RU2810906C2 - Pharmaceutical compositions, kits and methods of treating tumors - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following is described: a group of inventions including a pharmaceutical composition for treating a tumor in a subject and a kit for treating a tumor in a subject. The pharmaceutical composition contains a therapeutically effective amount of an exosome, a therapeutically effective amount of an oncolytic herpes simplex virus and a pharmaceutically acceptable carrier, wherein the exosome contains an inhibitory amount of a CTLA4-targeting siRNA and an exomotif operably linked to a seed sequence of the CTLA4-targeting siRNA to improve packaging of the CTLA4-targeting siRNA into ezkosome, and wherein the oncolytic herpes simplex virus expresses an immunostimulating agent or both an immunostimulating agent and an anti-PD-1 antibody.

EFFECT: invention expands the arsenal of agents for treating tumors in a subject.

40 cl, 3 dwg, 2 tbl, 1 ex

Description

Field of technology

The present invention relates to a pharmaceutical composition for the treatment of a tumor and, in particular, to a pharmaceutical composition containing a therapeutically effective amount of an exosome carrying a cytotoxic T lymphocyte antigen-4 (CTLA-4)-targeting microRNA (miRNA), and a therapeutically effective amount of an oncolytic simplex virus herpes (oHSV). The present invention also provides a kit containing an exosome carrying CTLA-4-targeting siRNA and oHSV, and methods of using a pharmaceutical composition and kit for treating a tumor.

State of the art

Cancer as a disease is a multifaceted enemy that can respond to prescribed treatment or can develop resistance to various types of therapy. A subpopulation of cells within tumors is resistant to conventional treatments and may be responsible for disease relapse.

Surgical treatment of cancer is a common local treatment option. In addition to some blood system malignancies such as leukemia, lymphoma, etc., various other malignancies include one or more distinct solid tumors that can be removed surgically. However, surgical treatment always carries certain risks and is often associated with the occurrence of other concomitant diseases or potential impairment of organ function.

To date, conservative cancer treatment methods (mainly traditional chemotherapy, targeted biological therapy and radiation therapy) have not yielded completely satisfactory results. Remaining challenges include poor target selectivity, drug resistance, inability to effectively influence the course of metastatic disease, and severe side effects. In contrast, immunological therapies, which generally stimulate the host immune system to mount a systemic response against tumors, currently have great clinical promise.

Oncolytic herpes simplex viruses (oHSV) are being widely investigated for the treatment of solid tumors. As a group, they offer many advantages over traditional cancer treatments. In particular, oHSVs typically contain a mutation that renders them susceptible to the inhibitory effects of certain aspects of innate immunity. As a consequence, they replicate in cancer cells in which one or more innate immune responses to infection are impaired, but do not replicate in normal cells with intact innate immune responses. oHSV is usually injected directly into a mass of tumor tissue where the virus can replicate. Due to the fact that the virus is injected into the target tissue and not systemically, there are no side effects typical of anti-cancer drugs. Viruses characteristically induce adaptive immune responses that reduce the possibility of repeated exposure. oHSV has been injected into tumors numerous times with no evidence of loss of efficacy or ability to induce adverse reactions, such as inflammatory responses. HSVs are large DNA viruses into whose genomes foreign DNA can be inserted and the expression of these genes can be regulated when introduced into tumors. Foreign genes suitable for use with oHSV are genes that help induce an adaptive immune response to the tumor.

A defect in overcoming the innate cellular immune response determines a number of tumors in which the oHSV virus, demonstrating its oncolytic ability, manifests itself as an anticancer drug. The more extensive the deletions, the more limited the range of cancer cells in which oHSV is effective, depending on the function of the deleted viral gene. The newest oHSV carries at least one cellular gene to enhance the anticancer activity of the virus.

The success of oHSV-based therapy depends on the degree of destruction of cancer cells. Early in the development of oHSV, it was recognized that HSV alone could not kill all cancer cells in a solid tumor, and it was unlikely that treatment with oHSV could effectively remove all cancer cells, although clinical studies have shown that tumor destruction by oHSV should resolve with the development of an adaptive immune response to the tumor. Further studies showed that the antitumor immune response generated by infected tumor cell remnants could be enhanced by the incorporation of cytokines. Comparison of oHSV without the cytokine gene with oHSV containing the immunostimulatory cytokine gene confirmed this hypothesis and ultimately led to the inclusion of the GM-CSF gene in oHSV developed for the treatment of melanoma.

Incorporation of genes encoding immunostimulatory cytokines enhances the immune response to tumors but does not effectively enhance T cell cytotoxicity, which is critical for antitumor effects. Tumors have inhibitory PD-1 and CTLA-4 signaling pathways, which silence the immune system. PD-1 is expressed on both activated T cells and other hematopoietic cells, and CTLA-4 is expressed on activated T cells, including regulatory T cells. Tumors activate inhibitory PD-1 and CTLA-4 signaling pathways to evade the host immune response.

Despite extensive study and analysis in preclinical and clinical settings, there remains an unmet need for the development of tumor therapies.

Short description

In one aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of an exosome carrying a CTLA-4-targeting siRNA and a therapeutically effective amount of an oncolytic herpes simplex virus (oHSV) expressing an immunostimulatory agent or an immunostimulatory agent and an anti-PD-1 antibody.

The exosome contains an exosome packaging-associated motif (hereinafter also referred to as an “exomotif”) operably linked, optionally via a linker, to a siRNA targeting CTLA-4. In one embodiment, the exosome contains an inhibitory amount of a CTLA-4-targeting siRNA, wherein the CTLA-4-targeting siRNA has a primer sequence that binds to CTLA-4 mRNA; and an exomotif operably linked to a CTLA-4-targeting siRNA seed sequence to enhance packaging of the CTLA-4-targeting siRNA into the excosome. In some embodiments, the exomotif is located downstream of and covalently linked to the seed sequence of the CTLA-4-targeting siRNA. In some embodiments, the exomotif is located downstream and is covalently linked to the CTLA-4 seed sequence by a linker. In some embodiments, an exomotif is produced by mutating one or more nucleic acids of a CTLA-4-targeting siRNA, excluding the seed sequence. In some embodiments, the exomotif is a double motif formed by the combination of two separate exomotifs. In some embodiments, the CTLA-4-targeting siRNA and the exomotif, if operably linked, have at least one or two nucleotides in common.

oHSV is a recombinant oncolytic herpes simplex virus type 1 (HSV-1) expressing an immunostimulatory agent or both an immunostimulatory agent and an anti-PD-1 antibody.

Another aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of an exosome carrying a CTLA-4-targeting siRNA, a therapeutically effective amount of oHSV, and a pharmaceutically acceptable carrier. The exosome contains an exosome packaging-associated motif operably linked, optionally via a linker, to the siRNA targeting CTLA-4. oHSV expresses an immunostimulatory agent or both an immunostimulatory agent and an anti-PD-1 antibody.

Another aspect of the invention relates to a tumor treatment kit containing an exosome carrying siRNA targeting CTLA-4 and oHSV expressing an immunostimulatory agent or both an immunostimulatory agent and an anti-PD-1 antibody. The kit may further contain instructions for using the exosome and oHSV to treat tumors.

A further aspect of the invention relates to a method of treating a tumor in a subject, comprising simultaneously administering to the subject a therapeutically effective amount of an exosome carrying siRNA targeting CTLA-4 and a therapeutically effective amount of oHSV expressing an immunostimulatory agent or both an immunostimulatory agent and anti-PD-1 antibody.

A further aspect of the invention relates to a method of increasing the effectiveness of oHSV therapy in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of an exosome carrying a CTLA-4-targeting siRNA of the invention in addition to the oHSV therapy.

Other aspects of the invention will be made available in the description below.

