Biological entities for the treatment of brain tumors

Abstract

Disclosed is a biological entity for treating brain tumors, particularly gliomas, and a vector comprising the biological entity. The biological entity is a construct comprising at least an anti-tumor transgene, which is composed of at least one GluA knockdown agent, preferably at least two GluA knockdown agents, and preferably further comprises a fusion protein, an anti-PD-1 antibody, an anti-CTLA-4 antibody, and an immune response promoter of IL12. The vector comprises a wild-type HSV-1 virus, and the biological entity replaces the ICP34.5 gene of the wild-type HSV-1 virus. The vector comprising the wild-type HSV-1 virus modified with the biological entity shows little negative effect on neurons, while showing positive effect on human glioblastoma cells, both when neuronal cells and glioblastoma cells are cultured separately and when co-cultured.

Description

Technical Field

[0001] 1. Field of Disclosure The present disclosure relates to novel biological entities for treating brain tumors, particularly gliomas.

[0002] 2. Consideration of Background Art Advanced Treatment of Gliomas Gliomas are the most common form of malignant brain tumors, and glioblastoma (GBM), the most malignant form among them, is one of the most lethal cancers. The average survival period of GBM patients is just over one year from diagnosis. Temodar, the current standard therapy in the primary treatment of glioblastoma, extended the overall survival period from 12.1 months to 14.6 months compared to radiation alone, as described in the full FDA prescribing information for Temodar (see Figure 1).

[0003] Over the past 20 years, a series of small-scale trials have been conducted to study oncolytic viruses in gliomas. Initially, the concept was that oncolytic viruses (OVs) were viruses engineered to infect tumor cells but not normal tissue, primarily by deleting the ICP34.5 gene of herpes simplex virus type 1 (HSV-1) (which needs to withstand the immune response produced by non-tumor cells) (see Mineta, T. et al. “Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas.” Nature Medicine 1, no. 9 (September 1995): 938-943). Because OVs need to be administered to the tumor site, the first major pharmaceutical efforts using OVs were in skin cancer. Biovex / Amgen's Imlygic, an engineered HSV-1 virus, was approved for skin cancer in 2015. Other viruses, including adenoviruses, vaccinia viruses, polioviruses, and coxsackieviruses, have also been engineered for anti-cancer purposes. However, to date, only HSV-1-based viruses have demonstrated clear clinical evidence for such treatments.

[0004] Randomized trials of oncolytic viruses have not been conducted in gliomas. However, the clinical community is hopeful due to evidence of tumor reduction and anti-tumor immune responses that appear to be caused by OV transmission in some patients. Qualitatively, OV studies conducted in gliomas are more numerous than in other tumors compared to all other studies. Several reasons may explain this: i) the blood-brain barrier poses challenges to the systemic delivery of conventional pharmacological agents; ii) the invasiveness of evaluation procedures makes intratumoral administration more acceptable; and iii) the severity of the prognosis provides a risk / benefit calculation that justifies the acceptance of intracerebral administration of experimental viruses.

[0005] In April 2021, an early clinical trial was published in The New England Journal of Medicine. Researchers at the University of Alabama at Birmingham treated patients with an OV construct created in 1995. (See Friedman et al. “Oncolytic HSV-1 G207 Immunotherapy for Pediatric High-Grade Gliomas”. N Engl. J Med 2021; 384:1613-1622) (See Figure 2).

[0006] DNAtrix, a single company, is moving its adenovirus OV study to Phase 3 for glioblastoma based on the response observed in a small number of patients in early clinical trials (see Lang et al. “Phase I Study of DNX-2401 (Delta-24-RGD) Oncolytic Adenovirus: Replication and Immunotherapeutic Effects in Recurrent Malignant Glioma.” Journal of Clinical Oncology 36, no. 14, 1419-1427 (May 2018)) (see Figure 3). Then, in June 2021, Japanese health authorities approved Teserpaturev (G47Δ), an oncolytic virus marketed by Todo and Daiichi Sankyo, as a treatment for malignant glioma.

