is to transfer to kill the target cells in vitro. The competent Rio virus can be recovered from blood cells rather than plasma, indicating that cell transport protects the virus for potential delivery. To the tumor. Analysis of the surgical specimens indicates that the Leovirus protein has a stronger selective performance in malignant cells than the tumor stroma or surrounding normal liver tissue. There is evidence that the viral factory is in the tumor and that the replication virus is recovered from the tumor (rather than the liver) in all four patients who have access to fresh tissue. Thus, despite the presence of neutralizing antibodies in the circulation, protective cell delivery is capable of selectively delivering the rib virus to the tumor after systemic administration. These findings provide novel preclinical and clinical pathways for enhancing in vivo immune avoidance and effective intravenous delivery of oncolytic viruses to patients in vivo.

Naturally occurring or genetically modified oncolytic viruses (OV) specifically target tumor cells for replication and cell death. In addition to its direct cytotoxic effects, OV can also stimulate therapeutic anti-tumor immune responses. Many OVs have now been promoted by preclinical and early clinical testing and do not indicate encouraging signs of greater toxicity and anti-tumor activity. Phase III studies of herpes simplex virus (OncoVex) have been completed in melanoma, and randomized trials using poxvirus (JX-594) to treat hepatocellular carcinoma have begun. The optimal route of administration for OV clinical applications remains unresolved. Direct intratumoral injection ensures that the virus effectively enters the tumor microenvironment to achieve immune activation and directly kills the cells, and bypasses the intravenous virus. In the circulation, the neutralizing antibody (NAB) is induced by the presence of baseline and/or when the drug is repeatedly administered. Concerns about activation. However, intratumoral injection is technically challenging and limits application to accessible tumor sites; in addition, systemic delivery is still more acceptable to clinicians. Direct injection of OncoVex into tumor deposits, while JX-594 has been used both by intratumoral and systemic approaches. Line pass. A recent report of intravenous JX-594 confirmed the successful delivery of the virus to the tumor, but in this phase I single infusion study, most patients were negative for anti-vaccinia NAB at the time of treatment.

Leovirus is a ubiquitous, non-genetically modified, non-pathogenic dsRNA virus that has anticancer activity mediated by both malignant cells that are directly targeted to the ras pathway and stimulates anti-tumor immunity. Clinical grade rio virus (type 3 Dearing; Reolysin®) has been tested intratumorally or intravenously, either alone or in combination with radiation therapy or chemotherapy, through phase I/II trials and is currently in phase III configuration with carboplatin ( Carboplatin) and paclitaxel combination were tested intravenously in head and neck squamous cell carcinoma. However, how the virus is transported from the blood to the tumor in the patient after intravenous injection and how this delivery can be improved has not been explored in humans.

This paper discloses a clinical study in which a single cycle of intravenous ribavirin monotherapy is administered to a patient, followed by a planned resection of colorectal cancer that has metastasized to the liver. Detailed analysis of continuous blood samples and excised tissue allows for the characterization of: i) the distribution, transport and replication of the Leo virus in different blood compartments; ii) the comparison of the Ley virus close to the tumor cells and the tumor stroma compared to normal liver; And iii) Comparison of the virus in the tumor with the replication in the normal liver to elucidate how the virus can evade antiviral immunity for selective delivery to the patient's tumor.

