HK1251017B - Construction of oncolytic herpes simplex viruses (ohsv) obligate vector and constructs for cancer therapy - Google Patents

Description

Oncolytic Herpes Simplex Virus (oHSV)-Specific Vectors and Their Construction for Cancer Therapy

Technical Field

The present invention generally relates to the use of oncolytic herpes simplex viruses (oHSV) to treat cancer. Specifically, the present invention relates to the preparation of obligate HSV vectors that can carry and express multiple genes encoding immune co-stimulatory molecules and/or immunotherapeutic agents. The present invention also relates to an innovatively designed genome that can serve as a vector for carrying and expressing multiple therapeutic genes for treating cancer.

Background Art

Oncolytic herpes simplex viruses (oHSVs) are being extensively investigated for the treatment of solid tumors. Oncolytic herpes simplex viruses offer numerous advantages over conventional cancer therapies (Markert et al., 2000; Russell et al., 2012; Shen and Nemunaitis, 2006). Specifically, oHSVs often contain mutations that render them susceptible to innate immune suppression. Consequently, they are able to replicate in cancer cells, but not in normal cells, where one or more innate immune responses to infection are impaired. These responses are intact in normal cells. oHSVs are typically delivered directly into the tumor mass, where they replicate. Because the virus is delivered to the target tissue rather than administered systemically, this anticancer agent is immune to side effects. A characteristic of viruses is their ability to induce adaptive immune responses, which limits their ability to be administered multiple times. oHSVs have been administered to tumors multiple times without evidence of loss of efficacy or induction of adverse reactions (such as inflammation). HSVs are large DNA viruses that can incorporate foreign DNA into their genomes and regulate the expression of these genes after administration to tumors. Suitable foreign genes for use with oHSV are those that can help induce an adaptive immune response against tumors.

Overcoming deficiencies in the cellular innate immune response determines the range of tumors that the virus can lyse as an anticancer agent. The more sequences deleted, the narrower the range of cancer cells that can be treated. The effectiveness of oHSV depends on the function of the deleted viral gene. Most recent oHSVs incorporate at least one cellular gene to support their anticancer activity (Cheema et al., 2013; Goshima et al., 2014; Markert et al., 2012; Walker et al., 2011).

It's easier to consider the structure of oHSV (called the backbone) and the foreign gene that's suitable for insertion. As mentioned above, the backbone structure determines the range of cancer susceptibility. The foreign gene causes the host to view cancer cells as legitimate targets for an adaptive immune response.

The HSV-1 genome consists of two covalently linked components, L and S. Each component consists of a unique sequence (UL for the _L component and US for the _S component), flanked by inverted repeats. The inverted repeats of the L component are called ab and b'a'. The inverted repeats of the S component are called a'c' and ca. The inverted repeats b'a' and a'c' constitute the internal inverted repeat region. The inverted repeat regions of the L and S components are known to contain two copies of five genes and a large amount of transcribed but non-protein-encoding DNA. These five genes encode proteins ICP0, ICP4, ICP34.5, ORF P, and OFR O, respectively.

Viruses currently being tested in cancer patients fall into three main categories. The first was designed based on the discovery that deletion of the ICP34.5 gene significantly attenuates viral toxicity (Andreansky et al., 1997; Chou et al., 1995; Chou et al., 1990; Chou and Roizman, 1992). To ensure safety for the treatment of malignant gliomas, G207 (the first virus tested in patients) was attenuated by further mutations in the gene encoding the viral ribonucleotide reductase (Mineta et al., 1995). However, G207 harboring both ICP34.5 and ribonucleotide reductase mutations was too weak to be effective and completely halted replication in cancer cells expressing wild-type protein kinase R (Smith et al., 2006).

The second design was based on the fact that if the Us11 viral protein is expressed early in infection, it can partially compensate for the effects of ICP34.5 deficiency and restore growth in cells expressing wild-type protein kinase R (Cassady et al., 1998a). The backbone design of this virus follows the results published by Cassady (Cassady et al., 1998b), in which the Us12 gene and the Us11 promoter are deleted. As a result, Us11 is expressed as an immediate-early gene (rather than a late gene).

The backbone of the third virus, originally called R7020 and later renamed NV1020, was modified by spontaneous mutation and initially tested as a live attenuated virus vaccine (Meignier et al., 1988; Weichselbaum et al., 2012). This variant lacks the internal inverted repeat region (composed of b'a' and a'c', encoding one copy of the genes ICP0, ICP4, ICP34.5, ORF P, and ORF O) and the genes encoding _UL56 and _UL24 . Furthermore, it possesses bacterial sequences and, because it was originally intended for vaccine use, contains genes encoding several HSV-2 glycoproteins. R7020 has been extensively tested in patients with liver metastases from colorectal cancer. In addition, it has been tested in models of head and neck epithelial squamous cell carcinoma, athymic nude mouse prostate cancer xenografts, and gallbladder tumors (Cozzi et al., 2002; Cozzi et al., 2001; Currier et al., 2005; Fong et al., 2009; Geevarghese et al., 2010; Kelly et al., 2008; Kemeny et al., 2006; Wong et al., 2001).

The success of oHSV therapy depends on the extent of cancer cell destruction. Early in the development of oHSV therapy, it was recognized that HSV alone could not kill all cancer cells in solid tumors. It was unlikely that oHSV treatment would effectively eliminate all cancer cells. In clinical trials, oHSV tumor destruction must include an adaptive immune response against the tumor. Further studies have shown that the anti-tumor immune response elicited by infected tumor cell fragments can be amplified by the addition of cytokines. Comparison of oHSV without cytokine genes with oHSV supplemented with immunostimulatory cytokines confirmed this hypothesis (Andreanski et al.) and ultimately led to the addition of GM-CSF to oHSV for the treatment of melanoma (Andtbacka et al., 2015).

The safety of oHSV depends on the deletion of genes that inactivate one or more viral functions that block the host's innate immune response to infection. Analysis of published data suggests that oHSVs in clinical trials to date are overly attenuated and could be improved (Miest and Cattaneo, 2014).

Adding genes encoding immunostimulatory cytokines enhances the immune response to tumors but does not effectively enhance T cell-induced cytotoxicity, which is crucial for antitumor effects. Tumors silence the immune system through the PD-1 and CTLA-4 inhibitory pathways. PD-1 is expressed on activated T cells and other hematopoietic cells, while CTLA-4 is expressed on activated T cells, including regulatory T cells (Fife and Pauken, 2011; Francisco et al., 2010; Keir et al., 2008; Krummel and Allison, 1995; Walunas et al., 1994). Tumors use the PD-1 and CTLA-4 inhibitory pathways to evade host immune responses. To maximize antitumor responses, neutralization of PD-1 with PD-1 antibodies and, in some cases, neutralization of CTLA4 on the surface of T cells to activate cytotoxic T cells is necessary (Topalian et al., 2015). Although systemic administration of single-chain antibodies against PD-1 or CTLA4 can effectively enhance the efficacy of oHSV, it is often accompanied by side effects and cannot be given repeatedly over a long period of time.

Therefore, there is an urgent need to develop a safe and more effective oHSV and combine it with immunotherapeutic agents.

Summary of the Invention

One aspect of the present invention relates to a modified herpes simplex virus type 1 (hereinafter also referred to as HSV-1, obligate vector, vector, HSV-1 virus) comprising a modified HSV-1 genome. The modification comprises deleting the sequence between the promoter of the _UL56 gene and the promoter of the _US1 gene of the wild-type HSV-1 genome, such that (i) one copy of all double-copy genes is deleted, and (ii) the sequences required for expression of all open reading frames (ORFs) present in the viral DNA after the deletion are intact.

Another aspect of the present invention provides an oncolytic herpes simplex virus type 1 (HSV-1) construct comprising (i) sequences required for expression of all single-copy open reading frames in the viral genome; (ii) a single copy of each of all double-copy genes in the viral genome; and (iii) a single copy of duplicative DNA encoding a non-coding RNA.

Another aspect of the present invention relates to a recombinant oncolytic herpes simplex virus type 1 (HSV-1), comprising (a) a modified HSV-1 genome, wherein the modification comprises a sequence deletion between the promoter of the _UL56 gene and the promoter of the _US1 gene of the wild-type HSV-1 genome, such that (i) one copy of all double-copy genes is deleted and (ii) the sequences required for expression of all open reading frames (ORFs) present in the viral DNA after the deletion are intact; and (b) an exogenous nucleic acid sequence encoding an immunostimulatory agent and/or an immunotherapeutic agent, wherein the exogenous nucleic acid sequence is stably incorporated into at least the deleted region of the modified HSV-1 genome.

