Methods and compositions for delivering immunotherapeutic agents that cross the blood-brain barrier to treat brain tumors

Abstract

The present invention provides a sequence that enhances the penetration of immunotherapeutic agents across the blood-brain barrier, a composition comprising the sequence, and a method of using the same for treating brain tumors, such as glioblastoma (GBM). [Solution] A brain-permeable AAV virus vector engineered to deliver a PD-L1 antibody systemically for the treatment of glioblastoma is described herein. A method for delivering an immunotherapy agent to cancer in a subject comprises the step of administering an adeno-associated virus (AAV) to a subject, comprising (i) a capsid protein comprising an amino acid sequence comprising at least four consecutive amino acids from a specific group of amino acid sequences, and (ii) a transgene encoding an immunotherapy agent, wherein, if necessary, cancer cells are present in the brain of the human subject.

Description

[Technical Field]

[0001] Claim of priority This application is based on U.S. Provisional Patent Application No. 62 / 959,625, filed on January 10, 2020. The author asserts the interests of the specification. The entirety of the foregoing is incorporated herein by reference.

[0002] Sequences that enhance the penetration of immunotherapeutic agents across the blood-brain barrier, compositions containing such sequences, and The method of use for treating brain tumors, such as glioblastoma (GBM), is described herein. It will be done. [Background technology]

[0003] Glioblastoma multiforme (GBM) is the most common and deadly brain tumor in adults, and overall survival is limited. The median period is only 15 months. 1 Approximately 12,000 new cases of GBM have been reported in the United States. It is diagnosed every year, and the incidence rate is 3.2 per 100,000 people. 2 GB Significant progress has been made in the understanding of M's histology, molecular landscape, and tumor microenvironment. in spite of 3~6 Since 2005, there has been little progress in treatment. One major obstacle to translating the inventors' extensive knowledge into effective treatment is G This is inefficient drug delivery to the BM tumor site. GBM tumors are structurally well angiogenic. Because 7 Intravenous administration, theoretically, can achieve a good tumor coverage, and is convenient. Therefore, it is a widely applicable route of drug administration. However, the blood-brain barrier (BBB) ​​and / Alternatively, designing drugs that can cross the blood-oncological barrier remains challenging. [Overview of the Initiative]

[0004] Glioblastoma is a highly fatal brain tumor that is difficult to treat using conventional methods. Systemic administration of cancer gene therapy represents a new treatment paradigm for treating glioblastoma. Therefore, to establish an intravascular gene delivery platform for glioblastoma gene therapy. For example, a brain engineered to deliver PD-L1 antibodies systemically for the treatment of glioblastoma. A permeable AAV virus vector is described herein.

[0005] Therefore, a method for delivering an immunotherapy agent to cancer in a target is provided herein. This method involves (i) array TVSALFK(array array 8); TVSALK(array array 8); 4); A small portion of KLASVT (sequence number 83); or KFLASVT (sequence number 84) Capsid proteins containing an amino acid sequence that includes at least four consecutive amino acids, and (i i) Adeno-associated virus (AAV) containing a transgene encoding an immunotherapy agent, The procedure includes administering the cancer cells to the brain of a human subject, if necessary.

[0006] In some embodiments, the amino acid sequence is TVSALK (SEQ ID NO: 4); TVSAL FK(sequence number 8); KLASVT(sequence number 83); or KFLASVT(sequence number 83); It contains at least five consecutive amino acids from 84).

[0007] In some embodiments, the amino acid sequence is TVSALK (SEQ ID NO: 4); TVSAL FK(sequence number 8); KLASVT(sequence number 83); or KFLASVT(sequence number 83); It contains at least six consecutive amino acids from 84).

[0008] Also provided herein are methods for delivering an immunotherapeutic agent to cancer in a subject. The method comprises (i) an adeno-associated virus (AAV) comprising a capsid protein comprising an amino acid sequence comprising at least four contiguous amino acids from the sequence 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 i) a transgene encoding an immunotherapeutic agent, and administering the AAV to a subject, wherein, optionally, the cancer cells are in the brain of a human subject.

[0009] In some embodiments, the targeting sequence comprises VPALR (SEQ ID NO: 1); VSALK (SEQ ID NO: 2); TVPALR (SEQ ID NO: 3); TVSALK (SEQ ID NO: 4); TVPMLK (SEQ ID NO: 12); TVPTLK (SEQ ID NO: 13); FTVSALK (SEQ ID NO: 5); LTV SALK (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).

[0010] In some embodiments, the transgene encoding an immunotherapeutic agent encodes an antibody that targets PD-1 or PD-L 1.

[0011] In some embodiments, the subject is a mammalian subject.

[0012] In some embodiments, the AAV is AAV9.

[0013] In some embodiments, AAV9 comprises AAV9 VP1.

[0014] In some embodiments, the targeting sequence is inserted at positions corresponding to amino acids 588 and 589 of AAV9 VP1, including SEQ ID NO: 85.

[0015] In some embodiments, the cells are within the brain of the subject, and the AAV is administered by parenteral delivery, intracerebral, or intrathecal delivery.

[0016] In some embodiments, the parenteral delivery is by intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular delivery.

[0017] In some embodiments, the intrathecal delivery is by lumbar injection, cisternal injection, or parenchymal injection.

[0018] In some embodiments, the method further comprises administering to the subject a chemotherapeutic agent, radiation, and / or surgical resection.

[0019] In some embodiments, the chemotherapeutic agent comprises temozolomide, lomustine, or a combination thereof.

[0020] 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 belongs. Methods and materials are described herein for use in the present invention, and other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, this specification, including definitions, will control.

[0021] ​​​​ Other features and advantages of the present invention are described in the following detailed description and drawings, as well as in the claims. It will become clear from the surrounding area. [Brief explanation of the drawing]