Patient, research procedure and sample collection

The study was discussed in a single center with patients with colorectal cancer who had metastases to the liver and who were scheduled to undergo radical resection and undergo routine planned resection. Written informed consent was obtained based on local institutional ethics and review approval. Clinical grade 3 Dearing Reolysin® was provided by Oncolytics Biotech (Calgary, Canada), but additional trials were initiated, operated and funded by the University of Leeds, UK. Eligibility criteria included ages 18 to 75 years; at least 4 weeks prior to entry into the study, any prior therapy was completed (2 out of 10 patients had neoadjuvant chemotherapy before surgery to reduce the burden of disease); Eastern Oncology Cooperative Group ( Eastern Cooperative Oncology Group, ECOG) Physical Status Score 2; life expectancy of at least 3 months; absolute neutrophil count 1.5×109/L; platelets 100×109/L; heme 9.0mg/dL; serum creatinine 1.5 times system normal limit (ULN), total bilirubin 1.5 times ULN; aspartate transaminase / alanine transaminase 2.5 times ULN; women with fertility were tested negative for pregnancy. Exclusion criteria include extensive liver disease requiring more extensive surgery than expanded hepatectomy; known brain metastases; known HIV, type B or C hepatitis infections; pregnancy or lactation; clinical major heart disease (New York Heart Association (New York) Heart Association) Category III or IV); dementia or mental state changes that would block informed consent. The study was approved by the appropriate Ethics and Biosafety Committee (EUDRACT No. 2007/000258-29; MREC 08/H1306/73). Prepare a single cycle of 1 x 10 ¹⁰ TCID ₅₀ Leo virus as described in Vidal et al. ( Clin. Cancer Res. 14:7127-7137 (2008)) and from day 1 to day 5, before surgery 6 Between day and 28 days, daily administration was given as an intravenous infusion over 60 minutes. Immediately before the first rio virus infusion and 1 hour after the first rio virus infusion; immediately before the infusion on days 3 and 5; 3 times before surgery and 1 month after surgery and 3 Get blood samples for months. The tumor and adjacent normal liver parenchyma were removed, the margin was preserved for histological diagnosis and confirmed to be adequately removed at the time of surgery.

Blood sample processing

By using Lymphoprep ^TM according to the manufacturer's instructions (Axis-Shield UK, Dundee, UK) density gradient centrifugation of anti-coagulated whole blood from the K3EDTA isolated PBMC. PBMC were frozen in a freezing medium (90% fetal calf serum (FCS; Biosera, Ringmer, UK) containing 10% DMSO (Sigma-Aldrich Co., Irvine, UK) at a cell density of 5 x 10 ⁶ /ml. Granulocytes were isolated from the same K3EDTA anti-coagulated whole blood using a 2-step procedure. Briefly, granulocytes were collected from the lower white blood cell interface after PBMC collection. Then according to the manufacturer's instructions, using Polymorphprep ^TM (Axis-Shield) was further purified granulocytes. The purified granulocytes were frozen in a freezing medium at a cell density of 1 × 10 ⁷ /ml. Platelets and red blood cells were isolated from K3EDTA anti-coagulated whole blood collected in a 6 ml vacuum blood collection tube (Greiner Bio-One Co., Ltd., Stonehouse, UK). The vacuum blood collection tube was centrifuged at 210 g for 10 minutes without interruption and the platelet-rich plasma (PRP) top layer was collected (retaining red blood cells for treatment alone), followed by further centrifugation at 210 g for 10 minutes to remove any contaminating white blood cells. The PRP was then centrifuged at 800 g for 10 minutes to collect platelets. The platelets were then washed twice in 5 ml of MACS buffer (no Ca ²⁺ Mg ²⁺ PBS (Oxoid, Basingstoke, UK) + 1% FCS + 0.2% EDTA (Sigma)), each of which was centrifuged at 800 g for 10 minutes. . Platelets were frozen in 1 ml of RNase-free water (Sigma). The red blood cells retained in the vacuum blood collection tube were centrifuged at 2000 g for 10 minutes. The upper white blood cell layer was removed and an aliquot of red blood cells was then collected and frozen. Whole blood was collected from serum in a serum clot activator vacuum blood collection tube (Greiner) and plasma was separated from whole blood collected in a K3EDTA vacuum blood collection tube. The vacuum blood collection tube was spun at 2000 g for 10 minutes, then serum and plasma were collected from the upper interface and frozen aliquots were taken. All samples were stored at -80 °C until needed.

Cell line and rio virus

Dube supplemented with 5% (v/v) FCS, 1% (v/v) glutamic acid (Sigma) and 0.5% (v/v) penicillin/streptomycin (5% DMEM) (Sigma) Murine fibroblast cell line L929 was cultured in Dulbecco's Modified Eagle's Medium (DMEM; Sigma). The cells were cultured at 37 ° C in a moisture-containing atmosphere containing 5% CO ₂ and routinely tested for mycoplasma infection and found to be negative for mycoplasma infection. Clinical grade 3 Dearings virus (Reolysin®) was titrated using a standard plaque assay protocol and stored in the dark at -80 °C for laboratory experiments.

