WO2026050737A1 - Method for treating cutaneous neurofibromas (cnfs) - Google Patents

Abstract

The present disclosure provides a method for treating cutaneous neurofibromas (cNFs) in patients with neurofibromatosis type 1 (NF1) using talimogene laherparepvec (T-VEC), an oncolytic herpes simplex virus 1 (oHSV1) vector, as an illustrative example. The method includes administering to a subject an effective amount of T-VEC, typically by intratumoral injection. T-VEC is genetically modified to enhance tumor selectivity and stimulate anti-tumor immune responses. The treatment involves an initial dose followed by subsequent doses at specified intervals. The method may include repeated administrations and combination with additional therapies for cNF or NF1. This approach offers a potential non-invasive alternative to current treatments for cNFs in NF1 patients.

Description

PATENT

ATTORNEY DOCKET NO. JHU4780-1WO

METHOD FOR TREATING CUTANEOUS NEUROFIBROMAS (CNFs) CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63/688,787, filed August 29, 2024. The content of the prior application is considered part of and is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present disclosure generally relates generally to a method for treating cutaneous neurofibromas (cNFs) and more specifically to using talimogene laherparepvec (T-VEC), an oncolytic herpes simplex virus 1 (oHSVl) vector, to treat cNFs in patients with neurofibromatosis type 1 (NFl).

BACKGROUND INFORMATION

[0003] Neurofibromatosis type 1 (NFl) is a common genetic disorder affecting approximately 1 in 3,000 individuals worldwide. It is caused by mutations in the NFl gene, which leads to the development of various tumors throughout the body. One of the hallmark features of NFl is the development of cutaneous neurofibromas (cNFs), benign tumors that arise from the nerve sheaths and typically appear during puberty or early adulthood.

[0004] While cNFs are not life-threatening, they can significantly impact the quality of life for NFl patients. These tumors often cause cosmetic concerns, pain, and itching, leading to psychological distress and social challenges for affected individuals. As NFl patients age, the number and size of cNFs tend to increase, exacerbating these issues over time.

[0005] Currently, there are no effective pharmacological treatments for cNFs. The primary management option is surgical removal, which presents several limitations. Surgical intervention is often impractical due to the large number of tumors that can develop in NFl patients. Additionally, the location of some tumors may make surgery challenging or risk significant scarring. Moreover, surgical removal does not prevent the development of new cNFs, meaning patients may require repeated procedures throughout their lives.

[0006] Other experimental approaches, such as laser therapy or electrodessication, have shown limited efficacy and may also result in scarring. The lack of effective, non-invasive treatment options represents a significant unmet medical need for NFl patients suffering from cNFs.

[0007] There is a critical need for novel therapeutic approaches that can effectively treat multiple cNFs simultaneously, prevent the development of new tumors, and avoid the

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ATTORNEY DOCKET NO. JHU4780-1WO limitations and risks associated with surgical intervention. An ideal treatment would be minimally invasive, capable of targeting multiple tumors, and have a favorable safety profile for long-term use.

[0008] The development of such a therapy would significantly improve the management of cNFs in NF1 patients, potentially enhancing their quality of life and reducing the psychological and social burdens associated with this condition. This unmet need drives the search for innovative treatments that can address the challenges posed by cNFs in the NF1 patient population.

SUMMARY OF THE INVENTION

[0009] The present disclosure is based on the seminal discovery that HSV1-AICP34.5 and T- VEC display anti -tumor efficacy in NF1 -associated tumors.

[0010] The present disclosure provides a method for treating cutaneous neurofibromas (cNFs) in patients with neurofibromatosis type 1 (NF1) using an oncolytic herpes simplex virus 1 (oHSVl) vector. This approach leverages the tumor-selective replication and immunostimulatory properties of talimogene laherparepvec (T-VEC) to target and eliminate cNFs, offering a potential non-invasive alternative to surgical removal.

[0011] In one embodiment, the present disclosure provides a method of treating a cutaneous neurofibroma (cNF) in a subject comprising administering to the subject an effective amount of an oncolytic herpes simplex virus 1 (oHSVl) vector, thereby treating the cNF.

[0012] In some aspects, the oHSVl vector comprises a deletion of the ICP34.5 gene from both long repeat (RL) regions of the HSV-1 genome and a deletion of the ICP47 gene.

[0013] In some aspects, the oHSVl vector comprises a deletion of the ICP34.5 gene from both long repeat (RL) regions of the HSV-1 genome, a deletion of the ICP47 gene, and an insertion of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene. [0014] In some aspects, the administration is by intratumoral injection.

[0015] In some aspects, the effective amount of the oHSV 1 vector is determined based on the size, location, and/or number of cNFs in the subject.

[0016] In one embodiment, the method involves administering an initial dose of about 10^A6 plaque-forming units (PFU)ZmL, followed by subsequent doses of greater than 10⁶ PFU/mL.

[0017] In some aspects, the subsequent doses are administered about three weeks after the initial dose and then about once every two weeks thereafter.

[0018] In some aspects, the total volume of oHSVl vector administration is up to about 4.0 mL per treatment session.

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[0019] In some aspects, the administration is performed on single or multiple lesions at a time.

[0020] In one embodiment, the subj ect has a mutation or deletion in the neurofi bromin (NF 1 ) gene on chromosome 17ql 1.2.

[0021] In some aspects, the effective amount of the oHSVl vector is sufficient to induce tumor cell lysis and immune cell infiltration in the cNF.

[0022] In one embodiment, the method includes repeating the administration at least once after an initial administration.

[0023] In some aspects, the administration of the oHSVl vector is combined with an additional therapy for cNF or NF1, such as surgical excision, radiotherapy, chemotherapy, or a targeted therapy.

[0024] In some aspects, the administration of the oHSVl vector results in a reduction or elimination of the cNF in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 illustrates the propagation efficacy of HSV1-AICP34.5 in NFl-related tumor cells in an in vitro propagation assay. The HSV1-AICP34.5-GFP (oHSVl -AICP47- AICP34.5^CMV'^GFP'^BGHpA) virus was incubated with the indicated MPNST- and pNF-derived cells, with Vero cells as control, in a dilution series (PFU/well) in 96-well plates. The plague formation was observed daily under a fluorescence microscope and the plague counts in the dilution series on day 3 were calculated and compared with that of Vero cells. A higher dilution factor indicates more potent propagation of oHSVl in a cell line.