[0007] In November 2020, a groundbreaking advance was made in the field of immunosuppressants (OVs) when Replimune, a company founded by the inventors of Imlygic, announced clinical data demonstrating the concept of RP2, the next generation of OVs. Its first-generation candidate, RP1, appeared to demonstrate efficacy in skin cancer when used in combination with immune checkpoint inhibitors, but its efficacy as a monotherapy had not been demonstrated. RP2 incorporates checkpoint inhibitor antibodies against PD-1 and CTLA-4 within its construct. Therefore, RP2 appears to be more potent than RP1, but it is a "loaded OV," that is, an OV that is an anticancer drug itself, but uses a virus as a delivery agent for the anticancer drug (see Aroldi, F. et al. “Initial results of a phase 1 trial of RP2, a first in class, enhanced potency, anti-CTLA-4 antibody expressing, oncolytic HSV as single agent and combined with nivolumab in patients with solid tumors”. Poster presented 2020 Society for Immunotherapy of Cancer Annual Meeting) (see Figure 4). A schematic diagram of Replimune (REPL)'s RP-1 is shown in Figure 5 (see Thomas, S. et al., “Development of a new fusion-enhanced oncolytic immunotherapy platform based on herpes simplex virus type 1.” Journal for ImmunoTherapy of Cancer 7, no. 1, 6-17 (December 2019)).

[0008] Oncorus, a publicly traded company, is currently testing an OV (Overdrive Gene) containing five immunostimulatory transgenes named ONCR-177 (see Figure 6). The ONCR-177 payload is said to be designed to stimulate a novel, productive antitumor response (see Kennedy, EM et al., “Design of ONCR-177 base vector, a next generation oncolytic herpes simplex virus type-1, optimized for robust oncolysis, transgene expression and tumor-selective replication.” Poster presented at the American Association for Cancer Research annual meeting 2019).

[0009] Calcium-permeable AMPA receptor (CPAR) In 2019, Venkataramani et al. published in Nature that calcium-permeable AMPA receptors (CPARs) contribute to the maintenance of tumor cells and their spread to glioma tumor cells via signaling at neuronal synapses (see Venkataramani, V. et al. “Glutamatergic synaptic input to glioma cells drives brain tumor progression.”, Nature 573, 532-538 (2019)) (Figure 7). CPARs are a subtype of AMPA glutamate receptors that have emerged as a key driver of changes in the brain over the past 10-15 years. Most often involved in learning and memory processes, CPARs are part of the physical expression of information storage in the hippocampus and are required for reward-driven behavior.

[0010] The inventors have contributed to elucidating the intracellular mechanism by which CPAR is transported from the neuronal cytoplasm to the synapse (see Tukey, DS et al., “Sucrose ingestion induces rapid AMPA receptor trafficking.” Journal of Neuroscience 33, No. 14, 6123-6132 (April 2013)) (see Figure 8), and have demonstrated that once CPAR is taken up into the synapse, CPAR itself can act as a driver of change (see Tukey, DS and Ziff, EB, “Ca2+-permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors and dopamine D1 receptors regulate GluA1 trafficking in striatal neurons”, Journal of Biological Chemistry 288, No. 49, 35297-35306 (December 2013)) (see Figure 9). Importantly, CPARs are also essential for synaptic changes between neurons and glial cells (see Ge, WP et al., “Long-term potentiation of neuron-glia synapses mediated by A2+-permeable AMPA receptors.” Science 312, No. 5779, 1533-1537 (June 2006)). Glial cells are neuronal supporting cells that give rise to gliomas due to genetic abnormalities.

[0011] overview This disclosure describes a novel biological entity for the treatment of brain tumors, specifically gliomas. This disclosure applies neuronal synaptic biology using a novel oncolytic virus (OV).

[0012] This disclosure provides a novel OV, herpes simplex virus 1 (HSV-1), which is engineered to selectively infect and replicate in tumor cells while expressing a transgene that stimulates an antitumor immune response in order to prevent or at least mitigate the spread of brain tumors by blocking neuronal-tumor synapses. The novel OV is obtained by deleting the ICP34.5 gene of HSV-1 and replacing it with a construct that may contain an antitumor transgene and is driven by the ICP47 gene promoter.

[0013] This novel ovulation therapy (OV) is administered via intratumoral injection and achieves better overall response rates and, consequently, better overall survival rates than current standard therapies / treatments. Current standard therapy is temozolomide monotherapy, and other OVs are currently under development for gliomas as described above. Slowing the spread of brain tumors through improved overall response rates represents an immeasurable benefit to patients' lives.