Another aspect of this invention is a viral vector comprising a single copy of all open reading frames (i.e., _UL 1 to _UL 56 and _US 1 to _US 12) and comprising an "a" sequence at the end of the genome, wherein the viral vector is an obligate vector, i.e., it cannot replicate in highly susceptible Vero cells by itself. The vector is capable of replication after insertion of DNA comprising (a) cellular DNA coding sequences or non-coding sequences or (b) viral DNA containing non-coding sequences. The obligate vector can tolerate a total DNA length of at least 15 KB or up to 22 KB.

Another aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of the recombinant oncolytic HSV-1 described herein and a pharmaceutically acceptable carrier. The composition can be formulated for intratumoral administration.

Another aspect of the present invention relates to a method for treating cancer, comprising administering an effective amount of the recombinant oncolytic HSV-1 or the pharmaceutical composition of the present invention to a subject in need thereof. Furthermore, the present invention also relates to the use of the recombinant oncolytic HSV-1 in a method for treating cancer.

Another aspect of the present invention relates to the use of the recombinant oncolytic HSV-1 or the pharmaceutical composition in the preparation of anticancer drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

These aspects and advantages as well as other aspects and advantages of the present invention will become apparent from the following detailed description taken in conjunction with the accompanying drawings.

Figure 1. Schematic representation of the HSV-1 virus. HSV-1, wild-type HSV-1 genome structure, showing the internal inverted repeat region b'a'-a'c' between bp 117005 and bp 132096; IMMV201, oHSV-1 genome structure, also known as the obligate vector; IMMV202, oHSV-1 expressing murine IL-12; IMMV203, oHSV-1 expressing IL-12; IMMV303, oHSV-1 expressing human CTLA-4 scFv; IMMV403, oHSV-1 expressing human PD-1 scFv.

Figure 2. Schematic representation of obligate vector-based oncolytic HSV-1 viruses expressing immunostimulatory and/or immunotherapeutic agents. IMMV502, oHSV-1 expressing human anti-PD-1 scFv and murine IL-12; IMMV504, oHSV-1 expressing murine anti-CTLA-4 scFv and murine IL-12; IMMV503, oHSV-1 expressing human anti-PD-1 scFv and human IL-12; IMMV505, oHSV-1 expressing human anti-CTLA-4 scFv and human IL-12; IMMV507, oHSV-1 expressing human anti-CTLA-4 scFv and human anti-PD-1 scFv; IMMV603, oHSV-1 expressing human anti-CTLA-4 scFv, human anti-PD-1 scFv, and human IL-12.

Figure 3. Expression of anti-PD-1 scFv from a secretion test construct. His-tagged scFv-anti-PD-1 was expressed under the CMV promoter along with signal peptide coding regions from various natural sources. Cell lysates and supernatants were collected, subjected to SDS-PAGE, and blotted with an anti-His antibody. Lane 1, GM-CSF signal peptide; lane 2, Gaussia luciferase signal peptide; lane 3, Hidden Markov Model 38 (HMM38) signal peptide; lane 4, antibody V gene signal peptide.

Figure 4. Affinity assay of scFv-anti-PD-1 targeting PD-1. ELISA assay of a CMV promoter-driven His-tagged scFv-anti-PD-1 with the HMM38 signal peptide. Supernatants were collected for ELISA and detected with an anti-His antibody.

Figure 5. In vitro cell growth and survival assay. (a) T24, human bladder cancer cells; (b) ECA109, human esophageal cancer cells; (c) CNE1, human nasopharyngeal cancer cells; (d) HCT116, human colon cancer cells; (e) Hep2, human laryngeal cancer cells; (f) MD-MB-231, human adenocarcinoma cells; (g) Hela, human epithelial adenocarcinoma cells; (h) A549, human lung epithelial cells; (i) H460, human non-small cell lung cancer cells.

DETAILED DESCRIPTION

definition

It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "recombinant oncolytic HSV-1" is understood to mean one or more recombinant oncolytic HSV-1 viruses. Therefore, the terms "a," "one or more," and "at least one" can be used interchangeably herein.

"Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing alignable positions in each sequence. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is related to the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, preferably less than 25% identity, with one of the sequences herein.

A polynucleotide or polynucleotide region having a particular percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same when comparing the two sequences. The alignment and percent homology or sequence identity can be determined using software programs known in the art.

As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to one or more antigens. The antibody can be a complete antibody and any antigen-binding fragment thereof or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide that is at least a portion of an immunoglobulin molecule that has antigen-binding biological activity. Such examples include, but are not limited to, a complementarity determining region (CDR) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region or any portion thereof, or at least a portion of a binding protein. The term antibody also includes a polypeptide or polypeptide complex that has antigen-binding ability when activated.

As used herein, the term "antibody fragment" or "antigen-binding fragment" is a portion of an antibody, such as F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, scFv, and the like. Regardless of the structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, Spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

The antibodies, antigen-binding polypeptides, or variants or derivatives thereof herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single chain antibodies, epitope-binding fragments (e.g., Fab, Fab', and F(ab') ₂ ), Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments comprising a VK or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies. The immunoglobulin or antibody molecule herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) of immunoglobulin molecules, or subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

"Specific binding" or "having specificity" generally refers to the binding of an antibody to an epitope via its antigen binding domain, and that the binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to the epitope via its antigen binding domain more easily than to a random, unrelated epitope. The term "specificity" is used herein to quantify the relative affinity with which an antibody binds to an epitope. For example, antibody "A" may be considered to have a higher specificity for a given epitope than antibody "B," or antibody "A" may be described as binding to epitope "C" with a higher specificity than to a related epitope "D."

As used herein, "cancer" or "tumor," as used interchangeably herein, refers to a group of diseases that can be treated according to the present disclosure and involve abnormal cell growth that may invade or spread throughout the body. Not all tumors are cancerous; benign tumors do not spread to other parts of the body. Possible signs and symptoms include: new lumps, unusual bleeding, prolonged coughing, unexplained weight loss, and changes in bowel movements, among others. There are over 100 different known cancers that affect humans. The present disclosure is preferably applicable to solid tumors. Non-limiting examples of tumors or cancers include gallbladder cancer, basal cell carcinoma, extrahepatic bile duct cancer, colon cancer, endometrial cancer, esophageal cancer, Ewing's sarcoma, prostate cancer, gastric cancer, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, laryngeal cancer, liver cancer, lung cancer, melanoma, mesothelioma, pancreatic cancer, rectal cancer, kidney cancer, thyroid cancer, malignant peripheral nerve cell tumor, malignant peripheral nerve sheath tumor (MPNST), skin and plexiform neurofibromas, leiomyomatoid tumors, fibromas, uterine fibroids, leiomyosarcomas, papillary thyroid cancer, undifferentiated thyroid cancer, The invention is used to treat esophageal cancer, lung cancer, prostate cancer, or bladder cancer.

As used herein, the term "treat" refers to both therapeutic treatment and prophylactic measures, the purpose of which is to prevent or slow (lessen) an undesirable physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, relief of symptoms, reduction in extent of disease, stabilization (i.e., non-worsening) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and disappearance of symptoms (whether partial or complete), whether detectable or undetectable. "Treatment" also means prolonging survival as compared to expected survival if not receiving treatment. Patients in need of treatment include those already suffering from the disease or condition, as well as those susceptible to the disease or condition, or those in whom the disease or condition is to be prevented.

"Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, livestock, farm animals, and zoo animals, sport animals, or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like.

As used herein, phrases such as "a patient in need of treatment" or "a subject in need of treatment" include subjects, such as mammalian subjects, who would benefit from administration of the disclosed antibodies or compositions for, eg, detection, diagnostic procedures, and/or therapy.

It will also be understood by those skilled in the art that the modified genomes disclosed herein can be modified so that they differ in nucleotide sequence from the modified polynucleotides from which they are derived. For example, a polynucleotide or nucleotide sequence derived from a specified DNA sequence can be similar, such as having a certain percentage identity to the starting sequence, for example, it can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the starting sequence.

