Methods and compositions for delivering immunotherapeutic agents across the blood-brain barrier to treat brain cancer

Abstract

The present application relates to sequences that enhance penetration of immunotherapeutics across the blood brain barrier (BBB), compositions comprising the same, and methods thereof for treating brain cancers such as glioblastoma (GBM). Further disclosed are a number of potential targeting peptide sequences identified as enhancing penetration across the BBB when inserted into the capsid of an adeno-associated virus (AAV).

Description

[0001] Priority Statement

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 959,625, filed January 10, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This article describes sequences that enhance the penetration of immunotherapeutic agents across the blood-brain barrier, compositions containing said sequences, and methods for using them to treat brain cancers such as glioblastoma (GBM). Background Technology

[0004] Glioblastoma multiforme (GBM) is the most common and deadliest brain tumor in adults, with a median overall survival of only 15 months. 1 In the United States, approximately 12,000 new cases of GBM are diagnosed each year, with an incidence rate of 3.2 cases per 100,000 people. 2 Despite significant advances in understanding the histology, molecular landscape, and tumor microenvironment of GBM. 3-6 However, there has been little progress in treatment since 2005. A key obstacle to translating our extensive knowledge of GBM into effective therapies is the low efficiency of drug delivery to GBM tumor sites. Intravenous administration is a convenient and widely applicable route of administration because GBM tumors are well-vascularized structurally. 7 In theory, good tumor coverage can be achieved. However, designing drugs that cross the blood-brain barrier (BBB) ​​and / or the blood-tumor barrier remains challenging. Summary of the Invention

[0005] Glioblastoma is an extremely deadly brain cancer that is difficult to treat with conventional methods. Systemic gene therapy for glioblastoma represents a novel therapeutic paradigm. This article describes the brain-penetrating AAV viral vector engineered to establish an intravascular gene delivery platform for glioblastoma gene therapy, for example, systemic delivery of PD-L1 antibodies for glioblastoma treatment.

[0006] Therefore, this article provides a method for delivering an immunotherapeutic agent to a cancer in a subject. The method includes administering an adeno-associated virus (AAV) to the subject, the AAV comprising (i) a capsid protein containing an amino acid sequence comprising at least four consecutive amino acids from the sequences TVSALFK (SEQ ID NO:8); TVSALK (SEQ ID NO:4); KLASVT (SEQ ID NO:83); or KFLASVT (SEQ ID NO:84), and (ii) a transgene encoding an immunotherapeutic agent, optionally wherein the cancer cells are in the brain of a human subject.

[0007] In some embodiments, the amino acid sequence comprises at least five consecutive amino acids from the sequences TVSALK (SEQ ID NO:4); TVSALFK (SEQ ID NO:8); KLASVT (SEQ ID NO:83); or KFLASVT (SEQ ID NO:84).

[0008] In some embodiments, the amino acid sequence comprises at least six consecutive amino acids from the sequences TVSALK (SEQ ID NO:4); TVSALFK (SEQ ID NO:8); KLASVT (SEQ ID NO:83); or KFLASVT (SEQ ID NO:84).

[0009] This article also provides a method for delivering an immunotherapeutic agent to a cancer in a subject. The method includes administering an adeno-associated virus (AAV) to the subject, the AAV comprising (i) a capsid protein comprising an amino acid sequence containing at least four consecutive amino acids from sequences V[S / p][A / m / t / ]L (SEQ ID NO:79), TV[S / p][A / m / t / ]L (SEQ ID NO:80), TV[S / p][A / m / t / ]LK (SEQ ID NO:81), or TV[S / p][A / m / t / ]LFK (SEQ ID NO:82), and (ii) a transgene encoding an immunotherapeutic agent, optionally wherein the cancer cells are in the brain of a human subject.

[0010] In some implementations, the target sequence includes VPALR (SEQ ID NO:1); VSALR (SEQ ID NO:2); TVPALR (SEQ ID NO:3); TVSALK (SEQ ID NO:4); TVPMLK (SEQ ID NO:12); TVPTLK (SEQ ID NO:13); FTVSLK (SEQ ID NO:5); LTVSLK (SEQ ID NO:6); TVSALFK (SEQ ID NO:8); TVPALFR (SEQ ID NO:9); TVPMLFK (SEQ ID NO:10) or TVPTLFK (SEQ ID NO:11).

[0011] In some embodiments, the transgene encoding the immunotherapeutic agent encodes an antibody targeting PD-1 or PD-L1.

[0012] In some implementations, the subject is a mammalian subject.

[0013] In some implementations, the AAV is AAV9.

[0014] In some implementations, the AAV9 includes AAV9 VP1.

[0015] In some embodiments, the target sequence is inserted at positions corresponding to amino acids 588 and 589 of AAV9 VP1 containing SEQ ID NO: 85.

[0016] In some embodiments, the cells are in the subject's brain, and the AAV is administered via parenteral, intracerebral, or intrathecal delivery.

[0017] In some implementations, the parenteral delivery is delivered via intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular delivery.

[0018] In some embodiments, the intrathecal delivery is via lumbar injection, cerebellomedullary cistern injection, or intraparenchymal injection.

[0019] In some embodiments, the method further includes administering chemotherapy, radiation, and / or surgical resection to the subject.

[0020] In some implementations, the chemotherapy includes temozolamide, lomustine, or a combination thereof.

[0021] Unless otherwise defined, 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 pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of conflict, the definitions herein shall prevail.

[0022] Other features and advantages of the invention will become apparent from the following detailed description, drawings, and claims. Attached Figure Description

[0023] Figure 1A-1C An exemplary strategy for engineering AAV9 by inserting a cell-penetrating peptide (CPP) into the capsid of AAV9 is described. Figure 1A This is a 3D model of the AAV9 virus. Individual CPPs inserted between amino acids 588 and 589 (VP1 number) in the capsid are shown on a 3x axis, where receptor binding may occur. Figure 1B The method for producing individual AAV cells is described. Three plasmids, including pRC (engineered or unengineered), pHelper, and pAAV, were co-transfected into HEK 293T cells, and AAV was harvested and purified using an iodixanol gradient. Figure 1C This is a plasmid diagram of an exemplary vector containing a sequence encoding an anti-PDL1 antibody.

[0024] Figure 2A-2B Describes representative images of mouse brain slices following intravenous administration of low-dose candidate AAV. Figure 2A ) and its quantitative analysis ( Figure 2B Mice with mixed genetic backgrounds were used. The candidate AAVs differed in their inserted CPPs (see Table 3), but all expressed nuclear red fluorescent protein (RFP) as a reporter protein. Candidate AAVs with low yields were excluded from further screening. The AAV dose was 1 × 10⁻⁶ per animal. 10 vg (viral genome). Figure 2A Each white dot in the image represents a cell labeled with RFP. Figure 2B middle, P < 0.05, relative to AAV9, ANOVA.

[0025] Figure 2C-2D Describes representative images of mouse brain slices following intravenous administration of AAV.CPP.11 and AAV.CPP.12 in repeated experiments. Figure 2C ) and its quantitative analysis ( Figure 2DAAV.CPP.11 and AAV.CPP.12 contain CPP BIP1 and BIP2, respectively (see Table 3). The dosage of AAV is increased to 1 × 10⁻⁶ per animal. 11 vg. Candidate AAV expresses nuclear red fluorescent protein (RFP) as a reporter protein. Figure 2C Each white dot in the image represents a cell labeled with RFP. Figure 2D middle, P < 0.05 P<0.01, relative to AAV9, ANOVA.

[0026] Figure 3A The optimization of the BIP targeting sequence is described to further engineer AAV9 towards better brain transduction. BIP1 (VPALR, SEQ ID NO:1), which enables AAV9 to transduce the brain more effectively (as in AAV.CPP.11), is derived from the rat protein Ku70. Human, mouse, and rat Ku70 proteins differ in their exact amino acid sequences. BIP2 (VSALK, SEQ ID NO:2), as in AAV.CPP.12, is a “synthetic” peptide associated with BIP1. Further engineering focuses on the VSALT sequence to minimize the species specificity of the ultimately engineered AAV. To generate new targeting sequences, amino acids of interest are added to the VSALT sequence, and in other cases, the positions of individual amino acids are switched. All the new sequences derived from BIP2 are then inserted back into the AAV9 capsid to generate new candidate AAVs for screening. The sequences appearing in sequence are SEQ ID NOs:69, 70, 71, 1-6, 72, 7, and 8.