TCID ₅₀ experiment

L929 was inoculated in a 96-well dish at 1×10 ⁴ and incubated for 24 hours, followed by an experiment. The supernatant was aspirated and a 10-fold dilution (starting at 1:10) of 100 μL of PBMC, granulocytes, platelets and plasma samples was added to L929 cells. Additional plating medium was added to each well 1-2 hours after infection/treatment and the cells were then incubated for 6 days. A 1:10 dilution of the reserve virus and 5% DMEM were cultured on L929 cells as positive and negative controls, respectively. CPE (i.e., cell virus induced cell death) was evaluated by examination under a light microscope to calculate the virus titer at TCID ₅₀ /ml (using Spearman-Karber statistic). Photomicrographs were also taken. L929 cell survival/cell death was also confirmed at the end of the analysis using the MTT assay described below.

MTT analysis

Cell viability was quantified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) analysis. 20 μL of 5 mg/ml MTT per well was added to the treated cells. After incubation at 37 ° C for 4 hours, the crystals were dissolved in DMSO and the absorbance was measured at 550 nm.

RNA detection

RNA was extracted from PBMC, granulocyte, platelet and plasma samples using the QIAamp virus mini-set and amplified using the OneStep RT-PCR kit (both from Qiagen, West Sussex, UK). The Ley virus σ3 cDNA targeting primer (Sigma) used was: forward 5'-GGGCTGCACATTACCACTGA (SEQ ID NO: 1) and reverse 5'-CTCCTCGCAATACAACTCGT (SEQ ID NO: 2) and the detection limit of 35 cycles was used. to evaluate. Samples were run on a 2% agarose gel and analyzed by the presence of a 300 bp PCR product. Includes positive (Leo virus RNA) and negative (RN-free water) controls. For virus detection after amplification (to assess whether functional replication is competent for the presence of the virus in the sample, But only at low levels), 1:10 sample dilutions (with positive and negative controls) were incubated on L929 cells as described above for TCID50 analysis, followed by collection of cells and supernatants and testing as described Ou virus RNA.

Neutralizing anti-Ley virus antibody detection

The modified antibody neutralizing assay as described in White et al. ( Gene Ther. 15: 911-920 (2008)) was used to detect antibody antibody titers in the samples.

Immunohistochemistry and fluorescence of Leovirus, caspase and tubulin

Immunohistochemical analysis was performed as described in the manufacturer's instructions using a Benchmark LT Automated Slide Stainer (Ventana Medical Systems, Tucson, AZ) as described in Comins et al. ( Clin. Cancer Res. 2010). Casparin-3 and tubulin antibodies were purchased from Abeam (Cambridge, MA) and the virus antibody was supplied by Oncolytics. The optimal dilution for detecting antibodies is 1:6000 (Rio virus); 1:50 (Casparin-3) and 1:100 (tubulin). All antigen acquisitions were performed for 30 minutes. Antigens were detected by contrast staining with hematoxylin using the Ultraview Universal DAB or Fast Red system (Ventana). Negative controls include internal controls that omit primary antibodies and cells/tissues that are known to be negative for the target. Using a Nuance microscope/computer based interface system (Cambridge Research Instrumentation, Hopkinton, MA), such as Nuovo et al. ( Nat. Protoc. 4: 107-115 (2009)) and Nuovo ( Methods , 52: 307-315 ( Co-expression analysis as described in 2010)) to explain co-localization signals. Briefly, the Nuance system parses the chromophore signals of different chromophores and converts these chromophore signals into fluorescent-based signals. This allows for a "fluorescence blend" combination to determine if a given cell has zero, one, or two or more signals.

Electron microscopy

Tissue samples were fixed in 3% glutaraldehyde (Sigma) for at least 4 hours and stored in 70% ethanol until further processing. The sample is then dehydrated by and incubated for 1 hour at -20 ℃ step embedded in LRWhiteTM (Sigma) 2 and ethanol 1: 1 mixture, followed by 1 hour incubation in three separate LRWhite ^TM at -20 ℃. Finally, the polymerization step (in gelatin capsules) was carried out at 37 ° C for 5 days. The 80-100 nm section was then cut and placed on a nickel grid. After blocking in PBS/2% bovine serum albumin (BSA), sections were incubated with 25 μl of a 1:800 dilution of anti-Ley virus Sigma 3 antibody (Developmental Studies Hybridoma Bank, Iowa, US) for 1 hour, followed by 1 hour. The cells were incubated with 25 μl of rabbit anti-mouse IgG antibody (Dako Cytomation, Stockport, UK) diluted 1:40 for 1 hour. Finally, 25 μl of protein-A immunogold particles (10 nm; Aurion, Wageningen, Netherlands) diluted 1:20 were incubated for 1 hour, followed by final staining with uranyl acetate for 20 minutes. Samples were observed using a Phillips/FEI CM10 transmission electron microscope operating at 80 kV. The image was captured on Kodak electronic imaging film (SO-163 type) using an exposure time of 2 seconds.