In addition, nucleotide or amino acid substitutions, deletions or insertions can be carried out to carry out conservative substitutions or changes in "non-essential" regions. For example, polypeptides or amino acid sequences derived from a specified protein may have one or more independent amino acid substitutions, insertions or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more single amino acid substitutions, insertions or deletions) and the remainder may be identical to the starting sequence. In certain embodiments, polypeptides or amino acid sequences derived from a specified protein may have 1 to 5, 1 to 10, 1 to 15 or 1 to 20 independent amino acid substitutions, insertions or deletions relative to the starting sequence.

Antibodies can be detectably labeled by coupling them to a chemiluminescent compound. The presence of the chemiluminescent-labeled antigen-binding polypeptide is then determined by detecting the presence of luminescence that occurs during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

Modified HSV-1 specific vector

On the one hand, the present invention provides an HSV-1 virus comprising a modified HSV-1 genome, also referred to as an HSV-1 obligate vector. The HSV-1 genome consists of two covalently linked components (called L and S). Each component consists of a unique sequence ( _UL for the L component and US for the _S component), flanked by inverted repeats. The inverted repeats of the L component are called ab and b'a'. The inverted repeats of the S component are called a'c' and ca. The inverted repeat region contains two copies of the transcription unit. At least five open reading frames with two copies are known in the art, and their proteins are named ICP0, ICP4, ICP34.5, ORF P, and ORF O, respectively. The inverted repeats b'a' and a'c'(b'a'-a'c') combine to form an internal inverted repeat region. Relatively, the inverted repeats ab and ca are referred to as external repeat regions in this article.

In one embodiment of the present invention, the modification comprises a deletion of a sequence between the promoter of the _UL56 gene and the promoter of the _US1 gene of the wild-type HSV-1 genome. In fact, the deleted sequence includes:

(a) A single copy of the transcription unit encoding at least five proteins (ICP0, ICP4, ICP34.5, ORF-O, and ORF-P), retaining all other open reading frames;

(b) a transcription unit entirely contained within the b'a'-a'c' sequence;

(c) Transcription units that begin in the unique region but extend into the deleted region.

In this article, the deletion is performed in a precise manner to ensure that the sequences required for the expression of all open reading frames (ORFs) present in the viral DNA after deletion are intact. In this case, "sequences required for the expression of all open reading frames present" include the ORF itself and the regulatory sequences (such as promoters and enhancers) required for expressing each ORF to ensure that the expression of the ORF is successful and that the translated protein is functional. "Complete" means that the sequence defined in this way is at least functional, but does not mean that the sequence must be 100% identical to the naturally occurring sequence. By including, for example, conservative substitutions or changes in "non-essential" regions, the sequence can have a slight change in nucleotide sequence compared to the naturally occurring sequence. In this case, the sequence can have 90%, 95%, 98% or 99% identity to the naturally occurring sequence.

Those skilled in the art will appreciate that the exact starting and ending positions of the nucleotides to be deleted in the present invention depend on the strain and genomic isoform of the HSV-1 virus and can be readily determined using techniques known in the art. It should be understood that this disclosure is not intended to be limited to any specific genomic isoform or strain of the HSV-1 virus. In one embodiment, the deletion results in the excision of nucleotides 117005 to 132096 from the genome. Those skilled in the art will also appreciate that other strains can also be used in the present invention, provided their genomic DNA has been sequenced. Sequencing technology is readily available in the literature and commercially. For example, in another embodiment, the deletion can be performed on HSV-1 strain 17, the genome of which can be obtained through GenBank Accession No. NC_001806.2. In another embodiment, the deletion can be performed on strain KOS 1.1, the genome of which can be obtained through GenBank Accession No. KT899744. In yet another embodiment, the deletion can be performed on strain F, the genome of which can be obtained through GenBank Accession No. GU734771.1.

In some embodiments, deletion is accurately carried out at a predetermined position, thereby starting from the promoter of the last known gene (such as _UL56 ) in the L component and ending with a DNA fragment of the promoter sequence of the first known gene (such as _US1 ) in the _S component. In this way, all ORFs of _UL1 to _UL56 genes in the _UL component and _US1 to _US12 in the US component and the sequences required for the expression of these ORFs are complete. The retention of the sequence required for the expression of all open reading frames (ORFs) present in the viral DNA after the precise excision and deletion has many advantages. "Retention" refers to that the modified vector comprises all genes in the unique sequence ( _UL and _US ) and only one copy of all double copy genes (such as the gene of ICPO, ICP4, ICP34.5, ORF P and ORF O). It should be noted that most of the deleted sequences do not encode proteins, but occupy the repeated non-coding sequences (such as, introns of ICPO, LAT domains, "a" sequences, etc.) in the deleted region. The obligate vectors of the present invention are also intended to comprise only one copy of the repetitive non-coding sequence.

The retention of all ORFs provides a more robust virus, both before and after the insertion of foreign genes, i.e., maximum resistance to environmental factors such as temperature, pressure, UV light, etc. It also maximizes the anti-cancer spectrum of the oncolytic HSV-1.

Various gene manipulation methods known in the art can be used to obtain modified HSV-1 vectors as described herein. For example, bacterial artificial chromosome (BAC) technology can be used. See, for example, Horsburgh BC, Hubinette MM, Qiang D, et al. Allele replacement: an application that permits rapid manipulation of herpes simplex virus type 1 genome. Gene Ther , 1999, 6(5):922-30. As another example, COS plasmids can be used in the present invention. See, for example, van Zijl M., Quint W, Briaire J, et al. Regeneration of herpes viruses from molecularly cloned subgenomic fragments. J Virol , 1988, 62(6):2191-5.

A key feature of the constructs described herein is that they act as obligate vectors. The definition applicable to such constructs is that they do not propagate in susceptible cells, but rather propagate following insertion of viral or cellular DNA sequences, thereby acting as vehicles for the expression of genes inserted into the vector sequences.

Recombinant oncolytic HSV-1 virus

The amount of exogenous DNA sequence that can be inserted into the wild-type virus is limited because it interferes with the packaging of DNA into viral particles. Precise deletions in designated regions provide ideal space for the insertion of exogenous DNA sequences. According to one embodiment of the present disclosure, the deletion removes at least 15 Kbp of the oncolytic viral vector, making it possible to accommodate a similar amount of exogenous DNA sequence. Other studies have shown that the wild-type genome can tolerate an additional 7 KB of DNA.

Therefore, on the other hand, the present disclosure provides a recombinant oncolytic herpes simplex virus type 1 (HSV-1), comprising (a) a modified HSV-1 genome, wherein the modification comprises a sequence deletion between the promoter of the _UL56 gene and the promoter of the _US1 gene of the wild-type HSV-1 genome, such that (i) one copy of all double-copy genes is deleted and (ii) the sequences required for expression of all open reading frames (ORFs) present in the viral DNA after the deletion are intact; and (b) an exogenous nucleic acid sequence encoding an immunostimulatory agent and/or an immunotherapeutic agent, wherein the exogenous nucleic acid sequence is stably incorporated into at least the deleted region of the modified HSV-1 genome.

In one embodiment, the recombinant oncolytic HSV-1 comprises an exogenous nucleic acid sequence encoding an immunostimulatory agent. In some embodiments, the immunostimulatory agent is selected from GM-CSF, IL 2, IL 5, IL 12, IL 15, IL 24, and IL 27. In one embodiment, the immunostimulatory agent is IL 12. In one embodiment, the immunostimulatory agent is human IL 12 or humanized IL 12. In one embodiment, the immunostimulatory agent is murine IL 12. In another embodiment, the immunostimulatory agent is IL 15.

In one embodiment, the recombinant oncolytic HSV-1 comprises an exogenous nucleic acid sequence encoding an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is selected from an anti-PD-1 agent and an anti-CTLA-4 agent. In one embodiment, the immunotherapeutic agent is an anti-PD-1 agent. In another embodiment, the immunotherapeutic agent is an anti-CTLA-4 agent.

In one embodiment, the exogenous nucleic acid sequence of the present invention is inserted into the region of the genome that has been deleted. In one embodiment, the exogenous nucleic acid sequence has a length similar to the region that has been deleted. In one embodiment, the length ratio of the exogenous nucleic acid sequence is 20% or shorter than the length in the region that has been deleted. In another embodiment, the length ratio of the exogenous nucleic acid sequence is 15%, 10%, 5%, 4%, 3%, 2% or 1% longer than the region that has been deleted.