Brief description of the figures

Figure 1. Panel A is a schematic diagram of the plasmid encoding siRNA targeting CTLA4. The plasmid consists of an enhanced green fluorescent protein (EGFP) coding sequence containing on its 3' untranslated region (3' UTR) a sequence coding for a designed miRNA targeting the CTLA-4 gene (miR-CTLA-4). Panel B shows the nucleotide sequence of siRNA targeting the mouse CTLA-4 gene. Nucleotides in bold italics and underlining indicate exosome packaging-associated motifs (EXO-motifs). Panel C shows the suppression of CTLA4 by the designed siRNAs. HEp-2 cells seeded in 24-well plates were cotransfected with 0.25 μg of plasmids expressing 10 DNA sequences encoding anti-CTLA-4 siRNAs (#1 - #10) or non-targeting siRNAs (NT), and 0.25 μg of plasmid encoding mouse histidine-tagged CTLA-4 (histidine-tagged CTLA-4). Cells were harvested 72 hours after transfection. The accumulation of CTLA4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured by a method known to one skilled in the art.

Figure 2. Characteristics of exosome carrying miR-CTLA-4. HEp-2 cells seeded in a T150 flask were transfected with 10 μg of the miR-CTLA-4-3# plasmid or a plasmid expressing non-target miRNA (NT), then incubated in serum-free medium. After 48 hours, the medium was collected and exosomes were purified as described in Materials and Methods. Purified exosomes were subjected to 2 sets of assays. First (panel A), equal numbers of cells in which exosomes formed and equal numbers of exosomes were dissolved, subjected to denaturing gel electrophoresis, and analyzed with antibodies to CD9, flotillin-1, and calnexin. Purified exosomes generally contained CD9, flotilla-1, but no calnexin. Size distribution of exosomes (panel B) formed by transfected cells was performed as described in Materials and Methods.

Figure 3. Effect of exosomes bearing miR-CTLA-4 administered alone (panel A) or simultaneously with T1012G (panel B), T2850 (panel C) or T3855 (panel D) on MFC tumor growth. MFC tumor cells were administered subcutaneously to C57BL/6J mice by injection into the right flank. In groups of 8 animals in which the MFC tumor averaged 80 mm ³ , 10 μg of exosome was injected into the tumor alone or simultaneously with 50 μl of 1 x 10 ⁷ PFU of T1012G, T2850, or T3855. All studies were performed simultaneously, but the results are presented in 4 panels. Tumor volumes are presented as the mean ± SEM of 8 animals in each group.

Detailed Description of the Invention

Definitions

It should be noted that "object/entity" with singular grammatical markers refers to one or more of these objects/entities; for example, "exosome" is understood to represent one or more exosomes. As such, singular grammatical markers, the terms “one or more” and “at least one” may be used interchangeably herein.

"Homology" or "identity" or "similarity" refers to the sequence similarity of two proteins or two nucleic acid molecules. Homology can be determined by comparing the position in each sequence, which can be aligned for comparison purposes. If a position in the sequence being compared is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence has less than 40% identity, but preferably less than 25% identity, with one of the sequences of the present invention.

A polynucleotide or region of a polypeptide (or polypeptide or region of a polypeptide) has a specified percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of “sequence identity” " with another sequence, meaning that in an alignment, that percentage of bases (or amino acids) is the same when comparing the two sequences. Such alignment and percentage of homology or sequence identity can be determined using software known in the art.

As used herein, the term “linker” refers to a short fragment of nucleotide sequence containing two or more nucleotides, which may be the same or different, the nucleotides being selected from the group consisting of adenine (A), guanine (G), thymine (T), and uracil (U).

As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or prophylactic measures aimed at preventing or slowing (reducing) an undesirable physiological change or disorder, such as tumor progression. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, minimization of disease severity, stabilization (i.e., no worsening) of a tumor, inhibition of tumor growth, reduction of tumor volume, delay or slowing of tumor progression, improvement of tumor status, or temporary relief, as well as remission (partial or complete), detectable or undetectable. Those who need treatment include those who already have a tumor, as well as those who are predisposed to the tumor.

By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis or therapy is desired. Mammalian subjects include humans, domesticated animals, farm and zoo animals, sporting or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows and so on. The subject herein is preferably a human.

As used herein, expressions such as “a patient in need of treatment” or “a subject in need of treatment” include subjects, such as mammalian subjects, that may benefit from administration of a composition of the present invention, used, for example, to determine, for diagnostic procedure and/or for treatment.

The present invention, among other things, utilizes antisense oligomers and similar molecules useful in modulating the function or effect of nucleic acid molecules encoding CTLA4. The hybridization of the oligomer of the present invention with its target nucleic acid is generally referred to as "antisense". Therefore, a preferred mechanism considered to be included in the implementation of certain preferred embodiments of the invention is referred to herein as "antisense inhibition". Such antisense inhibition typically relies on hybridization of oligonucleotide chains or segments to form hydrogen bonds, causing at least one chain or segment to be cleaved, degraded, or otherwise rendered nonfunctional. Therefore, antisense inhibition here preferably targets specific nucleic acid molecules and their functions.

RNA functions to be disrupted may include functions such as translocation of RNA to the site of protein translation, translocation of RNA to sites within the cell that are distant from the site of RNA synthesis, translation of protein from RNA, splicing of RNA to form one or more RNA species, and catalytic activity or complex formations involving RNA in which RNA may be involved or facilitated. One advantageous result of such disruption of target nucleic acid function is modulation of CTLA4 expression. In the context of the present invention, “modulation” and “modulation of expression” means reducing (inhibiting) the amount or levels of a nucleic acid molecule encoding a gene, such as DNA or RNA. Often the preferred target nucleic acid is mRNA.

In the context of this invention, "hybridization" means the pairing of complementary strands of oligomers. In the present invention, the preferred mechanism of pairing between complementary nucleoside or nucleotide bases (nucleic acid bases) of the chains of oligomeric compounds involves the formation of hydrogen bonds, which can occur by Watson-Crick, Hoogsteen or Hoogsteen feedback. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. Hybridization can occur under various circumstances.

In the present invention, the expression “stringent hybridization conditions” or “stringent conditions” refers to conditions under which a compound of the invention will hybridize to its target sequence but to a minimum number of other sequences. The stringency conditions are sequence dependent and will vary under different circumstances, and in the context of this invention, the “stringency conditions” under which oligomeric compounds hybridize to the target sequence are determined by the nature and composition of the oligomers and the studies by which they are studied.

The term “complementary” as used herein refers to the ability of precise pairing between two nucleic acid bases of an oligomeric compound. For example, if a nucleic base at a specific position of an oligonucleotide (oligomeric compound) is capable of forming a hydrogen bond with a nucleic base at a specific position of a target nucleic acid, the target nucleic acid being DNA, RNA or an oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the nucleic acid -target is considered a complementary position. An oligonucleotide and an additional DNA, RNA, or oligonucleotide molecule are complementary to each other if a sufficient number of complementary positions in each molecule are occupied by nucleic acid bases that can be hydrogen bonded to each other. Thus, “specifically hybridizing” and “complementary” are terms used to denote a sufficient degree of precise pairing or complementarity at a sufficient number of nucleic bases such that stable and specific binding occurs between the oligonucleotide and the target nucleic acid.

It is known in the art that the sequence of an antisense nucleotide does not need to be 100% complementary to its target nucleic acid in order to specifically hybridize. In addition, the oligonucleotide can hybridize to one or more segments such that intervening or adjacent segments are not involved in the hybridization event (eg, a loop structure or a hairpin structure). Preferably, the antisense compounds of the present invention have at least 70% or at least 75% or at least 80% or at least 85% sequence complementarity with the target region within the target nucleic acid, more preferably they have at least 90% sequence complementarity, and even more preferably, they have at least 95% or at least 99% sequence complementarity with the target sequence within the target nucleic acid sequence to which they are aimed. For example, an antisense compound in which 18 of the 20 nucleic bases of the antisense oligomer are complementary to the target region, and therefore will specifically hybridize, will have 90% complementarity. In the present example, the remaining non-complementary nucleic acid bases may be grouped or interspersed with complementary nucleic acid bases and need not be adjacent to each other or to the complementary nucleic acid bases. Thus, an 18 nucleobase long antisense oligomer containing four (4) non-complementary nucleic acid bases flanked by two regions of complete complementarity to the target nucleic acid would have an overall complementarity to the target nucleic acid of 77.8% and thus falls within the scope of the present invention. The percentage of complementarity of an antisense compound to a target nucleic acid region can be determined routinely using the BLAST (Base Local Alignment Search Tool) and PowerBLAST programs known in the art.

