CN117157104A - Targeting multiple T cell types using spherical nucleic acid vaccine architecture - Google Patents

Targeting multiple T cell types using spherical nucleic acid vaccine architecture Download PDF

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CN117157104A
CN117157104A CN202280029963.7A CN202280029963A CN117157104A CN 117157104 A CN117157104 A CN 117157104A CN 202280029963 A CN202280029963 A CN 202280029963A CN 117157104 A CN117157104 A CN 117157104A
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sna
antigen
oligonucleotide
oligonucleotides
seq
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查德·A·米尔金
迈克尔·霍普·特普连斯基
迈克尔·埃万盖洛普洛斯
王舒娅
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NORTHWEST UNIVERSITY
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NORTHWEST UNIVERSITY
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Priority claimed from PCT/US2022/022626 external-priority patent/WO2022212564A1/en
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Abstract

The present disclosure relates generally to Spherical Nucleic Acids (SNAs), i.e., nanostructures with a core surrounded by radial presentation of oligonucleotides, that can target multiple classes of immune cells. Methods of making and using the nanoparticles are also provided herein. In some aspects, the present disclosure provides a Spherical Nucleic Acid (SNA) comprising: (a) a nanoparticle core; (b) An oligonucleotide shell attached to an outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen.

Description

Targeting multiple T cell types using spherical nucleic acid vaccine architecture
Cross Reference to Related Applications
The present application claims the priority benefits of U.S. patent application Ser. No. 63/167,977, filed on 3/30 of 2021, and U.S. patent application Ser. No. 63/222,869, filed on 16 of 2021, 7, according to 35 U.S. C. ≡119 (e), which are incorporated herein by reference in their entirety.
Government support statement
The present invention was carried out with government support under grant numbers CA208783 and CA199091-03 awarded by the national institutes of health (National Institutes of Health). The government has certain rights in this invention.
Incorporation of electronically submitted materials by reference
The sequence listing as part of this disclosure is submitted concurrently with the specification as a text file. The text file containing the sequence listing, which was created at 2022, 3, 30 and was 9,503 bytes in size, is named "2021-087r_seqlising. The subject matter of the sequence listing is incorporated herein by reference in its entirety.
Technical Field
The present disclosure relates generally to Spherical Nucleic Acids (SNAs), i.e., nanostructures with a core surrounded by radial presentation of oligonucleotides, that can target multiple classes of immune cells. Methods of making and using the nanoparticles are also provided herein.
Disclosure of Invention
Spherical Nucleic Acids (SNAs) are potent immunotherapeutic agents against specific targets, which are capable of activating the immune system. SNAs comprising a dense oligonucleotide shell radially conjugated to a nanoparticle core have demonstrated that vaccine structure directly affects the function and ultimate success of therapy. This is a powerful concept called rational vaccinology and allows the same clinically used targets to be utilized to boost immune responses, but its architecture affects efficacy. The immune system is complex and thus vaccines require activation of many different types of cells to achieve a strong response and eventual tumor rejection. The present disclosure describes synthesis of SNAs by defining placement of multiple different targets that activate multiple different immune cell types and shows that the response can be enhanced and that the structure and placement of targets within SNAs largely determines vaccine efficacy.
Applications of the techniques described herein include, but are not limited to:
● Vaccine design
● Cancer immunotherapy
● Nanometer medicine
● Treatment of immune-related disorders (e.g., autoimmune disorders).
Advantages of the techniques described herein include, but are not limited to:
● Structural control of the vaccine, whereby presentation of the components to the cells is controlled;
● Defining placement of immune targets within the nanoparticle and controlling intracellular transport and release kinetics following immune cell uptake;
● Presentation enhances serum stability, cellular uptake, and co-delivery of immune activating and targeting components;
● Platform technology uses modularization to alter targets (e.g., cancer targets);
● Enhancing immune responses
Accordingly, in some aspects, the present disclosure provides a Spherical Nucleic Acid (SNA) comprising: (a) a nanoparticle core; (b) An oligonucleotide shell attached to an outer surface of the nanoparticle core, the oligonucleotideThe nucleotide shell comprises one or more immunostimulatory oligonucleotides; and (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen. In some embodiments, the first antigen is encapsulated in the nanoparticle core. In some embodiments, the second antigen is linked to one or more oligonucleotides in the oligonucleotide shell through a linker. In further embodiments, the second antigen is linked to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core via the linker. In still further embodiments, the second antigen is linked to an oligonucleotide that hybridizes to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker. In some embodiments, the second antigen is attached to the outer surface of the nanoparticle core via a linker. In some embodiments, the second antigen is encapsulated in the nanoparticle core. In further embodiments, the first antigen is linked to one or more oligonucleotides in the oligonucleotide shell through a linker. In some embodiments, the first antigen is linked to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker. In further embodiments, the first antigen is linked to an oligonucleotide that hybridizes to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker. In yet further embodiments, the first antigen is attached to the outer surface of the nanoparticle core via a linker. In some embodiments, SNAs of the present disclosure include a third antigen that is a major histocompatibility complex type I (MHC-I) antigen. In further embodiments, SNAs of the present disclosure comprise a fourth antigen that is a major histocompatibility complex type II (MHC-II) antigen. In some embodiments, the third antigen is encapsulated in the nanoparticle core. In further embodiments, the fourth antigen is linked to one or more oligonucleotides in the oligonucleotide shell through a linker. In still further embodiments, the fourth antigen passes through the A linker is attached to the oligonucleotide in the oligonucleotide shell that is attached to the nanoparticle core. In yet further embodiments, the fourth antigen is linked to an oligonucleotide that hybridizes to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker. In some embodiments, the fourth antigen is attached to the outer surface of the nanoparticle core via a linker. In some embodiments, the third antigen is linked to one or more oligonucleotides in the oligonucleotide shell through a linker. In some embodiments, the third antigen is linked to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker. In further embodiments, the third antigen is linked to an oligonucleotide that hybridizes to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker. In still further embodiments, the third antigen is attached to the outer surface of the nanoparticle core via a linker. In some embodiments, the fourth antigen is encapsulated in the nanoparticle core. In various embodiments, the first antigen and the third antigen are the same. In some embodiments, the first antigen and the third antigen are different. In some embodiments, the second antigen and the fourth antigen are the same. In further embodiments, the second antigen and the fourth antigen are different. In various embodiments, the MHC-I antigen is OVA257-264 (OVA 1) (SEQ ID NO: 7), GP100 (25-123) (KVPRNQDWL (SEQ ID NO: 11)), TC-1E6 (49-58) (VYDFAFRDLC (SEQ ID NO: 12)), TC-1E7 (49-57) (RAHYNIVTF (SEQ ID NO: 13)), PSMA (634-642) (SAVKNFTEI (SEQ ID NO: 14)), SPAS-1 (SNC 9-H8) (STHVNHLHC (SEQ ID NO: 15)), SIMS2 (237-245) (SLDLKLIFL (SEQ ID NO: 16)), PAP (115-123) (SAMTNLAAL (SEQ ID NO: 17)), B16T-1 (M27) (LCPGNKYEM (SEQ ID NO: 9)), TRP-1 (252-260) (ATGKNVCDV (SEQ ID NO: 18)), TRP-1 (252V 260M) (ATGKNVCDM (SEQ ID NO: 19)), TRP-1 (455-463) (TAPDNLGYA (SEQ ID NO: 20) 1 (SEQ ID NO: 20)), SPAS-1 (SEQ ID NO: 180) (463) or (463) and (SEQ ID NO: 180-35), tyrosine kinase (3) or (3) and (35-35) kinase (3) or (3) tyrosine kinase (3) 1-35 (37) MC38 Adpgk (ASMTNMELM (S) EQ ID NO: 23)), irgq-minimum (AALLNSAVL (SEQ ID NO: 24)), irgq-long peptide (KARDETAALLNSAVLGAAPLFVPPAD (SEQ ID NO: 25)), or combinations thereof. In various embodiments, the MHC-II antigen is OVA323-339 (OVA 2) (SEQ ID NO: 8), GP100: (46-58) (RQLYPEWTEAQRL (SEQ ID NO: 26)), TC-1E6 (43-57) (QLLRREVYDFAFRDL (SEQ ID NO: 27)), SIMS2 (240-254) (LKLIFLDSRVTEVTG (SEQ ID NO: 28)), PAP (114-128) (MSAMTNLAALFPPEG (SEQ ID NO: 29)), B16 MART-1 (M30) (VDWENVSPELNSTDQ (SEQ ID NO: 30)), TRP-1 (113-127) (CRPGWRGAACNQKIL (SEQ ID NO: 31)), TRP-1 (106-130) (SGHNCGTCRPGWRGAACNQKILTVR (SEQ ID NO: 32), li-Key (77-92) (LRMKLPKPPKPVSQMR (SEQ ID NO: 27)), tyrosine kinase (56-70), GP100 (44-59), GP100 (167-189), melan-A/MART-1 (102-111) (PAYEKLSAEQSPPPY (SEQ ID NO: 34)), melan-A/MART-1 (27-40) (AAGIGILTVILGVL (SEQ ID NO: 35)), melan-A/MART-1 (51-70) (RNGYRALMDKSLHVGTQCAL (SEQ ID NO: 36/33) (43-73), or some combinations thereof in (SEQ ID NO: 923-57), at least one of the one or more immunostimulatory oligonucleotides is a toll-like receptor (TLR) agonist. In further embodiments, each of the one or more immunostimulatory oligonucleotides is a toll-like receptor (TLR) agonist. In still further embodiments, the TLR is selected from the group consisting of: toll-like receptor 1 (TLR 1), toll-like receptor 2 (TLR 2), toll-like receptor 3 (TLR 3), toll-like receptor 4 (TLR 4), toll-like receptor 5 (TLR 5), toll-like receptor 6 (TLR 6), toll-like receptor 7 (TLR 7), toll-like receptor 8 (TLR 8), toll-like receptor 9 (TLR 9), toll-like receptor 10 (TLR 10), toll-like receptor 11 (TLR 11), toll-like receptor 12 (TLR 12) and toll-like receptor 13 (TLR 13). In some embodiments, the TLR is TLR9. In some embodiments, the immunostimulatory oligonucleotide comprises a CpG nucleotide sequence. In further embodiments, one or more of the oligonucleotides in the oligonucleotide shell comprises or consists of the sequence of 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 39). In some embodiments, the one or more oligonucleotides in the oligonucleotide shell comprise the sequence of 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID N O: 40) or consists of the same. In some embodiments, one or more of the oligonucleotides in the oligonucleotide shell comprises 5' -TCCATGACGTTCCTGACGTT (spacer-18 (hexaethylene glycol)) 2 The sequence of cholesterol-3' (SEQ ID NO: 41) or consists thereof. In further embodiments, one or more of the oligonucleotides in the oligonucleotide shell comprises 5' -TCGTCGTTTTGTCGTTTTGTCGTT (spacer-18 (hexaethyleneglycol)) 2 The sequence of cholesterol-3' (SEQ ID NO: 6) or consists thereof. In various embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the oligonucleotides in the oligonucleotide shell are immunostimulatory oligonucleotides. In various embodiments, the linker is an alkylene carbamate disulfide linker, a thiol linker, a disulfide linker, an alkylene amide thiosuccinimidyl linker, or a combination thereof. In further embodiments, the nanoparticle core is a micelle, liposome, polymer, lipid Nanoparticle (LNP), or a combination thereof. In various embodiments, the polymer is a polylactide, a polylactide-polyglycolide copolymer, a polycaprolactone, a polyacrylate, an alginate, albumin, silica, polypyrrole, polythiophene, polyaniline, polyethyleneimine, poly (methyl methacrylate), or chitosan. In some embodiments, the polymer is poly (lactic-co-glycolic acid) (PLGA). In some embodiments, the nanoparticle core is a liposome. In various embodiments, the liposome comprises a lipid selected from the group consisting of: 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1, 2-distearoyl-sn-glycero-3-phosphoric acid- (1 '-rac-glycerol) (DSPG), 1, 2-dioleoyl-sn-glycero-phosphoric acid- (1' -rac-glycerol) (DOPG), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-di- (9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dicetyl-sn-glycero-3-phosphoethanolamine (DPPE), and cholesterol. In some embodiments, one or more oligonucleotides in the oligonucleotide shell Is attached to the outer surface of the nanoparticle core via a lipid anchoring group. In further embodiments, the lipid anchoring group is attached to the 5 'or 3' end of the one or more oligonucleotides. In still further embodiments, the lipid anchoring group is tocopherol or cholesterol. In some embodiments, one or more oligonucleotides in the oligonucleotide shell are modified on their 5 'and/or 3' ends with Dibenzocyclooctyl (DBCO). In further embodiments, one or more oligonucleotides in the oligonucleotide shell are thiol-modified at their 5 'and/or 3' ends. In various embodiments, the oligonucleotide shell comprises a DNA oligonucleotide, an RNA oligonucleotide, or a combination thereof. In some embodiments, the oligonucleotide shells include DNA oligonucleotides and RNA oligonucleotides. In further embodiments, the oligonucleotide shell comprises single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, or a combination thereof. In some embodiments, one or more oligonucleotides in the oligonucleotide shell are modified oligonucleotides. In various embodiments, the oligonucleotide shells comprise from about 2 to about 200 oligonucleotides. In some embodiments, the oligonucleotide shell comprises from about 2 to about 100 oligonucleotides. In further embodiments, the oligonucleotide shell comprises about 150 oligonucleotides. In still further embodiments, the oligonucleotide shell comprises about 200 oligonucleotides. In various embodiments, the oligonucleotide shells comprise from about 10 to about 80 oligonucleotides. In some embodiments, the oligonucleotide shell comprises about 75 oligonucleotides. In various embodiments, each oligonucleotide in the oligonucleotide shell is about 5 to about 1000 nucleotides in length. In some embodiments, each oligonucleotide in the oligonucleotide shell is about 10 to about 50 nucleotides in length. In some embodiments, each oligonucleotide in the oligonucleotide shell is about 20 to about 30 nucleotides in length. In further embodiments, the SNA has a diameter of about 1 nanometer (nm) to about 500nm. In some embodiments, the SNA has a diameter of less than or equal to about 80 nanometers. In some embodiments, the SNA has a diameter of less than or equal to about 50 nanometers. In various embodiments, the oligonucleotide shell comprises a targeting oligonucleotide An inhibitory oligonucleotide, a non-targeting oligonucleotide, or a combination thereof. In further embodiments, the inhibitory oligonucleotide is an antisense oligonucleotide, a small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), an enzymatic DNA, or an enzymatic aptamer.
In some aspects, the present disclosure provides a composition comprising a plurality of SNAs as described herein. In some embodiments, at least two SNAs of the plurality of SNAs comprise different nanoparticle cores.
In some aspects, the present disclosure provides a pharmaceutical formulation comprising a plurality of SNAs as described herein or a composition as described herein, and a pharmaceutically acceptable carrier or diluent.
In a further aspect, the present disclosure provides a vaccine comprising the SNA, composition or pharmaceutical formulation of the present disclosure. In some embodiments, the vaccine comprises one adjuvant or additional adjuvants.
In some aspects, the present disclosure provides an antigen composition comprising SNA of the present disclosure or a pharmaceutical formulation of the present disclosure in the form of a pharmaceutically acceptable carrier, diluent, stabilizer, or preservative, wherein the antigen composition is capable of generating an immune response in a subject comprising antibody production, cytotoxic T cell activation, helper T cell activation, or protective immune response. In some embodiments, the immune response comprises an antibody response. In further embodiments, the antibody response is a neutralizing antibody response or a protective antibody response.
In some aspects, the present disclosure provides a method of inhibiting expression of a gene product, the method comprising hybridizing a polynucleotide encoding the gene product to an inhibitory oligonucleotide as described herein, wherein hybridization between the polynucleotide and the inhibitory oligonucleotide occurs over a length of the polynucleotide that is complementary to a degree sufficient to inhibit expression of the gene product. In various embodiments, expression of the gene product is inhibited in vivo or in vitro.
In some aspects, a method of generating an immune response in a subject, the method comprising administering to the subject an effective amount of SNA, composition, pharmaceutical formulation, vaccine, or antigen composition, each as described herein, thereby generating an immune response in the subject. In some embodiments, the immune response comprises an antibody response. In further embodiments, the antibody response is a total antigen-specific antibody response. In some embodiments, the antibody response is a neutralizing antibody response or a protective antibody response.
In some aspects, the present disclosure provides a method of immunizing a subject against one or more antigens, the method comprising administering to the subject an effective amount of SNA, composition, pharmaceutical formulation, vaccine, or antigen composition, each as described herein, thereby immunizing the subject against the one or more antigens. In some embodiments, the composition or the vaccine is a cancer vaccine.
In some aspects, the present disclosure provides a method of treating cancer, the method comprising administering to a subject an effective amount of SNA, composition, pharmaceutical formulation, vaccine, or antigen composition, each as described herein, thereby treating the cancer in the subject. In various embodiments, the cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, glioblastoma, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, lymphoma, non-Hodgkin's lymphoma, bone cancer, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, and human papilloma virus-induced cancer, or a combination thereof. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is colon cancer. In further embodiments, the cancer is a lymphoma. In some embodiments, the methods of the present disclosure further comprise administering an additional agent. In various embodiments, the additional agent is an anti-apoptosis protein 1 (PD-1) antibody, an anti-apoptosis ligand 1 (PD-L1) antibody, a cytotoxic T lymphocyte antigen 4 (CTLA-4) antibody, a T cell immunoglobulin and ITIM domain (TIGIT) antibody, or a combination thereof.
Drawings
FIG. 1 shows a Spherical Nucleic Acid (SNA) structure incorporating two antigen classes. (left panel) in the double antigen SNA (DA-SNA) 1, MHC-I (killer cd8+ T cells) antigen is encapsulated in the core of the liposome, while MHC-II (helper cd4+ T cells) antigen hybridizes to the adjuvant DNA shell. (right panel) DA-SNA2 is the opposite.
Fig. 2 depicts that SNA structures have demonstrated enhanced properties and can be used as a tool in immunotherapy.
Fig. 3 depicts the additional demonstrated enhanced properties of SNA structures that are particularly relevant to their use as tools in immunotherapy.
Figure 4 depicts SNA structures incorporating two categories of antigens defined in unique placements.
Fig. 5 depicts experimental results in which different double antigen SNAs activate cytokine production in different T cells.
FIG. 6 depicts experimental results in which the expression of memory markers in T cells varies based on DA-SNA structure.
Fig. 7 depicts experimental results showing that circulating antigen-specific immune cells differ based on dual antigen globular nucleic acid (DA-SNA) treatment.
FIG. 8 depicts experimental results showing that circulating immune cell memory profile is derived from DA-SNA treatment.
Figure 9 shows that using antigen to activate structural changes in both helper and cytotoxic T cells resulted in differences in efficacy. DA-SNA2 significantly alters vaccine efficacy and anti-tumor effects, delaying tumors.
Fig. 10 is a printed image showing that Spherical Nucleic Acid (SNA) allows elucidation of the effect of structural arrangement on immune response.
Fig. 11 shows that structural SNA changes alter tumor growth. 500,000 EG.7-OVA tumor cells were subcutaneously injected in right-hand C57BL6 female mice. On day 3, mice were initially treated weekly. A total of four vaccines were administered and tumors were measured every 2-3 days. N=9/group. Statistics were calculated on day 24 using one-way ANOVA followed by a base multiple comparison post test (Tukey multiple comparisons post-test), p <0.05; * P <0.01. With respect to fig. 9, fig. 11 additionally contains data showing the increased benefit provided by SNAs including both MHC-I and MHC-II antigens relative to SNAs including only MHC-I antigens. The presence of OVA1 antigen and absence of OVA2 antigen ("OVA 1-only SNA") did not delay tumor growth to the prolonged period of time delayed by DA-SNA 2.
Figure 12 shows that immune cell spleen populations varied as a result of vaccination. After a total of four weekly injections with SNA in C57BL6 female mice bearing eg7.ova tumors, the spleens were harvested and treated to identify cell populations. The relative ratio of CD8 to CD4 (helper) T cells is shown as a percentage of the population of (left panel) cd8+ (cytotoxic) T cells or (right panel). Statistics were calculated using a 1-way ANOVA on day 24 followed by a base multiple comparison post hoc test. * p <0.05; * P <0.01; * P <0.001; * P <0.0001.
Figure 13 shows that simultaneous DC activation with MHC I and II antigens increased T cell proliferation. Using an OVA system with identified helper (OVA 2) and cytotoxic (OVA 1) antigens, enhanced immune activation was observed when both classes of antigens were delivered to the same cell in one SNA. Delivery of different antigen types to the same cell (combination) elicits an approximately 2-fold increase in T cell proliferation compared to delivery of each antigen type on separate SNAs (alone). * p <0.05; * P <0.01.
FIG. 14 shows CD8 + T cells and CD4 + The gene expression profile of T cells is unique based on vaccination conditions. Principal Component Analysis (PCA) of transcriptomes of CD 8T cells and CD 4T cell populations isolated from spleen cells after in vivo vaccination with different treatments. PCA reduces the dimensionality of a genomic dataset consisting of a large number of related variables while preserving as much as possible of the variation present in the dataset. The grouping of similar colors (treatments) indicates that the treatments caused similar gene level changes. The maximum separation on the x-axis (PC 1) represents the most genetically dissimilar group (i.e., left panel, mixture and SNA containing OVA1H alone, and right panel, mixture and all three SNA groups). Square The next level of difference is the separation between the groups on the y-axis (PC 2).
FIG. 15 shows CD8 + The gene expression profile of T cells varies based on vaccination conditions. CD8 + The gene expression heatmap of the T cell population shows the only activated gene pathway based on vaccination treatment conditions. The black boxes highlight genes with significant variability across treatment groups.
FIG. 16 shows CD4 + The gene expression profile of T cells varies based on vaccination conditions. CD4 + The gene expression heatmap of the T cell population shows the only activated gene pathway based on vaccination treatment conditions. The black boxes highlight genes with significant variability across treatment groups.
FIG. 17 shows that DA-SNA 2 (see, e.g., FIG. 4 for a description of DA-SNA-1 and DA-SNA-2) produces a strong CD8 + T cell memory. Antigen placement within the double antigen SNA (DA-SNA) affects the immune response. DA-SNA 2 enhanced elevated CD8 compared to other vaccination treatments + Level of effector function. Both DA-SNA enhanced CD4 + Effect function.
FIG. 18 shows that DA-SNA 2 vaccination increases IFN- γ secretion upon antigen stimulation. Representative counts and images of IFN-gamma secreting spleen T cells at various ex vivo stimuli.
FIG. 19 shows that DA-SNA2 vaccination increases IFN-gamma secretion upon antigen stimulation. Counts of IFN-gamma secreting spleen T cells at various ex vivo stimuli. (representative images are shown in fig. 18).
FIG. 20 shows IFN-. Gamma.and CD107a production in CD8 in the treated group + There is an increase in cytotoxic T cells. CD8 + (left panel) T cells or CD4 + Intracellular IFN-gamma (left and right panels) and CD107a (middle panel) levels in T cells (right panel) indicate that IFN-gamma in DA-SNA2 vaccinated T cells is enhanced when ex vivo stimulation is performed differently. * P (P)<0.05。
Figure 21 shows that OVA 1-specific T cell memory was enhanced in SNA treatment. Tumor-bearing C57BL6 mice (n=6/group) treated with different groups (according to the same schedule as in fig. 11) were collected on day 17 to evaluate the memory immune response throughout the treatment. The amount of CD8 antigen specific (OVA 1) T cells as measured by dimer is shown (left panel). (right panel) among these specific T cells, the effector memory status of DA-SNA2 immunized mice was most enhanced. The asterisks directly above the bars are comparisons made for the PBS group. In addition, specific comparisons are shown. * P <0.05; * P <0.01; * P <0.001; * P <0.0001.