[0022] [Figure 1-1] Figures 1A-1C illustrate exemplary strategies for manipulating AAV9 by inserting cell-permeable peptides (CPPs) into its capsid. Figure 1A is a 3D model of the AAV9 virus. Individual CPPs inserted into the capsid between amino acids 588 and 589 (VP1 numbering) are shown on a triple axis where receptor binding likely occurs. Figure 1B illustrates a method for producing individual AAVs. Three plasmids containing pRC (manipulated or unmanipulated), p helper, and pAAV are co-transfected into HEK 293T cells, AAVs are recovered, and purified using an iodixanol gradient. Figure 1C is a vector diagram of an exemplary vector containing a sequence encoding an anti-PDL1 antibody. [Figure 1-2] (As stated above.) [Figure 2-1] Figures 2A-2B: These figures show representative images (Figure 2A) and quantitative analyses (Figure 2B) of mouse brain sections after intravenous administration of low-dose candidate AAVs. Mice with mixed genetic backgrounds were used. Candidate AAVs all express nuclear red fluorescent protein (RFP) as a reporter, although their inserted CPPs differ (see Table 3). Candidate AAVs with low production yields were excluded for further screening. The AAV dose was 1 × 10¹⁰ vg (viral genome) per animal. Each white dot in Figure 2A represents an RFP-labeled cell. In Figure 2B, *p<0.05, ANOVA for AAV9. [Figure 2-2] (As stated above.) [Figure 2-3]Figures 2C-2D: These figures show representative images (Figure 2C) and quantitative analyses (Figure 2D) of mouse brain sections after intravenous administration of AAV.CPP.11 and AAV.CPP.12 in repeated experiments. AAV.CPP.11 and AAV.CPP.12 contain CPP BIP1 and BIP2, respectively (see Table 3). The AAV dose was increased up to 1 × 10¹¹ vg per animal. Candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter. Each white dot in Figure 2C represents an RFP-labeled cell. In Figure 2D, *P<0.05, **P<0.01, ANOVA for AAV9. [Figure 2-4] (As stated above.) [Figure 3-1] Figure 3A: This figure shows the optimization of BIP targeting sequences for further manipulation of AAV9 toward better brain transduction. BIP1 (VPALR, SEQ ID NO: 1), which enables AAV9 to be transduced into the brain more efficiently (as in AAV.CPP.11), is derived from the rat protein Ku70. Human, mouse, and rat Ku70 proteins have different exact amino acid sequences. BIP2 (VSALK, SEQ ID NO: 2), as in AAV.CPP.12, is a "synthetic" peptide related to BIP1. Desiring to minimize species specificity of the final manipulated AAV, further manipulation focuses on the VSALK sequence. To generate a new targeting sequence, the amino acids of interest are added to the VSALK sequence, and in other cases, the positions of individual amino acids are swapped. All new BIP2-derived sequences are reinserted into the AAV9 capsid to generate new candidate AAVs for screening. The sequences listed in order are SEQ ID NOs: 69, 70, 71, 1-6, 72, 7, and 8. [Figure 3-2]Figures 3B-3C: This figure shows representative images (Figure 3B) of mouse brain sections after intravenous administration of multiple candidate AAVs and their quantitative analysis (Figure 3C). All candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter. The dose of AAV is 1 × 10¹¹ vg per animal. Each white dot in Figure 3B represents an RFP-labeled cell. AAV.CPP.16 and AAV.CPP.21 were identified as top hits with robust and widespread brain transduction. In Figure 3C, for AAV9, *P<0.05, **P<0.01, ***P<0.001, ANOVA. Figure 3D: This figure shows a quantitative analysis of transduction efficiency in the liver after intravenous administration of candidate AAVs. It represents the percentage of transduced hepatocytes. The dose of AAV is 1 × 10¹¹ vg per animal. For AAV9, ***P<0.001, ANOVA. [Figure 3-3] (As stated above.) [Figure 4-1] Figures 4A-4E: These figures show the screening of selected candidate AAVs in an in vitro spheroid model of the human blood-brain barrier. Figure 4A illustrates a spheroid containing human microvascular endothelial cells that form the barrier on its surface, as well as human pericytes and astrocytes inside the spheroid. Candidate AAVs were evaluated for their ability to permeate from the surrounding culture medium into the interior of the spheroid and transduce cells within. Figures 4B-4D show images of spheroids treated with AAV9 (Figure 4B), AAV.CPP.16 (Figure 4C), and AAV.CPP.21 (Figure 4D). Figure 4E shows the relative RFP intensity of spheroids treated with different AAVs. ***P<0.001, ANOVA, relative to AAV9. [Figure 4-2] (As stated above.) [Figure 4-3] (As stated above.) [Figure 5-1]Figures 5A-5B: These figures show representative images (Figure 5A) and quantitative analyses (Figure 5B) of brain sections after intravenous administration of AAV9, AAV.CPP.16, and AAV.CPP.21 in C57BL / 6J inbred mice. All candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter. The AAV dose is 1 × 10¹² vg per animal. Each white dot in Figure 5A represents an RFP-labeled cell. In Figure 5B, *P<0.05, ***P<0.001, ANOVA. [Figure 5-2] (As stated above.) [Figure 6-1] Figures 6A-6B: These figures show representative images (Figure 6A) and quantitative analyses (Figure 6B) of brain sections after intravenous administration of AAV9, AAV.CPP.16, and AAV.CPP.21 in BALB / cJ inbred mice. All candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter. The AAV dose is 1 × 10¹² vg per animal. Each white dot in Figure 6A represents an RFP-labeled cell. In Figure 6B, ***P<0.001, ANOVA. [Figure 6-2] (As stated above.) [Figure 7-1] Figures 7A-7B: These figures show representative images (Figure 7A) and quantitative analyses (Figure 7B) of brain sections after intravenous administration of high doses of AAV.CPP.16 and AAV.CPP.21 in C57BL / 6J inbred mice. Both candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter. The dose of AAV is 4 × 10¹² vg per animal. Each white dot in Figure 7A represents an RFP-labeled cell. In Figure 7B, *P<0.05, Student's test. [Figure 7-2] (As stated above.) [Figure 8-1]Figure 8A: This figure shows that AAV.CPP.16 and AAV.CPP.21 transduce mature neurons (labeled with NeuN antibody) across multiple brain regions in mice, including the cerebral cortex, midbrain, and hippocampus. Transduced neurons were co-labeled with NeuN antibody and RFP. 4 × 10¹² vg of AAV was administered intravenously to adult C57BL / 6J mice (6 weeks old). [Figure 8-2] (As stated above.) [Figure 8-3] Figure 8B: This figure shows that AAV.CPP.16 and AAV.CPP.21 exhibit enhanced ability compared to AAV9 in targeting spinal cord and motor neurons in mice. 4 × 10¹⁰ vg of AAV was administered intravenously to neonatal mice (1 day postnatal). Motor neurons in the anterior horn of the spinal cord were visualized using CHAT antibody staining. Co-localization of RFP and CHAT signals suggests specific transduction of motor neurons. [Figure 9-1] Figure 9A: This figure shows that AAV.CPP.16 exhibits enhanced ability compared to AAV9 in cardiac targeting in adult mice. 1 × 10¹¹ vg of AAV was administered intravenously to adult C57BL / 6J mice (6 weeks old). The percentage represents the ratio of RFP-labeled cells to all DAPI-stained cells. *P<0.05, Student's test. [Figure 9-2] Figure 9B: This figure shows that AAV.CPP.16 exhibits enhanced ability compared to AAV9 in targeting skeletal muscle in adult mice. 1 × 10¹¹ vg of AAV was administered intravenously to adult C57BL / 6J mice (6 weeks old). The percentage represents the ratio of RFP-labeled cells to all DAPI-stained cells. *P<0.05, Student's test. [Figure 9-3] Figure 9C: This figure shows that AAV.CPP.16 exhibits enhanced ability compared to AAV9 in targeting the dorsal root ganglia (DRG) in adult mice. 1 × 10¹¹ vg of AAV was administered intravenously to adult C57BL / 6J mice (6 weeks old). The percentage represents the ratio of RFP-labeled cells to all DAPI-stained cells. *P<0.05, Student's test. [Figure 10-1] Figure 10A: This figure shows that AAV.CPP.16 and AAV.CPP.21 exhibit enhanced ability to transduce brain cells in the primary visual cortex in non-human primates after intravenous administration, compared to AAV9. 2 × 10¹³ vg / kg of AAV-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 (shown in black) were visualized using antibody staining against AADC. The square regions in the left panel are enlarged as shown in the right panel. AAV.CPP.16 transduced significantly more cells than AAV9. AAV.CPP.21 also transduced more cells than AAV9, but its effect was less pronounced compared to AAV.CPP.16. [Figure 10-2] Figure 10B: This figure shows that AAV.CPP.16 and AAV.CPP.21 exhibit enhanced ability to transduce brain cells in the parietal cortex of non-human primates after intravenous administration, compared to AAV9. 2 × 10¹³ vg / kg of AAV-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 (shown in black) were visualized using antibody staining against AADC. The square regions in the left panel are enlarged as shown in the right panel. AAV.CPP.16 transduced significantly more cells than AAV9. AAV.CPP.21 also transduced more cells than AAV9, but its effect was less pronounced compared to AAV.CPP.16. [Figure 10-3]Figure 10C: This figure shows that AAV.CPP.16 and AAV.CPP.21 exhibit enhanced ability to transduce brain cells in the thalamus of non-human primates after intravenous administration, compared to AAV9. 2 × 10¹³ vg / kg of AAV-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 (shown in black) were visualized using antibody staining against AADC. The square regions in the left panel are enlarged as shown in the right panel. AAV.CPP.16 transduced significantly more cells than AAV9. AAV.CPP.21 also transduced more cells than AAV9, but its effect was less pronounced compared to AAV.CPP.16. [Figure 10-4] Figure 10D: This figure shows that AAV.CPP.16 and AAV.CPP.21 exhibit enhanced ability compared to AAV9 to transduce brain cells in the cerebellum after intravenous administration in non-human primates. 2 × 10¹³ vg / kg of AAV-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 (shown in black) were visualized using antibody staining against AADC. The square region in the left panel is enlarged as shown in the right panel. Both AAV.CPP.16 and AAV.CPP.21 transduce significantly more cells than AAV9. [Figure 11]Figures 11A-11B show that AAV.CPP.16 and AAV.CPP.21 do not bind to LY6A. LY6A acts as a receptor for AAV.PHP.B and its variants, including AAV.PHP.eB (as described in U.S. Patent No. 9102949 and U.S. Patent Application Publication No. 20170166926), and mediates the robust effect of AAV.PHP.eB when crossing the blood-brain barrier (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 293 cultured cells significantly increases the binding of AAV.PHP.eB to the cell surface (Figure 11A). In contrast, overexpression of LY6A did not increase viral binding to AAV9, AAV.CPP.16, or AAV.CPP.21 (Figure 11B). This suggests that AAV.CPP.16 or AAV.CPP.21 do not share LY6A as a receptor with AAV.PHP.eB. [Figure 12-1] Figures 12A-12C: These figures show that AAV.CPP.21 can be used to systemically deliver therapeutic genes to brain tumors in a mouse-like manner with glioblastoma (GBM). As shown in Figure 11A, intravenously administered AAV.CPP.21-H2BmCherry showed targeting of the tumor, particularly the tumor's spreading frontier (Figure 12A). In Figures 11B (image) and 11C (quantitative analysis), using AAV.CPP.21 to systemically deliver the “suicide gene” HSV.TK1, when combined with the prodrug ganciclovir, resulted in brain tumor reduction. HSV.TK1 converts ganciclovir, which is otherwise “dormant,” into a tumor killer. *P<0.05, Student’s test. [Figure 12-2] (As stated above.) [Figure 12-3] (As stated above.) [Figure 13-1]This figure shows that when AAV.CPP.21 was injected locally into the brains of adult mice, it produced more extensive and robust transduction of brain tissue compared to AAV9. Adult mice (>6 weeks old) were injected intracerebrally with AAV (1 × 10¹¹vg), and brain tissue was collected and examined 3 weeks after AAV injection. **P<0.01, Student's test. [Figure 13-2] (As stated above.) [Figure 14] Figure 14: A set of images comparing the delivery efficiency to the GBM tumor microenvironment in mouse models using AAV9 (top) and AAV.CPP16 (bottom). As can be seen in the inset (right), AAV.CPP16 provided a superior delivery effect. [Figure 15-1] Figures 15A-C show that AAV.CPP.16 anti-PD-L1 mediated immunotherapy extended survival time in a mouse GBM model. Figure 15A: Outline of the experimental protocol. Figure 15B: Survival time of animals treated as shown. Figure 15C: Long-term survival time of animals treated with AAV.CPP16-anti-PD-L1. LTS: Long-term survival time. [Figure 15-2] (As stated above.) [Figure 16-1] Figures 16A-C: These figures show that GBM tumors were eradicated in all long-term surviving mice. Figure 16A: H&E staining of both posterior and anterior brain sections at the tumor injection site. No residual GBM is present in any section. Figure 16B: Bioluminescence imaging 7 days after tumor transplantation, suggesting the success of the initial tumor transplant. Figure 16C: GBM tumor transplantation site with scar tissue. [Figure 16-2] (As stated above.) [Figure 17-1] Figures 17A-17B show the expression of HA-tagged anti-PD-L1 antibodies in GBM tumors as measured by Western blotting. 1e12vg of AAV or PBS was intravenously injected into mice 5 days after tumor transplantation. Tumor tissue was collected 14 days after IV injection. The intensity of HA tag staining (Figure 17A) was quantified as a measure of anti-PD-L1 antibody expression (Figure 17B). [Figure 17-2] (As stated above.) [Modes for carrying out the invention]