In one embodiment, the exogenous nucleic acid sequence has a length of less than about 18Kbp, about 17Kbp or about 16Kbp. In one embodiment, the exogenous nucleic acid sequence has a length greater than about 10Kbp, 11Kbp, 12Kbp, 13Kbp or 14Kbp. In one embodiment, the length of the exogenous nucleic acid sequence is between about 14Kbp and about 16Kbp. In one embodiment, the exogenous nucleic acid sequence has a length of about 15Kbp.

In some embodiments, the recombinant oncolytic HSV-1 comprises at least two exogenous nucleic acid sequences encoding immunostimulatory agents and/or immunotherapeutic agents. In some embodiments, the recombinant oncolytic HSV-1 comprises exogenous nucleic acid sequences encoding two different immunostimulatory agents. For example, in one embodiment, the recombinant oncolytic HSV-1 comprises exogenous nucleic acid sequences encoding IL-12 and GM-CSF. In another embodiment, the recombinant oncolytic HSV-1 comprises exogenous nucleic acid sequences encoding IL-15 and GM-CSF. In another embodiment, the recombinant oncolytic HSV-1 comprises exogenous nucleic acid sequences encoding IL12 and IL15.

In some embodiments, the recombinant oncolytic HSV-1 comprises exogenous nucleic acid sequences encoding two different immunotherapeutic agents. In one embodiment, for example, the recombinant oncolytic HSV-1 comprises exogenous nucleic acid sequences encoding an anti-PD-1 agent and an anti-CTLA-4 agent.

In some embodiments, the recombinant oncolytic HSV-1 comprises exogenous nucleic acid sequences encoding three different immunostimulatory and/or immunotherapeutic agents. For example, in one embodiment, the recombinant oncolytic HSV-1 comprises exogenous nucleic acid sequences encoding IL12, an anti-CTLA4 agent, and an anti-PD-1 agent.

When introducing more than one exogenous nucleic acid sequence encoding an immunostimulant and/or immunotherapeutic agent, it is preferred that the first exogenous nucleic acid sequence be inserted into the genomic deletion region. A second or additional exogenous nucleic acid sequence can be inserted into the genomic L component. In one embodiment, the second exogenous nucleic acid sequence is inserted between the _UL3 and _UL4 genes of the L component. In one embodiment, the second exogenous nucleic acid sequence is inserted between the _UL37 and _UL38 genes of the L component.

In one embodiment, the first exogenous nucleic acid sequence is inserted into the deleted region of the genome, and the second exogenous nucleic acid sequence is inserted between the _UL3 and _UL4 genes. In one embodiment, the first exogenous nucleic acid sequence is inserted into the deleted internal inverted repeat region of the genome, and the second exogenous nucleic acid sequence is inserted between the _UL37 and _UL38 genes of the L component. In one embodiment, the first exogenous nucleic acid sequence is inserted into the deleted internal inverted repeat region of the genome, the second exogenous nucleic acid sequence is inserted between the _UL3 and _UL4 genes, and the third exogenous nucleic acid sequence is inserted between the _UL37 and _UL38 genes of the L component.

In one embodiment, the first exogenous nucleic acid sequence encodes IL 12. In one embodiment, the second exogenous nucleic acid sequence encodes an anti-CTLA4 agent or an anti-PD-1 agent. In one embodiment, the third exogenous nucleic acid sequence encodes an anti-PD-1 agent or an anti-CTLA4 agent.

It is understood that the insertion of one or more exogenous nucleic acid sequences into the oncolytic HSV-1 genome does not interfere with the expression of native HSV-1 genes, and the exogenous nucleic acid sequences are stably integrated into the modified HSV-1 genome, so that functional expression of the exogenous nucleic acid sequences can be expected.

The recombinant gene of coding immunostimulant and/or immunotherapeutic agent contains nucleic acid of coded protein and the regulatory element for protein expression.Usually, regulatory element is present in the recombinant gene and is operably connected with nucleotide sequence to be expressed, and selects based on the host cell to be used for expression, and can comprise transcription promoter, ribosome bind site and terminator.In recombinant expression vector, " being operably connected " is intended to represent that interested nucleotide sequence is connected with the mode of regulating sequence in order to allow nucleotide sequence expression (for example, in in vitro transcription/translation system, express, or when virus is introduced into host cell, express in host cell).Term " regulating sequence " is intended to include promoter, enhancer and other expression control elements (for example polyadenylation signal).Regulating sequence is included in those regulating sequences of the constitutive expression of instructing nucleotide sequence in many types of host cells, and the regulating sequence (for example, tissue-specific regulating sequence) that only instructs nucleotide sequence expression in some host cell.

A kind of newly discovered regulatory sequence is insulator (insulator), it includes a class of DNA elements found on cell chromosomes, and it protects the gene in one region of chromosome from the regulatory influence of another region. Amelio et al. found a 1.5-kb region containing a cluster of CTCF motifs in the LAT region, and this region has insulator activity, particularly enhancer blocking and silencing effect (Amelio et al.. A Chromatin Insulator-Like Element in theHerpes Simplex Virus Type 1 Latency-Associated Transcript RegionBinds CCCTC-Binding Factor and Displays Enhancer-Blocking and Silencing Activities. Journal of Virology , Vol.80, No.5, Mar.2006, p.2358-2368).

A promoter is a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter can cause a high frequency of mRNA initiation. A suitable element for processing in eukaryotic cells is a polyadenylation signal. Antibody-related introns may also be present. Examples of expression cassettes for the production of antibodies or antibody fragments are well known in the art (e.g., Persic et al ., 1997, Gene 187:9-18; Boel et al ., 2000, J Immunol. Methods 239:153-166; Liang et al ., 2001, J. Immunol. Methods 247:119-130; Tsurushita et al ., 2005, Methods 36:69-83.).

Those of ordinary skill in the art can select suitable regulatory elements based on, for example, desired tissue specificity and expression levels. For example, cell type-specific or tumor-specific promoters can be used to restrict the expression of gene products to specific cell types. In addition to using tissue-specific promoters, local administration of viruses can achieve local expression and effects. Examples of non-tissue-specific promoters that can be used include early cytomegalovirus (CMV) promoters (U.S. Patent No. 4,168,062) and Rous sarcoma virus promoters. In addition, HSV promoters, such as HSV-1E promoters, can be used. In some embodiments, the promoter is selected from the promoters in the following table.

promoter Tumor or tissue target B-myb Glioma liver metastasis Nestin glioma CEA (carcinoembryonic antigen) colon cancer albumin Liver cancer DF3/MUC1 (Mucin 1) pancreatic cancer Caponin Leiomyosarcoma

For example, examples of tissue-specific promoters that can be used in the present technology include the prostate-specific antigen (PSA) promoter, which is specific for prostate cells; the desmin promoter, which is specific for muscle cells; the enolase promoter, which is specific for neurons; the beta globin promoter, which is specific for erythroid cells; the tau-globin promoter, which is also specific for erythroid cells; the growth hormone promoter, which is specific for pituitary cells; the insulin promoter, which is specific for pancreatic beta cells; the glial fibrillary acidic protein promoter, which is specific for astrocytes; the tyrosine hydroxylase promoter, which is specific for catecholaminergic neurons; the amyloid precursor protein promoter, which is specific for neurons; dopamine β-hydroxylase promoter, which is specific for noradrenergic and adrenergic neurons; tryptophan hydroxylase promoter, which is specific for serotonin/pinealocytes; choline acetyltransferase promoter, which is specific for cholinergic neurons; aromatic L-amino acid decarboxylase (AADC) promoter, which is specific for catecholaminergic/5-HT/D-type cells; proenkephalin promoter, which is specific for neurons/spermatogenic epididymal cells; reg (pancreatic stone protein) promoter, which is specific for colon and rectal tumors, as well as pancreatic and renal cells; and parathyroid hormone-related peptide (PTHrP) promoter, which is specific for liver and cecal tumors, as well as schwannomas, renal cells, pancreatic cells, and adrenal cells.