Figure 22 shows that OVA 2-specific T cell memory is enhanced in SNA treatment. Tumor-bearing C57BL6 mice (n=6/group) treated with different groups (according to the same schedule as in fig. 21) were collected on day 17 to evaluate the memory immune response throughout the treatment. The amount of CD4 antigen specific (OVA 2) T cells as measured by trimer is shown (left panel). (right panel) of these specific T cells, the effector memory status of DA-SNA 1 immunized mice was most enhanced, with slightly lower levels of DA-SNA2 and OVA1H only SNA groups. * P <0.05.
Figure 23 shows that DA-SNA delayed tumor growth when applied to clinically relevant melanoma tumors. 100,000B 16.F10 melanoma tumor cells were subcutaneously injected in right-hand C57BL6 female mice. On day 3, mice were initially treated weekly. A total of four vaccines were administered and tumors were measured every 2-3 days. N=6/group. Calculation statistics using one-way ANOVA at day 20 followed by a graph-based multiple comparison post hoc test, <0.05; * P <0.01.
Figure 24 shows that combination therapy with anti-PD-1 enhances the structurally driven SNA anti-tumor properties. 100,000B 16.F10 melanoma tumor cells were subcutaneously injected in right-hand C57BL6 female mice. On day 3, mice were initially treated weekly. A total of four vaccine administrations were performed with the anti-PD-1 checkpoint inhibitor injected at day 3 and day 6 post vaccine injection. Tumors were measured every 2-3 days. N=6/group. Calculation statistics using one-way ANOVA comparing the group with anti-PD-1 and subsequent base multiple comparison post-hoc test, <0.05; * P <0.01.
Figure 25 shows that combination therapy also positively affected animal survival. 100,000B 16.F10 melanoma tumor cells were subcutaneously injected in right-hand C57BL6 female mice. On day 3, mice were initially treated weekly. A total of four vaccine administrations were performed with the anti-PD-1 checkpoint inhibitor injected at day 3 and day 6 post vaccine injection. Tumors were measured every 2-3 days and animals were sacrificed when tumor size reached 1500mm 3. N=6/group. Statistics on tumor volumes were calculated using one-way ANOVA comparing the group with anti-PD-1 and a later base multiple comparison post hoc test. Survival statistics were compared using the Ji Heng-bristolo-Wilcoxon test (Gehan-Breslow-Wilcoxon test). * p <0.05, p <0.01.
Figure 26 shows how the delivery of two classes of antigens from a Spherical Nucleic Acid (SNA) vaccine alters how the antigen is processed in vitro. a) Double antigen SNA (DA-SNA) vaccines were synthesized to alter placement of MHC-I and MHC-II restricted antigens within the same nanoparticle structure. b) Delivery of both antigen classes on separate nanoparticles (dashed line, separate) or on a single DA-SNA (solid line, combination) did not affect CD86 or CD80 expressing CD11c + Fold change in population of DCs. c) CD8+ T cells specific for OVA1 antigen (left panel) or CD4 specific for OVA2 antigen generated from co-cultures of pulsed DC and naive spleen T cells + T cells. (left panel) T cells were hybridized with OVA2 alone and OVA2 hybridized and combined OVA1 alone and OVA2 hybridized (p=0.0025 and 0.0016, respectively) and OVA2 alone and OVA1 hybridized and combined OVA2 hybridized and OVA1 hybridized (p=0.0017 and 0.0094, respectively). T cells (right panel) hybridized to OVA2 with respect to combined OVA1 encapsulated (p= 0.0268) and combined OVA2 encapsulated (p=0.0285) hybridized to OVA 1. d) Antigen-specific CD8 + (left panel) or CD4 + (right panel) Median Fluorescence Intensity (MFI) of CD69 activation marker signals within a population of T cells. T cells hybridized with OVA1 relative to the combined OVA2 encapsulation (p=0.0047). e) Fold change in T cell proliferation from OT1 spleen cells specific for OVA1 antigen after co-culture with treatment pulsed DCs. (left panel) OVA 1-encapsulated and OVA 2-hybridized alone versus combined OVA 1-encapsulated and OVA 2-hybridized(p=0.0036); (right panel) OVA2 encapsulated and OVA1 hybridized alone (p=0.0306) versus combined OVA2 encapsulated and OVA1 hybridized. For all figures, the mean ± s.e.m., and statistical significance between the relevant comparisons are shown. Significance was calculated using one-way ANOVA with Sidak multiple comparison test, where n=3-4 replicates/group. ns = non-significant; * P is p <0.05；**p<0.01。
FIG. 27 is a schematic of individual SNA combinations administered as parallel comparisons with DA-SNA treatment to assess the effect of antigen distribution. Individual nanoparticles are prepared as single antigens hybridized on the surface and single antigens encapsulated within the liposome core.
FIG. 28 shows an example standard curve from a peptide assay for quantifying the amount of peptide encapsulated within liposomes. Fitting data points to a linear regression, R of the linear regression 2 Always at all times>0.98。
Fig. 29 shows an example standard curve from a Phosphatidylcholine (PC) assay for quantifying the concentration of liposomes by analyzing lipid content using the standards provided by the manufacturer. Fitting data points to a linear regression, R of the linear regression 2 Always at all times>0.98。
Fig. 30 shows ESI of four purified peptide-DNA conjugates used in example 2: m27, M30, OVA1 and OVA2, wherein the expected mass of those conjugates was confirmed.
Figure 31 shows Dynamic Light Scattering (DLS) of liposomes and SNAs. Size shifts of statistical significance between the encapsulated or DOPC liposomes and the DA-SNA 1 or 2 structures are shown, indicating SNA formation. The data show the mean ± s.e.m. from five independent measurements. * P <0.001; * P <0.0001.
FIG. 32 shows a gating strategy for the CD8 for FIG. 26C + 、OVA1-H-2k b Or CD4 + OVA2-H-2-Ia d The percentage of viable CD19 splenocytes that were double positive was quantified.
FIG. 33 shows the immune response after antigen placement within DA-SNA affected immunity. a) For C57BSchedule of two week one immunization of L/6 mice. Dosage is as follows: 6nmol of each antigen; 6nmol of adjuvant. b) CD8 in spleen after vaccination regimen + (left panel) or CD4 + (right panel) changes in cell population. (left panel) mixture relative to DA-SNA2 (p= 0.0414). c) Intracellular production of IFN-gamma pro-inflammatory cytokines (left panel) or CD107a degranulation markers (middle panel) upon ex vivo restimulation with peptide antigen was assessed. The quantification of T cells with multiple functions (double positive for both markers) was performed (right panel). DA-SNA2 significantly increased CD8 + Yields of all markers in T cells, while differences are in CD4 + The yields in T cells were finer, with both DA-SNAs increasing the levels observably to levels higher than mice immunized with the cocktail vaccine. (upper panels) mixture vs DA-SNA2 and DA-SNA 1 vs DA-SNA2 (all left panels: p= 0.0102 and 0.0124; middle panels: p=0.0188 and 0.0266; right panels: p=0.01 and 0.0119, respectively). d) By CD44 + Effect memory phenotype measured by CD 62L-markers by use of CD8 + DA-SNA 2 of T cells. CD4 + Effector function is most elevated for DA-SNA1 immunity. (left panel) mixture versus DA-SNA 2 and DA-SNA1 versus DA-SNA 2 (p=0.0016 and 0.0380, respectively). e) Representative counts and images of IFN- γ secreting spleen T cells at different ex vivo stimuli (left panel) and total Spot Forming Cells (SFC) as measured by ELISpot assay (right panel). For OVA1 ex vivo stimulation, the mixture was relative to DA-SNA 2 and DA-SNA1 was relative to DA-SNA 2 (p=respectively)<0.0001 and 0.0047). For OVA2 ex vivo stimulation, the mixture was relative to DA-SNA 2 and DA-SNA1 relative to DA-SNA 2 (p=respectively)<0.0001 and 0.0004). The mean ± s.e.m. is shown. n=3 mice/group. Statistical significance between the relevant comparisons is shown. Significance was calculated using one-way ANOVA and Sidak multiple comparison test for all plots. * P is p<0.05；**p<0.01；***p<0.001；****p<0.0001。
FIG. 34 shows the immunization of mice with vaccines of different structures inducing CD8 + T cells and CD4 + Specific differences in gene expression between T cells. a) From CD8 + (left panel) T cellsAnd CD4 + (right panel) Principal Component Analysis (PCA) plot of T cell whole transcriptome. b) CD8 due to different treatments performed + (left panel) T cells and CD4 + (right panel) changes in gene expression represented by a subset of log-fold change (LFC) of T cell populations. c) Selection of significantly enriched pathways calculated using GSEA analysis. The color of the square corresponds to that due to CD8 + (left panel) T cells and CD4 + (right panel) enrichment score for each pathway resulting from different treatments of T cells. d) CD8 + (upper panel) T cells and CD4 + (lower panel) genetic characteristics of T cells. Color refers to normalized (z-scored) gene expression levels. Selection of the relevant genes marked. e) CD8 between paired comparison of DA-SNA2 and DA-SNA1 + T cells (left panel) and CD4 + Volcanic plot of T cells (right panel). Red dots represent genes that are significantly expressed; positive LFC indicates up-regulation relative to DA-SNA1, DA-SNA2, while negative LFC indicates down-regulation relative to DA-SNA1, DA-SNA 2.
FIG. 35 shows DA-SNA immune activation for enhanced tumor suppression. a-C) subcutaneous inoculation of E.G7-OVA cells (5X 10) in right-hand C57BL/6 mice 5 And weekly immunization was started on day 3, a total of four vaccinations (6 nmol of adjuvant, 6nmol of each antigen) were performed. Mean tumor growth curves and animal survival are shown. Tumor volumes of DA-SNA2 (p=0.0121 and p=0.0084, respectively) compared to DA-SNA1 and the mixture on day 24. It will DA-SNA1 versus DA-SNA2 (p= 0.0219); animal survival of DA-SNA2 was compared against PBS and mixtures (p=0.0030 and p=0.0029, respectively). d) Tumor weight following the treatment schedule depicted in a (on day 15). PBS was compared against tumor weights of DA-SNA1 and DA-SNA2 (p=0.0064 and p=0.0034, respectively). e) Immune CD8 in spleen at the end of experiment + Evaluation of T cells (left panel). CD8 + /CD4 + T cell ratio (right panel). T cells of DA-SNA2 versus PBS (p=0.0297), mixture (p=0.0013) and DA-SNA 1 (p=0.0002) in the spleen. f-i) flow cytometry analysis of PBMCs isolated from tumor bearing mice on day 15 according to the schedule depicted in a. f) Has specific to OVA1 antigenHeterogenic CD8 + T cells. DA-SNA2 relative to the mixture (p= 0.0401). g) Effector memory CD8 within this antigen-specific T cell subset + T-cells (CD 44) + CD 62L-). DA-SNA2 relative to the mixture (p=0.0020). h) CD4 specific for OVA2 antigen + T cells. The mixture was relative to DA-SNA 1 (p=0.0229) and DA-SNA2 (p=0.0436). i) Effect memory CD4 + T-cells (CD 44) + CD 62L-). DA-SNA 1 was compared to the mixture (p=0.0001) and DA-SNA2 (p=0.0012). The data show the mean ± s.e.m. from two independent experiments (each experiment n=7-9). For panels b, d-g, I, significance was calculated using one-way ANOVA and a base multiple comparison test. Because of the significant difference in standard deviation between groups, figure h used the welch ANOVA (Welch ANOVA) followed by the Dunnett's multiple comparisons test multiple comparison test. Panel c was analyzed using a log rank test. * P is p <0.05；**p<0.01；***p<0.001。
FIG. 36 shows E.G7-OVA tumor growth curves from individual animals grown from each treatment. Average tumor growth values are depicted in figure 35B. The line was drawn on day 21 to facilitate comparison between different sets of spider images.
FIG. 37 shows the total image of E.G7-OVA tumors used to calculate tumor weight at day 15. The dashed white circle indicates that the mouse is tumor free. Scale bar: 1mm.
Figure 38 shows tumor suppression with immune checkpoint inhibitors using dual antigen immunotherapy. a-B) subcutaneous inoculation of B16-F10 cells in right-hand C57BL/6 mice (10) 5 And weekly subcutaneous immunization was provided starting on day 3, a total of four vaccinations (9 nmol of adjuvant, 9nmol of each antigen) were performed. Mean tumor growth curves and animal survival are shown. c) M27 antigen and memory CD8 in isolated PBMC + T cell marker 44+/62-specific CD8 + T cells. PBS versus DA-SNA 1 (p=0.0060) and DA-SNA 2 (p=0.0001). d-e) mice bearing B16-F10 tumors immunized subcutaneously weekly, 3 and 6 days after DA-SNA immunization, receiving intraperitoneal administration of DA-SNA in combination with an anti-PD-1 immune checkpoint inhibitor. Showing theMean tumor growth curves and animal survival are shown. Tumor growth on day 17 (p=0.0354), day 20 (p= 0.0319) and day 22 (p=0.0475) compared anti-PD-1 with respect to DA-sna2+ anti-PD-1. DA-SNA2+ anti-PD-1 was compared to PBS (P <0.0001 anti-PD-1 (P)<0.0001 Animal survival rate for comparison. f-k) flow cytometry analysis of PBMCs isolated from tumor bearing mice receiving the schedule shown in d on day 17. f) Circulation CD8 + Evaluation of T cells, and g) total effector memory CD8 + T-cells (CD 44) + /62L-). For f: DA-sna2+ anti-PD-1 versus PBS (p=0.0001), anti-PD-1 (p=0.0009), and DA-sna1+ anti-PD-1 (P)<0.0001). For g: DA-sna2+ anti-PD-1 versus PBS (p=)<0.0001 anti-PD-1 (P)<0.0001 DA-sna1+ anti-PD-1 (p=0.0090). DA-sna1+ anti-PD-1 versus PBS (p=0.0023) and anti-PD-1 (p=0.0203). h) M27 specific CD8 + CD19-T cells. DA-sna2+ anti-PD-1 versus PBS (p=0.0093), anti-PD-1 (p=0.0138), and DA-sna1+ anti-PD-1 (p=0.0052). I) For cycle CD4 + Quantification of T cells and j) pair effect memory CD4 + Evaluation of T cells (CD44+/62L-). For i: DA-sna2+ anti-PD-1 versus DA-sna1+ anti-PD-1 (p= 0.0372). For j: PBS versus DA-sna1+ anti-PD-1 (p= 0.0268) and DA-sna2+ anti-PD-1 (p=0.0349); anti-PD-1 versus DA-sna1+ anti-PD-1 (p=0.0067) and DA-sna2+ anti-PD-1 (p=0.0089). k) M30 specific cd4+/CD19-T cells. The data show the mean ± s.e.m. from two independent experiments (n=9-15). Significance was calculated using one-way ANOVA and a base multiple comparison test for all plots except b and e. Animal survival was analyzed using a log rank test. Asterisks in d represent statistically significant differences between DA-SNA2 and anti-PD-1 treated groups. n.s. =insignificant; n.d = undetected; * P is p <0.05，**p<0.01，***p<0.001 and p<0.0001。
FIG. 39 shows B16-F10 tumor growth curves from individual animals of each treatment group. Average tumor growth values are depicted in figure 38D.
Fig. 40 is a printed image depicting that vaccine design must combat heterogeneous tumor populations. CD4 + T cells can be in the absence of CD8 + The anti-tumor response is achieved in the case of T cells by: by secretion of cytokines such as interferon-gamma (Mumberg et al 1999; qin and Blankstein, 2000), or by activation and recruitment of effector cells such as macrophages and eosinophils (Greenberg, 1991; hung et al, 1998). However, CD4 + The primary role of T cells in the immune response against cancer is to CD8 + The cells become sensitized and maintain their proliferation.
Fig. 41 is a printed image depicting that vaccine design must combat heterogeneous tumor populations.
Figure 42 demonstrates that antigen targeting strategies that boost additional immune cells improve vaccine effectiveness.
Fig. 43 is a printed image showing various categories of potent vaccine delivery stimulating cells.
FIG. 44 shows the results of an experiment in which a-b) was subcutaneously inoculated with MC38 cells (5X 10) in right-hand C57BL/6 mice 5 And weekly immunization was started on day 3, a total of four vaccinations (6 nmol of adjuvant, 6nmol of each antigen) were performed. Mean tumor growth curves and animal survival are shown. c) Average tumor volume on day 24.
Detailed Description
In various aspects, the present disclosure provides Spherical Nucleic Acids (SNAs), i.e., nanostructures with nuclei surrounded by dense radial presentation of oligonucleotides, that are placed in a variety of different categories by incorporation of different antigen categories and discrete structures to target immune cells (e.g., T cells). Antigens may be incorporated where include, but are not limited to: encapsulated in a nanoparticle core, anchored to the surface, or conjugated to a complementary oligonucleotide strand hybridized to an adjuvant oligonucleotide SNA shell. In various embodiments, the multi-antigen targeted SNAs of the present disclosure define structural presentation of vaccine components to optimally activate a variety of different types of immune cells to achieve a synergistic immune response.
Spherical Nucleic Acids (SNAs) are nanomaterials that can improve the delivery and efficacy of vaccine components. Comprising a nanoparticle core and a radially oriented oligonucleotide (e.g., unmethylated cytosine-phosphate-guanine(CpG) motif DNA, which agonizes the SNA of the dense surface layer of toll-like receptor 9 (TLR 9) in Antigen Presenting Cells (APCs), is a modular structure with several advantages over conventional vaccines. It has high affinity target binding, rapid cellular uptake without transfection reagents, high biocompatibility, reduced nuclease degradation, and easy drainage to lymph nodes upon subcutaneous injection (Cutler, j.i.; auyeung, e.; mirkin, C.A. "spherical nucleic acid (Spherical Nucleic Acids)", journal of American society of chemistry (J.Am. Chem. Soc.) "2012, 134 (3), 1376-1391; the nucleic acid was prepared by the chemical society of U.S. Pat. No. 2, chernyak, N.P., narayan, S.P., mirkin, C.A., J.Chem.Touret.A., mirkat.C., burkhan, C.A., J.Chen.A., 2014,136 (28), 9866-9869, radovic-Moreno, A.F., chernyak, N., mader, C., nallagatla, S., R.S., han, L., walker, D.A., halo, T.L., merkle, T.J., mirkin, C.H., burkhan, C.A., green, S.M., spherical nucleic acid (Immunomodulatory Spherical Nucleic Acids) was prepared by the chemical society of U.S. Chemie.A., mild.A., mild.Natl., mild.A., mikroot.S. 35, J.A., J.S. 35, J.35, J.A., J.S., J.35, J.S., mikroog., J.E.A., mikroot.E., J.M., J.35, J.P.E., J.E., J.M., J.35, J.L., J.35, J.E., J.S., J.35, J.L., J.35, J.E., J.35, J.L.35, J.E., J.35, J.L., J.35, J.L.35, J., J.L., J., J.35, J., J.L.35, J.35, J.L.35, J.35, J.L., J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.used for J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.used for J.J.J.J.J.J.J.J.J.J.J.J.J.J.used.J.J.J.used for J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J J used J used J, "rational vaccinology with spherical nucleic acids (Rational Vaccinology with Spherical Nucleic Acids)" Proc.Natl.Acad.Sci.USA 2019,116 (21), 10473-10481. The ease of synthesis and inherent modularity thereof enable the design of compositionally identical structures with different arrangements of two key components, an oligonucleotide adjuvant (immune system activator) shell and an antigen (immune system target). Structural modifications have been shown to affect function and produce differences in vaccine architecture This has been demonstrated in a variety of tumor models such as model Ovalbumin (OVA) lymphoma tumors and HPV and in prostate cancer related systems targeting Prostate Specific Membrane Antigen (PSMA). However, to date, only one class of antigens that activate immune cells has been incorporated: cytotoxic CD8 + T cells.
Due to tumor heterogeneity and the ability to evolve and escape the immune system, it is important to not only produce targeted cytotoxic CD8 + Enhanced major histocompatibility complex I (MHC-I) of T cell responses also participate in synergistic interactions. In particular, CD8 + T cells and helper CD4 + T cells are both essential for long-term tumor rejection (Ostroumov, D.; fekete-Drimuz, N.; saborowski, M.; kuhnel, F.; woller, N.; CD4 and CD 8T lymphocytes interact in controlling tumor growth (CD 4 and CD 8T Lymphocyte Interplay in Controlling Tumor Growth) "cell and molecular life sciences (cell. Mol. Life Sci.)" "2018,75 (4), 689-713; shankaran, V.; ikeda, H.; bruce, A.T.; white, J.M.; swanson, P.E.; old, L.J.; schreiber, R.D." IFNγ and lymphocytes prevent the development of primary tumors and develop tumor immunogenicity (IFNγ and Lymphocytes Prevent Primary Tumour Development and Shape Tumour Immunogenicity) "" Nature (2001,410 (6832), 1107-1111). Thus, the present disclosure provides for the administration of MHC-I and-II antigens simultaneously to CD8 + T cells and CD4 + Both T cells are sensitized with SNA with enhanced vaccine efficacy. This is particularly important in melanoma where traditional treatments such as chemotherapy or radiation are less effective because of the high mutational burden and thus may easily escape the immune system. However, current clinical alternatives are ineffective in promoting a broad response that can handle mutations, are logically difficult or expensive, and do not consider the impact of structure on the immune response generated. SNA can act as a powerful cancer vaccine by controlling presentation of immunostimulatory cues, and targets multiple melanoma-associated antigens in an effort to reduce the likelihood of tumor immune escape. The present disclosure utilizes rational vaccinology methods toVaccine efficacy is enhanced by presenting multiple epitopes in a specific structural arrangement to stimulate both cytotoxic T cells and helper T cells.
Disclosed herein are these two classes of antigens (one class targeting CD8 + And another class targets CD4 + T cells) significantly alters the efficacy of the vaccine by inducing genetic, cellular and organism level changes. Using model MHC-I and-IIOVA antigens, it was observed that compositionally identical SNAs lead to different immune responses. One specific SNA structure in which MHC-I antigen hybridizes to the shell and MHC-II antigen is encapsulated in the core causes CD8 compared to the opposite case in which MHC-I antigen and MHC-II antigen are placed interchangeably + A 2-fold increase in effector cells (fig. 1) and resulted in a 3-fold increase compared to a simple mixture of both antigen and adjuvant. In addition, this SNA structure enhances antigen-specific IFN- γ secretion and intracellular IFN- γ production to levels 5-fold that from reverse SNA or simple mixtures. This was found to be a direct consequence of transcriptome changes, such as by exposure to large amounts of CD8 in the post-immunization spleen + T cells and CD4 + RNA-seq by T cells. Importantly, these trends translate into an in vivo melanoma system in which different SNAs induce significant differences in tumor growth.
The present disclosure demonstrates how tumor heterogeneity can be addressed by rational design of more complex vaccines. By incorporating this method and considering antigen placement in vaccine design, it is demonstrated herein that the efficacy of SNA vaccines can be altered. The materials and methods of the present disclosure are widely applicable in the field as they provide the opportunity to use nanostructures to present and coordinate the processing of a variety of immunostimulatory cues to immune cells. For example, the techniques described herein may be translated into other systems and biological knowledge as they provide information on the mechanical understanding of the structural basis of vaccine function. The technology described herein allows for an extensible, controllable, globally generated immune response. In the case of optimized structural presentation incorporating multiple targets as described herein, there is a more powerful response that, in the case of cancer, causes faster and complete remission before tumor immune escape.
As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
All language, such as "from," "to," "up to," "at least," "more than," "less than," etc., include the recited numbers and refer to ranges that can be subsequently broken down into sub-ranges.
The scope contains each individual member. Thus, for example, a group having 1-3 members refers to a group having 1, 2, or 3 members. Similarly, a group having 6 members refers to a group having 1, 2, 3, 4, or 6, etc. members.
"about" and "approximately" shall generally refer to an acceptable degree of error in a measured quantity given the nature or accuracy of the measurement. Exemplary degrees of error are within 20-25 percent (%) of the value or range of values, e.g., within 20, 10, 5, 4, 3, 2, or 1 percent.