[0027] Figure 3B-3C Representative images of mouse brain slices after intravenous administration of more candidate AAVs ( Figure 3B ) and its quantitative analysis ( Figure 3C All candidate AAVs expressed nuclear red fluorescent protein (RFP) as a reporter protein. The dosage of AAV was 1 × 10⁻⁶ per animal. 11 vg. Figure 3B The individual white dots represent RFP-labeled cells. AAV.CPP.16 and AAV.CPP.21 are identified as top hits due to their powerful and extensive brain transduction. Figure 3C middle, P < 0.05 P < 0.01, P<0.001, relative to AAV9, ANOVA.

[0028] Figure 3D A quantitative analysis describing the transduction efficiency in the liver after intravenous administration of the candidate AAV. The percentage of transduced hepatocytes is shown. The AAV dose was 1 × 10⁻⁶ per animal. 11 vg. P < 0.001, relative to AAV9, ANOVA.

[0029] Figures 4A-4E Describes the screening of selected candidate AAVs in an in vitro spherical model of the human blood-brain barrier. Figure 4A Describe the globular structures containing human microvascular endothelial cells that form a barrier on the surface, as well as the human pericytes and astrocytes within the globular structures. Evaluate the ability of candidate AAVs to penetrate from the surrounding media into the interior of the globular structures and transduce the internal cells. Figure 4B-4D Display AAV9 ( Figure 4B ), AAV.CPP.16 ( Figure 4C ) and AAV.CPP.21 ( Figure 4D A diagram of the processed spherical object. Figure 4E This shows the relative RFP intensity of spheres treated with different AAVs. P < 0.001, relative to AAV9, ANOVA.

[0030] Figures 5A-5B Describe representative images of brain slices after intravenous administration of AAV9, AAV.CPP.16, and AAV.CPP.21 in C57BL / 6J inbred mice. Figure 5A ) and its quantitative analysis ( Figure 5B All candidate AAVs expressed nuclear red fluorescent protein (RFP) as a reporter protein. The dosage of AAV was 1 × 10⁻⁶ per animal. 12 vg. Figure 5A Each white dot in the image represents a cell labeled with RFP. Figure 5B middle, P < 0.05 P < 0.001, ANOVA.

[0031] Figures 6A-6B Describes representative images of brain slices after intravenous administration of AAV9, AAV.CPP.16, and AAV.CPP.21 to BALB / cJ inbred mice. Figure 6A ) and its quantitative analysis ( Figure 6BAll candidate AAVs expressed nuclear red fluorescent protein (RFP) as a reporter protein. The dosage of AAV was 1 × 10⁻⁶ per animal. 12 vg. Figure 6A Each white dot in the image represents a cell labeled with RFP. Figure 6B middle, P < 0.001, ANOVA.

[0032] Figures 7A-7B Describes representative images of brain slices after intravenous administration of high doses of AAV.CPP.16 and AAV.CPP.21 in C57BL / 6J inbred mice. Figure 7A ) and its quantitative analysis ( Figure 7B Both candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter protein. The dosage of AAV was 4 × 10⁻⁶ per animal. 12 vg. Figure 7A Each white dot in the image represents a cell labeled with RFP. Figure 7B middle, P < 0.05, student test.

[0033] Figure 8A The transduction of adult neurons (labeled with NeuN antibody) by AAV.CPP.16 and AAV.CPP.21 in multiple brain regions, including the cortex, midbrain, and hippocampus, was demonstrated. The transduced neurons were co-labeled with NeuN antibody and RFP. 4 × 10⁴ N·m²⁻¹ were administered intravenously to adult C57BL / 6J mice (6 weeks old). 12 vg's AAV.

[0034] Figure 8B Describe the ability of AAV.CPP.16 and AAV.CPP.21 to enhance the targeting of spinal cord and motor neurons in mice compared to AAV9. 4 × 10⁴ cells were administered intravenously to newborn mice (day 1 after birth). 10 AAV of vg. Motor neurons in the ventral horn of the spinal cord were visualized using CHAT antibody staining. Co-localization of RFP and CHAT signals indicates specific transduction of motor neurons.

[0035] Figure 9A The ability of AAV.CPP.16 to target the heart of adult mice was described as enhanced compared to AAV9. In adult C57BL / 6J mice (6 weeks old), 1×10⁻⁶ AAV.CPP.16 was administered intravenously. 11 AAV of vg. Shows the percentage of RFP-labeled cells relative to all DAPI-stained cells. P < 0.05, student test.

[0036] Figure 9B The ability of AAV.CPP.16 to target skeletal muscle in adult mice, compared to AAV9, is described. In adult C57BL / 6J mice (6 weeks old), 1×10⁻⁶ skeletal muscle was administered intravenously. 11 AAV of vg. Shows the percentage of RFP-labeled cells relative to all DAPI-stained cells. P < 0.05, student test.

[0037] Figure 9C The ability of AAV.CPP.16 to target the dorsal root ganglion (DRG) in adult mice, as shown by comparison with AAV9, was described. 1 × 10⁻⁶ AAV.CPP.16 was administered intravenously to adult C57BL / 6J mice (6 weeks old). 11 AAV of vg. Shows the percentage of RFP-labeled cells relative to all DAPI-stained cells. P < 0.05, student test.

[0038] Figure 10A Describes the enhanced transduction of brain cells in the primary visual cortex by AAV.CPP.16 and AAV.CPP.21 relative to AAV9 after intravenous administration in nonhuman primates. (2 × 10⁻⁶) 13 Av / kg AAVs-CAG-AADC (as a reporter gene) was intravenously injected into 3-month-old cynomolgus monkeys with low levels of pre-existing neutralizing antibodies. AAV-transduced cells were visualized using antibody staining against AADC (shown in black). The square area in the left image is magnified, as shown in the right image. AAV.CPP.16 transduced significantly more cells compared to AAV9. AAV.CPP.21 also transduced more cells compared to AAV9, although its effect was less pronounced compared to AAV.CPP.16.

[0039] Figure 10B Describes the enhanced transduction ability of AAV.CPP.16 and AAV.CPP.21 relative to AAV9 in transducing brain cells in the parietal cortex following intravenous administration in nonhuman primates. (2 × 10⁻⁶) 13 vg / kg AAVs-CAG-AADC (as a reporter gene) were intravenously injected into 3-month-old cynomolgus monkeys with low levels of pre-existing neutralizing antibodies. AAV-transduced cells were visualized using antibody staining against AADC (shown in black). The square area in the left image is magnified, as shown in the right image. AAV.CPP.16 transduced significantly more cells compared to AAV9. AAV.CPP.21 also transduced more cells compared to AAV9, although its effect was less pronounced compared to AAV.CPP.16.

[0040] Figure 10CThis study depicts the enhanced transduction of brain cells in the thalamus by AAV.CPP.16 and AAV.CPP.21 relative to AAV9 following intravenous administration in nonhuman primates. (2 × 10⁻⁶) 13 vg / kg AAVs-CAG-AADC (as a reporter gene) were intravenously injected into 3-month-old cynomolgus monkeys with low levels of pre-existing neutralizing antibodies. AAV-transduced cells were visualized using antibody staining against AADC (shown in black). The square area in the left image is magnified, as shown in the right image. AAV.CPP.16 transduced significantly more cells compared to AAV9. AAV.CPP.21 also transduced more cells compared to AAV9, although its effect was less pronounced compared to AAV.CPP.16.

[0041] Figure 10D Describes the enhanced transduction of brain cells in the cerebellum by AAV.CPP.16 and AAV.CPP.21 relative to AAV9 after intravenous administration in nonhuman primates. (2 × 10⁻⁶) 13 Av / kg AAVs-CAG-AADC (as a reporter gene) was intravenously injected into 3-month-old cynomolgus monkeys with low levels of pre-existing neutralizing antibodies. AAV-transduced cells were visualized using antibody staining against AADC (shown in black). The square area in the left image is magnified, as shown in the right image. Both AAV.CPP.16 and AAV.CPP.21 transduced significantly more cells compared to AAV9.