Examples of promoters that function specifically in tumor cells include the stromelysin 3 promoter, which is specific for breast cancer cells; the surfactant protein A promoter, which is specific for non-small cell lung cancer cells; the secretory leukocyte protease inhibitor (SLPI) promoter, which is specific for SLPI-expressing cancers; the tyrosinase promoter, which is specific for melanoma cells; the stress-induced grp78/BiP promoter, which is specific for fibrosarcoma/tumorigenic cells; the AP2 adipogenic enhancer, which is specific for adipocytes; the alpha-1 antitrypsin transthyretin promoter, which is specific for hepatocytes; the interleukin-10 promoter, which is specific for glioblastoma multiforme; the c-erbB-2 promoter, which is specific for pancreatic, breast, gastric, ovarian, and non-small cell lung cells; the alpha-B-crystallin/heat shock protein 27 promoter, which is specific for brain tumor cells; and the alpha-B-crystallin/heat shock protein 27 promoter, which is specific for neuroblastoma cells. The basic fibroblast growth factor promoter specific for glioma and meningioma cells; the epidermal growth factor receptor promoter specific for squamous cell carcinoma, glioma and breast tumor cells; the mucin-like glycoprotein (DF3, MUC1) promoter specific for breast cancer cells; the mts1 promoter specific for metastatic tumors; the NSE promoter specific for small cell lung cancer cells; the somatostatin receptor promoter specific for small cell lung cancer cells; the c-erbB-3 and c-erbB-2 promoters specific for breast cancer cells; the c-erbB4 promoter specific for breast cancer and gastric cancer; the thyroglobulin promoter specific for thyroid cancer cells; the alpha-fetoprotein (AFP) promoter specific for liver cancer cells; the villin promoter specific for gastric cancer cells; and the albumin promoter specific for liver cancer cells. In another embodiment, the TERT promoter or the survivin promoter is used.

For example, in some embodiments, the exogenous nucleic acid sequence is operably linked to a promoter, such as a CMV promoter or an Egr promoter. In one embodiment, the nucleotide sequence encoding mIL12 is operably linked to the Egr promoter. In another embodiment, the nucleotide sequence encoding scFv-anti-hPD1 is operably linked to the CMV promoter.

Immunostimulants or immunosuppressants

In certain embodiments, the oHSV-1 of the present disclosure encodes one or more immunostimulatory agents (also known as immunostimulatory molecules), including cytokines (e.g., IL-2, IL4, IL-12, GM-CSF, IFNγ), chemokines (e.g., MIP-1, MCP-1, IL-8), and growth factors (e.g., FLT3 ligand).

Alternatively or in addition, the oHSV-1 of the present disclosure encodes one or more immunotherapeutic agents, such as a PD-1 binding agent (or anti-PD-1 agent) or a CTLA-4 binding agent (or anti-CTLA-4 agent), including antibodies or fragments thereof, such as an anti-PD1 antibody that specifically binds to PD-1 or an anti-CTLA-4 antibody that specifically binds to CTLA-4. The anti-PD-1 antibody can be a single-chain antibody that antagonizes PD-1 activity. In other embodiments, the oncolytic virus expresses an agent that antagonizes the binding of a PD-1 ligand to a receptor, such as an anti-PD-L1 antibody and/or PD-L2 antibody, a PD-L1 and/or PD-L2 decoy, or a soluble PD-1 receptor.

The PD-1 signaling pathway plays an important role in tumor-associated immune dysfunction. Infection and lysis of tumor cells can trigger a highly specific anti-tumor immune response that kills cells inoculated with the tumor as well as established, uninoculated tumor cells at a distance. Tumors and their microenvironment have formed mechanisms to evade, suppress, and inactivate natural anti-tumor immune responses. For example, tumors may downregulate target receptors, encapsulate themselves in a fibrous extracellular matrix, or upregulate host receptors or ligands involved in the activation or recruitment of regulatory immune cells. Natural and/or adaptive T regulatory cells (Treg) participate in tumor-mediated immunosuppression. Without wishing to be limited by theory, PD-1 blockade can inhibit Treg activity and improve the efficacy of tumor-reactive CTLs. Other aspects of this technology will be described in further detail below. PD-1 blockade can also stimulate anti-tumor immune responses by blocking the inactivation of T cells (CTLs and helper cells) and B cells.

On the one hand, the present technology provides an oncolytic virus carrying a gene encoding a PD-1 binding agent. Programmed cell death 1 (PD-1) is a 50-55 kDa type I transmembrane receptor (Ishida et al., 1992, Embo J. 11:3887-95) initially identified by subtractive hybridization of a mouse T cell line undergoing apoptosis. PD-1, a member of the CD28 gene family, is expressed on activated T cells, B cells, and myeloid lineage cells (Greenwald et al. , 2005, Annu. Rev. Immunol. 23:515-48; Sharpe et al. , 2007, Nat. Immunol. 8:239-45). Human and mouse PD-1 have approximately 60% amino acid identity, with four potential N-glycosylation sites and the conservation of residues defining the Ig-V domain. Two ligands for PD-1, PD ligand 1 (PD-L1) and ligand 2 (PD-L2), have been identified, both belonging to the B7 superfamily. PD-L1 is expressed on many cell types, including T cells, B cells, endothelial and epithelial cells, and antigen-presenting cells. In contrast, PD-L2 is expressed only on professional antigen-presenting cells, such as dendritic cells and macrophages.

PD-1 negatively regulates T cell activation, and this inhibitory function is associated with the immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (Parry et al. , 2005, Mol. Cell. Biol. 25:9543-53). Disruption of this inhibitory function of PD-1 can lead to autoimmunity. The opposite can also be harmful. In many pathological conditions, such as tumor immune escape and chronic viral infection, persistent negative signaling of PD-1 is involved in T cell dysfunction.

Host anti-tumor immunity is primarily influenced by tumor-infiltrating lymphocytes (TILs) (Galore et al. , 2006, Science 313:1960-4). Multiple lines of evidence indicate that TILs are regulated by the inhibitory effect of PD-1. First, PD-L1 expression has been demonstrated in many human and mouse tumor lines, and this expression can be further upregulated in vitro by IFN-γ (Dong et al. , 2002, Nat. Med. 8:793-800). Second, PD-L1 expression on tumor cells is directly associated with their resistance to lytic effects by anti-tumor T cells in vitro (Blank et al. , 2004, Cancer Res. 64:1140-5). Third, PD-1 knockout mice are resistant to tumor attack (Iwai et al ., 2005, Int. Immunol. 17:133-44), and T cells from PD-1 knockout mice are highly effective in tumor rejection when adoptively transferred to tumor-bearing mice (Blank et al., supra). Fourth, blocking PD-1 inhibitory signals by monoclonal antibodies can enhance host anti-tumor immunity in mice (Iwai et al. , supra; Hirano et al. , 2005, Cancer Res. 65:1089-96). Fifth, high PD-L1 expression in tumors (detected by immunohistochemical staining) is associated with poor prognosis in many human cancer types (Hamanishi et al. , 2007, Proc. Natl. Acad. Sci. USA 104:3360-5).

Oncolytic virus therapy is an effective method for shaping the host immune system by amplifying T or B cell populations specific for tumor-specific antigens (released after oncolysis). The immunogenicity of tumor-specific antigens depends largely on the affinity of host immune receptors (B cell receptors or T cell receptors) for antigen epitopes and the host tolerance threshold. High-affinity interactions will drive host immune cells into long-lasting memory cells through multiple rounds of proliferation and differentiation. Host tolerance mechanisms will offset this proliferation and expansion to minimize potential tissue damage caused by local immune activation. PD-1 inhibitory signals are part of this host tolerance mechanism, which can be supported by the following evidence. First, PD-1 expression is elevated in actively proliferating T cells, especially in T cells with a terminally differentiated phenotype (effector phenotype). Effector cells are generally associated with effective cytotoxic function and cytokine production. Second, PD-L1 is important for maintaining peripheral tolerance and locally limiting overactive T cells. Therefore, PD-1 inhibition using PD-1 binders expressed in the tumor microenvironment can be an effective strategy to increase the activity of TILs and stimulate effective and lasting anti-tumor immune responses.