A "subject" is a vertebrate organism. The subject may be a non-human mammal (e.g., a mouse, a rat, or a non-human primate), or the subject may be a human subject.
As used herein, the terms "administering", "administering" and the like refer to any mode of transferring, delivering, introducing or transporting SNA into a subject in need of treatment with such agents. Such modes include, but are not limited to, oral, topical, intravenous, intra-arterial, intraperitoneal, intramuscular, intratumoral, intradermal, intranasal, and subcutaneous administration.
As used herein, "treatment" and "treatment" refer to any reduction in the severity and/or onset of symptoms associated with a disease (e.g., cancer). Thus, "treatment" and "treatment" include both therapeutic measures and prophylactic measures. Those of ordinary skill in the art will appreciate that any degree of prevention or amelioration of a disease (e.g., cancer) is beneficial to a subject such as a human patient. The quality of life of a patient is improved by reducing the severity of symptoms in a subject to any degree and/or delaying the onset of symptoms.
As used herein, a "targeting oligonucleotide" is an oligonucleotide that directs SNA to a particular tissue and/or a particular cell type. In some embodiments, the targeting oligonucleotide is an aptamer. Thus, in some embodiments, SNAs of the present disclosure include an aptamer attached to the exterior of a nanoparticle core, wherein the aptamer is designed to bind to one or more receptors on the surface of a certain cell type.
As used herein, an "immunostimulatory oligonucleotide" is an oligonucleotide that can stimulate (e.g., induce or enhance) an immune response. Typical examples of immunostimulatory oligonucleotides are CpG motif-containing oligonucleotides, single-stranded RNA oligonucleotides, double-stranded RNA oligonucleotides and double-stranded DNA oligonucleotides. A "CpG motif" is a cytosine-guanine dinucleotide sequence. In any aspect or embodiment of the disclosure, the immunostimulatory oligonucleotide is a toll-like receptor (TLR) agonist (e.g., a toll-like receptor 9 (TLR 9) agonist).
The term "inhibitory oligonucleotide" refers to an oligonucleotide that reduces the production or expression of a protein, such as by interfering with translation of mRNA into a protein in the ribosome, or is sufficiently complementary to a gene or mRNA encoding one or more of the targeted proteins that specifically bind (hybridize) to the one or more targeted genes or mrnas, thereby reducing the expression or biological activity of the target protein. Inhibitory oligonucleotides include, but are not limited to, isolated or synthetic short hairpin RNAs (shRNA or DNA), antisense oligonucleotides (e.g., antisense RNA or DNA, chimeric antisense DNA or RNA), mirnas and miRNA mimics, small interfering RNAs (siRNA), DNA or RNA inhibitors of innate immune receptors, aptamers, enzymatic DNA or enzymatic aptamers.
The term "non-targeting oligonucleotide" refers to a specific activity contained in the oligonucleotide shell of SNA in some embodiments(e.g., immunostimulatory activity) is not relevant, but rather is used to reach a specific density of oligonucleotides on the outer surface of the SNA. Non-limiting examples of non-targeting oligonucleotides are oligonucleotides and/or homooligonucleotides comprising a scrambled nucleotide sequence (e.g., a poly thymine oligonucleotide (e.g., T) 20 ))。
An "antigen composition" is a composition of matter suitable for administration to a human or animal subject (e.g., in an experimental or clinical setting) capable of eliciting a specific immune response, e.g., against an antigen, such as one or more of the antigens described herein. In the context of the present disclosure, the term "antigen composition" will be understood to encompass compositions intended for administration to a subject or a population of subjects for the purpose of eliciting a protective or palliative immune response against an antigen, such as one or more of the antigens described herein.
As used herein, the term "dose" refers to a measured portion of any SNA of the present disclosure (e.g., SNA, antigen composition, pharmaceutical formulation as described herein) that is ingested (administered or received) by a subject at any time.
An "immune response" is a response of a cell of the immune system, such as a B cell, T cell or monocyte, to a stimulus such as SNA as described herein. The immune response may be a B cell response that elicits the production of specific antibodies, such as antigen-specific neutralizing antibodies. The immune response may also be a T cell response, such as CD4 + Helper T cell response or CD8 + Cytotoxic T cell response. B-cell responses and T-cell responses are aspects of "cellular" immune responses. As described herein, an "immune response" may also be a "treatment-based" response in which the immune system is sensitized while actively fighting a tumor. The immune response may also be a "humoral" immune response mediated by antibodies. In some cases, the response is specific for a particular antigen (i.e., an "antigen-specific response"). A "protective immune response" is an immune response that inhibits the deleterious function or activity of an antigen, or reduces symptoms (including death) caused by an antigen. The protection in this context is not necessarily requiredThe subject was fully protected from infection. A protective response is achieved when a subject is protected from developing a disease symptom, or when a subject experiences a disease symptom of lower severity. The protective immune response may be measured, for example, by an immunoassay using a serum sample from an immunized subject and testing serum antibodies for the ability to inhibit pseudovirus binding, such as: pseudovirus neutralization assay (or alternative virus neutralization assay), ELISA neutralization assay, antibody dependent cell-mediated cytotoxicity Assay (ADCC), complement Dependent Cytotoxicity (CDC), antibody dependent cell-mediated phagocytosis (ADCP), enzyme linked immunospot (ELISpot). In addition, vaccine efficacy can be tested by measuring B or T cell activation using flow cytometry (FACS) analysis or ELISpot assay after immunization. The protective immune response can be tested by measuring resistance to antigen challenge in vivo in animal models. In humans, protective immune responses can be demonstrated in population studies that compare measurements of symptoms, morbidity, mortality, etc. of treated subjects to measurements of untreated controls. The subject is exposed to an immunogenic stimulus, such as SNA described herein, which elicits a primary immune response specific for the stimulus, i.e., the exposure is "sensitized" to the immune response. Subsequent exposure to a stimulus, such as by immunization, may increase or "boost" the magnitude (or duration or both) of a particular immune response. Thus, increasing the magnitude of an antigen-specific response (e.g., by increasing the breadth of the antibody produced (i.e., in the case of administration of an enhancer of the immune system that is sensitized to the variant), by increasing antibody titer and/or affinity, by increasing the frequency of antigen-specific B cells or T cells, by inducing maturation effect functions, or a combination thereof) by administering, for example, an antigen composition of the present disclosure. "maturation and memory" of B cells and T cells can also be measured as an indicator of immune response.
An "adjuvant" refers to a substance that, when added to a composition comprising an antigen, non-specifically enhances or boosts the immune response to the antigen in a recipient upon exposure. In any aspect or embodiment of the disclosure, SNAs provided herein include an immunostimulatory oligonucleotide (e.g., and without limitation, a toll-like receptor (TLR) agonist) as an adjuvant, and include an antigen as described herein. In addition to TLR agonists AS described herein (e.g., cpG DNA, imiquimod for TLR7, motolimod for TLR8, MPLA4 for TLR4, poly (I: C) for TLR3, or a combination thereof), it is contemplated that additional adjuvants for use in accordance with the present disclosure include aluminum (e.g., aluminum hydroxide), lipid-based adjuvants AS01B, alum, MF59.
An "effective amount" or "sufficient amount" of a substance is that amount necessary to achieve a beneficial or desired result (including clinical results), and thus, the "effective amount" depends on the environment in which it is applied. For example, in the case of administration of SNAs of the present disclosure, an effective amount contains an antigen sufficient to elicit an immune response. In some embodiments, an effective amount of SNA is an amount sufficient to inhibit gene expression. An effective amount may be administered in one or more doses as further described herein. Efficacy may be shown in experimental or clinical trials, for example, by comparing the results obtained with the substance of interest with the results of experimental controls.
All references, patents, and patent applications disclosed herein are incorporated by reference for the subject matter that each refers to, which in some cases may encompass the entire document.
Spherical nucleic acid
Spherical Nucleic Acids (SNAs) include densely functionalized and highly oriented polynucleotides on the surface of nanoparticle cores. In various aspects, the present disclosure provides a Spherical Nucleic Acid (SNA) comprising: (a) a nanoparticle core; (b) An oligonucleotide shell attached to an outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen. In various embodiments, the nanoparticle core is a micelle, liposome, polymer, lipid Nanoparticle (LNP), or a combination thereof. In various embodiments, the polymer is a polylactide, a polylactide-polyglycolide copolymer, a polycaprolactone, a polyacrylate, an alginate, albumin, silica, polypyrrole, polythiophene, polyaniline, polyethyleneimine, poly (methyl methacrylate), or chitosan. In some embodiments, the polymer is poly (lactic-co-glycolic acid) (PLGA). The spherical structure of the polynucleotide shell confers unique advantages over traditional nucleic acid delivery methods, including independence from transfection agents entering almost all cells and resistance to nuclease degradation. In addition, SNAs can penetrate biological barriers, including the blood-brain barrier (see, e.g., U.S. patent application publication No. 2015/0031745, which is incorporated herein by reference in its entirety) and the blood-tumor barrier and epidermis (see, e.g., U.S. patent application publication No. 2010/023270, which is incorporated herein by reference in its entirety).
Liposomes are spherical self-enclosed structures of a range of different sizes comprising one or several hydrophobic lipid bilayers with hydrophilic cores. These lipid-based carriers range in diameter from 0.15 to 1 micron, which is significantly higher than the effective therapeutic range of 20 to 100 nanometers. Liposomes known as Small Unilamellar Vesicles (SUVs) can be synthesized in the size range of 20-50 nanometers, but encounter challenges such as instability and aggregation leading to interparticle fusion. This interparticle fusion limits the use of SUVs in therapeutic agents. In some aspects, a Liposomal Spherical Nucleic Acid (LSNA) comprises a liposomal core, an oligonucleotide shell attached to an outer surface of the liposomal core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; and a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen. Antigens contemplated for use in accordance with the present disclosure are described further below.
Liposome particles such as disclosed in International patent application No. PCT/US2014/068429, which is incorporated herein by reference in its entirety, are thus provided by the present disclosure. The liposome particles of the present disclosure have an at least substantially spherical geometry, an inner side and an outer side, and comprise a plurality of lipid groups. In various embodiments, the plurality of lipid groups comprises a lipid selected from the group consisting of: phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine lipid families. Lipids contemplated by the present disclosure include, but are not limited to: 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1, 2-distearoyl-sn-glycero-3-phosphate- (1 ' -rac-glycerol) (DSPG), 1, 2-dioleoyl-sn-glycero-3-phosphate- (1 ' -rac-glycerol) (DOPG), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-di- (9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dioleoyl-sn-phosphoethanolamine (DPPE), 1, 2-dioleoyl-glycero-3-phosphate- (1 ' -rac-glycerol) (DOPG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (dpp), stearoyl-3-phosphorylcholine (dpp), or a combination thereof. In various embodiments, at least one oligonucleotide in the oligonucleotide shell is linked to the exterior of the liposome core through a lipid anchoring group. In some embodiments, each oligonucleotide in the oligonucleotide shell is linked to the exterior of the nanoparticle core through a lipid anchoring group. In further embodiments, the lipid anchoring groups are attached to the 5 'or 3' end of at least one oligonucleotide. In still further embodiments, the lipid anchoring group is tocopherol or cholesterol. Thus, in various embodiments, at least one of the oligonucleotides in the oligonucleotide shell is an oligonucleotide-lipid conjugate comprising a lipid anchoring group, wherein the lipid anchoring group is adsorbed into a lipid bilayer. Thus, in some embodiments, all of the oligonucleotides in the oligonucleotide shell are oligonucleotide-lipid conjugates containing a lipid anchoring group, wherein the lipid anchoring group is adsorbed into a lipid bilayer. In various embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the oligonucleotides in the oligonucleotide shell are attached to the outside of the liposome core (e.g., adsorbed to the liposome core) via a lipid anchoring group. In various embodiments, the lipid anchoring groups include tocopherol, palmitoyl, dipalmitoyl, stearoyl, distearoyl, or cholesterol. Disclosed herein are methods of making oligonucleotides comprising lipid anchors. For example, the tocopherol modified with the oligonucleotide and the phosphoramidite is first provided, and then the oligonucleotide is exposed to the phosphoramidite modified tocopherol to produce the tocopherol modified oligonucleotide. Although not intended to be limiting, any chemistry known to those skilled in the art can be used to attach lipid anchors to oligonucleotides, including amide ligation or click chemistry.
Methods of preparing Liposomal SNA (LSNA) are described herein and are well known (see, e.g., wang et al, journal of the national academy of sciences of the united states of america 2019,116 (21), 10473-10481, which is incorporated herein by reference in its entirety).
The lipid nanoparticle spherical nucleic acid (LNP-SNA) comprises a lipid nanoparticle core decorated with oligonucleotide shells. The lipid nanoparticle core comprises an ionizable lipid, a phospholipid, a sterol, a lipid-polyethylene glycol (lipid-PEG) conjugate, and a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen. Antigens contemplated for use in accordance with the present disclosure are described further below. An oligonucleotide shell is attached to the outer surface of the lipid nanoparticle core, and in any aspect or embodiment of the disclosure, the oligonucleotide shell comprises one or more immunostimulatory oligonucleotides. The spherical architecture of the oligonucleotide shells gives unique advantages over traditional nucleic acid delivery methods, including independence of transfection reagents into almost all cells, resistance to nuclease degradation, sequence-based functions, targeting and diagnostics.
In some embodiments, the ionizable lipid is dioleylmethylene-4-dimethylaminobutyrate (DLin-MC 3-DMA), 2-dioleylene-4-dimethylaminoethyl- [1,3] -dioxolane (DLin-KC 2-DMA), C12-200, 1, 2-dioleoyl-3-dimethylammonium-propane (DODAP), similar lipid/lipid structures, or combinations thereof. In some embodiments, the phospholipid is 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-hexa-palmitoyl phosphatidylcholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), monophosphoryl lipid a (MPLA), or a combination thereof. In further embodiments, the sterol is 3β -hydroxycholesterol-5-ene (cholesterol), 9, 10-secholesterol-5, 7,10 (19) -trien-3 β -ol (vitamin D3), 9, 10-secergosterol-5, 7,10 (19), 22-tetralin-3 β -ol (vitamin D2), calcipotriol, 24-ethyl-5, 22-cholesten-3 β -ol (stigmasterol), 22, 23-dihydrostigmasterol (β -sitosterol), 3, 28-dihydroxy-lupeol (betulin), lupeol, ursolic acid, oleanolic acid, 24 α -methylcholesterol (campesterol), 24-ethylcholesterol-5, 24 (28) E-diene-3 β -ol (fucosterol), 24-methylcholest-5, 22-diene-3 β -ol (brassica seed), 24-methylcholest-5, 7, 22-trien-3 β -ol (stigmasterol), 9, 11-sitosterol, or any one or more of the foregoing amino acids. In some embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate comprises 2000 daltons (Da) polyethylene glycol. In further embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate is a lipid-PEG-maleimide. In still further embodiments, the lipid-PEG-maleimide is 1, 2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE) conjugated with 2000Da polyethylene glycol maleimide, 1, 2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE) conjugated with 2000Da polyethylene glycol maleimide, or a combination thereof.
In any aspect or embodiment of the disclosure, the oligonucleotide is attached to the exterior of the lipid nanoparticle core by covalent attachment of the oligonucleotide to a lipid-polyethylene glycol (lipid-PEG) conjugate. In various embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the oligonucleotides in the oligonucleotide shell are covalently attached to the exterior of the lipid nanoparticle core by the lipid-PEG conjugate. In various embodiments, one or more oligonucleotides in the oligonucleotide shell are attached to the exterior of the lipid nanoparticle core (e.g., adsorbed to the lipid nanoparticle core) through a lipid anchoring group as described herein. In various embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the oligonucleotides in the oligonucleotide shell are attached to the exterior of the lipid nanoparticle core (e.g., adsorbed to the lipid nanoparticle core) through a lipid anchoring group as described herein. In various embodiments, the lipid anchoring moiety is attached to the 5 'or 3' end of the oligonucleotide. In various embodiments, the lipid anchoring group is tocopherol, palmitoyl, dipalmitoyl, stearoyl, distearoyl, or cholesterol.
The SNA can range in size from about 1 nanometer (nm) to about 500nm, from about 1nm to about 400nm, from about 1nm to about 300nm, from about 1nm to about 200nm, from about 1nm to about 150nm, from about 1nm to about 100nm, from about 1nm to about 90nm, from about 1nm to about 80nm, from about 1nm to about 70nm, from about 1nm to about 60nm, from about 1nm to about 50nm, from about 1nm to about 40nm, from about 1nm to about 30nm, from about 1nm to about 20nm, from about 1nm to about 10nm, from about 10nm to about 150nm, from about 10nm to about 140nm, from about 10nm to about 130nm, from about 10nm to about 120nm, from about 10nm to about 110nm, from about 10nm to about 100nm, from about 10nm to about 90nm, from about 10nm to about 80nm, from about 10nm to about 70nm, from about 10nm to about 60nm, from about 10nm to about 40nm, from about 10nm to about 10nm, from about 10nm to about 40nm, from about 10nm to about 10nm, from about 10nm to about 40 nm. In a further aspect, the present disclosure provides a plurality of SNAs, each SNA comprising one or more oligonucleotides attached thereto. Thus, in some embodiments, the plurality of SNAs are about 10nm to about 150nm (average diameter), about 10nm to about 140nm in average diameter, about 10nm to about 130nm in average diameter, about 10nm to about 120nm in average diameter, about 10nm to about 110nm in average diameter, about 10nm to about 100nm in average diameter, about 10nm to about 90nm in average diameter, about 10nm to about 80nm in average diameter, about 10nm to about 70nm in average diameter, about 10nm to about 60nm in average diameter, about 10nm to about 50nm in average diameter, about 10nm to about 40nm in average diameter, about 10nm to about 30nm in average diameter, or about 10nm to about 20nm in average diameter. In some embodiments, the SNA diameter (or average diameter of the SNAs) is about 10nm to about 150nm, about 30 to about 100nm, or about 40 to about 80nm. In some embodiments, the size of the nanoparticles used in the method varies according to the needs of its particular use or application. The variation in size is advantageously used to optimize certain physical properties of SNAs, for example, the amount of surface area to which an oligonucleotide as described herein can be attached. It will be appreciated that the foregoing diameters of SNAs may apply to the diameter of the nanoparticle core itself, or to the diameter of the nanoparticle core and one or more oligonucleotides attached to the nanoparticle core.
Antigens
In various aspects, the Spherical Nucleic Acid (SNA) of the present disclosure comprises (a) a nanoparticle core; (b) An oligonucleotide shell attached to an outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen. As described herein, the number and location of MHC-I and MHC-II antigens affects the efficacy of SNA in its use as, for example, a vaccine. MHC-I antigen pair CD8 + T cell sensitization with MHC-II antigen to CD4 + T cell sensitization. In any aspect or embodiment of the disclosure, the SNA comprises at least one MHC-I antigen and at least one MHC-II antigen. MHC-I antigens contemplated by the present disclosure include, but are not limited to OVA 257-264 (OVA 1) (SEQ ID NO: 7), GP100 (25-33) (KVPRNQDWL (SEQ ID NO: 11)), TC-1E6 (49-58) (VYDFAFRDLC (SEQ ID NO: 12)), TC-1E7 (49-57) (RAHYNIVTF (SEQ ID NO: 13)), PSMA (634-642) (SAVKNFTEI (SEQ ID NO: 14)), SPAS-1 (SNC 9-H8) (STHVNHLHC (SEQ ID NO: 15)), SIMS2 (237-245) (SLDLKLIFL (SEQ ID NO: 16)), PAP (115-123) (SAMTNLAAL (SEQ ID NO: 17)), B16 MART-1 (M27) (LCPGNKYEM (SEQ ID NO: 9)), TRP-1 (252-260) (ATGKNVCDV (SEQ ID NO: 18)), TRP-1 (V260M) (ATGKNVCDM (SEQ ID NO: 19)), TRP-1 (455-463) (TAPDNLGYA (SEQ ID NO: 20)), TRP-1 (A M) (TAPDNLGYM (SEQ ID NO: 21) (180-188)), B16 MART-1 (M27) (LCPGNKYEM (SEQ ID NO: 9)), TRP-1 (252-260), TRP-1 (V260M) (455-455 (SEQ ID NO: 19)), tyrosine kinase (35) and tyrosine kinase (35) of the like, which may be selected from the group consisting of tyrosine kinase (35, 35 (35) TNMELM (SEQ ID NO: 23)), irgq-min (AALLNSAVL (SEQ ID NO: 24)), irgq-long peptide (KARDETAALLNSAVLGAAPLFVPPAD (SEQ ID NO: 25)), or combinations thereof. MHC-II antigens contemplated by the present disclosure include, but are not limited to OVA 323-339 (OVA 2) (SEQ ID NO: 8), GP100: (46-58) (RQLYPEWTEAQRL (SEQ ID NO: 26)), TC-1E6 (43-57) (QLLRREVYDFAFRDL (SEQ ID NO: 27)), SIMS2 (240-254) (LKLIFLDSRVTEVTG (SEQ ID NO: 28)), PAP (114-128) (MSAMTNLAALFPPEG (SEQ ID NO: 29)), B16 MART-1 (M30) (VDWENVSPELNSTDQ (SEQ ID NO: 30)), TRP-1 (113-127) (CRPGWRGAACNQKIL (SEQ ID NO: 31)), TRP-1 (106-130) (SGHNCGTCRPGWRGAACNQKILTVR (SEQ ID NO: 32), li-Key (77-92) (LRMKLPKPPKPVSQMR (SEQ ID NO: 27)), tyrosine kinase (56-70), GP100 (44-59), GP100 (167-189), melan-A/MART-1 (102-111) (PAYEKLSAEQSPPPY (SEQ ID NO: 34)), melan-A/MART-1 (27-40) (24 (SEQ ID NO: 35)), melan-A/MART-1 (51-70) (RNGYRALMDKSLHVGTQCAL (MeID NO: 36-51/73)), and (54lan-37) Melan-A/MART-1 (43-57) (IGCWYCRRRNGYRAL (SEQ ID NO: 38)) or a combination thereof.
The present disclosure contemplates that any configuration and combination of MHC-I antigens and MHC-II antigens may be used in SNA. Thus, in various aspects and embodiments, the present disclosure provides SNAs in which a) both MHC-I and MHC-II antigens are encapsulated in a nanoparticle core and no antigen is associated with the outside of the nanoparticle core; b) Both MHC-I antigen and MHC-II antigen are encapsulated in the nanoparticle core, and the MHC-I antigen and/or MHC-II antigen are otherwise associated with the outside of the nanoparticle core; c) Both MHC-I antigen and MHC-II antigen are located outside of the nanoparticle core and no antigen is encapsulated in the nanoparticle core; d) Both MHC-I antigen and MHC-II antigen are located on the outside of the nanoparticle core, and MHC-I antigen and/or MHC-II antigen are additionally encapsulated in the nanoparticle core; e) The MHC-I antigen is encapsulated in the nanoparticle core and the MHC-II antigen is associated with the outside of the nanoparticle core; and f) the MHC-II antigen is encapsulated in the nanoparticle core and the MHC-I antigen is associated with the outside of the nanoparticle core. Thus, in various embodiments, the MHC-I antigen and MHC-II antigen are encapsulated in the nanoparticle core, associated with the nanoparticle core on the outside of the nanoparticle core (by, e.g., covalent or non-covalent interactions), or any combination thereof. In any aspect or embodiment of the disclosure, the antigen associated with the nanoparticle core on the outside of the nanoparticle core is positioned distally of the nanoparticle core. Thus, in some embodiments, the antigen is attached to the end of the oligonucleotide in the oligonucleotide shell that is not attached to the nanoparticle core (e.g., if the oligonucleotide is attached to the nanoparticle core through its 3 'end, the antigen is attached to the 5' end of the oligonucleotide). Alternatively, in some embodiments, the antigen is attached to the end of the oligonucleotide in the oligonucleotide shell that is attached to the nanoparticle core (e.g., if the oligonucleotide is attached to the nanoparticle core by its 3 'end, the antigen is attached to the 3' end of the oligonucleotide). In further embodiments, the antigen is attached to the end of the oligonucleotide proximal to the nanoparticle core, wherein the oligonucleotide hybridizes to the oligonucleotide attached to the nanoparticle core. Alternatively, in some embodiments, the antigen is attached to the end of an oligonucleotide distal to the nanoparticle core, wherein the oligonucleotide hybridizes to the oligonucleotide attached to the nanoparticle core.