[0042] Figure 11A-11B AAV.CPP.16 and AAV.CPP.21 were not described to bind to LY6A. LY6A acts as a receptor for AAV.PHP.B and its variants, including AAV.PHP.eB (as described in US9102949 and US20170166926) and mediates a strong transBBB effect of AAV.PHP.eB in certain mouse strains (Hordeaux et al., Mol Ther 2019 27(5):912-921; Huang et al., 2019, dx.doi.org / 10.1101 / 538421). Overexpression of mouse LY6A in cultured 293 cells significantly increased the binding of AAV.PHP.eB to the cell surface ( Figure 11A Conversely, overexpression of LY6A does not increase viral binding to AAV9, AAV.CPP.16, or AAV.CPP.21. Figure 11B This indicates that AAV.CPP.16 or AAV.CPP.21 do not share LY6A as a receptor with AAV.PHP.eB.

[0043] Figures 12A-12CAAV.CPP.21 is described as being used for systemic delivery of therapeutic genes to brain tumors in a mouse model of glioblastoma (GBM). Figure 12A As shown, intravenously administered AAV.CPP.21-H2BmCherry showed targeting of the tumor mass, particularly the tumor expanding frontier. Figure 12A ).exist Figure 12B (Image) and Figure 12C In the quantitative analysis, when combined with the prodrug ganciclovir, the use of AAV.CPP.21 to deliver the "suicide gene" HSV.TK1 systemically induced the shrinkage of the brain tumor mass. HSV.TK1 transformed the previously "dormant" ganciclovir into a tumor-killing drug. P < 0.05, student test.

[0044] Figure 13 Compared to AAV9, AAV.CPP.21 elicited more extensive and potent brain transduction when injected locally into the brain of adult mice. In adult mice (>6 weeks old), intracerebral injection of AAV (1×10⁻⁶) was performed. 11 (vg), and brain tissue was harvested and examined 3 weeks after AAV injection. P < 0.01, student test.

[0045] Figure 14 This is a set of images comparing the delivery efficiency of AAV9 (top) and AAV.CPP16 (bottom) to the GBM tumor microenvironment in a mouse model. As shown in the inset (right), AAV.CPP16 provides greater delivery efficiency.

[0046] Figure 15A -C shows that AAV.CPP.16-antiPD-L1-mediated immunotherapy prolongs survival in a mouse GBM model. Figure 15A A schematic diagram of the experimental scheme. Figure 15B For example, the survival rate of animals subjected to treatment. Figure 15C Long-term survival rate of animals treated with AAV.CPP16-anti-PDL1. LTS: Long-term survival rate.

[0047] Figure 16A -C indicates that GBM tumors were eradicated in all long-term surviving mice. Figure 16A H&E staining of brain sections from the posterior and anterior portions of the tumor injection site. No residual GBM was found in any of the sections. Figure 16B Bioluminescence imaging 7 days after tumor implantation indicated that the initial tumor implantation was successful. Figure 16CGBM tumor implantation sites with scar-like tissue.

[0048] Figures 17A-17B This shows the expression of HA-labeled anti-PD-L1 antibody in GBM tumors, as measured by immunoblotting. In mice, 1 e12 vg of AAV or PBS was administered intravenously 5 days after tumor implantation. Tumor tissue was harvested 14 days after IV injection. The intensity of HA tag staining (…) Figure 17A The value was quantified as a measure of anti-PD-L1 antibody expression. Figure 17B ). Detailed Implementation

[0049] Difficulties associated with cross-BBB delivery have hindered the development of therapeutics for brain diseases, including cancer. Adeno-associated virus (AAV) has become an important research and clinical tool for delivering therapeutic genes to the brain, spinal cord, and eyes; see, for example, US9102949; US9585971; and US20170166926. Significant progress has been made in AAV-mediated gene therapy with the recent approvals of Luxturna and Zolgensma. The approval of Zolgensma for endovascular treatment of spinal muscular atrophy in patients under two years of age is particularly encouraging, as it demonstrates the feasibility of using a cross-BBB AAV vector for systemic gene therapy of the central nervous system (CNS). Despite success in young patients, the AAV serotype AAV9 used in Zolgensma has low cross-BBB efficiency, particularly in adults, which limits its application for other CNS diseases. 8,9 This article describes a next-generation, brain-penetrating AAV vector (AAV.CPP16) that is at least 5-10 times more potent than the current industry standard (AAV9) in both rodents and nonhuman primates, which can be used in a novel cross-BBB AAV platform for GBM cancer gene therapy.

[0050] Through rational design and targeted screening based on known cell-penetrating peptides (CPPs) (see, for example, Gomez et al., Bax-inhibiting peptides derived from Ku70 and cell-penetrating pentapeptides. Biochem. Soc. Trans. 2007; 35 (Pt 4):797-801), it has been found that targeting sequences, when engineered into the capsid of AAVs, improve gene delivery efficiency to the brain by up to three orders of magnitude. These methods have been used to engineer AAV vectors that significantly reduce tumor size in animal models of glioblastoma.

[0051] Furthermore, the brain is "immune privileged," which makes immunotherapy for GBM challenging. "Priming" the immune response aims to transform immunologically "cold" GBM tumors into immunogenic "hot" tumors. The method of this invention utilizes the vector described herein to deliver immunotherapeutic agents that can achieve this purpose, such as anti-PD-L1 antibodies. Not wishing to be bound by theory, it is believed that the AAV vector itself "primes" the immune system by increasing tumor infiltration of cytotoxic T cells, while the anti-PD-L1 antibody expressed at the tumor site and throughout the CNS activates previously "exhausted" T cells.

[0052] Target sequence

[0053] This method identifies many potential targeting peptides that enhance penetration through the BBB, for example, when inserted into the capsid of AAVs such as AAV1, AAV2, AAV8, or AAV9, or when chemically or by conjugating them as fusion proteins with biological agents such as antibodies or other large biomolecules.

[0054] In some embodiments, the targeting peptide comprises a sequence of at least 5 amino acids. In some embodiments, the amino acid sequence comprises at least 4, for example 5, consecutive amino acids of sequences VPALR (SEQ ID NO:1) and VSALR (SEQ ID NO:2).

[0055] In some embodiments, the targeting peptide comprises an X1X2X3X4X5 sequence, wherein:

[0056] (i) X1, X2, X3, X4 are any four distinct amino acids of V, A, L, I, G, P, S, T, or M; and

[0057] (ii) X5 is K, R, H, D or E (SEQ ID NO:73).

[0058] In some embodiments, the targeting peptide comprises a sequence of at least 6 amino acids. In some embodiments, the amino acid sequence comprises at least 4, for example 5 or 6 consecutive amino acids of the sequences TVPALR (SEQ ID NO:3), TVSALK (SEQ ID NO:4), TVPMLK (SEQ ID NO:12), and TVPTLK (SEQ ID NO:13).

[0059] In some embodiments, the targeting peptide comprises the sequence X1X2X3X4X5X6, wherein:

[0060] (i) X1 is T;

[0061] (ii) X2, X3, X4, and X5 are any four distinct amino acids of V, A, L, I, G, P, S, T, or M; and

[0062] (iii) X6 is K, R, H, D or E (SEQ ID NO:74).

[0063] In some embodiments, the targeting peptide comprises the sequence X1X2X3X4X5X6, wherein:

[0064] (i) X1, X2, X3, X4 are any four different amino acids derived from V, A, L, I, G, P, S, T, or M;

[0065] (ii) X5 is K, R, H, D, or E; and

[0066] (iii) X6 is E or D (SEQ ID NO:75).

[0067] In some embodiments, the targeting peptide comprises a sequence of at least 7 amino acids. In some embodiments, the amino acid sequence comprises at least 4, for example 5, 6, or 7 consecutive amino acids of the sequences FTVSALK (SEQ ID NO:5), LTVSALK (SEQ ID NO:6), TVSALFK (SEQ ID NO:8), TVPALFR (SEQ ID NO:9), TVPMLFK (SEQ ID NO:10), and TVPTLFK (SEQ ID NO:11). In some other embodiments, the targeting peptide comprises a sequence of X1X2X3X4X5X6X7, wherein:

[0068] (i) X1 is F, L, W or Y;

[0069] (ii) X2 is T;

[0070] (iii) X3, X4, X5, and X6 are any four distinct amino acids of V, A, L, I, G, P, S, T, or M; and

[0071] (iv) X7 is K, R, H, D or E (SEQ ID NO:76).