Self-organization to obtain replication competent virus

Tissue samples were cut into 5 mm cubes and subsequently dissociated into single cell suspensions using a cell dissociation sieve and a tissue grinder set (Sigma). The cells were then passed through a 70 [mu]m cell strainer (BD Biosciences, Oxford, UK) and any debris was removed by washing twice in PBS. A single cell suspension was then added to the semi-confluent L929 cells for 24-48 hours, then removed and replaced with 5% DMEM. After another 5-7 days of culture, the supernatant was collected and the Leovirus replication was determined by standard plaque assay using L929 cells. Briefly, samples (purified or diluted in serum-free medium) were added to L929 cells and incubated for 4 hours at 37 °C. The sample was gently removed, followed by the addition of a DMEM overlay medium supplemented with 5% (v/v) FCS and 1.6% (w/v) carboxymethylcellulose (Sigma) to each well. After 72 hours, the supernatant was collected (and stored for Western blotting) Analysis was performed), and then the cells were gently washed 3 times with PBS and fixed with 0.1% (v/v) glutaraldehyde (Sigma) for 10 minutes. The plaques were observed using 1% methylene blue (Sigma) and images were acquired using a Canon Ixus 100 digital camera.

Western ink dot

Samples were prepared in 2 x Laemmli buffer (collected from plaque assay as described above) and denatured at 95 °C for 5 minutes. Proteins were separated by electrophoresis on a 10% SDS polyacrylamide gel and transferred to a HybondTM C Super nitrocellulose membrane (Amersham Bio Sciences, Little Chalfont, Buckinghamshire, UK), followed by Odyssey blocking buffer at 4 °C. Block overnight (Li-Cor® Biosciences UK Ltd., Cambridge, UK). Membranes were probed sequentially with anti-Leovirus σ3 antibody (1:200 dilution), secondary IgG Alexa-Fluor 680 (1:5000 dilution; Invitrogen, Paisley, UK). The nitrocellulose membrane was observed at 700 nm on a Li-Cor® Odyssey infrared imager and analyzed using the Odyssey application software (v1.2). The presence of the Leo virus in the sample was confirmed by a bright band at 41 KDa.

Statistical Analysis

The p- value was calculated using two-way ANOVA and statistical significance was indicated by *p<0.05.