As described herein, in some aspects, the present disclosure provides a Spherical Nucleic Acid (SNA) comprising: (a) a nanoparticle core; (b) An oligonucleotide shell attached to an outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen. In some embodiments, the first antigen is encapsulated in the nanoparticle core. In further embodiments, the second antigen is linked to one or more oligonucleotides in the oligonucleotide shell through a linker. As used herein, an antigen that is "attached to one or more oligonucleotides in an oligonucleotide shell through a linker" may be attached in a variety of ways including, but not limited to a) the antigen is directly attached to an oligonucleotide that is attached to a nanoparticle core; and/or b) the antigen is linked to an oligonucleotide that hybridizes to an oligonucleotide linked to the nanoparticle core. In any aspect or embodiment of the disclosure, when an antigen is directly linked to an oligonucleotide linked to a nanoparticle core, the antigen is linked to a non-targeting oligonucleotide. Thus, in some embodiments, the second antigen is linked to the oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core via the linker. In further embodiments, the second antigen is linked to an oligonucleotide that hybridizes to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker. In still further embodiments, the second antigen is attached to the outer surface of the nanoparticle core by a linker. In some embodiments, the second antigen is encapsulated in the nanoparticle core. In further embodiments, the first antigen is linked to one or more oligonucleotides in the oligonucleotide shell through a linker. In various embodiments, the first antigen is linked to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core via the linker. In some embodiments, the first antigen is linked to an oligonucleotide that hybridizes to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker. In further embodiments, the first antigen is attached to the outer surface of the nanoparticle core via a linker. In some embodiments, the SNA comprises a third antigen that is a major histocompatibility complex type I (MHC-I) antigen. In further embodiments, the SNA comprises a fourth antigen that is a major histocompatibility complex type II (MHC-II) antigen. In some embodiments, the third antigen is encapsulated in the nanoparticle core. In further embodiments, the fourth antigen is linked to one or more oligonucleotides in the oligonucleotide shell through a linker. In still further embodiments, the fourth antigen is linked to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core via the linker. In some embodiments, the fourth antigen is linked to an oligonucleotide that hybridizes to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker. In some embodiments, the fourth antigen is attached to the outer surface of the nanoparticle core via a linker. In further embodiments, the third antigen is linked to one or more oligonucleotides in the oligonucleotide shell through a linker. In some embodiments, the third antigen is linked to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker. In further embodiments, the third antigen is linked to an oligonucleotide that hybridizes to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker. In still further embodiments, the third antigen is attached to the outer surface of the nanoparticle core via a linker. In some embodiments, the fourth antigen is encapsulated in the nanoparticle core. In various embodiments, the first antigen and the third antigen are the same. In some embodiments, the first antigen and the third antigen are different. In still further embodiments, the second antigen and the fourth antigen are the same. In some embodiments, the second antigen and the fourth antigen are different.
In some aspects, the present disclosure provides an SNA comprising a plurality of first antigens that are MHC-I antigens, a plurality of second antigens that are MHC-II antigens, or both. The first antigen and/or the second antigen may be encapsulated in the nanoparticle core, associated with the nanoparticle core on the outside of the nanoparticle core, or a combination thereof. In some embodiments, each antigen of the plurality of first antigens is the same. In further embodiments, the plurality of first antigens includes any combination of first antigens as described herein. In some embodiments, each antigen of the plurality of second antigens is identical. In further embodiments, the plurality of second antigens includes any combination of second antigens as described herein. Thus, SNA may include any combination of a first antigen and a second antigen as described herein.
In some aspects, the present disclosure provides a SNA comprising (a) a nanoparticle core; (b) An oligonucleotide shell attached to an outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; and (c) one or more MHC-I antigens encapsulated in the nanoparticle core, and one or more MHC-II antigens associated with the nanoparticle core on the outside of the nanoparticle core. In a further aspect, the present disclosure provides a SNA comprising (a) a nanoparticle core; (b) An oligonucleotide shell attached to an outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; and (c) one or more MHC-II antigens encapsulated in the nanoparticle core, and one or more MHC-I antigens associated with the nanoparticle core on the outside of the nanoparticle core. In some embodiments, some of the one or more MHC-I antigens are encapsulated in the nanoparticle core and are also associated with the nanoparticle core on the outside of the nanoparticle core. In some embodiments, some of the one or more MHC-II antigens are encapsulated in the nanoparticle core and are also associated with the nanoparticle core on the outside of the nanoparticle core. In further embodiments, some of the one or more MHC-I antigens and some of the one or more MHC-II antigens are encapsulated in the nanoparticle core and are also associated with the nanoparticle core on the outside of the nanoparticle core. In various embodiments, the one or more MHC-I antigens are all the same, while in some embodiments, the one or more MHC-I antigens comprise any combination of MHC-I antigens as described herein. In various embodiments, the one or more MHC-II antigens are all the same, while in some embodiments, the one or more MHC-II antigens comprise any combination of MHC-II antigens as described herein.
In a further aspect, the present disclosure provides a Spherical Nucleic Acid (SNA) comprising: (a) a nanoparticle core; (b) An oligonucleotide shell attached to an outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; (c) A first antigen that is a major histocompatibility complex type I (MHC-I) antigen, wherein the first antigen is encapsulated in the nanoparticle core, is linked to one or more oligonucleotides in the oligonucleotide shell by a linker, is linked to an outer surface of the nanoparticle core by the linker, or a combination thereof; and (d) a second antigen that is a major histocompatibility complex type II (MHC-II) antigen, wherein the second antigen is encapsulated in the nanoparticle core, is linked to one or more oligonucleotides in the oligonucleotide shell by a linker, is linked to an outer surface of the nanoparticle core by the linker, or a combination thereof.
In a further aspect, the present disclosure provides a Spherical Nucleic Acid (SNA) comprising: (a) a nanoparticle core; (b) An oligonucleotide shell attached to an outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; (c) A first antigen that is a major histocompatibility complex type I (MHC-I) antigen, wherein the first antigen is encapsulated in the nanoparticle core, is linked to one or more oligonucleotides in the oligonucleotide shell by a linker, is linked to an outer surface of the nanoparticle core by the linker, or a combination thereof; (d) A second antigen that is a major histocompatibility complex type II (MHC-II) antigen, wherein the second antigen is encapsulated in the nanoparticle core, is linked to one or more oligonucleotides in the oligonucleotide shell by a linker, is linked to an outer surface of the nanoparticle core by the linker, or a combination thereof; and (e) a third antigen that is a major histocompatibility complex type I (MHC-I) antigen, wherein the third antigen is encapsulated in the nanoparticle core, is linked to one or more oligonucleotides in the oligonucleotide shell by a linker, is linked to an outer surface of the nanoparticle core by the linker, or a combination thereof.
In some aspects, the present disclosure provides a Spherical Nucleic Acid (SNA) comprising: (a) a nanoparticle core; (b) An oligonucleotide shell attached to an outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; (c) A first antigen that is a major histocompatibility complex type I (MHC-I) antigen, wherein the first antigen is encapsulated in the nanoparticle core, is linked to one or more oligonucleotides in the oligonucleotide shell by a linker, is linked to an outer surface of the nanoparticle core by a linker, or a combination thereof; (d) A second antigen that is a major histocompatibility complex type II (MHC-II) antigen, wherein the second antigen is encapsulated in the nanoparticle core, is linked to one or more oligonucleotides in the oligonucleotide shell by a linker, is linked to an outer surface of the nanoparticle core by the linker, or a combination thereof; and (e) a third antigen that is a major histocompatibility complex type I (MHC-I) antigen, wherein the third antigen is encapsulated in the nanoparticle core, is linked to one or more oligonucleotides in the oligonucleotide shell by a linker, is linked to the outer surface of the nanoparticle core by the linker, or a combination thereof; and (f) a fourth antigen that is a major histocompatibility complex type II (MHC-II) antigen, wherein the fourth antigen is encapsulated in the nanoparticle core, is linked to one or more oligonucleotides in the oligonucleotide shell by a linker, is linked to an outer surface of the nanoparticle core by the linker, or a combination thereof.
In any aspect or embodiment of the disclosure, the Spherical Nucleic Acid (SNA) comprises: (a) a nanoparticle core; (b) An oligonucleotide shell attached to an outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen, wherein the first antigen is encapsulated in the nanoparticle core, and wherein the second antigen is linked to an oligonucleotide that hybridizes to the oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through a linker. In any aspect or embodiment of the disclosure, the method as described herein comprises administering to the subject a Spherical Nucleic Acid (SNA) comprising: (a) a nanoparticle core; (b) An oligonucleotide shell attached to an outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen, wherein the first antigen is encapsulated in the nanoparticle core, and wherein the second antigen is linked to an oligonucleotide that hybridizes to the oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through a linker.
In any aspect or embodiment of the disclosure, the Spherical Nucleic Acid (SNA) comprises: (a) a nanoparticle core; (b) An oligonucleotide shell attached to an outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen, wherein the second antigen is encapsulated in the nanoparticle core, and wherein the first antigen is linked to an oligonucleotide that hybridizes to the oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through a linker. In any aspect or embodiment of the disclosure, the method as described herein comprises administering to the subject a Spherical Nucleic Acid (SNA) comprising: (a) a nanoparticle core; (b) An oligonucleotide shell attached to an outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; and (c) a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen, wherein the second antigen is encapsulated in the nanoparticle core, and wherein the first antigen is linked to an oligonucleotide that hybridizes to the oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through a linker.
Oligonucleotides
The present disclosure provides Spherical Nucleic Acids (SNAs) comprising a nanoparticle core; an oligonucleotide shell attached to the exterior of the nanoparticle, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; and a first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen. In various embodiments, about or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the oligonucleotides in the oligonucleotide shell are immunostimulatory oligonucleotides. In various embodiments, the oligonucleotide shell comprises an inhibitory oligonucleotide, a targeting oligonucleotide, a non-targeting oligonucleotide, or a combination thereof. Oligonucleotides contemplated for use in accordance with the present disclosure include those oligonucleotides that are attached to the nanoparticle core by any means (e.g., covalent or non-covalent attachment). In various embodiments, the oligonucleotides of the present disclosure comprise DNA oligonucleotides, RNA oligonucleotides, modified forms thereof, or combinations thereof. In any aspect or embodiment described herein, the oligonucleotide is single-stranded, double-stranded or partially double-stranded. In any aspect or embodiment of the disclosure, the oligonucleotide comprises a detectable marker.
As described herein, the present disclosure also contemplates modified forms of oligonucleotides, including those oligonucleotides having at least one modified internucleotide linkage. In some embodiments, the oligonucleotide is wholly or partially a peptide nucleic acid. Other modified internucleotide linkages comprise at least one phosphorothioate linkage. Still other modified oligonucleotides include oligonucleotides comprising one or more universal bases. "universal base" refers to a molecule that is capable of substitution binding to either of A, C, G, T and U in a nucleic acid without significant structural instability by formation of hydrogen bonds. Oligonucleotides incorporating universal base analogues can function as probes in hybridization, for example. Examples of universal bases include, but are not limited to, 5 '-nitroindole-2' -deoxynucleosides, 3-nitropyrroles, inosine, and hypoxanthine.
As used herein, the term "nucleotide" or a plurality thereof is interchangeable with modified forms as discussed herein and otherwise known in the art. As used herein, the term "nucleobase" or a plurality thereof is interchangeable with modified forms as discussed herein and otherwise known in the art. Nucleotides or nucleobases include naturally occurring nucleobases A, G, C, T and U. Non-naturally occurring nucleobases include, for example and without limitation, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4-ethanolic cytosine, N' -ethanolic-2, 6-diaminopurine, 5-methylcytosine (mC), 5- (C3-C6) -alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, and the "non-naturally occurring" nucleobases described below: benner et al, U.S. Pat. No. 5,432,272 and Susan M.Freier and Karl-Heinz Altmann,1997, nucleic acids research (Nucleic Acids Research), vol.25, pages 4429-4443. The term "nucleobase" also includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Additional naturally and non-naturally occurring nucleobases include those disclosed in the following: U.S. Pat. No. 3,687,808 (Merigan et al), sanghvi in antisense research and applications (Research and Application), edited by S.T.Crooke and B.Lebleu, CRC Press (CRC Press), chapter 15 of 1993, englisch et al, 1991, german application chemistry International edition (Angewandte Chemie, international Edition), 30:613-722 (see, inter alia, pages 622 and 623 and Polymer science and engineering encyclopedia (Concise Encyclopedia of Polymer Science and Engineering), edited by J.I.Kroschwitz, han Willi father publication (John Wiley & Sons), 1990, pages 858-859, cook, anticancer drug design (Anti-Cancer Drug Design) 1991,6,585-607, each of which is hereby incorporated by reference in its entirety. In various aspects, oligonucleotides also comprise one or more "nucleobases" or "base units" that are a class of non-naturally occurring nucleotides, including compounds that can function as nucleobases, such as heterocyclic compounds, that include certain "universal bases" that are not nucleobases in the most classical sense but that function as nucleobases. The universal base comprises a 3-nitropyrrole, an optionally substituted indole (e.g., 5-nitroindole), and an optionally substituted hypoxanthine. Other desirable universal bases include pyrrole, diazole or triazole derivatives, including those known in the art.
Examples of oligonucleotides include those containing modified backbones or non-natural internucleotide linkages. Oligonucleotides having modified backbones include oligonucleotides that retain phosphorus atoms in the backbones as well as oligonucleotides that do not have phosphorus atoms in the backbones. Modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are considered to be within the meaning of "oligonucleotide".
Modified oligonucleotide backbones containing phosphorus atoms include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates (including 3' -alkylene phosphonates, 5' -alkylene phosphonates and chiral phosphonates), phosphonites, phosphoramidates (including 3' -phosphoramidates and aminoalkyl phosphoramidates), phosphorothioates having normal 3' -5' linkages, phosphorothioates, phosphoroselenophosphate and borane phosphates, 2' -5' linked analogues of these, and those having reversed polarity, wherein one or more internucleotide linkages are 3' to 3', 5' to 5' or 2' to 2' linkages. Oligonucleotides having reverse polarity, including a single 3' to 3' bond located at the most 3' internucleotide linkage, i.e., single reverse nucleoside residues (nucleotides missing or having hydroxyl groups substituted therefor) that may be abasic, are also contemplated. Salts, mixed salts and free acid forms are also contemplated. Representative U.S. patents teaching the preparation of the above phosphorus-containing bonds include U.S. Pat. nos. 3,687,808; 4,469,863; 4,476,301; no. 5,023,243; 5,177,196; 5,188,897; 5,264,423; U.S. Pat. No. 5,276,019; no. 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697; and 5,625,050, the disclosures of which are incorporated herein by reference.
Wherein no phosphorus is containedThe modified oligonucleotide backbone of atoms has a backbone formed by: short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatoms or heterocyclic internucleoside linkages. These backbones include backbones with morpholino linkages; a siloxane backbone; sulfide, sulfoxide, and sulfone backbones; formylacetyl and thiocarboxyacetyl backbones; methylene formylacetyl and thioformylacetyl backbones; a ribose acetyl backbone; a backbone comprising olefins; sulfamate backbone; methylene imino and methylene hydrazino backbones; sulfonate and sulfonamide backbones; an amide backbone; other mixed N, O, S and CH 2 A framework of the component parts. See, for example, U.S. Pat. nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; no. 5,489,677; 5,541,307; 5,561,225; 5,596,086; no. 5,602,240; 5,610,289; no. 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269; and 5,677,439, the disclosures of which are incorporated herein by reference in their entirety.
In still further embodiments, oligonucleotide mimics in which one or more of the sugars and/or one or more internucleotide linkages of a nucleotide unit are both replaced with a "non-naturally occurring" group. The bases of the oligonucleotides are maintained for hybridization. In some aspects, this embodiment contemplates Peptide Nucleic Acids (PNAs). In PNA compounds, the sugar backbone of the oligonucleotide is replaced by an amide containing backbone. See, for example, U.S. Pat. nos. 5,539,082; no. 5,714,331; and 5,719,262; and Nielsen et al, 1991, science 254:1497-1500, the disclosures of which are incorporated herein by reference.
In still further embodiments, oligonucleotides having phosphorothioate backbones are provided as well as oligonucleotides having heteroatom backbones and comprising: U.S. patent no-CH as described in 5,489,677 and 5,602,240 2 -NH-O-CH 2 -、-CH 2 -N(CH 3 )-O-CH 2 -、-CH 2 -O-N(CH 3 )-CH 2 -、-CH 2 -N(CH 3 )-N(CH 3 )-CH 2 -and-O-N (CH) 3 )-CH 2 -CH 2 -. Oligonucleotides having morpholino backbone structures described in U.S. Pat. No. 5,034,506 are also contemplated.
In various forms, the bond between two consecutive monomers in the oligonucleotide consists of 2 to 4, desirably 3 groups/atom selected from: -CH 2 -、-O-、-S-、-NR H -、>C＝O、>C＝NR H 、>C＝S、-Si(R") 2 -、-SO-、-S(O) 2 -、-P(O) 2 -、-PO(BH 3 )-、-P(O,S)-、-P(S) 2 -、-PO(R")-、-PO(OCH 3 ) -and-PO (NHR) H ) -, wherein R is H Selected from hydrogen and C 1-4 Alkyl, and R' is selected from C 1-6 Alkyl and phenyl. An illustrative example of such a bond is-CH 2 -CH 2 -CH 2 -、-CH 2 -CO-CH 2 -、-CH 2 -CHOH-CH 2 -、-O-CH 2 -O-、-O-CH 2 -CH 2 -、-O-CH 2 -ch= (containing R when used as bond for subsequent monomer 5 )、-CH 2 -CH 2 -O-、-NR H -CH 2 -CH 2 -、-CH 2 -CH 2 -NR H -、-CH 2 -NR H -CH 2 --、-O-CH 2 -CH 2 -NR H -、-NR H -CO-O-、-NR H -CO-NR H -、-NR H -CS-NR H -、-NR H -C(＝NR H )-NR H -、-NR H -CO-CH 2 -NR H -O-CO-O-、-O-CO-CH 2 -O-、-O-CH 2 -CO-O-、-CH 2 -CO-NR H -、-O-CO-NR H -、-NR H -CO-CH 2 -、-O-CH 2 -CO-NR H -、-O-CH 2 -CH 2 -NR H -、-CH＝N-O-、-CH 2 -NR H -O-、-CH 2 -O-n= (when used as bond for subsequent monomerWhen it contains R 5 )、-CH 2 -O-NR H -、-CO-NR H -CH 2 -、-CH 2 -NR H -O-、-CH 2 -NR H -CO-、-O-NR H -CH 2 -、-O-NR H 、-O-CH 2 -S-、-S-CH 2 -O-、-CH 2 -CH 2 -S-、-O-CH 2 -CH 2 -S-、-S-CH 2 -ch= (containing R when used as bond for subsequent monomer 5 )、-S-CH 2 -CH 2 -、-S-CH 2 -CH 2 --O-、-S-CH 2 -CH 2 -S-、-CH 2 -S-CH 2 -、-CH 2 -SO-CH 2 -、-CH 2 -SO 2 -CH 2 -、-O-SO-O-、-O-S(O) 2 -O-、-O-S(O) 2 -CH 2 -、-O-S(O) 2 -NR H -、-NR H -S(O) 2 -CH 2 -；-O-S(O) 2 -CH 2 -、-O-P(O) 2 -O-、-O-P(O,S)-O-、-O-P(S) 2 -O-、-S-P(O) 2 -O-、-S-P(O,S)-O-、-S-P(S) 2 -O-、-O-P(O) 2 -S-、-O-P(O,S)-S-、-O-P(S) 2 -S-、-S-P(O) 2 -S-、-S-P(O,S)-S-、-S-P(S) 2 -S-、-O-PO(R”)-O-、-O-PO(OCH 3 )-O-、-O-PO(O CH 2 CH 3 )-O-、-O-PO(O CH 2 CH 2 S-R)-O-、-O-PO(BH 3 )-O-、-O-PO(NHR N )-O-、-O-P(O) 2 -NR H H-、-NR H -P(O) 2 -O-、-O-P(O,NR H )-O-、-CH 2 -P(O) 2 -O-、-O-P(O) 2 -CH 2 -and-O-Si (R') 2 -O-; wherein-CH is taken into account 2 -CO-NR H -、-CH 2 -NR H -O-、-S-CH 2 -O-、-O-P(O) 2 -O-O-P(-O,S)-O-、-O-P(S) 2 -O-、-NR H P(O) 2 -O-、-O-P(O,NR H )-O-、-O-PO(R”)-O-、-O-PO(CH 3 ) -O-and-O-PO (NHR) N ) -O-, wherein R H Selected from hydrogen and C 1-4 Alkyl, and R' is selected from C 1-6 Alkyl and phenyl. Additional illustrative examples are provided below: mesmaeker et al, recent ideas of structural biology (Current Opinion in S)tructural Biology), 1995,5:343-355; and Susan M.Freier and Karl-Heinz Altmann, nucleic acids research 1997, volume 25, pages 4429-4443.
Still other modified forms of oligonucleotides are described in detail in U.S. patent application No. 20040219565, the disclosure of which is incorporated herein by reference in its entirety.
The modified oligonucleotides may also contain one or more substituted sugar moieties. In certain aspects, the oligonucleotide comprises one of the following at the 2' position: OH; f, performing the process; o-, S-or N-alkyl; o-, S-or N-alkenyl; o-, S-or N-alkynyl; or O-alkyl-O-alkyl; wherein alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C 1 To C 10 Alkyl or C 2 To C 10 Alkenyl and alkynyl groups. Other embodiments include O [ (CH) 2 ) n O]mCH 3 、O(CH 2 ) n OCH 3 、O(CH 2 ) n NH 2 、O(CH 2 ) n CH 3 、O(CH 2 ) n ONH 2 And O (CH) 2 ) n ON[(CH 2 ) n CH 3 ]2, wherein n and m are from 1 to about 10. Other oligonucleotides include one of the following at the 2' position: c (C) 1 To C 10 Lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkylaryl, arylalkyl, O-alkylaryl or O-arylalkyl, SH, SCH 3 、OCN、Cl、Br、CN、CF 3 、OCF 3 、SOCH 3 、SO 2 CH 3 、ONO 2 、NO 2 、N 3 NH2, heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl or RNA cleavage groups. In one aspect, the modification comprises 2 '-methoxyethoxy (2' -O-CH) 2 CH 2 OCH 3 Also known as 2'-O- (2-methoxyethyl) or 2' -MOE) (Martin et al, J.Swiss chemistry report (Helv.Chim. Acta), 1995,78,486-504), i.e., an alkoxyalkoxy group. Other modifications include 2' -dimethylaminooxyethoxy, i.e. O (CH) 2 ) 2 ON(CH 3 ) 2 Radicals, also known as 2'-DMAOE, and 2' -diMethylaminoethoxyethoxy (also known in the art as 2' -O-dimethyl-amino-ethoxy-ethyl or 2' -DMAEOE), i.e. 2' -O-CH 2 -O-CH 2 -N(CH 3 ) 2 。
Still other modifications include 2 '-methoxy (2' -O-CH) 3 ) 2 '-aminopropoxy (2' -OCH) 2 CH 2 CH 2 NH 2 ) 2 '-allyl (2' -CH) 2 -CH＝CH 2 ) 2 '-O-allyl (2' -O-CH) 2 -CH＝CH 2 ) And 2 '-fluoro (2' -F). The 2' -modification may be in the arabinose (upper) position or ribose (lower) position. In one aspect, 2 '-arabinose is modified to 2' -F. Similar modifications can also be made at other positions of the oligonucleotide, for example, at the 3 'position of the sugar on the 3' terminal nucleotide or in the 2'-5' linked oligonucleotide and in the 5 'position of the 5' terminal nucleotide. The oligonucleotide may also have a glycomimetic, such as a cyclobutyl moiety in place of the pentose sugar. See, for example, U.S. patent No. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, the disclosures of which are incorporated by reference in their entirety.