[0072] In some embodiments, the targeting peptide comprises the sequence X1X2X3X4X5X6X7, wherein:

[0073] (i) X1 is T;

[0074] (ii) X2, X3, X4, X5 are any four distinct amino acids of V, A, L, I, G, P, S, T, or M;

[0075] (iii) X6 is K, R, H, D, or E; and

[0076] (iv) X7 is E or D (SEQ ID NO:77).

[0077] In some embodiments, the targeting peptide comprises the sequence X1X2X3X4X5X6X7, wherein:

[0078] (i) X1, X2, X3, X4 are any four distinct amino acids of V, A, L, I, G, P, S, T, or M;

[0079] (ii) X5 is K, R, H, D or E;

[0080] (iii) X6 is either E or D; and

[0081] (iv) X7 is A or I (SEQ ID NO:78).

[0082] In some embodiments, the targeting peptide comprises the sequence V[S / p][A / m / t / ]L (SEQ ID NO:79), wherein uppercase letters are preferred at this position. In some embodiments, the targeting peptide comprises the sequence TV[S / p][A / m / t / ]L (SEQ ID NO:80). In some embodiments, the targeting peptide comprises the sequence TV[S / p][A / m / t / ]LK (SEQ ID NO:81). In some embodiments, the targeting peptide comprises the sequence TV[S / p][A / m / t / ]LFK (SEQ ID NO:82).

[0083] In some implementations, the targeting peptide is not composed of VPALR (SEQ ID NO:1) or VSALRK (SEQ ID NO:2).

[0084] Specific exemplary amino acid sequences, including the 5, 6, or 7-amino acid sequences mentioned above, are listed in Table 1.

[0085] Table 1 – Target Sequences

[0086]

[0087]

[0088] Targeting peptides including reverse sequences can also be used, such as KLASVT (SEQ ID NO:83) and KFLASVT (SEQ ID NO:84).

[0089] The targeted peptides disclosed herein can be modified according to methods known in the art for generating peptidomimetics. See, for example, Qvit et al., Drug Discov Today. Feb. 2017; 22(2): 454-462; Farhadi and Hashemian, Drug Des Devel Ther. 2018; 12: 1239–1254; Avan et al., Chem. Soc. Rev., 2014, 43, 3575-3594; Pathak et al., Indo American Journal of Pharmaceutical Research, 2015. 8; Kazmierski, WM, ed., Peptidomimetics Protocols, Human Press (Totowa NJ 1998); Goodman et al., ed., Houben-Weyl Methods of Organic Chemistry: Synthesis of Peptides and Peptidomimetics, Thiele Verlag (New York 2003); and Mayo et al., J. Biol. Chem., 278: 45746 (2003). In some cases, the modified peptide-like forms of the peptides and fragments disclosed herein exhibit enhanced in vivo stability compared to non-peptide-like peptides.

[0090] Methods for generating peptides include replacing one or more, or all, amino acids in a peptide sequence with D-amino acid enantiomers. Such sequences are referred to herein as “retro” sequences. In another method, the N-terminal to C-terminal order of amino acid residues is reversed, such that the N-terminal to C-terminal amino acid residue order of the original peptide becomes the C-terminal to N-terminal amino acid residue order of the modified peptide. Such sequences are referred to as “inverso” sequences.

[0091] Peptides can be in reverse and inverted forms, i.e., the “reverse-inverted” form of peptides disclosed herein. New peptides can be composed of D-amino acids arranged such that the N-terminal to C-terminal amino acid sequence in the peptide corresponds to the C-terminal to N-terminal amino acid sequence in the original peptide.

[0092] Other methods for preparing peptide analogs include replacing one or more amino acid residues in the peptide with chemically different but recognized functional amino acid analogs, i.e., artificial amino acid analogs. Artificial amino acid analogs include β-amino acids and β-substituted β-amino acids (“β-amino acids”). 3 -amino acids), phosphorus-containing analogues of amino acids such as -aminophosphonic acid and -Aminophosphonic acid, and amino acids having non-peptide bonds. Peptides can be generated using artificial amino acids, such as peptide oligomers (e.g., peptide amides or ester analogs), β-peptides, cyclic peptides, oligoureas, or oligocarbamate peptides; or heterocyclic molecules. Exemplary reverse-targeting peptides include KLASVT and KFLASVT, wherein the sequence comprises all D-amino acids. These sequences can be modified, for example, by biotinylation of the N-terminus and amidation of the C-terminus.

[0093] AAVs

[0094] Viral vectors used in this method and composition include recombinant retroviruses, adenoviruses, adeno-associated viruses, alpha viruses, and lentiviruses, which contain the target peptides described herein and optionally transgenes for expression in target tissues.

[0095] The preferred viral vector system for delivering nucleic acids in this method is adeno-associated virus (AAV). AAV is a tiny, non-enveloped virus with a 25 nm capsid. No disease associated with it or shown to be related to wild-type viruses is known. AAV has a single-stranded DNA (ssDNA) genome. AAV has been shown to exhibit long-term free transgene expression, and AAV has demonstrated excellent transgene expression in the brain, particularly in neurons. AAV vectors containing as few as 300 base pairs can be packaged and integrated. The spatial restriction of the exogenous DNA is approximately 4.7 kb. AAV vectors, such as those described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985), can be used to introduce DNA into cells. AAV vectors have been used to introduce various nucleic acids into different cell types (see, for example, Hermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51:611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790). (1993). Numerous AAV variants are available (over 100 have been cloned), and AAV variants have been identified based on desired properties. In some embodiments, the AAV is AAV1, AAV2, AAV4, AAV5, AAV6, AV6.2, AAV7, AAV8, AAV9, rh.10, rh.39, rh.43, or CSp3; for CNS use, in some embodiments, the AAV is AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, or AAV9. As an example, AAV9 has been shown to be relatively efficient across the blood-brain barrier. Using this method, the AAV capsid can be genetically engineered to increase penetration across the BBB or to increase penetration into specific tissues by inserting the target sequence as described herein into the capsid protein, for example, between amino acids 588 and 589 in the AAV9 capsid protein VP1.

[0096] An exemplary wild-type AAV9 capsid protein VP1 (Q6JC40-1) sequence is shown below:

[0097]

[0098]

[0099] Therefore, this document provides AAVs comprising one or more of the target peptide sequences described herein, for example, AAVs comprising capsid proteins containing the target sequences described herein, for example, capsid proteins comprising SEQ ID NO:1, wherein the target peptide sequence has been inserted into the sequence, for example, between amino acids 588 and 589.

[0100] Immunotherapy Genetically Modified Organisms

[0101] In some embodiments, the AAV also includes a transgenic sequence (i.e., a heterologous sequence) encoding an immunotherapeutic agent, such as that described herein or known in the art. The transgenic sequence is preferably linked to a sequence that promotes / drives the expression of the transgenic sequence in the target tissue.

[0102] Exemplary transgenes used as immunotherapeutic agents include transgenes encoding immune checkpoint inhibitory antibodies or antigen-binding fragments thereof, such as single-chain variable region fragments (scFv) antibodies that act as checkpoint inhibitors.

[0103] Examples of immunotherapy include, but are not limited to, adoptive T-cell therapies or cancer vaccine formulations designed to induce T lymphocytes to recognize cancer cells, and checkpoint inhibitors such as anti-CD137 antibodies (e.g., BMS-663513), anti-PD1 antibodies (e.g., nivolumab, pembrolizumab / MK-3475, pitilizumab (CT-011)), anti-PDL1 antibodies (e.g., BMS-936559, MPDL3280A), or anti-CTLA-4 antibodies (e.g., ipilimumab; see, for example, Krüger et al., (2007) Histol Histopathol. 22(6): 687-96; Eggermont et al., (2010) Semin Oncol. 37(5): 455-9; Klinke (2010) Mol. Cancer. 9: 242; Alexandrescu et al., (2010) J. Immunother. 33(6): 570-90; Moschella et al., (2010) Ann NY Acad Sci. 1194: 169-78; Ganesan and Bakhshi, (2010) Natl. Med. J. India 23(1): 21-7; and Golovina and Vonderheide, (2010) Cancer J. 16(4): 342-7.