[0026] FIGs. 2A-2C illustrate efficacy of HSV1-AICP34.5 and T-VEC in an NF1 xenograft mouse model. FIG. 2A shows representative images of mice bearing dermally implanted ST8814 tumors treated with T-VEC by intratumoral injection. FIG. 2B demonstrates the tumor volume reduction in ST8814 xenograft tumors treated with HSV1-AICP34.5 strain compared to untreated contralateral tumors. FIG. 2C illustrates the tumor volume reduction in ST8814 xenograft tumors treated with T-VEC compared to untreated contralateral tumors.

[0027] FIGs. 3A-3B illustrate histological confirmation of viral propagation in NF1 xenograft tumors. FIG. 3A shows histological confirmation of HSV1-AICP34.5-GFP propagation in dermal ST8814 xenograft tumors through H&E and anti-GFP staining. FIG. 3B demonstrates histological confirmation of T-VEC propagation in dermal ST8814 xenograft tumors through H&E and anti-ICP27 staining.

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[0028] FIG. 4 is a schematic illustrating oHS V 1 vector inj ection along multiple tracks within the lesion to obtain as wide a dispersion as possible.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The present disclosure is based on the seminal discovery that HSV1-AICP34.5 and T- VEC display anti -tumor efficacy in NF1 -associated tumors.

[0030] The present disclosure provides a method for treating cutaneous neurofibromas (cNFs) in patients with neurofibromatosis type 1 (NF1) using talimogene laherparepvec (T- VEC), an oncolytic herpes simplex virus 1 (oHSVl) vector, as an illustrative example. This approach offers a potentially more effective, and less invasive alternative to current treatment options for cNFs.

[0031] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular aspects only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0032] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0034] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” include one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0035] As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

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[0036] The term “cutaneous neurofibromas” or “cNFs” refer to benign tumors that develop on or just under the skin, most commonly associated with Neurofibromatosis Type 1 (NF1).

[0037] The terms “therapeutically effective amount”, “effective dose,” “therapeutically effective dose”, “effective amount,” or the like refer to that amount of the subject compound that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. Generally, the response is either amelioration of symptoms in a patient or a desired biological outcome (e.g., treating cutaneous neurofibroma). The effective amount can be determined as described herein. [0038] The terms “subject” and “patient” may be used interchangeably. As used herein, in some aspects, a subject is a mammal. In some aspects, the subject is a human. In some aspects, the subject is a mammal, e.g., a human, diagnosed with a disease or disorder. In some aspects, the subject is a mammal, e.g., a human, at risk of developing a disease or disorder.

[0039] The terms “administration of’ and or “administering” should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment. In some aspects, “administering” may refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art.

[0040] Administration routes can be enteral, topical or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrastemal , oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, and nebulization. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration. Examples of routes of administration may include but are not limited to inhalation, otic, buccal, conjunctival, dental, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraabdominal, intraamniotic, intraarterial, intraarticular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavemous, intracavitary, intracerebroventricular, intracistemal, intracorneal, intracoronal, intracoronary, intracorpous cavemaosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intrahippocampal, intraileal,

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ATTORNEY DOCKET NO. JHU4780-1WO intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, retrobulbar, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, vaginal, infraorbital, intratumoral, intraparenchymal, intrathecal, intraventricular, stereotactic administration, or any combination thereof.

[0041] As used herein, the terms “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a disease or disorder resulting from the administration of one or more therapies. Treating may be determined by assessing whether there has been a decrease, alleviation and/or mitigation of one or more symptoms associated with the underlying disorder such that an improvement is observed with the patient, despite that the patient may still be afflicted with the underlying disorder. The term “treating” includes both managing and ameliorating the disease. The term “oncolytic herpes simplex virus 1” or “oHSVl” refers to a genetically modified virus used in cancer therapy to selectively infect and destroy tumor cells while sparing normal tissue. The virus causes infected tumor cells to burst (oncolysis), releasing tumor antigens. Examples of oHSVl include but are not limited to T- VEC, G47A, HSV1716, NV1020, rQNestin34.5v2, VC2, HF 10, and MGH2. Common genetic modifications in oHSV-1 include but are not limited to deletion of ICP34.5, deletion of ICP47, insertion of cytokine genes (e.g., human granulocyte-macrophage colony -stimulating factor (GM-CSF), IL-12, IL-15), and tumor-specific promoters.

[0042] T-VEC is a genetically modified form of herpes simplex virus type 1 (HSV-1). The genetic modifications include deletion of the ICP34.5 gene from both long repeat (RL) regions of the HSV-1 genome, which enhances tumor selectivity and attenuates neurovirulence; deletion of the ICP47 gene, which enhances antigen presentation and stimulates the anti-tumor immune response; and insertion of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, which further enhances the anti-tumor immune response.

[0043] The deletion of ICP34.5 disables the ability of the virus to replicate in normal cells, where the PKR-eIF-2A antiviral mechanism is intact. However, in cancer cells, particularly

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ATTORNEY DOCKET NO. JHU4780-1WO those with an activated RAS-MEK-ERK pathway (as is the case in NF1 -deficient cells), this antiviral mechanism is often impaired, allowing for selective viral replication. The deletion of ICP47 enhances the presentation of viral antigens, making infected tumor cells more visible to the immune system. The insertion of the GM-CSF gene leads to local production of this cytokine, which recruits and activates dendritic cells and T cells, further amplifying the antitumor immune response.

[0044] In some aspects, the administration is by intratumoral injection. The effective amount of the oHSVl vector may be determined based on the size, location, and/or number of cNFs in the subject. The method may involve administering an initial dose of about 10⁶ plaque-forming units (PFU)ZmL, followed by subsequent doses of greater than 10⁶ PFU/mL. These subsequent doses may include, but are not limited to, concentrations of 10⁷ PFU/mL, 10⁸ PFU/mL, and higher.