In some aspects, the modification of the sugar comprises a Locked Nucleic Acid (LNA) in which the 2' -hydroxyl group is attached to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. In certain aspects, the bond is a methylene (-CH) bridging the 2 'oxygen atom and the 4' carbon atom 2 -) n A group wherein n is 1 or 2.LNA and its preparation are described in WO 98/39352 and WO 99/14226.
Modified nucleotides are described in EP 1 072 679 and WO 97/12896, the disclosures of which are incorporated herein by reference. Modified nucleobases include, but are not limited to, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-deaza-adenine and 3-deaza-adenine. Further modified bases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimidine [5,4-b ] [1,4] benzoxazin-2 (3H) -one), phenothiazine cytidine (1H-pyrimidine [5,4-b ] [1,4] benzothiazin-2 (3H) -one), G-cams, such as substituted phenoxazine cytidine (e.g., 9- (2-aminoethoxy) -H-pyrimidine [5,4-b ] [1,4] benzo-oxazine-2 (3H) -one), carbazole cytidine (2H-pyrimidine [4,5-b ] indol-2-one), pyridoindole cytidine (H-pyridine [3',2':4,5] pyrrole [2,3-d ] pyrimidine-2-one). Modified bases may also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanine, 2-aminopyridine, and 2-pyridone. Additional nucleobases include the nucleobases disclosed in U.S. Pat. No. 3,687,808, pages 858-859 of the polymeric science and engineering encyclopedia, kroschwitz, J.I. edition, the nucleobases disclosed in John Wili parent-child publishing company, 1990, englisch et al, 1991, the nucleobases disclosed in German application chemistry, international edition, 30:613, and the nucleobases disclosed in Sanghvi, Y.S., chapter 15, antisense research and application, pages 289-302, crooke, S.T. and Lebleu, B.edition, CRC Press, 1993. Some of these bases are useful for increasing binding affinity and include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. It has been shown that 5-methylcytosine substitution increases the stability of the nucleic acid duplex to 0.6-1.2 ℃ and in some way binds to 2' -O-methoxyethyl sugar modification. See U.S. Pat. No. 3,687,808, U.S. Pat. No. 4,845,205; no. 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; no. 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,750,692; and 5,681,941, the disclosures of which are incorporated herein by reference.
Methods for preparing polynucleotides of predetermined sequence are well known. See, e.g., sambrook et al, molecular cloning: laboratory Manual (Molecular Cloning: ALaboratory Manual) (2 nd edition 1989) and F.Eckstein (eds.) (oligonucleotides and analogues (Oligonucleotides and Analogues)), 1 st edition (Oxford university Press, new York, 1991). Solid phase synthesis methods are preferred for both polyribonucleotides and polydeoxyribonucleotides (well known methods of synthesizing DNA can also be used for synthesizing RNA). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases can also be incorporated into polynucleotides. See, for example, U.S. patent No. 7,223,833; katz, journal of American society of chemistry, 74:2238 (1951); yamane et al, journal of the American society of chemistry, 83:2599 (1961); kosturko et al, biochemistry, 13:3949 (1974); thomas, journal of American society of chemistry, 76:6032 (1954); zhang et al, journal of the American society of chemistry, 127:74-75 (2005); and Zimmermann et al, journal of the American society of chemistry, 124:13684-13685 (2002).
In various aspects, the oligonucleotides of the disclosure, or modified versions thereof, are typically from about 5 nucleotides to about 1000 nucleotides in length. More specifically, the oligonucleotides of the present disclosure are about 5 to about 1000 nucleotides in length, about 5 to about 900 nucleotides in length, about 5 to about 800 nucleotides in length, about 5 to about 700 nucleotides in length, about 5 to about 600 nucleotides in length, about 5 to about 500 nucleotides in length, about 5 to about 450 nucleotides in length, about 5 to about 400 nucleotides in length, about 5 to about 350 nucleotides in length, about 5 to about 300 nucleotides in length, about 5 to about 250 nucleotides in length, about 5 to about 200 nucleotides in length, about 5 to about 150 nucleotides in length, about 5 to about 100 nucleotides in length, about 5 to about 90 nucleotides in length, about 5 to about 80 nucleotides in length, about 5 to about 70 nucleotides in length, about 5 to about 60 nucleotides in length, from about 5 to about 50 nucleotides in length, from about 5 to about 45 nucleotides in length, from about 5 to about 40 nucleotides in length, from about 5 to about 35 nucleotides in length, from about 5 to about 30 nucleotides in length, from about 5 to about 25 nucleotides in length, from about 5 to about 20 nucleotides in length, from about 5 to about 15 nucleotides in length, from about 5 to about 10 nucleotides in length, from about 10 to about 1000 nucleotides in length, from about 10 to about 900 nucleotides in length, from about 10 to about 800 nucleotides in length, from about 10 to about 700 nucleotides in length, from about 10 to about 600 nucleotides in length, from about 10 to about 500 nucleotides in length, from about 10 to about 450 nucleotides in length, from about 10 to about 400 nucleotides in length, from about 10 to about 350 nucleotides in length, the oligonucleotide may be about 10 to about 300 nucleotides in length, about 10 to about 250 nucleotides in length, about 10 to about 200 nucleotides in length, about 10 to about 150 nucleotides in length, about 10 to about 100 nucleotides in length, about 10 to about 90 nucleotides in length, about 10 to about 80 nucleotides in length, about 10 to about 70 nucleotides in length, about 10 to about 60 nucleotides in length, about 10 to about 50 nucleotides in length, about 10 to about 45 nucleotides in length, about 10 to about 40 nucleotides in length, about 10 to about 35 nucleotides in length, about 10 to about 30 nucleotides in length, about 10 to about 25 nucleotides in length, about 10 to about 20 nucleotides in length, about 10 to about 15 nucleotides in length, about 18 to about 28 nucleotides in length, about 15 to about 26 nucleotides in length, and specifically the oligonucleotide may be centered to a small extent. In further embodiments, the oligonucleotides of the present disclosure are from about 5 to about 100 nucleotides in length, from about 5 to about 90 nucleotides in length, from about 5 to about 80 nucleotides in length, from about 5 to about 70 nucleotides in length, from about 5 to about 60 nucleotides in length, from about 5 to about 50 nucleotides in length, from about 5 to about 40 nucleotides in length, from about 5 to about 30 nucleotides in length, from about 5 to about 20 nucleotides in length, from about 5 to about 10 nucleotides in length, and in particular all oligonucleotides centered in size to the extent that the oligonucleotides are capable of achieving a desired result. In various embodiments, therefore, the oligonucleotides of the disclosure are or are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 51, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides. In a further embodiment of the present invention, the oligonucleotides of the disclosure are less than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides. In various embodiments, the oligonucleotide shell attached to the exterior of the nanoparticle core of the SNA comprises a plurality of oligonucleotides all having the same length/sequence, while in some embodiments, the plurality of oligonucleotides comprises one or more oligonucleotides having a different length and/or sequence relative to the length and/or sequence of at least one other oligonucleotide in the plurality of oligonucleotides. For example and without limitation, in some embodiments, the oligonucleotide shell comprises a plurality of immunostimulatory oligonucleotides, wherein one immunostimulatory oligonucleotide has a sequence that is different from at least one other immunostimulatory oligonucleotide of the plurality of immunostimulatory oligonucleotides.
In some embodiments, one or more oligonucleotides in the oligonucleotide shell comprise (GGX) n A nucleotide sequence or consisting of, wherein n is 2-20 and X is a nucleobase (A, C, T, G or U). In some embodiments, (GGX) n The nucleotide sequence is located on the 5' end of one or more oligonucleotides. In some embodiments, (GGX) n The nucleotide sequence is located on the 3' end of one or more oligonucleotides. In some embodiments, one or more oligonucleotides in the oligonucleotide shell comprise (GGT) n A nucleotide sequence or consisting of, wherein n is 2-20. In some embodiments, (GGT) n The nucleotide sequence is located on the 5' end of one or more oligonucleotides. In some embodiments, (GGT) n The nucleotide sequence is located on the 3' end of one or more oligonucleotides.
In some embodiments, the oligonucleotides in the oligonucleotide shells are targeting oligonucleotides, such as aptamers. Thus, all features and aspects of the oligonucleotides described herein (e.g., length, type (DNA, RNA, modified forms thereof), optional presence of spacers) are also applicable to the aptamer. An aptamer is an oligonucleotide sequence that can evolve to bind to a variety of target analytes of interest. The aptamer may be single-stranded, double-stranded or partially double-stranded.
Spacer. In some aspects and embodiments, one or more oligonucleotides in the oligonucleotide shell that are linked to the nanoparticle core of SNA include a spacer. As used herein, "spacer" means a moiety used to increase the distance between the nanoparticle core and the oligonucleotide, or between individual oligonucleotides when attached in multiple copies to the nanoparticle core, or to improve synthesis of SNA. Thus, it is contemplated that the spacer is located between the oligonucleotide and the nanoparticle core.
In some aspects, the spacer, when present, is an organic moiety. In some aspects, the spacer is a polymer, including but not limited to a water-soluble polymer, a nucleic acid, a polypeptide, an oligosaccharide, a carbohydrate, a lipid, ethylene glycol, or a combination thereof. In any aspect or embodiment of the disclosure, the spacer is an oligo (ethylene glycol) -based spacer. In various embodiments, the oligonucleotide comprises 1, 2, 3, 4, 5, or more spacer (e.g., spacer-18 (hexaethylene glycol)) moieties. In further embodiments, the spacer is an alkane-based spacer (e.g., C12). In some embodiments, the spacer is an oligonucleotide spacer (e.g., T5). The oligonucleotide spacer may have any sequence that does not interfere with the ability of the oligonucleotide to bind to the nanoparticle core or to the target. In certain aspects, the bases of the oligonucleotide spacer are all adenylates, all thymidylates, all cytidylates, all guanylate, all uridylates, or all some other modified bases.
In various embodiments, the spacer is at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, 5 to 10 nucleotides, 10-30 nucleotides, or even more than 30 nucleotides in length.
SNSurface Density A. Typically, at least about 0.5 picomoles/cm 2 The surface density of the oligonucleotides of (2) will be sufficient to provide a stable SNA. In further embodiments, at least about 1 picomole/cm 2 1.5 picomole/cm 2 Or 2 picomoles/cm 2 The surface density of the oligonucleotides of (a) will be sufficient to provide a stable SNA (e.g., LSNA or LNP-SNA). In some aspects, SNAs of the present disclosure have a surface density of at least 15 picomoles/cm 2 . Also provided are methods wherein the oligonucleotide is linked to the nanoparticle core of SNA at the following surface densities: about 0.5 picomole/cm 2 To about 1000 picomoles/cm 2 Or about 2 picomoles/cm 2 To about 200 picomoles/cm 2 Or about 10 picomoles/cm 2 To about 100 picomoles/cm 2 . In some embodiments, the surface density is about 1.7 picomoles/cm 2 . In some embodiments, the surface density is about 2 picomoles/cm 2 . In further embodiments, the surface density is at least about 0.5 picomoles/cm 2 At least about 0.6 picomole/cm 2 At least about 0.7 picomole/cm 2 At least about 0.8 picomole/cm 2 At least about 0.9 picomole/cm 2 At least about 1 picomole/mc 2 At least about 1.5 picomoles/cm 2 At least about 2 picomoles/cm 2 At least 3 picomoles/cm 2 At least 4 picomoles/cm 2 At least 5 picomoles/cm 2 At least 6 picomoles/cm 2 At least 7 picomoles/cm 2 At least 8 picomoles/cm 2 At least 9 picomoles/cm 2 At least 10 picomoles/cm 2 At least about 15 picomoles/cm 2 At least about 19 picomoles/cm 2 At least about 20 picomoles/cm 2 At least about 25 picomoles/cm 2 At least about 30 picomoles/cm 2 At least about 35 picomoles/cm 2 At least about 40 picomoles/cm 2 At least about 45 picomoles/cm 2 At least about 50 picomoles/cm 2 At least about 55 picomoles/cm 2 At least about 60 picomoles/cm 2 At least about 65 picomoles/cm 2 At least about 70 picomoles/cm 2 At least about 75 picomoles/cm 2 At least about 80 picomoles/cm 2 At least about 85 picomoles/cm 2 At least about 90 picomoles/cm 2 At least about 95 picomoles/cm 2 At least about 100 picomoles/cm 2 At least about 125 picomoles/cm 2 At least about 150 picomoles/cm 2 At least about 175 picomoles/cm 2 At least about 200 picomoles/cm 2 At least about 250 picomoles/cm 2 At least about 300 picomoles/cm 2 At least about 350 picomoles/cm 2 At least about 400 picomoles/cm 2 At least about 450 picomoles/cm 2 At least about 500 picomoles/cm 2 At least about 550 picomoles/cm 2 At least about 600 picomoles/cm 2 At least about 650 picomoles/cm 2 At least about 700 picomoles/cm 2 At least about 750 picomoles/cm 2 At least about 800 picomoles/cm 2 At least about 850 picomoles/cm 2 At least about 900 picomoles/cm 2 At least about 950 picomoles/cm 2 At least about 1000 picomoles/cm 2 Or larger. In further embodiments, the surface density is less than about 2 picomoles/cm 2 Less than about 3 picomoles/cm 2 Less than about 4 picomoles/cm 2 Less than about 5 picomoles/cm 2 Less than about 6 picomoles/cm 2 Less than about 7 picomoles/cm 2 Less than about 8 picomoles/cm 2 Less than about 9 picomoles/cm 2 Less than about 10 picomoles/cm 2 Less than about 15 picomoles/cm 2 Less than about 19 picomoles/cm 2 Less than about 20 picomoles/cm 2 Less than about 25 picomoles/cm 2 Less than about 30 picomoles/cm 2 Less than about 35 picomoles/cm 2 Less than about 40 picomoles/cm 2 Less than about 45 picomoles/cm 2 Less than about 50 picomoles/cm 2 Less than about 55 picomoles/cm 2 Less than about 60 picomoles/cm 2 Less than about 65 picomoles/cm 2 Less than about 70 picomoles/cm 2 Less than about 75 picomoles/cm 2 Less than about 80 picomoles/cm 2 Less than about 85 picomoles/cm 2 Less than about 90 picomoles/cm 2 Less than about 95 picomoles/cm 2 Less than about 100 picomoles/cm 2 Small and smallAt about 125 picomoles/cm 2 Less than about 150 picomoles/cm 2 Less than about 175 picomoles/cm 2 Less than about 200 picomoles/cm 2 Less than about 250 picomoles/cm 2 Less than about 300 picomoles/cm 2 Less than about 350 picomoles/cm 2 Less than about 400 picomoles/cm 2 Less than about 450 picomoles/cm 2 Less than about 500 picomoles/cm 2 Less than about 550 picomoles/cm 2 Less than about 600 picomoles/cm 2 Less than about 650 picomoles/cm 2 Less than about 700 picomoles/cm 2 Less than about 750 picomoles/cm 2 Less than about 800 picomoles/cm 2 Less than about 850 picomoles/cm 2 Less than about 900 picomoles/cm 2 Less than about 950 picomoles/cm 2 Or less than about 1000 picomoles/cm 2 。
Alternatively, the density of SNA-linked oligonucleotides is measured by the number of SNA-linked oligonucleotides. Regarding the surface density of oligonucleotides linked to SNAs of the present disclosure, SNAs as described herein are contemplated to include or consist of about 1 to about 2,500, or about 1 to about 500 oligonucleotides on their surface. In various embodiments, SNAs comprise from about 10 to about 500, or from about 10 to about 300, or from about 10 to about 200, or from about 10 to about 190, or from about 10 to about 180, or from about 10 to about 170, or from about 10 to about 160, or from about 10 to about 150, or from about 10 to about 140, or from about 10 to about 130, or from about 10 to about 120, or from about 10 to about 110, or from about 10 to about 100, or from 10 to about 90, or from about 10 to about 80, or from about 10 to about 70, or from about 10 to about 60, or from about 10 to about 50, or from about 10 to about 40, or from about 10 to about 30, or from about 10 to about 20, or from about 75 to about 200, or from about 75 to about 150, or from about 100 to about 200, or from about 200 oligonucleotides in the oligonucleotide shell attached to the nanoparticle core. In some embodiments, SNAs comprise from about 80 to about 140 oligonucleotides in an oligonucleotide shell attached to a nanoparticle core. In further embodiments, SNAs comprise at least about 5, 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 oligonucleotides in the oligonucleotide shell attached to the nanoparticle core. In further embodiments, SNAs consist of 5, 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 oligonucleotides in the oligonucleotide shell attached to the nanoparticle core. In still further embodiments, the oligonucleotide shells attached to the nanoparticle core of SNA comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 60, 70, 75, 80, 90, 100, 150, 160, 170, 175, 180, 190, 200, or more oligonucleotides. In some embodiments, the oligonucleotide shells attached to the nanoparticle cores of SNAs consist of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 75, 80, 90, 100, 150, 160, 170, 175, 180, 190, or 200 oligonucleotides. In some embodiments, the oligonucleotide shells comprise from about 10 to about 80 oligonucleotides. In some embodiments, the oligonucleotide shell comprises or consists of about 75 oligonucleotides.
Joint. The present disclosure provides compositions and methods wherein an antigen associates and/or links with the surface of SNA through a linker. In various embodiments, the joint may be a cleavable joint, a non-cleavable joint, a traceless joint, and combinations thereof.
The linker links the antigen to the oligonucleotides in the SNAs disclosed, or connects the antigen to the oligonucleotides in the SNAs disclosedSurface ligation of SNAs (i.e., antigen-linker-oligonucleotides or antigen-linkers). The oligonucleotide may be hybridized to another oligonucleotide linked to the SNA, or may be linked to the SNA directly (e.g., via a lipid anchor group). Some specifically contemplated linkers include alkylene carbamates, alkylene aryl carbamates, alkylene amide disulfides, alkylene amide aryl disulfides, thiol linkers, and alkylene amide succinimidyl linkers. In some cases, the linker comprises-NH-C (O) -O-C 2-5 alkylene-S-S-C 2-7 alkylene-or-NH-C (O) -C 2-5 alkylene-S-S-C 2-7 Alkylene-. The carbon alpha of the-S-moiety may be branched, for example, -CHX-S-or-S-che-or a combination thereof, wherein X and Y are independently Me, et or iPr. The carbon alpha of the antigen may be branched, e.g. -CHX-C 2-4 alkylene-S-S-, wherein X is Me, et or iPr. In some cases, the linker is-NH-C (O) -O-CH 2 -Ar-S-S-C 2-7 Alkylene-, and Ar is meta-or para-substituted phenyl. In some cases, the linker is-NH-C (O) -C 2-4 alkylene-N-succinimidyl-S-C 2-6 Alkylene-.
Additional linkers include SH linkers, SM linkers, SE linkers, and SI linkers. The present disclosure contemplates multiple junctions (e.g., disulfide cleavage, linker cyclization, and dehybridization) that can be used to modulate antigen release, and the kinetics of antigen release at each junction can be controlled. For example, spatial packing around disulfides can reduce S N 2 rate of reaction; the increased length of the alkyl spacer or the steric bulk attached to the alkyl spacer may affect the rate of ring closure; and mismatched nucleotide sequences reduce melting temperature (T m ) While locked nucleic acid increases T m 。
Use of SNA in immunomodulation
Toll-like receptors (TLRs) are a class of proteins expressed in sentry cells that play a key role in the regulation of the innate immune system. The mammalian immune system uses two general strategies to combat infectious diseases. Pathogen exposure rapidly triggers an innate immune response characterized by the production of immunostimulatory cytokines, chemokines and multi-reactive IgM antibodies. The innate immune system is activated by exposure to pathogen-associated molecular patterns (PAMPs) expressed by different groups of infectious microorganisms. PAMPs are recognized by members of the Toll-like receptor family. TLR receptors, such as TLR 8 and TLR 9, that respond to specific oligonucleotides are located within specific intracellular compartments called endosomes. Regulatory mechanisms such as, but not limited to, TLR 8 and TLR 9 receptors are based on DNA-protein interactions.
As described herein, synthetic immunostimulatory oligonucleotides containing CpG motifs similar to those present in bacterial DNA stimulate a similar response to TLR receptors. Thus, the CpG oligonucleotides of the present disclosure are capable of acting as TLR agonists. Other TLR agonists contemplated by the present disclosure include, but are not limited to, single stranded RNAs and small molecules (e.g., R848 (requisimid)). Thus, immunomodulatory (e.g., immunostimulatory) oligonucleotides have a variety of potential therapeutic uses, including the treatment of diseases (e.g., cancer).
Thus, in some embodiments, methods of modulating toll-like receptors using SNAs as described herein are disclosed. The methods up-regulate Toll-like receptor activity through the use of TLR agonists. The methods comprise contacting a cell having a toll-like receptor with SNA of the present disclosure, thereby modulating activity and/or expression of the toll-like receptor. The modulated toll-like receptor comprises one or more of the following: toll-like receptor 1, toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 6, toll-like receptor 7, toll-like receptor 8, toll-like receptor 9, toll-like receptor 10, toll-like receptor 11, toll-like receptor 12 and/or toll-like receptor 13.
Method of inducing an immune response
The disclosure also includes methods for eliciting an immune response in a subject in need thereof, the methods comprising administering to the subject an effective amount of SNA of the disclosure (e.g., formulated as an antigen composition). In various embodiments, administration of SNAs of the present disclosure (e.g., formulated as compositions, pharmaceutical formulations, or antigen compositions) to a subject increases the amount of neutralizing antibodies to an antigen produced in the subject relative to the amount of neutralizing antibodies to an antigen produced in a subject not administered SNAs. In further embodiments, the increase is a 2-fold increase, a 5-fold increase, a 10-fold increase, a 50-fold increase, a 100-fold increase, a 200-fold increase, a 500-fold increase, a 700-fold increase, or a 1000-fold increase.
In further embodiments, SNAs of the present disclosure activate human peripheral blood mononuclear cells and generate an antibody response to one or more antigens as described herein. In some embodiments, the antibody response is a total antigen-specific antibody response. In further embodiments, administration of SNAs of the present disclosure (e.g., formulated as compositions, pharmaceutical formulations, or antigen compositions) to a subject increases the amount of total antigen-specific antibodies to an antigen produced in the subject relative to the amount of total antigen-specific antibodies to an antigen produced in a subject not administered SNAs. In further embodiments, the increase is a 2-fold increase, a 5-fold increase, a 10-fold increase, a 50-fold increase, a 100-fold increase, a 200-fold increase, a 500-fold increase, a 700-fold increase, or a 1000-fold increase. The "total antigen-specific antibody response" is a measure of all antibodies (including neutralizing and non-neutralizing antibodies) that bind to a particular antigen.
The immune response elicited by the methods of the present disclosure generally comprises an antibody response, preferably a neutralizing antibody response, maturation and memory of T cells and B cells, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and T cell-mediated response, such as CD4 + 、CD8 + . The immune response generated by SNA as disclosed herein generates an immune response and preferably treats a disease (e.g., cancer) as described herein. Methods for assessing antibody responses following administration of an antigen composition (immunization or vaccination) are known in the art and/or described herein. In some embodiments, the immune response comprises a T cell-mediated response (e.g., a peptide-specific response, such as a proliferative response or a cytokine response). In any aspect or embodiment of the disclosure, the immune response comprises both a B cell response and a T cell responseAnd then the other is a member. The antigen composition may be administered in a variety of suitable ways, such as intramuscular injection, subcutaneous injection, intradermal administration, and mucosal administration, such as oral administration or intranasal administration. Additional modes of administration include, but are not limited to, intravenous, intraperitoneal, intranasal, intravaginal, intrarectal, and oral administration. The present disclosure also contemplates combinations of different routes of administration in an immunized subject, e.g., simultaneous intramuscular and intranasal administration.