[0104] Exemplary anti-PD-1 antibodies that can be used in the methods described herein include those that bind to human PD-1; exemplary PD-1 protein sequences are provided in NCBI Registry No. NP_005009.2. Exemplary antibodies are described in U.S. Patent Nos. 8,008,449; 9,073,994; and U.S. Publication No. 2011 / 0271358, including, for example, PF-06801591, AMP-224, BGB-A317, BI754091, JS001, MEDI0680, PDR001, REGN2810, SHR-1210, TSR-042, pembrolizumab, nivolumab, and avelumab. ), cimiplimab, spartalizumab, camrelizumab, sintilimab, pidilizumab, tislelizumab, toripalimab, AMP-224, AMP-514 and atezolizumab.

[0105] Exemplary anti-CD40 antibodies that can be used in the methods described herein include those that bind to human CD40; exemplary CD40 protein precursor sequences are provided in NCBI accessions NP_001241.1, NP_690593.1, NP_001309351.1, NP_001309350.1 and NP_001289682.1. Exemplary antibodies include those described in International Publications WO2002 / 088186; WO2007 / 124299; WO2011 / 123489; WO2012 / 149356; WO2012 / 111762; WO2014 / 070934; U.S. Publications 2013 / 0011405; 2007 / 0148163; 2004 / 0120948; 2003 / 0165499; and U.S. Patent No. 8,591,900; including, for example, dacetuzumab, lucarumumab, bleselumab, teneliximab, ADC-1013, CP-870,893, and Chi Lob. 7 / 4, HCD122, SGN-4, SEA-CD40, BMS-986004, and APX005M. In some implementations, the anti-CD40 antibody is a CD40 agonist, not a CD40 antagonist.

[0106] Exemplary anti-PD-L1 antibodies that can be used in the methods described herein include those that bind to human PD-L1; exemplary PD-L1 protein sequences are provided in NCBI accessions NP_001254635.1, NP_001300958.1 and NP_054862.1. Exemplary antibodies are described in US Publication No. 2017 / 0058033; International Publication Nos. WO2017 / 118321A1; WO2016 / 061142A1; WO2016 / 007235A1; WO2014 / 195852A1 and WO2013 / 079174A1, including, for example, BMS-936559 (MDX-1105), FAZ053, KN035, atezolizumab (Tecentriq, MPDL3280A), avelumumab (Bavencio), durvalumab (Imfinzi, MEDI-4736), envafolimab (KN035), CK-301, CS-1001, SHR-1316 (HTI-1088), and CBT-502. (TQB-2450), BGB-A333, and BMS-986189. Non-antibody peptide inhibitors, such as AUNP12 and CA-170, can also be used. See also Akinleye & Rasool, Journal of Hematology & Oncology, 12:92 (2019) doi:10.1186 / s13045-019-0779-5.

[0107] In some embodiments, the immunotherapeutic agent is an antigen-binding portion of an anti-PD-L1 antibody or contains an antigen-binding portion of an anti-PD-L1 antibody, such as a single-chain variable region fragment (scFv) antibody targeting the human PD-L1 protein (PD-L1.Hu); an exemplary sequence encoding the anti-PDL1 antibody scFv is shown in SEQ ID NO: 105 or a portion thereof, such as lacking one, two or more of the signal peptide, HA-tag and Myc-tag, for example containing amino acids (aa) 31-513 of SEQ ID NO: 105:

[0108] Exemplary anti-PDL1 scFv sequence (signal peptide (aa 1-21); HA-tag, aa 21-30; Myc-tag, aa514-523)

[0109] .

[0110] The following are exemplary anti-PD-L1 nucleic acid sequences (signal peptide (nt 1-63); HA-tag, nt 64-90); Myc-label Signed, nt 1540-1569 )

[0111] .

[0112] Other antibodies, and methods for generating nucleic acids encoding these antibodies, are known in the art; see, for example, Li et al., Int J Mol Sci. 2016 Jul; 17(7): 1151; Engeland et al., Mol Ther. 2014 Nov; 22(11): 1949–1959 and the references above.

[0113] The virus may also include one or more sequences that promote transgene expression, such as one or more promoter sequences; enhancer sequences, such as a 5' untranslated region (UTR) or a 3' UTR; polyadenylation sites; and / or isolator sequences. In some embodiments, the promoter is a brain tissue-specific promoter, such as a neuron-specific or glial-specific promoter. In some embodiments, the promoter is a promoter selected from the following genes: neuronal nucleus (NeuN), glial fibrillary acidic protein (GFAP), MeCP2, adenomatous polyposis (APC), ionized calcium-binding aptamer 1 (Iba-1), synaptic protein I (SYN), calcium / calmodulin-dependent protein kinase II, tubulin α I, neuron-specific enolase, and platelet-derived growth factor β chain. In some embodiments, the promoter is a pancellular promoter, such as a cytomegalovirus (CMV), β-glucuronidase (GUSB), ubiquitin C (UBC), or Rous sarcoma virus (RSV) promoter. Post-transcriptional response elements (WPREs) of marmot hepatitis virus can also be used. In some embodiments, a human signal or leader sequence, such as an IgK leader sequence, is used. In some embodiments, a human signal sequence is used instead, as shown in the table below (adapted from novoprolabs.com / support / articles / commonly-used-leader-peptide-sequences-for-efficient-secretion-of-a-recombinant-protein-expressed-in-mammalian-cells-201804211337.html):

[0114]

[0115] Barash et al., Biochem Biophys Res Commun. 2002 Jun 21;294(4):835-42. In some embodiments, secretory sequences that promote antibody secretion are used, for example, as described in von Heijne, J Mol Biol. 1985 July 5;184(1):99-105; Kober et al., Biotechnol. Bioeng. 2013; 110: 1164–1173; Tsuchiya et al., Nucleic Acids Research Supplement No. 3 261-262 (2003).

[0116] In some embodiments, the AAV also has one or more additional mutations that increase delivery to target tissues such as the CNS or decrease extra-tissue targeting, such as mutations that decrease liver delivery when delivery to the CNS, heart, or muscle is intended (e.g., as described in Pulicherla et al., (2011) Mol Ther 19:1070-1078); or add other targeting peptides, such as those described in Chen et al., (2008) Nat Med 15:1215-1218 or Xu et al., (2005) Virology 341:203-214 or US9102949; US9585971; and US20170166926. See also Gray and Samulski (2011) “Vector design and Considerations for CNS applications”, Gene Vector Design and Application to Treat Nervous System Disorders ed. Glorioso J., ed., (Washington, DC: Society for Neuroscience;) 1–9, available at sfn.org / ~ / media / SfN / Documents / Short%20Courses / 2011%20Short%20Course%20I / 2011_SC1_Gray.ashx.

[0117] How to use

[0118] The methods and compositions described herein can be used to deliver immunotherapeutic compositions to tissues, such as the central nervous system (brain), heart, muscles, dorsal root ganglia, or spinal cord (peripheral nervous system). In some embodiments, the method includes delivery to specific brain regions, such as the cortex, cerebellum, hippocampus, substantia nigra, or amygdala. In some embodiments, the method includes delivery to neurons, astrocytes, and / or glial cells.

[0119] In some embodiments, the methods and compositions such as AAV are used to deliver a nucleic acid sequence encoding an immunotherapeutic agent to a subject with brain cancer. Brain cancer includes gliomas (e.g., glioblastoma multiforme (GBM)), metastatic cancers (e.g., those from lung cancer, breast cancer, melanoma, or colon cancer), meningiomas, pituitary adenomas, and acoustic neuromas. Therefore, the methods may include systemic administration, such as intravenous administration, to a subject diagnosed with brain cancer, of an AAV (e.g., AAV9) containing a targeting peptide as described herein and encoding an immunotherapeutic agent (e.g., AAV9 with CPP 16 inserted, also referred to herein as AAV.CPP16).

[0120] In some embodiments, the method further includes co-administration of a chemotherapy agent. In some embodiments, the chemotherapy agent is a toxin or cytotoxic drug, including but not limited to temozolomide, lomustine, or combinations thereof. See, for example, Herrlinger et al., Lancet. 2019 Feb 16; 393(10172):678-688. The method may also include administration of radiation, surgical resection, or both.

[0121] Pharmaceutical Compositions and Administration

[0122] The methods described herein include using a pharmaceutical composition containing an AAV as an active ingredient, said AAV comprising (i) a targeting peptide and (ii) a sequence encoding an immunotherapeutic agent.