[0045] The term “initial dose” or “initial priming dose” refers to the administration of a small, sub-therapeutic dose of oHSVl vector. The initial priming dose may be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, and 20% of the full therapeutic dose. In some aspects, the priming dose is administered 1 to 10 minutes before the administration of the full therapeutic dose. In some aspects, the dose of oHSV vector is up to 0.05 ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.5 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, or 10 mL of 10⁶ pfu/mL to prime the tumors and the immune system.

[0046] In some aspects, the subsequent doses are administered about three weeks after the initial dose and then about once every two weeks thereafter. The total volume of oHSVl vector administration may be up to about 10 mL per treatment session, and the administration may be performed on single or multiple lesions at a time. In some aspects, the total volume of oHSVl vector is up to 0.05 ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.5 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, or 10 mL of 10⁶ pfu/mL.

[0047] In some aspects, the treatment cycle is administration of oHSV vector each 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38, 39 days, or 40 days. In some aspects, the oHSV vector is administered on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28 of cycle 1, and then every 1, 2, or 3 weeks after that for a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 treatment sessions.

[0048] The injection volume varies with lesion size as shown in Table 1.

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[0049] Table 1: Injection Volume of T-VEC Required per Size of Lesion.

[0050] In some aspects, biomarkers are measured in tissue or blood from the subject. In some aspects the biomarker comprises extracellular matrix proteins, inflammation biomarkers, INF- gamma, and/or percent of target tumors with response >50% reduction in surface area. In some aspects, extracellular matrix proteins in the subject are modulated compared to levels of extracellular matrix proteins in a subject not suffering from cNF. In some aspects, the levels of inflammation in the subject are modulated compared to levels of inflammation in a subject not suffering from cNF. In some aspects, levels of INF-gamma in the subject are modulated compared to levels of INF-gamma in a subject not suffering from cNF. In some aspects, levels of percent of target tumors with response >50% reduction in surface area in the subject is increased compared to percent of target tumors with response >50% reduction in surface area in a subject not suffering from cNF. In some aspects the extracellular matrix protein includes COL1A1, COL6A1, or a combination thereof. In some aspects, the inflammation biomarker includes IL-6, TNF-alpha, GM-CSF, INF-gamma, or a combination thereof.

[0051] The term “extracellular matrix protein” refers to key structural and functional components of the complex network of molecules that provide physical scaffolding and biochemical support to surrounding cells in tissues. Examples of extracellular matrix proteins include but are not limited to collagens, elastin, fibronectin, laminin, proteoglycans, glycosaminoglycans (GAGs), and Tenascin.

[0052] The term “inflammation biomarker” refers to measurable substances in the body that indicate the presence, intensity, and progression of inflammation. Examples of inflammation biomarkers include but are not limited to C-reactive protein (CRP), high-sensitivity CRP (hs- CRP), serum amyloid A (SAA), ferritin, fibrinogen, haptoglobin, alpha- 1 antitrypsin, procalcitonin (PCT), pro-adrenomedullin (Pro-ADM), tumor necrosis factor-alpha (TNF-a), interleukin- 1 (IL- la, IL- 1 ), interleukin-6 (IL-6), interleukin-8 (IL-8 / CXCL8), interleukin- 10 (IL-10), interleukin-17 (IL-17), interferon-gamma (IFN-y), CCL2 (MCP-1), CCL3 (MIP-

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ATTORNEY DOCKET NO. JHU4780-1WO la), CCL5 (RANTES), CXCL10 (IP-10), CXCL12 (SDF-1), matrix metalloproteinases (MMP-1, MMP-3, MMP-9), tissue inhibitors of metalloproteinases (TIMPs), ICAM-1, VCAM-1, E-selectin, P-selectin, suPAR, sCD14, sCD163, neopterin, calprotectin, prostaglandins (e.g., PGE2), GM-CSF, INF-gamma, leukotrienes (e.g., LTB4), lipoxins, resolvins, erythrocyte sedimentation rate (ESR), white blood cell count (WBC), and neutrophil- to-lymphocyte ratio (NLR).

[0053] In some aspects, treated tumor size is reduced by 10 %, 15 %, 20 %, 25%, 30 %, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more in its surface area.

[0054] In some aspects, the subject has a mutation or deletion in the neurofibromin (NF1) gene on chromosome 17ql 1.2. The effective amount of the oHSVl vector is sufficient to induce tumor cell lysis and immune cell infiltration in the cNF.

[0055] The method may include repeating the administration at least once after an initial administration. In some embodiments, the administration of the oHSVl vector may be combined with an additional therapy for cNF or NF1, such as surgical excision, radiotherapy, chemotherapy, or a targeted therapy.

[0056] The administration of the oHSVl vector is expected to result in a reduction or elimination of the cNF in the subject. This may be observed as a decrease in tumor size, a change in tumor consistency, or complete disappearance of the tumor.

[0057] T-VEC works through a dual mechanism of action. It directly causes oncolysis by selectively replicating in tumor cells, leading to their lysis and death. Additionally, it stimulates anti-tumor immunity. The lysis of tumor cells releases tumor-associated antigens, and the GM- CSF produced by T-VEC recruits and activates antigen-presenting cells, enhancing the antitumor immune response.

[0058] Based on extensive experience with T-VEC in treating advanced melanoma, this therapy has proven to be generally safe with minimal risks. However, as with any medical treatment, potential side effects should be monitored and managed appropriately. Common side effects may include local inflammation at the injection site, flu-like symptoms, and fatigue.

[0059] Following T-VEC administration, patients should be monitored for both treatment efficacy and potential side effects. Tumor response can be assessed through physical examination, photography, and imaging studies if appropriate. Long-term follow-up is important to assess the durability of the treatment effect and to monitor for any late-onset side effects or recurrence of tumors.

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[0060] The methods disclosed herein offer anovel approach to treating cNFs in NF1 patients. By leveraging the tumor-selective and immunostimulatory properties of T-VEC, this treatment strategy has the potential to provide significant benefits to patients suffering from this challenging condition. The combination of direct tumor cell killing, immune stimulation, and the ability to treat multiple lesions simultaneously makes T-VEC a promising candidate for the treatment of cNFs in NF1 patients.