[0023] Difficulties associated with delivery across the blood-brain barrier (BBB) ​​make it challenging for therapeutic agents to treat brain disorders, including cancer. Adeno-associated virus (AAV) has hindered the development of therapeutic genes in the brain, spinal cord, and eyes. It emerged as an important research and clinical tool for delivering [the substance]. For example, U.S. 910 U.S. Patent No. 2949; U.S. Patent No. 9585971; and U.S. Patent Application Publication No. 20 See Specification No. 170166926. Gene therapy mediated by AAV is The recent approvals of Luxturna and Zolgensma have significantly Progress has been made. Approval of Zolgensma for endovascular treatment in patients with spinal muscular atrophy under 2 years of age has been granted. Therefore, AAV vectors that surpass BBB for systemic gene therapy of the central nervous system (CNS) It is particularly promising because it demonstrates the feasibility of using it, especially in young patients. Nevertheless, AAV9, the AAV serotype used in Zolgensma, is particularly prevalent in adults. It suffers from low efficiency when crossing blood-brain barrier (BBB), which limits its application to other CNS diseases. doing 8、9 Current industrial standards in both rodents and non-human primates (i.e.) Achieving at least 5-10 times enhancement compared to AAV9, for GBM cancer gene therapy Next-generation brain permeability that can be used for new BBB-transcending AAV platforms. AAV vectors (i.e., AAV.CPP16) are described herein.

[0024] Rational design and targeted screening based on known cell-permeable peptides (CPPs) (For example, Gomez et al., Bax-inhibiting peptides derived from Ku70 and cell-pen See etrating pentapeptides. Biochem. Soc. Trans. 2007;35(Pt 4):797-801. When manipulated into the AAV capsid via ), the efficiency of gene delivery to the brain can be increased by up to three orders of magnitude. Improved targeting sequences have been discovered. These methods have been used in animal models of glioblastoma. It was used to manipulate AAV vectors to dramatically reduce tumor size.

[0025] Furthermore, the brain possesses "immune privileges" that make immunotherapy for GBM difficult. To transform "cold" GBM tumors into immunogenic "hot" GBM tumors, the immune response is " It is desirable to "rhyme". This method is precisely an immunotherapy agent that can achieve this, for example Theoretically, the vector described herein is used to deliver the anti-PD-L1 antibody. While we do not wish to be bound by it, the AAV vector itself is a tumor of cytotoxic T cells. It "primes" the immune system by increasing tumor infiltration, but the tumor site and the whole Anti-PD-L1 antibodies expressed in the CNS as a whole are "depleted" by other means. It is thought to activate cells.

[0026] Targeted sequence This method applies to, for example, AAV1, AAV2, AAV8, or AAV9. When inserted into the capsid, or expressed chemically or as a fusion protein, Furthermore, when conjugated to biological factors, such as antibodies or other large biomolecules, We identified several potentially targeted peptides that enhance blood-brain barrier (BBB) ​​permeability.

[0027] In some embodiments, the targeted peptide comprises a sequence of at least five amino acids. In this embodiment, the amino acid sequence consists of at least four amino acids, for example, sequence VPALR(sequence number) It contains five consecutive amino acids, including (1) and VSALK (SEQ ID NO: 2).

[0028] In some embodiments, the targeted peptide includes the sequence X1X2X3X4X5, (i) X1, X2, X3, X4 are any of the following: V, A, L, I, G, P, S, T, or M These are four non-identical amino acids. (ii) X5 is K, R, H, D, or E (Sequence ID 73).

[0029] In some embodiments, the targeted peptide includes a sequence of at least six amino acids. In the embodiment of this part, the amino acid sequence consists of at least four amino acids, for example, the sequence TVPALR(sequence Number 3), TVSALK (SEQ ID NO: 4), TVPMLK (SEQ ID NO: 12), and TVP Contains 5 or 6 consecutive amino acids of TLK (SEQ ID NO: 13).

[0030] In some embodiments, the targeted peptide includes the sequence X1X2X3X4X5X6, (i) X1 is T, (ii) X2X3X4X5 is any one of V, A, L, I, G, P, S, T, or M These are four non-identical amino acids, (iii) X6 is K, R, H, D, or E (Sequence ID 74).

[0031] In some embodiments, the targeted peptide includes the sequence X1X2X3X4X5X6, (i) X1X2X3X4 are any four from V, A, L, I, G, P, S, T, or M These are not identical amino acids. (ii) X5 is K, R, H, D, or E, (iii) X6 is either E or D (Sequence ID 75).

[0032] In some embodiments, the targeted peptide includes a sequence of at least seven amino acids. In one embodiment, the amino acid sequence consists of at least four amino acids, for example, the sequence FTVSALK( Column number 5), LTVSALFK (sequence number 6), TVSALFK (sequence number 8), TVPA LFR (SEQ ID NO: 9), TVPMLFK (SEQ ID NO: 10), and TVPTLFK (SEQ ID NO: 9), It contains five, six, or seven consecutive amino acids of number 11). In some other embodiments The targeted peptide contains the sequence X1X2X3X4X5X6X7. (i) X1 is F, L, W, or Y, (ii) X² is T, (iii) X3, X4, X5, X6 are from among V, A, L, I, G, P, S, T, or M These are any four non-identical amino acids, (iv) X7 is K, R, H, D, or E (Sequence ID 76).

[0033] In some embodiments, the targeted peptide includes the sequence X1X2X3X4X5X6X7. , (i) X1 is T, (ii) X2, X3, X4, X5 are from among V, A, L, I, G, P, S, T, or M Any four non-identical amino acids, (iii) X6 is K, R, H, D, or E, (iv) X7 is either E or D (Sequence ID 77).

[0034] In some embodiments, the targeted peptide includes the sequence X1X2X3X4X5X6X7. , (i) X1, X2, X3, X4 are any of the following: V, A, L, I, G, P, S, T, or M These are four non-identical amino acids. (ii) X5 is K, R, H, D, or E, (iii) X6 is either E or D, (iv) X7 is either A or I (Sequence ID 78).

[0035] In some embodiments, the targeted peptide is a sequence of V[S / p][A / m / t / ]L ( It includes column number 79), and uppercase letters are preferred in that position. In some embodiments, targeted peptide The sequence "Do" includes the sequence TV[S / p][A / m / t / ]L (sequence number 80). Some implementation forms In this state, the targeted peptide is the sequence TV[S / p][A / m / t / ]LK (SEQ ID NO: 81) ) includes. In some embodiments, the targeted peptide is TV[S / p][A / m / t / ]L Includes the FK. sequence (sequence number 82).