result Patient, study design and toxicity

Ten patients were recruited to this transformational biological endpoint clinical trial. All patients are scheduled to undergo resection of colorectal cancer liver metastases as part of standard clinical care for curative purposes. The clinical features of the patients are shown in Table 1 and are illustrated in Figure 1 for a trial of a single cycle of intravenous ribovirus prior to planned surgery. Treatment with the Leo virus is well tolerated, with the most common side effects being flu-like symptoms, consistent with previous clinical experience. No grade 3 or 4 toxicity And 1/2 grade toxicity included nausea (1 patient), constipation (1 patient), headache (1 patient), fever (6 patients), myalgia (3 patients), stiffness (1 patient), Leukopenia (1 patient), insomnia (1 patient), hallucination (1 patient), and hypotension (1 patient) (Table 1). Of the 3 patients, less than the planned 5 doses of the Leo virus were given. In one patient, a single infusion was omitted due to clinical concerns about a decrease in white blood cell count, and in the other two cases, only one and three treatments were given, respectively, because the patient himself was concerned that flu-like symptoms might interfere with planned surgery. This causes it to reject subsequent infusions. Surgery was performed between 6 and 28 days after the last rio virus treatment. This timing is determined by clinical factors (specifically, the availability of the guard bed); the surgical delay is by no means due to the toxicity of the Leo virus. The endpoints of this trial were: i) tracking the progression of NAB response to the Ley virus after treatment; ii) detecting different blood compartments (plasma, peripheral blood mononuclear cells (PBMC), granulocytes, platelets, and red blood cells) Viral; iii) assessment of tumor and normal liver virus; and iv) monitoring of toxicity, specifically the toxicity associated with planned surgery. Despite the presence of neutralizing antibodies, the Leo virus genome can be detected in plasma rather than replicating competent viruses after systemic delivery. It was first confirmed that anti-Leovirus NAB was present in patients at baseline (Figure 2A), and consistent with other intravenous monotherapy trials, it was found that after intravenous treatment, titers increased, with peaks roughly at the time of surgery (Figure 2B). ). The following assay was used to test the presence of the virus initially in plasma. First, RT-PCR of RNA ("pure" RT-PCR) was performed directly to find the viral genome. Next, the acquisition of replication competent virus was evaluated by adding plasma to Leo virus-sensitive L929 cells in the TCID50 assay. This quantifies viral titer by demonstrating the cytopathic effect (CPE) and cytotoxicity of serial dilutions of the sample against L929 cells. In addition, RNA extracted from L929 cells was subjected to "amplification" RT-PCR at the end of this TCID50 analysis to confirm an increase in the intensity of the bright band of the genome consistent with viral replication. A clear bright band was observed by plasma RT-PCR 1 hour after the first infusion, At the same time, in all patients except 2 patients, the Ley virus genome was not detected at a later time point (Fig. 2C). In patients 3 and 4, bright bands were also observed on days 3 and 5 (i.e., samples taken immediately before the third and fifth infusions, respectively). However, when plasma was tested with L929 cells in a virus amplification assay, no replication of the virus was observed, as evidenced by CPE/cytotoxic or positive amplification RT-PCR signals. Therefore, the free virus in the circulation can be easily detected in plasma, specifically detected early in the treatment period, but the productive infection and cell killing are presumed to exist at baseline before the first rio virus infusion. NAB was functionally neutralized.

Peripheral blood mononuclear cells carry the Leo virus with replication and target killing function

PBMC were isolated from patient blood samples and tested for input (purified) and amplified (replicated) viruses. Figure 3A shows that according to RT-PCR, Clarion virus can be detected in PBMC 1 hour after the first infusion, as evidenced by a weak band that can be detected only in some patients. However, significantly different from plasma, this signal was significantly amplified in all patients after 7 days of culture of PBMC on L929 cells, consistent with viral replication to target L929 cells in vitro and replication in target L929 cells. This amplified signal was also detected at a later time point in Patient 3 (Day 5; Day 3 sample not available) and Patient 4 (Day 3 and Day 5); in all other patients, all later The time points are all negative. At the end of the 7-day PBMC/L929 co-culture, replicated cytotoxic virus was confirmed on the patient's PBMC by CPE of L929 cells (Fig. 3B) and L929 killing as measured by MTT assay (Fig. 3C). Finally, the viral titer (TCID ₅₀ /ml) against PBMC was calculated as shown in Figure 3D. Interestingly, patients 3 and 4 were the only cases in which replication was detected in the amplification assay 1 hour after the first infusion time (Fig. 3B, C, D), as evidenced by pure PCR. The same patient with positive plasma and a later time point (Figure 2B). In summary, these data show that despite the baseline NAB (Figure 2A), the PBMC in the patient carries the virus after intravenous delivery. Significantly different from plasma, a functionally replicating PBMC-associated virus can be delivered in vitro to a target L929 cell, thus providing a protective delivery mechanism that allows the rio virus to reach the tumor systemically in a patient.

The granulocytes and platelets in the patient, but not the red blood cells, also carry the Leo virus. The same analysis as described for PBMC was used to study the ability of other parts of the blood cells collected from the patient to carry the Leo virus. In particular, granulocytes, platelets, and red blood cells are tested because blood components may be able to bind and/or carry viruses (granulocytes and platelets are only available from patients 7 to 10). Figures 4 and 5 show that in three of the four patients, similar to PBMC, both granulocytes and platelets carry oncolytic functional viruses, such as pure and amplified RTPCR by L929 cells (Fig. 4/5A) Confirmed by analysis of CPE (Fig. 4/5B) and target killing (Fig. 4/5C). Viral titers are shown in Figure 4/5D, which also shows that the rio virus was detected only in granulocytes and platelets 1 hour after the first infusion (in all cases, at a later point in time, For granulocytes and platelets, the corresponding pure and amplified RT-PCR analysis was also negative). Furthermore, in any sample, at any point in time, the Leo virus was not detected in red blood cells, but immediate RT-PCR was the only technically feasible analysis for these samples. Thus, despite the presence of NAB, in patients, granulocytes and platelets as well as PBMC (but not red blood cells) can potentially carry the rib virus to tumor targets.