[0061] The following examples are provided to further illustrate the aspects of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLES

[0062] The following examples are provided to further illustrate aspects of the present invention but are not intended to limit the scope of the invention.

EXAMPLE 1

In Vitro Propagation of HSV1-AICP34.5 in NFl-Related Tumor Cells

[0063] This example describes the propagation efficacy of HSV1-AICP34.5 in NFl-related tumor cells, demonstrating the potential of oncolytic herpes simplex virus vectors for treating NF 1 -associated tumors.

[0064] A panel of NFl-related tumor cell lines was used, including plexiform neurofibroma (pNF) cells and malignant peripheral nerve sheath tumor (MPNST) cells derived from NF1 patients. The ST8814 cell line was included as a representative MPNST cell line. HSV1- AICP34.5 (oHSVl- AICP47- AICP34.5^CMV-^GFP-^BGHP^A), a precursor to T-VEC, was used in this study. Cells were seeded in 96-well plates and incubated with various dilutions of HSV1- AICP34.5, starting from 1 plaque-forming unit (PFU) per well and increasing by a factor of 10. After 3 days of incubation, the formation of GFP-positive plaques was observed and counted in the well with the lowest PFU.

[0065] As shown in FIG.l, HSV1-AICP34.5 demonstrated consistent propagation across all tested NF1 tumor cell lines. The JH-2-002 cell line exhibited the highest viral propagation among the tested lines. The dilution factors, calculated as the number of plaques divided by the PFU in the well, indicated the propagation potential of the oHSVl vector in each cell line.

[0066] These results suggest that NFl-related tumor cells, including both benign and malignant types, are highly susceptible to infection and propagation by oHSVl vectors like T- VEC. This susceptibility is likely due to the hyperactive Ras pathway in NFl-deficient cells,

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ATTORNEY DOCKET NO. JHU4780-1WO which supports viral replication. The consistent propagation across different cell lines indicates that T-VEC could potentially be effective against a wide range of NF 1 -associated tumors, including cNFs. This broad applicability is crucial for developing a comprehensive treatment strategy for NF1 patients who often present with multiple tumor types.

EXAMPLE 2

In Vivo Efficacy of HSV1-AICP34.5 and T-VEC in an NF1 Xenograft Mouse Model [0067] This example illustrates the anti-tumor efficacy of HSV1-AICP34.5 and T-VEC in a mouse model of NF 1 -associated tumors, providing evidence for their potential as a treatment for cutaneous neurofibromas.

[0068] NSG mice were used to create a xenograft model. Each mouse was implanted with two ST8814 tumors, one on each body side, into the dermis to mimic the natural location of cNFs. Tumors on the right side were treated with either oHSVl-AICP34.5 (oHSVl-AICP47- ICP34.5^CMV-^{GFP BGH}P^A, HSV1- AICP34.5-GFP) or AICP34.5) or T-VEC via intratumoral injections at a dose of 10⁸ PFU per week. Contralateral tumors served as controls. Tumor volumes were measured using calipers and calculated using the formula V = 0.5 x L x W²' [0069] As illustrated in FIG. 2A, representative images of mice bearing dermally implanted ST8814 tumors treated with T-VEC show visible tumor regression on the treated side compared to the untreated contralateral side. These images were taken at different time points posttreatment to demonstrate the progression of tumor regression.

[0070] FIG. 2B shows the quantitative analysis of tumor volume reduction in ST8814 xenograft tumors treated with the oHSVl-AICP34.5 (OHSV1-AICP47- ICP34.5^{CMV GFP BGH}P^A, HSV1- AICP34.5-GFP or AICP34.5) strain. The graph plots tumor volume over time, comparing treated tumors to untreated contralateral tumors. A significant reduction in tumor size was observed in the treated tumors, with complete tumor eradication occurring within 11 to 14 days following the initial treatment.

[0071] Similarly, FIG. 2C illustrates the tumor volume reduction in ST8814 xenograft tumors treated with T-VEC. The graph shows a comparable pattern of tumor regression to that seen with oHSVl-AICP34.5 (OHSV1-AICP47- ICP34.5^{CMV GFP BGH}P^A, HSV1-AICP34.5-GFP or AICP34.5), demonstrating the potent anti-tumor effect of T-VEC against NF 1 -related tumors in vivo.

[0072] In both cases, treated tumors showed a significant reduction in size, while control tumors on the contralateral side exhibited progressive enlargement. These results demonstrate the potent anti -tumor effect of oHSVl vectors, including T-VEC, against NF 1 -related tumors

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ATTORNEY DOCKET NO. JHU4780-1WO in vivo. The rapid and complete eradication of tumors suggests that T-VEC could potentially offer a fast-acting and highly effective treatment for cNFs. The stark contrast between treated and control tumors underscores the specificity of the treatment, indicating that T-VEC could selectively target NF 1 -associated tumors while sparing surrounding healthy tissue.

EXAMPLE 3

Histological Confirmation of Viral Propagation in NF1 Xenograft Tumors

[0073] This example describes the histological analysis of NF1 xenograft tumors treated with HSV1-AICP34.5 and T-VEC, confirming the selective propagation of these oncolytic viruses in tumor tissues.

[0074] Tumors treated with HSV1-AICP34.5-GFP or T-VEC, along with surrounding tissues, were collected and prepared for histological analysis. The tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 jim thickness. For HSV1- AICP34.5-GFP treated tumors, anti-GFP staining was performed using a rabbit polyclonal anti- GFP antibody (Abeam, ab6556) followed by a biotinylated goat anti-rabbit secondary antibody and DAB detection. For T-VEC treated tumors, anti-ICP27 staining was used as a marker for HSV1 vector propagation, using a mouse monoclonal anti-ICP27 antibody (Abeam, ab31631) followed by a biotinylated horse anti-mouse secondary antibody and DAB detection. Hematoxylin and eosin (H&E) staining was performed on adjacent sections for morphological analysis.