[0123] Pharmaceutical compositions typically contain pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" includes saline solutions, solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents that are compatible with drug administration.

[0124] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular, or injection or infusion administration. Thus, delivery can be systemic or local.

[0125] Methods for preparing suitable pharmaceutical compositions are known in the art; see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions for parenteral application may include the following components: sterile diluents, such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for tonicity adjustment, such as sodium chloride or glucose. The pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0126] Pharmaceutical compositions suitable for injectable applications may include sterile aqueous solutions (water-soluble) or dispersions, and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL... TM (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should flow readily for easy injection. It should be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. For example, appropriate flowability can be maintained by using coatings such as lecithin, maintaining the desired particle size in the case of dispersions, and using surfactants. Antimicrobial activity can be achieved using various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride. Extended absorption of the injectable composition can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0127] A sterile injectable solution can be prepared by incorporating the desired amount of the active compound with one or a combination of the ingredients listed above into a suitable solvent, followed by filtration and sterilization. Typically, a dispersion is prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other desired ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying, which yields powders of the active ingredient and any other desired ingredients from a previously sterile filtered solution.

[0128] In one embodiment, a therapeutic compound is prepared together with a carrier that protects the therapeutic compound from rapid elimination from the body, such as as a controlled-release formulation, including implants and microencapsulated drug delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques or are commercially available, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeting selected cells with monoclonal antibodies against cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0129] The pharmaceutical composition may be included in a kit, container, package, or dispenser along with the instructions for use.

[0130] Example

[0131] The invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0132] Materials and methods

[0133] The following materials and methods were used in the following embodiments.

[0134] 1. The generation of capsid variants

[0135] To generate capsid variant plasmids, a DNA fragment encoding the cell-penetrating peptide (Table 3) was synthesized using the CloneEZ seamless cloning technology (GenScript) and inserted into the backbone of the AAV9 Rep-cap plasmid (pRC9) between amino acid positions 588 and 589 (VP1 amino acid number). CPPs BIP1 (VPALR, SEQ ID NO:1) and BIP2 (VSALK, SEQ ID NO:2), and their derivatives such as TVSALK (SEQ ID NO:4) in AAV.CPP.16 and TVSALFK (SEQ ID NO:8) in AAV.CPP.21, derived from the Ku70 protein, have the following sequences:

[0136]

[0137] In addition, the VP1 protein sequences of AAV9, AAV.CPP.16, and AAV.CPP.21 are shown below:

[0138]

[0139]

[0140] 2. Production of recombinant AAV

[0141] Recombinant AAV was packaged using a standard three-plasmid co-transfection protocol (pRC plasmid, pHelper plasmid, and pAAV plasmid). pRC9 (or a variant thereof), pHelper, and pAAV carrying a transgene (e.g., nuclear-directed RFPH2B-mCherry driven by the ubiquitous EF1a promoter) were co-transfected into HEK 293T cells using polyethyleneimine (PEI, Polysciences). The rAAV vector was collected from serum-free medium at 72 and 120 hours post-transfection, and from cells at 120 hours post-transfection. AAV particles in the medium were concentrated using PEG precipitation with 8% PEG-8000 (wt / vol). The cell pellet containing viral particles was resuspended and lysed by sonication. Viral vectors derived from a combination of PEG precipitates and cell lysates were treated with DNase and RNase at 37°C for 30 min, followed by purification using ultracentrifugation (VTi 50 rotor, 40,000 rpm, 18°C, 1 h) via an iodixanol gradient (15%, 25%, 40%, and 60%). rAAV was then concentrated using a Millipore Amicon filter unit (UFC910008, 100K MWCO) and prepared in DuPont phosphate-buffered saline (PBS) containing 0.001% Pluronic F68 (Gibco).

[0142] 3. AAV titration method

[0143] Viral titers were determined by quantitative PCR using DNase-resistant genome copy number. pAAV-CAG-GFP was digested with PVUII (NEB) to generate free ends of the plasmid ITR, which were used to generate a standard curve. Viral samples were incubated with DNase I to remove contaminating DNA, followed by treatment with sodium hydroxide to dissolve the viral capsid and release the viral genome. Quantitative PCR was performed using the ITR forward primer 5'-GGAACCCCTAGTGATGGAGTT (SEQ ID NO:91) and the ITR reverse primer 5'-CGGCCTCAGTGAGCGA (SEQ ID NO:92). Vector titers were normalized relative to rAAV-2 reference standard materials (RSMs, ATCC, catalog number: VR-1616, Manassas, VA).

[0144] 4. Administration of AAV in mice

[0145] For intravenous administration, adult mice (6 weeks and older) were administered AAV diluted in sterile saline (0.2 ml) via tail vein injection. Animals were kept alive for three weeks before euthanasia for tissue harvesting. For intracranial injection, AAV diluted in PBS (10 μl) was injected using a Hamilton syringe at coordinates from the anterior fontanelle: right 1.0 mm, posterior 0.3 mm, depth 2.6 mm. All animal studies were conducted at an IACUC-approved AAALAC-accredited facility.

[0146] 5. Mouse tissue processing

[0147] Anesthetized animals underwent cardiac perfusion with cold phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA). Tissue was post-fixed overnight in 4% PFA, then soaked in 30% sucrose solution for two days, subsequently embedded in OCT and flash-frozen. Typically, 80µm thick brain sections were excised for natural fluorescence imaging, and 40µm thick brain sections were used for IHC.

[0148] 6. In vitro human BBB spherical model

[0149] Hot 1% agarose (w / v, 50 μL) was added to 96-well plates for cooling / solidification. Primary human astrocytes (Lonza Bioscience), human brain microvascular pericytes (HBVP, ScienCell Research Laboratories), and human brain microvascular endothelial cells (hCMEC / D3; Cedarlane) were then seeded onto the agarose gel at a 1:1:1 ratio (1500 cells per type). Cells were cultured at 37°C in a 5% CO2 incubator for 48–72 hours to spontaneously assemble multicellular BBB spheroids. A multicellular barrier, mimicking the BBB, was reported to form around the spheroids. AAVs-H2B-mCherry were added to the medium, and after 4 days, all spheroids were fixed with 4% PFA. The spheroids were then transferred to Nunc Lab-Tek II thin-glass 8-well coverslips (Thermo Scientific) and imaged using a Zeiss LSM710 confocal microscope. Examine the RFP signal intensity within the spherical body and use it as a "reading".

[0150] 7. Administration of AAV in non-human primates (NHPs)

[0151] All NHP studies were conducted by a CRO at an AAALAC-accredited facility approved by IACUC. Cynomolgus monkeys were pre-screened for little to no pre-existing neutralizing antibodies against AAV9 (titers <1:5). AAV diluted in PBS / 0.001% F68 was injected intravenously (via cephalic or femoral vein) using a peristaltic pump. After 3 weeks, the animals were perfused with PBS, followed by 4% PFA. Tissue was then collected and processed for paraffin embedding and sectioning.

[0152] 8. Immunohistochemistry

[0153] Mouse tissue sections were float-stained using primary antibodies diluted in PBS containing 10% donkey serum and 2% Triton X-100. The primary antibodies used included: chicken anti-GFP (1:1000); rabbit anti-RFP (1:1000); mouse anti-NeuN (1:500); rat anti-GFAP (1:500); goat anti-GFAP (1:500); and mouse anti-CD31 (1:500). Secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 fluorophores were applied at a 1:200 dilution to the host species of the primary antibody.

[0154] For paraffin sections of NHP tissue, DAB staining was performed to visualize cells transduced by AAV-AADC. Rabbit anti-AADC antibody (1:500, Millipore) was used as the primary antibody.

[0155] 9. AAV binding test

[0156] HEK 293T cells were cultured at 37°C in a 5% CO2 incubator. One day after seeding HEK 293T cells into 24-well plates at a density of 250,000 cells per well, the LY6A cDNA plasmid was transiently transfected into the cells using a transfection mixture of 200 μL DMEM (31053028; Gibco), 1 μg DNA plasmid, and 3 μg PEI. Forty-eight hours post-transfection, the cells were chilled on ice for 10 minutes. The medium was then replaced with 500 μL of ice-cold serum-free DMEM containing rAAVs-mCherry with an MOI of 10,000. After incubation on ice for 1 hour, cells presumed to be AAV-bound were washed three times with cold PBS, followed by genomic DNA extraction. Viral particles binding to cells were quantified by qPCR using mCherry-specific primers and normalized to the HEK 293T genome using human GCG as a reference.