Systemic Leuvirus is preferentially delivered to tumor cells

The Leo virus was first examined by immunohistochemistry of the σ3 capsid protein to remove the tumor and surrounding normal liver (as a marginal resection of the metastasis). In nine of the 10 patients (but not in the 3 tested control patients who had not been resected), the Leo virus protein was detected. Tumor staining scores were either non-existent (1 patient), weak (3 patients) or strong (6 patients). Figure 6A shows representative data from one patient with weak tumor staining and one patient with strong tumor staining. In swollen In all 9 cases in which the tumor was positive for the virus, colorectal metastasis was stained more strongly than the tumor stroma (in Figure 6A, the black arrow is opposite the red arrow) or the surrounding liver, which is transmitted to the malignant cell than to the malignant cell. Non-malignant cells, and/or replication in malignant cells are consistent with greater selectivity for replication in non-malignant cells. A certain degree of staining of normal liver tissue was observed. In 5 patients, weak positive staining of hepatocytes was observed (in which there was a strong tumor staining in 4 patients and weak tumor staining in 1 patient), while liver tissue samples were scored in the other 5 cases. Negative (including a single case where the tumor is negative). Figure 6B shows a representative example of weak hepatocyte staining and a negative case. The presence of the Ley virus protein in the excised tumor was further confirmed by electron microscopy (Fig. 6C). In addition, in four patients with sufficient tissue for analysis, the Leovirus was co-localized with Casparin 3 (Fig. 6D), which is consistent with tumor cell apoptosis (5% vs. 30%) Virus-positive cells were co-stained with Casparase, whereas in a single case without the apparent Leu virus, no Casperase was observed. Changes in nuclear and cytoplasmic degradation in infected tumor cells (marked with arrows in Figure 6D) are also consistent with apoptosis of lymphoid-associated cells in Casparin 3 positive cells. Therefore, in all 10 cases except one case, systemically administered Leovirus was observed in the excised tissue, in which tumor cell staining was always stronger than tumor-associated stroma or excised adjacent normal liver.

Copying the competent Leo virus can be obtained by resecting the tumor instead of the liver tissue

Detection of the Leovirus capsid protein indicates that the virus was successfully delivered to the target tissue, but it did not clarify whether the virus is or has replication function. To further elucidate the problem of specific replication of the Ley virus in tumors, sections were stained to co-localize the Leovirus and tubulin as an indirect marker for replication in the virus factory. Co-localization was observed in 4 of the 6 evaluable tumors (i.e., having sufficient tissue available). In these cases, 15%, 30%, 40%, and 40% of the virus showed tumor Cells were also counted as tubulin positive, with co-staining confined to tumors as opposed to stromal cells (a representative patient is shown in Figure 7A). Next, an attempt was made to obtain the copy-winning Leo virus directly from the excised sample. Initially, no CPE or viral plaques were observed when the frozen/thawed lysate from the tumor and liver was pulsed onto L929 cells. However, for patients 7 to 10, a single liver or tumor cell suspension is designed to obtain tissue by direct access from the operating room, and the suspension is treated directly as described herein to increase virus detection sensitivity. experiment. This improved technique avoids any viral loss during the preparation of the lysate (due to freezing and thawing and/or retention of the virus in the aggregated cell debris) and also utilizes the Leo virus to deliver the target to the target from the intact infected cell compared to the free virus. Enhanced. Under these conditions, fresh tumor (but not liver) cells from all four test patients did produce virus, as evidenced by plaques on L929 cells as shown in Figure 7B. According to Western blots, Figure 7C confirms that these plaques are Rio virus and the liver sample is negative again. Thus, after intravenous delivery, replication of the competent Leo virus can be obtained from the patient's tumor rather than the liver.

Surgical outcome

All 10 patients continued their planned surgery, with no delay attributable to the Leo virus treatment, and no unexpected or excessive surgical complications (eg, blood loss, discharge time) compared to non-test patients undergoing similar procedures ).

Figure 1 illustrates a clinical trial diagram.