[0036] In some embodiments, the targeted peptide is VPALR (SEQ ID NO: 1) or VSARK (Sequence ID 2) is incorrect.

[0037] Specific example amino acid sequences containing the aforementioned 5, 6, or 7 amino acid sequences are: These are listed in Table 1.

[0038] [Table 1-1]

[0039] [Table 1-2]

[0040] Targeted peptides containing inverted sequences, e.g., KLASVT (SEQ ID NO: 83) and K FLASVT (sequence number 84) may also be used.

[0041] The targeted peptides disclosed herein are the technical components for producing peptide mimetic products. It can be modified according to methods known in the field. For example, Qvit et al., Drug Discov Today. 2017. Feb; 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 Am erican Journal of Pharmaceutical Research, 2015. 8; Kazmierski, WM, ed., Pepti Domimetics Protocols, Human Press (Totowa NJ 1998); Goodman et al., eds., Houben -Weyl Methods of Organic Chemistry: Synthesis of Peptides and Peptidomimetics, T hiele Verlag (New York 2003); and Mayo et al., J. Biol. Chem., 278:45746 (2003) See also. In some cases, modifications of the peptides and fragments disclosed herein. The peptide mimetic type, compared to the non-peptide mimetic peptide, is superior in vivo. It exhibits enhanced stability.

[0042] A method for producing peptide mimes involves one of the amino acids in the peptide sequence or This includes, for example, substituting all of them with D-amino acid enantiomers. The sequence is referred to herein as the “retro” sequence. Alternatively, the N-terminus of the original peptide. The order of amino acid residues from the C-terminus to the N-terminus in the modified peptide mimeograph is changed. The order of amino acid residues from the N-terminus to the C-terminus is reversed so that the order of amino acid residues at the ends is the same. Such sequences can be called "Inverso" sequences.

[0043] Peptide mimes include both retro and inverso types, i.e., those disclosed herein. It may be a "retro-inverso" type of peptide. The new peptide mimetic is a peptide The order of amino acid residues from the N-terminus to the C-terminus in the mimic is the same as the C-terminus in the original peptide. It is composed of D amino acids arranged to correspond to the order of amino acid residues at the N-terminus. ru.

[0044] Other methods for producing peptide mimes include one or more amino acids in a peptide. No acid residues are chemically different but recognized functional analogs of amino acids, i.e., artificial amino acids. This includes substitution with amino acid analogs. Artificial amino acid analogs include β-amino acids and β-amino acids. β-amino acid ("β 3 -amino acids), ∀-aminophosphonic acid and ∀-aminophosph It contains phosphite analogs of amino acids such as phosphate, as well as amino acids having non-peptide bonds. Artificial amino acids are peptoid oligomers (e.g., peptoidamides or es). (Tel analogs), β-peptides, cyclic peptides, oligoureas or oligocarbamates It can be used to create peptide mimes such as peptides; or heterocyclic molecules. (Example) The retro-inverso-targeted peptide mimetic includes KLASVT and KFLASVT. Furthermore, the sequence contains all D amino acids. These sequences, for example, have biotides at the amino terminus. It can be modified by ionization and carboxyl-terminus amidation.

[0045] AAV The viral vectors used in this method and composition are those specified herein. Recombinant peptides and, if necessary, transgenes for expression in target tissues. Retroviruses, adenoviruses, adeno-associated viruses, alphaviruses, and lentigines It contains thivirus.

[0046] A preferred viral vector system useful for nucleic acid delivery in this method is adeno-associated virus It is an AAV (Anthotropic Aerosol). An AAV is a small non-enveloped wafer with a 25nm capsid. It is a wild-type virus. No diseases associated with the wild-type virus are known or have not been demonstrated. AAVs have a single-stranded DNA (ssDNA) genome. AAVs have long-term episomal induction. It has been shown to promote input gene expression, and AAV is superior in the brain, especially in nerve cells. The transgene expression was demonstrated. The vector, containing only 300 base pairs of AAV, was packaged... It can be sizing and incorporated. The space for exogenous DNA is approximately 4.7kb. It is restricted. (As described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985)) AAV vectors like the one shown can be used to introduce DNA into cells. Acids have been introduced into different cell types using AAV vectors (e.g., Hermonat et al.) 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 See 268:3781-3790 (1993). Many alternative AAV variants exist (1 (More than 00 have been cloned), AAV variants are identified based on desired features. In some embodiments, the AAVs are AAV1, AAV2, AAV4, AAV5, AAV6, AV6.2, AAV7, AAV8, AAV9, rh.10, rh.39, rh .43 or CSp3; in use with CNS, in some embodiments, AAV is A These are AV1, AAV2, AAV4, AAV5, AAV6, AAV8, or AAV9. As an example, AAV9 has been shown to cross the blood-brain barrier with a certain degree of efficiency. Using this method, the AAV capsid is converted to a capsid protein, for example, amino acid 588 The targets described herein for the AAV9 capsid protein VP1 between 589 and 589 Insertion of morphogenetic sequences increases the ability to cross the blood-brain barrier (BBB) ​​or penetrate into specific tissues. They can be genetically engineered in this way.

[0047] An example of the wild-type AAV9 capsid protein VP1 (Q6JC40-1) sequence is as follows: As stated above:

[0048] [ka]

[0049] Therefore, AAV containing one or more of the targeted peptide sequences described herein For example, a capsid protein containing a targeting sequence as described herein, for example, sequence number AAV containing capsid protein 1 is provided herein, and a targeted peptide sequence For example, it is inserted into the sequence between amino acids 588 and 589.

[0050] Immunotherapy Induction Genes In some embodiments, AAV also includes, for example, the techniques described herein or said techniques. Transgene sequences (i.e., heterologous sequences) that encode immunotherapy agents, as is well known in the field of technology. The introduced gene preferably promotes / drives the expression of the introduced gene in the target tissue. It is concatenated into the array.

[0051] An exemplary transgene for use as an immunotherapy agent is an immune checkpoint inhibitor. The body or its antigen-binding fragments, for example, single-strand variable molecules that act as checkpoint inhibitors. Includes those encoding fragment (scFv) antibodies.

[0052] Examples of immunotherapy include, but are not limited to, inducing T lymphocytes to recognize cancer cells. Adoptive T-cell therapy or cancer vaccine formulations designed to do so, and anti-CD137 antibodies (e.g.) For example, BMS-663513), anti-PD1 antibodies (e.g., nivolumab, pembrolizumab) / MK-3475, pizilizumab (CT-011), anti-PDL1 antibody (e.g., BMS) -936559, MPDL3280A), or anti-CTLA-4 antibody (e.g., ipilumi ipilumimab; for example, Kruger 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. 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 This includes checkpoint inhibitors (see -7).

[0053] Exemplary anti-PD-1 antibodies that may be used in the methods described herein include human PD-1 This includes those that bind to it, and an example PD-1 protein sequence is NCBI accession number NP_ Provided under 005009.2. An example antibody is shown in U.S. Patent No. 8,008,449. The Book; Specification No. 9,073,994; and U.S. Patent Application Publication No. 2011 / 02713 This is described in Specification No. 58, for example, PF-06801591, AMP-224, B GB-A317, BI754091, JS001, MEDI0680, PDR001, R EGN2810, SHR-1210, TSR-042, pembrolizumab, nivolumab, Avelumab, semiprimab, spartalizumab, camrelizumab, cintilimab, pizili Zumab, tislerizumab, tripalimab, AMP-224, AMP-514, and ate It contains zolizumab.

[0054] Exemplary anti-CD40 antibodies that may be used in the methods described herein include human CD40 Includes those that bind, and the exemplary CD40 protein precursor sequence is NCBI Accession Number NP_001241.1, NP_690593.1, NP_001309351.1, N Provided in P_001309350.1 and NP_001289682.1. Exemplary. Antibodies are mentioned in International Publication No. 2002 / 088186; International Publication No. 2007 / Brochure No. 124299; International Publication No. 2011 / Brochure No. 123489; International Pamphlet No. 2012 / 149356; International Pamphlet No. 2012 / 111762 Brochure; International Publication No. 2014 / 070934; U.S. Patent Application Publication No. 2 Specification No. 013 / 0011405; Specification No. 2007 / 0148163; Specification No. 200 Specification No. 4 / 0120948; Specification No. 2003 / 0165499; and U.S. Patent This includes those described in Specification No. 8,591,900, for example, dasetuzumab, Lucatumumab, breserumab, teneriximab, ADC-1013, CP-870, 89 3, Chi Lob 7 / 4, HCD122, SGN-4, SEA-CD40, BMS- This includes 986004 and APX005M. In some embodiments, an anti-CD40 antibody is used. It is a CD40 agonist, not a CD40 antagonist.