Administration may involve single dose or multi-dose schedules. Multiple doses may be used in the primary immunization schedule and/or in the boost immunization schedule. In a multi-dose schedule, the various doses may be administered by the same or different routes, such as parenteral sensitization and mucosal enhancement, mucosal sensitization and parenteral enhancement, or subcutaneous sensitization and subcutaneous enhancement. Administration of more than one dose (typically two doses) may be particularly useful for immunogenic primary subjects or subjects in a low-reactivity population (e.g., subjects with diabetes or chronic kidney disease (e.g., dialysis patients)). In various embodiments, the second dose is administered about or at least about 2 weeks after the first dose. The multiple doses will typically be administered at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, or about 16 weeks). In some embodiments, the multiple doses are administered one month, two months, three months, four months, or five months apart. The antigen compositions of the present disclosure may be administered to a patient substantially simultaneously with other vaccines (e.g., during the same medical consultation or visit by a healthcare professional).
Use of LNP-SNA for treating a condition
In some aspects, SNAs of the present disclosure are used to treat a disorder. Thus, in some aspects, the present disclosure provides a method of treating a disorder, the method comprising administering to a subject (e.g., a human subject) in need thereof an effective amount of SNA of the present disclosure, wherein the administration treats the disorder. In some aspects, the present disclosure provides methods of treating cancer, the methods comprising administering to a subject (e.g., a human subject) an effective amount of SNA of the present disclosure, thereby treating cancer in the subject. In various embodiments, the cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, glioblastoma, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, lymphoma, non-hodgkin's lymphoma, bone cancer, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, and human papillomavirus-induced cancer, or a combination thereof.
Additional pharmaceutical agents
In any aspect or embodiment of the disclosure, the additional agent is administered separately from the SNA of the disclosure. Thus, in various embodiments, the therapeutic agent is administered before, after, or concurrently with SNAs of the present disclosure to treat a disorder (e.g., cancer).
In some aspects, SNAs provided herein optionally further comprise an additional agent or agents thereof. In various embodiments, the additional agent is simply associated with the oligonucleotide in the oligonucleotide shell that is attached to the exterior of the nanoparticle core of SNA, and/or the additional agent is associated with the nanoparticle core of SNA, and/or the additional agent is encapsulated in the nanoparticle core of SNA. In some embodiments, the additional agent is associated with the end of the oligonucleotide in the oligonucleotide shell that is not linked to the nanoparticle core (e.g., if the oligonucleotide is linked to the nanoparticle core through its 3 'end, the additional agent is associated with the 5' end of the oligonucleotide). Alternatively, in some embodiments, the additional agent is associated with the end of the oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core (e.g., if the oligonucleotide is linked to the nanoparticle core by its 3 'end, the additional agent is associated with the 3' end of the oligonucleotide). In some embodiments, the additional agent is covalently associated with an oligonucleotide in the oligonucleotide shell that is attached to the exterior of the nanoparticle core of SNA. In some embodiments, the additional agent is non-covalently associated with an oligonucleotide in the oligonucleotide shell that is attached to the exterior of the nanoparticle core of SNA. However, it is to be understood that the present disclosure provides SNAs wherein one or more additional agents are covalently and non-covalently associated with the oligonucleotide in the oligonucleotide shell that is attached to the exterior of the lipid nanoparticle core of the SNA. It is also understood that non-covalent association comprises hybridization, protein binding, and/or hydrophobic interactions.
Additional agents contemplated by the present disclosure include, but are not limited to, proteins (e.g., therapeutic proteins), growth factors, hormones, interferons, interleukins, antibodies or antibody fragments, small molecules, peptides, antibiotics, antifungals, antivirals, chemotherapeutics, or combinations thereof. In some embodiments, the additional agent is an anti-apoptosis protein 1 (PD-1) antibody.
As used herein, the term "small molecule" refers to a chemical compound or drug or any other low molecular weight organic compound, whether natural or synthetic. By "low molecular weight" is meant that the molecular weight of the compound is less than 1500 daltons, typically between 100 daltons and 700 daltons.
Use of SNA in gene regulation
In some aspects of the disclosure, an oligonucleotide associated with SNA (e.g., LNP-SNA, LSNA) of the disclosure inhibits expression of a gene. Thus, in some embodiments, SNAs perform both vaccine and gene suppression functions. In such aspects, the oligonucleotide shell attached to the outer surface of the nanoparticle core comprises one or more immunostimulatory oligonucleotides and one or more inhibitory oligonucleotides designed to inhibit expression of the target gene.
Methods provided herein for inhibiting expression of a gene product include those methods wherein expression of a target gene product is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% compared to expression of the gene product in the absence of SNA. In other words, the methods provided encompass methods that result in substantially any degree of inhibition of expression of a target gene product.
The extent of inhibition is determined in vivo by a body fluid sample or biopsy sample or by imaging techniques well known in the art. Alternatively, the extent of inhibition is typically determined in a cell culture assay as a predictable measure of the extent of inhibition that can be expected in vivo by the use of a particular type of SNA and specific oligonucleotides.
In various aspects, the methods comprise using an inhibitory oligonucleotide that is 100% complementary (i.e., perfectly matched) to the target polynucleotide, while in other aspects, the oligonucleotide is at least (meaning greater than or equal to) about 95% complementary to the polynucleotide over the length of the oligonucleotide, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20% complementary to the length of the polynucleotide to the extent that the oligonucleotide is capable of achieving a desired degree of inhibition of the target gene product.
The percent complementarity is determined by the length of the oligonucleotide. For example, given an antisense compound in which 18 of the 20 nucleotides of the inhibitory oligonucleotide are complementary to a 20 nucleotide region in a target polynucleotide having a total length of 100 nucleotides, the oligonucleotide will be 90% complementary. In this example, the remaining non-complementary nucleotides can be clustered or interspersed with complementary nucleobases, and need not be adjacent to each other or to the complementary nucleotides. The percentage of complementarity of an inhibitory oligonucleotide to a region of a target nucleic acid can be determined using the BLAST program (basic local alignment search tool) and the PowerBLAST program known in the art (Altschul et al J. Mol. Biol.) 1990,215,403-410; zhang and Madden, genome Res., 1997,7,649-656).
The oligonucleotides used in such methods are RNA or DNA. The RNA may be an inhibitory oligonucleotide, such as an inhibitory RNA (RNAi) that performs a regulatory function, and in various embodiments is selected from the group consisting of: small inhibitory RNAs (sirnas), single-stranded RNAs (ssrnas), and ribozymes. Alternatively, the RNA is a microrna that performs a regulatory function. In some embodiments, the DNA is antisense DNA. In some embodiments, the RNA is piwi-interacting RNA (piRNA).
The following examples are given merely to illustrate the present disclosure and do not limit its scope in any way.
Examples
Example 1
Methods of synthesizing SNAs of the present disclosure. In some embodiments, a multi-antigen targeted SNA vaccine is synthesized using a liposome core. Lipid membranes containing DOPC (1, 2-dioleoyl-sn-glycero-3-phosphorylcholine) were hydrated with PBS containing solubilized peptide antigen. It was then extruded, dialyzed overnight to remove excess unencapsulated peptide, and characterized using DLS, pierce assay (for peptide quantification) and PC assay (for lipid quantification). Separately, peptide-conjugated complementary DNA (for adjuvants) was synthesized. This process may require a number of chemistries, but involves disulfide interactions between the peptide (either method 1-cysteine or method 2-linked linker) and the complementary DNA (blocked by a thiol). In the case of cysteine (method 1), the peptide was first activated with Aldrithiol at room temperature for at least 1 hour. This product was then mixed with thiol-terminated complementary DNA in a 5:1 molar ratio overnight. In method 2, the N-terminus of the peptide on the resin was reacted with the NHS ester terminated linker overnight at room temperature. This product was excised from the resin, deprotected and purified before conjugation to thiol-terminated complementary DNA at a molar ratio of 10:1. In either method, the final product was purified by polyacrylamide gel electrophoresis and analyzed using ESI. The purified product was mixed 1:1 with cholesterol-capped DNA adjuvant and lyophilized. It was resuspended in duplex buffer and slowly cooled to duplex (70 ℃ C. For 10 min, 25 ℃ C. For 1.5 hours, 4 ℃ C. Overnight). The duplex was then mixed with peptide encapsulated in liposomes in equimolar ratio and the liposomes backfilled (up to 75 strands per liposome) to form SNAs.
Example 2
Cancer vaccines must activate multiple immune cell types to be effective against invasive tumors, but design considerations for these multi-target vaccines have not been explored. Here, the effect of structural presentation of various antigenic peptides on the immune response at the transcriptome, cellular and organism level is demonstrated. Spherical Nucleic Acid (SNA) nanostructures were used to study differences in antigen processing, cytokine production and memory, which stem from the spatial distribution and nanoscale placement of the two antigen classes for activating the two T cell types. A single double antigen SNA (DA-SNA) elicits 30% and a double increase in antigen-specific T cell activation and proliferation, respectively, compared to two single antigen SNAs. Antigen placement within DA-SNA alters immune gene expression and tumor growth: encapsulation of helper and externally conjugated cytotoxic T cell antigens (referred to as DA-SNA 2) increases the anti-tumor gene pathway, delaying tumor in mice with lymphoma. When combined with an anti-PD-1 checkpoint inhibitor in clinically relevant melanoma, DA-SNA2 inhibited the tumor and increased circulating T cell memory. This example demonstrates the importance of implementing the structural control provided by the modular nanoscale architecture to synthesize a multi-antigen vaccine with improved efficacy.
Vaccination is an attractive strategy for cancers that express both tumor-associated antigens and neoantigens. For melanoma, there has been an increasing effort to develop vaccines targeting identified tumor-associated proteins (e.g., gp100, MAGE-A3, MART-1, NY-ESO-1). 1-4 However, while these vaccines elicit some benefit (i.e., increase activated melanoma-specific T cells), many vaccines are designed to activate only one immune cell type. Tumors may have significant heterogeneity and high mutational burden 5,6 This makes it easy to evade immune monitoring. 7 Thus, vaccines that primarily activate one immune cell type are inadequate, requiring vaccines containing antigens that target multiple cell types to induce complete tumor remission.
A common method of eliciting a versatile immune response is to administer: 1) "long peptides" whose sequences cover multiple epitopes to activate both cytotoxic T cells and helper T cells, or 2) multiple "minimal" peptide antigens, each of which is unique to a subset of T cells. 8-11 However, many of these ongoing efforts involve peptide pools delivered in saline as a mixture with or without an adjuvant.
This example explores the involvement ofAnd vaccine design space for multiple cell-targeted antigens. By using structural changes in antigen placement, the impact of the generated immune response is elucidated and used to drive the success of the translation work. The antigen used activates cytotoxic (cd8+) T cells to effectively kill the tumor and activates helper (cd4+) T cells to synergistically interact to achieve long-term tumor rejection. 17,18 Cd4+ T cells maintain tumor-directed cd8+ functionality by recruiting them to tumor sites and enhancing their proliferation and effector functions. 19-23 Cancer vaccine pathways with cytotoxic and helper T cell antigens can activate different classes of T cells by presenting cytotoxic antigens on MHC-I receptors and helper antigens on MHC-II receptors. (see FIGS. 40-43). See also hos, a. For a natural review: drug discovery (Nat. Rev. Drug discovery.) 2016,15 (4), 235-247. Thus, the vaccine described herein allows for the precise structural placement of Major Histocompatibility Complex (MHC) -I and-II restricted antigen targets (cd8+ and cd4+ activation, respectively) for most effective sensitization to the immune system.
Here, spherical Nucleic Acids (SNAs) were used to elucidate the effect of nanoscale structures on the multi-antigen immune process. SNAs comprise a nanoparticle core (e.g., a liposome) with a dense radially arranged oligonucleotide surface. SNA is due to its biocompatibility, 24 Ability to rapidly enter cells in large amounts, 25,26 Potent immune activation when toll-like receptor 9 (TLR 9) agonist DNA is used as a shell 27 And the modularity of nanoscale placement that enables the use of well-known chemically defined components is a powerful tool to explore these complex relationships. 28-30 In this example, it was demonstrated how the structure of SNA vaccines carrying multiple immune cell targeting antigens greatly influences immune activation. Altering the location of antigen types within SNAs regulates immune cell pathways at the transcriptome level, enhances the production and secretion of cytokines and memory markers at the cellular level, and slows tumor growth at the biological level. Overall, these changes define vaccine efficacy against invasive B16-F10 melanoma tumor models and importantly elucidate the multiple antigen placement for conversion to other therapeutic agentsDesign insights of the device.
Results and discussion
The experiments described in this example aim to determine the optimal antigen processing conditions for a multi-antigen SNA vaccine to produce potent cytotoxicity and helper T cell responses. In particular, it was investigated how delivery of two antigen classes (MHC-I and-II restricted) to Dendritic Cells (DCs) would alter in vitro processing. DCs are critical professional antigen presenting cells that induce signaling for effective T cell sensitization. Previous literature has demonstrated the potential to enhance DC activation by delivering both cytotoxic and helper antigens simultaneously, 31,32 None of the literature has a platform that can understand the best way to present such antigens. 14 It is assumed that delivering both antigen classes simultaneously on the same nanoparticle, rather than on different nanoparticles, enhances activation of both T cell types and the structural location of the antigen significantly affects vaccine performance.
Processing of multiple antigens in vitro based on antigen distribution on SNA. To test this theory, a double antigen SNA vaccine (DA-SNA) was designed and synthesized that contained MHC-I and-II restricted antigens in different nanoscale positions (based on placement of each antigen, referred to as DA-SNA 1 and DA-SNA2 (see, e.g., fig. 1, 4, and 26A)). Due to the modularity of SNAs, there are a number of different locations within SNA constructs where antigens can be placed. For this work, an encapsulation and hybridization arrangement was selected for antigen placement and compared to each other. To assess the distribution of antigens and how delivery on different nanoparticles affected immune activation, formulations containing two separate SNAs, each presenting only one antigen class in the same location as the DA-SNA vaccine, were synthesized (fig. 27). For single SNA delivering a single antigen, the formulation is referred to as "single" and for DA-SNA containing a double antigen, it is referred to as "combination".
For the synthesis of DA-SNA, peptides from one antigen class were encapsulated into 50nm 1, 2-dioleoyl-sn-glycerol-3-phosphorylcholine (DOPC) liposomes during their formation (FIGS. 28 and 29). In parallel, another is formed using disulfide bond formationPeptides of the antigen class were conjugated to a strand complementary to the CpG motif adjuvant DNA shell of SNA ("CpG complement") (fig. 30). The DNA and peptide sequences used in this example can be found in tables 1 and 2. Hybrid duplex formation is achieved by slow cooling of the CpG complement with antigen attached to the complementary 3' -cholesterol-terminated CpG strand. Cholesterol anchors the duplex to the surface of the liposome. The products of this hybridization are added to liposomes to obtain equimolar amounts of each antigen. The liposome surface was backfilled with antigen-free non-targeting DNA to obtain 75 total DNA strands per liposome, which corresponds to 1.6 picomoles/cm 2 At which SNA-related properties making it useful for biology are observed (see fig. 2, 3 and 10). 16 SNAs containing a single encapsulated antigen or a single hybridized antigen were synthesized following previous protocols ("individual" formulations). 16 SNA formation was confirmed using dynamic light scattering (fig. 31).
Table 1: the sequence of the DNA used in this example.
1 Calculation using an OligoAnalyzer tool of IDT (https:// www.idtdna.com/calc/Analyzer (Integrated DNA technology OligoAnalyzer, 2021))
( PO = phosphate backbone; PS = phosphorothioate backbone; sp18=18-O-dimethoxytrityl hexaethylene glycol, 1- [ (2-cyanoethyl) - (N, N-diisopropyl) ] -phosphoramidite (Glen Research) No. 10-1918; cholesterol=3' -cholesteryl-TEG CPG (galen research company No. 20-2975); thiol=3' -thiol-modifier C3S-S CPG (galen research company No. 20-2933) )
Table 2: the sequences of the peptides used in this example.
After synthesis of these different structures, MHC of Ovalbumin (OVA) was usedModel antigens restricted by-I and-II (called OVA1 and OVA2, respectively) were evaluated for activation in DCs. Murine bone marrow derived DCs and delivered antigens were used as two separate SNA constructs with a combined DA-SNA vaccine to characterize the activation of DC cues and the ability to sensitize T cells. Specifically, CD11c, which expresses the innate co-stimulatory markers CD86 and CD80 + The percentage of DC did not change significantly due to structural arrangement (fig. 26B). This may be due to the fact that delivery of the adjuvant is equivalent in the combined formulation and the separate formulation and indicates that hybridization with CpG does not interfere with TLR9 activation. In addition, antigen is presented to naive spleen T cells to boost antigen-specific T cells (which are indicators of the ability of the immune system to recognize tumor antigens) 33 Is not affected by the way the antigen is arranged; naive T cells were able to clonally expand into OVA1 and OVA2 specific T cells (fig. 26C); gating strategy in fig. 32. In fact, approximately 0.6-0.7% of the population of live CD 19-pairs OVA1-H-2kb dimer and CD8 + The marker is double positive. Similar trends were observed for OVA 2-specific T cell differentiation, with approximately 0.9-1.1% containing OVA2-H-2-Iad tetramer and CD4 + Double positive markers for antibodies. Only DA-SNA structures, rather than formulations alone, were able to significantly raise the amount of antigen-specific T cells to an amount above the T cell control baseline; this suggests that the delivery of both antigens to the DCs in combination results in greater T cell differentiation. This is even more evident when assessing the expression of the early activation marker CD69 in any antigen-specific T cell population. When the antigen was delivered in combination as one DA-SNA, the amount of CD69 signal increased (as measured by Median Fluorescence Intensity (MFI)), with DA-SNA 2 structure being better than all groups tested (fig. 26D). Furthermore, the use of OT1 splenocytes specific for OVA1 antigen, delivered as DA-SNA structure, translated into a two-fold increase in T cell proliferation, an important step in anti-tumor response, regardless of antigen placement (fig. 26E, fig. 13). Based on these findings, DA-SNA was used in all subsequent experiments, rather than SNA formulation alone.
In vivo activation by DA-SNA. Based on these promising in vitro findings using DA-SNA, C57BL/6 mice were kept in vivoImmunization to describe how differences in placement of MHC-I and-II restricted antigens within a DA-SNA vaccine affect immune activation. Mice were given a total of three injections (6 nmol per DNA and each peptide; FIG. 33A). On day 35, spleen cells were collected to evaluate the elevated specific immune response against both peptide antigens. Five weeks later, DA-SNA2 immunized CD8 when compared to using a simple mixture containing peptide antigen and adjuvant DNA (referred to as a "mixture", FIG. 33B) + The level is significantly elevated. CD4 + The levels did not vary significantly between treatment groups, but a reduction of approximately 8% was observed due to DA-SNA2 immune helper T cells (fig. 33B). DA-SNA2 is the only vaccine that is able to significantly increase the production of the key pro-inflammatory cytokine IFN-gamma as well as the degranulation marker CD107a after ex vivo restimulation with OVA1 peptide. DA-SNA2 immunization also produced a greater percentage of multifunctional spleen CD8 + T cells (approximately 17%, fig. 33C, fig. 5, fig. 20). Furthermore, this is associated with effector memory CD8 + The percentage increase of T cells correlated (CD 44 + CD62L-, about 48%) (fig. 33D, fig. 6, fig. 17). CD4 with OVA2 peptide compared to the mixture treatment + Ex vivo stimulation by T cells showed an overall increase in these same parameters for both DA-SNAs, further demonstrating the importance of delivering both antigens in combination with an adjuvant to immune cells; no significant differences were observed between the two DA-SNA constructs (fig. 33C-D, fig. 5, fig. 6). When IFN- γ secretion was assessed by enzyme-linked immunosorbent spot (ELISPot) assay, an increase in Spot Forming Cells (SFC) of both DA-SNA structures was observed compared to the spot forming cells of the mixture, and the greatest enhancement was seen for DA-SNA2 (FIGS. 33E, 18 and 19). Furthermore, when stimulated ex vivo with OVA1 or OVA2, splenocytes immunized with DA-SNA2 produced at least twice as many SFCs as DA-SNA 1, demonstrating the efficacy of this arrangement of antigens on DA-SNA to respond ex vivo to MHC-I or-II restricted antigenic cues. Taken together, these results underscore that placement of MHC-I restricted antigens in the hybridized architecture optimizes presentation of DC processing to effectively activate CD8 + T cell response with simultaneous encapsulation of MHC-II restricted antigens in the nucleus to induce CD4 + Moderate enhancement of Activity, asWhile retaining the cytotoxic function.
The mechanism of DA-SNA-induced immune activation and transmission is understood by RNA sequencing. To understand how different DA-SNAs and mixture treatments induce such different immune responses in vivo, spleen CD8 was post-immunization + And CD4 + T cells were collected and isolated and batch RNA sequencing (RNAseq) was performed. Principal Component Analysis (PCA) revealed overall CD8 in mice immunized with the mixture formulation + And CD4 + The T cell gene expression profile was most similar to that from naive mice, suggesting that this is responsible for the low overall activation (fig. 34A). The DA-SNA2 immunized mice were most different from the naive mice in their CD8+ transcriptome, while DA-SNA1 and 2 were most different from the naive mice in their CD4 + The gene expression profile in T cells was similarly different (fig. 14). This suggests that DA-SNA2 induced CD8 + T cell function was significantly increased, but CD4 between the two DA-SNAs was observed in FIG. 33 + The rationale for the similarity of T cell function levels. Furthermore, differentially regulated genes of DA-SNA2 immunized mice exhibited greater absolute Log Fold Changes (LFCs) in both T cell types than other treatments, with the number of differentially regulated genes due to DA-SNA2 immunization at least doubled compared to the number of differentially regulated genes of DA-SNA1 (fig. 34B). Differentially regulated genes were enriched in pathways involved in inflammatory responses and pro-inflammatory cytokine upregulation, chemotaxis, and migration of key immune cell populations (fig. 34C, fig. 14, fig. 15, and fig. 16). While some of the enriched pathways from the DA-SNA2 treatment are common to the mixture treatment and others are common to the DA-SNA1 treatment, overall, extensive activation of DA-SNA2 architecture induction at the transcriptome level is associated with enhanced immune output.
Relevant genetic features were identified for adaptive and innate immune activation and function across all treatments and included, for example, CXCR3, TNFSF9, and GZMK (fig. 34D). These genes have particular relevance in T cell effector function and trafficking, antigen presentation, and production of cytotoxic T cells, and helper T cell cytolytic function, respectively.Specific comparisons of DA-SNA 2 relative to DA-SNA 1 showed unique nanoscale-induced genetic differences that were induced simply by altering placement of antigen classes (FIG. 34E). CD8 between two DA-SNAs + And CD4 + A total of 452 and 229 overlapping significant genes were detected in T cells, respectively. Specifically, DA-SNA 2 induces IL2RA, CD44, XCL1 at CD8 compared to DA-SNA 1 + In T cells and LAG3, CCR7, CCL9 in CD4 + Higher expression in T cells. Finally, comparison of the genetic signature across all immunization treatments highlights the substantial impact of vaccine structure and in particular nanoscale antigen placement on genome and expression pattern. These results emphasize the detected immunological measurements, providing a mechanism to emphasize the pathways leading to T cell activation and sustained responses, and details the framework for vaccine design using the structural considerations of interest.