[0075] As shown in FIG. 3A, tumors treated with oHSVl-AICP34.5-GFP showed positive GFP staining, indicating successful viral infection and propagation. The H&E staining revealed areas of tumor cell lysis and immune cell infiltration, consistent with the oncolytic activity of the virus. Importantly, the surrounding healthy skin tissue showed no signs of infection or damage, demonstrating the tumor-selective nature of the viral vector.

[0076] FIG. 3B demonstrates the histological confirmation of T-VEC propagation in dermal ST8814 xenograft tumors. The positive anti-ICP27 staining in the tumor tissues indicated effective viral propagation and infection. The H&E staining again showed areas of tumor cell destruction and immune cell infiltration. As with HSV1-AICP34.5-GFP, the surrounding healthy skin tissue showed no signs of T-VEC infection or damage.

[0077] These histological findings provide crucial evidence of the selective propagation of oHSVl vectors, including T-VEC, in NFl-related tumor tissues. The presence of viral markers throughout the tumor tissue suggests efficient viral spread, which is essential for complete

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1623029679.2 PATENT ATTORNEY DOCKET NO. JHU4780-1WO tumor eradication. The lack of viral markers in surrounding healthy tissue confirms the tumor- selective nature of these viral vectors, a key safety feature for clinical application.

[0078] The selective propagation in tumor tissues while sparing normal cells is particularly important for treating cNFs, which are often numerous and located in cosmetically sensitive areas. This selectivity could allow for the treatment of multiple cNFs simultaneously without damaging surrounding healthy skin, potentially leading to better cosmetic outcomes and improved quality of life for NF1 patients.

[0079] Overall, these results provide strong preclinical evidence supporting the potential of T-VEC as a safe and effective treatment for cNFs in NF1 patients. The combination of efficient tumor cell killing, selective propagation, and the ability to stimulate anti-tumor immune responses makes T-VEC a promising candidate for clinical development in this indication.

EXAMPLE 4

Feasibility and Tolerability of oHSV vector for the Treatment of Cutaneous Neurofibromas in Adults with NF1

[0080] The study objective is described in Table 2 below.

[0081] Table 2: Study Objectives

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EXAMPLE 5 Study Design

General

[0082] This is a single institutional feasibility and tolerability study of oHSV vector monotherapy in adults with NF1 and cNFs in need of treatment. Each 28-day treatment cycle will be defined as intralesional administration of oHSV vector administered on Days 1 and 21 of each cycle for 4 cycles (8 treatments).

[0083] All patients must be at least 18 years of age or older with at least 8 cNFs (maximum of 15) measuring 5 mm at minimum in longest diameter, Kamofsky index of 80% or higher, and have adequate hematologic, renal and hepatic function. After providing written informed consent to participate in the study, patients will be evaluated for study eligibility during the screening visits. During screening, a complete medical history will be documented, and a complete physical examination will be conducted.

[0084] Measurement of vital signs and Kamofsky performance status will be obtained. Laboratory samples will be collected to assess hematologic, renal, and hepatic function. We will also collect a sample to evaluate HSV1 DNA PCR, HSV1 IgG and IgM antibodies, these will be CLIA certified laboratory tests at baseline and at the end of the study.

[0085] Patients should be treated until week 16 (4 months), even in the presence of new lesions to allow for delayed immune-based anti -tumor effects to occur. After 16 weeks, patients will remain on study for 30-days to allow post-treatment assessment, as defined in this protocol (Section 6.2). If a participant achieves a complete response (cutaneous neurofibromas resolve and are no longer visible) for all of the target tumors before the end of the planned treatment, then the study drug will be stopped at that point, but the patient will be seen at the 30 day mark post last treatment in all cases.

[0086] Efficacy assessment will be based on tumor measurement by physical measurement, photographs of superficial lesions, and representative biopsy of residual masses suspected. After cycles 2, 4 and at EOT, a thorough assessment of the clinical response status will be made, particularly to determine if any evidence of biological activity of treatment can be observed.

Screening Period

[0087] The following assessments and procedures will be conducted at the screening visit which is to occur within 28 days before Day 1 of Cycle 1 :

• Provision of written informed consent

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• Completed medical history, including demographics, cancer history and associated therapies

• Complete physical exam

• Vital signs, including systolic and diastolic blood pressure, respiratory and heart rates, and temperature

• Kamofsky performance level

• Photographs of all visible lesions on the target area (i.e. cutaneous lesions and subcutaneous lesions providing a visible protrusion from the skin surface)

• Laboratory tests: o Serum -hCG (for women of childbearing potential only) o Complete blood count with differential o Comprehensive metabolic panel o PT/INR, and PTT o HSV-1 antibody

• Skin lesions will be measured by calipers or ruler and 3D imaging

• Documentation and collection of concomitant medications and other supportive therapy

• Skindex assessment

• Global Aesthetic Improvement Scale (GAIS)

• Ablon Visibility Index (AVI)

• Baseline and 30-day post-treatment INF-Gamma levels

Body Regions for Evaluation

[0088] Participants will be examined to identify cNFs for treatment and cNFs that will not be treated but will be monitored for abscopal effect. Eligible participants will have at least 5 cNFs for treatment with the goal of using 4 mL of oHSV vector as determined in Table 1 (Section 2.3d). 2D photographs of the cNFs selected for treatment will be taken on cycle 1 day 1 prior to the first treatment and the photos will be uploaded to the patients’ chart to ensure treatment of the same lesions during serial assessments.

[0089] Measurable cNFs are defined as single (as opposed to composite or sometimes called “collision” tumors) surrounded by normal skin. Each cNF must measure > 0.5cm by the longest diameter, but smaller than 5 cm.

[0090] There is an additional requirement for removal of a total of 6 cNF tumors non-clinical histologic evaluation and biomarker assessment:

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1623029679.2 PATENT ATTORNEY DOCKET NO. JHU4780-1WO o 2 cNF at baseline prior to treatment o 3 treated cNFs and 1 untreated cNFs collected during the follow up visit 30 days after treatment completion (+/- 5 days).

[0091] All excised cNFs must be at least 5 mm in size in their largest diameter. See laboratory manual for details of cNF removal and tissue handling. cNFs that are not excised will be assessed longitudinally to determine if oHSV vector has biological activity in them.