[0014] Novel oncolytic agents (OVs) preferably inhibit the transport of AMPA receptors and, consequently, glioma cell-neuronal synaptic transport in glioma cells by at least genetic interference between the proteins GluA1 and GluA2, which are major subunits of the AMPA receptor including CPAR, thereby slowing tumor cell proliferation. Novel OVs are designed to enhance the efficacy of loaded oncolytic constructs in gliomas by delivering GluA1 and GluA2 knockdown agents in an efficient manner to achieve localized synaptic GluA1 and GluA2 knockdown. [Brief explanation of the drawing]

[0015] Further details, features, and benefits of this disclosure will become apparent from the following description of the exemplary embodiments shown in the figures. [Figure 1] This figure shows the Kaplan-Meier curves comparing overall survival rates for radiotherapy alone versus radiotherapy plus temodal. [Figure 2] This figure shows the Kaplan-Meier curves of overall survival for patients treated with the OV Construct G207, which was created in 1995. [Figure 3] This figure shows brain scans of patients A, B, and C who responded to an adenovirus oncolytic reagent developed by DNAtrix, along with a graph showing the change in tumor size over time (%). [Figure 4] This figure shows the antitumor activity and response dynamics of RP2 oncolytic HSV as monotherapy and in combination with nivolumab in patients with solid tumors. [Figure 5] This is a schematic diagram of the RP-1 construct. [Figure 6] This figure shows the design of an ONCR-177-based vector, which is an HSV-1 containing five immunostimulatory transgenes within its construct. [Figure 7]Figure 7a shows representative time series of GB cells (green) and cerebral microvessels (red) under control conditions (arrows, 5 independent experiments with 4 mice) versus high-dose isoflurane conditions (4 independent experiments with 4 mice). Figure 7b shows the infiltration rate of GB cells (n=254 control cells vs. n=143 isoflurane cells in 4 S24 PDX mice). Figures 7c and 7d show representative time series of unresponsive and responsive S24 PDX cells (neuronal ChR2 stimulation (red line); 9 independent experiments with 6 mice) (cells measured at 0 hours (red arrow) and 5 hours (green arrow)). Figure 7e shows S24 PDX Figure 7f shows the invasion rate of GB cells (unresponsive (NR), n=164 cells in 5 mice; responsive (R), n=53 mice). Figure 7g shows a representative time series of glioma invasion by cells expressing Glu-2A-DN-GFP along with tdTomato (arrow) compared to glioma cells expressing tdTomato alone. Figure 7h shows that the dominant-negative GluR2 subunit significantly slows the tumor invasion rate in an animal model of malignant glioma. Figure 7h shows the difference between Glu-2A-DN-GFP and tdTomato (arrow) or tdTomato. Figure 7 shows representative images of S24 xenografts containing GB cells expressing , Figure 7i shows the change in cell density over 14 days (n=13 regions in 5 mice), Figure 7j shows the glioma region at day 0 and 14 under control conditions and after treatment with the AMPAR antagonist perampanel, Figure 7k shows the cell density at day 14 vs day 0 under control conditions vs perampanel (PER) conditions in two different cell lines, S24 and BG5, and Figures 7f and g show prior art that is considered relevant to the purposes of this disclosure. [Figure 8]Figure 8a shows electron micrographs illustrating the induction of multi-step GluA1 transport by sucrose intake, thereby demonstrating that preventing intracellular GluA1 transport prevents synaptic strengthening. Figure 8b shows electron micrographs prepared from animals (3 animals per test group) in water, sucrose / water, and sucrose. Figures 8c-f show that repeated sucrose intake increases intra-synaptic and PSD GluA1, while acute sucrose intake induces rapid GluA1 transport to the extra-synaptic membrane. (Note: Data in Figures 8c-e are presented as the average number of particles per spine.) [Figure 9] This figure demonstrates confirmation of AMPA-induced GluA1 transport through genetic blockade, suggesting that synapses possessing CPARs have a mechanism that induces feedforward synaptic strengthening. [Figure 10] This figure shows the construction and validation of a human GluR1 / 2-expressing virus. Oncolytic HSV-1 expressing human gluR1-P2A-gluR2 was constructed by driving transgene expression from the ICP47 HSV-1 promoter. To construct HSV-1 expressing hGlur1 / 2, infectious viral DNA of the parent virus, gamma 34.5 double deletion virus, was used. Lysates of U20S cells co-transfected with infectious viral DNA and plasmid DNA (containing the UL26 / 27 flanking sequence and the gluR1 / 2 and eGFP expression transgene sequences) were collected after 4 days and infected fresh monolayers. Recombinant plaques were visualized using eGFP expression. PCR from purified viral genomic DNA of 10-fold purified plaques (A) shows a correct 528 bp band, and immunoblotting (B) shows the gluR2 protein expressed in infected SF-295 glioblastoma cells. [Figure 11]Schematic diagram of a vector containing a wild-type HSV-1 virus modified with a preferred biological entity according to the present invention, wherein the biological entity replaces the ICP34.5 gene of the wild-type HSV-1 virus. [Figure 12] Figure showing the components and explanations of each element of the biological entity shown in Figure 11, as well as both preferred embodiments and options. [Figure 13] Figure showing the genetic map of the present invention comprising a wild-type HSV-1 virus modified with the biological entity used in Experiment 1 below. [Figure 14] Figure visually showing the cytotoxic effect of human gluR1 / 2-expressing HSV-1 used in Experiment 1 below, where SF-295 glioblastoma cells were infected with a multiplicity of infection (MOI) of 1 of the wild-type or gluR1 / 2-expressing HSV-1 of the present invention, and HSV-1 expressing hgluR1 / 2 causes extensive cytotoxic effects in SF-295 cells. [Figure 15] Figure showing, in the form of a bar graph, the visual results shown in Figure 14, specifically, the index of cell viability (metabolism) evaluated using the CellTiter-glo kit (Promega), where the fold change was calculated as the relative value to untreated control cells, and the values are the mean standard deviation of three independent experiments. ***p < 0.0005, ****p < 0.00005.