In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or their mimetics, chimeras, analogs and homologues. The term includes oligonucleotides containing naturally occurring nucleobases, sugars and covalent internucleoside (backbone) bonds, as well as oligonucleotides containing non-natural moieties that are functionally similar. Such modified or substituted oligonucleotides are often preferred over native forms due to desirable properties such as, for example, improved cellular uptake, improved affinity for the target nucleic acid, and increased stability in the presence of nucleases.

As used herein, the term "microRNA", "miRNA" or "miR" (miR) refers to RNA that functions to post-transcriptionally regulate gene expression, typically by binding to complementary sequences in the tri-prime (3') untranslated regions (3'- UTR) of messenger RNA (mRNA) transcripts, which usually results in gene silencing. miRNAs are usually small regulatory RNA molecules, for example, consisting of 21 or 22 nucleotides. The terms microRNA, miRNA, and miR are used interchangeably.

As used herein, the term “tumor” refers to malignant tissue containing transformed cells that grow uncontrollably (ie, a hyperproliferative disease). Tumors include leukemias, lymphomas, myelomas, plasmacytomas, etc.; and solid tumors. Examples of solid tumors that can be treated according to the invention include, but are not limited to the listed: sarcomas and carcinomas, such as melanoma, fibrosarcoma, mixeds, liposarcoma, chondrosarcoma, osteogeneous sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangiodotheliosarcarcoma, synovilia OMA, Mesotelioma, Tumor Ewing's, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma , medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, gastric carcinoma and gastric cardia carcinoma.

As used herein, the term “CTLA4” refers to “cytotoxic T-lymphocyte-associated protein 4,” which is one of many co-inhibitory molecules that are capable of suppressing T cell activation by inhibitory co-stimulation and transmitting inhibitory signals to T cells. The amino acid sequence of CTLA4 is available from NCBI under accession numbers NP_033973.2 or NP_001268905.1. CTLA4 is also known as Ctla-4, Cd152, or Ly-56. The NCBI accession number for the CTLA4 sequence is NC_000067.6 and the gene identifier is 12477. The human CTLA4 gene encodes a 233 amino acid protein that belongs to the immunoglobulin superfamily. CTLA4 consists of a single V-type domain flanked on either side by two hydrophobic regions. CTLA4 can also alter the structure of immune synapses, which play a key role in T cell proliferation and CTLA4 differentiation. CTLA4 polymorphisms are associated with susceptibility to many diseases, including type I diabetes, primary biliary cirrhosis, and Graves' disease.

The term "IL-12" as used herein refers to "interleukin 12", which is a cytokine with potent antitumor effects. Thus, IL-12 induces the TH-1 type of immune response, which can provide a sustainable antitumor effect. It has been noted that IL-12 has anti-angiogenic activity in vivo, which may also contribute to its antitumor effect. Finally, IL-12 has been noted to stimulate the production of high levels of interferon gamma (IFN-γ), which has multiple immunoregulatory effects, including the ability to stimulate the activation of cytotoxic T lymphocytes (CTL), natural killer cells and macrophages, as well as induce/enhance the expression major histocompatibility complex (MHC) class II antigens. IFN-γ plays a significant role in the process of inducing the migration of T cells into tumor sites. Increased intratumoral IFN-γ levels correlate with decreased tumor mass volume.

Programmed cell death receptor 1 (PD-1) is a 50-55 kDa type I transmembrane receptor originally identified by subtractive hybridization of an apoptotic mouse T cell line (Ishida et al., 1992, Embo J. 11:3887- 95). As a member of the CD28 gene family, PD-1 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). The amino acid sequence of human and mouse PD-1 is approximately 60% identical, with retention of four potential N-glycosylation sites and residues that define the IgG-V domain. PD-1 negatively modulates T cell activation, and this inhibitory function is associated with the immunoreceptor tyrosine inhibitory motif (ITIM) of its cytoplasmic domain (Parry et al., 2005, Mol. Cell. Biol. 25:9543-53). Disruption of this inhibitory function of PD-1 can lead to autoimmune reactions.

As used herein, the term “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 the whole antibody and any antigen binding fragment or individual chain thereof. Thus, the term "antibody" includes any protein or peptide containing a molecule that includes at least a portion of an immunoglobulin molecule having antigen binding biological activity. Examples thereof include, but are not limited to, a heavy or light chain complementarity determining region (CDR) 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 one part of the binding protein. The term antibody also includes polypeptides or polypeptide complexes that, when activated, have the ability to bind an antigen.

By “therapeutically effective amount” is meant that the oncolytic virus and/or exosome of the present invention is administered in an amount that is sufficient to effect “treatment” as described above. The amount that will be therapeutically effective in treating a particular individual disorder or condition will depend on the symptoms and severity of the disease, and can be determined by standard clinical techniques. In addition, in vivo or in vitro assays may not necessarily be used to assist in determining optimal dose ranges. The exact dosage to be used in a formulation will also depend on the route of administration and the severity of the disease or disorder, and should be determined in accordance with the judgment of the practitioner and the circumstances of each patient. Effective doses can be extrapolated from dose-response curves obtained in in vitro test systems or animal models.

siRNAs targeting CTLA4

The terms "CTLA4-targeting siRNA", "CTLA4-targeting siRNA" and "CTLA4-targeting siRNA", which are used interchangeably herein, refer to small non-coding RNA (microRNA or miRNA) configured to target or specifically bind with the mRNA encoding the CTLA4 protein in such a way that transcription, translation and, as a consequence, the expression of CTLA4 are disrupted, reduced or eliminated. As described above, siRNA does not necessarily bind to the target mRNA with 100% specificity. It is known that miRNA has a seed sequence (2-8 nucleotides from the 5' end), which determines the specificity of binding to the target mRNA, while the remaining nucleotides are not necessarily exactly complementary to the target mRNA. Thus, in one embodiment, the siRNA has a seed sequence from any of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the siRNA targeting CTLA4 blocks expression of the CTLA4 protein in the cell after its delivery to the tumor cell.

Exosomes carrying siRNA targeting CTLA4

Exosomes are small vesicles of relatively universal size that are formed from cell membranes. For example, exosomes can have a diameter of from about 30 to about 100 nm. They contain several key proteins (e.g. CD9, CD63, CD81, CD82, annexation, flotillin, etc.) and, in addition, they package proteins, mRNAs, long non-coding RNAs and miRNAs. Exosomes transport substances from cell to cell. Upon entry into recipient cells, the contents of the exosome are released into the cytoplasm.

In some embodiments, the siRNA targeting CTLA4 is delivered into the cell via an exosome. Thus, one embodiment of the invention provides an exosome carrying any of the CTLA4-targeting siRNAs as described above. The present invention uses a fragment of nucleotide sequence, referred to herein as an "exomotif", to promote or stimulate the packaging of siRNA into an exosome. In one embodiment, the exomotif is selected from any of the sequences defined in Table 1.