[0092] If all eligibility criteria are met, the participant will be enrolled.

Treatment Period

[0093] There will be a total of 8 treatment sessions. Treatment visit #2 is three weeks after treatment visit #1. All subsequent treatment visits (treatments #3-8) will be 2 weeks apart. All participants will receive study drug until:

1 ) Completion of the pre-specified 4 cycles (8 treatment sessions); or

2) Earlier if the participant has complete resolution of all the treated cNFs; or

3) At any point if cessation of study treatment is recommended due to intolerance or withdrawal of consent from the study; or

4) Evidence that the treated cNFs are increasing in size; or

5) Or at any time at investigator’s discretion due to non-compliance with study observations and requirements, or administrative reasons.

[0094] The treatment period ends with the administration of the last (8^th) dose. The study ends 30 days after the last dose. Prior to Cycle 1, 2, 3 and 4 as well as 30 days post-treatment, all participants will complete the following patient-reported outcome (PRO) measures:

• Skindex assessment

• Global impression of change (by study participant and by clinician)

• Ablon Visibility Index

Safety Follow Up Period

[0095] The post-study follow-up (FU) visit will be performed at 30 days (±5 days) after the last dose of study drug, except for participants who died, withdrew consent and objected to further data collection, or were lost to follow up.

[0096] The follow-up visits will include physical examination, laboratory and PRO assessments.

Example 6

Treatment Plan

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Treatment Overview

[0097] Study treatment will only be administered to eligible patients under the supervision of the investigator or identified sub-investigator(s). Patients will be treated on an outpatient basis. The patient should be considered clinically stable by the investigator before discharge from the study center.

Drug Dosing and Agent Administration

[0098] oHSV vector will be administered by trained site staff on scheduled study visits. oHSV vector will be administered on Days 1 and 21 of cycle 1, and then every 2 weeks after that for a total of 8 treatment sessions.

[0099] On Day 1 of Cycle 1 (C1D1), the first dose of oHSV vector will be up to 4mL of 106 pfu/mL to prime the tumors and the immune system. The injection site may be pre-treated with atopical anesthetic agent such as EMLA cream. oHSV vector should be injected along multiple tracks within the lesion to obtain as wide a dispersion as possible (FIG. 4).

[0100] An injectable local anesthetic may be used if needed which should not be injected directly into the lesion but is injected around the lesion. The injection site will then be swabbed with alcohol.

[0101] When removing the needle, withdraw it from the lesion slowly to avoid leakage of oHSV vector at the insertion point.

[0102] oHSV vector will be inj ected, and the treated area will be covered with a dry occlusive dressing after treatment (an absorbent pad and an occlusive cover - e.g. Tegaderm® or Tegaderm® with absorbent pad).

[0103] Patients must remain under close medical supervision for 2 hours (i.e., vital signs taken 1 and 2 hours) after the first injection (Day 1, Cyclel) of oHSV vector.

[0104] After dose 1, all subsequent injections will utilize a dose of 108 pfu/mL every 2 weeks (+ 3 days).

[0105] The dose (volume) delivered to the tumor(s) will be dependent on the size of the cNF and will be determined according to the following algorithm:

[0106] Up to 0. ImL for tumors up to 0.5cm longest dimension.

[0107] Up to 0.5mL for tumors of 0.5 to 1.5cm longest dimension.

[0108] Up to l.OmL for tumors of 1.5 to 2.5cm longest dimension.

[0109] Up to 2.0mL for tumors of 2.5 to 5cm longest dimension.

[0110] Up to 4.0mL for tumors >5cm longest diameter

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[0111] The maximum volume of oHSV vector given on any one treatment day will be 4mL (equivalent to one reconstituted vial of either 106 pfu/mL for the first treatment day or 108 pfu/mL for subsequent treatment days. All dosing procedures will follow the FDA approved dosing guidelines (https://www.imlygichcp.com/dosing).

[0112] Small unit syringes (e.g., 0.5 mL insulin syringes) with a detachable 18-26 G needle for oHSV vector withdrawal and a 22-26 G detachable needle will be used for injection.

[0113] No patient will be dosed with more than 8 injection days (treatment sessions) on this protocol; see section 3.4 for more details regarding treatment period.

Dose Modifications

[0114] If for some reason the study drug is not administered on a scheduled study visit, all assessments should be completed if possible, and the dose noted as missed. If dosing of the next cycle is delayed more than 4 weeks due to an AE related to study treatment, then treatment should be discontinued.

[0115] Dose reductions are not permitted, other than with respect to a reduction in the volume injected due tumor response. If a DLT occurs, oHSV vector administration should be delayed until the DLT has resolved to at least CTCAE Grade 1. If dosing is delayed by more than 4 weeks (e.g., one treatment cycle) due to the occurrence of a DLT or AE that is considered related to the study drug, then the patient should be taken off study.

[0116] All visits should be made on the scheduled day whenever possible. Where the patient cannot attend (e.g., public holiday), the visit should occur within three (+/-3) days following the planned visit. If a delay of more than three days is anticipated, permission should be sought from the study chair.

Dose Limiting Toxici es (DLT)

[0117] All adverse events will be described and graded according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. A copy of the CTCAE version 5.0 can be downloaded from the CTEP web site (National Institutes of Health, n.d.).

[0118] Dose-limiting toxicity will be evaluated during the first 35 days after the initial administration of oHSV vector; this will be considered the DLT evaluation period. A DLT is defined as any of the following adverse events occurring during the DLT evaluation period that are at least possibly related to oHSV vector. Dose limiting toxicities must have an attribution of possible, probable, or definite. For patients experiencing a DLT, the study treatment will be stopped. If the patient recovers (< grade 1, or tolerable grade 2) treatment may be resumed.

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[0119] Hematological toxicities will be considered dose limiting if any of the following occur:

[0120] Absolute neutrophil count of < 1500/mm3.

[0121] Platelets < 100,000/mm3.

[0122] Absolute lymphocyte count of <1000/mm3

[0123] Febrile neutropenia (ANC <1000/mm3 with a single temperature of 38.3 degrees C (101 degrees F) or a sustained temperature of >38 degrees C (100.4 degrees F) for more than one hour).