[0016] Description of Preferred Embodiments As described above, Figures 1 - 9 show prior art attempts to treat glioblastoma using various models and biological entities, as well as an explanation of AMPA receptor trafficking related to the present invention. Those skilled in the art are likely to know the details and analysis of these prior art attempts, so no detailed description of any of them is presented here. Those skilled in the art who may not know the details of these prior art attempts can easily examine the studies described in the figures by investigating the literature specified in the background of the above invention.

[0017] Figure 10 shows the composition of the present invention in which the HSV-1 ICP34.5 gene is deleted and replaced with a biological entity composed of the C-termini of the GluR1 and GluR2 AMPA receptor subunits. Panel a of Figure 10 shows that the biological entity is intact in the virus. Panels b and c of Figure 10 show that the C-terminus of the AMPA receptor subunit GluR2 is specifically expressed by the virus of the present invention.

[0018] Figure 11 shows a schematic diagram of a vector containing a wild-type HSV-1 virus modified with a biological entity according to the present invention, which replaces the ICP34.5 gene of the wild-type HSV-1 virus. The ICP34.5 gene of the wild-type HSV-1 virus is deleted and replaced with a construct according to Figure 11 that contains an anti-tumor transgene driven by the ICP34.5 promoter of the wild-type HSV-1 virus. The anti-tumor transgene contains two AMPA receptor subunit interfering agents or GluA knockdown agents. The anti-tumor transgene preferably further contains a fusogenic protein, an anti-PD-1 antibody, an anti-CTLA-4 antibody and an IL12 construct. The first AMPA receptor subunit interfering agent contains the C-terminal fragment of GluA1 and is designed to prevent the transport of GluA1 to neuronal synapses and / or extrasynaptic membranes. The second GluA knockdown agent may contain the C-terminal fragment of GluA2 designed to prevent the transport of GluA2 to neuronal synapses and / or extrasynaptic membranes, or may contain an N-terminal antibody against GluA1 that can block CPAR synaptic transmission in adjacent cells after tumor cell lysis.

[0019] Figure 12 shows the components of the preferred biological entities of the present invention and their general and detailed descriptions. Figure 12 also shows variations and options of each component, including preferred options. In general, the HSV-1 virus is preferred because more is known about it and its strain is used in Replimune's RP1-3 product. The strain used in the RP1-3 product is a preferred virus to use. As mentioned above, the novel OV contains at least gene knockdowns of the proteins GluA1 and GluA2. The first GluA knockdown agent contains a C-terminal fragment of GluA1 and is designed to prevent the transport of GluA1 to neuronal synapses and / or extrasynaptic membranes. The second GluA knockdown agent contains a C-terminal fragment of GluA2 and is designed to prevent the transport of GluA2 to neuronal synapses and / or extrasynaptic membranes. For neuronal protection, deletion of both copies of the ICP34.5 gene is designed to prevent viral replication in terminally differentiated cells such as neurons. Regarding infectivity, HSV-1 has already shown remarkable targeting of nervous system cells most similar to GBM cells, including oligodendrocytes and their precursors. Incorporation of GALV fusion proteins enhances infectivity. Preferably, two checkpoint inhibitors are provided: a sequence encoding an anti-PD-1 antibody, preferably a PD-1 blocker (similar to Oncorus), and a sequence encoding an anti-CTLA-4 antibody, preferably a CTLA-4 blocker (similar to Replimune). Finally, preferably, an immunostimulant IL12 incorporating a sequence encoding the IL12 cytokine is incorporated.