[0055] Exemplary anti-PD-L1 antibodies that may be used in the methods described herein include human PD- This includes L1-binding proteins, and the exemplary PD-L1 protein sequence is given by NCBI Accession Number NP_001254635.1, NP_001300958.1, and NP_0548 Provided in 62.1. An exemplary antibody is described in U.S. Patent Application Publication No. 2017 / 0058033. Specification No.; International Publication No. 2017 / 118321A1 Pamphlet; International Publication No. 2016 Pamphlet No. / 061142A1; International Publication No. 2016 / 007235A1 Pamphlet Pamphlet No. 2014 / 195852A1; and Pamphlet No. 201 It is listed in the 3 / 079174A1 pamphlet, for example, BMS-936559 (MDX-1105), FAZ053, KN035, Atezolizumab (Tecentriq, M PDL3280A), avelumab (Bavencio), durvalumab (Imfinzi, ME DI-4736), Embafolimab (KN035), CK-301, CS-1001, SHR-1316 (HTI-1088), CBT-502 (TQB-2450), BGB -A333 and BMS-986189 are included. Non-antibody peptide inhibitors, for example, AUNP12 and CA-170 may also be used. (Akinleye & Rasool, Journal of Hematologia) See also gy & Oncology 12:92 (2019) doi:10.1186 / s13045-019-0779-5.

[0056] In some embodiments, the immunotherapy agent is the antigen-binding moiety of an anti-PD-L1 antibody, for example, human A single-stranded variable fragment (scFv) antibody against the PD-L1 protein (PD-L1.Hu) Exemplary sequences encoding the anti-PDL1 antibody scFv, which include or contain them, are distributed As shown in column number 105, or for example, signal peptides, HA-tags, and My Lack of one, two, or more C-tags, for example, amino acids (aa) in SEQ ID NO: 105 This is the section that includes 31-513.

[0057] Exemplary anti-PDL1 scFv sequence (signal peptide (aa1~21); HA-tag aa21~30; Myc-tag, aa514~523)

[0058] [ka]

[0059] The following is an example of an anti-PD-L1 nucleic acid sequence (signal peptide (nt1~63); HA- The tags are nt64~90; Myc-tags are nt1540~1569.

[0060] [ka]

[0061] Other antibodies, and methods for producing nucleic acids encoding such antibodies, are part of the art. This is publicly known in the field, for example, Li et al., Int J Mol Sci. 2016 Jul; 17(7): 1151; Engel and et al., Mol Ther. 2014 Nov; 22(11): 1949-1959, and refer to the above references. I want to.

[0062] The virus also promotes the expression of one or more sequences, for example, one or multiple promoter sequences; enhancer sequences, for example, the 5' untranslated region (UTR) Alternatively, it may include a 3'UTR; a polyadenylation site; and / or an insulator sequence. In some embodiments, the promoter is a brain tissue-specific promoter, for example, a nerve cell promoter. It is a cell-specific or glial-specific promoter. In certain embodiments, the promoter - includes nerve nuclei (NeuN), glial fibrillary acidic proteins (GFAP), MeCP2, and the large intestine. Adenomatous polyposis (APC), ionized calcium-binding adapter molecule 1 (Iba-1), synapse Protein kinase I (SYN), calcium / calmodulin-dependent protein kinase II, tube Select from phosphate alpha I, nerve cell-specific enolase, and platelet-derived growth factor beta chain. It is the promoter of the gene being promoted. In some embodiments, the promoter is a pancellular promoter Lomotors, for example, cytomegalovirus (CMV), beta-glucuronidase (GU) SB), ubiquitin C (UBC), or Roussarcoma virus (RSV) promoter Yes, the woodchuck hepatitis virus post-transcriptional response element (WPRE) may also be used. In some embodiments, a human signal or leader sequence, for example, an IgK leader sequence, is used. It is used. In some embodiments, the human signal sequence is replaced as shown in the table below. Used for (see novoprolabs.com / support / articles / commonly-used-leader-pept ide-sequences-for-efficient-secretion-of-a-recombinant-protein-expressed-in-mamm (Edited from alian-cells-201804211337.html).

[0063] [Table 2]

[0064] In some embodiments, for example, see von Heijne, J Mol Biol. 1985 Jul 5;184(1):99-105; Kober et al., Biotechnol. Bioeng. 2013; 110: 1164-1173; Tsuchiya et al., Nucleic As described in Acids Research Supplenzent No. 3 261-262 (2003), antibodies A secretory sequence that promotes secretion is used.

[0065] In some embodiments, AAV also increases delivery to target tissue, such as the CNS. Or, one or more further mutations that reduce off-targeting of the tissue, For example, a mutation that reduces hepatic delivery when CNS, cardiac, or muscle delivery is intended. (For example, as described in Pulicherla et al. (2011) Mol Ther 19:1070-078); also For example, Chen et al. (2008) Nat Med 15:1215-1218 or Xu et al., (2005) Virol ogy 341:203-214 or U.S. Patent No. 9102949; U.S. Patent No. 958597 Specification No. 1; and as described in U.S. Patent Application Publication No. 20170166926 It also includes the addition of other targeted peptides. See also sfn.org / ~ / media / SfN / Documents / Gray a is available at Short%20Courses / 2011%20Short%20Course%20I / 2011_SC1_Gray.ashx. nd Samulski (2011) “Vector design and considerations for CNS applications,” in Gene Vector Design and Application to Treat Nervous System Disorders ed. Glorio See also pp. 1-9. (Washington, DC: Society for Neuroscience;) J., editor.

[0066] How to use The methods and compositions described herein involve the application of immunotherapy compositions to tissues, such as the central nervous system. To be delivered to the meridian (brain), heart, muscles, or dorsal root ganglia or spinal cord (peripheral nervous system) It can be used for certain purposes. In some embodiments, the method can be used for specific brain regions, such as the cerebral cortex and cerebellum. , including delivery to the hippocampus, substantia nigra, or amygdala. In some embodiments, the method involves nerve cells, This includes delivery to astrocytes and / or glial cells.

[0067] In some embodiments, methods and compositions, for example, AAV, encode an immunotherapy agent. It is used to deliver nucleic acid sequences to subjects with brain tumors. Brain tumors include gliomas. For example, glioblastoma multiforme (GBM), metastasis (for example, lung cancer, breast cancer, melanoma, or tumors) This includes (from intestinal cancer), meningiomas, pituitary adenomas, and acoustic neuromas. Therefore, the method is AAVs comprising targeted peptides described herein and encoding immunotherapy agents (e.g., AAV9) (For example, CPP16 is also referred to herein as AAV.CPP16) The inserted AAV9 is administered systemically, for example, intravenously, to patients diagnosed with brain tumors. It may include the following.

[0068] In some embodiments, the method also includes the co-administration of a chemotherapeutic agent. In terms of form, the chemotherapy agents are not limited to temozolamide, lomustine, or those. These are toxins or cytotoxic drugs, including combinations of these substances. For example, Herrlinger et al., Lancet. See 2019 Feb 16;393(10172):678-688. Methods also include radiation, surgical resection, This may include administering either or both.

[0069] Pharmaceutical composition and method of administration The methods described herein include (i) a targeted peptide and (ii) an active ingredient. This includes the use of a pharmaceutical composition containing an AAV that includes a sequence encoding an immunotherapy agent.

[0070] Pharmaceutical compositions typically contain pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to a carrier suitable for drug administration, such as saline solution or a solvent. This includes dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc.

[0071] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes include parenteral administration, such as intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular, or injection. This may include infusion administration. Therefore, delivery may be systemic or local. It's fine to target it.

[0072] Methods for formulating appropriate pharmaceutical compositions are publicly known in the art, for example, Remingto n: The Science and Practice of Pharmacy, 21st ed., 2005; and the series Drugs and d the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY) Please refer to the following book. For example, the solutions or suspensions used for parenteral administration are as follows: The following ingredients may be included: Water for injection, physiological saline, non-volatile oil, polyethylene glyco Sterile diluents such as glycerin, propylene glycol, or other synthetic solvents; benzyl Antimicrobial agents such as alcohol or methylparaben; ascorbic acid or sodium bisulfite Antioxidants such as ammonium; chelating agents such as ethylenediaminetetraacetic acid; acetates, citrates, etc. Alternatively, buffering agents such as phosphates and isotonic modifiers such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral formulations are available in ampoules, disposable syringes, or glass or plastic containers for multiple uses. It can be sealed in an administration vial.