Figure 2 shows that the neutralizing antibody (NAB) is present at baseline and increases after treatment, while the viral genome can only be transiently detected in plasma after infusion. (A) The baseline NAB content of the samples collected from each patient prior to the first viral infusion was tested. The curve shows neutralization of serial dilutions of L929 cells induced by Leovirus, as measured by MTT assay performed at 72 hours. L929 cells were treated with either Rio virus (+rio virus) or not (UN) as positive and negative controls, respectively, and anti-Ley virus multiple goat antibodies (Ab3054) were used at an initial dilution of 1:1000. A standard curve for analysis. (B) NAB titer of end-stage virus in each patient over time (NA indicates that the sample is not available for analysis). (C) Evaluation of plasma virus RNA of the patient by RT-PCR over time. Including Leo virus RNA or RNase-free water as positive and negative controls, respectively.

Figure 3 shows that despite the presence of circulating NAB, PBMC transiently carries the Leo virus after infusion, which can replicate in the target cells in vitro and kill the target cells. (A) The patient's PBMC's Leovirus RNA ("Pure") was assessed directly by RTPCR on the first day after infusion or by the RT-PCR after 7 days of re-amplification with L929 cells. "Augmentation"). Including Leu virus RNA and RNase-free water as positive and negative controls, respectively, and the Leovirus 1:10 dilution or 5% DMEM cultured on L929 cells as amplification positive and negative control (AMP), respectively. The later time points of patients 3 and 4 are also shown. (B) patient PBMC assessment of functional analysis of reovirus TCID _50, in which L929 cells and incubated with serial dilutions of PBMC 7 days and observing CPE. The micrographs show the patient results from all day PBMCs after infusion and the patient results at later time points of patients 3 and 4 for all samples. The Leovirus 1:10 dilution or 5% DMEM (UN) was incubated on L929 cells as positive and negative controls, respectively. Rounded cells and unused (red) medium represent CPE. (C) The virus-induced cell killing of the PBMC samples on day 1 was further confirmed by MTT assay in the TCID ₅₀ assay. * indicates statistical significance of p < 0.05 relative to untreated controls. (D) Virus titer over time in each patient's PBMC sample, expressed as TCID ₅₀ /ml, as determined by Spearman-Karber statistics. (NA indicates that the sample is not available for analysis).

Figure 4 shows that granulocytes similarly carry the replication competent Leo virus after infusion. (A) Using the pure sample and the amplified sample for the PBMC in Fig. 3A, the ribosomal RNA of the granulocytes from the patient 1 to 10 after the infusion was evaluated by RT-PCR. (B) Functional Rio virus from granulocytes from patients 7 to 10 was evaluated in the TCID ₅₀ assay for PBMC in Figure 3B. Micrographs show granulocyte dilution on day 1 after infusion; rounded cells and unused (red) medium indicate CPE. (C) Leovirus-induced cell killing of granulocytes from patients 7 to 10 was further confirmed by MTT assay in the TCID ₅₀ assay. * indicates statistical significance of p < 0.05 relative to untreated controls. (D) Viral titers over time from granulocytes of patients 7 to 10, expressed as TCID 50/ml, as determined by Spearman-Karber statistics. (NA indicates that the sample is not available for analysis). ).

Figure 5 shows that the platelets also carry the Leo virus after the infusion. (A) Platelet virulence RNA from day 7 after infusion of patients 7 to 10 was assessed by RT-PCR using both purified and amplified samples for the PBMC in Figure 3A. (B) Functional Rio virus from platelets 7 to 10 of patients was evaluated in the TCID ₅₀ analysis for PBMC in Figure 3B. Micrographs show platelet dilution on day 1 after infusion; rounded cells and unused (red) medium indicate CPE. (C) Leovirus-induced cell killing of platelets from patients 7 to 10 was further confirmed by MTT assay in the TCID ₅₀ assay. * indicates statistical significance of p < 0.05 relative to untreated controls. (D) Viral titers over time from platelets 7 to 10 of patients, expressed as TCID _50/ ml, as determined by Spearman-Karber statistics. (NA indicates that the sample is not available for analysis).