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a member of the immunoglobulin (Ig) superfamily of proteins. The Ig superfamily is a group of proteins that share key structural features of the variable (V) or constant (C) domains of Ig molecules. Members of the Ig superfamily include, but are not limited to, immunoglobulins themselves, major histocompatibility complex (MHC) class molecules (i.e., class I and class II MHC), and TCR molecules. T cells require two types of signals from antigen-presenting cells (APCs) for activation and subsequent differentiation into effector functions. First, there is an antigen-specific signal generated by the interaction between the TCR on the T cell and the MHC molecule presenting the peptide on the APC. Second, there is an antigen-independent signal mediated by the interaction of CD28 with members of the B7 family (B7-1 (CD80) or B7-2 (CD86)). CTLA-4's integration into the context of the immune response is initially quite evasive. Mouse CTLA-4 was first identified and cloned by Brunet et al. as part of a quest to identify molecules preferentially expressed on cytotoxic T lymphocytes (Brunet et al. Nature 328:267-270 (1987)). Human CTLA-4 was discovered by Dariavach et al. and cloned shortly thereafter (Dariavach et al. Eur. J. Immunol. 18:1901-1905 (1988)). Mouse and human CTLA-4 molecules share approximately 76% overall sequence homology and near-complete sequence identity in their cytoplasmic domains (Dariavach et al. Eur. J. Immunol . 18:1901-1905 (1988)).

Researchers began to delineate the role of CTLA-4 in T cell stimulation in 1993 and culminated in 1995. Using monoclonal antibodies against CTLA-4, Walunas et al. (Walunas et al. Immunity 1:405-13 (1994)) first provided evidence that CTLA-4 could act as a negative regulator of T cell activation.

In cancer, Kwon et al. PNAS USA 94:8099-103 (1997) established a syngeneic mouse prostate cancer model and examined two different approaches designed to induce anti-prostate cancer responses through enhanced T cell costimulation: (i) direct costimulation provided by transducing prostate cancer cells expressing B7.1 ligands and (ii) in vivo antibody-mediated blockade of T cell CTLA-4, which prevents T cell downregulation. It has been shown that in vivo antibody-mediated blockade of CTLA-4 enhances anti-prostate cancer immune responses. In addition, Yang et al. Cancer Res 57:4036-41 (1997) studied whether blocking CTLA-4 function leads to enhanced anti-tumor T cell responses at different stages of tumor growth. Based on in vitro and in vivo results, they found that CTLA-4 blockade in tumor-bearing individuals enhanced the ability to generate anti-tumor T cell responses, but the manifestation of this enhancement was limited to the early stages of tumor growth in their model. In addition, Hurwitz et al. Proc Natl Acad Sci USA 95:10067-71 (1998) studied that the generation of T cell-mediated anti-tumor responses depends on T cell receptor engagement of major histocompatibility complex/antigen and the connection of B7 to CD28. Certain tumors (such as SM1 breast cancer) are difficult to treat by anti-CTLA-4 immunization. Therefore, by using a combination therapy of a CTLA-4 blocker and a vaccine composed of SM1 cells expressing granulocyte-macrophage colony-stimulating factor, regression of parental SM1 tumors was observed, but single treatment was ineffective. This combination therapy has long-lasting immunity to SM1 and is dependent on CD4(+) and CD8(+) T cells. These findings suggest that CTLA-4 blockade works at the level of host-derived antigen-presenting cells.

Anti-PD-1 and anti-CTLA-4 agents

On the one hand, the present technology provides an oncolytic virus comprising an exogenous nucleic acid encoding an anti-PD-1 agent and/or an anti-CTLA-4 agent. In some embodiments, the anti-PD-1 agent or anti-CTLA-4 agent contains an antibody variable region that provides specific binding to a PD-1 or CTLA-4 epitope. The antibody variable region can be present in, for example, a complete antibody, an antibody fragment, and a recombinant derivative of an antibody or antibody fragment. The term "antibody" refers to an immunoglobulin that can be natural, partially synthesized, or fully synthesized. Therefore, the anti-PD-1 agent or anti-CTLA-4 agent of the present technology includes any polypeptide or protein having a binding domain that specifically binds to a PD-1 or CTLA-4 epitope.

Different types of antibodies have different structures. IgG can be used to illustrate the different regions of an antibody. An IgG molecule contains four polypeptide chains: two long heavy chains and two shorter light chains interconnected by disulfide bonds. The heavy and light chains each contain a constant region and a variable region. The heavy chain consists of a heavy chain variable region ( _VH ) and a heavy chain constant region (CH1, CH2, and CH3). The light chain consists of a light chain variable region ( _VL ) and a light chain constant region ( _CL ). Within the variable region are three hypervariable regions that are responsible for antigen specificity. (See, for example, Breitling et al. , Recombinant Antibodies , John Wiley & Sons, Inc. and Spektrum Akademischer Verlag, 1999; and Lewin, Genes IV , Oxford University Press and Cell Press, 1990.)

The hypervariable regions are often referred to as complementarity determining regions ("CDRs") and are located between more conserved flanking regions called framework regions ("FWs"). From the NH2 terminus to the COOH terminus there are four (4) FW regions and three (3) CDRs: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The amino acids associated with the framework regions and CDRs can be numbered and aligned using the methods described by Kabat et al. , Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991; C. Chothia and AM Lesk, J Mol Biol 196(4):901 (1987); or B. Al-Lazikani, et al. , J Mol Biol 273(4):27, 1997. For example, framework regions and CDRs can be identified taking into account the Kabat and Chothia definitions. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The two heavy chain carboxyl regions are linked by a disulfide bond to form the constant region of the Fc region. The Fc region is important for providing effector functions (Presta, Advanced Drug Delivery Reviews 58:640-656, 2006). Each of the two heavy chains that make up the Fc region extends through a hinge region to a different Fab region.

Anti-PD-1 or anti-CTLA-4 agents typically contain antibody variable regions. Such antibody fragments include, but are not limited to, (i) Fab fragments, which are monovalent fragments consisting of _VH , _VL , _CH , and _CH domains; (ii) _Fab2 fragments, which are bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) Fd fragments consisting of _VH and _CH1 domains; (iv) Fv fragments consisting of the _VH and _VL domains of a single antibody arm; (v) dAb fragments, which contain either a _VH or _VL domain; (vi) scAbs, which are antibody fragments containing _VH and _VL and either _C1 or _CH1 ; and (vii) artificial antibodies based on protein scaffolds, including, but not limited to, fibronectin type III polypeptide antibodies (e.g., see U.S. Patent No. 6,703,199). In addition, although the two domains _VL and _VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, so that they can be made into a single protein chain, in which the _VL and _VH regions are paired to form a monovalent molecule, called a single-chain Fv (scFv). Therefore, the antibody variable region can be present in a recombinant derivative. Examples of recombinant derivatives include single-chain antibodies, diabodies, triabodies, tetrabodies and minibodies. Anti-PD-1 agents or anti-CTLA-4 agents can also contain one or more variable regions that recognize the same or different epitopes.

In some embodiments, the anti-PD-1 agent or anti-CTLA-4 agent is encoded by an oncolytic virus produced using recombinant nucleic acid technology. Different anti-PD-1 agents can be produced by different technologies, including, for example, single-chain proteins (such as scFv) comprising VH and VL regions connected by a linker sequence, as well as antibodies or fragments thereof; and multi-chain proteins containing _VH and _VL regions on separate polypeptides. Recombinant nucleic acid technology involves constructing a nucleic acid template for protein synthesis. Suitable recombinant nucleic acid technology is well known in the art. (See, for example, Ausubel, Current Protocols in Molecular Biology, John Wiley, 2005; Harlow et al., Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). Recombinant nucleic acids encoding anti-PD-1 antibodies or anti-CTLA-4 antibodies can be expressed in cells infected with oncolytic viruses and released into the tumor microenvironment after viral lysis. Cells actually serve as factories for encoding proteins.

Nucleic acids comprising one or more recombinant genes encoding either or both of the _VH or _VL regions of an anti-PD-1 or anti-CTLA-4 agent can be used to produce complete proteins/polypeptides that bind to PD-1/CTLA-4. For example, a single gene can be used to encode a single-chain protein (e.g., scFv) comprising a _VH and _VL region connected by a linker, or multiple recombinant regions can be used to produce _VH and _VL regions to provide a complete binding agent.

Exemplary anti-PD-1 antibodies or anti-CTLA-4 antibodies, or fragments or derivatives thereof, that can be used in the present disclosure are available in the art, see, for example, WO 2006/121168, WO 2014/055648, WO 2008/156712, US 2014/0234296, or U.S. Pat. No. 6,984,720.

The recombinant oHSV-1 in the present disclosure delivers immune-enhancing proteins precisely in the tumor where they are needed (rather than systemically). In addition, by reducing protein production in the tumor, and likely also reducing protein uptake, cytotoxic manifestations may be greatly reduced or absent.