[0020] Figure 14 visually shows the effects of treating plated SF-295 human glioblastoma cells with either a vehicle control, wild-type HSV-1, or an HSV-1 vector containing the biological entity of the present invention.

[0021] Figure 15 shows the results from Figure 14 in the form of a bar graph. In Figure 15, "+" and "-" indicate the presence or absence of the specified virus.

[0022] Examples The human GluR1 / 2-expressing virus used in the experiment was constructed and validated as follows. Oncolytic HSV-1 expressing human gluR1-P2A-gluR2 was constructed by driving transgene expression from the ICP47 HSV-1 promoter (see Figure 13). Infectious viral DNA of the parent virus, gamma 34.5 double deletion virus, was used to construct HSV-1 expressing Glur1 / 2. Lysates of U20S cells co-transfected with infectious viral DNA and plasmid DNA (containing UL26 / 27 flanking sequences and gluR1 / 2 and eGFP-expressing transgene sequences) were collected after 4 days, infected fresh monolayers, and recombinant plaques were visualized using eGFP expression. PCR from purified viral genomic DNA of 10-fold plaque-purified viral plaques showed a correct 528 bp band. Immunoblotting showed the gluR2 protein expressed in infected SF-295 glioblastoma cells. The nucleic acid sequence of the entire GluR-P2A-GluR2 biological entity is as follows: [ka]

[0023] Experiment 1 SF-295 human glioblastoma cells were plated using high-glucose DMEM supplemented with 10% FBS as a support medium. The experiment lasted for two days. At the end of the experiment, SF-295 cells proliferated significantly in both uninfected plated cells and cells infected with wild-type HSV-1 virus, visually demonstrating virtually no difference in cellular metabolism between the two groups (see Figure 13). On the other hand, plated cells infected with an HSV-1 vector containing the biological entity of the present invention showed a significant decrease in cellular metabolism compared to the other two plated cells. These visual results were confirmed by calculating the effect of wild-type HSV-1 containing the ICP34.5 gene or HSV-1 containing GluR1 / 2 of the present invention replacing the ICP34.5 gene in wild-type HSV-1, and these results are presented in the form of a bar graph (see Figure 15).

[0024] Experiment 2 Human neurons were plated onto two plates using an appropriate culture medium. One plate was infected with wild-type HSV-1 virus, while the other plate was inoculated with an HSV-1 vector containing the biological entity of the present invention. Observations were made on days 0, 3, 10, and 21. After 21 days, there was no visual difference in the survival rate of the human neurons plated on both plates. This indicates that the biological entity of the present invention does not have any visually detectable adverse effects on human neurons.

[0025] Experiment 3 Rat neurons at 1 day postnatal are grown in appropriate medium for 7 days. On day 7, SF-295 human glioblastoma cells are plated onto the neurons in appropriate medium at ratios of 1:1, 1:2, 1:5, and 1:10 (glioblastoma cells:rat neurons). Seven days after the addition of SF-295 cells, the co-culture is treated with either a vehicle control, wild-type HSV-1 virus, or HSV-1 virus containing the biological entity of the present invention. Two days after infection, the co-culture treated with the virus containing the biological entity of the present invention showed a significant decrease in SF-295 infiltration rate and cellular metabolism compared to the co-culture treated with the wild-type virus, and the number and size of dendritic spines, as measured by confocal microscopy, were significantly reduced in the co-culture treated with the virus of the present invention compared to the wild-type virus.

Claims

1. comprising at least one antitumor transgene containing two GluA knockdown agents, The two GluA knockdown agents mentioned above target AMPA receptor subunits including GluR1 and GluR2, and The two GluA knockdown agents are HSV-1 viruses used to kill human glioblastoma cells, each containing the C-terminal fragment of GluA1 and the C-terminal fragment of GluA2.

2. The HSV-1 virus according to claim 1, further comprising a fusion protein and an immune response promoter in the form of an anti-PD-1 antibody, an anti-CTLA-4 antibody, and an IL-12 construct.

3. The formulation comprises wild-type HSV-1 virus and at least one antitumor transgene containing two GluA knockdown agents. The two GluA knockdown agents mentioned above target AMPA receptor subunits including GluR1 and GluR2, and The two GluA knockdown agents are vectors used to kill human glioblastoma cells, each containing the C-terminal fragment of GluA1 and the C-terminal fragment of GluA2.

4. The vector according to claim 3, wherein the antitumor transgene further comprises a fusion protein and an anti-PD-1 antibody, an anti-CTLA-4 antibody, and an IL-12 immune response promoter.

5. The vector according to claim 3, wherein the vector replaces the ICP34.5 gene of the wild-type HSV-1 virus.

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