In some embodiments, exomotives are used in combination. For example, two or more exomotifs defined in the table are combined to form a double exomotif. Motifs can be combined linearly by linking the 5' end of one exome to the 3' end of another exome. In this context, if the first nucleotide of the 5' end of one exomotif is identical to the last nucleotide of the 3' end of another exomotif, one of the identical nucleotides can be excluded. For example, "GGAG" (SEQ ID NO: 21) is combined with "GGAC" (SEQ ID NO: 22) to form the double exomotif "GGAGGAC" (SEQ ID NO: 48). If the first nucleotide of the 5' end of one exomotive is different from the last nucleotide of the 3' end of another exomotive, the two exomotifs can be connected by a linker or directly by a covalent bond. For example, "GGAC" (SEQ ID NO: 22) can be combined with "GGAG" (SEQ ID NO: 21) linker "TG" to form the double exomotif "GGACUGGGAG" (SEQ ID NO: 49), "GGAC" (SEQ ID NO: 22) can also be combined with "GGAG" (SEQ ID NO: 21) by a covalent bond to form the double exomotif "GGACUGGGAG" (SEQ ID NO: 51). The present invention also contemplates a triple or more exomotif, for example, an exomotif containing three or more motifs from SEQ ID NO: 21 to SEQ ID NO: 47. Thus, the term “exomotif” as used herein includes nucleotide sequences that are capable of stimulate or promote packaging of siRNA into an exosome, including any of the single exomotifs from SEQ ID NO: 21 through SEQ ID NO: 47, as well as any double (for example, any of SEQ ID NO: 48-52), triple or more exomotifs, obtained using a combination of individual motifs.

In the present invention, the exomotif is operably linked to the siRNA seed sequence. The term “operably linked” refers to a functional relationship between a regulatory sequence (eg, an exomotif) and a nucleic acid sequence (eg, an siRNA seed sequence) resulting in promoting or facilitating packaging of the siRNA into an exosome. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in operative relationship to a second nucleic acid sequence. Functionally related RNA sequences may be adjacent to each other or may be associated with a linker.

In some embodiments, the exomotif is located downstream of the siRNA seed sequence. In some embodiments, the exomotif is located upstream of the siRNA seed sequence. In some embodiments, the miRNA seed sequence is flanked on both sides by exomotifs. In one embodiment of the invention, the exomotif is operably linked to the siRNA seed sequence. In one embodiment of the invention, an exomotif is obtained by mutation of one more nucleotide sequence of the siRNA, with the exception of the seed sequence. In one embodiment, the CTLA4-targeting siRNA with an exomotif comprises the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In one embodiment, the CTLA4-targeting siRNA with The exomotif is the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

In some embodiments, in which the exomotif is located downstream of the siRNA seed sequence, the last nucleotide at the 3' end of the seed surface and the first nucleotide at the 5' end of the exomotif are the same nucleotide, for example, a guanine nucleotide "G". For example, SEQ ID NO: 6 shows that the exomotif and seed sequence share a guanine nucleotide "G". In some embodiments, in which the exomotif is located downstream of the siRNA seed sequence, the last two nucleotides at the 3' end of the seed sequence and the first two nucleotides at the 5' end of the exomotif are the same nucleotides, e.g., guanine "GG" nucleotides. For example, SEQ ID NO: 8 shows that the exomotif and seed sequence share two guanine nucleotides "GG".

In some embodiments, the exomotif is located downstream of the miRNA seed sequence and is linked to the miRNA seed sequence by a linker, such as "GC". For example, SEQ ID NO: 7 indicates that the exomotif and the seed sequence are linked by a "GC" linker.

In addition to the seed sequence and exomotif, the miRNA also includes an additional nucleic acid sequence to enhance (amplify) the target region of the mRNA. These additional nucleic acids are typically found downstream of the exomotive and are several nucleotides in length, for example 1 to 10 nucleotides. The additional nucleic acid sequences are preferably complementary to the corresponding segment of the target mRNA, however, as described above, the complementarity does not have to be 100%.

Methods for transferring siRNA into an exosome are available in the art, such as cotransfecting a cell with an siRNA expression vector and a plasmid encoding CTLA4, as described in the Example. Exosome isolation, identification, and characterization are technically feasible in the state of the art. Some proteins, such as CD9, CD63, CD81, CD82, annexation, flotillin, etc., can be used as a marker of exosomes. Other methods for packaging siRNA into exosomes may also be applicable to the present invention.

The exosome of the present invention contains an inhibitory amount of siRNA targeting CTLA4. An inhibitory amount means an amount sufficient to inhibit the expression of CTLA4 protein after the siRNA in question has been delivered to the tumor cell.

The table below lists the siRNA nucleic acid sequences, seed sequences and siRNA motifs used in the Example of the invention.

Oncolytic herpes simplex virus (oHSV)

Oncolytic herpes simplex virus (oHSV) as used herein refers to any oncolytic herpes simplex virus type 1 (HSV-1) known in the art, designed, useful for, or effective in killing a tumor cell. In addition, the oHSV used in the present invention may also be genetically engineered (modified) such that one or more characteristics of the naturally occurring oHSV are removed. In addition or alternatively, naturally occurring oHSV can be genetically engineered to introduce one or more exogenous fragments of coding sequences into the viral genome so as to confer one or more additional functions to the virus, such as immunotherapeutic or immunostimulatory properties.

One skilled in the art will appreciate that the precise starting and ending positions of the nucleotides to be deleted in accordance with the present invention depend on the strains and isomers of the HSV-1 virus genome and can be readily determined by methods known in the art. In some embodiments, the deletion causes excision of nucleotides 117005 to 132096 in the genome. In some embodiments, the oHSV is selected from strain 17 (GenBank accession no. NC 001806.2), strain KOS 1.1 (GenBank accession no. KT899744), or strain F (GenBank accession no. GU734771.1) of HSV-1. In some embodiments, oHSV is HSV-1 strain F.

In some embodiments, oHSV is a genetically engineered HSV-1 F strain expressing an immunostimulatory agent that is selected from GM-CSF, IL-2, IL-5, IL-12, IL-15, IL-24, and IL-27. In some embodiments, oHSV is a genetically engineered HSV-1 F strain that expresses only IL-12. In some embodiments, oHSV is a genetically engineered HSV-1 F strain expressing both IL-12 and anti-PD-1 antibody.

Methods and means of therapy

In one aspect of the invention, there is provided a method of treating a tumor in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of an exosome carrying siRNA targeting CTLA-4 and a therapeutically effective amount of oHSV expressing an immunostimulatory agent or both an immunostimulatory agent and anti-PD-1 antibody.

In one aspect of the invention, there is provided a method of increasing the effectiveness of oHSV therapy in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of an exosome carrying a CTLA-4-targeting siRNA of the invention in addition to the oHSV therapy. In some embodiments, oHSV expresses an immunostimulatory agent or both an immunostimulatory agent and an anti-PD-1 antibody. In some embodiments, oHSV expresses only IL-12. In some embodiments, oHSV expresses IL-12 and an anti-PD-1 antibody.

In some embodiments, in the methods of the invention, administration of exosomes carrying siRNA targeting a CTLA-4 inhibitor and oHSV expressing an immunostimulatory agent or both an immunostimulatory agent and an anti-PD-1 antibody is accomplished by administering to the subject a pharmaceutical composition containing a therapeutically effective amount an exosome carrying siRNA targeting CTLA-4, and a therapeutically effective amount of oHSV expressing an immunostimulatory agent or both an immunostimulatory agent and an anti-PD-1 antibody, and a pharmaceutically acceptable carrier.

In some embodiments, in the methods of the invention, administration of exosomes carrying siRNA targeting CTLA-4 and oHSV expressing an immunostimulatory agent or both an immunostimulatory agent and an anti-PD-1 antibody is accomplished by administering to the subject exosomes carrying siRNA targeting CTLA-4, and oHSV expressing an immunostimulatory agent or both an immunostimulatory agent and an anti-PD-1 antibody, separately. In some embodiments, exosomes carrying siRNA targeting CTLA-4 are administered before, simultaneously with, or after administration of oHSV expressing an immunostimulatory agent or both an immunostimulatory agent and an anti-PD-1 antibody. In such cases, it is contemplated that both agents may be administered to the subject with an interval of approximately 12 to 72 hours between administrations. However, in some situations it may be desirable to significantly increase the treatment period, with periods ranging from several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7) between appropriate administrations. or 8).