DA-SNA structure driven tumor suppression and immune activation. To assess the therapeutic efficacy and immunological impact of DA-SNA, a murine e.g7-OVA lymphoma cancer model was used, as it stably expressed OVA protein, thereby expressing the OVA1 and OVA2 epitopes used above. 34 Briefly, C57BL/6 mice were subcutaneously vaccinated with E.G7-OVA cells and immunized weekly with DA-SNA or a mixture formulation (6 nmol of each of OVA1 antigen and OVA2 antigen, 6nmol of adjuvant DNA) (FIG. 35A). Tumor-bearing mice immunized with DA-SNA 2 showed approximately 3-fold reduction in tumor growth compared to the control group (saline-treated) and the mixture group five days after the second immunization (day 15), and showed a difference in tumor growth of more than 16-fold when compared to saline-treated mice 22 days after tumor inoculation (fig. 35B, fig. 9, and fig. 36). Importantly, the DA-SNA 1 treatment did not effectively prevent tumor growth compared to the mixture, unlike the DA-SNA 2 treatment. The reduction of tumor growth by approximately 7-fold on day 24 compared to the mixture and DA-SNA 1, highlights the significant impact of antigen localization and the resultant conversion to a significant prolongation of animal survival (median survival in days: pbs=27; mixture=24; DA-sna1=28; DA-sna2=35) (fig. 35C, fig. 11). To further investigate the physical effect of treatment on tumor growth, the following was followed on day 15 The same treatment protocol resected the tumor from the mice and the tumor was subsequently weighed (fig. 35D and 37). Interestingly, at this time, both SNA groups showed a significant decrease in tumor weight when compared to PBS-treated mice in the tumor growth curve, indicating that DA-SNA1 was able to boost the anti-tumor immune response, but not as long lasting as the immune response generated by DA-SNA 2.
Spleens were collected and evaluated for CD8 + And CD4 + T cell changes to elucidate differences due to treatments that might contribute to tumor reduction (fig. 35E, fig. 12). Spleen-produced CD8 from DA-SNA2 treated mice when compared to other treatment groups + The percentage of T cells is significantly higher and also shows CD8 + T cells and CD4 + The ratio of T cells is also generally higher. To assess the immunological differences contributing to tumor suppression, circulating Peripheral Blood Mononuclear Cells (PBMCs) of tumor-bearing C57BL/6 mice were assessed on day 15, at which time a difference in tumor growth was first observed, and at which time the effects of DA-SNA2 treatment began to block tumor growth, while the effects of other treatments were negligible. Notably, DA-SNA2 treated mice exhibited circulating antigen-specific CD8 + The highest level of T cells (fig. 35F, fig. 7, fig. 21). Further evaluation by CD8 + A memory phenotype of a subpopulation of lymphocytes. In this case, DA-SNA2 treatment significantly increased the effector memory phenotype to OVA 1-specific circulating CD8 + More than 60% of T cells (fig. 35G, 8, 22). Antigen-specific CD4 for DA-SNA treated mice + T cells were also significantly elevated (fig. 35H, fig. 7, fig. 21). As expected, since previously herein was directed to CD4 + Transcriptome profile and immunological parameters of T cell exploration, the differences between the two DA-SNA groups were negligible. Although there is not enough OVA 2-specific CD4 + T cells accurately describe the memory phenotype in this sub-population, but when treated with DA-SNA1, the entire CD4, compared to treatment with DA-SNA2 + T cells exhibit enhanced effector memory status (approximately 30% CD4 + T cells), which treatment with DA-SNA2 resulted in approximately 10% CD4 + T cell maturation (fig. 35I, fig. 8).
Structural influence of antigen placement in clinically relevant melanoma models. Findings in the model system to date on DA-SNA structure were used to assess the ability of dual antigen placement to affect B16-F10 melanoma tumor growth. This model has been established to have highly invasive and enhanced immunosuppressive properties. Select and select 35 Recently reported 36 The M27 and M30 neoepitopes from the MART-1 protein containing mutations present only in tumors served as MHC-I and-II antigens, respectively. Initially, weekly vaccinated C57BL/6 mice with DA-SNA1 or DA-SNA2 vaccinated with B16-F10 tumor cells showed a significant percentage of inhibition of tumor growth on day 17 (68% and 48%, respectively) compared to saline treated mice (fig. 38A-B, fig. 23). Indeed, circulating effector memory antigen-specific CD8 was observed in mice treated with either DA-SNA relative to saline treatment + A statistically significant approximately 4-fold increase in T cells (fig. 38C). Thus, an antigen-specific immune response is generated, but the negligible effect of this antigen-specific immune response on tumor growth indicates that there may be a highly immunosuppressive tumor environment that inhibits the therapeutic potential of these T cells.
Because of these observations and the inherent aggressiveness of this tumor model, DA-SNA treatment was combined with immune checkpoint inhibitor anti-PD-1 (which is an FDA approved treatment for advanced melanoma) to overcome immunosuppression of tumors. 37,38 Notably, when anti-PD-1 was administered in combination with DA-SNA, a significant decrease in tumor growth was observed for animals treated with the combination DA-sna2+ anti-PD-1 therapy, starting 17 days as early as after tumor inoculation, while no significant decrease in tumor growth was observed for mice of the other treatment groups (fig. 38D). See fig. 24, 25 and 39. This translates into a 40% increase in median survival of these mice compared to saline-treated or anti-PD-1 monotherapy-treated groups (fig. 38E). Furthermore, comparison between the DA-SNA combination treatment groups revealed a significant increase in overall median survival of the combined DA-sna2+ anti-PD-1 animals (p= 0.0507). For separating from peripheral blood Evaluation of isolated immune cells further highlights this significant structure-induced difference, where DA-SNA2 appears to act synergistically with checkpoint inhibitors. Circulating CD8 was observed in animals treated with the combination DA-sna2+ anti-PD-1 compared to all other groups + T cells increased significantly (fig. 38F). Interestingly, when assessing total CD8 in these circulating PBMCs + When the population of effector memory T cells, a significant increase was detected for the two combined DA-sna+anti-PD-1 groups, with DA-sna2+anti-PD-1 at approximately 60% of circulating CD8 + Effector memory phenotypes were generated in T cells (fig. 38G). Furthermore, only this combined DA-sna2+ anti-PD-1 treatment was able to induce potent antigen-specific CD8 + T cell response (fig. 38H). For CD4 + Observations of T cell circulation revealed a similarly high increase due to combined DA-sna2+ anti-PD-1 therapy (fig. 38I), but both combined DA-sna+ anti-PD-1 groups significantly increased the effector memory phenotype to CD4 + Approximately 28% of the T cell population (fig. 38J). The combination of DA-sna2+ anti-PD-1 treatment with anti-PD-1 monotherapy increased antigen-specific CD4 compared to DA-sna1+ anti-PD-1 treatment + Levels of T cell production, but no significant differences were observed between the groups (fig. 38K).
Conclusion(s)
While extensive research has generally explored the importance of adjuvants and antigens in the generation of powerful new immunotherapies, to date, no research institution has explored the importance of the structural presentation of multiple antigens within a particular construct and their role in eliciting an effective and desirable immune response. Overall, the data presented herein show that antigen placement is as important as antigen selection in terms of vaccine efficacy. Indeed, in a specific embodiment, therapeutic benefit against tumors is significantly altered when the placement of MHC-I and-II restricted antigens in two compositionally nearly identical vaccines is altered; one vaccine is effective and the other is relatively ineffective. The sources of these differences may be due to antigen localization affecting the processing pathways they undergo in immune cells and their residence time in different cell compartments. By altering the processing pathways and these signaling kinetics, this affects the immune response generated at the genetic, cellular and organism level. The encapsulated MHC-II and hybridized MHC-I restricted antigens upregulate genes specific for inflammatory responses, chemotaxis and migration of critical immune cells that together affect immune cell activity. These structurally defined genetic differences are transformed into immune behavior under repeated in vivo immunization, and ultimately define tumor growth profiles for both models of e.g7-OVA lymphoma and clinically relevant B16-F10 melanoma mouse tumor systems. This is a key demonstration of the impact of vaccine antigen localization in multiple cellular processes.
This example demonstrates that the ability to optimize antigen presentation to match the desired signaling profile is critical to the production of a potent vaccine, where small vaccine changes in antigen placement significantly increase cell-cell communication, cross-talk, and cell synergy.
Materials and methods
Materials and animals: all reagents were purchased from commercial sources and used as received unless otherwise indicated. Oligonucleotides were synthesized as follows. Peptides were purchased from gold srey corporation (GenScript) or from the university of Northwestern peptide synthesis center (Northwestern s Peptide Synthesis core). Chemicals were purchased from suppliers listed in brackets. C57BL/6 mice and C57BL/6-Tg (TcraTcrb) 1100Mjb/J (OT-1, 003831) female mice were purchased from Jackson laboratories (Jackson Laboratory) at an age of 8-12 weeks. Mice were used according to guidelines and regulations in all countries and places, and the protocol performed was approved by the university of northwest institutional animal use committee (institutional animal use committee at Northwestern University, IUCAC). E.G7-OVA and B16-F10 cells were purchased from ATCC. Antibodies were purchased and cloned as provided in table 3.
Table 3: antibody charts of antibodies used in this example.
Oligonucleotide synthesis and purification: oligonucleotide Using Standard phosphoramidite on ABI 394 synthesizerSynthesis was performed using phosphate or phosphorothioate backbones as indicated (table 1). After synthesis, the chain was deprotected using a 1:1 solution of 37% ammonium hydroxide/40% methylamine at 55 ℃ for 35 minutes, but with the exception of the dye, where it was deprotected using 37% ammonium hydroxide at Room Temperature (RT) overnight. The chains were then purified on reverse phase HPLC using C18 or C4 (for chains containing dyes or cholesterol) columns and peaks were collected as fractions. Dimethoxytrityl (DMT) was removed from the product chain by: incubation was performed for 1 hour in 20% aqueous acetic acid at RT, followed by three washes with ethyl acetate to remove DMT. The final product was lyophilized and resuspended in deionized water (di h 2 O). The concentration was measured using UV-vis absorption at 260nm, with the extinction coefficient calculated by IDT OligoAnalyzer on-line tool (listed in table 1).
Oligonucleotide-peptide conjugate synthesis and purification: will be di H 2 The thiol-functionalized oligonucleotides in O are reduced to produce free thiols for future reactions. Reduction was performed using dithiothreitol (100 mM, DTT, sigma) dissolved in Phosphate Buffered Saline (PBS) at pH 8.5 at RT for 45 min at a final concentration of 100 mM. The solution was treated with diH in a 3kDa molecular weight cut-off (MWCO) spin filter (Amicon) 2 O was washed at least three times. For the OVA peptide conjugate, the peptide was purchased as a resin and washed three times with Dimethylformamide (DMF) and acetone, then 5 μmol was reacted overnight at RT with a solution of ethyl 2- (2-pyridyldithio) succinimidyl carbonate (SDEC, prepared using previous scheme 39) in DMF (10 equivalents relative to initial peptide loading on solid support) and N, N-diisopropylethylamine (5 equivalents). The beads were then washed three times with DMF and acetone each and dried in air, followed by deprotection with 95% trifluoroacetic acid (2.5% triisopropylsilane, 2.5% di h2 o) for 1 hour at RT. TFA was purged with nitrogen and the beads were redissolved in DMF and filtered through glass wool. The peptide product was precipitated by addition of about 5-6 times diethyl ether and maintained at-20 ℃ for 1-2 hours for further precipitation. Centrifuging the solution (2,000Xg, 3 minutes) to pellet the peptide, and granulating the peptideCollected, dried and dissolved in DMF. The reduced DNA (0.5. Mu. Mol) was reacted with peptide (5. Mu. Mol) dissolved in 70-75% DMF in water at RT overnight, the total volume of reaction being about 1.5mL. For MART-1 peptide conjugates, the peptide was activated for 30 min at RT with gentle stirring using 2,2' -dithiodipyridine (150. Mu. Mol) dissolved in 10 equivalents DMF. The activated peptide was then washed three times in diethyl ether, pelleted by centrifugation (2,000Xg, 3 min) and allowed to dry. The reduced DNA (0.3. Mu. Mol) was reacted with peptide (1.5. Mu. Mol) dissolved in about 70% DMF in water overnight at RT, the total volume of the reaction being about 1.5mL.
After peptide conjugation, the solution was centrifuged at 17,000Xg for 2 minutes to pellet any broken peptide, and the supernatant was transferred to a 3kDa MWCO spin filter for use with diH 2 O is washed 3-4 times. Concentrating the volume to<500. Mu.L, and the solution was purified by preparative scale denaturation (8M urea) on a 15% PAGE gel (no more than 0.5. Mu. Mol based on DNA loaded onto a single gel). The gel was run at 175V for 30 minutes, then at 350V for about 3 hours, and then imaged using a UV lamp to cut the desired strips. The excised gel bands were crushed and the product was collected by washing three times with 1x Tris/boric acid/EDTA (TBE) buffer every about 4 hours. The product mass was confirmed by electrospray ionization mass spectrometry (ESI-MS) and the concentration was measured by UV-vis at 260nm assuming the extinction coefficient of DNA.
SNA synthesis: SNA was synthesized with minor modifications as reported previously. 16,40 Briefly, 50mg of 1, 2-dioleoyl-sn-glycerol-3-phosphorylcholine (DOPC, angstrom Wen Di polar lipid company (Avanti Polar Lipids)) was hydrated with 3-4mL of PBS or peptide-containing PBS for use in encapsulated liposomes. (Note: the peptide-containing solution contained OVA1:1mg/mL, dissolved in PBS containing approximately 100. Mu.L of 1M NaOH; OVA2:1mg/mL, dissolved in PBS containing approximately 60. Mu.L of 1M NaOH; M27:2.25mg/mL, dissolved in PBS containing approximately 60. Mu.L of 1M NaOH; M30:1mg/mL, dissolved in PBS containing approximately 100. Mu.L of 1M NaOH). The solution was freeze-thawed 20 times in liquid nitrogen and then sonicated at 37-40 ℃. Using aperture Polycarbonate filters of 200nm, 100nm, 80nm and 50nm, liposomes were extruded using continuous high pressure extrusion (northern lipid company (Northern Lipids Inc))); liposomes were passed through each pore size three times. After extrusion, liposomes were concentrated to approximately 2-3mL using a 100kDa MWCO spin filter and dialyzed against 3.5L PBS overnight to remove unencapsulated peptides. Liposome concentration was determined using a Phosphatidylcholine (PC) assay kit (sigma, MAK049-1 KT) assuming 50-nm liposomes contained 18,140 lipids per liposome. 24 In the case of liposome encapsulated peptides, the peptide concentration was determined as follows: using Pierce TM Fluorescence assay kit (sameiser's science and technology (thermo fisher), 23290), 1% Sodium Dodecyl Sulfate (SDS) was added to rupture the liposomes and release the peptide for quantification, and peptide dissolved in 1% SDS was used as a standard curve. The peptide loading in each liposome was calculated by dividing the peptide concentration by the liposome concentration. The amount of each antigen in each particle is in the range of about 25-40, depending on the encapsulation yield per batch, which is adjusted using different starting concentrations.
Purified oligonucleotide-peptide conjugates were mixed with complementary 3' -cholesterol-capped CpG DNA at a 1:1 molar ratio and treated with Centrivap overnight. The next day, approximately 20-40 μl duplex buffer (IDT) was added, and the solution was slowly cooled to duplex the strands according to the following procedure: the temperature is 70 ℃ for 10 minutes, 23 ℃ for 1.5 hours, and 4 ℃ for more than or equal to 1 hour. The duplex is added to the solution of the synthesized liposome in an amount equimolar to the peptide encapsulated within the liposome. To obtain up to 75 strands per liposome, the remaining space was filled with 3' cholesterol-capped T20 DNA. This mixture was incubated overnight at 37℃and then stored at 4 ℃.
Cell culture: all cells were maintained at 5% CO at 37 ℃ 2 In an incubator. E.G7-OVA cells and DCs were cultured with RPMI 1640 medium (Ji Boke company (Gibco), 11875093) (referred to herein as RPMI+/+) containing 10% heat-inactivated fetal bovine serum (HI-FBS) and 1% penicillin-streptomycin. DME containing 10% HI-FBS and 1% penicillin-streptomycin was usedB16-F10 was treated with M medium (Ji Boke Co., 11965092).
Bone marrow derived dendritic cell (BMDC) collection: bone marrow cells were collected from mice according to the previous protocol; 39 briefly, erythrocytes were lysed with 2-3mL of ACK lysis buffer (Ji Boke, A1049201) for about 4 min and plated at 10-cm with 40ng/mL GM-CSF (hundred Biol, 576304) prior to use 2 The cells were allowed to stand on the dish for 5-7 days to differentiate DCs from the population.
BMDC activation and cross-sensitization of T cells in vitro: from 10-cm 2 Cells were collected in cell culture dishes and DCs were isolated from the mixture using a magnetic biotin positive selection kit (stem cell technology, inc (Stemcell Technologies), 17665). Using CD11c + The biotin-labeled antibodies were used to select DCs (bai biosystems) and after isolation, purified DCs were counted using a Vi-CELL BLU CELL viability analyzer. For DC activation, 6X 10 cultures were grown with SNA treatment at a final volume of 200. Mu.L 4 And DC. After 22 hours in the incubator, the cells were washed with PBS to end the treatment, stained with 0.5. Mu.L of each antibody (L/D, CD11c, CD86 and CD 80) per tube at 4℃for 15 minutes, washed with PBS, and fixed with 100. Mu.L of fixation buffer (BAOCHINE, 420801). To assess T cell specificity and activation, 1.6x10 was used 5 The individual purified DCs were pulsed with SNA in an incubator for 30 minutes at a final volume of 200. Mu.L. After 30 min pulse, cells were washed twice with rpmi+/+ to remove any residual SNA from the cell solution, and cells were resuspended in 500 μl rpmi+/+. Meanwhile, spleen cells were isolated from the naive mice. After spleen dissociation and erythrocyte lysis, cells were counted and resuspended to 3×10 in warmed rpmi+/+ 6 A concentration of individual cells/mL, and 100 μl of this cell solution was transferred to each well in a 96-well round bottom plate. To each well 100 μl of treated DC (3.3x10 4 Individual cells) such that the ratio of DC to splenocytes is 1:9. Cells were co-cultured for three days in an incubator, after which the cells were washed with PBS and directed against DimerX mouse H-2kb:ig fusion protein (bidi, 552944) or according to the manufacturer's instructionsOVA2 tetramer (Proimmune Co.). In addition to the peptide-specific TCR markers, the stained antibodies comprise L/D, CD or CD4, CD19 and CD69. After staining, the cells were fixed with 100 μl of fixation buffer. To assess T cell proliferation, 2.6x10 cells were taken 5 The individual purified DCs were pulsed with SNA in an incubator for 30 minutes at a final volume of 200. Mu.L. After 30 min pulse, cells were washed twice with rpmi+/+ to remove any residual SNA from the cell solution, and cells were resuspended in 266.6 μl rpmi+/+. Meanwhile, spleen cells were isolated from C57BL/6-Tg (TcraTcrb) 1100Mjb/J (OT-1) mice (Jackson laboratories, 003831). After spleen dissociation and erythrocyte lysis, cells were counted and resuspended to 4×10 in PBS 7 Concentration of individual cells/mL to proliferate the dye eFluor with cells TM 450 (eBioscience, inc. (eBioscience), 65-0842-85) was stained according to the manufacturer's instructions. After staining, cells were washed, counted, and resuspended to 3×10 in rpmi+/+ 6 Concentration of individual cells/mL; 100. Mu.L of this cell solution was transferred to each well in a 96-well round bottom plate. To each well 33.3 μl of treated DC (3.3x10 4 Individual cells) such that the ratio of DC to splenocytes was 1:9 and each well was brought to a final concentration of 200 μl with medium. Cells were co-cultured for three days in an incubator, after which the cells were washed with PBS, stained for CD8 (0.5 μl antibody per tube), washed, and immediately on a flow cytometer using eFluor TM Dilutions of 450 were analyzed as a measure of T cell proliferation. All samples were analyzed using a bi company A3 Symphony flow cytometer using data analyzed on FlowJo.
In vivo immunization to measure five weeks cumulative immune response: female C57BL/6 mice were immunized subcutaneously three times per two weeks with different treatments. The treatment comprises the following steps: simple mixtures (mixture, 6nmol of each peptide and 6nmol CpG 1826DNA) or any DA-SNA (1826, 6nmol per OVA peptide and CpG). The injected treatment volume was kept at 100. Mu.L. One week after the final immunization, mice were sacrificed and spleens were harvested for subsequent immune assessment.
And (3) acquisition procedure: the removed spleens were collected and temporarily held in place3-5mL RPMI+/+ until all spleens are collected, then the spleens are passed through a 70 μm cell strainer with constant PBS flow. The cells were centrifuged at 1,200rpm for 5 min, after which the supernatant was removed and the cells were resuspended in 2-3mL ACK lysis buffer (Ji Boke company, a 1049201) for 4 min. To dilute the lysis buffer, PBS was then added to a final volume of 30mL and the cells were counted, followed by centrifugation to 1X 10 8 Individual cells mL -1 Is resuspended in RPMI+/+ medium.
IFN-gamma cytokine production: t cells were re-stimulated ex vivo to assess antigen-specific intracellular IFN- γ production. Will be 4×10 6 Individual spleen cells were incubated at 37℃with 5% CO 2 The incubator was incubated with 450. Mu.L of RPMI+/+ medium containing: OVA1 peptide or OVA2 peptide (10. Mu.g/mL), monensin (2. Mu.M), brefeldin A (5. Mu.g/mL), and CD107a antibody (0.5. Mu.L). After 4 hours incubation, the cells were centrifuged at 1,200rpm for 5 minutes, aspirated and washed with 600. Mu.L of PBS, followed by staining with surface antibodies (0.5. Mu.L each of samples: L/D, CD, CD 4) for 15 minutes at 4 ℃. Cells were washed with 600 μl PBS, centrifuged at 1,200rpm for 5 min, aspirated, and resuspended in 100 μl Cytofix fixation and permeabilization solution (bidi, 554722) at 4 ℃ for 20 min. After that, the cells were washed with 600. Mu.L of Perm/Wash Buffer (Bidi Co., 554723), centrifuged at 1,200rpm for 5 minutes, aspirated, and resuspended in 100. Mu.L of Perm/Wash Buffer containing the intracellular antibody IFN-. Gamma.0.5. Mu.L of each sample. Samples were stored at 4 ℃ prior to flow cytometry analysis.
T cell memory phenotyping: t cells were evaluated for effector memory phenotype. Will be 3X 10 6 Individual spleen cells were washed with 600. Mu.L of PBS and stained with surface antibodies (0.5. Mu.L each of each sample: L/D, CD8, CD4, CD44 and CD 62L) for 15 minutes at 4 ℃. Cells were washed with 600 μl PBS, centrifuged at 1,200rpm for 5 min, aspirated and resuspended in 100 μl of fixation buffer (hundred forward biosystems, 420801) and stored at 4 ℃ prior to flow cytometry analysis.
ELISpot assay: using commercially available mouse INF-. Gamma.ELISPOThe T-kit (bi di company, 551083) was subjected to ELISpot analysis according to the manufacturer's instructions. Briefly, the provided clean plates were coated with capture antibody overnight at 4 ℃. Thereafter, the plates were washed with rpmi+/+ medium and then blocked with 200 μl rpmi+/+ medium for 2 hours at room temperature. Removal of the blocking buffer by pipetting, taking care not to dry the wells, and rapid application of the solution containing 2X 10 5 100. Mu.L of RPMI+/+ from each spleen cell was replaced. An additional 100. Mu.L of antigen, non-specific peptide, medium (negative control) or positive control solution (antigen and non-specific peptide added to a final concentration of 5. Mu.g/mL; positive control was prepared as a mixture of anti-CD 3 antibody and anti-CD 28 antibody at a final concentration of 2. Mu.g/mL for each antibody) was added to each well. The solution was kept at 37℃in an incubator with 5% CO2 for 48 hours. After this incubation, the plates were washed and detection antibodies, enzyme conjugates and chromogenic substrates were added according to the manufacturer's instructions. The dried plates were imaged and analyzed using a CTL immunoblotter imager.