[0157] 10. Glioblastoma mouse model

[0158] All experiments were conducted according to protocols approved by the Animal Care and Use Committee (IACUC) of Brigham and Women's Hospital and Harvard Medical School. Syngeneically immunocompetent C57BL / 6 female mice weighing 20 ± 1 g (Envigo) were used. GL261-Luc (100,000 mouse glioblastoma cells) resuspended in 2 μl phosphate-buffered saline (PBS) was injected intracranially using a 10 μl syringe (80075; Hamilton) with a 26-gauge needle. The implantation site was located using a stereotactic frame (anterior fontanelle coordinates, in mm: right 2, anterior 0.5, cortical depth 3.5). Seven days later, a single 200 μl dose of AAV-HSV-TK1 (1E+12 viral genome, IV) was administered, followed by daily ganciclovir (50 mg / kg) for 10 days.

[0159] Example 1. Modification of AAV9 capsid

[0160] To identify peptide sequences that enhance the penetration of biomolecules or viruses across the blood-brain barrier, AAV peptide display technology was used to insert the individual cell-penetrating peptides listed in Table 3 into the AAV9 capsid between amino acids 588 and 589 (VP1 number), such as... Figure 1A As shown in the diagram. Insertion is performed by modifying the RC plasmid, one of three co-transfection plasmids used for AAV packaging; Figure 1B An exemplary schematic diagram of the experiment is shown. Individual AAV variants were generated and screened separately. See Materials and Methods #1-3 for more details.

[0161] Table 3

[0162]

[0163] #, SEQ ID NO:

[0164] Syn, synthetic

[0165] Example 2. First round of in vivo screening

[0166] AAV expressing nuclear RFP (H2B-RFP) was intravenously injected into adult mice with a mixed C57BL / 6 and BALB / c genetic background. Three weeks later, brain tissue was harvested and sectioned to visualize RFP-labeled cells. Figure 2A and 2C The white dots in the image are respectively located in Figure 2B and 2D(Medium-quantitative). CPP BIP1 and BIP2 were inserted into the capsids of AAV.CPP.11 and AAV.CPP.12, respectively. See Materials and Methods #4-5 for more details.

[0167] Example 3. Optimization of the modified AAV9 cap.

[0168] AAV.CPP.11 and AAV.CPP.12 were further engineered by optimizing the BIP targeting sequence. The BIP insert was derived from protein Ku70 (see full sequence for details). Figure 3A Materials / Methods #1). The BIP sequence VSALT, chosen as the source of "synthesis," was selected as the focus of this study to minimize the potential species specificity of the engineered AAV vector. AAVs were generated, and their brain transduction efficiency was tested separately compared to AAV9 (see [link to study]. Figure 3B -C). In Figure 3D The figure shows the percentage of cellular transduction in mouse livers 3 weeks after IV injection of certain AAV variants that deliver the reporter gene RFP. See Materials and Methods #1-5 for more details.

[0169] Example 4. In vitro model – BBB penetration screening

[0170] The ability of several AAV variants to cross the human BBB was screened using an in vitro globular BBB model. The globular structures were contained in human microvascular endothelial cells, as well as human pericytes and astrocytes, which form a surface barrier. The ability of AAVs carrying nuclear RFP as a reporter protein to penetrate from the surrounding media into the globular structures and transduce the internal cells was evaluated. Figure 4A A schematic diagram of the experiment is shown. Figure 4B -D shows the results for wtAAV9, AAV.CPP.16, and AAV.CPP.21, respectively, which, along with other peptides, [are shown in the original text]. Figure 4E Quantitative analysis was performed. In this model, peptides 11, 15, 16, and 21 produced the greatest penetration into the globule. For more details, see Materials and Methods #6.

[0171] Example 5. In vivo BBB penetration screening

[0172] Following the experiments described in Example 2 above, AAV.CPP.16 and AAV.CPP.21 were selected for further evaluation in an in vivo model. All AAVs carry nuclear RFP as a reporter protein. Intravenous administration was performed on adult C57BL / 6J mice (… Figure 5A White spots in brain slices, Figure 5B Medium-quantitative) and BALB / c adult mice ( Figure 6A White spots in brain slices, Figure 6BAfter quantification, both showed an enhanced ability to transduce brain cells relative to AAV9.

[0173] High doses of AAV.CPP.16 and AAV.CPP.21 (4 × 10⁻⁶ per mouse) 12 VG and IV administration induced widespread brain transduction in mice. Both AAVs carry nuclear RFP as a reporter protein (VG, IV). Figure 7A White spots in brain slices, Figure 7B (Medium-quantitative).

[0174] Example 6. In vivo distribution of modified AAV

[0175] like Figure 8A As shown, AAV.CPP.16 and AAV.CPP.21 preferentially target neurons (tagged with NeuN antibody) in multiple brain regions in mice, including the cortex, midbrain, and hippocampus. Both AAVs carry nuclear RFP as a reporter protein.

[0176] AAV.CPP.16 and AAV.CPP.21 also showed enhanced targeting of spinal cord and motor neurons in mice compared to AAV9. All AAVs carried nuclear RFP as a reporter protein and were administered intravenously to newborn mice (4 × 10⁻⁶). 10 (vg). Motor neurons were visualized using CHAT antibody staining. Figure 8B In this study, the colocalization of RFP and CHAT signals indicates specific transduction of motor neurons.

[0177] The relative potency of AAV-CAG-H2B-RFP and AAV.CPP.16-CAG-H2B-RFP in various tissues of mice was also evaluated. Intravenous injection of 1×10 11 vg. The number of transduced cells was normalized to the total number of DAPI-labeled cells. Results showed that AAV.CPP.16 targeted the mouse heart ( Figure 9A ); skeletal muscle ( Figure 9B ) and dorsal root ganglion ( Figure 9C It is more effective than AAV9 in terms of organization.

[0178] Example 7. BBB Penetration in a Non-Human Primate Model

[0179] 2×10 13 vg / kg AAVs-CAG-AADC (as a reporter gene) were intravenously injected into 3-month-old cynomolgus monkeys. AAV-transduced cells were visualized using antibody staining against AADC (shown in black). Figure 10AAs shown in Figure D, after intravenous administration to non-human primates, AAV.CPP.16 and AAV.CPP.21 exhibited enhanced transduction capabilities in brain cells compared to AAV9. AAV.CPP.16 showed enhanced transduction capabilities in the primary visual cortex (…). Figure 10A ), apical cortex ( Figure 10B ),thalamus( Figure 10C ), and cerebellum ( Figure 10D Significantly more cells were transduced in ) than in wtAAV9. See Materials and Methods #7-8 for more details.

[0180] Example 8. AAV.CPP.16 and AAV.CPP.21 do not bind to LY6A.

[0181] LY6A acts as a receptor for AAV.PHP.eB and mediates a robust transBB-bound pathway of AAV.PHP.eB in certain mouse strains. Overexpression of mouse LY6A in cultured 293 cells significantly increased the binding of AAV.PHP.eB to the cell surface (see [link to LY6A]). Figure 11A Conversely, overexpression of LY6A does not increase viral binding to AAV9, AAV.CPP.16, or AAV.CPP.21 (see [link to relevant documentation]). Figure 11B This indicates that AAV.CPP.16 or AAV.CPP.21 does not share LY6A as a receptor with AAV.PHP.eB. See Materials and Methods #9 for more details.

[0182] Example 9. Delivery of therapeutic proteins to the brain using AAV.CPP.21

[0183] AAV.CPP.21 was used for systemic delivery of the "suicide gene" HSV.TK1 in a mouse model of brain tumors (Materials and Methods #10). HSV.TK1 transforms the previously dormant ganciclovir into a tumor-killing drug. AAV.CPP.21-H2BmCherry was administered intravenously. Figure 12A (The lower left and middle right images) show the targeted tumor mass, especially the tumor's extended boundaries. Figure 12B As shown in -C, when combined with the prodrug ganciclovir, systemic delivery of the "suicide gene" HSV.TK1 using AAV.CPP.21 induced shrinkage of the brain tumor mass. These results suggest that AAV.CPP.21 can be used for systemic delivery of therapeutic genes into brain tumors. See Materials and Methods #10 for more details.