[0073] Pharmaceutical compositions suitable for injection use are sterile aqueous solutions (if water-soluble) or dispersions and sterilization. It may contain sterile powder for immediate preparation of bacterial injection solutions or dispersion systems. Intravenous administration. Regarding appropriate carriers, physiological saline, bacteriostatic water, Cremophor EL (trademark) ( Contains BASF, Parsippany, NJ, or phosphate-buffered saline (PBS) In all cases, the composition must be sterile and readily injectable. It should be a fluid. It should be stable under manufacturing and storage conditions. The carrier must be protected from contamination by microorganisms such as bacteria and fungi. For example, water, ethanol, polyols (for example, glycerol, propylene glycol, A solvent containing (and liquid polyethylene glycol, etc.) and a suitable mixture thereof The dispersion medium may also be used. Appropriate fluidity can be achieved by using a coating such as lecithin. In the case of dispersions, this is achieved by maintaining the required particle size, and by using surfactants. It can be maintained. Inhibition of microbial action can be achieved with various antimicrobial and antifungal agents, such as parabens. This is achieved by chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Obtain. Often, isotonic agents, such as sugars, mannitol, sorbitol, and other polyhydric alcohols. It is preferable that the composition contains ol and sodium chloride. The sustained absorption of the injectable composition is Absorption retarders, such as aluminum monostearate and gelatin, are included in the composition. It can be brought about by this.

[0074] The sterile injection solution may contain, as necessary, one or a combination of the components listed above. It can be prepared by incorporating the required amount of the active compound into a solvent, followed by sterile filtration. Generally, a dispersion system contains a basic dispersion medium and other necessary components from those listed above. It is prepared by incorporating the active compound into a sterile vehicle. A sterile injection solution is prepared. For sterile powders used for this purpose, preferred preparation methods are vacuum drying and freeze-drying. In addition to the active ingredient, any further desired components from the previously sterile filtered solution may be added. This powder is obtained.

[0075] In one embodiment, the therapeutic compound is delivered via implants and microencapsulation systems. A carrier that protects therapeutic compounds from rapid elimination from the body, such as controlled-release formulations containing methyl phosphate. It is prepared from: ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, Biodegradable, biocompatible polymers such as polyorthoesters and polylactic acid are used. Such formulations may be prepared using standard techniques, or, for example, Alza Corporation and Nova Pharmaceutical Commercially available from s, Inc.: Liposome suspension (monochrome for cellular antigens). (Containing liposomes that target cells selected by a single antibody) is also pharmaceutically acceptable. These can be used as carriers. These are, for example, as described in U.S. Patent No. 4,522,811. As described, it can be prepared according to methods known to those skilled in the art.

[0076] The pharmaceutical composition is packaged in a kit, container, pack, or disc along with instructions for administration. It may be included in Pensar. [Examples]

[0077] The present invention does not limit the scope of the invention as described in the claims, and applies to the following embodiments. It is described in the following.

[0078] material and method The following materials and methods were used in the following examples.

[0079] 1. Generation of capsid variants To generate capsid variant plasmids, cell-permeable peptides (Table 3) are used. Synthesize the DNA fragments to be cloned (GenScript), and CloneEZ seamless clone Using GenScript technology, amino acid positions 588 and 589 (VPA) Insertion into the main chain of the AAV9 Rep-cap plasmid (pRC9) between (mino acid numbering) CPP BIP1(VPALR, Array No. 1) and BIP2(VSALK, Array No. 1) were used. Number 2), as well as their derivatives, for example, TVSAL in AAV.CPP.16 K (SEQ ID NO: 4) and TVSALFK (SEQ ID NO: 8) in AAV.CPP.21 are It is derived from the Ku70 protein, and its sequence is provided below:

[0080] [ka]

[0081] Furthermore, VPs regarding AAV9, AAV.CPP.16, and AAV.CPP.21 The protein sequence is provided below:

[0082] [ka]

[0083] [ka]

[0084] 2. Manufacturing of recombinant AAV Standard 3 plasmid cotransfection protocol (pRC plasmid, p he Recombinant AAVs are packaged using Ruper plasmids and pAAV plasmids. The introduced gene (for example, a nuclear index driven by the ubiquitous EF1a promoter) was introduced. pRC9 (or nucleus-directed RFP H2B-mCherry) (Its variant), p helper, and pAAV, polyethyleneimine (PEI, Po HEK 293T cells were co-transfected using (lysciences). The rAAV vector was tested for serum-free conditions 72 and 120 hours after transfection. The substance was recovered from the culture medium and also from the cells 120 hours after transfection. AAV particles in the culture medium were precipitated using PEG precipitation with 8% PEG-8000 (weight / volume). It was concentrated using this method. The cell pellet containing the virus particles was resuspended and dissolved by sonication. The virus vector, combined from PEG precipitate and cell lysates, was dissolved in 30 minutes. The mixture is treated with DNase and RNase at 37°C, followed by ultracentrifugation (VTi Iodixanol gradient (50 rotors, 40,000 rpm, 18°C, 1 hour) It was purified by (15%, 25%, 40%, and 60%). Then, rAAV was M Ilipore Amicon filter unit (UFC910008, 100K M) Concentrated using WCO) and 0.001% Pluronic F68 (Gibco) The formulation was prepared with Dulbecco's phosphate-buffered saline (PBS).

[0085] 3.AAV titration We will measure the viral titer by measuring the DNase-resistant genome copy using quantitative PCR. Further determination was made. pAAV-CAG-GFP was digested with PVIII(NEB) and plasmid Free ends for ITR were generated and used to generate the standard curve. The virus sample was DNase Incubate with I to remove contaminating DNA, then treat with sodium hydroxide and use Ruscapsid was lysed to release the viral genome. Quantitative PCR was performed using an ITR forward. Doprimer 5'-GGAACCCCTAGTGATGGAGTT (SEQ ID NO: 91) ITR Reverse Primer 5'-CGGCCTCAGTGAGCGA (SEQ ID NO: 92) The procedure was performed using rAAV-2 reference standards (RSM, ATCC). Normalized to catalog number: VR-1616, Manassas, VA).

[0086] 4. Administration of AAV in mice For intravenous administration, AAV diluted with sterile saline (0.2 ml) was administered to adult mice. The drug was administered by tail vein injection to animals (over 6 weeks of age). The animals were then kept alive for 3 weeks. The animal was euthanized and tissue was collected. For intracerebral injection, AA was diluted with PBS (10 ul). Place point V at a coordinate 1.0 mm to the right, 0.3 mm posterior, and 2.6 mm deep from the cross suture, Hamilt Injection was administered using a syringe. All animal studies were conducted under AA standards approved by IACUC. The procedure was carried out at an ALAC-certified facility.

[0087] 5. Processing of mouse tissue Anesthetized animals were perfused intracardiacly with cold phosphate-buffered saline (PBS), followed by 4% Paraformaldehyde (PFA) was perfused intracardiacly. The tissue was then post-cured overnight with 4% PFA. After fixing, it was immersed in a 30% sucrose solution for two days, then embedded in an OCT and snagged. The tissue was frozen. Typically, 80 μm thick brain sections were cut for spontaneous fluorescence imaging. Brain sections 40 μm thick were cut for intravascular coagulation (IHC).

[0088] 6. In vitro human BBB spheroid model Place hot 1% agarose (weight / volume, 50 µl) into a 96-well plate and let it cool. Then, the primary human astrocytes (Lonza) were placed on this agarose gel. Bioscience), Human brain microvascular pericytes (HBVP, ScienCell R esearch Laboratories), and human brain microvascular endothelial cells (hCM) EC / D3 (Cedarlane) in a 1:1:1 ratio (1500 cells of each type) The cells were seeded and incubated in a 5% CO2 incubator at 37°C for 48–72 hours. This allowed for the spontaneous construction of multicellular BBB spheroids. In the surrounding area, it has been reported that a multicellular barrier mimicking the blood-brain barrier (BBB) ​​is formed. AAV-H2B-mCherry was added to the culture medium, and after 4 days, all spheroids were removed. Fixed using %PFA in a Nunc Lab-Tek II thin glass 8-well chamber. - Transfer to a cover glass (Thermo Scientific) and use a Zeiss LSM7 Image was obtained using a 10-confocal microscope. The intensity of the RFP signal within this spheroid was measured. I researched it and used it as a "lead-out."

[0089] 7. AAV administration in non-human primates (NHPs) All NHP trials are conducted by CROs at AAALAC-accredited facilities approved by IACUC. The experiment was conducted in cynomolgus monkeys, which have almost no neutralizing antibodies against AAV9 or have We pre-screened for whether or not it would occur (<1:5 titer). PBS / 0.001%F AAV diluted with 68 is administered using a peristaltic pump (via the cephalic vein or femoral vein). It was administered intravenously. Three weeks later, the animals were perfused transcardially with PBS, followed by transcardial perfusion of 4% PFA. The tissue was then perfused. Subsequently, the tissue was collected and processed for paraffin embedding and sectioning. did.