Sequencing of RNA in batches: isolation of CD4 from Whole splenocytes from separate treatment groups using a magnetic positive selection kit (Stem cell technology Co., ltd., 18952 and 18953) + And CD8 + T cells. From these isolated cell populations, use is made ofThe Plus miniprep kit (Qiagen) was combined with QIAshredder (Qiagen) for RNA extraction according to the manufacturer's instructions. RNA concentrations were quantified using NanoDrop 8000 (sameire femoro technologies) and RNA samples were stored at-80 ℃ prior to further use. Sequencing was performed at NUSeq core facility at northwest university. Briefly, the quality of the total RNA sample was checked using the RNA integrity value (RIN) generated by the agilent biological analyzer 2100 (Agilent Bioanalyzer 2100). RNA amounts were determined using a Qubit fluorometer. Kit (Illumina TruSeq Stranded mRNA Library Preparation Kit) was prepared from a TruSeq chain mRNA library from Emamna to obtain a high quality RNA sample (RIN) of 125ng>7) Preparing a sequencing library. The kit procedure, comprising mRNA purification and fragmentation, cDNA synthesis, and,3' -end adenylation, because of Mener company adapter ligation, library PCR amplification and verification. The library was sequenced using a HiSeq 4000 sequencer from Emamna, resulting in a single 50bp read at a depth of 2000-2500 ten thousand reads per sample. The quality of reads in FastQC format was evaluated using FastQC. Reads were trimmed using cutadapt to remove the because Mena adaptors from the 3' end. 41 The trimmed reads were aligned with the mouse (Mus musculus) genome (mm 10) using STAR. 42 Using htseq-count 43 The read count for each gene was calculated in combination with the mm10 gene annotation file obtained from Ensembl (http:// useast. Ensembl. Org/index. Html). Normalized and differential expression was calculated using DESeq2 with the Wald test. 44 The cut-off value used to determine significantly differentially expressed genes was an FDR-adjusted p-value of less than 0.05 using the Benjamini-Huo Jiba grid method (Benjamini-Hochberg method).
Gene Set Enrichment Analysis (GSEA): GSEA45 was performed to see if differentially expressed genes were associated with differentially enriched pathways. Based on log obtained from DeSeq2 analysis 10 The converted nominal P-value, genes detected using RNA sequencing were ranked and compared to naive T-cells. Pathway enrichment analysis was performed using GSEA software (version 4.0.3) and following the protocol of reiand et al. 46 The gene set is obtained from a molecular characterization database and comprises reactiomes, KEGG. The ordered list was remapped using the CHIP platform from the sub-feature database, which uses the mouse gene symbols to remap to human orthologs (version 7.1). If FDR of a term <0.05, the term is defined as differentially enriched. A subset of the strongly enriched pathways were selected for visualization in R using the pheeatmap package (version 1.0.12). This option includes FDR<0.05, and with CD8 + And CD4 + The immune response in T cells has a correlation.
Gene expression profile: selecting e.g. by FDR p-value<Genes whose expression was significantly altered in both SNA treatment groups as defined in 0.05 were used for visualization in the form of a heat map. Conversion of Gene expression scoring in FPKM to Cross-processingZ-scores for the groups and the gene expression values were clustered using K-means clustering. Combining the two conditions of interest in pairs to obtain the primary CD4 + Or CD8 + T cells were set as controls. Using volcanic diagrams to map e.g. pass FDR p values<0.05 and log2 fold change>0.5 (Up-Regulation) or log 2 Multiple of change<0.05 (Down) defined gene visualizations that were up-or down-regulated between groups. In vivo efficacy study: female C57BL/6 mice of 8-12 weeks of age were obtained from Jackson laboratories. By subcutaneous (s.c.) injection 5×10 in the right side animals 5 E.G7-OVA or 10 5 Individual B16-F10 cells were tumor seeded. Immunization was administered by s.c. injection in the abdomen at doses of 6nmol (OVA 1/2) or 9nmol (M27/30) of each antigen and CpG. Immunization was administered by s.c. injection at the abdomen as listed in the treatment schedule provided in the corresponding figures. For combination therapy with immune checkpoint inhibitor anti-PD-1, 100 μg of anti-mouse PD-1 was administered to mice by intraperitoneal injection (clone RMP1-14, bioXcell inc (BioXcell)). Tumor growth was measured on a predetermined day and the volume was calculated using the following equation: tumor volume = length x width 2 X 0.5. When the tumor volume reaches 2,000mm 3 (E.G7-OVA) or 1,500mm 3 (B16-F10) euthanasia was performed on the animals at the time of or when the animals were moribund.
Immune activation of PBMCs: for the collection of Peripheral Blood Mononuclear Cells (PBMCs), animals were vaccinated with cancer cells as described above. The treatments were performed according to the same schedule and animals were euthanized on either day 15 (E.G 7-OVA) or day 17 (B16-F10). Blood was collected into EDTA-lined collection tubes (bi di company) by cardiac puncture and simply mixed by inversion. Erythrocytes were lysed and washed using ACK lysis buffer (Ji Boke company) and the remaining cells were subsequently stained with antibodies to L/D, CD4, CD8, CD19, CD44 and CD62L using the methods described above.
Whole organ immune assessment: tumor weight and spleen cell assessment was performed on C57BL/6 mice bearing E.G7-OVA tumors on the right. Three days after tumor inoculation, a first immunization was administered followed by an additional dose after 7 days (day 10). Tumors and spleens were resected from animals on day 15 and subsequently analyzed. To produce a single cell spleen cell solution, the spleen was mechanically forced through a 70 μm cell strainer while remaining hydrated in PBS solution. The cells were then centrifuged at 1200rpm for 5 minutes. The pellet was then resuspended in ACK lysis buffer (sameidie technologies) for 4 minutes to lyse the erythrocytes, and then neutralized in PBS, followed by centrifugation. After centrifugation, cells were labeled with the following antibodies: CD4, CD8 and CD19.
Statistical analysis: statistics were calculated using GraphPad Prism 8 software and the specific statistical analysis used was highlighted in the corresponding pictorial illustration. Because of the lack of assumptions that can be made based on large differences in standard deviation between groups, comparisons between multiple groups employ one-way ANOVA withOr a atlas comparison test, or a Welch ANOVA with Dennity multiple comparison test. Statistics on animal survival were calculated using a log rank test. The outliers of fig. 35E-F and fig. 38H, J were identified using the ROUT method with Q set to 10% or 1%, respectively. For all cases, the p-values are plotted as follows: * P is p<0.05；**p<0.01；***p<0.001；****p<0.0001; n.s. indicates that significance was not determined, and n.d. indicates that no value was detected. The animals were assigned to each group, the immunization administered and the measurements for the tumor study were all performed blindly. The values in the figures are plotted as mean ± s.e.m., and this value as well as the sample size are indicated in the corresponding picture descriptions.
Example 3
This example describes experiments performed in a colon cancer model using SNA constructs according to the present disclosure.
Female C57BL/6 mice of 8-12 weeks of age were obtained from Jackson laboratories. By subcutaneous (s.c.) injection 5×10 in the right side animals 5 Tumor inoculation was performed on individual MC38 colon cancer cells. Immunization was administered by s.c. injection in the abdomen at a dose of each antigen and CpG 6nmol (Adpgk 1/2). Immunization by passing through abdomen as listed in the provided treatment scheduleThe sections were administered s.c. injection. Tumor growth was measured on a predetermined day and the volume was calculated using the following equation: tumor volume = length x width 2 X 0.5. When the tumor volume reaches 2,000mm 3 (MC 38) the animals are euthanized at the time of death or when the animals are moribund. See fig. 44.
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Claims (93)

1. A Spherical Nucleic Acid (SNA), comprising:
(a) A nanoparticle core;
(b) An oligonucleotide shell attached to an outer surface of the nanoparticle core, the oligonucleotide shell comprising one or more immunostimulatory oligonucleotides; and
(c) A first antigen that is a major histocompatibility complex type I (MHC-I) antigen and a second antigen that is a major histocompatibility complex type II (MHC-II) antigen.
2. The SNA of claim 1, wherein the first antigen is encapsulated in the nanoparticle core.
3. The SNA of claim 1 or claim 2, wherein the second antigen is linked to one or more oligonucleotides in the oligonucleotide shell through a linker.
4. The SNA of claim 3, wherein the second antigen is linked to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker.
5. The SNA of claim 3, wherein the second antigen is linked to an oligonucleotide that hybridizes to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker.
6. The SNA of claim 1 or claim 2, wherein the second antigen is attached to the outer surface of the nanoparticle core by a linker.
7. The SNA of claim 1 or claim 2, wherein the second antigen is encapsulated in the nanoparticle core.
8. The SNA of any one of claims 1 or 3-7 wherein the first antigen is linked to one or more oligonucleotides in the oligonucleotide shell through a linker.
9. The SNA of claim 8, wherein the first antigen is linked to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker.
10. The SNA of claim 8, wherein the first antigen is linked to an oligonucleotide that hybridizes to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker.
11. The SNA of any one of claims 1 or 3-7, wherein the first antigen is attached to an outer surface of the nanoparticle core via a linker.
12. The SNA according to any one of claims 1 to 11, comprising a third antigen which is a major histocompatibility complex type I (MHC-I) antigen.
13. The SNA according to any one of claims 1 to 12, comprising a fourth antigen which is a major histocompatibility complex type II (MHC-II) antigen.
14. The SNA of claim 12 or claim 13, wherein the third antigen is encapsulated in the nanoparticle core.
15. The SNA of claim 13 or claim 14 wherein the fourth antigen is linked to one or more oligonucleotides in the oligonucleotide shell by a linker.
16. The SNA of claim 15, wherein the fourth antigen is linked to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker.
17. The SNA of claim 15, wherein the fourth antigen is linked to an oligonucleotide that hybridizes to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker.
18. The SNA of claim 13 or claim 14, wherein the fourth antigen is attached to the outer surface of the nanoparticle core by a linker.
19. The SNA of any one of claims 12, 13 or 15-18, wherein the third antigen is linked to one or more oligonucleotides in the oligonucleotide shell through a linker.
20. The SNA of claim 19, wherein the third antigen is linked to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker.
21. The SNA of claim 19, wherein the third antigen is linked to an oligonucleotide that hybridizes to an oligonucleotide in the oligonucleotide shell that is linked to the nanoparticle core through the linker.
22. The SNA of any one of claims 12, 13 or 15-18, wherein the third antigen is attached to the outer surface of the nanoparticle core by a linker.
23. The SNA of any one of claims 13, 14 or 19-22, wherein the fourth antigen is encapsulated in the nanoparticle core.
24. The SNA of any one of claims 12-23, wherein the first antigen and the third antigen are the same.
25. The SNA of any one of claims 12-23, wherein the first antigen and the third antigen are different.
26. The SNA of any one of claims 13-25 wherein the second antigen and the fourth antigen are the same.
27. The SNA of any one of claims 13-25 wherein the second antigen and the fourth antigen are different.
28. The SNA of any one of claims 1-27, wherein the MHC-I antigen is OVA 257-264 (OVA 1) (SEQ ID NO: 7), GP100 (25-M27) (SEQ ID NO: 11), TC-1E6 (49-58) (SEQ ID NO: 12), TC-1E7 (49-57) (SEQ ID NO: 13), PSMA (634-642) (SEQ ID NO: 14), SPAS-1 (SNC 9-H8) (SEQ ID NO: 15), SIMS2 (237-245) (SEQ ID NO: 16), PAP (115-123) (SEQ ID NO: 17), B16 MART-1 (M27) (SEQ ID NO: 9), TRP-1 (252-260) (SEQ ID NO: 18), TRP-1 (252V 260M) (SEQ ID NO: 19), TRP-1 (455-463) (SEQ ID NO: 20), TRP-1 (455A M) (SEQ ID NO: 21), TRP-2 (180-188) (SEQ ID NO: 22), melan-A/MART- (127-135), kinase (1-9), PAP (369), adk (SEQ ID NO: 24), tyrosine (377-25-q) or a combination thereof。
29. The SNA of any one of claims 1-28, wherein the MHC-II antigen is OVA 323-339 (OVA 2) (SEQ ID NO: 8), GP100: (46-58) (SEQ ID NO: 26), TC-1E6 (43-57) (SEQ ID NO: 27), SIMS2 (240-254) (SEQ ID NO: 28), PAP (114-128) (SEQ ID NO: 29), B16 MART-1 (M30) (SEQ ID NO: 30), TRP-1 (113-127) (SEQ ID NO: 31), TRP-1 (106-130) (SEQ ID NO: 32), li-Key (77-92) (SEQ ID NO: 33), tyrosine kinase (56-70), GP100 (44-59), GP100 (167-189), melan-A/MART-1 (102-111) (SEQ ID NO: 34), melan-A/MART-1 (27-40) (SEQ ID NO: 35), melan-A/MART-1 (51-70) (SEQ ID NO: 36), melan-A/MART-1 (51-73) (SEQ ID NO: 37), melan-A/T-1 (43-57) (SEQ ID NO: 38), or a combination thereof.
30. The SNA of any one of claims 1-29, wherein at least one of the one or more immunostimulatory oligonucleotides is a toll-like receptor (TLR) agonist.
31. The SNA of any one of claims 1-30, wherein each of the one or more immunostimulatory oligonucleotides is a toll-like receptor (TLR) agonist.
32. The SNA according to claim 30 or claim 31 wherein the TLR is selected from the group consisting of: toll-like receptor 1 (TLR 1), toll-like receptor 2 (TLR 2), toll-like receptor 3 (TLR 3), toll-like receptor 4 (TLR 4), toll-like receptor 5 (TLR 5), toll-like receptor 6 (TLR 6), toll-like receptor 7 (TLR 7), toll-like receptor 8 (TLR 8), toll-like receptor 9 (TLR 9), toll-like receptor 10 (TLR 10), toll-like receptor 11 (TLR 11), toll-like receptor 12 (TLR 12) and toll-like receptor 13 (TLR 13).
33. The SNA of any one of claims 30-32 wherein the TLR is TLR9.
34. The SNA of any one of claims 1-33, wherein the immunostimulatory oligonucleotide comprises a CpG nucleotide sequence.
35. The SNA of any one of claims 1-34, wherein one or more oligonucleotides in the oligonucleotide shell comprises or consists of the sequence of 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 39).
36. The SNA of any one of claims 1-35, wherein one or more oligonucleotides in the oligonucleotide shell comprises or consists of the sequence of 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 40).
37. The SNA of any one of claims 1-36 wherein one or more oligonucleotides in the oligonucleotide shell comprises 5' -tccatgacgttctctctgaccgtt (spacer-18 (hexaethylene glycol)) 2 The sequence of cholesterol-3' (SEQ ID NO: 41) or consists thereof.
38. The SNA of any one of claims 1-37 wherein one or more oligonucleotides in the oligonucleotide shell comprises 5' -tcgtcgttttgtcgttttttgtcgtt (spacer-18 (hexaethylene glycol)) 2 The sequence of cholesterol-3' (SEQ ID NO: 6) or consists thereof.
39. The SNA of any one of claims 1-38, wherein at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the oligonucleotides in the oligonucleotide shell are immunostimulatory oligonucleotides.
40. The SNA of any of claims 1-39 wherein the linker is an alkylene carbamate disulfide linker, a thiol linker, a disulfide linker, an alkylene amide thiosuccinimidyl linker, or a combination thereof.
41. The SNA of any of claims 1-40, wherein the nanoparticle core is a micelle, liposome, polymer, lipid Nanoparticle (LNP), or a combination thereof.
42. The SNA of claim 41, wherein the polymer is a polylactide, a polylactide-polyglycolide copolymer, a polycaprolactone, a polyacrylate, an alginate, albumin, silica, polypyrrole, polythiophene, polyaniline, polyethyleneimine, poly (methyl methacrylate), or chitosan.
43. The SNA of claim 42 wherein the polymer is poly (lactic-co-glycolic acid) (PLGA).
44. The SNA of any of claims 1-43, wherein the nanoparticle core is a liposome.
45. The SNA of claim 44 wherein the liposome comprises a lipid selected from the group consisting of: 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1, 2-distearoyl-sn-glycero-3-phosphoric acid- (1 '-rac-glycerol) (DSPG), 1, 2-dioleoyl-sn-glycero-phosphoric acid- (1' -rac-glycerol) (DOPG), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-di- (9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dicetyl-sn-glycero-3-phosphoethanolamine (DPPE), and cholesterol.
46. The SNA of any one of claims 1-45, wherein one or more oligonucleotides in the oligonucleotide shell are attached to the outer surface of the nanoparticle core by a lipid anchoring group.
47. The SNA of claim 46, wherein the lipid anchoring group is linked to the 5 'end or the 3' end of the one or more oligonucleotides.
48. The SNA of claim 46 or claim 47, wherein the lipid anchoring group is tocopherol or cholesterol.
49. The SNA of any of claims 1-48 wherein one or more oligonucleotides in the oligonucleotide shell are modified at their 5 'and/or 3' ends with Dibenzocyclooctyl (DBCO).
50. The SNA of any one of claims 1-49 wherein one or more oligonucleotides in the oligonucleotide shell are thiol-modified at their 5 'and/or 3' ends.
51. The SNA of any one of claims 1-50 wherein the oligonucleotide shell comprises a DNA oligonucleotide, an RNA oligonucleotide, or a combination thereof.
52. The SNA of any one of claims 1-51 wherein the oligonucleotide shells comprise DNA oligonucleotides and RNA oligonucleotides.
53. The SNA of any one of claims 1-52, wherein the oligonucleotide shell comprises single stranded DNA, double stranded DNA, single stranded RNA, double stranded RNA, or a combination thereof.
54. The SNA of any one of claims 1-53 wherein one or more oligonucleotides in the oligonucleotide shell are modified oligonucleotides.
55. The SNA of any one of claims 1-54 wherein the oligonucleotide shells comprise from about 2 to about 200 oligonucleotides.
56. The SNA of any one of claims 1-55 wherein the oligonucleotide shell comprises from about 2 to about 100 oligonucleotides.
57. The SNA of any one of claims 1-55 wherein the oligonucleotide shell comprises about 150 oligonucleotides.
58. The SNA of any one of claims 1-55 wherein the oligonucleotide shell comprises about 200 oligonucleotides.
59. The SNA of claim 55 or claim 56 wherein the oligonucleotide shells comprise from about 10 to about 80 oligonucleotides.
60. The SNA of claim 55, claim 56, or claim 59, wherein the oligonucleotide shell comprises about 75 oligonucleotides.
61. The SNA of any one of claims 1-60 wherein each oligonucleotide in the oligonucleotide shell is about 5 to about 1000 nucleotides in length.
62. The SNA of claim 61 wherein each oligonucleotide in the oligonucleotide shell is about 10 to about 50 nucleotides in length.
63. The SNA of claim 61 or claim 62, wherein each oligonucleotide in the oligonucleotide shell is about 20 to about 30 nucleotides in length.
64. The SNA of any of claims 1-63 wherein the SNA has a diameter of from about 1 nanometer (nm) to about 500nm.
65. The SNA of any of claims 1-64 wherein the SNA has a diameter of less than or equal to about 80 nanometers.
66. The SNA of any of claims 1-65 wherein the SNA has a diameter of less than or equal to about 50 nanometers.
67. The SNA of any one of claims 1-66, wherein the oligonucleotide shell comprises a targeting oligonucleotide, an inhibitory oligonucleotide, a non-targeting oligonucleotide, or a combination thereof.
68. The SNA of claim 67 wherein the inhibitory oligonucleotide is an antisense oligonucleotide, small interfering RNA (siRNA), aptamer, short hairpin RNA (shRNA), enzymatic DNA, or enzymatic aptamer.
69. A composition comprising a plurality of SNAs according to any one of claims 1-68.
70. The composition of claim 69, wherein at least two SNAs of the plurality of SNAs comprise different nanoparticle cores.
71. A pharmaceutical formulation comprising a plurality of SNAs according to any one of claims 1 to 68, or a composition according to claim 69 or claim 70, and a pharmaceutically acceptable carrier or diluent.
72. A vaccine comprising the SNA of any one of claims 1-68, the composition of claim 69 or claim 70, or the pharmaceutical formulation of claim 71.
73. The vaccine of claim 72, comprising an adjuvant.
74. An antigen composition comprising the SNA of any one of claims 1-68, or the pharmaceutical formulation of claim 71, in a pharmaceutically acceptable carrier, diluent, stabilizer, or preservative form, wherein the antigen composition is capable of generating an immune response in a subject comprising antibody production, cytotoxic T cell activation, helper T cell activation, or a protective immune response.
75. The antigen composition of claim 74, wherein the immune response comprises an antibody response.
76. The antigen composition of claim 75, wherein the antibody response is a neutralizing antibody response or a protective antibody response.
77. A method of inhibiting expression of a gene product, the method comprising hybridizing a polynucleotide encoding the gene product to the inhibitory oligonucleotide according to claim 67 or claim 68, wherein hybridization between the polynucleotide and the inhibitory oligonucleotide occurs over a length of the polynucleotide that is complementary to a degree sufficient to inhibit expression of the gene product.
78. The method of claim 77, wherein expression of said gene product is inhibited in vivo or in vitro.
79. A method of generating an immune response in a subject, the method comprising administering to the subject an effective amount of the SNA of any one of claims 1-68, the composition of claim 69 or claim 70, the pharmaceutical formulation of claim 71, the vaccine of claim 72 or claim 73, or the antigen composition of any one of claims 74-76, thereby generating an immune response in the subject.
80. The method of claim 79, wherein the immune response comprises an antibody response.
81. The method of claim 80, wherein the antibody response is a total antigen-specific antibody response.
82. The method of claim 80, wherein the antibody response is a neutralizing antibody response or a protective antibody response.
83. A method of immunizing a subject against one or more antigens, the method comprising administering to the subject an effective amount of SNA of any one of claims 1-68, composition of claim 69 or claim 70, pharmaceutical formulation of claim 71, vaccine of claim 72 or 73, or antigen composition of any one of claims 74-76, thereby immunizing the subject against the one or more antigens.
84. The method of claim 83, wherein the composition or the vaccine is a cancer vaccine.
85. A method of treating cancer, the method comprising administering to a subject an effective amount of SNA according to any one of claims 1-68, composition according to claim 69 or claim 70, pharmaceutical formulation according to claim 71, vaccine according to claim 72 or 73, or antigen composition according to any one of claims 74-76, thereby treating the cancer in the subject.
86. The method of claim 84 or claim 85, wherein the cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, endometrial cancer, glioblastoma, renal cancer, leukemia, liver cancer, lung cancer, melanoma, lymphoma, non-hodgkin's lymphoma (non-hodgkin's lymphoma), bone cancer, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, and human papillomavirus-induced cancer, or a combination thereof.
87. The method of claim 86, wherein the cancer is melanoma.
88. The method of claim 86 or claim 87, wherein the cancer is colon cancer.
89. The method of any one of claims 86-88, wherein the cancer is lymphoma.
90. The method of any one of claims 79 to 89, further comprising administering an additional agent.
91. The method of claim 90, wherein the additional agent is an anti-apoptosis protein 1 (PD-1) antibody, an anti-apoptosis ligand 1 (PD-L1) antibody, a cytotoxic T lymphocyte antigen 4 (CTLA-4) antibody, a T cell immunoglobulin, and an ITIM domain (TIGIT) antibody, or a combination thereof.
92. The method of any one of claims 77-91, wherein the SNA is the SNA of claim 5.
93. The method of any one of claims 77-91, wherein the SNA is the SNA of claim 10.
CN202280029963.7A 2021-03-30 2022-03-30 Targeting multiple T cell types using spherical nucleic acid vaccine architecture Pending CN117157104A (en)

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