[0184] Example 10. Intracerebral administration of AAV.CPP.21

[0185] In addition to systemic application (e.g., in Example 2), AAV was locally applied to the brain of mice as described herein. AAV9-H2B-RFP and AAV.CPP.21-H2B-RFP ( Figure 13 Intracerebral injection of AAV.CPP.21 resulted in more extensive and higher-intensity RFP signals in brain slices treated with AAV.CPP.21 compared to those treated with AAV9. See Materials and Methods #4 for more details.

[0186] Example 11. Systemic delivery of AAV.CPP.16 into the glioblastoma tumor microenvironment

[0187] AAV, as described herein, was delivered into the brain of an orthotopically immunogenic mouse glioblastoma model (GL261 model) using systemic administration (e.g., in Example 2). (As described in Materials and Methods #10). Figure 14 As shown, AAV.CPP16 is far superior to AAV9, delivering large quantities to the tumor and surrounding microenvironment.

[0188] To determine whether this improved delivery efficiency translates into improved therapeutic efficacy, various treatments were administered to a mouse GBM model. Figure 15A A schematic diagram of the experimental protocol is provided. The results are as follows: Figure 15B As shown in -C, AAV.CPP.16-anti-PD-L1 mediated immunotherapy significantly prolonged survival in a mouse GBM model. Figure 15B As shown, one of the eight mice treated with AAV9-anti-PD-L1 survived long-term, while six of the eight mice treated with AAV.CPP.16-anti-PD-L1 survived long-term (over 100 days). Figure 15C The study showed that all six long-term survivors (five treated with AAV.CPP.16-anti-PD-L1 plus one treated with AAV9-anti-PD-L1; one long-term survivor treated with AAV.CPP.16-anti-PD-L1 died during a re-challenge surgery due to technical reasons) remained alive 200 days after tumor implantation. Therefore, intravenous injection of AAV.CPP.16, an antibody expressing an antibody targeting mouse PD-L1, eradicated GBM tumors in 75% of mice, while untreated mice died within one month of tumor implantation.

[0189] Long-term surviving mice were sacrificed at 200 days, and their brains were examined. Figure 16A As shown, there is no evidence of residual tumor. Figure 16B The image shows a bioluminescent image of one of the mice with extended survival, showing the presence of tumor cells 7 days after implantation. Figure 16CThe initial tumor implant showed no residual tumor and only residual glial scar tissue, indicating complete tumor eradication.

[0190] Furthermore, immunohistochemistry showed that CB8+ cytotoxic T cells were also present at the GBM tumor site, further demonstrating the immune response.

[0191] Example 12. Expression of HA-labeled anti-PD-L1 antibody in GBM tumors

[0192] Figures 17A-17B The expression of HA-tagged anti-PD-L1 antibody in GBM tumors, as measured by immunoblotting, is shown in the figure. In mice, 1 e12 vg of AAV or PBS was administered intravenously 5 days after tumor implantation. Tumor tissue was harvested 14 days after IV injection. The intensity of HA tag staining (…) Figure 17A The value was quantified as a measure of anti-PD-L1 antibody expression. Figure 17B ).

[0193] References

[0194] 1. Stupp, R. et al. Radiotherapy plus concomitant and adjuvanttemozolomide for glioblastoma. The New England journal of medicine 352, 987-996 (2005).

[0195] 2. Ostrom, QT et al. CBTRUS Statistical Report: Primary brain and other central nervous system tumors diagnosed in the United States in 2010-2014. Neuro-oncology 19, v1-v88 (2017).

[0196] 3. Brennan, CW et al. The somatic genomic landscape of glioblastoma. Cell 155, 462-477 (2013).

[0197] 4. Lim, M., Xia, Y., Bettegowda, C. & Weller, M. Current state ofimmunotherapy for glioblastoma. Nature reviews. Clinical oncology 15, 422-442(2018).

[0198] 5. Perry, A. & Wesseling, P. Histologic classification of gliomas.Handbook of clinical neurology 134, 71-95 (2016).

[0199] 6. Wen, P. Y. & Kesari, S. Malignant gliomas in adults. The NewEngland journal of medicine 359, 492-507 (2008).

[0200] 7. Jain, R. K. et al. Angiogenesis in brain tumours. Nature reviews.Neuroscience 8, 610-622 (2007).

[0201] 8. Deverman, B. E., Ravina, B. M., Bankiewicz, K. S., Paul, S. M. &Sah, D. W. Y. Gene therapy for neurological disorders: progress andprospects. Nature reviews. Drug discovery (2018).

[0202] 9. Hudry, E. & Vandenberghe, L. H. TherapeuticAAVGene Transfer to theNervous System: A Clinical Reality. Neuron 101, 839-862 (2019).

[0203] 10. Batista, A. R. et al. Ly6a differential expression inBBBisresponsible for strain specific CNS transduction profile of AAV-PHP.B. Humangene therapy (2019).

[0204] 11. Hordeaux, J. et al. The GPI-Linked Protein LY6A Drives AAV-PHP.BTransport across the Blood-Brain Barrier. Molecular therapy : the journal ofthe American Society of Gene Therapy 27, 912-921 (2019).

[0205] 12. Huang, Q. et al. Delivering genes across the blood-brain barrier:LY6A, a novel cellular receptor for AAV-PHP.B capsids. PloS one 14, e0225206(2019).

[0206] 13. Hordeaux, J. et al. The Neurotropic Properties of AAV-PHP.B AreLimited to C57BL / 6J Mice. Molecular therapy : the journal of the AmericanSociety of Gene Therapy 26, 664-668 (2018).

[0207] 14. Matsuzaki, Y. et al. Intravenous administration of the adeno-associated virus-PHP.B capsid fails to upregulate transduction efficiency inthe marmoset brain. Neuroscience letters 665, 182-188 (2018).

[0208] 15. Nakashima, H. et al. Modeling tumor immunity of mouseglioblastoma by exhausted CD8(+) T cells. Scientific reports 8, 208 (2018).

[0209] 16. Reul et al., Tumor-Specific Delivery of Immune CheckpointInhibitors by EngineeredAAVVectors. Front Oncol. 2019 Feb 14;9:52.

[0210] 17. Engeland et al., CTLA-4 and PD-L1 Checkpoint Blockade EnhancesOncolytic Measles Virus Therapy. Molecular Therapy 22(11):1949–1959 Nov.2014.

[0211] Other implementation methods

[0212] It should be understood that although the invention has been described in conjunction with a detailed description, the foregoing description is intended to be illustrative and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are also within the scope of the appended claims.

Claims

1. Use of adeno-associated virus (AAV) in the preparation of a reagent for delivering an antibody or antigen-binding fragment thereof that binds to PD-L1 to a cancer in a subject by means of administering the adeno-associated virus (AAV) to the subject, the adeno-associated virus (AAV) comprising (i) a capsid protein containing a targeting sequence, the targeting sequence being the amino acid sequence TVSALFK (SEQ ID NO:8) or TVSALK (SEQ ID NO:4), wherein the targeting sequence is inserted at positions 588 and 589 corresponding to amino acids 588 and 589 of AAV9 VP1 comprising SEQ ID NO:85, and (ii) a transgene encoding an antibody or antigen-binding fragment thereof that binds to PD-L1, wherein the cancer is glioblastoma.

2. The use according to claim 1, wherein the subject is a mammalian subject.

3. The use according to claim 2, wherein the AAV is administered via parenteral delivery, intracerebral delivery, or intrathecal delivery.

4. The use according to claim 3, wherein the parenteral delivery is via intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular delivery.

5. The use according to claim 3, wherein the intrathecal delivery is via lumbar injection, cerebellomedullary cistern injection, or intraparenchymal injection.

6. The use according to claim 1, further comprising administering chemotherapy, radiation and / or surgical resection to the subject.

7. The use according to claim 6, wherein the chemotherapy comprises temozolomide, lomustine, or a combination thereof.

8. The use according to claim 1, wherein the antibody binding to PD-L1 or its antigen-binding fragment comprises amino acids 31-513 of SEQ ID NO:

105.

9. The use according to claim 1, wherein the antibody binding to PD-L1 or its antigen-binding fragment comprises atezolizumab, avelumab, durvalumab, or emfellimab.

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