[0124] Any other grade 4 hematological toxicity or any hematologic toxicity that prevents administration of the next planned treatment session by more than 3 weeks.

[0125] Non-hematologic DLTs are now defined as:

[0126] Grade >3 non-hematological toxicities lasting for more than 3 days and attributed as at least possibly related to study drug will be considered dose limiting.

[0127] Specific toxicities of interest considered a DLT include:

[0128] Herpetic infection that is severe or medically significant or hospitalization or prolongation of existing hospitalization indicated or IV intervention indicated or limiting self- care ADL Injection site complications including necrosis, severe tissue damage, operative indication indicated Skin infection that requires IV antibiotic treatment or an invasive intervention indicated Sepsis, with positive blood culture with signs or symptoms and treatment is indicated Grade 3 or 4 immune system disorders as listed in the CTCAE version 5, this includes immune system disorder reactions involving major organ (e.g., colitis, anemia, myocarditis, kidney) that is severe or medically significant and re-quires hospitalization as limiting self-care ADL If DLT occurs, oHSV vector administration should be delayed until the DLT has resolved to at least CTCAE Grade 1. If dosing is delayed by more than 4 weeks (i.e., one treatment cycle) due to a DLT or AE considered related to the study drug, the patient should not receive additional doses and be monitored until resolution to at least grade 1 AE.

[0129] Participants who withdraw from the study for reasons other than an AE, prior to completing 4 cycles of treatment, will be replaced up to three times for an enrollment up to a maximum of 10 evaluable participants (maximum number of participants to be enrolled).

Concomitant Medications and Supportive Care

[0130] All medications and supportive therapies that are administered during the study, including the start and stop date(s) and indication, must be recorded. Concomitant medications

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Permited Medications

[0131] The following medications/supportive therapies may be administered if needed, at the investigator’s discretion, during the study:

• Anti-pyretic

• Analgesics

• Anti-emetic

• Anti-diarrheal

• Anti-allergic measures

• Broad-spectrum antibiotics

• Oral or systemic steroid medication use at a dose of <10mg/day of prednisone or equivalent (steroids with low systemic absorption (e.g., triamcinolone hexacetonide) injected into a joint space is allowed).

• Intermittent use of topical steroids outside of the treatment target area

• Injectable anesthetics, local or topical

Excluded Concomitant Medications

[0132] The following medications are not permitted during the study (i.e., treatment phase and during response evaluation):

• Other investigational drugs

• Concurrent anti-neoplastic therapies

• Oral or systemic steroids with use of > 10 mg of prednisone daily or equivalent. See section 4.3 above for allowed steroid use.

• Any antiviral drugs (including antiherpetics), oral or topical

Withdrawal of Patients from Treatment

[0133] oHSV vector is to be permanently discontinued for patients meeting any of the following criteria:

• The investigator decides that the patient should be withdrawn from the treatment due to a serious adverse event or a clinically significant laboratory value.

• The patient, for any reason, needs treatment with another therapeutic agent for the study disease. In this case, discontinuation from the treatment occurs immediately upon introduction of the new agent.

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• The treatment exhibits unacceptable toxicity, e.g., persistent Grade 3 for more than 4 weeks or any Grade 4

• Any toxicities Grade 2 or greater that, in the investigator's opinion, would require a dose delay of more than 4 weeks or discontinuation of therapy.

• The patient becomes pregnant or fails to use adequate birth control (for those patients who can conceive).

• Intercurrent medical illness that, in the judgment of the investigator, would make continued intervention dangerous for the patient.

• In addition, the investigator may withdraw patients from treatment if their condition worsens from initiation of treatment.

[0134] In addition, patients will be informed that they have the right to withdraw from the study at any time for any reason. The investigator also has the right to withdraw patients from the study for any of the following reasons:

• The patient is noncompliant with study procedures and/or refusal of therapy.

• The patient withdraws consent. The consequence of withdrawal of all consent by a patient will be that no new information will be collected from that patient and added to the existing data or database. However, every effort will be made to follow all patients for safety.

• Administrative reasons.

[0135] All patients should be encouraged to continue, if possible, with the scheduled study and follow-up visits. If a patient is withdrawn, they should complete the End of Treatment (EOT) visit, 30 days after their last oHSV vector treatment. All SAEs and AEs considered related to study treatment will be followed until resolution or at minimum 30 days after they were initially reported. The reason(s) for a patient’s withdrawal from the study are to be recorded in the patient’s records.

Example 7

Table of Scheduled of Activities

[0136] Table 1: Overview of Treatment Schedule

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Example 8

Pharmaceutical Information of oHSV Vector

Study Drug

[0137] Proper name: talimogene laherparepvec

[0138] Tradename: oHSV vector

[0139] Other names: oHSV vector

[0140] Indication: For the local treatment of unresectable cutaneous, subcutaneous, and nodal lesions in patients with melanoma recurrent after initial surgery.

[0141] oHSV vector is a weakened form of Herpes Simplex Virus Type I, which is commonly called the cold sore virus. It is injected directly into tumors on the skin, beneath the skin, or in your lymph glands (Amgen Oncology, 2023).

[0142] Recommended starting dose is up to a maximum of 4mL of oHSV vector at a concentration of 10⁶ (1 million) pfu/mL. Subsequent doses should be administered up to 4mL of oHSV vector at a concentration of 10⁸ (100 million) pfu/mL.

[0143] Dosage Forms and Strengths: Injection: 10⁶ pfu/mL, 10⁸ pfu/mL in single-use vials. (Amgen Oncology, 2023).

Formulation

[0144] oHSV vector (Talimogene laherparepvec, oHSV vector) is an attenuated version of the wild type (wt) herpes simplex virus type-1 (HSV-1) genome engineered to express human granulocyte macrophage colony-stimulating factor (hGM-CSF). This product is generated by modifying two regions of the wt HSV-1 new isolate JS1 : 1) deletion of the ICP34.5 and ICP47 genes and 2) incorporation of the hGM-CSF expression cassette into the ICP34.5 loci. oHSV vector is supplied as a sterile, single use, preservative-free frozen liquid in a cyclic olefin polymer (COP) plastic resin vial for intralesional injection. Each vial contains l.OmL deliverable volume at a nominal potency of 10⁶ pfu/mL or 10⁸ pfu/mL. The drug product is supplied in 2mL vials, each containing a recoverable product volume of ImL, and is stored at -80°C ±10°C until use. (Amgen Oncology, 2023).