[0090] 8. Immunohistochemistry Primary antibody diluted in PBS containing 10% donkey serum and 2% Triton X-100. Floating staining of mouse tissue sections was performed using the following method. The primary antibody used was Niwa Bird anti-GFP (1:1000); rabbit anti-RFP (1:1000); mouse anti-NeuN ( 1:500); Rat anti-GFAP (1:500); Goat anti-GFAP (1:500); Mau Includes anti-CD31 (1:500). Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 fluorophores The jugated secondary antibody was applied to the host species of the primary antibody at a dilution of 1:200.

[0091] For paraffin sections of NHP tissue, perform DAB staining and then AAV-AADC staining. More transduced cells were visualized. Rabbit anti-AADC antibody (1:500, Milli (pore) was used as the primary antibody.

[0092] 9. AAV binding assay HEK293T cells were cultured in a 5% CO2 incubator at 37°C. HEK293 to a 24-well plate at a cell density of 250,000 cells per well One day after T cell seeding, 200 µl of DMEM (31053028; Gibco), 1 Using a transfection mixture of ug of DNA plasmid and 3ug of PEI Then, these cells were transiently transfected with the LY6A cDNA plasmid. 48 hours after infection, the cells were placed on ice and cooled for 10 minutes. Then, Prepare the culture medium with 500 µl of ice-cold serum containing rAAV-mCherry at an MOI of 10,000. The medium was changed to Lee DMEM. After incubation on ice for 1 hour, presumably Cells with AAV bound to their surface were washed three times with cold PBS, and then the genomic DNA was isolated. The virus particles bound to the cell were queried using a primer specific to mCherry. Quantification was performed using PCR, and HEK293T was measured using human GCG as a reference. Normalized for Nomura.

[0093] 10. Mouse model of glioblastoma All experiments were conducted at Brigham and Women's Hospital and At Harvard Medical School, Animal Care and In accordance with the protocol approved by the Use Committees (IACUC) The procedure was performed. Syngeneic immune-responsive C57BL / 6 female mammograms weighing 20+ / -1g (Envigo) were administered. A mortar was used. GL261- was resuspended in 2 μl of phosphate-buffered saline (PBS). Luc (100,000 mouse glioblastoma cells) was injected using a 26-gauge needle (80075; Ham It was injected intracranially using a 10 μl syringe equipped with an ilton. Stereotactic fixation frame Using the map, the position of the transplant site was determined (coordinates from the cross suture (mm): 2 to the right, 0.5 Anteriorly, at a depth of 3.5 to the cerebral cortex. 7 days later, 200 ul of AAV-HSV-TK1 ( Administer 1E+12 viral genomes (IV) once, followed by ganciclovir (50 mg / kg The drug was administered daily for 10 days.

[0094] [Example 1] AAV9 Capsid Modification Peptide sequences that may enhance the penetration of biomolecules or viruses across the blood-brain barrier. AAV peptide display technology was used to identify the following: (Listed in Table 3) Individual cell-permeable peptides, as illustrated in Figure 1A, are amino acids 588 and 5 It was inserted into the AAV9 capsid between 89 (VP1 numbering). This insertion was performed on the AAV package. RC plasmid is one of three plasmids that are co-transfected for aging. This was carried out by modifying Sumido, and Figure 1B shows an exemplary schematic diagram of this experiment. The AAV variants were manufactured and screened separately. For further details, Please refer to Materials and Methods #1-3.

[0095] [Table 3]

[0096] [Example 2] The first round of in vivo screening AAV expressing nuclear RFP (H2B-RFP) was intravenously injected into adult mice with a mixed C57BL / 6 genetic background and B ALB / c genetic background. Three weeks later, brain tissues were collected, sectioned, and RFP-labeled cells were revealed (white dots in Figures 2A and 2C , quantified in Figures 2B and 2D, respectively). CPP BIP1 and BIP2 were inserted into the capsids of AAV.CPP.11 and AAV.CPP.12, respectively. For further details, see Materials and Methods #4-5.

[0097] [Example 3] Optimization of the modified AAV9 capsid AAV.CPP.11 and AAV.CPP.12 were further engineered by optimizing the BIP targeting sequence. The BIP insert was derived from the protein Ku70 (for the complete sequence, see Figure 3A and Materials / Methods #1). The "synthetic" origin BIP sequence VSALK was selected as the research focus to minimize the potential species specificity of the engineered AAV vector . AAVs were produced and tested separately for brain transduction efficiency compared to AAV9 (see Figures 3B-C). The percentage of cell transduction in mouse liver three weeks after IV injection of several AAV variants delivering the reporter gene RFP is shown in Figure 3D. For further details, see Materials and Methods #1-5 . .

[0098] [Example 4] In vitro model - BBB penetration screening​​​​ Some AAV variants were identified using an in vitro spheroid BBB model. They then screened for the ability to surpass human blood-brain barriers (BBB). This spheroid was on the surface It includes human microvascular endothelial cells that form a barrier, as well as human pericytes and astrocytes. AAV containing nuclear RFP as a spheroid is permeated from the surrounding culture medium into the interior of this spheroid. We evaluated its ability to transduce cells internally. Figure 4A shows an overview of the experiment. Figures 4B-D show wt AAV9, AAV.CPP.16, and AAV.CPP.21 The results for each are shown, and these and other peptides are quantified in Figure 4E. In this model, Peptides 11, 15, 16, and 21 cause maximum penetration into the spheroid. For further details, please refer to Materials and Methods #6.

[0099] [Example 5] In vivo blood-brain barrier penetration screening In the experiment conducted as described above with respect to Example 2, in the in vivo model For further evaluation, we selected AAV.CPP.16 and AAV.CPP.21. All AAVs had a nuclear RFP as reporters. Both were C57BL / 6J. Somatic mice (white dots in the brain section in Figure 5A, quantified in Figure 5B) and BAL In B / c adult mice (white dots in the brain section in Figure 6A, quantified in Figure 6B), Furthermore, it showed enhanced ability to transduce brain cells after intravenous administration compared to AAV9.

[0100] High doses of AAV.CPP.16 and AAV.CPP.21 (4x per mouse) 10 12Vg and IV administration resulted in widespread brain transduction in mice. Both AAVs had nuclear RFP as a reporter (white dots in the brain sections in Fig. 7A, quantified in Fig. 7B).

[0101] [Example 6] In vivo distribution of modified AAVs As shown in Fig. 8A, AAV.CPP.16 and AAV.CPP.21 preferentially targeted neurons (labeled with NeuN antibody) across multiple brain regions in mice, including the cerebral cortex, midbrain, and hippocampus. Both AAVs had nuclear RFP as a reporter and were intravenously administered to neonatal mice (4 × 10

[0102] AAV.CPP.16 and AAV.CPP.21 also showed enhanced ability over AAV9 in targeting spinal cord and motor neurons in mice. All AAVs had nuclear RFP as a reporter and were intravenously administered to neonatal mice (4 × 10 vg). Motor neurons were visualized using CHAT antibody staining. Co-localization of the RFP 1 0 signal and CHAT signal in Fig. 8B suggested specific transduction of motor neurons and were used to evaluate the relative ability of AAV-CAG-H2B-RFP and AA V.CPP.16-CAG-H2B-RFP to transduce various tissues in mice. 1 × 10

[0103] vg was intravenously injected. The number of transduced cells was normalized to the number of all cells labeled by DAPI nuclear staining. This result showed that AAV.CPP.16 had enhanced targeting ability in heart tissue in mice (Fig. 9A); skeletal muscle tissue (Fig. 9B), and dorsal root ganglion tissue (Fig. 9C) 11 vg was intravenously injected. The number of transduced cells was normalized to the number of all cells labeled by DAPI nuclear staining. This result showed that AAV.CPP.16 had enhanced targeting ability in heart tissue in mice (Fig. 9A); skeletal muscle tissue (Fig. 9B), and dorsal root ganglion tissue (Fig. 9C) (Fig. 9A); skeletal muscle tissue (Fig. 9B), and dorsal root ganglion tissue (Fig. 9C) showed efficiency compared to AAV9.