In some embodiments, exosomes carrying siRNA targeting CTLA-4 are administered concomitantly with the administration of oHSV expressing an immunostimulatory agent or both an immunostimulatory agent and an anti-PD-1 antibody. In some embodiments, exosomes carrying siRNA targeting CTLA-4 are administered in the form of a pharmaceutical composition comprising a therapeutically effective amount of exosomes carrying siRNA targeting CTLA-4 and a pharmaceutically acceptable carrier, a oHSV, expressing an immunostimulatory agent or as an immunostimulatory agent. agent and the anti-PD-1 antibody are administered in the form of a pharmaceutical composition containing a therapeutically effective amount of oHSV expressing the immunostimulating agent or both the immunostimulating agent and the anti-PD-1 antibody, and a pharmaceutically acceptable carrier. In such embodiments, a pharmaceutical composition comprising a therapeutically effective amount of exosomes carrying siRNA targeting CTLA-4 and a pharmaceutically acceptable carrier, as well as a pharmaceutical composition containing a therapeutically effective amount of oHSV expressing an immunostimulatory agent or both an immunostimulatory agent and an anti- The PD-1 antibody and the pharmaceutically acceptable carrier may be packaged in one kit.

In certain embodiments of the invention, any pharmaceutical composition is administered parenterally or non-parenterally, i.e. intratumorally, intravenously, intramuscularly, percutaneously or intradermally. In some embodiments, any pharmaceutical composition is preferably administered intratumorally.

In some embodiments, a method of treating a tumor is aimed at increasing the antitumor efficacy of oHSV therapy, for example, by inhibiting tumor growth and/or reducing the volume of tumors. Accordingly, in some embodiments, a method of treating a tumor is provided, comprising administering to a subject in need thereof a therapeutically effective amount of an exosome in combination with a therapeutically effective amount of oHSV, as described above. In certain embodiments of the invention, a method of treating a tumor prevents the onset, progression and/or recurrence of symptoms associated with the tumor. Thus, in some embodiments, a method of preventing tumor-related symptoms in a subject includes administering a therapeutically effective amount of exosome and a therapeutically effective amount of oHSV, as described above, to a subject in need thereof.

In some embodiments, oHSV is a genetically engineered HSV-1 F strain expressing an immunostimulatory agent that is selected from GM-CSF, IL-2, IL-5, IL-12, IL-15, IL-24, and IL-27. In some embodiments, oHSV is a genetically engineered HSV-1 F strain that expresses only IL-12. In some embodiments, oHSV is a genetically engineered HSV-1 F strain expressing both IL-12 and anti-PD-1 antibody.

The methods according to the invention involve the treatment of various tumors, in particular solid tumors. Examples of solid tumors that can be treated according to the invention include, but are not limited to, sarcomas and carcinomas such as melanoma, fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, tumor Ewing's, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma , medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, gastric carcinoma and gastric cardia carcinoma.

Pharmaceutical compositions and kits

In one aspect of the invention, a pharmaceutical composition is provided comprising a therapeutically effective amount of an exosome, a therapeutically effective amount of oHSV as described above, and a pharmaceutically acceptable carrier. The pharmaceutical composition is used for the prevention and treatment of a tumor in a subject. The pharmaceutical composition may be formulated in a suitable pharmaceutically acceptable carrier or excipient.

In another aspect of the invention, there is provided a first pharmaceutical composition comprising a therapeutically effective amount of an exosome, as described above, and a pharmaceutically acceptable carrier. In addition, a second pharmaceutical composition is provided containing a therapeutically effective amount of oHSV as described above and a pharmaceutically acceptable carrier. In such an aspect, a kit is provided comprising a first pharmaceutical composition and a second pharmaceutical composition in a single package. The kit may further include a specification for use that can be referred to by the physician during clinical use.

In some embodiments, oHSV is a genetically engineered HSV-1 F strain expressing an immunostimulatory agent that is selected from GM-CSF, IL-2, IL-5, IL-12, IL-15, IL-24, and IL-27. In some embodiments, oHSV is a genetically engineered HSV-1 F strain that expresses only IL-12. In some embodiments, oHSV is a genetically engineered HSV-1 F strain expressing both IL-12 and anti-PD-1 antibody.

Under normal conditions of storage and use, such preparations/compositions contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid enough to be easily administered by syringe. It must be stable during manufacture and storage, and must be protected from contamination by microorganisms such as bacteria and fungi.

The carrier may be a solvent or dispersion medium including, for example, water, ethanol, polyhydric alcohol (eg, glycerin, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, and/or vegetable oils. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, maintaining the desired particle size in the case of dispersion and using surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases it will be preferable to include isotonic substances such as sugars, sodium chloride or phosphate buffered saline. Prolonged absorption of injectable compositions can be achieved by using absorption delaying agents in the compositions, for example, aluminum monostearate and gelatin.

For parenteral administration in aqueous solution, for example, the solution must optionally contain a buffer and the liquid vehicle must first be made isotonic with a sufficient amount of salt or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this regard, those skilled in the art will know a sterile aqueous medium that can be used for the present invention. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or administered by injection at the intended infusion site (see, for example, Remington's Pharmaceutical Sciences, 15th edition, pages 1035- 1038 and 1570-1580). Some dosage changes will be inevitable depending on the condition of the subject being treated. The person responsible for administering the drug will in any event determine the appropriate dose for the individual subject. Moreover, for administration to humans, drugs must meet the standards of sterility, pyrogenicity, general safety and purity as required by the US Food and Drug Administration.

Sterile injection solutions are prepared by incorporating the active compounds in the required quantity into an appropriate diluent together with a number of other ingredients listed above, if necessary, followed by sterilization by filtration. Typically, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle that contains a base dispersion medium and the necessary other ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, yielding a powder of the active ingredient plus any additional desired ingredient, from a solution thereof previously sterilized by filtration.

The compositions of the present invention can be formulated in neutral form or as a salt. Pharmaceutically acceptable salts include acid addition salts (formed by free amino groups of proteins) which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups can also be prepared with inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like. Once formulated, the solutions will be administered in a manner consistent with the dosage form and in amounts that are therapeutically effective. The compositions are readily administered in a variety of dosage forms, such as injection solutions, drug release capsules, and the like.

As used herein, the term “carrier” includes any and all solvents, dispersion media, carrier media, diluents, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except to the extent that any conventional vehicle or agent is incompatible with the active ingredient, it is intended for use in therapeutic compositions. Also, auxiliary active ingredients can be included in the composition.

The expression "pharmaceutically acceptable" refers to molecules and compositions that do not cause an allergic or similar adverse reaction when administered to humans. The preparation of an aqueous composition that contains protein as an active ingredient is well known in the art. Typically, such compositions are prepared as injectable preparations, either as liquid solutions or suspensions; Solid forms suitable for dissolution in liquid or suspension prior to injection may also be prepared.

Examples

We describe the development of adjuvant therapies involving exosomes engineered to carry and release siRNA into tumor cells, designed to specifically target mRNAs encoding the CTLA-4 checkpoint.

Exosomes are extracellular vesicles defined for therapeutic purposes by their size and protein content. They package RNA and proteins in the cells in which they are produced and deliver cargo to the cells they act on. In the studies described in this report, the desired exosome content was siRNA.

miRNAs are powerful tools that can, in principle, be used to control the replication of individual protein-coding RNAs. The objectives were to develop siRNAs that could block the replication of cytotoxic T-lymphocyte-associated protein 4 and that could be delivered to infected cells using exosomes. Ten siRNAs were designed to target the mRNA encoding CTLA4. Of these 10 miRNAs, miR-CTLA4-1#, miR-CTLA4-2#, miR-CTLA4-3#, and miR-CTLA4-6#, when transfected into susceptible cells, effectively blocked the accumulation of CTLA4. In order to facilitate the packaging of miRNAs into exosomes, we introduced into the sequence miR-CTLA4-3# a motif responsible for packaging into exosomes. Similar to our previous study, miR-CTLA4-3# could be packaged into exosomes and successfully delivered by exosomes to susceptible cells, in which it remained stable for at least 72 hours. Moreover, miR-CTLA4-3# delivered to tumors by exosomes effectively reduced CTLA4 expression.