[0145] oHSV vector is provided in single-use vials of ImL each, in two different concentrations: 10⁶ pfu/mL for initial dose only and 10⁸ pfu/mL for all subsequent doses. oHSV vector is administered by injection into cutaneous, subcutaneous, and/or nodal lesions that are visible, palpable, or detectable by ultrasound guidance (Amgen Oncology, 2023).

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[0146] Cutaneous Neurofibroma (cNF)-Skindex Questionnaire. Cutaneous neurofibroma (cNF)-Skindex questionnaire consists of 18 items, each ranging from ‘0’ / ‘Never bothered’ to ‘67‘ Always bothered’ (Fertitta et al., 2022; Fertitta et al., 2023).

Example 9

Global Aesthetic Improvement Scale (GAIS)

[0147] To be completed by participants and clinicians.

[0148] Table 3: Global Aesthetic Improvement Scale (GAIS)

Example 10 Ablon Visibility Index

[0149] To be completed by clinicians.

[0150] Visibility: Ratings are based on appearance of the person fully dressed and how readily symptoms could be perceived in impersonal interaction. It should be noted, however, that many persons who display no tumors on areas visible in normal street clothes have numerous tumors or cafe-au-lait (darkened) spots on the torso which would be apparent in physically intimate situations and may gravely affect sexual behavior.

[0151] 1. Mild: Essentially no visible tumors outside of normal clothing areas; gait and posture appear unremarkable when casually observed (this allows for heavy coating of neurofibromas on the body and some minor skeletal symptoms).

[0152] 2. Moderate: Some tumors on neck, face, hands, mild scoliosis or other skeletal features without noticeable limp.

[0153] 3. Severe: Numerous tumors on face, optic glioma (tumor) that has affected sight and eye socket, severe scoliosis, or skeletal features with noticeable limp.

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[0154] Severity: Ratings of a combination of clinical and cosmetic implications which may potentially affect lifestyle, mobility and/or threaten life.

[0155] 1. Mild: Symptoms such as neurofibromas or mild learning disorders which do not threaten physical or social life.

[0156] 2. Moderate: Symptoms may compromise lifestyle but are not severely threatening. Numerous external or internal neurofibromas, mild scoliosis, controlled learning disorders.

[0157] 3. Severe: Hundreds or thousands of visible neurofibromas which threaten functioning; blinding optic glioma; severe scoliosis or other skeletal features; serious internal neurofibromas; or malignancies.

[0158] While preferred aspects of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the aspects of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered accordingly.

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Claims

PATENT ATTORNEY DOCKET NO. JHU4780-1WO WHAT IS CLAIMED IS:

1. A method of treating a cutaneous neurofibroma (cNF) in a subject comprising administering to the subject an effective amount of an oncolytic herpes simplex virus 1 (oHSVl) vector, thereby treating the cNF.

2. The method of claim 1, wherein the oHSVl vector comprises a deletion of ICP34.5 gene from both long repeat (RL) regions of HSV-1 genome and a deletion of ICP47 gene.

3. The method of claim 2, wherein the oHSVl vector further comprises an insertion of human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene into the oHSVl vector.

4. The method of any one of claims 1 to 3, wherein the administration is by intratumoral injection.

5. The method of any one of claims 1 to 4, wherein the effective amount of the oHSVl vector is determined based on size, location, and/or number of cNFs in the subject.

6. The method of any one of claims 1 to 5, wherein administering the effective amount of the oHSVl vector comprises administering an initial dose of about 10⁶ plaque-forming units (PFU)ZmL.

7. The method of any one of claims 1 to 6, further comprising administering subsequent doses of greater than 10⁶ PFU/mL.

8. The method of claim 7, wherein subsequent doses are administered about three weeks after the initial dose.

9. The method of any one of claims 1 to 8, wherein subsequent doses are administered about once every two weeks thereafter.

10. The method of any one of claims 1 to 9, wherein total volume of the oHSVl vector administered is up to about 4.0 mL per treatment session.

11. The method of any one of claims 1 to 10, wherein the administration is performed on single or multiple lesions at a time.

12. The method of any one of claims 1 to 11, wherein the subject has a mutation or deletion in neurofibromin (NF1) gene on chromosome 17ql 1.2.

13. The method of any one of claims 1 to 12, wherein the effective amount of the oHSVl vector is sufficient to induce tumor cell lysis and immune cell infiltration in the cNF.

14. The method of any one of claims 1 to 13, wherein the administration is repeated at least once after an initial administration.

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15. The method of any one of claims 1 to 14, wherein the administration is combined with an additional therapy for cNF or NF1.

16. The method of claim 15, wherein the additional therapy is surgical excision, radiotherapy, chemotherapy, or a targeted therapy.

17. The method of any one of claims 1 to 16, wherein the administration results in a reduction or elimination of the cNF in the subject.

18. The method of any of claims 1 to 17, wherein the vector is T-VEC.

19. The method of claim 1, wherein levels of extracellular matrix proteins in the subject are modulated compared to levels of extracellular matrix proteins in a subject not suffering from cNF.

20. The method of claim 19, wherein the extracellular matrix protein is COL1A1, COL6A1, or a combination thereof.

21. The method of claim 1, wherein levels of inflammation biomarkers in the subject are modulated compared to levels of inflammation biomarkers in a subject not suffering from cNF.

22. The method of claim 21, wherein the inflammation biomarker comprises IL-6, TNF-alpha, GM-CSF, INF-gamma, or a combination thereof.

23. The method of claim 1, wherein levels of INF-gamma in the subject are modulated compared to levels of INF-gamma in a subject not suffering from cNF.

24. The method of claim 1, wherein levels of percent of target tumors with response >50% reduction in surface area in the subject is increased compared to percent of target tumors with response >50% reduction in surface area in a subject not suffering from cNF.

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