[0104] [Example 7] BBB Penetration in Non-Human Primate Models 2×10 13 vg / kg of AAVs-CAG-AADC (as a reporter gene) was intravenously injected into 3-month-old cynomolgus monkeys. AAV-transduced cells (shown in black) were visualized using antibody staining against A ADC. As shown in FIGS. 10A-D, AAV.C PP.16 and AAV.CPP.21 showed enhanced ability to transduce brain tissue after intravenous administration in non-human primates compared to AAV9. AAV.CPP.16 transduced significantly more cells in the primary visual field (FIG. 10A), parietal cortex (FIG. 10B), thalamus (FIG. 10C), and cerebellum (FIG. 10D)

[0105] [Example 8] AAV.CPP.16 and AAV.CPP.21 do not bind to LY6A LY6A functions as a receptor for AAV.PHP.eB and mediates the robust effect of AAV.PHP.eB on BBB crossing 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 FIG. 11A). Conversely, overexpression of LY6A did not increase virus binding for AAV9, AAV.CPP.16, or AAV.CPP.21 (see FIG. 11B). This suggests that AAV.CPP.16 or AAV.C PP.21 do not share LY6A as a receptor with AAV.PHP.eB For further details, please refer to Materials and Methods #9.

[0106] [Example 9] Delivery of therapeutic proteins to the brain using AAV.CPP.21 Using AAV.CPP.21, the "suicide gene" HS was developed in a mouse model of brain tumors. V.TK1 was delivered whole-body (Materials and Methods #10). HSV.TK1 was delivered by other methods. This converts the "dormant" ganciclovir into a tumor-killing agent. AAV is administered intravenously. CPP.21-H2BmCherry (Figure 12A, lower left and center right panels) is a tumor. In particular, it was shown that the tumor can target the frontier where it spreads. This is shown in Figures 12B-C. Sea urchins using AAV.CPP.21 for systemic delivery of the "suicide gene" HSV.TK1 Therefore, when used in combination with the prodrug ganciclovir, it resulted in a reduction in brain tumor size. These results indicate that using AAV.CPP.21 to deliver therapeutic genes systemically to brain tumors This demonstrates that it is achievable. For further details, see Materials and Methods #10.

[0107] [Example 10] Intracerebral administration of AAV.CPP.21 In addition to systemic administration (for example, in Example 2), the AAV described herein may be administered to the mouth It was administered locally to the brain. AAV9-H2B-RFP and AAV.CPP.21-H2 Intracerebral injection of B-RFP (Figure 13) was performed on brain sections treated with AAV9, Extensive and high-intensity RFP signals were observed in brain sections treated with .CPP.21. For further details, please refer to Materials and Methods #4.

[0108] [Example 11] Systemic delivery of AAV.CPP.16 to the glioblastoma tumor microenvironment Using systemic administration (for example, in Example 2), the AAV described herein is administered in the same manner. Delivery to the brain of a glioblastoma model in a mouse with a local immune response (GL261 model) (material and (As described in Method #10). As shown in Figure 14, AAV.CRP16 is used in tumors and It far outperformed AAV9 in terms of significant delivery to both the surrounding microenvironment and the surrounding microenvironment.

[0109] To determine whether this increased delivery efficiency leads to improved therapeutic effects, Various treatments were administered to the GBM model. Figure 15A provides an overview of the experimental protocol. The results are shown in Figures 15B-C, AAV.CPP.16 anti-PD-L1 mediated immunotherapy. However, it demonstrated a significant extension of the survival period in the mouse GBM model. This is shown in Figure 15B. One of the eight mice treated with AAV9 anti-PD-L1 survived for a long period, but A Six out of eight mice treated with AV.CPP.16 anti-PD-L1 survived for a long period (100 days or more). (Longer). Figure 15C shows that all 6 individuals that survived for a long period were (AAV.CPP.16-antiP Five cattle were treated with D-L1, and one cat was treated with AAV9-anti-PD-L1; AAV.CPP. One of the long-term survivors treated with 16-anti-PD-L1 died due to technical reasons. (The patient died during the second attempt surgery), but this shows that he survived 200 days after tumor transplantation. Therefore, intravenous administration of an antibody that targets mouse PD-L1 in AAV.CPP.16 Injection eradicated GBM tumors in 75% of mice, but untreated mice still had tumors. He died within a month of the transplant.

[0110] Long-term surviving mice were sacrificed on day 200, and their brains were examined. As shown in Figure 16A. No evidence of tumor was found. Figure 16B shows the results from one of the mice whose survival time was extended. The bioluminescence image taken shows that tumor cells were present 7 days after transplantation. Figure 16C This indicates that the initial tumor transplant lacked residual tumor tissue, and only gliosis scar tissue was present. This demonstrates complete tumor eradication.

[0111] Furthermore, immunohistochemistry revealed the presence of CB8+ cytotoxic T cells in GBM tumor sites. And further evidence regarding the immune response was presented.

[0112] [Example 12] Expression of HA-tagged anti-PD-L1 antibodies in GBM tumors HA-tagged anti-PD-L in GBM tumors as measured by Western blotting The expression of antibody 1 is shown in Figures 17A-17B. 5 days after tumor transplantation in mice, antibody 1e12v A dose of AAV or PBS was administered intravenously. Tumor tissue was collected 14 days after the IV injection. The intensity of HA tag staining (Figure 17A) was quantified as a measure of anti-PD-L1 antibody expression (Figure 17A). 17B).

[0113] References

[0114] [Table 4-1]

[0115] [Table 4-2]

[0116] Other Embodiments The present invention is described in conjunction with its detailed description, but the above description is in accordance with the attached claims. The scope of this invention, as defined by the range, is intended to be illustrative and not limited. It must be understood that this has not been done. Other aspects, advantages, and modifications are described below. It is within the scope of the request.

Claims

1. A method for delivering an immunotherapy agent to cancer in a target, wherein (i) the sequence TVSALFK ( SEQ ID NO: 8); TVSALK (SEQ ID NO: 4); KLASVT (SEQ ID NO: 83); or K Amino acids containing at least four consecutive amino acids from FLASVT (SEQ ID NO: 84) (ii) a capsid protein containing the sequence and a transgene encoding an immunotherapy agent The step includes administering adeno-associated virus (AAV) to the subject, and if necessary, The method wherein cancer cells are present in the brain of a human subject.

2. The aforementioned amino acid sequences are: TVSALK (SEQ ID NO: 4); TVSALFK (SEQ ID NO: 8) ); KLASVT (SEQ ID NO: 83); or KFLASVT (SEQ ID NO: 84) The method according to claim 1, comprising at least five consecutive amino acids.

3. The aforementioned amino acid sequences are: TVSALK (SEQ ID NO: 4); TVSALFK (SEQ ID NO: 8) ); KLASVT (SEQ ID NO: 83); or KFLASVT (SEQ ID NO: 84) The method according to claim 1, comprising at least six consecutive amino acids.

4. A method for delivering an immunotherapy agent to cancer in a target, comprising: (i) sequence 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 (Sequence No. 81), or TV[S / p][A / m / t / ]LFK. (Sequence ID 82) contains at least four consecutive amino acids (ii) a capsid protein containing a mino acid sequence and a transgene encoding an immunotherapy agent. The procedure includes administering adeno-associated virus (AAV) containing the above to the subject, as necessary. The method wherein cancer cells are present in the brain of a human subject.

5. The target sequences are VPALR (SEQ ID NO: 1); VSALK (SEQ ID NO: 2); TVPALR (Sequence ID 3); TVSALK (Sequence ID 4); TVPMLK (Sequence ID 12); TVP TLK (SEQ ID NO: 13); FTVSALK (SEQ ID NO: 5); LTVSALK (SEQ ID NO: 6) ); TVSALFK (Sequence No. 8); TVPALFR (Sequence No. 9); TVPMLFK ( The method according to claim 4, comprising SEQ ID NO: 10) or TVPTLFK (SEQ ID NO: 11).

6. The transgene encoding the immunotherapy agent targets PD-1 or PD-L1. The method according to any one of claims 1 to 5, which encodes a body.

7. The method according to claim 6, wherein the subject is a mammal.

8. The method according to claim 7, wherein the AAV is AAV9.

9. The method according to claim 8, wherein the AAV9 includes AAV9 VP1.

10. The aforementioned targeting sequence is amino acids 588 and 58 of AAV9 VP1, which includes SEQ ID NO:

85. The method according to claim 9, which is inserted at the position corresponding to 9.

11. The cells are located within the brain of the subject, and the AAV is delivered parenterally, intracerebral, or intrathecally. The method according to claim 7, administered by delivery.

12. The parenteral delivery is by intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular delivery, according to the claim. The method described in 11.

13. Claim 12 states that the intrathecal delivery is by lumbar injection, cisterna magna injection, or intraparenchymal injection. The method.

14. The step of administering chemotherapeutic agents, radiation, and / or surgical resection to the subject further The method according to any one of claims 1 to 13, including the method described in the above.

15. Claims that the chemotherapeutic agent comprises temozolamide, lomustine, or a combination thereof. The method described in 14.

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