RNA incorporation into exosomes is sequence dependent and is facilitated by the action of hnRNPA2B1, a component of exosomes. hnRNPA2B1 sorts into exosomes RNAs containing one of two known motifs responsible for packaging into exosomes (EXO-motifs). The key function of hnRNPA2B1 is to regulate the transport of mRNA to axons in nerve cells, which is mediated by the binding of a 21-nucleotide RNA sequence called an RNA transport sequence (RTS). These sequences contain both EXO motifs.

It was found that the tumor in mice is relatively resistant to the oncolytic effects of murine T1, T2 and T3 series of oHSV. The T1 series oHSV is an HSV-1 F strain that does not express the immunoregulatory agent (hereinafter also referred to as “T1012G”). The T2 series of oHSV is an F strain of HSV-1 that expresses only IL-12 (hereinafter also referred to as “T2850”). The T3 series of oHSV is an HSV-1 F strain that simultaneously expresses IL-12 and anti-PD-1 antibodies (hereinafter also referred to as “T3855”).

The examples show that the antitumor efficacy of T2850 and especially T3855 can be enhanced and the volume of tumors can be reduced by delivering to tumors a composition containing T3855 (or T2850) and exosomes carrying siRNA designed to target the mRNA encoding CTLA-4.

Materials and methods

Model using syngeneic mice. Syngeneic Balb/c mice were used for the MFC tumor model.

Plasmids expressing miR-CTLA-4. SiRNA target sequences against mouse CTLA4 were designed using Life Technologies' BLOCK-iT™ RNAi Designer software and synthesized by Ige Biotechnology (Guangzhou, China). The synthesized miRNA fragments were digested using the restriction enzymes BamHI and XhoI and cloned into the corresponding sites of the control plasmid pcDNA6.2-GW/EmGFP-miR-neg (mvitrogen).

The miRNA sequences are presented below:

Underlining indicates the mature miRNA sequence.

Expression plasmid carrying mouse histidine-tagged CTLA4 (mCTLA4-his) was purchased from YouBio Biotechnology (Changsha, China).

Cell Lines HEp-2 cells were obtained from the American Type Culture Collection and were cultured in Dulbecco's modified Eagle's minimal essential medium (Life Technologies) supplemented with 5% (v/v) fetal bovine serum (FBS) as per routine practice. MFC (mouse proventriculus carcinoma) cells were kindly provided by JOINN Laboratories, Inc. (Beijing, China). B16 (murine melanoma) were kindly provided by Shenzhen International Biomedical Research Institute (Shenzhen, China).

Antibodies. The antibodies used in this study were anti-His-tag (catalog number: 66005-1-Ig, Proteintech Group) and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (catalog number: # 2118, Cell Signaling Technology).

Exosome isolation. HEp-2 cells (5 × 10 ^b ) were transfected using 10 μg of plasmids expressing miR-CTLA-4. After 4 h of incubation, the cells were washed three times with PBS to eliminate potential exosome contamination in the serum, and the cells were cultured in serum-free medium for the next 48 h. The supernatant was collected, mixed with the recommended dose of Total Exosome Isolation kit reagent (Thermo Fisher part number: 4478359), stored overnight at 4°C and then centrifuged for 1 hour. The centrifugation-pelleted exosomes were then resuspended in 200 ml PBS or lysed in radioimmunoprecipitation assay (RIPA) buffer and then quantified by the bicinchoninic acid (BCA) method using the Enhanced BCA Protein Assay Kit (Beyotime Biotechnology, China) according to the instructions. manufacturer. The protein content in the exosome was determined by calibration using a standard curve, which was obtained by plotting the absorbance at a wavelength of 562 nm compared with the standard concentration of bovine serum albumin.

Exosome size assays and quantification The size distribution of exosomes was analyzed using the qNano system (Izon, Christchurch, New Zealand). Izon's qNano technology (www.izon.com) was used to detect extracellular vesicles passing through a nanopore by single-molecule electrophoresis. In practice, this makes it possible to obtain accurate particle sizes of vesicles for exosome sizes from 75 to 150 mn without averaging particle sizes. Purified exosomes were diluted 1:10 in PBS with 0.05% polysorbate-20, shaken vigorously, and measured using an NP150 nanopore aperture (A45540) according to the manufacturer's instructions. Data processing and analysis were performed using Izon Control Suite version 3.3 software (Izon Science).

Immunoblot analysis. Detection of CTLA-4, histidine-tagged proteins, GAPDH, and proteins involved in exosome formation using immunoblot analysis. Cells were harvested and lysed using RIPA lysis buffer (Beyotime) supplemented with 1 mM protease inhibitor phenylmethylsulfonyl fluoride (PMSF) (Beyotime) and phosphatase inhibitor (Beyotime). Cell lysates were heat-denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore). Proteins were detected by incubation with the appropriate primary antibody followed by a horseradish peroxidase-conjugated secondary antibody (Pierce) and ECL reagent (Pierce) and filmed or imaged using the ChemiDoc Touch Imaging System (Bio-Rad) and processed using ImageLab software. The densities of the corresponding layers were quantified using ImageJ software.

oHSV design. An exemplary oHSV design (such as T2850 and T3855) includes a recombinant oncolytic herpes simplex virus type 1 (HSV-1) containing (a) a modified HSV-1 genome, wherein the modification contains a deletion between the U.56 gene promoter and the U.56 gene promoter of the genome wild-type HSV-1 such that (i) one copy of all duplicated genes is missing and (ii) the sequences required for expression of all present open reading frames (ORFs) in the viral DNA after deletion are intact: and (b) heterologous nucleic acid sequences acids encoding an immunostimulating and/or immunotherapeutic agent, wherein the heterologous nucleic acid sequence is stably integrated into at least a remote region of the modified HSV-1 genome. When only one heterologous nucleic acid sequence encoding an immunostimulatory or immunotherapeutic agent is inserted, the heterologous nucleic acid sequence is preferably incorporated into a remote region of the genome. When incorporating more than one heterologous nucleic acid sequences encoding immunostimulatory and/or immunotherapeutic agents, the first heterologous nucleic acid sequences are preferably inserted into a remote region of the genome. The second or subsequent heterologous nucleic acid sequences may be inserted into the L component of the genome.

A more detailed description of the design and properties of oncolytic herpes simplex virus (oHSV) is available in WO 2017/181420.

results

Design and production of exosomes containing miR-CTLA-4 (miR-CTLA-4 exo)

The goal of the first series of experiments was to design and produce exosomes containing siRNA targeting CTLA4. To do this, we first designed 10 miRNAs, designated as miR-CTLA4-l#, miR-CTLA4-2#, miR-CTLA4-3#, miR-CTLA4-4#, miR-CTLA4-5#, miR-CTLA4-6 #, miR-CTLA4-7#, miR-CTLA4-8#, miR-CTLA4-9# and miR-CTLA4-10#. The sequence of each miRNA is shown in Figure 1, which includes additional sequences downstream of the miRNA seed sequence, which are the exosome packaging-related motifs (exomotifs). As shown in Figure 1A, the siRNA was cloned downstream of the open reading frame encoding EGFP into a siRNA expression vector called “control plasmid pcDNA6.2-GW/EmGFP-miR-neg” as described in Materials and Methods.

Then, for miRNA analysis, HEp-2 cells were cotransfected with miRNA expression vectors (miRmCTLA4-l#, miRmCTLA4-2#, miRmCTLA4-3#, miRmCTLA4-4#, miRmCTLA4-5#, miRmCTLA4-6#, miRmCTLA4-7#, miRmCTLA4-8#, miRmCTLA4-9#, miRmCTLA4-10#.), described above, and a plasmid encoding C-terminal histidine-tagged CTLA4 (mCTLA4-His). As shown in Figure 1 B, of the 10 constructs, miR-CTLA4-3# was most effective in suppressing CTLA4 accumulation. The results show that CTLA4 accumulation is suppressed by miR-CTLA4-3# with higher efficiency. miR-CTLA4-l#, miR-CTLA4-2#, miR-CTLA4-4#, miR-CTLA4-5#, miR-CTLA4-6# and miR-CTLA4-7# showed a moderate effect, while non-targeting (NT ) plasmids miR-CTLA4-8#, miR-CTLA4-9# and miR-CTLA4-10# had no effect on CTLA4 accumulation (Figure 1B). Thus, miR-CTLA4-3# was selected for further studies.