Exemplary anti-PD-1 scFv and anti-CTLA-4 scFv sequences

Composition

Oncolytic viruses can be prepared in suitable pharmaceutically acceptable carriers or excipients. Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions and sterile powders (U.S. Patent number 5,466,468) for the temporary preparation of sterile injection solutions or dispersions. In all cases, the preparation must be sterile and must be fluid for easy injection. Must be stable under production and storage conditions and must prevent contamination by microorganisms such as bacteria and fungi.

The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol etc.), a suitable mixture thereof and/or a vegetable oil. For example, by using a coating such as lecithin, by maintaining the required particle size and by using a surfactant to maintain suitable fluidity in the case of dispersion. The effect of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal etc. In many cases, it is preferred to include isotonic agents, such as sugar or sodium chloride. The absorption of the injectable composition can be prolonged by using an agent (for example, aluminum monostearate and gelatin) that prolongs absorption in the composition.

For parenteral administration in aqueous solution, for example, the solution should be appropriately buffered (if necessary) and the liquid diluent should first be made isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this regard, according to the present disclosure, the sterile aqueous medium that can be used will be known to those skilled in the art. For example, a dose can be dissolved in 1mL of isotonic NaCl solution and added to 1000mL of subcutaneous perfusion solution, or injected at the recommended infusion position (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Depending on the condition of the subject being treated, some changes will inevitably occur in the dosage. In any case, the person responsible for administration will determine the appropriate dosage for the individual subject. In addition, for human administration, the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA biological product standards.

Sterile injectable solutions are prepared by combining the active compound in the required amount with the various other ingredients listed above as needed in a suitable solvent and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques, which produce a powder containing the active ingredient and any additional desired ingredients from a previously sterile filtered solution.

Compositions disclosed herein can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (forming acid addition salts with the free amino groups of proteins), including salts formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). The salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. When formulated, the solution will be used in a manner compatible with the dosage formulation and in an amount effective for treatment. The preparation is easy to administer in various dosage forms (such as injectable solutions, drug release capsules, etc.).

As used herein, "carrier" includes any and all solvents, dispersion media, carriers, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. Such media and agents for pharmaceutically active substances are well known in the art. In addition to any conventional media or agents that are incompatible with the active ingredient, it is contemplated that other media or agents may be used in the therapeutic compositions. Supplementary active ingredients may also be incorporated into the compositions.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar adverse reaction when administered to a human. The preparation of aqueous compositions containing proteins as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in a liquid prior to injection can also be prepared.

therapy

Another aspect of the present disclosure provides a method for treating or alleviating cancer, comprising administering to a subject in need thereof an effective amount of a recombinant oncolytic HSV-1 virus or a pharmaceutical composition comprising a recombinant oncolytic HSV-1 virus as described above. Similarly, the present disclosure provides an oncolytic HSV-1 virus for use in a method for treating or alleviating cancer as described above.

In certain embodiments, the recombinant oncolytic HSV-1 virus or pharmaceutical composition is administered intratumorally. In one embodiment, the HSV-1 virus or pharmaceutical composition is injected directly into the tumor mass in the form of an injectable solution.

In some embodiments, oncolytic viruses carrying genes encoding immunostimulants and/or immunotherapeutics can be combined with other agents for the effective treatment of cancer. For example, the treatment of cancer can be implemented with oncolytic viruses and other anticancer therapies (such as anticancer agents or surgery). In this technology, it is expected that oncolytic virus therapy can be used in combination with chemotherapeutics, radiotherapeutics, immunotherapeutics or other biological interventions.

"Anticancerous" agents can negatively affect the cancer in a subject, for example, by killing cancer cells, inducing apoptosis, reducing cancer cell growth rate, reducing metastasis incidence or number, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cell, promoting the immune response to a cancer cell or tumor, preventing or inhibiting cancer progression, or increasing the life span of a cancer patient. Anticancer agents include biologics (biotherapy), chemotherapeutics, and radiotherapeutics. More generally, these other compositions will be provided in a combined amount that effectively kills or inhibits cell proliferation. The process may involve contacting cells with an expression construct and an agent or multiple factors simultaneously. This can be achieved by contacting cells with a single composition or pharmacological preparation comprising two agents, or by contacting cells with two different compositions or preparations simultaneously, wherein one composition comprises an expression construct, and the other comprises a second agent.

In some embodiments, an oncolytic virus carrying a gene encoding an immunostimulant and/or immunotherapeutic agent is combined with an adjuvant. In one embodiment, the adjuvant is an oligonucleotide comprising an unmethylated CpG motif. The unmethylated dinucleotide CpG motif in bacterial deoxyribonucleic acid (DNA) has the advantage of stimulating several immune cells to secrete cytokines to enhance innate immunity and adaptive immunity.

Viral therapy can be carried out within a few minutes to a few weeks before or after other drug treatments.In the embodiment in which other medicaments and oncolytic viruses are applied to cells respectively, it will generally be ensured that a considerable period of time is not spaced between each delivery time, so that medicament and virus can still advantageously apply the combined effect on cells.In such a case, it is expected that cells can be contacted with two therapies within about 12-24 hours apart from each other.However, in some cases, when each application is spaced apart for a few days (2,3,4,5,6 or 7 days) to a few weeks (1,2,3,4,5,6,7 or 8 weeks), it may be necessary to significantly extend the time for treatment.

Construction of oHSV expressing immunostimulatory or immunotherapeutic genes

Construction of a modified oHSV-1 specific vector (IMMV201)

In the obligate vector IMMV201, generated using bacterial artificial chromosome (BAC) technology, a CMV promoter cassette containing three stop codons, ATGCAGGTGCAGTAATAGTAA, was inserted into the T-Easy vector. A prototype (P)-aligned HSV-1 was used. The following gene cassette, flanked by nucleotides 117,005 upstream and 132,096 downstream, was PCR amplified from the HSV-1 viral genome using two sets of primers, (GAAGATCTAATATTTTTATTGCAACTCCCTG, CTAGCTAGCTTATAAAAGGCGCGTCCCGTGG) and (GCTCTAGATTGCGACGCCCCGGCTC, CCTTAATTAAGGTTACCACCCTGTAGCCCCGATGT), respectively. The cassette was then inserted into a plasmid containing the CMV promoter and three stop codons. This gene replacement plasmid was then constructed into pKO5, generating pKO-CMV-STOP. IMMV201 was obtained by electroporating pKO-CMV-STOP into Escherichia coli RecA+ carrying BAC HSV.

BAC-IMMV201 is unable to propagate the virus in mammalian cells

2-3 μg of the BAC-IMMV201 constructed above was transfected into Vero cells at 70% confluency using OptiMEM (Life Technologies, Inc.) according to its instructions. The cells were incubated for 4 hours in a 37°C, 5% _CO2 incubator. Following incubation, the medium was replaced with 4 ml of fresh complete growth medium (5% newborn calf serum/DMEM). No viral plaques appeared within 3-4 days. The experiment was repeated three times, and no viral plaques were observed.

Construction of oHSV-1 expressing a single immunostimulatory or immunotherapeutic gene (IMMV202, 203, 303, 403)

The CMV promoter gene cassette driving immunostimulatory genes (mouse IL12, human IL12) or immunotherapy genes (human PD-1 scFV (SEQ ID No. 1 or 3), human CTLA-4 scFV (SEQ ID No. 5)) was obtained by electroporating the pKO gene cassette into Escherichia coli RecA + carrying IMMV201 (as shown in Figure 1).

Construction of oHSV-1 expressing two immunostimulatory genes or immunotherapy genes (IMMV502, 503, 504, 505, 507)

The CMV promoter cassette driving the immunostimulatory gene (IL12) and immunotherapeutic genes (PD-1 scFV, CTLA-4 scFV) was further inserted between the UL3 and UL4 genes in the IMMV202, 203, 303, 403 vectors to generate recombinant oHSV expressing a combination of the immunostimulatory gene (IL12) and the immunotherapeutic gene (as shown in Figure 2).

Construction of oHSV-1 (IMMV603) expressing one immunostimulatory gene and two immunotherapeutic genes

By inserting CTLA-4 scFV between the UL37 and UL38 genes of the IMMV503 vector (shown in Figure 2), IMMV603 was constructed to express all three immunostimulatory cDNAs encoding human IL12, PD-1 scFV, and CTLA-4 scFV, as well as the immunotherapeutic cDNA.