Figure 6 shows metastatic colorectal tumor cells selectively delivered to the liver by intravenous ribovirus. (A) Immunohistochemical image showing the performance of the Ley virus protein (red staining) in the resected colorectal liver metastasis (magnification 400 times). A representative case showing weak (left) and strong (right) staining indicates that the signal is always strong in malignant cells (black arrows) relative to the tumor stroma (red arrow). (B) Immunohistochemical image (magnification 400 times) of the performance of the Ley virus protein (red staining) in normal liver resected at the margins around the colorectal liver metastasis. A representative case of each of the weak (left) and negative (right) stains is shown. (C) Representative electron microscopy images showing immunogold staining of the Ley virus σ3 capsid (marked by the black dot of the arrow) in the colorectal liver metastasis. The scale bar represents 500 nm. (D) RGB image analysis of the resected colorectal liver metastases (magnification 400 times) using the Nuance system. Images from a representative patient and showed Leu virus staining (red) and Casparin-3 staining (brown) (left image; arrows indicate changes in nuclear and cytoplasmic degradation in tumor cells infected with Ley virus) . The image on the right shows RGB images converted to: Fluorescent Green (Casparin-3), Fluorescent Red (Leo Virus), and Yellow (total performance).

Figure 7 shows that the replication competent Leo virus can be obtained from tumor tissue. (A) RGB image analysis of the resected colorectal liver metastases (magnification 200 times) using the Nuance system. Images from a representative patient and showed: (top left) Leo virus staining (red) and tubulin staining (brown) (malignant cells are marked by black arrows and tumor stroma is marked by red arrow); (top right) RGB Image converted to fluorescent red (Leo virus); (bottom left) RGB image converted to fluorescent green (tubulin) and (bottom right) Leo virus and tubulin co-expression (yellow) (B) from patient Fresh from 7 to 10 Single cell suspensions of tumor and liver tissue were excised and incubated with L929 cells for 24-48 hours, then removed and replaced with 5% DMEM. Supernatants were collected after another 5-7 days of culture and the Leovirus replication was assessed using standard plaque assay. Photographs of plaques from tumor samples show representative wells for each of the following dilutions: 1: 2500 supernatant dilution (patient 7), pure supernatant (patients 8 and 9), and 1:1200 dilution (patient 10) . Photographs of liver samples show representative negative wells from pure supernatants from all patients. Separate stocks of Leovirus 1 x 10-6 dilution or 5% DMEM were included as positive and negative controls, respectively. (C) Leovirus σ3 protein of the culture supernatant from the tumor plaque assay performed in (B) was evaluated by Western blots. The presence of the Leo virus in all four samples was confirmed by a bright band at 41 KDa.

Claims (12)

A modified non-Le lives virus comprising a Leovirus σ1 protein, wherein the Leo virus σ1 protein replaces the naturally-attached protein of the Non-Ley virus, and wherein the modified virus does not comprise the naturally-attached protein of the non-Leovirus Any part of it. The non-Ley virus of claim 1, wherein the Leovirus σ1 protein is attached to a carrier cell that protects the virus from neutralizing antibodies during delivery in vivo. The non-Ley virus of claim 2, wherein the Leovirus σ1 protein is attached to a carrier cell that protects the virus from neutralizing antibodies during delivery to a tumor in vivo. The non-Leovirus according to any one of claims 1 to 3, wherein the virus is an adenovirus, a poxvirus, a herpes simplex virus, a paramyxovirus or a parapoxvirus. The method of any one of claims 1 to 4 wherein the non-Leovirus is an oncolytic virus. A method for preparing a non-Leovirus protected against a neutralizing antibody, comprising replacing the naturally-attached protein of the non-Leovirus with a Leovirus σ1 protein, wherein the full-length sequence of the naturally-attached protein of the non-Leovirus Aovirus σ1 protein replacement. The method of claim 6, wherein the replacement of the naturally-attached protein of the non-Ley virus with the Leovirus σ1 protein allows the non-riovirus to adhere to a carrier cell that protects the virus from neutralizing antibodies during delivery in vivo. . A method of treating a cell proliferative disorder in a mammal comprising administering to the individual an effective amount of a virus according to item 5 of the scope of the patent application under conditions which result in substantial dissolution of the cells of the proliferative disorder. The method of claim 8, wherein the cell proliferative disorder is a tumor. The method of any one of clauses 1 to 8, wherein the sigma-1 protein is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. The method of any one of clauses 1 to 10 wherein the sigma-1 protein is under the control of its natural promoter. The method of any one of clauses 1 to 10, wherein the sigma-1 protein is under the control of a promoter of the naturally occurring protein.