In the next step, we constructed exosomes encoding the selected miR-CTLA-4. HEp-2 cells seeded in a T150 flask were transfected with 10 μg of a plasmid encoding miR-CTLA-4-3# or a plasmid expressing a nontarget miRNA (NT). After 48 hours, the extracellular medium was collected and exosomes were purified as described in Materials and Methods.

Characteristics of exosomes carrying miR-CTLA-4.

Several experiments have been carried out to characterize the exosome carrying miR-CTLA-4. First, equal numbers of cells in which exosomes formed and equal numbers of exosomes were dissolved, subjected to denaturing gel electrophoresis, and analyzed using antibodies to CD9, flotillin-1, and calnexin. As expected, the result (Figure 2A) shows that exosomes contain CD9 and flotillin-1, but not calnexin.

Exosomes purified from HEp-2 cells transfected with 10 μg of a plasmid encoding miR-CTLA or a plasmid expressing a non-targeting miRNA (miR-NT) were then measured for size by nanoparticle trajectory analysis using Izon's qNano technology. The result (Figure 2B) shows that the diameter of exosomes obtained in transfected cells averages 100–200 nm.

Coadministration of exosomes bearing miR-CTLA-4 and oHSV into implanted MFC tumors enhanced the oncolytic activity of T3, but not T1 or T2 oHSVs.

In the first step of this series of experiments, replicate HEp-2 cultures were transfected with the miR-CTLA4-3# plasmid, and the cells were cultured for 48 hours. The HEp-2-derived exosome (miR-CTLA-4 exo) was then purified as described in the Materials and Methods section.

In the second step, oHSV T1012G, T2850, and T3855 were constructed as described in Materials and Methods. Mouse forestomach carcinoma cells (MFC) were then injected into C57BL/6J mice (8 groups of 8 mice) subcutaneously via right flank injection to form a tumor. When tumors reached an average size of 80 mm ³ , they were intratumorally injected with a single dose of 1 × 10 ⁷ PFU of T1012G (panel B), T2850 (panel C), or T3855 (panel D) alone or in combination with 10 Pg miR-CTLA exosomes -4.

At the final stage, tumor volumes were measured every 3 or 4 days until 26 days after injection. The result shows that the tumor volume in each group gradually increased after injection. Figure 3A shows that the tumor volume of the miRNA-CTLA4 exo-injected mouse increased at almost the same rate as the control group, and at day 26 post-injection, the tumor volume of the miRNA-CTLA4 exo-injected mouse was larger. than in the control group. Figure 3B shows that the tumor volume in the control group, T1012G and T1012G+miRNA-CTLA4 groups gradually increased after injection. At 26 days post-injection, the tumor volume of the mouse injected with T1012G + miRNA-CTLA4 exo was smaller than that of the control group and the T1012G group. In Figure 3C, tumor volume in the T2850 and T2850 + miRNA-CTLA4 exo groups increased more slowly than in the control group. 26 days after injection, tumor volume in the T2850 and T2850 + miPvNA-CTLA4 exo groups was smaller than in the control group. And the tumor volume in the T2850 + miRNA-CTLA4 exo group was not significantly different from the tumor volume in the T2850 only group. In Figure 3D, the tumor volume of the T3855 + miRNA-CTLA4 exo group increased the slowest, followed by the T3855 group and finally the control group. At 26 days after injection, the tumor volume in the control group was the largest, followed by the T3855 group in volume, and the smallest tumor volume was in the T3855 + miRNA-CTLA4 exo group.

The results of this series of experiments show that T2850 and T3855 can effectively inhibit tumor growth, and simultaneous intratumoral administration of T3855 and miRNA-CTLA4 exo enhances the antitumor efficacy of T3855 and inhibits tumor growth.

The results show that miR-CTLA4-3# targets CTLA-4 and suppresses the expression of CTLA-4. We also showed that miR-CTLA4-3# is packaged into exosomes and purified exosomes contained CD9, flotilla-1, but did not contain calnexin. Exosomes produced using Hep-2 cells transfected with the miR-CTLA4-3# plasmid or non-targeting siRNA (NT) average 100-200 nm in diameter. The results presented here also show that oHSV expressing IL-12 alone or both IL-12 and anti-PD-1 antibody can effectively inhibit tumor growth. In conclusion, we have shown that simultaneous intratumoral administration of an exosome carrying siRNA targeting CTLA-4 and oHSV expressing both IL-12 and anti-PD-1 antibody enhances the antitumor efficacy of oHSV and inhibits tumor growth.

It should be understood that while the present invention has been specifically disclosed by way of preferred embodiments and optional features, modifications, improvements, and changes may be made to the teachings embodied herein by those skilled in the art, and that such modifications, improvements, and changes are considered to be within the scope of the present invention. The materials, methods and examples presented here represent preferred embodiments of the invention, are illustrative and are not intended to limit the scope of the invention.

All publications, patent applications, patents and other sources referenced herein are expressly incorporated by reference in their entirety to the same extent as if each had been individually incorporated by reference. In the event of a conflict, this specification, including definitions, will control. The disclosures described illustratively herein may be appropriately practiced in the absence of any element or elements, limitations or limitations not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. should be understood broadly and without limitations. In addition, the terms and expressions used herein are used as terms of description and not limitation, and in the use of such terms and expressions it is not intended to exclude any equivalents of the features shown and described, or portions thereof, but it is recognized that various modifications are possible in within the scope of the claimed invention.

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<120> PHARMACEUTICAL COMPOSITIONS, KITS AND METHODS FOR TREATING TUMORS

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<210> 26

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 26

gggc 4

<210> 27

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 27

ugag 4

<210> 28

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 28

ugac 4

<210> 29

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 29

ugcg 4

<210> 30

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 30

ugcc 4

<210> 31

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 31

ugggg 4

<210> 32

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 32

uggc 4

<210> 33

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 33

cgag 4

<210> 34

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 34

cgac 4

<210> 35

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 35

cgcg 4

<210> 36

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 36

cgcc 4

<210> 37

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 37

cggg 4

<210> 38

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 38

cggc 4

<210> 39

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 39

cccu 4

<210> 40

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 40

cccg 4

<210> 41

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 41

ccca 4

<210> 42

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 42

uccu 4

<210> 43

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 43

uccg 4

<210> 44

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 44

ucca 4

<210> 45

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 45

GCCU 4

<210> 46

<211> 4

<212> DNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 46

gccg 4

<210> 47

<211> 4

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 47

gcca 4

<210> 48

<211> 7

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 48

ggaggac 7

<210> 49

<211> 10

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 49

ggacugggag 10

<210> 50

<211> 7

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 50

ggagggag 7

<210> 51

<211> 8

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 51

ggacggag 8

<210> 52

<211> 10

<212>RNA

<213> Artificial Sequence

<220>

<223> exo-motif

<400> 52

ggagggcggag 10

<210> 53

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> sequences of miR-CTLA4-1#

<400> 53

aaccttcagt ggagttggcg agttttggcc actgactgac tcgccaacca ctgaaggtt 59

<210> 54

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> sequences of miR-CTLA4-2#

<400> 54

cttcagtgga gttggcgagc agttttggcc actgactgac tgctcgccct ccactgaag 59

<210> 55

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> sequences of miR-CTLA4-3#

<400> 55

actgtgctgc ggaggacaaa tgttttggcc actgactgac atttgtcccg cagcacagt 59

<210> 56

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> sequences of miR-CTLA4-4#

<400> 56

cgacattcac ggaggagaat agttttggcc actgactgac tattctcccg tgaatgtcg 59

<210> 57

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> sequences of miR-CTLA4-5#

<400> 57

gaacctcacc ctccaaggac tgttttggcc actgactgac agtccttggg gtgaggttc 59

<210> 58

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> sequences of miR-CTLA4-6#

<400> 58

atccaaggac tgggagctgt tgttttggcc actgactgac aacagctcag tccttggat 59

<210> 59

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> sequences of miR-CTLA4-7#

<400> 59

actcatgtac cctccgccat agttttggcc actgactgac tatggcgggg tacatgagt 59

<210> 60

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> sequences of miR-CTLA4-8#

<400> 60

ggcaacggga ggcggagtta tgttttggcc actgactgac ataactccct cccgttgcc 59

<210> 61

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> sequences of miR-CTLA4-9#

<400> 61

gggcaacggg acggagattt agttttggcc actgactgac taaatctctc ccgttgccc 59

<210> 62

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> sequences of miR-CTLA4-10#