In vitro tests

Expression of PD-1 scFV

In a series of experiments described herein, 2 × 10 ⁶ H293T cells were transfected with mock or plasmid containing cDNA encoding a His-tagged scFV-anti-PD-1 driven by a CMV promoter and signal peptide coding regions from various natural sources. Cell lysates and supernatants were collected 46 hours after transfection and then subjected to SDS-PAGE and immunoblotting with anti-His antibodies. 40 μL of 2 mL supernatant and 30 μL of 200 μL cell lysate were loaded on a 12% PAGE gel. The amount of PD-1 scFV accumulated in the cell culture supernatant (Figure 3) reflects the efficiency of different signal peptides.

Binding affinity of PD-1 scFV to PD-1

2 × 10 ⁶ H293T cells were transfected with mock or plasmid containing cDNA encoding a His-tagged scFV-anti-PD-1 driven by a CMV promoter and the HMM38 signal peptide. Supernatants were collected 46 hours after transfection and then subjected to ELISA assays using anti-His antibodies (Figure 4). Secreted PD-1 scFV bound to PD-1 in a dose-dependent manner.

In vitro cell viability growth assay

5 x 10 ³ of the following human tumor cells seeded in 96-well plates were infected with mock (negative control) or IMMV507 (oHSV-1 expressing PD-1 and CTLA-4 antibodies) at 0.01, 0.1, 1, 10, and 100 PFU per cell, respectively. Cell viability was measured using a CCK-8 kit every 24 hours for up to 96 hours ( Figure 5 ). Absorbance was measured at 450 nm using a microplate reader (BiotekEpoch).

The tumor cell lines used in these studies are: T24, human bladder carcinoma; ECA109, human esophageal carcinoma; CNE1, human nasopharyngeal carcinoma; HCT116, human colon carcinoma; Hep2, human laryngeal carcinoma; MD-MB-231, human breast carcinoma; Hela, human epithelial adenocarcinoma; A549, human lung epithelial cell line; and H460, human non-small cell lung carcinoma.

IMMV507 killed tumor cells in a dose-dependent manner. After exposure to oHSV-1 virus, the number of tumor cells decreased over time.

Those skilled in the art will readily appreciate that the present invention is well suited to achieving the objects and advantages mentioned and inherent therein. The methods, variations, and compositions described herein as presently representative preferred embodiments are merely exemplary and are not intended to limit the scope of the invention. Variations and other uses will occur to those skilled in the art, but are also encompassed within the true spirit of the invention as defined by the scope of the claims.

The invention illustratively described herein may suitably be practiced in the absence of any element or elements, or limitation or limitations, not specifically disclosed herein. The terms and expressions used are used as descriptions and not limitations, and there is no intention to exclude any equivalents of the features shown or described, or portions thereof, when using these terms and expressions, but it is recognized that various modifications are possible within the scope of the invention. Therefore, it is to be understood that although the invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

In addition, where features or aspects of the invention are described in terms of Markush groups or other groupings of alternatives, those skilled in the art will recognize that the invention is also described in terms of any individual member or subgroup of members of that Markush group or other group.

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Claims (22)

1. A type I herpes simplex virus (HSV-1) vector comprising a modified wild-type HSV-1 genome, wherein the modification comprises a deletion resulting in the removal of nucleotides 117005 to 132096 in the F strain genome, such that the vector comprises: (a) Only one copy of each of all double-copy genes. (b) The sequences required to express all single-copy open reading frames (ORFs) in the HSV-1 genome, and (c) A single copy of a repetitive DNA encoding non-coding RNA. The sequences required for expressing all single-copy open reading frames (ORFs) in the HSV-1 genome are complete and include the open reading frames themselves as well as the regulatory sequences required for expressing each open reading frame, and the regulatory sequences include promoters. 2. The HSV-1 vector according to claim 1, wherein the double-copy gene comprises genes encoding ICP0, ICP4, ICP34.5, ORF P and ORF O. 3. The HSV-1 vector according to claim 1, wherein the vector does not reproduce in susceptible cells. 4. The HSV-1 vector according to claim 1, wherein the vector only proliferates in susceptible cells after the viral DNA or cellular DNA sequence has been inserted. 5. The HSV-1 vector according to claim 1, wherein the repetitive DNA encoding non-coding RNA comprises an intron of ICP0, a LAT domain, and an "a" sequence. 6. A recombinant oncolytic type I herpes simplex virus (HSV-1), comprising (a) The type I herpes simplex virus (HSV-1) vector according to any one of claims 1 to 5; and (b) An exogenous nucleic acid sequence encoding an immunostimulant and/or an immunotherapeutic agent, wherein the exogenous nucleic acid sequence is stably added to at least a deleted region of the HSV-1 vector and does not interfere with the expression of the native HSV-1 gene. 7. The recombinant oncolytic HSV-1 according to claim 6, wherein the immunostimulant is selected from GM-CSF, IL 2, IL 12, IL 15, IL 24 and IL 27. 8. The recombinant oncolytic HSV-1 according to claim 6, wherein the immunotherapeutic agent is an anti-PD-1 agent and/or an anti-CTLA-4 agent. 9. The recombinant oncolytic HSV-1 according to claim 6, wherein the recombinant oncolytic HSV-1 comprises an exogenous nucleic acid sequence encoding IL-12, an anti-PD-1 agent, and/or an anti-CTLA-4 agent. 10. The recombinant oncolytic HSV-1 according to claim 6, wherein the recombinant oncolytic HSV-1 comprises a first exogenous nucleic acid sequence encoding IL-12 and a second exogenous nucleic acid sequence encoding an anti-PD-1 agent or an anti-CTLA-4 agent. 11. The recombinant oncolytic HSV-1 according to claim 6, wherein the recombinant oncolytic HSV-1 comprises a first exogenous nucleic acid sequence encoding an anti-PD-1 agent and a second exogenous nucleic acid sequence encoding an anti-CTLA-4 agent. 12. The recombinant oncolytic HSV-1 according to claim 6, wherein the recombinant oncolytic HSV-1 comprises a first exogenous nucleic acid sequence encoding IL-12, a second exogenous nucleic acid sequence encoding an anti-PD-1 agent, and a third exogenous nucleic acid sequence encoding an anti-CTLA-4 agent. 13. The recombinant oncolytic HSV-1 of claim 6, wherein the recombinant oncolytic HSV-1 further comprises a promoter sequence operatively linked to the exogenous nucleic acid sequence. 14. The recombinant oncolytic HSV-1 according to claim 10 or 11, wherein the first exogenous nucleic acid sequence is inserted into the deleted region of the modified HSV-1 genome, and the second exogenous nucleic acid sequence is inserted between the UL3 and UL4 genes of the UL component of the modified HSV-1 genome. 15. The recombinant oncolytic HSV-1 of claim 12, wherein the first exogenous nucleic acid sequence is inserted into the deleted region of the modified HSV-1 genome, the second exogenous nucleic acid sequence is inserted between the UL3 and UL4 genes of the UL component of the modified HSV-1 genome, and the third exogenous nucleic acid sequence is inserted between the UL37 and UL38 genes of the UL component of the modified HSV-1 genome. 16. The recombinant oncolytic HSV-1 of claim 12, wherein the first exogenous nucleic acid sequence is inserted into the deleted region of the modified HSV-1 genome, the second exogenous nucleic acid sequence is inserted between the UL37 and UL38 genes of the UL component of the modified HSV-1 genome, and the third exogenous nucleic acid sequence is inserted between the UL3 and UL4 genes of the UL component of the modified HSV-1 genome. 17. The recombinant oncolytic HSV-1 according to claim 13, wherein the promoter sequence is a CMV promoter or an Egr promoter. 18. The recombinant oncolytic HSV-1 according to claim 6, wherein the immunostimulant and/or immunotherapeutic agent is humanized. 19. The recombinant oncolytic HSV-1 of claim 8, wherein the anti-PD-1 agent or the anti-CTLA-4 agent respectively comprises an antibody variable region providing specific binding to the PD-1 or CTLA-4 epitope. 20. The recombinant oncolytic HSV-1 according to claim 8, wherein the anti-PD-1 agent or anti-CTLA-4 agent is an intact antibody, an antibody fragment, or a recombinant derivative of the antibody or antibody fragment. 21. A pharmaceutical composition comprising an effective amount of the recombinant oncolytic HSV-1 of any one of claims 6 to 20 and a pharmaceutically acceptable carrier. 22. The pharmaceutical composition of claim 21, wherein the composition is formulated for intratumoral administration.