<400> 62

tgactgtgct gcggcggaca agttttggcc actgactgac ttgtccgcca gcacagtca 59

<---

Claims (48)

1. A pharmaceutical composition for treating a tumor in a subject, comprising (a) a therapeutically effective amount of exosome, (b) a therapeutically effective amount of oncolytic herpes simplex virus; and (c) a pharmaceutically acceptable carrier, wherein the exosome contains an inhibitory amount of a CTLA4-targeting siRNA and an exomotif operably linked to a seed sequence of the CTLA4-targeting siRNA to enhance packaging of the CTLA4-targeting siRNA into the exosome, and wherein the oncolytic herpes simplex virus expresses an immunostimulating agent or both an immunostimulating agent and an anti-PD-1 antibody. 2. The composition according to claim 1, characterized in that the seed sequence of the CTLA4-targeting siRNA contains any of the nucleic acid sequences from SEQ ID NO: 1 to SEQ ID NO: 4. 3. The composition according to claim 1 or 2, characterized in that the exomotif is selected from the group consisting of the nucleic acid sequence from SEQ ID NO: 21 to SEQ ID NO: 49. 4. The composition according to claim 1, characterized in that the exomotif is located downstream and is covalently linked to the seed sequence of the CTLA4-targeting siRNA. 5. The composition according to claim 1, characterized in that the exomotif is obtained by mutation of one or more nucleic acids of the CTLA4-targeting siRNA, with the exception of the seed sequence. 6. The composition of claim 1, characterized in that the exomotif is a dual motif formed by combining two separate exomotifs, wherein any of the two separate exomotifs is selected from the group consisting of the nucleic acid sequence of SEQ ID NO: 21 to SEQ ID NO: 47. 7. The composition according to claim 1, characterized in that the dual motif has the nucleic acid sequence SEQ ID NO: 48. 8. The composition according to claim 1, characterized in that the CTLA4-targeting siRNA and the exomotif, if operably linked, contain at least one common nucleotide or two common nucleotides or are connected through a linker. 9. The composition according to claim 8, characterized in that the linker consists of two or more nucleotides selected from the group consisting of adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) . 10. The composition according to claim 9, characterized in that the linker is -GC-. 11. The composition according to claim 1, characterized in that the CTLA4-targeting siRNA and the exomotif, if operably linked, have the nucleic acid sequence SEQ ID NO: 7. 12. The composition according to claim 1, characterized in that the immunostimulating agent is selected from GM-CSF, IL-2, IL-5, IL-12, IL-15, IL-24 and IL-27. 13. The composition according to claim 12, characterized in that the immunostimulating agent is IL-12. 14. The composition according to claim 1, characterized in that the oncolytic herpes simplex virus expresses both IL-12 and anti-PD-1 antibody. 15. The composition according to claim 1, characterized in that the oncolytic herpes simplex virus is HSV-1 expressing IL-12 and anti-PD-1 antibody. 16. The composition according to claim 15, characterized in that HSV-1 is strain F of HSV-1. 17. The composition according to claim 16, characterized in that the fragment of the nucleotide sequence from 117005 to 132096 of the native backbone is deleted. 18. The composition according to claim 1, characterized in that the tumor is a malignant tumor. 19. The composition according to claim 18, characterized in that the malignant tumor is selected from the group consisting of melanoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Yu tumor Inga, Leiomiosarcoma, rabdomyosarcoma, colon carcinomas, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, plane cell carcinoma, basal cell carcinoma, adenocarcinoma, carcinoma of the ceiling glands, carcinomas of the sebaceous glands, pillular cellular cellular cells, and papillary carcinoma, papillary cell Enocarcin, cystadenocardicinoma, medical carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma , craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, gastric carcinomas and carcinomas of the gastric cardia. 20. The composition according to claim 1, characterized in that the subject is a person. 21. A kit for treating a tumor in a subject, comprising (a) a therapeutically effective amount of an exosome, wherein the exosome contains an inhibitory amount of a CTLA4-targeting siRNA and an exomotif operably linked to a seed sequence of the CTLA4-targeting siRNA to enhance packaging of the CTLA4-targeting siRNA into the exosome, (b) a therapeutically effective amount of an oncolytic herpes simplex virus, wherein the oncolytic herpes simplex virus expresses an immunostimulatory agent or both an immunostimulatory agent and an anti-PD-1 antibody, and optionally (c) instructions for use. 22. The kit according to claim 21, characterized in that the seed sequence of the CTLA4-targeting siRNA contains any of the nucleic acid sequences of SEQ ID NO: 1 through SEQ ID NO: 4. 23. The kit according to claim 21 or 22, characterized in that the exomotive is selected from the group consisting of the nucleic acid sequence of SEQ ID NO: 21 through SEQ ID NO: 49. 24. The kit according to claim 21, characterized in that the exomotif is located downstream and is covalently linked to the seed sequence of the CTLA4-targeting siRNA. 25. The kit according to claim 21, characterized in that the exomotif is obtained by mutation of one or more nucleic acids of the CTLA4-targeting siRNA, excluding the seed sequence. 26. The kit of claim 21, wherein the exomotif is a dual motif formed by combining two separate exomotifs, wherein any of the two separate exomotifs is selected from the group consisting of the nucleic acid sequence of SEQ ID NO: 21 to SEQ ID NO: 47. 27. The kit according to claim 21, characterized in that the dual motif has the nucleic acid sequence SEQ ID NO: 48. 28. The kit according to claim 21, characterized in that the CTLA4-targeting siRNA and the exomotif, if operably linked, contain at least one common nucleotide or two common nucleotides or are connected through a linker. 29. The set according to claim 28, characterized in that the linker consists of two or more nucleotides selected from the group consisting of adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) . 30. The kit according to claim 29, characterized in that the linker is -GC-. 31. The kit of claim 21, wherein the CTLA4-targeting siRNA and the exomotif, if operably linked, have the nucleic acid sequence of SEQ ID NO: 7. 32. The kit according to claim 21, characterized in that the immunostimulating agent is selected from GM-CSF, IL-2, IL-5, IL-12, IL-15, IL-24 and IL-27. 33. The kit according to claim 32, characterized in that the immunostimulating agent is IL-12. 34. The kit according to claim 21, characterized in that the oncolytic herpes simplex virus expresses both IL-12 and anti-PD-1 antibody. 35. The kit according to claim 21, characterized in that the oncolytic herpes simplex virus is HSV-1 expressing IL-12 and an anti-PD-1 antibody. 36. The kit according to claim 35, characterized in that HSV-1 is strain F of HSV-1. 37. The kit according to claim 36, characterized in that the fragment of the nucleotide sequence from 117005 to 132096 of the native backbone is deleted. 38. The kit according to claim 21, characterized in that the tumor is a malignant tumor. 39. The set according to claim 38, characterized in that the malignant tumor is selected from the group consisting of melanoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing tumor , Leiomiosarcoma, rabdomyosarcoma, colon carcinomas, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, plane cell carcinoma, basal cell carcinoma, adenocarcinoma, carcinoma of the ceiling glands, carcinomas of the sebaceous glands, pillular cellular cellular cells, and papillary carcinoma, papillary cell Enocarcin, cystadenocardicinoma, medical carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma , craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, gastric carcinomas and carcinomas of the gastric cardia. 40. The set according to claim 21, characterized by the fact that the subject is a person.